DEVELOPING BRAIN IN DANGER : CRITICAL

PERIODS OF VULNERABILITY FROM IN-UTERO

TO ADOLESCENCE

EDITED BY : Carla Cannizzaro, Miriam Melis and Sophie Laye

PUBLISHED IN: F
 rontiers in Neuroscience, Frontiers in Genetics and
Frontiers in Behavioral Neuroscience
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Frontiers in Neuroscience 1 February 2022 | Developing Brain in Danger : Critical Periods

 DEVELOPING BRAIN IN DANGER : CRITICAL
PERIODS OF VULNERABILITY FROM IN-UTERO
TO ADOLESCENCE

Topic Editors:
Carla Cannizzaro, University of Palermo, Italy
Miriam Melis, University of Cagliari, Italy
Sophie Laye, INRA Centre Bordeaux-Aquitaine, France

Citation: Cannizzaro, C., Melis, M., Laye, S., eds. (2022). Developing Brain in
Danger : Critical Periods of Vulnerability From In-utero to Adolescence.
Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88974-617-0

Frontiers in Neuroscience 2 February 2022 | Developing Brain in Danger : Critical Periods

 Table of Contents
05 Exposure of Developing Male Rats to One or Multiple Noise Sessions and
Different Housing Conditions: Hippocampal Thioredoxin Changes and
Behavioral Alterations
Sonia Jazmín Molina, Gustavo Ezequiel Buján,
Monserrat Rodriguez Gonzalez, Francisco Capani,
Maria Eugenia Gómez-Casati and Laura Ruth Guelman
24 Δ9-Tetrahydrocannabinol During Adolescence Attenuates Disruption of
Dopamine Function Induced in Rats by Maternal Immune Activation
Salvatore Lecca, Antonio Luchicchi, Maria Scherma, Paola Fadda,
Anna Lisa Muntoni and Marco Pistis
32 Folic Acid and Risk of Preterm Birth: A Meta-Analysis
Bingbing Li, Xiaoli Zhang, Xirui Peng, Shan Zhang, Xiaoyang Wang and
Changlian Zhu,
46 Sex-Dimorphic Interactions of MAOA Genotype and Child Maltreatment
Predispose College Students to Polysubstance Use
Paula J. Fite, Shaquanna Brown, Waheeda A. Hossain, Ann Manzardo,
Merlin G. Butler and Marco Bortolato
56 Environmental Tobacco Smoke During the Early Postnatal Period of Mice
Interferes With Brain 18F-FDG Uptake From Infancy to Early
Adulthood – A Longitudinal Study
Larissa Helena Torres, Caroline Cristiano Real, Walter Miguel Turato,
Lídia Wiazowski Spelta, Ana Carolina Cardoso dos Santos Durão,
Tatiana Costa Andrioli, Lorena Pozzo, Peterson Lima Squair, Marco Pistis,
Daniele de Paula Faria and Tania Marcourakis
67 Targeting the Stress System During Gestation: Is Early Handling a
Protective Strategy for the Offspring?
Valentina Castelli, Gianluca Lavanco, Anna Brancato and Fulvio Plescia
79 Expression of Behavioral Phenotypes in Genetic and Environmental
Mouse Models of Schizophrenia
Razia Sultana and Charles C. Lee
91 Gender Differences in the Outcome of Offspring Prenatally Exposed to
Drugs of Abuse
Francesco Traccis, Roberto Frau and Miriam Melis
105 Environmental Enrichment During Adolescence Mitigates Cognitive
Deficits and Alcohol Vulnerability due to Continuous and Intermittent
Perinatal Alcohol Exposure in Adult Rats
Anna Brancato, Valentina Castelli, Gianluca Lavanco and Carla Cannizzaro
119 Hypothalamic Gene Expression and Postpartum Behavior in a Genetic Rat
Model of Depression
Wendy Luo, Patrick H. Lim, Stephanie L. Wert, Stephanie A. Gacek,
Hao Chen and Eva E. Redei
130 Postnatal Antioxidant and Anti-inflammatory Treatments Prevent Early
Ketamine-Induced Cortical Dysfunctions in Adult Mice
Maria Bove, Paolo Tucci, Stefania Dimonte, Luigia Trabace,
Stefania Schiavone and Maria Grazia Morgese

Frontiers in Neuroscience 3 February 2022 | Developing Brain in Danger : Critical Periods

 142 Oculomotor Behavior as a Biomarker for Differentiating Pediatric Patients
With Mild Traumatic Brain Injury and Age Matched Controls
Melissa Hunfalvay, Nicholas P. Murray, Claire-Marie Roberts, Ankur Tyagi,
Kyle William Barclay and Frederick Robert Carrick
151 Folic Acid Fortification Prevents Morphological and Behavioral
Consequences of X-Ray Exposure During Neurulation
Kai Craenen, Mieke Verslegers, Zsuzsanna Callaerts-Vegh, Livine Craeghs,
Jasmine Buset, Kristof Govaerts, Mieke Neefs, Willy Gsell, Sarah Baatout,
Rudi D’Hooge, Uwe Himmelreich, Lieve Moons and
Mohammed Abderrafi Benotmane
172 Ontogenetic Oxycodone Exposure Affects Early Life Communicative
Behaviors, Sensorimotor Reflexes, and Weight Trajectory in Mice
Elena Minakova, Simona Sarafinovska, Marwa O. Mikati, Kia M. Barclay,
Katherine B. McCullough, Joseph D. Dougherty, Ream Al-Hasani and
Susan E. Maloney
186 The Long-Term Effects of Neonatal Inflammatory Pain on Cognitive
Function and Stress Hormones Depend on the Heterogeneity of the
Adolescent Period of Development in Male and Female Rats
Irina P. Butkevich, Viktor A. Mikhailenko, Elena A. Vershinina and
Gordon A. Barr
203 The Impact of Adolescent Alcohol Exposure on Nicotine Behavioral
Sensitization in the Adult Male Neonatal Ventral Hippocampal Lesion Rat
Emily D. K. Sullivan, Liam N. Locke, Diana J. Wallin, Jibran Y. Khokhar,
Elise M. Bragg, Angela M. Henricks and Wilder T. Doucette,
214 High-Salt Diet in the Pre- and Postweaning Periods Leads to Amygdala
Oxidative Stress and Changes in Locomotion and Anxiety-Like Behaviors
of Male Wistar Rats
Pedro Ernesto de Pinho Tavares Leal, Alexandre Alves da Silva,
Arthur Rocha-Gomes, Tania Regina Riul, Rennan Augusto Cunha,
Christoph Reichetzeder and Daniel Campos Villela

Frontiers in Neuroscience 4 February 2022 | Developing Brain in Danger : Critical Periods

 ORIGINAL RESEARCH
published: 13 August 2019
doi: 10.3389/fnbeh.2019.00182

Exposure of Developing Male Rats to

One or Multiple Noise Sessions and
Different Housing Conditions:
Hippocampal Thioredoxin Changes
and Behavioral Alterations
Sonia Jazmín Molina 1 , Gustavo Ezequiel Buján 1,2 , Monserrat Rodriguez Gonzalez 2 ,
Francisco Capani 3,4 , Maria Eugenia Gómez-Casati 5 and Laura Ruth Guelman 1,2 *
1
Centro de Estudios Farmacológicos y Botánicos (CEFyBO, UBA-CONICET), Facultad de Medicina, Consejo Nacional de
Investigaciones Científicas y Técnicas, Universidad de Buenos Aires, Buenos Aires, Argentina, 2 Facultad de Medicina,
Cátedra de Farmacología, Universidad de Buenos Aires, Buenos Aires, Argentina, 3 Consejo Nacional de Investigaciones
Científicas y Técnicas, Instituto de Investigaciones Cardiológicas (ININCA, UBA-CONICET), Facultad de Medicina,
Universidad de Buenos Aires, Buenos Aires, Argentina, 4 Instituto de Ciencias Biomédicas, Facultad de Ciencias de la Salud,
Universidad Autónoma de Chile, Santiago de Chile, Chile, 5 Facultad de Medicina, Instituto de Farmacología, Universidad de
Buenos Aires, Buenos Aires, Argentina

Exposure of developing rats to noise has shown to induce hippocampal-related

behavioral alterations that were prevented after a week of housing in an enriched
environment. However, neither the effect of repeated exposures nor its impact on key
endogenous antioxidants had been studied yet. Thus, the aim of the present work
was to reveal novel data about hippocampal oxidative state through the measurement
Edited by:
of possible age-related differences in the levels of hippocampal thioredoxins in rats
Sophie Laye, exposed to noise at different developmental ages and subjected to different schemes
INRA Centre Bordeaux-Aquitaine, and housing conditions. In addition, the possibility that oxidative changes could underlie
France
hippocampal-related behavioral changes was also analyzed. Developing male Wistar rats
Reviewed by:
Eddy A. Van Der Zee, were exposed to noise for 2 h, either once or for 5 days. Upon weaning, some animals
University of Groningen, Netherlands were transferred to an enriched cage for 1 week, whereas others were kept in standard
Eleni Paizanis,
cages. One week later, auditory and behavioral assessments, as well as measurement of
University of Caen Normandy, France
hippocampal thioredoxin, were performed. Whereas no changes in the auditory function
*Correspondence:
Laura Ruth Guelman were observed, significant behavioral differences were found, that varied according to
[email protected] the age, scheme of exposure and housing condition. In addition, a significant increase in
Trx-1 levels was found in all noise-exposed groups housed in standard cages. Housing
Received: 03 May 2019
Accepted: 23 July 2019 animals in an enriched environment for 1 week was effective in preventing most of
Published: 13 August 2019 these changes. These findings suggest that animals become less susceptible to undergo
Citation: behavioral alterations after repeated exposure to an environmental challenge, probably
Molina SJ, Buján GE, Rodriguez
Gonzalez M, Capani F, Gómez-Casati
due to the ability of adaptation to an unfavorable condition. Moreover, it could be
ME and Guelman LR hypothesized that damage to younger individuals could be more easily prevented by
(2019) Exposure of Developing Male a housing manipulation.
Rats to One or Multiple Noise
Sessions and Different Housing Keywords: noise, thioredoxin, behavior, hippocampus, enriched environment
Conditions: Hippocampal Thioredoxin
Changes and Behavioral Alterations. Abbreviations: HC, Hippocampus; PND7, Rats exposed to noise at 7 days of age; PND15, Rats exposed to noise at
Front. Behav. Neurosci. 13:182. 15 days of age; N1/N5, Exposure schemes: 1 day and 5 days, respectively; St, Standard cage; EE, Enriched environment;
doi: 10.3389/fnbeh.2019.00182 Trx-1, Trx-2, Thioredoxin-1 and Thioredoxin-2, respectively; CNS, Central Nervous System.

Frontiers in Behavioral Neuroscience | www.frontiersin.org 51 August 2019 | Volume 13 | Article 182

Molina et al. Behavioral Alterations in Noise-Exposed Developing Rats

INTRODUCTION It should be highlighted that brain is more susceptible to

oxidative damage when compared with other tissues for different
Data from the literature have shown that exposure to noise could reasons. First, it consumes higher oxygen amounts; second, it
be capable to induce damage to the auditory system (Frenzilli has more iron content; third, it has high levels of unsaturated
et al., 2004; Gourévitch et al., 2014) as well as to structures fatty acids and finally, it has lower activities of antioxidant
of different extra-auditory tissues, such as brain structures enzymes such as superoxide dismutase and catalase. The high
(prefrontal cortex and hippocampus), cardiac tissues or adrenal vulnerability can be observed after hypoxia (Romero et al., 2015;
and thyroid glands (Trapanotto et al., 2004; Manikandan et al., Ten and Starkov, 2012), ionizing radiation exposure (Caceres
2006; Uran et al., 2010; Gannouni et al., 2013; Molina et al., 2016a; et al., 2009, 2010) and different nervous system disorders (Chen
Miceli et al., 2018). However, whereas exposure to occupational et al., 2010; Ma et al., 2012). Of importance, it has been reported
noise seems to be one of the main causes of disabling hearing that an environmental threat such as noise was able to induce an
loss, limited data are available concerning the effects of noise oxidative imbalance in different tissues (Cassarino and Bennett,
exposure on everyday lives of the ordinary population (Kopke 1999; Sathyasaikumar et al., 2007; Samson et al., 2008; Uran et al.,
et al., 2007). Actually, people living in big cities should be aware 2010, 2012; Massaad and Klann, 2011; Molina et al., 2016b).
that they might be involuntarily exposed to high levels of noise A study of Zheng and Ariizumi (2007) showed an increase in
coming from different sources. The urban traffic, the use of noisy oxidative stress and a suppression of the immune function after
household appliances or the attendance to concerts venues and noise exposure during 28 days, whereas Cheng et al. (2011)
discotheques might be examples of some of the many health- found that only 1 week of moderate noise was capable to induce
threatening environments. oxidative stress in different structures of mice brain. Cui and Li
It is well known that several environmental challenges (2013) reported an increase in brain oxidative stress, as well as
increase the production of reactive oxygen species (ROS) alterations of spatial memory in adult animals exposed to noise.
in different tissues, which may overwhelm the endogenous Finally, several behavioral and biochemical changes were found
antioxidant defenses and trigger a disturbance in the redox in extra-auditory tissues of noise-exposed animals, including
homeostasis (Erkal et al., 2006; Halliwell, 2006). In particular, impairment of hippocampal-dependent reference and working
it has been reported that exposure to noise was able to induce spatial memory as well as changes in hippocampal antioxidant
changes in the cochlear oxidative state (Yamane et al., 1995; enzymes activities (Manikandan et al., 2006; Rabat et al., 2006)
Yamasoba et al., 1998; Dehne et al., 2000; Yamashita et al., 2004; and a decrease in the number of hippocampal neurons (Jáuregui-
Fetoni et al., 2015). In addition, Ohlemiller et al. (1999) reported Huerta et al., 2011).
a significant increase in ROS cochlear levels 1 h after exposure Thioredoxins (Trx) are part of an endogenous family of
to noise, even when the acoustic stimulus is no longer present oxido-reductases, recognized as the major reductant among a
and Tamura et al. (2012) found that oxidative stress might be variety of antioxidant enzymes (Lillig and Holmgren, 2007; Lillig
induced in the Corti organ of the inner ear after noise exposure et al., 2008; Romero et al., 2015). Even though the Trx family
in a rodent animal model. Finally, Kurioka et al. (2014) reported includes various proteins, the main Trx isoforms are the cytosolic
an increase in mitochondrial ROS production and excitotoxicity Trx-1 and the mitochondrial Trx-2 (Lillig et al., 2008; Aon-
in the cochlea of rats exposed to noise. Bertolino et al., 2011; Godoy et al., 2011). Trx-1 is a regulator
ROS are unstable molecular species that contain one or more of cellular functions that take place in response to redox signals
unpaired electrons that make them highly reactive (Halliwell, and modulates various signaling pathways. Different literature
1992). Hydrogen peroxide (H2 O2 ), superoxide anion (O2 •− ) data show an increase in Trx-1 when an oxidative imbalance
or hydroxyl radicals (OH• ) are ROS that have the ability is induced in different nervous areas of animals subjected to
to damage cellular lipids, proteins and to mitochondrial and neonatal hypoxia (Romero et al., 2015), intended to maintain a
nuclear genome through oxidative mechanisms, leading to reduced environment to protect cells and tissues from oxidative
mutations and cellular death (Halliwell and Gutteridge, 1991; damage (Silva-Adaya et al., 2014). In addition, Cunningham
Halliwell, 1992, 2006; Harman, 1992; Uttara et al., 2009; Massaad et al. (2015) showed that Trx-1 overexpression extended lifespan
and Klann, 2011; Hanschmann et al., 2013). Although these of transgenic mice by protecting against oxidative stress and
species are persistently generated during aerobic respiration as Wu et al. (2015) found that a treatment with Trx-1 siRNA
derivatives of redox reactions and considering that even low induced behavioral deficits. Therefore, it could be hypothesized
amounts are required to regulate certain signaling pathways, that under physiological conditions the balance between
an imbalance between the production of ROS and the system ROS generation and antioxidant activity is highly controlled.
of endogenous antioxidants (i.e., a disproportionate increase in However, when an injury is going on, an activation of the
ROS levels and/or excessive decrease in antioxidant enzymes endogenous antioxidant defense systems can primarily occur as
activities) might lead to cell damage (Jones, 2006). In fact, an attempt to counteract the oxidative process. Nevertheless, the
although the classic definition of oxidative stress focuses on endogenous antioxidant system often can fail in restoring redox
an imbalance between pro- and anti-oxidative molecules in a homeostasis and the defense activity might result insufficient to
given structure, at present this definition has been approached prevent damage.
to a new concept in which oxidative stress is defined as Last, a non-pharmacological neuroprotective strategy, the
the disruption of normally occurring redox signaling events enriched environment (Laviola et al., 2008) has shown to be an
(Jones, 2006). effective tool that could be protective against different central

Frontiers in Behavioral Neuroscience | www.frontiersin.org 62 August 2019 | Volume 13 | Article 182

Molina et al. Behavioral Alterations in Noise-Exposed Developing Rats

nervous system (CNS) injuries (Lores-Arnaiz et al., 2006). It (PND) 0. In average, 10 pups per litter were delivered and only
consists of a cage larger than the standard, which contains male rats (usually 4–6 per litter) were used for the different
different toys, ramps and wheels. Although we have reported that experimental procedures.
EE was able to prevent noise-induced behavioral alterations in To prevent from litter effects, no more than one animal from
PND28 animals exposed at PND7 and PND15 to noise for 2 h each litter was used to measure each parameter.
(Molina et al., 2016a), data in animals exposed for 5 days have After behavioral and auditory experiments at PND28, animals
not been obtained yet. were euthanized under a CO2 chamber for final disposal. Those
Unfortunately, data obtained from developing animals animals assigned to western blot experiments were sacrificed
exposed to noise are very scarce in the literature. The results through guillotine decapitation, the brain was exposed and the
from our laboratory showed different behavioral, biochemical hippocampus was subsequently dissected.
and histological alterations when immature rats were exposed PND7 and PND15 littermates were randomly assigned to four
to noise. In addition, housing in an enriched environment has experimental groups: sham (control) at PND7, sham (control)
demonstrated to be an effective neuroprotective tool when at PND15, noise-exposed at PND7 and noise-exposed at PND15
rats were exposed to noise for a single day (Molina et al., (n = 84 each group). In turn, within each group, animals received
2016a). However, a comparison between the effects of single one of the following exposure schemes: single (N1) or five
or repeated exposures to noise, at different developmental consecutive daily sessions (N5; n = 42 each group). Finally,
ages and/or housing conditions, as well as a possible each subgroup was divided into standard (St) or enriched (EE)
relationship with the hippocampal oxidative state, has not cages housing, conforming 16 experimental groups (n = 21 each
been made yet. group). To reduce confounding factors, animals within each
Thus, the main hypothesis was that hippocampal thioredoxins group were randomly assigned to the different measurements,
might be responsible, at least in part, of the behavioral being different those animals used for behavioral experiments
changes induced in developing rats after exposure to noise. (with some rats performing two behavioral tests, usually seven for
Therefore, the aim of the present work was to reveal novel data OF and elevated plus maze (EPM) and other seven animals for
about hippocampal oxidative state through the measurement IA) western blot experiments (four rats for each group) and
of possible age-related differences in the levels of hippocampal auditory assessment (three rats for each group). Figure 1 depicts
Trx-1 and Trx-2, the major members of the thioredoxin family the experimental groups used.
of endogenous antioxidants, in animals exposed to noise at All littermates were kept with their dams until weaning,
7 and 15 days according to different schemes. In addition, the at 21 days of age. Then, rats were separated and were put
possibility that oxidative changes could underlie hippocampal- in groups of 2–3 in standard and 3–4 in enriched cage for
related behavioral changes was also analyzed. Finally, the impact 1 week with food and water ad libitum, on 12 h light-dark
of housing conditions on noise-induced changes was additionally cycles (lights on at 7 A.M.) at 21 ± 2◦ C and mashed cornflower
evaluated. To discard hearing alterations, the auditory pathway for bedding.
function was assessed. Animals were handled and sacrificed according to the
Institutional Committee for the Use and Care of Laboratory
MATERIALS AND METHODS Animal rules (CICUAL, School of Medicine, University of
Buenos Aires, Argentina). The present experimental protocol
Animals was approved by this Committee and registered with the number
Healthy male and female albino Wistar rats were obtained 53679/16. The CICUAL adheres to the rules of the ‘‘Guide for
from the animal facilities of the Biochemistry and Pharmacy the Care and Use of Laboratory Animals’’ (NIH; 2011 revision)
School, University of Buenos Aires, Argentina. A total of and to the EC Directive 86/609/EEC (2010 revision) for animal
30 multiparous females and 10 males were used for mating experiments.
procedures. Pregnant rats were isolated and left undisturbed To avoid circadian rhythm alterations, noise exposures were
until delivery. The day of birth was designated as postnatal day performed in the intermediate phase of the light cycle, between

FIGURE 1 | Experimental design. Sham: non-exposed animals; N1: single noise exposure; N5: five-daily noise exposure. St: standard housing. EE: enriched
environment.

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Molina et al. Behavioral Alterations in Noise-Exposed Developing Rats

10 A.M. and 2 P.M. All experiments were performed in 2 days to ensure continued novelty. Rats were maintained
PND28 animals. in their housing condition (St or EE) for 1 week, prior to
behavioral studies.
Noise Exposure
For this procedure, the computer software TrueRTA was chosen Auditory Pathway Assessment (ABR)
to produce white noise, using a bandwidth from 20 Hz to The auditory brainstem responses (ABRs) are sound-evoked
20,000 Hz in octave bands. For sound amplification, an active potentials generated by neuronal circuits in the ascending
2 way monitor (SKP, SK150A, 40 W RMS per channel) was auditory pathways and consequently require functional integrity
used, located 30 cm above the animal cage, placed in an of hair cells, as well as their afferent neurons.
‘‘ad hoc’’ wooden sound chamber of 1 m × 1 m × 1 m fitted PND28 animals were anesthetized with ketamine (100 mg/kg,
with a ventilated top as reported by Cui et al. (2009). Before i.p.) and xylazine (20 mg/kg, i.p.) and placed in an acoustically
exposure, noise intensity was measured with an omnidirectional electrically shielded chamber maintained at 30◦ C. Methods for
measurement condenser microphone (Behringer ECM 8000) by measuring ABRs were essentially as described (Kujawa and
positioning the microphone in the sound chamber at several Liberman, 2009; Maison et al., 2013). Briefly, acoustic stimuli
locations and taking an average of the different readings. Animals were delivered through an acoustic system consisting of two
were kept in their home cages and the entire litter was assigned miniature dynamic earphones used as sound sources and an
to the same group so that they were not handled throughout the electret condenser microphone coupled to a probe tube to
exposure period. Sham animals were placed in the same chamber, measure sound pressure near the eardrum. Digital stimulus
but without being exposed to noise. Given that experimental generation and response processing were handled by digital I-O
animals were still being breastfed and mothers had to be boards from National Instruments driven by custom software
transiently removed for the period in which the pups were being written in LabVIEW. ABRs were recorded with needle electrodes
exposed to noise, this action was carried out also in non-exposed inserted at vertex and pinna with a ground reference near the tail.
sham animals in order to discard possible changes that could be Auditory responses were evoked with 5 ms tone pips, amplified
attributed to mother separation. (10,000×), filtered to six different frequencies (0.1–3 kHz), and
Based on previous publications of our laboratory (Uran acquired on a computer. The sound level was raised in 10 dB steps
et al., 2010) with further modifications (Molina et al., 2016a), and ‘‘threshold’’ was defined as the lowest SPL level at which a
PND7 and PND15 animals were exposed for 2 h to white noise at wave is detected.
95–97 dB SPL (20–20,000 Hz), either a single day (N1) or for five To avoid potential data misinterpretation, animals assigned
consecutive days (N5). Background noise level ranged between to ABR assessments were not subjected to further behavioral or
50 and 55 dB SPL, being within the harmless interval suggested biochemical evaluations and were euthanized in a CO2 chamber
by the WHO guidelines (NIOSH, 1998) and by others (Campeau for final disposal.
et al., 2002; Sasse et al., 2008). Lighting was provided by a 20 W
lamp located in the upper left corner of the sound chamber. In Behavioral Assessment
addition, the chamber had a sound attenuation system made with PND28 animals were used for all behavioral experiments. To
CelotexTM . control for variables that could significantly alter physiological
The intensity and duration of noise used in the present work and behavioral indicators of stress (Walf and Frye, 2007), animals
were chosen considering its potential translational value, as it remained in their home cage and placed in a separate area
could be comparable to the intensity and duration perceived of the main housing room for 30 min prior to the behavioral
in various workplaces, mainly induced by different machines, assessments. Thereafter, they were individually housed for 5 min
data that can be found even in the earliest WHO report in the same area and finally were taken to the adjacent
(WHO, 1999). testing room, which had identical environmental conditions, for
additional 3 min to complete the acclimation period, prior to the
Enriched Environment (EE) behavioral assessments.
At weaning (PND21), a subset of animals was housed in an
EE with 3–4 animals residing together whereas a subset of Open Field Task (OF)
2–3 was accommodated in standard cages (St). In contrast to An open field device was used to analyze habituation memory
St, conventional top-wired, stainless steel rectangular cages of and exploratory activity, behaviors known to depend on the
40 cm × 25 cm × 16 cm, EE consisted of 54 cm × 40 cm × 41 cm hippocampus (Vianna et al., 2000; Barros et al., 2006). In this
plastic cages with two levels, containing two connecting ramps. task, the reduction of locomotor activity triggered by a repeated
Different plastic toys and tunnels, as well as a running exposure to the same environment can be taken as a measure
wheel, were placed in the cage. A palatable food, such as of preservation of habituation memory (Vianna et al., 2000;
Froot Loopsr , was added regularly in small quantities in Pereira et al., 2011). In addition, the activity in the first session
addition to the conventional balanced food. It should be of the OF can be used to assess changes in emotionality induced
highlighted that the minimal sugar and fat amounts of the by exposure to a novel environment. In consequence, vertical
Froot Loopsr offered are much below those contained in a exploratory activity can be quantified by recording the number of
‘‘cafeteria diet,’’ known to induce per se metabolic and behavioral rearing and climbing, holding on the hind legs. The activity was
changes (Zeeni et al., 2015). The objects were changed every recorded using a camcorder (Handycam DCR-DVD810, Sony).

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Molina et al. Behavioral Alterations in Noise-Exposed Developing Rats

- Apparatus: OF device consists of a 50 cm × 50 cm × 50 cm of associative memory (Ennaceur and Delacour, 1988; Izquierdo
wooden box, with a floor divided into 25 equal squares by and Medina, 1997).
black lines.
- Apparatus: the apparatus consists of a box
- First session: rats were withdrawn from the cage, placed on
(60 cm × 60 cm × 40 cm), divided into two compartments:
the center rear quadrant of the OF box and allowed to freely
one is illuminated while the other is equipped with a
explore the box for 5 min. The number of crossed lines as well
removable cover to allow it to be dark, as described by
as the number of rearing and climbing, were recorded over
Roozendaal (2002). A removable partition divides the two
the session.
compartments. The floor of the dark compartment consists
- Second session: after 1 h inter-trial in their home cages,
of a stainless steel grid at the bottom, through which a
animals were acclimatized to the behavioral room and allowed
continuous current could be delivered.
to explore the apparatus for another 5 min. The number of
- Habituation session: the rat was placed into the lit box and
crossed lines was recorded and compared with the number
allowed to freely explore the apparatus. Either after passing
crossed in the first session to evaluate habituation to the device
three times to the dark side or after 3 min staying in the dark
(Barros et al., 2000).
side, the rat was removed from the apparatus. After 10 min, the
Elevated Plus Maze (EPM) rat was placed again in the lit side and when it entered the dark
This task was used to evaluate anxiety-related behaviors that side, the doors were closed and the rat was retained for 10 s on
depend on the integrity of the hippocampus (Montgomery, 1955; this side.
Brenes et al., 2009; Violle et al., 2009). - Training session (T1): after 1 h, each rat was placed in the lit
Anxiety-related behaviors are calculated as the number of compartment, facing away from the dark compartment; the
entries to the open arms as well as the latency required to access latency to move into the dark compartment was recorded.
the open arms. When an increase in the first and a decrease When the rat stepped with all four paws in the dark
in the latter are observed, it could be stated that a decrease in compartment, a foot shock (1.2 mA, 2 s) was delivered. The
anxiety-like behaviors could have occurred. rat was quickly removed from the apparatus and returned to
Additionally, some ethological parameters can be evaluated its home cage.
using this task (Carobrez and Bertoglio, 2005), designated as - Retention session (T2): retention test was performed 1 h after
risk assessment behavior because they have been associated the training session by following a similar procedure, except
to detection and analysis of threats or threatening situations for the fact that no footshock was delivered. The ratio between
(Rodgers and Cole, 1993). One of these parameters is called the latency to move into the dark compartment in the retention
head dipping (HD). As closed arms and center platform and the training sessions (T2 and T1, respectively) was taken
were designated as ‘‘protected’’ areas (i.e., offering relative as a measure of associative memory retention (T2/T1).
security), the percentage of head-dipping in closed arms (%HD
in closed arms) was calculated as the percentage of these
Western Blot Experiments
behaviors displayed in or from the protected areas. Therefore,
The levels of the Trx-1 and Trx-2 were determined in
this parameter describes the action of the animal when it is
hippocampal homogenates of rats from all experimental
positioned on a closed arm and, at the junction with the open
groups through Western blot experiments. To prevent from
arm, stretches the head over the ledge of an open arm and
confounding influences, those animals destined to western blot
bends down.
experiments were not previously used for behavioral or auditory
- Apparatus: the wooden apparatus consists of four arms of measurements. Animals were euthanized through guillotine
equal dimensions (50 cm × 10 cm) and raises 50 cm above decapitation, brain was exposed, and hippocampus dissected.
the floor. Two arms, enclosed by walls 40 cm high, are Briefly, tissues were homogenized in ice-cold lysis buffer (25 mM
perpendicular to the two other opposed open arms. Hepes, 6 mM MgCl, 1 mM EDTA, mix of protease inhibitors)
- Session: rats were placed in one of the closed arms, facing and centrifuged at 10,000 g. The supernatants were analyzed
the center of the maze, and were recorded for 5 min using a for total protein concentration using Bradford solution, with
camcorder (Handycam DCR-DVD810, Sony). The number of bovine serum albumin (BSA) as standard. According to the
entries to open arms, the latency to reach the open arms, as determined protein concentration, the samples were diluted
well as the percent of HD in closed arms, were calculated. Only with sample buffer solution (6×: 0.346 M SDS, 30% glycerol,
few rats randomly distributed across experimental groups fell 6% 2-mercaptoethanol, 0.179 mM bromophenol blue, 0.998 M
when they walked along the open arms; these animals were Tris–HCl, pH 6.8) in order to have 10 µg of tissue/ml. Therefore,
excluded from the study. homogenates were preincubated with 1 µl DTT 1 M per 10 µl
of sample for 30 min at room temperature and then heated to
94◦ C for 10 min. Then, samples were run on 14% polyacrylamide
Inhibitory Avoidance Task (IA) gels under denaturing conditions. The samples were electro-
Inhibitory avoidance task was used to measure the memory of an transferred to PVDF membranes which were blocked with 5%
aversive experience through the simple avoidance of a location in non-fat milk and 1% BSA and incubated overnight with the
which the unpleasant experience occurred. This task is thought to primary antibody at 4◦ C [Trx-1 and Trx-2 rabbit antibodies, used
depend heavily on the dorsal hippocampus and is a reliable index in a dilution of 1:1,000, were a generous gift of Dr. Lillig from

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Molina et al. Behavioral Alterations in Noise-Exposed Developing Rats

University of Greifswald, Germany; sc-32233 GAPDH (load

control) rabbit antibody from Santa Cruz Biotech. was used in
a dilution of 1:5,000]. After that, samples were incubated at room
temperature with the secondary anti-rabbit HRP-conjugated
antibody (sc-2768 Santa Cruz Biotech., diluted 1:5,000) for 2 h
under shaking, scanned densitometrically by the Image Quant
analyzer and quantified using ImageJ software.

Statistical Analysis
Normality test was performed for each group (KS-test).
Significant differences between groups were analyzed through
one-, two- or three-way analysis of variance (ANOVA) tests with
LSD post hoc comparisons using the Infostat/L software. When
the normality tests failed, a non-parametric analysis was made,
using the Kruskal–Wallis test. Different letters (a, b, c, d) were
used to depict significant differences between the means, being
significantly different one bar from another when they have no
common letters. For example, if a bar received an ‘‘a’’ score
and another a ‘‘b’’ score, it means that they differ statistically
with p < 0.05. Considering the large number of groups to be
compared, with the consequent difficulties in data interpretation,
the differences between the results of PND7 and PND15-exposed
animals were analyzed separately. A probability < 0.05 was
accepted as significant.
When interactions were significant, a simple effect
analysis was performed, through which one-way ANOVA
analyses were performed. The results were expressed as mean
values ± standard error of the mean (SEM) and graphs were
performed with Prism Graphpad software v5.

RESULTS
Auditory Function
No significant changes in ABRs thresholds in any of the
frequencies tested were observed in PND28 animals exposed
to noise at PND7 and PND15 [non-parametric Kruskall–Wallis
test, H < 4 and p > 0.05 (NS) for all frequencies, Figures 2A,B].

Open Field (OF) Task

(i) The number of lines crossed in two sessions of 5 min in an FIGURE 2 | Auditory brainstem responses (ABRs) in PND28 animals
OF, separated by an interval of 1 h, was taken as an index of exposed at (A) PND7 and (B) PND15. Sham: non- exposed animals; N1:
short-term habituation to a new environment. single noise exposure; N5: five-daily noise exposure. St: standard housing.
EE: enriched environment. Data represent the mean ± standard error of the
Data show that exposure to noise at PND7, according to N1 mean (SEM) of the ABR, n = 3 for each group.
and N5 schemes, induced a decrease in the number of lines
crossed in the second session of the OF when compared with
the first session, both in standard or in enriched conditions, p < 0.01. Post hoc comparisons: first vs. second session: all
that resulted similar to what was observed in sham animals, groups, p < 0.05].
when evaluated at PND28 [Figure 3A, N1: Three-way ANOVA, In addition, most groups showed a decrease in the lines
F (7,65) = 7.77, p < 0.01. Between factors: exposure (sham or crossed in the second session of the OF when the animals were
noise), F (1,65) = 0.22, NS; housing (St or EE), F (1,65) = 5.4, exposed at PND15 according to N1 scheme [Three-way ANOVA,
p < 0.05; within factor: session (first or second), F (1,65) = 43.88, F (7,47) = 9.65, p < 0.01. Between factors: exposure (sham or
p < 0.01. post hoc comparisons: first vs. second session: noise), F (1,47) = 9.49, p < 0.01; housing (St or EE), F (1,47) = 3.67,
all groups, p < 0.01. Figure 3B, N5: Three-way ANOVA, NS; within factor: session (first or second), F (1,47) = 38.91,
F (7,67) = 6.29, p < 0.01. Between factors: exposure (sham or p < 0.01]. However, given that a significant interaction between
noise), F (1,67) = 0.04, NS; housing (St or EE), F (1.67) = 0.23, exposure and session was found (F (1,47) = 10.14, p < 0.01), a
NS; within factor: session (first or second) F (1,67) = 41.79, simple effect analysis was performed. Data show that whereas

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FIGURE 4 | Number of lines crossed in the first and second session of the
FIGURE 3 | Number of lines crossed in the first and second session of the
OF by PND28 animals exposed to noise at PND15. (A) PND15 animals
OF by PND28 animals exposed to noise at PND7. (A) PND7 animals exposed
exposed according to N1; (B) PND15 animals exposed according to N5.
according to N1; (B) PND7 animals exposed according to N5. Sham: non-
Sham: non-exposed animals; N1: single noise exposure; N5: five-daily noise
exposed animals; N1: single noise exposure; N5: five-daily noise exposure.
exposure. St: standard housing. EE: enriched environment. Different letters
St: standard housing. EE: enriched environment. Different letters (a, b, c, d)
(a, b, c, d) symbolize significant differences with p < 0.05. Data represent the
symbolize significant differences with p < 0.05. Data represent the
mean ± SEM of the number of lines crossed in the first and second session
mean ± SEM of the number of lines crossed in the first and second session
of the OF, n = 7 for each group.
of the OF, n = 7 for each group.

significant differences were observed between the first and PND15, except for animals exposed to noise at PND15 according
second session in most groups, non-significant differences were to N1 scheme and housed in St conditions.
observed in animals exposed to noise according to N1 scheme (ii) The number of forelimb elevations (i.e., rearing and
and housed in St conditions (Figure 4A, Sham: Two-way climbing) made in the first session of the OF task was taken as
ANOVA, F (3,21) = 9.34, p < 0.01, post hoc comparisons: first an index of exploratory activity.
session vs. second session, St and EE, p < 0.05. Noise: Two-way Data show a significant main effect in this parameter
ANOVA, F (3,25) = 8.21, p < 0.01. Post hoc comparisons: first [Figure 5A, Three-way ANOVA, F (7,85) = 3.42, p < 0.01. Between
session vs. second session, St, NS; EE, p < 0.05). Finally, factors: exposure (sham or noise), F (1,85) = 0.57, NS; housing
when animals exposed at PND15 according to N5 scheme were (St or EE), F = 13.08, p < 0.01; within factors: scheme of
evaluated, significant differences were observed between the lines exposure (N1 or N5) F (1,85) = 0.55, NS]. As the interaction
crossed in the first and second session of the OF in all groups between exposure and housing was significant (F (1,85) = 8.43,
[Figure 4B, N5: Three-way ANOVA, F (7,59) = 5.81, p < 0.01. p < 0.01), a simple effect analysis was performed [St: Two-way
Between factors: exposure (sham or noise), F (1,59) = 5.68, ANOVA, F (3,46) = 2.09, NS. Between factor: scheme (N1 or
p < 0.05; housing (St or EE), F (1,59) = 5.97, p < 0.05; within N5), NS; within factor: exposure (sham or noise), p < 0.05.
factor: session (first or second) F (1,59) = 26.81, p < 0.01. Post EE: Two-way ANOVA, F (3,38) = 1.53, NS]. The results show a
hoc comparisons: first vs. second session: St (sham and noise), significant increase in animals exposed at PND7 according to N1
p < 0.01; EE (sham and noise), p < 0.05]. and housed in St conditions when compared to their respective
In summary, results show a significant decrease in the number controls. In contrast, no changes were observed after EE housing
of lines crossed in the second session of the OF when compared of N1-exposed rats. Finally, exploration activity of N5-exposed
with the first session in most groups, both exposed at PND7 and animals (both after St and EE housing) remained unaltered [post

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housed in EE. In contrast, a significant increase was observed in

animals repeatedly exposed to noise, both after St or EE housing.

Elevated Plus Maze (EPM) Task

Open arms-related parameters measured in the EPM, such as the
decrease in the latency to enter and an increase in the number
of entries, are thought to be associated with a reduction of
anxiety-like behaviors. HD in an open arm might be related with
risk assessment behaviors.
Latency to Enter to the Open Arms in the Elevated
Plus Maze (EPM) Task
Figure 6A shows a significant main effect on the latency to enter
the open arms of the EPM when animals exposed at PND7 were
evaluated [Three-way ANOVA, F (7,52) = 10.69, p < 0.01; between
factors: exposure (sham or noise), F (1,52) = 23.35, p < 0.01;
housing (St or EE), F (1,52) = 15.60, p < 0.01; within factors:
scheme of exposure (N1 or N5), F (1,52) = 30.63, p < 0.01]. The
results show a significant decrease in animals exposed to noise
according to N1 scheme, both in St and EE housing conditions,
without changes when exposure was done according to N5 (post
hoc comparisons: sham vs. noise, St: N1, p < 0.05; N5, NS. EE:
N1, p < 0.05; N5, NS).
When PND15 animals were exposed, a significant main
effect was observed [Figure 6B, Three-way ANOVA,
F (7,55) = 5.85, p < 0.01; between factors: exposure (sham or
noise), F (1,55) = 5.59, p < 0.01; housing (St or EE), F (1,55) = 0.45,
NS; within factor: scheme (N1 or N5), F (1,55) = 3.25, NS]. As
a significant interaction was observed between exposure and
FIGURE 5 | Number of elevations (climbing and rearing) made by scheme (F (1,55) = 28.12, p < 0.01), a simple effect analysis was
PND28 animals exposed to noise at PND7 and PND15 in the OF task. (A)
performed. A significant increase in the latency to open arms
PND28 animals exposed at PND7; (B) PND28 animals exposed at PND15.
Sham: non- exposed animals; N1: single noise exposure; N5: five-daily noise
was found in noise-exposed animals according to N1, both in
exposure. St: standard housing. EE: enriched environment. Different letters (a, St and EE housing [Two-way ANOVA, F (3,28) = 7.07, p < 0.01;
b, c) symbolize significant differences with p < 0.05. Data represent the between factor: exposure (sham or noise), F (1,28) = 19.61,
mean ± SEM of the number of elevations (climbing and rearing) made in the p < 0.01; within factor: housing (St or EE), F (1,28) = 1.40, NS.
OF task, n = 7 for each group.
Post hoc comparisons: sham vs. noise: St and EE, p < 0.05].
As a significant main effect was observed after N5 scheme
[Two-way ANOVA, F (3,26) = 4.67, p < 0.01; between factor:
hoc comparisons: sham vs. noise: N1: St, p < 0.05; EE, NS; N5 (St exposure (sham or noise), F (1,26) = 8.18, p < 0.01; within factor:
and EE), NS]. housing (St or EE), F (1,26) = 0.03, NS] and an interaction was
On the other hand, a significant main effect was found in observed (F (1,26) = 5.79, p < 0.05), a simple effect analysis was
animals exposed at PND15 (Figure 5B, Three-way ANOVA, performed, which showed a significant decrease in noise-exposed
F (7,77) = 2.69, p < 0.05). As the interaction between exposure and animals housed in St cages when compared with their respective
scheme was significant (F (1,77) = 12.65, p < 0.01), a simple effect controls (p < 0.05).
analysis was performed [N1: Two-way ANOVA, F (3,38) = 1.29, In summary, results show a decrease in the latency to enter
NS. N5: Two-way ANOVA, F (3,37) = 6.35, p < 0.01. Between to the open arms in noise-exposed animals according to N1 at
factor: exposure (sham or noise), F (1,37) = 17.29, p < 0.01; within PND7 and an increase in this parameter when animals were
factor: scheme (St or EE), F (1,37) = 1.74, NS, post hoc comparisons: exposed to N1 at PND15, after St and EE housing conditions.
sham vs. noise: N1: St, NS; EE, p < 0.05; N5 (St and EE), p < 0.05]. On the other hand, no changes were found when exposure was
In summary, results show an increase in the number of done according to N5 at PND7 whereas a significant decrease
forelimb elevations in animals exposed at PND7 according to was observed when animals were exposed to N5 at PND15 and
N1 scheme housed in St when compared to the sham group. housed in St, without changes when animals were housed in EE.
In contrast, no changes were observed when these animals were
housed in EE or in groups exposed to N5 scheme (both after St Number of Entries to the Open Arms in the Elevated
and EE housing). On the other hand, results show that whereas Plus Maze (EPM) Task
no changes were observed in this parameter after exposure of Figure 6C shows a significant main effect on the number of
PND15 animals to noise according to N1 scheme and St housing, entries to the open arms of the EPM in animals exposed at
a significant decrease was observed when exposed animals were PND7 (Three-way ANOVA, F (7,55) = 4.19, p < 0.01). As some

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FIGURE 6 | Anxiety-related behaviors measured in the elevated plus maze (EPM; latency and number of entries to open arms) of PND28 animals exposed to noise
at PND7 and PND15 in the EPM task. Latency to open arms (in seconds): (A) PND28 animals exposed at PND7; (B) PND28 animals exposed at PND15. Entries to
open arms: (C) PND28 animals exposed at PND7; (D) PND28 animals exposed at PND15. Sham: non- exposed animals; N1: single noise exposure; N5: five-daily
noise exposure. St: standard housing. EE: enriched environment. Different letters (a, b, c) symbolize significant differences with p < 0.05. Data represent the
mean ± SEM of latency (seconds) or entries to open arms made in the EPM task, n = 7 for each group.

interactions were significant (between exposure and housing: contrast, no changes were observed in animals exposed according
F (1,55) = 18.31, p < 0.01; between exposure, housing and scheme: to N5 scheme [Between factors: exposure (sham or noise) or
F (1,55) = 4.47, p < 0.05) a simple effect analysis was performed housing (St or EE), NS; within factors: scheme of exposure
[St: Two-way ANOVA, F (3,28) = 3.82, p < 0.05. Between factor: (N1 or N5), NS]. As some interactions were significant (between
exposure (sham or noise), p < 0.01; within factor: scheme (N1 or exposure and scheme: F (1,60) = 12.19, p < 0.01; between exposure,
N5), NS. Post hoc comparisons: sham vs. noise: St, p < 0.05; EE, scheme and housing: F (1,60) = 15.21, p < 0.01), a simple effect
p < 0.01. EE: Two-way ANOVA, F (3,26) = 8.61, p < 0.01. Between analysis was performed [St: Two-way ANOVA, F (3,30) = 5.15,
factor: exposure (sham or noise), p < 0.01; within factor: scheme p < 0.01. Between factor: exposure (sham or noise), NS; within
(N1 or N5), NS]. factor: scheme (N1 or N5), F (1,30) = 8.81, p < 0.01. EE: Two-way
Data show a significant increase in St-housed animals exposed ANOVA, F (3,29) = 4.85, p < 0.01. Between factor: exposure (sham
according to N1 scheme and a decrease when exposed animals or noise), NS. Within factor: scheme (N1 or N5), F (1,29) = 5.83,
were housed in EE (post hoc comparisons: sham vs. noise: St, p < 0.05. Post hoc comparisons: sham or noise: St: N1, p < 0.05;
p < 0.05; EE, p < 0.01). No changes were observed in rats N5, NS. EE: N1, p < 0.05; N5, NS]. As a significant interaction
exposed for 5 days [between factors: exposure (sham or noise) was found between exposure and scheme, both within St and
or housing (St or EE), NS; within factors: scheme of exposure EE-housed animals (St: F (1,30) = 4.63, p < 0.05; EE: F (1,29) = 8.56,
(N1 or N5), NS]. However, as the interaction between exposure p < 0.01), simple effect analysis were performed. Data show
and scheme in rats housed within EE group was significant a significant decrease in noise-exposed animals according to
(F (1,26) = 13.27, p < 0.01), a simple effect analysis was performed. N1 scheme, both in St and EE conditions (p < 0.05).
In summary, results show a significant increase in noise-exposed In summary, results show significant differences in the
animals according to N1 (p < 0.05) and no changes in rats number of entries to the open arms in noise-exposed animals
exposed according to N5. when compared to their controls according to N1 scheme,
Figure 6D shows a significant main effect on the number without changes after N5 noise-exposure scheme. When animals
of entries to the open arms of the EPM in animals exposed were exposed to N1 at PND7, an increase in this parameter in
at PND15 (Three-way ANOVA, F (7,60) = 4.77, p < 0.01). A St-housed animals and a decrease when animals were housed
decrease in this parameter was observed in animals housed in in EE were observed. Moreover, when animals were exposed at
St and EE conditions and exposed according to N1 scheme. In PND15 a decrease was observed, both for St and EE housing.

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housing (St or EE), p < 0.01. Post hoc comparisons, sham vs.
noise, N1: St, p < 0.05; EE, NS. N5: St and EE, NS].
Finally, non-significant differences were observed between N1
and N5 noise-exposed and the corresponding sham group in
PND15 animals, both in standard and EE conditions (Figure 7B,
Three-way ANOVA, F (7,63) = 1.08, NS).
In summary, results show no significant changes in %HD in
closed arms in most groups, both exposed at PND7 and PND15,
except from animals exposed to noise at PND7 according to
N1 scheme and housed in St conditions, which showed an
increase in this parameter when compared to their sham group.

Inhibitory Avoidance (IA) Task: Ratio

Between the Latency to Enter the Dark
Compartment in the Retention and the
Training Sessions
In the IA task, T1 is defined as the time required to enter the
dark compartment (i.e., the side in which an electric shock,
an aversive stimulus, was delivered) in the training session and
T2 is the time required to enter the same compartment in the
retention session, after an interval of 1 h. The ratio T2/T1 is
the relationship between the seconds measured in the retention
and the training sessions and might be taken as an index of
associative memory. Figure 8A shows a significant main effect in
the T2/T1 ratio in rats exposed at PND7 (Three-way ANOVA,
F (7,50) = 5.49, p < 0.01).Whereas non-significant differences
were induced after exposure to noise under standard conditions
according to N1 scheme when compared with sham animals, a
FIGURE 7 | Percentage of head dipping (HD) in closed arms in the EPM
significant increase was observed under EE housing [between
task made by PND28 animals exposed to noise at PND7 and PND15. (A)
PND28 animals exposed at PND7; (B) PND28 animals exposed at PND15.
factors: exposure (sham or noise), F (1,50) = 9.92, p < 0.01;
Sham: non- exposed animals; N1: single noise exposure; N5: five-daily noise housing (St or EE), NS; within factors: scheme of exposure
exposure. St: standard housing. EE: enriched environment. Different letters (a, (N1 or N5), NS]. In contrast, an increase in this ratio was
b) symbolize significant differences with p < 0.05. Data represent the observed after repeated exposures to noise of animals housed
mean ± SEM of the % of HD in closed arms made in the EPM task, n = 7 for
in standard cages, without changes observed after housing in
each group.
EE when compared with the corresponding sham rats. As some
interactions were significant (between housing and scheme:
F (1,50) = 5.50, p < 0.05; between exposure, scheme and housing:
Head Dipping (HD) F (1,50) = 20.32, p < 0.01), a simple effect analysis was performed
When HD was analyzed, a significant main effect was observed [St: Two-way ANOVA, F (3,24) = 5.36 p < 0.01. Between factor:
when animals were exposed at PND7 (Figure 7A, Three-way exposure (sham or noise), NS; within factor: scheme (N1 or N5),
ANOVA, F (7,56) = 8.38, p < 0.01). Data show a significant NS. EE: Two-way ANOVA, F (3,25) = 6.85, p < 0.01. Between
increase in percentage of HD in closed (protected) arms factor: exposure (sham or noise), F (1,25) = 7.71, p < 0.01; within
(%HD in closed arms) between animals exposed to noise factor: scheme (N1 or N5), NS]. As the interaction between
at PND7 according to N1 scheme and housed in standard exposure and scheme was significant both in St and EE animals
conditions and sham animals, without changes when animals (St: F (1,24) = 10.43, p < 0.01; EE: F (1,25) = 10.36, p < 0.01), simple
were housed in EE. No changes were observed when animals effect analyses were performed. Post hoc comparisons show a
were exposed according to N5 scheme in comparison with the significant increase in the T2/T1 ratio of N5, St-housed, noise-
corresponding sham group [Between factors: exposure (sham or exposed animals (p < 0.05) and in N1, EE-housed, noise-exposed
noise), NS; housing (St or EE), F (1,56) = 36.86, p < 0.01; within animals (p < 0.05).
factors: scheme of exposure (N1 or N5), F (1,56) = 13, p < 0.01]. Finally, noise exposure at PND15 induced a significant main
As the interaction between exposure and scheme was significant effect in the T2/T1 ratio (Figure 8B, Three-way ANOVA,
(F (1,56) = 5.29, p < 0.05), a simple effect analysis was performed F (7,53) = 2.79, p < 0.01). Although a significant increase was
[N1: Two-way ANOVA, F (3,29) = 6.62, p < 0.01. Between factor: observed in St housed animals exposed according to N1 scheme,
exposure (sham or noise), p < 0.05. Within factor: housing (St or five consecutive daily exposures did not produce changes in this
EE), p < 0.01. N5: Two-way ANOVA, F (3,26) = 7.26, p < 0.01. parameter. Housing in an EE induced a significant increase only
Between factor: exposure (sham or noise), NS; within factor: when rats were exposed once daily, for five consecutive days,

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without changes observed in animals exposed according to

N5 scheme.
In summary, data showed an increase in the ratio between
the latency to enter the dark compartment in the retention
and the training sessions in noise-exposed animals housed in St
conditions according to N5 at PND7 and N1 at PND15, when
compared with the respective controls, without changes in these
groups after housing in an EE. On the other hand, an increase
in noise-exposed animals was observed when compared to their
sham groups, only when animals were housed in EE, according
to N1 at PND7 and N5 at PND15, without changes when housed
in standard cages.

Hippocampal Trx1 and Trx2 Levels

Figure 9A shows that noise exposure at PND7 induced a
significant increase in hippocampal Trx-1 levels, when exposed
according to both N1 or N5 schemes [Three-way ANOVA,
F (7,42) = 2.82, p < 0.05. Between factors: exposure (sham or
noise), F (1,42) = 6.08, p < 0.05; housing (St or EE), F (1,42) = 4.17,
p < 0.05; within factors: scheme (N1 or N5), NS], that remained
similar to the corresponding sham levels when animals were
housed in an EE. As interaction between exposure and housing
was significant (F (1,42) = 8.32, p < 0.01), a simple effect analysis
was performed [St: Two-way ANOVA, F (3,20) = 5.47, p < 0.01.
Between factor: exposure (sham or noise), F (1,20) = 16.05,
p < 0.01; within factor: scheme (N1 or N5), NS. EE: Two-way
ANOVA, F (3,21) = 0.27, NS. Between factor: exposure (sham
or noise), NS; within factor: scheme (N1 or N5), NS. Post hoc
comparisons: St: sham vs. noise: N1 and N5, p < 0.05. EE: N1 and
N5, NS].
Similarly, animals exposed to noise at PND15 showed a
significant increase, according to N1 or N5 schemes and housed
in St conditions, that remained similar to the corresponding
FIGURE 8 | Ratio between the latency to enter the dark compartment (in
sham values when housed in EE [Figure 9B, Three-way ANOVA,
seconds) in the retention session and the training session (T2/T1) in the
inhibitory avoidance (IA) task in PND28 animals exposed to noise at F (7,36) = 2.67, p < 0.05. Between factors: exposure (sham or
PND7 and PND15. (A) PND28 animals exposed at PND7; (B) noise), F (1,36) = 4.18, p < 0.05; housing (St or EE), F (1,36) = 4.76,
PND28 animals exposed at PND15. Sham: non-exposed animals; N1: single p < 0.05; within factors: scheme (N1 or N5), NS]. As interaction
noise exposure; N5: five-daily noise exposure. St: standard housing. EE: between exposure and housing was significant (F (1,36) = 9.72,
enriched environment. Different letters (a, b) symbolize significant differences
p < 0.01), a simple effect analysis was performed [St: Two-way
with p < 0.05. Data represent the mean ± SEM of T2/T1in the IA task,
n = 7 for each group. ANOVA, F (3,19) = 3.47, p < 0.05. Between factor: exposure (sham
or noise), F (1,19) = 10.41, p < 0.01; within factor: scheme (N1 or
N5), NS. EE: Two-way ANOVA, F (3,16) = 0.40, NS. Between
without changes observed after N1 scheme [between factors: factor: exposure (sham or noise), NS; within factor: scheme
exposure (sham or noise), F (1,53) = 7.84, p < 0.01; housing (N1 or N5), NS. Post hoc comparisons: sham vs. noise: St: N1 and
(St or EE), NS; within factors: scheme (N1 or N5), NS]. As N5, p < 0.05. EE: N1 and N5, NS].
the interaction between exposure, housing and scheme was Figure 9C shows a significant main effect on Trx-2 in animals
significant (F (1,53) = 7.89, p < 0.01), a simple effect analysis exposed at PND7, although significant differences between
was performed [St: Two-way ANOVA, F (3,26) = 3.30, p < 0.05. sham and noise-exposed animals were observed only after five
Between factors: exposure (sham or noise), F (1,26) = 2.42, NS; repeated exposures in standard housing [Three-way ANOVA,
within factor: scheme (N1 or N5), NS. EE: Two-way ANOVA, F (7,36) = 6.05, p < 0.01. Between factors: exposure (sham or
F (3,26) = 3.20, p < 0.05. Between factors: exposure (sham or noise), F (1,36) = 3.17, NS; housing (St or EE), F (1,36) = 1.80, NS;
noise), F (1,26) = 5.62, p < 0.05; within factor: scheme (N1 or within factors: scheme (N1 or N5), F (1,36) = 28.17, p < 0.01.
N5), NS. Post hoc comparisons: sham vs. noise, EE: N1, NS; N5, Post hoc comparisons: sham vs. noise: N1: St and EE, NS; N5: St,
p < 0.05]. As the interaction between exposure and scheme was p < 0.05; EE, NS]. In contrast, non-significant main effects were
significant in St animals (F (1,26) = 4.16, p < 0.05), a simple effect observed in animals exposed at PND15 (Figure 9D, Three-way
analysis was performed. Data show a significant increase in the ANOVA, F (7,34) = 1.34, NS). However, as a significant interaction
T2/T1 ratio of N1 St-housed, noise-exposed animals (p < 0.05), was found between exposure, housing and scheme (F (1,34) = 4.43,

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FIGURE 9 | Hippocampal Thioredoxin (Trx) levels.Trx-1: (A) PND28 animals exposed at PND7; (B) PND28 animals exposed at PND15. Trx-2: (C) PND28 animals
exposed at PND7; (D) PND28 animals exposed at PND15. Sham: non- exposed animals; N1: single noise exposure; N5: five-daily noise exposure. St: standard
housing. EE: enriched environment. Different letters (a, b) symbolize significant differences with p < 0.05. Data represent the mean ± SEM of the levels of
hippocampal Trx-1 or Trx-2, n = 4 for each group.

p < 0.05), a simple effect analysis was performed. In addition, were compared. The housing in an enriched environment, a
although no changes were induced in rats housed both in St non-pharmacological strategy of neuroprotection, was effective
and EE conditions (Two-way ANOVA, St: F (3,17) = 0.40, NS; EE: in preventing some of these changes that differed between the
F (3,16) = 2.47, NS), a significant interaction was found between different groups. Finally, non-significant changes in auditory
exposure and scheme in rats housed in EE (F (1,16) = 4.42, function were found in neither group.
p < 0.05), with a significant decrease only in rats exposed
according to N1 scheme (p < 0.05). Auditory Pathway Evaluation
In summary, results show an increase in hippocampal No changes in the auditory thresholds were induced, neither
Trx1 levels in all noise-exposed animals when compared to their when the rats were exposed at PND7 nor at PND15, supporting
respective sham groups, according to both schemes (N1 and results in other animal models (Pienkowski and Eggermont,
N5) and ages of exposure (PND7 and PND15), when animals 2012; Gourévitch et al., 2014). The fact that auditory system
were housed in St condition, without changes after EE housing. become mature at approximately PND12, could explain why
Moreover, data show an increase in hippocampal Trx2 levels in there were no significant changes in the auditory threshold of
PND7 noise-exposed animals only after five repeated exposures animals exposed at PND7, considering that auditory pathway
in standard housing when compared to their sham group, was not functional at the age of exposure. For this reason, the
without changes after EE housing. Finally, although no changes observation of damage when exposure was done at PND7, an age
in Hippocampal Trx2 levels were induced in rats exposed to noise at which the auditory pathway is still immature, might suggest
according to N1 scheme and housed both in St and EE conditions that moderate noise exposure can produce the behavioral and
at PND15, a significant decrease was observed in animals exposed biochemical effects through a direct rather than an indirect
to noise according to N1 and housed in EE when compared to the mechanism, as hypothesized by Säljö et al. (2011). Otherwise,
sham group. it is possible that in the case of rats exposed at PND15, which
already had a functional auditory pathway, the intensity of noise
used was not high enough to generate an effect on the auditory
DISCUSSION thresholds at PND28. However, it should not be discarded that
animals’ auditory system could be affected after PND 12 and
Present results show that exposure of 7 and 15-days-old animals prior to PND28, age at which animals were evaluated.
to moderate levels of white noise (95–97 dB SPL, 2 h), using
single or repeated session’s exposures, was capable to trigger Behavioral Assessment
hippocampal-related behavioral alterations as well as oxidative- The behavioral alterations found in PND15 animals exposed
related molecular changes when evaluated after several days, according to N5 scheme differed from those observed in
that differed according to the scheme used. In addition, animals PND7 rats subjected to the same noise scheme, as was previously
were not uniformly affected when different ages of exposure found for animals exposed at PND7 and PND15 according

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Molina et al. Behavioral Alterations in Noise-Exposed Developing Rats

to N1 scheme (Uran et al., 2010, 2012, 2014; Molina et al., observed in both groups, it could be suggested that greater
2016a). Even more, when exposed animals were housed in an exploration might be associated with decreased anxiety-like
EE, prevention of most behavioral alterations was observed behavior, supporting Kalouda and Pitsikas (2015) and Wright
in all groups. These data suggest that a prompt housing et al.’s (2011) results. In addition, it could be claimed that an
intervention, soon after single or multiple exposures to an increase in exploratory activity with the consequent collection
environmental potentially hazardous agent, could be effective to of information from the environment can favor the habituation
avoid unfavorable effects, mainly if it is implemented in early and adaptability of these animals. Furthermore, an increase in
stages of development (Smith et al., 2018; Gong et al., 2018). novelty anxiety triggered by the new environment might affect
It is important to highlight that habituation memory refers exploration and habituation (Leussis and Bolivar, 2006), because
to behavioral changes that could be triggered in response to shared mechanisms might be involved (Izquierdo and Medina,
repeated exposure to novelty (Leussis and Bolivar, 2006). In 1997; Salomons et al., 2012).
addition, fear conditioning (i.e., inhibitory avoidance) implies Conversely, animals with impairment in habituation memory
a predictive relationship between a stimulus and an event (i.e., those exposed at PND15N1) did not exhibit changes
(Ennaceur and Delacour, 1988). Interestingly, both depend exploratory activity. Even more, the increase in anxiety-related
on the hippocampal integrity (Vianna et al., 2000; Leussis observed in animals exposed at PND15N1 might be related to
and Bolivar, 2006). Finally, exploration is a behavior that can a deficit in habituation memory (Venero et al., 2005; Barzegar
be measured in the OF and is triggered by novel stimuli: et al., 2015). Furthermore, rats exposed at PND15N1 could have
consists of behavioral acts and postures that permit an animal an increased fear response, which would imply that these animals
to collect information about new aspects of the environment would have greater emotional reactivity.
(Barros et al., 2006). However, there are some debate in the However, whereas housing in an EE prevented the changes
literature (Ennaceur, 2014), as several authors suggested that in exploration, as observed in PND7N1 rats, no prevention was
as the anxiety-like behavior decreases, the animals increase the observed when animals exposed at PND15N5 were evaluated.
exploration of the environment (Escorihuela et al., 1999; Prut These data suggest that there would seem to be a window of
and Belzung, 2003; Lever et al., 2006; Kalouda and Pitsikas, opportunity to intervene using a neuroprotection strategy that
2015), whereas others postulated that it may be interpreted as depends on the developmental stage at which the injury took
an anxiogenic-like behavior (Barnett and Cowan, 1976; Lamprea place (Smith et al., 2018; Gong et al., 2018).
et al., 2008).
Emotional Reactivity: Anxiety-Like Behavior and Risk
Habituation Memory
Assessment Behavior
When a rodent is placed in a novel environment, it begins to
It should be considered that decreased anxiety-like behavior
form an internal representation of the surrounding spatial
could be interpreted as a behavioral improvement. However,
information. Once this hippocampal-dependent map is
it could not be true in the wild, because certain minimal
‘‘complete,’’ the animal decreases the exploration of the
anxiety levels might be required to cope with eventual dangerous
environment because it would be considered habituated to the
situations. In contrast, although low or moderate levels of anxiety
new context (O’Keefe and Nadel, 1979; Leussis and Andersen,
may be positive for learning and memory processes, it has been
2008). Given that impairment in this parameter was observed
shown that high levels could lead to a cognitive deficit (Silva and
only in PND15N1 animals and considering that the deficit
Brandão, 2000).
was not evident when younger animals were exposed to noise,
A decrease in the entries to open arms of the EPM might
habituation memory might be used as a marker of vulnerability.
be taken as a sign of an increase in anxiety-like behavior, as
Therefore, as the auditory system becomes active between
observed in animals exposed at PND15N1 and supported by
PND7 and PND15 (de Villers-Sidani et al., 2008; Säljö et al.,
Angrini and Leslie (2012). Conversely, an increase in the entries
2011), it could be postulated that more immature animals could
might imply a decrease in anxiety-like behavior, as observed
be refractory to the damaging effects of noise on this type of
in PND7N1 and supported by Eraslan et al. (2015). Therefore,
memory, probably due to the impossibility of noise to affect
it could be suggested that not only the developmental stage at
CNS by means of a functional auditory system. As no effect was
which the animals are exposed to the environmental agent but
observed when PND15 animals were exposed to noise for 5 days,
also the scheme of exposure come into play to determine the
it could be suggested that repeated exposures might trigger
development of emotional alterations. The lack of change of
adaptive mechanisms intended to counteract potential damage
anxiety-like behavior in animals subjected to five daily noise
(Febbraro et al., 2017; Scott et al., 2017). The ability of EE to
sessions (PND7N5 and PND15N5) could be explained by a
prevent noise-induced changes in PND15N1 animals might
possible compensation that could be triggered as a consequence
depend on the same adaptive mechanisms.
of the repeated exposure to the environmental challenge.
Exploratory Activity %HD in closed arms is a significant behavioral dimension
Significant differences among groups were observed in whose biological function is to inform behavioral strategies
exploratory activity, with an increase in those exposed to in potentially dangerous situations (Carobrez and Bertoglio,
noise at PND7N1 and PND15N5 and without changes in the 2005). Noise was capable to increase this parameter only when
other groups. As a decrease in the latency and/or an increase animals were exposed at PND7N1. Actually, animals with
in the number of entrances to open arms of the EPM was also decreased anxiety-like behaviors would be less cautious and

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could be more exposed to potential hazards. As a decrease this statement, demonstrating that anxiety and fear response
in anxiety-like behaviors was observed in the group exposed could depend on different CNS structures (Kjelstrup et al., 2002;
at PND7N1, the finding of an increase in risk assessment Pentkowski et al., 2006).
behavior might not support this hypothesis. This result implies In consequence, it could be suggested that the longer latency
that at an early developmental age noise exposure increased to enter into the dark compartment might be related to an
the consciousness against potential dangers, such as the open increased emotional reactivity, a non-adaptive response, as
environment of the OF task (Rodgers and Cole, 1993). Neither suggested by Costanzi et al. (2011) that might be also related
repeated exposure sessions nor maturation was able to induce to the increase in anxiety-related behavior, as observed in
changes, suggesting that this unique defensive behavior in humans (Michael et al., 2007; Ponomarev et al., 2010). Therefore,
mammals that reduces the chances of the animal to being although fear is essential for survival, destined to learn about a
harmed might be more important in helpless animals and tend to potential danger, the lack of behavioral flexibility might expose
disappear with the advancement of CNS maturation. In contrast, individuals to environmental changes that might affect not only
the increases in this behavior observed in PND7N1 animals hippocampus but also other structures-related behaviors (Barros
can be effectively prevented by housing in EE, suggesting that et al., 2000; Izquierdo et al., 2016).
animals exposed to noise at earlier ages could be handled through In addition, whereas EE was able to prevent the noise-
the modification of rearing conditions when subjected to a induced changes in the associative memory of PND7N5 and
threatening situation. PND15N1 groups, this housing condition induced an
Interestingly, EE has shown ‘‘per se’’ to increase %HD improvement in the performance of noise-exposed PND7N1 and
when non-exposed PND7N1 rats were tested, when compared PND15N5 groups, suggesting that differences in environmental
with the respective groups housed in standard conditions, stimulation could favor different behavioral phenotypes in the
indicating that these ethological readings might be altered presence of an unfavorable previous condition, such as exposure
through an environmental intervention, supporting results of to noise.
Pietropaolo et al. (2004a) using a mice model of housing in
an enriched environment. Usually, an increase in this risk EE as a Neuroprotective Strategy
assessment behavior is correlated with a decrease in anxiety-like The EE has shown to be an effective tool to protect against
behaviors (Cole and Rodgers, 1995). In contrast, non-exposed CNS injury (Lores-Arnaiz et al., 2006), obtaining benefits on
PND15 animals housed in EE cages showed unchanged %HD learning and memory (Schrijver et al., 2002; Baraldi et al., 2013)
when compared with those animals housed under standard as well as on anxiety-like behaviors (Friske and Gammie, 2005;
conditions, suggesting that the age of exposure is critical to Lima et al., 2014).
driving this emotional output. It should be highlighted that short periods of housing in
an enriched environment appeared to be enough to produce
Associative Memory brain changes in young, but not in adults rats, suggesting
Associative memory can be evaluated through the IA task by that in rodent species adolescence is a highly sensitive period
means of the ratio between the seconds taken to enter the dark likely to be modified by environmental challenges (Spear, 2000).
compartment in the retention and the training sessions (T2/T1, Actually, only 1 week of EE used in the present experimental
Roozendaal, 2002). Although all animals retained associative model as a neuroprotective strategy contrasts with the long
memory in this task, the performance in the associative memory periods required to be protective when adult animals are the
task was increased in rats exposed at PND7N5 and PND15N1, experimental subjects, supporting this hypothesis.
suggesting that these animals would have a more detailed In addition, housing in EE generated changes on its own in
representation of the traumatic event, as reported by Atucha and some behavioral parameters. For example, behavioral differences
Roozendaal (2015). Again, the lack of change in the other groups were observed between control groups depending on the type
might be related to either immature associative mechanisms of housing in parameters such as anxiety-like behavior, %HD
(PND7N1) or to adaptive mechanisms (PND15N5) that could be and exploratory activity. In addition, in some cases, exposed
triggered by repeated exposures, intended to counteract potential animals presented changes in their behavior when compared
damage, as observed in different stress models (Febbraro et al., with their respective sham group only when they were housed in
2017; Scott et al., 2017). However, as memory retention has an EE, whereas no differences were observed when housed under
been observed in both groups, it should not be discarded standard conditions. Supporting these observations, several
that PND7N5 and PND15N1 rats experimented an increase in authors found behavioral changes in untreated animals after
fear sense instead of an improvement in associative memory housing in EE, even during short periods (van Praag et al., 1999;
(i.e., there seems not to be a memory acquisition trouble). It Nithianantharajah and Hannan, 2006; Mitra and Sapolsky, 2012;
must be underlined that fear can be distinguished from anxiety Sampedro-Piquero and Begega, 2017). It has been postulated
as it occurs in response to threats perceived as imminent, while that beneficial effects of EE could be due to the novelty and
anxiety could occur in response to potential or sustained threats increased social contact and exercise, which are rewarding for
(Izquierdo et al., 2016). In other words, a greater fear sense animals as well as efficacious in supplying for their ethological
that could explain the increase in T2 in the IA task could be needs (Pietropaolo et al., 2004b; Crofton et al., 2015).
distinguished from the anxiety in response to a potential danger Therefore, it could be suggested that housing for a week in an
that occurs in the EPM test. There is also evidence supporting EE was able to generate behavioral changes by itself, as well as to

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unmask differences between exposed animals and their controls, and that the alterations induced by repeated exposures might be
which highlight the importance of the interaction with the more effectively compensated in younger animals.
environment that surrounds the animal, given that differences in Therefore, these findings suggest that after repeated exposure
environmental stimulation may favor the development of certain to an environmental challenge animals become less susceptible
behavioral phenotypes (Nithianantharajah and Hannan, 2006; to noise-induced behavioral changes, probably due to the ability
Mychasiuk et al., 2012). of adaptation to an unfavorable condition. Moreover, it could be
hypothesized that damage to younger individuals could be more
Is Hippocampal Oxidative State Involved? easily prevented by an environmental manipulation.
Finally, it is known that the balance of cellular oxidative The knowledge of the mechanisms involved in the damage,
status might be affected after several insults (Bendix et al., as well as the strategies aimed to prevent them, is of clinical
2012; Sies, 2015). A significant increase in the antioxidant relevance considering noise exposure as a public health problem
enzymes activities might indicate that a prior increase in ROS that is increasing in urbanized societies.
production could have been triggered, suggesting that the brain
endogenous antioxidant defense system is capable of being DATA AVAILABILITY
activated in response to excessive ROS generation. As some
changes in hippocampal oxidative status were observed after The datasets generated for this study are available on request to
noise exposure of PND15N1 animals (Uran et al., 2010, 2014), the corresponding author.
the measurement of Trx, an endogenous antioxidant often
involved in brain injuries, could be taken as a marker of ETHICS STATEMENT
damage in the present model that could underlie behavioral
changes. However, although a similar increase in hippocampal This study was carried out in accordance with the Institutional
Trx-1 levels was found in all groups, dissimilar changes in the Committee for the Use and Care of Laboratory Animal rules
behavioral parameters in each group were observed. This lack of (CICUAL, School of Medicine, University of Buenos Aires,
correlation suggests that this endogenous antioxidant could not Argentina). The present experimental protocol was approved by
be the main responsible for the behavioral changes. Although, this Committee and registered with the number 53679/16. The
Trxs are a part of the vast antioxidant machinery, these key CICUAL adheres to the rules of the ‘‘Guide for the Care and
enzymes are frequently altered in oxidative-related pathologies. Use of Laboratory Animals’’ (NIH; 2011 revision) and to the EC
Nevertheless, other markers should be measured to confirm Directive 86/609/EEC (2010 revision) for animal experiments.
these findings.
AUTHOR CONTRIBUTIONS
CONCLUSION SM performed the experiments, with the collaboration of GB
and MR. MG-C performed auditory measurements. LG wrote the
In conclusion, noise exposure using single or repeated session’s
manuscript, with the collaboration of FC.
schemes was capable to trigger hippocampal-related behavioral
alterations as well as oxidative-related molecular changes in
animals exposed at PND 7 and PND15 and evaluated after FUNDING
several days that differed according to the scheme used and
This work was supported by UBACYT 20020160100005BA
the age of exposure. Housing in an enriched environment, a
(Universidad de Buenos Aires, UBA) and PIP 00323 (Consejo
non-pharmacological strategy of neuroprotection, was effective
Nacional de Investigaciones Científicas y Técnicas, CONICET)
in preventing some of these changes. In addition, an oxidative
grants to LG. SM has a post-graduate CONICET fellowship and
imbalance might be triggered in the hippocampus of rats from all
GB has a post-graduate UBA fellowship.
groups, without changes in the auditory function.
The different ages of exposure, as well as the different
schemes applied, might predispose animals to undergo different ACKNOWLEDGMENTS
alterations: more behavioral alterations were observed in younger
animals, exposed for a single day. Therefore, it could be suggested We thank Marcela Vázquez, Eduardo Nieves and Enzo Cuba for
that immature animals might be more vulnerable to noise impact their helpful assistance in the care of laboratory animals.

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Molina et al. Behavioral Alterations in Noise-Exposed Developing Rats

Yamasoba, T., Harris, C., Shoji, F., Lee, R. J., Nuttall, A. L., and Miller, J. M. Conflict of Interest Statement: The authors declare that the research was
(1998). Influence of intense sound exposure on glutathione synthesis conducted in the absence of any commercial or financial relationships that could
in the cochlea. Brain Res. 804, 72–78. doi: 10.1016/s0006-8993(98) be construed as a potential conflict of interest.
00660-x
Zeeni, N., Bassil, M., Fromentin, G., Chaumontet, C., Darcel, N., Tome, D., et al. Copyright © 2019 Molina, Buján, Rodriguez Gonzalez, Capani, Gómez-Casati and
(2015). Environmental enrichment and cafeteria diet attenuate the response Guelman. This is an open-access article distributed under the terms of the Creative
to chronic variable stress in rats. Physiol. Behav. 139, 41–49. doi: 10.1016/j. Commons Attribution License (CC BY). The use, distribution or reproduction in
physbeh.2014.11.003 other forums is permitted, provided the original author(s) and the copyright owner(s)
Zheng, K. C., and Ariizumi, M. (2007). Modulations of immune functions are credited and that the original publication in this journal is cited, in accordance
and oxidative status induced by noise stress. J. Occup. Health 49, 32–38. with accepted academic practice. No use, distribution or reproduction is permitted
doi: 10.1539/joh.49.32 which does not comply with these terms.

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 BRIEF RESEARCH REPORT
published: 06 September 2019
doi: 10.3389/fnbeh.2019.00202

∆9-Tetrahydrocannabinol During
Adolescence Attenuates Disruption
of Dopamine Function Induced in
Rats by Maternal Immune Activation
Salvatore Lecca 1†‡ , Antonio Luchicchi 1†‡ , Maria Scherma 1 , Paola Fadda 1,2 ,
Anna Lisa Muntoni 2 and Marco Pistis 1,2 *
Edited by:
Sophie Laye, 1
Department of Biomedical Sciences, Division of Neuroscience and Clinical Pharmacology, University of Cagliari, Monserrato,
INRA Centre Bordeaux-Aquitaine, Italy, 2 Section of Cagliari, Neuroscience Institute, National Research Council of Italy (CNR), Monserrato, Italy
France

Reviewed by:
Caroline E. Bass,
The combination of prenatal, such as maternal infections, and postnatal environmental
University at Buffalo, United States insults (e.g., adolescent drug abuse) increases risks for psychosis, as predicted
Matthew Wanat,
by the two-hit hypothesis of schizophrenia. Cannabis abuse during adolescence is
University of Texas at San Antonio,
United States widespread and is associated with increased risk of psychoses later in life. Here, we
*Correspondence: hypothesized that adolescent ∆9 -tetrahydrocannabinol (THC) worsens the impact of
Marco Pistis prenatal maternal immune activation (MIA) on ventral tegmental area (VTA) dopamine
[email protected]
cells in rat offspring. Additionally, since substance abuse disorder is particularly prevalent
†
These authors have contributed among schizophrenia patients, we also tested how VTA dopamine neurons in MIA
equally to this work
‡
offspring respond to acute nicotine and cocaine administration. We used a model of
Present address:
Salvatore Lecca, neurodevelopmental disruption based on prenatal administration of the polyriboinosinic-
Department of Fundamental polyribocytidilic acid [poly (I:C)] in rats, which activates the maternal immune system
Neuroscience, University of
Lausanne, Lausanne, Switzerland
by mimicking a viral infection and induces behavioral abnormalities and disruption of
Antonio Luchicchi, dopamine transmission relevant to psychiatric disorders in the offspring. Male offspring
Department of Anatomy and were administered THC (or vehicle) during adolescence (PND 45–55). Once adult (PND
Neurosciences, Division of Clinical
Neuroscience, VU Medical Center, 70–90), we recorded the spontaneous activity of dopamine neurons in the VTA and
Amsterdam UMC, Amsterdam, their responses to nicotine and cocaine. MIA male offspring displayed reduced number,
Netherlands
firing rate and altered activity pattern of VTA dopamine cells. Adolescent THC attenuated
Specialty section:
This article was submitted to
several MIA-induced effects. Both prenatal [poly (I:C)] and postnatal (THC) treatments
Motivation and Reward, a section of affected the response to nicotine but not to cocaine. Contrary to our expectations,
the journal Frontiers in Behavioral
adolescent THC did not worsen MIA-induced deficits. Results indicate that the impact
Neuroscience
of cannabinoids in psychosis models is complex.
Received: 14 June 2019
Keywords: dopamine neurons, maternal immune activation, cannabinoids, adolescence, electrophysiology,
Accepted: 19 August 2019
schizophrenia
Published: 06 September 2019

Citation:
Lecca S, Luchicchi A, Scherma M, INTRODUCTION
Fadda P, Muntoni AL and Pistis M
(2019) ∆9 -Tetrahydrocannabinol Environmental factors, such as prenatal exposure to a variety of infectious agents and consequent
During Adolescence Attenuates
maternal immune activation (MIA), can lead to aberrant brain development, emerging in
Disruption of Dopamine Function
Induced in Rats by Maternal
pathological phenotypes, such as autism and schizophrenia (Hornig et al., 2018). An association
Immune Activation. between MIA and increased risks of developing psychiatric disorders in offspring later in
Front. Behav. Neurosci. 13:202. life has been reported by preclinical investigations and epidemiological studies in humans
doi: 10.3389/fnbeh.2019.00202 (Meyer et al., 2011).

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Lecca et al. Adolescent ∆9 -THC in Maternal Immune Activation

In utero exposure to polyriboinosinic-polyribocytidylic acid tobacco smoking (Winterer, 2010) and stimulant use disorder
(Poly I:C), a double-stranded synthetic RNA that activates an (Hunt et al., 2018), is particularly prevalent among schizophrenia
innate immune response, induces MIA in rodents by mimicking patients we also tested how VTA dopamine neurons in MIA
a viral infection and has been shown to induce schizophrenia- offspring treated with THC and their controls respond to acute
or autism-like phenotypes in rodents (Zuckerman et al., nicotine and cocaine administration.
2003). Hence, offspring display behavioral abnormalities, e.g.,
impairment in recognition memory, in social interactions and in
MATERIALS AND METHODS
sensorimotor gating as well as alterations in brain regions key
in the neuropathology of psychoses, such as the dopaminergic All procedures were performed in accordance with the European
ventral tegmental area (VTA; Patterson, 2002, 2009; Meyer legislation EU Directive 2010/63 and were approved by the
et al., 2005; Boksa, 2010). Indeed, previous studies reported an Animal Ethics Committee of the University of Cagliari and
increase in the number of TH-immunoreactive neurons in the by Italian Ministry of Health (auth. n. 658/2015-PR). Animals
VTA, TH-positive terminals in the striatum (Meyer et al., 2008; were housed in groups of three to six in standard conditions
Winter et al., 2009; Vuillermot et al., 2010), increases in evoked of temperature (21 ± 1◦ C) and humidity (60%) under a 12 h
striatal dopamine release ex vivo (Zuckerman et al., 2003) and light/dark cycle (lights on at 7:00 A.M.) with food and water
enhanced dopamine levels in the prefrontal cortex and lateral available ad libitum. We made all efforts to minimize animal
globus pallidus (Winter et al., 2009). In our previous studies discomfort and to reduce the number of animals used.
we observed a marked alteration of VTA dopamine neuron
activity (reduced firing rate, reduced number of spontaneously Prenatal Treatment
active cells and altered firing pattern) in male but not female Female Sprague–Dawley rats (Envigo, Italy) were mated at
offspring coupled with disruption of sensorimotor gating and the age of 3 months. The first day after the copulation was
of cognitive and social behavior, and increase in dopamine defined as gestational day 1 (GD 1). MIA was induced at GD
levels in the nucleus accumbens (Luchicchi et al., 2016; 15, following the procedure described by Zuckerman et al.
De Felice et al., 2019). (2003). Dams were anesthetized with isoflurane 2% and a
Besides the prenatal period, adolescence is also a critical single dose of Poly I:C (4.0 mg/kg, i.v.; InvivoGen, San Diego,
window of enhanced vulnerability. During adolescence the brain CA, USA) or an equivalent volume of endotoxin-free saline
is particularly susceptible to perturbations, such as exposure to solution was administered in the lateral vein of the tail. To
drugs of abuse, which can disrupt cognitive, emotional, and social assess the efficacy of Poly I:C injection, all pregnant rats were
maturation (Crews et al., 2007). Cannabis is the most widely weighed for the first 3 days after the administration of either
used illegal drug during adolescence and its consumption might Poly I:C or saline to evaluate weight loss as underlined by
induce neurobiological changes that affect adult brain function previous investigations (Zuckerman et al., 2003; Wolff and
(Rubino and Parolaro, 2016). Bilkey, 2010). After weaning, male offspring were housed
The dopamine system is particularly sensitive to with littermates and maintained undisturbed until adolescent
cannabinoids. Both ∆9 -tetrahydrocannabinol (THC) and treatment (PND 45–55) and experiments in adulthood (PND
synthetic cannabinoids induce increases in firing rate of 70–90). Male rats were randomly assigned to the experimental
mesolimbic and mesocortical VTA dopamine cells (Diana procedures and care was taken to avoid assigning more than three
et al., 1998; Gessa et al., 1998) and in extracellular dopamine animals from the same litter to the same experimental group
levels in terminal regions (Tanda et al., 1997). Accordingly, in (Kentner et al., 2019).
humans, THC reduces [11 C]raclopride binding in the ventral
striatum, consistent with a modest increase in dopamine Adolescent Treatment
release (Bossong et al., 2009, 2015) and exacerbates psychotic Male rats were intraperitoneally injected with THC (THC-Pharm
symptoms (Mason et al., 2009). We and others reported that GmbH) or vehicle (1% ethanol, 2% Tween 80 and saline) at
adolescent THC administration induced long-lasting changes in PND 45, in the mid-adolescence period. Increasing doses of THC
the response to dopamine cells to drugs of abuse and enhanced (2.5 mg/kg, PND 45–47; 5 mg/kg, PND 48–51; 10 mg/kg, PND
behavioral responses and self-administration (Pistis et al., 2004; 52–55) or vehicle were given twice/day for 11 consecutive days.
Scherma et al., 2016) which might extend across generations Theses doses of THC were chosen according to the literature
(Vassoler et al., 2013). Moreover, adolescent administration of (Scherma et al., 2016). Body weight and food intake were
cannabinoids is associated with schizophrenia-like deficits in monitored for the entire period of treatment.
adult rodents (Rubino et al., 2009; Leweke and Schneider, 2011).
Considering that in humans early marijuana intake is In vivo Electrophysiological Experiments
associated with increased risk of psychoses later in life In vivo electrophysiology experiments were carried out at PND
(Arseneault et al., 2004; Fergusson, 2004; Degenhardt and 70–90. This age window, which corresponds to the young
Hall, 2006), our hypothesis is that cannabinoid administration adulthood in humans, was selected as it is the most vulnerable
during adolescence in male rats exposed to MIA would worsen age for the onset of schizophrenia (Häfner, 2003). Moreover,
the outcome, as the two-hits hypothesis of schizophrenia studies on the ontogeny of MIA-induced deficits showed that
(genetic/prenatal plus postnatal environment factors) predicts. these are evident at PND 70 (Romero et al., 2010; Vuillermot
Additionally, since substance abuse disorder, specifically heavy et al., 2010).

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In vivo electrophysiological recordings were performed as where appropriate. Post hoc multiple comparisons were made
described previously (Melis et al., 2008, 2009; Luchicchi et al., using the Sidak’s test. Data were analyzed using GraphPad Prism
2016). At PND 70–90, male rats were anesthetized with urethane (San Diego, CA, USA). The significance level was established at
(1.3 g/kg, i.p.) and placed in the stereotaxic apparatus (Kopf, P < 0.05.
Tujunga, CA, USA) with their body temperature maintained at
37 ± 1◦ C by a heating pad. RESULTS
For the placement of a recording electrode, the scalp was
retracted, and one burr hole was drilled above the parabrachial In agreement with previous studies (Zuckerman et al., 2003), rat
pigmented nucleus (PBP) of the posterior VTA (AP, 5.8–6.2 mm dams underwent a significant weight loss in the 24 h following
posterior from Bregma, L, 0.4–0.6 mm lateral from midline) Poly I:C systemic administration (−4.9 ± 2.8 g n = 8; vs.
according to the Atlas of Rat Brain (Paxinos and Watson, 2007). controls +7.5 ± 2.4 g n = 7; P < 0.01, Student’s t-test; data
We selected this subregion as it contains the largest density of not shown). This weight loss indicates that Poly I:C treatment
dopamine cells as compared to the more medial portions of the induced a flu-like syndrome in treated rats (Kentner et al.,
posterior VTA. 2019). However, Poly I:C treatment did not affect litter size
Extracellular single-unit activity of dopamine neurons located (controls: 11.6 ± 1.8 pups, n = 8; Poly I:C: 12.4 ± 1.2 pups, n = 7,
in the VTA (V, 7.0–8.0 mm from the cortical surface) was P = 0.72, Student’s t-test). As our previous studies determined
recorded with glass micropipettes filled with 2% Pontamine that detrimental effects induced by MIA were only evident in
sky blue (PSB) dissolved in 0.5 M sodium acetate (impedance males (De Felice et al., 2019), we evaluated the effect of pubertal
2.5–5 MΩ). The population spontaneous activity of VTA THC solely in male rats. Hence, prenatal Poly I:C or vehicle male
dopamine cells was determined in 6–9 predetermined tracks offspring were randomly assigned to the adolescent THC or
separated by 200 µm each other. Putative VTA dopamine vehicle groups, taking care that no more than three animals from
neurons were selected when all criteria for identification the same litter were assigned to the same experimental group or
were fulfilled: firing rate <10 Hz and duration of action procedure (Kentner et al., 2019). Therefore, electrophysiological
potential >2.5 ms as measured from start to end (Grace and experiments were carried out in four experimental
Bunney, 1983). At the end of the experimental session, inhibition groups: vehicle-vehicle, vehicle-THC, Poly I:C-vehicle and
of spontaneous activity by dopamine receptor agonists and Poly I:C-THC.
subsequent reversal by dopamine receptor antagonists was tested. We next determined if Poly I:C prenatal and THC postnatal
Bursts were defined as the occurrence of two spikes at interspike treatments affect spontaneous activity of dopamine cells, by
interval <80 ms, and terminated when the interspike interval carrying out a population sample in the VTA. For these
exceeded 160 ms (Grace and Bunney, 1984). The electrical experiments we utilized n = 14 vehicle-vehicle (from 6 litters),
activity of each neuron was recorded for 2–3 min. Single-unit n = 19 Poly I:C-vehicle (from 8 litters), n = 8 vehicle-THC (from
activity was filtered (bandpass 0.1–10,000 Hz) and individual 4 litters) and n = 10 Poly I:C-THC (from 5 litters) male offspring.
action potentials were isolated and amplified (Neurolog System, The number of cells/track (Figure 1A), which is an index
Digitimer, Hertfordshire, UK), displayed on a digital storage of population activity of dopamine neurons in the VTA, was
oscilloscope (TDS 3012, Tektronics, Marlow, UK) and digitally significantly reduced by Poly I:C treatment in vehicle-treated
recorded. Experiments were sampled on-line and off-line with but not in THC-treated male offspring [two-way ANOVA:
Spike2 software (Cambridge Electronic Design, Cambridge, UK) effect of Poly I:C, F (1,45) = 9.49, P < 0.01; effect of THC,
by a computer connected to CED 1401 interface (Cambridge F (1,45) = 14.78, P < 0.001; interaction between treatments,
Electronic Design, Cambridge, UK). At the end of recording F (1,45) = 0.66, P > 0.05; post hoc Sidak’s test: significant
sessions, DC current (15 mA for 5 min) was passed through the effect only between vehicle-vehicle and Poly I:C-vehicle rats
recording micropipette in order to eject PSB for marking the (t (45) = 3.2, P < 0.05, Figure 1A)]. The firing rate was reduced
recording site. Brains were then rapidly removed and frozen in by Poly I:C treatment in both vehicle- and THC-treated rats
isopentane cooled to −40◦ C. The position of the electrodes was (Figure 1B; two-way ANOVA: effect of Poly I:C, F (1,454) = 13.53,
microscopically identified on serial 60 µm sections stained with P < 0.01; effect of THC, F (1,454) = 1.98, P > 0.05; interaction
Neutral Red. between treatments, F (1,454) = 0.10, P > 0.05). The percentage
In separate experiments where the effects of nicotine and of spikes per burst was reduced by both Poly I:C and THC
cocaine were assessed, after 5 min of stable baseline activity, treatments (Figure 1C) [two-way ANOVA: effect of Poly I:C,
cocaine (Akzo Pharma Division Diosynth, Oss, Netherlands) was F (1,386) = 26.28, P < 0.001; effect of THC, F (1,386) = 9.92, P < 0.01;
administered i.v. at exponentially increasing cumulative doses interaction between treatments, F (1,386) = 4.67, P < 0.05; post
(0.25–2 mg/kg) every 2 min or nicotine [(-)-nicotine hydrogen hoc Sidak’s test: significant effect between vehicle-vehicle and
tartrate), Sigma-Aldrich, Italy] at a bolus dose of 0.2 mg/kg. all other groups: (t (386) = 5.5, t (386) = 3.9, t (386) = 6.1 for
vehicle-vehicle vs. Poly I:C-vehicle, vehicle-THC, Poly I:C-THC,
Statistical Analysis respectively, P < 0.001 for all comparisons, Figure 1C)]. The
Averaged data from different experiments are given as number of spikes per burst (Figure 1D) was significantly reduced
mean ± SEM. Data were checked for outliers (ROUT test) by Poly I:C treatment only in vehicle-treated rats [two-way
and statistical significance was assessed using Student’s t-test, ANOVA: effect of Poly I:C, F (1,334) = 20.85, P < 0.0001;
one-way ANOVA, two-way ANOVA and two-way ANCOVA, effect of THC, F (1,334) = 6.32, P < 0.05; interaction between

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Lecca et al. Adolescent ∆9 -THC in Maternal Immune Activation

reduced by Poly I:C treatment in vehicle-treated but not in

THC-treated offspring [two-way ANOVA: effect of Poly I:C,
F (1,338) = 7.87, P < 0.01; effect of THC, F (1,338) = 3.26,
P > 0.05; interaction between treatments, F (1,338) = 4.25,
P < 0.05; post hoc Sidak’s test: significant effect only
between vehicle-vehicle and Poly I:C-vehicle rats (t (338) = 3.6,
P < 0.01, Figure 1F)].
In summary, in Poly I:C-vehicle male offspring we detected a
reduced number of spontaneously active cells, lower frequency,
shorter bursts, a lower number of action potentials per burst,
when compared with vehicle-vehicle offspring. Adolescent THC
treatment in vehicle-THC rats did not exert significant effects,
except for the percentage of spikes in burst, which was reduced
when compared to vehicle-vehicle offspring, whereas in Poly I:C-
THC offspring, THC reversed the effects of MIA on cells/track
index, mean spikes/burst, mean burst duration and mean intra-
burst frequency. Our data indicate that dopamine cells in
prepubertal THC-treated offspring are less affected by MIA when
compared with Poly I:C-vehicle rats.
Considering that we recorded several neurons from each
individual rat and that each cell was considered as an
independent replicate, a two-way ANCOVA was carried out with
treatments as factors and individual subjects as covariate, to
exclude that differences among individual rats had significant
effects. The results indicated that individual subjects had no
significant effect overall (two-way ANCOVA P > 0.05 for
all parameters).
We next examined the response of VTA dopamine cells to a
nicotine challenge and to cumulative doses of cocaine.
Figure 2A shows that pre- and postnatal treatments affect
the response of VTA dopamine cells to nicotine (0.2 mg/kg,
FIGURE 1 | Effects of maternal immune activation (MIA) and adolescent
∆9 -tetrahydrocannabinol (THC) administration on ventral tegmental area (VTA) i.v.). The dose of nicotine was selected as it approximately
dopamine neuron activity in vivo. Adolescent THC administration prevented corresponds to the i.p. dose of nicotine (0.4 mg/kg) that induces
the Poly I:C-induced decrease in the number of spontaneously active VTA a robust conditioned place preference and, consistently, induces
dopamine neurons (A) but not the decrease in firing rate (B). Graphs show also a strong increase in firing rate of VTA dopamine cells
the effect of poly IC and THC (or vehicles) in the percentage of spikes in burst
(C), mean burst duration (D), mean number of spikes in bursts (E) and
in control animals (Melis et al., 2008; Mascia et al., 2011;
intra-burst frequency (F). Superimposed colored diamonds show the Sagheddu et al., 2019). Spontaneous activity of VTA neurons
averages for each individual rat. Both Poly I:C and THC, or their combination, was recorded for 5 min then a bolus dose of nicotine was
induced a reduction in the percentage of spikes in bursts (C), whereas THC injected intravenously. In vehicle-vehicle rats nicotine induced
prevented alterations induced by Poly I:C in the other electrophysiological a robust increase in firing rate, amounting to ∼165% of baseline
parameters (D,E). The number of cells for each group is: veh-veh, n = 156;
Poly I:C-veh, n = 121; veh-THC, n = 101; Poly I:C-THC, n = 117. The
(F (4,7) = 6.7, P < 0.05, one-way ANOVA), which remained stable
horizontal blue line represents the mean. Statistical analysis was conducted across the recording time. On the other hand, nicotine did not
with two-way ANOVA (Poly I:C and THC treatments as factors) and Sidak’s significantly affect firing rate of VTA cells either in Poly I:C-
multiple comparison test. Asterisks on graphs represent the result of the vehicle, vehicle-THC nor in Poly I:C-THC rats (F (4,5) = 3.6,
Sidak’s multiple comparison test: ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
F (4,5) = 2.8, F (4,5) = 0.4, respectively, P > 0.05, one-way ANOVA
for all comparisons). When curves were compared across groups,
treatments, F (1,334) = 9.30, P < 0.01; post hoc Sidak’s test: two-way ANOVA revealed a significant interaction between
significant effect only between vehicle-vehicle and Poly I:C- factors (time and treatments; F (12,88) = 2.10, P < 0.05) and
vehicle rats (t (334) = 5.4, P < 0.0001, Figure 1D)]. The mean post hoc analysis indicates that nicotine-induced effects were
burst duration (Figure 1E) was also significantly reduced by significantly different in Poly I:C-THC rats when compared to
Poly I:C treatment in vehicle-treated but not in THC-treated rats the vehicle-vehicle group (t (110) = 2.5, P < 0.05, Sidak’s multiple
[two-way ANOVA: effect of Poly I:C, F (1,367) = 3.58, P > 0.05; comparison test).
effect of THC, F (1,367) = 3.42, P > 0.05; interaction between It is well established that cocaine inhibits dopamine neurons
treatments, F (1,367) = 5.62, P < 0.05; post hoc Sidak’s test: via increased somatodendritic dopamine release acting on
significant effect only between vehicle-vehicle and Poly I:C- D2 autoreceptors (Einhorn et al., 1988). As illustrated in
vehicle rats (t (367) = 3.2, P < 0.01, Figure 1E)]. Similarly, Figure 2B, we confirmed that cocaine (0.25, 0.5, 1.0 and
the mean intraburst frequency (Figure 1F) was significantly 2.0 mg/kg, i.v., expressed as the final cumulative doses at

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doses of cocaine onto VTA dopamine cells (F (12,64) = 0.63,

P = 0.8, two-way ANOVA).

DISCUSSION
The present findings confirm our previous studies that
MIA, evoked by maternal exposure to Poly I:C, induces
harmful effects in offspring, namely disruption of dopamine
cell electrophysiological activity: (i) reduced number of
spontaneously active cells; (ii) decrease in their firing rate;
and (iii) profound alterations in their firing pattern (Luchicchi
et al., 2016; De Felice et al., 2019). We and other groups
showed that changes in dopamine transmission translate into
abnormal behavior such as disrupted sensorimotor gating,
deficits in cognition and social interactions (Zuckerman et al.,
2003; Meyer et al., 2011; Luchicchi et al., 2016). The risk to
develop schizophrenia has often been hypothesized with models
requiring two hits in order to induce the clinical phenotype: an
early priming in a genetically/prenatally predisposed individual
and a second, likely environmental, insult (Davis et al., 2016).
Consistent with this scenario, combining exposure to prenatal
immune challenge and peripubertal stress in mice was shown
to induce synergistic pathological effects on adult behavior and
neurochemistry (Giovanoli et al., 2013, 2016).
Cannabis exposure during adolescence is consistently
associated with an increased risk to develop schizophrenia
later in life and with an earlier onset of the disease (Arseneault
et al., 2004; Fergusson, 2004; Degenhardt and Hall, 2006).
Preclinical findings consistently indicate that adolescent
FIGURE 2 | Effects of nicotine and cocaine on firing rate of VTA dopamine cannabinoid agonist intake induces long-term behavioral
neurons in prenatal Poly I:C and adolescent THC treated offspring and their
controls. (A) Representative firing rate histograms of VTA dopamine neurons
impairment and depressive-like signs (Rubino et al., 2009;
recorded from vehicle-vehicle, Poly I:C-vehicle, vehicle-THC and Poly I:C-THC Rubino and Parolaro, 2016). Therefore, it may represent a risk
rats showing the effects of a bolus dose of nicotine (0.2 mg/kg, i.v.). Arrows factor for developing psychotic-like symptoms in adulthood
indicate the times of nicotine injection. The graph shows that the combination (Rubino et al., 2008).
of prenatal Poly I:C and adolescent THC prevented nicotine-induced increase
Thus, our hypothesis was that exposure to THC during
of firing rate (vehicle-vehicle, n = 8; Poly I:C-vehicle, n = 6; vehicle-THC,
n = 6 and Poly I:C-THC, n = 6; two-way ANOVA and Sidak’s test, ∗ P < 0.05).
adolescence might exacerbate the disruption in VTA dopamine
(B) Representative firing rate histograms of VTA dopamine neurons recorded cell activity observed in offspring following MIA. Contrary to our
from vehicle-vehicle, Poly I:C-vehicle, vehicle-THC and Poly I:C-THC rats expectations, adolescent THC did not induce effects in prenatal
showing the effects of cumulative doses of cocaine (0.25–2.0 mg/kg, i.v.). vehicle-treated animals, apart from a decrease in the bursting
Arrows indicate the times of cocaine injections (0.25, 0.25, 0.5, 1.0 mg/kg).
activity of dopamine cells, whereas in Poly I:C-treated offspring
The bottom graph displays the dose–response curves of the effect of
cumulative doses of cocaine on the firing rate of VTA DA neurons recorded it attenuated several alterations induced by MIA. Notably, MIA
from vehicle-vehicle (n = 5), Poly I:C-vehicle (n = 6), vehicle THC (n = 4) and with Poly I:C was shown to induce in rats persistent increases
Poly I:C-THC (n = 4). Results are presented as mean ± SEM of firing rate in cannabinoid CB1 receptor expression in adulthood in sensory
expressed as a percentage of baseline levels. cortex and hypothalamus assessed by PET (Verdurand et al.,
2014). These findings indicate that prenatal Poly I:C leads
to region-specific long-term alterations in the integrity of the
each point, as we injected 0.25, 0.25, 0.5 and 1 mg/kg, endocannabinoid system that mirror those observed in patients
i.v.), dose-dependently reduced firing rate of dopamine cell with schizophrenia in post-mortem and in vivo PET studies
to approximately 50% in vehicle-vehicle rats (F (4,5) = 17.29, (Köfalvi and Fritzsche, 2008). It is tempting to speculate that
P < 0.001, one-way ANOVA). This inhibitory effect was THC in adolescence might induce changes in CB1 receptor
similar to the control group and statistically significant also expression that, in our model, counteract those induced by MIA.
in Poly I:C-vehicle (F (4,5) = 4.5, P < 0.05, one-way ANOVA), As an example, in MIA-exposed male offspring we observed a
vehicle-THC (F (4,3) = 29.9, P < 0.01, one-way ANOVA) decrease in the probability of glutamate and GABA release onto
and Poly I:C-THC rats (F (4,3) = 81.6, P < 0.01, one-way dopamine cells, indexed by an increase in the paired-pulse ratio
ANOVA). The comparison across groups revealed that neither of excitatory and inhibitory currents coupled with a reduced
Poly I:C nor THC treatments, or their interaction with the frequency of miniature inhibitory and excitatory postsynaptic
doses of cocaine, changed the inhibitory effect of cumulative currents (De Felice et al., 2019). As the release of GABA and

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glutamate is tightly regulated by 2-arachidonoylglicerol (2-AG) reason for this effect requires further investigation. It can be
acting on presynaptic CB1 receptors (Melis et al., 2004, 2014), we speculated that a reduced response to nicotine in both THC-
can speculate that this reduced neurotransmitter release might or MIA-exposed rats might be relevant for the high prevalence
be caused by an increased expression or activity of CB1 receptors of heavy tobacco smoking reported in both cannabis abusers
on GABA or glutamate terminals, consistent with the study by or schizophrenia patients (Kalman et al., 2005; Swendsen
Verdurand et al. (2014). Alternatively, one possibility is that of et al., 2010), as higher nicotine doses might be required to
an enhanced biosynthesis of 2-AG by DAG-lipase in dopamine attain positive subjective effects. On the other hand, the
cells or reduced degradation by MAG-lipase. How adolescent inhibitory effect of cocaine did not change among the four
THC might reverse these changes is not known. One intriguing experimental groups.
possibility is that adolescent subchronic THC might induce Our results, together with other previous studies, confirm that
a long-lasting tolerance by reducing expression or activity of the effects of adolescent cannabinoid exposure in MIA-exposed
CB1 receptors, as shown in our previous studies (Pistis et al., individuals are more complex than expected and that the
2004; Dudok et al., 2015). combination of prenatal and postnatal insults (the double hit
To the best of our knowledge, this is the very first study hypothesis of schizophrenia) in neurodevelopmental models of
carried out in a neurodevelopmental schizophrenia model with schizophrenia needs to be further explored.
the phytocannabinoid THC and not with synthetic cannabinoids
(Gomes et al., 2014; Aguilar et al., 2018). Interestingly, in DATA AVAILABILITY
line with our findings, the study by Gomes et al. (2014)
reported that administration of the synthetic cannabinoid The datasets generated for this study are available on request to
WIN55212 during adolescence did not exacerbate the behavioral the corresponding author.
and electrophysiological changes in methylazoxymethanol
acetate (MAM)-treated rats but attenuated the enhanced
locomotor response to amphetamine. On the other hand, in the
ETHICS STATEMENT
study by Aguilar et al. (2018), pubertal exposure to WIN55212 or The animal study was reviewed and approved by Animal Ethics
to the fatty acid amide hydrolase (FAAH) inhibitor URB597, Committee of the University of Cagliari and by Italian Ministry
which increases endogenous anandamide levels, augmented of Health (auth. n. 658/2015-PR).
the proportion of second-generation MAM rats that develop
schizophrenia-like deficits. In both studies, the synthetic
cannabinoid treatment was able to increase the number of AUTHOR CONTRIBUTIONS
spontaneously active dopamine cells in vehicle-treated animals.
Although we observed a trend toward an increase in the cell/track AL and SL contributed to the acquisition of animal data,
index in vehicle-THC offspring (Figure 1A), this effect did not performed data analysis, contributed to interpretation of results
reach statistical significance. These divergent results with our and provided critical revision of the manuscript. MS and
study might be due to different pharmacology of the cannabinoid PF assisted with acquisition of animal data, analysis and
agonists used (full vs. partial agonist), to the different length and interpretation of findings. PF and AM provided critical revision
protocol of adolescent cannabinoid treatment (11 vs. 25 days, of the manuscript for important intellectual content. MP was
continuous vs. intermittent), or to different neurodevelopmental responsible for the study concept and design and drafted the
models (MAM vs. MIA). manuscript. All authors critically reviewed the content and
Epidemiological studies confirm that schizophrenia patients approved the final version for publication.
show enhanced prevalence of substance use disorders,
particularly concerning nicotine dependence, psychostimulant FUNDING
and cannabis abuse (Kalman et al., 2005; Swendsen et al.,
2010). In animal models of psychiatric disorders, responses to We thank Regione Autonoma della Sardegna for bursaries for
psychostimulant or nicotine is altered: locomotor response to young researchers awarded to AL and SL. This work was
psychostimulants is enhanced in neurodevelopmental models supported by Fondazione Di Sardegna (Progetti cofinanziati
of schizophrenia (Gomes et al., 2014; Aguilar et al., 2018), Università di Cagliari, Bando 2017) to MP and by POR
whereas nicotine is more self-administered and ameliorated SARDEGNA FSE 2014–2020—Asse III ‘‘Istruzione e formazione,
cognitive deficits in a lipopolysaccharide MIA model of Obiettivo Tematico: 10, Obiettivo Specifico: 10.5,’’ Azione
schizophrenia (Waterhouse et al., 2018). Here we tested if dell’accordo di Partenariato: 10.5.12 ‘‘Avviso di chiamata per il
prenatal and/or postnatal treatments affected responses of finanziamento di Progetti di ricerca’’—Anno 2017 to MS.
VTA dopamine neurons to nicotine and cocaine. We found
a blunted effect of nicotine on VTA dopamine cells in all ACKNOWLEDGMENTS
groups when compared to vehicle-vehicle animals, although
this difference reached a statistical significance only in Poly We thank Marta Tuveri and Dr. Barbara Tuveri for their skillful
I:C-THC offspring. These results suggest that adolescent assistance. We thank the European Brain Research Institute
THC and MIA, or the combination of both factors, induce (EBRI)/National Research Council of Italy (CNR) collaborative
persistent changes in neuronal response to nicotine. The agreement (AM).

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Lecca et al. Adolescent ∆9 -THC in Maternal Immune Activation

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activation of mesolimbic dopamine transmission by a common mu1 opioid original publication in this journal is cited, in accordance with accepted academic
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2048 with these terms.

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 SYSTEMATIC REVIEW
published: 28 November 2019
doi: 10.3389/fnins.2019.01284

Folic Acid and Risk of Preterm Birth:

A Meta-Analysis
Bingbing Li 1 , Xiaoli Zhang 1 , Xirui Peng 1 , Shan Zhang 1 , Xiaoyang Wang 1,2 and
Changlian Zhu 1,3*
1
Henan Key Laboratory of Child Brain Injury, Third Affiliated Hospital and Institute of Neuroscience of Zhengzhou University,
Zhengzhou, China, 2 Perinatal Center, Institute of Neuroscience and Physiology, University of Gothenburg, Sahlgrenska
Academy, Gothenburg, Sweden, 3 Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology,
University of Gothenburg, Sahlgrenska Academy, Gothenburg, Sweden

The results from epidemiologic studies linking blood folate concentrations, folic acid
supplementation, or dietary folate to the risk of preterm birth are inconsistent. In this
study, we aimed to summarize the available evidence on these associations. A systematic
search of the PubMed/MEDLINE, Google Scholar, Web of Science, and Cochrane Library
databases up to October 20, 2018 was performed and reference lists of retrieved articles
Edited by:
Sophie Laye,
were screened. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) for the
INRA Centre highest vs. the lowest levels of folate concentrations, folic acid supplementation, and
Bordeaux-Aquitaine, France dietary folate were calculated using random-effects models. Subgroup analyses and
Reviewed by: univariate meta-regression were performed to explore the sources of heterogeneity. Ten
Mladen-Roko Rasin,
Rutgers, The State University of New studies (six prospective cohort studies and four case-control studies) were included on
Jersey, United States folate concentrations, 13 cohort studies were included about folic acid supplementation,
Masahiro Tsuji,
Kyoto Women’s University, Japan
and 4 cohort studies were included regarding dietary folate intake. Higher maternal folate
*Correspondence:
levels were associated with a 28% reduction in the risk of preterm birth (OR 0.72, 95%
Changlian Zhu CI 0.56–0.93). Higher folic acid supplementation was associated with 10% lower risk of
[email protected] preterm birth (OR 0.90, 95% CI 0.85–0.95). In addition, a significant negative association
was observed between dietary folate intake and the risk of preterm birth (OR 0.68, 95%
Specialty section:
This article was submitted to CI 0.55–0.84), but no significant relation was seen between dietary folate and the risk
Neurogenesis, of spontaneous preterm birth (OR 0.89, 95% CI 0.57–1.41). In the subgroup analysis,
a section of the journal
Frontiers in Neuroscience
higher maternal folate levels in the third trimester were associated with a lower risk of
Received: 03 September 2019
preterm birth (OR 0.58, 95% CI 0.36–0.94). To initiate taking folic acid supplementation
Accepted: 12 November 2019 early before conception was adversely associated with preterm birth risk (OR 0.89, 95%
Published: 28 November 2019
CI 0.83–0.95). In conclusion, higher maternal folate levels and folic acid supplementation
Citation:
were significantly associated with a lower risk of preterm birth. The limited data currently
Li B, Zhang X, Peng X, Zhang S,
Wang X and Zhu C (2019) Folic Acid available suggest that dietary folate is associated with a significantly decreased risk of
and Risk of Preterm Birth: A preterm birth.
Meta-Analysis.
Front. Neurosci. 13:1284. Keywords: folate levels, folic acid supplementation, dietary folate intake, meta-analysis, preterm birth, preterm
doi: 10.3389/fnins.2019.01284 brain injury, sequelae of preterm birth

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Li et al. Folic Acid and Preterm Birth

INTRODUCTION data from five randomized trials and reported no statistically

significant effects (Saccone and Berghella, 2016), while the other
Preterm birth (PTB) and its associated complications, which identified nine observational studies and showed a decreased
include brain injury, retinopathy of prematurity, cerebral palsy, risk of PTB when initiating folic acid supplementation after
and developmental disabilities, are among the most serious conception (Zhang et al., 2017). Additional larger cohort studies
global health issues. These complications directly affect the have been published since then that might enhance the statistical
child’s quality of life and are a huge burden both socially and power (Liu et al., 2016; Zheng et al., 2016), and thus an updated
economically (Goldenberg et al., 2008; Song et al., 2016; WHO, meta-analysis is needed.
2018). Thus, prevention of PTB is a global priority (Blencowe In this study, we aimed to evaluate the available evidence on
et al., 2013). Recent estimates of the incidence of PTB in most the associations between blood folate levels, dietary folate intake,
Europe countries range from 3.7 to 7.5% of live births (Poulsen and folic acid supplementation and the risk of PTB.
et al., 2015). In the United States, the incidence is higher at about
9.62% (The Lancet, 2016). In Australia, the incidence was at 8.7%
according to the latest annual report in 2015 (Hoh et al., 2019). MATERIALS AND METHODS
In China, the incidence was at ∼7% in 2016 (Chen et al., 2018).
Literature Search
However, despite ongoing research, there has been no significant
We conducted a literature search of PubMed (Medline), Google
reduction in PTB rates. This might be a result of an inadequate
Scholar, Web of Science, and the Cochrane Library from their
understanding of the pathological processes contributing to PTB.
inception through October 2018. The search terms included
PTB is considered a multifactorial syndrome, with almost 70%
“folic acid,” “vitamin B9,” “folate,” “folate status,” “folate levels,”
of PTBs resulting from spontaneous labor and/or rupture of
“folate concentrations,” “serum folate,” “red blood cell folate,”
membranes and the remainder from iatrogenic causes. Hence,
“folic acid consumption,” “folic acid supplementation,” “folic
it can be broadly categorized into spontaneous PTB (sPTB) and
acid intake,” “food folate,” or “dietary folate” combined with
indicated PTB (Goldenberg et al., 2008). It has been recognized
“preterm delivery,” “premature birth,” or “preterm birth.” We
that there are numerous biological mechanisms that vary between
adhered to the Meta-analysis Of Observational Studies in
individuals and that might lead to PTB (Frey and Klebanoff,
Epidemiology (MOOSE) guidelines when undertaking this study
2016). Therefore, the identification of modifiable risk factors is
(Stroup et al., 2000).
of great importance for PTB management and prevention.
Maternal nutrition is an important determinant of the
duration of pregnancy and fetal growth, and thereby influences Inclusion Criteria
pregnancy outcomes. Experimental data from animal studies Articles were included if (1) the study design was observational,
suggest that maternal nutritional status such as folate status (2) the population was healthy women who had the intention
might play a role in PTB (Zhao et al., 2013; Scholl and Chen, to become pregnant or who were pregnant, (3) the exposure of
2015). Folate is an essential B vitamin that plays a role in interest was folate levels or dietary folate intake or folic acid
DNA synthesis and cell division to support growth and fetal supplementation, (4) the outcome of interest was preterm birth,
development (Lucock, 2000). During pregnancy, there is an which was defined as delivery at <37 weeks gestation, (5) the
increased demand for folate due to the rapid fetal growth. association between folate levels or dietary folate intake or folic
A previous study reported that pregnant women had a 5- to acid supplementation and risk of PTB was evaluated, and (6)
10-fold higher folate requirement than non-pregnant women adjusted risk estimates [relative risks (RRs), hazard risks (HRs),
(Antony, 2007). Blood folate levels, including serum/plasma or or odds ratios (ORs)] with their corresponding 95% confidence
red blood cell (RBC) folate, are considered reliable indicators of intervals (CIs) or standard errors were reported. Additionally,
folate status (World Health Organization, 2015). Some recent we excluded reviews, editorials, non-human studies, randomized
epidemiological studies have indicated that low blood folate clinical trials, and letters without sufficient data. We excluded
levels during pregnancy are associated with an increased risk of randomized clinical trials without folic acid in a control group
PTB (Bergen et al., 2012; Chen et al., 2014), while some other because of limited publications and ethical issues. When multiple
studies have shown no association between blood folate levels reports based on the same study were published, only the most
and PTB (Dunlop et al., 2012; Heeraman, 2016). In addition, recent or complete report was used.
a meta-analysis has not yet been conducted to summarize the
epidemiological evidence on this association. Data Extraction
Folate cannot be synthesized by the body, and humans are We extracted the following data from the included articles: the
entirely dependent on dietary sources or dietary supplements name of the first author, year of publication, country, study
for their folate supply. There have been a large number of design, sample size, follow-up period, study period, assessment
studies describing the association between folate intake and methods of folate levels, or dietary folate intake or folic acid
preterm birth (Vahratian et al., 2004; Lassi et al., 2013; Li et al., supplementation, ascertainment of PTB, ORs and corresponding
2014; Mantovani et al., 2014; Martinussen et al., 2015; Zheng 95% CIs, and the confounding factors used for adjustment
et al., 2016); however, their results are conflicting. Two meta- of the ORs. The ORs with more adjusted confounders were
analyses from 2015 assessed the possible association between folic chosen when studies had different models for the calculation of
acid supplementation and the risk of PTB. One incorporated estimated risks.

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Quality Assessment provide similar estimates of risk when the incidence of outcome
Two reviewers independently performed the quality assessment is very low (<10%) (Greenland, 1987), we chose ORs as the
using the Newcastle-Ottawa Scale (for cohort and case-control common effect size and combined HRs and RRs with ORs in the
studies; Stang, 2010), which is a nine-point system including meta-analysis. The statistical analyses for the overall association
the selection process of studies (0–4 points), the comparability between folate levels, folic acid supplementation/dietary folate
of studies (0–2 points), and the identification of the exposure intake, and PTB risk were based on comparisons of the highest
and the outcomes of the study participants (0–3 points). The category with the lowest. If the original studies did not provide
quality of articles was first evaluated according to the established corresponding data, the OR and its 95% CI were recalculated.
questions, which were scored as 1 if the item was considered The ORs and corresponding 95% CIs were pooled using the
in the study or 0 if the item was not considered or if it was DerSimonian and Laird random-effects model (DerSimonian
impossible to determine whether it was considered or not. We and Laird, 1986), which considers both within-study and
assigned scores of 0–3, 4–6, and 7–9 points for low, moderate, between-study variations. The summary measures were
and high-quality studies, respectively (Supplementary Table 1). presented as forest plots where the sizes of the data markers
(squares) correspond to the inverse of the variance of the natural
Statistical Analysis logarithm of the OR from each study and the diamond indicates
Because most of included studies reported risk estimates as ORs, the pooled OR. Statistical heterogeneity among studies was
and because it was previously reported that ORs, HRs, and RRs quantified using the I 2 statistic (Higgins et al., 2003).

FIGURE 1 | Flow chart for study selection (through October 20, 2018).

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To further evaluate the effects of heterogeneity, univariate reported probabilities (P-values) were two-sided, with P < 0.05
meta-regression analyses were performed examining the effects considered statistically significant, except for the Cochran’s Q
of several key study characteristics. Stratified analyses by statistic in the heterogeneity test, in which the significance level
specimen gestational age, sample type, initiation time of folic was 0.10.
acid supplementation, and dosage of folic acid were conducted
to assess their impact on our estimates. Sensitivity analyses
were employed to find potential origins of heterogeneity RESULTS
and to examine the influence of various exclusions on the
combined OR. Funnel plots were used to assess small- Characteristics of the Studies
study effects. Publication bias was assessed through the visual A total of 25 articles were included. The process of study
inspection of funnel plots and with tests of Begg rank selection is depicted in Figure 1. A total of 9 studies assessed the
correlation (Begg and Mazumdar, 1994). P < 0.05 was association between blood folate levels and the risk of PTB, 12
considered to be representative of a statistically significant examined folic acid supplementation and the risk of PTB, and 2
publication bias. Forest plots were created to assess the overall studies assessed the association between dietary folate intake and
association between folate levels, dietary folate, folic acid the risk of PTB. Moreover, 1 study examined the association of
supplementation, and PTB. folic acid supplementation and dietary folate and the risk of PTB,
All statistical analyses were performed with STATA version and 1 study assessed the association of blood and dietary folate
12.0 software (Stata Corporation, College Station, TX, US). All with the risk of PTB.

FIGURE 2 | Forest plot of the meta-analysis of PTB risk in relation to blood folate levels, comparing the highest category with the lowest. The solid diamonds and
horizontal lines indicate the study-specific ORs and 95% CIs. The size of the gray area reflects the study-specific statistical weight. The hollow diamonds represent the
pooled ORs and 95% CIs of each subgroup and the overall population. The vertical solid line shows the OR of 1, and the vertical red dashed line represents the
combined effect estimate.

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Blood Folate Levels and Risk of PTB and PTB (Begg’s test, P = 0.592). A sensitivity analysis by
For the overall risk of PTB in relation to blood folate levels, omitting one study at a time did not dramatically influence
we included 6 prospective cohort studies (Scholl et al., 1996; the pooled ORs, suggesting that the combined OR was valid
Siega-Riz et al., 2004; Bodnar et al., 2010; Bergen et al., 2012; and credible.
Dunlop et al., 2012; Chen et al., 2014) and 4 case-control studies Subgroup analysis of specimen gestational age suggested no
(Ronnenberg et al., 2002; Martí-Carvajal et al., 2004; Furness association between blood folate levels in the first (OR = 0.68,
et al., 2012; Heeraman, 2016). A total of 7 studies measured 95% CI 0.27–1.66, P = 0.393) and second trimester (OR = 0.82,
folate levels in plasma/serum, and 3 studies measured folate 95% CI 0.63–1.07, P = 0.139) and the risk of PTB. However, the
levels in RBCs. Among these studies, 3 were sampled in the first blood folate level in the third trimester was significantly inversely
trimester, 3 in the second trimester, and 3 in the third trimester. associated with the risk of PTB (OR = 0.58, 95% CI 0.36–0.94,
The main characteristics of these studies are summarized in P = 0.026; Figure 3A).
Supplementary Tables 2, 3. Subgroup analysis showed that higher plasma/serum folate
Overall, a significant negative association between blood was associated with 30% lower risk of PTB (OR = 0.70,
folate levels and PTB risk was observed. The pooled OR (95% 95% CI 0.52–0.93, P = 0.014), while RBC folate was not
CI) for PTB risk in individuals with the highest level of associated with the risk of PTB (OR = 0.89, 95% CI 0.45–1.79,
blood folate compared with the lowest level was 0.72 (95% P = 0.75; Figure 3B).
CI, 0.56–0.93) with a moderate to high degree of statistical
heterogeneity (I 2 = 68.6%; Figure 2). For the 6 cohort studies,
the negative association was consistent and the combined OR Folic Acid Supplementation and Risk of
was 0.68 (95% CI, 0.50–0.92) with a high heterogeneity (I 2 = PTB
78%). For the 4 case-control studies, no significant association Of the 13 cohort studies that assessed folic acid supplementation
was found between blood folate levels and the risk of PTB and the overall risk of PTB (Scholl et al., 1997; Vahratian et al.,
(OR 0.88, 95% CI 0.50–1.56). Heterogeneity was observed 2004; Catov et al., 2007, 2011; Timmermans et al., 2009; Alwan
between studies (I 2 = 41%). To examine this heterogeneity, et al., 2010; Czeizel et al., 2010; Papadopoulou et al., 2013; Li et al.,
we conducted meta-regression analyses with type of design, 2014; Martinussen et al., 2015; Liu et al., 2016; Zheng et al., 2016;
gestational age of the specimen, geographical region, year of Baron et al., 2017), 6 studies reported two separate outcomes
publication, and sample type as the independent variables. As stratified by initiation time of supplementation (preconception
shown in Table 1, no significant differences were found among and postconception). In this case, each of studies could be
these groups. No evidence of publication bias was observed considered as two independent reports. Thus, there were 19
when assessing the association between maternal folate levels independent reports included in this meta-analysis. Among
these studies, 4 used folic acid alone and the rest used folic
acid containing multivitamins. A total of 9 stated the exact
TABLE 1 | Blood folate levels and the risk of preterm birth analyzed by univariate content of folic acid supplements, and 7 out of the 9 studies
meta-regression model. used folic acid at 400 µg daily (as suggested by WHO), and
2 used high doses of folic acid of more than 1,000 µg daily.
Covariate Number of β-coefficient P-value
studies
Overall, the lowest category (reference category) observed in
the included studies ranged from 0 to 200 µg daily, and the
Type of design highest category ranged from any folic acid/folic acid-containing
Cohort study, Case-control 10 0.266 0.429 supplements consumption to ≥1,000 µg daily. Because we used
study the categories reported by the studies, these categories were not
Specimen gestational age mutually exclusive. None of the studies described the mutual
First trimester vs. Second 10 −0.021 0.927 effects associated with multivitamins. The main characteristics of
trimester vs. Third trimester these studies are summarized in Supplementary Table 4.
vs. preconception
The results combining the ORs comparing the highest and
Geographical region
lowest category of folic acid supplementation for the risk of PTB
Asia vs. US vs. Europe vs. 10 0.084 0.597
are shown in Figure 4. An inverse association was found (OR
Australia
= 0.90, 95% CI 0.86–0.95), and low to moderate heterogeneity
Sample size
across the studies was found (I 2 = 30.1%). To examine this
≥1,000 vs. <1,000 10 0.444 0.083
heterogeneity, we conducted meta-regression analyses with
Year of publication
initiation time of supplementation, geographical region, source
≥2010 vs. <2010 10 0.089 0.756
of cohorts, and ascertainment of PTB, sample size, year of
Type of control
publication and dosage of folic acid intake. As shown in Table 2,
Hospital vs. Population 10 0.418 0.077
no significant differences were observed among these subgroups.
Sample type
Visual inspection of the funnel plot showed little asymmetry for
Serum/Plasma vs. Red 10 0.216 0.534
studies on folic acid supplementation and PTB risk (Figure 5).
blood cells
No evidence of publication bias was found across the included
The β-coefficient represents the change in log OR per unit increase in the relevant variable. studies (Begg’s test, P = 0.284), and sensitivity analyses using

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FIGURE 3 | Forest plot of the meta-analysis of PTB risk in relation to blood folate stratified by gestational age of the specimen (A) and sample type (B), comparing the
highest category with the lowest. The diamonds and horizontal lines indicate the subgroup-specific ORs and 95% CIs. The size of the gray area reflects the
study-specific statistical weight. The vertical solid line shows the OR of 1.

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FIGURE 4 | Forest plot of the meta-analysis of PTB risk in relation to folic acid supplementation, comparing the highest category with the lowest. The diamonds and
horizontal lines indicate the corresponding ORs and 95% CIs. The size of the gray area reflects the study-specific statistical weight. The vertical solid line shows the
OR of 1, and the vertical red dashed line represents the combined effect estimate. The suffix “a” or “b” after the studies indicates two separate outcomes stratified by
the initiation time of supplementation (preconception and post-conception) in the same study.

a fixed-effect model or omitting one study at a time did not µg was not significantly associated with the risk of PTB (OR =
substantially alter the pooled results. 0.95, 95% CI: 0.74–1.20, P = 0.65; Figure 6B).
Subgroup analysis of initiation time of folic acid
supplementation showed that initiating folic acid supplements Dietary Folate Intake and Risk of PTB
before conception was associated with a significant decreased Of the 4 cohort studies that assessed dietary folate intake and
risk of PTB (OR = 0.87, 95% CI: 0.84–0.91, P < 0.001), while risk of PTB (Siega-Riz et al., 2004; Shaw et al., 2011; Sengpiel
starting folic acid supplementation at post-conception was et al., 2014; Liu et al., 2016), 1 study reported two separate
associated with a marginal decreased risk of PTB (OR = 0.90, outcomes stratified by the initiation time of supplementation
95% CI: 0.80–1.00, P = 0.049; Figure 6A). (preconception and post-conception). Thus, there were 5
In the analysis stratified by dose of folic acid intake, a independent reports, including 95,448 participants, in our meta-
statistically significant protective effect was noted between folic analysis. Out of these 5 studies, 2 studies reported the outcome
acid supplementation at a daily dosage of <1,000 µg and PTB of sPTB in addition to overall PTB, 2 studies just reported sPTB,
risk (OR = 0.90, 95% CI: 0.85–0.95, P < 0.001). However, taking and 1 study just reported overall PTB. In total, 3 studies reported
folic acid supplementation at a daily dosage of more than 1,000 the overall risk of PTB in relation to dietary folate intake, and 4

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TABLE 2 | Folic acid supplementation and the risk of preterm birth analyzed by influence the development of and maintenance of uteroplacental
univariate meta-regression model. circulation (Baker et al., 2017), which subsequently triggers
Covariate Number of β-coefficient P-value
poor pregnancy outcomes including PTB (Engel et al., 2006;
studies Bailey, 2009). It has been observed that folate transporters,
which transfer folate from maternal circulation to the fetus, are
Time of FA intake present at lower concentrations in preterm placentas compared
Preconception vs. 19 0.014 0.829 to term placentas (Castaño et al., 2017). Second, folate is also
Postconception vs.
Periconception
a cofactor in the metabolism of homocysteine, which might be
Geographical region
a contributing factor for placental vascular disease (Van der
Asia vs. US vs. Europe 18* −0.0008 0.98 Molen et al., 2000), and epidemiological studies have shown
Sample size that elevated homocysteine concentrations are associated with
≥1,000 vs. <1,000 19 −0.012 0.877 PTB (Bergen et al., 2012; Chen et al., 2014). Third, folate status
Year of publication during pregnancy might play an anti-inflammatory role. Many
≥2010 vs. <2010 19 −0.054 0.565 cases of PTB are associated with an abnormal inflammatory
Source of Cohort response, which is often caused by intrauterine infection and
Hospital vs. Population 19 0.012 0.875 inflammation (Goldenberg et al., 2000). In a mouse model of
Definition of GA lipopolysaccharide-induced PTB, folate reduced the levels of
LMP vs. Ultrasound vs. Both 19 −0.009 0.977 circulating biomarkers of inflammation, including interleukin
Dose of intake (IL)-6 and keratinocyte-derived cytokine in the amniotic fluid of
Moderate vs. High 19 0.067 0.571
mice (Zhao et al., 2013).
*One study did not provide data for geographical region. The β-coefficient represents the
change in log OR per unit increase in the relevant variable. FA, folic acid; LMP, the last
normal menstrual period; GA, gestational age. Blood Folate Levels and Risk of PTB
We found an inverse association between blood folate levels in
studies reported sPTB. The main characteristics of these studies the third trimester and the risk of PTB, while no significant
are summarized in Supplementary Tables 5, 6. association was observed in the first and second trimester. This
For the 3 studies about overall PTB, there was a significant might be explained by the following mechanisms. Placental
inverse association observed between dietary folate intake and dysfunction is one of the risk factors for PTB (Romero et al.,
the overall risk of PTB, and the pooled OR and 95% CI for PTB 2014), and it was observed that maternal blood folate levels
when comparing the highest with the lowest levels of dietary decreased from the fifth month of pregnancy and plasma
folate intake was 0.68 (95% CI 0.55–0.84) with a moderate homocysteine concentrations increased in later pregnancy
heterogeneity (I 2 = 67.8%; Figure 7A). For the 4 studies about (Wang et al., 2016). Thus, we hypothesized that inadequate
sPTB, a non-significant association was observed. The summary third trimester maternal folate levels impact fetal development
OR and 95% CI was 0.89 (95% CI 0.57–1.41) with a high by adversely affecting placental function during the period of
heterogeneity (I 2 = 88.1%; Figure 7B). We did not perform maximal fetal development. Previous studies have reported that
subgroup analysis or sensitivity analysis due to the small number the persistence of placental dysfunction from the 24th week of
of studies. pregnancy is associated with increased risks of adverse pregnancy
outcomes (López-Quesada et al., 2004; Gaillard et al., 2013).
DISCUSSION On the other hand, folate might also be indirectly involved
in placental development through its role in the homocysteine
Principal Findings of This Study cycle. Folate deficiency may disrupt the function of the enzymes
The current meta-analysis is the first time to assess the association in homocysteine metabolism and lead to an increase in
of PTB risk with blood folate levels and dietary folate intake. We homocysteine levels. It was reported that elevated homocysteine
found that blood folate levels, folic acid supplementation, and concentration is also associated with oxidative stress, arteriolar
dietary folate intake were negatively associated with the overall constriction, endothelial damage, and placental thrombosis, all of
risk of PTB. Furthermore, we found that dietary folate intake was which increase the risk of pregnancy complications (Maged et al.,
not significantly associated with the risk of sPTB. 2017). Moreover, folate is an important methyl-group vitamin,
Compared to the previous meta-analysis on folic acid and maternal plasma folate levels are associated with offspring
supplementation and risk of PTB, which included 9 studies, DNA methylation, which is in turn related to fetal development.
this updated meta-analysis included 13 studies and increased It was reported that maternal folate levels in late pregnancy are
the sample size from 306,695 to 562,068 participants, which more important than folate levels in early pregnancy for overall
increased the statistical power of our analysis. Moreover, fetal growth (Sulaiman et al., 2017). Thus, an inverse association
the included studies used modern methods of multivariate was evident between maternal folate levels in the third trimester
adjustment rather than raw data. and the risk of PTB, and maternal folate levels in the third
Evidence from biological studies supports a role of folate trimester might be an indirect predictor of PTB.
in PTB. First, folate contributes to oocyte maturation and In general, the RBC folate concentration is generally
early placentation (Jongbloet et al., 2008; Koukoura et al., considered to reflect folate status during the preceding 3–4
2012). Folate deficiency may lead to poor placentation and months, and plasma or serum folate is a short-term measure

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FIGURE 5 | Funnel plot for studies of folic acid supplementation in relation to PTB risk. The vertical solid line represents the summary effect estimates, and the dotted
lines are pseudo 95% CIs.

reflecting fluctuation of dietary/supplement intakes over the past (Pickell et al., 2011), and it has been shown that starting folic
month (He et al., 2016). Theoretically, the two different folate acid supplementation before conception significantly increases
indicators are likely to be one source of heterogeneity. Although maternal RBC folate concentrations and prevents the decline in
no evidence was found that the sample type might have affected serum folate concentration after pregnancy, and this might be
results, our meta-analysis suggested that there was no significant beneficial to fetal growth (Bailey, 2009).
association between RBC folate and PTB risk, but a negative There have been concerns that high folic acid intake might
association was identified between plasma/serum folate and PTB be linked to abnormal embryonic development and long-term
risk. Additionally, the higher I 2 for the plasma/serum subgroup negative health outcomes in the offspring of mice (Dwarkanath
compared to RBCs suggests that different methods for measuring et al., 2013) and humans (Shaw et al., 2004), therefore, it
blood folate might be potential reasons for the heterogeneity, is necessary to evaluate the association between high folic
and the difference between serum/plasma and RBCs might had acid intake and the risk of PTB. However, a meta-analysis
affected the I 2 results to some degree. of randomized controlled trials found that higher folic acid
supplementation had no significant reduction of PTB risk
(Saccone and Berghella, 2016). Another meta-analysis found that
Folic Acid Supplementation and Risk of high folic acid reduced the risk of PTB (Zhang et al., 2017).
PTB In this meta-analysis, we found that folic acid supplementation
In accordance with a previous meta-analysis of observational was effective in reducing the risk of PTB only if the daily
studies, we found that folic acid supplementation reduced dose was <1,000 µg (moderate dose group), and folic acid
the risk for PTB. Moreover, we found that starting folic supplementation ≥1,000 µg per day (high dose group) did not
acid supplementation before conception was more effective influence the risk of PTB. The cutoff of 1,000 µg of folic acid
in reducing the risk of PTB compared with post-conception. supplementation was chosen because most prenatal vitamins
A population-based mega-cohort study came to the same contain <1,000 µg folic acid (Bailey, 2009).
conclusion as us (Nijhout et al., 2004). This seems biologically
plausible given that folate has a half-life of 100 days (Tamura Dietary Folate Intake and Risk of PTB
and Picciano, 2006). It has been well-established that folate In this meta-analysis, the included 3 studies were unable to
concentrations in the circulation decline as pregnancy advances differentiate between spontaneous preterm birth and iatrogenic

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FIGURE 6 | Forest plot of the meta-analysis of PTB risk in relation to folic acid supplementation stratified by initiation time (A) and dose of folic acid intake (B),
comparing the highest category with the lowest. The diamonds and horizontal lines indicate the subgroup-specific ORs and 95%CIs. The size of the gray area reflects
the study-specific statistical weight. The vertical solid line shows the OR of 1. The suffix “a” or “b” after the studies indicates two separate outcomes stratified by
initiation time of supplementation (preconception and post-conception) in the same study.

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FIGURE 7 | Forest plot of the meta-analysis of PTB risk (A) and sPTB risk (B) in relation to dietary folate, comparing the highest category with the lowest. The
diamonds and horizontal lines indicate the corresponding ORs and 95% CIs. The size of the gray area reflects the study-specific statistical weight. The vertical solid
line shows the OR of 1, and the vertical red dashed line represents the combined effect estimate.

preterm birth and thus the two were equated. We found that Limitations of the Study
dietary folate intake showed a strong inverse association with There were several limitations in the present meta-analysis.
the overall risk of PTB, which was consistent with a previous a First, we did not include RCTs in the current meta-
cross-sectional study (Deniz et al., 2018). As for the 4 studies analysis. Because of ethical issues, few RCTs have been
specifically examining sPTB, dietary folate intake showed no conducted that have studied the association between folic
significant reduction in the risk of sPTB. These results should be acid supplementation and PTB compared to placebo or to
interpreted with caution due to the limited data, however, and no supplementation. Second, there were not enough studies
future studies are required to address these issues. to explore the dose-response trend of blood folate levels and

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folic acid supplementation in relation to PTB risk. Third, AUTHOR CONTRIBUTIONS

only a relatively small number of studies on the association
between dietary folate intake and PTB risk have been published, BL conceptualized and designed the study, collected and
so conclusions should be drawn with caution. Moreover, organized the data, and drafted the initial manuscript. XZ
we defined folic acid supplementation as folic acid alone collected and organized the data, reviewed the included articles,
or folic acid-containing vitamins, and this might have led and conducted the analyses. XP and SZ collected and organized
to the introduction of clinical heterogeneity. We were also the data and reviewed the included articles. XW conceptualized
unable to assess the mutual effect of multivitamins because and designed the study and critically reviewed and revised
of insufficient information. In addition, major risk factors for the manuscript. CZ conceptualized and designed the study,
PTB, including socioeconomic status, lifestyle factors, and coordinated and supervised data collection, and critically
adverse health behaviors were difficult to control for in the reviewed and revised the manuscript. All authors read and
included studies. approved the final manuscript.

FUNDING
CONCLUSIONS
This work was supported by the National Key Research and
In summary, the results of the present meta-analysis suggest Development Program of China (2018YFC1004604), the
that higher folate levels and folic acid supplementation are National Nature Science Foundation of China (31761133015,
significantly associated with a lower overall risk of overall PTB. U1704281), the Swedish Research Council (2015-02845,
Dietary folate intake seemed to be significantly associated with a 2015-06276), Swedish Governmental grants to scientists
decreased risk of overall PTB and was not associated with risk of working in health care (ALFGBG-717791, ALFGBG-
sPTB. However, this should be interpreted with caution because 429801), and the Henan Provincial Science and Technology
of the small number of studies. Subgroup analyses indicated that Department (171100310200).
higher maternal folate levels in late pregnancy are associated with
lower PTB risk and that initiating folic acid supplementation ACKNOWLEDGMENTS
early before conception has a significant protective effect
against PTB. The authors thank Dr. Yong Gan, lecturer, School of Public
Therefore, considering the increasing numbers of preterm Health, Tongji Medical College, Huazhong University of Science
infants and recent reports on the neuroprotective effect of folate and Technology, Wuhan, China, for his assistance in developing
intake during pregnancy (Julvez et al., 2009), pregnant women the statistical strategy.
should reinforce and start folate intake early before conception
in order to reduce the risk of PTB and subsequent risks SUPPLEMENTARY MATERIAL
for long-lasting neurodevelopmental impairments. Additionally,
moderately decreased folate levels in late pregnancy might The Supplementary Material for this article can be found
increase the risk of PTB, and this will help with clinical risk online at: https://www.frontiersin.org/articles/10.3389/fnins.
stratification and patient counseling. 2019.01284/full#supplementary-material

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pregnancy increases β-cell numbers at 16 days of life in growth-restricted twin Conflict of Interest: The authors declare that the research was conducted in the
lambs. Am. J. Physiol. Endocrinol. Metab. 313, E381–E90. doi: 10.1152/ajpendo. absence of any commercial or financial relationships that could be construed as a
00033.2017 potential conflict of interest.
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Clin. Nutr. 83, 993–1016. doi: 10.1093/ajcn/83.5.993 Copyright © 2019 Li, Zhang, Peng, Zhang, Wang and Zhu. This is an open-access
The Lancet (2016). The unfinished agenda of preterm births. Lancet 388:2323. article distributed under the terms of the Creative Commons Attribution License (CC
doi: 10.1016/S0140-6736(16)32170-5 BY). The use, distribution or reproduction in other forums is permitted, provided
Timmermans, S., Jaddoe, V. W., Hofman, A., Steegers-Theunissen, R. P., and the original author(s) and the copyright owner(s) are credited and that the original
Steegers, E. A. (2009). Periconception folic acid supplementation, fetal growth publication in this journal is cited, in accordance with accepted academic practice.
and the risks of low birth weight and preterm birth: the Generation R Study. Br. No use, distribution or reproduction is permitted which does not comply with these
J. Nutr. 102, 777–785. doi: 10.1017/S0007114509288994 terms.

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 ORIGINAL RESEARCH
published: 17 January 2020
doi: 10.3389/fgene.2019.01314

Sex-Dimorphic Interactions of MAOA

Genotype and Child Maltreatment
Predispose College Students to
Polysubstance Use
Paula J. Fite 1,2*, Shaquanna Brown 1,2, Waheeda A. Hossain 1,3, Ann Manzardo 1,3,
Merlin G. Butler 1,3 and Marco Bortolato 1,4*
1 Consortium for Translational Research on Aggression and Drug Abuse (ConTRADA), University of Kansas, Lawrence, KS,

United States, 2 Clinical Child Psychology Program, University of Kansas, Lawrence, KS, United States, 3 Departments of
Psychiatry and Behavioral Sciences and Pediatrics, University of Kansas Medical Center, Kansas City, KS, United States,
4 Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT, United States

Edited by:
Polysubstance use (PSU) is highly prevalent among college students. Recent evidence
Carla Cannizzaro, indicates that PSU is based on gene x environment (G×E) interactions, yet the specific
University of Palermo, biosocial factors underlying this problem remain elusive. We recently reported that lifetime
Italy
use of tobacco and cannabis in college students is influenced by the interaction of the X-
Reviewed by:
Nela Pivac, linked MAOA (monoamine oxidase A) gene and child maltreatment. Building on these
Rudjer Boskovic Institute, premises, here we evaluated whether the same G×E interaction may also predict PSU in
Croatia
Yong-Kyu Kim,
this population. Students of a large Midwestern university (n = 470; 50.9% females) took
Howard Hughes Medical part in a computer survey for substance use, as well as childhood trauma exposure, using
Institute (HHMI), United States the Child Trauma Questionnaire (CTQ). DNA was extracted from their saliva samples and
*Correspondence: genotyped for MAOA variable-number of tandem repeat (VNTR) variants. Findings
Paula J. Fite
pfi[email protected] indicated that the highest number of substances were used by male students
Marco Bortolato harboring low-activity MAOA alleles with a history of childhood emotional abuse. In
[email protected]
contrast, female homozygous high-activity MAOA carriers with a history of emotional
Specialty section:
and physical abuse reported consumption of the greatest number of substances. Our
This article was submitted to results indicate that PSU among college students is influenced by the interaction of MAOA
Behavioral and Psychiatric Genetics,
and child maltreatment in a sex-specific fashion. Further studies are warranted to
a section of the journal
Frontiers in Genetics understand the mechanisms of sex differences in the biosocial interplays underlying
Received: 27 September 2019 PSU in this at-risk group.
Accepted: 02 December 2019
Published: 17 January 2020 Keywords: polysubstance use, MAOA, child maltreatment, sex differences, gene × environment interactions

Citation:
Fite PJ, Brown S, Hossain WA,
Manzardo A, Butler MG and INTRODUCTION
Bortolato M (2020)
Sex-Dimorphic Interactions of MAOA Polysubstance use (PSU) is a major health concern that has garnered much attention from clinicians
Genotype and Child Maltreatment
and researchers, due to its robust association with substance use disorders and other negative
Predispose College Students to
Polysubstance Use.
outcomes throughout the lifespan (McCabe et al., 2006; Trenz et al., 2012; Moss et al., 2014). Recent
Front. Genet. 10:1314. surveys have ascertained that PSU risk is particularly high among college students (Gledhill-Hoyt
doi: 10.3389/fgene.2019.01314 et al., 2000; Johnston et al., 2004; Mohler-Kuo et al., 2003; Barrett et al., 2006; National Center on

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1 January 2020 | Volume 10 | Article 1314
Fite et al. Gene–Environment Interactions in Polydrug Use

Addiction and Substance Abuse at Columbia University, 2007; 2011). Low-activity uVNTR alleles (hereafter designated as
O'Grady et al., 2008) with alcohol, tobacco, and cannabis being MAOA-L), for example, are associated with a younger age of
the three most widely used substances in this population (Lipari onset of alcohol dependence (Vanyukov et al., 1995; Vanyukov
and Jean-Francois, 2016). Indeed, these drugs share similar et al., 2004) and antisocial alcoholism (Samochowiec et al., 1999)
trajectories of use among emerging adults, with high rates of in males. A history of maltreatment predisposes female carriers of
comorbidity (Jackson et al., 2008) and simultaneous high-activity alleles (MAOA-H) or male MAOA-L carriers to a
consumption (Martin et al., 1992; Baggio et al., 2014). greater risk of alcohol use (Nilsson et al., 2011). In alignment with
Vulnerability to PSU, and more generally to substance use these findings, we found that greater lifetime tobacco use was
disorders and related behavioral phenotypes (including predicted by the interaction of childhood maltreatment and
externalizing psychopathology), is strongly influenced by both MAOA-L variants in males and MAOA-H alleles in females
genetic (Uhl et al., 2001; Dick et al., 2009) and environmental (Fite et al., 2018).
factors. Several genes implicated in the predisposition to Given these premises, the present study tested the hypothesis
substance use disorders have been shown to be related to that the same gene x environment (G×E) interactions may
monoamine neurotransmitters, such as serotonin, dopamine, predispose to PSU in college students and analyzed whether
and norepinephrine (Guo et al., 2007; Ducci and Goldman, the influence of these biosocial interplays may follow a sex-
2012); these molecules are known to serve a pivotal role in the dimorphic pattern.
pathophysiology of drug abuse (Volkow et al., 2007; Fitzgerald,
2013; Müller and Homberg, 2015). Early-life adversity, and
particularly child maltreatment is another well-known variable
associated with high risk of PSU (Galaif et al., 2001; Leeb et al.,
METHODS
2008; Goldstein et al., 2013; Cohen et al., 2017). It has been Participants
estimated that ~70% of adolescents receiving substance abuse Participants were 470 students (239 females and 231 males; see
treatment have a history of trauma (Funk et al., 2003), and that Table 1) enrolled in undergraduate psychology courses at a large
maltreated children are 300% more likely to develop substance Midwestern university. Recruitment was based on SONA, an
abuse (Kilpatrick et al., 2003). According to recent conceptual online system that allows students to electronically sign up to
frameworks, the pathogenic influence of child maltreatment and participate in active studies at the university. Most students
other forms of early stress on PSU is moderated by genetic (71.1%) identified as Caucasians, attended the first year of
factors (Vink, 2016). However, only limited data are available on college (61.1%) and reported that their parents had a higher
the specific interactions of heritable factors and child educational level than high school (80.9% of fathers and 79.7%
maltreatment with respect to PSU predisposition. of mothers).
We recently showed that, among college students, tobacco
and cannabis consumption is influenced by the interaction of Procedures
child maltreatment and the gene MAOA, the X-linked gene All study procedures were approved by the researchers'
encoding for monoamine oxidase A (Fite et al., 2018). In line Institutional Review Board. All participants were instructed to
with our report, Stogner and Gibson (2013) also documented abstain from eating for 1 h before the study, and refrain from the
that the interplay of this gene with lifetime stress increases the use of any drug (including prescription medicines and
risk for initiation to alcohol and cannabis use in male caffeinated beverages) for at least 3 h before the study. Upon
adolescents. Monoamine oxidase A catalyzes the degradation arrival, they were given a complete summary of the study and
of serotonin, norepinephrine and dopamine (Bortolato et al., provided informed consent. Subsequently, participants rinsed
2008). The best-characterized MAOA functional polymorphism their mouth with water and, ten minutes later, were instructed to
is a 30-bp variable number tandem repeat located in its promoter give 2 ml of saliva in a tube for genetic analyses. Then, they
region (uVNTR) (Sabol et al., 1998). The six alleles of this provided demographic information, including their age and race/
genotype feature different numbers of repeats (2, 3, 3.5, 4, 5, ethnicity, and completed a Qualtrics online survey in about 1 h.
and 6) (Huang et al., 2004), in association with different At the end of the study, participants were compensated with a $5
transcriptional efficiency and enzyme activity. The two- and debit card for the saliva sample and 3 SONA credits for the
three-repeat variants, which are associated with low activity survey. To keep the identity of participants anonymous, survey
(Sabol et al., 1998; Deckert et al., 1998; Denney et al., 1999), responses and saliva samples were assigned a unique ID without
confer a greater risk for externalizing psychopathology in male any identifying information.
carriers with a history of maltreatment (Caspi et al., 2002; Kim-
Cohen et al., 2006; Williams et al., 2009; Fergusson et al., 2011). Questionnaires
A large body of evidence has documented that MAOA uVNTR The survey included the following questionnaires:
variants exert a sex-dimorphic influence on the overall risk and
specific clinical manifestations of alcohol use disorders, both per 1. the Child Trauma Questionnaire (CTQ), a standardized self-
se and in interaction with early-life adversity (Samochowiec et al., report instrument for the retrospective assessment of trauma
1999; Schmidt et al., 2000; Vanyukov et al., 2004; Guindalini exposure during childhood (Bernstein and Fink, 1998). The
et al., 2005; Herman et al., 2005; Ducci et al., 2008; Nilsson et al., CTQ consists of 5 subscales of trauma (physical abuse,

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Fite et al. Gene–Environment Interactions in Polydrug Use

TABLE 1 | Participant demographics and descriptive statistics.

Overall Sample (n = 470) Males (n = 231) Females (n = 239)

M (SD) Age 18.95 (1.19) 19.14 (1.25) 18.76 (1.10)

Year in school
% 1st year student 61.1 55.8 66.1
% 2nd year student 27.4 29.4 25.5
% 3rd year student 8.9 11.7 6.3
% 4th year student 1.9 2.6 1.3
% 5th year or more student 0.7 0.5 0.8
Race/Ethnicity
% Caucasian 71.1 72.7 69.5
% African American 3.6 3.0 4.2
% Hispanic/Latino 6.2 4.8 7.5
% Native American 1.3 .9 1.7
% Asian 10.6 10.4 10.9
% Mixed or other 7.2 8.2 6.2
Medical History
% Psychological Disorder 13.2 10.4 15.9
% Current Illness/Injury 3.4 3.5 3.3
% Currently Medications 43.4 25.1 61.1
Parental Education at birth
% Fathers greater than high school 80.9 81.0 78.4
% Mothers greater than high school 79.7 83.8 78.2

emotional abuse, sexual abuse, physical neglect, and emotional for comparability between males and females, MAOA-LL and
neglect) with multiple items based on a 5-point Likert scale MAOA-LH female participants were combined (n = 165), in
format. Mean scores for each subscale, as well as an overall agreement with previous functional studies on sex-dimorphic
child maltreatment score, were calculated. The physical effects of MAOA uVNTR variants (Fan et al., 2003; Meyer-
neglect subscale yielded the lowest reliability coefficient (a = Lindenberg et al., 2006; Frazzetto et al., 2007; Buckholtz et al.,
0.56) in the current sample; internal consistencies for the 2008; Dannlowski et al., 2009) The validity of this approach was
remaining four subscales were good (with all a's > 0.81); confirmed by analyzing the interactions of MAOA genotype
2. A substance use questionnaire. based on three items from the variants (MAOA-LL, MAOA-HH, and MAOA-LH) and
Center for Substance Abuse Prevention (CSAP) Student maltreatment types in female participants. The results of these
Survey (Pentz et al., 1989), a self-report instrument assessing analyses indicated that MAOA-LH genotype operated consistent
lifetime tobacco (i.e., “Have you ever smoked a cigarette, even with the MAOA-LL genotype in its interaction with
just a few puffs, or used chewing tobacco, snuff, or dip), maltreatment types to predict PSU. All genotypic and
alcohol (i.e., “Have you ever had a drink of alcohol?”), and phenotypic data are presented as Supplementary Materials.
cannabis use (i.e., “Have you ever tried marijuana?”). The
number of substances used by each participant was calculated Data Analysis
(ranging from 0 to 3). Of the original 500 students recruited for the study, MAOA
genotyping could not be performed for 11 participants, while 11
MAOA uVNTR Variants Genotyping participants were missing CTQ and/or substance use data. We
DNA extracted and MAOA-uVNTR genotyping were performed further excluded 8 participants (4 males and 4 females) carrying
as previously described (Fite et al., 2018). All laboratory 5-repeat uVNTR alleles. Based on power tables (Aiken and West,
procedures were carried out by personnel blind to the 1991), it was determined that the current sample had adequate
demographic and psychological characteristics of the subject power (a = 0.80) to detect moderate to large, but not small,
(other than gender). All genotype data of participants are MAOA × maltreatment interaction effects for males and females.
shown in Table 2. Given that the MAOA gene is located on No differences in sex or age (ps > 0.48) or in child maltreatment
the X chromosome, males were designated as either low-activity scores (ps > 0.16) were found in the comparison between the
(MAOA-L) or high-activity (MAOA-H) hemizygous, depending participants included in and excluded from the analyses.
on the number of repeats of their allelic variant (2 and 3 vs 3.5 Multiple regression models were used to evaluate proposed
and 4, respectively). Conversely, females were either associations. Substance use count was the dependent variable
homozygous for either allele (MAOA-LL or MAOA-HH) or in each model, with sex, MAOA variant, and maltreatment types
heterozygous carriers (MAOA-LH). In line with previous included as independent variables. All five maltreatment types
studies on MAOA (Byrd and Manuck, 2014), carriers of 5- were included in each model to evaluate unique associations.
repeat uVNTR alleles were excluded from the analyses, as the Three-way interactions (e.g., sex × MAOA variant ×
actual functional significance of this variant remains maltreatment type) were then evaluated one at a time to
controversial (Sabol et al., 1998; Deckert et al., 1998). To allow determine if child maltreatment-MAOA interactive effects

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Fite et al. Gene–Environment Interactions in Polydrug Use

TABLE 2 | Genotypic data of all participants. Genotypes containing 5-repeat variants were not included in either MAOA low-activity (MAOA-L) or high-activity (MAOA-H)
allele groups. For more details, see text.

MALES

Number of repeats Number Percentage

MAOA-L 2 1 0.43%
3 93 39.57%
MAOA-H 3.5 10 4.26%
4 127 54.04%
Excluded genotypes 5 4 1.70%

FEMALES

Number of repeats Number Percentage

MAOA-LL 2-2 1 0.41%

2-3 1 0.41%
3-3 42 17.28%
MAOA-LH 2-3.5 0 0%
2-4 1 0.41%
3-3.5 2 0.82%
3-4 118 48.56%
MAOA-HH 3.5-3.5 0 0%
3.5-4 4 1.65%
4-4 70 28.81%
Excluded genotypes 2-5 0 0%
3-5 0 0%
3.5-5 0 0%
4-5 4 1.65%

depended on sex. All independent variables were mean centered TABLE 3 | Three-way interaction regression analyses. Significant results are
indicated in bold.
prior to analyses to aid in the interpretation of interaction effects.
Statistically significant interactions were probed based on sex SU Count
(male vs. female) and for MAOA variants to determine the
B p
nature of the interactions, consistent with standard procedures
(Aiken and West, 1991). Sexual Abuse 0.43 0.39
Emotional Neglect 0.40 0.15
Physical Abuse 1.37 0.00
Emotional Abuse 0.58 0.04
RESULTS Physical Neglect 0.54 0.26
Any Maltreatment 1.36 0.00
Approximately 11.5% of the sample had not used any substance,
28.9% of the sample had used one substance, 23.2% had used two
substances, and 36.4% of the sample had used three substances. The statistically significant three -way interactions with any
Based on the clinical cutoff scores recommended by Bernstein maltreatment type, physical abuse, and emotional abuse were
and Fink (1998), ~46.5% of the sample reported at least low levels further evaluated by conducting tests of the simple slopes
of one or more maltreatment types. This percentage is consistent (Table 4). Specifically, the models were conditioned at MAOA-H
with previous data on undergraduate, emerging adult samples and MAOA-L for both males and females to determine the patterns
(Reichert and Flannery-Schroeder, 2014). of associations. For MAOA-L males, there was a marginally
Regression analyses indicated a significant three-way interaction statistically trend for any maltreatment type (B = 0.42, p = 0.08)
when examining any experience of maltreatment (B = 1.36, p = 0.00; (Figure 1A) and statistically significant effect for and emotional
see Table 3). Additionally, a significant three -way interaction was abuse (B = 0.38, p = 0.03) (Figure 1C) to be positively associated
found for physical abuse (B = 1.37, p = 0.00) as well as emotional with the number of substances used. However, an association
abuse (B = 0.58, p = 0.04). However, no significant three -way between physical abuse and number of substances used was not
interactions were found for any other child maltreatment type: found (B = 0.26, p = 0.17) (Figure 1B). For MAOA-H males, any
physical neglect (B = 0.54, p = 0.26), emotional neglect (B = 0.40, p = maltreatment type was marginally statistically negatively associated
0.15), or sexual abuse (B = 0.43, p = 0.39). Additionally, no (B = -0.42, p = 0.07) (Figure 1A) and physical abuse was statistically
significant two-way interactions between maltreatment variables negatively associated (B = -0.33, p = 0.03) with the number of
and MAOA alleles were evident (ps > 0.12). substances used (Figure 1B). Emotional abuse (B = -0.05, p = 0.77)

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TABLE 4 | Simple-slope analyses of three-way interactions. SE, standard error. *p < 0.05; +p < 0.09.

Males Females

MAOA – L MAOA – H MAOA – LL+ MAOA-LH MAOA – HH

B SE B SE B SE B SE

Any Maltreatment 0.42+ 0.24 –0.42+ 0.23 0.00 0.16 0.52* 0.26
Physical Abuse 0.26 0.19 –0.33* 0.15 –0.25 0.19 0.54* 0.24
Emotional Abuse 0.38* 0.17 –0.05 0.17 0.19 0.14 0.34* 0.17

FIGURE 1 | Associations between child maltreatment types and substance use count for male and female carriers of MAOA uVNTR variants. (A) Overall
associations with child maltreatment scores. (B) Associations with physical abuse scores. (C) Associations with emotional abuse scores.

was statistically unrelated to number of substances used for remain mostly elusive; if confirmed by future studies, our results
MAOA-H males (Figure 1C). may point to MAOA as a key molecular basis for PSU.
In contrast, for female carriers of low-activity MAOA variants The present findings extend our previous report of sex-
(MAOA-LL and MAOA-LH), there was no association evident dimorphic influences of G×E interactions in the lifetime use of
between any maltreatment type (B = 0.00, p = 0.99) (Figure 1A), tobacco (Fite et al., 2018) among college students. Furthermore,
physical abuse (B = -0.25, p = 0.18) (Figure 1B), or emotional these results are consistent with previous evidence indicating sex
abuse (B = 0.19, p = 0.17) (Figure 1C) and number of substances differences in the interactive influence of these G×E interactions
used. For homozygous MAOA-H females, there was a statistically with respect to antisocial conduct (Nikulina et al., 2012; Stogner and
significant positive association between any maltreatment type Gibson, 2013; Byrd and Manuck, 2014; Harro and Oreland, 2016)
(B = 0.52, p = 0.04) (Figure 1A), and physical abuse (B = 0.54, p = and alcohol use (Nilsson et al., 2011). The interaction of MAOA
0.03) (Figure 1B), and emotional abuse (B = 0.34, p = 0.04) alleles and child maltreatment can be interpreted from the
(Figure 1C) and number of substances used. perspective of the diathesis-stress model, which postulates that
the predisposition to specific neurobehavioral deficits is the result
of a synergistic combination of genetic and environmental
untoward factors (Zuckerman, 1999). Another alternative
DISCUSSION interpretation follows the differential susceptibility hypothesis,
which posits that specific genetic variables may sensitize to both
The results of the current study showed that, in a sample of students the positive and the negative influence of early experiences (Ellis
enrolled in a large Midwestern university, PSU was predicted by the et al., 2011). This possibility is partially supported by Belsky and
interaction of MAOA uVNTR allelic variants, sex, and specific child colleagues (Belsky et al., 2009; Belsky and Beaver, 2011), who
maltreatment types. The highest number of substances used was have conceptualized that MAOA variants may act as plasticity
found in MAOA-L male and MAOA-HH female carriers with a factors in the predisposition to substance use and other
history of emotional abuse (as well as physical abuse in women). To psychopathological conditions.
our knowledge, this is the first report documenting a key role of In line with previous data (Armour et al., 2014), the current
MAOA as a mediator of child maltreatment with respect to PSU. results highlight the importance of examining specific
While previous studies have shown the importance of G×E maltreatment types in relation to PSU. Our findings suggest
interactions in PSU (Vaughn et al., 2009; Rende, 2011), the that, although physical abuse and emotional abuse interact with
specific genetic factors implicated in such biosocial interplays MAOA variants to predict PSU even when statistically controlling

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for the other maltreatment types, no interaction effects were Trentacosta and Shaw, 2009; Gillespie et al., 2018), as well as
found for sexual abuse, physical neglect, or emotional neglect. in the role of G×E interactions in PSU (Vaughn et al., 2009). The
This evidence is partially consistent with a previous study by effects of MAOA on ACC activation patterns are sex-dimorphic;
Nikulina and colleagues (2012) suggesting that MAOA does not specifically, MAOA-L male and MAOA-H female carriers with a
serve as a protective or risk factor for substance use outcomes history of early stress display impairments in the activation of the
among individuals who have experienced childhood sexual abuse. ACC in response inhibition (Holz et al., 2016), a process directly
However, in contrast with our results, the results of that related to self-regulation (Posner and Rothbart, 1998; Blair and
investigation showed that alcohol use was not predicted by the Ursache, 2011; Hofmann et al., 2012). It should be noted that
interaction of MAOA with either physical abuse or neglect. Given functional deficits of the ACC are associated with a reduction in
that the participants of that study ranged between 31 and 51 years inhibitory control (Bush et al., 2000; Chan et al., 2011), as well as
of age, it is possible that the discrepancy with those results may a facilitation of ventral striatal responses to incentive stimuli,
reflect age differences; accordingly, the moderating effect of which in turn increases drug use propensity (Holmes et al., 2016;
MAOA on child maltreatment and negative outcomes has been Koyama et al., 2017). Notably, these deficits may be particularly
hypothesized to be age-dependent (Huizinga et al., 2006). overt in young individuals (and therefore highly relevant in the
Alternatively, these divergent findings may result from other age range of college students), due to their incomplete
differences between our studies, including the substance use myelination of the ACC as well as the development of the
outcomes (i.e., PSU vs alcohol abuse) and measurement of child dopaminergic system, which further exacerbates their proclivity
maltreatment (i.e., self-report vs official records). Nevertheless, to engage in impulsive and risky actions and heightens their
research shows that experiences of child maltreatment are reward sensitivity (Casey et al., 2008; Steinberg, 2008). At least in
associated with decreased propensity for reward selection, females, the presence of MAOA-H alleles may further reduce
which could be due to lower reward sensitivity (Guyer et al., dopamine levels, ultimately promoting the ontogeny of reward
2006). In turn, this dual risk might increase the risk of PSU. Thus, deficiency syndrome (Blum, 2017; Blum et al., 2018). From this
child maltreatment types, physical abuse and emotional abuse perspective, these results suggest that the interaction of MAOA-L
may be more saliently associated with blunted reward sensitivity. alleles in males and MAOA-H in females and early-life
The existence of sex-dimorphic G×E interactions involving maltreatment may interfere with the development of inhibitory
MAOA uVNTR alleles has been attested in other control in emerging adulthood, ultimately increasing PSU risk.
psychopathological states. For example, male carriers of Several limitations of this study should be acknowledged.
MAOA-L alleles with a history of child maltreatment have a F i r s t , o u r a n a l y s e s f o c u s e d e xc l u s i v e l y o n M A O A
significantly higher risk of antisocial, aggressive, and violent polymorphisms, yet several studies point to the importance of
behavior (Caspi et al., 2002; Kim-Cohen et al., 2006; Beaver many other genes in the vulnerability to PSU, such as those
et al., 2010; Aslund et al., 2011; Fergusson et al., 2011; Fergusson encoding for dopamine receptor 2 and 4 as well as dopamine and
et al., 2012; Byrd and Manuck, 2014; Godar et al., 2016). Notably, serotonin transporters (Blum et al., 2010); further studies are
the same G×E interaction has been reproduced in mouse models, needed to evaluate the potential interaction of child
further supporting the biological nature of this biosocial interplay maltreatment with these vulnerability factors. Second, although
(Godar et al., 2019). Conversely, female carriers of MAOA-H rich literature has documented that MAOA variants interact with
alleles with a positive history for early-life adversity display a childhood maltreatment to increase the propensity for
higher proclivity for antisocial and violent responses (Sjöberg externalizing behaviors, our findings need to be replicated in
et al., 2007; McGrath et al., 2012; Verhoeven et al., 2012). It has larger samples from multiple colleges and with less skewed ethnic
been hypothesized that this effect may reflect the enhancement of distribution. Indeed, our sample comprised of predominantly
emotional reactivity during adolescence (Byrd et al., 2018). Caucasian youth, which may limit the generalizability of current
Furthermore, these effects may reflect sex- and genotype- results. Second, this study relied solely on self-reports of
specific differences in the effects of MAOA on monoamine constructs, with a low internal consistency associated with our
metabolism (Jönsson et al., 2000; Aklillu et al., 2009). Notably, measure of physical neglect. Future research examining
aggression and delinquency have been extensively linked to PSU, associations in other samples (e.g., clinical and criminal) using
particularly in boys (McCormick and Smith, 1995; Mason and multiple, psychometrically sound assessments of constructs
Windle, 2002; Martinotti et al., 2009). This concurrence strongly would be useful for establishing generalizability of findings.
suggests that the G×E interaction of MAOA genotype and child Finally, our research combined two- and three-repeat variant
maltreatment may predispose to a broad set of externalizing carriers in the MAOA-L group; however, previous studies,
responses, ranging from antisocial personality to PSU however, have shown that, in males, two-repeat alleles resulted
propensity. In line with this interpretation, neuroimaging in much lower levels of promoter activity as well as stronger
studies have pointed to MAOA as a key molecule to influence phenotypic effects than the three-repeat genotype (Sabol et al.,
the function of the anterior cingulate cortex (ACC) (Passamonti 1998; Guo et al., 2008). Notably, two-repeat variants have shown
et al., 2008). This region plays a major role in the regulation of to increase antisocial phenotypes, including the propensity to
self-regulation (Posner et al., 2007), the key domain implicated in engage in particularly violent conduct (such as shooting and
the ontogeny of antisocial behavior (Gardner et al., 2008; stabbing), in African-American males (Beaver et al., 2013; Beaver

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Fite et al. Gene–Environment Interactions in Polydrug Use

et al., 2014). Unfortunately, given that only one male participant AUTHOR CONTRIBUTIONS
was found to carry the two-repeat alleles, our analyses were not
sufficiently powered to differentiate across specific genotypes; PF conceptualized the project, supervised data collection and
however, future studies will be needed to verify whether specific analysis, and drafted the first draft of the manuscript. SB helped
differences may be identified with respect to the interaction of with data collection and analysis and helped draft the first
specific variants with early maltreatment. version of the manuscript. WH performed genotyping analyses
Despite these limitations, the current study contributes to the and drafted part of the method sections. AM and MGB
growing literature indicating sex differences in genetic risk of contributed to the conceptualization of the study and reviewed
MAOA in addition to the importance of the interactive genotyping data. MB conceptualized the project, reviewed data
influences of genetic and environmental risk for PSU. Further, analysis, and wrote the final version of the manuscript. All
findings indicate the importance of evaluating specific authors have read and approved the final version of
maltreatment types to better understand MAOA and the manuscript.
maltreatment interactive risks for substance use.

ACKNOWLEDGMENTS
DATA AVAILABILITY STATEMENT The present manuscript was supported by a University of Kansas
Strategic Initiatives Grant (to PF and MB), the National Institute
All datasets generated for this study are included in the article/
of Mental Health (NIH R01 MH104603 to MB), and the ALSAM
Supplementary Material.
Foundation (to MB).

ETHICS STATEMENT SUPPLEMENTARY MATERIAL

The studies involving human participants were reviewed and The Supplementary Material for this article can be found online
approved by IRB University of Kansas. The patients/participants at: https://www.frontiersin.org/articles/10.3389/fgene.2019.
provided their written informed consent to participate in this study. 01314/full#supplementary-material

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Dopamine in drug abuse and addiction: results of imaging studies and these terms.

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 ORIGINAL RESEARCH
published: 29 January 2020
doi: 10.3389/fnins.2020.00005

Environmental Tobacco Smoke

During the Early Postnatal Period of
Mice Interferes With Brain 18F-FDG
Uptake From Infancy to Early
Adulthood – A Longitudinal Study
Larissa Helena Torres 1,2 , Caroline Cristiano Real 3 , Walter Miguel Turato 1 ,
Lídia Wiazowski Spelta 1 , Ana Carolina Cardoso dos Santos Durão 1 ,
Tatiana Costa Andrioli 1 , Lorena Pozzo 4 , Peterson Lima Squair 4 , Marco Pistis 5 ,
Daniele de Paula Faria 3 and Tania Marcourakis 1*
Edited by: 1
Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo,
Carla Cannizzaro, São Paulo, Brazil, 2 Departamento de Alimentos e Medicamentos, Faculdade de Ciências Farmacêuticas, Universidade
University of Palermo, Italy Federal de Alfenas, Alfenas, Brazil, 3 Laboratory of Nuclear Medicine (LIM-43), Departamento de Radiologia e Oncologia,
Reviewed by: Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil, 4 Instituto de Pesquisas Energéticas e Nucleares,
Ramon Guirado, São Paulo, Brazil, 5 Department of Biomedical Sciences and CNR Institute of Neuroscience, Faculty of Medicine
University of Valencia, Spain and Surgery, University of Cagliari, Cagliari, Italy
Yongsoo Kim,
Penn State Health Milton S. Hershey
Medical Center, United States Exposure to environmental tobacco smoke (ETS) is associated with high morbidity and
*Correspondence: mortality, mainly in childhood. Our aim was to evaluate the effects of postnatal ETS
Tania Marcourakis exposure in the brain 2-deoxy-2-[18 F]-fluoro-D-glucose (18 F-FDG) uptake of mice by
[email protected]
positron emission tomography (PET) neuroimaging in a longitudinal study. C57BL/6J
Specialty section: mice were exposed to ETS that was generated from 3R4F cigarettes from postnatal
This article was submitted to day 3 (P3) to P14. PET analyses were performed in male and female mice during
Neurogenesis,
a section of the journal
infancy (P15), adolescence (P35), and adulthood (P65). We observed that ETS exposure
Frontiers in Neuroscience decreased 18 F-FDG uptake in the whole brain, both left and right hemispheres, and
Received: 23 September 2019 frontal cortex in both male and female infant mice, while female infant mice exposed
Accepted: 07 January 2020
to ETS showed decreased 18 F-FDG uptake in the cerebellum. In addition, all mice
Published: 29 January 2020
showed reduced 18 F-FDG uptake in infancy, compared to adulthood in all analyzed
Citation:
Torres LH, Real CC, Turato WM, VOIs. In adulthood, ETS exposure during the early postnatal period decreased brain 18 F-
Spelta LW, dos Santos Durão ACC, FDG uptake in adult male mice in the cortex, striatum, hippocampus, cingulate cortex,
Andrioli TC, Pozzo L, Squair PL,
Pistis M, de Paula Faria D and
and thalamus when compared to control group. ETS induced an increase in 18 F-FDG
Marcourakis T (2020) Environmental uptake in adult female mice when compared to control group in the brainstem and
Tobacco Smoke During the Early
cingulate cortex. Moreover, male ETS-exposed animals showed decreased 18 F-FDG
Postnatal Period of Mice Interferes
With Brain 18 F-FDG Uptake From uptake when compared to female ETS-exposed in the whole brain, brainstem, cortex,
Infancy to Early Adulthood – left amygdala, striatum, hippocampus, cingulate cortex, basal forebrain and septum,
A Longitudinal Study.
Front. Neurosci. 14:5.
thalamus, hypothalamus, and midbrain. The present study shows that several brain
doi: 10.3389/fnins.2020.00005 regions are vulnerable to ETS exposure during the early postnatal period and these

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1 January 2020 | Volume 14 | Article 5
Torres et al. ETS Modifies Brain 18 F-FDG Uptake

effects on 18 F-FDG uptake are observed even a long time after the last exposure.
This study corroborates our previous findings, strengthening the idea that exposure to
tobacco smoke in a critical period interferes with brain development of mice from late
infancy to early adulthood.
Keywords: environmental tobacco smoke, passive smoke, neuroimaging, positron emission tomography,
18 F-FDG uptake, glucose metabolism, longitudinal study, brain

INTRODUCTION use and 18 F-FDG uptake in the occipital gyrus and parietal–
temporal junction (Costello et al., 2010). However, there is
Exposure to environmental tobacco smoke (ETS), one of the most still a lack of studies evaluating the effects of tobacco smoke
common indoor pollutants, is composed of both mainstream and on glucose metabolism during the brain development period.
sidestream smoke. Approximately 40% of children in the world Thus, our aim was to investigate the effects of ETS during the
are exposed to ETS, which is related to allergic reactions in the early postnatal period on glucose metabolism in a longitudinal
short-term, while it is associated to acute myocardial infarction, preclinical study, by 18 F-FDG PET imaging during mice infancy,
lung cancer, and chronic obstructive pulmonary disease in the adolescence, and adulthood.
long term (Oberg et al., 2011).
Clinical studies show that ETS leads to behavioral disorders
and deleterious effects on the brain. The exposure to ETS is MATERIALS AND METHODS
related to attention deficits and hyperactive behavior during
childhood (Pagani, 2014), while maternal smoke during lactation Animals
causes sleep and wake disruption (Banderali et al., 2015). Also, C57BL/6 mice were obtained from the animal facility of the
paternal smoke in the early postnatal period of childhood School of Medicine of University of São Paulo and were housed
has been linked with perinatal mortality, respiratory disease, at 20–22◦ C with a 12 h/12 h light/dark cycle with water and
neurobehavioral problems, decreased academic performance, commercial pellet food for small rodents from Nuvital (Nuvilab
and brain tumors (Plichart et al., 2008; Hwang et al., 2012). CR-1; Colombo, Brazil) ad libitum. All of the procedures were
Adolescents exposed to tobacco smoke during prenatal period approved by the Ethics Committee of the School of Medicine
show distinct brain function in the working memory and (027/14) and the School of Pharmaceutical Sciences (P446/14),
alterations in the brain volume, especially in the cerebellum (de University of São Paulo.
Zeeuw et al., 2012; Bennett et al., 2013). In rodents, exposure to
mainstream smoke during a critical period of brain development Experimental Design
leads to hyperactivity and aggressive behavior (Yochum et al., The size of each litter was randomly adjusted to six to seven
2014), while exposure to ETS disturbs cognitive functions, pups within the first day after delivery, as previously described
synaptic proteins, and myelination process from late infancy to by Torres et al. (2015b). The C57BL/6 pups were exposed to
early adulthood (Torres et al., 2015a,b). ETS as described by Lobo-Torres et al. (2012). Briefly, the pups,
Positron emission tomography (PET) is a molecular imaging together with their mothers, were subjected to two exposure
technique that enables studying brain function in vivo. The 2- sessions per day of 1-h each (1 h at 8 a.m. and 1 h at 5 p.m.)
deoxy-2-[18 F]-fluoro-D-glucose (18 F-FDG) has been widely used to a mixture of mainstream and sidestream tobacco smoke from
to evaluate changes in cerebral glucose metabolism. 18 F-FDG reference cigarettes 3R4F (College of Agriculture, University of
is required in metabolically active tissues, and the metabolic Kentucky). The exposure was performed from the 3rd (P3)
activity of a brain region is directly proportional to the amount to the 14th (P14) days of life, within a chamber measuring
of 18 F-FDG that accumulates in this region (Sokoloff et al., 1977; 564 × 385 × 371 mm. The levels of CO in the chamber during
Welch et al., 2013). the exposure (470.2 ± 90.93 ppm) and measurements of the
Relatively few studies evaluated the effects of tobacco smoke exposure biomarkers (COHb: 21.62 ± 1.80%; plasma nicotine:
on brain glucose metabolism by PET imaging and focuses 139.94 ± 13.02 ng/mL; plasma cotinine: 113.65 ± 16.78 ng/mL)
on dependence by nicotine in humans. In a context of were similar to previous studies from our group (Torres et al.,
tobacco craving and exposure to cues that are related to 2015a,b). Control subjects were exposed to the same experimental
tobacco, heavy smokers showed rise in glucose metabolism in conditions but inhaled compressed air only.
the anterior cingulate gyrus, orbitofrontal cortex, dorsolateral Based on Vanhove et al. (2015), the number of animals
prefrontal cortex, anterior insula, and sensorimotor cortex required for imaging studies for changes about 20–25% is four
(Brody et al., 2002). In addition, smokers treated with bupropion, to six animals. Thus, in the present study, we opted to use five
a norepinephrine and dopamine reuptake inhibitor, showed animals of each sex in each group. Nonetheless, we used the
decrease in glucose metabolism in the anterior cingulate cortex isogenic C57Bl/6 mice in order to reduce intra-animal variability.
(Brody et al., 2004). Costello et al. (2010) reported that bupropion After the exposure period, 19 animals were used from P15
and practical group counseling reduce glucose metabolism in to P65 to evaluate the regional brain metabolism of the animals
the posterior cingulate gyrus, with association between cigarette with 18 F-FDG on PET/CT during infancy (P15; n = 5 females

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ETS-exposed and n = 4 females control; n = 5 males ETS-exposed n = 5 males control), and adulthood (P65; n = 5 females ETS-
and n = 5 males control), adolescence (P35; n = 4 females ETS- exposed and n = 4 females control; n = 5 males ETS-exposed and
exposed and n = 4 females control; n = 5 males ETS-exposed and n = 5 males control) in a longitudinal study.

FIGURE 1 | 18 F-FDG uptake in the whole brain (A), left hemisphere (B), right hemisphere (C), frontal cortex (D), and cerebellum (E) for female and male infant (n = 5
females ETS-exposed and n = 4 females control; n = 5 males ETS-exposed and n = 5 males control), adolescent (n = 4 females ETS-exposed and n = 4 females
control; n = 5 males ETS-exposed and n = 5 males control), and adult (n = 5 females ETS-exposed and n = 4 females control; n = 5 males ETS-exposed and n = 5
males control) mice exposed to ETS during the early postnatal period. Three-way mixed ANOVA with repeated measures (groups × VOIs) and post hoc paired t-test
(Bonferroni) with multiple comparison correction. Continuous bar: p < 0.05. Dashed bar: trend toward statistical significance.

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18 F-FDG-PET/CT Imaging test with multiple comparison correction was performed to

Positron emission tomography/CT images were acquired test 18 F-FDG uptake differences between the time points
using a protocol modified from Welch et al. (2013). Briefly, and groups for each VOI (Figure 1). PET imaging of adult
animals received about 19 MBq (18.86 ± 2.70) of 18 F-FDG animals was analyzed by a two-way ANOVA, considering
intraperitoneally (i.p.). After 65 (67 ± 5) min of injection groups as between and VOIs (whole brain, brainstem, cortex,
(biodistribution period of the radiotracer), animals were cerebellum, amygdala, striatum, hippocampus, cingulate cortex,
anesthetized with isoflurane (2% in O2 ) and positioned in an basal forebrain and septum, thalamus, hypothalamus, and
equipment bed with the brain in the center of the field of view midbrain) as within-subject factors. Bonferroni post hoc with
(FOV). The scanner used was an Albira PET-SPECT-CT (Bruker multiple comparison correction was performed to test 18 F-
Biospin, Valencia, Spain), for small animals. The static PET FDG uptake differences between the groups for each VOI
image was acquired for 50 min with 94.4 mm of trans-axial (Figure 3). The data were analyzed using SPSS Statistics 20
FOV. A CT scan was acquired immediately after, with 400 Software, Armonk, NY: IBM Corp., United States and data
projections, 45 kVp, and 400 µA and magnification factor of were plotted using GraphPad Prism 6 Software, La Jolla, CA,
1.46. After acquisitions, PET images were reconstructed using United States. Results are presented as mean ± standard error.
maximum-likelihood expectation–maximization (MLEM), with Differences with a probability of 95% (p < 0.05) were considered
12 iterations, and corrected for radioactive decay, scatter, and statistically significant.
random, but not for attenuation. CT was reconstructed using
filtered back projection (FBP) algorithm.
RESULTS
PET Image Analysis
Positron emission tomography image analysis was performed ETS During the Early Postnatal Period
with PMOD 3.4 software (PMODTM Technologies Ltd., Decreased Brain 18 F-FDG Uptake in
Switzerland). The scans were manually co-registered to: (1) own Infant Mice
animal CT for analysis in the different animals’ age and (2) to Positron emission tomography scan data of glucose uptake for
a T2 weighted MRI template (available in the PMOD software) male and female infant, adolescent, and adult mice exposed to
in the adult animals’ analysis to facilitate the identification of ETS during the early postnatal period were analyzed by a three-
different brain regions. way mixed ANOVA with repeated measures (groups × VOIs)
In the analysis of the brain in the different ages, manual
volumes of interest (VOIs) were drawn in the PET images fused
to the CT (Zovein et al., 2004). Due to the small size of the TABLE 1 | Detailed description of the statistical analysis of 18 F-FDG uptake in
infant animals’ brain, to allow comparison with adolescent and infancy, adolescence, and adulthood mice in distinct brain regions.
adult mice, the VOIs for all ages were defined as whole brain,
VOI 18 F-FDG uptake
left and right brain hemispheres, frontal cortex, and cerebellum,
always using the skull defined by the CT as a border line.
ETS-exposed ETS-exposed vs. control
When analysis was restricted to adult animals, PET image was
co-registered to the MRI template and different brain regions Male vs. female Male Female
considered in the analysis (whole brain, brainstem, cortex,
Infancy
cerebellum, left and right amygdala left and right striatum, left
Whole brain ns ↓ p = 0.020 ↓ p = 0.013
and right hippocampus, cingulate cortex, basal forebrain and
Left hemisphere ns ↓ p = 0.010 ↓ p = 0.026
septum, thalamus, hypothalamus, and left and right midbrain).
Right hemisphere ns ↓ p = 0.013 ↓ p = 0.036
The 18 F-FDG uptake is presented as a standardized uptake
Frontal cortex ns ↓ p = 0.010 ↓ p = 0.031
value (SUV) which is calculated as radioactivity concentration
Cerebellum ↓ p = 0.014 ns ↓ p = 0.021
(kBq/cc) divided by the ratio between injected dose (kBq) and Adolescence
animal body weight (g). Whole brain ns ns ns
Left hemisphere ns ns ns
Statistical Analysis Right hemisphere ns ns ns
As in young animals it is difficult to analyze small brain Frontal cortex ns ns ns
areas, due to the limited PET imaging spatial resolution, and Cerebellum ns ns ns
in order to compare infancy, adolescence, and adulthood, Adulthood
we analyzed brain glucose metabolism in the following brain Whole brain ↓ p = 0.060 ↓ p = 0.019 ↑ p = 0.068
areas: whole brain, left and right brain hemispheres, frontal Left hemisphere ↓ p = 0.060 ↓ p = 0.011 ↑ p = 0.072
cortex, and cerebellum. Thus, we performed a three-way mixed Right hemisphere ↓ p = 0.015 ↓ p = 0.024 ns
ANOVA with repeated measures, considering groups as between Frontal cortex ↓ p = 0.026 ↓ p = 0.015 ns
and time and VOIs (whole brain, frontal cortex, cerebellum, Cerebellum ↓ p = 0.040 ↓ p = 0.062 ↑ p = 0.029
right hemisphere, and left hemisphere) in infancy, adolescence, Each p-value is adjusted for multiple comparisons. ↓, decreased 18 F-FDG uptake;
and adulthood as within-subject factors. Bonferroni post hoc ↑, increased 18 F-FDG uptake.

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and Bonferroni post hoc test with multiple comparison in the whole brain, left and right hemisphere and frontal
correction. We found a significant effect for the factors VOIs cortex, compared to the control group. Females showed
(F 4 ,56 = 33.7333; p < 0.00010) and time (F 2 ,8 = 10.99; p < 0.0001), decreased 18 F-FDG uptake in the cerebellum (Figure 1 and
and a significant VOIs × time interaction (F 8 ,112 = 4.592, Table 1). When mice reached adulthood, males showed a
p < 0.0001). We also had a significant VOIs × time × group reduction in 18 F-FDG uptake when compared to controls
interaction (F 24 ,112 = 1.753; p < 0.05). in all VOIs analyzed (Figure 1 and Table 1). However, in
The post hoc analysis revealed that all mice showed reduced the females a different pattern occurred, as they had higher
18 F-FDG uptake in infancy, compared to adulthood in all 18 F-FDG uptake in the whole brain, left hemisphere, and

analyzed VOIs. Regarding the females of ETS group, the 18 F- cerebellum, when compared to the control group (Figure 1
FDG uptake was also lower during infancy when compared and Table 1). When both ETS-exposed groups were compared,
to adolescence. As detailed at Table 1, in infancy, both male the post hoc analysis showed a reduction in 18 F-FDG uptake
and female mice ETS-exposed had lower 18 F-FDG uptake in the males in all the VOIs evaluated when compared to

FIGURE 2 | Representative PET/CT brain images of male and female mice exposed to ETS during the early postnatal period and the control group. (A) CT images in
different anatomical planes in the top left, and CT with the drawn volumes of interest (VOIs) used for quantification in the top right (whole brain, left hemisphere, right
hemisphere, frontal cortex, and cerebellum). (B) PET images fused to the CT images of infant (Day 15), adolescent (Day 35), and adult (Day 65) male and female
mice of the control and ETS groups.

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females (Figure 1 and Table 1). Figure 2 shows a representative significance in the cortex, and right hippocampus (Figure 3
brain PET/CT scans of mice exposed to ETS during the and Table 2). Regarding adult female mice exposed to ETS, we
early postnatal period and the control group during infancy, observed an increase in glucose uptake when compared with
adolescence, and adulthood. female mice from the control group in the brainstem, with trend
toward statistical significance in the cingulate cortex. Figure 4
ETS During the Early Postnatal Period shows brain PET/CT average scans of female and male adult
mice exposed to ETS during the early postnatal period and
Decreased 18 F-FDG Uptake in Adult Male
the control group.
Mice in Distinct Brain Regions
Positron emission tomography scan data of glucose uptake
for male and female adult mice exposed to ETS during the
DISCUSSION
early postnatal period were analyzed by two-way ANOVA
(VOIs × treatment) with Bonferroni post hoc test with p-values To the best of our knowledge, this is the first study
corrected for multiplicity. We found a significant effect for VOIs that investigated the effects of ETS exposure during brain
(F 15 ,240 = 7.101, p < 0.0001) and treatment (F 3 ,240 = 59.5, development on 18 F-FDG uptake in the brain of mice. By
p < 0.0001) factors; however, the interaction between them was PET imaging, we observed that ETS exposure during the early
not significant (F 45 ,240 = 0.033, p > 0.999; see Table 2 for detailed postnatal period decreased brain 18 F-FDG uptake in both male
description of the statistical analysis). and female infant mice and in adult male mice in distinct
The post hoc analysis showed that exposure to ETS during brain regions and increased 18 F-FDG uptake in adult female
the early postnatal period decreased 18 F-FDG uptake in adult mice in the brainstem and cingulate cortex. In addition, male
male mice when compared with adult female mice in the whole ETS-exposed mice showed decreased 18 F-FDG uptake when
brain, brainstem, left amygdala, left and right striatum, left and compared to female ETS-exposed. These results are in accordance
right hippocampus, cingulate cortex, basal forebrain and septum, with previous studies of our group and with studies that show
thalamus, hypothalamus, and left and right midbrain. There was that exposure to tobacco smoke during brain development
a trend of statistical significance in the cortex (Figure 3 and can affect the central nervous system. Exposure to tobacco
Table 2). We also observed that adult male mice exposed to ETS smoke extract during gestational period of Sprague–Dawley rats
showed a decrease in glucose metabolism when compared with decreased nicotinic and serotonin receptors in different brain
male mice from the control group in the left and right striatum, regions (Slotkin et al., 2017). In addition, postnatal exposure
left hippocampus, cingulate cortex, and thalamus (Figure 3 to tobacco smoke leads to impairment in the myelination,
and Table 2). It was also detected a trend toward statistical learning, and memory, and induces oxidative stress and lower
brain-derived neurotrophic factor (BDNF) and synaptic proteins
levels (Stangherlin et al., 2009; Lobo-Torres et al., 2012;
TABLE 2 | Detailed description of the statistical analysis of 18 F-FDG uptake in Torres et al., 2015a,b).
adult mice in distinct brain regions. The exposure biomarkers of the present study were similar
18 F-FDG
to Obot et al. (2004), Amos-Kroohs et al. (2013), and Torres
VOI uptake
et al. (2015a,b, 2019a,b). Nwosu and Kum-Nji (2018) suggested
ETS-exposed ETS-exposed vs. control
that the classification as passive or active smoking could be
done according to serum cotinine levels. Thus, serum cotinine
Male vs. female Male Female levels between 0.05 and 10 ng/mL can be considered as passive
smokers and >10 ng/mL as active smoking. Although cotinine
Whole brain ↓ p = 0.033 ns ns
concentration of the present study could be considered as active
Brainstem ↓ p = 0.020 ns ↑ p = 0.021
smoker by Nwosu and Kum-Nji (2018), the classification was
Cortex ↓ p = 0.058 ↓ p = 0.058 ns
Cerebellum ns ns ns
based in children and adolescent under 17 years old and not
Left amygdala ↓ p = 0.012 ns ns
in rodents. Moreover, the authors did not mention how long
Right amygdala ns ns ns after tobacco smoke exposure the blood was collected. In the
Left striatum ↓ p = 0.003 ↓ p = 0.038 ns present study, due to the weaker binding affinity of CO for mouse
Right striatum ↓ p = 0.019 ↓ p = 0.033 ns hemoglobin when compared to that of human hemoglobin
Left hippocampus ↓ p = 0.001 ↓ p = 0.024 ns (Watson et al., 1987), blood collection for the quantification
Right hippocampus ↓ p = 0.008 ↓ p = 0.053 ns of the biological markers was performed immediately after
Cingulate cortex ↓ p = 0.0003 ↓ p = 0.028 ↑ p = 0.069 the ETS exposure. Thus, the cotinine levels data reflect the
Basal forebrain and spetum ↓ p = 0.049 ns ns peak of cotinine, as the half-life of plasma nicotine in rodents
Thalamus ↓ p = 0.0007 ↓ p = 0.022 ns is 0.9–1.1 h.
Hypothalamus ↓ p = 0.033 ns ns Neuronal activity requires high energy, mainly in the
Left midbrain ↓ p = 0.0008 ns ns level of synaptic connections and signaling transduction
Right midbrain ↓ p = 0.0007 ns ns pathways (Sokoloff, 1999). In this scenario, 18 F-FDG brain
Each p-value is adjusted for multiple comparisons. ↓, decreased 18 F-FDG uptake; uptake correlates with brain metabolic activity, since we can
↑, increased 18 F-FDG uptake. predict brain function through the relationship between energy

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FIGURE 3 | 18 F-FDG uptake for adult mice (n = 5 females ETS-exposed and n = 4 females control; n = 5 males ETS-exposed and n = 5 males control) in the whole
brain (A), brainstem (B), cortex (C), cerebellum (D), left amygdala (E), right amygdala (F), left striatum (G), right striatum (H), left hippocampus (I), right hippocampus
(J), cingulate cortex (K), basal forebrain and septum (L), thalamus (M), hypothalamus (N), left midbrain (O), and right midbrain (P). Two-way ANOVA with Bonferroni
post hoc test and adjusted p-values for multiple comparisons. Continuous bar: p < 0.05. Dashed bar: trend toward statistical significance.

consumption and neuronal activity (Shulman et al., 2004). Small the ninth year of life there is a plateau, followed by a
animal PET imaging in longitudinal studies allows in vivo steady decline until adulthood, in the second decade of life,
quantification of brain metabolic activity in the same animal when the prefrontal cortex has completed its maturation
during different periods of life making possible to analyze (Kennedy and Sokoloff, 1957). During normal aging, brain
how brain behave in different stages and how xenobiotics passes through structural and function changes in white
might be able to interfere in the homeostatic state. Our and gray matters, reflecting in a declined metabolic activity
data showed decreased 18 F-FDG uptake in infancy, suggesting (Moeller et al., 1996).
that ETS exposure is affecting brain neuronal activity during It is interesting to note that even a long time after the
this important period of brain development. In fact, it is last exposure, adult mice showed changes in 18 F-FDG uptake
known that in humans, childhood is a period in which in distinct brain regions, which were sex dependent. Adult
the central nervous system is under active development, male mice exposed to ETS during brain development showed
maturation, and with open critical periods of synaptic plasticity, decreased 18 F-FDG uptake compared with adult female mice or
with the higher levels of metabolism, reaching a peak on with male controls in different brain regions. In fact, sex seems to
the fourth year of life (Chugani and Phelps, 1991). Until be an important factor in brain response to ETS. A clinical study

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rhythmogenesis only in female mice (Garcia et al., 2013). These

data are relevant, since post-hypoxic depression is involved in
the pathogenesis of sudden infant death syndrome (SIDS). About
60% of the children affected by SIDS are male (Richardson
et al., 2010). The exposure to ETS is related to higher risk
of SIDS, a syndrome that has no known mechanism, but it
requires immature cardiorespiratory control and impairments in
sleep arousal (Mitchell and Milerad, 2006; Moon et al., 2016).
The brainstem is associated with respiratory and cardiovascular
responses, therefore is related to pathogenesis of SIDS and
it is susceptible to ETS exposure. Previous studies showed
that exposure to ETS in the early postnatal period decreased
myelin-specific proteins and alter receptors and enzymes of
the endocannabinoid system in the brainstem (Torres et al.,
2015a, 2019a). The present study corroborates these findings
since we observed that ETS exposure decreased 18 F-FDG
uptake in ETS-exposed male mice compared with female mice
and increased 18 F-FDG uptake in ETS-exposed female mice
compared with control group.
Environmental tobacco smoke exposure during the early
postnatal period induced a similar result in the left amygdala,
which have a key role in the acquisition of memory related to
fear conditioning (Fujisaki et al., 2004). Amygdala is associated
to emotional experience, including fear and anxiety (De Bellis
et al., 2000; de Oliveira et al., 2013). In fact, pediatric generalized
anxiety disorder was associated to higher amygdala volumes (De
Bellis et al., 2000). Active smokers have significantly reduced
amygdala volumes compared with non-smokers (Luhar et al.,
2013). In line with these results, preclinical studies have shown
that the exposure to ETS during postnatal periods leads to
anxiety-like behavior in a short- and long-term withdrawal
(Abreu-Villaça et al., 2015; de la Peña et al., 2016; Torres et al.,
2019b). Taken together, these data suggest that the anxiety
behavioral disorders related to tobacco smoke may be associated
with amygdala alterations.
FIGURE 4 | PET average images of female and male adult mice exposed to
In the present study, we observed that exposure to ETS
ETS during the early postnatal period and the control group. On the top, a
MRI template used for drawing volumes of interest (whole brain, brainstem, decreased 18 F-FDG uptake in striatum of adult male mice.
cortex, cerebellum, left amygdala, right amygdala, left striatum, right striatum, This data are consistent with studies that revealed that
left hippocampus, right hippocampus, cingulate cortex, basal forebrain and ETS exposure during a critical period induced oxidative
septum, thalamus, hypothalamus, left midbrain, and right midbrain). The first stress, decreased synaptic proteins levels and BDNF, and
two rows represent PET average images fused to MRI template and the third
row represent the difference of PET average image fused to MRI template
modified elements of the endocannabinoid system in the
between control group and ETS exposed mice. striatum (Lobo-Torres et al., 2012; Torres et al., 2019a,b). The
striatum, constituted by the caudate nucleus and putamen,
is involved in motor, cognitive, and limbic functions, and
is involved in the neurobiology of addiction (Burton et al.,
showed that nicotine administered by patch induced increased 2015). Addictive drugs increase dopamine levels in mesolimbic
brain 18 F-FDG uptake in females than males during a Continuous system, especially in the dorsal and ventral striatum/nucleus
Performance Task or the Bushman Competition and Retaliation accumbens (Hyman et al., 2006). Romoli et al. (2019)
Task tests (Fallon et al., 2005). A previous study of our group reported that exposure to nicotine during the early postnatal
also showed that the effects of ETS exposure during the early period increased nicotine consumption during adulthood, effect
postnatal period are sex-dependent, as infant female mice showed mediated by dopaminergic neurons in the midbrain, that
poorer performance in learning and memory tests than males contains dopaminergic neurons that are located in the ventral
(Torres et al., 2015b). tegmental area and in the substantia nigra, regions that are
Garcia et al. (2013) revealed that sex is determinant for important for the development of drug addiction (Björklund and
post-hypoxic depression and recovery. Persistent post-hypoxic Dunnett, 2007; Romoli et al., 2019).
depression is more recurrent in male mice than female Weinstein et al. (2010) observed that smokers showed
and glucose supplementation improves post-hypoxic recovery increased craving scores after watching a videotape with smoking

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Torres et al. ETS Modifies Brain 18 F-FDG Uptake

scenes, which were associated with brain 18 F-FDG uptake in the ETHICS STATEMENT
ventral striatum, anterior cingulate, orbitofrontal cortex, middle
temporal lobe, hippocampus, insula, midbrain, and thalamus The animal study was reviewed and approved by the Ethics
(Weinstein et al., 2010). In addition, Domino et al. (2000) showed Committee of the School of Medicine (027/14) and the School
that nicotine increases regional cerebral blood flow, evaluated by of Pharmaceutical Sciences (P446/14), University of São Paulo.
PET, in the thalamus, pons, primary visual cortex, and cerebellum
of tobacco smokers. These individuals also showed reduction
in the hippocampal area (Domino et al., 2000; Hanlon et al., AUTHOR CONTRIBUTIONS
2014). We observed that ETS decreased glucose metabolism in
ETS-exposed male mice compared with female mice and with LT and TM conceived and designed the project. LT, LS, AS, and
control group in the hippocampus, one of the main areas that TA performed all the experiments related to exposure to tobacco
is involved in learning and memory. Indeed, ETS exposure smoke and biomarkers quantification under TM supervision.
during the early postnatal period decreased synaptic proteins LT, WT, LP, and PS performed the experiments related to
and BDNF in hippocampus and induced impairment in the PET/CT image acquisition. WT, CR, and DP performed the
learning and memory from late infancy to early adulthood image processing and analysis. LT, WT, MP, CR, DP, and TM
(Torres et al., 2015b). wrote and edited the manuscript. All authors contributed to
It is important to point out the limitations of the present the manuscript revision, and read and approved the submitted
protocol. In order to evaluate the long-lasting effect of version of the manuscript.
tobacco smoke exposure during the early postnatal period,
we used a longitudinal study to measure 18 F-FDG uptake
from infancy to adulthood. Although longitudinal studies have
the advantage of evaluating the same animal throughout life, FUNDING
this type of protocol does not allow other measures to be
This work was supported by the São Paulo Research
performed, since euthanasia is only done when the animal
Foundation (Fundação de Amparo à Pesquisa do Estado de
reaches adulthood.
São Paulo – FAPESP, Grants 2013/08881-9 and 2013/12769-7);
In summary, we showed that several brain regions are
Coordination for the Improvement of Higher Education
vulnerable to ETS exposure during the early postnatal period
Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível
and these effects on 18 F-FDG uptake were observed even a long
Superior – Brazil – CAPES – Finance Code 001); and the National
time after the last exposure. This study corroborates our previous
Counsel of Technological and Scientific Development (Conselho
studies, supporting the hypothesis that exposure to tobacco
Nacional de Desenvolvimento Científico e Tecnológico – CNPq
smoke in a critical period interferes with brain development of
Grant 405353/2016-2). TM is a research fellow of CNPq.
mice from late infancy to early adulthood.

DATA AVAILABILITY STATEMENT ACKNOWLEDGMENTS

The datasets generated for this study are available on request to The authors gratefully acknowledge Fábio Luís de Souza Duran
the corresponding author. for his technical assistance.

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function in mice. Toxicol. Appl. Pharmacol. 266, 366–374. doi: 10.1016/j.taap. Conflict of Interest: The authors declare that the research was conducted in the
2012.11.024 absence of any commercial or financial relationships that could be construed as a
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J. T., and Richardson, J. R. (2014). Prenatal cigarette smoke exposure
causes hyperactivity and aggressive behavior: role of altered catecholamines Copyright © 2020 Torres, Real, Turato, Spelta, dos Santos Durão, Andrioli, Pozzo,
and BDNF. Exp. Neurol. 254, 145–152. doi: 10.1016/j.expneurol.2014. Squair, Pistis, de Paula Faria and Marcourakis. This is an open-access article
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K., et al. (2004). Postnatal hypoxic-ischemic brain injury alters mechanisms original author(s) and the copyright owner(s) are credited and that the original
mediating neuronal glucose transport. Am. J. Physiol. Regul. Integr. Comp. publication in this journal is cited, in accordance with accepted academic practice. No
Physiol. 286, R273–R282. use, distribution or reproduction is permitted which does not comply with these terms.

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 ORIGINAL RESEARCH
published: 31 January 2020
doi: 10.3389/fnbeh.2020.00009

Targeting the Stress System During

Gestation: Is Early Handling a
Protective Strategy for the Offspring?
Valentina Castelli 1 , Gianluca Lavanco 2,3,4 , Anna Brancato 5 and Fulvio Plescia 5 *
1
Department of Biomedicine, Neuroscience and Advanced Diagnostics, University of Palermo, Palermo, Italy, 2 INSERM
U1215, Neuro Centre Magendie, Bordeaux, France, 3 University of Bordeaux, Bordeaux, France, 4 Department of Biomedical
and Biotechnological Sciences, Section of Pharmacology, University of Catania, Catania, Italy, 5 Department of Health
Promotion, Mother and Child Care, Internal Medicine and Medical Specialties “Giuseppe D’Alessandro”, University of
Palermo, Palermo, Italy

The perinatal window is a critical developmental time when abnormal gestational

stimuli may alter the development of the stress system that, in turn, influences
behavioral and physiological responses in the newborns. Individual differences in
Edited by:
stress reactivity are also determined by variations in maternal care, resulting from
Miriam Melis,
University of Cagliari, Italy environmental manipulations. Despite glucocorticoids are the primary programming
Reviewed by: factor for the offspring’s stress response, therapeutic corticosteroids are commonly
Muriel Koehl, used during late gestation to prevent preterm negative outcomes, exposing the
Institut National de la Santé et de la
offspring to potentially aberrant stress reactivity later in life. Thus, in this study, we
Recherche Médicale (INSERM),
France investigated the consequences of one daily s.c. injection of corticosterone (25 mg/kg),
Michele Navarra, from gestational day (GD) 14–16, and its interaction with offspring early handling,
University of Messina, Italy
consisting in a brief 15-min maternal separation until weaning, on: (i) maternal
*Correspondence:
Fulvio Plescia
behavior; and (ii) behavioral reactivity, emotional state and depressive-like behavior in
[email protected] the adolescent offspring. Corticosterone plasma levels, under non-shock- and shock-
Specialty section: induced conditions, were also assessed. Our results show that gestational exposure
This article was submitted to Emotion to corticosterone was associated with diminished maternal care, impaired behavioral
Regulation and Processing, a section
reactivity, increased emotional state and depressive-like behavior in the offspring,
of the journal
Frontiers in Behavioral Neuroscience associated with an aberrant corticosterone response. The early handling procedure,
which resulted in increased maternal care, was able to counteract the detrimental
Received: 03 December 2019
effects induced by gestational corticosterone exposure both in the behavioral- and
Accepted: 15 January 2020
Published: 31 January 2020 neurochemical parameters examined. These findings highlight the potentially detrimental
Citation: consequences of targeting the stress system during pregnancy as a vulnerability factor
Castelli V, Lavanco G, Brancato A for the occurrence of emotional and affective distress in the adolescent offspring.
and Plescia F (2020) Targeting the
Stress System During Gestation: Is
Maternal extra-care proves to be a protective strategy that confers resiliency and
Early Handling a Protective Strategy restores homeostasis.
for the Offspring?
Front. Behav. Neurosci. 14:9. Keywords: prenatal exposure, glucocorticoid, early handling, stress reactivity, depressive-like
doi: 10.3389/fnbeh.2020.00009 behavior, emotionality

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Castelli et al. Prenatal Glucocorticoids and Early Handling

INTRODUCTION dams spend less time in activities directed towards the pups
rather than in self-oriented behaviors (Patin et al., 2002; Smith
Numerous studies across a wide range of species have et al., 2004; Boero et al., 2018). After birth, the infant is
shown that prenatal exposure to different conditions such as dependent on the primary caregiver, not only for nursing and
infections, nutritional deficiencies, teratogenic substances, and protection but also for the normal development of emotional
emotional distress, predisposes the newborns to a spectrum behavior (Bella et al., 2018). Indeed, deficiency of motherly
of different disorders characterized by deficits in cognitive care during infancy affects the development of stress reactivity,
functioning, motor, and visuospatial abilities and to the genesis contributing to the raising of the individual distinctness in
of chronic systemic diseases (Cannizzaro et al., 2002, 2005, emotional responses (Cannizzaro et al., 2005, 2006b). On the
2006b, 2007, 2008; Hellemans et al., 2010; Leggio et al., other hand, early handling procedures are able to significantly
2014; Sarro et al., 2014; Martines et al., 2016; Moukarzel affect the development of the offspring’s emotional behavior and
et al., 2018). Notably, maternal stress during pregnancy could HPA axis physiology. In particular, extensive research has shown
dispose of the offspring toward vulnerability to neurobehavioral that brief periods of maternal separation of the pups during
disorders. As mediators of the stress response, glucocorticoids the nursing stage result in offspring decreased adrenal reactivity
are among the main primary programming factors conveying in response to stressors (Liu et al., 1997; Cannizzaro et al.,
maternal stress to the fetus via the placenta (Zarrow et al., 2005, 2006b, 2007; Plescia et al., 2014b), as well as fear-oriented
1970; Schmidt et al., 2019), through the activation of the behavior and emotionality (Cannizzaro et al., 2005, 2006b). The
glucocorticoid receptors (GR), whose ontogenetic pattern has majority of these behavioral and neuroendocrine studies have
been detected in human from the early prenatal life stages been carried out on the adult progeny exposed to repeated
(Kitraki et al., 1997; Diaz et al., 1998; Kemp et al., 2016). prenatal stress. However, none of them has yet investigated
Thus, glucocorticoids, by a receptor-mediated regulatory role the influence of the gestational exposure to corticosterone
during ontogenic development, could affect normal brain on emotional behaviors in adolescence, which emerges as a
neurogenesis (Cintra et al., 1993). In this regard, prospective ‘‘critical’’ phase in the development of stress responsiveness
animal- and retrospective human studies have revealed that (Cannizzaro et al., 2006b).
antenatal glucocorticoid administration in late gestation can Thus, given these premises, the aim of the current study was to
lead to lifelong alterations on brain structures and functionality investigate the consequences of prenatal corticosterone exposure
and may produce long-lasting modifications in the maturation on maternal and offspring outcomes, during a timeframe when
of the hypothalamic-pituitary-adrenal (HPA) axis (Heim et al., a relatively high expression of GR in multiple brain areas
1997; French et al., 1999, 2004; Sloboda et al., 2005; de of the pups occurs (Cintra et al., 1993). In particular, we
Vries et al., 2007; Charil et al., 2010; Fowden and Forhead, assessed maternal behavior, behavioral reactivity, emotionality
2015). Indeed, exposure to glucocorticoids during pregnancy, and depressive-like behavior in the adolescent male offspring
reducing negative-feedback on HPA axis, increases cortisol employing, respectively, the open field test (OFT), the acoustic
release in the progeny (Alexander et al., 2012): this leads startle reflex (ASR) and the forced swim test (FST). Offspring
to a slower recovery from stressors, reducing coping strategy corticosterone plasma levels, under non-shock- and shock-
in aversive situations (Welberg et al., 2001; Plescia et al., induced conditions were evaluated as a measure of HPA axis
2013). This evidence represents a key issue in the therapeutic activity. Early handling procedure, as a brief maternal separation,
administration of antenatal corticosteroids, which are commonly was also carried out as a putative protective strategy able to
used when at risk of preterm delivery to ensure the survival restore homeostasis.
of the preterm infant (Singh et al., 2012). Indeed, last-trimester
administration of synthetic glucocorticoids also ‘‘programs’’ MATERIALS AND METHODS
outcomes comparable to those elicited by prenatal stress in
humans (Seckl et al., 2000). Accordingly, treating pregnant Animals and Pharmacological Treatment
rodents with synthetic glucocorticoids leads to offspring with Wistar rats (Harlan, Udine, Italy) housed with free access to food
similar HPA axis- and behavioral changes as prenatally stressed and water were maintained on a 12 h on/off cycle (8:00–20:00 h)
offspring (Schmidt et al., 2019). at a constant temperature (22 ± 2◦ C) and humidity (55 ± 10%).
The gestational experiences may also affect the maternal- Pairs of primiparous females of 120 days of age were mated
infant dyad (Tarullo et al., 2017; Reck et al., 2018). Indeed, with one male of 150 days of age. The day on which sperm was
pregnant women who experience social and emotional stress detected in the vaginal smear was designed as gestational day
may divest themselves of maternal bonding (Baker et al., (GD) 1. Pregnancy was determined by weighing and palpation.
2008; Azhari et al., 2019). Importantly, these conditions appear The pregnant dams’ weight on GD 14 was approximately 300 g.
to have a major impact on child cognitive, emotional and From GD 14 through GD 16, a period of time during which
physical development (Cogill et al., 1986; Bhagwanani et al., corticosterone can interact with GR expressed in the last week
1997; Smith et al., 2004). Alterations in maternal caregiving of gestation, the dams received a single daily subcutaneous
behavior after maternal stress, or exogenous administration injection of corticosterone (Ct; Sigma–Aldrich, Italy; 25 mg/kg)
of glucocorticoids, occur also in rodent models (Darnaudéry or vehicle (Vh; 100 mM DMSO in 0.9% saline solution) in a
et al., 2004; Koehl et al., 2012; Jafari et al., 2017; Gemmel volume of 1 ml/kg. The pregnant dams were individually housed
et al., 2018). For instance, acutely and repeatedly stressed in standard rat cages (40 cm × 60 cm, 20 cm in height) for

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Castelli et al. Prenatal Glucocorticoids and Early Handling

at least 7 days before delivery. All litters born within a 2-day data were recorded using dichotomous scores (0/1): score
period were reduced to ten pups (five males and five females) 0 was assigned when the behavior was not shown in
Forty male pups in total were used in our investigations; they the interval of observation; score 1 was assigned when
were divided into the following experimental 10-rat (five rats the behavior was performed. Thus, a daily score ranged
per litter) groups: vehicle-non-handled (Vh); corticosterone- between 0 and 12. In order to gain a comprehensive
non-handled (Ct); vehicle-handled (Vh-H); corticosterone- framework of the behavioral measurements, a daily index of
handled (Ct-H). At weaning time, postnatal day (PND) 22, overall maternal behavior (MB-I) was calculated as follows:
rats were randomly assigned two per cage accordingly to each (maternal score) − (non-maternal score)/(maternal score) +
experimental condition. The experiments were performed on (non-maternal score). The index ranges from −1 (totally
adolescent rats—from PND 32 to 43. On the test day, each non-maternal behaviors) to +1 (totally maternal behaviors;
group of rats was brought into the laboratory and allowed to Brancato et al., 2016).
acclimate for at least 60 min prior to the experimental session.
The experiments were performed in a sound isolated room Open Field Test
between 9:00 and 14:00 and the animals were tested randomly, Locomotor activity and explorative behavior were assessed in
regardless of the group they belonged to. Animal performance the open-field arena with a contrast-sensitive, video tracking
during the different experimental sessions was recorded on the system, ANY MAZE (Ugo Basile, Gemonio, Italy), in a mean
computer and then analyzed by an experimenter unaware of light intensity (100 lx) illuminated room (Brancato et al., 2014).
the different treatments. All the experiments were conducted The apparatus consisted in a square box (44 × 44 × 20 cm)
in accordance with the regulations of the Committee for the and produced a quality-quantitative mapping of the ambulatory
Protection and Use of Animals of the University of Palermo, patterns, measuring simultaneously: total distance traveled
Italy, in accordance with current Italian legislation on animal (TDT) in centimeters, number of transition from peripheral to
experimentation (D.L. 26/2014) and the European Directive central squares of the arena (NCT) and amount of time spent
(2010/63/EU) on the care and use of laboratory animals. All on the central areas (ATC) in second. The 5-min recording and
efforts were made to minimize the number of animals used and measurement of each experimental session started after 1-min
possible distress. habituation in the arena, to allow the rats to acclimatize, and was
displayed on a personal computer (Cacace et al., 2011). The test
Early Handling and Pups Body Weight was performed at PND 32.
Half of Ct- and Vh-treated litters remained undisturbed during Acoustic Startle Reflex Test
the post-weaning period (i.e., non-handled, Ct and Vh groups), The ASR provides a useful readout of the neural processing
and half of prenatally Ct- and Vh-treated litters underwent early that might underpin an organism’s response to an emotional
handling procedure (Ct-H and Vh-H groups), from PND 2 until context or stressor (Hoffman, 2016; Hantsoo et al., 2018). The
PND 21. Early handling procedure consisted of removing the ASR response was measured using a Responder-X apparatus
dam from the nest for 15 min during which she was temporarily (Columbus Instruments, USA) at PND 34. The peak amplitude
placed in a separate cage. Simultaneously, pups were moved into of the responses was recorded and displayed on a personal
a different room and individually placed into sawdust-containing computer. A 10-min test session started by placing the rat in a
small plastic cups for 15 min. In the end, mothers and pups were 28 cm long, 16 cm wide, 15 cm high device with a stainless-steel
brought together in their home cages. Early handling procedure grid floor, into a ventilated, sound-attenuated and darkroom,
was performed in the same room, at the same time (10:00 h) and in which the animal was left undisturbed for the first 5 min
by the same experimenter. From PND 2 to PND 21 pups’ body period and was subsequently subjected to the startle stimulus for
weight was also evaluated. 5 min. The startle stimulus consisted of a 110 dB, 8 kHz tone
superimposed on a continuous 50 dB white noise background;
Maternal Behavior Assessment the stimulus duration was 200 ms, with a fixed 10-s interval.
Dam’s behavior in the presence of the offspring was assessed Sound levels in the test room were measured with a Bruel and
by direct periodic observations under undisturbed conditions Kjaer 2209 sound level meter. The maximum force exerted by
in their home cages (Capone et al., 2005), from PND 2 to the rat on-grid floor during the 200 ms period was designated as
PND 21. Each animal was subjected to four assessments a peak amplitude. The amplitude of ASR was measured in units,
day, during the diurnal time (9:00 am, 11:30 am, 01:30 pm, over the range of 60–550 g (1 unit = 2.1 g of force); maximum
and 03:00 pm) when animals behave more maternally (Ader output was 255 units. The experimental session consisted of
and Grota, 1970); instantaneous 20-s sampling was repeated 10 trials.
three times at each time, for a total of 12 instantaneous
observations per animal per day (3 observations × 4 times Forced Swim Test
per day × 20 days = 240 observations per dam). The 20-s We employed the FST as described by Porsolt et al. (1977) with
time of observation allows for an exact identification of some modifications, in order to test depressive-like behavior at
the on-going behavioral patterns: retrieval, nursing (arched- PND 38. The test was composed of a pre-test stage (15 min)
back, blanket, passive), pup care (licking, anogenital licking), and, 24 h later, of a test stage (5 min), for both pre-test
dam self-care (self-grooming, eating, drinking), and others and test sessions, conducted under low illumination (12 lx),
(rearing, moving, resting, standing out of nest). Original the animals were placed inside a transparent Plexiglas cylinder

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Castelli et al. Prenatal Glucocorticoids and Early Handling

(50 cm high, 20 cm inside diameter) filled with tap clean post hoc comparisons were calculated with Tukey’s multiple
water at 23 ± 1◦ C, adjusting the water depth according to the comparison post-test (α = 0.05). Data are reported as mean ± SD.
rat’s size, so that it cannot touch the bottom of the container Statistical significance was set at p < 0.05.
with its hind legs (Yankelevitch-Yahav et al., 2015). A video
camera was placed above the tank and connected to a video RESULTS
recorder to register each stage for subsequent scoring. An
experimenter, unaware of the different treatments, scored the Pups Body Weight
specific behavioral parameters from the videotape. Behavioral Rats’ body weight was recorded from PND 2 to PND 21 in
categories considered were as follows: immobility time, defined order to obtain data related to the influence of a single daily
as floating in the water, making only the movement necessary to corticosterone administration and early handling procedure on
keep the head above water; swimming time, defined as making weight gain during the pre-weaning period. No significant
swimming motions and moving around the cylinder. Following differences in number, weight, morbidity or mortality were
either pre-test stage or test stage, the rats were dried with a observed among the different experimental groups. The results
towel and kept warm on a heating pad for 30 min in their of a three-way ANOVA performed on body weight as a
home cages. dependent variable, and days, prenatal corticosterone exposure
and early handling as independent variables are shown in
Stress Procedure Table 1. The table indicates that: the factors days, prenatal
At PND 43, rats from each experimental group were individually corticosterone treatment, and early handling were significant.
placed in a cage with an electrified grid floor through which Moreover, the interaction between days- and prenatal treatment-
shock could be delivered. The session started immediately after with early handling was significant. The results of Tukey’s
placing the rat into the shock-delivering apparatus. Rats (five per multiple comparisons test performed on each single day showed
group) received an inescapable mild footshock (0.6 mA for 3 s) a reduction in body weight in Ct treated rats on days 10, 11 and
every 20 s, along 1 min. Control animals (non-stressed, five per 12 (q = 6.29, p = 0.04340; q = 6.25; p = 0.0463; q = 6.219,
group) were placed into the apparatus for the same time but were p = 0.0495), with respect to Vh groups; and a decrease in body
not shocked (Cannizzaro et al., 2006b). weight on days 19 and 21 (q = 5.825, p = 0.0270; q = 6.341;
p = 0.0380) in Ct with respect to Ct-H groups (Figure 1). No
Plasma Corticosterone Assay statistical difference was observed when Ct-H was compared to
Rats were killed by decapitation 30 min after being placed into Vh and Vh-H groups.
the shock-delivering apparatus. Trunk blood was collected into
heparinized tubes. After centrifugation at 3,000 rpm at 4◦ C for Dams Spontaneous Maternal Behavior
5 min, plasma samples were separated and stored at −80◦ C prior In order to evaluate the impact of gestational manipulation by
to assay. Plasma corticosterone concentration was assayed in corticosterone, the influence of a daily 15-min early handling
duplicate using the RIA kit for rats (IDS Limited, Boldon, UK). procedure on dams spontaneous behavior, retrieval, nursing
The inter-and intra-assay coefficient of variation was 8% and 3% (arched-back, blanket, passive), pup care (licking, anogenital
respectively, with a detection limit of 0.5 ng/mL. All measures licking, digging), dam self-care (self-grooming, eating, drinking),
were in the linear range of the standard curve (0.5–62.5 ng/mL). and other behaviors (rearing, moving, resting, standing out of
nest) were scored. Results from a two-way ANOVA performed
Statistical Analysis on MB-I as dependent variable and prenatal corticosterone
Statistical data from bodyweight were carried out by a three-way and early handling as independent variables, showed that: the
ANOVA followed by Tukey’s test post-test (α = 0.05). factor prenatal corticosterone (F (1,76) = 19.77; p < 0.0001) early
Statistical analysis of the data from the OFT, ASR, FST, handling (F (1,76) = 64.58; p < 0.0001) and their interaction
maternal behavior scores and from non-shock- and shock- (F (1,76) = 8.646; p = 0.0043) were significant. In detail, post hoc
induced corticosterone plasma levels were carried out using analysis conducted by Tukey’s multiple comparison post-test
a two-way ANOVA for unpaired measures. When necessary, highlighted a significant lower maternal behavior in Ct treated

TABLE 1 | Pups body weight: results of three-way ANOVA performed on body weight as dependent variable and days (1), prenatal treatment with corticosterone (2),
and early handling (3) as independent variables.

Source of Variation DF SS MS F P-level

1-days 19 922,580 48,557 89.39 <0.001

2-prenatal treatment 1 54,686 54,686 100.7.83 <0.001
3-early handling 1 77,176 77,176 142.1.33 <0.001
1:2 19 9,384 492 0.9057 =0.5775
1:3 19 17,917 943 1.736.79.1 =0.0468
2:3 1 42,968 42,968 0.6584 <0.001
1:2:3 19 6,795 357.7 =0.8477
Residuals 80 43,457 543.2

Pups’ body weight (g) was expressed as the weight for the entire litter.

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interaction were significant for TDT (F (1,36) = 91.79, p < 0.0001;

F (1,36) = 66.16, p < 0.0001; F (1,36) = 8.866, p = 0.0052), and
ATC (F (1,36) = 6.388; p = 0.0160; F (1,36) = 87.15; p < 0.0001;
F (1,36) = 4.494; p = 0.0410). Post hoc analysis conducted by
Tukey’s multiple comparison post-test showed that prenatal
Ct induced a decrease in both TDT and in ATC (q = 12.56;
p < 0.001; q = 4.647; p < 0.0116) when compared to Vh
groups. On the contrary, the early handling procedure induced
an increase in TDT and in ATC in both Vh (q = 5.156, p < 0.0044;
q = 7.216, p < 0.001) and Ct (q = 17.71, p < 0.001; = 11.46,
p < 0.001) treated rats when compared to respective controls
(Figure 3). No statistical difference was observed when Ct-H was
compared to Vh-H group. No statistical differences were found
on a number of transitions from peripheral to central squares of
the arena.
FIGURE 1 | Graph showing the effect of prenatal corticosterone on body
weight from postnatal day 2 until 21. Each value represents the mean ± SD Acoustic Startle Reflex Test
of 10 rats. *p < 0.05 vs. Vh, ◦ p < 0.05 vs. Vh-H. The effects of prenatal corticosterone exposure and the influence
of early handling procedure on the response to an anxiety-
inducing intense stimulus, were evaluated measuring startle
amplitude in the ASR test. The results of a two-way ANOVA
performed on the peak amplitude as dependent variable, and
prenatal corticosterone and early handling as independent
variables, indicated that: the factor prenatal corticosterone
(F (1,36) = 29.48; p < 0.0001) early handling (F (1,36) = 503.4;
p < 0.0001) and their interaction (F (1,36) = 78.57; p < 0.0001)
were significant. In detail, Tukey’s multiple comparison post-test
analysis showed that the prenatal treatment with Ct induced
an increase in startle amplitude (q = 14.29; p < 0.001) when
compared to Vh. Interestingly, early handling was able to reduce
startle amplitude in both Vh (q = 13.57; p < 0.001) and in Ct
(q = 31.30; p < 0.001) treated rats, when compared to respective
controls. No statistical difference was observed when Ct-H was
compared to Vh-H group (q = 16.70; p > 0.05; Figure 4).

Forced Swim Test

Rats were tested in the Porsolt test in order to evaluate the effects
FIGURE 2 | Maternal Behavior Index (MB-I). Influence of a daily 15-min early
of prenatal exposure to corticosterone and the influence of early
handling procedure on dams spontaneous behavior (retrieval, nursing, pup handling procedure on depressive-like behavior. Rats were first
care, dam self-care). Each value represents the mean of ± SD of exposed to the pre-stage and, 24 h after, underwent the 5-min
20 measures. ***p < 0.001, **p < 0.01, vs. Vh, +++ p < 0.001 vs. Ct. stage test, when immobility-, swimming-time were recorded.
A two-way ANOVA performed on time spent in immobility,
dams (q = 7.386, p < 0.0001) with respect to Vh group. Moreover, swimming, as a dependent variable, and prenatal corticosterone
early handling was able to increase dams maternal behavior and early handling as independent variables. The results indicate
in both Vh-H (q = 5.096, p < 0.0031) and Ct-H (q = 10.98, that prenatal corticosterone, early handling and their interaction
p < 0.0001) groups, when compared to respective control groups were significant for both immobility (F (1,36) = 5.327; p = 0.0269);
(Figure 2). (F (1,36) = 299.9; p < 0.0001); (F (1,36) = 10.81; p = 0.0023)
and swimming (F (1,36) = 7.119; p = 0.0114); (F (1,36) = 242.9;
Open Field Test p < 0.0001); (F (1,36) = 11.32; p = 0.0018). Tukey’s multiple
Rats were tested in the OFT in order to assess the influence comparison post-test analysis showed that the prenatal treatment
of prenatal corticosterone exposure and early handling on with corticosterone induced an increase in immobility time in
behavioral reactivity. Results obtained by a two-way ANOVA Ct (q = 5.596; p < 0.0019) with respect to Vh, and a significant
performed on total distance travelled, number of transition from decrease on immobility time in both Vh-H (q = 14.03; p < 0.001)
peripheral to central squares of the arena, and amount of time and in Ct-H (q = 20.6; p < 0.001) when compared respectively
spent on the central areas as dependent variables, and prenatal with Vh and Ct groups (Figure 5A). In agreement with these
corticosterone and early handling as independent variables, results, post hoc analysis showed a significant decrease in Ct
showed that prenatal corticosterone, early handling and their (q = 6.033; p = 0.0008) compared to Vh-rats and an increase in

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FIGURE 4 | Effects of prenatal corticosterone exposure and early handling

procedure on the peak amplitude in acoustic startle reflex (ASR). Each value
represents the mean ± SD of 10 rats. ***p < 0.001 vs. Vh,
+++
p < 0.001 vs. Ct.

non-shock-induced conditions. The results of Tukey’s multiple

comparisons test showed that non-shock-induced corticosterone
plasma levels increased in Ct-exposed offspring compared to
vehicle group (q = 4.426; p < 0.0174) and that early handling
reduced corticosterone plasma levels in both Vh-H (q = 20.59,
p < 0.0001; q = 24.84, p < 0.0001) and in Ct-H (q = 26.23,
p < 0.001; q = 28.67, p < 0.001) when compared with respective
control groups (Figure 6A).
When rats were exposed to shock-induced stress conditions
in order to evaluate the corticosterone plasma levels under
stressful conditions, the results of a two-way ANOVA performed
respectively on the levels of corticosterone as dependent
variables, and prenatal corticosterone treatment, early handling
as independent variables showed a significant effect for early
FIGURE 3 | Open field test (OFT). Effects of prenatal corticosterone handling (F (1,36) = 913.3; p < 0.0001) and interaction between
exposure and early handling procedure on total distance traveled (TDT), and corticosterone treatment and early handling (F (1,36) = 4.325;
amount of time spent (ATC) in the central area of the arena. Each value
p = 0.0447), but no for prenatal corticosterone treatment
represents the mean ± SD of 10 rats. ***p < 0.001, **p < 0.01, vs. Vh,
+++
p < 0.001 vs. Ct. (F (1,36) = 2.584; p = 0.1167). In detail, Tukey’s multiple
comparison post-test analysis showed that; shock exposure
did not modify corticosterone levels in Ct-exposed offspring
swimming time in both Vh-H (q = 12.22; p < 0.001) and in Ct-H (q = 0.472, p = 0.9870), and that early handling reduced
(q = 18.95; p < 0.001) with respect to their non-handled controls, corticosterone plasma levels in both Vh-H (q = 28.14, p < 0.0001)
and (Figure 5B). and in Ct-H (q = 32.30, p < 0.0001) when compared with
respective control groups (Figure 6B).
Corticosterone Plasma Levels
The effects of prenatal exposure to corticosterone, early
handling and their mutual influence on corticosterone DISCUSSION
plasma levels in rats under non-shock- or shock-induced
stress conditions were also investigated. A two-way ANOVA In agreement with previous animal studies, we here show
performed on the levels of corticosterone under non-shock- that exposure to corticosterone, during the 3rd week of rat
induced conditions as dependent variables, and prenatal gestation, can affect maternal care and program an abnormal
corticosterone treatment, early handling as independent neuroendocrine and behavioral profile of the adolescent
variables indicate that: prenatal corticosterone (F (1,36) = 5.311; offspring that resembles a vulnerable phenotype for affective
p = 0.0271) early handling (F (1,36) = 515.9; p < 0.0001) and their disorders (French et al., 1999; Shoener et al., 2006). Notably,
interaction (F (1,36) = 4.502; p = 0.004), were significant under early handling as a brief maternal separation during the

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Castelli et al. Prenatal Glucocorticoids and Early Handling

FIGURE 5 | Effects of prenatal corticosterone exposure and early handling procedure on immobility (A) and swimming (B) in the Forced swim test (FST). Each value
represents the mean ± SD of 10 rats. ***p < 0.001, *p < 0.05 vs. Vh, +++ p < 0.001 vs. Ct.

FIGURE 6 | Effects of prenatal corticosterone exposure and early handling procedure on: non-shock- (A) and shock-induced (B) corticosterone plasma levels.
Each value represents the mean ± SD of five rats. ***p < 0.001, *p < 0.05 vs. Vh, +++ p < 0.001 vs. Ct.

early stages of postnatal life, promoted an increase in 2004). Although the reduced body weight of the offspring
maternal care and counterbalanced the detrimental effects as a consequence of gestational corticosterone exposure is
induced by the prenatal glucocorticoid manipulation in all the still not fully clear, Iwasa et al. (2014) suggested a possible
investigated parameters. alteration of serum leptin and hypothalamic neuropeptide Y
(NPY) mRNA levels, two peptides playing pivotal roles in the
Effects of Prenatal Exposure to regulation of appetite and calories intake, as well as in the
Corticosterone modulation of emotionality (Velísek, 2006; Iwasa et al., 2014;
The first evidence following the manipulation of the intrauterine Plescia et al., 2014a).
environment by corticosterone injection from GD 14 to A critical outcome of glucocorticoid exposure in early life is
16 was a reduction in weight during the pre-weaning time. the programming of emotional and affective homeostasis. In the
Our data are in accordance with studies showing that rat, in utero glucocorticoids, either from an exogenous source
glucocorticoid treatment during pregnancy reduces offspring or via maternal extra-release, induce a decrease in behavioral
birth weight and body weight throughout adolescence (Smith reactivity in the open field and an increase in anxiety-like
and Waddell, 2000; Manojlovi ć-Stojanoski et al., 2012) as behavior in the elevated plus-maze in the offspring (Harris
well as the reduction on birth weight appears more evident and Seckl, 2011). These alterations may be associated with an
when glucocorticoids are administered during the 3rd week of impairment in offspring’s capacity to cope under a stressful
gestation and not earlier, indicating a late gestational window situation in adolescence (Vallée et al., 1997; Dickerson et al.,
of sensitivity to glucocorticoids (Nyirenda et al., 1998; Seckl, 2005; Harris and Seckl, 2011), enhancing the risk of emerging

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psychological disorders (Casey et al., 2010). Accordingly, our Plasma corticosterone levels in the non-shock-induced group
data demonstrate that the prenatal Ct-treatment during a do not differ from levels in the shock-induced group. This
sensitive time window, was able to induce an overall impairment may be for that non-shocked group plasma corticosterone levels
of locomotor activity in the adolescent offspring, as shown by a do not reflect baseline activity of the HPA axis, but rather
reduction in TDT and in the exploration of the central areas of HPA axis reactivity in response to novelty of the electrified
the arena. The reduction in behavioral reactivity might reflect grid floor cage (Friedman et al., 1967; Bassett et al., 1973).
an increased emotional response to the novel environment. Furthermore, differently from plasma corticosterone levels in
Consistently, adolescent rats exposed in utero to corticosterone shock-induced condition, we found that corticosterone release
exhibited an increase in the peak amplitude of the ASR, as after shock administration did not differ between prenatally
a proof of their negative emotional state (Lang et al., 1990; exposed adolescent offspring and Vh group. This can be due
Lang, 1995; Bradley and Sabatinelli, 2003; McMillan et al., to an altered drive of the HPA axis programming that may
2012). Indeed, The ASR, a reflexive movement occurring after result from the combination of in utero Ct-treatment and stress
sudden exposure to loud noise, represents a valid behavioral exposure in adolescence. Indeed, we may speculate that prenatal
model to study the emotional response of the animals. An corticosterone treatment was able to reduce the density of
increase in the amplitude of the ASR is ascribed to a rise corticosteroid receptors that, through the attenuation of HPA
in emotionality, which mirrors a higher sensitivity of the axis feedback sensitivity, set the release of corticosterone to
animals towards an anxiogenic environment (Hijzen et al., a ceiling set point already at basal conditions (Pornsawad,
1995; Cannizzaro et al., 2002). These results are in accordance 2013). This might prevent the physiological rise in stress-
with our data on the FST the most commonly used assay to related glucocorticoid release as we have observed in this study
test the efficacy of chronic antidepressant treatments (Detke and might represent a vulnerable factor for the development
et al., 1997). Our findings indicate that prenatal Ct treatment of emotional and affective disorders (Harris and Seckl, 2011;
was able to increase immobility time and decrease swimming Constantinof et al., 2016).
in the adolescent offspring, promoting the occurrence of a
depressive-like phenotype (Yankelevitch-Yahav et al., 2015). Effects of Early Brief Maternal Separation
Indeed, over-exposure to glucocorticoids and impaired GR In most mammalian species, the maternal environment
signaling can result in degeneration and functional impairment represents the developmental context within which mothers
of brain regions critically involved in mood processing and shape socio-emotional maturation of the progeny, serving
contribute to the induction of depressive symptoms later in life as essential external regulators of infant physiology,
(Anacker et al., 2011; Brancato et al., 2017; Di Liberto et al., 2017; neurodevelopment, and behavioral responses. Thus,
Shishkina and Dygalo, 2017). manipulating quality and consistency of maternal care during the
During development, there is a relatively high expression early stages of life can influence and, also revert developmental
of GR from midgestation onwards (Diaz et al., 1998), which processes that set emotional and physiological responses in
are essential for normal brain development and offspring adulthood (Drury et al., 2016).
survival (Kapoor et al., 2008). In the rat, antenatal stress or Numerous studies have shown that at least some of
maternal administration of glucocorticoids during this time the long-term effects of early-life exposure to an adverse
window results in offspring with decreased expression of environment are mediated by low levels of parent-child linking
GR mRNA in specific brain areas involved in glucocorticoid and decreased parental investment during early childhood.
feedback such as the hippocampus, hypothalamus, and pituitary For instance, poor parental ties are usually associated with
(Levitt et al., 1996; Liu et al., 2001). This reduction could increased risk for several psychological vulnerabilities, whereas
promote pups grow up with altered negative feedback response, an increase in parental care improved behavioral outcomes,
manifested as a chronic elevation of corticosterone (Maccari and cognitive performance and also boost resiliency to stress (Canetti
Morley-Fletcher, 2007). Indeed, the behavioral outcomes here et al., 1997; Meaney, 2001; Kaffman and Meaney, 2007).
observed are supported by the results from plasma corticosterone Accordingly, early handling procedure, consisting in a short
level assessment in non-shock-induced conditions. Specifically, maternal separation of the mother from the pups, represents a
prenatally exposed adolescent offspring showed an increase particular event for the dam that is able to produce higher level
in non-shock-induced plasma corticosterone levels, in line of interest by the mother in the offspring and, in turn, elicits more
with findings in rodents and non-human primates (Welberg maternal care upon reunion (Rees and Fleming, 2001; Kosten
et al., 2001; de Vries et al., 2007; Rakers et al., 2017). It and Kehoe, 2010; Zimmerberg and Sageser, 2011; Own and
has been shown previously that differences in HPA axis Patel, 2013; Orso et al., 2018). These observations are consistent
activity are associated with differences in locomotor activity with those obtained in the present research where the effects on
in response to novelty (Gancarz et al., 2012). Prenatal stress the maternal-infant dyad were investigated. Indeed, our results
induces a prolonged corticosterone secretion, which is negatively show that early handling procedure produced an increase in
correlated with lower levels of explorative behavior in the open maternal care, as shown by a higher MB-I, that in turn, improved
field (Rosecrans, 1970; Iuvone and Van Hartesveldt, 1976; Vallée the response to stressful situations and reduced emotionality
et al., 1997). Moreover, a significant correlation between plasma in the offspring. Specifically, when compared to non-handled
corticosterone levels and the behavioral scores in the FST was counterparts, briefly maternal separated adolescent rats showed
observed (Morley-Fletcher et al., 2003). increased locomotor activity, reduced avoidance of the center of

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the arena in the open field, and decreased peak amplitude in ASR. later in life, contributing to the programming of a vulnerable
At the same time, early-handled offspring displayed a reduction phenotype to emotional- and affective-like disorders. This
in immobility time and an increase in swimming time in the FST, issue is particularly relevant due to the common practice
together with a reduction in corticosterone plasma levels, under of multiple administrations of glucocorticoids to pregnant
non-shock- and shock-induced conditions. women during late gestation to ensure the survival of the
The mitigated emotional profile observed in early handled preterm newborns. Even though synthetic glucocorticoids,
rats in this study may be dependent upon modifications of the such as dexamethasone (DEX) or betamethasone, have been
developing HPA axis (Kaffman and Meaney, 2007). In particular, extensively used rather than cortisol or hydrocortisone (Jobe,
the effect of early handling on behavioral reactivity and 2003; Oliveira et al., 2006; Singh et al., 2012), the natural
emotionality may be due to a dampening of HPA axis response glucocorticoid is increasingly considered as an alternative
in the progeny that better cope with the task administered therapy during pregnancy (Crowther et al., 2019). Therefore, a
(Cannizzaro et al., 2006b). Indeed, maternal behaviors, such long-term follow-up in children who were treated in utero with
as licking, grooming and arched-back, lead to increased GR glucocorticoids is strongly recommended. As expected, we here
mRNA expression in the brain, glucocorticoid negative feedback show that enhanced maternal care plays a primary role in setting
sensitivity, and decreased hypothalamic corticotropin-releasing pro-adaptive behavioral and neuroendocrine responses and
factor mRNA levels (Meaney, 2001; Edelmann et al., 2016). may re-route aberrant trajectories during neurodevelopment,
Taken together these data suggest that postnatal maternal care emphasizing the role of an optimal mother-infant dyad as a
is able to affect the magnitude of the HPA axis response protective factor for healthy development of the offspring.
to stress, ‘‘hardening’’ the pups which display a blunting in
corticosterone release and in emotional profile (Meaney et al.,
DATA AVAILABILITY STATEMENT
1985, 1988; Liu et al., 1997). On the other hand, the variations
in the early postnatal environment can interact with the effects The datasets generated for this study are available on request to
of prenatal exposure to stressors in a complex, mutually the corresponding author.
interacting process (Cannizzaro et al., 2006b). Indeed, whether
early exposure to corticosterone is associated with elevation of
non-shock-induced conditions corticosterone release and with a ETHICS STATEMENT
vulnerable phenotype for emotional and affective disturbances,
The animal study was reviewed and approved by the Committee
early handling procedure induces opposite modifications in the
for the Protection and Use of Animals of the University
stress-behavioral responses and corticosterone release that are
of Palermo.
associated to the occurrence of a ‘‘rescued’’ profile. Although
we believe that the rodent model used in this study will
be helpful to identify physiological mechanisms underlying AUTHOR CONTRIBUTIONS
the neuroendocrine functional response to stress induced by
early handling in prenatal corticosterone condition, this issue FP has formulated evolution of overarching research goals and
deserves further insight in future researches on many distinct aims and has coordinated the research activity planning and
players which may take part to the interplay between maternal execution. He has provided statistical analyzses, has written
care and the regulation of the HPA axis, such as oxytocin the article and has acquired the financial support for the
(Cannizzaro et al., 2006a; Kojima et al., 2012; Cox et al., 2015; project leading to this publication. VC has carried out research
Zinni et al., 2018). However, it is evident that increasing the and investigation activity, performed the experiments, and has
intensity of maternal care, could serve as a source for the collaborated on the writing of the manuscript. GL and AB
enhancement of neuronal plasticity able to promote adaptive has carried out research and investigation activity, performed
behavioral responses. the experiments.

CONCLUSION FUNDING
These findings highlight a brief prenatal exposure to This research was supported by a grant from Funding for Basic
glucocorticoids during the 3rd week of gestation as a signal Activities Related to Research (FFABR), Italian National Agency
able to produce behavioral and neuroendocrine abnormalities for the Evaluation of Universities and Research Institutes.

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stress enhances adult anxiety and alters amygdala gene expression related to absence of any commercial or financial relationships that could be construed as a
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2013.10.064
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of DNA methylation of murine placenta in a sex-dependent manner and
is associated to anxiety-like behavior in adulthood. Transl. Psychiatry 9:23. Copyright © 2020 Castelli, Lavanco, Brancato and Plescia. This is an open-access
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 ORIGINAL RESEARCH
published: 28 February 2020
doi: 10.3389/fnbeh.2020.00029

Expression of Behavioral Phenotypes

in Genetic and Environmental Mouse
Models of Schizophrenia
Razia Sultana* and Charles C. Lee

Neural Systems Laboratory, Department of Comparative Biomedical Sciences, Louisiana State University School of
Veterinary Medicine, Baton Rouge, LA, United States

Schizophrenia is a neuropsychiatric disorder characterized by multifactorial etiology

involving complex interactions among genetic and environmental factors. “Multiple-
hit” models of the disorder can explain its variable incidence and prevalence in
related individuals. Hence, there is a dire need to understand these interactions
in the emergence of schizophrenia. To test these factors in the emergence of
schizophrenia-like behaviors, we employed a genetic mouse model of the disorder
(harboring the DISC1 mutation) along with various environmental insults, such as
early life stress (maternal separation of pups) and/or pharmacological interventions
Edited by: (ketamine injections). When assessed on a battery of behavioral tests, we found that
Sophie Laye,
INRA Centre Bordeaux-Aquitaine,
environmental interventions affect the severity of behavioral phenotypes in terms of
France increased negative behavior, as shown by reduced mobility in the forced swim and tail
Reviewed by: suspension tests, and changes to positive and cognitive symptoms, such as increased
Aniko Korosi, locomotion and disrupted PPI along with reduced working memory, respectively. Among
University of Amsterdam,
Netherlands
the various interventions, the genetic mutation had the most profound effect on
Vanja Nagy, behavioral aberrations, followed by an environmental intervention by ketamine injections
Institute of Molecular Biotechnology
and ketamine-injected animals that were maternally separated during early postnatal
(OAW), Austria
days. We conclude that although environmental factors increased the prevalence of
*Correspondence:
Razia Sultana aberrant behavioral phenotypes, genetic background is still the predominant influence
[email protected] on phenotypic alterations in these mouse models of schizophrenia.
Specialty section:
Keywords: DISC1 (disrupted-in-schizophrenia 1), maternal separation (MS), NMDAR hypofunction, ketamine
This article was submitted to injections, schizophrenia-like psychoses, gene-environment (G-E) interaction
Pathological Conditions, a section of
the journal Frontiers in Behavioral
Neuroscience INTRODUCTION
Received: 16 October 2019 Schizophrenia is a neuropsychiatric disorder whose etiology encompasses the interaction of several
Accepted: 07 February 2020 genetic and environmental factors. Heritability of the disorder is as high as 80% (Sullivan et al.,
Published: 28 February 2020
2003), with considerable ecogenetic variation in the prevalence of the disease among related
Citation: individuals (Ettinger et al., 2004). Such variation correlates with the degree of genetic relatedness
Sultana R and Lee CC of affected individuals; prevalence in first degree relatives (4%–8%), second-degree relatives
(2020) Expression of Behavioral
(2%–3.5%), and children of affected individuals (one parent affected, 13.6%; and both the parents
Phenotypes in Genetic and
Environmental Mouse Models of
affected, 37%) is indicative of the genetic heritability of the disorder (Salleh, 2004). The most
Schizophrenia. pronounced variations exist in twin studies with a concordance of 50% (Cardno and Gottesman,
Front. Behav. Neurosci. 14:29. 2000), suggesting a multifactorial etiology for schizophrenia and related disorders beyond
doi: 10.3389/fnbeh.2020.00029 genetic predisposition.

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Sultana and Lee Genetic and Environmental Mouse Models of Schizophrenia

Epidemiological studies of the disease show pronounced schizophrenia, major depression (47%), adolescent misconduct
interactions between genetic and environmental factors, which (9.5%), bipolar and minor depression, respectively (4.7%;
can explain variable degrees of onset, prevalence, and severity of Hennah et al., 2006). Furthermore, a frameshift mutation of
disorders in different individuals with a genetic predisposition the DISC1 gene in an American family was associated with
for the disorder (Karl and Arnold, 2014). Among the schizophrenic and schizotypic affective disorders (Sachs et al.,
environmental factors, maternal separation, early life stress, 2005; Zhang et al., 2006). In animal studies, such as in the 129S
drug abuse, and season and place of birth are related to the inbred strain of mouse (with a spontaneous, native truncation in
clinical presentation of schizophrenia (Tsuang, 2000; Tsuang the C-terminal of DISC1), L100 and Q31l mutations of the gene
et al., 2001; Morgan and Fisher, 2006). Such an interplay of result in schizophrenia-like phenotypes (Clapcote and Roder,
genetics and environment has given rise to ‘‘multiple-hit’’ models 2006; Clapcote et al., 2007; Niwa et al., 2010; Sultana et al., 2019).
of schizophrenia and associated psychotic disorders, where With its relative prevalence and concordant disease expression,
both genes and environment are important factors for disease DISC1 mutations are an important genetic factor in the etiology
expression in humans, as well as in animal models of psychotic of schizophrenia pathogenesis.
disorders (Bayer et al., 1999; Maynard et al., 2001; McGrath et al., Despite the advances in understanding the genetic and
2003; Feigenson et al., 2014). environmental factors involved in the etiology of schizophrenia
Several candidate genes have been associated with and a plethora of molecular interactions of DISC1 protein at
schizophrenia through genome-wide association (GWA) presynaptic, synaptic and/or postsynaptic sites (Hikida et al.,
and single nucleotide polymorphisms (SNPs) studies (McClellan 2012; Weng et al., 2018; Barnett et al., 2019) it remains
et al., 2007; Gejman et al., 2010). Among these, the DISC1 unclear the degree to which the DISC1 gene interacts with
(Disrupted in Schizophrenia 1) gene confers a 2% risk of environmental stressors and how such interactions impact
schizophrenia in carriers (Callicott et al., 2005; Song et al., the disease presentation in affected individuals. Therefore to
2008; Williams et al., 2009). DISC1 is a scaffolding protein understand the behavioral impact of DISC1 interactions with
that interacts with several genes, such as NDE, NUDEL, PDE4, environmental insults, in the present study, we utilized a
ATF4 and PCM etc. (Blackwood et al., 2001; Brandon et al., 2009; mouse model of schizophrenia (Jones et al., 2011) to test the
Porteous and Millar, 2009; Bradshaw and Porteous, 2012; Teng effects of an environmental stressor (maternal separation) and/or
et al., 2018). In particular, its interaction with PDE4 in dendritic pharmacological intervention (ketamine; Table 1) on the severity
spines serves as a molecular brake to maintain levels of cAMP of behavioral phenotypes in genetically predisposed animals
to restore synaptic connectivity in the PFC (Soares et al., 2011). (with DISC1 mutation) compared with controls. We found
Due to its synaptic localization with PDE4 and HCN channels that although environmental variables increased the number of
it plays a vital role in maintaining working memory and other animals exhibiting aberrant behaviors, the genetic composition
related behavioral phenotypes (Niwa et al., 2010; Gamo et al., of the animals was still the major driver in the expression of
2013; Paspalas et al., 2013). schizophrenia-related phenotypes.
Several human and animal studies have further demonstrated
the role of mutations in the DISC1 gene that lead to differential MATERIALS AND METHODS
disease phenotypes with variable prevalence (Gottesman and
Shields, 1976; Munafò et al., 2005; Van Winkel et al., 2010; Animal Care and Housing
Uher, 2014). In humans, a focused study of a Scottish family A total of 12 animals were used in each group: 129SvEv
with this mutation, 33.3% of individuals exhibited symptoms of (129S:∆DISC1) a DISC1 mutation model and C57BL/6J

TABLE 1 | Description of all the intervention groups used.

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(control), and the intervention groups described below. Mice performed in the order as we have described in our prior study
were obtained from the Jackson Laboratory (Bar Harbor, (Sultana et al., 2019). The following tests were included.
ME, USA). These animals were assessed on a battery of
behavioral tests when 8 weeks old, with the least stressful tests Open Field Test for Thigmotaxis and
performed first (Sultana et al., 2019). Initially, control and Overall Activity
129S:∆DISC1 animals were characterized by behavior without The total distance traveled in the apparatus was calculated and
any interventions. Animals were housed in a temperature and used as a measure of overall activity (Foshee et al., 1965). In
humidity-controlled room with a 12 h light/dark cycle with lights addition, this test was also used as a measure of anxiety-like
on at 7:00 am and food and water provided ad libitum. All behavior in terms of thigmotaxis, i.e., time spent near the
the experiments were conducted according to NIH guidelines periphery of the chamber (Simon et al., 1994; Seibenhener and
and were approved by the Institutional Animal Care and Use Wooten, 2015; Walz et al., 2016). Thus, we also determined the
Committee (IACUC) of the Louisiana State University School of percent time the test animal spent at the periphery vs. center
Veterinary Medicine. (Sultana et al., 2019) during the total 5 min test duration.

Animal Models and Interventions Sociability and Novelty

Maternal Separation (MS; Early Life Environmental As a measure of social interaction, sociability and social novelty
Stress) were tested as previously described (Kaidanovich-Beilin et al.,
Maternal separation of newborns, young children have shown 2011). On the test day, animals were assessed for sociability, as
a strong correlation with psychotic disorder precipitation in defined by the percent time that the test animal spent socializing
human subjects (Mäki et al., 2003; Paksarian et al., 2015). with stranger 1 (S1) i.e., (S1/S1+E) ∗ 100. Social novelty was
Maternal separation in mouse and rat pups, in particular, has assessed as the percent time spent with stranger 2 (S2) as
been used to model and study schizophrenia (Lehmann et al., (S1/S1+S2) ∗ 100.
2000; Fabricius et al., 2008). In this study, maternal separation
Modified Porsolt Forced Swimming Test
of pups was performed with slight modifications to previously
As a metric of despair, we utilized the modified Porsolt forced
described procedures (Roceri et al., 2002; Ellenbroek and Riva,
swimming test, derived from the procedure of Can et al.
2003). The pups were separated from dams for 4 h a day
(2012a,b). The camera (1080 HD, Logitech, Newark, CA, USA)
from postnatal day (PND) 3 to PND12 (critical period of brain
was positioned with a side view of the beaker to record the
development at these stages; Rice and Barone, 2000) daily from
leg movements of the animal. Scoring of the movements was
10:00 a.m. to 2:00 p.m. and weaned at PND21. The animals were
done as previously described by Can et al. (2012a,b). Percent
tested after PND60.
mobility time was calculated from a total 4 min testing period,
NMDAR Hypofunction (i.p. Ketamine Injection) following an initial 2 min acclimation period which was later
NMDAR hypofunction is a convergent molecular deficit found excluded from calculations. Measurements included when the
in instances of schizophrenia. To induce a pharmacologically animal was actively struggling to escape from the water container,
targeted behavioral deficit, we used a previously established whereas the propelling movement was not considered in the
model of schizophrenia (Ogundele and Lee, 2018) To induce mobility calculations.
NMDAR hypofunction similar to molecular findings in
schizophrenia, both 129S:∆DISC1 and control animals were
Tail Suspension Test
This test was used as another metric of negative behavior,
injected with a subanesthetic dose of ketamine (30 mg/kg) for
animals were suspended by a custom holder and percent mobility
5 days from day 45–50, as described previously (Becker et al.,
during suspension was assessed (Can et al., 2012b). The total test
2003; Frohlich and Van Horn, 2014; Ogundele and Lee, 2018)
duration was 6 min, but the latter 4 min were analyzed to remove
and were tested behaviorally starting at 5–7 days following the
any bias involving acclimation.
last ketamine injection.
Maternal Separation (PND3–PND12) With i.p. Stress Calls
Ketamine Injection During Adulthood When interpreted contextually, in certain psychotic disorders
Both 129S:∆DISC1 and control animals were separated like schizophrenia and schizotypic affective disorders, ultrasonic
maternally (from PND3-PND12; 4 h a day). In adulthood, they vocalization (USV) patterns can provide an indicator of
were injected with ketamine (i.p. 30 mg/kg), as described above. the affective state of the animal (Knutson et al., 2002;
These animals were then tested under the behavioral test battery Schwarting and Wöhr, 2012; Mun et al., 2015). Stress calls
as follows. were recorded simultaneously to the tail-suspension test, as
described previously. An AT125 bat call recorder (Binary
Behavioral Test Battery Acoustics, Carlisle, PA, USA) and digital recording software
All behavioral experiments were performed by the same SPECT’R (Binary Acoustics, Carlisle, PA, USA) was used. Calls
investigator during the late morning. The behavioral procedures were analyzed offline using SCAN’R software (Binary Acoustics,
have been described in detail in our previous work (Sultana et al., Carlisle, PA, USA). Calls above 30 kHz (typical of adult mice)
2019). One set of experiments was performed per day over a with a minimum duration of 5 ms were considered and analyzed.
16 days period with resting days in between. Experiments were The number of calls per 6 min period was calculated along with

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the mean and maximum frequency and the duration of each call. prevalence of this behavior in human schizophrenia patients
The entire 6 min period was analyzed since no difference was (Temmingh and Stein, 2015).
found when examining these data compared to the 4 min period
post-acclimation. Exploratory Behavior and Activity
While in the open field, the total distance covered by all groups
Y-Maze Test was assessed. Control animals traveled a significantly greater
Working memory includes the ability to rapidly form a distance as compared to other groups, i.e., DISC1 animals
memory trace and the exclusion of old information from that alone and with interventions (p ≤ 0.001; Figure 1B).
which is currently valid. This task was used as a tool to Environmental interventions on the DISC1 background
assess schizophrenia-related cognitive impairment. Videos were did not affect exploratory behavior when compared to the
recorded using a Logitech HD 1180 camera and later analyzed DISC1 without intervention animals. Moreover, control
with ANY-maze (ANY-maze, Wood Dale, IL, USA). Percent animals with maternal separation, ketamine injection
entries into the correct arm were calculated using the formula showed significantly decreased exploration (p ≤ 0.01 and
described previously (Sultana et al., 2019). p ≤ 0.001, respectively). These results support the interpretation
that all the environmental interventions affect animal behavior
Habituation to Acoustic Startle and Pre-pulse
differently depending on the genetic background and their
Inhibition (PPI)
specific interactions.
As a measure of pre-attentive deficits, this test is also used to
assess sensorimotor gating in human subjects with schizophrenia Sociability and Novelty
(Swerdlow et al., 2006). Following the protocol described by Control animals exhibited significantly higher time
Valsamis and Schmid (2011), responses to acoustic startle stimuli socializing compared with other groups, with the exception
were used to measure habituation and pre-pulse inhibition (PPI). of maternally separated (MS) controls (where different
The apparatus and protocols were followed as described in our groups differed at p-values control+ketamine at p ≤
prior study (Sultana et al., 2019). For stimulus delivery and 0.001, control+MS+ketamine at p ≤ 0.01, DISC1 at
recording of the startle signal, Audacity software 2.2.2 (Carnegie p ≤ 0.01, along with DISC1+MS, DISC1+ketamine,
Mellon University, Pittsburgh, PA, USA) was used. The startle DISC1+MS+ketamine at p ≤ 0.001, respectively; see
data were exported into Excel (Microsoft, Redmond, WA, USA) Figure 1C). The intervention groups did not exhibit an
using Python. Further analysis was done using Excel followed by intragroup difference in percent time with S1 (stranger 1) when
statistical analysis with GraphPad Prism 5 (LaJolla, CA, USA). compared amongst themselves, as shown in Figure 1C.
The data were expressed as mean ± SEM. These results indicate that environmental interventions
equally affect social withdrawal in all the models, with a
Statistical Analysis higher degree of social isolation in genetic mutation animals
ANOVA followed by Tukey’s post hoc test for multiple
(significant at p ≤ 0.01 in all DISC1 background groups).
comparisons was used to determine significant differences
However, environmental interventions on a DISC1 mutation
among groups. All the data were expressed as mean ± SEM. A
background did not change the sociability behavior of these
p-value < 0.05 was considered statistically significant. Statistical
animals significantly.
analysis was performed using GraphPad Prism 5 (GraphPad
Social novelty (i.e., percent time with S2) exhibited a
Software, La Jolla, CA, USA).
different outcome (Figure 1D), with control animals differing
from the DISC1 (p ≤ 0.05), maternally separated DISC1
RESULTS (p ≤ 0.01), MS+DISC1 injected with ketamine at adulthood
(p ≤ 0.01), and the DISC1+ ketamine injection animals
Thigmotaxis, Anxiety-Related Behavior exhibited an increased social novelty vs. DISC1 animals alone
As noted previously, time spent by animals along the walls (p ≤ 0.01), suggesting a complex interplay in DISC1 animals
of the open field provides an index of anxiety (Sultana et al., with pharmacologically induced NMDAR hypofunction (using
2019). Here, we calculated the percent time that animal groups ketamine; Figure 1D). Additionally, other environmental
spent at the periphery. We found that control animals spent a interventions in DISC1 animals did not differ significantly
significantly higher time in the center vs. control+MS+ketamine from those with no interventions. Control animals did not
(p ≤ 0.05; see Figure 1A), while animals with the DISC1 mutation show a significant difference when environmental interventions
showed a variable degree of time at the periphery differing from were imposed on this background, indicating that the genetic
control animals (DISC1 alone at p ≤ 0.001 and DISC1+ketamine mutation in DISC1 animals influenced their behavior towards
at p ≤ 0.01). Different interventions on the control background social novelty.
did not affect this behavior, except in the control+MS+ketamine
group (at p ≤ 0.05; Figure 1A). The variable degree of Y Maze: As an Index of Working Memory
anxiety-related behavior was intriguing, since not all the In this behavioral task, control animals exhibited significantly
interventions on the DISC1 and/or control group background higher percent time in the novel /correct arm (previously
exhibited this behavior (37.5% of the population among all blocked arm) as compared to DISC1 (DISC1 alone and with
the groups tested here), compared to schizophrenia subjects, interventions at p ≤ 0.001) and other groups (control+MS
anxiety-related behavior is not the endophenotype, with a 38% and control+MS+ketamine at p ≤ 0.001 and control+MS at

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FIGURE 1 | Three-chambered test for (A) percent time spent at the center of open field. (B) Total distance traveled throughout the apparatus. (C) Sociability. (D)
Novelty. All data presented here are presented as mean ± SEM, where a∗ compared to control at p ≤ 0.001; a# as compared to control at p ≥ 0.05; a as
compared to control at p ≤ 0.01 and b with above symbols show comparison of groups with DISC1 mutation animals.

p ≤ 0.01; Figure 2). It is important to note that environmental

interventions on a DISC1 mutation background did not
significantly disrupt the results of the working memory task
when compared to DISC1 animals without interventions. These
results demonstrate that working memory is affected in all the
control animal groups with various environmental interventions,
exhibiting the effect of these stressors on the hippocampus
(Malhotra et al., 1997; de Azeredo et al., 2017). Percent novel arm
entries did not show any significant difference among control vs.
intervention groups (data not shown).

Porsolt Forced Swim Test (FST)

Mobility during forced swim test (FST) can be used as a measure
of the degree of despair in animal models of behavioral disorders
(Can et al., 2012a). When test groups were compared for mobility
timing in FST, we found a variation in the number of animals
exhibiting depressive behavior within each group, whereas FIGURE 2 | Y-maze task for working memory: showing percent time each
when statistically compared, there was a significant reduction group spent in the novel arm. All data presented here are presented as
mean ± SEM, where a∗ as compared to control at p ≤ 0.001; a# as
in mobility timing (DISC1, DISC1+MS, DISC1+MS+ketamine
compared to control at p ≤ 0.05.
and control+ketamine, control+MS+ketamine at p ≤ 0.001;
and at p ≤ 0.01 for control+MS; as shown in Figure 3). The
environmental factors over a DISC1 mutation background Holmes, 2007), ketamine injections, and combinations of
did not show a significant reduction in mobility time both interventions affect the animals of each group to a
when compared to DISC1 mutation animals without variable degree, these groups still exhibit an overall depressed
intervention. Although maternal separation (Millstein and phenotype (Elk et al., 1986).

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other groups (see Figure 5B, control and DISC1 with ketamine,
control and DISC1 with MS+ketamine, and DISC1 alone) did
not show habituation but instead exhibited sensitization to
the acoustic stimulus (Figures 5B,D). The animals exhibited
increased startle, indicating that pharmacologically induced
NMDAR hypofunction might be affecting the ability of the brain
to habituate to the repeated acoustic stimulus.

Prepulse Inhibition (PPI)

Similar to the habituation test, the PPI responses exhibited
a similar pattern of inhibition to different inter-stimulus
interval (ISI) and pre-pulse intensity combinations. We found
that control animals showed inhibition to all trial (ISI-PP
intensity) combinations (see Figures 6A–D), significantly
differing from control+MS+ketamine (p ≤ 0.001), DISC1 (p
≤ 0.05) and DISC1+ketamine (p ≤ 0.01; at ISI of 30 ms with
FIGURE 3 | Forced Swim Test (FST) for depressive behavior: exhibiting the
intensity of 75 dB depicted as 30_75 shown in Figure 6A),
percent mobility time by all the groups. All the presented here are presented
as mean ± SEM, where a* as compared to controls at p ≤ 0.001; a# as
control+ketamine (p ≤ 0.05) and control+MS+ketamine [p
compared to controls at p ≤ 0.05. ≤ 0.01; at 30 (ISI)_85 (Prepulse intensity); Figure 6B], and
control vs. control+ketamine, control+MS+ketamine [p ≤
0.05 at 100 ms (ISI)_75 dB (Prepulse Intensity); Figure 6C
Tail Suspension Test and Stress Calls and p ≤ 0.001 at 100 ms (ISI)_85 dB (Prepulse Intensity)
The tail suspension test was used as another measure of despair combinations; Figure 6D]. Control animals also differed from
and resulted in a similar outcome as the FST. Control animals DISC1+MS [p ≤ 0.01 at 100 ms (ISI)_85 dB (Prepulse Intensity;
showed the highest mobility, which was significantly different Figure 6D)]. Thus, unlike habituation to acoustic startle
from other groups (at p ≤ 0.001 for all the groups and p stimulus, maternal deprivation alone in these animals did not
≤ 0.01 for DISC1+MS+Ketamine vs. control; see Figure 4A). cause aberrations in PPI (Ellenbroek and Cools, 2002). Both
DISC1 mutation animals vs. DISC1 with other interventions control and DISC1 animals with ketamine injections exhibited
did not show significant differences amongst themselves. Thus, aberrations in PPI behavior, showing an impact of NMDAR
various environmental stressors led to decreased mobility in hypofunction on sensorimotor gating mechanism of brain
the groups which was not significantly different from each circuitry (Cilia et al., 2007).
other (Figure 4).
While the animals were suspended, we also recorded the
USV emitted by these animals, as a measure of calls produced
DISCUSSION
under stress. We found that DISC1 mutation animals produced Schizophrenia is a neuropsychiatric disorder associated with
fewer calls compared to control animals (p ≤ 0.01; Figure 4B; multiple genetic and environmental etiologies (Tsuang et al.,
Zimmerberg et al., 2003; Yin et al., 2016). Control animals 2001). Although no single gene or environmental factor is known
also differed significantly from DISC1, with maternal separation to be completely causal (Choi et al., 2009; van de Leemput et al.,
and DISC1 maternally separated with ketamine injection (p 2016; Howes et al., 2017), interactions among multiple factors
≤ 0.01; Figure 4B). Affective vocalizations differed to varying increase the emergence of the disorder. We focused our studies
degrees in control animals that were maternally separated and/or on two different genetic backgrounds, control group (C57BL/6J)
treated with ketamine (for traces of USVs see also Figure 4C). which does not have a genetic predisposition to schizophrenia
On the other hand, DISC1 animals with interventions did not or schizotypic disorders, and the test group that is genetically
exhibit a reduction in calls when compared to DISC1 animals predisposed (129S strain with C-terminal truncation of DISC1
without interventions. gene) on a behavioral test battery (Brixey et al., 1993; Krystal
et al., 1994; Ellenbroek and Riva, 2003; Koike et al., 2006).
Habituation to Acoustic Startle Response We have previously observed that this 129S: ∆DISC1 strain
(ASR) differs behaviorally from several other common inbred and
Habituation to acoustic startle response (ASR) was measured outbred mouse strains (Sultana et al., 2019). Our prior findings
to determine sensorimotor gating and pre-attentive deficits. As indicate that the inherent DISC1 mutation in the 129SvEv
previously described, the first test block measured habituation mice has a penetrant effect on behavioral phenotype above
(Sultana et al., 2019). We found that maternal separation of various genetic backgrounds. Other mouse strains (Balb/c,
the control pups and DISC1 pups did not affect habituation CBA/J, etc.) could potentially serve as an appropriate behavioral
to ASR vs. control, exhibiting habituation of 65%, 53% and control strain here, since they are all similar behaviorally and
42% (respectively for control, control+MS and DISC1+MS; distinct from the 129SvEv strain, putatively as a result of the
Figures 5A,D). The magnitude of habituation was not DISC1 genetic mutation. This is supported by findings that
significantly different in these animal groups. However, all DISC1 mutations on the same background strain do not differ

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FIGURE 4 | Tail Suspension Test (TST) for depressive behavior: (A) exhibiting the percent mobility time by all the groups. (B) Stress calls recorded/6 min period
while under tail suspension. (C) Representative stress calls in all the groups; with y-axis showing frequency/call and x-axis showing duration (6 min). All data
presented here are presented as mean ± SEM, where a∗ as compared to control at p ≤ 0.001; a# as compared to control at p ≤ 0.05. a as compared to control at
p ≤ 0.01 and b with above symbols shows comparison of groups with DISC1 mutation animals.

when compared across strains (Lee et al., 2011). Nevertheless, novelty, overall activity (Figure 1), USV (Figure 4B), and
the comparisons employed here do add a potential caveat to our habituation to acoustic startle (Figure 5) on control animals
results, which must be considered in their interpretation. suggesting that genetic predisposition might be necessary for
DISC1 mutations affect behavior by its interactions with this stressor to contribute to disease pathology to exhibit above
pathways such as PDE4, upregulation of SK2 (calcium-activated mentioned behavioral syndrome.
small potassium channels at the PSD; Sultana et al., 2018), In many of the behavioral tasks, environmental interventions
and HCN (Paspalas et al., 2013). These interactions take place on the DISC1 background did not increase the severity of
at the dendritic synaptic densities, where NMDARs acts as a behavioral phenotypes. We suggest that the severity of symptoms
convergence point for DISC1 and its interacting partners such in many of the behavioral task have a lower/upper limit.
as PDE4. NMDAR hypofunction alone or in combination with However, an interesting finding from our data is that the
DISC1 mutations aggravate behavioral phenotypes, as discussed animals harboring the DISC1 mutation often exhibit a bimodal
below (see also Figures 1–3). distribution in the expression of behavioral phenotypes, which
We assessed the effects of genetic predisposition is not found following environmental interventions. Thus, we
(DISC1 mutation), environmental factors (maternal separation propose that the effects of the environmental interventions
and ketamine injections), and interactions of these factors may not necessarily be on the magnitude of the behavioral
(see Table 1). Among these factors, our results suggest that effects, but rather the probability that these animals may develop
genetic factors play the predominant role in the presentation schizophrenia-related behavioral phenotypes.
of behavioral phenotypes associated with the disease, while As models to study schizophrenia, these interventions are
pharmacological intervention (ketamine injections) and argued to have faced, construct and/or predictive validity
maternal separation showed incremental effects, particularly (Jones et al., 2011). The DISC1 mutation is known to associate
on the genetically predisposed animals. Interestingly, maternal with a neurological disorder in about 33.3% of the large Scottish
separation did not show a significant effect in terms of sociability population where the mutation is present, with members of the

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FIGURE 5 | Habituation to acoustic startle response (ASR). (A) Groups showing habituation; control (black), control+MS (green) and DISC1+MS (pink). (B) Groups
that did not show habituation. (C) Merge showing all the groups with and without habituation. (D) Percent habituation in different groups towards the last tone. All
data presented here are presented as mean ± SEM, where a∗ as compared to control at p ≤ 0.001; a# as compared to control at p ≤ 0.05; a as compared to
control at p ≤ 0.01 and b with above symbols shows comparison of groups with DISC1 mutation animals.

family exhibiting schizophrenia, bipolar and major depression Genetic mutation and environmental stress affect the
disorders etc. In the present study, DISC1 mutation animals behavioral emergence of schizophrenia. However, when there is
exhibited decreased sociability, novelty (Figure 1), mobility time a combination of factors, we found that genetic background has
in forced swim and tail suspension test (Figures 3, 4A). When the biggest influence as shown by reduced sociability and novelty
compared with prior studies, animal models of DISC1 mutation in the DISC1 mutation background animals (Figures 1C,D),
produced using various methods, e.g., shRNA, use of chemicals, when compared with the same insults on the control background
backcrossing 129S on C57BL6 background, report similar results highlighted in terms of anxiety-like behavior, stress calls and
to our findings (Jaaro-Peled, 2009; Johnstone et al., 2011; habituation to ASR as well as PPI (Figures 1A, 4B, 5, 6).
Tomoda et al., 2016). Moreover, disruption of sensorimotor Environmental factors such as pharmacological interventions
gating has been observed in various models of schizophrenia that cause direct NMDAR hypofunction (ketamine injections)
including the DISC1 genetic mutation (Tomoda et al., 2017). results in similar behavioral outcomes (such as reduced
Additionally, NMDAR hypofunction is a key finding in sociability and mobility in FST and TST) for both control and
human postmortem studies of schizophrenia and bolsters the DISC1 mutation animals, showing that NMDAR hypofunction
glutamate dysfunction theory of schizophrenia pathogenesis is a convergence point for the molecular mechanism behind
(Snyder and Gao, 2013). Furthermore, it is known that core symptoms of schizophrenia. On the other hand, maternal
DISC1 and NMDARs interact dynamically with each other separation of pups leads to more negative symptoms, i.e., reduced
(Wang and Zhu, 2014), such that DISC1 dependent changes in mobility in FST and TST and spatial memory, but it does
NMDAR synaptic responses are speculated to affect cognition in not affect the social recognition behavior of these animals
individuals with schizophrenia (Ramsey et al., 2011; Wei et al., (Figures 1C,D). All other combinations of interventions
2014). Behaviorally, previous results from our lab also found that influenced the behavioral phenotype to a variable degree with
there is a reduced interaction of test mice in terms of sociability, DISC1+MS+ketamine animals showing more aberrant behaviors
social novelty, reduced spatial/working memory (Ogundele and when compared with the control+MS+ketamine group, clearly
Lee, 2018), similar to our results in ketamine-treated animals indicating the effects of genetic predisposition.
(control as well as DISC1 mutation background). We also found Overall, our study supports the most recent theories of gene-
that ketamine injection in the DISC1 mutation animals resulted environment interactions and their effects on the behavioral
in increased hypo-frontality, leading to enhanced negative signs, phenotype of nervous disorders, such as schizophrenia.
such as more depressed behavior (Figures 3, 4), as indicated by Interactions between multiple components affect behaviors
decreased mobility in FST and TST as well as the reduced number at various levels for positive (aberrant PPI, reduced habituation
of stress calls in these animals. to acoustic startle) and negative symptoms (decreased mobility

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Sultana and Lee Genetic and Environmental Mouse Models of Schizophrenia

FIGURE 6 | Pre-pulse Inhibition (PPI). Prepulse inhibition at different inter-stimulus interval and pulse intensity. (A) 30_75. (B) 30_85. (C) 100_75. (D)
100_85, respectively. All data presented here are presented as mean ± SEM, where a* as compared to control at p ≤ 0.001; a# as compared to control at p ≤ 0.05;
a as compared to control at p ≤ 0.01 and b with above symbols shows comparison of groups with DISC1 mutation animals.

timing on FST and TST tests; Figures 3, 4), including cognitive as are any potential epigenetic changes that these stressors may
tasks with learning and memory deficits (as shown in Y bring about in healthy individuals (Roth et al., 2009).
maze test; Figure 2; Ellenbroek and Cools, 2002; Powell
and Miyakawa, 2006; Gómez-Sintes et al., 2014). We also
DATA AVAILABILITY STATEMENT
emphasize the finding that, although DISC1 mutation animals
with various environmental interventions did not change The datasets generated for this study are available on request to
the severity of the behavioral profile of these animals when the corresponding author.
compared to DISC1 mutation alone, the prevalence of animals
exhibiting aberrant behavioral phenotype increased due to
gene-environmental interaction. We propose then that this effect ETHICS STATEMENT
could be due to environmental intervention acting as a second
The animal study was reviewed and approved by Institutional
hit to increase the chances of disease development in genetically
Animal Care & Use Committee (IACUC) of the Louisiana State
predisposed animals with the DISC1 mutation.
University School of Veterinary Medicine.

CONCLUSION
AUTHOR CONTRIBUTIONS
These behavioral changes suggest several aberrant molecular
interactions must be occurring at the cellular, subcellular and/or CL designed the study, edited and finalized the manuscript. RS
extracellular levels. Here, we have first attempted to assess the designed, executed the experiments, collected data, statistically
combination of environment and genetics in the development analyzed and wrote the manuscript.
of behavioral phenotype. The results of our present study
suggest that the DISC1 genetic mutation predominates over the FUNDING
environmental factors used in our study in the presentation of
schizophrenia-like behavioral phenotypes. The molecular and This study was funded by the National Institute of Health
neural factors that lead to these behaviors remain to be examined, (R03MH104851 & R03 NS 109682).

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Conflict of Interest: The authors declare that the research was conducted in the
schizophrenia-1. Biol. Psychiatry 75, 414–424. doi: 10.1016/j.biopsych.2013.
absence of any commercial or financial relationships that could be construed as a
06.009
potential conflict of interest.
Weng, Y.-T., Chien, T., Kuan, I. I., and Chern, Y. (2018). The TRAX, DISC1, and
GSK3 complex in mental disorders and therapeutic interventions. J. Biomed. Copyright © 2020 Sultana and Lee. This is an open-access article distributed
Sci. 25:71. doi: 10.1186/s12929-018-0473-x under the terms of the Creative Commons Attribution License (CC BY). The use,
Williams, J. M., Beck, T. F., Pearson, D. M., Proud, M. B., Cheung, S. W., and distribution or reproduction in other forums is permitted, provided the original
Scott, D. A. (2009). A 1q42 deletion involving DISC1, DISC2, and TSNAX author(s) and the copyright owner(s) are credited and that the original publication
in an autism spectrum disorder. Am. J. Med. Genet. A 149A, 1758–1762. in this journal is cited, in accordance with accepted academic practice. No use,
doi: 10.1002/ajmg.a.32941 distribution or reproduction is permitted which does not comply with these terms.

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12 February 2020 | Volume 14 | Article 29
 MINI REVIEW
published: 05 June 2020
doi: 10.3389/fnbeh.2020.00072

Gender Differences in the Outcome

of Offspring Prenatally Exposed to
Drugs of Abuse
Francesco Traccis, Roberto Frau and Miriam Melis*
Department of Biomedical Sciences, Division of Neuroscience and Clinical Pharmacology, University of Cagliari, Cagliari, Italy

Despite great efforts to warn pregnant women that drugs of abuse impact development
of the embryo and the fetus, the use of legal and illegal drugs by childbearing women
is still a major public health concern. In parallel with well-established teratogenic effects
elicited by some drugs of abuse, epidemiological studies show that certain psychoactive
substances do not induce birth defects but lead to subtle neurobehavioral alterations
in the offspring that manifest as early as during infancy. Although gender differences
in offspring susceptibility have not been fully investigated, a number of longitudinal
studies indicate that male and female progeny exposed in utero to drugs of abuse show
different vulnerabilities to deleterious effects of these substances in cognitive, executive,
and behavioral domains. Here, we briefly review the existing literature focusing on
gender differences in the neurobehavioral consequences of maternal exposure to
drugs of abuse. Overall, the data strongly indicate that male exposed progeny are
Edited by: more susceptible than female to dysfunctions in cognitive processing and emotional
Gregg Stanwood, regulation. However, insights into the mechanisms determining this natural phenomenon
Florida State University, United States
are not currently available. Our analysis prompts future investigations to implement
Reviewed by:
Gordon Alfred Barr, clinical studies including the influence of gender/sex as a biological variable in the
Children’s Hospital of Philadelphia, outcome of offspring prenatally exposed to drugs of abuse.
United States
Devon L. Graham, Keywords: development, drugs of abuse, gender, neuropsychiatric, prenatal, sex, vulnerability
Florida State University, United States
*Correspondence:
Miriam Melis INTRODUCTION
[email protected]
As a rule, drugs should not be used during pregnancy unless prescribed, because many can be
Specialty section: toxic to the placenta or the developing fetus. Yet, the use of drugs, including prescription or
This article was submitted to non-prescription drugs, medicinal herbs, and licit (tobacco and alcohol) or illicit drugs, during
Emotion Regulation and Processing, pregnancy keeps increasing (SAMHSA, 2011). Indeed, objective measurements of xenobiotics in
a section of the journal meconium, amniotic fluid, and cord blood indicate widespread fetal exposure to such agents
Frontiers in Behavioral Neuroscience
during their intrauterine life (for an excellent review see Barr et al., 2007). Such exposure
Received: 30 December 2019 may induce developmental adaptations that can be interpreted as derangements from normal
Accepted: 21 April 2020
development, which not only interfere with the immediate viability of the fetus but may also
Published: 05 June 2020
result in the individual’s adverse health outcome in the short and long term (Hales and Barker,
Citation: 2001; Barker, 2007). Hence, the “developmental origin of health and disease” hypothesis (Barker,
Traccis F, Frau R and Melis M
2007) stems from epidemiological studies showing that malnutrition, exposure to xenobiotics
(2020) Gender Differences
in the Outcome of Offspring Prenatally
(e.g., environmental chemicals and prescription, legal, and illegal drugs), infective diseases, or
Exposed to Drugs of Abuse. stress during specific periods of development might increase the risk of disorders later in life.
Front. Behav. Neurosci. 14:72. This hypothesis also stresses the importance of investigating the mechanisms of fetal exposure to
doi: 10.3389/fnbeh.2020.00072 xenobiotics and further in general to adverse intrauterine and perinatal factors.

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Traccis et al. Gender Difference in Drug Exposure

In this minireview, we will provide an up-to-date analysis drugs but includes prescription and over-the-counter drugs.
of the evidence for a sex differential in the susceptibility Approximately 60% of pregnant women take prescription drugs
to the consequences of maternal drug use on neurocognitive and about 13% of them use herbal supplements. Furthermore,
and behavioral development of the offspring. Research has the infographics based on the National Survey on Drug Use and
pointed to gender differences in these sequelae, since exposed Health (SAMHSA, 2018) show that 5.4% of pregnant women
males often appear more vulnerable than exposed females. have used illicit drugs in the past 30 days, while 9.9 and
Insights into the neurobiological mechanisms underlying the 11.6% reported past-month alcohol or cigarette smoking use,
sex bias observed in certain neurobehavioral outcomes remain respectively. To complicate this issue, many women take drugs
unidentified. At this stage, we could only make inferences when they are not aware of being pregnant.
from animal studies, although they do not allow for a precise Regardless of their legal status, all drugs cross and/or alter the
understanding of the underpinnings, especially in the context of placental barrier, reach the fetus, and affect infant development.
sex differences. In particular, many factors might moderate the Additionally, multiple drugs also pass into mother’s breast milk,
reported sex dichotomy, including individual (e.g., species, strain, thus resulting in prolonged drug exposure of the newborn.
age) and experimental (e.g., design, drug, dosage, route, regimen) According to the United States Centers for Disease Control and
variables, and objective endpoints (e.g., behavioral paradigm, Prevention, almost 3% of newborns have birth defects because
experimental technique). Here, we attempt to integrate the of genetic, environmental, or other unknown causes (Parker
gender difference results across drugs used by pregnant women. et al., 2010). Among environmental factors, drug use is the
Such integration could be useful for physicians and healthcare major cause leading to birth defects ranging from fetal growth
providers when caring for a pregnant substance abusing woman. reductions to medical complications such as preterm birth and
Interspecies extrapolations will be carefully avoided to ensure infections. Furthermore, the progeny prenatally exposed to drugs
sound conclusions. The authors refer to excellent preclinical of abuse develop neurobehavioral phenotypes that manifest
studies’ reviews (Bruin et al., 2010; Schneider et al., 2011; during infancy and persist to adolescence and young adulthood.
Ross et al., 2015; Gkioka et al., 2016; Comasco et al., 2018; Research on the effects of prenatal alcohol, tobacco, opioids,
Scheyer et al., 2019). stimulants, and cannabis indicates an association between fetal
exposure to these substances and deficits in cognitive and
behavioral domains. However, in humans, the role of fetal sex
SUBSTANCE USE IN WOMEN on functional consequences of prenatal exposure to drugs of
abuse remains grossly understudied. Here we present data on
The historical gap in substance use prevalence between men and illicit psychostimulants, opioids, cannabis, nicotine, and alcohol
women has gradually narrowed in the past decade, particularly in an attempt to provide a clear picture of neurobehavioral
among adolescents (Keyes et al., 2008; Seedat et al., 2009; outcomes in male and female progeny. When gender differences
Steingrimsson et al., 2012; EMCDDA, 2019). While women still have not been examined, our interpretation is limited to the
exhibit lower rates of drug use disorder than men, prevalence overall outcome.
rates indicate that the number of female drug abusers is on the
rise. A recent snapshot of the European drug use situation shows
that women account for one-quarter of the general population Effects of in utero Exposure to
with drug issues and around one-fifth of all first-time drug abuse Psychostimulants
treatment seekers (EMCDDA, 2019). Gender differences are clear Psychostimulants, including cocaine and methamphetamine,
in the pattern of use at each stage of the addiction cycle. Women are the illicit drugs most commonly used by childbearing
typically begin to use substances later in life (Greenfield et al., women, though no recent estimate of their consumption during
2010; Keyes et al., 2010), misuse prescription drugs (e.g., opioids) pregnancy is known. Despite their well-described neurotoxic
(McHugh et al., 2013), and their rate of consumption increases effects on central nervous system (CNS) development, only very
more rapidly than that of men (Greenfield et al., 2010; Keyes few studies have addressed the negative neurobehavioral sequalae
et al., 2010). Women also exhibit higher prevalence rates of on human offspring, particularly when gender is included as an
comorbidity with other psychiatric disorders as well as of relapse additional biological variable (Table 1 and Figure 1).
(Wilcox and Yates, 1993; Conway et al., 2006; Back et al., 2011; Longitudinal studies of long-term consequences of cocaine
Khan et al., 2013). use during pregnancy on the offspring focusing on emotional
regulation, behavior, and cognition suggest that female gender
is a protective factor (Singer et al., 2004; Dennis et al., 2006;
DRUG USE DURING PREGNANCY AND Accornero et al., 2007; Bennett et al., 2008; Ackerman et al.,
BREASTFEEDING: EFFECTS ON MALE 2010; Bridgett and Mayes, 2011). Male progeny exhibit stronger
AND FEMALE OFFSPRING impairment in inhibitory response, whereas females exhibit only
mild alterations that disappear with age (Carmody et al., 2011).
The consumption of drugs in childbearing women has been Accordingly, male offspring exhibit greater emotion regulation
progressively increasing. Women abusing recreational drugs problems and externalizing symptoms (e.g., aggressive and
before pregnancy tend to continue the use even during risky behaviors); lower intellectual capabilities; and deficits in
gestation (Forray, 2016), and this use is not limited to illegal attention, short-term memory, and problem solving compared

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Traccis et al.
TABLE 1 | Detailed information on the studies covered in this minireview examining gender as a variable.

Author year Substance Type of Experimental Neuro Prenatal Socioeconomic Age Performed tasks Gender results
study groups developmental polysubstance status
outcome exposure
Prenatal psychostimulant exposure
Lu et al., Meth Cross- Methamphetamine Cognitive Yes Matched for 7–15 year Wechsler Intelligence Impaired verbal learning
2009 amphetamine sectional exposed vs alcohol abilities (methamphetamine, socioeconomic Scale for Children, 4th capacities in
study exposed vs control alcohol) status Edition (WISC-4), California methamphetamine and
Verbal Learning Test for alcohol exposed
Children (CVLT-C)
Diaz et al., Meth Longitudinal Exposed vs control Cognitive Yes Matched for 7.5 year Conners’ Parent Rating Significantly higher
2014 amphetamine study abilities (methamphetamine, socioeconomic Scale–Revised: Short cognitive problems scores
alcohol, cannabis, status Form (CPRS-R:S) in exposed children
tobacco)
Piper et al., Meth Cross- Methamphetamine Cognitive Yes Matched for 7–9 year Wechsler Abbreviated Exposed children show
2011 amphetamine sectional and polysubstance Abilities (methamphetamine, socioeconomic Scale of Intelligence, deficit in executive
study exposed vs alcohol, tobacco, status Conners’ Continuous functions (e.g., behavioral
Unexposed cannabis) Performance Test II, regulation and
Behavioral Rating metacognition) and spatial
Inventory of Executive memory
Function, the CMS Family
Pictures and Dot Location
tests, the Spatial Span test
from WISC-IV-Integrated,
and a recently developed
spatial learning and
memory measure (Memory
Island)
Kiblawi et al., Meth Longitudinal Exposed vs ADHD risk Yes Controlled for 5 year Conners’ Kiddie KCPT scores suggest
2013 amphetamine unexposed (methamphetamine, low Continuous Performance higher ADHD risk for
alcohol, cannabis, socioeconomic Test (K-CPT) exposed children
tobacco) status
LaGasse Meth Longitudinal Exposed vs ADHD risk Yes (methadone, Adjusted for 3–5 year Child Behavior Checklist Higher prevalence of ADHD
et al., 2012 amphetamine study unexposed alcohol and low symptoms in exposed
tobacco, cannabis) socioeconomic males than girls
status
LaGasse Meth Longitudinal Exposed vs Behavioral Yes (methadone, Adjusted for 3–5 year Child Behavior Checklist More externalizing
et al., 2012 amphetamine study unexposed problems alcohol and low problems and aggressive

Gender Difference in Drug Exposure

tobacco, cannabis) socioeconomic behavior in exposed males
June 2020 | Volume 14 | Article 72

status than girls

Bennett Cocaine Longitudinal Exposed vs Cognitive Yes (cocaine, Measured as 4, 6, Stanford-Binet IV Lower composite IQ score
et al., 2008 study unexposed abilities alcohol, tobacco, environmental 9 year intelligence test (abstract/visual and verbal
cannabis) risk reasoning, short-term
memory) in exposed boys
but not girls
(Continued)

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Traccis et al.
TABLE 1 | Continued

Author year Substance Type of Experimental Neuro Prenatal Socioeconomic Age Performed tasks Gender results
study groups developmental polysubstance status
outcome exposure
Dennis et al., Cocaine Longitudinal Exposed vs Cognitive Yes (cocaine, Measured as 5 year The impossible pulley task More difficulties in problem
2006 study unexposed abilities alcohol, tobacco, environmental solving and altered
cannabis) risk reactivity/ regulating
behavior in exposed males
than females
Singer et al., Cocaine Longitudinal Exposed vs Cognitive Yes (cocaine, Measured as 0–4 year Wechsler Preschool and Mild but significant
2004 study non-exposed abilities alcohol, tobacco, caregiving Primary Scales of difficulties in cognitive
cannabis) environmental Intelligence-Revised abilities (visual-spatial and
risk arithmetic skills) in exposed
males
Mayes et al., Cocaine Longitudinal Exposed vs Cognitive Yes (cocaine, Measured as 0–3 year Bayley Scales of Infant Lower BSID-II mental
2003 study non-drug and non- abilities alcohol, tobacco, environmental Development (BSID-II) performance in cocaine
cocaine-exposed cannabis) risk exposed children
compared to both non-drug
and non-cocaine-exposed
children
Accornero Cocaine Longitudinal Exposed vs Deficit in Yes Matched for 5–7 year Continuous performance Deficits in attention
et al., 2007 study unexposed attention and socioeconomic tests (CPTs) processing in exposed
inhibition status offspring
response
Karmel and Cocaine Longitudinal Exposed vs Attention and Yes (cocaine, ND 0–1 year Visual looking preferences Arousal-modulated
Gardner, study unexposed arousal alcohol, tobacco) attention deficit in exposed
1996 male and female infants
Bennett Cocaine Longitudinal Exposed vs Neurobehavioral Yes (cocaine, Adjusted for 10 year Youth Risk Behavior Highest scores for
et al., 2007 study unexposed problems alcohol, tobacco, low Survey aggression, substance use,
cannabis) socioeconomic high-risk behavior in
status exposed males
Nordstrom Cocaine Longitudinal Exposed vs Neurobehavioral Yes (cocaine, Controlled for 6–7 year Achenbach Teacher Delinquent behavior and
Bailey et al., study unexposed problems alcohol) socioeconomic Report Form (TRF) clinically significant
2005 (aggressive status externalizing behavior
behavior) scores in exposed boys
Sood et al., Cocaine Historical Alcohol exposed vs Neurobehavioral Yes (cocaine, Controlled for 6–7 year Caregiver reported Higher aggressive (in
2005 prospective cocaine and/or problems alcohol) socioeconomic Achenbach Child Behavior females) and delinquent

Gender Difference in Drug Exposure

study alcohol exposed status Checklist (CBCL) behaviors (in males) scores
June 2020 | Volume 14 | Article 72

in exposed offspring
Bendersky Cocaine Longitudinal Exposed vs Neurobehavioral Yes (cocaine, Measured as 5 year ACHENBACHChild Aggressive behavior in
et al., 2006 study unexposed problems alcohol, tobacco, environmental Behavior Checklist (CBCL), exposed male offspring
cannabis) risk LRRH Reinisch Revision,
Teacher Rating of
Aggression (TRA)
(Continued)

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Traccis et al.
TABLE 1 | Continued

Author year Substance Type of Experimental Neuro Prenatal Socioeconomic Age Performed tasks Gender results
study groups developmental polysubstance status
outcome exposure
Prenatal opioid exposure
Nygaard Heroin Longitudinal Heroin exposed vs Cognitive Yes (heroin, Controlled for 1–3 year Bayley Scales II (Mental Significantly and stably
et al., 2015 study polysubstance abilities benzodiazepines, low Development Index, MDI) lower levels of cognitive
exposed alcohol) socioeconomic functioning in male progeny
status
Nygaard Heroin Longitudinal Heroin exposed vs Cognitive Yes (heroin, Controlled for 4 year McCarthy Scales of Significantly and stably
et al., 2015 study polysubstance abilities benzodiazepines, low Children’s Abilities lower levels of cognitive
exposed alcohol) socioeconomic functioning in male progeny
status
Nygaard Heroin Longitudinal Heroin exposed vs Cognitive Yes (heroin, Controlled for 8 year The Wechsler Intelligence Significantly lower cognitive
et al., 2015 study polysubstance abilities benzodiazepines, low Scale for scores in both exposed
exposed alcohol) socioeconomic Children—Revised males and females
status
Nygaard Heroin Longitudinal Heroin exposed vs Cognitive Yes (heroin, Matched for 17– Wechsler Abbreviated Significantly worse
et al., 2017 study polysubstance abilities tobacco, socioeconomic 21 year Scale of Intelligence cognitive performances in
exposed benzodiazepines, status (WASI), The Rey Complex male and female exposed
alcohol, Figure Test (RCFT), The offspring compared to
psychopharmaca) California Verbal Learning controls
Test – 2nd Ed (CVLT-II),
Wechsler Adult Intelligence
Scale 3rd Ed(WAIS-III)
Suffet and Methadone Longitudinal Exposed males vs Cognitive ND ND 0–2 year Bayley Scales (Mental Significantly lower cognitive
Brotman, study exposed females abilities Development Index, MDI) scores in both male and
1984 female exposed offspring
Ornoy et al., Heroin Longitudinal Exposed vs Inattention/ Yes (heroin, Compared for 8 year (5– The Conners and Highest rate of ADHD in
2001 study unexposed hyperactivity methadone, socioeconomic 12 year) Achenbach questionnaires both heroin exposed boys
phenotype or benzodiazepines status and the Pollack Taper test and girls
risk for ADHD and other
psychoactive
drugs)
Prenatal tobacco exposure
Moe and Tobacco Longitudinal Exposed vs Cognitive Yes (tobacco, Compared for 1–3 year Bayley Scales II (Mental Lower Mental
Slinning, study unexposed abilities opioids, alcohol, socioeconomic Development Index, MDI) Developmental Scores in
2001 cannabis, status exposed male infants
psychostimulants,

Gender Difference in Drug Exposure

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and more)
Kotimaa Tobacco Longitudinal Exposed vs control Hyperactivity Yes (tobacco and Adjusted for 8 year Children’s Behavior Hyperactivity in males and
et al., 2003 study phenotype alcohol) socioeconomic Questionnaire (Rutter B2) females prenatally exposed
and ADHD status to nicotine
risk
Willoughby Tobacco Epidemiological Exposed vs control Attention, Yes (tobacco, Adjusted for 0–1 year Infant Behavior Record Significantly lower cognitive
et al., 2007 Study reactivity, alcohol) socioeconomic (IBR) performances in exposed
irritability status males
(Continued)

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Traccis et al.
TABLE 1 | Continued

Author year Substance Type of Experimental Neuro Prenatal Socioeconomic Age Performed tasks Gender results
study groups developmental polysubstance status
outcome exposure
Cornelius Tobacco Longitudinal Exposed vs Inattention/ Yes (tobacco, Controlled for 6 year Child Behavior Checklist, Increased activity and
et al., 2007 study unexposed hyperactivity cannabis, alcohol) socioeconomic RouthActivity Scale, and attention problems in both
offspring from phenotype status the SNAP male and female
teenager mothers exposedoffspring
Gatzke-Kopp Tobacco Longitudinal Exposed vs Neurobehavioral Yes (tobacco, Controlled for 7–15 year Child Behavior Checklist Exposed offspring shows
and study unexposed problems, alcohol, cannabis, socioeconomic (CBCL; Achenbach, 1991), more severe ADHD
Beauchaine, ADHD and amphetamines, status Child Symptom Inventory symptoms and cognitive
2007 cognitive heroin) (CSI) behavioral problems
abilities
Langley et al., Tobacco Cross- Exposed with ADHD ND Measured as 7–8 year Clinical diagnosis Maternal smoking in
2007 sectional ADHD vs diagnosis environmental pregnancy and high
study unexposed with risk environmental risk,
ADHD independently influence the
clinical presentation of the
ADHDphenotype without
sex-vulnerability
Hutchinson Tobacco Longitudinal Exposed vs ADHD risk ND Confounding 3 year SDQs Higher risk for conduct and
et al., 2010 study unexposed and factor hyperactivity–inattention
neurobehavioral problems in males whose
problems mothers persistently
smoked throughout
pregnancy
Wakschlag Tobacco Longitudinal Exposed vs Neurobehavioral Yes (tobacco, Controlled for 10 year The Diagnostic Interview Exposed boys, but not
and Hans, study unexposed problems alcohol, opioids, socioeconomic for Children and girls, are significantly more
2002 (conduct cannabis) status Adolescents (DICA) likely to develop conduct
disorder) disorder symptoms
Fergusson Tobacco Longitudinal Exposed vs Neurobehavioral Yes (tobacco, Adjusted for 16– Composite International More severe conduct
et al., 1998 study unexposed problems alcohol, illicit drugs) socioeconomic 18 year Diagnostic Interview and disorders symptoms in
(conduct status the Self-Report male adolescents than in
disorder) Delinquency Inventory females prenatally exposed
to tobacco
Prenatal alcohol exposure
Richardson Alcohol Longitudinal Exposed vs control Cognitive Yes (alcohol, Controlled for 10 year Wisconsin Card Sorting Significantly lower cognitive
et al., 2002 study abilities cannabis, tobacco, low Test, Wide Range scores (learning and

Gender Difference in Drug Exposure

cocaine) socioeconomic Assessment of Memory memory) in both male and
June 2020 | Volume 14 | Article 72

status and Learning (WRAML), female exposed offspring

Trail Making
Howell et al., Alcohol Longitudinal Exposed vs control Cognitive Yes (alcohol, Controlled for 15 year Wechsler Intelligence Significantly lower IQ score
2006 study abilities cannabis, tobacco, low Scale for Children and mathematical abilities
cocaine) socioeconomic (WISC-III), Wechsler in both male and female
status Individual Achievement exposed offspring
Test (WIAT)
(Continued)

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Traccis et al.
TABLE 1 | Continued

Author year Substance Type of Experimental Neuro Prenatal Socioeconomic Age Performed tasks Gender results
study groups developmental polysubstance status
outcome exposure
Kelly et al., Alcohol Longitudinal Exposed vs control Cognitive Yes (alcohol, Adjusted for 3 year British Ability Scale (BAS), Significantly lower cognitive
2009 study abilities tobacco) low Bracken School Readiness scores in males born to
socioeconomic Assessment (BSRA) heavy-drinking mothers
status compared to exposed
females
Willford et al., Alcohol Longitudinal Exposed vs control Cognitive Yes (alcohol, Controlled for 14 year Children’s Memory Scale Deficits in learning,
2004 study abilities cannabis, tobacco, low short-term and long-term
cocaine) socioeconomic memory, specifically in the
status verbal domain, in both
exposed males and females
Coles et al., Alcohol Longitudinal Exposed vs control Inattention/ Yes (alcohol, Controlled for 15 year Continuous performance Deficits in sustained
2002 study hyperactivity cannabis, tobacco) low task (CPT) attention, processing in the
phenotype socioeconomic visual and auditory modality
status in exposed progeny
Herman Alcohol Cross- FASD offspring with ADHD ND Controlled for 6–16 year ADHD diagnosis Higher prevalence of ADHD
et al., 2008 sectional ADHD vs FASD diagnosis low diagnosis in exposed males
study offspring socioeconomic than females
status
Kelly et al., Alcohol Longitudinal Exposed vs control Behavior Yes (alcohol, Adjusted for 3 year Parent-report version of Exposed males were more
2009 study problems tobacco) low the Strengths and likely to have clinically
(hyperactivity, socioeconomic Difficulties Questionnaire relevant high total
conduct, peer status (SDQ) difficulties, hyperactivity,
problems) conduct and peer problems
compared to girls
Prenatal cannabis Exposure
Noland et al., Cannabis Longitudinal Exposed vs control Cognitive Yes (cannabis, Controlled for 4 year Picture deletion task (PDT), Higher omission error rates
2005 study abilities tobacco, alcohol, low continuous performance in exposed offspring
cocaine) socioeconomic task (CPT), Wechsler
status Preschool and Primary
Scales of
Intelligence-Revised
(WPPSI-R)
El Marroun Cannabis Longitudinal Exposed vs control Attention Yes (cannabis, ND 1–2 year Child Behavior Checklist Prenatal cannabis is

Gender Difference in Drug Exposure

et al., 2011 study problems tobacco, alcohol) associated with attention
June 2020 | Volume 14 | Article 72

problems specifically in
exposed girls
Richardson Cannabis Longitudinal Exposed vs control Cognitive Yes (alcohol, Controlled for 10 year Continuous performance Deficit in memory and
et al., 2002 study abilities, cannabis, tobacco, low test learning, together with
attention and cocaine) socioeconomic higher impulsivity score in
impulsivity status both males and females
(Continued)

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A thorough description of each study is presented with designed study type, experimental groups enrolled, outcomes, occurrence of poly-drug use, socioeconomic status, age at assessment, type of task used, and
to female offspring (Bennett et al., 2002, 2007, 2008; Delaney-

exposed offspring of heavy

girls during early childhood
Black et al., 2004; Nordstrom Bailey et al., 2005; Sood et al., 2005;

(aggressive behavior) in
Externalizing problems
Bendersky et al., 2006; Dennis et al., 2006; Carmody et al., 2011).

Delinquent behavior in
In contrast, no gender dichotomy was found in the occurrence
Gender results

marijuana users
of attention deficit/hyperactivity disorder (ADHD) phenotypes
from infancy to preadolescence (Karmel and Gardner, 1996;
Mayes, 1996; Mayes et al., 1998, 2003; Accornero et al., 2007).
With regard to methamphetamine, the most frequent
outcomes reported in newborns occur during the first year of
life and include motor dysfunctions (e.g., disorganized behaviors

scale and Child Behavior

Child Behavior Checklist

Self-Report Delinquency with poor quality of movement), which tend to disappear

with development in boys (LaGasse et al., 2012; Shah et al.,
Performed tasks

2012; Zabaneh et al., 2012; Kiblawi et al., 2014), whereas

they persist throughout adolescence in girls (Eriksson and
Checklist

Zetterström, 1994; Cernerud et al., 1996). In contrast, other

neurobehavioral problems (e.g., anxious/depressive phenotypes,
emotional problems) appear during late infancy and childhood
and do not exhibit sex bias (LaGasse et al., 2012). Similarly,
1–2 year

14 year

impairments in cognitive skills occur equally in both female and

gender results. Gender effect is displayed as pink and blue for female and male progeny, respectively. Gender differences not observed are white.
Socioeconomic Age

male offspring (Lu et al., 2009; Piper et al., 2011; Diaz et al.,
2014). However, deficits in inhibitory control and ADHD-like
symptoms are prevalent in boys (LaGasse et al., 2012; Kiblawi
et al., 2013).
Matched
status

Effects of Prenatal Exposure to Opioids

ND

Regardless of the efforts aimed at discouraging opioid use,

prevalence rates show an increasing trend in pregnant women
tobacco, alcohol)

tobacco, alcohol)
polysubstance

(Haight et al., 2018). However, gender was not considered in most

Yes (cannabis,

Yes (cannabis,

of the human studies on the effects of heroin, methadone, and

exposure
Prenatal

other prescription opioids.

Children born to mothers who use opioids during gestation
suffer from the so-called neonatal opioid withdrawal syndrome
developmental

(NOWS) (Gomez-Pomar and Finnegan, 2018), characterized

by several signs and symptoms (e.g., tremors, sleep problems,
(aggressive

(delinquent
Behavioral

Behavioral
outcome

problems

problems
behavior)

behavior)

hyperactive reflexes, vomiting, dehydration, and respiratory

Neuro

problems), which are more severe in boys than in girls (Jansson

et al., 2007, 2010). Maternal consumption of methadone—the
gold standard for opioid maintenance therapy—is associated
Exposed vs control

Exposed vs control

with poorer cognitive performance and lower IQ scores in

Experimental

exposed males when compared to females during infancy, an

age-dependent effect (Suffet and Brotman, 1984; Nygaard et al.,
groups

2015). However, no gender difference is found in symptoms

related to ADHD and aggressive behavior up to preadolescence
(Ornoy et al., 2001).
Longitudinal

Longitudinal
Type of

Effects of Maternal Tobacco

study

study

study

Nicotine and its related tobacco products are the most studied
substances in relation to long-term neurobehavioral outcomes
in offspring exposed to tobacco during pregnancy. Despite the
Substance

Cannabis

Cannabis

limitations due to several environmental confounding factors,

a high degree of consistency exists for the association of
TABLE 1 | Continued

maternal smoking and cognitive and behavioral problems (for an

exhaustive review, see England et al., 2017). From these studies
Author year

emerge a male bias toward diverse behavioral and cognitive

et al., 2011
El Marroun

Day et al.,

domains, depending on age: at 6–8 months, males appear

2011

more vulnerable to deficits in cognitive and executive functions

(e.g., inattention) and in motor functions and to alterations in

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Traccis et al. Gender Difference in Drug Exposure

FIGURE 1 | Heatmaps of neurobehavioral outcomes collectively identified as cognitive deficits (including executive function), ADHD risk, and behavioral problems,
with missing information on gender differences shown in gray. Outcome intensities are displayed as colors ranging from pink (female) to blue (male) shown in the key.
Gender differences were not observed in executive functions during childhood in offspring prenatally exposed to cocaine in one study only (Accornero et al., 2007);
therefore, the heatmap displays blue as a result of the larger number of consistent studies reporting that boys are more susceptible to exhibit deficits in cognitive
abilities when compared to girls (see text and also Table 1).

reactivity (Moe and Slinning, 2001; Wakschlag and Hans, 2002; stress-related cues (Haley et al., 2006). In contrast, neuroimaging
Willoughby et al., 2007). From infancy through childhood, boys studies do not reveal sex differences in long-term abnormalities of
appear at risk for ADHD (Kotimaa et al., 2003; Cornelius et al., brain morphology because the reduction in both size and volume
2007; Willoughby et al., 2007; Agrawal et al., 2010; Hutchinson of frontal, temporal, cingulate, and striatal regions of offspring
et al., 2010); however, only during infancy do they display prenatally exposed to alcohol did not differ between genders
less positive mood (Pickett et al., 2008) than females; during (Eckstrand et al., 2012; Treit et al., 2013; De Guio et al., 2014).
childhood and adolescence, males present more externalizing and These findings suggest that such psychopathological traits cannot
disruptive behaviors (e.g., conduct disorders, antisocial behavior) be attributed to these structural changes.
than females (Wakschlag et al., 1997; Fergusson et al., 1998;
Hutchinson et al., 2010). Conversely, parental tobacco exposure Effects of in utero Cannabis Exposure
is associated with nicotine dependence and high consumption of In line with the data on general population, the rates of cannabis
tobacco only in adolescent girls (Rydell et al., 2012). Although use among pregnant women have markedly increased, with
the risk of developing ADHD symptoms in nicotine-exposed prevalence rates reaching 75% between 2002 and 2016 (Brown
progeny is high during adolescence, no gender differences were et al., 2017). Despite this alarming scenario, a few studies
found (Gatzke-Kopp and Beauchaine, 2007; Agrawal et al., 2010; have assessed the long-term neurobehavioral repercussions
Sourander et al., 2019). of maternal cannabis use on the offspring, though gender
differences were not consistently examined: the Ottawa Prenatal
Effects of Maternal Alcohol Prospective Study (OPPS), the Maternal Health Practices and
Despite the widely described dose-dependent teratogenic effect Child Development Project (MHPCDP), the Generation R study,
of alcohol (Kodituwakku, 2007; Ornoy and Ergaz, 2010), and Adolescent Brain Cognitive Development (ABCD) study. The
approximately 10% of women aged between 15 and 44 years OPPS study included gender as a confounding factor, and it
consume alcohol during pregnancy, with 3% exhibiting a binge- described a number of long-lasting neurobehavioral alterations,
drinking pattern (SAMHSA, 2011). Irrespective of the amount ranging from heightened tremors and startle responsiveness to
and pattern of consumption, a wealth of clinical evidence deficits in executive function (e.g., attention, cognitive flexibility,
describes that prenatal alcohol exposure markedly impairs problem solving, impulse control) (Fried and Makin, 1987;
cognitive, behavioral, and motor functions of offspring (Mattson Fried and Smith, 2001). Similarly, gender was not examined
et al., 1998; Coles et al., 2002; Richardson et al., 2002; Willford when assessing performance in memory, verbal, and perceptual
et al., 2004; Riley and McGee, 2005; Howell et al., 2006). Maternal processes as well as the first clinical signs of impulsivity at
moderate to heavy drinking produces a group of pathological childhood (Smith et al., 2006). However, when the same authors
conditions termed fetal alcohol spectrum disorder (FASD). subsequently included the gender factor on clinical signs that
Epidemiological studies report sexual dichotomy in FASD, with persisted at young adulthood, such as deficits in executive
prevalence rates and severity being higher in male than in female function tasks that require impulse control, they found no gender
patients (May et al., 2007; Astley, 2010; Thanh et al., 2014; differences (Smith et al., 2004). In the MHPCD study, the
but see May et al., 2014; Fox et al., 2015). A sex bias is also authors seldom included “gender” in their analysis. However,
described for other psychopathological traits, such as elevated they reported (1) significant sleep disturbances and deficits
rates of ADHD in 6- to 16-year-old boys but not girls (Coles et al., in mental development as well as in short-term memory and
2002; Herman et al., 2008). Boys also exhibit altered responses to verbal reasoning at both 9 months and 3 years of age; (2)
stress, measured as larger changes in cortisol levels induced by deficits in attention and memory, increased anxiety/depressive

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symptoms, impulsivity, hyperactivity, and aggression at 6 and neurobiological underpinnings of drug-exposed phenotypes. Of
14 years of age (Richardson et al., 1989, 2002; Dahl et al., 1995; similar importance is the need to study the association between
Leech et al., 2006; Day et al., 2011). Gender at 10 years of the perturbations of in utero–placental exchange and adverse
age did not affect cognitive deficits (Richardson et al., 2002). mental health outcome later in life. Indeed, increasing evidence
In contrast, the Generation R study showed that girls but not points to the role of the placenta in fetal programming, which
boys at 18 months of age exhibited increased scores on an is altered in response to prenatal insults and contributes to
aggressive behavior scale that persisted through childhood (El psychopathology (Burton et al., 2010; Khalife et al., 2012;
Marroun et al., 2011). Notably, this sex bias disappears during Roescher et al., 2014; Park et al., 2018; Kratimenos and Penn,
adolescence. Also, during infancy girls appear to be at risk for 2019). Notably, the placenta influences in a sex-dependent
the development of ADHD, a susceptibility that is age dependent manner the outcome for offspring who were exposed to perinatal
(Table 1 and Figure 1). Remarkably, although from Generation R malnutrition and stressors (Walsh et al., 2019). However, research
and ABCD studies maternal cannabis use has been associated to into whether the gender bias results from sex differences
proneness to psychosis in middle to late childhood, significantly in placental structure and functions or its genes, proteins,
earlier than the typical onset of first psychotic episode (Bolhuis and steroids is surprisingly lacking. Hence, future research
et al., 2018; Fine et al., 2019; Paul et al., 2019), again gender should aim at disentangling how sex impacts neurobiology
was not considered. Importantly, an independent investigation from the transfer of maternal drug concentrations across
showed that prenatal marijuana exposure has an equally negative the placenta to the effect on placental gene transcription or
effect on sustained attention of the offspring from childhood to expression of discrete transporters (e.g., ATP-binding cassette
adolescence (Noland et al., 2005). carriers) in the cord. In fact, to date, such investigations
have been performed only to relate maternal drug use and
placental perturbations to fetal growth and other morphological
CONCLUSION abnormalities (Janssen et al., 2015).
Substance (ab)use screening protocols, including
The literature here examined reveals gender differences in questionnaires and urine toxicology testing, should be established
immediate and long-term negative consequences of maternal worldwide as routine to identify pregnant women using drugs.
drug use on both cognition and behavior. When gender was Public health interventions regarding the awareness of the
included as a variable, irrespective of the drug used, male progeny harm associated with maternal drug use, and special programs
appear more vulnerable to cognitive deficits and at risk of ADHD to enter treatment and/or increase spontaneous quit rates,
from infancy through childhood (Table 1 and Figure 1). Notably, should be implemented (Jantzen et al., 1998; Forray, 2016 and
these gender differences tend to disappear with age. However, references therein; Patrick et al., 2017). Progress on tailored,
we cannot depict a clear picture for internalizing problems, safe, and acceptable pharmacotherapies to restore proper
drug use, and motor function deficits due to the paucity of neurodevelopmental trajectories of the progeny should be
data. Regarding the problems in the behavioral domain (i.e., incentivized. Additional preventative outreach programs should
externalizing problems), the current scenario is clearer: girls be implemented to raise community awareness and support
exposed in utero to cannabis are more vulnerable than boys and to provide access to treatment for the children who are
up until adolescence, but this conclusion cannot be extended prenatally exposed to drugs. Finally, future investigations should
to other drugs. Remarkably, this is in contrast to what is often be implemented to include the influence of sex as a biological
reported in rodent studies (Fernandez-Ruiz et al., 1998; Hurd variable (for guidelines please refer to (Clayton, 2018; Mannon
et al., 2019; Scheyer et al., 2019; de Salas-Quiroga et al., 2020), et al., 2020) in the outcome of offspring prenatally exposed
where female sex often acts as a protective factor. Nevertheless, to drugs of abuse.
the advantage of animal studies is to dissect the effects of genetic,
biological, and/or environmental risk factors. The establishment
of a biological causality between prenatal drug exposure and AUTHOR CONTRIBUTIONS
repercussions on the progeny from animal investigations is
pivotal. These mechanistic insights along with the observations All authors participated in the conceptualization, design, and
reported in human studies may help in developing therapeutic preparation of the manuscript.
interventions, on a gender-specific basis, which would ultimately
result in more effective treatment outcome.
The longitudinal studies examined have often considered FUNDING
different factors that might have contributed to gender
differences, including socioeconomic status, lifestyle indicators, The present study was supported by the University of Cagliari
stressful life events, social support (or lack thereof), and (RICCAR 2019 to MM).
psychiatric comorbidity. In this regard, an additional degree
of complexity arises from the evidence that single drug use
is virtually non-existent. At this stage, we cannot certainly ACKNOWLEDGMENTS
resolve this issue in human studies, as it deserves as much
attention as neuroimaging and omics analyses to reveal We thank William T. Dunn III for proofreading this minireview.

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birth outcomes. Proc. Natl. Acad. Sci. U.S.A. 116, 23996–24005. doi: 10.1073/
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 ORIGINAL RESEARCH
published: 25 September 2020
doi: 10.3389/fnbeh.2020.583122

Environmental Enrichment During

Adolescence Mitigates Cognitive
Deficits and Alcohol Vulnerability due
to Continuous and Intermittent
Perinatal Alcohol Exposure in
Adult Rats
Edited by: Anna Brancato 1 *, Valentina Castelli 2 , Gianluca Lavanco 3,4,5 and Carla Cannizzaro 1
Pedro Bekinschtein, 1
Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties of Excellence “G.
CONICET Institute of Cognitive and
D’Alessandro”, University of Palermo, Palermo, Italy, 2 Department of Biomedicine, Neuroscience and Advanced Diagnostics,
Translational Neuroscience (INCYT),
University of Palermo, Palermo, Italy, 3 INSERM U1215, NeuroCentre Magendie, Bordeaux, France, 4 University of Bordeaux,
Argentina
Bordeaux, France, 5 Department of Biomedical and Biotechnological Sciences, Section of Pharmacology, University of
Reviewed by: Catania, Catania, Italy
Ricardo Marcos Pautassi,
Medical Research Institute Mercedes
and Martín Ferreyra (INIMEC), Perinatal alcohol exposure affects ontogenic neurodevelopment, causing physical
Argentina and functional long-term abnormalities with limited treatment options. This study
Romana Šlamberová,
Charles University, Czechia investigated long-term consequences of continuous and intermittent maternal alcohol
*Correspondence:
drinking on behavioral readouts of cognitive function and alcohol vulnerability in
Anna Brancato the offspring. The effects of environmental enrichment (EE) during adolescence
[email protected]
were also evaluated. Female rats underwent continuous alcohol drinking (CAD)—or
Specialty section: intermittent alcohol drinking paradigm (IAD), along pregestation, gestation, and lactation
This article was submitted to
periods—equivalent to the whole gestational period in humans. Male offspring were
Learning and Memory,
a section of the journal reared in standard conditions or EE until adulthood and were then assessed for
Frontiers in Behavioral Neuroscience declarative memory in the novel object recognition test; spatial learning, cognitive
flexibility, and reference memory in the Morris water maze (MWM); alcohol consumption
Received: 14 July 2020
Accepted: 25 August 2020 and relapse by a two-bottle choice paradigm. Our data show that perinatal CAD
Published: 25 September 2020 decreased locomotor activity, exploratory behavior, and declarative memory with respect
Citation: to controls, whereas perinatal IAD displayed impaired declarative memory and spatial
Brancato A, Castelli V, Lavanco G
and Cannizzaro C
learning and memory. Moreover, both perinatal alcohol-exposed offspring showed
(2020) Environmental Enrichment higher vulnerability to alcohol consummatory behavior than controls, albeit perinatal
During Adolescence Mitigates
IAD rats showed a greater alcohol consumption and relapse behavior with respect
Cognitive Deficits and Alcohol
Vulnerability due to Continuous and to perinatal-CAD progeny. EE ameliorated declarative memory in perinatal CAD,
Intermittent Perinatal Alcohol while it mitigated spatial learning and reference memory impairment in perinatal-IAD
Exposure in Adult Rats.
Front. Behav. Neurosci. 14:583122.
progeny. In addition, EE decreased vulnerability to alcohol in both control and perinatal
doi: 10.3389/fnbeh.2020.583122 alcohol-exposed rats. Maternal alcohol consumption produces drinking pattern-related

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Brancato et al. Enrichment Mitigates Perinatal Alcohol-Related Deficits

long-term consequences on cognition and vulnerability to alcohol in the offspring.

However, increased positive environmental stimuli during adolescence may curtail the
detrimental effects of developmental alcohol exposure.
Keywords: alcohol, perinatal binge alcohol drinking, perinatal continuous alcohol drinking, declarative memory,
spatial memory, alcohol vulnerability, environmental enrichment

INTRODUCTION access procedure in the two-bottle choice paradigm, consisting

of cycles of drinking and abstinence, leads to a rapid increase in
Perinatal exposure to alcohol can affect in utero voluntary alcohol consumption, in comparison with continuous
neurodevelopment, causing both physical and functional access to alcohol (Carnicella et al., 2014). Rats exposed to this
long-term alterations (Dejong et al., 2019). Despite procedure consume the most abundant amount of their daily
pre-conceptional alcohol cessation is recommended, alcohol total intake within the first hour of availability of the alcohol
drinking during pregnancy is prevalent worldwide, especially bottle, reaching intoxicating blood alcohol levels in a short period
in Europe (Popova et al., 2017). One of the best predictors of of time (about mg/dl after the first 30 min–1 h, Simms et al.,
alcohol use throughout the perinatal period is the pattern of 2008; Carnicella et al., 2009; Loi et al., 2014). This procedure
alcohol use before pregnancy; indeed, women who report binge models a voluntary binge-like drinking pattern (Crabbe et al.,
or heavy drinking prior to pregnancy likely maintain it during 2011; Sprow and Thiele, 2012; Sabino et al., 2013; Carnicella
pregnancy and throughout lactation (Davidson et al., 1981; et al., 2014; Spear, 2018; Jeanblanc et al., 2019) and, as such, may
Ethen et al., 2009; Mallard et al., 2013; Anderson et al., 2014; represent a valuable tool to model drinking trajectories during
Kitsantas et al., 2014), increasing the risk for growth deficits, pregnancy and lactation. Interestingly, when female rats are
facial dysmorphology, and behavioral and neurocognitive exposed to a long-term binge-like intermittent alcohol drinking
abnormalities in the progeny (Viljoen et al., 2005; May et al., (IAD) paradigm, they display a significant decrease in alcohol
2007; Urban et al., 2008). Aside from the more severe fetal consumption during pregnancy and resume excessive alcohol
alcohol syndrome (FAS), ‘‘fetal alcohol spectrum disorders’’ consumption during the lactation period (Brancato et al., 2016).
(FASD) have been recently characterized as a broad range of Thus, in the present study, we aimed at investigating whether
deficits observed in the child when exposed to alcohol at any the binge-like IAD paradigm, resulting in higher and irregular
time prenatally (Dejong et al., 2019). Those alterations involve peaks of blood alcohol levels in the dams, could lead to
memory, attention, affective and social behavior, abnormal distinct long-term consequences on cognition and vulnerability
responses to stress and natural rewards (American Psychiatric to alcohol abuse in the offspring, with respect to continuous
Association, 2013), and susceptibility to drug and alcohol abuse alcohol drinking (CAD), which produces steady lower peaks
later in life (Baer et al., 2003; Alati et al., 2006; Glantz and of blood alcohol levels, even in the face of overall high levels
Chambers, 2006). of exposure. On the other hand, even the exposure to low to
While the consequences related to heavy prenatal alcohol moderate blood alcohol concentrations can cause significant
exposure are generally acknowledged, the assessment of the neuronal damage, when it occurs during the neurodevelopmental
neurobehavioral alterations potentially produces by low-to- window such as throughout gestation (Patten et al., 2014).
moderate alcohol exposure in humans displays mixed results Therefore, it follows that according to time, dosage, and duration
(Kelly et al., 2013; Flak et al., 2014; Kilburn et al., 2015). This may of perinatal alcohol exposure, different developmental alterations
be due to a number of methodological issues—most of the studies thus, may occur.
focus on physical malformations—and confounding variables, The long-term cognitive effects of perinatal alcohol exposure,
such as the unreliable self-reports about the degree of alcohol either continuous or intermittent, were assessed in the adult
exposure (number of drinks per week rather than amount at offspring, through a multidimensional behavioral battery,
one session) and the underestimation of subtle neurobehavioral including declarative memory in the novel object recognition
deficits which may appear later in life (Conover and Jones, 2012). test, spatial learning, cognitive flexibility, and reference memory
Preclinical models of maternal alcohol drinking can enhance in the Morris water maze (MWM). Vulnerability to excessive
our understanding of the adverse outcomes secondary to alcohol drinking, in terms of rate of voluntary alcohol
developmental alcohol exposure. Indeed, fetal alcohol exposure consumption and relapse behavior after a period of forced
in humans can be modeled by perinatal alcohol exposure in rats, abstinence, was assessed using a two-bottle ‘‘alcohol vs. water’’
since the full gestational period in rodents is equivalent to the choice drinking paradigm.
first and second trimesters in humans, while the first 10 postnatal It is worth noting that treatment strategies to prevent or
days in rats correspond to the third trimester in humans (Patten mitigate perinatal alcohol-related deficits are currently very
et al., 2014). Besides, high levels of alcohol consumption can limited (Murawski et al., 2015). In this regard, growing evidence
be induced in Sardinian alcohol-preferring and Wistar female supports a beneficial role of the exposure to positive stimuli
rats by manipulating the schedule of alcohol access (Loi et al., during sensitive time windows of brain development. Indeed,
2014; Brancato et al., 2016). First developed and characterized the environmental-enrichment (EE), experimental paradigm
in male rats (Wise, 1973; Simms et al., 2008), the intermittent consisting of housing conditions that include novelty, social

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interaction and exercise, enhances sensory, cognitive, and gestational day 1 (GD1), eight female rats were randomly
motor stimulation, which, in turn, translates into increased selected from each experimental group (n = 12), housed in
neuroplasticity in brain regions critical for emotional regulation, standard maternity cages, filled with wood shavings. Dams were
cognitive functions and reward sensitivity (Bayat et al., 2015; inspected twice daily for delivery until the day of parturition,
Crofton et al., 2015; Morera-Herreras et al., 2019). However, considered as postnatal day 0 (PND 0); dams and litters were kept
conflicting evidence is reported when the effect of EE was in a nursery (24 ± 2◦ C) and not separated until weaning, in order
evaluated toward motivational effects of drugs of abuse, to model the human condition and avoid confounding factors
including alcohol (Nithianantharajah and Hannan, 2006; Solinas (Subramanian, 1992; Wilson et al., 1996; Santangeli et al., 2016).
et al., 2009; Pautassi et al., 2017; Rae et al., 2018). Thus, while it is Mean alcohol consumption at 1 h and 24 h by CAD and IAD
critical to identify maternal alcohol consumption as a primary rat dams during pre-conception period, gestation, and lactation
target to prevent fetal consequences, we investigated whether was recorded and reported as g/kg ± SEM. After weaning, two
EE during adolescence could prevent or mitigate the effects of male rats from each litter of the three drinking groups were
perinatal alcohol exposure on behavioral readouts of cognitive randomly assigned to either the standard (SE) or enriched (EE)
function and alcohol vulnerability. rearing environment, so that the experimental groups of
rat offspring were perinatal water-exposed controls
(p-CTRL SE, n = 8); perinatal continuous alcohol-exposed rats
MATERIALS AND METHODS (p-CAD SE, n = 8); perinatal intermittent alcohol-exposed
rats (p-IAD SE, n = 8); perinatal water-exposed controls + EE
Animals, Perinatal Alcohol Exposure, (p-CTRL EE, n = 8); perinatal continuous alcohol-exposed rats
and Rearing Conditions + EE (p-CAD EE, n = 8); and perinatal intermittent alcohol-
The methods used for perinatal alcohol exposure and breeding exposed rats + EE (p-IAD EE, n = 8). In detail, from PND
have been previously reported in detail (Brancato et al., 2018). 21 onward, the rats reared in SE conditions were housed in pair
Briefly, adult female Wistar rats (200–220 g, Envigo, Italy) in standard rat cages and left undisturbed by the experimenters
were housed individually in standard rat cages (40 × 60 cm, except for weekly cage change, whereas the EE rats were group-
20 cm in height), with ad libitum access to water and food, in housed (8/cage) in large cages (60 × 45 × 76 cm) with pet toys,
a temperature- (22 ± 2◦ C) and humidity- (55 ± 5%) controlled pots, hideouts, ropes, running wheel, ladder, tunnel and plastic
room, on a 12-h light/dark cycle (08:00–20:00). boxes, etc., which were relocated or changed daily to create
Rats were gently handled for 3 min per day for a week before novelty (Griva et al., 2017).
the experimental procedures, when they were randomly assigned Experiments were approved by the Committee for the
to one of the three experimental groups, according to the Protection and Use of Animals of the University of Palermo,
two-bottle choice self-administration paradigm: water drinking in accordance with the current Italian legislation on animal
controls (CTRL), CAD, and IAD. Female rats underwent the experimentation (D.L. 26/2014) and the European directive
self-administration procedure during pre-gestation (12 weeks), (2010/63/EU) on care and use of laboratory animals. Every
gestation (3 weeks), and post-gestation (3 weeks) periods, effort was made to minimize the number of animals used and
accordingly to the respective home-cage two-bottle ‘‘alcohol vs. their sorrow.
water’’-choice-drinking paradigm.
Indeed, CTRL rats were given two bottles of tap water. CAD Behavioral Procedures
rats were given a 24-h free choice between one bottle of alcohol The offspring were tested for behavioral reactivity in the
(20% v/v) and one of tap water, 7 days per week; IAD rats were open-field test at PND 55, for declarative memory in the
given 24-h alcohol (20% v/v) access during 3 days per week, novel object recognition test at PND 56–58, and for spatial
i.e., on Monday, Wednesday, and Friday, while they received learning, memory, and cognitive flexibility in MWM from
two bottles of tap water on the intervening days. PND 60 to PND 65. Afterward, they were assessed for alcohol
Plastic bottles (120 ml; metal cap 0.8-mm-diameter hole, vulnerability, in terms of rate of voluntary alcohol consumption
Tecniplast, Italy) were filled every day with 100 ml of 20% in the induction and relapse-like phases of the two-bottle choice
alcohol (daily prepared from alcohol 96◦ (Carlo Erba Reagents, drinking paradigm, from PND 66 to PND 143.
Italy) diluted with tap water) and presented at lights-off in an The experiments were carried out in a sound-isolated room
alternative left–right position in order, to avoid side preference. between 9:00 and 14:00. On the test days, rats were acclimatized
Rats were weighed daily, and alcohol and water intake was to the testing room for 60 min before the experimental session.
measured 1 h after lights-off and the day after, immediately Rats’ performance was recorded and monitored in an adjacent
before lights-off, by weighing the bottles. Possible fluid spillage room. The equipment was thoroughly cleaned in between each
was monitored by using multiple bottles filled with water and test, to avoid that rats’ behavior was affected by the detection of
alcohol 20%, allocated in empty cages interspersed in the racks other rats’ scent.
(Loi et al., 2014).
At the end of the 12-week two-bottle choice drinking Open-Field Test
paradigm, each female rat was housed with a single breeder. Behavioral reactivity in a novel environment was tested
The day when pregnancy was confirmed by vaginal smear in the open-field test. The open-field arena is a Plexiglas
(Cannizzaro et al., 2008; Plescia et al., 2014b), designed as square box (44 × 44 × 44 cm where locomotor activity

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Brancato et al. Enrichment Mitigates Perinatal Alcohol-Related Deficits

and explorative behavior were measured) by employing an SE. A removable transparent escape platform (10 cm × 10 cm)
automatic video-tracking system (AnyMaze, Ugo Basile, was positioned in the middle of the quadrant, with the center
Italy), in a mean light- (100 lx) illuminated chamber. Each 30 cm away from the wall and 1.5 cm below the water level,
experimental session lasted 5 min (Cannizzaro et al., 2016). and not visible to the swimming rat. The pool was placed in
The video-tracking system produces a quali-quantitative an experimental room, decorated with several extra-maze cues
mapping of the motor pattern, measuring total distance (e.g., bookshelves and posters), and not modified throughout
traveled (TDT, m), as a measure of locomotor activity in a the entire experimental period. The experimental room was
novel environment. illuminated by a white light (60 W). The paths taken by the
animals in the pool were monitored by a video camera mounted
Novel Object Recognition Test in the ceiling and recorded by the automatic video-tracking
Declarative learning and memory were tested in the novel system (ANY MAZE, Ugo Basile, Italy).
object recognition test, as previously described (Brancato et al.,
2020). On day 1, a 5-min habituation session was performed at Experimental Design
10.00 a.m., in order to let the animals freely explore the arena Place Learning (Days 1–3)
(44 × 44 × 44 cm) in a dim light-illuminated room. Twenty-four The Place learning task was employed to assess spatial learning
hours after the habituation session, rats underwent a 5-min and consisted of training the rats to escape from the water and
training session when they were presented with two identical, reach the hidden platform placed in the SE zone, where it was
nontoxic objects (i.e., two red metal cans) which were placed maintained throughout the experimental session. The rat was
against a wall in the open-field arena. To prevent coercion to introduced into the pool facing the wall of each quadrant, in the
explore the objects, rats were released against the center of the following order of starting points: NE, SW, NW, SE. Each rat
opposite wall with its back to the objects. The time spent on underwent four trials a day, along 3 days, and was allowed to
exploring each object was recorded by using the AnyMaze video- swim until the escape on the platform for a maximum of 90 s;
tracking system (Stoelting Europe); a 2-cm2 area surrounding escape latency was recorded as a measure of spatial learning and
the objects was defined such that nose entries were recorded memory and reported as mean value of the four trials performed
as time exploring the object. After the training session, animals on each day of the experiment.
were placed in their home cage for the retention interval. Then, If the escape platform was reached, the rat was allowed
animals were returned to the arena for the test session, 24 h after to remain 15 s on it to reinforce the information on the
the training session. During the 5-min test session, the arena visual–spatial cues in the environment. If the rat did not find the
was equipped with two objects, one was identical to the one escape platform within 90 s, the experimenter guided gently the
presented in the training session (i.e., familiar); the other was rat to the platform and allowed it to stay on it for 15 s. During the
a novel object (a yellow hard plastic cup/ a green hard plastic 5-min intertrial interval, rats were placed into their home cages
pepper). Objects were randomized and counterbalanced across and warmed under a heating lamp.
animals. Objects and arena were thoroughly cleaned at the end
of each experimental session. Time spent on exploring familiar New Place Learning (Days 4–5)
and novel objects was recorded during both training and test The new place learning task was aimed at assessing rats’ cognitive
sessions. The recognition index (RI%), i.e., the percentage of flexibility. On the first day of task, the position of the escape
time spent on investigating the novel object, out of the total platform was moved to the opposite quadrant (NW) compared
object investigation time [RI % = Time novel object /(Time novel to the place learning session. In this task, the rat was required
object + Time familiar object)%], is a measure of novel object to learn the new location of the platform during four trials,
recognition and the main index of recognition memory. If RI% and escape latency was recorded as a measure of new spatial
is higher than 50%, it indicates that the rat spent more time information acquisition, i.e., reversal learning. On the second
investigating the novel object, thus recalling the memory of the day, the position of the platform was maintained in the same
familiar one. quadrant as in the first day of the new place learning task.
The escape latency was recorded as a measure of acquisition
Morris Water Maze
and retrieval of the spatial information necessary to reach
Spatial learning, cognitive flexibility, and reference memory were
the platform location. Starting points, trial duration, inter-trial
assessed in the MWM, by employing place learning, new place
interval, reinforcement time on the platform, and any other
learning, and probe tasks (Cacace et al., 2011, 2012; Plescia et al.,
experimental condition were the same as in the previous days.
2015) as described in detail below.
Apparatus Probe Test (Day 6)
The MWM apparatus consisted of a circular, light-blue Twenty-four hours after the last place learning session, rats were
swimming pool with a diameter of 160 cm, and walls 70 cm returned to the water maze for the probe test, aiming at assessing
high. It was filled with tap water to a depth of 50 cm. The water reference memory at the end of learning. The hidden platform
temperature was carefully maintained at 23 ± 2◦ C, and no agent was removed from the pool, and rats were allowed to swim freely
was added to make the water opaque. The pool was divided for 90 s. The amount of time spent in the quadrant where the
into four quadrants of equal size by two imaginary diagonal platform was previously located (target quadrant) was used as an
lines running through the center, designated NW, NE, SW, and index of the rat’s spatial reference memory.

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Two-Bottle “Alcohol vs. Water” Choice EE Prevents Alcohol-Induced Alteration in

Drinking Paradigm Behavioral Reactivity in p-CAD Offspring
The offspring underwent the two-bottle ‘‘alcohol vs. water’’- Two-way ANOVA on log-transformed TDT data, including
choice drinking paradigm (modified from Cacace et al., 2011) perinatal alcohol exposure and rearing conditions as statistical
and were given 24-h free choice between one bottle of alcohol factors, highlights a significant main effect of perinatal alcohol
(10% v/v) and one of tap water, 7 days per week, for 8 weeks exposure (F (2,42) = 0.6.412, p = 0.01742). The Tukey multiple-
(induction period), followed by a 2-week relapse period, after comparison test indicates that p-CAD SE offspring showed a
7 days of alcohol deprivation. 10% alcohol was daily prepared by significant decrease in locomotor activity with respect to p-CTRL
diluting alcohol 96◦ (Carlo Erba Reagents, Italy) with tap water. SE rats (q = 5.184, df = 42, p = 0.0019) and p-IAD SE rats
Plastic bottles (120 ml; metal cap 0.8 mm diameter hole, (q = 4.752, df = 42, p = 0.0047). No significant difference was
Tecniplast, Italy) were filled with 100 ml solution every day and observed among EE offspring (Figure 1A).
presented at lights-off in an alternative left–right position, to
avoid side preference. Alcohol and water intake were measured
by weighing the bottles. Possible fluid spillage was monitored
EE Rescues Recognition Memory
by using multiple bottles filled with water and 10% alcohol, in p-CAD Offspring
positioned in empty cages interspersed in the cage racks (Loi The results of the two-way ANOVA on the total time spent
et al., 2014). Rats’ body weight was daily monitored, and rats’ on the exploration of the two identical objects during the
consummatory behavior was measured, in terms of g/kg of sample phase reveal a significant main effect of perinatal alcohol
alcohol consumed along the drinking paradigm. exposure (F (2,42) = 11.21, p = 0.0001) and EE (F (1,42) = 9.658,
p = 0.0034). Tukey’s post hoc test shows that p-CAD SE offspring
spent significantly less time exploring the objects than p-CTRL
Statistical Analysis SE rats (q = 4.378, df = 42, p = 0.0382) and p-IAD SE rats
Statistical analysis was performed using Prism 8, GraphPad
(q = 4.797, df = 42, p = 0.0178). EE rats showed increased
Software, LLC, and IBM Statistical Package for the Social Sciences
total exploration than SE offspring, with no significant pattern
(SPSS) Statistics software (IBM, Armonk, NY, USA). Data
influence (Figure 1B).
were assessed for variance and normality by employing the
Data analysis from familiar- and novel-object exploration
Brown–Forsythe test and D’Agostino–Pearson omnibus K2 test,
during the test session included perinatal alcohol exposure
respectively, and for sphericity, by the Mauchly test. When data
and environmental rearing conditions as the between-subject
showed equal variance and normal distribution, the analysis
factors and object as the within-subject factor. The results
included two-way analysis of variance (ANOVA), followed by
indicate a significant main effect of object (F (1,42) = 62.673,
Tukey’s multiple-comparison test to assess simple effects of
p < 0.001), perinatal alcohol exposure (F (2,42) = 8.501, p < 0.001),
the two different perinatal alcohol exposures, and repeated-
and rearing environment (F (1,42) = 13.489, p < 0.001) and a
measure ANOVA using the generalized linear model, with
significant interaction between perinatal alcohol exposure and
Bonferroni correction for pairwise comparisons. When data did
rearing environment (F (2,42) = 4.796, p = 0.013), object and
not show normal distribution or sphericity, log-transformation
perinatal alcohol exposure (F (2,42) 13.506, p < 0.001), and
and Geisser–Greenhouse correction were employed. Data are
object, perinatal alcohol exposure, and rearing environment
reported as mean ± SEM. Statistical significance was set at
(F (2,42) = 14.367, p < 0.001). Pairwise comparisons with
p < 0.05.
Bonferroni correction show that both p-CAD SE and p-IAD SE
rats displayed a significant decrease in the exploration of the
novel object, when compared to p-CTRL SE offspring (p < 0.001;
RESULTS p < 0.001; Figure 1C). In addition, while p-CTRL EE rats
showed decreased exploration of the novel object, with respect
Perinatal Alcohol Exposure and to p-CTRL SE offspring (p < 0.001), p-CAD EE rats increased
Developmental Data the exploration of the novel object with respect to their SE
Alcohol intake of CAD and IAD dams is reported in Table 1. counterparts (p < 0.001; Figure 1C).
Alcohol consumption did not affect maternal weight gain, litter When RI% values from the test session were analyzed,
size or pup birth weight, compared to controls. two-way ANOVA, considering perinatal alcohol exposure and
EE as statistical factors, showed a significant main effect of
TABLE 1 | Mean alcohol consumption (g/kg) of continuous alcohol drinking perinatal alcohol exposure (F (2,42) = 6.812, p = 0.0027) and
(CAD) rats and intermittent alcohol drinking (IAD) rats at pre-conception,
EE (F (1,42) = 4.577, p = 0.0383) and a significant interaction
gestation, and lactation time.
(F (2,42) = 6.348, p = 0.0039). In detail, Tukey’s post hoc test
CAD IAD indicates a significant decrease in RI% of p-CAD SE- (q = 6.188,
Period 1h 24 h 1h 24 h df = 42, p = 0.0010) and p-IAD SE rats (q = 4.835, df = 42,
Pre-conception 0.8 ± 0.2 3.5 ± 0.1 3.4 ± 0.2 8.1 ± 0.3 p = 0.0165), with respect to p-CTRL SE rats. EE rescued the RI%
Gestation 2.1 ± 0.2 3.4 ± 0.4 2.6 ± 0.3 5.4 ± 0.6 deficit in p-CAD rats (q = 5.581, df = 42, p = 0.0038), whereas
Lactation 3.1 ± 0.3 5.6 ± 0.6 3.6 ± 0.4 8.5 ± 0.4
no significant difference was observed between SE and EE p-IAD
Data refer to mean ± SEM of n = 8 female rats along the alcohol drinking paradigm. progeny (q = 1.121, df = 42, p = 0.9673; Figure 1D).

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FIGURE 1 | Effects of perinatal alcohol exposure and rearing conditions on locomotor activity and declarative memory. (A) In the open-field test, p-CAD SE
offspring showed a significant decrease in locomotor activity (**p < 0.01 vs. p-CTRL SE, ∧∧ p < 0.01 vs. p-IAD SE). (B) In the sample phase of the NOR test, p-CAD
SE progeny showed a significant decrease in total object exploration (*p < 0.05 vs. p-CTRL SE; ∧ p < 0.05 vs. p-IAD SE). (C) During the test phase of the NOR test,
p-CAD SE and p-IAD SE rats displayed decreased exploration of the novel object, which was increased by environmental enrichment (EE) only in p-CTRL and p-CAD
rats (◦◦◦ p > 0.001 vs. novel–p-CTRL SE; ***p > 0.001 vs. respective SE groups). (D) p-CAD SE and p-IAD SE rats showed decreased object discrimination in terms
of recognition index. EE during adolescence was able to ameliorate the declarative memory performance in p-CAD offspring (*p < 0.05; **p < 0.01 vs. p-CTRL
SE,  p < 0.01 vs. p-CAD SE). Each bar represents the mean ± SEM of n = 8 rats. p-CTRL, perinatal control; p-CAD, perinatal continuous alcohol drinking; p-IAD,
perinatal intermittent alcohol drinking; SE, standard rearing environment; EE, enriched rearing environment. TDT, total distance travelled; NOR, novel object recognition.

EE Mitigates Spatial Learning and Memory Cognitive Flexibility in the New Place Learning Task
Deficits in p-IAD Offspring Statistical analysis on escape latency during the new place
Spatial Learning in the Place Learning Task learning task, when the platform was moved to the NW quadrant,
Data analysis performed on escape latency during the place included perinatal alcohol exposure and rearing environment as
learning task, when the offspring were trained to find the hidden the between-subject factors and days as the repeated-measure
platform over 3 days, considered perinatal alcohol exposure factor. The results reveal a significant main effect of days
and rearing environment as the between-subject factors, and (F (1,42) = 14.541, p < 0.001); perinatal alcohol exposure, rearing
days as the repeated-measure factor. The results indicate a environment and their interactions displayed no significant effect
significant main effect of days (F (2,84) = 80.256, p < 0.0001) (Figures 2A–D).
and rearing environment (F (1,42) = 5.636, p = 0.022) and a Spatial Reference Memory in the Probe Task
significant interaction between days and rearing environment Data analysis performed on time spent in each of the MWM
(F (2,84) = 12.319, p < 0.001), perinatal alcohol exposure and quadrants during the probe task included perinatal alcohol
rearing environment (F (2,42) = 5.048, p = 0.011), and among exposure and rearing environment as the between-subject factors
day, perinatal alcohol exposure, and rearing environment and quadrant as the within-subject factor. The results show
(F (4,84) = 4.54, p = 0.002). Pairwise comparisons with Bonferroni a significant main effect of quadrant (F (2.250,94.499) = 85.652,
correction show that p-IAD SE rats displayed increased escape p < 0.001) and a significant interaction between quadrant and
latency with respect to p-CTRL SE (p = 0.003) and p-CAD perinatal alcohol exposure (F (4.5,94.499) = 3.889, p = 0.004),
SE (p = 0.004) offspring on day 1; in addition, p-CAD quadrant and rearing environment (F (2.25,94.499) = 3.051,
EE rats showed a significant decrease in escape latency p = 0.046), and perinatal alcohol exposure and rearing
with respect to p-CAD SE (p = 0.005) on day 3, whereas environment (F (2,42) = 4.667, p = 0.015). Pairwise comparisons
p-IAD EE offspring displayed a significantly decreased latency with Bonferroni correction indicate that p-IAD SE offspring
with respect to p-IAD SE rats (p < 0.001) on day 1 spent significantly less time in the NW quadrant (p = 0.003),
(Figures 2A–D). and longer time in the NE quadrant (p = 0.042) than p-CTRL

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FIGURE 2 | Effects of perinatal alcohol exposure and rearing conditions on spatial learning, cognitive flexibility, and reference memory. (A) p-IAD SE offspring
showed a significant impairment in spatial learning (**p > 0.01 vs. p-CTRL SE; ∧∧ p < 0.01 vs. p-CAD SE). (B) p-CTRL rats exposed to EE during adolescence did
not differ from their SE counterparts. EE ameliorated the spatial learning performance in (C) p-CAD and (D) p-IAD offspring (**p < 0.01; ***p < 0.001 vs. respective
SE counterparts). In addition, (E) p-IAD SE rats showed a reference memory deficit in the probe task, which was rescued by EE (*p < 0.05; **p < 0.01 vs. p-CTRL
SE;  p < 0.05 vs. p-IAD SE). Each dot and each bar represent the mean ± SEM of n = 8 rats. p-CTRL, perinatal control; p-CAD, perinatal continuous alcohol
drinking; p-IAD, perinatal intermittent alcohol drinking; SE, standard rearing environment; EE, enriched rearing environment. MWM, Morris water maze; NW,
nord—west quadrant; NE, nord—east quadrant; SW, sud—west quadrant; SE, sud—east quadrant.

SE rats. On the other hand, p-IAD EE rats spent increased time Moreover, p-IAD SE rats showed increased alcohol consumption
in the NW quadrant with respect to p-IAD SE rats (p = 0.028; on weeks 2 (p < 0.001), 3 (p < 0.001), 5 (p < 0.001), 6 (p < 0.001)
Figure 2E). 7 (p < 0.001), and 8 (p < 0.001) with respect to p-CTRL SE rats,
along with increased alcohol intake on weeks 1 (p < 0.001), 2
(p < 0.001), and 8 (p < 0.001) with respect to p-CAD SE rats
EE in Adolescence Blunts Long-Time (Figure 3A).
Alcohol Vulnerability in p-CAD EE modified alcohol consumption in p-CTRL rats, with a
and p-IAD Offspring significant increase on week 2 (p = 0.001) and a decrease on
Induction Period week 4 (p = 0.001), when compared with the SE rearing condition
Data analysis performed on mean alcohol intake along (Figure 3B). Similarly, the enriched rearing environment
the 8 weeks of the two-bottle choice paradigm included increased alcohol intake in p-CAD offspring on week 2
perinatal alcohol exposure and rearing environment as the (p = 0.007) and significantly decreased it afterward, on weeks 3
between-subject factors and weeks as the repeated-measure (p = 0.005), 4 (p = 0.016), 6 (p < 0.001), and 7 (p = 0.001) with
factor. The results show a significant main effect of weeks respect to p-CAD SE rats (Figure 3C).
(F (2.37,99.521) = 14.091, p < 0.001), rearing environment On the other hand, EE decreased alcohol intake in p-IAD
(F (1,42) = 18.554, p < 0.001), and perinatal alcohol exposure progeny on weeks 1 (p = 0.021), 5 (p = 0.010), 6 (p < 0.001), 7
(F (2,42) = 13.807, p < 0.001) and a significant interaction (p < 0.001), and 8 (p < 0.001) when compared with p-IAD SE
between perinatal alcohol exposure and rearing environment counterparts (Figure 3D).
(F (2,42) = 10.225, p < 0.001), weeks and perinatal alcohol
exposure (F (4.739,99.521) = 6.668, p < 0.001), weeks and rearing Relapse Period
environment (F (2.37,99.521) = 9.576, p < 0.0001), and among The analysis of data from mean alcohol intake over the 2 weeks
weeks, perinatal alcohol exposure, and rearing environment of the relapse paradigm included perinatal alcohol exposure
(F (4.739,99.521) = 7.559, p < 0.001). Pairwise comparisons and rearing environment as the between-subject factors and
with Bonferroni correction indicate that p-CAD SE offspring weeks as the repeated-measure factor. The results indicate a
displayed decreased alcohol intake on week 1 (p = 0.012) significant main effect of weeks (F (1,42) = 76.57, p < 0.001),
and increased alcohol consumption on week 6 (p < 0.001), 7 rearing environment (F (1,42) = 17.112, p < 0.001), and perinatal
(p = 0.003) and 8 (p = 0.011) with respect to p-CTRL SE rats. alcohol exposure (F (2,42) = 12.215, p < 0.001) and a significant

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FIGURE 3 | Effects of perinatal alcohol exposure and rearing conditions on alcohol consummatory behavior. (A) Apart from the first week of the two-bottle choice
paradigm, both p-CAD- and p-IAD SE offspring showed increased alcohol intake with respect to p-CTRL SE rats; moreover, p-IAD SE rats displayed increased
alcohol consumption with respect to p-CAD SE offspring (# p < 0.05; ## p < 0.01; ### p < 0.001 p-CAD SE vs. p-CTRL SE; ***p < 0.001 p-IAD SE vs. p-CTRL SE;
∧∧∧
p < 0.001 p-IAD SE vs. p-CAD SE). EE exposure during adolescence. (B) Altered alcohol consumption in p-CTRL rats in (C) p-CAD rats and (D) decreased
alcohol consumption p-IAD offspring (*p < 0.05; **p < 0.01; ***p < 0.001 vs. respective SE). (E) When offspring were assessed for alcohol deprivation effect during
the relapse-like weeks, p-IAD-SE offspring showed higher alcohol intake with respect to p-CTRL SE and p-CAD SE rats; ***p < 0.001 vs. p-CTRL SE; ∧∧∧ vs.
p-CAD SE). EE exposure during adolescence (F) decreased alcohol deprivation effect in p-CTRL offspring, (G) did not alter alcohol-related behavior in p-CAD rats,
and (H) prevented alcohol deprivation effect in p-IAD offspring (**p < 0.01; ***p < 0.001 vs. respective SE). Each dot represents the mean ± SEM of n = 8 rats.
p-CTRL, perinatal control; p-CAD, perinatal continuous alcohol drinking; p-IAD, perinatal intermittent alcohol drinking; SE, standard rearing environment; EE,
enriched rearing environment.

interaction between perinatal alcohol exposure and rearing readouts, including behavioral reactivity, declarative and spatial
environment (F (2,42) = 18.4513, p < 0.001), weeks and perinatal learning and memory, and alcohol vulnerability.
alcohol exposure (F (2,42) = 5.371, p = 0.008), weeks and Moreover, we also exposed the offspring to an enriched
rearing environment (F (1,42) = 24.567, p < 0.001), and among rearing environment during adolescence, in order to evaluate
weeks, perinatal alcohol exposure, and rearing environment whether sensorimotor stimulation and social interaction at that
(F (2,42) = 5.648, p = 0.007). Pairwise comparisons with Bonferroni age could result in a rescue strategy able to mitigate or prevent
correction indicate that p-IAD SE offspring showed higher perinatal alcohol-induced adverse effects.
alcohol intake than p-CTRL SE and p-CAD SE rats on week In human studies, records on maternal blood alcohol levels
1 (p < 0.001; p < 0.001) and week 2 (p < 0.001; p < 0.001; are generally not available; however, estimates suggest that blood
Figure 3E). alcohol levels of over 200 mg/dl may be responsible for the severe
EE significantly decreased alcohol consumption in p-CTRL FAS phenotype, while lower levels (80 mg/dl) may produce
rats on week 1 (p = 0.008) with respect to their SE counterparts milder forms of FASD (Maier and West, 2001). In addition,
(Figure 3F) whereas no difference was observed between p-CAD high peaks of blood alcohol concentrations, rather than steady
SE and EE offspring (Figure 3G). On the other hand, p-IAD levels, as a result of both dose and pattern of alcohol exposure
EE offspring displayed significantly lower alcohol intake on both (i.e., binge-drinking vs. daily) during the brain developmental
week 1 and 2 (p < 0.001; p < 0.001), when compared to p-IAD time-window, are associated with increased neurotoxicity (Ieraci
SE rats (Figure 3H). and Herrera, 2007; Parnell et al., 2009).
In our experimental conditions, female rats were trained
to voluntarily consume 20% alcohol in the drinking water
DISCUSSION prior to pregnancy (Patten et al., 2014) and consumed relevant
amounts throughout pregnancy and lactation. In particular,
The present study aimed at evaluating the long-term CAD dams showed a mean daily alcohol consumption of
consequences of maternal continuous- and binge-like 3.4 ± 0.4 g/kg during pregnancy, and 5.6 ± 0.6 g/kg during
intermittent alcohol drinking, from pre-conceptional time lactation, resulting in a daily low-to-moderate perinatal exposure
to lactation, on the adult male offspring’s cognitive behavioral for p-CAD offspring (Marquardt and Brigman, 2016). On the

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other hand, IAD dams engaged in a binge-like drinking pattern In detail, alcohol exposure (dose range from 4.00 to 5.25 g/kg)
by every-other-day intermittent alcohol access to 20% alcohol, during the developmental equivalent of the second and/or third
which resulted in mean alcohol consumption of 5.4 ± 0.6 g/kg trimesters in humans did not impair recognition memory in rats
during pregnancy and 8.5 ± 0.4 during the postpartum period. (Jablonski et al., 2013; Tattoli et al., 2001; MacIlvane et al., 2016),
In particular, the IAD dams’ mean alcohol intake during but when Sprague–Dawley female rats were given continuous
the lactation period, measured after the first hour following unlimited access to alcohol from pre-conceptional period until
alcohol presentation, is suggestive of an intermittent exposure weaning time, the offspring failed to discriminate the novel object
to intoxicating blood alcohol concentrations for p-IAD offspring in the object recognition test (Dandekar et al., 2019; Sanchez
(>80 mg/dl, Loi et al., 2014). This evidence is particularly et al., 2019). Interestingly, maternal binge-like drinking during
relevant since the intermittent pattern of exposure causes high both gestation and lactation was reported to decrease recognition
peaks of blood alcohol concentrations during lactation in the memory along with the expression of brain-derived neurotrophic
rat dams, and this time window corresponds to the third factor (BDNF), the main neurotrophin involved in learning and
developmental trimester in humans (Patten et al., 2014). memory (Montagud-Romero et al., 2019). Notably, even low
Our first data on the behavioral sequelae of perinatal alcohol levels of alcohol administered by oral gavage from GD 10–16 are
exposure show pattern-related consequences on behavioral able to exert a disruption in object recognition in the NOR,
reactivity. In detail, p-CAD rats displayed a decrease in but not in object-place location; accordingly, this was associated
locomotor activity in the novel environment of the open field, with alterations in BDNF expression in the perirhinal cortex—a
with respect to p-CTRL and p-IAD rats, whereas p-IAD rats brain area which plays a crucial role in object discrimination-
showed no alteration in total distance traveled, in comparison —rather than in the hippocampus, which is more involved in
to p-CTRL. These results confirm early findings from this place location (Plescia et al., 2014c; Terasaki and Schwarz, 2017).
laboratory showing that perinatal long-term continuous In our experimental conditions, perinatal alcohol exposure
exposure to alcohol decreased behavioral reactivity in the induced memory deficits regardless of the drinking pattern,
adolescent male offspring (Brancato et al., 2018). While suggesting an impairment in the regional circuitries
moderate- and heavy-alcohol exposure during early-middle underpinning declarative memory and that deserve attention
pregnancy either increased behavioral reactivity (Riley et al., from a translational point of view. However, it should not be
1993; Abel and Berman, 1994; Thomas et al., 2004; Kim overlooked that the recognition memory performance displayed
et al., 2013) or did not affect locomotion (Dursun et al., 2006; by p-CAD rats could have been affected by their low behavioral
Hellemans et al., 2010; Brady et al., 2012), the exposure to reactivity and object exploration, rather than a pure deficit in
moderate alcohol concentration throughout gestation and the declarative memory formation.
early postnatal period decreased locomotion in mice (Kleiber On the other hand, when offspring were tested for spatial
et al., 2011). Our data further suggest that the developmental learning and memory in the MWM, spatial navigation of p-CAD
effects of alcohol on locomotion and behavioral reactivity are rats did not differ from control offspring, with no difference in
affected not only by the dose and timing but also by the pattern spatial learning, in terms of latency to find the hidden platform
of alcohol exposure. over the 3 days of place learning, and in cognitive flexibility,
In accordance with our first evidence, the analysis of the along the 2 days of new place learning task. The evidence of no
behavior in the sample phase of the novel object recognition test impairment in spatial reference memory supports the presence
revealed that p-CAD rats showed a significant decrease in the of regular spatial learning abilities in p-CAD progeny, since they
exploration of the two identical objects when compared to both searched the platform in the target quadrant during the probe
p-CTRL and p-IAD, while p-IAD rats explored the objects at trial, 24 h after the last new place learning session.
the same extent as the control group did. Similarly, a decrease Taken together, our data are in line with previous reports
in exploration during the sample phase of the novel object demonstrating that chronic prenatal exposure to low-to-
recognition test was reported in Sardinian alcohol-preferring moderate doses of alcohol is sufficient to induce decreased
rats exposed to 3% alcohol from day 15 of gestation to day behavioral reactivity in the open field (Kleiber et al., 2011) and
7 after parturition (Tattoli et al., 2001) and interpreted as an declarative memory deficits in the novel object recognition test
altered responsiveness to situations requiring adaptation to novel (Dandekar et al., 2019), together with a detrimental impact on
environmental stimuli (Colombo et al., 1995). the neuroimmune function of the perirhinal cortex (Terasaki
On the other hand, the analysis of the test phase of the novel and Schwarz, 2017). On the other hand, repeated low-dose
object recognition test suggested a deficit in declarative explicit prenatal alcohol exposure does not produce detrimental effects
memory, since both prenatal alcohol-exposed groups displayed a on pyramidal cells within the dorsal hippocampus or does not
significant decrease in discrimination of the novel object: indeed, impair spatial learning and memory performance in the MWM
they spent the same time in the exploration of the familiar and (Cullen et al., 2014).
the novel object, and this led to a significant decrease in the When interpreting these data, the stressful nature of the
recognition index with respect to control offspring. MWM task needs to be taken into account. The training in
Preclinical findings have provided inconsistent evidence the MWM task increases the neuroendocrine stress response
on the consequences of perinatal alcohol exposure on object in rats, inducing high serum corticosterone concentrations that
discrimination, and the discrepancies are likely dependent on may affect the cognitive response in accordance with the positive
different times of exposure and blood concentrations. role of glucocorticoids on learning and memory consolidation

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(Aguilar-Valles et al., 2005). It is reported that chronic alcohol the first weeks of the two-bottle choice paradigm. In addition,
exposure during pregnancy induces a reduction in ACTH basal while p-CAD-rats did not show higher consumption of alcohol
levels while corticosterone secretion is not modified; besides, the after a week of deprivation with respect to control offspring,
exposure to some stressors induces an increase in corticosterone p-IAD progeny displayed a pronounced relapse behavior, when
and CRH secretion, more than in controls (Lu et al., 2018). compared to both p-CAD and p-CTRL progenies. The alcohol
It is therefore reasonable to hypothesize that the stressful deprivation effect is a reliable proxy of increased motivation to
contingency of the MWM may boost the coping strategies of seek and consume alcohol, loss of control, and relapse (Spanagel
p-CAD progeny, by an ‘‘ad hoc’’ compensatory response of the and Hölter, 2000; Martin-Fardon and Weiss, 2013), and our
HPA axis that makes p-CAD performance as ‘‘fair’’ as controls’ data indicate that the perinatal exposure to a drinking pattern
(Franks et al., 2020). that promotes high peaks of blood alcohol level is discretely
On the contrary, p-IAD offspring displayed a spatial learning crucial in conferring a permanent vulnerability to alcohol
impairment, in terms of increased latency to reach the hidden abuse, whose occurrence can be detected since adolescence
platform in the place learning test on day 1, compared to (Brancato et al., 2018).
control offspring. In addition, p-IAD rats showed reference This evidence supports clinical data showing that prenatally
memory deficits, since they spent less time in the target alcohol-exposed offspring display increased vulnerability to the
quadrant in the probe trial, compared to p-CTRL groups. The rewarding properties of alcohol (Barbier et al., 2008) and risk
impairment in spatial learning and reference memory in the for alcohol abuse and drug dependence later in life (Baer
water maze tasks is suggestive of hippocampal dysfunction et al., 2003; Alati et al., 2006). This phenotype may result from
likely resulting from the perinatal exposure to the binge-like morpho-functional alterations in the ventral tegmental area, such
alcohol drinking in the intermittent access. Indeed, despite some as decreased number of dopamine neurons and spontaneous
inconsistencies about alcohol-induced developmental effects action potentials, reduced size of their cell bodies, increased
on BDNF expression in the rat hippocampus (Feng et al., activated microglia (Shen et al., 1999; Aghaie et al., 2020),
2005; Ceccanti et al., 2012), binge-like alcohol exposure from and persistent expression of immature excitatory synapses onto
one-to-third trimester-equivalent causes significant deficits in dopaminergic neurons (Wang et al., 2006). As far as the pattern-
hippocampal and cortical neuroplasticity, resulting in alterations related behavioral abnormalities observed in this study concern,
in dendritic arborization, adult, neurogenesis, neuroimmune we could speculate that a dysregulation in the HPA axis may
activation in the hippocampus, and spatial learning impairment critically impact memory performance especially in the stressful
(Blanchard et al., 1987; Christie et al., 2005; An and Zhang, setting of the MWM and may predispose to alcohol vulnerability
2013; Harvey et al., 2019). Thus, due to the strong correlation (Brancato et al., 2014; Maniaci et al., 2015; Lee et al., 2018).
between BDNF, hippocampal function and HPA axis reactivity, Notably, enriched rearing conditions ameliorated the
it is possible to interpret the current data on the basis of a pattern- behavioral performance of p-CAD rats in the novel object
specific effect exerted by the perinatal exposure to IAD on the recognition test, likely remodeling p-CAD rats’ behavioral
stress axis response. reactivity, decreasing emotionality and restoring those perceptive
To our knowledge, the study by Wieczorek et al. (2015) is and attentive skills that make them able to overcome the
the only one focusing on HPA axis and behavioral sequelae cognitive impairment resulting from the perinatal continuous
of prenatal binge-like alcohol exposure. According to their alcohol exposure. Accordingly, previous evidence showed that
findings, male mice exposed to an early binge-like dose of alcohol EE in early adulthood can recover cognitive impairment due
on gestational day 7 showed no difference in corticosterone to alcohol exposure during adolescence (Rico-Barrio et al.,
levels with respect to controls, whereas they observed a 2019). On the other hand, in our experimental conditions,
blunted ACTH response to an acute stressor. Thus, it is declarative memory performance of p-IAD EE rats was not
reasonable to hypothesize that the exposure to intermittent different from their SE counterparts’ one, suggesting that the
alcohol drinking, which produces the cycling repetition of abnormalities in declarative memory formation due to the
intoxications and withdrawals (Plescia et al., 2014a), when perinatal intermittent exposure to alcohol are not rescued by
‘‘brain growth spurt’’ and synaptogenesis occur (Patten et al., the EE. Besides, the repeated exposure to environmental stimuli
2014), may impair spatial learning and memory in the MWM has been reported to decrease the incentive value of novelty
through a pronounced alteration in the neurodevelopmental (Cain et al., 2006; Garcia et al., 2017), suggesting that a lower
programming of corticosteroid signaling in the hippocampus interest in the novel object may explain its lower exploration
(Conrad et al., 1999). by the EE offspring. Interestingly, our data show that EE
The complex relationship between stress and alcohol is mitigated the spatial learning and reference memory deficits
bidirectional, and the dysregulation of the stress response is a induced by the perinatal intermittent alcohol paradigm. These
well-known risk factor for alcohol abuse vulnerability (Lee et al., findings are in agreement with previous reports, indicating
2018). The present data extend our previous findings and show that enriched environment attenuates hippocampal-dependent
that perinatal alcohol exposure is able to produce an alcohol- memory impairment induced by prenatal alcohol exposure,
prone phenotype in adult rats in a pattern-related fashion. While via an increase in hippocampal BDNF (Tipyasang et al., 2014;
p-CAD offspring increased their alcohol intake with respect Di Liberto et al., 2017). The interpretation of the effects of
to controls in the long-term, p-IAD rats showed a higher EE upon alcohol vulnerability in the first weeks of the two-
vulnerability to alcohol consummatory behavior starting from bottle-choice paradigm is not univocal since we observed

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mixed effects in the control offspring. In this regard, EE has are interesting questions to address in further cross-fostering
been reported to promote the formation of conditioned place experiments. Moreover, studies including female offspring
preference to alcohol in adolescent mice, likely recruiting are needed to explore sex differences in the developmental
the oxytocin signaling (Pautassi et al., 2017; Rae et al., 2018). effects of alcohol, and their underlying mechanisms. Overall,
However, the postweaning exposure to EE substantially subsequent developmental periods, such as adolescence, provide
rescued the increased vulnerability induced by perinatal a window of opportunity for inducing positive experience-
alcohol exposure in p-CAD and p-IAD offspring. Indeed, EE based neuroplasticity in brain regions critical for emotional
decreased alcohol consumption in p-CAD and p-IAD rats, regulation, cognitive functions, and reward sensitivity, which
with respect to standard housing, during the last weeks of the allow curtailing the lifetime consequences of developmental
self-administration paradigm. Thus, the increase in alcohol alcohol exposure.
consumption, as time goes by, is a hallmark feature of early
stages of the addiction cycle and represents a substantial risk DATA AVAILABILITY STATEMENT
factor predicting the development of alcohol addiction (Crabbe
et al., 2011). The raw data supporting the conclusions of this article will be
In addition, our data show that the enriched rearing made available by the authors, without undue reservation.
environment decreases the deprivation effect after a week
of forced abstinence, in p-IAD offspring and in p-CTRL
rats. These observations are consistent with previous reports ETHICS STATEMENT
showing that exposure to enriched environmental conditions
The animal study was reviewed and approved by Committee for
mitigates VTA dopamine neurons’ dysfunction due to perinatal
the Protection and Use of Animals of the University of Palermo.
alcohol exposure (Wang et al., 2018; Aghaie et al., 2020)
and, overall, decreases the occurrence of an addictive-like
phenotype (Galaj et al., 2020). Notably, the effect of the EE AUTHOR CONTRIBUTIONS
against the development of excessive alcohol intake seems to be
protective when the exposure occurs during adolescence, while AB: experimental procedures and data analysis and contribution
its protective role is limited when EE occurs during adulthood to writing. VC and GL: experimental procedures and
(Rodríguez-Ortega et al., 2018). To date, circumstantial evidence contribution to writing. CC: experimental design, data
suggests that its protective effect against alcohol drinking interpretation, and writing. All authors contributed to the
is due to decreased CRH signaling in the amygdala and its article and approved the submitted version.
downstream target (Sztainberg et al., 2010). Whether CRH
abnormalities may be the primum movens for the occurrence FUNDING
of the dysfunctional phenotype consequent to perinatal alcohol
exposure observed in this study, and at what extent alteration in This research was supported by a grant from PO. FESR
maternal care can contribute to alcohol developmental effects, 2007/2013 (grant number: R4D18+D18+0000) to CC.

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2003.10.002
Tipyasang, R., Kunwittaya, S., Mukda, S., Kotchabhakdi, N. J., and Copyright © 2020 Brancato, Castelli, Lavanco and Cannizzaro. This is an
Kotchabhakdi, N. (2014). Enriched environment attenuates changes in open-access article distributed under the terms of the Creative Commons Attribution
water-maze performance and BDNF level caused by prenatal alcohol exposure. License (CC BY). The use, distribution or reproduction in other forums is permitted,
EXCLI J. 13, 536–547. provided the original author(s) and the copyright owner(s) are credited and that the
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Cape Province: prevalence and risk factors. S. Afr. Med. J. 98, 877–882. with these terms.

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 ORIGINAL RESEARCH
published: 22 October 2020
doi: 10.3389/fnbeh.2020.589967

Hypothalamic Gene Expression and

Postpartum Behavior in a Genetic
Rat Model of Depression
Wendy Luo 1 , Patrick H. Lim 1 , Stephanie L. Wert 1 , Stephanie A. Gacek 1 , Hao Chen 2
and Eva E. Redei 1 *
1
Department of Psychiatry and Behavioral Sciences, Feinberg School of Medicine, Chicago, IL, United States, 2 Department
of Pharmacology, Addiction Science, and Toxicology, University of Tennessee Health Science Center, Memphis, TN,
United States

Postpartum depression is a complex illness that often occurs in genetically predisposed

individuals. Closely related inbred rat strains are a great resource to identify novel
causative genes and mechanisms underlying complex traits such as postpartum
behavior. We report differences in these behaviors between the inbred depression
model, Wistar Kyoto (WKY) More Immobile (WMI), and the isogenic control Wistar
Kyoto Less Immobile (WLI) dams. WMI dams showed significantly lower litter survival
rate and frequency of arched back and blanket nursing, but increased pup-directed
Edited by:
Sophie Laye,
licking, grooming, and retrieval during postpartum days (PPD) 1–10, compared to control
INRA Centre Bordeaux-Aquitaine, WLIs. This increased pup-directed behavior and the frequency of self-directed behaviors
France
segregated during selective breeding of the progenitor strain of WKY, which is also a
Reviewed by: depression model. These behaviors are manifested in the WMIs in contrast to those
Monika Sadananda,
Mangalore University, India of WLIs. Furthermore, habitual differences in the self-directed behavior between light
Shanaz Tejani-Butt, and dark cycles present in WLIs were missing in WMI dams. Hypothalamic transcript
University of the Sciences,
United States
levels of the circadian rhythm-related gene Lysine Demethylase 5A (Kdm5a), period 2
(Per2), and the maternal behavior-related oxytocin receptor (Oxtr), vasopressin (Avp),
*Correspondence:
Eva E. Redei and vasopressin receptor 1a (Avpr1a) were significantly greater in the post-weaning WMI
[email protected] dams at PPD 24 compared to those of WLIs, and also to those of WMI dams whose litter
Specialty section: died before PPD 5. Expression correlation amongst genes differed in WLI and WMI dams
This article was submitted to and between the two time-points postpartum, suggesting genetic and litter-survival
Individual and Social Behaviors,
a section of the journal differences between these strains affect transcript levels. These data demonstrate that
Frontiers in Behavioral Neuroscience the genetically close, but behaviorally disparate WMI and WLI strains would be suitable
for investigating the underlying genetic basis of postpartum behavior.
Received: 31 July 2020
Accepted: 25 September 2020 Keywords: Wistar Kyoto More Immobile, oxytocin receptor, vasopressin, vasopressin receptor 1a, lysine
Published: 22 October 2020 demethylase 5A, period 2
Citation:
Luo W, Lim PH, Wert SL, Gacek SA,
Chen H and Redei EE
(2020) Hypothalamic Gene
INTRODUCTION
Expression and Postpartum Behavior
in a Genetic Rat Model of
Maternal behavior has long-term effects on the brain development of offspring, and depressive
Depression. disorders impair maternal behaviors. One of the largest risk factors for depressive episodes
Front. Behav. Neurosci. 14:589967. in the perinatal period is depression before pregnancy (Rich-Edwards et al., 2006; Grant
doi: 10.3389/fnbeh.2020.589967 et al., 2008; Topiwala et al., 2012; Perani and Slattery, 2014). While most animal models

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Luo et al. Postpartum Characteristics of Depression Model

of postpartum depression focus on mirroring the group of of its receptors with subsequent effects on the transcription of
women who are experiencing depression for the first time their target genes and their receptors. Clinical trials involving
in their life during postpartum (Perani and Slattery, 2014; estrogen as a potential treatment for negative maternal behaviors
Putnam et al., 2017; Eid et al., 2019), the present study employs are ongoing; for example, one study investigated the potential
a genetic model of depression-like behavior and its isogenic use of transdermal estradiol as a treatment for postpartum
control strain to begin to investigate characteristics of these depression (Wisner et al., 2015). In addition to hormonal
dams during postpartum, modeling the risk factor of depression regulation, several genes and their pathways have been shown to
before pregnancy. influence maternal behavior, including those related to oxytocin
The genetic rat model of depression-like behaviors, the (Oxt) and vasopressin (Avp). Oxytocin is released within various
Wistar Kyoto (WKY) More Immobile (WMI) rat strain, brain regions including the paraventricular nucleus (PVN),
was bi-directionally selectively bred from the parental WKY supraoptic nucleus, septum, hippocampus, and olfactory bulb
strain. The WKY rat strain is a well-established model (Bosch and Neumann, 2012). In humans, mothers with higher
for depression as its behavior mirrors symptoms of human oxytocin expression show increased maternal touch and contact
major depression and anxiety, including despair-like behavior, with their children (Pratt et al., 2015). In rats, Oxt release
excessive anxiety, learned helplessness, disturbed sleep patterns, leads to mothers fostering more positive interactions with their
and hypoactivity (Paré and Redei, 1993; Paré, 1994a,b; Dugovic offspring (Leng et al., 2008), and central administration of
et al., 2000; Redei et al., 2001; Solberg et al., 2001, 2004; Oxt receptor antagonist can block the onset of maternal care,
Malkesman et al., 2005; Baum et al., 2006). Chronic treatments and reduce pup-directed behaviors (Champagne et al., 2001;
with antidepressants, electroshock administration (model for Pedersen and Boccia, 2003). The medial preoptic area seems to
electroconvulsive therapy), and deep-brain stimulation can all be a key brain region of vasopressin (Avp) actions on maternal
reverse these depression-like behaviors (Jeannotte et al., 2008; care. It receives vasopressinergic input from the suprachiasmatic
Falowski et al., 2011; Kyeremanteng et al., 2012). The WKY nucleus, and diurnal changes in local Avp release have been
strain was developed as the normotensive control for the described (Kalsbeek and Buijs, 2002). Vasopressin regulates
Spontaneously Hypertensive Rat (SHR) strain. Louis and Howes maternal care and it seems to occur via vasopressin receptor
(Louis and Howes, 1990) demonstrated that the WKY strain was 1a (Avpr1a; Pedersen et al., 1994; Bosch et al., 2007, 2010). As
distributed to different vendors and universities between F12 and many of the Oxt- and Avp-relevant brain regions are within
F17 generations of inbreeding. the hypothalamus, which is also intimately involved in affecting
The fact that the WKY rats showed genetic and behavioral maternal behaviors (Fang et al., 2018), we focused on the
differences (Kurtz et al., 1989; Paré and Kluczynski, 1997) expression of these neuropeptides and their receptors in the
motivated the bi-directional selective breeding using FST whole hypothalamus in this study.
immobility as a functional selector (Will et al., 2003). The The purpose of this study is to determine whether a genetic
WMIs, now at their 44th generation and completely inbred model of depression, the WMI rat, shows alterations in maternal
after >35 generations of full-sib breeding, show despair-like functioning compared to its isogenic control strain. Here,
behaviors and greater sensitivity to stress compared to their we explore postpartum behaviors and expression of relevant
isogenic control counterparts, the WKY Less Immobile (WLI) hypothalamic genes in these strains of inbred rats.
rats (Will et al., 2003; Andrus et al., 2012; Lim et al., 2018b). While
the WMIs show higher immobility behavior in the forced swim
test, which was the original functional selector for this strain, MATERIALS AND METHODS
WLI males and females present immobility behavior comparable
to that of other control strains. In our lab, Sprague–Dawley Animals and Behavioral Assessments
and Fischer 344 male rats show immobility very similar to All animal procedures were approved by the Institutional Animal
that of WLI males (Solberg et al., 2003; Wilcoxon et al., 2005; Care and Use Committee of Northwestern University. The
Andrus et al., 2012; Mehta et al., 2013; Mehta-Raghavan et al., 40th generation WLI and WMI inbred strains were housed
2016). WLI female immobility is similar to that of Wistar and under temperature and humidity-control with food and water
F344 females (Kokras et al., 2018). Through the WMI genetic ad libitum on a 12:12 LD cycle, lights on at 06:00 h. During the
model of depression and its WLI control strain, any observed experiment, red lights were on between 18:00 and 23:00 h to allow
behavioral and transcriptomic differences may be directly or video recording during the dark phase.
indirectly related to the identified <5,000 sequence variations Females of both strains (18 for WLI and 28 for WMI) were
between the two strains (Chen et al., 2017; Bryant et al., 2020). mated for this study. Maternal behavior of dams (six WLI and
During the early postnatal period, adaptive changes occur in six WMI) that birthed litters that survived to wean (postpartum
the mothers’ (dam) neuroendocrine system, which enables them day 24: PPD 24) was monitored and recorded daily. Maternal
to provide appropriate maternal care. These adaptive changes behavior was observed for 10 days PPD 1–PPD 10, as described
are pivotal and brought about by the hormonal alterations before (Ahmadiyeh et al., 2004), which was a modification
due to parturition (Levy, 2016). The decrease in estrogen of previous studies (Myers et al., 1989; Francis et al., 2003).
and/or progesterone at parturition may directly affect maternal Behaviors were automatically recorded for an hour under light
behaviors (Hauser and Gandelman, 1985; Glynn et al., 2016; (11:00–14:00 h) and dark (18:00–23:00 h) conditions, starting
Murakami, 2016). Changes in estrogen levels alter the expression at PPD 1, the day after birth. Behavioral analyses were scored

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manually. Blind scoring was not feasible as the WMI strain had primer sequences can be found in Supplementary Table 1. Five
many more mating pairs (to account for litter loss) and, therefore, ng of cDNA was amplified in a 20 µl reaction using SYBR Green
the experimenter was aware of which strain was giving birth at Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) in
any given time. The following behaviors were scored every 3 min: the QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher
arched-back nursing; blanket nursing (mother lies over pups); Scientific, Waltham, MA, USA). Triplicates of reactions were
proximity to pups, which is either passive nursing (mother is on performed and reached threshold amplification within 34 cycles.
the side or back with pups feeding) or just resting with pups very Target transcript levels were quantified relative to Gapdh, a
close by; licking/grooming of pups and pup retrieval; no contact housekeeping gene previously demonstrated to show similar
(mother leaves pup alone more than half a cage length away); or expression across strains and conditions, and to a general WLI
self-directed behaviors (eating and drinking; Ahmadiyeh et al., male hypothalamic calibrator using the 2(−∆∆CT) method.
2004). Arched back nursing and blanket nursing categories were
combined, and also licking/grooming of pups and pup retrieval Statistical Analysis
behaviors. Behavioral measures were shown as a frequency of Data are presented as mean ± standard error of the mean. All
observations for each hour of monitoring. statistical analyses were performed using GraphPad Prism v8.0
Litter deaths were observed during a previous 8-month (GraphPad Software, La Jolla, CA, USA). Behavioral observations
mating period, and results from this period prompted the were analyzed across postpartum days for the light and the dark
investigation of maternal behavior in this study. Litter death was phase by two-way ANOVA with repeated measures or mixed
also recorded in the current study. Although we have not made effect models, followed by Sidak’s post hoc analysis for multiple
a quantitative assessment, most pups that died before weaning comparisons. Cohen’s d values were calculated by using the
either died from cannibalism by the mother or showed signs of Cohen’s d = (M2 − M1)/SDpooled equation, where M1 and M2 are
undernourishment with no milk in their stomachs. means of the groups to be compared. Gene expression differences
Dams were euthanized, either before PPD 5 after their litters were analyzed by two-way ANOVA followed by Tukey’s multiple
died (four WLI and six WMI) or at PPD 24 (six WLI and comparison test. Pearson correlation of the gene expression
six WMI) after the pups were weaned, during lights on at data was corrected for multiple comparisons. Technical outliers,
11:00–14:00 h by fast decapitation. when multiple days of behavioral observations were lost or when
RT-qPCR data were marked by the program as technical outliers,
Brain Dissection and RNA Isolation were omitted from the analysis.
Hypothalami were dissected with a brain matrix according ANOVAs, mixed effect analyses, and Cohen’s d effect sizes
to Paxinos coordinates (anterior-posterior, −0.30 to −4.16; are described in the results, while post hoc analyses are shown
medial-lateral, 0–2.2; dorsal-ventral, −0.40 to −2.8) and were on the figures.
temporarily stored in RNAlater (Ambion, Austin, TX, USA)
at −80◦ C. Tissue samples were homogenized using a TRIzol RESULTS
reagent (Ambion, Austin, TX, USA) and total RNA from
each hypothalamic sample was isolated with Direct-zol RNA Litter Statistics
MiniPrep Kit (Zymo Research, Irvine, CA, USA) following the Body weights of adult female WLIs were significantly higher
manufacturer’s protocol. Once isolated, 1 µg of the total RNA compared to those of same age WMI females before mating
was reverse transcribed using the SuperScript VILO cDNA (175.3 ± 2.3 g vs. 134.7 ± 1.6 g; p = 1.9e-15).
Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). WLI and WMI litters differed significantly in their rate of
All of these methods have been described previously (Raghavan survival to weaning (p = 0.044; Table 1). While the WLI litters
et al., 2017; Lim et al., 2018a,b; Meckes et al., 2018). had a survival rate of 50%, their WMI counterparts had a survival
rate of less than half, 21.4%. This survival rate is in agreement
Real-Time Reverse with the one observed during a previous 8-month mating period
Transcription-Polymerase Chain Reaction (13 WLI litters survived out of 22 total litters for a 59% rate
(RT-qPCR) compared to 10 WMI litters survived from a total of 37 litters,
For each experimental group, RT-qPCR was performed to 27% survival rate; p = 0.014). Additionally, WMI litters seemed to
compare the hypothalamic target gene expression levels between be smaller at birth compared to WLI litters, based on the counts
strains (WLI vs. WMI). Primers for each target gene were without disturbing the cage. Due to the high pup mortality, the
designed using Applied Biosystems Primer Express software exact number of pups at birth was not determined since we did
(version 3.0, PE Applied Biosystems, Foster City, CA, USA); the not want to disturb the dams and litters after birth. WMI litters

TABLE 1 | Litter characteristics.

Strain Litters survived/total; % Days survived by litters Number of pups at Male : female ratio at
survival not weaned weaning weaning

WLI 9/18, 50% 5.22 ± 2.17 8.50 ± 0.68 4.41 ± 0.55 : 4.08 ± 0.41
WMI 6/28∗ , 21.4% 3.375 ± 0.63 5.92 ± 0.47∗∗ 2.83 ± 0.44∗ : 3.08 ± 0.35

Values are mean ± SEM; ∗ p < 0.05, ∗∗ p < 0.01 strain differences.

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had significantly fewer pups than the WLI litters at weaning validated using Sanger Sequencing (Supplementary Figure 1).
(t (22) = 3.13, p = 0.005; Cohen’s d = 1.28), and the numbers were Therefore, the hypothalamic expression of Kdm5a and the
uneven between the sexes with less male WMI pups survived transcribed clock genes period 1 and 2 (Per1, Per2) was
compared to WLI males (males, t (22) = 2.29, p = 0.036; Cohen’s measured. Transcript levels of Kdm5a differed significantly
d = 0.91; females, t (22) = 1.82, p = 0.082; Cohen’s d = 0.74; between WLI and WMI females, regardless of postpartum
Table 1). days (Kdm5a, F (1,15) = 68.77, p < 0.0001), with WMI dams
showing higher expression (Figure 2). Transcript levels of Per1
Maternal Behavior and Per2 also differed significantly between WLI and WMI
Maternal and self-directed behaviors were assessed for 10 days females (Per1, F (1,15) = 4.63, p = 0.048; Per2, F (1,15) = 8.05,
postpartum and their distribution is shown in Figure 1A. The p = 0.013), with WMI dams showing higher expression
figure shows the percentage of different behaviors in both strains, (Figure 2).
measured as the average frequency observed during the 1-h Hypothalamic transcript levels of target genes are shown
observation period in the light and the dark phase. A significant in Figure 3. Interestingly, while Oxt expression tended to
strain difference was observed in arched-back and blanket differ significantly between WLI and WMI dams only by litter
nursing (strain, F (1,18) = 7.77, p = 0.012), with no differences survival (litter survival by strain, F (1,11) = 7, 15, p = 0.022),
between light and dark phases (Figure 1B). Large effect sizes expression of Oxtr showed both a clear strain and a strain
were detected for the strain comparisons (Cohen’s d, light: 1.23; by litter survival effect (strain, F (1,13) = 4.80, p = 0.047;
dark: 1.28). Similarly, there were strain differences in licking, litter survival by strain, F (1,13) = 5.38, p = 0.037). The Avp
grooming, and pup retrieval (F (1,19) = 4.61, p = 0.045; Figure 1B), system showed major differences between the strains and
with WMIs showing the greater of these pup-directed behaviors. litter survival. Specifically, expression of both Avp and Avpr1a
The effect sizes revealed that this strain difference originated showed strain differences (Avp, F (1,12) = 12.54, p = 0.004;
more from the dark phase comparison (Cohen’s d, light: 0.76; Avpr1a, F (1,15) = 7.01, p = 0.018). However, while there were
dark: 1.24). Both strains of dams showed a lower frequency of no litter survival and strain by litter survival effects for Avp
these pup-directed behaviors during the dark phase (time of day, expression, Avpr1a expression was greater in PPD 24 WMI
F (1,19) = 8.79, p = 0.008). Self-directed behaviors such as eating, hypothalamus compared to all other groups (litter survival,
drinking, and self-grooming, also differed by strain and time of F (1,15) = 8.64, p = 0.010; strain by litter survival, F (1,15) = 5.62,
day (strain, F (1,19) = 6.12, p = 0.023; time of day F (1,19) = 10.29, p = 0.032). In contrast, hypothalamic transcript levels of Avpr1b
p = 0.005; Figure 1B). Interestingly, the diurnal change in this did not differ between the strains but showed a significant
behavior was only seen in the WLI dams (strain × time of association with litter survival (F (1,13) = 21.56, p = 0.0005).
day, F (1,19) = 6.19, p = 0.022). This is confirmed by the large Expression of Esr1 differed significantly between the WLI
effect size in strain comparisons at the dark phase (Cohen’s d, and WMI hypothalamus (F (1,12) = 5.24, p = 0.041), but the
light: 0.02; dark: 1.59). expression of Esr2 only showed a difference by litter survival
Figure 1C shows self-directed behaviors across the 10 days (F (1,15) = 7.97, p < 0.013).
observation period for both WLI and WMI dams. WLI dams Heatmaps of the correlations between hypothalamic gene
showed day by day differences in self-directed behaviors across expressions are shown in Supplementary Figure 2 for WLIs
the observation period, and also between the light and dark and WMIs. The heatmaps illustrate the differential pattern
phases (time of day, F (1,3) = 23.42, p = 0.017; days × time of day, of correlations between the strains and between the days
F (9,27) = 2.45, p = 0.035). Specifically, self-directed behaviors were postpartum. Unique strain differences in a significant correlation
significantly greater in the dark phase than the light phase on are present in WLIs regardless of PPD, such as the correlation
PPD 5–7 and 10 (PPD 5, t (30) = 3.56, p = 0.013; PPD 6, t (30) = 4.07, between hypothalamic expression of Oxtr and Avpr1a and
p = 0.003; PPD 7, t (30) = 3.56, p = 0.013, and PPD 10, t (30) = 4.07, Kdm5a and Per2. These correlations are not present in WMIs,
p = 0.003). In contrast, there were no significant differences in while the Avp, Per1, Kdm5a, and Avpr1b correlations are
self-directed behaviors across the days of observation in WMIs, unique to PPD 24 WMI hypothalamus. Correlations unique to
but light, dark phase differences tended to occur postpartum postpartum days or litter survival regardless of strain indicate
day 5 (days × time of day, F (9,35) = 2.12, p = 0.054; PPD 5, that in the PPD <5 groups, hypothalamic expression of Oxt
t (39) = 3.07, p = 0.038). correlated with Avp, while at PPD 24, Avpr1a correlated with
Esr2 expression.
Hypothalamic Target Gene Expression
The observation of the lack of circadian rhythm in the DISCUSSION
self-directed behaviors of WMI dams prompted us to
examine any potential causative genetic differences in circadian The WMI genetic animal model of depression has shown
rhythm-regulating genes between WLI and WMI. The Lysine major differences during postpartum compared to their isogenic
Demethylase 5A (Kdm5a or Jumonji/ARID domain-containing controls, the WLI strain that does not indicate depression-like
protein1A: JARID1A) is connected to the circadian epigenome behavior. Many of these characteristics segregated from the WKY
(Masri and Sassone-Corsi, 2013). We first identified the parent phenotype during selective breeding with most behavioral
Arg745Cys variation in the Kdm5a gene between WMI and phenotypes of WMIs being the same as the WKYs, while
WLI strains from whole-genome sequencing data. This was WLIs being different. Particularly, WMI dams have reduced

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FIGURE 1 | Maternal and self-directed behaviors of Wistar Kyoto Less Immobile (WLI) and Wistar Kyoto More Immobile (WMI) dams as observed during
postpartum day 1–10. (A) Percentage of average frequency/hr of each category of maternal behaviors in the light and the dark phases of the day are shown.
(B) Frequency per hour of observation of arched-back and blanket nursing is greater in WLI dams compared to WMIs, regardless of the time of day. Pup-directed
behaviors, such as licking, grooming, and retrieval are greater in WMI than WLI dams and differ between the light and the dark phases of the day, the latter being
less. Self-directed behaviors do not differ between WLI and WMI dams during the light phase, but during the dark phase (when nocturnal animals are more active),
WMI dams spend significantly less time eating and drinking compared to WLIs. (C) Self-directed behaviors are greater in the dark phase in WLIs across the 10 days
observation, with only one time of day difference in WMIs. Data are presented as mean ± standard error of the mean. *p < 0.05, ∗∗ p < 0.01.

litter survival, smaller litter size and decreased arched-back rhythm disturbances observed in patients with depression
and blanket nursing, but increased licking, grooming retrieval compared to healthy controls (Mondin et al., 2017; Ozcelik
behaviors toward their pups than WLI dams over the and Sahbaz, 2020). Hypothalamic expression of Kdm5a, Per1,
first 10 days postpartum. WMIs self-directed behaviors are and Per2 is greater in WMIs than WLIs, both in dams
decreased and WMIs show no diurnal rhythm in these whose litter died before postpartum day 5 and in those
behaviors while WLI dams do. This is similar to the biological whose litter survived to wean. Hypothalamic transcript levels

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FIGURE 2 | Hypothalamic gene expression of Kdm5a, Per1, and Per2 in WLI and WMI dams who lost their litter before postpartum day 5 (PPD < 5) and in those
whose litter were weaned at PPD 24. Hypothalamic transcript levels were measured by quantitative RT-PCR and shown as Relative Quantification (RQ). Data are
presented as mean ± standard error of the mean. N = 4–6 per group. ∗ P < 0.05 and ∗∗ P < 0.01 represent post hoc comparisons.

of Oxtr, Avp, and Avpr1a are also increased in the WMI Altered maternal-child interactions are reported when
dams compared to their isogenic control WLIs, but only women suffer from postpartum depression and comorbid
in dams whose litter survived to wean. Another parallel to anxiety. Decreased breastfeeding, or premature cessation of
the human condition is that these gene expression increases it, has been observed in women with depression before or
are similar to what was found in the hypothalamus of during pregnancy (Wallenborn et al., 2018; Jordan et al., 2019).
depressed patients (Meynen et al., 2006; Wang et al., 2008). Genetic rodent models with depression-like behavior show
Since WMI dams show characteristics similar to depressed some of the characteristics of these altered interactions, as
patients and decreased nursing, but increased pup-directed seen with the decreased nursing of WMIs compared to WLIs.
behaviors, we propose that the dams of the WMI genetic Although maternal behavior has been examined in many inbred
animal model of depression are a potential animal model of and outbred strains of rats and mice (Perani and Slattery,
postpartum depression. 2014), findings from the Flinders Sensitive Line (FSL) and the
In prior studies, differences in maternal care were not related WKY rats, two different genetic animal models of depression
to basic measures of reproductive success, such as litter survival (Lavi-Avnon et al., 2005; Braw et al., 2009), are particularly
to weaning (Champagne et al., 2003). The parental strain of relevant to the current study. FSL dams do not differ from
the WLI and WMI, the WKY rat, has an average litter size of control Sprague Dawley dams in their maternal behavior, while
8.16 pups which is similar to litter sizes of the control WLI WKY dams perform more pup-directed activities and fewer
dams and other rat strains (Gill Iii et al., 1979). However, self-directed activities compared to Wistar controls (Braw et al.,
after the establishment of these new inbred strains, the litter 2009). This again implies that the WKY parental phenotype of
size for the WMI dams became close to half of that of the pup-directed and self-directed activities are segregated together
WLI dams. Furthermore, although WMI litters were birthed and present in the WMI strain, differently from that of
at the same rate as WLI litters, they showed a significantly the litter size phenotype. Interestingly, increased pup-directed
lower survival rate than WLI litters. One of the potential behavior could represent the ‘‘helicopter parenting’’ maternal
causes of these findings could be aberrant maternal behavior phenotype observed in mothers with postpartum depression
of the WMI dam, as more nourishment-directed maternal and anxiety (Perani and Slattery, 2014). Even more specifically,
behaviors are thought to increase litter survival (Weber et al., maternal anxiety is associated with higher maternal control
2016). While dams of both strains show limited arched-back and intrusiveness in the mother-infant interaction (Stein et al.,
and blanket nursing behaviors during the observation period, 2012; Parfitt et al., 2013; Hakanen et al., 2019). Since both
WMI dams spent significantly less time with arched-back and WKY (Solberg et al., 2003; Baum et al., 2006) and WMI
blanket nursing of the pups than WLIs. In contrast, WMI females show concurrent increased depression and anxiety-like
dams spent more time licking, grooming, and retrieval of the phenotypes (Mehta et al., 2013) and more pup-directed
pups than WLIs. Although arched-back, blanket nursing and activities compared to their respective controls (Wistar and
licking, grooming and retrieval of the pups often co-occur, WLI; Braw et al., 2009), it is feasible that their behavior
previous studies have found them to vary independently. For is also associated with maternal anxiety. This is similar to
example, undernourished pups elicit increased licking, grooming findings in which women with postpartum depression very
from dams regardless of the dam’s ability or frequency of often show comorbid anxiety (Farr et al., 2014). The main
nursing (Lynch, 1976). It has been suggested that these two predictor for depressive, anxiety, or psychotic diseases after
behaviors have distinct developmental roles with distinct effects delivery is an antenatal episode of the illness (Perani and
on offspring biobehavioral outcomes, and also that arched-back Slattery, 2014). Thus, WKY and WMI females do have risk
nursing is typically not a significant predictor of offspring factors for showing characteristics of postpartum depression
outcomes (Jensen Peña and Champagne, 2013). after delivery.

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FIGURE 3 | Hypothalamic gene expression of Oxt, Oxtr, Avp, Avpr1a and Avpr1b, and Esr1 and Esr2 in WLI and WMI dams who lost their litter before postpartum
day 5 (PPD < 5) and in those whose litter were weaned at PPD 24. Hypothalamic transcript levels were measured by quantitative RT-PCR and shown as Relative
Quantification (RQ). Data are presented as mean ± standard error of the mean. N = 4–6 per group. # < 0.10, ∗ P < 0.05, and ∗∗ P < 0.01 represent
post hoc comparisons.

The decreased self-directed behavior of the WMI dams a similar phenotype, suggesting that it may be a common
became very apparent in this study because we observed maternal phenotype in animal models of depression (Shiromani and
behavior during both the light and the dark phase for 10 days Overstreet, 1994). Some data demonstrate that patients with
postpartum. This is in contrast to some other studies in which depression may have an altered sensitivity to light (Duncan,
spot check observations were conducted on PPD 4 and 9 only 1996). Additionally, greater biological rhythm disturbances have
(Braw et al., 2009), or behaviors were examined on PPD been observed in patients with depression compared to healthy
3–4 and 17–18 only in the light phase (Lavi-Avnon et al., controls (Mondin et al., 2017; Ozcelik and Sahbaz, 2020). Sleep
2005). Nocturnal animals eat and drink more during the dark, disturbances have also been observed in WKYs (Dugovic et al.,
just as the WLI dams did, but the disturbed rhythm of WMIs 2000; Dasilva et al., 2011), very similar to those described in
recalls a similar phenomenon in the WKYs. The WKY rats were depressed patients (Rosenwasser and Wirz-Justice, 1997). Thus,
found to be less responsive to light, which may cause possible the lack of diurnal rhythm of self-directed behaviors in the
alterations in daily rhythm patterns of this strain (Rosenwasser, WMI dams suggests that this phenotype segregated with the
1993; Solberg et al., 2001). Interestingly, the FSL has shown depression-like behavior.

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Because the WLI and WMI strains are isogenic, we could associated with higher levels of oxytocin binding in relevant
peruse the sequences for possible causative sequence variations brain regions (Champagne et al., 2001; Curley et al., 2012),
that may be associated with the lack of diurnal rhythm including the paraventricular nucleus of the hypothalamus
observed in the WMI dams. The confirmed nonsynonymous (Bayerl et al., 2016). Furthermore, the central administration
sequence variation in the coding region of lysine-specific of an Oxtr antagonist reduces high levels of pup licking,
demethylase 5A (Kdm5a/Jarid1a) elevated this gene to grooming, and arched-back nursing in dams (Champagne et al.,
a possible causative gene, although the single nucleotide 2001). Thus, increased pup licking, grooming behavior of the
polymorphism (SNP) was in the WLI strain. Kdm5a forms a WMI dam is in concordance with their increased hypothalamic
complex with transcription factors Clock and Bmal1, which expression of Oxtr, but not with their decreased arched-back
results in transcriptional activation of the Period genes and and blanket nursing compared to that of WLIs. Administration
maintenance of circadian oscillations (DiTacchio et al., 2011). of Avp increases pup grooming in rats (Caldwell et al., 1986;
Interestingly, Kdm5a and Per2 expression were increased Elkabir et al., 1990), while antagonism of Avpr1a does not
in the WMI compared to WLI, regardless of litter survival affect arched back nursing and pup retrieval, but decreases
or postpartum days. Increased hypothalamic expression of other types of nursing (Bayerl et al., 2016). Thus again, the
Kdm5a and Per2 may contribute to the lack of circadian increased Avp expression in the WMI hypothalamus is as
rhythm seen in the WMI’s behavior. Constitutive expression per a component of the licking, grooming behavior of WMI
of Per2 abolishes diurnal rhythm (Chen et al., 2009), and dams, but not with the rest of the observed behavior. These
Kdm5a is known to activate Per2 expression (DiTacchio et al., seeming contradictions suggest that discrete components of
2011). Thus, the lack of diurnal variation in self-directed maternal behavior are influenced by different neuropeptidergic
behaviors of the WMI dam could be related to the increased mechanisms or differing neurocircuitry (Bosch and Neumann,
expression of Per2 in the hypothalamus. Whether the increased 2012). The complexity of the association between maternal
Per2 is also a molecular characterization of the WMI’s behavioral differences and these neuropeptidergic mechanisms
depressive behavior is not known, but antidepressants are is exaggerated by the findings that the administration of an
known to reduce Per2 expression in experimental models Avpr1a antagonist reduces anxiety/depression-like behavior in
(Orozco-Solis et al., 2017). preclinical studies (Wigger et al., 2004). Furthermore, AVP and
Alternatively, the SNP in the Kdm5a gene in the WLIs AVPR1A expressions are higher in the human hypothalamus of
could have a loss or gain of function effect. The gain of depressed patients compared to that of controls (Meynen et al.,
function could enhance the circadian rhythm of WLI in 2006; Wang et al., 2008). Therefore, the increased hypothalamic
their self-directed behavior, and the Kdm5a-induced negative expression of Avp and Avpr1a could be the manifestation of the
regulation of transcription by RNA polymerase II could depression-like behavior of the WMI dams.
suppress the expression of the target genes in WLIs, and The most thought-provoking findings of the present study,
not in WMIs. However, there were no correlations between such as the increased licking, grooming, and retrieval but
the expression of Kdm5a and other genes except for Per2 decreased arched-back and blanket nursing and self-directed
in the WLIs. In contrast, Kdm5a expression correlated with behaviors of the WMI dams, seem to parallel postpartum
Avp, but only in the WMIs, suggesting a loss of function behavior of its progenitor, the WKY strain, which is considered
effect of the WLI SNP. These proposed mechanisms do an animal model of depression. In contrast, these behaviors
not explain the lack of diurnal rhythm in the WMIs are very different in the WLIs, where the inbred strain does
self-directed behavior, as no single regulator can. However, not show depression-like behavior. Thus, some postpartum
they implicate that genetic manipulations in these strains behaviors segregated together with depression-like behavior
could, potentially, identify causative variations affecting between the two strains during selective breeding. Genetic
maternal behavior. studies using the WKY and another strain with phenotypic
The observed increases in the hypothalamic expression of differences are possible, but less likely to produce causative
Oxtr, Avp, and Avp1ra in the WMI dams compared to WLIs and genes due to the nature of these quantitative trait loci
also to those WMIs whose litter died are difficult to interpret studies (Solberg et al., 2004). In contrast, exploring the
in the context of their accepted role in maternal behavior. A underlying genetic basis of these behaviors in the isogenic
large body of literature supports the role of neuroendocrine WLI and WMI strains using the reduced complexity cross
processes in the induction and regulation of maternal behavior approach would be more meaningful, as we described it
(Bridges, 2015). Both neuropeptides, Oxt and Avp, have been recently (Bryant et al., 2020). The loss of diurnal patterns
known to have an impact on maternal behavior (Pedersen and in self-directed behaviors and increased hypothalamic
Prange, 1979; Pedersen et al., 1982; Bosch and Neumann, 2008; Avp and Avpr1a expression are resonant to findings in
Bayerl and Bosch, 2019). Their receptors, Avpr1a and Oxtr, human depressed patients. Future studies could investigate
have also been implicated in maternal behaviors (Donaldson whether attenuating the WMI’s depression-like behavior
and Young, 2008) with complementary expression patterns before gestation, by antidepressant treatment (Will et al.,
in the ventromedial hypothalamus (Raggenbass, 2008). Several 2003) or by environmental enrichment (Mehta-Raghavan
rat and mice studies showed a correlation between Oxtr et al., 2016), would equalize litter survival, maternal and
and Avpr expression, similar to what is seen in the WLI self-directed behaviors, and neuropeptide receptor expression
dams’ hypothalami. However, more maternal care has been in the hypothalamus of WMI and WLI dams. Since the

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greatest risk factor for postpartum depression is a history AUTHOR CONTRIBUTIONS

of depression before pregnancy (Putnam et al., 2017),
the WLI and WMI rat strains could be valuable tools The study was designed by EER and WL. Experimental work was
to investigate the molecular and genetic underpinning of carried out by WL, PHL, SLW, SAG, and HC. Data analysis was
this disorder. carried out by WL and EER. The manuscript was drafted by WL,
EER, and HC. All authors contributed to the article and approved
the submitted version.
DATA AVAILABILITY STATEMENT
FUNDING
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation. This work was supported by the Davee Foundation to EER.

SUPPLEMENTARY MATERIAL
ETHICS STATEMENT
The Supplementary Material for this article can be found
The animal study was reviewed and approved by Northwestern online at: https://www.frontiersin.org/articles/10.3389/fnbeh.
University Institutional ACUC. 2020.589967/full#supplementary-material.

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depression: clinical and experimental models,’’ in Physiology and Pharmacology potential conflict of interest.
of Biological Rhythms, eds P. H. Redfern and B. Lemmer (Berlin: Springer),
457–486. Copyright © 2020 Luo, Lim, Wert, Gacek, Chen and Redei. This is an open-access
Shiromani, P. J., and Overstreet, D. (1994). Free-running period of article distributed under the terms of the Creative Commons Attribution License
circadian rhythms is shorter in rats with a genetically upregulated central (CC BY). The use, distribution or reproduction in other forums is permitted,
cholinergic system. Biol. Psychiatry 36, 622–626. doi: 10.1016/0006-3223(94) provided the original author(s) and the copyright owner(s) are credited and that the
90075-2 original publication in this journal is cited, in accordance with accepted academic
Solberg, L. C., Ahmadiyeh, N., Baum, A. E., Vitaterna, M. H., Takahashi, J. S., practice. No use, distribution or reproduction is permitted which does not comply
Turek, F. W., et al. (2003). Depressive-like behavior and stress reactivity are with these terms.

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 ORIGINAL RESEARCH
published: 04 November 2020
doi: 10.3389/fnins.2020.590088

Postnatal Antioxidant and

Anti-inflammatory Treatments
Prevent Early Ketamine-Induced
Cortical Dysfunctions in Adult Mice
Maria Bove † , Paolo Tucci † , Stefania Dimonte, Luigia Trabace, Stefania Schiavone* and
Maria Grazia Morgese
Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy

Early brain insult, interfering with its maturation, may result in psychotic-like
disturbances in adult life. Redox dysfunctions and neuroinflammation contribute to
long-term psychiatric consequences due to neurodevelopmental abnormalities. Here,
we investigated the effects of early pharmacological modulation of the redox and
inflammatory states, through celastrol, and indomethacin administration, on reactive
oxygen species (ROS) amount, levels of malondialdehyde (MDA) and antioxidant
Edited by:
enzymes (superoxide dismutase 1, SOD1, glutathione, GSH, and catalase, CAT), as
Carla Cannizzaro, well as of pro-inflammatory cytokines (tumor necrosis factor-alpha, TNF-α, interleukin-6,
University of Palermo, Italy
IL-6, and interleukin-1 beta, IL-1β), in the prefrontal cortex of adult mice exposed
Reviewed by:
to a neurotoxic insult, i.e. ketamine administration, in postnatal life. Early celastrol or
Maria L. De Ceballos,
Consejo Superior de Investigaciones indomethacin prevented ketamine-induced elevations in cortical ROS production. MDA
Científicas (CSIC), Spain levels in ketamine-treated mice, also administered with celastrol, were comparable with
Cristiano Chiamulera,
University of Verona, Italy
the control ones. Indomethacin also prevented the increase in lipid peroxidation following
*Correspondence:
early ketamine administration. Whereas no significant differences were detected in
Stefania Schiavone SOD1, GSH, and CAT levels between ketamine and saline-administered mice, celastrol
[email protected]
elevated the cortical amount of these antioxidant enzymes and the same effect was
† These authors have contributed
induced by indomethacin per se. Both celastrol and indomethacin prevented ketamine-
equally to this work
induced enhancement in TNF-α and IL-1β levels, however, they had no effects on
Specialty section: increased IL-6 amount resulting from ketamine exposure in postnatal life. In conclusion,
This article was submitted to
Neurogenesis,
our data suggest that an early increase in cortical ROS scavenging and reduction of
a section of the journal lipid peroxidation, via the enhancement of antioxidant defense, together with inhibition
Frontiers in Neuroscience of neuroinflammation, may represent a therapeutic opportunity against psychotic-like
Received: 31 July 2020 disturbances resulting, later in life, from the effects of a neurotoxic insult on the
Accepted: 28 September 2020
Published: 04 November 2020 developing brain.
Citation: Keywords: celastrol, indomethacin, ketamine, prefrontal cortex, redox, inflammation, animal models
Bove M, Tucci P, Dimonte S,
Trabace L, Schiavone S and
Morgese MG (2020) Postnatal
Antioxidant and Anti-inflammatory
INTRODUCTION
Treatments Prevent Early
Ketamine-Induced Cortical
Early insults affecting the central nervous system (CNS) during crucial phases of its maturation
Dysfunctions in Adult Mice. have been reported to induce neurodevelopmental abnormalities. This has been associated with
Front. Neurosci. 14:590088. increased risk of developing psychotic-like disturbances in adult life (Hussain and Murray,
doi: 10.3389/fnins.2020.590088 2015). In this pathological process, the prefrontal cortex (PFC), characterized by highly

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Bove et al. Celastrol and Indomethacin in Brain Dysfunctions

vulnerable cellular populations, has been described as unlike ibuprofen, might be neuroprotective against the long
one of the most consistently implicated brain regions term effects of cerebral insults, such as ventricular hemorrhage
(Selemon and Zecevic, 2015). (Favrais et al., 2014).
Multiple molecular mechanisms underlying long-term Postnatal administration of subanesthetic doses of ketamine,
psychiatric consequences of early brain insults have been a NMDA receptor (NMDA-R) antagonist, is a reliable tool
proposed. Among them, dysfunctions of the antioxidant to mimic in rodents an early insult interfering with brain
enzymes, such as superoxide dismutase 1 (SOD1), glutathione maturation (Frohlich and Van Horn, 2014). In this regard,
(GSH), and catalase (CAT), have been described (Cabungcal NMDA-Rs reach their maximum expression in the first 2 weeks
et al., 2013). The expression and activity of these enzymes of postnatal life. Hence, inhibition of these receptors in
physiologically occur during key neurodevelopmental phases. this period is associated with increased neuronal damage
Indeed, SOD1, expressed primarily in cortical neurons (Peluffo (Bubenikova-Valesova et al., 2008). Indeed, ketamine exposure
et al., 2005), has been shown to reach a peak in the second during CNS development has been shown to cause a down-
postnatal week (Ceballos-Picot et al., 1992). Similarly, CAT regulation of NMDA-Rs located in the PFC resulting in
activity in rodent developing CNS has been found to be psychiatric-like symptoms in rat adult offspring (Ren et al.,
higher than in the mature brain (Del Maestro and McDonald, 2019). Furthermore, early genetic ablation or ketamine-induced
1987; Hamby-Mason et al., 1997), with a maximum observed blockade of NMDA-Rs of cortical parvalbumin-expressing
from postnatal day (PND) 5 to PND 10 (Del Maestro and GABAergic interneurons can induce in adult animals persistent
McDonald, 1987; Aspberg and Tottmar, 1992). Moreover, GSH behavioral deficits, reminiscent of cognitive and negative
has been reported to increase and modify the redox state of psychotic symptoms (Jeevakumar et al., 2015). In addition, we
the cells toward a more reduced condition starting from PND have demonstrated that ketamine administration at PNDs 7,
10 until PND 30 (Galkina et al., 2017). Altered antioxidant 9 and 11 caused psychotic-like neurochemical and behavioral
defense in the brain may result in increased levels of reactive alterations in adult mice (Schiavone et al., 2019, 2020).
oxygen species (ROS) and consequent lipid peroxidation in Here, we assessed the effects of early pharmacological
neurons. One of the final products of this biochemical process modulation of the redox and inflammatory states, through
is malondialdehyde (MDA). Enhanced amount of this highly celastrol and indomethacin administration, on possible alteration
reactive compound has been reported in the PFC of young mice of ROS production, lipid peroxidation, as well as SOD1, GSH,
perinatally exposed to a neurotoxic insult (Del Rio et al., 2005; and CAT levels in the PFC, induced by the exposure to an early
Tsikas, 2017). The natural compound celastrol, derived from neurotoxic trigger, i.e., ketamine, in postnatal life. Moreover,
the root of Tripterygium wilfordii, pharmacologically modulate celastrol or indomethacin effects on possible ketamine-induced
ROS amount and antioxidant defense system. It has shown changes of proinflammatory cytokines levels, i.e., TNF-α, IL-6,
to be effective for a broad range of pathological conditions, and IL-1β were also assessed in the same brain region.
including neurodegenerative disorders (Kiaei et al., 2005; Paris
et al., 2010; Choi et al., 2014), cerebral ischemia (Li et al., 2012;
Jiang et al., 2018) and traumatic brain injury (Eroglu et al., MATERIALS AND METHODS
2014). Moreover, it has been reported to prevent psychotic-
like behavioral alterations, oxidative stress and inflammatory Animals
imbalance in adult mice exposed to a neurotoxic insult in their Mice were housed at constant room temperature (22 ± 1◦ C)
postnatal life (Schiavone et al., 2019). and relative humidity (55 ± 5%), under a 12 h light/dark
Neuroinflammation is a crucial contributor of long-term cycle (lights on from 7:00 AM to 7:00 PM). They had free
psychiatric consequences of early neurodetrimental insults. In access to food and water. Experimental procedures involving
particular, the developing brain is characterized by increased animals and their care were performed in conformity with the
vulnerability to proinflammatory cytokines, such as Tumor institutional guidelines of the Italian Ministry of Health (D.Lgs. n.
necrosis factor (TNF)-α, interleukin-6 (IL-6), and interleukin-1 26/2014), the Guide for the Care and Use of Laboratory Animals:
beta (IL-1β) (Hagberg and Mallard, 2005). In this regard, levels of Eight Edition, the Guide for the Care and Use of Mammals
TNF-A were enhanced in the cerebellum of adult mice postnatally in Neuroscience and Behavioral Research (National Research
exposed to a neurotoxic insult (Schiavone et al., 2019). In Council, 2004), the Directive 2010/63/EU of the European
addition, non-steroidal anti-inflammatory drugs (NSAIDs) have Parliament and of the Council of 22 September 2010 on the
been shown to exert protective effects on neurodevelopmental protection of animals used for scientific purposes, as well as the
processes. This occurs via the inhibition of the synthesis of ARRIVE guidelines. The experimental protocol was approved
inflammatory mediators at systemic level and via cyclooxygenase by the Italian Ministry of Health (approval number 679/2017-
(COX) inhibition at blood brain barrier site (Favrais et al., PR, protocol n. B2EF8.17) Animal welfare was daily monitored
2007). Among NSAIDs, indomethacin, a non-selective inhibitor throughout the experimental phase. All efforts were made to
of COX 1 and 2, has been shown to readily pass the blood minimize the number of animals used, as well as their suffering.
brain barrier (Parepally et al., 2006; Novakova et al., 2014). It
also exerted neuroprotective effects in newborn rodents exposed Experimental Protocol
to hypoxic-ischemic insult (Tutak et al., 2005; Taskin et al., A total of five C57/Bl6 male mice of 8–10 weeks of age, weighting
2009). Accordingly, clinical evidence showed that indomethacin, 25–30 g, and ten age and weight-matched adult females (Envigo,

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Bove et al. Celastrol and Indomethacin in Brain Dysfunctions

San Pietro al Natisone, Italy) were mated (one male and two was resuspended in 5 ml PBS 1× and put in ice for 10 min.
females per cage). Male pups were divided into the following After a 1-h incubation at 37◦ C, samples were analyzed in 96-well
experimental groups, according to the different treatments they microplate by using a fluorometer (Filter Max F5, Multi-Mode
received at PNDs 7, 9, and 11: Microplate Reader, excitation length 475 nm, emission length
535 nm). Results were expressed as µmol DCF/mg of tissue.
(1) Saline (10 ml/kg i.p.);
(2) Ketamine (Sigma-Aldrich Corporation, Saint Louis, MO, MDA Assay
United States; 30 mg/kg i.p., dissolved in saline) (Sorce MDA assay was performed by using a commercially available kit
et al., 2010; Jeevakumar et al., 2015); (Sigma-Aldrich, Milano, Italy) as previously described (Fan et al.,
(3) Celastrol (Sigma Aldrich, Milano, Italy; 1 mg/kg i.p., 2019), according to the manufacturer’s instructions. Each sample
dissolved in 50% DMSO/PBS) (Paris et al., 2010; Schiavone and standard analysis was performed in duplicate to avoid intra-
et al., 2019); assay variations.
(4) A 50% DMSO/PBS solution (5 ml/kg i.p.);
(5) Ketamine (30 mg/kg i.p., dissolved in saline, injected in the Enzyme-Linked Immunosorbent Assays
right side of the peritoneum) and celastrol (1 mg/kg i.p., Samples were homogenized in 10 volumes of PBS with
dissolved in 50% DMSO/PBS, injected in the left side of the protease inhibitors, as previously described (Schiavone et al.,
peritoneum) (Schiavone et al., 2019)-indicated throughout 2019). Commercially available Enzyme-Linked Immunosorbent
the text as “ketamine + celastrol”; Assay (ELISA) kits were used for measurement of SOD1
(6) Indomethacin [Promedica, Parma, Italy, 10 mg/kg i.p., (La (Wuhan Fine Biotech Co., Ltd.-FineTest, Wuhan, China),
Vitola et al., 2018), dissolved in saline]; GSH (Biomatik Life Science Products and Service, Ontario,
(7) Ketamine (30 mg/kg i.p., dissolved in saline, injected Canada), CAT (Wuhan Fine Biotech Co., Ltd.-FineTest, Wuhan,
in the right side of the peritoneum) and indomethacin China), TNF-A (MyBiosource, San Diego, CA, United States),
(10 mg/kg i.p., dissolved in saline, injected in the left side IL-6 (MyBiosource, San Diego, CA, United States) and IL-
of the peritoneum) – indicated throughout the text as 1ß (MyBiosource, San Diego, CA, United States) in the
“ketamine + indomethacin”. PFC, according to the manufacturer’s instructions. All samples
For ethical reasons, in keeping with the pursuing of 3R and standards were analyzed in duplicate to avoid intra-
requirements foreseen by the Directive 2010/63/EU of the assay variations.
European Parliament, as well as of the Council of 22 September
2010 on the protection of animals used for scientific purposes, the Blindness of the Study
ARRIVE guidelines, and also based on our previous experience Data analysis was performed by researchers who were blind with
(we did not detect any differences between a double with respect respect to the treatment conditions. The blindness of the study
to single injection of vehicles), the group consisting of double- was maintained until data analysis ended.
vehicle injection was omitted from the experimental protocol.
All pups were grown until adulthood (10 weeks of age). At Statistical Analysis
this time point, they were euthanized by cervical dislocation Statistical analysis was performed by using GraphPad 5.0 software
for PFC collection. for Windows. Data were checked for normality by using Bartlett’s
test and then analyzed by One Way ANOVA, followed by
Tukey’s post hoc test or Kruskal-Wallis test, followed by Dunn’s
PFC Collection
multiple comparison test. For all tests, a p-value < 0.05 was
The PFC of 10-weeks mice was collected by using the Mouse
considered as statistically significant. Results are expressed as
Brain Matrix, making coronal sections of 1 mm of thickness
means ± mean standard error (SEM). No significant differences
and dissecting it from the obtained brain slices according to the
in all the considered parameters were detected between saline and
Mouse Brain in Stereotaxic Coordinates, 3rd Edition, Franklin
50% DMSO/PBS-treated animals. Therefore, graphs only include
and Paxinos (2015). Immediately after, tissues were frozen in
results related to saline-treated animals.
isopentane and stored at −80◦ C, until biomolecular analyses
were performed (Bove et al., 2018).
RESULTS
ROS Measurement
Reactive oxygen species measurement in PFC was performed Effects of Early Celastrol or
as previously described (Baek et al., 2018; Pirozzi et al., 2020), Indomethacin Administration on ROS
by using the fluorogenic dye 20 ,70 dichlorofluorescein diacetate
(Sigma Aldrich, Milano, Italy) (Kirkland et al., 2007). Briefly,
Production and Lipid Peroxidation in the
tissue was homogenized in PBS 1× (pH = 7.4) according to the PFC of Adult Mice Treated With
following proportion: 500 µl of PBS 1× for 2,5 mg of tissue. The Ketamine in Postnatal Life
dye was added to the sample with a final concentration of 5 µM To assess possible effects of early celastrol or indomethacin
and incubation was performed for 15 min at 37◦ C. Samples were administration on ROS production and lipid peroxidation
than centrifuged for 10 min at 4◦ C and 12,500 rpm. The pellet induced by ketamine exposure in postnatal life, we quantified

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ROS, and MDA levels in the PFC of adult mice. No significant

differences in cortical ROS production were detected between
controls and celastrol-injected animals. Ketamine exposure in
postnatal life resulted in increased ROS production compared to
saline-treated mice which was prevented by ketamine + celastrol
administration (Figure 1A, One way ANOVA, followed by
Tukey’s Multiple Comparison post hoc test, F (3,12) = 5.742,
p < 0.05). While no significant differences in MDA levels
were observed between animals administered with saline and
the celastrol group, adult mice that received ketamine in
early postnatal life, showed significant MDA elevations in
the considered brain region. Early celastrol administration
was able to prevent ketamine-induced lipid peroxidation
(Figure 1B, Kruskal-Wallis test, followed by Dunn’s multiple
comparison test, Kruskal-Wallis statistic = 9.573, p < 0.05).
Early ketamine + indomethacin administration prevented
ketamine-induced elevation in ROS levels (Figure 2A, One way
ANOVA, followed by Tukey’s Multiple Comparison post hoc
test, F (3,13) = 9.642, p < 0.05, and p < 0.001). Indomethacin
treatment per se was able to significantly lower MDA levels
with respect to both controls and ketamine-exposed mice. Lipid
peroxidation in mice early receiving ketamine + indomethacin
was decreased compared to ketamine-treated mice but did not
reach the same levels than the ones detected in mice early exposed
to indomethacin alone (Figure 2B, One way ANOVA, followed
by Tukey’s Multiple Comparison post hoc test, F (3,18) = 35.10,
p < 0.01 and p < 0.001).

Effects of Early Celastrol or

Indomethacin Administration on FIGURE 1 | Effects of early celastrol administration on ROS production and
Antioxidant Enzyme Expression in the MDA levels in the PFC of adult mice, administered with ketamine in postnatal
life. (A) ROS production (µmol DCF/mg of tissue) in the PFC of 10 weeks
PFC of Adult Mice Treated With mice receiving saline (Sal, n = 4) or ketamine (Ket, n = 4) or celastrol (Cel,
Ketamine in Postnatal Life n = 4) or ketamine + celastrol (Ket + Cel, n = 4) at PNDs 7, 9, and 11. One
To investigate the possible impact of early celastrol or way ANOVA, followed by Tukey’s Multiple Comparison post hoc test,
F (3,12) = 5.742, *p < 0.05 Ket vs Sal, # p < 0.05 Ket + Cel vs Ket. (B) MDA
indomethacin administration on antioxidant enzyme expression levels (nmol/mg tissue) in the PFC of 10 weeks mice receiving saline (Sal,
following ketamine exposure in postnatal life, we quantified levels n = 5) or ketamine (Ket, n = 8) or celastrol (Cel, n = 5) or ketamine + celastrol
of SOD1, CAT, and GSH in the PFC of adult mice. Whereas (Ket + Cel, n = 6) at PNDs 7, 9, and 11. Kruskal-Wallis test, followed by Dunn’s
comparable SOD1 amount was detected among saline, ketamine multiple comparison test, Kruskal-Wallis statistic = 9.573 *p < 0.05 Ket vs Sal.
and celastrol-treated animals, significant increased expression of
this antioxidant enzyme was observed in ketamine + celastrol-
treated animals with respect to both saline and ketamine
groups (Figure 3A, One way ANOVA, followed by Tukey’s life, alone or in combination with ketamine, was able to elevate
Multiple Comparison post hoc test, F (3,14) = 5.318, p < 0.05). GSH expression with respect to controls and ketamine-treated
Early indomethacin treatment per se resulted in an increased mice (Figure 4B, One way ANOVA, followed by Tukey’s Multiple
SOD1 expression with respect to both saline and ketamine- Comparison post hoc test, F (3,16) = 25.59, p < 0.01, p < 0.001).
administered mice (Figure 4A, One way ANOVA, followed by Whereas early ketamine exposure did not affect CAT levels in
Tukey’s Multiple Comparison post hoc test, F (3,12) = 7.715, the PFC of adult mice, increased levels of this antioxidant enzyme
p < 0.05 and p < 0.01). were detected in ketamine + celastrol-treated animals compared
No significant differences in GSH levels were detected between to ketamine groups (Figure 3C, One way ANOVA, followed
early ketamine and saline-treated animals. Increased amount by Tukey’s Multiple Comparison post hoc test, F (3,13) = 5.095,
of this enzyme was observed in celastrol-treated animals with p < 0.05). Levels of this enzyme were enhanced following early
respect to the saline and ketamine groups and in animals exposed administration of indomethacin compared to saline or ketamine-
to ketamine + celastrol compared to ketamine and celastrol- treated mice and this was prevented by ketamine + indomethacin
treated mice (Figure 3B, One way ANOVA, followed by Tukey’s injection (Figure 4C, One way ANOVA, followed by Tukey’s
Multiple Comparison post hoc test, F (3,16) = 29.89, p < 0.01, and Multiple Comparison post hoc test, F (3,15) = 7.200, p < 0.05 and
p < 0.001). Administration of indomethacin in early postnatal p < 0.01).

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FIGURE 2 | Effects of early indomethacin administration on ROS production

and MDA levels in the PFC of adult mice, administered with ketamine in
postnatal life. (A) ROS production (µmol DCF/mg of tissue) in the PFC of 10
weeks mice receiving saline (Sal, n = 4) or ketamine (Ket, n = 4) or
indomethacin (Ind, n = 5) or ketamine + indomethacin (Ket + Ind, n = 4) at
PNDs 7, 9, and 11. One way ANOVA, followed by Tukey’s Multiple
Comparison post hoc test, F (3,13) = 9.642, *p < 0.05 Ket vs Sal,
### p < 0.001 Ket + Ind vs Ket. (B) MDA levels (nmol/mg tissue) in the PFC of

10 weeks mice receiving saline (Sal, n = 5) or ketamine (Ket, n = 8) or FIGURE 3 | Effects of early celastrol administration on SOD1, GSH, and CAT
indomethacin (Ind, n = 4) or ketamine + indomethacin (Ket + Ind, n = 5) at levels in the PFC of adult mice, administered with ketamine in postnatal life.
PNDs 7, 9, and 11. One way ANOVA, followed by Tukey’s Multiple (A) SOD1 levels (ng/ml) in the PFC of 10 weeks mice receiving saline (Sal,
Comparison post hoc test, F (3,18) = 35.10, **p < 0.01 Ket vs Sal, n = 5) or ketamine (Ket, n = 5) or celastrol (Cel, n = 4) or ketamine + celastrol
***p < 0.001 Ind vs Sal, ### p < 0.001 Ind vs Ket and Ket + Ind vs Ket, (Ket + Cel, n = 4) at PNDs 7, 9, and 11. One way ANOVA, followed by Tukey’s
◦◦ p < 0.01 Ket + Ind vs Ind. Multiple Comparison post hoc test, F (3,14) = 5.318, *p < 0.05 Ket + Cel vs
Sal, # p < 0.05 Ket + Cel vs Ket. (B) GSH levels (µg/ml) in the PFC of
10 weeks mice receiving saline (Sal, n = 3) or ketamine (Ket, n = 7) or celastrol
(Cel, n = 3) or ketamine + celastrol (Ket + Cel, n = 7) at PNDs 7, 9, and 11.
One way ANOVA, followed by Tukey’s Multiple Comparison post hoc test,
Effects of Early Celastrol or F (3,16) = 29.89, ***p < 0.001 Cel vs Sal, ### p < 0.001 Cel vs Ket and
Indomethacin Administration on Ket + Cel vs Ket, §§ p < 0.01 Ket + Cel vs Cel. (C) CAT levels (pg/ml) in the
PFC of 10 weeks mice receiving saline (Sal, n = 4) or ketamine (Ket, n = 5) or
Pro-inflammatory Cytokines in the PFC celastrol (Cel, n = 3) or ketamine + celastrol (Ket + Cel, n = 5) at PNDs 7, 9,
of Adult Mice Treated With Ketamine in and 11. One way ANOVA, followed by Tukey’s Multiple Comparison post hoc
test, F (3,13) = 5.095, # p < 0.05 Ket + Cel vs Ket.
Postnatal Life
To evaluate possible effects of early celastrol or indomethacin
administration on pro-inflammatory cytokines following
ketamine exposure in postnatal life, we quantified levels of PNDs 7, 9, and 11 resulted in TNF-A elevations, which were
TNF-A, IL-1ß, and IL-6 in the PFC of adult mice. Whereas no prevented by early celastrol administration (Figure 5A, One way
significant differences in TNF-A amount was detected among ANOVA, followed by Tukey’s Multiple Comparison post hoc
saline and celastrol treatments, ketamine administration at test, F (3,14) = 4.708, p < 0.05). Indomethacin, both per se and

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FIGURE 5 | Effects of early celastrol administration on TNF-A and IL-1ß levels

FIGURE 4 | Effects of early indomethacin administration on SOD1, GSH, and in the PFC of adult mice, administered with ketamine in postnatal life.
CAT levels in the PFC of adult mice, administered with ketamine in postnatal (A) TNF-A levels (pg/ml) in the PFC of 10 weeks mice receiving saline (Sal,
life. (A) SOD1 levels (ng/ml) in the PFC of 10 weeks mice receiving saline (Sal, n = 4) or ketamine (Ket, n = 5) or celastrol (Cel, n = 4) or ketamine + celastrol
n = 5) or ketamine (Ket, n = 5) or indomethacin (Ind, n = 3) or (Ket + Cel, n = 5) at PNDs 7, 9, and 11. One way ANOVA, followed by Tukey’s
ketamine + indomethacin (Ket + Ind, n = 3). One way ANOVA, followed by Multiple Comparison post hoc test, F (3,14) = 4.708; *p < 0.05 Ket vs Sal;
Tukey’s Multiple Comparison post hoc test, F (3,12) = 7.715, *p < 0.05 Ind vs # p < 0.05 Ket + Cel vs Ket. (B) IL-1ß levels (pg/ml) in the PFC of 10 weeks

Sal, ## p < 0.01 Ind vs Ket. (B) GSH levels (µg/ml) in the PFC of 10 weeks mice receiving saline (Sal, n = 3) or ketamine (Ket, n = 4) or celastrol (Cel,
mice receiving saline (Sal, n = 3) or ketamine (Ket, n = 7) or indomethacin (Ind, n = 3) or ketamine + celastrol (Ket + Cel, n = 4) at PNDs 7, 9, and 11. One
n = 5) or ketamine + indomethacin (Ket + Ind, n = 5). One way ANOVA, way ANOVA, followed by Tukey’s Multiple Comparison post hoc test,
followed by Tukey’s Multiple Comparison post hoc test, F (3,16) = 25.59, F (3,10) = 5538, ***p < 0.001 Ket, Cel and Ket + Cel vs Sal; ### p < 0.001 Cel
***p < 0.001 Ind vs Sal, **p < 0.01 Ket + Ind vs Sal, ### p < 0.001 Ind and and Ket + Cel vs Ket. (C) IL-6 levels (pg/ml) in the PFC of 10 weeks mice
Ket + Ind vs Ket. (C) CAT levels (pg/ml) in the PFC of 10 weeks mice receiving receiving saline (Sal, n = 5) or ketamine (Ket, n = 5) or celastrol (Cel, n = 3) or
saline (Sal, n = 4) or ketamine (Ket, n = 5) or indomethacin (Ind, n = 5) or ketamine + celastrol (Ket + Cel, n = 5) at PNDs 7, 9, and 11. One way
ketamine + indomethacin (Ket + Ind, n = 5). One way ANOVA, followed by ANOVA, followed by Tukey’s Multiple Comparison post hoc test,
Tukey’s Multiple Comparison post hoc test, F (3,15) = 7.200, *p < 0.05 Ind vs F (3,14) = 20.08, *p < 0.05 Ket vs Sal; **p < 0.01 Cel vs Sal, ### p < 0.001
Sal,## p < 0.01 Ind vs Ket, p < 0.05 Ind + Ket vs Ind. Cel vs Ket, §§§ p < 0.001 Ket + Cel vs Cel.

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concomitantly administered with ketamine, was able to decrease

TNF-A levels compared to controls and ketamine-exposed mice
(Figure 6A, One way ANOVA, followed by Tukey’s Multiple
Comparison post hoc test, F (3,13) = 45.20, p < 0.05, and
p < 0.001).
Cortical levels of IL-1ß in adult mice were enhanced following
ketamine administration in early life compared to saline-treated
animals. Celastrol treatment was able to significantly decrease
amount of these pro-inflammatory cytokines in the PFC and
this was also observed when it was administered with ketamine
(Figure 5B, One way ANOVA, followed by Tukey’s Multiple
Comparison post hoc test, F (3,10) = 5538, p < 0.001). Despite
IL-1ß amount was significantly lowered by indomethacin per
se compared to both saline and ketamine-treated mice, levels
of this cytokine following early ketamine + indomethacin
administration were comparable to the ones detected in the
saline group, although still significantly decreased with respect to
ketamine-exposed mice (Figure 6B, One way ANOVA, followed
by Tukey’s Multiple Comparison post hoc test, F (3,11) = 39.92,
p < 0.01, and p < 0.001).
Early ketamine treatment resulted in increased IL-6 levels
in the PFC of adult mice. Pups that received only celastrol or
indomethacin showed, at adulthood, a significant decrease of this
cytokine compared to both saline and ketamine-administered
animals. However, in mice concomitantly injected with ketamine,
no significant differences were detected compared to animals that
received saline or ketamine in early life (Figure 5C, One way
ANOVA, followed by Tukey’s Multiple Comparison post hoc test,
F (3,14) = 20.08, p < 0.05, p < 0.01, and p < 0.001 and Figure 6C,
One way ANOVA, followed by Tukey’s Multiple Comparison
post hoc test, F (3,13) = 18.41, p < 0.05, p < 0.01, and p < 0.001).

DISCUSSION
In this work, we demonstrated that administration of
subanesthetic doses of ketamine at PNDs 7, 9, and 11 caused
increased ROS production in the PFC of adult mice. Supporting FIGURE 6 | Effects of early indomethacin administration on TNF-A and IL-1ß
levels in the PFC of adult mice, administered with ketamine in postnatal life.
these findings, previous observations, obtained on the same
(A) TNF-A levels (pg/ml) in the PFC of 10 weeks mice receiving saline (Sal,
animal model, showed both early and persistent increased levels n = 4) or ketamine (Ket, n = 5) or indomethacin (Ind, n = 4) or
of 8-hydroxydeoxyguanosine (8OHdG), an indirect marker of ketamine + indomethacin (Ket + Ind, n = 4) at PNDs 7, 9, and 11. One way
oxidative stress, and NOX2, a ROS-producing enzyme, in the ANOVA, followed by Tukey’s Multiple Comparison post hoc test,
same brain region (Schiavone et al., 2020). Accordingly, it has F (3,13) = 45.20, *p < 0.05 Ket vs Sal, ***p < 0.001 Ind and Ket + Ind vs Sal,
### p < 0.001 Ind and Ket + Ind vs Ket. (B) IL-1ß levels (pg/ml) in the PFC of
been reported that, although oxidative stress contributes to the
10 weeks mice receiving saline (Sal, n = 3) or ketamine (Ket, n = 4) or
physiological postnatal brain development in rodents, the effects indomethacin (Ind, n = 4) or ketamine + indomethacin (Ket + Ind, n = 4) at
of increased ROS levels on the CNS, following an external insult, PNDs 7, 9, and 11. One way ANOVA, followed by Tukey’s Multiple
might be revealed later in life (Wilhelm et al., 2016). Moreover, Comparison post hoc test, F (3,11) = 39.92, **p < 0.01 Ket and Ind vs Sal,
### p < 0.001 Ind and Ket + Ind vs Ket. (C) IL-6 levels (pg/ml) in the PFC of
antioxidant treatment with N-acetyl cysteine in mice could
10 weeks mice receiving saline (Sal, n = 5) or ketamine (Ket, n = 5) or
prevent cognitive and behavioral dysfunctions at adulthood, indomethacin (Ind, n = 4) or ketamine + indomethacin (Ket + Ind, n = 3) at
resulting from ketamine administration at PNDs 7, 9, and 11 PNDs 7, 9, and 11. One way ANOVA, followed by Tukey’s Multiple
(Phensy et al., 2017). Comparison post hoc test, F (3,13) = 18.41, *p < 0.05 Ket vs Sal, **p < 0.01
Here, we also observed enhanced lipid peroxidation after Ind vs Sal, ### p < 0.001 Ind vs Ket, ◦◦ p < 0.001 Ket + Ind vs Ind.
early ketamine exposure. In good agreement with this finding,
ketamine-induced increase in MDA content in the cortex of
young rodents, associated with elevations in levels of indirect have been described during early postnatal development, with
markers of oxidative stress were previously reported (Cheung a physiological decrease in adult animals (Galkina et al., 2009).
and Yew, 2019). Interestingly, changes in brain lipid peroxidation Indeed, during the neonatal period, brain has been reported

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to have low peroxidation potential corresponding to the by ketamine. Indeed, it might be hypothesized that celastrol
rapid phase of cell proliferation (Pushpendran et al., 1994). administration may inhibit ketamine-induced increase in NOX2
Dysfunctions of this process, induced by an external trigger, expression observed in the PFC of mice pups and, consequently,
such as the exposure to NMDA-R antagonists, have been the persistent elevations of this enzyme in adult life (Schiavone
shown to result in the persistence of high levels of lipid et al., 2020). On the other side of the redox balance, celastrol
peroxidation at adulthood, contributing to the development of exposure in the early phases of postnatal life might result in the
psychotic-like neuropathological and behavioral dysfunctions recovery of the physiological role of the antioxidant system in
in rodents (de Carvalho Cartágenes et al., 2019), as well as response to ketamine-induced oxidative stress elevations. Hence,
neuropsychiatric disorders in humans (Joshi and Pratico, 2014; ketamine-induced increase of ROS production and consequent
Romano et al., 2017). lipid peroxidation might be prevented by the synergistic action
An important finding of our study consists in the lack of these two hypothesized mechanisms.
of significant differences in cortical amounts of antioxidant We also showed that early administration of indomethacin
enzymes between early ketamine-treated mice and controls. per se could decrease MDA levels in the PFC of adult mice
This result might appear contrasting with preclinical evidence treated with ketamine in early life. This finding might appear in
describing, instead, a decreased activity of SOD and CAT in contrast with a previous report showing an aggravation of lipid
the PFC of ketamine-treated rats (de Oliveira et al., 2009), peroxidation, in terms of increased MDA levels, in newborn rats
as well as a reduction of GSH concentration (Abdel-Salam with hypoxic-ischemic cerebral injury following indomethacin
et al., 2015). However, in these previously published studies, administration (Taskin et al., 2009). However, the result obtained
ketamine exposure occurred in adult life. Furthermore, the lack in our experimental conditions might be explained by both the
of differences in levels of antioxidant enzymes observed in our different time point at which this parameter (adult life) was
experimental conditions might be also interpreted as a long- evaluated and the kind of insult impacting on the developing
term dysfunction, induced by early ketamine exposure, of the brain. Indeed, in this regard, previous evidence reported a
physiological roles of the antioxidant system in controlling ROS beneficial effect of indomethacin in reducing peripheral and
damage and in regulating ROS signaling (Wang et al., 2018). central MDA levels following rat exposure to other neurotoxic
Here, we also demonstrated that early celastrol treatment insult, such as CCl4 (Kadiiska et al., 2005). Moreover, cortical
prevented ketamine-induced increased lipid peroxidation increase of lipid peroxidation, induced in rats by an infectious
and ROS production in the PFC of adult mice. Different insult, was also found to be reduced following intraperitoneal
mechanisms of action have been proposed to describe celastrol indomethacin administration (Guzman et al., 2018). However,
pharmacological effects. Among these, the induction of the we cannot totally exclude that indomethacin per se could induce
expression of neuroprotective factors, the block of ROS-induced an increase in MDA levels in the early phases of postnatal
cellular apoptosis and the decrease of oxidative stress have been life, thus stimulating in the developing brain the activation of
reported (Chen et al., 2018). In particular, the reduction of neuroprotective mechanisms that may result in the decreased
oxidative stress also occurs via the inhibition of the NADPH lipid peroxidation we observed in adult mice.
oxidase NOX enzymes with an increased potency against NOX1 Interestingly, indomethacin per se induced elevations in
and NOX2 isoforms, resulting in the lack of the functional levels of all the considered antioxidant enzymes compared
association between the cytosolic subunits and the membrane to both controls and early ketamine-exposed mice. Thus,
flavocytochrome of these enzymes (Jaquet et al., 2011; Tarafdar it might be hypothesized that increased antioxidant defense
and Pula, 2018). The effect of celastrol on ketamine-induced might be a possible mechanism underlying indomethacin effects
increase in MDA and ROS levels observed in the present study on cortical lipid peroxidation. Supporting our hypothesis,
is in line with previous evidence obtained on the same animal COX inhibition has been reported to significantly improve
model, where celastrol administration in postnatal life was found antioxidant defense both at peripheral and central levels (Kumar
to prevent ketamine-induced elevations in cerebellar expression et al., 2011; Ahmed et al., 2014). Concomitant administration
of 8OHdG, a marker of oxidative damage to DNA, and of the of ketamine and indomethacin could prevent the cortical
ROS producing enzyme NADPH oxidase NOX1 (Schiavone increase in ROS amount and in MDA levels induced by
et al., 2019). Intriguingly, in our experimental conditions, the early ketamine exposure, suggesting a neuroprotective role of
reduction of cortical ROS amount and lipid peroxidation induced this compound against the impact that a neurodetrimental
by early celastrol administration was accompanied by increased insult might have on the developing brain. In support of
levels of SOD1, GSH and CAT with respect to mice receiving only this hypothesis, indomethacin has been reported to prevent
ketamine in postnatal life. Supporting our hypothesis, previous the loss of neurogenesis markers following a neurotoxic
preclinical evidence have reported that celastrol could attenuate insult, i.e., ethanol in adolescent rodents (Vetreno et al.,
oxidative damage by increasing levels and activity of SOD, GSH, 2018). Furthermore, indomethacin was able to regulate the
glutathione peroxidase, glutathione reductase, and CAT (Shaker peripheral expression of neurotrophins, such as BDNF and
et al., 2014; Wang et al., 2014; Boran et al., 2019; Gao et al., 2020). NGF (Kemi et al., 2006; Hochstrasser et al., 2013). Hence,
Although still speculative, the effects of celastrol on cortical it can be hypothesized that this might also happen at
ROS levels and lipid peroxidation, at least in our experimental central level following an early neurotoxic insult affecting CNS
conditions, might be explained by a concomitant action of this development. Moreover, clinical evidence reported the use of
compound on ROS production and their degradation, finally indomethacin as neuroprotective strategy to prevent the later
resulting in the recovery of the redox balance, early altered consequences of neonatal brain injury (Favrais et al., 2014),

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via the strengthening of the immature blood-brain barrier promotion of postnatal murine CNS development, most likely
(Sims, 2012). being perturbations in its levels the cause of long-lasting and
In this manuscript, it is also showed that ketamine irreversible damage (Storer et al., 2018). However, we cannot
administration in early life stages caused an enhancement of totally exclude that a possible effects of celastrol or indomethacin
proinflammatory cytokines, i.e., TNF-α, IL-1ß, and IL-6, at on ketamine-induced dysfunctions of IL-6 levels might have
adulthood. In good agreement with our findings, previous been detected at different time points from ketamine exposure,
preclinical and clinical reports highlighted a crucial role of early such as during mice adolescence or later than 10 weeks of
and persistent cortical neuroinflammation in the development life. Further investigations are certainly needed in this sense,
of psychotic-like symptoms in rodents (Schiavone et al., 2017; also considering the physiological link existing between IL-6
Ben-Azu et al., 2019; Kogan et al., 2019) as well as of and anti-inflammatory cytokines (Ropelle et al., 2010), as
schizophrenia in humans (Zhang et al., 2016; Barron et al., well as the role of the pro-inflammatory/anti-inflammatory
2017). Importantly, it has been reported that dysregulation of the balance in neurodevelopmental-related mental disturbances
redox, immune and glutamatergic systems, induced by NMDA-R (Ratnayake et al., 2013).
antagonists, including ketamine, especially when it occurs during With respect to a possible translation of the results of the
brain development, represents a “central hub” in schizophrenia present study to clinics, a limitation consists in the fact that, in
pathophysiology (Steullet et al., 2016). In line with this concept, animals, pharmacological treatments were initiated at the same
in the same animal model, together with increased levels of pro- time of ketamine administration. Indeed, this same therapeutic
inflammatory cytokines, we do observe increased cortical ROS strategy cannot be directly translated into the clinical setting,
amount and lipid peroxidation and we previously demonstrated because of the impossibility to identify in humans the exact time
early and persistent increase of oxidative damage to DNA as well of the neurotoxic insult.
as alterations of NADPH expression in the same brain region In conclusion, our data suggest that both the enhancement
(Schiavone et al., 2020). of antioxidant defense, reducing cerebral oxidative stress and
In our experimental conditions, together with an effect inhibition of inflammatory pathways, may represent a suitable
of celastrol per se on IL-1ß and of indomethacin per se therapeutic approach preventing psychotic-like disturbances
on TNF-α and IL-1ß, we also found that early celastrol or resulting from the impact of neurotoxic insult during crucial
indomethacin administrations were able to prevent ketamine- phases of brain maturation.
induced elevations in TNF-α and IL-1ß in PFC of adult mice,
suggesting a possible protective role of these two compounds
against the possible long-lasting detrimental effects exerted by DATA AVAILABILITY STATEMENT
early neuroinflammation on the developing brain (Franceschini
The raw data supporting the conclusions of this article will be
and Zusso, 2019). This result should also be considered in the
made available by the authors, without undue reservation.
light of previous evidence showing a reduction of microglia
activation following celastrol (Dai et al., 2019) or indomethacin
(Lopes et al., 2016) administration, as well as a strict ETHICS STATEMENT
interrelation between TNF-α and IL-1ß and redox dysregulation
in CNS disorders (Fischer and Maier, 2015; Schiavone and The animal study was reviewed and approved by the Italian
Trabace, 2017). Indeed, it might be hypothesized that ketamine Ministry of Health (approval number 679/2017-PR, protocol
administration in postnatal life may cause microglia activation, n. B2EF8.17).
with consequent release of TNF-α and IL-1ß which, in turn,
induce ROS production, further sustaining neuroinflammation
and neuronal damage. In addition, the possible inhibition of AUTHOR CONTRIBUTIONS
ketamine-induced enhancement of microglial NADPH oxidase
MB, PT, LT, SS, and MGM designed the research. MB, PT, SD,
NOX2 by celastrol administration might also play a key role
SS, and MGM performed the research. MB, PT, SS, and MGM
in this process. Hence, in this regard, it has been highlighted
analyzed the data. MB, PT, LT, and SS wrote the manuscript. MB,
that NOX2 activation in microglia exerts neurotoxic effects via
PT, SD, LT, SS, and MGM revised the manuscript. All authors
extracellular ROS production as well as the initiation of microglia
contributed to the article and approved the submitted version.
redox signaling, finally resulting in the amplification of the pro-
inflammatory response (Surace and Block, 2012).
Despite the decrease in cortical IL-6 levels detected following FUNDING
celastrol or indomethacin treatments alone compared to both
controls and ketamine-exposed mice, these two compounds, This work was supported by PRIN 2017 code 2017AY8BP4 to
administered concomitantly with ketamine, could not prevent SS from Italian Ministry of Education, University and Research
elevations of this pro-inflammatory cytokine in postnatal life. (MIUR), PRIN 2017 code 2017YZF7MA to LT from MIUR,
This result should be considered in the light of the physiological and Zardi Gori Foundation Fellowship 2019 to MB. The open
expression of IL-6 and its receptor in rodent cortex during access fee was paid with a contribution from 5 × 1000 IRPEF
postnatal development (Gadient and Otten, 1994), as well funds in favour of the University of Foggia, in memory of
as of the central role reported for this cytokine in the Gianluca Montel.

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 ORIGINAL RESEARCH
published: 12 November 2020
doi: 10.3389/fnbeh.2020.581819

Oculomotor Behavior as a Biomarker

for Differentiating Pediatric Patients
With Mild Traumatic Brain Injury and
Age Matched Controls
Melissa Hunfalvay 1 , Nicholas P. Murray 2* , Claire-Marie Roberts 3 , Ankur Tyagi 1 ,
Kyle William Barclay 4 and Frederick Robert Carrick 5,6,7
1
RightEye, LLC, Bethesda, MD, United States, 2 Department of Kinesiology, East Carolina University, Greensville, NC,
United States, 3 Health and Social Sciences, University of the West of England, Bristol, United Kingdom, 4 Case Western
Reserve University, Cleveland, OH, United States, 5 Centre for Mental Health Research in Association with University
of Cambridge, Cambridge, United Kingdom, 6 College of Medicine, University of Central Florida, Orlando, FL, United States,
7
MGH Institute of Health Professions, Boston, MA, United States

Edited by: Importance: Children have the highest incidence of mild traumatic brain injury (mTBI)
Sophie Laye, in the United States. However, mTBI, specifically pediatric patients with mTBI, are
INRA Bordeaux-Aquitaine, France
notoriously difficult to detect, and with a reliance on traditional, subjective measurements
Reviewed by:
Hoonkyo Suh,
of eye movements, the subtle but key oculomotor deficits are often missed.
Cleveland Clinic, United States
Objective: The purpose of this project is to determine if the combined measurement
Andrew David Greenhalgh,
The University of Manchester, of saccades, smooth pursuit, fixations and reaction time represent a biomarker for
United Kingdom differentiating pediatric patients with mild traumatic brain injury compared to age
*Correspondence: matched controls.
Nicholas P. Murray
[email protected] Design: This study used cross-sectional design. Each participant took part in a suite of
tests collectively labeled the “Brain Health EyeQ” to measure saccades, smooth pursuit,
Specialty section:
This article was submitted to fixations and reaction time.
Pathological Conditions,
a section of the journal Participants: The present study recruited 231 participants – 91 clinically diagnosed
Frontiers in Behavioral Neuroscience with a single incident mTBI in the last 2 days as assessed by both the Glasgow Coma
Received: 09 July 2020 Scale (GCS) and Graded Symptoms Checklist (GSC), and 140 age and gender-matched
Accepted: 07 October 2020
Published: 12 November 2020
controls (n = 165 male, n = 66 female, M age = 14.20, SD = 2.78).
Citation: Results: One-way univariate analyses of variance examined the differences in
Hunfalvay M, Murray NP,
performance on the tests between participants with mTBI and controls. ROC curve
Roberts C-M, Tyagi A, Barclay KW
and Carrick FR (2020) Oculomotor analysis examined the sensitivity and specificity of the tests. Results indicated that
Behavior as a Biomarker together, the “Brain Health EyeQ” tests were successfully able to identify participants
for Differentiating Pediatric Patients
With Mild Traumatic Brain Injury
with mTBI 75.3% of the time, providing further validation to a growing body of literature
and Age Matched Controls. supporting the use of eye tracking technology for mTBI identification and diagnosis.
Front. Behav. Neurosci. 14:581819.
doi: 10.3389/fnbeh.2020.581819 Keywords: eye-tracking, oculomotor, mTBI, concussion, pediateric case

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Hunfalvay et al. Oculomotor Behavior of mTBI and Controls

INTRODUCTION sensory stimulus and the response to it (Land and Tatler, 2009;
Leigh and Zee, 2015; Lange et al., 2018). Each of these different
Mild traumatic brain injury (mTBI) occurs about once every eye movements activates different parts of the brain (Wong, 2007;
15 s, and the excessive frequency of these injuries costs the Møllenbach et al., 2013).
United States more than $77 billion dollars annually (Langlois The Saccadic system focuses on the rapid movements of the
et al., 2004; Prins et al., 2013). Ninety percent of TBI’s are fovea between fixation points (Wong, 2007). Several different
classified as mild (Langlois et al., 2004; Howell et al., 2018). brain structures are involved in the regulation of saccades,
Clinical diagnosis of mTBI is determined by the American including the brain stem, pons, midbrain, and cerebral cortex
Congress of Rehabilitation Medicine (ACRM) definition in which (Wong, 2007). Burst neuron circuits in the brainstem are
“a patient with a mTBI is a person who has had a traumatically responsible for the motor signals that control the extraocular
induced physiological disruption of brain function, as induced by muscles in the eyes that generate saccades (Wong, 2007). There
one of the following: a loss of consciousness, any memory loss, is a division of labor between the pons and the midbrain, with the
any alteration of mental state, and/or focal neurological deficits pons primarily involved in generating horizontal saccades and
(Bazarian et al., 2005).” the midbrain primarily involved in generating vertical saccades
Pediatric head injury is extremely common (Schunk and (Wong, 2007). In addition, because eye movements are closely
Schutzman, 2012). mTBI is the most common form of head related to cognitive behaviors in higher mammals, the cerebral
injury accounting for 75–85% of these injuries (Goldstein and cortex also plays an important role in the function of saccades
Levin, 1987). Children have the highest incidence of mTBI. both directly through the burst neuron circuit, and via the
In the United States, mTBI occurs in 692 of 100,000 children superior colliculus (Wong, 2007).
younger than 15 years of age (Guerrero et al., 2000). Identification The smooth pursuit system is what allows humans to
of pediatric mTBI differs from adult mTBI due to age-related predictively track moving objects (Wong, 2007; Møllenbach
anatomical and physiological differences, pattern of injuries et al., 2013). Because the complete smooth pursuit pathway is
based on the physical ability of the child, and difficulty in so complex, it is not yet completely understood (Wong, 2007).
neurological evaluation in children (Araki et al., 2017). Evidence First, visual information is relayed from the striate cortex to
suggests that children exhibit a specific pathological response the extrastriate areas, which contain specialized neurons that
to TBI with distinct accompanying neurological symptoms encode both eye and object movement (Wong, 2007). These
(Araki et al., 2017). extrastriate areas have connections to the brain stem, which
An important factor contributing to this epidemic is communicates information to the cerebellum. This explains why
the fact that concussions are often hard to diagnose and researchers have recently found functional similarity between the
therefore treat (Howell et al., 2018). Most symptoms are saccadic and smooth pursuit systems (Wong, 2007). Pursuits are
relatively subjective and easily attributed to other conditions controlled primarily by a network of cortical areas, including
(Howell et al., 2018). Therefore, it is essential to build on the frontal eye field and other structures such as the superior
established means of mTBI detection that are both objective colliculus and basal ganglia (Wong, 2007). Vertical smooth
and reliable (Howell et al., 2018). Currently, there are three pursuits and horizon pursuits have similar pathways differing
accepted branches to mTBI diagnosis: neurological, vestibular, only at a spot in the pons, the y-group, and the cerebellum
and oculomotor (Sussman et al., 2016). In the past, most (Wong, 2007).
of the oculomotor assessment was carried out subjectively Fixations hold a stationary object on the fovea while the
through examination by clinicians, with objective measurements head is not moving and prevent the image from fading (Wong,
of symptoms, rare (Bedell and Stevenson, 2013). Research 2007; Leigh and Zee, 2015). This process is active and involves
suggests that subjective measurements of eye movements are a network of brain regions, including the parietal eye field,
more likely to miss subtle deficits, which makes the need for V5 and V5A areas, supplemental eye field, and dorsolateral
reliable, objective symptom detection increasingly important. prefrontal cortex (Wong, 2007). The brain stem and part of the
One uniquely powerful method of objectively measuring eye basal ganglia and the superior colliculus are involved, although
movements can be achieved through eye-tracking technology specific functions are not localized to one area. Instead, they are
(Bedell and Stevenson, 2013). Eye-tracking can be used to study distributed across several (Munoz, 2002; Wong, 2007). Fixations
neurological function, oculomotor assessment and can detect operate like a simple negative feedback loop in which the drifting
abnormalities in neurocircuitry and map oculomotor dysfunction movements of the eye (not the actual target) trigger the tracking
to damaged sites (Bedell and Stevenson, 2013; Lai et al., 2013; mechanism to return the eye to the target (Leigh and Zee, 2015).
Johnson et al., 2015). This behavior explains the constant microsaccades characteristic
Oculomotor assessment can be further divided into the of fixations; it’s simply the gaze repeatedly returning to the target
measurement of four specific types of eye movements. These (Leigh and Zee, 2015).
include saccades, smooth pursuits, fixations, and reaction time Reaction time (RT) is a measure of attention (Zomeren and
(Land and Tatler, 2009; Leigh and Zee, 2015; Lange et al., 2018). Brouwer, 1994). However, the applications of RT assessment are
Saccades are short and fast eye movements between fixed points; much more numerous than just measuring attention. RT has
smooth pursuits use predictive tracking to stabilize moving been found in numerous studies to be a marker of CNS damage
targets, fixations are even smaller movements that focus an image and neuropathology, including mTBI (Knopman, 1991; Murtha
on the fovea, and reaction time is the time elapsed between a et al., 2002; Lange et al., 2018). RT can also be used to evaluate

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a person’s motor skill or to determine how well they interact them. Several studies exist that consider the impact mTBI has
with their environment. RT itself is the time elapsed between the on reaction time (MacFlynn et al., 1984; Hetherington et al.,
presentation of stimuli and the behavioral response (Shelton and 1996; Hugenholtz et al., 1998). mTBI patients have been found
Kumar, 2010). RT assessments can be split up into simple reaction to have reduced processing speed as it relates to reaction time,
time (SRT), choice reaction time (CRT) and discriminate reaction along with increased reaction time overall (Suh et al., 2006b;
time (DRT) (Lange et al., 2018). SRT is a single response to a Lange et al., 2018).
single stimulus, CRT is multiple responses to multiple stimuli Between the four eye-movements being considered, there are
and DRT is a single response to one of the multiple stimuli a plethora of studies the look at the impact of mTBI, however,
(Lange et al., 2018). Traditional measurements of RT often fail to none exist that consider all these components together. Nor is
account for eye-specific RT metrics, including saccadic latency, there much research conducted specifically on the oculomotor
visual speed, and visual processing speed (Lange et al., 2018). behavior of pediatric patients with mTBI. Nevertheless, these
Eye-tracking does measure these values, and this greater level metrics can distinguish between mTBI and Controls, and so
of detail provides valuable information during RT assessment it stands to reason that all together, they represent a superior
(Lange et al., 2018). method of mTBI detection. Of the four factors considered,
Currently, pediatric mTBIs are diagnosed using a variety fixations especially are in need of more research. Further
of measures such as level of consciousness and length of investigation is also necessary to determine how the four metrics
post-traumatic amnesia (Maruta et al., 2010; Levin and Diaz- interact with each other, and how the combined ability to
Arrastia, 2015). The Glasgow Coma Score (GCS) is commonly distinguish mTBI differs from the individual capacities. The
used to evaluate consciousness on a 13–15 scale for mTBI purpose of this study was to compare Brain Health EyeQ
that accounts for a motor response, verbal response, and eye- score (a composite of saccades, smooth pursuits, fixations, and
opening ability (Arbour et al., 2016). However, the GCS is reaction time) of pediatric patients with clinically diagnosed
widely used but not necessarily the best measure of pediatric mTBI and age matched controls. A secondary purpose was to
mTBI (Ghaffarpasand et al., 2013). Furthermore, clinicians do examine the reaction time responses in a choice and discriminate
not usually use imagining for pediatric mTBI cases (Oakes, reaction time task.
2018). Therefore, The Graded Symptoms Checklist (GSC)
in the Standardized Assessment of Concussion (SAC) was
also used as a secondary clinical tool for measurement of MATERIALS AND METHODS
mTBI as recommended by the Journal of the American
Medical Association Pediatrics clinical guidelines (Adjorlolo, Participants
2018; Lumba-Brown et al., 2018a,b). Though numerous, current Data from two-hundred and thirty-one participants were
methods of concussion detection are often subjective or lacking in analyzed. One hundred and sixteen were clinically diagnosed
their oculomotor components (Ventura et al., 2015). Eye tracking as having a mTBI within 2 days of the assessment. Twenty-five
is capable of delivering precise and objective measurements to of these participants were excluded (see procedure), leaving 91
assist in mTBI diagnosis, and this is why it is so important to total participants with mTBI. One-hundred and forty participants
consider (Komogortsev and Karpov, 2013). were age and gender matched controls. Participants were between
Compromised saccades, smooth pursuits, fixations, and the ages of 6–18 years (M = 14.20, SD = 2.78); 165 were males
reaction time have all been linked to mTBI. Numerous studies (71.4%), 66 were females (28.6%). Of the 231 participants, 68.8%
have found compromised saccades in patients with mTBI such were White, 3.0% were Hispanic, 0.4% were Asians, 7.4% were
as prolonged latencies and directional errors on memory- Black, and 20.4% opted not to report ethnicity. The groups were
guided and antisaccades tasks and impaired self-paced saccades matched by age (see Table 1).
(Williams et al., 1997; Heitger et al., 2002; Johnson et al., 2015;
DiCesare et al., 2017). Both vertical and horizontal saccades Clinical Diagnosis of mTBI for Pediatric Patients
have been shown to differ in patients with mTBI, and saccades All participants had been clinically assessed by Board Certified
of patients with mTBI have been found especially deficient neurologists with at least 5 years’ experience in diagnosing
under conditions of high cognitive load (Ettenhofer et al., TBIs. Clinical diagnosis of mTBI was based on the American
2018; Hunfalvay et al., 2019). Several studies have also found Congress of Rehabilitation Medicine (ACRM) definition of mTBI
deficits in smooth pursuits in patients with mTBI (Heitger (Mild Traumatic Brain Injury Committee, 1993). All participants
et al., 2009; Hoffer et al., 2017). Patients with mTBI have been were examined using the GCS and scored between 13 and
shown to have both reduced prediction and more position 15 on the scale. However, the GCS is widely used but not
errors (Suh et al., 2006a,b; Armstrong, 2018). mTBI patients
have also been found to have increased error and variability in
TABLE 1 | Demographic data by age and gender.
gaze position and reduced smooth pursuit velocity in tracking
tests (Maruta et al., 2014). Another study found that fixational Group (n) Mean Age (±SD) Females Males
errors for mTBI patients were abnormally high with evidence
Control (140) 14.31 (2.48) 39 101
of increased drift, saccadic intrusions, and nystagmus (Ciuffreda
mTBI (91) 14.13 (2.97) 27 64
et al., 2004). Though fixations do not have as much focus in
current literature, this is only further reason to continue to study n, Number; SD, Standard Deviation.

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necessarily the best measure of pediatric mTBI (Ghaffarpasand Reaction Time Tasks
et al., 2013). Furthermore, clinicians do not usually use imagining In addition to the BHEQ, we examined separately Choice
for pediatric mTBI cases (Oakes, 2018). Therefore, The Graded Reaction Time (CRT) and Discriminate Reaction Time (DRT; see
Symptoms Checklist (GSC) in the Standardized Assessment of Lange et al., 2018 for further details). In brief, the CRT test, the
Concussion (SAC) was also used as a secondary clinical tool participant viewed three stimuli and was asked to provide one
for measurement of mTBI as recommended by the Journal of of three responses. In the DRT test, the participant viewed three
the American Medical Association Pediatrics clinical guidelines stimuli and was required to respond to only one stimulus.
(Lumba-Brown et al., 2018a,b). Using results from Grubenhoff
et al. (2010) and the American Academy of Neurology concussion Procedure
grading scale pediatric patients (6–18 years of age) were evaluated Participants were recruited through RightEye clinical providers.
as having mTBI if their GSC score was between 7.7 and 19.3 (Kelly The study was conducted in accordance with the tenets of the
et al., 1991; Grubenhoff et al., 2010). According to Grubenhoff Declaration of Helsinki. The study protocols were approved
et al. (2010) this yielded a 95% confidence interval for case- by the Institutional Review Board of East Carolina University.
patients with an AAN grade 1 TBI (7.7–10.7) or grade 2 TBI The nature of the study was explained to the participants
(11.5–19.3) (Grubenhoff et al., 2010). Therefore, participants in and all participants provided written consent to participate.
the mTBI group in this study scored between 13–15 on the GCS Participants were excluded from the study they had more than
and 7.7–19.3 on the GSC. a single discrete episode of mTBI (n = 21). Following informed
consent, participants were asked to complete a prescreening
Apparatus questionnaire and an acuity vision screening where they were
The RightEye tests were presented on a Tobii I15 vision 1500 required to identify four shapes at 4 mm in diameter. If any of
monitor fitted with a Tobii 90 Hz remote eye tracker and a the prescreening questions were answered positively and any of
Logitech (model Y-R0017) wireless keyboard and mouse. The the vision screening shapes were not correctly identified, then
participants were seated in a stationary (non-wheeled) chair that the participant was excluded from the study (n = 3). Participants
could not be adjusted in height. They sat in front of a desk in were excluded from the study if they reported any of the following
a quiet, private room. Participants’ heads were unconstrained. conditions, which may have prevented successful test calibration
The accuracy of the Tobii eye tracker was 0.4◦ within the during the prescreening process: this included vision-related
desired headbox of 32 cm × 21 cm at 56 cm from the screen. issues such as extreme tropias, phorias, static visual acuity of
For standardization of testing, participants were asked to sit >20/400, nystagmus, cataracts or eyelash impediments or if they
in front of the eye-tracking system at a distance of 56 cm had consumed drugs or alcohol within 24 h of testing (n = 1)
(ideal positioning within the virtual headbox range of the (Han et al., 2010; Holmqvist and Nystrom, 2011; Renard et al.,
eye tracker). 2015; Kooiker et al., 2016; Niehorster et al., 2017). Participants
were also excluded if they were unable to pass a nine-point
The Brain Health EyeQ Score (BHEQ) calibration sequence. Less than 1% of the participants fell into
The Brain Health EyeQ Score (BHEQ) includes a combination of these categories.
saccade, pursuit, fixation and simple reaction time oculomotor Qualified participants who successfully passed the nine-
variables. A total of 58 metrics make-up the testing model. point calibration sequence completed the eye-tracking tests. The
Weights range from 0.1 to 13% across metrics. More about the calibration sequence required participants to fixate one at a time
individual tests and metrics can be found in published papers on nine points displayed on the screen. The participants had to
mentioned above (Lange et al., 2018; Hunfalvay et al., 2019; successfully fixate on at least eight out of nine points on the screen
Murray et al., 2019). The metrics associated with the BHEQ score to pass the calibration sequence. Written instructions on screen
all passed reliability standards (Murray et al., 2019). Extreme and animations were provided before each test to demonstrate
gradient boosting (XGB) was used for the classification task appropriate behavior required in each of the tests. The testing
using the Rworker GitHub repository R language version 3.5.2. lasted less than 5 min to complete.
The efficacy of the model was evaluated using accuracy of
classification. This model also outputs the importance (weights) Data Analysis
that each variable has on the classification accuracy. These The differences in the groups (control, mTBI) were analyzed
weights were then applied to the respective metrics (variables) on clinically verified data using JMP PRO 14.0 (SAS Institute,
to calculate the percentile value of a participant compared to Cary, NC, United States). The comparison was evaluated using
his/her peers within the same age group. The percentiles are then one-way univariate ANOVAs on the Brain Health EyeQ score,
aggregated over all metrics that collapse into specific tests to Choice Reaction Time measures (saccadic latency, visual speed,
calculate overall scores and percentile on that test; for example, processing speed, and reaction time), and Discriminate RT
all metrics that create circular smooth pursuit (CSP), horizontal measures (saccadic latency, visual speed, processing speed, and
smooth pursuit (HSP), and visual smooth pursuit (VSP) tests reaction time). The alpha level was set at p < 0.05 and
were used to calculate overall percentile and score for the test. Omega squared (ω2 ) was used to determine effect size. In
Results revealed pursuit test weighting 60.93% (CSP: 8.4%; HSP: addition, a series of ROC curve analysis were plotted for the
40.4%; VSP: 12.13%); self-paced saccade test weighted 24.95% Oculomotor variables. Significant area under the curve (AUC)
(horizontal saccade (HS): 15.57%; vertical saccade (VS): 9.38%); with 95% confidence intervals (p < 0.05) was used to indicate
and fixation test contributed 14.2% weighting of the model. the ability of each variable to differentiate concussed participants

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from non-concussed. We set our criteria for a satisfactorily

accurate area under the curve (AUC) to the standard of least of
0.7 (Adjorlolo, 2018). We calculated cut-off points, sensitivity,
specificity, and positive and negative predictive value (PPV
and NPV, respectively) for each significant AUC. Optimal cut-
off points were determined by visually assessing which score
combines maximum sensitivity and specificity.

RESULTS
The ANOVA results for Brain Health EyeQ Score demonstrated FIGURE 2 | Dot Graph and probability curve for Control group (blue) and TBI
a significant main effect for Group [F(1,229) = 21.906; p < 0.001, group (red).
ω2 = 0.89]. The data revealed a significant difference between
mTBI group (M = 53.98, SD = 20.75) and the Control group
(M = 67.52, SD = 21.92; Figure 1). Further a logistic regression Discriminate Reaction Time (DRT)
analysis was conducted to evaluate how well the criterion variable The ANOVA results for Discrimination Reaction Time
BHEQ predicted mTBI status (see Figure 2). The mTBI status test demonstrated a significant main effect for Saccade
was significantly related to the BHEQ, χ2 = 27.31; p < 0.0001, Latency [F(1,226) = 9.483; p < 0.01, ω2 = 0.35]
Nagelkerke R2 = 0.185. and Processing Speed [F(1,219) = 15.63; p < 0.001,
ω2 = 0.62]. Similar to Choice Reaction Time test, both
Choice Reaction Time (CRT) Visual Processing Speed [F(1,226) = 3.544; p = 0.061,
The ANOVA results for Choice Reaction Time test demonstrated ω2 = 0.011] and Reaction Time [F(1,218) = 0.164;
a significant main effect for Saccade Latency [F(1,229) = 19.53; p = 0.686, ω2 = 0.004] did not differentiate between mTBI
p < 0.001, ω2 = 0.074] and processing speed [F(1,226) = 4.17; and Control groups in the Discriminate Reaction Time
p < 0.05, ω2 = 0.44]. Further, we examined Visual Speed test (Table 3).
[F(1, 226) = 0.182; p = 0.670, ω2 = −0.003] and Reaction
Time [F(1,224) = 0.342; p = 0.559, ω2 = 0.003] which ROC Curve Analysis
demonstrated non-significant differences between Control and Among the RightEye variables, ROC curves were significant
mTBI groups (Table 2). (p < 0.0001) for Brain Health EyeQ score; DRT Saccade

FIGURE 1 | Mean Differences (with standard error) comparing BHEQ score between mTB and Control.

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TABLE 2 | Mean and standard deviation for choice reaction time variables.

Group (n) Saccade latency* Processing speed* Visual speed Reaction time

Control 364.95 (139.83) 609.44 (227.56) 149.01 (143.20) 1123.93 (383.98)

mTBI 288.35 (109.41) 669.91 (203.61) 141.10 (126.54) 1095.77 (304.76)

*p < 0.05.

TABLE 3 | Mean and standard deviation for discriminate reaction time variables.

Group (n) Saccade latency* Processing speed* Visual speed Reaction time

Control 336.81 (108.39) 379.39 (152.68) 142.32 (154.34) 856.98 (290.43)

mTBI 286.62 (136.58) 478.01 (218.24) 106.46 (117.56) 873.75 (316.35)

*p < 0.001.

TABLE 4 | Summarization of outcomes at the ROC curve analysis including: area under the curve (AUC) with standard error (S.E.), p values; cut-off points; sensitivity and
specificity percentages; positive and negative predictive values (PPV and NPV), respectively.

Variables AUC S.E. p Cut-off Sensitivity Specificity PPV NPV

BHEQ 0.704* 0.00618 0.0001 63 75.3% 68.0% 73.7% 81.2%

BHEQ subscale analysis
Fixation Stability 0.640 0.1346 0.0003 5.11 66.3% 67.8% 57.2% 75.4%
Horizontal Saccade Efficiency 0.560 0.0263 0.2691 7.31 59.0% 86.8% 30.6% 33.1%
Vertical Saccade Efficiency 0.597 0.210 0.0688 5.27 85.5% 81.1% 40.3% 65.1%
CSP Saccade percentage 0.68 0.273 0.0027 4.29 84.5% 71.8% 43.3% 73.3%
VSP Saccade percentage 0.55 0.018 0.2218 5.10 57.2% 11.5% 29.8% 31.5%
HSP Saccade percentage 0.42 0.17 0.698 18.45 98.6% 94.5% 40.3% 85.3%
Reaction Time Tasks
DRT Saccade Latency 0.724* 0.00170 0.0039 259 58.8 % 86.4% 75.0% 75.2%
DRT Processing Speed 0.692* 0.00093 0.0004 365 73.2 % 60.7% 76.3% 76.6%
CRT Saccade Latency 0.716* 0.00138 0.0001 248 53.6% 91.4% 81.3% 74.0%
CRT Processing Speed 0.623 0.00062 0.045 578 64.9% 55.7% 70.4% 69.6%

*Represents an acceptable probability that the test differentiates mTBI from no TBI.
Cut-off points (or thresholds) distinguish between a “positive” and a “negative” mTBI result and represents maximum balance between sensitivity and specificity
within each test.
Sensitivity represents confidence that a person has a mTBI or the true positive rate and specificity represents the accuracy of the test or the true negatives.

Latency, DRT Processing Speed, CRT Saccade Latency, CRT detects such differences by examining all the major oculomotor
Processing Speed CRT (Table 4 and Figure 3). ROC curves behaviors (fixations, pursuits, and saccades). Furthermore, the
were not significant or produced low AUC score for the BHEQ score showed a significant 0.7 AUC with a sensitivity
remaining DRT and CRT variables (Reaction Time and of 75.3%. These scores indicate that the BHEQ score has a
Visual Speed). balance of sensitivity and specificity and represents the ability
to discriminate whether a specific condition is present or not
present. It is important to note the sensitivity and specificity
DISCUSSION are based on determining appropriate cut-off points which
distinguish between a “positive” and a “negative” outcome. We
The purpose of this article was to examine the oculomotor utilized our data to determine these appropriate cut-scores,
behavior of pediatric patients with clinically diagnosed mTBI however, with lower cut-off scores based on minimal clinically
versus controls. This was done using a combination of saccade, important differences would result in better sensitivity and
pursuit, fixation and reaction time oculomotor variables that specificity in the measure. Furthermore, BHEQ did better overall
together made up a BHEQ Score. Results revealed a significant considering AUC, p-value, sensitivity, and specificity of the
difference between groups, with the mTBI group showing sub-measures including pursuit test, self-paced saccade test,
lower (poorer) oculomotor behavior than the control group. and fixation test and the BHEQ score has more precision in
A mean difference of 13.54% (67.52–53.98) was found. This distinguishing those with mTBI and without mTBI.
result shows that oculomotor behavior of those with mTBI is It is well known that independent tests, such as saccades
poorer, as they scored lower than those of the control group. It tests show differences between those with mTBI and those
also shows that the BHEQ linear combination score effectively without (Hunfalvay et al., 2019). The same is true for

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FIGURE 3 | Receiver Operator Characteristic analysis predicting mTBI Status for all significant variables.

pursuit eye movements (Suh et al., 2006b). However, to show a high specificity 86.4 and 91.4%, respectively. Furthermore,
date, there has not been one combination score of all the they showed high positive predictive values (75.0 and 81.3%).
major eye movements that a clinician can review as part DRT and CRT Processing Speed showed high sensitivity 73.2
of the clinical workflow to determine if there is a global and 64.9%, respectively. Taken together, these metrics indicate a
oculomotor difference for a patient compared to an age matched high predictive value, sensitivity and specificity for differentiating
control. One global score, one standard of reference in clinical patients with and without mTBI. Such results further validate
practice, is an important benchmark for which to determine if the use of eye movements as a biomarker for identification of
further, more in-depth examination is required. Furthermore, mTBI. Limitations of this study include an unequal distribution
the RightEye test only require 5 min to complete the test of males and females in the sample populations. Past research has
and are not impacted by acute eye fatigue during the test found conflicting evidence of gender differences in mTBI groups
(Murray et al., 2019). (Farace and Alves, 2000; Brickell et al., 2017) and future research
A secondary purpose of this article was to examine choice is needed. A second limitation is that 24.7% of cases that are
and discriminate reaction time tests and associated oculomotor potentially missed. However, mTBI describes a broad term that
variables between the two groups. Two variables, saccadic latency, describes a vast array of injuries and this test indicates visual
and processing speed were found to be significantly different in motor impairment due to mTBI. Potentially, the missed cases
both the CRT and DRT test. mTBI group had faster saccadic are result from other symptoms or impairments and additional
latency and slower processing speed than the Control group. measures are needed to account for the diversity of mTBI
This is consistent with past research where saccadic latency and especially in pediatric patients. A third limitation is the limited
processing speed where found to show differences between mTBI age group of pediatric patients only. Lastly, very nature of mTBI
versus controls and mTBI versus athlete groups (Lange et al., is complicated injury with completed tautology.
2018). Interestingly the previous research showed much larger This study was the first to examine a combined Brain
standard deviations even with a larger sample size (N = 651) Health EyeQ score in mTBI pediatric patients. Future research
compared to the current research (N = 91). It is possible that should examine adults, specifically those over 65 who are the
the 10-day time limit for mTBI patients in the current study second largest group of persons who incur mTBIs and is
reduced the variability in results. Nevertheless, the same results describe as the “silent epidemic” in older adults according to
were replicated. Both CRT and DRT Saccadic Latency values Thompson et al. (2006). In conclusion, the results of this study

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show that (a) oculomotor behavior differs between pediatric ETHICS STATEMENT
patients with mTBI and age matched controls; (b) the BHEQ
score, that combines the major categories of oculomotor The studies involving human participants were reviewed and
behavior, differentiates pediatric patients with mTBI from approved by East Carolina University IRB. Written informed
controls, and (c) the CRT and DRT tests results were replicated consent to participate in this study was provided by the
from past research supporting the need for RT to be part of a participants’ legal guardian/next of kin.
mTBI assessment (Lange et al., 2018).

DATA AVAILABILITY STATEMENT AUTHOR CONTRIBUTIONS

The raw data supporting the conclusions of this article will be All authors listed have made a substantial, direct and intellectual
made available by the authors, without undue reservation. contribution to the work, and approved it for publication.

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05695.x
Mild Traumatic Brain Injury Committee (1993). Definition of mild traumatic brain Conflict of Interest: MH is Co-founder and Chief Science Officer of RightEye,
injury. J. Head Trauma Rehabil. 8, 86–87. headquartered in Bethesda, MD, United States. However, MH did not collect or
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of digitized eye tracking tests. Vis. Dev. Rehabil. 5, 269–284. doi: 10.31707/ conflict of interest.
vdr2019.5.4.p269
Murtha, S., Cismaru, R., Waechter, R., and Chertkow, H. (2002). Increased Copyright © 2020 Hunfalvay, Murray, Roberts, Tyagi, Barclay and Carrick. This
variability accompanies frontal lobe damage in dementia. J. Int. Neuropsychol. is an open-access article distributed under the terms of the Creative Commons
Soc. 8, 360–372. doi: 10.1017/s1355617702813170 Attribution License (CC BY). The use, distribution or reproduction in other forums
Niehorster, D. C., Cornelissen, T. H. W., Holmqvist, K., Hooge, I. T. C., and is permitted, provided the original author(s) and the copyright owner(s) are credited
Hessels, R. S. (2017). What to expect from your remote eye-tracker when and that the original publication in this journal is cited, in accordance with accepted
participants are unrestrained. Behav. Res. Methods 50, 1–15. doi: 10.1016/j.cps. academic practice. No use, distribution or reproduction is permitted which does not
2009.07.003 comply with these terms.

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 ORIGINAL RESEARCH
published: 08 January 2021
doi: 10.3389/fnbeh.2020.609660

Folic Acid Fortification Prevents

Morphological and Behavioral
Consequences of X-Ray Exposure
During Neurulation
Edited by: Kai Craenen 1,2† , Mieke Verslegers 1*† , Zsuzsanna Callaerts-Vegh 3† , Livine Craeghs 1,2† ,
Sophie Laye, Jasmine Buset 1 , Kristof Govaerts 4† , Mieke Neefs 1 , Willy Gsell 4† , Sarah Baatout 1† ,
INRA Centre
Rudi D’Hooge 3† , Uwe Himmelreich 4† , Lieve Moons 1,2† and
Bordeaux-Aquitaine, France
Mohammed Abderrafi Benotmane 1†
Reviewed by:
1
Chul Hoon Kim, Radiobiology Unit, Interdisciplinary Biosciences, Institute for Environment, Health and Safety, Belgian Nuclear Research
Yonsei University, South Korea Centre (Studiecentrum voor Kernenergie; Centre d’étude de l’énergie nucléaire), Mol, Belgium, 2 Laboratory of Neural Circuit
Thiago C. Moulin, Development and Regeneration, Animal Physiology and Neurobiology Section, Department of Biology, Faculty of Science,
Uppsala University, Sweden Katholieke Universiteit Leuven, Leuven, Belgium, 3 Laboratory of Biological Psychology, Faculty of Psychology and
Educational Sciences, Katholieke Universiteit Leuven, Leuven, Belgium, 4 Molecular Small Animal Imaging Center, Biomedical
*Correspondence:
MRI Unit, Department of Imaging and Pathology, Katholieke Universiteit Leuven, Leuven, Belgium
Mieke Verslegers
[email protected]

† ORCID: Previous studies suggested a causal link between pre-natal exposure to ionizing
Kai Craenen radiation and birth defects such as microphthalmos and exencephaly. In mice, these
orcid.org/0000-0001-6368-6394
defects arise primarily after high-dose X-irradiation during early neurulation. However,
Mieke Verslegers
orcid.org/0000-0001-8616-234X the impact of sublethal (low) X-ray doses during this early developmental time window
Zsuzsanna Callaerts-Vegh on adult behavior and morphology of central nervous system structures is not known.
orcid.org/0000-0001-9091-2078
Livine Craeghs
In addition, the efficacy of folic acid (FA) in preventing radiation-induced birth defects
orcid.org/0000-0003-0441-799X and persistent radiation-induced anomalies has remained unexplored. To assess the
Kristof Govaerts
efficacy of FA in preventing radiation-induced defects, pregnant C57BL6/J mice were
orcid.org/0000-0002-7106-7566
Willy Gsell X-irradiated at embryonic day (E)7.5 and were fed FA-fortified food. FA partially prevented
orcid.org/0000-0001-7334-6107 radiation-induced (1.0 Gy) anophthalmos, exencephaly and gastroschisis at E18, and
Sarah Baatout
orcid.org/0000-0001-8110-751X
reduced the number of pre-natal deaths, fetal weight loss and defects in the cervical
Rudi D’Hooge vertebrae resulting from irradiation. Furthermore, FA food fortification counteracted
orcid.org/0000-0003-2243-2106
radiation-induced impairments in vision and olfaction, which were evidenced after
Uwe Himmelreich
orcid.org/0000-0002-2060-8895 exposure to doses ≥0.1 Gy. These findings coincided with the observation of a reduction
Lieve Moons in thickness of the retinal ganglion cell and nerve fiber layer, and a decreased axial
orcid.org/0000-0003-0186-1411
Mohammed Abderrafi Benotmane
length of the eye following exposure to 0.5 Gy. Finally, MRI studies revealed a volumetric
orcid.org/0000-0002-8985-1578 decrease of the hippocampus, striatum, thalamus, midbrain and pons following 0.5 Gy
Specialty section: irradiation, which could be partially ameliorated after FA food fortification. Altogether, our
This article was submitted to study is the first to offer detailed insights into the long-term consequences of X-ray
Pathological Conditions,
a section of the journal exposure during neurulation, and supports the use of FA as a radioprotectant and
Frontiers in Behavioral Neuroscience antiteratogen to counter the detrimental effects of X-ray exposure during this crucial
Received: 23 September 2020 period of gestation.
Accepted: 08 December 2020
Published: 08 January 2021 Keywords: radiation, radioprotectant, folic acid, birth defect, anophthalmos, agnathia, exencephaly, hyposmia

Frontiers in Behavioral Neuroscience | www.frontiersin.org 151

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Craenen et al. Folic Acid Prevents Radiation-Induced Neuropathologies

INTRODUCTION prevent (congenital) morphological and functional defects that

arise from irradiation during brain development.
Exposure to ionizing radiation during embryonic development Folic acid (FA), a synthetic vitamin, is generally known to
has been linked to an increased risk of birth defects. The prevent NTDs [reviewed in Imbard et al. (2013)], in addition
type and severity of these defect are predominantly determined to other defects such as heart defects and some skeletal defects
by the developmental stage during which exposure occurred (Kappen, 2013). Besides, FA has been suggested to prevent
(Craenen et al., 2017). Epidemiological studies on Ukrainian the development of age-related neurodegenerative diseases and
cohorts illustrated an increased prevalence of neural tube defects overall cognition (Craenen et al., 2020a). Several countries
(NTDs) and eye defects (EDs) in regions severely contaminated enforce staple food fortification, whereas others support FA
with radioactive Cs-137 isotopes following the Chernobyl nuclear supplementation during pregnancy (Imbard et al., 2013). Of note
accident. Although there are no accurate dose estimates, uptake is that FA supplementation/fortification initiatives are currently
of radioactive isotopes is known to be particularly high in lacking in high-risk areas, such as those severely contaminated
pregnant women living in these regions (Wertelecki et al., 2016). with radioisotopes from the Chernobyl disaster. Although FA
Initially, it was observed that the more recent Fukushima Daiichi food fortification can prevent some defects such as NTDs, its
nuclear power plant accident elicited no increase in birth defects efficacy depends on the causative teratogens or mutations. For
and pre-natal mortality due to environmental radioisotope example, BMS-189453 (a synthetic retinoid) causes anomalies
contamination (Fujimori et al., 2014), but subsequent papers such as NTDs and heart defects that can be prevented with FA
debated this conclusion (Mangano and Sherman, 2015; Scherb fortification (Cipollone et al., 2009), whereas arsenate-induced
et al., 2016). In contrast to these more recent observations, NTDs do not appear to be responsive (Ferm and Hanlon, 1986).
reports after the atomic bombings in Japan only mentioned an Interestingly, many of the hallmark consequences of ionizing
increased incidence of microcephaly and intellectual disability radiation exposure, including oxidative stress, DNA damage,
(Plummer, 1952; Neel and Schull, 1956). It is likely that the cell cycle arrest, cell death and epigenetic alterations, might be
discrepancy in health effects of the nuclear accidents and atomic countered by FA (Heyer et al., 2000; Martin et al., 2014; Reisz
bombings stems from differences in dose, dose rate, exposure et al., 2014).
duration and radiation type. The above highlights the need to This study is the first to offer an in-depth analysis
increase our knowledge about the effects of pre-natal irradiation of the morphological and behavioral consequences of
on biological structures and functions. irradiation during neurulation in mice. To this end, we
Exposure to ionizing radiation during pregnancy most used a multidisciplinary approach, including an extensive
commonly occurs during clinical radiodiagnostic or therapeutic behavioral test battery and imaging techniques such as spectral
procedures (Mettler et al., 2009). Although medical practitioners domain optical coherence tomography (SD-OCT) and magnetic
advise against irradiation during pregnancy, it may be resonance imaging (MRI). In addition, we assessed the efficacy
unavoidable in medical urgencies (Lazarus et al., 2009). In of FA food fortification in preventing fetal malformations as well
terms of radiation protection, conventional shielding methods as adult functional and morphological defects resulting from
are currently being used to partially mitigate the fetal dose X-ray exposure.
(Chatterson et al., 2014; Moore et al., 2015; Owrangi et al., 2016).
However, depending on the dose or the developmental stage
during which exposure occurs, these conventional shielding
strategies may not suffice. Animal studies have shown that the
MATERIALS AND METHODS
neurulation period in the early embryo is especially radiosensitive Animals and FA Fortification
with regard to the pathogenesis of radiation-induced NTDs and All animal experiments were conducted in line with the relevant
ED (Russell, 1950, 1956; Di Majo et al., 1981; Heyer et al., 2000; guidelines and were approved by the Institutional Ethical
Craenen et al., 2017, 2020b), but also in terms of cognitive Committees of SCK-CEN/VITO (ref. 02–012) and the Animal
disabilities and altered vision. Indeed, a decreased visual acuity Welfare Committee of the KU Leuven University, and are in strict
in atomic-bomb survivors, irradiated in the first trimester, and accordance with the European Communities Council Directive
born from mothers with acute radiation syndrome (≤2 km of 22 September 2010 (2010/63/EU). C57BL6/J mice (Janvier, Bio
from hypocenter) has been reported (Burrow et al., 1964). Services, The Netherlands) were housed in individually ventilated
Yet, most experimental work has focused on health risks after cages, under standard laboratory conditions (12-h light/dark
radiation exposure during neurogenesis, coinciding with the cycle) and fed ad libitum. One week before coupling, animals
second trimester of human pregnancy (Plummer, 1952; Neel and designated for the macroscopic fetal study were placed on a
Schull, 1956; Verreet et al., 2015, 2016a,b). Furthermore, there control Teklad (Carfil Quality, Oud-Turnhout, Belgium) diet (3.5
are currently no anti-teratogens or radio-protectants available to mg/kg FA), a FA fortified diet (8 mg/kg FA) or an extra-FA
fortified diet (12 mg/kg FA). The FA concentrations within the
final customized food products were investigated in compliance
Abbreviations: EPM, Elevated plus maze; E, embryonic day; ED, eye defect; with ISO 17025. We selected the dose of 8 mg/kg because it
FA, folic acid; MRI, magnetic resonance imaging; MWM, Morris water maze;
NF+GCL, nerve fiber + retinal ganglionic cell layer; NTD, neural tube defect; NS,
was observed that this is an effective concentration to achieve
non-social odor; RARE, rapid acquisition relaxation enhancement; RM, repeated antiteratogenic effects in mice (Gray and Ross, 2009; Harris,
measures; S, social odor; SD-OCT, spectral domain optical coherence tomography. 2009). A dose of 12 mg/kg was included based on the assumption

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TABLE 1 | Sample sizes.

Control diet High FA diet High FA diet

(8 mg/kg) (12 mg/kg)

0.0 Gy 0.1 Gy 0.5 Gy 1.0 Gy 0.0 Gy 0.1 Gy 0.5 Gy 1.0 Gy 1.0 Gy

n N n N n N n N n N n N n N n N n N

Macroscopic 126 15 n.a. n.a. 116 18 n.a. n.a. n.a. 114 19 107 16
Skeletal 9 3 n.a. n.a. 9 3 n.a. n.a. n.a. 9 3 9 3
Behavior/OCT 10 4 13 6 12 6 n.a. 12 4 11 6 12 6 n.a. n.a.
MRI 9 4 12 6 5 3 n.a. 5 2 7 5 7 4 n.a. n.a.

N, number of litters; n, number of fetuses.

that some teratogens require higher doses of FA (Gray and Ross,

TABLE 2 | Overview of test order and age at time of testing.
2009; Harris, 2009).
Animals designated for the behavioral tests and MRI were Protocol Age range (weeks)
limited to the control diet or the 8 mg/kg FA diet, and were
kept on their respective diets until they were euthanized. Timed Optokinetic tracking response W5–W7
couplings were performed during a 2-h period at the start of the Optical coherence tomography W5–W7
light phase (7:30 a.m.−9:30 a.m.) to attain synchronous timing Cage activity W7–W9
of embryonic development. The day of coupling was identified Open field W7–W9
as E0. At E7.5, animals were placed in a Plexiglas holder and Social exploration W8–W10
transported to the irradiation installation. Mice intended for Elevated plus maze W8–W10
the macroscopic study were either sham-irradiated or irradiated Accelerating rotarod W8–W10
with 1.0 Gy of X-rays. Animals used for behavioral testing and Odor habituation/dis-habituation W9–W11
MRI were sham-irradiated or received a sub-lethal dose of 0.1 MRI W9–W11
or 0.5 Gy of X-rays at E7.5. Irradiation was performed using an Morris Water Maze W10–W13
X-strahl 320 kV (0.14 Gy/min, inherent filtration: 0.21 mmAl, Passive avoidance W12–W14
additional filtration: 3.8 mm Al + 1.4 mm Cu + DAP, tube
voltage: 250 kV, tube current: 12 mA,) in accordance to ISO
4037. The number of animals used for the macroscopic, skeletal,
behavioral and MRI experiments is depicted in Table 1, unless
habituation/dis-habituation assay. Finally, two tests for memory
otherwise specified.
were included: the Morris water maze (MWM) and passive
Macroscopic Scoring and Skeletal avoidance, to test spatial and fear-related memory, respectively.
Stainings
The dissections, macroscopic scorings and alcian blue/alizarin Optokinetic Tracking Response
red skeletal stainings were performed at E18 as previously Using a virtual-reality chamber (OptoMotry, Cerebral
described (Craenen et al., 2017). For the skeletal analyses, E18 Mechanics, Medicine Hat, AB, Canada), the optokinetic
fetuses were randomly selected from the macroscopic study. The tracking response was assessed (De Groef et al., 2016; Van
axial skeleton was analyzed, with a focus on the vertebrae and the Hove et al., 2016). The animal was placed on the center of an
ribs. A subdivision was made between atlas, cervical, thoracic, elevated platform within the optokinetic installation, where a
lumbar, sacral and caudal vertebrae, whilst also differentiating vertical sine wave pattern was displayed on the monitors. Using a
between true, false and floating ribs and sternum. real-time camera system, visual acuity was scored manually using
a staircase procedure, composed of random spatial frequencies
Behavioral Tests (100% contrast, 12◦ per second speed).
Starting at week (W)5 and ending at W14, behavioral tests were
performed on male mice in the order described below (Table 2).
All experiments were performed under blinded conditions. To Cage Activity
assess visual acuity, optokinetic tracking was performed. We The impact of ionizing radiation exposure on ambulatory
included cage activity to assess global activity, during both light behavior was investigated over a 23 h time-period, starting at 4
and dark-phase, and assessed explorative and social behavior p.m. until 3:30 p.m. the next day (Verreet et al., 2016a). During
with the open field and social exploration tests. The elevated this period, animals were individually housed in transparent
plus maze (EPM) was included to ascertain anxiety, whereas cages (20 × 26 cm) with minimal bedding, chow and water and
the accelerating rotarod was used to identify issues in motility. placed in a laboratory-built activity logger with three infrared
Next, to explore olfactory performance we used the odor beams. Beam breaks were recorded over 30 min time bins.

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Open Field and Social Exploration Morris Water Maze

To assess exploration and social interaction, a transparent In order to assess whether FA and sub-lethal pre-natal doses of
Plexiglas arena (50 × 50 cm) was used (Stroobants et al., 2008; X-rays during neurulation affected adult spatial learning, MWM
Bollen et al., 2015; Callaerts-Vegh et al., 2015). The arena was was performed. Animals were tested in a circular pool (diameter
homogenously illuminated and equipped with an Any-maze 150 cm, height 30 cm), filled with opacified non-toxic water
(Dublin, Ireland) tracking system. For the open field test, animals as previously described (Latif-Hernandez et al., 2016; Verreet
were placed in the empty arena for 1 min of acclimatization, et al., 2016a). For the acquisition trials, a see-through acrylic
immediately followed by a 10 min test phase with active tracking. platform was consistently placed in the same quadrant, 1 cm
The social exploration experiment was identical to the open field below the water surface. The pool was located in the center of a
test, except that in the center of the arena a small cage with two homogeneously-lit room, with invariable visual cues. Acquisition
same-sex strange mice was placed. training was performed over a period of 5 days, followed by a 2-
day resting period, followed again by 5 days of training. During
Elevated Plus Maze each training day, every mouse was subjected to four trials.
In order to investigate anxiety, animals were subjected to EPM The trial interval was approximately 15-min and the quadrant-
testing as was previously described (Verreet et al., 2015). The starting positions varied in a semi-random order for every trial.
cross-shaped EPM consisted of two perpendicular open and If the animal was unable to find the platform within 120 s, it was
closed arms (21 × 5 cm). Five infrared detectors were installed placed on the platform for 10 s and subsequently removed from
on the EPM: 2 at the exits out of the closed arms and two at the basin. On day 5 of the acquisition trials and 2 days after
the entrances to the open arms (entries/exits) and one along the the last acquisition trial, probe trials were performed. During
length of the open arms (time spent on the open section). The these probe trials, the platform was removed from the basin
animal was placed in a closed arm and after 1 min of adaptation, and mice were subjected to a single probe trial of 100 s, where
beam breaks were recorded for 10 min. the starting position was opposite to the target quadrant. Using
an automated video capture and tracking system (EthoVision,
Noldus, The Netherlands), various parameters such as trajectory
Accelerating Rotarod and swim speed were recorded. We observed floating behavior
General motor function and balance following in utero X-ray (swim velocity <5 cm/s, more than 30 s per swim) in all groups,
exposure during neurulation were assessed using an accelerating except the control diet + 0.0 Gy group. However, for the path
rotarod (Ugo Basile, Italy), as was described previously (Verreet length analysis to determine if the animals covered the same track
et al., 2015). Initially, the animals underwent two adaptation trials during learning, we included all animals due to the low animal
(2 min each), each at a constant speed of 4 rpm. In turn, the numbers per group. Non-responders (floating >35% of test time)
mouse was subjected to four subsequent test trials, where during were excluded for the reference memory test. As such, a reduced
each 5 min trial the rotation speed gradually increased from 4 to number of animals was included for the reference memory test,
40 rpm. Latency was recorded when the mouse lost its footing as compared to Table 1. More specifically, for this analysis in
and fell off the rotating beam. particular we included under control diet condition 29 animals
(9 for 0.0 Gy, 11 for 0.1 Gy and 9 for 0.5 Gy), and 27 under high
Odor Habituation and Dis-Habituation FA condition (10 for 0.0 Gy, 9 for 0.1 Gy and 8 for 0.5 Gy).
To assess the interaction of mice with olfactory cues, i.e.,
habituation and dis-habituation, the animals were subjected to an Passive Avoidance
odor discrimination test as was described previously (Yang and We investigated fear-aggravated learning and memory using a
Crawley, 2009; Yang et al., 2012; Arbuckle et al., 2015). Animals passive avoidance set-up (Lo et al., 2013). Animals were placed
were individually placed in a fresh cage with a small amount in a brightly lit compartment and the door leading into a dark
of bedding, followed by 30 min of acclimatization with a dry adjacent chamber was opened after 5 s. Latency to enter the dark
cotton swab fixed to the cover grid (tip ∼5 cm from bottom). chamber was timed starting immediately after opening of the
Next, the animals were exposed to a sequence of 15 subsequent dark chamber and stopped when the animal had all four paws
odor exposures (2 min each): Three trials with water, three trials on the electric grid in the dark room. Next, the door separating
with grape (non-social odor 1 = NS1), three trials with banana the two compartments was closed and a shock (0.3 mA, 2 s) was
(NS2), three trials with social odor one (S1) and three trials administered. The next day, the procedure was repeated, albeit
with social odor two (S2). For the preparation of the NS odor without the administration of an electric shock.
tests, respectively 1:100 diluted grape extract (SAFC, W26820-8-
K methyl anthranilate ≥98%) and 1:100 diluted banana extract In vivo Imaging
(Acros Organics, AC269481000 n-Butyl propionate >99%) on Optical Coherence Tomography
cotton tips was used. For the S odors, cotton tips were dipped To assess retinal development and thickness, SD-OCT was
in water and moved in a cross-pattern through the bedding of used as was previously discussed (Van Hove et al., 2016). The
soiled cages of same-sex mice. During each trial, sniffing-time animal was anesthetized by intraperitoneal (ip) injection of 75
was recorded manually whenever the subject’s nose was within mg/kg body weight ketamine (Anesketin, Eurovet, Bladel, The
a 2 cm radius of the cotton swab. The inter-session interval never Netherlands) and 1 mg/kg medetomidine (Domitor, Pfizer, NY,
exceeded 2 min. USA). Shortly before imaging, pupils were dilated using topical

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Craenen et al. Folic Acid Prevents Radiation-Induced Neuropathologies

0.5% tropicamide (0.5% Tropicol, Thea Pharma, Wetteren, comparisons) post-hoc tests. To assess dishabituation, paired t-
Belgium). Next, SD-OCT was performed using an Envisu testing was done, whilst two-way ANOVA + Sidak was utilized
R2210 (Bioptigen, Morrisville, NC, USA) via 100 serial B- to investigate habituation. To perform inter-dose and inter-
scan lines with each line consisting of 1,000 A-scans, in a diet comparisons, one-way ANOVA + Dunnet was used in
1.4 × 1.4 mm field. Afterwards, ip injection of atipamezol (1 conjunction with the first trial of the different odors. For all
mg/kg, Antisedan, Pfizer) was applied to reverse the anesthesia. statistical tests, a p-value of 0.05 was considered statistically
Thickness of the retina was investigated using InVivoVue Diver significant. All values are represented as mean ± SEM.
software (Bioptigen).
RESULTS
Magnetic Resonance Imaging
For MRI we used female mice, which originated from the FA Reduces the Prevalence of
same litters as the behavioral test mice. When the female mice Radiation-Induced Anophthalmos,
were on average W10, in vivo MR imaging of the brain was Exencephaly and Agnathia
performed using a 7 T Bruker Biospec 70/30 MRI scanner (30 cm First, we examined the prevalence of radiation-induced EDs
horizontal bore with actively shielded gradients (200 mT/m), and the prevention thereof with FA fortification. The prevalence
Bruker Biospin, Ettlingen, Germany). All data were acquired of left-eye anophthalmos (Figure 1A), microphthalmos
using a quadrature volume coil (72 mm internal diameter, (Figure 1B) and iris anomaly (Figure 1C) was significantly
transmit, actively decoupled) in combination with a dedicated increased following X-irradiation (respectively, 28.26 ± 4.72,
mouse brain surface receive coil (Bruker Biospin). To obtain 23.56 ± 4.28, and 17.92 ± 3.39 %). In contrast, X-irradiation
high resolution 3D images of the entire mouse brain, image did not affect the prevalence of the left eye open phenotype
acquisition and animal anesthesia was performed similar to (3.03 ± 1.40%) (Figure 1D). Similar observations were made
previously described experiments (Verreet et al., 2016a). In brief, for the right eye (respectively 42.41 ± 5.93, 29.32 ± 3.93, and
after the acquisition of localizer scans morphological 3D MR 18.59 ± 4.35%) (Figures 1E–H). Here, the right eye also showed
imaging was performed using a rapid acquisition relaxation an increase of the open phenotype (4.55 ± 1.89%). Of interest,
enhancement (RARE) T2-weighted sequence with a RARE factor we revealed a partial prevention of radiation-induced left-eye
of 16 and a repetition time and echo time of 1,000 ms and anophthalmos with both the 8 mg/kg FA (9.02 ± 3.40%) and 12
67 ms, respectively. The field of view was 24 × 15 × 8.3 mm mg/kg FA (10.62 ± 2.74%) diets (Figure 1A). No such rescue
with a matrix of 256 × 160 × 88, resulting in an isotropic was observed for the right eye (Figure 1E).
resolution of 94 µm. The total acquisition time was 16 min. The In addition, we determined the number of fetuses with
methodology of image post-processing and the labeled template exencephaly, agnathia, gastroschisis and cleft palate. X-
was based on previously published work (Verreet et al., 2016a). irradiation increased the prevalence of exencephaly (15.26 ±
Briefly, we first corrected for image intensity inhomogeneity 3.95%) and agnathia (17.88 ± 4.17%) when the animals were fed
using the N4 bias field correction algorithm (Tustison et al., the control diet, whilst 8 and 12 mg/kg FA provided significant
2010) using an in-house developed MeVislab pipeline (MeVis prevention of both exencephaly (respectively, 4.89 ± 2.10 and
Medical Solutions, Germany). Images were affinely registered 4.43 ± 2.03%) (Figure 2A) and agnathia (respectively, 5.41 ±
to the template used in Verreet et al. (2015, 2016a) to obtain 1.86 and 1.56 ± 1.56%) (Figure 2B). Furthermore, irradiation
brain masks for each animal, which were isotropically dilated also increased the number of fetuses affected by gastroschisis in
by 2 voxels. These brain masks were applied to the raw data mothers on the control diet (11.3 ± 2.83%), but here no rescue
and the bias field correction was repeated. Finally, images were was observed with the FA fortified diets (Figure 2C). Finally,
non-rigidly registered to the template using the Fast Free-Form X-ray exposure at E7.5 did not affect the occurrence of cleft
Deformation algorithm implemented in Niftyreg (Modat et al., palate in the fetuses, regardless of the diet (Figure 2D).
2010). Template labels were propagated to the individual study
images using the transformations obtained from this step, and
quantified using an in-house developed Python script (Python
FA Counteracts the Effects of X-Ray
2.7, Python Software Foundation). Exposure on Pre-natal Survival
In the next part of our study, we investigated the impact of
X-irradiation during neurulation on the number of implants,
Statistics pre-natal deaths and fetal weight. Neither X-irradiation nor
Statistical analyses were performed with GraphPad Prism 7.02 FA fortification affected the total number of conceptuses per
(GraphPad Software, San Diego, CA, USA). To analyze the pregnant female (Figure 3A). In terms of late fetal deaths (E18
data on the macroscopic and skeletal defects, the Kruskal-Wallis fetuses with no signs of life), an increase was observed after
methodology was used, in combination with Dunn’s post-hoc irradiation in mothers on the control diet, while this increase
testing. Data on pre-natal viability were assessed using two- was prevented with 8 and 12 mg/kg FA diets (Figure 3B).
way ANOVA and Dunnet testing for multiple comparisons. Furthermore, we found an increase in resorptions (implantation
For most behavioral tests, MRI and SD-OCT, we used two- site at E18, which holds no developed fetus, and shows evident
way ANOVA (with pairing where required) in combination with embryonic-stage death) after 1.0 Gy irradiation, with a notable
Dunnet (inter-dose comparisons) and (Holm-)Sidak (inter-diet rescue after 8 mg/kg, but not after 12 mg/kg FA fortification

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FIGURE 1 | Prevalence of various eye defects observed at E18, following 1.0 Gy X-ray exposure at E7.5, and prevention with FA fortification. Radiation significantly
increased the risk for left eye anophthalmos; a defect that could in turn be prevented by both FA fortified diets (8 mg/kg and 12 mg/kg FA) (A). Although both left eye
(Continued)

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Craenen et al. Folic Acid Prevents Radiation-Induced Neuropathologies

FIGURE 1 | microphthalmos (B) and iris anomaly (C) were induced by X-irradiation, no rescue effect of FA was observed on these phenotypes. The left eye open
phenotype was not increased in prevalence following irradiation (D). Although defects of the right eye, including anophthalmos (E), microphthalmos (F), iris anomaly
(G) and open eye (H) were more prevalent following irradiation, we observed no significant prevention of these defects by FA. Data are represented as mean ± SEM,
*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

FIGURE 2 | Prevalence of radiation-induced birth defects at E18, affecting the head and abdomen, and the prevention with FA. Irradiation at E7.5 significantly
increased the risk for exencephaly (A), agnathia (B) and gastroschisis (C). The first two of these radiation-induced defects could be partially prevented by FA food
fortification (both 8 and 12 mg/kg) (A,B). Radiation had only minimal impact on the prevalence of cleft palate (D). Data are represented as mean ± SEM, *p ≤ 0.05,
**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

(Figure 3C). Finally, irradiation resulted in a marked fetal weight excessive cartilage (Figures 4B,C). At the thoracic level, the
loss at E18 (Figure 3D), which was not rescued following FA most common vertebral defects included fusions and excessive
fortification. Of note, sham-irradiated fetuses gained weight cartilage, whereas ribs were often missing (Figures 4D,E). Here,
when placed on the 12 mg/kg FA diet, as compared to sham- we also observed impaired ossification of the ribs (Figure 4F)
irradiated animals on the control diet (Figure 3D). Altogether, and split ossification centers within the vertebrae (Figure 4G).
we were able to demonstrate a preventive role of FA for radiation- Of note, irradiation also increased the incidence of a tilted
induced late fetal deaths and resorptions. sternum (Figures 4H,I). To a lesser extent, radiation lead to
tilted vertebrae, displaced ribs, hooked (i.e., bent) ribs and
Axial Skeletal Defects and Prevention With short-length ribs (Supplementary Figures 1A–C). 8 mg/kg FA
FA fortification prevented the occurrence of radiation-induced
To assess general teratogenicity of X-ray exposure on the axial defects in the cervical region, whilst the 12 mg/kg diet group
skeleton, alcian blue/alizarin red staining was utilized, a common also showed a strong trend (henceforth defined as P = 0.05–0.08)
methodology to assess the sub-macroscopic teratogenicity of toward prevention (Figure 4, cervical vertebrae). Surprisingly, a
chemical and physical agents (Young et al., 2000). X-irradiation combination of 8 mg/kg FA and 1.0 Gy increased the number of
at E7.5 increased the number of defects within the vertebrae, defects within the caudal vertebrae, as compared to the 1.0 Gy
specifically in the atlas, the cervical vertebrae and the thoracal irradiated animals that were fed the control diet (Figure 4A,
vertebrae when the animals were fed the control diet (Figure 4A, caudal vertebrae). Furthermore, a trend was observed for the
atlas, cervical and thoracal vertebrae). Within the cervical rescue of defects within the true and false ribs following
region, radiation primarily resulted in fused vertebrae and fortification with the 8 mg/kg and 12 mg/kg diets (Figure 4A

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FIGURE 3 | Impact of 1.0 Gy X-ray exposure at E7.5 and FA on the number of conceptuses (A), late fetal deaths (B), resorptions (C) and fetal weight (D). Neither
radiation nor FA diet (8 and 12 mg/kg FA) had an impact on the number of conceptuses at E18 per litter (A). Irradiation strongly increased the rate of late fetal deaths
when mothers were fed the control diet, whilst FA fortification prevented this (B). A significant increase in resorptions was observed for irradiated mice on the control
diet, which was in turn prevented only by the 12 mg/kg FA diet (C). FA fortification significantly increased fetal weight, whilst irradiation decreased fetal weight (D).
Data are represented as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

true and false ribs). Overall, FA fortification partially prevented (Figure 5B), midbrain [F (2, 37) = 4.654; P = 0.0157] (Figure 5E)
skeletal defects within the cervical and thoracic vertebrae, whilst and the pons [F (2, 37) = 3.792; P = 0.0318] (Figure 5F), which
a trend toward prevention could be observed within the true and supports an FA-dependent rescue of radiation-induced size
false ribs. decrease. Furthermore, X-irradiation resulted in a trend toward
a volumetric decrease of the posterior cerebral cortex [F (2, 37) =
2.731; P = 0.0783] (Figure 5G) and the basal ganglia [F (2, 37)
Abnormal Adult Brain Morphology = 2.768; P = 0.0758] (Figure 5H). Unexpectedly, FA food
Following Pre-natal X-Ray Exposure fortification reduced the size of the basal ganglia [F (1, 37) = 4.961;
We performed volumetric MRI analyses to assess whether the P = 0.0321) (Figure 5H) and the striatum [F (1, 37) = 7.067; P =
adult brain is structurally affected following irradiation at E7.5. 0.0115] (Figure 5C). A trend toward FA-induced size decrease
Here we also assessed whether FA fortification could prevent was also observed for the anterior commissure [F (1, 37) = 3.796;
any radiation-induced anomalies with inclusion of the 8 mg/kg P = 0.0590] (Figure 5I).
FA diet, which was based on the rescue effect we observed
in view of the radiation-induced fetal defects. We observed
no differences in the volumes of whole brain, the olfactory Irradiation Impairs Vision and Olfaction,
system, the frontal cortex, the corpus callosum, the amygdala, Which Is Ameliorated by FA Fortification
the cerebellum and the corpora quadrigemina in response to In order to determine whether X-irradiation during neurulation
radiation and/or FA (Supplementary Table 1). In contrast, other can affect visual acuity later in life, and whether these effects
brain regions were affected by the radiation dose and the diet. could be countered by FA, a virtual optokinetic drum was
Ventricles appeared significantly enlarged following irradiation used. Here, we observed that radiation decreased visual acuity
[F (2, 37) = 6.125; P = 0.0050] (Figure 5A), although no [Figure 6A, F (2, 64) = 10.02; P = 0.0002], whilst FA increased
significance was reached when comparing individual radiation visual performance as compared to animals on the control diet
doses. We also found a radiation-induced reduction in volume [Figure 6A, F (1, 64) = 6.565; P = 0.0128]. Furthermore, the
of the hippocampus (Figure 5B), striatum (Figure 5C), thalamus impairment in acuity elicited by 0.1 Gy was alleviated by FA
(Figure 5D), midbrain (Figure 5E) and pons (Figure 5F) when (Figure 6A). SD-OCT analysis did not show any changes in total
the mothers were irradiated with 0.5 Gy. Of interest, an retinal thickness following X-ray exposure or FA fortification
interaction effect between irradiation and the diet could be (Figure 6B). Yet, a more detailed investigation revealed that the
established for the hippocampus [F (2, 37) = 4.654; P = 0.0157) nerve fiber and retinal ganglionic cell layer (NF + GCL) thickness

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to elicit a protective effect on the radiation-induced decrease in

eye diameter (Figure 6D). Altogether, we showed a radiation-
induced decrease in visual acuity starting from a low dose of
0.1 Gy onward, together with a reduced NF and GCL layer
thickness and eye diameter following 0.5 Gy. FA prevented the
decreased visual acuity elicited by 0.1 Gy, and rescued the 0.5
Gy-induced decrease in axial eye size.
To investigate whether X-ray exposure during neurulation
has an impact on olfactory performance and discrimination,
and whether radiation-induced differences can be rescued by
FA, we performed an olfaction-dependent habituation and
dis-habituation test. When presented with a novel odor, mice
will show specific approach and sniffing behavior (dishabituation,
see Table 3), which increases further in the presence of S odors,
and diminishes over the three time bins of 2 min (habituation).
To compare the rate of habituation and dishabituation between
the groups, we calculated the difference in sniffing time between
(a) within the same odor of trial 1 and trial 3 (habituation)
and (b) between odors from old to new odor (dishabituation).
Habituation was observed in all conditions for all odors,
indicating a normal loss of interest for odors over time
(Figures 7A,B, Table 3). Similarly, approach behavior to a novel
odor was observed in both sham and irradiated animals and was
not affected by FA enrichment (Figures 7A,B, Table 3). However,
irradiation reduced the total amount of time spent sniffing
NS odors under control diet conditions compared to sham-
irradiated animals (Figure 7C). Two-way ANOVA for factor diet
(control diet or FA) and dose (sham, 0.1 and 0.5 Gy) during
the NS odor presentation, indicated a significant effect for diet
[F (1, 63) = 4.078; P = 0.048] and for dose [F (2, 63) = 3.908; P =
0.025] without significant interaction. Post-hoc analysis revealed
a significant difference between sham- and 0.5 Gy-irradiation in
the control diet group, indicating a reduced approach time to
NS odors after irradiation. This reduced approach was alleviated
when given the high FA diet. Of note, this reduced sniffing
time is not due to an inability to approach, since presentation
of S odors increased the sniffing time, but is possibly due to a
FIGURE 4 | Prevalence and categories of axial skeletal defects at E18,
decrease in attractiveness or detection of the odor itself. High
following 1.0 Gy at E7.5 and prevention by FA. (A) Radiation significantly FA diet normalized the sniffing time and approach to novel
increased the number of defects in the atlas, cervical and thoracal vertebrae, odors to baseline levels (Figures 7B,C), which is indicative of a
while an insignificant trend could be observed in the ribs. FA fortification with 8 protective role for FA. A similar trend was observed for S odors,
mg/kg prevented defects within the cervical vertebrae, with a trend toward
however, the two way ANOVA did not indicate a significant
prevention apparent in the true and false ribs. (B–I) In control animals, the
arches of the cervical vertebrae only sporadically demonstrated an anomaly
effect of either factors. To conclude, irradiation in conjunction
(B), whereas irradiation lead to a notable presence of vertebral fusions with the control diet resulted in hyposmia (i.e., a decreased
(affecting two or three arches, shown by an arrow ← and arrowhead ◭, sense of smell) for the NS odors, or a reduced interest in NS
respectively) and excessive cartilage ≪ (C). In controls, both the thoracic odors. These anomalies were alleviated when the diet was fortified
vertebrae and ribs never showed any anomalies (D), but irradiated fetuses
with FA.
often lacked ribs (arrowhead ◭) and depicted excessive cartilage (double
arrowhead ≪) and fusions (arrow ←) in the vertebrae (E). In addition, the ribs
also showed delayed ossification (arrowhead ◭) (F) and the vertebral bodies
showed split ossification centers (arrowhead ◭) (G). Finally, radiation also
No Changes in Activity and Motor
promoted the presence of a tilted sternum (H,I). Data are represented as Performance Following Irradiation of
mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Asterisks
indicate a significant change as compared to the control FA + 1.0 Gy group of
Animals on the Control Diet
the respective skeletal region. General arousal and changes in circadian activity was assessed in
the 23 h cage test. Under control diet conditions, radiation had no
effect on the spontaneous activity, and all animals displayed the
was decreased following 0.5 Gy irradiation [Figure 6C, F (2, 63) typical increase in night-time activity. Here, repeated measures
= 7.618; P = 0.0011], which was not alleviated following the (RM) ANOVA indicated a significant effect for time [F (47, 1504)
FA-rich diet. On the other hand, the high FA diet was shown = 38.34; P < 0.0001], but not for radiation dose (Figure 8A).

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FIGURE 5 | Volumetric analyses of various brain regions after pre-natal irradiation at E7.5. Ventricles were significantly increased after irradiation (A), whereas the
hippocampus (B), striatum (C), thalamus (D), midbrain (E) and pons (F) were significantly smaller following a dose of 0.5 Gy in animals on the control diet. According
to two way ANOVA, the radiation factor was significant in decreasing size of the posterior cerebral cortex (G). FA by itself decreased the size of the basal ganglia (H)
and the anterior commissure (I). Data are represented as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01.

Animals on the high FA diet also showed a typical increase in Radiation Did Not Affect Overall Cognition,
night-time activity [F (47, 1504) = 50.38; P < 0.0001). In addition, but FA Adversely Altered Social Behavior
the high FA diet increased night-time activity in mice exposed to Open Field and Social Exploration
a low dose of 0.1 Gy [F (2, 32) = 5.063; P = 0.0123) (Figures 8B,C). The open field test was used to assess exploratory behavior
When animals were fed the high FA diet, repeated-measures in a novel and stressful environment. The animals are dark-
(RM) ANOVA revealed a significant interaction effect between adapted and then placed in a brightly illuminated open field
radiation and diet during the dark period [F (2, 64) = 3.485; for 10 min. Anxious animals will spend their time close to the
P = 0.0366], and post-hoc analysis indicated that only 0.1 Gy walls and will not enter the open center zone. We observed that
was significantly different from the sham-irradiated group (P all groups spent most of their time close to the wall, and there
= 0.0191). This interaction effect was also observed in the was no effect of radiation nor of FA enriched diet on the time
overall duration of the experiment [F (2, 64) = 4.127; P = 0.0206] spent in the periphery (Figure 9A). Center visits were frequent
(Figure 8D). (Figure 9B) but overall rather short (Figure 9C). Furthermore,
Balance and coordination was tested on the accelerating the distance the animals traveled over 10 min was similar in all
rotarod, but radiation had no effect on motor coordination, and groups (Figure 9D). Likewise, other parameters, such as latency
we also saw no effect of FA diet (Supplementary Figure 2). to enter the center, walking speed or distance traveled in the

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FIGURE 6 | Visual acuity, retinal morphology, eye size and the impairment thereof by X-irradiation during neurulation. Radiation decreased visual acuity in adult mice
(W5-W7) starting from 0.1 Gy, according to the optomotor test (A). 8 mg/kg FA increased the irradiation dose required for a significant effect on optokinetic response
to 0.5 Gy (A). Irradiation or FA diet did not affect total retinal thickness, according to OCT analysis (B), but a more detailed analysis showed that 0.5 Gy significantly
reduced thickness of the nerve fiber + ganglion cell layer (C). Using the MRI images for eye-size measurements, we identified a reduced axial length following 0.5 Gy,
which was in turn ameliorated by FA fortification (D). Data are represented as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

TABLE 3 | Differences in sniff time used to assess habituation and dishabituation. the test mouse. We observed similar distance covered in all
groups (Figure 10A), but FA fortification decreased the time
Control diet 8 mg/kg FA
spent in the center as compared to animals on the control
Dose 0.0 Gy 0.1 Gy 0.5 Gy 0.0 Gy 0.1 Gy 0.5 Gy diet [Figure 10B, F (1, 63) = 4.316; P = 0.0418]. Factors such as
Habituation: difference in sniff time within same odor center distance, center entries, mean speed, time in periphery
Time (s) −26.2 −21.8 −18.7 −24.7 −22.5 −23.0 and latency to enter the center were unaltered (respectively,
± 3.2 ± 3.7 ± 2.1 ± 2.2 ± 3.2 ± 3.3 Supplementary Figures 4A–E).
Dishabituation: difference in sniff time from old to new odor
Time (s) 30.6 13.0 20.4 30.9 25.3 27.4
± 4.0 ± 3.9 ± 3.2 ± 4.1 ± 4.0 ± 3.2

To quantify habituation (reduction in sniffing time to the same odor) and dishabituation
Elevated Plus Maze
(changes in sniffing time from old to new odor), we calculated the difference between tx1 - The EPM is considered the typical test to assess anxiety related
tx = ∆t, and averaged the values across all odors for each animal. All ∆t were different exploration. We observed no significant difference in total beam
from 0 and there was no effect of radiation and/or FA. Data are represented as mean
breaks between the different groups. Two-way ANOVA indicated
± SEM.
no effect of factor radiation [F (2, 63) = 0.4912; P = 0.6142]
nor of diet [F (1, 63) = 2.206; P = 0.1424) on total beam breaks
center were also not different between the groups (respectively, (Figure 11A). Open arm visits (Figure 11B) and open arm dwell
Supplementary Figures 3A–C). We used a modified setup to (Figure 11C), both readouts for anxiety, were similar in all
evaluate social approach: two stranger mice were placed in groups. In general, neither radiation exposure nor diet had an
the center of the arena and provide an attraction point for impact on anxiety-related activity.

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FIGURE 7 | Odor habituation and dis-habituation in adult mice (W9–W11) following pre-natal irradiation at E7.5, under control or FA fortified diet. All animals show a
typical approach behavior toward novel odors (dishabituation) and reduction in sniffing over time toward the same odor (habituation) (A,B). Under control diet
condition, irradiated animals show reduced sniffing time to novel odors (A). In contrast, under high FA diet, sniffing time is at sham level (B). The total amount of
sniffing time toward a non-social odor, was reduced in irradiated animals under control diet, while high FA fortification increased the sniffing time to sham levels (C).
Data are represented as mean ± SEM, *p ≤ 0.05 vs. sham (post hoc), $ p ≤ 0.05 control vs. FA diet (two-way ANOVA).

Morris Water Maze high FA diet revealed no interaction between diet and latency
During place learning, under control diet, all groups learned [Supplementary Figure 5B, F (1, 18) = 0.1248; P = 0.7280]. These
in a similar way to locate the hidden platform (Figure 12A). data suggest that neither radiation nor high FA diet has an impact
RM ANOVA indicated an effect for day [F (9, 270) = 41.4; P < on passive avoidance learning.
0.001], but not for radiation dose and no interaction. In contrast,
under FA conditions, RM ANOVA indicated an effect for day
[F (9, 288) = 37.0; P < 0.001], and an effect of dose [F (2, 288) DISCUSSION AND CONCLUSION
= 4.34; P = 0.022), without interaction (Figure 12B). Post-hoc
analysis indicated, as compared to sham-irradiated animals, a Radiation-Induced Anophthalmos,
significantly longer path length in 0.5 Gy-irradiated animals only Exencephaly and Gastroschisis Are
on the first 2 days [q(3) = 4.0; P = 0.013, and q(3) = 3.6; P Prevented by FA
= 0.029] (Figure 12B). However, this finding was considered X-irradiation at E7.5 induced various congenital eye defects,
biologically irrelevant due to the low number of days affected. exencephaly, agnathia and gastroschisis in the offspring.
The probe trials were interspersed after day 5 and 10. Target Interestingly, the right eye appeared more susceptible toward
quadrant preference was evaluated by comparing time spent in radiation-induced anophthalmos as compared to the left eye.
the target quadrant with chance level (25%). Under control diet This observation is in line with our previous study (Craenen
conditions, 0.1 Gy-irradiated mice showed a clear lack of target et al., 2017) and could be explained by the used mouse strain with
quadrant preference even after 2 weeks of training, which was a C57BL6/J genetic background. C57BL6/J mice have a strong
also true for 0.5 Gy-irradiated animals during the first probe natural tendency toward developing asymmetrical eye defects,
trial (Figure 12C). When the diet was FA fortified, all radiation- with a bias toward right-eye anophthalmos/microphthalmos
dose groups demonstrated significant target quadrant preference (Smith et al., 1994). Alternatively, in the developing embryo
during the second trial (Figure 12C), suggestive for a FA-induced there are various gestational stages that demonstrate left-
amelioration of reference memory and supporting FA to have a right asymmetry (e.g., various signaling mechanisms). Even
role in learning and memory. Nonetheless, these results are to the developing eye is known to exhibit such developmental
be interpreted with caution due to the relatively low numbers of asymmetry (Levin, 2005), hypothetically allowing potential
animals being included in the analysis. teratogens such as ionizing radiation to interfere with the left-
right axis during embryogenesis. Hence, to assess why radiation
Passive Avoidance induces an asymmetric eye phenotype, it is of interest to compare
The effect of pre-natal X-ray exposure and FA on amygdala our results to a different mouse strain, and to more closely explore
and hippocampal dependent fear-related memory formation was the molecular mechanisms along the embryonic left-right axis
tested using the passive avoidance set-up. Animals on the control after X-irradiation.
diet [F (1, 30) = 86.87; P < 0.0001] and on the high FA diet [F (1, 30) In this study, we demonstrated for the first time that FA
= 219.7; P < 0.0001] demonstrated an increased latency to enter fortification (8 mg/kg FA and 12 mg/kg FA) prevents radiation-
the dark chamber after the shock (Supplementary Figure 5A). induced anophthalmos, exencephaly and agnathia. Already in the
A comparison between animals on the control diet and on the 1960’s a link between FA intake and the incidence of congenital

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FIGURE 8 | Cage activity in adult mice (W7–W9) and the impact of X-irradiation at E7.5 and FA food fortification. When the control diet was fed, no effect of pre-natal
radiation exposure on cage activity was observed (A). When animals were fed the FA diet, irradiation with 0.1 Gy at E7.5 significantly increased activity during the dark
period, as tested in adult 7–9 week old mice (B). (C,D) A summarized total of beam breaks during the dark period (C) and the overall experiment (D) confirmed the
observations made in (A,B). Data are represented as mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

EDs was suggested. For instance, maternal FA-deficiency was a FA-deficient diet in mice could lead to anomalies such as
shown to increase the risk of EDs in rats (Armstrong and anophthalmos and microphthalmos (Maestro-de-las-Casas et al.,
Monie, 1966). A later study supported these findings, where 2013). Furthermore, ethanol-induced retinal anomalies were

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FIGURE 9 | Effect of embryonic X-irradiation and FA fortification on exploration behavior in adult mice, according to the open field test. Neither E7.5 irradiation, nor
diet had an impact on the time spent in the periphery (A), entries into the center (B), time spent in the center (C) and the total distance traveled (D). Data are
represented as mean ± SEM.

FIGURE 10 | Social exploration in an adapted open field test was unaffected by 1.0 Gy exposure at E7.5, but was impaired by FA fortification. Neither radiation, nor
FA diet had an impact on exploration behavior in adult mice, according to the total distance traveled in the arena (A). In contrast, FA fortification resulted in animals
spending less time in the center, in close proximity to the unknown mouse (B). Data are represented as mean ± SEM, *p ≤ 0.05.

rescued with FA supplementation in zebrafish (Muralidharan anophthalmos and microphthalmos. Yet, the authors conceded
et al., 2015). In contrast, an epidemiological study could that several caveats such as a small case population and the lack
not determine a link between FA intake and the risk for of clinical analyses of key biomarkers may have impaired proper

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FIGURE 11 | Effect of embryonic X-irradiation and FA fortification on anxiety, tested in the elevated plus maze. Neither irradiation at E7.5 nor diet had an effect on the
total number of beam breaks (A), beam breaks in the open arms (B) or time spent in the open arms (C) by adult animals. Data are represented as mean ± SEM.

FIGURE 12 | Spatial place learning and reference memory of adult mice in the MWM, following X-ray exposure at E7.5 and/or FA food fortification (8 mg/kg FA). All
groups learned to located the hidden platform under control diet (A) and FA fortification (B). Reference memory was assessed during interspersed probe trials (C). In
FA enriched diet, all animals displayed significant preference to the target quadrant above chance level (25%). Data are represented as mean ± SEM, *p ≤ 0.05 to
chance level (25 %).

effect estimation (Shaw et al., 2007). The second category of eye and gastroschisis. The iris anomaly observed in our study
radiation-induced birth defects that appears folate-responsive, was characterized by a strongly decreased pupil size, with the
is exencephaly. The prevention of exencephaly by FA is well- most severe cases having no apparent pupil at all (Smith et al.,
described in literature, albeit not with ionizing radiation as the 1994; Craenen et al., 2017). In accordance, in a previous study
effecting teratogen, but with e.g., ethanol (Yanaguita et al., 2007) on hyperthermia-induced iris anomalies, no protective effect
and glucose (Wentzel and Eriksson, 2005; Oyama et al., 2009). Of of FA could be found (Czeizel et al., 2011). Yet, there are
note, international FA food fortification initiatives have already to our knowledge no other publications that have previously
successfully decreased the incidence of NTDs (Blom et al., 2006). investigated the efficacy of FA in preventing open eye anomalies.
The third category of radiation-induced birth defects, that is Hereto, it might be worthwhile to investigate the protective effect
partially preventable by FA, is agnathia. In humans, agnathia of thyroxine supplementation on radiation-induced open eyes,
is a very rare congenital disorder, commonly classified within as some success was already made with this hormone (Juriloff,
the otocephaly family of disorders (incidence <1/70 000 births) 1985). With regard to gastroschisis, it remains severely debated
(Gekas et al., 2010; Herman et al., 2012; Jagtap et al., 2015; whether these defects can be prevented with FA, with efficacy
Sergouniotis et al., 2015). To our knowledge, the only published strongly depending upon the acting teratogen (Godwin et al.,
observation where FA fortification could prevent agnathia was in 2008; Paranjothy et al., 2012; Yang et al., 2016).
Twisted gastrulation mutant mice, which have a high penetrance
of midline facial defects and jaw defects (Billington et al., 2013).
Even though our study is the first to demonstrate the prevention
Reduced Fetal Weight and Increased
of X-ray-induced anophthalmos, exencephaly, and agnathia, the Pre-natal Death Following Irradiation Are
rescue is only partial. It would be of interest to further explore Ameliorated by FA
the efficacy of other radioprotectant compounds, potentially in Aside from gross macroscopic defects, other aspects of pre-
combination with FA, in preventing these defects. natal development were also assessed. Irradiation significantly
In contrast to the defects discussed above, we observed no reduced fetal weight at E18, as was also observed in a previous
folate-responsiveness of radiation-induced iris anomalies, open study (Craenen et al., 2017), which was not notably ameliorated

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by FA. This stands in contrast to the teratogen ethanol, where Persistent Radiation-Induced Defects in
embryotoxic weight loss can be prevented by FA (Xu et al., the Adult Nervous System and the
2006). However, a potentially adverse outcome observed after
Preventive Role of FA
fortification of the highest dose of FA was the increase of
Because pre-natal exposure to ionizing radiation can induce gross
fetal weight at E18. Epidemiological studies already reported
congenital central nervous system defects (e.g., microphthalmos
that increased fetal weight is a consequence of FA fortification
and anophthalmos) at moderate to high X-ray doses (0.5–1.0 Gy)
(Tamura and Picciano, 2006; Balarajan et al., 2013; Li et al., 2016;
(Craenen et al., 2017), we decided to explore whether this can
Ramakrishnan et al., 2016), which is linked to type 2 diabetes and
elicit functional and morphological neurological defects that
adult obesity (Curhan et al., 1996; Johnsson et al., 2015). Both
persist into adult age.
the number of late fetal deaths and resorptions were increased
We used in vivo MRI to investigate whether X-ray exposure
following irradiation, which is consistent with previous work
during neurulation has an effect on adult brain and eye
(Pampfer and Streffer, 1988; Kim et al., 2001; Craenen et al.,
morphology. Although we observed no global microcephaly, as
2017). These cases of pre-natal mortality were reduced by FA
was shown after irradiation during neurogenesis (Verreet et al.,
fortification, which is in line with epidemiological studies that
2015, 2016a), volumetric analyses unveiled a decreased volume
focused on fetal loss (Andersen et al., 2010) and miscarriage
of some dedicated brain areas. More specifically, we observed
(Byrne, 2011).
that 0.5 Gy significantly reduces the size of the hippocampus,
striatum, thalamus, midbrain and pons. These structures are
involved in various mechanisms, ranging from cognition to
Radiation-Induced Fetal Morphological visual acuity. For example, the thalamus is known for its
Defects, and Prevention With FA importance in processing and relaying visual information (Tyll
Since the mid-twentieth century, it has already been known that et al., 2011). The pons and midbrain are also involved in visual
exposure to (high) doses of ionizing radiation during pregnancy functioning, as anomalies within these brainstem regions can
can result in a variety of axial and appendicular skeletal result in both horizontal and vertical gaze palsy (Strupp et al.,
defects (Jarmonenko, 1988). These studies focused mostly on 2014; Lin et al., 2018). Furthermore, we observed that irradiation
severe spinal defects such as spina bifida, which are known with 0.5 Gy decreased the axial length of the adult eye, which can
to result from exposure to external radiation sources such as be relayed directly to an increased incidence of radiation-induced
neutrons and γ-rays (A-bombs) and internal contamination microphthalmia (Verma and Fitzpatrick, 2007; Craenen et al.,
from e.g., depleted uranium (commonly used in munitions) 2017), and can be associated to an increased risk of refractive
[reviewed by Hindin et al. (2005)]. In our study, pre-natal errors (Bhardwaj and Rajeshbhai, 2013). Supporting the MRI-
irradiation had the most detrimental impact on the cervical based findings, SD-OCT revealed a decreased thickness of the
and thoracic vertebrae. This was also observed in a study by NF+GCL layer in the adult eye, following 0.5 Gy at E7.5, which
Russell, who used 2.0 Gy of X-rays at E7.5 (Russell, 1956). might lead to a decreased visual acuity (Moster et al., 2016).
A contrasting study described more malformations in the Concomitant with these radiation-induced alterations in the
ribs of CRI mice than in the vertebrae, following 2.0 Gy-γ- brain and the observed eye anomalies, we indeed observed a
irradiation at E7.5 (Kim et al., 2001). This difference might decreased visual acuity following E7.5 irradiation. Interestingly,
be mouse strain dependent, or might result from variations this was also observed in 0.1 Gy-irradiated animals, that did
in radiation dose and type. In further support of our study, not show a decreased eye size and NF+GCL thickness nor a
a dose-dependent induction of skeletal malformations after reduction in brain volumes, suggesting that other mechanisms
irradiation (0.5 Gy to 4.0 Gy) at E11.5 was observed (Kim et al., might also be involved. The morphological defects underlying the
2001). It would therefore be interesting to further investigate radiation-induced loss of visual acuity may thus extend beyond
this dose-dependency and the existence of a dose-threshold in changes in eye structure and warrants further investigation.
our experimental set-up. Intriguing was the presence of split Even though pre-natal irradiation had no marked impact
spinal ossification centers after irradiation, which could lead on the olfactory system in the adult brain, behavioral tests for
to open vertebral arches and potentially spina bifida occulta olfaction were included in the test battery. This decision was
in later life (Regnier et al., 2002). Altogether, we are the first based on a previous study that showed transient transcriptional
to explore in such detail developmental defects in the axial disturbances in the embryonic head following 1.0 Gy irradiation
skeleton after irradiation at E7.5, and to demonstrate that FA at E7.5 that were related to the development of the olfactory
fortification can significantly reduce the risk for radiation- epithelium (Craenen et al., 2020b). This is an important
induced skeletal defects. observation, as the olfactory system starts to develop during this
Although we observed an increase in axial skeletal defects after neurulation period (Treloar et al., 2010). Besides, the overall
1.0 Gy irradiation, it appears that the sub-lethal doses (Craenen process of olfaction extends well-beyond the olfactory lobe
et al., 2017) used for the behavioral assays (≤0.5 Gy) might have (Lehmkuhl et al., 2014), with congenital anomalies within the
been too low to elicit any functional detriment. Indeed, in terms peripheral olfactory system (e.g., the olfactory epithelium) having
of motor performance, none of the behavioral tests could identify been linked to hyposmia (Bergman et al., 2010). We are the
a clear impairment following irradiation, as is discussed in more first to demonstrate a decreased olfactory acuity in adult mice
detail below. following 0.5 Gy at E7.5. In particular, we could demonstrate a

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more pronounced anosmia for NS than S odors, which could prevented by FA food fortification. In the context of radiation
be attributed to the functional importance of social odors protection, our study supports the use of FA fortification to
and/or chemical differences between the respective odorants increase the dose threshold required to elicit adult brain and
(Sinding et al., 2017). Since defects at this level might explain eye anomalies.
the observed hyposmia, it is of interest for future studies to
more closely investigate this complex structure following pre- Potential Mechanisms Underlying
natal X-irradiation.
FA-Mediated Radioprotection
In contrast to the morphological and sensory anomalies
Although the exact mechanism through which FA elicits its
discussed above, we observed no effect of irradiation on
radioprotective role is currently unknown, it is still of interest
cognition. Previous behavioral screenings demonstrated that
to highlight several likely modes of action. A first hallmark
irradiation of mice during neurogenesis had a marked effect
consequence of ionizing radiation exposure is the generation
on memory-based performance (Verreet et al., 2015, 2016a).
of reactive oxygen and nitrogen species, which can in turn
However, when we irradiated animals during neurulation, no
damage various cellular structures. The detrimental impact of
evidence for impaired memory formation or retention could
excessive oxidative stress in the developing embryo and pregnant
be observed, both for spatial and fear-dependent learning.
mother has been repeatedly addressed in literature. Indeed,
The observation that 0.5 Gy irradiation decreases hippocampal
it appears that a disturbed redox status is a recurrent theme
volume may appear paradoxical in that sense, but this volumetric
in the etiology of birth-defects caused by various chemicals,
decrease might not be directly related to a loss in function.
including thalidomide, phenytoin and ethanol (Dennery, 2007).
Although irradiated animals had a notable loss of visual acuity,
FA is known to have antioxidative properties in vitro, which is
the defect may have been too small to influence MWM
suggestive of its potential radioprotective effect, but it remains
performance, which depends on large and distinct visual cues
unclear whether this antioxidative role persists at a systemic level
(Lindner et al., 1997; Brown and Wong, 2007; Phillips et al., 2013;
in vivo. A second hallmark consequence of irradiation is DNA-
Vorhees and Williams, 2014).
damage (Reisz et al., 2014), which can theoretically be repaired
Many of the radiation-induced anomalies that were observed
more efficiently with an increased access to one-carbon donors
in adult mice could be (in part) prevented by FA fortification.
such as FA. A third hallmark consequence of irradiation includes
FA fortification by itself had only a minimal impact on the adult
epigenetic alterations, in particular DNA methylation. Folates
tests. For instance, we observed a decreased social exploration
fulfill an important role in DNA methylation (Crider et al., 2012).
in mice on the FA-fortified diet and also identified a lower
As the key one-carbon donor behind the methylation process,
volume of the basal ganglia (i.e., caudate putamen and adjacent
it stands to reason that changes in the folate pool would affect
structures). This might be related to the social impairments in
this epigenetic process and potentially reverse radiation-induced
these mice, since a decreased basal ganglia volume was already
DNA hypomethylation. The fourth potential mode-of-action lies
linked with autism-spectrum disorder (Barua et al., 2014), but
in radiation-induced changes in the transcriptome and proteome.
whether FA fortification is a risk factor for abnormal social
We previously demonstrated that X-irradiation (1.0 Gy) at E7.5
behavior remains controversial [reviewed in Wiens and DeSoto
in mice reduced the expression of Lhx2, a key transcription factor
(2017)]. Furthermore, this volumetric loss might have played a
for eye, brain and olfactory development (Craenen et al., 2020b).
role in the changes in cage activity as well, because the basal
Furthermore, mutations in genes associated with Lhx2 are known
ganglia are involved in the regulation of the sleep-wake cycle (Qiu
to cause birth defects such as exencephaly (Barbera et al., 2002).
et al., 2010) and general activity (Portmann et al., 2014).
As such, it is of interest to assess whether the radiation-induced
It is important to note that, depending on the causative factor,
suppression of Lhx2 transcription/translation can be alleviated
congenital defects may respond in a dose-dependent manner
by FA fortification. Although this study does not address any of
to FA fortification, with higher doses of FA yielding lower
the aforementioned modes-of-action directly, an exploration of
defect prevalences (Gray and Ross, 2009). Yet, in our study, the
the mechanisms that might be involved in the antiteratogenic
radioprotective role of FA did not appear dose-responsive and
and radioprotective effect of folic acid is warranted. Such novel
no added benefit was noted following 12 mg/kg FA fortification.
insights might contribute to developing even more efficient
Hence, we decided to limit the studies in adult animals to the
means to protect the unborn child from genotoxic hazards such
8 mg/kg FA diet. Radiation-induced volumetric decreases of
as radiation.
the hippocampus, striatum, thalamus, midbrain and pons were
prevented with FA fortification. In addition, FA rescued visual
acuity loss following a dose of 0.1 Gy, but not 0.5 Gy. Yet, not all CONCLUSION
morphological eye anomalies were prevented by FA, for instance
the radiation-induced reduction of NF+GCL thickness. Finally, FA food fortification is effective at partially preventing the
radiation-induced hyposmia for NS odors was alleviated by AF. embryotoxic effects of X-ray exposure. Specifically severe defects
To the best of our knowledge, were are the first to highlight this such as anophthalmos, exencephaly and agnathia were responsive
radioprotective/antiteratogenic character of FA. In all, we can to FA. In addition, late fetal deaths, the incidence of resorptions,
conclude that X-ray exposure during neurulation affects the adult fetal weight and skeletal defects within the cervical and thoracal
nervous system at both a morphological and functional level, vertebrae were all negatively affected by 1.0 Gy X-irradiation at
from a dose of 0.1 Gy onward, and that these defects can be in part E7.5, which was in turn partially countered by FA. Behavioral

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studies demonstrated that X-ray exposure to sub-lethal doses Considering both the promising results and the limitations of
(≤0.5 Gy) at E7.5 resulted in a decrease of visual acuity and this study, the authors support larger (epidemiological) studies
olfactory performance in the habituation/dishabituation test. (with lower fetal radiation doses) to explore the use of FA as a
The impaired visual performance was supported by radiation- radioprotectant in humans.
induced loss of NF+GCL thickness and a decreased eye diameter,
at least for the highest dose of 0.5 Gy. We can conclude from DATA AVAILABILITY STATEMENT
our MRI data that irradiation during neurulation has more
site-specific consequences than irradiation during neurogenesis The raw data supporting the conclusions of this article will be
(Verreet et al., 2015, 2016a). As such, it would be of interest to made available by the authors, without undue reservation.
follow up this study with more sensitive behavioral tests, tailored
more specifically to those brain regions that are decreased in ETHICS STATEMENT
volume following X-irradiation.
The increasing exposure of humans to ionizing radiation The animal study was reviewed and approved by Institutional
is a contemporary topic that deserves proper investigation. Ethical Committees of SCK-CEN/VITO (ref. 02–012) and the
The heightened exposure to ionizing radiation finds its roots Animal Welfare Committee of the KU Leuven.
in the clinical environment, nuclear disasters, war or terrorist
activities and natural sources such as Radon gas. With this AUTHOR CONTRIBUTIONS
research paper, the authors wish to address and promote novel
radioprotection strategies such as FA fortification and the future MB and MV mentored KC, LM supervised KC as University
implementation thereof in high-risk groups that currently do promotor. KC, MV, LM, and MB planned the experiments and
not have access to FA-fortified staple foods (or FA supplements). offered guidance regarding experimental design. KC performed
Included in these risk-groups are e.g., pregnant patients who the majority of the lab work and wrote the manuscript. LC, JB,
require radiodiagnostics or radiotherapy and pregnant women and MN contributed significantly to lab work. KG processed
living in radioisotope-contaminated regions. The fetal doses the MRI images and WG assisted with the MRI image capture
that can be expected during clinical exposure events (including procedures. ZC-V and RD’H offered invaluable guidance and
conventional radiotherapy, computed tomography and nuclear assistance regarding the behavioral tests. MB, MV, LM, KG, SB,
medicine) range from 0.01 to 43.9 mGy (Lazarus et al., 2009). ZC-V, RD’H, and UH reviewed the initial manuscript and offered
These doses are lower than those used in this study, as we crucial feedback that contributed to the submitted paper. KC was
opted to reduce the number of animals required to observe a joint PhD student of SCK CEN and KU Leuven funded through
significant radiation effects. As such, it is difficult to make a a scholarship of SCK CEN. All authors contributed to the article
direct extrapolation from the animal research presented here and approved the submitted version.
to the human exposure scenarios listed above. Nonetheless, as
a proof of concept this study demonstrates the potential for FUNDING
using FA fortification to protect the unborn child against ionizing
radiation. Protecting the unborn child from the detrimental KC was funded by an SCK CEN/KU Leuven joint doctoral grant.
effects of ionizing radiation will improve their quality of
life, by preventing radiation-induced birth defects and sensory SUPPLEMENTARY MATERIAL
deprivation. Although our study in mice indicates that ad libitum
FA food fortification at 8 mg/kg is sufficient to provide a The Supplementary Material for this article can be found
radioprotective effect, the optimal concentration for humans online at: https://www.frontiersin.org/articles/10.3389/fnbeh.
remains to be studied in the context of radiation protection. 2020.609660/full#supplementary-material

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combined supplementation of folic acid and Vitamin B12 suppresses ethanol-
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doi: 10.1016/j.reprotox.2005.12.004 Neefs, Gsell, Baatout, D’Hooge, Himmelreich, Moons and Benotmane. This is an
Yanaguita, M. Y., Gutierrez, C. M., Ribeiro, C. N. M., Lima, G. A., Machado, open-access article distributed under the terms of the Creative Commons Attribution
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with folic acid supplementation in saccharose. Childs Nerv. Syst. 24, 99–104. provided the original author(s) and the copyright owner(s) are credited and that the
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21 January 2021 | Volume 14 | Article 609660
 ORIGINAL RESEARCH
published: 22 February 2021
doi: 10.3389/fnbeh.2021.615798

Ontogenetic Oxycodone Exposure

Affects Early Life Communicative
Behaviors, Sensorimotor Reflexes,
and Weight Trajectory in Mice
Elena Minakova 1† , Simona Sarafinovska 2,3,4† , Marwa O. Mikati 3,5,6,7,8 , Kia M. Barclay 5,6,7,8 ,
Katherine B. McCullough 2,3 , Joseph D. Dougherty 2,3,9 , Ream Al-Hasani 5,6,7,8* ‡ and
Susan E. Maloney 3,9* ‡
1
Department of Pediatrics, Washington University in St. Louis, St. Louis, MO, United States, 2 Department of Genetics,
Edited by: Washington University in St. Louis, St. Louis, MO, United States, 3 Department of Psychiatry, Washington University in St.
Carla Cannizzaro, Louis, St. Louis, MO, United States, 4 Medical Scientist Training Program, Washington University in St. Louis, St. Louis, MO,
University of Palermo, Italy United States, 5 Division of Biology and Biomedical Sciences, Washington University in St. Louis, St. Louis, MO,
United States, 6 Department of Anesthesiology, Washington University in St. Louis, St. Louis, MO, United States,
Reviewed by: 7
Washington University Pain Center, Washington University in St. Louis, St. Louis, MO, United States, 8 Center for Clinical
Patrizia Romualdi,
Pharmacology, St. Louis College of Pharmacy, University of Health Sciences and Pharmacy, St. Louis, MO, United States,
University of Bologna, Italy 9
Intellectual and Developmental Disabilities Research Center, Washington University In St. Louis, St. Louis, MO,
William Kenkel,
United States
University of Delaware, United States
Florence Iris Roullet,
McMaster University, Canada
Nationwide, opioid misuse among pregnant women has risen four-fold from 1999 to
*Correspondence:
2014, with commensurate increase in neonates hospitalized for neonatal abstinence
Ream Al-Hasani
[email protected] syndrome (NAS). NAS occurs when a fetus exposed to opioids in utero goes into rapid
Susan E. Maloney withdrawal after birth. NAS treatment via continued post-natal opioid exposure has been
[email protected]
suggested to worsen neurodevelopmental outcomes. We developed a novel model to
† These authors have contributed

equally to this work and share first

characterize the impact of in utero and prolonged post-natal oxycodone (Oxy) exposure
authorship on early behavior and development. Via subcutaneous pump implanted before breeding,
‡ These authors have contributed C57BL/6J dams were infused with Oxy at 10 mg/kg/day from conception through
equally to this work and share senior
pup-weaning. At birth, in utero oxy-exposed pups were either cross-fostered (paired
authorship
with non-Oxy exposed dams) to model opioid abstinence (in utero Oxy) or reared by
Specialty section: their biological dams still receiving Oxy to model continued post-natal opioid exposure
This article was submitted to
Emotion Regulation and Processing,
(prolonged Oxy). Offspring from vehicle-exposed dams served as cross-fostered (in
a section of the journal utero Veh) or biologically reared (prolonged Veh) controls. In utero Oxy exposure resulted
Frontiers in Behavioral Neuroscience in sex-dependent weight reductions and altered spectrotemporal features of isolation-
Received: 19 October 2020 induced ultrasonic vocalization (USV). Meanwhile, prolonged Oxy pups exhibited
Accepted: 29 January 2021
Published: 22 February 2021 reduced weight and sex-differential delays in righting reflex. Specifically, prolonged
Citation: Oxy female offspring exhibited increased latency to righting. Prolonged Oxy pups also
Minakova E, Sarafinovska S, showed decreases in number of USV calls and changes to spectrotemporal USV
Mikati MO, Barclay KM,
McCullough KB, Dougherty JD,
features. Overall, ontogenetic Oxy exposure was associated with impaired attainment
Al-Hasani R and Maloney SE (2021) of gross and sensorimotor milestones, as well as alterations in communication and
Ontogenetic Oxycodone Exposure affective behaviors, indicating a need for therapeutic interventions. The model developed
Affects Early Life Communicative
Behaviors, Sensorimotor Reflexes, here will enable studies of withdrawal physiology and opioid-mediated mechanisms
and Weight Trajectory in Mice. underlying these neurodevelopmental deficits.
Front. Behav. Neurosci. 15:615798.
doi: 10.3389/fnbeh.2021.615798 Keywords: opioid, behavior, in utero, post-natal, oxycodone, neonatal abstinence syndrome

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1 February 2021 | Volume 15 | Article 615798
Minakova et al. Oxycodone Exposure Affects Early Life Neurodevelopment

INTRODUCTION effects on developmental outcomes (Slamberová et al., 2005;

Niu et al., 2009). Opioids exert their pharmacologic effects
In the past two decades, illicit drug use and prescription opioid by activating the endogenous opioid system. While opioids
use in the United States have risen to epidemic proportions, with are prescribed for their analgesic effects, acute activation of
the United States Department of Health declaring a public health the µ-opioid receptor (MOR) by these medications has also
emergency in 2017. The United States Department of Health been associated with feelings of euphoria, award reinforcement,
states that 46,802 people died from opioid overdose in 2018 and and increased socio-emotional processing, which are linked
an estimated 2 million people have an opioid use disorder (ASPA, to the drugs’ potential for misuse (Vanderschuren et al.,
2017). The public health crisis is largely driven by increased 1995). Despite the rising incidence of Oxy misuse, there is
misuse of the prescription opioids hydrocodone, oxycodone a paucity of literature evaluating the effects of Oxy, a µ-
(Oxy), and methadone (Kenan et al., 2012; Haight, 2018). and κ-agonist, on early developmental behaviors. κ-agonists
As a result of the opioid epidemic, the national prevalence are of particular interest because over-activation of κ-opioid
of opioid use disorder among pregnant women has more receptors (KOR) by dynorphin upregulation has been implicated
than quadrupled, from 1.5 to 6.5 per 1,000 deliveries, from in withdrawal physiology and depressed mood in humans, along
1999 through 2014 (Haight, 2018). Consequently, there with decreased social play in juvenile rodents (Vanderschuren
has been a significant increase in the number of neonates et al., 1995; Li et al., 2016). In addition, most rodent opioid
hospitalized for neonatal abstinence syndrome (NAS), a exposure models begin exposure mid-pregnancy, which may
constellation of withdrawal symptoms affecting the nervous explain inconsistently documented or absent developmental
system, gastrointestinal tract, and respiratory system following in changes (Richardson et al., 2006). To address the above
utero exposure to opioids (Wiles et al., 2014). Currently, medical concerns, we are adopting an ontogenetic model in which
management of NAS involves keeping the infant swaddled in opioid exposure spans preconception through early offspring
a low-stimulation environment with promotion of maternal- development. This new model also enables us to better
infant bonding (Wiles et al., 2014). In cases of moderate-severe understand the ontogenetic impact of short- and long-term
NAS, neonatal withdrawal is managed by opioid replacement opioid exposure on early development in the absence of
therapy to alleviate withdrawal symptomatology (Wiles et al., confounding factors present in clinical observational studies.
2014). Overall, clinical studies have not addressed whether Specifically, we evaluated the effects of in utero Oxy exposure
long-term neurobehavioral outcomes are improved by managing on early developmental and behavioral outcomes in male and
withdrawal or whether continued post-natal exposure to opioids female offspring of C57BL/6J mouse dams. We implemented
and adjunct agents used for withdrawal management worsen a cross-fostering approach that allows us to compare the
long-term outcomes (Hudak et al., 2012). neurodevelopmental impact of continued post-natal opioid
Epidemiological evidence suggests that in utero opioid exposure (prolonged Oxy) to the impact of exposure only until
exposure is associated with lower birth weight and adverse birth (in utero Oxy) by pairing opioid exposed pups with
neurodevelopmental outcomes in childhood, including non-oxy exposed dams.
cognitive deficits, attention deficit hyperactivity disorder Overall, we observed differences in the spectrotemporal
(ADHD), aggression, impaired language development, and features of affective vocalizations and sex-based differences in
decreased social maturity (Hunt et al., 2008; Azuine et al., weight gain trajectories in offspring exposed to in utero Oxy.
2019; Conradt et al., 2019). However, large epidemiological Continued post-natal Oxy exposure (prolonged Oxy) further
studies evaluating long-term behavioral outcomes of children impacted weight, communicative behavior, and sensorimotor
exposed to in utero opioids have been difficult to perform due reflexes. Our findings suggest that pups with continued post-
to confounding environmental variables including genetic and natal opioid exposure showed worse overall developmental
epigenetic factors, quality of caregiving, continued parental outcomes compared to pups following opioid cessation at
substance abuse with its impact on the maternal-infant dyad, birth, which may have implications regarding the safety
and other socioeconomic variables which can significantly of continued opioid treatment as mitigation for clinical
affect neurodevelopmental outcomes (Lutz and Kieffer, 2013). NAS symptomology.
Consequently, the development of ontogenetic rodent models
of opioid exposure is necessary to enable investigation of the
biological mechanisms mediating deficits as well as testing MATERIALS AND METHODS
alternative treatment avenues for post-natal withdrawal.
To date, there have been a limited number of rodent studies Animals
evaluating early life developmental milestones following in utero Animal Ethics, Selection, and Welfare
opioid exposure. Current literature on early developmental All procedures using mice were approved by the Washington
effects of in utero opioid exposure in pre-clinical models University Institutional Care and Use Committee and conducted
demonstrates decreased birth weight following methadone and in accordance with the approved Animal Studies Protocol.
buprenorphine exposure (Kunko et al., 1996; Hung et al., C57BL/6J mice (Jackson Laboratory, stock #: 000664) were
2013; Chiang et al., 2015). Increased latency to right has housed in individually ventilated translucent plastic cages (IVC)
been observed following in utero morphine exposure and measuring 36.2 × 17.1 × 13 cm (Allentown) with corncob
is suggestive of different classes of opioids having variable bedding and ad libitum access to standard lab diet and

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Minakova et al. Oxycodone Exposure Affects Early Life Neurodevelopment

TABLE 1 | Litter and group size, including number of pups at the level of litter, them to the nest of a lactating foster dam with two of her own
group, and experiment.
pups of the same approximate age (Figure 1B; Lohmiller and
Litter ID Group No. of pups Swing, 2006). The remaining litters were reared by the biological
dam and exposed to post-natal vehicle (prolonged Veh) or Oxy
1 Prolonged Oxy 5 (prolonged Oxy) through lactation (Figures 1A,B). To control
2 Prolonged Oxy 4 for litter effects, each group included multiple, independent litters
3 Prolonged Oxy 3 (Table 1). All mice were weaned at P21 and group-housed by
4 Prolonged Oxy 6 sex with random assignment for drug/dam. A subset of the
5 Prolonged Oxy 6 in utero Oxy mice required saline injections at P23-P25 due
Total: 24 to skin tenting, hunched posture, and significant weight loss
6 In utero Oxy 5 concerning for dehydration. Following saline injections, recovery
7 In utero Oxy 6 was noted in two of the three affected mice with one associated
8 In utero Oxy 6 mortality. Experimenters were all female and blinded to group
9 In utero Oxy 6 designations during testing.
10 In utero Oxy 6
Total: 29 Drug Dosage
11 Prolonged Veh 6
The dosage of Oxy (Sigma-Aldrich, Saint Louis, MO,
12 Prolonged Veh 6
United States; Lot#: SLBX4974) administration was guided
13 Prolonged Veh 6
by previous literature with concentrations ranging from 0.5 to
14 Prolonged Veh 6
33 mg/kg/day (Enga et al., 2016; Sithisarn et al., 2017; Zanni
15 Prolonged Veh 6
et al., 2020). Based on this dosage range, we generated our
Total: 30
own dosage curve through continuous Oxy administration to
16 In utero Veh 5
pregnant dams at 5, 10, or 15 mg/kg/day using the subcutaneous
17 In utero Veh 6
Alzet 2006 model pump (Durect Corporation, Cupertino,
18 In utero Veh 3
CA, United States; Lot #: 10376-17). We chose the dose of
19 In utero Veh 6
10 mg/kg/day administered at 0.15 ul/h to pregnant dams
20 In utero Veh 5
as increased concentrations at 15 mg/kg/day resulted in
21 In utero Veh 3
lower litter success rate (vehicle, 5 and 10 mg/kg/d: 100%
Total: 28
success rate; 15 mg/kg/d: 80% success rate). We chose to
Total pups: 111
administer a consistent dose of opioids to the dam throughout
pregnancy and lactation to model the current management
of pregnant mothers with opioid use disorder. Women with
water. Animals were kept at 12/12 h light/dark cycle, and opioid use disorder are now encouraged to enroll in opioid
room temperature (20–23◦ C) and relative humidity (50%) were medication-assisted treatment programs with the goal of
controlled automatically. attaining a steady-state drug level. Maintaining a consistent
Adult male and female mice were used for breeding cohorts level of opioid administration during pregnancy in mothers
as described below. Sample sizes were determined by power with opioid use disorder limits adverse maternal and fetal
analyses (f = 0.40, α = 0.05, 1-β = 0.80). A total of 24 dams were consequences associated with fluctuations related to higher
housed in pairs and randomly selected to receive either the Oxy opioid concentrations. Increases in opioid dosage can result
or Vehicle (Veh) treatment infusion. In addition, another set of in maternal respiratory depression and lethal overdose and
pair-housed, drug-naïve dams served as foster dams. The total may decrease fetal heart rate and variability (Krans et al., 2015;
sample size was 111 pups (Table 1). Since an inexperienced dam Rosenthal and Baxter, 2019).
can exhibit poor maternal behavior with her first litter, all females
were first bred to an age-matched male at post-natal day (P) 60. Surgery and Drug Delivery System
Following weaning of the first litter, treatment dams underwent Female dams were anesthetized at P95 with isoflurane (5%
surgical subcutaneous pump placement at P95 followed by a 1- induction, 2% maintenance, 0.5 l/min) and placed in the
week recovery period (Figure 1A). Afterward, each female dam mouse adapter (Stoelting, Wood Dale, IL, United States). Body
was placed into an individual cage containing a male for breeding. temperature was maintained at 37◦ C using a heating pad. The
Foster dams were bred at the same time and remained untreated dorsum of the back was shaved and a ∼1 cm horizontal incision
throughout pregnancy. Following 20 days of co-habitation, cages was made below the scapulae with subsequent formation of
were checked daily for pups. Upon detection, dam and litter a subcutaneous pocket. The Alzet pump was implanted and
were moved to a new cage, without the male, and culled to 6– continuously infused with either Oxy or sterile 0.9% NaCl (Veh)
8 pups per litter with equal males and females when possible. over a period of 60 days. The pump duration allowed for adequate
To evaluate the behavioral impact of early opioid cessation in post-surgical recovery time, breeding, and administration of
the developing offspring, half of the litters (in utero Oxy and in treatment through weaning of offspring at P21. In addition, the
utero Veh) were cross-fostered at this time to drug-naïve dams use of a subcutaneous pump limited unwanted maternal stress
by removing the pups from their biological dam and transferring that can occur with daily injections.

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Minakova et al. Oxycodone Exposure Affects Early Life Neurodevelopment

A OXY/VEH PUMP BIRTH USV RIGHTING WEANING

{
PLACEMENT & FOSTERING REFLEX
EXPERIENTIAL EXPERIMENTAL
BREEDING BREEDING WEIGHT

Dam P60 P95 P102 P122 P143

Pup E0 P0 P5 P7 P9 P11 P14 P21 P30
In utero
Prolonged Maternal Treament

B REARED BY FOSTER NON-EXPOSED DAMS

In utero Oxy P21
BIRTH
& FOSTERING

DEVELOPMENTAL
ASSESSMENT

OXYCODONE Pup Pup

EXPOSED E0 P0
DAMS (E0-P21) Prolonged Oxy
(n = 12) REARED BY OXY-EXPOSED DAMS

P21

REARED BY FOSTER NON-EXPOSED DAMS

P21
In utero Veh
BIRTH
& FOSTERING

DEVELOPMENTAL
ASSESSMENT

VEHICLE Pup Pup

EXPOSED E0 P0
DAMS (E0-P21) Prolonged Veh
(n = 12) REARED BY VEH-EXPOSED DAMS

P21

FIGURE 1 | Ontogenetic model of prolonged and limited maternal Oxy exposure. (A) Schematic of the paradigm for maternal Oxy exposure, including duration of in
utero and prolonged maternal treatment, dam ages at experimental manipulations in yellow, and pup age for behavioral tests in purple. (B) Outline of the four
different experimental groups, including Oxy or Veh exposure, and rearing dam exposure status.

Behavioral Testing the start of testing. We maintained an average pup surface

Maternal Isolation-Induced Ultrasonic Vocalization body temperature of 34◦ C prior to placement into the USV
Recording recording chamber, as low pup body temperature increases USV
Neonates with NAS often exhibit excessively high-pitched production (Branchi et al., 2001). The surface body temperature
crying, irritability, and prolonged periods of inconsolability of all pups was assessed via a non-contact HDE Infrared
(Anbalagan and Mendez, 2020). Affective characteristics of Thermometer prior to placement into the recording chamber,
ultrasonic vocalizations (USVs) in rodents are generally thought and no differences in body surface temperature were observed
to communicate different emotional states, such as aggression between groups. The recording chamber was maintained at room
or pain. USV quantity, duration, pitch, frequency, and loudness temperature (22–23◦ C). For recording, pups were individually
(dB) of the calls allow for the assessment of call characteristics removed from the home cage and placed into an empty
following in utero Oxy exposure (Vivian and Miczek, 1993a,b). standard mouse cage (28.5 × 17.5 × 12 cm) inside a sound-
USV recordings were performed on P5, P7, P9, and P11 attenuating chamber (Med Associates). USVs were recorded
(Figure 1A). Dams were removed from the home cage and via an Avisoft UltraSoundGate CM16 microphone placed 5 cm
placed into a clean IVC for the duration of testing. The away from the top of the cage, Avisoft UltraSoundGate 116H
home cage with the pups in nest was placed into a warming amplifier, and Avisoft Recorder software (gain = 3 dB, 16 bits,
box (Harvard Apparatus) set to 34◦ C for 10 min prior to sampling rate = 250 kHz). Pups were recorded for 3 min,

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Minakova et al. Oxycodone Exposure Affects Early Life Neurodevelopment

after which they were weighed and returned to home cages. TABLE 2 | Test statistics from hierarchical linear models.
Frequency sonograms were prepared from USV recordings
Variable Factor Output p-value
in MATLAB [frequency range = 25–120 kHz, Fast Fourier
Transform (FFT) size = 512, overlap = 50%, time resolution Weight (g) Sex F (1, 107) = 10.219 p = 0.002
1.024 s, frequency resolution = 488.2 Hz]. Individual calls Drug F (1, 109) = 5.448 p = 0.021
and other spectrotemporal features were identified from the Age F (9, 901) = 785.001 p = 0.000
sonograms adapted from validated procedures (Holy and Guo, Litter Size F (1,102) = 9.922 p = 0.002
2005; Maloney et al., 2018a,b). Sex × Dam × Drug × Age F (67, 685) = 4.232 p = 1.2916E−22
Righting Dam F (1,24) = 4.630 p = 0.042
Developmental Reflexes and Milestones Assessment reflex Sex × Dam × Drug F (4,74) = 2.518 p = 0.048
Mice were evaluated for achievement of physical and behavioral Litter Size F (1,26) = 9.335 p = 0.005
milestones from early development through early juvenile stage. Number of Dam F (1,156) = 7.015 p = 0.009
Weight was measured at 10 time points: P5, P7, P9, P11, USV calls Drug F (1,151) = 12.000 p = 0.001
P14, P23, P25, P27, and P30. A visual inspection of normal Dam × Drug F (1,159) = 5.223 p = 0.024
physical milestone attainment was performed with evaluation for Litter Size F (1, 159) = 3.064 p = 0.082
detached pinnae at P5 and eye opening at P14. Righting reflex Pitch range Drug F (1,169) = 8.456 p = 0.004
was assessed at P14 as follows: each mouse was placed prone onto (Hz) Dam × Drug F (1,169) = 13.528 p = 0.000315
its abdomen and quickly pronated 180◦ to its back in a smooth Age × Dam × Drug × Sex F (24, 319) = 1.385 p = 0.097
motion. The time for the mouse to right itself with all four paws Mean pitch Drug F (1,160) = 29.552 p = 1.9953E−7
positioned underneath the abdomen was recorded (Zanni et al., (Hz) Dam × Sex F (1,160) = 5.373 p = 0.022
2020). Each mouse underwent three timed trials, which were Drug × Sex F (1,160) = 5.354 p = 0.022
averaged for analysis. Sex × Dam × Drug F (1,160) = 8.365 p = 0.004
Sex × Dam × Drug × Age F (24, 301) = 1.617 p = 0.036
Statistical Analyses Sex F (1, 161) = 3.190 p = 0.076
SPSS (IBM, v.25) was used for all statistical analyses. Data Peak Dam F (1,163) = 5.632 p = 0.019
were screened for missing values, influential outliers, fit between power (dB) Drug F (1,171) = 10.327 p = 0.002
distributions and the assumptions of normality and homogeneity Dam × Drug F (1,172) = 12.399 p = 0.001
of variance. Variables that violated assumptions of normality Litter Size F (1,161) = 6.313 p = 0.013
(including number of USV calls, mean pitch, pitch range, and
Showing significant main and interaction effects. Age was grand mean-centered
peak power) were square root-transformed. Data were analyzed for analysis, where included. Litter size was included as a covariate.
using hierarchical linear models with sex clustered within litters
and age clustered within individual pups. Fixed factors were
dam, drug, sex and, where appropriate, age. Age was also treated groups for pinnae detachment at P5 or eye opening by P14. In
as a random repeated effect and was grand mean-centered for our analysis of weight, we found male mice weighed significantly
analysis. Interactions between the fixed factors are reported when more than females in all groups at all ages, and therefore, weight
significant. If an interaction effect was significant, p-values were data are segregated by sex (Figures 2C,D) from the full factorial
obtained from the hierarchical linear model for simple main linear mixed model including sex, drug, and duration as factors.
effects and reported for differences between different levels within Prolonged Oxy exposure led to an overall decrease in mean
the interaction. If sex had a significant main effect, findings are weight compared to prolonged Veh exposure, which was more
shown segregated by sex. As litter size can influence behavior and pronounced in male offspring. Prolonged Oxy-exposed male
litter cannot be separated from drug treatment in this study, all offspring exhibited significantly reduced weights compared to
models included litter size as a covariate. Probability value for prolonged Veh offspring post-weaning at P25, P27, and P30, with
all analyses was p < 0.05. Test statistics and analysis details are non-significant reductions at P23 (Figure 2E). Female prolonged
provided in Table 2. The datasets generated for this study are Oxy offspring showed significantly reduced weight compared
available upon reasonable request to the corresponding author. to prolonged Veh controls at P21, P23, and P25, with non-
significant reductions at P27 and P30 (Figure 2F). These data
indicate that overall prolonged Oxy exposure reduces weight
RESULTS
across development in male and female offspring, with the effect
on weight gain compounding once potentially compensatory
Oxycodone Impacted Developmental maternal care is lost after weaning.
Weight Trajectories Differentially by Sex We then evaluated the potential effects of early opioid
and Exposure Duration cessation (in utero Oxy) on weight gain in male and female
We examined the effects of Oxy administration on gross and offspring and once more found male offspring susceptible to Oxy
sensorimotor development in mice from birth throughout the effects. In contrast to prolonged exposure, an overall reduction in
early juvenile stage (Figures 2A,B). To evaluate general health weight was not observed with in utero Oxy exposure compared
and gross development, we assessed the appearance of physical to in utero Veh exposure. However, in utero Oxy males showed
milestones and weight. No differences were observed between a precipitous decrease in weight gain trajectory after weaning

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Minakova et al. Oxycodone Exposure Affects Early Life Neurodevelopment

A BIRTH
CROSS-FOSTERING WEANING
WEIGHT

E0 P0 P5 P7 P9 P11 P14 P21 P30

In utero
Prolonged Maternal Treament

B G IN UTERO OXY VS VEH

(.001)
(1.8E-6) H IN UTERO OXY VS VEH

(.001)

Pup Group (.042)

Weight (g)
Prolonged Veh

Weight (g)

Weight (g)
In utero Veh
Prolonged Oxy
In utero Oxy

WEANING
WEANING

WEANING
C MALES D FEMALES I OXY PROLONGED
(.002)
J OXY PROLONGED
VS IN UTERO (.026)
VS IN UTERO
(.053)

Weight (g)

Weight (g)
Weight (g)
Weight (g)

(.049)

WEANING
WEANING

WEANING
WEANING
(.011)
E PROLONGED OXY VS VEH
(.002)
F PROLONGED OXY VS VEH K VEH PROLONGED L VEH PROLONGED
(.011) (.089) VS IN UTERO (.063) VS IN UTERO (.035)

(.027) (.075) (.064)

(.075) (.022) (.033)
(.041) (.042)

Weight (g)

Weight (g)
Weight (g)

(.010) (.057)
Weight (g)

WEANING
WEANING

WEANING
WEANING

FIGURE 2 | Prolonged and in utero Oxy exposure, as well as cross-fostering, decreases weight gain after weaning. (A) Schematic of the treatment paradigm for
maternal Oxy exposure and weight measurements throughout development. (B) Line graph of weight in all offspring (sex, p = 0.002; drug, p = 0.021; age,
p < 0.000; sex × dam × drug × age, p = 1.2916E- 22 ). (C,D) Line graph of weight in (C) male and (D) female offspring. (E,F) Prolonged Oxy exposure, relative to
prolonged Veh exposure, led to significantly lower weights post-weaning in (E) male (P23, p = 0.075; P25, p = 0.027; P27, p = 0.011; P30, p = 0.002) and (F)
female offspring (P21, p = 0.010; P23, p = 0.041; P25, p = 0.022; P27, p = 0.075; P30, p = 0.089). (G,H) In utero Oxy exposure, relative to in utero Veh exposure,
led to significantly lower weights post-weaning in (G) male offspring only (P23, p = 0.042; P25, p = 0.001; P27, p = 0.001; P30, p = 0.000018), with no effects in (H)
female offspring. (I,J) In utero Oxy exposure with cross-fostering, relative to prolonged Oxy exposure, led to decreased weight in adolescence in (I) male pups only
(P5, p = 0.049; P25, p = 0.053; P27, p = 0.026; P30, p = 0.002), with no effects in (J) female offspring. (K,L) In utero Veh exposure with cross-fostering, relative to
prolonged Veh exposure, led to decreased weight post-weaning in (K) male (P27, p = 0.063; P30, p = 0.011) and (L) female offspring (P21, p = 0.057; P23,
p = 0.042; P25, p = 0.033; P27, p = 0.064; P30, p = 0.035). Closed circles depict mean weight, with litter size as a covariate (p = 0.002), while open circles depict
individual weights. Gray vertical line indicates date of weaning.

from the foster dam at P23, P25, P27, and P30 as compared to We have shown so far that Oxy exposure, compared to Veh,
in utero Veh controls (Figure 2G). Female in utero Oxy offspring decreased post-weaning weight following both long and short
showed no difference in weight across development compared to exposures. We next sought to determine how the duration of
in utero Veh controls (Figure 2H). Clinically, male infants are Oxy exposure influences weight by assessing the weight gain
more susceptible to NAS (Charles et al., 2017), so the precipitous trajectory differences between prolonged and in utero Oxy pups.
decrease in weight gain trajectory in the in utero Oxy male Comparisons between the male mice showed in utero Oxy pups
offspring may be associated with withdrawal symptomatology initially weighed more than prolonged Oxy pups at P5. However,
unmasked by cessation of care under a foster dam. after the in utero Oxy male pups were weaned at P21, their
We also examined weight trajectories between in utero and weights decreased relative to the prolonged Oxy group at P27 and
prolonged vehicle-exposed groups. In vehicle-exposed males, P30 (Figure 2I). No differences in weight gain between in utero
cross-fostering was associated with decreased weights in the in and prolonged groups were detected in female Oxy-exposed pups
utero Veh group at P30, with a trend toward decreased weight at (Figure 2J). Overall, early Oxy cessation was associated with
P27, relative to the prolonged Veh group (Figure 2K). Of interest, increased weights at very early post-natal ages, followed by weight
cross-fostered female pups (in utero Veh) weighed less compared loss in males at weaning.
to prolonged Veh female pups post-weaning at P23, P25, and P30
(Figure 2L). These findings suggest that cross-fostering alone can
influence post-weaning weight trajectories in a sex-dependent Prolonged Oxycodone Exposure Altered
manner. However, it is noteworthy that the decrease of weight Sensorimotor Reflex in Female Offspring
gain trajectories in the male in utero Oxy group persisted above Only
and beyond the observed decreased weights in male in utero Veh Righting reflex at P14 was examined as an assessment of
controls, indicating in utero Oxy exposure affects weight gain sensorimotor milestones, early gross locomotor abilities, and
when controlling for cross-fostering (Figure 2G). general strength (Figures 3A,B). In males, no difference in

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A BIRTH RIGHTING
CROSS-FOSTERING REFLEX WEANING

E0 P0 P5 P7 P9 P11 P14 P21 P30

In utero
Prolonged Maternal Treament

B .

In utero In utero Prolonged Prolonged

oxy veh oxy veh

C MALES D FEMALES
. .
(.036)

.
(.032) . (.024)

. .

. .

. .

. .

. .

In utero In utero Prolonged Prolonged In utero In utero Prolonged Prolonged

oxy veh oxy veh oxy veh oxy veh

FIGURE 3 | Oxy exposure delays maturing of sensorimotor reflexes. (A) Schematic of the treatment paradigm for maternal Oxy exposure and righting reflex
measurement throughout development. (B) Mean latency to right in all offspring (sex × dam × drug × age, p = 0.048; dam, p = 0.042). (C,D) Mean latency to right
in (C) male and (D) female offspring. Prolonged Oxy exposure led to significantly longer latency to right relative to in utero Oxy (p = 0.032) in (C) male pups and
relative to prolonged Veh (p = 0.024) in (D) female pups. Male pups exposed to prolonged Veh also show longer latency to right relative to in utero Veh (p = 0.036).
Mean latencies, with litter size as a covariate (p = 0.005), while open circles depict individual latencies. Error bars represent standard error.

latency to right was noted between the prolonged Oxy or Oxycodone Exposure Disrupts Early
prolonged Veh groups (Figure 3C). An increased latency to right Communicative Behaviors
was demonstrated in prolonged Oxy male pups compared to in Language delays have been demonstrated in toddlers with
utero Oxy. However, cross-fostering may have a confounding prenatal opioid exposure (Conradt et al., 2019). Therefore,
effect on the righting reflex in male pups, since prolonged we assessed early affective and communicative behaviors by
Veh males also exhibited an increased latency to right relative evaluating maternal isolation-induced USVs. USVs are an
to in utero Veh males. In females, prolonged Oxy pups affective and communicative response that elicits maternal search
exhibited significantly increased latency to right relative to and retrieval, lactation, and caretaking behaviors (Haack et al.,
prolonged Veh controls (Figure 3D). No significant differences 2009; Maloney et al., 2018a). As a result, characterization of
in the righting reflex were observed in the in utero Oxy or quantity and quality of USV calls has been used in the rodent
in utero Veh female offspring. Together, these data indicate literature as a model for investigating early communicative
that females, but not males, are susceptible to the effects of deficits (Enga et al., 2016). Here, we quantified USV production
prolonged developmental Oxy exposure on attainment of the and spectrotemporal features to examine the influence of Oxy
sensorimotor reflex. on early communicative behaviors during the first 2 weeks of

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Minakova et al. Oxycodone Exposure Affects Early Life Neurodevelopment

life (Figure 4A). Overall, we detected a highly significant effect compared to prolonged Veh females (Figure 5C). However,
of continued Oxy exposure on USV production. Specifically, female in utero Oxy offspring did exhibit USVs with significantly
prolonged Oxy pups produced significantly fewer USVs relative lower mean pitch relative to in utero Veh. A similar non-
to prolonged Veh pups and in utero Oxy pups (Figure 4B), which significant reduction in mean pitch was observed in USVs
persisted from P5 through P11 (Figure 4C), an age at which the produced by in utero Oxy males relative to in utero Veh
C57Bl/6J strain used here still has a detectable call rate (Rieger males (Figure 5B). Thus, in utero Oxy exposure was associated
and Dougherty, 2016). While we did not see as substantial of a with changes in affective components of communication, the
peak at P7/P9 as we normally see in these experiments, the effect significance of which warrants further investigation. Since opioid
of prolonged Oxy was consistent regardless. withdrawal has been associated with high-pitched crying and
Beyond call numbers, spectrotemporal USV features such increased agitation, we also assessed for alterations in USV peak
as duration, pitch frequency, and power (loudness) inform power. In utero Oxy exposure resulted in louder USV calls
of an affective component to USV characteristics (Wöhr and compared to those produced by prolonged Oxy pups and in utero
Schwarting, 2013). In previous analyses of USV spectrotemporal Veh controls (Figure 5D). The increased peak power, or loudness,
features in mouse models of intellectual and developmental in in utero Oxy pup calls only may be temporally related to onset
disorder risk factors and early drug exposure models, we and of withdrawal after drug cessation at P0, relative to the prolonged
others have demonstrated the vulnerability of these features Oxy pups which are weaned off the drug at P21.
to genetic and early environmental insults (Dougherty et al., Overall, prolonged Oxy pups demonstrated significant
2013; Maloney et al., 2018a,b; Kopp et al., 2019). We examined decreases in number of USVs along with a narrower pitch
call features including call duration, pitch range and mean, frequency range and mean pitch in male pups. In utero Oxy
peak power, and fraction of calls with a pitch jump. Prolonged pups produced a similar number of USVs compared to controls,
Oxy administration narrowed the USV pitch range compared yet those calls were louder than controls and prolonged Oxy
to USVs produced by in utero Oxy pups and prolonged Veh calls, and lower in mean pitch when produced by females.
controls (Figure 5A). Prolonged Oxy exposure also led to a Together, our ontogenetic model of in utero Oxy exposure
highly significant reduction in mean pitch of USVs in prolonged demonstrates some alterations in loudness of affective calls,
Oxy male pups compared to in utero Oxy and prolonged Veh while the prolonged Oxy exposure further shows alterations in
males (Figure 5B). Interestingly, USVs produced by prolonged number and spectrotemporal features of early communicative
Oxy female pups did not show a significant difference in pitch and affective behaviors.

A USV
BIRTH
{

CROSS-FOSTERING WEANING Pup Group

Prolonged Veh
E0 P0 P5 P7 P9 P11 P14 P21 P30
In utero In utero Veh
Prolonged Maternal Treament Prolonged Oxy
In utero Oxy
B (.0001) C
(.002)

In utero In utero Prolonged Prolonged P5 P7 P9 P11

oxy veh oxy veh

FIGURE 4 | Prolonged Oxy exposure decreases pup USV call production. (A) Schematic of the treatment paradigm for maternal Oxy exposure and USV
measurements throughout development. (B) Cumulative means number of USV calls (dam, p = 0.009; drug, p = 0.001; dam × drug, p = 0.024). Prolonged Oxy
exposure led to decreased number of calls relative to prolonged Veh (p = 0.000126) and relative to in utero Oxy exposure (p = 0.002). (C) Line graph of mean call
number at all time points. Thick bars and closed circles depict mean call number, with litter size as a covariate, and open circles depict individual call numbers. Error
bars represent standard error.

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Minakova et al. Oxycodone Exposure Affects Early Life Neurodevelopment

A
(.005)
B MALES
(.015)
(.000012)
(.059) (4.5E-9)

In utero In utero Prolonged Prolonged In utero In utero Prolonged Prolonged

oxy veh oxy veh oxy veh oxy veh
C FEMALES D
(.001)
(.016) (.000002)

In utero In utero Prolonged Prolonged In utero In utero Prolonged Prolonged

oxy veh oxy veh oxy veh oxy veh

FIGURE 5 | Prolonged and in utero Oxy exposure affect spectrotemporal features of pup USV calls. (A) Cumulative means of pitch range (Hz) of USV calls (drug,
p = 0.004; dam × drug, p = 0. 000315; sex × dam × drug × age, p = 0.097). Prolonged Oxy exposure led to lower pitch range relative to prolonged Veh
(p = 0.000012) and relative to in utero Oxy exposure (p = 0.005). (B,C) Cumulative mean pitch (Hz) in (B) male and (C) female offspring (drug, p = 1.9953E- 7 ;
sex × dam, p = 0.022; sex × drug, p = 0.022; sex × dam × drug, p = 0.004; sex × dam × drug × age, p = 0.036; sex, p = 0.076). Prolonged Oxy exposure in (B)
male pups led to decreased pitch relative to prolonged Veh (p = 4.4816E- 9 ) and relative to in utero Oxy (p = 0.015). In utero Oxy exposure led to a marginal decrease
in pitch relative to in utero Veh (p = 0.059) in (B) male pups and a significant decrease in (C) female pups (p = 0.016). (D) Cumulative means of peak power (dB)
(dam, p = 0.019; drug, p = 0.002; dam × drug, p = 0.001). In utero Oxy exposure leads to significantly higher peak power relative to prolonged Oxy exposure
(p = 0.001) and relative to in utero Veh (p = 0.000002). Thick bars depict means, with litter size as a covariate, while open circles depict individual measurements.
Error bars represent standard error.

DISCUSSION post-natal exposure on early development. However, many

infants with NAS experience decreased bonding time and
Here we present a novel model to investigate the ontogenetic skin–skin contact with the biological mother secondary to
impact of in utero only versus prolonged mitigating opioid socioeconomic barriers and are cared for by healthcare staff
exposure on early neurodevelopmental outcomes, while for NAS. This type of setting may result in an environmental
controlling for confounding factors present in clinical stressor to the neonate due to inconsistent maternal contact,
observational studies. The utilization of a biological dam frequent alteration of caregivers, along with a higher incidence
and cross-foster dam in our novel model of ontogenetic rodent of foster care placement following hospital discharge in neonates
exposure was based on an attempt to parallel opioid exposure with maternal history of drug use (Brundage and Levine,
through a method most consistent with clinical management 2019). As a result, we rationalized a cross-foster approach
and observations. Active maternal bonding and breast-feeding would be a feasible model for evaluating the effects of in utero
has been shown to decrease hospitalization length, decrease rates opioid exposure on developmental impact in the in utero Oxy
of pharmacotherapy administration, and decrease NAS severity group.
in the NICU (Rosenthal and Baxter, 2019). The prolonged Oxy In utero Oxy exposure decreased weight gain trajectory
group remained with the biological dam and was weaned off following weaning from foster dams, in male offspring.
Oxy through lactation, allowing for the assessment of continued Further, in utero Oxy-exposed male and female pups

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Minakova et al. Oxycodone Exposure Affects Early Life Neurodevelopment

showed alterations in the spectrotemporal features of USVs. feeding behaviors secondary to Oxy exposure, since the opioid
Meanwhile, offspring with prolonged Oxy exposure until system has a strong role driving food intake homeostasis
weaning at P21 demonstrated poorer neurodevelopmental (Valbrun and Zvonarev, 2020).
outcomes compared to mice exposed only until birth. Early Oxy cessation significantly decreased weight gain
Notably, continued post-natal Oxy exposure was associated trajectory in a sex-specific manner not observed in the prolonged
with decreased weight gain trajectory, impaired motor Oxy exposure cohort. Interestingly, in utero Oxy exposure led
reflexes, and abnormal early communication behaviors. to significantly higher weight at P5 in males, though both
Both male and female offspring in prolonged Oxy groups show comparable averages at P21. Female weights
exposure groups had decreased weight gain following following in utero Oxy exposure do not show any significant
weaning at P21, with delayed latency to right observed weight differences from controls. Males exposed to Oxy in
in females. In addition, prolonged Oxy-exposed offspring utero showed a rapid, significant and persistent decrease in
had significantly reduced USV production and alterations weight gain following weaning. Since the in utero exposure
in spectrotemporal features reflecting affective and early group was cross-fostered at birth to a non-drug exposed
communicative impairment. dam, normal weight gain trajectory was potentially maintained
through adequate maternal care from the foster dam. A further
explanation could be the “two-hit” hypothesis in which early
Oxycodone Exposure Impairs Attainment life susceptibility, such as abstinence and withdrawal following
of Physical and Motor Development in utero Oxy exposure, compounded with the post-natal stress
Decreased fetal growth can be used as a general indicator of of weaning precipitated a weight loss phenotype (Nederhof
harmful in utero drug exposures (Haight, 2018). The association and Schmidt, 2012; Peña et al., 2019). Perhaps, males are
between birth weight and decreased infant survival is highly more sensitive to early life stressors and may have long-
robust, though the underlying biological mechanisms are not term consequences from in utero opioid exposure compared
always clearly understood (Basso et al., 2006; Yazdy et al., to females. Male human neonates are more at risk for
2015). We did not obtain birth weights at P0 in order developing NAS compared to females, so long-term changes
to minimize animal handling which can reduce behavioral in opioid circuitry governing feeding behaviors could explain
and hormonal reactivity to stress and confound behavioral the abnormal weight trajectory in male mouse offspring post-
testing results (Luchetti et al., 2015). Initial weight assessment weaning (Charles et al., 2017). Prospective human studies
occurred at P5 with no observed effect of in utero or evaluating the long-term effects of in utero opioid and effects
prolonged Oxy exposure relative to Veh controls. Overall, on weight trajectory during childhood through adulthood have
human literature shows low birth weight in the setting of not been performed to our knowledge. The altered weight
maternal methadone use during pregnancy, but no evidence of trajectory findings in both the in utero Oxy and prolonged Oxy
low birth weight following in utero exposure to other opioids suggest a potential role of in utero opioid exposure on long-
including codeine, tramadol, hydrocodone, or Oxy (Yazdy et al., term impact on growth that requires further evaluation in the
2015). Thus, our findings are consistent with existing Oxy human literature.
human literature that shows no reported association between In the vehicle-treated groups, cross-fostered pups showed
Oxy and low birth weight in neonates (Kelly et al., 2011; decreased weight gain trajectory after weaning relative to pups
Yazdy et al., 2015). reared by a biological dam. Our observation of decreased
Interestingly, female offspring exposed to continued Oxy, weight gain following weaning in Veh-exposed cross-fostered
relative to Veh controls, showed a significant decrease in pups may be related to potential alterations in emotionality
weight after weaning at P21, which persisted through P25, and stress responses secondary to confounders involved with
early juvenile development in mice. Neurodevelopmental cross-fostering, such as early handling (Luchetti et al., 2015).
processes occurring at these ages in the rodent occur in the Regardless, the effect of cross-fostering on weight did not mask
human at approximately 2–3 years and pre-pubertal juvenile our ability to identify effects of in utero Oxy exposure on
ages, respectively (Semple et al., 2013). Male weights, after weight in males. Indeed, the effect of in utero Oxy exposure
prolonged exposure, showed a significant decline on P25– on weight occurred at additional younger ages and with a
P30 as compared to Veh controls. Since these offspring were larger magnitude than in utero Veh exposure and persisted
separated from the dam partially through a period during when controlling for effects of litter and cross-fostered dam
which they are naturally weaning from nursing (König status. Similarly, in females, weight reduction was observed
and Markl, 1987), it is unclear whether the acute onset of following prolonged Oxy exposure compared to prolonged Veh
decreased weight gain after separation from the dam at P21 controls, and in utero Veh exposure compared to the prolonged
is related to Oxy withdrawal symptomatology or not. It Veh exposure control group. Despite the independent effect
is certainly plausible. However, human studies of prenatal on weight by cross-fostering, this method was valuable in
opioid exposure have described decreased adaptive behaviors allowing us to cease Oxy exposure at birth and thus observe
during infancy through toddlerhood (Conradt et al., 2019). effect of Oxy limited to in utero development. Furthermore,
Cessation of care from the biological dam may potentially have these findings highlight the importance of including proper
uncovered deficiencies in self-care of offspring. The decreased cross-foster control groups in study designs for accurate
weight gain post-weaning could also stem from maladaptive interpretation of results.

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Prenatal opioid exposure has also been associated with delays Ontogenetic Oxycodone Exposure May
in attainment of motor milestones in children. A meta-analysis Delay Early Communicative Behaviors
by Yeoh et al. (2019) detected significant delays in motor
outcomes in children aged 0–6 years that experienced prenatal and Alter Spectrotemporal Features of
opioid exposure. We assessed the righting reflex at P14, the USVs
beginning of the visual critical period, and an age at which mice Currently, studies exploring the effects of prenatal opioid
should be fully ambulatory (Williams and Scott, 1954; Hooks exposure on language development in children demonstrate
and Chen, 2007; Feather-Schussler and Ferguson, 2016). The equivocal results (Conradt et al., 2019). Previous work has
righting reflex corrects the orientation of the body from an off identified language development impairments following in utero
axis position (Troiani et al., 2005). Proper execution of the reflex exposure to methadone or heroin (Conradt et al., 2019).
requires a combination of visual, vestibular, and somatosensory However, several of these analyses did not control for important
system inputs to make appropriate postural adjustments through confounders such as socioeconomic status or maternal use
neural pathways within the brain and cerebellum. Females in of other substances. In general, large epidemiological studies
the prolonged Oxy exposure group had significantly increased evaluating impact of prenatal opioid exposure on language
latency to right compared to Veh controls. There was no development have been difficult to perform due to various
significant difference in righting reflex latency between females confounding environmental variables including quality of
exposed to Oxy and Veh in utero. Hence, only continued post- caregiving, parental education level, and socioeconomic factors.
natal Oxy exposure seems to result in delayed sensorimotor Despite the advantage of limiting confounds through the use
development. Previously, increased latency to right has been of rodent models, and indications that communicative circuits
demonstrated with in utero morphine exposure in both male are conserved between rodents and humans (Arriaga et al.,
and female rat pups, but rodent studies evaluating effects of 2012), there have been minimal rodent studies evaluating the
opioids on the righting reflex have been limited (Slamberová effects of prenatal opioid exposure on early communicative
et al., 2005). Though the exact mechanism of action is unclear, behaviors to date. Isolation-induced USVs are a strongly
significant evidence in the literature demonstrates selective conserved adaptive response of young rodent pups to elicit
vulnerability of cerebellar granule neuroblasts to opioids, along maternal caregiving responses (Haack et al., 2009). Our observed
with opioids’ negative effects on neuronal somatosensory cortex collective decrease in mean USV production following prolonged
development (Seatriz and Hammer, 1993; Hauser et al., 2003). Oxy exposure, compared to in utero Oxy exposure and
Multiple studies have further linked opioid exposure to increased Veh controls, is suggestive of impaired early communication.
apoptosis and decreased differentiation of Purkinje cells in Previous studies have shown evidence for neuropharmacological
the cerebellum (Hauser et al., 2003). Additionally, perinatal modulation of USVs through alteration of mood or arousal
morphine treatment in rats decreased total number of neurons state (Vivian and Miczek, 1991, 1993b; Maloney et al., 2018a).
in the somatosensory cortex (Seatriz and Hammer, 1993). In addition to serving as an analgesic, Oxy is also a sedative
Thus, it is possible multiple circuits are mediating the effect. and an anxiolytic agent, which may decrease USV calling
Overall, the sex-specificity of the effect is also interesting. This in the prolonged Oxy exposure pups by actively suppressing
observed sex bias could be related to sex-specific dimorphic USV circuitry secondary to reduced reactivity to surrounding
alterations in catecholamine levels in the cerebellum as shown environmental stressors and decreased respiratory rate (Rao
in a previous study of prenatal morphine exposure (Vathy and Desai, 2002). Furthermore, the interplay between pup
et al., 1995). Interestingly, cross-fostering resulted in a shorter communication and maternal care is complicated. In vocally
latency to exhibit the righting reflex in males compared to impaired pups, decreased USV production has been shown
male pups reared by biological dams. This reflex is dependent to result in maternal neglect, because without calls the dams
on vestibular inputs that sense head movement but lacks cannot locate the pups outside of the nest (Hernandez-
cortical involvement. The righting reflex is frequently used Miranda et al., 2017). Thus, maternal care may be reduced in
in studies evaluating anesthesia reversal, sepsis survival or response to decreased USVs calling by prolonged Oxy-exposed
traumatic brain injuries (Gao and Calderon, 2020). In general, pups. Maternal care has also been reported to be attenuated
an increased latency to right is associated with decreased following morphine exposure during pregnancy, with a study
arousal or underlying neurological impairment but a decreased reporting increased time to pup retrieval, decreased nursing
latency has not been shown to correlate with any particular and cleaning of pups, and increased maternal self-care time
pathophysiological condition or stressed anxiety state (Gao (Slamberová et al., 2001). This reduced maternal care could
and Calderon, 2020). Decreased latency to right is unlikely further disrupt neurodevelopment of the pup, and thus be a
to be linked to a behavioral or neurological impairment but possible indirect influence on later adult behaviors. However,
could be secondary to a non-pathological increased arousal currently the literature on maternal care following Oxy exposure
state due to cross-fostering in these litters. Overall, the shorter is conflicting. Two recent rodent studies found no changes in
latency seen to right in the cross-fostered group is likely maternal behavior or maternal motivation following dam Oxy
not indicative of altered development of sensorimotor reflexes. exposure (Watters et al., 2020; Zanni et al., 2020). Notably,
However, future studies will be necessary to delineate the in our study, in utero Oxy exposed offspring demonstrated
mechanisms underlying this behavioral phenotype, and its sex- comparable USV means to Veh controls. Normal USV call
specific expression. production in the in utero Oxy offspring at P5 suggests that,

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Minakova et al. Oxycodone Exposure Affects Early Life Neurodevelopment

in the prolonged exposure group, Oxy reduces USV production Oxy cessation can be appropriately modeled in rodents. If so,
by acute suppression of USV circuits. Finally, the adequate testing of novel agents for treatment of withdrawal symptoms
weight trajectories before weaning in the Oxy group further will be possible, with the goal of limiting continued post-natal
indicate appropriate dam care. Hence, we would hypothesize opioid exposure given potential long-term side effects of early
that maternal care likely did not result in the behavioral post-natal opioid administration on neurodevelopment (Attarian
deficits observed. Still, to our knowledge, the direct impact of et al., 2014). Finally, few mouse models of in utero opioid
Oxy exposure on maternal behaviors has not been examined. exposure currently exist, with the majority of the literature
Furthermore, there is currently scant literature considering utilizing rat perinatal opioid models. Thus, our model will
the effect of ongoing Oxy exposure on maternal behavior in facilitate genetic manipulations using established cutting-edge
rodents, with even more limited studies utilizing a mouse genetic tools available in the mouse to broaden understanding
model. This warrants an individual study to assess reciprocity of of the mechanisms mediating consequences of early opioid
interactions between pup and dam. Overall, deficits in observed exposure on neurodevelopment.
USV production could be the result of a combination of
factors, including acute drug effects on circuits, influence of
dam care, and opioid-mediated effects on neural development
and communication. DATA AVAILABILITY STATEMENT
Affective characteristics of USVs in rodents are generally
The raw data supporting the conclusions of this article will be
thought to communicate different emotional states, such as
made available by the authors, without undue reservation.
aggression or pain. Rodent USVs are particularly interesting
as they occur only in salient situations such as exposure to
painful stimuli, maternal behavior, sexual behavior, or aggression.
As has been well-characterized in rats, affective features of ETHICS STATEMENT
rodent USVs may be reflected by alterations in duration,
pitch, frequency, and loudness (dB) of the calls (Vivian and The animal study was reviewed and approved by the
Miczek, 1993a,b). In our developmental cohort, pups exposed Institutional Animal Care and Use Committee of Washington
to prolonged Oxy demonstrated decreased mean pitch (in males University in St. Louis.
only), and narrower pitch range. Interestingly, a previous study
administered morphine to adult rats and observed decreased
USV pitch, duration, and rate (Vivian and Miczek, 1993a). The
decreased pitch and pitch range could be a result of Oxy’s AUTHOR CONTRIBUTIONS
depressive effects on respiration, or of Oxy’s anxiolytic drug
EM contributed to the conceptualization, methodology,
properties which may dampen USV circuity. For the in utero
investigation, data curation, writing (original draft preparation
Oxy exposure group, we predicted increased USV production
and editing), and funding acquisition for this project.
along with increased duration, pitch, frequency, and amplitude
SS contributed to the methodology, validation, formal
of USVs secondary to discomfort associated with withdrawal.
analysis, writing (original draft preparation and editing),
The assumption is based on the current human description
and visualization for this project. MM, KB, and KM contributed
of NAS characterized by human neonates exhibiting prolonged
to the methodology for this project. JD contributed to the
periods of high-pitched crying and inconsolability secondary to
conceptualization, methodology, validation, resources, writing
withdrawal (Anbalagan and Mendez, 2020). However, human
(draft editing), supervision, project administration, and
studies do not formally characterize the spectrotemporal features
funding acquisition for this project. RA-H contributed to
of crying in infants with NAS, so the description of a high-
the conceptualization, methodology, validation, writing
pitched cry may be subjective in nature. We found that that
(draft editing), supervision, project administration, and
continued opioid exposure during the post-natal period in the
funding acquisition for this project. SM contributed to the
prolonged Oxy group resulted in less USV calls compared to
conceptualization, methodology, data curation, resources,
both the in utero Oxy and vehicle controls. We did not expect
writing (draft editing), validation, supervision, project
the prolonged Oxy group to be undergoing withdrawal at this
administration, and funding acquisition for this project. All
time, thus we did not hypothesize an increase in call rate in this
authors contributed to the article and approved the submitted
group. A reduction in call rate is likely more consistent with an
version.
acute dampening of the circuits mediating USV production by
the continued presence of Oxy in these pups during recordings.
Indeed, reduced call rates were observed in rat pups following
injection of µ- or δ-opioid receptor agonists (Carden et al., FUNDING
1991). Future studies are needed to evaluate the impact of early
suppression of these social communicative circuits by opioid Support for this study was provided by the NIH/National
agonists on social behavior consequences at older ages. Center for Advancing Translational Sciences (NCATS) grant
Our novel Oxy administration paradigm enables future ULITR002345 at Washington University School of Medicine
exploration of withdrawal periods, to determine if NAS following (RA-H and SM), a Seed grant 20-186-9770 was funded by

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Minakova et al. Oxycodone Exposure Affects Early Life Neurodevelopment

the Center for Clinical Pharmacology (CCP), Washington ACKNOWLEDGMENTS

University School of Medicine and St. Louis College of
Pharmacy (RA-H, JD, EM, and SM), and the NICHD/NIH (P50 This manuscript has been released as a pre-print at bioRxiv
HD103525, IDDRC@WUSTL). (Minakova et al., 2020).

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administration during pregnancy attenuates maternal behavior. absence of any commercial or financial relationships that could be construed as a
Psychoneuroendocrinology 26, 565–576. doi: 10.1016/s0306-4530(01)00 potential conflict of interest.
012-9
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the supine position and preparatory eye movements. Acta Otolaryngol. 125, Dougherty, Al-Hasani and Maloney. This is an open-access article distributed
499–502. doi: 10.1080/00016480510036448 under the terms of the Creative Commons Attribution License (CC BY). The use,
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jocmr4066 distribution or reproduction is permitted which does not comply with these terms.

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 ORIGINAL RESEARCH
published: 21 July 2021
doi: 10.3389/fnbeh.2021.691578

The Long-Term Effects of Neonatal

Inflammatory Pain on Cognitive
Function and Stress Hormones
Depend on the Heterogeneity of the
Adolescent Period of Development in
Male and Female Rats
Irina P. Butkevich 1* , Viktor A. Mikhailenko 1 , Elena A. Vershinina 2 and Gordon A. Barr 3,4
1
Laboratory of Ontogenesis of the Nervous System, Pavlov Institute of Physiology, Russian Academy of Sciences, Saint
Petersburg, Russia, 2 Department of Information Technologies and Mathematical Modeling, Pavlov Institute of Physiology,
Russian Academy of Sciences, Saint Petersburg, Russia, 3 Department of Anesthesiology and Critical Care Medicine,
The Children’s Hospital of Philadelphia and the Perelman School of Medicine, Philadelphia, PA, United States, 4 Department
of Psychology, University of Pennsylvania, Philadelphia, PA, United States

Exposure to stress at an early age programs the HPA axis which can lead to cognitive
Edited by:
Carla Cannizzaro,
deficits in adults. However, it is not known whether these deficits emerge in adulthood or
University of Palermo, Italy are expressed earlier in life. The aims of the study were to investigate (1) the immediate
Reviewed by: effects of early injury-induced stress in one-day-old (P1) and repeated stress on at P1
Federica Filice, and P2 rat pups on plasma corticosterone levels; and (2) examine the subsequent
Université de Fribourg, Switzerland
Bruno Jacson Martynhak, long-term effects of this early stress on spatial learning and memory, and stress
Federal University of Paraná, Brazil reactivity in early P26-34 and late P45-53 adolescent male and female rats. Intra-plantar
*Correspondence: injection of formalin induced prolonged and elevated levels of corticosterone in pups and
Irina P. Butkevich
[email protected]
impaired spatial learning and short- and long-term memory in late adolescent males
and long-term memory in early adolescent females. There were sex differences in late
Specialty section: adolescence in both learning and short-term memory. Performance on the long-term
This article was submitted to
Pathological Conditions,
memory task was better than that on the short-term memory task for all early adolescent
a section of the journal male and female control and stressed animals. Short-term memory was better in the
Frontiers in Behavioral Neuroscience
late age control rats of both sexes and for formalin treated females as compared with
Received: 06 April 2021
the early age rats. These results are consistent with an impaired function of structures
Accepted: 21 June 2021
Published: 21 July 2021 involved in memory (the hippocampus, amygdala, prefrontal cortex) after newborn pain.
Citation: However, activation of the HPA axis by neonatal pain did not directly correlate with
Butkevich IP, Mikhailenko VA, spatial learning and memory outcomes and the consequences of neonatal pain remain
Vershinina EA and Barr GA (2021) The
Long-Term Effects of Neonatal
are likely multi-determined.
Inflammatory Pain on Cognitive
Keywords: neonatal pain, corticosterone, adolescence, spatial memory, sex differences, spatial learning
Function and Stress Hormones
Depend on the Heterogeneity of the
Abbreviations: The HPA axis, the hypothalamo-pituitary-adrenocortical system; the HPG axis, the hypothalamus-pituitary-
Adolescent Period of Development gonadal system; MWM, the Morris water maze; CFA, complete Freund’s adjuvant; GD, gestational day; P1, P2, postnatal
in Male and Female Rats. day1, postnatal day 2; PVN, paraventricular nucleus; CA1 hippocampus; GR, glucocorticoid receptor; CRH, corticotrophin-
Front. Behav. Neurosci. 15:691578. releasing hormone; ACTH, adrenocorticotropic hormone; NMDA receptor, the N-methyl-D-aspartate receptor; PFC,
doi: 10.3389/fnbeh.2021.691578 prefrontal cortex.

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Butkevich et al. Neonatal Pain Effects Cognition in Adolescence

INTRODUCTION cortex, hippocampus and amygdala (Brenhouse and Andersen,

2011), reorganization in the hormonal, neurotransmitter, and
The longitudinal analysis of outcomes of pain for premature reproductive systems (McCormick et al., 2004, 2016; Romeo
infants in a neonatal clinic found that pain in these infants and McEwen, 2006; Romeo, 2010; McCormick, 2011), and
resulted in impaired cognitive function in the child and behavioral and cognitive maturation (McCormick and Mathews,
adolescent (Haley et al., 2006; Grunau et al., 2009; Doesburg 2010; McCormick et al., 2012; Siddiqui and Romeo, 2019). The
et al., 2013; Vinall et al., 2014; Chau et al., 2017). Whereas the trajectory of these processes in adolescence can be altered by
long-term influence of non-pain stress (e.g., maternal separation, neonatal stress, including clinically necessary painful procedures
mother and offspring isolation, early handling or the limited (Amaral et al., 2015; Chen et al., 2016; Mooney-Leber and
nesting model) on cognitive behavior and memory in animal Brummelte, 2017; Xia et al., 2020), which can modify behavior
models is abundant in the literature (Krugers and Joëls, 2014; and cognitive abilities in adolescents (Mooney-Leber and
Schroeder et al., 2018; Bonapersona et al., 2019; Cordier et al., Brummelte, 2020; see Williams and Lascelles, 2020 for a review).
2021), the long-term consequences of pain stress have been Thus, early-life pain can give rise to numerous clinical, social,
understudied (Khawla et al., 2017), especially considering it educational problems in adolescents which may differ from those
prevalence in the clinical setting (Ranger and Grunau, 2014; of adults.
Mooney-Leber and Brummelte, 2017) and the strong connection Based on behavioral and the nervous system maturation,
between neonatal pain and disturbances of central nervous adolescence in rats ranges from 28-48 days (P28-48) with adults
system maturation (Schwaller and Fitzgerald, 2014; Brewer and defined as P60 and older (Spear, 2000). A more granular view
Baccei, 2020; Williams and Lascelles, 2020). The neonatal period of this critical period of development divides adolescence into
is a critical period of development due to the rapidly changing three sub-periods: early P21-P34, middle P34-P46 and late P46-
neurobiological processes, which result in altered sensitivity to P59 (Tirelli et al., 2003). These epochs within adolescence vary
external stimuli and a high level of plasticity of the nervous somewhat depending on which criteria are used (morphogenetic,
system (Lupien et al., 2009). The nervous system during the behavioral, neurohormonal, and neural). Much attention has
neonatal period is highly sensitive to painful stimuli (Goksan been paid to the different neurobiological systems within the
et al., 2015; Williams and Lascelles, 2020) with the critical period age-related intervals of adolescence (McCormick et al., 2016,
for later effects on adult pain ending around the end of the 2020; Bailey et al., 2020; Marcolin et al., 2020; Gore-Langton
first week of life in the rat (Ren et al., 2004). It is known that et al., 2021). One of the main factors determining the timing
repeated pain in the neonatal period disrupts the balance between of adolescence is the maturation of the hypothalamic-pituitary-
the processes of excitation and inhibition in the central nervous adrenal system (the HPA axis), and its feedback mechanisms,
system (Brewer and Baccei, 2020), modifies brain development which continue to mature during adolescence. Glucocorticoids,
(Schwaller and Fitzgerald, 2014), programming the HPA axis the secretion of which is controlled by the HPA axis, affect
(Mooney-Leber and Brummelte, 2017), therefore modifying brain development, including neurogenesis, synaptogenesis, and
multiple types of behavior (Williams and Lascelles, 2020). cell death (McEwen, 2000). The development of the HPA axis
Moreover, there is a close neuroanatomical and physiological during adolescence may be modified by stress experienced
interaction between pain and the HPA axis, which is regulated early in life (Van Bodegom et al., 2017). The timing of
in part by the hypothalamus, amygdala, hippocampus, prefrontal adolescence can also be determined by sexual maturation and
cortex (PFC), and thalamus (Ulrich-Lai and Herman, 2009; its relationship with the HPA axis (McCormick and Mathews,
Victoria et al., 2013; Timmers et al., 2019). The features of 2007). The pubertal onset of sexual maturation, determined
this interaction in response to damaging stimuli at an early age by vaginal opening in female rats and preputial separation in
are poorly understood. Because there are multiple physiological male rats, occurs earlier in females (P35 ± 2) than in males
systems affecting pain and the HPA axis in the neonatal period (P42 ± 2) (McCormick and Mathews, 2007, 2010), making
(Mooney-Leber and Brummelte, 2017, 2020; Van Bodegom et al., it important to include males and females at different stages
2017) the data on the effect of pain on the HPA axis, both in of adolescence in studies. The peripheral steroid hormone of
the clinic and in animals, are incomplete (Walker et al., 2003; the HPA axis, cortisol in humans, corticosterone in rodents,
Victoria et al., 2013; Victoria and Murphy, 2016). Moreover, the plays an important role in learning and memory (Akirav et al.,
effects of stress and pain on the HPA are age dependent, even in 2004). The effects of inflammatory pain on the secretion of
infancy (Van Bodegom et al., 2017). Further research is needed corticosterone in newborns, and the consequences of these effects
to clarify the relationship between neonatal pain and the HPA on the development of brain structures involved in cognitive
axis, since there is a multifaceted relationship between the type of function and in the formation of HPA axis before puberty
pain, severity, gender, and age of pain exposure and the response are still largely unknown. Repeated prick needles of the pad
of the HPA axis. of hind paws have been used as a pain stressor in newborn
In adolescence, problems of neurodevelopment and behavior rodents (Anand et al., 1999; Walker et al., 2003; Nuseir et al.,
caused by infant stressful influences first appear. To correct the 2015, 2017; Ranger et al., 2019); however, the results are often
behavior during the adolescent period, it is important to know the mixed. For example, repeated needle pricks to the pad of the
features of the age-related intervals of an adolescent development hind paws of newborn rodents did not change the level of
period. Adolescence is characterized by intensive processes of corticosterone in adult rats (Anand et al., 1999; Walker et al.,
synaptogenesis and myelinization, especially in the prefrontal 2003), but did decrease stress reactivity of the HPA axis in

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adolescence, and impaired the ability to retain spatial memory The aim here was to investigate the immediate effect of
in prepubertal male rats (Chen et al., 2016). In prepubertal formalin-induced pain in one-day-old and two-day-old (P1, P2)
mice, similar injury impaired spatial learning and short-term rat pups on corticosterone level in blood plasma and the long-
(Nuseir et al., 2015) and long-term (Nuseir et al., 2017) memory, term effects of early-life pain stress on spatial learning and
whereas in adult mice, it impaired short-term memory, but memory in the Morris water maze, and stress reactivity of the
did not alter spatial learning ability and long-term memory HPA axis in male and female rats of early P26-34 and late P45-53
(Ranger et al., 2019). age groups of adolescence.
We are aware of only a few rodent studies that investigated
the effect of neonatal inflammatory pain on memory. For
instance, inflammatory pain caused by the intraplantar injection MATERIALS AND METHODS
of carrageenan (1%) on the day of birth (P0), resulted in spatial
memory deficits in adult rats (Henderson et al., 2015), and also Animals
changed the regulation of the HPA axis (Victoria et al., 2013); Subjects were the offspring of Wistar rats (parents: males, n = 35
complete Freund’s adjuvant on P1 did not affect short-or long- and females n = 62) from the biocollection of Pavlov Institute of
term memory in male or female rats on P60, but resulted in Physiology of the Russian Academy of Sciences. After two days
spatial learning deficits in males (Amaral et al., 2015). Formalin- of adapting to new quarters, the rats were mated, and a vaginal
induced pain in newborn rats, which produces less prolonged smear was examined next morning to verify insemination. The
pain than does carrageenan or CFA, impaired visual-spatial days of insemination and birth were considered as gestational
learning and memory in the radial 8-arm maze, which uses food day (G) 0 and postnatal day (P) 0, respectively. Pregnant dams
reinforcement, in adult rats (Anand et al., 2007). were housed four per cage, then individually after the 17th
There are many models of neonatal pain, each with their day of pregnancy. All animals were maintained under standard
advantages and disadvantages. All are meant to model the conditions (12 h light, 12 h dark, lights on at 08:00, 20–22◦ C)
experience of the infant in the NICU who experiences many in standard plastic rat cages with food and water available
skin breaking experiences each day (Grunau et al., 2006). The ad libitum. The birth of offspring was checked at 8, 13, 17 and
four most common are repeated needle stabs, or carrageenan, 20 h. A day after the birth of the offspring, litters were reduced to
CFA or formalin injection, although others exist (e.g., paw 8 rat pups (4 males and 4 females if possible). From each mother,
incision; local capsaicin treatment). Formalin has the advantage one male and one female were included in the experiment; in two
of producing a reliable short-lived behavioral response (<1 h) cases, two rats of each sex from one dam were used. In the latter
and accompanying edema (Anand et al., 1999), without long- case, data from these two animals were averaged to produce a
term immune activation or substantial disruption of mother-pup “litter” mean (see the statistics section below). The remaining rats
interactions, therefore limiting the duration of pain and allowing in a litter were used in other experiments. All procedures were
precise control over the age of injury. approved by the Local Ethics Committee for Animal Experiments
We previously showed that inflammatory pain on P1and of the I. P. Pavlov Institute of Physiology, Russian Academy of
P2 did not alter spatial learning in 33-day-old adolescent Sciences (Saint Petersburg, Russia) and followed the guidelines
female rats (Butkevich et al., 2020). A more granular study of published by the Committee for Research and Ethical Issues of
the consequences of inflammatory painful effects on cognitive the IASP on ethical standards for investigations of experimental
abilities and the stress-hormonal system in adolescence is pain in animals.
especially important, since neurobiological and behavioral
changes vary greatly at different stages of adolescence and Neonatal Inflammatory Pain
these changes, caused by stressful effects at an early age, may On the first and second day of life (P1 and P2) offspring of both
be manifested specifically at one or more of these stages. sexes were injected with the inflammatory agent formalin (2.5%,
Identifying this unique developmental pattern would help 0.5 µl) into the pad of the left hind paw; as a control, a single prick
direct efforts to ameliorate any untoward effects of early stress needle or saline injection was used. In preliminary experiments,
pain more precisely. in which P1 and P2 rat pups were subjected to a single needle
Most of the basic research has been and is being done on males prick (n = 5 in each adolescent age and sex group) or a single
(Chen et al., 2016; Xia et al., 2020). When both sexes are tested, injection of saline solution into the pad of the left hind paw (n = 5
females have been found to be more vulnerability to the noxious in each adolescent age and sex group), the animals were tested
influences at an early age by some (Gildawie et al., 2021), whereas at P26-34 and P45-53. No differences were evident in spatial
others report increased vulnerability in males (Van Dammen learning and memory in the Morris water maze (MWM) or in
et al., 2020). Androgens in adults inhibit the activity of the HPA, the stress reactivity of corticosterone between the pricked and
whereas estrogens, on the contrary, increase it (Handa et al., 1994; saline animals in both age and sex groups (see Supplementary
McCormick et al., 2002). Pain-related sex differences are present Figures 1–4), so needle prick rats were used as a control for
from birth (Verriotis et al., 2018; Gursul et al., 2019). Pain stress is formalin injection in the MWM experiments and saline injection
often presented in the neonatal clinic, and it is important to know as control for the corticosterone assays in newborn rat pups.
how consequences of pain stress are manifested in different term In addition, the rats designed to determine basal corticosterone
intervals of adolescence in order to correct the behavior in this levels were handled at the same time as Control or Formalin rats
period of postnatal development. but otherwise untreated. P1 and P2 in rats roughly correspond

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to extreme prematurity in human (gestation weeks 24), based on occurs within one day of testing, and is calculated by taking
development of the brain and pain system in rats and humans the difference between the latency in the first and last tests and
(Dobbing, 1981; Fitzgerald et al., 1988). All animals in each averaging this difference for all days of spatial learning. The
litter were randomized to the inflammatory pain (Formalin) and savings index is the measure of how well, on the first test of each
Control. Formalin rats were labeled with a weak solution of picric day, the rats remember what was learned on the previous day.
acid along the back, control rats remained unmarked; remaining This value is calculated as the difference between the latency in
rats designed for other experiments, had their heads painted the last test of a given day and the latency in the first test of the
with picric acid. next day and averaged over all days of spatial learning. Thus, the
savings index reflects consolidating and storing memory and/or
Morris Water Maze (MWM) its retrieval process.
A modified version of the MWM was used to assess spatial
learning, spatial short-term and long-term memory (Morris, Spatial Memory Assessment
1981; Vorhees and Williams, 2014). The MWM consisted of a On the fifth and last training day, the rats were exposed only to
round tank (120 cm diameter, 72 cm deep) filled to 40 cm with the first four training trials, and then dried and returned to their
opaque with chalk-clouded water (24 ± 2◦ C) to eliminate the cages in a different room. After one hour, each rat was placed back
platform’s visibility. The tank, located in a room with several into the pool and the spatial short-term memory was examined
strongly contrasting extra maze cues, was visually divided into without the platform. This short-term assessment reflects a
four equal quadrants West (W), South (S), East (E) and North combination of reference and working memory (Vorhees and
(N). A steel platform (39 cm height, 12 cm diameter) was placed Williams, 2014). The NW quadrant start location was used for
in the SW quadrant approximately 40 cm from the side wall, and short- and long-term memory assessment. Long-term spatial
its location fixed for all the animals during all training days. The memory, memory retention, was examined 96 h after the short-
investigator was visible to the rats, and her location was constant term memory study (34th and 53d days of life in the early and late
throughout all experiments. The installation was illuminated by age groups), by placing each animal sequentially in water without
two lamps (250 W), the light from which was directed to the a platform. Animals were allowed to swim freely for 60 s in the
ceiling to obtain soft diffused lighting. water tank without the platform. The latency to locate the spot
where the hidden platform had been previously and the amount
Spatial Learning Assessment of time the animal spent in the target quadrant (SW quadrant)
Spatial learning tests were conducted at two age groups during were the scores for the short- and long-term memory probe
the adolescent period: early (P26-34) (formalin rats n = 15 males trials. Behaviors in the short- and long-term memory tests were
and n = 12 females, and control rats n = 16 males and n = 14 recorded using a webcam with automatic focusing (Microsoft
females) and late (P45-53) (Formalin rats n = 15 males and n = 15 5WH-00002) and also visually in real time.
females, and Control rats n = 16 males and n = 16 females).
Rats tested at the early age were with their mother until the
end of the experiments (on the 34th day of life). The rats of Blood Collection and Corticosterone
the late age group were weaned also at 34 days, and males and Determination
females placed in different cages, 3–4 per cage. Both cohorts were There were 56 newborn rat pups (basal, n = 19; saline (control)
tested identically. n = 18; formalin, n = 19) used for determination of corticosterone
In the MWM, each rat was trained for 5 consecutive days to in the blood. Blood samples were collected by rapid decapitation
locate a platform with eight training trials per day, divided into without anesthesia. Basal samples were taken at 9 AM. To
four trials with an interval between them of 4 minutes. For each determine the effect of inflammatory pain in newborn rats on the
training trial, the rat was placed in the water facing the tank wall, activity of the HPA axis, blood samples were collected following
in the first training trial into the NW quadrant, then consistently decapitation of one female and one male into a single test tube
in the SW, SE and NE quadrants. The rat was allowed to search (the volume of blood from one rat neonate is very small, so we
for the platform for 60 s. Failing that, the experimenter placed combined samples from one male and one female in one test
the rat on a platform, where it remained for 20 s to learn its tube) 30 minutes after subcutaneous injection of formalin (2.5%,
location relative to the extra-maze visual cues. Then the rat was 0.5 µl) or saline into the pad of the left hind paw. The time course
transferred to a dry cage with paper towels as bedding for 15 s, of the effects of the formalin treatment on corticosterone levels
after which the training trials were continued. The latency (from were evaluated one day and seven days after injection of formalin
when the rat was submerged in the water tank to when it located or saline. In early adolescent rats (basal, n = 8 males and n = 8
the platform) was recorded in each trial. If the rat did not locate females; control (needle prick), n = 10 males and n = 9 females;
the platform during a trial, it was assigned the maximum trial formalin, n = 10 males and n = 8 females) and late adolescent
duration 60 s as the score for that trial. The average latency in rats (basal, n = 7 males and n = 11 females; control (needle
the first four training trials and the average latency in second four prick), n = 6 males and n = 6 females; formalin, n = 6 males and
training trials were used as measures of learning. n = 6 females), corticosterone reactivity to the forcing swimming
In addition to latency, we used the index of acquisition and the was determined 30 min after the long-term spatial memory test
savings index as additional measures of spatial learning during in MWM. Here, unlike the corticosterone assay in newborn
training tests (Whiting and Kokiko-Cochran, 2016; Tucker et al., rat pups, blood of males and females was collected in separate
2018). The index of acquisition describes the learning that test tubes by rapid decapitation without anesthesia. Following

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collection, blood was centrifuged, and blood plasma was stored platform decreased on the fifth training day in all the animals
in a freezer (−20◦ C). Corticosterone was determined in duplicate (Figure 1). However, there were differences in spatial learning
by immune-enzyme analysis, using standard kits (“Xema-Medica between P26-P34 and P45-P53 rats. For early adolescent period
Co” Cat No: K210R; Russia); the intra-assay coefficient was 3.8. (P26-P34) (Figures 1A,B) post hoc analysis found no differences
in the latency to find the platform between the Control rats
Statistical Analysis (males n = 16, females n = 14) and Formalin rats (males n = 15,
Mixed ANOVA was used for spatial learning Analysis I for the females n = 12) in either training trial on each of the 5 training
first four training trials and Analysis II for the second four days. Neonatal formalin pain did not alter the latency to find the
training trials, the within-subjects factor was day of testing platform. For late adolescent period (P45-P53) (Figures 1C,D)
(days 1,2,3,4,5), and between-subjects factors were age (26– post hoc analysis found that neonatal pain significantly increased
30 days/45–49 days), sex (males/females), exposure (formalin the latency to find the platform in Formalin males (n = 15)
P1 & P2 or needle prick at P1 & P2); when Mauchly’s Test of compared to the latency in Control males (n = 16) (Figure 1C).
Sphericity was significant, we used Greenhouse-Geisser method. Age and sex differences in the latency to find the platform
A mixed ANOVA was used for comparison of the first four are presented in Figure 2. The latency was longer in P26-34
and second four training trials, Analysis III, the within-subjects rats of both sexes. Age differences were found in Control males
factors for day number (1,2,3,4 on each of four days), between- (Figures 2A,C) and Control females (Figures 2B,D) in the first
subjects factors were the same as Analyses I and II. For the four (Figures 2A,B) and second four (Figures 2C,D) training
index of acquisition, three-way analysis of variance ANOVA trials. It was found, that in the first training day, that characterizes
was used; the factors were age, sex, exposure. For the savings the greatest response to the stimulus in MWM (Vorhees and
index, three-way (factors: age, sex, exposure) and two-way Williams, 2014), neonatal pain significantly increased the latency
(factors: age, exposure) analyses of variance ANOVA were used. to find the platform in P45-53 males in both four training trials to
For memory, mixed ANOVA was used, within-subjects factors: the levels of the younger males, whereas in females, similar effects
memory (short-time memory, STM/long-term memory LTM) for of formalin were not evident. The greater latency to find the
latency (time to find platform location) and for time the animal platform in the late adolescent period in Formalin P45-53 males
spent in the target quadrant, and between-subjects factors age compared to the latency in Formalin P45-53 females indicates
(30 and 34 days for STM and 49 and 53 days for LTM), sex their greater vulnerability to the effects of neonatal inflammatory
(males/females) and exposure (formalin P1 & P2/needle prick pain on spatial learning in males than in females (Figure 2C).
P1 & P2). For corticosterone in newborns, a one-dimensional
two-factor analysis of variance ANOVA was used. The dependent The Index of Acquisition (Figure 3A) and
variables were corticosterone, day of test (30 minutes, first day, the Savings Index (Figure 3B) for the
seventh day) and exposure (basal level, saline, formalin); for
corticosterone of adolescent rats, three-way univariate analysis of
Latency
We used the index of acquisition and the savings index as
variance ANOVA was used, and the between factors were day of
supplemental ways to assess the effectiveness of spatial learning.
test (34 days/53days), sex (males/females), exposure (basal/needle
The index of acquisition (Figure 3A) and the savings index
prick/formalin). Comparison of corticosterone separately for
(Figure 3B) support the data presented in Figures 1, 2, indicating
males and females did not reveal sex differences; therefore, a
that the spatial learning within a day was more successful (latency
variant of paired comparisons was made for males and females in
was shorter) in Control P45-53 rats than in Control P25-34 rats
total. Post hoc comparisons were made with Bonferroni multiple
(p < 0.05, males and females). The savings index (Figure 3B)
comparisons test. For two cases, when two rats of each sex from
demonstrated that Control P45-53 females, compared to Control
one dam were used, we averaged the data from littermates (where
P25-34 females, (p < 0.05), recalled better on the first trial of
they were 2 from a litter) to create a single data point per litter.
each day what was learned on the last trial of the previous day.
We ran the analysis of variance for litters per group, subjected to
Neonatal formalin pain neutralized these age-related differences.
the same exposure. The analysis showed that in the majority of
In the early age group of rats of both sexes, neither index
the groups of the rats there is no principal differences between
showed effects in the formalin treated animals. Indeed, the index
the results of dimension in different litters, this indicates that the
of acquisition and the savings index indicated that formalin
litters are uniform for the most part and we re-ran the statistical
treated P45-53 females showed deficits in the both learning
analysis this way.
and memory (the latency was greater) compared to Control
females of the same age. These data are not consistent with
RESULTS those data in Figure 1. It is likely that the scoring mechanism
underlying this alternative method determines these differences,
Details of the analyses of main experimental data are in Table 1 as reflected in the sex differences in the first four and second four
and summarized below. training trials.

Spatial Learning, Latency to Find the Short- and Long-Term Spatial Memory,
Platform Latency to Find the Platform (Figure 4)
Both Control and Formalin rats in both age groups and of In latency to the target quadrant, there were no age or sex
both sexes showed spatial learning, since the latency to find the differences in the treated groups for either the short- and

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TABLE 1 | Details of the statistical analyses.

Spatial learning, latency to find the platform

Main effects for early and late adolescent periods of development for the
first four training trials (Analysis I)
Within subjects effects F(3,1, 342,4) = 224, ***p < 0,001, η2 = 0,670,
day F(3,1, 342,4) = 5,724, ***p = 0,001, η2 = 0,049,
day X age: Note. Mauchly’s Test of Sphericity p < 0,001,
Between subjects effects Greenhouse-Geisser method Df1 = 3,084, Df2 = 342,359.
age F(1,111) = 20,208, ***p < 0,001, η2 = 0,154.
Main effects for early and late adolescent periods of development for the
second four training trials (Analysis II)
Within subjects effects F(2,5, 272,4) = 64,1, ***p < 0,001, η2 = 0,366,
day F(2,5, 272,4) = 8,9, ***p < 0,001, η2 = 0,074,
day X age: Note. Mauchly’s Test of Sphericity p < 0,001,
Between subjects effects Greenhouse-Geisser Df1 = 2,454, Df2 = 272,378.
age F(1,111) = 20,5, *** p < 0,001, η2 = 0,156,
exposure F(1,111) = 4,3, * p = 0,041, η2 = 0,037,
age X sex F(1,111) = 4,7, *p < 0,032, η2 = 0,041.
Analysis III (try1-2)
Age and sex differences in the latency to find the platform
Effect of training day F(4, 108) = 71, ***p < 0,001, η2 = 0,725
Effect of training day in X age X sex F (4,108) = 2,6, p = 0,04, η2 = 0,088,
Tests of between-subjects effects F(1, 111) = 12,2, **p = 0,001, η2 = 0,099, F(1, 111) = 25,9,
Age ***p < 0,001, η2 = 0,189, F(1, 111) = 15,9, ***p < 0,001,
Posthoc analysis η2 = 0,126 (the first, second and third day respectively)
The index of acquisition for the latency p = 0.042, p = 0.041, p = 0.022, the first, third and fourth
age training days
age X exposure F(1,111) = 4,304, p = 0,040, η2 = 0,037
The savings index for the latency F(1,111) = 4,416, p = 0,038, η2 = 0,038;
age X exposure F(1,111) = 4,046, p = 0,046, η2 = 0,035
Short- and long-term spatial memory, latency to find the platform
and time spent in target quadrant
Within subjects effects
memory
memory X age F(2, 107) = 20,5, ***p < 0,001, η2 = 0,277,
memory X exposure F(2, 107) = 19,48, ***p < 0,001, η2 = 0,267
Between subjects effects F(2, 107) = 7,32, ***p = 0,001, η2 = 0,120;
age F(2, 107) = 5,87, ***p = 0,004, η2 = 0,099
exposure. F(2, 107) = 3,76, *p = 0,026, η2 = 0,06
Comparison between short- and long-term memory
Within subjects effects F(1, 108) = 8,9, **p = 0,003, η2 = 0,076,
latency F(1, 108) = 13, ** p < 0,001, η2 = 0,107,
memory X exposure
Within subjects effects
time spent in target quadrant F(1, 108) = 37, ***p < 0,001, η2 = 0,255
memory X age F(1, 108) = 38,4 ***p < 0,001, η2 = 0,263;
memory X exposure F(1, 108) = 3,4 p = 0,068, η2 = 0,031,
memory X age X sex F(1, 108) = 3,1 p = 0,079, η2 = 0,028;
Between subjects effects, latency
age F(1, 108) = 4,51, *p = 0,036, η2 = 0,040
Between subjects effects,
time spent in target quadrant F(1, 108) = 9,17, **p = 0,003, η2 = 0,078,
age F(1, 108) = 7,4, **p = 0,008, η2 = 0,064.
exposure
In newborn rats, main effects for corticosterone
day of test,
exposure, F(2,47) = 75,6, p < 0,001, η2 = 0,763;
day of test X exposure, F(2,47) = 26,3, p < 0,001, η2 = 0,528;
In adolescent rats, main effects for corticosterone F(4,47) = 9,66, p < 0,001, η2 = 0,451.
day of test,
exposure F(1,83) = 27,45, ***p < 0,001, η2 = 0,249
day of test X exposure F(2,83) = 66,28 ***p < 0,001, η2 = 0,615,
F(2,83) = 10,7 ***p < 0,001, η2 = 0,205.

long-term memory measures. However, the formalin treated more quickly in the long-term compared to the short-term
males, but not females, showed long-term memory deficits at memory task (p = 0.018 and p = 0.006, males and females
both ages (Figures 4B,B1) with no differences in the short-term respectively) as did P45-53 females (p = 0.003) (Figures 4A,B).
memory task (Figure 4A). Control P26-34 rats of both sexes Thus, neonatal pain impaired long-, but not short-term memory,
found the quadrant where the platform had been previously in the males, but not females, in both ages. Formalin pain

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FIGURE 1 | Mean (±SEM) latency to find the platform in the first four training trials for 5 days and second four training trials for four training days of spatial learning in
Control and Formalin male and female rats of early (P26-34) and late (P45-53) age groups. Panels (A,B) show data for male and female rats at the early age group.
Panels (C,D) show data for male and female rats at the late age group. The abscissa shows the first and second training four trials (1 and 2) in each of the five
training days. + p < 0.05, ++ p < 0.01, +++ p < 0.001 significant differences in Control rats between the first four training trials and the second four training trials
each day. ∗ p < 0.05, ∗∗ p < 0.01 Formalin rats vs Control rats.

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FIGURE 2 | Age and Sex effects in the mean (±SEM) latency to find the platform in Control and Formalin male and female rats in the first four training trials during
five training days [early P26-34 and late P45-53 age groups (A,B)] and in the second four training trials during four training days [early P26-34 and late P45-53 age
groups (C,D)]. ∗ p < 0.05, ∗∗ p < 0.01 age differences in Formalin rats, + p < 0.05, ++ p < 0.01 age differences in Control rats. # p < 0.05 sex differences in P45-P48
Formalin rats. Abscissa, training days. The number of the rats in the groups corresponds to the number of rats in Figure 1.

neutralized the differences in latency between short-term and task than did the same-age Control males (Figures 5A,A1).
long-term memory, which were found in Control animals. There were age differences in the short-term memory task in
Control males and females and Formalin females (Figure 5A).
In the long-term memory task, less time was spent in target
Short- and Long-Term Spatial Memory, quadrant in Formalin P45-53 males and Formalin P26-34 females
the Time Spent in Target Quadrant as compared to the time in Control rats of the same ages
(Figure 5) (Figures 5B,B1). There were differences between short-and term-
For the time in the target quadrant, Formalin P45-53 male rats long memory performance in P26-34 animals, in both Control
spent less time in target quadrant in the short-term memory and Formalin rats of both sexes (Figures 5A,B). The time spent in

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Butkevich et al. Neonatal Pain Effects Cognition in Adolescence

FIGURE 3 | The index of acquisition (A) and the savings index (B) for latency to find the platform during spatial learning in male and female rats of early (P26-34) and
late (P45-53) age groups. The data are: Mean (±SEM) latency differences between the first and last training trials of each of the five training days of spatial learning
(A). Mean (±SEM) latency differences between the last training trial of a given day and the first training trial of the next day during the five-day spatial learning (B).
Both indices illustrate a decrease of latency to find the platform with age in Control rats; neonatal formalin-induced pain leveled the age differences. ∗ p < 0.05
Formalin vs Control (A,B); + p < 0.05 Control P45-49 rats vs Control P26-34 rats (A,B). The number of the rats in the groups corresponds to the number of rats in
Figures 1, 2.

the target quadrant was less in the short-term memory than in the pups. Corticosterone was elevated quickly and that elevation was
long-term memory task. Sex differences were found in the short- maintained for at least 24 h compared to basal levels and saline
term memory test since females spent more the time in the target injection controls. This suggests that neonatal inflammatory pain
quadrant in Formalin P45-53 rats than did males (Figure 5A). could modify the development of the HPA. We hypothesized
that this could lead to changes in stress reactivity and cognitive
Corticosterone Determination in abilities in adolescent rats. Indeed, there were differences in
Newborn Rats (Figure 6) and Adolescent adolescent rats’ plasma corticosterone in response to a swim
Rats (Figure 7) stress and in aspects of spatial learning and memory depending
on whether early and late age rats were tested. In the early
In newborn rats, 30 min after formalin injection corticosterone
P26-34 and late P45-53 age groups, the effects of repetitive
levels were higher in the Control and Formalin pups compared
neonatal peripheral inflammatory pain on spatial learning, short-
to basal levels, and higher in the Formalin pups than in
term and long-term memory, strongly indicate a pronounced
Control pups (Figure 6). Likewise, twenty-four hours after
heterogeneity of the effects of early pain during the adolescent
formalin injection, the Formalin pups had a higher corticosterone
period of rat development. However, the long-term effects of
level than the Control and the basal pups. Seven days after
early formalin injury and subsequent HPA activation cannot
formalin injection, there were no significant differences in the
explain the adolescent effects in any simple way.
corticosterone levels among the three groups of pups.
In neonates, the HPA axis rapidly develops and responds to
In adolescent rats, there were no sex differences in
strong stressors (Wood and Walker, 2015). We had previously
corticosterone levels and therefore male and female data were
shown that even during the stress hyporesponsive period of the
combined. Thirty min after forced swimming, which rats were
HPA axis development, formalin-induced pain caused a gradual
subjected to after long-term memory testing, corticosterone levels
over an hour an increase in plasma corticosterone levels in 7-
were higher compared to basal levels in both the Control and
day-old male rats; twenty-four hours after injection of formalin
Formalin groups in early and late ages (Figure 7). Age differences
corticosterone still exceeded the basal value (Butkevich et al.,
were found in both sexes in the Control and Formalin rats with
2013). We also showed that neonatal formalin-induced pain
greater corticosterone levels in early age group.
caused an increase in an inflammatory pain response, depression-
like behavior, and impairment of learning in adolescent male
DISCUSSION rats (Butkevich et al., 2016). But the question remains open as
to whether the altered endogenous cortisol following early pain
The goal of this work was to examine the acute and subacute stress may play a role in learning and memory performance, as
effects of an injury to the pad of the hind paw on plasma has been suggested in children (Mooney-Leber and Brummelte,
corticosterone, a marker of stress reactivity, in newborn rat 2017), especially in the prepubertal period in both sexes.

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FIGURE 4 | Mean (±SEM) latency to find the platform (A,B) for short-term (A) and long-term (B) spatial memory in the Formalin or Control male and female rats for
the early (P26-34) and late (P45-53) age groups. Differences in latency between short-term and long-term memory were found in the Control male and female rats of
the early age group and in females of the late age group. Formalin vs Control rats; differences between short- and long-term memory: in latency, ∧ p < 0.05,
&& p < 0.01, in Control P26-34 males and females, and $$ p < 0.001, in Control P45-53 females. The number of the rats in the groups corresponds to the number of

rats in the groups in Figures 1, 2. The graphs on the right illustrate significant results of statistical analysis. ∗ p < 0.05 significant effect of exposure.

Weaning is stressful for the offspring. In the present work, age group showed differences between Formalin and Controls
weaning occurred on P34 in both age groups. Experiments with on the first training day. Differences in spatial learning between
rats of the early group were conducted before weaning, the pups age groups in Control and Formalin females only appeared in
after the experiment were in the home nest with their mother, the second four trials. These sex differences may be due to
while the pups of the late group were without the mother, but different rates of adolescent sexual maturation which occurs later
with their sisters or brothers. If weaning took place in P25, as in males (∼P42 ± 2), than in females (∼P35 ± 2) (McCormick
is usual in our laboratory (Butkevich et al., 2017), then testing and Mathews, 2007, 2010). This suggests that sex hormones can
with P26 would be more stressful for the early group of rats than be one of the reasons for these differences between males and
testing with P45 for the late group of rats. So, the rats had weaning females. Another reason for these differences may be the different
at a later age. The rats of the late age group had enough time reactivity of the HPA axis in males and females. However, no
(11 days) for adaptation of life without the mother. In the MWM, differences in corticosterone reactivity were found between sexes
the early age Control rats were capable of spatial learning, which after assessing long-term memory. Interestingly, when using
is consistent with the literature (Vorhees and Williams, 2014), other metrics for spatial learning, the index of acquisition and the
but which is in contrast to earlier work that found that effective savings index (Whiting and Kokiko-Cochran, 2016), we found an
strategies for spatial learning in the Morris water maze appear increase in the latency to find the platform, which is impairment
relatively late in adolescence (P42) (Schenk, 1985). Probably, the of cognition, in Formalin P45-53 females, as compared to Control
differences in the line of rats (Wistar and Hooded rats) and the P45-53 females, but not in males of the same age group. The two
testing methodology (in Schenk, 1985, Hooded rats were allowed different metrics measuring learning and memory can explain
to swim only for 30 s) underlie these differences. Formalin treated this difference. The index of acquisition – measure of the
males and females of both age groups also demonstrated spatial learning within one day of testing, and is calculated by taking
learning, as evidenced by the gradual decrease in the latency the difference between the latency in the first and last tests and
of finding the platform during five training days. However, averaging this difference for all days of learning. The savings
compared to controls, older male but not younger male formalin- index is the measure of how well, on the first test of each
induced neonatal pain rats took longer to find the platform for day, the rats remember what was learned on the previous day.
both four training trials. This difference (35%) was particularly This value is calculated as the difference between the latency in
pronounced on the first training day and it is first training day the last test of a given day and the latency in the first test of
that is an important criterion for the learning process (Vorhees the next day and averaged over all days of learning. However,
and Williams, 2014). In contrast to males, females of neither the absence of differences in the latency to find the platform

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FIGURE 5 | Mean (±SEM) time in target quadrant (A,B) for short-term (A) and long-term (B) spatial memory in the Formalin or Control male and female rats for the
early (P26-34) and late (P45-53) age groups. Differences in time in target quadrant between short-term and long-term memory were found in Control and Formalin
males and females of early age groups. In all cases, the time in target quadrant was shorter in the short-term memory (A,B). +++ p < 0.001, age differences in
Control rats; ### p < 0.001, age differences in Formalin rats. Differences between short- and long-term memory: in time in target quadrant, ∧∧∧ p < 0.001,
&&& p < 0.001 in Control P26-34 males and females, vvv p < 0.001, αα p < 0.01, in Formalin P26-34 males and females; 0 p < 0.05, sex differences in P45-53

Formalin rats. The number of the rats in the groups corresponds to the number of rats in Figures 1, 2. The graphs below illustrate significant results of statistical
analysis. ∗ p < 0.05, ∗∗ p < 0.01 significant effect of exposure. Graphs (A1) and (B1) illustrate the significant outcomes of the statistical analyses.

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in the late age group. Differences identified in memory processes

using latency to find the platform and the duration to stay in the
target quadrant indicate participation of different brain structures
in these behavioral characteristics of memory.
We are aware of only a few rodent studies that investigated the
effect of neonatal inflammatory pain on memory. For instance,
formalin-induced pain in newborn rats impaired visual-spatial
learning and memory in the radial 8-arm maze, which uses food
reinforcement, in adult rats (Anand et al., 2007). Inflammatory
pain caused by the intra-plantar injection of carrageenan (1%)
on the day of birth, P0, resulted in spatial memory deficits also
in adult rats (Henderson et al., 2015), and dysregulated the HPA
axis (Victoria et al., 2013). Complete Freund’s adjuvant on P1
did not affect short-or long-term memory in male or female rats
on P60, but resulted in spatial learning deficits in males (Amaral
et al., 2015). Therefore, although there are some inconsistencies,
in general early experiences of painful injury can disrupt adult
spatial learning/memory processes. When assessed, the single
injection of carrageenan on the day of birth activated the infant
HPA axis in rat pups (Victoria et al., 2014). Daily needle pricks in
each paw at 6-h intervals until P7 (Chen et al., 2016), decreased
serum corticosterone in P24, had no effect at P45 and increased
corticosterone in adult rats. Thus, these early insults can have
long-term effects on subsequent HPA axis function. However,
we know of no comparable data for testing the effects of early
inflammatory injury in adolescence.
We measured HPA reactivity in response to forced swimming
in the rats after testing in the MWM, and found no differences
in corticosterone levels in adolescent rats between Formalin
and Control rats of either sex. Importantly, both Control and
Formalin rats at the early age showed greater corticosterone
levels compared to those of the late age group. The forced swim
test is known to stimulate the HPA activity in rats (Mathews
et al., 2008), and HPA axis reactivity is modified by previous
FIGURE 6 | Mean (±SEM) corticosterone levels in blood plasma in neonatal
pups under basal conditions, or 30 min, 24 h and 7 days after injection of
stress history, especially during critical periods of rapid brain
Formalin (2.5%, 0.5 µl) or Control into the pad of the left hind paw. ∗ p < 0.05, development (reviewed in Meaney and Szyf, 2005). Stress at
∗∗ p < 0.01, Formalin vs saline; + p < 0.05, +++ p < 0.001, Formalin vs basal; an early age changes adaptive behavior. For example, we have
∧∧ p < 0.01, saline vs basal.
previously shown that the formalin test preceding the forced
swim test sharply reduced the immobility time only in 7-day-old
rat pups that had been prenatally stressed but not control pups
between Formalin and Control rats in the early P26-34 age group (Mikhailenko et al., 2010). Our long-term experience with the
was the same as the results obtained using these indices and forced swim test indicates that the severe physical and emotional
analysis I and II. stress experienced by the rat in this test can obviate the effects
When assessing memory by the latency to find the platform, of other varied types of stress. It is important to note, that the
neonatal pain caused deficits only in long-term memory in males absence of differences in the reactivity of the HPA axis between
in both age groups, whereas when assessing memory by the time Formalin and Control rats in our current study could be a
spent in the target quadrant, neonatal pain decreased it in males consequence of the cumulative effects of testing in MWM and
of the late age group in both short- and long-term memory and forced swimming on the activity of the HPA axis. The interaction
also in females of early age group. Only control rats of both of different types of stress, especially during critical periods of
sexes of the early age group showed differences between short- development, can lead to unexpected results (Sokołowski et al.,
and long-term memory in both latency and time spent in the 2020). Especially interesting and of practical importance is the
target quadrant, with shorter latency and longer time spent in consequence of suppressing the adverse effects of one stress by
the target quadrant in long-term memory. In the time spent in another adverse stress (Van Bodegom et al., 2017). Our data using
the target quadrant, Formalin rats of the early age group, showed the formalin pain stress in newborns showed suppression of the
the similar behavior. Note, age differences were found only in expected pronociceptive effect of prenatal stress in the formalin
short-term memory in Control rats of both sexes and Formalin test in adolescent rats, but did not reduce depressive-like behavior
females, and only in the target quadrant, with a longer parameter (Butkevich et al., 2020).

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FIGURE 7 | Mean (±SEM) corticosterone levels in blood plasma in response to forced swimming in Control and Formalin rats at the age of P34 and P53 after testing
in the Morris Water Maze. + p < 0.05, ++ p < 0.01, +++ p < 0.001, Formalin vs basal; ∧∧ p < 0.01, ∧∧∧ p < 0.001 Control vs basal; & p < 0.05, age differences
between Control rats; vv p < 0.01, age differences between Formalin rats. The number of the rats in the groups corresponds to the number of rats in Figures 1, 2.

Our present experiments have shown that the activation of the depend on the balance in maturation of the HPA and the
HPA axis by neonatal pain has no direct relationship with spatial hypothalamus-pituitary-gonadal (the HPG) axis. Neonatal pain,
learning and memory in rats in adolescence. Other physiological by disrupting the processes of inhibition or excitation in the
systems besides the HPA axis may be involved in the effects of central nervous system, could modify the synchronization of
inflammatory pain in newborns, such as the immune system, development of the HPA and HPG systems, which closely interact
which responds to inflammation and stress and can affect brain and affect the neuroplasticity of learning and memory. The
neurons and cognitive function. The immune system closely hippocampus, medial prefrontal cortex, and amygdala, brain
interacts with the HPA axis (Gaillard, 2003). Sex differences structures implicated in the control of the HPA axis (Herman
in microglia, neuroimmune cells, begin to emerge during the et al., 2003) and cognition (Euston et al., 2012; Méndez-Couz
prenatal organizational period for sexual differentiation of the et al., 2014), mature rapidly during adolescence (Spear, 2000) and
brain (Schwarz et al., 2012). Immunocompetent cells of the can influence sensitivity of the HPA axis to sex hormones and
brain express steroid hormone receptors and are regulated by alter cognitive abilities.
hormones and activation of the immune system is determined The relatively long-lasting high level of corticosterone evoked
by sex hormones (Lombardo et al., 2021). Moreover, the by formalin-induced pain in newborn rat could impair the
immune system acts as a regulator of sex differences in brain development of the PVN. In the newborn rat, the PVN and CA1
development and behavior (Nelson and Lenz, 2017; VanRyzin of the hippocampus contain GR mRNA expression (Pryce, 2008).
et al., 2018). The immune and sexual systems interact with The CRH hippocampal system regulates neurogenesis in the
the HPA axis (Bereshchenko et al., 2018). One can suggest hippocampus which is involved in spatial learning and memory
that the sex differences reported here following neonatal pain (Koutmani et al., 2019). Elevated levels of glucocorticoids have

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also been shown to impair working and reference memory that long-term memory performance was more robust than
(Stylianakis et al., 2018). CRH neurosecretory systems release short-term memory. The differences found in spatial memory
glutamate, in addition to neuropeptides, into the pericapillary performance in MWM in P25-34 and P45-53 rats provide strong
space of hypophysial portal vessels, and there is expression of evidence of the heterogeneity in the development of cognitive
the mRNA for vesicular glutamate transporter-2 in the rat CRH processes in the two age groups of the adolescence. These
neurons in the PVH (Hrabovszky et al., 2005). Glutamatergic behavioral changes suggest that neonatal pain causes changes
neurons are one of the main links in the processes of learning in various structures and neurotransmitters involved in spatial
and memorization (review, Mooney-Leber and Brummelte, short-term and long-term memory only in P45-53 rats. The effect
2017). Excessive levels of glucocorticoids enhance the release of stress at an early age on memory and the HPA axis, as well
of glutamate, causing neurotoxicity, which enhances apoptosis, as brain structures involved in memory processes in adulthood
as shown in the hippocampus and other brain regions during are well studied (Krugers and Joëls, 2014; Schroeder et al., 2018;
the first postnatal week in the rat (Lu et al., 2003; Dührsen Bonapersona et al., 2019; Cordier et al., 2021), but information
et al., 2013). The role of glutamate during development has been on the effects of neonatal pain stress on memory and the
primarily associated with the NMDA receptor, which is present participation of the HPA axis in this process is very meager. Our
at P0 in the rats (Behuet et al., 2019). During normal early work is the first, as far as we know, aimed at studying the effects of
development when the NMDA receptor containing the NR2B early-life inflammatory pain on spatial learning and memory, and
subunit in the hippocampus of the newborn rat is activated, the HPA reactivity at different age intervals within the adolescent
the corresponding channel remains in the open position much period. It was also important in our study to include male and
longer than in the mature receptor. In addition, neurons female rats, as very few studies have included rats of both sexes in
with such receptors develop long-term potentiation, a form of adolescence, and our results show clear differences in the effects
activity-dependent synaptic strengthening, more quickly, which in males and females that might be accounted for by different
contributes to memory strengthening. The selective loss of NR2B developmental trajectories during adolescence. The limitation of
protein and subsequent synaptic dysfunction weakens prelimbic the work was that we analyzed corticosterone not after MWM,
PFC function during development and may underlie early but after the further stress of the forced swim, to determine the
cognitive impairments (Gulchina et al., 2017). We hypothesize reactivity of the HPA axis in Formalin and Control rats. We
that the impairment of the NR2B subunit caused by increased also conducted that assay only once, and thus did not evaluate
corticosterone in rats with neonatal pain may be associated with the dynamics of corticosterone change. It will be interesting to
the abnormalities in spatial memory that we found. investigate changes in these behavioral and endocrine systems in
Short and long exposures to corticosterone differentially tune adult rats exposed to inflammatory neonatal pain to determine if
NMDAR signaling in hippocampus by altering the expression the age and sex differences that we identified here continue into
and synaptic presence of NMDAR subunits, allowing adaptations adulthood or are unique features of the adolescent period.
of glutamate synapses (Mikasova et al., 2017). Taking into
account the different roles of metabotropic and inotropic
glutamatergic and GABAergic receptors in the effect of stress
on learning and memory, as well as the mechanism of co-
DATA AVAILABILITY STATEMENT
transmission of glutamate with GABAergic neurons (Trudeau The raw data supporting the conclusions of this article will be
and Mestikawy, 2018), it is possible that these complex made available by the authors, without undue reservation.
relationships are involved both in the differences we found in
the effect of early pain stress on cognitive abilities in adolescent
rats, and in the absence of differences in the reactivity of the
HPA axis to stress in the adolescent Formalin and Control rats. ETHICS STATEMENT
It is known that the serotonergic, the HPA axis, glutamatergic,
and GABAergic systems are all involved in nociception and are All procedures were approved by the Local Ethics Committee for
affected by stress (Goudet et al., 2009; Quintero et al., 2011; Animal Experiments of the I. P. Pavlov Institute of Physiology,
Bannister et al., 2017; Hernández-Vázquez et al., 2019). Formalin- Russian Academy of Sciences (Saint Petersburg, Russia) and
induced neonatal pain effects various neurotransmitter systems, followed the guidelines published by the Committee for Research
disrupts the balance between excitation and inhibition in the and Ethical Issues of the IASP on ethical standards for
central nervous system, modifies the development of functional investigations of experimental pain in animals.
activity of the HPA axis, and thus affects the neurophysiological
mechanisms underlying cognitive processes.
In conclusion, we found that activation of the HPA axis by AUTHOR CONTRIBUTIONS
neonatal pain did not directly correlate with spatial learning
and memory in adolescence, and therefore the consequences of IB and VM: experimental design. IB, VM, and EV: collection of
newborn pain remain are likely multi-determined. Neonatal pain data and conduction of statistical analyses. IB, VM, EV, and GB:
impaired spatial learning and long- and short-term memory in interpretation and analysis of data, participation in the drafting
late adolescent males and long-term memory in early adolescent and revising of the manuscript, and reviewing and approving the
females. The comparative analysis of the memory scores revealed final submitted manuscript.

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Butkevich et al. Neonatal Pain Effects Cognition in Adolescence

FUNDING to thank Ekaterina Tul’kova the collaborator of Laboratory of

regulation of neural function of brain (Elena Rybnikova) for the
This study was supported partly by the Russian Foundation for analysis of the corticosterone level in the blood plasma.
Basic Research (grant no. 17-04-00214-a).

SUPPLEMENTARY MATERIAL
ACKNOWLEDGMENTS
The Supplementary Material for this article can be found
We are grateful to our collaborators Elena Lavrova, Natal’ya online at: https://www.frontiersin.org/articles/10.3389/fnbeh.
Ulanova for their help with behavioral testing. We would like 2021.691578/full#supplementary-material

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Tirelli, E., Laviola, G., and Adriani, W. (2003). Ontogenesis of behavioral absence of any commercial or financial relationships that could be construed as a
sensitization and conditioned place preference induced by psychostimulants in potential conflict of interest.
laboratory rodents. Neurosci. Biobehav. Rev. 27, 163–178. doi: 10.1016/s0149-
7634(03)00018-6 Copyright © 2021 Butkevich, Mikhailenko, Vershinina and Barr. This is an open-
Trudeau, L. E., and Mestikawy, S. (2018). Glutamate cotransmission in cholinergic, access article distributed under the terms of the Creative Commons Attribution
GABAergic and monoamine systems: contrasts and commonalities. Front. License (CC BY). The use, distribution or reproduction in other forums is permitted,
Neural. Circuits 12:113. doi: 10.3389/fncir.2018.00113 provided the original author(s) and the copyright owner(s) are credited and that the
Tucker, L. B., Velosky, A. G., and McCabe, J. T. (2018). Applications of the morris original publication in this journal is cited, in accordance with accepted academic
water maze in translational traumatic brain injury research. Neurosci. Biobehav. practice. No use, distribution or reproduction is permitted which does not comply
Rev. 88, 187–200. doi: 10.1016/j.neubiorev.2018.03.010 with these terms.

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 ORIGINAL RESEARCH
published: 11 November 2021
doi: 10.3389/fnbeh.2021.760791

The Impact of Adolescent Alcohol

Exposure on Nicotine Behavioral
Sensitization in the Adult Male
Neonatal Ventral Hippocampal
Lesion Rat
Emily D. K. Sullivan 1,2* , Liam N. Locke 3 , Diana J. Wallin 1,2 , Jibran Y. Khokhar 1,4 ,
Elise M. Bragg 1 , Angela M. Henricks 1,5 and Wilder T. Doucette 1,2
1
Department of Psychiatry, Dartmouth-Hitchcock Medical Center, Lebanon, NH, United States, 2 Geisel School of Medicine,
Dartmouth College, Hanover, NH, United States, 3 Department of Psychological and Brain Sciences, Dartmouth College,
Hanover, NH, United States, 4 Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada, 5 Department
of Psychology, Washington State University, Pullman, WA, United States

Edited by:
Miriam Melis, Nicotine and alcohol use is highly prevalent among patients with serious mental
University of Cagliari, Italy
illness, including those with schizophrenia (SCZ), and this co-occurrence can lead to
Reviewed by:
a worsening of medical and psychiatric morbidity. While the mechanistic drivers of co-
Anna K. Radke,
Miami University, United States occurring SCZ, nicotine use and alcohol use are unknown, emerging evidence suggests
Jerome Jeanblanc, that the use of drugs during adolescence may increase the probability of developing
INSERM U1247 Groupe
de Recherche sur l’Alcool et les
psychiatric disorders. The current study used the neonatal ventral hippocampal lesion
Pharmacodépendances (GRAP), (NVHL) rat model of SCZ, which has previously been shown to have enhanced
France
nicotine behavioral sensitization and, following adolescent alcohol, increased alcohol
*Correspondence:
consumption. Given how commonly alcohol is used by adolescents that develop SCZ,
Emily D. K. Sullivan
[email protected] we used the NVHL rat to determine how exposure to adolescent alcohol impacts the
development of nicotine behavioral sensitization in adulthood. Male Sprague-Dawley
Specialty section:
rats underwent the NVHL surgery or a sham (control) surgery and subsequently, half
This article was submitted to
Motivation and Reward, of each group was allowed to drink alcohol during adolescence. Nicotine behavioral
a section of the journal sensitization was assessed in adulthood with rats receiving subcutaneous injections of
Frontiers in Behavioral Neuroscience
nicotine (0.5 mg/kg) each day for 3 weeks followed by a nicotine challenge session
Received: 18 August 2021
Accepted: 12 October 2021 2 weeks later. We demonstrate that all groups of rats became sensitized to nicotine
Published: 11 November 2021 and there were no NVHL-specific increases in nicotine behavioral sensitization. We
Citation: also found that NVHL rats appeared to develop sensitization to the nicotine paired
Sullivan EDK, Locke LN,
context and that adolescent alcohol exposure blocked this context sensitization. The
Wallin DJ, Khokhar JY, Bragg EM,
Henricks AM and Doucette WT (2021) current findings suggest that exposure to alcohol during adolescence can influence
The Impact of Adolescent Alcohol behaviors that manifest in the adult NVHL rat (i.e., context sensitization). Interestingly,
Exposure on Nicotine Behavioral
Sensitization in the Adult Male nicotine behavioral sensitization levels were not altered in the NVHL groups regardless
Neonatal Ventral Hippocampal of adolescent alcohol exposure in contrast to prior reports.
Lesion Rat.
Front. Behav. Neurosci. 15:760791. Keywords: adolescent alcohol, NVHL, co-occurring disorders, mental illness, smoking, nicotine behavioral
doi: 10.3389/fnbeh.2021.760791 sensitization

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Sullivan et al. Adolescent Alcohol in NVHL Rat

INTRODUCTION of one drug yields sensitization to another drug (Kalivas and

Stewart, 1991; Steketee and Kalivas, 2011).
Smoking is highly prevalent among patients with serious mental Neonatal ventral hippocampal lesion rats have also been
illness and this co-occurrence leads to medical and psychiatric shown to increase alcohol consumption in adulthood after
morbidity (Green et al., 1999, 2008; Mallet et al., 2019) as well as voluntary adolescent alcohol intake (Jeanblanc et al., 2015).
an increased mortality risk (Tran et al., 2009; McGinty et al., 2012; Alcohol remains one of the most commonly used drugs by
Dickerson et al., 2018). Specifically, patients with schizophrenia adolescents [Johnston et al., 2020; Substance Abuse and Mental
(SCZ) have higher smoking rates than the general population Health Services Administration (SAMHSA), 2020], and as such,
(de Leon and Diaz, 2005) with lifetime prevalence reported at combining the NVHL developmental insult with adolescent
60–90% (Volkow, 2009). In one investigation studying patients alcohol exposure can be used to study the complex dynamics
with SCZ or bipolar disorder, current smokers showed worse between adolescent drug use, SCZ, and increased smoking. In the
cognitive functioning and had poorer functional outcomes than present study, we used the NVHL rat to determine how exposure
past or never smokers. These effects were observed regardless to adolescent alcohol affects nicotine behavioral sensitization
of diagnosis, however, the patients with SCZ were twice as in adulthood. In humans, alcohol use during adolescence has
likely to be smokers compared to those with bipolar disorder been linked to increased substance use in adulthood (Ellickson
(Depp et al., 2015). Moreover, in a recent study, 31% of current et al., 2003; Grant et al., 2006; Ryan et al., 2019), therefore,
smokers were readmitted to a psychiatric hospital within 1 year we hypothesized NVHL animals with alcohol exposure would
of discharge compared to 26% of never smokers (Kagabo et al., demonstrate increased nicotine behavioral sensitization.
2019). Collectively, these studies indicate a correlation between
smoking in patients with a serious mental illness and increased
psychiatric morbidity and mortality. MATERIALS AND METHODS
The underlying causes of co-occurring mental illness and
substance use disorders are largely unknown. However, there is Subjects and Housing
evidence indicating that genetic factors combined with prenatal Lactating Sprague-Dawley female rats (n = 4) with 10 male
and/or postnatal developmental insults (including the use of pups each were ordered from Charles River (Wilmington, MA,
drugs during adolescence; Khokhar et al., 2017), contribute to United States) and arrived on the pups’ postnatal day (PD) 2. We
the development of these disorders. A number of studies suggest specifically chose to use the outbred rat strain Sprague-Dawley
cannabis use (Fergusson et al., 2003), and tobacco smoking (Gage in order to maximize the genetic and epigenetic variability, as
and Munafo, 2015; Kendler et al., 2015) may be associated with any behavioral signals would likely be more generalizable to other
increased psychotic symptoms. Additionally, for many patients rats. Additionally, as reports indicate that the prevalence rates of
substance use precedes psychosis, with reports finding that any current tobacco product use is higher in men than women
substance use rates among patients with first episode psychosis in the general population (Higgins et al., 2015; Cornelius et al.,
are 30–70% (Abdel-Baki et al., 2017). Thus it is important 2020) and in patients with SCZ (Kelly and McCreadie, 1999;
to study substance use during adolescence and its potential Ohi et al., 2018), we used male rats. All rats were housed on
role in contributing to an individual’s risk of developing a a reverse 12-h light cycle with ad libitum access to food and
psychiatric diagnosis. water. All experiments were carried out in accordance with the
One developmental insult used in rats that results in several National Institutes of Health Guide for the Care and Use of
dysregulated behavioral endophenotypes is the neonatal ventral Laboratory Animals (NIH Publications No. 80–23) and were
hippocampal lesion (NVHL). NVHL rats display symptoms approved by the Institutional Animal Care and Use Committee
resembling those occurring across psychiatric disorders, though of Dartmouth College.
they are often used as a model of SCZ (Lipska et al., 1993,
1995; Sams-Dodd et al., 1997; Brady et al., 2010; Gruber et al., Neonatal Ventral Hippocampal Lesion
2010; Placek et al., 2013). Moreover NVHL rats self-administer Surgery
drugs, including nicotine, at a higher rate than normal rats Neonatal ventral hippocampal lesion or sham (control) surgeries
(Chambers and Self, 2002; Berg et al., 2011; Sentir et al., 2020), as were carried out following previously published guidelines
well as demonstrate enhanced nicotine behavioral sensitization (Chambers and Lipska, 2011). On PD 7 when pups weighed
(Berg and Chambers, 2008). Behavioral sensitization is the between 15 and 20 g, they were anesthetized via hypothermia and
progressive increase of drug-induced locomotion with repeated then placed on a Styrofoam platform attached to a stereotaxic
exposure to a drug (Robinson and Berridge, 1993) and is a frame (Kopf Instruments, Tujunga, CA, United States). Half
phenomenon documented in both humans and animals (Kalivas of the pups (NVHL; n = 20) were bilaterally injected with
and Stewart, 1991; Robinson and Berridge, 2008). This behavior 0.3 µl excitotoxic ibotenic acid [10 µg/µl ibotenic acid (Tocris,
is indicative of neuroadaptations occurring in motivation related Minneapolis, MN, United States) in artificial cerebrospinal fluid
brain regions underlying drug-wanting and craving (Robinson (aCSF)] into the ventral hippocampi (from bregma: AP −3.0 mm,
and Berridge, 2008) and can be affected by perturbations ML ± 3.5 mm, DV −5.0 mm). The remaining pups (Sham;
occurring in adolescence (McCormick et al., 2004; Mathews et al., n = 20) were injected with 0.3 µl of aCSF at the same coordinates.
2008; McCormick, 2010; Garcia et al., 2017). Furthermore, cross- After surgery, wounds were closed with surgical glue (VetOne
sensitization has also been shown, where the repeated exposure Surgical Adhesive, Boise, ID, United States) and the pups were

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Sullivan et al. Adolescent Alcohol in NVHL Rat

warmed on a heating pad until their activity level was restored, were given a 2 week washout period where they remained in
at which time they were returned to their home cages. In order their home cage. Following the washout period, rats underwent
to control for litter/dam effects, half of each litter underwent the a challenge session where, again, they had a 30 min preinjection
NVHL surgery and the other half was sham-operated. Rats were period, followed by an injection of nicotine (0.5 mg/kg), and
weaned on PD 21 and housed individually. One sham rat did not remained in the arena for a 60 min postinjection period
recover after surgery. (Figure 1). Every pre- and post-injection session was videotaped
using a Defeway Security camera system (Shenzhen, China)
Alcohol Drinking in Adolescence and analyzed using Noldus EthoVision XT tracking software
We followed the methods from previously published studies (Wageningen, Netherlands) for distance traveled (cm), velocity
(Jeanblanc et al., 2015; Khokhar and Todd, 2017), but briefly, (cm/s), and location within the chamber (i.e., center zone).
half of each group [NVHL with alcohol exposure (NVHL AE); One NVHL AE rat had to be euthanized after completing the
sham with alcohol exposure (Sham AE)] was given free access 15 sessions but prior to the challenge session due to seizures.
to 10% v/v ethanol (EtOH) in water solution in their home cage One NVHL no AE rat died before completing the nicotine
for 24 h per day from PD 28–42. Alcohol intake, water intake, behavioral sensitization paradigm. Final numbers for the four
and body weights were measured daily and the position of the groups were: NVHL AE = 10; Sham AE = 10; NVHL no AE = 8;
alcohol and water bottles was alternated each day to prevent and Sham no AE = 9.
development of a side preference. At the end of PD 42, the alcohol
bottle was removed, and the rats had access to water only for the
Anxiety-Like Behavior
duration of the study.
Anxiety-like behavior was assessed using latency to center,
frequency in center, and total duration in center zone for
Nicotine Sensitization preinjection and postinjection on days 1, 5, 10, 15, and challenge.
Nicotine behavioral sensitization began on PD 60 and was The center zone (20 cm × 20 cm) was created using EthoVision
performed during the active cycle (the time when the animal XT arena settings by dividing the arena floor into nine equal-sized
rooms are dark between 0700 and 1,900 h). Nicotine bitartrate zones. The rat was considered in the center zone if the center
dihydrate (MilliporeSigma, Burlington, MA, United States; tracking point (while using three-point tracking) was within 2 cm
0.5 mg/mL) was dissolved in 0.9% sterile saline, adjusted to 7.4 of the defined center zone. In sessions where the rat never entered
pH, and administered with a volume of 1 mL/kg bodyweight. the center zone, the variable latency to center was recorded as
Locomotor activity was assessed in four open field arenas the maximum number of seconds for that session (i.e., 1,800 or
(60 cm × 60 cm × 33 cm) located in an animal behavior 3,600 s for preinjection and postinjection, respectively).
room, separate from the rats’ housing room. The lights were
turned on in the behavior room during nicotine behavioral
sensitization (average light intensity was 297.7 lux), and the Lesion Verification
paradigm was conducted so each round of four animals was At the end of the experiment, rats were euthanized by CO2
comprised of both NVHL and sham rats. The arena used for overdose, brains were extracted and flash frozen using 2-
each individual rat remained consistent throughout the entire methylbutane on dry ice. Tissue was stored at −20◦ C prior to
experiment and in between each round of four animals, the being sectioned at 40 µm using a Leica Biosystems CM1850
arenas were thoroughly cleaned. cryostat (Buffalo Grove, IL, United States) and stained with
The injection series occurred Monday through Friday for thionin. Lesion size was verified using an AmScope light
three consecutive weeks (15 sessions). During each session, rats microscope (Irvine, CA, United States). Lesions include cell loss,
were first placed in the arena for 30 min (i.e., preinjection). cellular disorganization, and ventricle enlargement (Figure 2).
After 30 min, each rat was given a subcutaneous (s.c.) injection NVHL rats with extra-hippocampal damage or unilateral damage
of nicotine (0.5 mg/kg in 1 mL/kg) and returned to the same were excluded from analysis. One NVHL rat was removed due to
arena for 60 min (i.e., postinjection). After the 15th session, rats an exceedingly large lesion.

FIGURE 1 | Experimental timeline. NVHL or sham surgery was performed on postnatal day (PD) 7. Half of each group received access to 10% ethanol (EtOH) in their
home cage from PD 28–42. Nicotine sensitization began on PD 60 and occurred Monday through Friday for three consecutive weeks (15 sessions). Each session
had a 30 min preinjection phase before the rat received a subcutaneous injection of 0.5 mg/kg nicotine followed by a 60 min postinjection phase. After a 2 week
washout, all rats had a challenge session on PD 96.

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Sullivan et al. Adolescent Alcohol in NVHL Rat

of nicotine on anxiety and to account for an increase in total

distance traveled after nicotine, the ratio of center crosses
to total distance traveled was calculated for preinjection and
postinjection on challenge day. A RMANOVA was used to assess
the preinjection and postinjection ratio between the four groups.

RESULTS
Adolescent Alcohol Intake
As shown in Figure 3, RMANOVA revealed that alcohol intake
during adolescence did not differ between NVHL AE and Sham
FIGURE 2 | Representative image of a NVHL (left) and sham (right) brain. AE groups [F (1,16) = 0.068, p = 0.798].
The arrow points to the NVHL lesion in the ventral hippocampus.
Nicotine Sensitization
Three-way RMANOVA revealed a significant treatment∗ day
Data Analyses interaction [F (3.869,127.671) = 73.656, p < 0.001] indicating
Alcohol Intake that distance traveled during postinjection was greater than
The alcohol intake (g EtOH/kg bodyweight) for each group preinjection, demonstrating nicotine behavioral sensitization.
(NVHL AE or Sham AE) was averaged for each day. A repeated There was also a significant treatment∗ day∗ group interaction
measures analyses of variance (RMANOVA) was used to [F (11.606,127.671) = 2.763, p = 0.003]. Two-way RMANOVA
compare the average alcohol intake between the groups across revealed a significant group effect in distance traveled during
alcohol exposure time. the preinjection phase across the 15 nicotine sessions [F
(3,33) = 4.639, p = 0.008; Figure 4A]. Bonferroni post hoc analyses
Nicotine Sensitization showed that the NVHL no AE group traveled significantly further
Total distance traveled during preinjection and postinjection was than every other group: Sham no AE (p = 0.027), NVHL AE
calculated for each day and averaged across groups. To account (p = 0.021), and Sham AE (p = 0.027). A similar pattern emerged
for individual differences in locomotor activity and to determine when focusing on the postinjection phase. Two-way RMANOVA
the level of nicotine behavioral sensitization, the distance traveled showed a significant group effect across the 15 nicotine sessions
during the first 30 min of postinjection was compared to that [F (3,33) = 10.206, p < 0.001; Figure 4B]. Bonferroni post hoc
days’ preinjection for each rat and expressed as a percentage analyses showed that the NVHL no AE rats traveled significantly
change. A three-way RMANOVA was run with day (day 1–15) further following nicotine injection than Sham no AE (p < 0.001),
and treatment (preinjection or postinjection) as within-subject NVHL AE (p = 0.009), and Sham AE (p < 0.001) rats.
factors and group (NVHL AE, Sham AE, NVHL no AE, Sham Looking at the challenge day, an ANOVA indicated a
no AE) as the between group factor. Two-way RMANOVAs were significant difference in distance traveled between groups during
subsequently used to determine group differences in preinjection, the preinjection phase [F (3,32) = 7.864, p < 0.001; Figure 4C].
postinjection, and percentage change. ANOVAs were used to Bonferroni post hoc showed that the NVHL no AE group traveled
compare distance traveled between the groups on challenge day. significantly further than Sham no AE (p = 0.002), NVHL AE
If the assumption of sphericity was violated, the Greenhouse- (p = 0.012), and Sham AE (p = 0.001) groups. Additionally,
Geisser correction was used. Any significant effects were further an ANOVA showed a significant difference in distance traveled
analyzed using Bonferroni post hoc tests. between groups during the postinjection phase on the challenge

Velocity
A three-way RMANOVA was run with day (day 1, 5, 10,
15) and treatment (preinjection or postinjection) as within-
subject factors and group as the between group factor. Two-
way RMANOVAs were used to compare preinjection and
postinjection average velocity between the groups. ANOVAs were
used to compare preinjection and postinjection velocity between
the groups on challenge day.

Anxiety-Like Behavior
Repeated measures analyses of variances were used to compare
preinjection and postinjection latency to center, center frequency,
FIGURE 3 | Adolescent alcohol intake. Alcohol Intake (g EtOH/kg bodyweight)
and total center duration over days 1, 5, 10, and 15.
from PD 28–42 did not differ between NVHL AE and Sham AE rats. Data is
ANOVAs were used to compare groups during preinjection shown as group mean ± SEM.
and postinjection on challenge day. To determine the effect

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Sullivan et al. Adolescent Alcohol in NVHL Rat

FIGURE 4 | (Continued)
AE group only traveled significantly further than the Sham no AE group.
(E) NVHL no AE rats had significantly elevated average velocity during the
preinjection phase on days 1, 5, 10, and 15. (F) NVHL no AE rats had
significantly elevated average velocity during the postinjection phase on days
1, 5, 10, and 15. (G) Average velocity of the NVHL no AE rats remained
elevated during the preinjection phase on the challenge day. (H) During the
postinjection phase, NVHL no AE rats only had significantly increased velocity
compared to Sham no AE rats. Data is shown as group mean ± SEM.
∗ p ≤ 0.05; ∗∗ p ≤ 0.01; and ∗∗∗ p ≤ 0.001.

day [F (3,32) = 4.051, p = 0.015; Figure 4D]. Bonferroni

post hoc analyses indicated that the NVHL no AE group traveled
significantly further than the Sham no AE group (p = 0.012).
However, the NVHL no AE group was no longer significantly
different from the NVHL AE or the Sham AE group following
the injection of nicotine.
Since the NVHL no AE group showed significantly greater
distance traveled in both the preinjection and postinjection
phase, the level of nicotine behavioral sensitization when
controlling for any nicotine induced context sensitization
was determined by calculating the percentage change in
distance traveled from the preinjection phase to the first
30 min of the postinjection phase on each day for each
rat. RMANOVA revealed no group differences in the level
of nicotine behavioral sensitization across the 15 sessions [F
(3,33) = 0.380, p = 0.768; Figure 5], however, a significant
effect of day using the Greenhouse-Geisser correction again
indicates that all groups did become sensitized to nicotine
[F (3.879,128.006) = 16.872, p < 0.001]. An ANOVA on
percentage change in distance traveled on the challenge day
showed no differences between groups [F (3,32) = 2.259, p = 0.1;
Figure 5].

Velocity
Three-way RMANOVAs revealed significant treatment∗ day,
day∗ group, and group∗ treatment interactions. Two-way
RMANOVAs showed a group effect in average velocity during
the preinjection [F (3,33) = 5.165, p = 0.005; Figure 4E] and
postinjection [F (3,33) = 7.949, p < 0.001; Figure 4F] phase
across days 1, 5, 10, and 15. Bonferroni post hoc analyses showed
that NVHL no AE rats moved with greater average velocity
during both preinjection and postinjection sessions compared to
Sham no AE (p = 0.027 and p = 0.002 for pre- and post-injection,
respectively), NVHL AE (p = 0.006 and p = 0.013), and Sham AE
(p = 0.023 and p = 0.001) groups.
During the challenge day, ANOVA showed a significant
difference in average velocity between groups during the
FIGURE 4 | Total distance traveled and average velocity. (A) Total distance preinjection phase [F (3,32) = 7.397, p = 0.01; Figure 4G]
traveled before an injection of 0.5 mg/kg nicotine across the 15 sensitization and the postinjection phase [F (3,32) = 4.154, p = 0.014;
sessions. (B) Total distance traveled after an injection of 0.5 mg/kg nicotine
across the 15 sensitization sessions. The NVHL no AE group showed
Figure 4H]. Bonferroni post hoc analyses indicated that during
significantly greater distance traveled compared to the Sham no AE, NVHL the preinjection phase NVHL no AE rats had greater average
AE, and Sham AE groups during both the preinjection and postinjection velocity than all other groups (Sham no AE [p = 0.001],
phases. (C) On the preinjection phase of the challenge day, the NVHL no AE NVHL AE [p = 0.018], NVHL no AE [p = 0.003]). However,
group showed significantly more distance traveled than the other three
during the postinjection phase, NVHL no AE rats had
groups. (D) During the postinjection phase on the challenge day, the NVHL no
(Continued)
significantly increased velocity compared to only Sham no AE
rats (p = 0.025).

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Sullivan et al. Adolescent Alcohol in NVHL Rat

FIGURE 5 | Percentage change in distance traveled. The level of nicotine

sensitization was determined by calculating the percentage change in
distance traveled from the preinjection phase to the first 30 min of the
postinjection phase across each of the initial 15 sessions and on the challenge
day for each rat. No group differences were observed but a significant effect
of day across the 15 sessions indicates that all groups became sensitized to
nicotine. Data is shown as group mean ± SEM.

Anxiety-Like Behavior
Repeated measures analyses of variances found no significant
differences between groups in latency to center zone
(Supplementary Figures 1A,B), frequency in center
(Figures 6A,B), and total duration in center (Supplementary
Figures 1E,F) in both preinjection and postinjection phases
across days 1, 5, 10, and 15.
Similarly, ANOVAs found no significant differences between
groups in latency to center zone (Supplementary Figures 1C,D)
and total duration in center (Supplementary Figures 1G,H)
during preinjection and postinjection on the challenge day.
However, there was a significant difference between groups in
frequency in center during the preinjection phase on challenge
day [F (3,32) = 5.518, p = 0.004; Figure 6C]. Bonferroni post hoc FIGURE 6 | Center zone frequency. (A) Frequency of center zone crosses
analysis showed that NVHL no AE rats entered the center zone before the injection of nicotine on days 1, 5, 10, and 15. (B) Frequency of
more frequently than Sham no AE (p = 0.027), NVHL AE center zone crosses after the injection of nicotine on days 1, 5, 10, and 15.
No significant group differences were observed. (C) NVHL no AE rats had
(p = 0.028), and Sham AE (p = 0.004). Following the nicotine
significantly increased frequency of center crosses during preinjection on the
injection on challenge day, the significant differences between challenge day. (D) Following the nicotine injection on challenge day, no group
groups in center frequency no longer remained (Figure 6D). differences were observed. (E) There was a significant increase in the ratio of
To assess the effect of nicotine on anxiety-like behaviors frequency of center crosses to distance traveled following nicotine on the
and to account for an increase in locomotive behavior after challenge day with no group differences. Data is shown as group
mean ± SEM. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; and ∗∗∗ p ≤ 0.001.
nicotine, the ratio of number of center crosses to total distance
traveled was calculated for preinjection and postinjection on
the challenge day. RMANOVA revealed a significant increase
in the center crosses-to-distance ratio during the postinjection between NVHL and sham rats, regardless of adolescent alcohol
phase [F (1,32) = 37.161, p < 0.001] with no significant group exposure. Importantly though, all groups did sensitize to
differences (Figure 6E). nicotine, as demonstrated by a significant treatment∗ day
interaction in distance traveled and the significant increase in
percentage change across the 15 sessions (Figure 5).
DISCUSSION The NVHL no AE group showed a significant increase
in distance traveled during the preinjection phase of the 15
Here, we sought to determine the effects that alcohol exposure sessions (Figure 4A), in addition to a significant increase in
during adolescence would have on nicotine behavioral velocity (Figure 4E). While previous studies have found that
sensitization in the NVHL model of SCZ. The results suggest postpubertal NVHL rats show spontaneous hyperlocomotion
that adolescent alcohol exposure from PD 28–42 did not alter the (Lipska et al., 1993; Sams-Dodd et al., 1997), as well as increased
amount of nicotine behavioral sensitization. When controlling locomotor response to a novel environment (Berg and Chambers,
for baseline differences in distance traveled, there were no 2008), we saw no group differences in distance traveled during
differences in the amount of nicotine behavioral sensitization the preinjection phase on Day 1. However, we did observe

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that the NVHL no AE group showed significant increases in the PFC (Laplante et al., 2004a). Another study indicated that
distance traveled during the preinjection phase across days once tail-pinch stress resulted in a significantly greater increase
nicotine injections began, suggesting the development of context in PFC acetylcholine release in the NVHL rats, which was
sensitization, a phenomenon that has previously been reported subsequently blocked by D1 and D2 antagonists (Laplante et al.,
in the literature in normal rats. Rats treated with nicotine 2004b). Interestingly, nAChRs have been shown to be involved
(0.6 mg/kg) for 9 days showed an increase in locomotor activity in alcohol-related behaviors where blocking nAChRs partially
compared to saline treated animals in the 30 min prior to prevented alcohol-induced locomotor activity (Blomqvist
drug administration (Kosowski and Liljequist, 2005). Similarly, et al., 1992). While some preclinical studies suggest that
an environment repeatedly paired with nicotine (0.6 mg/kg) moderate lengths of alcohol exposure (15–17 days) do not alter
acquired the ability to elicit increases in activity even in the nicotinic receptor binding (de Fiebre and Collins, 1993; Ribeiro-
absence of nicotine (Walter and Kuschinsky, 1989; Bevins et al., Carvalho et al., 2009), chronic alcohol treatment (28 weeks)
2001). These data are the first to report that the NVHL rat has in rats produced long-lasting reductions in acetylcholine
enhanced context sensitization, possibly pointing to an increase levels, acetylcholinesterase activity, choline uptake, and
in the salience of nicotine, and a shift of that salience from acetylcholinesterase-positive neurons (Arendt et al., 1988,
nicotine to the context, in this group. Furthermore, it appears 1989). Similarly, non-human primates chronically treated with
that adolescent alcohol exposure impairs the formation of context alcohol for 4 weeks had decreased nAChR availability in cortical
sensitization, possibly by dampening the salience of nicotine, and thalamic regions (Cosgrove et al., 2010). Though the NVHL
in the NVHL AE rat. An increase in the salience of nicotine AE and Sham AE rats in the current study were only exposed to
is supported by previous work demonstrating that NVHL rats 14 days of alcohol, there is potential that alcohol exposure during
have increased nicotine seeking behavior during extinction than adolescence could alter nAChR function.
their sham counterparts (Berg et al., 2013; Rao et al., 2016; Sentir Using percentage change as a measure of the amount of
et al., 2020). Thus the current results lend further support to the nicotine behavioral sensitization, we found that there were no
NVHL rat as a model to better understand SCZ and the increased differences between NVHL and sham groups, regardless of
prevalence of nicotine use. whether they received alcohol during adolescence. Our results
While additional research is needed to elucidate the exact contrast previously published results showing that NVHL rats
mechanism, one potential reason for the reduction in context (without adolescent alcohol exposure) have enhanced nicotine
sensitization seen in the NVHL AE group when compared to behavioral sensitization (Berg and Chambers, 2008). A possible
the NVHL no AE group, may be the impact that alcohol has explanation for the discrepancies between these studies is the
on developing brain regions. Clinical studies show that alcohol post-weaning housing conditions of the animals. The rats in
use during adolescence impacts the volume of several brain the current study were singly housed so that alcohol intake
regions such as the prefrontal cortex (PFC; De Bellis et al., during adolescence could be determined for each individual.
2005), nucleus accumbens (Thayer et al., 2012), hippocampus (De The rats used in the previously published study (Berg and
Bellis et al., 2000; Nagel et al., 2005; Medina et al., 2007), and Chambers, 2008) were pair housed after weaning. Several studies
amygdala (Wilson et al., 2015). Preclinical studies corroborate have shown that housing conditions can influence not only
these findings with alcohol causing numerous anatomical and the locomotor response to a novel environment, but also the
functional alterations, including decreases in neurogenesis and behavioral response to drugs, including nicotine. Rats housed
region-specific brain damage and cell death (Crews et al., in isolation show increased locomotor response in a novel
2000; Spear, 2015, 2016). As many of these brain regions environment compared to those housed in pairs (Garcia et al.,
play a role in incentive salience, it is possible that disrupted 2017) and those housed in groups (Smith et al., 1997; Cheeta
cortical development stemming from alcohol exposure during et al., 2001). Furthermore, rats housed in isolation have enhanced
adolescence dampened the salience of nicotine in the NVHL AE sensitization to the locomotor effects of repeated administration
group which prevented the development of context sensitization. of amphetamine (Smith et al., 1997). Additionally, female
Another neurobiological mechanism that may underlie rats that underwent chronic social stress during adolescence
behavioral changes within the NVHL rat are disruptions in (isolation for 1 h each day and then housed with a new
nicotinic acetylcholine receptor (nAChR) function. Extensive partner) show increased locomotor sensitization in response to
literature exists demonstrating that patients with SCZ have amphetamine (Mathews et al., 2008) and nicotine (McCormick
disrupted nAChR function and decreased receptor density et al., 2004). Therefore it is reasonable that isolated housing
(Freedman et al., 1995; Breese et al., 2000; Durany et al., led to an increase in the nicotine behavioral sensitization of the
2000; D’Souza and Markou, 2011; Esterlis et al., 2014). These sham groups, and combined with a potential ceiling effect in the
results are supported by a preclinical study showing that nicotine behavioral sensitization of the NVHL groups, any group
NVHL rats have a 12% reduction in nAChR binding in the differences were masked.
PFC compared to their sham counterparts (Berg et al., 2015). Using measures related to the center zone of the open
Furthermore, additional cholinergic alterations exist in this field arena as a proxy for anxiety-like behaviors (latency to
model. In vivo acetylcholine release was hyper reactive to enter the center zone and the duration of time spent in the
both peripheral and local administration of a dopamine (D)1 center zone), we found no differences between NVHL and
agonist in NVHL rats, and receptor autoradiography showed sham groups, regardless of alcohol exposure (Supplementary
an increase in muscarinic (M)1 -like receptor binding sites in Figure 1). The NVHL no AE group did have a significantly

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Sullivan et al. Adolescent Alcohol in NVHL Rat

increased number of center entrances compared to the other unlikely that repeated saline injections in our hands would cause
groups, but only during the preinjection phase during the behavioral sensitization.
challenge day (Figure 6C). With no other increases in anxiety- In this study, we found that NVHL rats demonstrated
like behaviors and the significant increase in both total distance an apparent sensitization to the nicotine paired context, and
traveled and average velocity, it is likely that the significantly adolescent alcohol exposure prevented the formation of this
elevated center frequency in the NVHL no AE rats was due to context sensitization in the NVHL AE rats. We found that
their increased context sensitization. Previous studies assessing exposure to alcohol during adolescence did not impact the
anxiety-related behaviors in the NVHL rat have found mixed amount of nicotine behavioral sensitization in adulthood.
results based on the method used to measure anxiety. In one Surprisingly, NVHL rats (regardless of alcohol exposure) did
study, male NVHL rats demonstrated persistent anxiety as not show increased nicotine behavioral sensitization as had been
adolescents and adults compared to control rats, spending less previously reported, potentially due to post-weaning housing
time in the central zone of an open field task (Sams-Dodd conditions. Nicotine treatment had an anxiolytic effect during the
et al., 1997). However, several other studies found that male postinjection phase of the challenge day, however, there were no
and female NVHL rats spend more time in the open arm of an group differences. Future studies could more specifically test the
elevated plus maze, suggesting less anxiety (Wood et al., 2003; impact of social isolation on nicotine behavioral sensitization and
Beninger et al., 2009). Although the current results found that the development of context sensitization in the NVHL rat, as well
there were no group differences in anxiety-like behavior using as expand this work to females.
an open field task, future studies assessing anxiety in the NVHL
rat should take into account locomotor differences that may
confound the results. DATA AVAILABILITY STATEMENT
In order to assess the effect of nicotine treatment on anxiety-
like behaviors and to control for an increase in movement after The raw data supporting the conclusions of this article will be
an injection of nicotine, the ratio of number of center crosses made available by the authors, without undue reservation.
to total distance traveled was calculated for preinjection and
postinjection on the challenge day. The significant increase in ETHICS STATEMENT
this ratio during postinjection demonstrates that nicotine had
an anxiolytic effect on all groups (Figure 6E). This is consistent The animal study was reviewed and approved by Institutional
with some previous literature showing that 7 days of nicotine Animal Care and Use Committee of Dartmouth College.
treatment (Irvine et al., 2001) or chronic nicotine administered
via drinking water (Onaivi et al., 1994) both resulted in anxiolytic
effects on elevated plus maze behaviors. AUTHOR CONTRIBUTIONS
One limitation of the current study was not using both
sexes, though there is variability in the literature as to whether ES contributed to planning the experimental design and wrote
a sex difference in nicotine behavioral sensitization exists. the manuscript. ES, LL, and EB collected the behavioral data. ES
Nevertheless, some studies have found that female rats show and DW performed the NVHL surgeries and DW contributed
more locomotor activity in response to nicotine behavioral to editing the manuscript. AH contributed to data analysis
sensitization (Booze et al., 1999; Harrod et al., 2004), while and editing the manuscript. EB contributed to the histological
others find that sex does not have marked influences on this assessment. JK and WD contributed to planning the experimental
behavior (Kanyt et al., 1999; Ericson et al., 2010). A limited design and editing the manuscript. All authors read and approved
number of studies have used both male and female NVHL rats the final manuscript.
to assess cognitive abilities (Chambers et al., 1996; Beninger
et al., 2009), neurotransmitter release (Beninger et al., 2009),
and expression of G-protein coupled receptor kinases (Bychkov
FUNDING
et al., 2011). However, to our knowledge, no work has This work was supported through Alan I. Green’s endowed fund
been done exploring sex differences in nicotine behavioral as the Raymond Sobel Professor of Psychiatry at Dartmouth,
sensitization specifically in the NVHL rat. Another limitation an NIAAA training grant (F31AA028413; ES), the Hitchcock
of the current study was the absence of a saline injected Foundation (AH and DW), a NIDA T32 grant (T32DA037202;
group which would serve to control for any handling and AH and DW), a NIDA R21 grant (R21DA044501; JK, DW, and
injection stress. While this is an important control group, many EB), and a NIMH K08 grant (K08MH117347; WD). Funds were
previous studies have demonstrated that repeated subcutaneous received from the Dartmouth College Library Fund to Support
or intraperitoneal injections of saline do not increase locomotor Open Access Publication.
activity (McCormick et al., 2004; Kosowski and Liljequist, 2005;
Varvel et al., 2007; Marin et al., 2009; Gomez et al., 2012;
Hamilton et al., 2014; Carrara-Nascimento et al., 2020; Trujillo ACKNOWLEDGMENTS
and Heller, 2020). Additional studies corroborate these findings
specifically in NVHL rats (Conroy et al., 2007; Berg and The authors would like to thank the late Alan I. Green for his
Chambers, 2008; Chambers et al., 2013). Therefore, it is highly remarkable mentorship. His commitment to supporting trainees

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8 November 2021 | Volume 15 | Article 760791
Sullivan et al. Adolescent Alcohol in NVHL Rat

helped to foster a love of science among many. His guidance will preinjection phase on days 1, 5, 10, and 15. (B) There were no significant group
be profoundly missed. differences in latency to center zone during the postinjection phase on days 1, 5,
10, and 15. (C) There were no significant group differences in latency to center
zone during preinjection phase on the challenge day. (D) There were no significant
group differences in latency to center zone during postinjection phase on the
SUPPLEMENTARY MATERIAL challenge day. (E) There were no significant group differences in the total duration
of time spent in the center zone during the preinjection phase on days 1, 5, 10,
and 15. (F) There were no significant group differences in the total duration of time
The Supplementary Material for this article can be found
spent in the center zone during the postinjection phase on days 1, 5, 10, and 15.
online at: https://www.frontiersin.org/articles/10.3389/fnbeh. (G) There were no significant group differences in the total duration of time spent
2021.760791/full#supplementary-material in the center zone during the preinjection phase of the challenge day. (H) There
were no significant group differences in the total duration of time spent in the
Supplementary Figure 1 | Latency to and duration in center zone. (A) There center zone during the postinjection phase of the challenge day. Data is shown as
were no significant group differences in latency to center zone during the group mean ± SEM.

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an incentive-sensitization theory of addiction. Brain Res. Rev. 18, 247–291. potential conflict of interest.
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of addiction: some current issues. Philos. Trans. R. Soc. B. 363, 3137–3146. and do not necessarily represent those of their affiliated organizations, or those of
doi: 10.1098/rstb.2008.0093 the publisher, the editors and the reviewers. Any product that may be evaluated in
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Alcohol use by Youth. Pediatrics 144:e20191357. doi: 10.1542/peds.2019-1357 endorsed by the publisher.
Sams-Dodd, F., Lipska, B. K., and Weinberger, D. R. (1997). Neonatal lesions
in the rat ventral hippocampus result in hyperlocomotion and deficits in Copyright © 2021 Sullivan, Locke, Wallin, Khokhar, Bragg, Henricks and Doucette.
social behaviour in adulthood. Psychopharmacology 132, 303–310. doi: 10.1007/ This is an open-access article distributed under the terms of the Creative Commons
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Sentir, A. M., Bell, R. L., Engleman, E. A., and Chambers, R. A. (2020). is permitted, provided the original author(s) and the copyright owner(s) are credited
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 ORIGINAL RESEARCH
published: 04 January 2022
doi: 10.3389/fnbeh.2021.779080

High-Salt Diet in the Pre- and

Postweaning Periods Leads to
Amygdala Oxidative Stress and
Changes in Locomotion and
Anxiety-Like Behaviors of Male
Wistar Rats
Edited by:
Pedro Ernesto de Pinho Tavares Leal 1,2 , Alexandre Alves da Silva 1,2 ,
Carla Cannizzaro,
University of Palermo, Italy
Arthur Rocha-Gomes 2 , Tania Regina Riul 2,3 , Rennan Augusto Cunha 1 ,
Christoph Reichetzeder 4* and Daniel Campos Villela 1*
Reviewed by:
Kaichi Yoshizaki, 1
Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal dos Vales do Jequitinhonha e Mucuri,
Aichi Developmental Disability Center, Diamantina, Brazil, 2 Laboratório de Nutrição Experimental – LabNutrex, Departamento de Nutrição, Universidade Federal
Institute for Developmental Research, dos Vales do Jequitinhonha e Mucuri, Diamantina, Brazil, 3 Programa de Pós-Graduação em Ciências da Nutrição,
Japan Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina, Brazil, 4 Department of Nutritional Toxicology,
María Carolina Fabio, Institute of Nutritional Science, University of Potsdam, Potsdam, Germany
Medical Research Institute Mercedes
and Martín Ferreyra (INIMEC),
Argentina High-salt (HS) diets have recently been linked to oxidative stress in the brain, a fact that
Valentina Castelli, may be a precursor to behavioral changes, such as those involving anxiety-like behavior.
University of Palermo, Italy
However, to the best of our knowledge, no study has evaluated the amygdala redox
*Correspondence:
Daniel Campos Villela status after consuming a HS diet in the pre- or postweaning periods. This study aimed
[email protected] to evaluate the amygdala redox status and anxiety-like behaviors in adulthood, after
Christoph Reichetzeder
inclusion of HS diet in two periods: preconception, gestation, and lactation (preweaning);
[email protected]
and only after weaning (postweaning). Initially, 18 females and 9 male Wistar rats
Specialty section: received a standard (n = 9 females and 4 males) or a HS diet (n = 9 females and 5
This article was submitted to
Pathological Conditions,
males) for 120 days. After mating, females continued to receive the aforementioned
a section of the journal diets during gestation and lactation. Weaning occurred at 21-day-old Wistar rats and
Frontiers in Behavioral Neuroscience the male offspring were subdivided: control-control (C-C)—offspring of standard diet
Received: 17 September 2021 fed dams who received a standard diet after weaning (n = 9–11), control-HS (C-HS)—
Accepted: 15 November 2021
Published: 04 January 2022 offspring of standard diet fed dams who received a HS diet after weaning (n = 9–11),
Citation: HS-C—offspring of HS diet fed dams who received a standard diet after weaning (n = 9–
Leal PEdPT, da Silva AA, 11), and HS-HS—offspring of HS diet fed dams who received a HS diet after weaning
Rocha-Gomes A, Riul TR, Cunha RA,
Reichetzeder C and Villela DC (2022)
(n = 9–11). At adulthood, the male offspring performed the elevated plus maze and
High-Salt Diet in the Pre- open field tests. At 152-day-old Wistar rats, the offspring were euthanized and the
and Postweaning Periods Leads amygdala was removed for redox state analysis. The HS-HS group showed higher
to Amygdala Oxidative Stress
and Changes in Locomotion locomotion and rearing frequency in the open field test. These results indicate that this
and Anxiety-Like Behaviors of Male group developed hyperactivity. The C-HS group had a higher ratio of entries and time
Wistar Rats.
Front. Behav. Neurosci. 15:779080.
spent in the open arms of the elevated plus maze test in addition to a higher head-
doi: 10.3389/fnbeh.2021.779080 dipping frequency. These results suggest less anxiety-like behaviors. In the analysis of

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Leal et al. Early Life High-Salt Effects

the redox state, less activity of antioxidant enzymes and higher levels of the thiobarbituric
acid reactive substances (TBARS) in the amygdala were shown in the amygdala of
animals that received a high-salt diet regardless of the period (pre- or postweaning).
In conclusion, the high-salt diet promoted hyperactivity when administered in the pre-
and postweaning periods. In animals that received only in the postweaning period,
the addition of salt induced a reduction in anxiety-like behaviors. Also, regardless
of the period, salt provided amygdala oxidative stress, which may be linked to the
observed behaviors.
Keywords: high-sodium, open-field, elevated plus-maze, pre-natal, post-natal, redox state

INTRODUCTION production of nitrogen and oxygen-free radicals (Kitiyakara

et al., 2003; Huang et al., 2017; Zheng et al., 2019). Also, it is
Sodium chloride (NaCl), also known worldwide as salt, is one of highlighted that a high-salt diet causes oxidative stress in the
the most widely used condiments in food processing (Steffensen hippocampus, hypothalamus, and cerebellum, important brain
et al., 2018). It is estimated that current salt intake averages are regions for behavior and cognition (Bai et al., 2017; Ge et al.,
6 g/day in most countries (86% greater than the optimal amount), 2017; Stocher et al., 2018). However, to the best of our knowledge,
with varying usages ranging from food preservation to flavor there are no studies evaluating the amygdala redox status
enhancement (Afshin et al., 2019; Tan et al., 2021). Excessive use after administration of a high-salt diet, either before weaning
of salt in the diet is responsible for the development mainly of (preweaning) or after weaning (postweaning). Noteworthy, the
cardiovascular diseases (Huang et al., 2020; Neal et al., 2021), but amygdala is a major brain region in the interpretation of
also stomach cancer (Ge et al., 2012), kidney diseases (Garofalo environmental threats, possibly related to anxiety-like and fear
et al., 2018), and osteoporosis (Fatahi et al., 2018). Moreover, behaviors in rodents (Calhoon and Tye, 2015; Wilson et al., 2015;
recent data indicates that high-salt diets were directly related dos Santos et al., 2017).
to approximately three million deaths in 1 year, being classified Therefore, this study aimed to evaluate the effects of the high-
as one of the top 3 dietary risk factors for health (Bill and salt diet on amygdala redox status and anxiety-like behaviors
Foundation, 2019; He et al., 2020). at adulthood, considering: (1) the inclusion of the salt in the
In addition to the known harmful health effects, the use preconception, gestation, and lactation periods (preweaning) and
of high-salt diets has recently been linked to cerebrovascular (2) the addition of salt in the diet only after weaning until
diseases and cognitive impairment in humans (Heye et al., adulthood (postweaning). The main hypothesis was that the
2016). Studies in rodents that used dietary or water salt high-salt diet may result in amygdala oxidative stress regardless of
supplementation (2–8%) confirm these findings, reporting the period, which, in turn, would promote changes in anxiety-like
impaired cognition, aggravation of cerebral ischemic injury, and behaviors at adulthood.
high-stress responsivity (Ge et al., 2017; Faraco et al., 2018,
2019; Mitchell et al., 2018; Gilman et al., 2019a; Zhang et al.,
2020). Importantly, preclinical studies suggest that the maternal MATERIALS AND METHODS
high-salt diet can also induce changes in locomotion, inhibition,
and anxiety in the offspring, when fed in the preconception, Ethics
gestation, or lactation periods (Mcbride et al., 2008; Mecawi This experimental protocol was approved by the Ethics
and Almeida, 2017; Dingess et al., 2018). During these periods, Committee on the Use of Animals of Universidade Federal dos
the offspring is highly susceptible to dietary salt, which may Vales do Jequitinhonha e Mucuri (CEUA-UFVJM) (protocol
impact on development, potentially leading to lifelong changes 025/2018). These are also in agreement to the ethical principles
in metabolism and behavior. These changes are related to the of the National Institutes of Health Guide for the Care and Use
Developmental Origin of Health and Disease (DOHaD), which of Laboratory Animals (NIH Publications No. 80-23). All the
proposes that adversities in early life can result in persistent rats (Wistar—Rattus norvegicus) were obtained from Laboratório
changes in physiology, leading to an increased risk of developing de Pós-Graduação e Pesquisa (LPP-UFVJM) and housed in
diseases in adulthood (O’Donnell and Meaney, 2016; Klein et al., conditions of natural moisture, temperature of 22 ± 2◦ C
2018; de Souza et al., 2020a). (controlled by an air conditioner), and a 12-h cycle of light
One of the main possible mechanisms for behavioral changes and darkness, with the light cycle beginning at 7:00 am. All the
caused by salt consumption is related to the oxidative stress animals had free access to potable water and their respective diets.
(Santisteban and Iadecola, 2018; He et al., 2020). Evidence
indicates that a high-salt diet can reduce nitric oxide (NO) Experimental Design
production (Dong et al., 2011; Kouyoumdzian et al., 2016; Initially, 18 female and 9 male Wistar rats aged 21 days were
Zheng et al., 2019), suppress the activity of antioxidant enzymes used. The animals were housed in 3 per box according to
(Kitiyakara et al., 2003; Huang et al., 2017), and increase the sex in order to randomly receive the diets for a duration of

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120 days: control (C): received standard diet (laboratory chow arm (closed or open) and the time spent in them were evaluated
for rodents: Nuvilab CR-1, Quimtia S/A, Paraná, Brazil) (n = 9
R
(Teixeira et al., 2020). In addition, to analyze the risk assessment
females and 4 males) or high-salt (HS) diet: received laboratory of animal, the frequency of head-dipping (the head flexes below
chow with added salt (4% NaCl non-iodized, Mossoró —purity R
the edge of the open arms), rearing (frequency with which the
96.04% bought at the local store) (n = 9 females and 5 males). animal stands on its hind legs), and grooming (frequency of
Copulation was evaluated every morning and confirmed by the time which the animal spent licking or scratching itself while
presence of sperm in the vaginal smear, which was considered stationary) was recorded (Plescia et al., 2015; Guedine et al., 2018;
the beginning of gestation. All the animals received food and Riul and Almeida, 2020).
water ad libitum. After this period, the nulliparous female rats The OF test is widely used to check locomotion of animal
(141 days old) were placed for mating with males (1 male to 3 through distance covered, but is also used to evaluate anxiety-
females) during the dark cycle (7:00 pm to 7:00 am) every day. like behaviors over the conflict between exploring a new
Parents during mating (males and females) and dams during environment and exposed to an open arena (Montgomery, 1955).
gestation and lactation continued receiving the aforementioned The OF is a square wooden arena, with total dimensions of
diets (control or HS). At birth, the litters were culled to eight pups 70 cm × 70 cm × 50 cm (dimensions of central zone of the
(6 males and 2 females). arena: 35 cm × 35 cm), being subdivided into 16 quadrants
In the postweaning period, only male offspring were used, (17.5 cm × 17.5 cm). Each animal was placed in the center of the
housed 3 animals per box. Male offspring was randomly allocated OF and free exploration was allowed for 10 min (Teixeira et al.,
to receive either control (laboratory chow Nuvilab CR-1) or HS
R
2020). The parameters of center zone entries frequency (defined
diets (laboratory chow with added salt 4% NaCl non-iodized). when the animal inserted the four paws in the central zone), time
Therefore, the offspring were subdivided into the following spent in the center zone, distance covered (quadrants), rearing,
groups: control-control (C-C)—offspring of standard diet fed and grooming frequency were observed (Teixeira et al., 2020;
dams who received a standard diet after weaning (n = 9–11), Rocha-Gomes et al., 2021a).
control-high-salt (C-HS)—offspring of standard diet fed dams
who received a HS diet (laboratory chow with added salt at 4% Redox State
NaCl non-iodized) after weaning (n = 9–11), HS-C—offspring The animals were euthanized by decapitation when they
of HS diet fed dams who received a standard diet after weaning were at 152 day-old. The whole brain was rapidly removed
(n = 9–11), and HS-HS—offspring of HS diet fed dams who (<1 min) and submerged on cold (4◦ C) phosphate-buffered
received a HS diet (laboratory chow with added salt at 4% NaCl saline (PBS) (50 mM; pH 7.0), followed by the amygdala
non-iodized) after weaning (n = 9–11). dissection (Paxinos and Watson, 2014). After, the tissues were
The male offspring received the aforementioned diets until homogenized in cold PBS (4◦ C; 50 mM; pH 7.0) and centrifuged
adulthood (141 day-old), when behavioral tests were carried out. at 750 × g for 10 min at 4◦ C (Melo et al., 2019). Both
Approximately, 1–2 animals from each litter were used for the the sides of the amygdala were used for the analysis of the
behavioral and redox status analyses, in order to reduce litter total antioxidant capacity, activity of antioxidant enzymes, and
effects. The experimental design is shown in Figure 1. oxidative stress marker.
The total antioxidant capacity was evaluated using the ferric
Offspring Behavior reducing antioxidant power (FRAP) method (Benzie and Strain,
All the tests were performed in an isolated room (130 lux) and 1996). The assay is based on the ability of the antioxidant
in a double-blind manner. The offspring performed the elevated compounds of the sample to reduce the ferric-tripyridyltriazine
plus maze (EPM) (141 day-old) and open field (OF) (151 day-old) complex to ferrous tripyridyltriazine, monitored at 550 nm.
tests, both during the morning period (7:00–12:00 am). A camera Ferrous sulfate (FeSO4 ) was used as standard and the results were
(Sony Handycam ) was positioned above the arena and two
R
reported as nM of FeSO4 /mg protein (Freitas et al., 2019).
independent, blinded, and experienced studies later evaluated the For the activity of the antioxidant enzyme superoxide
randomly arranged videos. Between the performances of the two dismutase (SOD), a solution containing 50 mM potassium
behavioral tests (EPM and OF), the animals were kept with their dihydrogen phosphate (KH2 PO4 ) and 1 mM diethylene-
respective diets in the conditions mentioned previously. All the triamine-pentaacetic acid (DTPA) was added to the tissue
equipment used was cleaned with 70% ethanol between each test homogenate. Following this, 0.2 mM of pyrogallol was added and
to eliminate olfactory cues. its oxidation was measured at 420 nm for 250 s at interval of 10 s.
The EPM test is based on the aversion to open and high The results were defined as one unit (U) of SOD per mg protein
spaces of the rodents and is a classic test for assessing anxiety-like in the sample (U/mg protein) (Marklund and Marklund, 1974;
behaviors (Pellow et al., 1985; de Souza et al., 2020b). The EPM is Melo et al., 2019).
made of wood, with two closed arms (50 cm × 10 cm × 40 cm) Catalase (CAT) activity was assessed by metabolizing
perpendicular to two open arms (50 cm × 10 cm), besides a hydrogen peroxide (Nelson and Kiesow, 1972). To perform
central area (10 cm × 10 cm), raised 50 cm high from the floor. this test, 5 µl of hydrogen peroxide (0.3 M) was added to a
Each rat was placed individually in the central area of the EPM solution containing potassium phosphate buffer (50 mM; pH
with its head facing toward one closed arm and its movements 7.0; 25◦ C) and 30 µl of sample. The readings were performed in
were filmed for 10 min (Teixeira et al., 2020). The ratio of entries a microplate reader every 15 s for 1 min (at 25◦ C). CAT activity
(considered as the animal inserting all the four paws) in each was expressed in 1E/min/mg of protein (Freitas et al., 2019).

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FIGURE 1 | Representation of the experimental design. Control (standard diet; laboratory chow Nuvilab R CR-1) or high-salt (HS) (laboratory chow with added salt
4% NaCl non-iodized) diets were provided for parents at 21 day-old. They received their respective diets for 120 days, until they reached adulthood. During the
periods of mating, gestation, and lactation, the dams remained on the aforementioned diets. The preconception, gestation, and lactation periods were classified as
“preweaning.” At 21 day-old, the offspring were weaned and received until adulthood the control or HS diets. The period from weaning to adulthood was classified
as “postweaning.” Therefore, the offspring were subdivided into the groups: control-control (C-C), offspring of standard diet fed dams who received a standard diet
after weaning; control-HS (C-HS), offspring of standard diet fed dams who received a HS diet after weaning; HS-C, offspring of HS diet fed dams who received a
standard diet after weaning; and HS-HS, offspring of HS diet fed dams who received a HS diet after weaning.

Glutathione S-transferase (GST) activity was estimated postweaning (received standard or HS diets only after weaning).
spectrophotometrically as previously described (Habig et al., The Newman–Keuls was used as a post hoc test when appropriate
1974). The assay occurred according to the formation of (p < 0.05). Data with non-normal distributions were analyzed by
glutathione conjugated with 2,4-dinitrochlorobenzene (molar the Kruskal–Wallis test with the Dunn’s post hoc test. Results are
coefficient extinction: ε340 = 9.6 mmol × L−1 × cm−1 ). expressed as a mean and SEM.
One unit of GST activity was defined as the amount of
the enzyme that catalyzed the formation of one µmol of
product × min−1 × mL−1 (Rocha-Gomes et al., 2021a). RESULTS
The lipid peroxidation evaluation was performed using the
thiobarbituric acid reactive substances (TBARS) method and is In the EPM test, the ratio of entries in the open arms showed a
classified as an oxidative stress marker (Ohkawa et al., 1979). significant difference in the preweaning factor [F PRE(1 ,36) = 9.19,
A solution containing acetic acid (2.5 M; pH 3.4), thiobarbituric p < 0.01]. The offspring who received a HS diet until weaning
acid (0.8%), and sodium dodecyl sulfate (8.1%) was added to entered less in the open arms compared to the offspring of
the tissue sample for 90 min at 95◦ C. The TBARS formation standard diet fed dams (p < 0.01). In addition, an interaction
was evaluated at 532 nm using malondialdehyde (MDA) (1,1,3,3- in the factors pre- and postweaning was observed [F PRE ×
tetramethoxypropane) as the standard. The results are expressed POST(1 ,36) = 4.93, p < 0.05]. The C-HS group showed higher ratio
in nmol MDA/mg protein (Freitas et al., 2019). of entries in the open arms compared to the C-C (p < 0.05),
All the redox analyses were performed in triplicate using HS-C (p < 0.01), and HS-HS (p < 0.01) groups (Figure 2A).
a plate reader (UV/Visible U-200 L Spectrophotometer). Similarly, the ratio of time spent in the open arms showed a
Protein content was quantified using bovine serum albumin difference in the preweaning factor [F PRE(1 ,36) = 4.36, p < 0.05].
(BSA) (1 mg/ml) as the standard (Bradford, 1976). The The offspring who received a HS diet until weaning spent less
results of the redox state were corrected for the amount of time in the open arms compared to the offspring of standard diet
protein in the samples. fed dams (p < 0.05). Also, an interaction in the factors pre- and
postweaning was observed [F PRE × POST(1 ,36) = 4.28, p < 0.05].
Statistical Analysis The C-HS group spent more time in the open arms in relation to
Statistical analysis was performed with Statistica software the C-C (p < 0.05) and HS-HS (p < 0.05) groups (Figure 2B).
(version 10.0, StatSoft , Hamburg, Germany). Graphics were
R
For the head-dipping frequency, a significant difference in the
made using the GraphPad Prism version 7.0 (GraphPad,
R
preweaning factor could be seen [F PRE(1 ,36) = 12.52, p < 0.01].
La Jolla, CA, United States). Sample normality was evaluated The offspring who received a HS diet until weaning showed
using the Shapiro–Wilk test. Data with normal distribution lower head-dipping frequency compared to the offspring of
were analyzed using the two-way ANOVA, with the factors: standard diet fed dams (p < 0.01). Moreover, a difference in
preweaning (received standard or HS diets until weaning) and the interaction of pre- and postweaning factors was observed

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[F PRE × POST(1 ,36) = 1.97, p < 0.05]. The C-HS group performed animals that received a HS diet in, both, the pre- and postweaning
head-dipping more frequently compared to the HS-C (p < 0.01) period (HS-HS group). In addition, animals that received the
and HS-HS (p < 0.01) groups (Figure 2C). No differences were HS diet only after weaning displayed a decrease in anxiety-like
found in the evaluation of rearing (p = 0.06) and grooming behaviors (C-HS group). Furthermore, both the groups showed
frequency (p = 0.73) in the EPM test (Figures 2D,E). amygdala oxidative stress, which may explain the behavioral
In the evaluation of the time spent in the OF central zone, changes observed.
a difference was found with respect to the preweaning diet The HS-HS group received the HS diet in both the periods
[F PRE(1 ,32) = 5.12, p < 0.05]. The offspring who received a (pre- and postweaning) resulting in adulthood hyperactivity
HS diet until weaning remained more time in the central zone measured by higher locomotion in the OF test. Also, this group
of the OF test compared to the offspring of standard diet fed presented an increase in the rearing frequency, which can be
dams (p < 0.05) (Figure 3B). The total distance covered in classified as a vertical exploration, confirming a high activity
the OF test showed a difference in the interaction of pre- and (Borta and Schwarting, 2005; Wardwell et al., 2020). Interestingly,
postweaning diets [F PRE × POST(1 ,32) = 16.59, p < 0.01]. The with a similar protocol, Mcbride et al. (2008) observed that
HS-HS group reported higher locomotion in relation to the C-C both the male Wistar rats treated with HS diet (4% NaCl) in
(p < 0.05), C-HS (p < 0.01), and HS-C (p < 0.01) groups the pre- (preconception and gestation periods) and postnatal
(Figure 3D). The rearing frequency in the OF test showed a periods (lactation) had increased locomotion in the OF test.
difference in the interaction of pre- and postweaning diets [F PRE In combination, these data indicate that a HS diet can induce
× POST(1 ,32) = 0.16, p < 0.05]. The C-HS and HS-C groups hyperactivity in rodents. Moreover, these animals were more
accomplished lower numbers of rearing in relation to the C-C and sensitive to the stimulating effect on locomotion produced by
HS-HS groups (p < 0.05) (Figure 3E). No differences were shown the administration of amphetamine compared to the group
in the evaluation of latency to escape of center zone (p = 0.27) and that received a standard diet (Mcbride et al., 2008). This result
grooming frequency (p = 0.35) in the OF test (Figures 3A,C,F). leads to the assumption that a HS diet of this study could also
In the amygdala redox state evaluation, a difference with sensitize offspring to the effects of amphetamines. Interestingly,
respect to the postweaning diet factor was shown for SOD we have previously showed that cafeteria or calorie-restricted
analysis [F POST(1 ,20) = 29.92, p < 0.001]. The offspring who diets during lactation and postlactation can alter anxiety and
received a HS diet after weaning reported less SOD activity locomotion of offspring after ephedrine (psychostimulant drug)
compared to the offspring of standard diet fed dams (p < 0.001). application, reaffirming the role of diets in sensitization to
In addition, an interaction in the pre- and postweaning diets was some drugs by mechanisms that are not yet clearly established
observed [F PRE × POST(1 ,20) = 0.13, p < 0.05]. The C-HS and (Rocha-Gomes et al., 2021b).
HS-HS groups showed less SOD activity compared to the C-C Curiously, the spontaneously hypertensive rats (SHR) model
and HS-C groups (p < 0.01) (Figure 4B). For GST activity, a consistently exhibits hyperactivity in the OF test (Botanas
difference was shown in the postweaning diet [F POST(1 ,20) = 5.69, et al., 2016; Aparicio et al., 2019; Chen et al., 2019). This
p < 0.05]. The offspring who received a HS diet after weaning model was initially developed for the study of deleterious
displayed less GST activity compared to the offspring of standard effects of cardiovascular diseases. However, due to its behavioral
diet fed dams (p < 0.05). Also, an interaction in the pre- and characteristics of hyperactivity, high impulsivity, and learning
postweaning diets was found [F PRE × POST(1 ,20) = 0.42, p < 0.05]. disabilities, SHR rats are also used as a model of attention-
The HS-HS group reported less GST activity with respect to the deficit/hyperactivity disorder (ADHD) (Leffa et al., 2019). It
C-C group (Figure 4D). In the TBARS evaluation, a difference is important to note that the excessive salt consumption is
in the postweaning diet was observed [F POST(1 ,20) = 5.21, recognized as a risk factor for the development of arterial
p < 0.05]. The offspring who received a HS diet after weaning hypertension (Valenzuela et al., 2021). In addition, rodents on
reported higher TBARS compared to the offspring of standard HS diets during the pre- or postnatal periods can develop
diet fed dams (p < 0.05). Moreover, an interaction of pre- hypertension in adulthood (Contreras et al., 2000; Swenson et al.,
and postweaning diets was observed [F PRE × POST(1 ,20) = 2.14, 2004). Although we did not use the SHR model in this study
p < 0.05]. The C-HS, HS-C, and HS-HS groups showed the and did not check the blood pressure of animals, we speculated
higher TBARS levels compared to the C-C group (p < 0.05) in relation to the similarities between the results presented by
(Figure 4E). No differences were reported in the FRAP (p = 0.16) the HS-HS group and the SHR model. It is possible that a HS
and CAT (p = 0.57) evaluations (Figures 4A,C). diet in the HS-HS group has programmed the mechanisms for
controlling blood pressure and also induced hyperactive behavior
in adulthood, similar to that observed in the studies with the
DISCUSSION SHR model. Therefore, a hyperactivity phenotype is suggested for
the HS-HS group. However, further studies are needed to assess
High-salt diets are consumed worldwide and are associated whether the phenotype presented by this group may have any
with cardiovascular morbidity and mortality. Noteworthy, HS relation to ADHD.
intake has also been linked to behavioral changes in rodents. Furthermore, one of our main hypotheses was that a
This study evaluated differential effects of HS and standard diet HS diet could promote changes in anxiety-like behavior at
combinations given in the pre- or postweaning period. In this adulthood. In this study, the C-HS group reported less anxiety-
study, an increase in locomotion was showed in the group of like behavior in the EPM test, due to the higher ratio of

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FIGURE 2 | Ratio of entries (A) and time spent in the open arms (B); head-dipping (C), rearing (D), and grooming (E) frequency in the elevated plus maze test. C-C,
offspring of standard diet fed dams who received a standard diet after weaning; C-HS, offspring of standard diet fed dams who received a HS diet after weaning;
HS-C, offspring of HS diet fed dams who received a standard diet after weaning; and HS-HS, offspring of HS diet fed dams who received a HS diet after weaning.
Data are shown as mean and SEM; n = 9–11; &p < 0.05 (preweaning factor); *p < 0.05, **p < 0.01 (interaction of the pre- and postweaning factors) using the
ANOVA and the Newman–Keuls tests.

entries and time spent in the open arms, in addition to the in situations that would be naturally aversive to rodents,
higher head-dipping frequency (Souto et al., 2020). Gilman as in the EPM test. This has important implications; as by
et al. (2019a) observed that after a short exposure to a HS exposing themselves to open or higher spaces, these animals
diet (4% NaCl; during 7 days), rodents reduced behavioral may be more exposed to risky conditions or even increasing
inhibition under relatively low-threat conditions. In particular, their visibility to predators (Gilman et al., 2019a). This result
this means that a HS diet can decrease anxiety-like behavior of a higher activity in potentially aversive situations was

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FIGURE 3 | Entries frequency (A), time spent at the central zone (B), latency to leave the center zone (C), distance covered (D), rearing (E), and grooming frequency
(F) in the open field test. C-C, offspring of standard diet fed dams who received a standard diet after weaning; C-HS, offspring of standard diet fed dams who
received a HS diet after weaning; HS-C, offspring of HS diet fed dams who received a standard diet after weaning; and HS-HS, offspring of HS diet fed dams who
received a HS diet after weaning. Data are shown as mean and SEM; n = 9. &p < 0.05 (preweaning factor); *p < 0.05, **p < 0.01 (interaction of the pre- and
postweaning factors) using the ANOVA and the Newman–Keuls tests.

found in male mice (C57BL/6J) using other paradigms after inflammation (Mitchell et al., 2018; Gilman et al., 2019b),
consuming a HS diet (4% NaCl; during 7 days) such as the possibly establishing a link to a HS intake, low anxiety-like
forced swim test (Mitchell et al., 2018; Gilman et al., 2019b). behavior, and cellular damages in a specific brain region.
In addition, the abovementioned studies observed amygdala Although we cannot distinguish anxiety-like from impulsive

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FIGURE 4 | The ferric reducing antioxidant power (FRAP) total antioxidant capacity (A), superoxide dismutase (SOD) (B), catalase (CAT) (C), glutathione
S-transferase (GST) activity (D), and the thiobarbituric acid reactive substances (TBARS) (E) concentration in the amygdala. C-C, offspring of standard diet fed dams
who received a standard diet after weaning; C-HS, offspring of standard diet fed dams who received a HS diet after weaning; HS-C, offspring of HS diet fed dams
who received a standard diet after weaning; and HS-HS, offspring of HS diet fed dams who received a HS diet after weaning. Data are shown as mean and SEM;
n = 6. #p < 0.05 (postweaning factor); *p < 0.05, **p < 0.01 (interaction of the pre- and postweaning factors) using the ANOVA and the Newman–Keuls tests.

behaviors, increased exploratory (horizontal and vertical) activity EPM (with no statistical difference) tests remain to be clarified
in new environments is a characteristic of impulsive behavior, in future studies.
which may also be caused by alterations in specific brain areas It is well established that experiences of mother during
related to decision-making in adverse situations (Almeida et al., preweaning periods can modify the developmental health
1993). However, the reasons why the C-HS group had a lower trajectory of her offspring. However, in some cases, no significant
frequency of rearing in the OF and a lower tendency in the deleterious effects are observed, as demonstrated in the EPM test

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by the groups that received a HS diet in the preweaning period previously published data are raised. The nuclear factor erythroid
(HS-C and HS-HS). These observations are combined with the 2-related factor 2 (Nrf2) is a transcription factor that regulates
Predictive Adaptive Response (PAR) hypothesis, which argues the expression of several proteins, among them some involved
that some changes that occur in early life in response to aversive in antioxidant defense system of cells. For example, antioxidant
stimuli are important to provide an advantage later in life. enzymes such as CAT, SOD, and GST are produced after
The PAR hypothesis predicts that these changes occur through activating the Nrf2 pathway (Iranshahy et al., 2018; Liu et al.,
epigenetic programming, which may also bring specific costs in 2020). Previously, Liu et al. (2020) showed a downregulation
the adult environment, making the animal maladapted on certain of the Nrf2 expression in renal tissue of rats receiving a HS
occasions (Raubenheimer et al., 2012; St-Cyr and McGowan, diet. Similarly, Wang et al. (2020) reported high levels of
2018). However, further studies are suggested to assess epigenetic reactive oxygen species and low activity of SOD and CAT in
changes that may be related to the results obtained here. the hippocampus of HS diet rats. This result indicates that
Reactive oxygen and nitrogen species can be considered as the downregulation of the Nrf2 pathway can occur not only
essential for the full development of neuronal functions when at the systemic level, but also in the brain after consuming
occurring in low or moderate amounts. However, at excessive a HS diet. In addition, a HS diet can provide a reduction
levels, they are harmful and can lead to oxidative/nitrosative in NO production (Kouyoumdzian et al., 2016; Zheng et al.,
stress, causing damage to proteins, lipids, and nucleic acids (da 2019). In situations where there is oxidative stress of the tissue,
Silva et al., 2014; Salim, 2017). In turn, this can lead to the release reactive oxygen species can inactivate NO (NO + O2− →
of inflammatory signals, resulting in neuroinflammation, loss of ONOO− ). The radical ONOO− is a very powerful oxidant
function, and, consequently, in behavioral changes (Hatanaka and nitrosating agent. Thus, besides generates a toxic molecule
et al., 2016; Cirulli et al., 2020; Dias et al., 2020; Maciel August (ONOO− ), this reaction decreases the NO availability. NO
et al., 2020). Previous studies in rodents have shown that HS plays an important role as a vasodilator, thus reducing it also
diets caused an imbalance in the brain redox state, with decreased contributing to arterial hypertension (Modlinger et al., 2004;
cognition (Liu et al., 2014; Ge et al., 2017; Faraco et al., 2019) and Vaziri and Rodríguez-Iturbe, 2006). Notably, hypertension is
increased reactivity to stressful situations (Bai et al., 2017; Dingess strongly linked to oxidative stress (González et al., 2014; Ahmad
et al., 2018). Moreover, a HS diet in the preconception, gestation, et al., 2017; Guzik and Touyz, 2017; Small et al., 2018).
and lactation periods has been shown to negatively influence the This study has some limitations. First, to better understand
redox state of the cerebellum, hypothalamus, and hippocampus the relationship between amygdala oxidative stress and observed
of the offspring (Stocher et al., 2018). These findings indicate behavioral changes, it is necessary in the future the use of
a role of salt-rich diets with respect to the brain redox status, drugs that alter the production of reactive oxygen and nitrogen
being able to induce oxidative stress in regions of fundamental species and the evaluation of the Nrf2 expression. Second, the
importance for behavior and cognition. assessment of inflammation in the amygdala would be important
The brain is very vulnerable to the excessive reactive oxygen to understand the real impact of a HS diet at the cellular level and
and nitrogen species production, due to its high O2 consumption the extent of tissue damage. Also, it is also important to evaluate
and modest antioxidant defenses (Bakunina et al., 2015; Salim, serotonin levels in this brain region, since its concentration in the
2017). In addition, regions such as the hippocampus and the amygdala is directly related to anxiety-like behaviors. Third, the
amygdala have been reported as the most susceptible to oxidative use of females is necessary, since sexual dimorphism is common
stress, consequently being more prone to functional decline in behavioral assessment studies. Females could have different
(Bouayed et al., 2009; Salim, 2017). In this study, amygdala responses due to other developmental vulnerabilities, altered
oxidative stress was observed, due to high levels of the TBARS neuroendocrine regulation, or placental and epigenetic different
(C-HS, HS-C, and HS-HS groups), in addition to the low activity effects. Fourth, the evaluation of other tests related to anxiety-
of SOD (C-HS and HS-HS groups) and GST (HS-HS group) like (light-dark box and hole-board tests) and hyperactivity (SHR
antioxidant enzymes. It is important to note that the amygdala model; use of drugs that affect locomotion) behaviors must be
plays a key role in the interpretation of environmental threats. performed to better understand the outcomes of this model. Fifth,
Sensory stimuli are received in the amygdala that imbues them finally, the next studies should assess blood pressure and heart
with emotional value and processing the outcomes as negative rate, in an attempt to establish a link between these physiological
or positive valence, directly influencing anxiety-like behaviors responses and the observed behaviors.
mainly through the serotonergic system (Calhoon and Tye, 2015; In summary, this study demonstrated negative effects of a HS
dos Santos et al., 2017; de Lima et al., 2020). It is possible that diet on the amygdala redox state. In addition, a HS diet promoted
diet-associated amygdala oxidative stress may be related to the hyperactivity when administered in the combination of pre-
behavioral alterations observed in the EPM and the OF tests; and postweaning periods and decreased anxiety-like behaviors
however, no clear patterns linking behavioral and redox readouts when offered only in the postweaning period. To the best of
were noticeable in this study. Future studies are needed to better our knowledge, this is the first study that indicates damage to
characterize this hypothetical relationship by also analyzing the amygdala in addition to behavior changes, regardless of the
potential mediators that could serve as a link between changes period in which salt is added to the diet. This fact is highlighted,
in amygdala redox status and behavior. due to the large consumption of salt in the world (Steffensen et al.,
In relation to the mechanism by which a HS diet can trigger 2018), its relationship with the development of cardiovascular
oxidative stress of brain tissues, some suggestions based on diseases (Bill and Foundation, 2019; He et al., 2020), and with

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Leal et al. Early Life High-Salt Effects

the hypotheses of behavioral changes and cognitive deficits after dos Vales do Jequitinhonha e Mucuri (CEUA-UFVJM;
HS consumption also in humans (Heye et al., 2016; Abdoli, 2017; protocol 025/2018).
Afroz and Alviña, 2019).

CONCLUSION AUTHOR CONTRIBUTIONS

A HS diet promoted hyperactivity when administered in the pre- All authors listed have made a substantial, direct, and intellectual
and postweaning periods. In animals that received only in the contribution to the work, and approved it for publication.
postweaning period, the addition of salt induced a reduction in
anxiety-like behaviors. Regardless of the administration period,
salt provided amygdala oxidative stress, which may be linked to
the observed behaviors. FUNDING
This study was supported by the Coordenação de
DATA AVAILABILITY STATEMENT Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
(Financial code 001), Fundação de Amparo á Pesquisa do
The original contributions presented in the study are included
Estado de Minas Gerais (FAPEMIG) (Financial code APQ-00791-
in the article/supplementary material, further inquiries can be
21), and Conselho Nacional de Desenvolvimento Científico
directed to the corresponding author/s.
e Tecnológico (CNPq). We want to thank the University
of Potsdam for supporting the initiation of this study via
ETHICS STATEMENT a KoUP cooperation grant. Furthermore, we acknowledge
the support of Deutsche Forschungsgemeinschaft (German
The animal study was reviewed and approved by Ethics Research Foundation) and Open Access Publication Fund of
Committee on the Use of Animals of Universidade Federal Potsdam University.

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