Annual Research Review: Critical Windows - The Microbiota - Gut-Brain Axis in Neurocognitive Development

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Journal of Child Psychology and Psychiatry **:* (2019), pp **–** doi:10.1111/jcpp.13156
Annual Research Review: Critical windows – the

microbiota–gut–brain axis in neurocognitive
development
Caitlin S. M. Cowan,1 Timothy G. Dinan,1,2 and John F. Cryan1,3
1 2
APC Microbiome Ireland, University College Cork, Cork; Department of Psychiatry and Neurobehavioural Science,
University College Cork, Cork; 3Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland
The gut microbiota is a vast, complex, and fascinating ecosystem of microorganisms that resides in the human
gastrointestinal tract. As an integral part of the microbiota–gut–brain axis, it is now being recognized that the
microbiota is a modulator of brain and behavior, across species. Intriguingly, periods of change in the microbiota
coincide with the development of other body systems and particularly the brain. We hypothesize that these times
of parallel development are biologically relevant, corresponding to ‘sensitive periods’ or ‘critical windows’ in the
development of the microbiota–gut–brain axis. Specifically, signals from the microbiota during these periods are
hypothesized to be crucial for establishing appropriate communication along the axis throughout the life span. In
other words, the microbiota is hypothesized to act like an expected input to calibrate the development of the
microbiota–gut–brain axis. The absence or disruption of the microbiota during specific developmental windows
would therefore be expected to have a disproportionate effect on specific functions or potentially for regulation of
the system as a whole. Evidence for microbial modulation of neurocognitive development and neurodevelopmental
risk is discussed in light of this hypothesis, finishing with a focus on the challenges that lay ahead for the future
study of the microbiota–gut–brain axis during development. Keywords: Cognitive development; early-life
experience; environmental exposures; neurodevelopmental disorders; child development.
body’s largest immune interface (Gensollen et al.,

Introduction
2016). The gut microbiota is therefore the subject of
Can the brain understand the brain? Can it under- the vast majority of research regarding the impact of
stand the mind? Is it a giant computer, or some the microbiota on brain and behavior. In this review,
other kind of giant machine, or something more? we focus on the gut microbiota as a key regulator of
David H. Hubel brain and behavior in early life. We will first examine
The past decade has seen this idea of ‘something what is known about the developmental trajectory of
more’ expand to the trillions of bacteria within the the microbiota and propose that there are sensitive
gut, the microbiota (Cryan et al., 2019; Rhee et al., periods in the microbiota–gut–brain axis with con-
2009). Indeed, there has been an explosion of sequences for neurocognitive development. We will
research investigating the microbiota and its contri- then provide evidence for this hypothesis based on
bution to all aspects of human health and disease, studies of both humans and nonhuman animals,
including psychological well-being and neurodevel- examine potential mechanisms for interactions
opment. But what exactly is the microbiota? It is between the developing microbiota and neurodevel-
defined as the collection of microorganisms, includ- opment, and finally discuss challenges for transla-
ing bacteria, viruses, fungi, and archaea, which tional research in this area.
reside in a particular niche (note that the ‘micro-
biome’, although often used interchangeably with Development of the microbiota
‘microbiota’, refers to both the community and its
collective genome). It has been estimated that the Using the latest sequencing technologies, a clearer
human microbiota contains 3.8 9 1013 bacteria, picture is beginning to emerge of how the gut
more than the number of human cells in the body microbiota develops over the life span and how this
(Sender et al., 2016), with its genome outnumbering influences the host (see Box 1 for further information
human genes by up to 150:1 (Qin et al., 2010). The on how the microbiota is measured). Whether micro-
largest of these microbial communities resides bial colonization occurs in utero is currently the
within the gastrointestinal tract, with the gut micro- subject of heated debate (Perez-Mu~ noz et al., 2017;
biota playing a key role in extracting nutrients from Walker et al., 2017). What is clear is that the
the diet and training the immune response at the microbiota is rapidly populated at birth (Ferretti
et al., 2018).
This initial seeding and the subsequent develop-
Conflict of interest statement: See Acknowledgements for full
ment of the microbiota are dependent on many
disclosures. factors (see Box 2 and Figure 1), but there are some
© 2019 Association for Child and Adolescent Mental Health

Published by John Wiley & Sons Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK and 350 Main St, Malden, MA 02148, USA
2 Caitlin S. M. Cowan, Timothy G. Dinan, and John F. Cryan
Box 1 Measuring the microbiota
The most commonly used techniques for measuring the microbiota are high-throughput sequencing
methods such as 16S rRNA sequencing and shotgun metagenomics. Using these methods, we can identify
the composition of the microbiota (i.e., who is in there, based on the genetic read-out) and the functional
potential of the community (i.e., what are they doing? see Bastiaanssen et al., 2019; Claesson et al., 2017 for
summaries of these techniques, written for clinicians). Different measures of diversity are usually used to
describe and compare the output of these analyses, with the two main metrics being alpha diversity and beta
diversity.
Alpha diversity. Refers to diversity within a given microbial community. Counting the number of species
present is one simple way to estimate diversity (more species = more ‘richness’ more diversity). Some
measures of alpha diversity also take into account the ‘evenness’ of a community, which refers to the
balance of different species (similar numbers of each species = more evenness more diversity). Other
measures take into account the phylogenetic relatedness of the community members (more closely related
species less diversity). Examples of common alpha diversity metrics include the Chao1, Shannon, and
Simpson’s Indices.
Increased alpha diversity is commonly interpreted as an indicator of a healthier microbiota. This is based on
the assumption that diversity increases the capacity of the community to perform a broader range of
functions and resist invasion from pathogens. However, more diversity is not always better and we should be
particularly wary of such simplistic interpretations in the context of development.
Beta diversity. Refers to the differences between two (or more) microbiota. As for alpha diversity, different
metrics may take into account different factors such as phylogenetic relatedness. Beta diversity is usually
shown using a principal component analysis (PCA) plot or similar data simplification method to represent
the complex microbial communities in just 2–3 dimensions. In these plots, the further apart two
communities are, the more different they are and thus the higher their beta diversity.
clear trends across maturation. At least in vaginally Intriguingly, periods of change in the microbiota
delivered babies, the majority of early gut colonizers coincide with times of rapid development in other
are transmitted from the maternal microbiota (from bodily systems and particularly the brain (Borre
various sites, but predominantly the maternal vagi- et al., 2014). This parallel development is likely to be
nal and stool microbiota; Ferretti et al., 2018). After biologically relevant, and we hypothesize that these
the first few days, there is a slow increase in the developmental windows correspond to sensitive peri-
number and diversity of species represented, shifting ods in the microbiota–gut–brain axis.
from mostly aerobic or facultative species (that can
survive in oxygenated environments) toward more
Sensitive periods
anaerobic species (Ferretti et al., 2018; Timmerman
et al., 2017). The early microbiota seems to be geared Sensitive periods (often used synonymously with the
toward extracting nutrients in order to support the terms ‘critical periods’ and ‘critical windows’) are
rapid development of the brain and body of the host defined as specific developmental windows during
(Hollister et al., 2015; Koenig et al., 2011). which a system exhibits heightened plasticity and is
There is another rapid burst in the development of particularly responsive to certain environmental
the microbiota at weaning, as the infant shifts from cues. These cues (also known as ‘expected inputs’)
exclusive breastfeeding or formula intake to a solid are used to tune the system in a highly efficient
diet (a pattern observed across species, including manner. The classic experiments by Nobel Prize
humans and rodents; Al Nabhani et al., 2019; winners Hubel and Wiesel showed that deprivation
Guevarra et al., 2018; Koenig et al., 2011; Stewart of light input into one eye permanently changes the
et al., 2018; Yatsunenko et al., 2012). According to activity of neurons in the cerebral cortex of cats, but
some studies, weaning is followed by a period of only during a narrow developmental time window
relative stability in the microbiota (Stewart et al., (Hubel & Wiesel, 1970). Later exposure to light was
2018; Yatsunenko et al., 2012). However, other insufficient to reverse the changes that occurred
results suggest there is ongoing change and plastic- during the sensitive period, while depriving an eye of
ity across middle childhood and even well into ado- light outside the sensitive period had little effect on
lescence (for a review, see Derrien et al., in press). neuronal activity or vision. The existence of such
These investigations indicate a pattern of gradual sensitive periods optimizes system development
development from middle childhood toward an when certain experiences (like exposure to light)
adult-like profile (Agans et al., 2011; Hollister reliably occur during certain stages of development
et al., 2015). (during the time of first eye opening).

Critical windows in the microbiota–gut–brain axis 3
Box 2 Factors that shape the developing microbiota
Starting even before birth, there are many factors that shape the developing microbiota. These include, but
are not limited to, genetics, stress, mode of birth, diet, infection or disease, medication (including
antibiotics), and environmental factors. Overall, the responsive nature of the microbiota offers a potential
advantage when it comes to targeting disease.
Genetic factors. Studies of the relationship between host genetics and the microbiota have discovered and
validated many heritable gut bacteria in humans (Goodrich et al., 2016; Goodrich et al., 2014; Lim et al.,
2017; Turpin et al., 2016). However, a large proportion of the microbiota is not heritable and environmental
factors explain far more of the variation in the human microbiota (Rothschild et al., 2018).
Stress. Stress influences all levels of the microbiota–gut–brain axis, particularly in the early stages of
development. Both prenatal and postnatal stress are established risk factors for the development of
psychological and neurodevelopmental disorders (Kessler et al., 2010; Kim et al., 2015), as well as
gastrointestinal problems and/or microbiota alterations (Callaghan et al., in press; Michels et al., 2019;
Pohl et al., 2015). These observations in humans are reflected in experimental animal models of early-life
stress, which have long been used to model both psychological and gastrointestinal disorders (O’Mahony
et al., 2011). Moreover, a number of recent studies have demonstrated links between prenatal stress
(including maternal anxiety) or early-life adversity (i.e., postnatal stress) and alterations in the human
microbiota across the life span (Callaghan et al., in press; D’Agata et al., 2019; Hantsoo et al., 2019;
Hemmings et al., 2017; Hu et al., 2019; Labus et al., 2017; Michels et al., 2019; Zijlmans et al., 2015).
Mode of delivery at birth. Parturition acts as the first large-scale exposure to environmental microbes, and
mode of delivery has a profound effect on the initial composition of the infant microbiota (Stewart et al.,
2018). Vaginally delivered babies are exposed to microbes resident in the maternal birth canal, whereas
babies born via Cesarean section exhibit a microbiota profile that more closely resembles adult skin and/or
hospital microbiota (Dominguez-Bello et al., 2010). This effect of delivery mode emerges in the transitional
stool rather than the meconium (i.e., the first stool after birth), but dissipates within a few weeks/months of
birth (Chu et al., 2017; Hill et al., 2017; Stewart et al., 2018).
Diet. During infancy, one of the most robust microbiota findings is that formula-fed and breastfed infants
exhibit divergent profiles, with cessation of breastfeeding accelerating the development of the microbiota
(Stewart et al., 2018). In cases of childhood malnutrition, the maturation of the microbiota is delayed, an
effect that is only partially or temporarily restored by dietary intervention (Smith et al., 2013; Subramanian
et al., 2014). Diet continues to play an important role in determining microbiota composition throughout
life; even short manipulations of diet in adulthood can have rapid effects on the microbiota (David et al.,
2014; Johnson et al., 2019). This is intriguing given that diet tends to change over the life span, with
adolescents being particularly prone to poor food choices, in combination with drug and alcohol
experimentation, at a time when their microbiota and brains are still developing (Flannery et al., 2019;
McVey Neufeld et al., 2016).
Medical factors. Bacterial or viral infections, gastrointestinal problems (e.g., diarrhea), and antibiotic use are
obvious sources of variation in the microbiota (Mortensen et al., 2018). However, other nonantibiotic
medications (including many targeting neurological or psychological problems) have also been shown to
alter the growth of certain gut bacteria in vitro as well as overall microbiota composition in rodents (Cussotto
et al., 2018; Maier et al., 2018). In adult humans, bariatric surgery results in substantial changes to the
microbiota (Guo et al., 2018). There is also a rich literature showing that premature infants, who typically
undergo multiple medical procedures in the early stages of life, exhibit microbiota alterations (Chernikova
et al., 2018; La Rosa et al., 2014, but see also, Stewart et al., 2018).
Environmental factors. Many facets of day-to-day life contribute to microbial exposure and therefore to the
microbiota. Urbanization (encompassing reduced contact with the natural environment and changes in
diet), pet ownership, and increasing use of antibiotics and disinfectants are all factors that alter microbial
exposure and have been associated with altered microbiota (Ayeni et al., 2018; De Filippo et al., 2017;
Stewart et al., 2018; Tun et al., 2017; Tun et al., 2018).
Sensitive periods have now been described for for these higher order functions, there is not just one
other, more complex brain functions and behaviors critical window but a cascading series of sensitive
such as language (Werker & Hensch, 2015) and periods with intricately linked timing, each dependent
emotional learning (Alberini & Travaglia, 2017; on the preceding experiences. Furthermore, the tim-
Gogolla et al., 2009; Hartley & Lee, 2015). Particularly ing of onset and offset of the sensitive period is less

Sensitive periods in microbiota–gut–brain axis

development. We hypothesize that signals from
the microbiota are required for certain functions,
acting like an expected input (similar to the role of
Birth Mode Medication
& Disease the light in the original Hubel and Wiesel experi-
ments) to calibrate the microbiota–gut–brain axis.
The absence or disruption of the microbiota during
specific developmental windows would therefore
have a disproportionate effect on specific functions
Diet Stress that are relevant to the time window of disruption
and potentially for regulation of the system as a
whole. In addition, external cues such as changes in
diet and exposure to environmental microbes may be
Environment Genetics considered expected inputs for the developing micro-
biota (see Box 2). Multiple sensitive periods likely
occur across the development of the microbiota–gut–
Figure 1 Many factors affect the developing microbiota and may brain axis, just as there are multiple sensitive
therefore alter the developmental trajectory of the microbiota–
gut–brain axis. See Box 2 for further discussion
periods within each domain of neurodevelopment
(see Figure 2). While these ideas are still in a
preliminary phase of testing, the current evidence
well-defined for these higher order functions, at least points to their validity, as we will endeavor to show
in part due to the extended development of the throughout this review.
supporting brain regions (Tottenham, 2017). Even The microbiota itself seems to be especially sensi-
the most well-defined sensitive periods exhibit a tive to disruption during early life. Although its
gradual decline in plasticity, subthreshold responses composition continues to be malleable to some
to the expected inputs into adulthood, and potential to extent throughout life, it has been hypothesized that
be reopened by pharmacological or environmental early colonizers, or ‘founder species’, in the gut have
manipulations (Morishita & Hensch, 2008). This a disproportionate influence on the ultimate steady-
latter property has great therapeutic application, state composition of the microbiota (Litvak & Baum-
offering hope for effective intervention even after the ler, 2019). The nature of the maturational trajecto-
closure of a sensitive period. ries of the microbiota also makes it more responsive
Sensory function Learning & memory
Language Higher order cognition

Neurodevelopmental
Plasticity
ASD
ADHD
Schizophrenia
Birth Weaning Adolescence Adulthood

Age / Development
Microbiota
Plasticity
Microbiota
?
Figure 2 Sensitive periods, or critical windows of heightened plasticity, occur in a cascading fashion across human development, with
multiple critical windows for each functional domain (shown on the upper axis). The timeframes for peak plasticity in the microbiota (in
the early postnatal period, at weaning, and possibly again at adolescence; lower axis) overlap with these trajectories and also coincide
with peaks in the onset of neurodevelopmental disorders (shown in black). Note that, compared to the brain, the microbiota exhibits a
relatively high level of ongoing plasticity beyond the developmental period

to external influences during development. The

Evidence for microbial modulation of
postnatal microbiota is relatively volatile, gaining
neurocognitive development
stability across maturation (Koenig et al., 2011;
Behavioral and cognitive development
Stewart et al., 2018; Yee et al., 2019), perhaps due
to the increasing diversity described above in the Human studies. Two studies, including a recent
section on Development of the microbiota. Periods of investigation of a large Finnish cohort, have observed
microbiota instability also occur during later phases complex, sex-specific relationships between the
of development, with a clear shift at weaning and an microbiota composition and very early temperament,
understudied transition considered likely during the including extraversion, regulation, and fear reactivity
adolescent period (Figure 2). Given the dearth of (Aatsinki et al., 2019; Christian et al., 2015). Gas-
longitudinal studies examining patterns of micro- trointestinal distress, which is associated with dis-
biota development beyond infancy, there may very ruption of the microbiota, has been shown to predict
well be other sensitive periods in between (see future anxiety across childhood, even when account-
Derrien et al., in press for a discussion of the ing for initial anxiety (Callaghan et al., in press). The
preschool and primary school years). evidence for microbiota modulation of psychological
There is additional evidence that the microbiota is or behavioral outcomes in pediatric populations with
particularly influential on the gut–brain axis during a physical illness is limited and mixed (Kan et al.,
development. It has recently been shown that the 2019). However, there are some promising studies
natural course of microbiota maturation at weaning is suggesting that modulation of the microbiota can
crucial for development of the immune system, one of improve behavioral symptoms of neurodevelopmental
the key gut–brain pathways (Al Nabhani et al., 2019). disorders (see section on Microbiota and neurodevel-
Weaning induces a sudden change in the composition opmental disorders).
of the microbiota, with an associated spike in inflam- With respect to cognitive development in humans,
matory activation that was termed the ‘weaning reac- we are aware of just one study that has directly
tion’. Using several elegant methods to alter the timing measured both the microbiota and cognitive perfor-
of weaning or suppress the weaning reaction, it was mance, which is part of the ongoing University of
demonstrated that such disruptions to the develop- North Carolina Early Brain Development Study
mentally appropriate timing of the weaning reaction (Carlson et al., 2018; Gao et al., 2019). In that cohort
led to pathological imprinting of the immune system, of 89 infants, the composition of the fecal microbiota
increasing risk for various inflammatory disorders in at one year of age predicted cognitive performance on
response to later immune challenges. the Early Learning Composite (a global cognitive
Taking a further step into the microbiota–gut– index) of the Mullen scale at 2 years. This effect was
brain axis, there are now several examples that driven by differences in the receptive and expressive
support the idea of critical windows for microbiota language domains. Lower alpha diversity at 1 year of
modulation of brain and behavior in animals. Stud- age was also predictive of better cognitive perfor-
ies where germ-free rodents have been recolonized mance, as indicated by negative correlations between
with ‘normal’ microbiota (i.e., from specific patho- alpha diversity and the overall Early Learning Com-
gen-free animals) at different ages have shown that posite, expressive language scale, and visual recep-
postweaning recolonization is more effective at tion scale at 2 years of age.
restoring germ-free deficits than recolonization later Finally, there have been some preliminary human
in life, at least for specific aspects of brain or immune developmental studies examining both neural/cog-
function and behavior (Buffington et al., 2016; Diaz nitive and behavioral outcomes in relation to early-
Heijtz et al., 2011; Olszak et al., 2012; Sudo et al., life microbiota manipulations. It has been reported
2004). For example, social deficits in germ-free mice that antibiotic use in the first year of life has a
were reversed by recolonization at weaning but not detrimental effect on overall IQ and reading ability
by recolonization 4 weeks later in adulthood (Buff- during the primary school years (Slykerman et al.,
ington et al., 2016). Still other functions in germ-free 2017). Such early-life antibiotic exposure was also
animals cannot be restored by recolonization even at associated with greater behavioral difficulties, more
the time of weaning, hinting that the window for oppositional behavior, and more symptoms of ADHD
microbial influence on these functions is already and depression. However, the strength of these
closed by the age of weaning (e.g., Chu et al., proposed microbiota–cognition and microbiota–be-
2019; Clarke et al., 2013). In support of the trans- havior relationships is difficult to ascertain as the
lational value of the sensitive period hypothesis, two researchers did not directly examine the microbiota
studies of childhood antibiotic exposure have nor collect data on the reasons for antibiotic use. The
reported that exposures in the first year of life, but same research group later conducted a placebo-
not at later time-points, have a negative impact on controlled trial of two probiotic strains administered
cognitive development (Slykerman et al., 2017, throughout pregnancy and the first two years of life.
2019; although see section on Behavioral and cog- In that sample, which exhibited high rates of antibi-
nitive development for a discussion of limitations in otic use (>80% in the first 2 years, similar across
these studies). treatment groups), there were no significant effects of

either probiotic on cognitive or behavioral outcomes that this relationship has an evolutionary basis,
at 11 years of age (Slykerman et al., 2018), although driven by the mutual benefits of social interaction
the effect of early-life antibiotics was replicated for both host and microbiota (Stilling et al., 2014).
(Slykerman et al., 2019). However, again, this study That is, social interaction encourages propagation
had several limitations; there was no justification and transfer of microbes to different hosts and
provided for the choice of probiotic strains, the simultaneously increases diversity in the microbiota
doses, or the methods of administration, nor was of any particular individual, with potential benefits
there a measure of the microbiota across treatment for satisfying the nutritional and health needs of the
(problems that are common in this area of research). host.
Finally, a recent study of premature infants in a
Neonatal Intensive Care Unit (NICU) also examined Animals – learning and memory. Germ-free stud-
the effect of perinatal antibiotics administered either ies have demonstrated that rodents reared without a
to the mother during gestation or to the infant during commensal microbiota exhibit exaggerated condi-
the NICU stay (Firestein et al., 2019). In this study, tioned fear responding (Hoban et al., 2018), but
which excluded cases of confirmed sepsis to reduce impairments in fear extinction, object recognition
the confounding effect of infection, perinatal antibi- memory and working memory on a spontaneous
otic exposure was associated with increased atten- alternation test in adulthood (Chu et al.,
tional problems at 4–5 years of age. Antibiotic- 2019; Gareau et al., 2011; Luk et al., 2018). Antibi-
exposed children also exhibited higher delta power otic administration from adolescence or adulthood
on an electroencephalogram (EEG), a pattern has a similar effect on the novel object task, impair-
observed in ADHD and interpreted as a delay in ing recognition memory (Desbonnet et al., 2015;
maturation. Finally, an intervention that encouraged Fr€ohlich et al., 2016). The effects of antibiotic
physical contact (and therefore transmission of administration on spatial memory performance are
microbes) between parent and infant during the more mixed, with one developmental study finding
NICU stay reduced the risk for both behavioral and that early-life antibiotic treatment did not alter
neural alterations (Firestein et al., 2019). In addition spatial memory in adulthood (O’Mahony et al.,
to this preliminary evidence for microbiota modula- 2014), despite other evidence that chronic antibi-
tion of developmental outcomes in humans, there otics impair spatial memory in adult rats (Hoban,
have been many more studies using animal models Moloney, et al., 2016; Wang et al., 2015). These
to explore different aspects of cognitive and behav- differences may be attributable to differences in the
ioral development. antibiotic regimen and rodent species; further devel-
opmental studies would be useful to understand the
Animals – social behavior and cognition. Perhaps discrepancy.
the strongest evidence for microbial modulation of Dietary interventions have shown promise in
cognition and behavior in animal models comes reducing the impact of stress or other microbiota
from studies of social interactions (Sherwin et al., perturbations on learning and memory outcomes
2019). Germ-free rodents typically exhibit deficits in across the life span. In adult rodents, probiotic
both sociability and memory for social stimuli supplementation has been used to reverse spatial
(indexed by preference for a novel social partner; memory deficits following stress, infection, or antibi-
Buffington et al., 2016; Desbonnet et al., 2014; otic treatment (Gareau et al., 2011; Liang et al.,
Sgritta et al., 2019; Stilling et al., 2018; but see also 2015; Wang et al., 2015), and even to provide
Arentsen et al., 2015). Similar effects are observed benefits in healthy animals in terms of spatial
following antibiotic depletion of the microbiota dur- memory, object recognition memory, and long-term
ing development (Degroote et al., 2016; Desbonnet fear memory (Savignac et al., 2014). During adoles-
et al., 2015), while administration of certain probi- cence, dietary supplementation with omega-3
otic species can enhance social behavior in various polyunsaturated fatty acids and vitamin A restored
murine models of autism (Buffington et al., 2016; cecal microbiota composition and novel object recog-
Hsiao et al., 2013; Sgritta et al., 2019) and inflam- nition impairments following chronic social instabil-
mation-induced social withdrawal (D’Mello et al., ity stress (Provensi et al., 2019). Earlier in
2015). In fruit flies (Drosophila melanogaster), both development, specific probiotic strains (Lactobacillus
developing and mature individuals rely on micro- rhamnosus and Lactobacillus helveticus) rescued the
biota-derived volatile compounds as social signals, expected developmental patterns of conditioned fear
influencing food and mating preference (Sharon behavior in infant rats exposed to early-life maternal
et al., 2010; Venu et al., 2014). This is reminiscent separation stress (Callaghan et al., 2016; Cowan,
of the associations between microbiota composition Callaghan, & Richardson, 2016). Stressed infants
and extraversion observed in human infants, as exhibit more persistent fear memories and are more
described above (Aatsinki et al., 2019; Christian vulnerable to relapse following fear extinction, but
et al., 2015). Thus, there is a robust relationship both these behavioral abnormalities are reversed by
between social behavior and the microbiota probiotic treatment (an effect demonstrated in two
observed across species. It has been hypothesized separate laboratories; Cowan, Callaghan, Kan, &

Richardson, 2016; Peng et al., 2019). Remarkably, structural brain changes are not limited to the
the probiotic treatment is even effective to prevent neuronal architecture; germ-free and antibiotic-in-
the generational transmission of these stress-induced microbiota depletion also leads to changes in
duced behavior changes (Callaghan et al., 2016). microglia maturation (more immature microglia;
Erny et al., 2015; Thion, Low, et al., 2018) and
levels of myelination (hypermyelination of the pre-
Brain development
frontal cortex; Gacias et al., 2016; Hoban, Stilling,
Morphology. Studies of the microbiota’s role in et al., 2016), while white matter integrity was
human structural brain development are still rare. associated with diet-induced changes in the gut
In adults, a cluster analysis identified different microbiota in rats (Ong et al., 2018).
microbiota compositions that were associated with
different white matter and gray matter signatures, Function and activity. In adult humans, several
including regional volume differences in the right brain imaging studies have shown that probiotic
hippocampus, left nucleus accumbens, right ante- bacteria can alter brain activity in response to emo-
rior occipital sulcus, and cerebellum (Tillisch et al., tional stimuli (Pinto-Sanchez et al., 2017; Tillisch
2017). Alpha diversity in the microbiota has also et al., 2013; Wang et al., 2019), while certain micro-
been linked to microstructure of the hypothalamus, biota compositions are associated with different pat-
caudate nucleus, and hippocampus of obese adults, terns of brain activity (Tillisch et al., 2017). Metabolic
while the relative abundance of a specific phylum, function of the microbiota (specifically synthesis of
Actinobacteria, correlated with microstructure vari- phenylalanine, a dopamine precursor) was associated
ables in the hypothalamus, thalamus, and amygdala with decreased ventral striatal activity during reward
(Fernandez-Real et al., 2015). In children, there has anticipation in a group of young adults with or without
been just one study examining brain structure and ADHD (Aarts et al., 2017).
gut microbiota, which reported positive associations To our knowledge, there are only two published
between alpha diversity at 1 year of age and volumes studies examining microbial modulation of brain
of the left precentral gyrus, left amygdala, and right function or activity in children, both of which were
angular gyrus at 2 years (Carlson et al., 2018). The published in the last year. In the first, functional
same study also reported specific regional brain connectivity of various brain networks was assessed
volume differences based on a cluster analysis of in sleeping 1-year-olds (Gao et al., 2019). Alpha
the microbiota. diversity of the fecal microbiota correlated with func-
A more experimental approach was taken in a tional connectivity in three separate networks: amyg-
fecal microbiota transplant study from preterm dala-thalamic, anterior cingulate cortex-right
babies in the Neonatal Intensive Care Unit (NICU; anterior insula, and supplemental motor area-left
Lu et al., 2018). Germ-free mice colonized by the parietal cortex. Further, this latter cluster was asso-
microbiota of babies who showed poor growth in the ciated with performance on a cognitive assessment at
NICU exhibited indicators of delayed brain develop- 2 years of age, perhaps providing a partial mecha-
ment with respect to markers of neuronal differen- nism for the previously described association
tiation, oligodendrocyte development, and between the microbiota and cognitive performance
myelination in the cerebral cortex compared with (Carlson et al., 2018). The second study was a recent
recipients of the high-growth microbiota. The micro- pilot conducted in 5- to 11 year-old children exposed
biota from low-growth babies also affected various to early adversity (orphanage rearing) and age-
neurotransmission pathways and increased neu- matched controls (Callaghan et al., in press). In this
roinflammation while lowering circulating levels of cohort, levels of certain bacterial taxa correlated with
growth hormones. prefrontal cortex activation to emotional faces. Some
Such extensive changes in the structural develop- of these taxa were less prevalent in the children with a
ment of the brain are similarly observed in animal history of early adversity, supporting the idea that the
models of microbiota depletion. Germ-free mice microbiota acts as a link between early traumatic
exhibit alterations in gross morphology, including experiences and alterations in both neurodevelop-
expansion of the amygdala and hippocampus ment and psychological risk.
(Luczynski et al., 2016). Morphology of germ-free This study fits nicely with observations in animal
mice also differs at the neuronal level in a region- models of early-life stress. Following maternal sep-
specific manner, with hypertrophy of neurons in the aration, a rodent model of early-life stress known to
amygdala and periaqueductal gray but shorter, less disturb the microbiota (O’Mahony et al., 2009), rat
complex neurons in the anterior cingulate cortex pups exhibit accelerated maturation of conditioned
and hippocampus (Luczynski et al., 2016, 2017). fear responding (Callaghan & Richardson, 2013) and
These structural differences, at least in the hip- a corresponding increase in activation of the pre-
pocampus, are likely related to observations of frontal cortex during expression and inhibition of
increased rates of hippocampal neurogenesis in both conditioned fear (Cowan et al., 2019). Probiotic
germ-free (Ogbonnaya et al., 2015) and antibiotic- supplementation during the stressful period was
treated mice (M€ ohle et al., 2016). Microbiota-related sufficient to reverse the effects on both behavior and

prefrontal cortex activation (Cowan, Callaghan, &

Metabolism and nutrient availability
Richardson, 2016; Cowan et al., 2019).
Outside the context of stress, there is a strong and Perhaps the most parsimonious hypothesis to
growing evidence base for microbial modulation of explain microbial modulation of neurodevelopment
brain function or activity in various animal models is that the microbiota is a key source of essential
(for a comprehensive review, see Cryan et al., 2019). nutrients and energy for the growing brain. Certainly,
For example, transcriptomic analysis of the amyg- the microbiota is known to convert food components
dala of germ-free animals revealed differential gene that would otherwise be indigestible into products
expression, exon usage, and RNA editing (Stilling with nutritional or biological value (see Rowland
et al., 2015). A recent investigation of fear extinction et al., 2018; Sela & Mills, 2010, for reviews on this
in microbiota-depleted mice observed significant topic, the latter in infants), and nutrition itself is a
changes in gene expression, neuronal activity, and well-established modulator of cognitive outcomes.
dendritic spine remodelling in the medial prefrontal Breastfeeding, a dietary factor that influences micro-
cortex (Chu et al., 2019). In addition, there are a biota maturation (Ho et al., 2018), has long been
number of reports of alterations in various neuro- purported to improve cognitive outcomes (Johnstone
transmitter systems by manipulations of the micro- et al., 1999), although more recent data indicate that
biota, notably in regard to serotonin and BDNF this effect is largely accounted for by confounding
(Bercik et al., 2011; Clarke et al., 2013; Desbonnet variables such as maternal intelligence or education
et al., 2015; Diaz Heijtz et al., 2011; Gareau et al., levels (Walfisch et al., 2013). On the other hand, both
2011; Neufeld et al., 2011; Sudo et al., 2004). specific nutrient deficiencies and low overall diet
Together with the studies of brain morphology, this quality are associated with negative long-term
body of work highlights the far-reaching influence of impacts on cognitive development (Freeman et al.,
the microbiota when it comes to brain development. 1980; Tandon et al., 2016), and nutritional interven-
tions in vulnerable populations have been shown to
improve cognitive outcomes during childhood (e.g.,
Digging deeper into mechanism: pathways for Freeman et al., 1980; Isaacs et al., 2009; Lucas et al.,
a microbial influence on neurodevelopment 1998). It has been argued that these effects need to be
There are now a number of known communication considered in terms of the developing microbiota
pathways within the microbiota–gut–brain axis, (Goyal et al., 2015). In support of this argument, a
including the vagus nerve, HPA axis, spinal cord, recent cohort study in Dutch primary school children
immune system, and peripheral transport of metabo- found that the strength of the relationship between
lites, among others. These have been reviewed in preschool diet and metabolic phenotype was depen-
detail elsewhere (e.g., Cryan et al., 2019). Here, we will dent on microbiota composition (Zhong et al., 2019).
provide a brief and simplified overview of just some of Childhood malnutrition delays maturation of the
these pathways with a specific developmental focus microbiome (Smith et al., 2013; Subramanian et al.,
(see Figure 3). 2014), and fecal microbiota transplant from
Immune system
Blood-Brain
barrier function
Peripheral immune cells
Spinal
Endothelial Astrocyte
cell cord
Cytokines Microglia
Vagus
nerve
Tight Basement
junction membrane
Epithelial Mucous Milk

cell layer
Microbiota Metabolites
Intestinal barrier
function Nutrient extraction
Figure 3 The microbiota–gut–brain axis consists of multiple pathways that allow bidirectional communication between the microbiota
and the brain. During development, some of the key pathways include nutrient extraction, immune signaling, and barrier function, as
well as neuronal and hormonal signaling along the spinal cord, vagus nerve, and hypothalamic–pituitary–adrenal (HPA) axis

undernourished children into rodents or pigs indi- microbiota, at least in animal models. The micro-
cates that this altered microbiota plays a causal role biota closely regulates development of the gastroin-
in the stunting and metabolic problems associated testinal tract, with germ-free animals exhibiting
with undernutrition (Charbonneau et al., 2016; profound structural and functional alterations in
Smith et al., 2013). These microbiota-dependent the intestinal barrier (for a review, see Sommer &
problems were resolved in both animal models by B€ackhed, 2013). These include alterations in the
supplementation with sialylated milk oligosaccha- expression of tight junction proteins and mRNA
rides (Charbonneau et al., 2016). These compounds (claudin-1 and occludin), elongated microvilli, and
are typically found in breastmilk, act as prebiotics for loss of the mucous layer, all of which may contribute
the microbiota, and were low in the breastmilk of to an observed increase in intestinal permeability
mothers with malnourished children. Another recent (Hayes et al., 2018). More surprisingly, the micro-
trial of ‘microbiota-directed complementary food’ biota has also been implicated in the development of
found promising effects compared with conventional the BBB (Braniste et al., 2014). Germ-free animals
food treatments in both animal models and malnour- exhibit a striking increase in BBB permeability,
ished children (Gehrig et al., 2019). The microbiota- starting in utero and continuing into adulthood. Loss
targeted intervention supported microbiota matura- of BBB integrity in GF animals was accompanied by
tion as well as biomarkers of growth, neurodevelop- reduced expression of the tight junction proteins
ment, and immune function in malnourished occludin and claudin-5. Both intestinal and BBB
children. Thus, there appears to be an important permeability can be restored in GF animals by
interaction between nutrient availability, the micro- recolonization in adulthood, pointing to a life-long
biota, and metabolic development whereby feeding connection between barrier function and the gut
the microbiota allows the microbiota to feed the microbiota (Braniste et al., 2014; Hayes et al., 2018).
developing body and brain.
Immune system
Barrier function
A growing literature is exploring the ‘neuro-immune
Between the microbiota and the brain, there are two axis’ as its own bidirectional network or as a compo-
major barriers: the gastrointestinal barrier and the nent of the microbiota–gut–brain axis (Irwin & Cole,
blood–brain barrier (BBB). The permeability of these 2011; Kraneveld et al., 2014). Apart from regulating
barriers is particularly relevant for transmission of barrier function, inflammatory signals alter neural
microbiota-derived metabolites and neurotransmit- activity through several mechanisms, including
ters along the microbiota–gut–brain axis; the more interactions with the HPA axis and vagus nerve as
permeable the membrane, the more signals will be well as the direct action of cytokines in the brain.
transmitted. There are striking similarities between Cytokines are produced by many different cell types,
the two barriers (Daneman & Rescigno, 2009). First, including glia and neurons in the brain, and can also
the main function of both is to protect against invading be actively transported across the blood–brain bar-
pathogens and toxins. Second, they share some broad rier following peripheral secretion. The levels of
structural similarities, being composed of a cellular cytokines in the central nervous system can alter
layer that forms the main physical barrier alongside metabolism of various neurotransmitter systems
immune cells (particularly T cells at the gut barrier, (including serotonin, dopamine, and glutamate;
microglia in the brain) that guard against pathogens Miller et al., 2013). In this manner, aberrant cytokine
(although there are also obvious structural differ- levels can disrupt the function of many important
ences, including the presence of a mucosal layer in the neurocircuits, including those involved in motivation
gastrointestinal tract). Third, despite these structural and emotion (Miller et al., 2013). Certain cytokines
boundaries and the importance of separating the inner can also act as growth factors and/or activate gene
and outer environments at these critical interfaces, pathways involved in various basic cell functions
neither barrier is completely impenetrable. Rather, (survival, migration, proliferation, differentiation,
both are selectively permeable to certain biological and apoptosis), which are particularly vulnerable to dis-
chemical elements. In a healthy state, this allows ruption during development (Deverman & Patterson,
nutrients or signaling molecules from the gastroin- 2009). It is therefore unsurprising that abnormal
testinal milieu to flow into the bloodstream and/or cytokine production (e.g., following maternal infec-
pass between the brain and body. However, if these tion during pregnancy or in the case of allergy) is a
tightly regulated systems break down, increased bar- risk factor for neurodevelopmental disorders (Garay
rier permeability can cause vulnerabilities to various & McAllister, 2010; van Sadelhoff et al., 2019). Thus,
forms of pathology. Fourth, both barriers continue to we can conclude that appropriate cytokine signaling
develop in the postnatal period and are considered and immune function are essential for neurodevel-
most vulnerable during this early developmental stage opment.
(van Elburg et al., 2003; Saunders et al., 2012). Extending the neuro-immune axis to the gut
Finally, both the gastrointestinal and blood–brain microbiota, there is a growing understanding that
barriers are known to be regulated by the the microbiota plays a critical role in the

development of the immune system. The gastroin-

The vagus nerve
testinal tract represents the largest immune inter-
face in the body, and exposure to gut microbes is an The vagus nerve is considered to be the most direct
important part of the process for training the route for signals from the microbiota to reach the
immune system to distinguish harmful and innocu- brain (F€ulling et al., 2019). It is the longest cranial
ous stimuli and to subsequently mount appropriate nerve and consists of many branches that innervate
responses to these different elements (Gensollen the visceral organs, including the gastrointestinal
et al., 2016). Germ-free animals exhibit profound tract, with both afferent (sensory) and efferent (mo-
alterations in both innate and adaptive immunity tor) neurons. These fibers continue to develop across
(Strauch et al., 2005), while specific commensal the first year of life in humans, with an increase in
bacteria can regulate the maturation of and balance myelinated fibers (Pereyra et al., 1992). Signals from
between different types of T cells in vitro and in vivo gastrointestinal vagal afferents reach the brain at the
(Baba et al., 2008; Ivanov et al., 2009; Sudo et al., nucleus stria terminalis, which acts as a ‘relay
2002). Microbiota manipulations can alter both station’ through its projections to other brain
circulating and central levels of cytokines (Burokas regions, including many relevant to behavior (e.g.,
et al., 2017; Luczynski et al., 2017; O’Mahony et al., hypothalamus, amygdala, ventral tegmental area).
2005; Sudo et al., 2004) as well as microglia devel- Lesion of the vagus nerve prevents the behavioral
opment (Erny et al., 2015; Thion, Low, et al., 2018). effects of certain probiotics (Bravo et al., 2011;
Moreover, there is a growing realization of a link Sgritta et al., 2019). Most recently, it was shown
between immune/allergic sensitivity and alterations that vagotomy at weaning prevented the rescue of
in the microbiota–gut–brain axis (Berni Canani et al., social behavior by probiotic treatment in a genetic
2019). Finally, in keeping with the sensitive period mouse model of autism (Sgritta et al., 2019).
hypothesis, there also seem to be critical windows for
both microbiota–immune interactions (as described
in the section on Sensitive periods in microbiota- Clinical significance
gut-brain axis development; Al Nabhani et al., 2019) The above indications of the sensitivity of the micro-
and neural–immune interactions (for a review, see biota–gut–brain axis during early development, with
Thion, Ginhoux, & Garel, 2018). long-term effects on host health, have implications
for various clinical conditions. Here, we will focus on
neurodevelopmental disorders, although there is
Hypothalamic–pituitary–adrenal (HPA) axis
also substantial evidence that many other psycho-
One of the seminal studies to investigate the role of the logical disorders have developmental origins (Kessler
microbiota in brain development focused on the et al., 2007; Lee et al., 2014) and are modulated by
hypothalamic–pituitary–adrenal (HPA) axis stress the microbiota (for reviews, see Bastiaanssen et al.,
response (Sudo et al., 2004). The study demonstrated 2019; Foster & McVey Neufeld, 2013).
that adult germ-free mice have an exaggerated HPA
response to restraint stress, including elevated levels
Microbiota and neurodevelopmental disorders
of ACTH and corticosterone (since replicated by
Clarke et al., 2013). This effect could be attenuated The most concrete evidence for a role of the micro-
or exacerbated by colonization with specific probiotic biota in any neurodevelopmental disorder is found in
or pathogenic bacteria, respectively (Sudo et al., the study of autism spectrum disorders (ASD). A
2004). Recolonization via fecal microbiota transfer large proportion of individuals with ASD report
from healthy mice also attenuated the HPA stress comorbid gastrointestinal problems (Chaidez et al.,
response. In keeping with our sensitive period hypoth- 2014; Parracho et al., 2005) and several observa-
esis, this strategy was only effective when the recol- tional studies have reported differences in the micro-
onization occurred by 6 weeks of age (Sudo et al., biota between children with ASD and neurotypical
2004). In terms of stress experienced during develop- controls (Adams et al., 2011; Finegold et al., 2010;
ment, the microbiota has been shown to mediate the Kang et al., 2013, 2018; Parracho et al., 2005).
effects of early-life stress in the maternal separation Emerging experimental studies corroborate a more
model. Whereas conventionally colonized mice exhibit causal link between ASD symptoms and the micro-
exaggerated anxiety- and depressive-like behaviors biota, although all have been small studies and
following maternal separation stress, germ-free mice caution must therefore be taken in their interpreta-
are not affected by maternal separation with regard to tion. Fecal microbiota transplant from a small num-
these behaviors (De Palma et al., 2015). Treatment ber of autistic individuals into germ-free mice led to
with specific bacteria (Lactobacillus rhamnosus and an emulation of aspects of autism (social deficits and
Lactobacillus helveticus) was also effective at revers- increased repetitive behavior; Sharon et al., 2019).
ing the effects of maternal separation stress on Three open-label pilot studies have demonstrated
glucocorticoid production during development, both promising effects of antibiotics (Sandler et al., 2000),
under basal conditions and in response to an acute probiotics (Shaaban et al., 2017), or fecal microbiota
stressor (Gareau et al., 2007; Peng et al., 2019). transplants from healthy donors (Kang et al., 2017,

2019) as strategies to reduce symptom severity in fecal microbiota from patients into germ-free mice
ASD. Another three studies have used double-blind, induced hyperactivity and exaggerated startle
placebo-controlled designs to assess different micro- responses as well as changes in hippocampal gluta-
biota interventions. The initial trial of a 12-week matergic function, but no changes in social behavior
probiotic treatment for 3- to 16-year-old children or prepulse inhibition (Zheng et al., 2019). In
with ASD was plagued by a high dropout rate, but humans, probiotic interventions have thus far not
still indicated some benefit of the treatment for bowel shown any effect on symptom severity in this com-
function and perhaps for behavioral symptoms as plex neurodevelopmental disorder (see Ng et al.,
well (Parracho et al., 2010). Similar benefits for 2019, for a systematic review of the 3 available
gastrointestinal and behavioral symptoms were studies, all with negative results).
observed in a dietary intervention that assessed an
exclusion diet with or without a prebiotic supple-
Challenges for translation from animals to humans
ment (Grimaldi et al., 2018). Both the diet and the
prebiotic led to alterations in microbiota composition The state of the research has not yet reached the stage
that correlated with fecal metabolite content. Inter- where recommendations can be made for the use of
estingly, behavioral changes were only observed in microbiota-based medicines or diagnostics in clinical
the group that received both the exclusion diet and settings. One of the chief limiting factors is the relative
the prebiotic, whereas the changes in gastrointesti- dearth of studies in humans and particularly in
nal symptoms were driven by the exclusion diet. children. As discussed above, the evidence points to
Finally, a preliminary trial (N = 75) of postnatal a link between the microbiota and both neurocogni-
probiotic supplementation for the first 6 months of tive and psychological development but, with few
life found that treatment had a prophylactic effect, exceptions, this evidence has been generated from
reducing the risk for ASD at 13 years of age (P€ artty animal models and/or correlational studies. While
et al., 2015). this is a common problem across many scientific
In addition to reducing risk for ASD, postnatal fields, there are a few challenges that become acutely
probiotic treatment (Lactobacillus rhamnosus GG) evident when considering the study of the microbiota–
also reduced risk for attention deficit hyperactivity gut–brain axis during development.
disorder (ADHD; P€ artty et al., 2015). Diet has long The study of such a complex whole-body system
been considered a contributing factor to ADHD that incorporates multiple organs presents a number
symptomatology, and a Western-style diet (high in of challenges in itself. First, the complexity of inter-
processed meat, refined grains, fats, and sugars) actions demands collaboration between experts in
during adolescence has been linked to the disorder each system. There is a need for input from gas-
(Howard et al., 2011). Many dietary interventions troenterologists, child psychologists/psychiatrists,
have been trialed in ADHD with varying degrees of microbiologists, and bioinformaticians to design
success, the most effective being elimination diets appropriate experiments and ensure that the data
that exclude artificial food dyes (Jacobson & are correctly analyzed. Second, this multiple-sys-
Schardt, 1999; Pelsser et al., 2011). More direct tems problem compounds the species differences
examinations of the microbiota have shown that within each individual system, which raises the
individuals with ADHD have a different microbiota barrier for translation of results from animal models.
profile compared with age-matched healthy controls While rodents are the most common model system
(Aarts et al., 2017; Prehn-Kristensen et al., 2018). partly because they exhibit many similarities to
Using a hypothesis-driven approach, it was identi- humans in terms of both the brain and the gastroin-
fied that alterations in the predicted functionality of testinal system, there are also clear differences in
the microbiota along a dopamine pathway were each component of the microbiota–gut–brain axis
associated with activity during a reward-based task across species (Nguyen et al., 2015). For instance,
(Aarts et al., 2017). These findings suggest that it is the brain and gastrointestinal tract are both simpler
worthwhile to further evaluate the links between at a gross morphological level in rodents, while
microbiota, diet, and symptomatology in ADHD, humans also have several distinct features such as
especially in childhood. the relative dominance of the cortical regions in the
Finally, there has been much speculation about brain and the relatively small size of the cecum.
the role of the microbiota in schizophrenia. This Considering these systems across development
relationship is particularly difficult to disentangle introduces additional complexity due to variation in
due to the almost ubiquitous use of antipsychotic the developmental timelines across species. The
medications, which are known to alter the micro- trajectory of neurodevelopment is very different for
biota (Cussotto et al., 2018; Maier et al., 2018). humans and rodents; the rodent brain at birth is
Nonetheless, some studies have identified differ- similar to that of a very premature infant (approxi-
ences in the oral and fecal microbiota of patients mately the start of the third trimester; Clancy et al.,
with schizophrenia, including particular taxa that 2007). Another clear example is the timing of wean-
correlated with symptom severity (Castro-Nallar ing, a key event that we have discussed as relevant to
et al., 2015; Zheng et al., 2019). Transplant of the potential critical windows of microbiota–gut–brain

development but which happens at different stages prematurity on cognitive performance were stronger
of brain development in rodents and humans. Young in boys, who also showed greater improvement with
rodents begin to ingest solid food around postnatal a dietary intervention (Lucas et al., 1998). In the
day (P) 16 and are fully weaned by P21 (Londei et al., other (Aatsinki et al., 2019), associations between
1988). For human children, the World Health Orga- microbiota composition and temperament were
nization recommends introduction of semi-solid stronger in boys (Aatsinki et al., 2019, although this
foods from 6 months of age, with a completely solid sex effect was not observed in a smaller, less well-
diet at 18–24 months (World Health Organization, powered study examining the same variables; Chris-
2005). However, equating P16 in a rodent to tian et al., 2015). Thus, the preliminary findings hint
6 months in a human does not necessarily match that males may be more vulnerable to the effects of
on to estimates of neurodevelopment, which can vary microbiota manipulations on neurodevelopment, in
wildly depending on the metric of interest. For keeping with the pattern of sex differences observed
example, some place a P16 rodent at the equivalent for neurodevelopmental disorders.
of 2–3 human months based on physical character- The sheer size of the datasets generated by
istics (Clancy et al., 2007), or more than 2 human sequencing technologies can be bewildering to most
years based on the development of attachment experimental researchers. The vast majority of
relationships and fear neurocircuitry (Callaghan microbiota members are not well-characterized at
et al., 2019). Thus, it will be important to consider the species or strain level, leaving large gaps in our
the specific developmental trajectories of different knowledge regarding their function and relative
aspects of neurodevelopment when attempting to importance. Databases are developing at a rapid
predict how studies of the microbiota in model rate in an attempt to close these gaps, but this
species will translate to humans. requires vigilant updating and maintenance of the
Another factor that warrants further attention is databases and techniques used to analyze them,
the impact of sex. Certainly, there are clear sex impeding direct comparison between studies. When
differences in the prevalence of neurodevelopmental choosing methods to manipulate the microbiota in
disorders, with ASD, ADHD, and schizophrenia humans, more consideration needs to be given to
being more common in males. The microbiota con- choices of probiotic, dosage titration (particularly
tribution to such sex differences in neuropsycholog- across development), method of administration, and
ical function is largely unknown, despite strong target population (with some evidence that micro-
arguments to support further investigation and some biota-based interventions are more useful when
intriguing preliminary findings (Audet, 2019; there is a pre-existing imbalance in the microbiota–
Jasarevi
c et al., 2016). In rodents, there are now gut–brain axis; Ng et al., 2018).
several examples where more pronounced effects of
early microbiota disruption have been observed in
males compared with females (Clarke et al., 2013; Conclusions
Desbonnet et al., 2014; Hoban, Stilling, et al., 2016; The field is beginning to narrow the gaps in terms of
Jasarevi
c et al., 2017, but see also complex sex- our understanding of the pathways that allow com-
specific effects described by Luk et al., 2018). For munication along the microbiota–gut–brain axis.
example, prenatal stress has more profound effects More work is needed to fine-tune this knowledge
on the developing male microbiota (Ja sarevi
c et al., and understand the boundary conditions for when
2015, 2017), while germ-free males but not females specific pathways are most important. This mecha-
exhibit hypermyelination of the prefrontal cortex nistic approach needs to be complemented with
(Hoban, Stilling, et al., 2016) and disruption of the more translational studies to test the relevance of
hippocampal serotonergic system (Clarke et al., evidence produced by animal models for humans. In
2013). Similarly, maternal germ-free status had a particular, studies examining sex differences, clini-
substantially greater impact on the microglia of cal populations, and prospective longitudinal
males during embryonic development (>1,000 genes cohorts are needed to assess the dynamic contribu-
differentially expressed in male microglia vs. 20 tion of the microbiota to neuropsychological and
genes in females), although this sex difference was clinical outcomes. Based on the evidence for poten-
reversed in adulthood (Thion, Low, et al., 2018). tial sensitive periods in the microbiota–gut–brain
Following a fecal microbiota transplant from chil- axis, investigations focused on early development
dren with ASD, stronger behavioral deficits were could be a fruitful approach for gaining traction in
observed in male recipient mice than in females harnessing the potential of the microbiota for treat-
(Sharon et al., 2019). In healthy children, two ment of psychological or cognitive problems.
studies tested for but did not detect sex differences
in the composition of the developing microbiota
(Carlson et al., 2018; Stewart et al., 2018). However, Acknowledgements
interactions between sex and microbiota-relevant This work was supported, in part, by research grants
outcomes have been reported in at least two obser- from Science Foundation Ireland (SFI) to APC Micro-
vational studies. In the first, the effects of biome Ireland through the Irish Government’s National

Development Plan (Grant no. SFI/12/RC/2273_P2). that they have no competing or potential conflicts of
C.S.M.C. is supported by a European Union H2020 interest.
Marie Skłodowska-Curie Individual Fellowship (Grant
no. 797592). The authors would like to thank Ryan
Carceller for his assistance with figure design. T.G.D. Correspondence
and J.F.C. have received research support from Mead John F. Cryan, Department of Anatomy and Neuro-
Johnson, Cremo, 4D Pharma, Suntory Wellness, Nutri- science, University College Cork, Rm 3.86, Western
cia, and DuPont. The remaining author has declared Gateway Building, Cork, Ireland; Email: [email protected]
Key points
The gut microbiota, or the collection of microorganisms that inhabit the gastrointestinal tract, have been
shown to affect cognitive and behavioral outcomes across species.
The microbiota–gut–brain axis is made up of many pathways through which the microbiota and brain can
communicate in a bidirectional manner.
We propose that developmental transitions in the microbiota may represent sensitive periods during which
the microbiota–gut–brain axis is most vulnerable, but also most malleable to intervention.
More work is needed to test the translational value of evidence gathered from animal models regarding the
potential for targeting the microbiota in neurodevelopmental and psychological disorders, particularly in
developing populations.
Bastiaanssen, T.F.S., Cowan, C.S.M., Claesson, M.J., Dinan,

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Journal of Child Psychology and Psychiatry **:* (2019), pp **–** doi:10.1111/jcpp.13156

Annual Research Review: Critical windows – the

body’s largest immune interface (Gensollen et al.,

© 2019 Association for Child and Adolescent Mental Health

Box 1 Measuring the microbiota

© 2019 Association for Child and Adolescent Mental Health

Box 2 Factors that shape the developing microbiota

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Sensitive periods in microbiota–gut–brain axis

Sensory function Learning & memory

Language Higher order cognition

Birth Weaning Adolescence Adulthood

© 2019 Association for Child and Adolescent Mental Health

to external influences during development. The

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

prefrontal cortex activation (Cowan, Callaghan, &

Epithelial Mucous Milk

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

development of the immune system. The gastroin-

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

Bastiaanssen, T.F.S., Cowan, C.S.M., Claesson, M.J., Dinan,

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

© 2019 Association for Child and Adolescent Mental Health

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Journal of Child Psychology and Psychiatry **:* (2019), pp – doi:10.1111/jcpp.13156