Cortical Auditory Deafferentation Induces Long-Term Plasticity in The Inferior Colliculus of Adult Rats: Microarray and QPCR Analysis

ORIGINAL RESEARCH ARTICLE
NEURAL CIRCUITS
published: 26 November 2012
doi: 10.3389/fncir.2012.00086
Cortical auditory deafferentation induces long-term

plasticity in the inferior colliculus of adult rats: microarray
and qPCR analysis
Cheryl Clarkson, M. Javier Herrero-Turrión and Miguel A. Merchán*
Instituto de Neurociencias de Castilla y León, Universidad de Salamanca, Salamanca, Spain
Edited by: The cortico-collicular pathway is a bilateral excitatory projection from the cortex to the infe-
Eric D. Young, Johns Hopkins
rior colliculus (IC). It is asymmetric and predominantly ipsilateral. Using microarrays and
University, USA
RT-qPCR we analyzed changes in gene expression in the IC after unilateral lesions of the
Reviewed by:
Avril Genene Holt, Wayne State auditory cortex, comparing the ICs ipsi- and contralateral to the lesioned side. At 15 days
University, USA after surgery there were mainly changes in gene expression in the IC ipsilateral to the lesion.
Richard Altschuler, University of Regulation primarily involved inflammatory cascade genes, suggesting a direct effect of
Michigan, USA
degeneration rather than a neuronal plastic reorganization. Ninety days after the cortical
*Correspondence:
lesion the ipsilateral IC showed a significant up-regulation of genes involved in apoptosis
Miguel A. Merchán, Laboratorio de
Neurobiología de la Audición, Facultad and axonal regeneration combined with a down-regulation of genes involved in neurotrans-
de Medicina, Departamento de mission, synaptic growth, and gap junction assembly. In contrast, the contralateral IC at
Biología Celular y Patología, Instituto 90 days post-lesion showed an up-regulation in genes primarily related to neurotransmis-
de Neurociencias de Castilla y León,
sion, cell proliferation, and synaptic growth.There was also a down-regulation in autophagy
Universidad de Salamanca, C/Pintor
Fernando Gallego, 1, Salamanca and neuroprotection genes. These findings suggest that the reorganization in the IC after
37007, Spain. descending pathway deafferentation is a long-term process involving extensive changes in
e-mail: [email protected] gene expression regulation. Regulated genes are involved in many different neuronal func-
tions, and the number and gene rearrangement profile seems to depend on the density of
loss of the auditory cortical inputs.
Keywords: brain injury, gene expression profiling, corticofugal projection, long-term post-lesion, adult lesion
plasticity
INTRODUCTION and Suga, 1998, 2000; Ma and Suga, 2005; Rutkowski and Wein-
The inferior colliculus (IC) is an obligatory relay station for berger, 2005), adaptation after sensory deafferentation (Holt et al.,
almost all ascending and descending auditory projections and 2005, 2006; Illing et al., 2005; Illing and Reisch, 2006; Rubio, 2006),
it is a key nucleus for excitatory and inhibitory auditory input and auditory cortex lesion (Druga and Syka, 2001; Bowen et al.,
convergence (Malmierca and Merchán, 2004). The descending 2003; Rybalko et al., 2006; Clarkson et al., 2010a,b,c).
cortico-collicular projection acts as a filter for neuronal responses In the IC partial deafferentation of the ascending auditory
(Sun et al., 2007). It participates in a positive feedback loop which, pathway reduced sound-evoked inhibitory collicular responses
in combination with lateral inhibition, sharpens, and adjusts the (Semple and Kitzes, 1985; Bledsoe et al., 1995; McAlpine et al.,
tuning of neurons in the auditory pathway (Zhang et al., 1997; 1997; Vale and Sanes, 2002). Whole cell patch recordings in brain
Jen and Zhang, 1999). The descending auditory corticofugal pro- slices showed that bilateral deafness induced a decrease in lat-
jection is glutamatergic and therefore excitatory (Feliciano and eral lemniscus neurotransmitter release and synaptic strength
Potashner, 1995). It is bilateral, but is denser on the ipsilateral side variations in both excitatory and inhibitory IC synapses (Vale
(Saldana et al., 1996; Bajo et al., 2007). Corticofugal deactivation and Sanes, 2002). It is known that changes in the balance of
results in unbalanced excitatory/inhibitory IC afferent projections, excitation and inhibition trigger synaptic plasticity, adapting bio-
which induce alterations in the amplitude and latencies of IC neu- physical membrane properties (synaptic strength and axon con-
ronal responses (Nwabueze-Ogbo et al., 2002; Popelar et al., 2003), ductance properties) or modifying the synthesis and trafficking
indicating that this projection affects directly the activity of IC of receptors (Perez-Otano and Ehlers, 2005; Turrigiano, 2012).
neurons. Holt et al. (2005) suggested unbalanced excitation and inhibi-
The developmental auditory system exhibits great ability for tion as a basis for long-term (90 days) changes in adult IC gene
network reorganization induced by sensory deprivation (Kandler, expression after inactivation of the ascending auditory pathway
2004; Keuroghlian and Knudsen, 2007). In adults, this capability by bilateral cochleotomy. Ascending lemniscal connections to the
to induce plastic changes is reduced but still present. In the audi- IC include both excitatory and inhibitory fibers (Riquelme et al.,
tory pathway, plastic changes in the adult have been tested using 2001). Therefore ascending deafferentation included a loss of both
acoustic stimulation (Norena and Eggermont, 2006), training- excitatory and inhibitory inputs. However, the descending path-
behavioral methods (Edeline and Weinberger, 1991a,b, 1992; Gao way is solely excitatory, and its absence should lead to a strong
Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 1

Clarkson et al. Midbrain gene expression lesion plasticity
unbalance of excitation and inhibition with consequences in gene quantify the percentage of auditory cortices affected by ablation
expression, which are currently unknown. (Clarkson et al., 2010a,b).
We have previously used an auditory cortical ablation model in
adult rats to analyze long-term plastic changes in the IC (Clarkson RNA ISOLATION
et al., 2010a,b,c). We demonstrated that restricted cortical abla- Collected ICs were homogenized and total RNA was purified using
tion in acoustically stimulated animals strongly decreased c-Fos TRIZOL® (Gibco BRL, Gaithersburg, MD, USA). RNA quality
immunoreactivity in IC neurons at 15 days post-lesion, with a sig- was assessed and quantitated by Agilent 2100 Bioanalyzer soft-
nificant recovery taking place after 90 days (Clarkson et al., 2010a). ware (Agilent Technologies, Palo Alto, CA, USA) associated with
It is well known that c-Fos immunoreactivity is an anatomical a RNA 6000 Nano kit. A RNA integrity number (RIN) >8.0 were
marker of neuronal activity (Bullitt, 1995), and that its alter- found in all samples. In addition, further RNA purification using
ation may be related to changes in neuronal transcription (Kovacs, an RNeasy Mini Kit for RNA clean-up (Qiagen Sciences, Maryland,
2008). Based on our c-Fos results (Clarkson et al., 2010a), we sug- USA) was performed.
gest that time-dependent activation of IC neurons after long-term
cortical deprivation may be a consequence of the reorganization MICROARRAY, DATA, AND ONTOLOGICAL ANALYSIS
of auditory pathway projections. In the Central Nervous System, Microarray analyses were performed at the Cancer Research Cen-
mechanisms of neural plasticity underlying long-term network ter (Centro de Investigacion del Cancer – CIC) at the University of
reorganization such as collateral sprouting and pruning, cell death, Salamanca (Spain). Labeling and hybridizations were performed
neurotransmitter regulation, or glial functional differences, are according to Affymetrix protocols. One hundred to three hundred
reflected in changes in levels of gene expression (Wieloch and nanograms of total RNA were amplified and labeled using the
Nikolich, 2006). To gain insights into long-term plastic changes WT Sense Target labeling and control reagents kit (Affymetrix
after descending IC deafferentation, we have used in our audi- Inc., Santa Clara, CA, USA), and hybridized to Rat Gene 1.0
tory cortex ablation animal model (Clarkson et al., 2010a,b,c) ST Array (Affymetrix). Washes and scans were performed using
a Gene Chip Microarray technology, validated by quantitative GeneChip System of Affymetrix (GeneChip Hybridization Oven
reverse transcription real-time PCR (RT-qPCR). 640, GeneChip Fluidics Station 450, and GeneChip Scanner 7G).
Following image analysis, microarray data were imported into
GeneSpring GX 7.3 (Agilent Technologies). RMA (Robust Multi-
MATERIALS AND METHODS array Analysis), a method for normalizing and summarizing
EXPERIMENTAL ANIMALS
probe-level intensity measurements, was used. For this analysis,
All experiments were performed according to national (R.D.
all genes that did not change between samples were excluded.
1201/2005) and EU regulations (DOCE L 222; 24-08-1999) for
We compared the experimental samples with naïve controls; all
use and care of animals in research. Nine male rats (Wistar albino,
our samples passed a high data quality control, showing a high
Charles River Laboratories) weighing 230 g and 12 weeks of age at
homogeneity intra-group (Datasheet 1 in Supplementary Mate-
the beginning of experiments were used. Animals were free of ear
rial). Potential differential expression was determined with a
infection and for a quick assessment of normal hearing we used in
one-way analysis of variance ANOVA (variances not assumed
all cases bilateral finger friction test. For DNA microarrays analysis
to be equal) and subsequently an unpaired t -test, p < 0.05, fil-
we used both IC (ipsilateral and contralateral) from three animals
tered for 1.5-fold was made in order to search differences in
in each group (naïve control, 15 days post-lesion and 90 days post-
the gene expression (control samples were used as basal levels).
lesion). For RT-qPCR three replicates from control and 90 days
Further processing including functional analysis and overrepre-
post-lesion group (both ICs) were randomly selected and run in
sentation calculations based on Gene Ontology (GO) Annotation
triplicate twice for each gene product (25 genes).
Tool and publication data from Database for Annotation, Visu-
alization, and Integrated Discovery were made with GeneSpring
SURGERY AND AUDITORY CORTEX LESION LOCALIZATION GX 7.3 and Database for Annotation, Visualization, and Inte-
Animals were anesthetized with ketamine chlorhydrate (30 mg/Kg, grated Discovery (DAVID) Bioinformatics Resources 6.7 (http:
Imalgene® 1000, Rhone Méreuse, Lyon, France) and xylazine //david.abcc.ncifcrf.gov/; Dennis et al., 2003; Huang et al., 2009).
chlorhydrate (5 mg/Kg Rompun®, Bayer, Leverkusen, Germany). Only genes with a Fold Change (FC) >1.5 (up or down), were
Unilateral ablation by aspiration of the left auditory cortices (pri- considered for analysis (Datasheet 1 in Supplementary Material).
mary – Au1, dorsal – AuD, and ventral – AuV areas), including From this group of genes we centered our attention in cate-
cortical layers V and VI, was carried out under stereotaxic control gories reported altered in the IC after auditory cortical lesion
using a stereotaxic frame (David Kopf Ins., Tujinga, CA, USA) fol- or are related directly to post-lesional plasticity (genes shown in
lowing a procedure described in detail elsewhere (Clarkson et al., Figures 2–4).
2010b).
Following the appropriate number of days post-lesion, ablated, QUANTITATIVE REVERSE TRANSCRIPTION REAL-TIME PCR
and naïve control animals were deeply anesthetized with sodium Total RNA (2 µg), primed with oligo-dT, was reverse-transcribed
pentobarbital (60 mg/kg) and decapitated. After quickly exposing into cDNA at 37˚C for 2 h using the first-strand cDNA synthesis kit
the brain stem, both ICs were removed and the brain was stored (Promega Corporation, Madison, WI, USA). In all cases, a reverse
overnight in a solution of 4% paraformaldehyde in phosphate transcriptase negative control was used for testing genomic DNA
buffer (PB) 0.1 M, pH 7.4. Finally, the brains were cryoprotected contamination. RT-qPCR was carried out on a real-time detec-
in 30% sucrose in 0.1% PB and serially sectioned at 40 µm to tion instrument (ABI Prism 7300 system) in 96-well optical plates

using TaqMan Universal PCR Master Mix and Assay on Demand and neurotransmitter transporters (glutamate, γ-aminobutyric
primers and probes. Probe sets used are listed in Datasheet 2 in acid (GABA), and glycine) and different types of channels (chlo-
Supplementary Material. Reaction components included: 2X Taq- ride, potassium, sodium, and calcium). Other functional cate-
Man Universal Master Mix with UNG, 450 nM unlabeled PCR gories notably detected include genes encoding proteins impli-
primers, 125 nM FAM dye-labeled TaqMan MGB probe, and 1 µL cated in neural/synaptic plasticity, axonal growth/degeneration,
cDNA reaction product in a 10 µL total reaction volume. PCR myelin organization, and regulation, sprouting, neuroprotection,
conditions were as follows: 2 min at 50˚C, 10 min at 95˚C and immune response, regulation of apoptosis, autophagy, and cell
40 cycles of 15 s at 95˚C and 1 min at 60˚C. Relative quantities proliferation, migration, and differentiation.
were calculated using the 2−∆∆Ct analysis method (Schmittgen All unilateral ablations were restricted in depth to the cortical
and Livak, 2008) with GAPDH (Glyceraldehyde-3-phosphate dehy- gray matter (including layer 6) and their extension mainly affected
drogenase: Rn99999916_s1) as the endogenous control. 2−∆∆Ct the primary and secondary auditory cortices with an average of
values were analyzed using One-way analysis of variance (ANOVA; 69.74 + 4.7% for 15 days post-lesion group and 74.19 + 9.4% in
p < 0.05) with a post hoc Student Newman-Keuls test (p < 0.05) the group 90 days post ablation (Figure 1).
was performed for statistical analysis.
CONTROL VS. 15 DAYS POST-LESION IN THE IPSILATERAL IC AND
RESULTS CONTROL VS. 15 DAYS POST-LESION IN THE CONTRALATERAL IC
We analyzed differences in gene expression profiling in the IC in Microarray comparisons between control and lesioned cases after
the short (15 days) and long-term range (90 days) after unilateral 15 days showed that the genomic profile in the IC ipsilateral to the
auditory cortex ablation. In addition, as this is an anatomically side of the lesion was slightly affected with 19 genes significantly
asymmetric projection in terms of innervation density, we also up-regulated (FC > 1.5 and p < 0.05) and 14 genes significantly
studied the differences in gene expression between the ipsi- and down-regulated (Figure 2A and Datasheet 1 in Supplementary
contralateral IC at each time post-lesion. Comparisons between Material). The IC contralateral to the side of the lesion also fol-
control and deafferented groups showed gene products corre- lowed a similar trend with 16 genes up-regulated and three down-
sponding to a total of 24,070 probes (of the 27,342 total probes regulated (Figure 2B and Datasheet 1 in Supplementary Material).
on the arrays) which were confidently detected based on signal After examining the potential function for each significantly
intensity at a fixed value above background level (See Material and affected gene per lesioned side we found that after 15 days post-
Methods, Robust Multi-array Analysis, RMA). lesion, the ipsilateral IC which had lost a denser cortical projection
To enhance biological interpretation of the differentially had a major up-regulation in several genes involved in inflam-
expressed genes we performed function enrichment analysis for matory pathways (Serglycin, Srgn; Podocalyxin, Podx; Complement
these genes using the functional classification tool “DAVID Bioin- Component 3, C3; and Claudin 5, Cldn5) and stem cell regenera-
formatics.” Our results indicated that, although there were many tion (Prominin 1, Prom1). On the other hand, one gene directly
over-represented biological function group of genes as shown in related with neuroprotection (Latexin, Lxn) was clearly down-
Datasheet 3 in Supplementary Material, the majority of them were regulated (Figure 2C). Furthermore, the contralateral IC that
related to a few functional categories. In our study the most rel- had lost a weaker projection showed an up-regulation in genes
evant categories included genes related to neurotransmission and involved in inflammation processes (NADH dehydrogenase sub-
signal propagation such as receptors for glutamate, glycine, acetyl- unit 6 | Cytochrome c oxidase subunit 3, Nd6|Cox3) and synaptic
choline, or serotonin. This classification also involves enzymes growth (Spinster, Spns; Figure 2D).
FIGURE 1 | Location and percentage of auditory cortical lesion for all Watson (2005). We used like reference for the coordinates of the vertical axis
cases used after 15 and 90 days post-lesion. On the left, the diagram the distance in mm from rhinal fissure. Right side, superimposition of each
shows the representation in our cases of primary and secondary auditory lesion contours over auditory cortex diagram. AuC, Primary auditory cortex;
cortices (Clarkson et al., 2010a) for all interaural levels drawn by Paxinos and AuV, Ventral auditory cortex; AuD, Dorsal auditory cortex.

FIGURE 2 | Differential gene expression between control and lesioned (down-regulation) sectors indicate the percentage of genes whose change
cases in the inferior colliculus 15 days after ablation of the auditory was greater than 1.5-fold. Notice for both ICs a predominant up-regulation in
cortex. The insets show in black the unilateral auditory cortical lesion area and comparison to down-regulation of gene expression. (C,D): Bar graph showing
in blue the location of cortical projection fields in both IC. Notice that dark functional analysis of the most representative genes for comparison between
blue means a higher density of terminals and light blue a weaker cortical controls group and IC ipsilateral (C) and contralateral (D) IC to the cortical
projection density. (A,B) Number of regulated genes in the IC ipsilateral (A) lesion. No changes *: Number of genes without significantly expression
and contralateral (B) to the cortical lesion. The blue sector indicates the genes changes. Acx-Lesion, Auditory cortex lesion; IC, Inferior colliculus; ND6/COX3,
whose change was 1 < FC < 1.5. The red (up-regulation) and green NADH dehydrogenase subunit 6 | cytochrome c oxidase subunit 3.
CONTROL VS. 90 DAYS POST-LESION IN THE IPSILATERAL IC AND Fis1; and Cytochrome c oxidase subunit VIIb, Cox7b) and down-
CONTROL VS. 90 DAYS POST-LESION IN THE CONTRALATERAL IC regulation in an anti-apoptotic gene (Gelsolin, Gsn). At the same
The analysis of gene expression after 90 days post-lesion, on both time, the NADH dehydrogenase, subunit 6 |cytochrome c oxi-
the ipsi- and contralateral sides, showed greater changes compared dase III (Nd6|Cox3) gene involved in inflammatory response
to control than did the 15-days group. The ipsilateral side (90 days) also showed up-regulation at this time post-lesion. Addition-
displayed up-regulation of 322 genes and down-regulation in ally, genes involved in synaptic growth, such as Myelin-associated
108 genes (Figure 3A and Datasheet 1 in Supplementary Mate- glycoprotein (Mag ) and Cadherin 13 (Cdh13), also displayed
rial). In the contralateral IC, 146 genes were up-regulated and down-regulated expression, accompanied by up-regulation in one
64 genes were down-regulated (Figure 3B and Datasheet 1 in gene related to axonal degeneration (Calpain 7, Capn7 ). Fur-
Supplementary Material). thermore, Gap junction proteins α1 and β6 (Gja1 and Gjb6),
In particular, after 90 days post-lesion, gene function analy- also named Connexin 43 and 30, respectively, showed down-
sis showed that the ipsilateral IC had significant adjustments regulation in gene expression. Finally, relevant genes for cal-
in genes involved in neurotransmission and signal propagation. cium regulation like (Nucleobindin 1, Nucb1) and calcium
Specifically, five were clearly down-regulated: Calcium channel – sensors like Synaptotagmin XI (Syt11) were down-regulated
voltage-dependent – γ3, Cacng3; Potassium inwardly rectifying (Figure 3C).
channel, subfamily J, member 16, Kcnj16; Adducin 1 (alpha), Ninety days after the cortical lesion, the contralateral IC
Add1; Claudin 11, Cldn11; and Calsyntenin 3, Clstn3, and one exhibited larger changes in the expression of genes related with
was up-regulated (Sodium channel and clathrin linker 1, Sclt1; neurotransmission than at 15 days, showing an up-regulation
Figure 3C). We also found up-regulation in the expression of in Dopamine receptor D1 interacting protein gene (Drd1ip, also
genes potentially involved in apoptotic processes (Programmed named Calcyon); Synaptogyrin 3 (Syngr3); Synaptic vesicle related
cell death 10, Pdcd10; Growth arrest-specific 2, Gas2; Fission 1, protein (SV2); Opioid receptor-like 1 (Oprl1); Potassium voltage

FIGURE 3 | Differential gene expression between control and lesioned shows functional analysis of the most representative genes for
animals in the inferior colliculus 90 days after ablation of the auditory comparison between control group and ipsilateral (C) and contralateral IC
cortex. The insets show in black the unilateral auditory cortical lesion area (D) to the cortical lesion. ATPase-6, CO-III, NADH dehydrogenase subunit
and in blue the location of cortical projection fields in both IC. Notice that 6 | cytochrome c oxidase subunit 3; COX subunit VIIb, Cytochrome c
dark blue means a higher density of terminals and light blue a weaker oxidase subunit VIIb; KCNJ16, Potassium inwardly rectifying channel,
cortical projection density. (A,B) Number of regulated genes in the IC subfamily J, member 16; RAB24, RAB24, member RAS oncogene family;
ipsilateral (A) and contralateral (B) to the cortical lesion. The blue sector Kcna6, potassium voltage gated channel, shaker related subfamily,
indicates the genes whose change was 1 < FC < 1.5. The red member 6; NPDC1, neural proliferation, differentiation and control, 1;
(up-regulation) and green (down-regulation) sectors indicate the Nrdg4, N-myc downstream regulated gene 4; SV2a, synaptic vesicle
percentage of genes whose change was greater than 1.5-fold. Bar graph glycoprotein 2a; Drd1ip, dopamine receptor D1 interacting protein.
gated channel, shaker related subfamily, member 6 (Kcna6 ), and IPSI- VS. CONTRALATERAL CHANGES AT 15 AND 90 DAYS POST-LESION
Sodium channel, type IV, beta gene (Scn4b; Figure 3D). Fur- Unbalanced cortical inputs to the IC induced by unilateral cortical
thermore, Neuregulin 3 (Nrg3) and Sodium channel and clathrin ablation seem to induce complex gene regulation patterns which
linker 1 (Sclt1) genes involved in neurotransmission were down- are better appreciated by comparing the IC ipsi – and contralateral
regulated. Interestingly, we found up-regulation in genes related to the ablation side. In naïve control group, we did a compar-
to neural plasticity like Opioid receptor, sigma 1 (Oprs1); Hip- ison between ipsilateral and contralateral IC profiling and no
pocalcin (Hpca); P55; Synaptogyrin family (Syngr1 and 3), and significant FC in gene expression were found. Moreover, both ICs
also in genes that regulate synaptic growth, like Spinster, and ipsi- and contralateral to the cortical lesion showed quantitatively
cell proliferation, such as N-myc downstream regulated gene 4, slight alterations in gene expression after short-term post-lesion
(Ndrg4), and Neural proliferation, differentiation and control, 1 (15 days), with changes in 14 genes (Datasheet 2 in Supplementary
(Npdc1; Figure 3D). In contrast, we found at this time/side Material). Of these genes, two have a known function as mediators
down-regulation in Latexin, which is involved in neuroprotec- of inflammatory processes and exhibit a down-regulation in the
tion. Mixes of genes related with autophagy were also affected, contralateral IC.
with an up-regulation in RAB24, member RAS oncogene fam- At long-term post-lesion (90 days), a much larger adjustment
ily and a down-regulation in Scrapie responsive gene 1 (Scrg1; in gene expression was found, with 1659 probes significantly
Figure 3D). altered, i.e., 802 genes up-regulated and 857 genes down-regulated

(Figure 4A and Datasheet 1 in Supplementary Material). Analy- synthetase (Qars); for GABA: Glutamic acid decarboxylase 1 (Gad1)
sis of genes function showed changes in 54 genes related with and 4-aminobutyrate aminotransferase (Abat ); for glycine: Glycyl-
neurotransmission. These changes covered a wide range of neuro- tRNA synthetase (Gars); and for endocannabinoid mobilization
transmission processes, with up-regulation of glutamate receptor through metabotropic glutamate receptors (mGluRs): Dyacylglyc-
genes (Glutamate receptor, ionotropic, N-methyl d-aspartate; Grin1, erol lipase alpha (Dagla)]. The neurotransmitter transporters also
or Nmdar1), glycine receptor (Glycine receptor alpha 1, Glra1), were affected. Two glutamate transporters genes were up-regulated
acetylcholine receptor subunits (Cholinergic receptor, nicotinic, Solute carrier family 17 (sodium-dependent inorganic phosphate
beta polypeptide 2; Chrnb2), a serotonin receptor subunit (5- cotransporter) member 6, Slc17a6, or also named Vesicular gluta-
hydroxytryptaine-serotonin receptor 1A, Htr1a) and other receptor- mate transporter 2 (VGlut2); and Solute carrier family 1 (Glial high
like Oprs1. We also found up-regulation in five neurotrans- affinity glutamate transporter) member 3, Slc1a3 or GLAST. Two
mitter synthesis enzyme genes [for glutamate: Glutaminyl-tRNA GABA transporters (Solute carrier family 6 – member 11 and family
FIGURE 4 | Continued

FIGURE 4 | Several of the most representative differentially expressed higher density of terminals and light blue a weaker cortical projection density.
genes in the comparison between 90 days post-lesion in the ipsilateral IC Graph bar showing functional analysis of the most representative genes in
vs. 90 days post-lesion in the contralateral IC. The inset shows in black the each functional category. (A) Neurotransmission (NTS). (B) Axonal growth,
unilateral auditory cortical lesion area and in blues the location of cortical anti-apoptosis, sprouting, synaptic plasticity, cell migration and differentiation,
projection fields in both inferior colliculus. Notice that dark blue means a myelin, apoptosis, postsynaptic density, Ca2+ and Gap junctions.
32 – member 1, Slc6a11, and Slc32a1, respectively), one glycine trafficking as well, such as those for α-amino-3-hydroxy-5-methyl-
transporter [Solute carrier family 6 member 9, Slc6a9] and one pro- 4-isoxazolepropionic acid (AMPA) receptors: N-ethylmaleimide
line transporter [Solute carrier family 6 member 7, Slc6a7 ] were also sensitive fusion protein (Nsf) and Contactin associated protein 1
up-regulated. Up-regulation affected genes related with receptor (Cntnap1). Also for acetylcholine receptors: SEC14-like 2 and for

sodium channel internalization with Sodium channel and clathrin Differentially regulated genes confirmed by RT-qPCR belong to
linker 1 (SLCT1). Moreover, the Contactin 1 (Cntn1) gene that a majority of functional categories already described (neurotrans-
regulates traffic and synaptic content of AMPA receptors showed mission, neural/synaptic plasticity, axonal growth/degeneration,
down-regulation after 90 days post-lesion (Figure 4A). myelin organization and regulation, sprouting, neuroprotection,
In addition, voltage-operated and other non-ligand gated ion regulation of apoptosis, and autophagy). Thus, as shown in
channel genes were also up-regulated. These included three chlo- Table 1, altered gene expression of the majority of genes, but not
ride channel genes (Clcn2, 6, and 7 ), five potassium channels all, were confirmed by RT-qPCR.
[K + inwardly rectifying channel subfamily J – members 2 and 9 Microarray analysis identified many neurotransmission-related
(Kcnj2 and 9); K + voltage gated channel shaker related subfamily genes with significant increases in expression at 90 days after uni-
β3 (Kcnab3); K + channel subfamily K – member 1 (Kcnk1); and lateral auditory cortex ablation (Figure 4A). From these genes,
Hyperpolarization activated cyclic nucleotide-gated K + channel 2 12 were further assessed with RT-qPCR and showed comparable
(Hcn2)] and a sodium channel [sodium channel voltage-gate, type changes. A serotonin receptor (Htr1a), a glutamate receptor (Grin1
IIβ (Scn2b)]. Additionally, a channel for potassium [K + channel or NMDAR1), an enzyme for GABA synthesis (Glutamic acid
subfamily T – member 2 (Kcnt2)], another for calcium regulation decarboxylase 1, Gad1), a gene involved in the regulation of AMPA
(Tctex1 domain containing 2, Tctex1d2) and one receptor for sub- receptors (N-ethylmaleimide sensitive fusion protein, Nsf), a gluta-
stance P (Tachykinin 1, Tac1) were down-regulated (Figure 4A). mate transporter Slc17a6 (=VGlut2), and two GABA transporters
It is important that the synaptic vesicular machinery was also (Slc32a1 and Slc6a11) showed significant increases of 4.09-, 1.81-,
affected, with an up-regulation of genes involved in neurotrans- 2.13-, 1.48-, 1.79-, 1.52-, and 1.87-fold at 90 days in the contra-
mitter release, as well as calcium sensors for exocytosis, such as (vs. ipsilateral) IC, respectively. Increased gene expression at the
Syngr1 and 3, the Synaptotagmin family (Syt1, 2, 11, and 12) and same time point was seen for a cholinergic receptor (Chrnb2),
SV family (SV2a and c), and genes related with synaptic vesicle NMDA receptor associated protein (Grina), Neuronal pentraxin 2
plasticity such as Synaptophysin and genes associated with synap- (Nptx2), and the glutamate transporter Slc1a3 (=GLAST ) using
tic vesicle docking and fusion (Syntaxin binding protein 1, Stxbp1, microarrays analysis, whereas RT-qPCR did not confirm such a sig-
and Syntaxin 16, Stx16 ; Figure 4A). nificant increase. Conversely, Nsf and VGlut2 had increased levels
Furthermore, as shown in Figure 4B, we found up- or down- of expression at 90 days in the ipsilateral IC and genes like Grin1
regulation in genes involved in axonal growth and guidance, and Gad1 also had increased levels of expression at the same time
such as cortactin (Cttn) and Dyacylglycerol lipase, alpha (Dagla). point in the contralateral IC by RT-qPCR analysis, whereas the
At the same time, we detected changes in genes involved in microarray data did not show a significant change. Also, RT-qPCR
myelin organization and regulation as Peripheral myelin protein for Nptx2 and a GABA transporter (Slc32a1) showed a 1.74- and
22 (Pmp22) and Developmentally regulated protein TPO1 (Tpo1), 1.57-fold decrease in expression at 90 days in the ipsilateral IC after
Myelin-associated glycoprotein (Mag ), and Oligodendrocyte-myelin unilateral auditory cortex ablation, whereas microarrays did not
glycoprotein (Omg ). We also found both types of regulation in show significant changes. In contrast, increased gene expression
genes involved in sprouting [dopamine receptor D1 interacting was seen at 90 days in the contralateral IC for the Sodium chan-
protein (Drd1ip), Sprouty homolog 3 (Spry-3), and Rho-associated nel, type IV, beta (Scn4b) using microarrays analysis, whereas the
coiled-coil containing protein kinase 1 (Rock1)], in synaptic plastic- RT-qPCR did not show a significant increase.
ity [Amyloid beta (A4) precursor protein (App), Neuronal pentraxin Microarray results for the neurogenesis-related genes or neu-
2 (Nptx2) and Calcium/calmodulin-dependent protein kinase II, β ronal differentiation, such as Amyloid beta (A4) precursor protein
Camk2b] and in postsynaptic density reorganization (p55, and (App), Neural proliferation, differentiation and control, 1 (Npdc1),
Tubulins γ2, α1A, and β2C). In addition, even after 90 days post- and N-myc downstream regulated gene 4 (Ndrg4), showed an
lesion, we still found regulations (up or down) in apoptotic genes, increase in expression at 90 days in the contralateral IC vs. control
such as Cathepsins B and D (Ctsb and Ctsd), Growth arrest-specific and vs. ipsilateral IC, respectively, after unilateral auditory cortex
2 (Gas2) and Programmed cell death 10 (Pdcd10), but at the same ablation. On the other hand, decreased gene expression at 90 days
time we found an increase in anti-apoptotic gene expression [N- in the contra vs. ipsilateral IC was seen in the Diazepam binding
myc downstream regulated gene 4 (Ndrg4) and Gelsolin (Gsn)]. inhibitor (Dbi) using microarrays analysis, while the RT-qPCR did
This regulation was accompanied by changes in genes involved in not show a significant increase. In contrast, this same gene showed
cell migration and differentiation [e.g., Neural proliferation, differ- 1.54-fold increase in expression at 90 days in the contralateral IC by
entiation and control, 1 (Npdc1) and Diazepam binding inhibitor RT-qPCR, whereas the microarray data did not show a significant
(Dbi)] and glial connexins [Gap junction membrane channel pro- change at this time point.
tein alpha 12 (Gja12), and gap junction protein, alpha 1 (Gja1 or Other genes such as the enzyme involved in endocannabi-
Cnx43)]. noid mobilization Dyacylglycerol lipase alpha (Dagla), glial con-
nexin Gja1 (=Cnx43), Apoptosis-inducing factor mitochondrion-
RT-qPCR ANALYSIS associated 3 (Aifm3), p55, and Gelsolin (Gsn) assessed by RT-qPCR
RT-qPCR analysis was used to confirm a subset of gene expression showed expression levels which were also increased at 90 days in
changes observed in the microarray analysis. Genes chosen for the contra vs. ipsilateral IC, whereas Myelin-associated glycopro-
RT-qPCR confirmation were mainly selected 90 days after cortical tein (Mag ), Rho-associated coiled-coil containing protein kinase 1
ablation, based on ontological categories with potential functional (Rock1), Syt1, and Scrapie responsive gene 1 (Scrg1) did not show
roles. significant changes. Moreover, the gene encoding the gap junction

Clarkson et al.
Table 1 | Comparison of gene expression at 90 days after unilateral auditory cortex ablation: Microarray vs. RT-qPCR data.
Transcripts Gene description Gene Control vs. Control vs. 90dpl-ipsi vs. Array PCR ID
cluster ID symbol 90dpl-Ipsi 90dpl-Contra 90dpl-contra
Microarray RT-qPCR Microarray RT-qPCR Microarray RT-qPCR
Frontiers in Neural Circuits

(1) (2) (1) (2) (1) (2)
Neurotransmission: 10812879 5-hydroxytryptamine Htr1a NS NS NS NS 1.746 4,093 ± 1,23* Rn00561409_s1

receptors, binding (serotonin) receptor 1A
proteins, 10824517 Cholinergic receptor, nicotinic, Chrnb2 NS NS NS NS 1.954 NS Rn00570733_m1
transports and beta polypeptide 2 (neuronal)
metabolism, 10843400 Glutamate receptor, Grin1 = NS NS NS 2,509 ± 0,45* 1.663 1,814 ± 0,33* Rn01436038_m1
transport_ ionotropic, N-methyl NMDAR1
channels d-aspartate 1
10897146 Glutamate receptor, Grina NS NS NS NS 1.967 NS Rn00596289_g1
ionotropic, N-methyl
d-aspartate-associated
protein 1 (glutamate binding)
10836734 Glutamic acid decarboxylase 1 Gad1 NS NS NS 3,607 ± 0,45* 1.871 2,135 ± 0,27* Rn00690300_m1
10748118 N-ethylmaleimide sensitive Nsf NS 1,537 ± 0,03* NS 2,277 ± 0,10* 2.028 1,481 ± 0,01* Rn00612444_m1
fusion protein
10760290 Neuronal pentraxin 2 Nptx2 NS −1,745 ± 0,07* NS NS 1.584 NS Rn01756377_m1
www.frontiersin.org
10909621 Sodium channel, type IV, beta Scn4b NS NS 1.683 NS NS NS Rn01418017_m1
10821824 Solute carrier family 1 (glial Slc1a3 = NS NS NS NS 1.814 NS Rn00570130_m1
high affinity glutamate Glast
transporter), member 3
10707325 Solute carrier family 17 Slc17a6 = NS 2,375 ± 0,29* NS 4,270 ± 0,71* 1.89 1,798 ± 0,30* Rn00584780_m1
(sodium-dependent inorganic VGlut2
phosphate cotransporter),
member 6
10841774 Solute carrier family 32 Slc32a1 NS −1,570 ± 0,07* NS NS 1.75 1,524 ± 0,05* Rn00824654_m1
(GABA vesicular transporter),
member 1
10857916 Solute carrier family 6 Slc6a11 NS NS NS NS 1.848 1,871 ± 0,32* Rn00577664_m1
(neurotransmitter transporter,
GABA), member 11
(Continued)
November 2012 | Volume 6 | Article 86 | 9

Midbrain gene expression lesion plasticity
Clarkson et al.
Table 1 | Continued
Transcripts Gene description Gene Control vs. Control vs. 90dpl-ipsi vs. Array PCR ID
Frontiers in Neural Circuits

cluster ID symbol 90dpl-Ipsi 90dpl-Contra 90dpl-contra
Microarray RT-qPCR Microarray RT-qPCR Microarray RT-qPCR

(1) (2) (1) (2) (1) (2)
Neurogenesis_ 10752811 Amyloid beta (A4) precursor App NS NS NS 2,232 ± 0,30* 1.848 1,697 ± 0,23* Rn00570673_m1
neuronal protein
differentiation 10931154 diazepam binding inhibitor Dbi NS NS NS 1,543 ± 0,11* −1.778 NS Rn00821402_g1
10834225 Neural proliferation, Npdc1 NS NS 1.585 1,971 ± 0,16* 1.773 1,632 ± 0,13* Rn01438701_g1
differentiation and control, 1
10809100 N-myc downstream regulated Ndrg4 NS NS 1.581 2,371 ± 0,13* 1.858 1,347 ± 0,07* Rn00582990_m1
gene 4
Axonal growth_ 10728676 Diacylglycerol lipase, alpha Dagla NS −1,44 ± 0,10* NS NS 1.509 1,546 ± 0,15* Rn01454304_m1
sprouty_axonal 10720813 Myelin-associated Mag NS NS NS NS 1.811 NS Rn00567868_m1
branching glycoprotein
10803158 Rho-associated coiled-coil Rock1 NS NS NS NS −1.509 NS Rn00579490_m1
containing protein kinase 1
Synaptic vesicular 10902232 Synaptotagmin I Syt1 NS NS NS NS 1.51 NS Rn00436862_m1
www.frontiersin.org
exocytosis_
calcium sensor
GLIA 10830189 Gap junction protein, alpha 1 Gja1 = −1.749 −1,842 ± 0,16* NS 1,590 ± 0,05* 1.593 2,928 ± 0,09* Rn01433957_m1
Cnx43
Cell death_ apop- 10755728 Apoptosis-inducing factor, Aifm3 NS NS NS 3,588 ± 0,16* 1.868 2,367 ± 0,11* Rn01405066_m1
tosis_autophagy mitochondrion-associated 3
10894498 P55 P55 NS NS 1.52 2,676 ± 0,34* 1.684 1,831 ± 0,24* Rn01509468_g1
10791500 Scrapie responsive gene 1 Scrg1 NS NS −2.141 NS −1.79 NS Rn00583743_m1
10835757 Gelsolin Gsn −1.527 NS NS NS 1.854 2,033 ± 0,01* Rn01438922_m1
(1)Change in intensity, expressed as fold change.

(2)Expressed as fold change (2−∆∆Ct ): mean ± SD.
NS, not statistically significant. Red indicates up-regulated; Green indicates down-regulated.
*p < 0.05.
November 2012 | Volume 6 | Article 86 | 10

Midbrain gene expression lesion plasticity
protein Cnx43 and p55 showed 1.84-fold decrease and 2.68-fold of signaling pathways and transcriptional mechanisms to orches-
increase in expression at 90 days post-lesion in the ipsilateral and trate long-lasting changes in their physiology through the synaptic
contralateral IC (vs. control), respectively, after unilateral auditory activity-regulated transcription of new gene products (Lyons and
cortex ablation. A number of the RT-qPCR confirmation analy- West, 2011). After auditory cortical lesions, these long-term tran-
sis (Dagla at 90 days in the ipsilateral IC, and Cnx43 and Aifm3 scriptional profiling changes in the bilateral ICs are an indication
at 90 days in the contralateral IC) which did not reach statisti- of adult synaptic plasticity triggers, which are dependent on both
cal significance, demonstrated expression profiles similar to those post-lesioned time and density deafferentation.
observed in the microarray. On the other hand, Gsn at 90 days Our results are a first look at the entire post-lesional auditory
in the ipsilateral IC and Scrg1 at 90 days in the contralateral IC, plasticity process. To assess an IC reorganization after cortical abla-
showed changes in gene expression levels in microarrays, whereas tion both genomic and proteomic analysis are necessary. Genomic
the RT-qPCR did not show a significant increase. results cannot be extrapolated to protein production since it is
As shown in Table 1, we performed RT-qPCR analysis on 24 known that there exist some mismatches between genomic and
genes at 90 days in the contra vs. ipsilateral IC after unilateral proteomic data in both brain cell culture (Howley et al., 2012)
auditory cortex ablation and 15 of them showed similar changes and most specifically in auditory system nuclei (Bush and Hyson,
in expression levels using microarrays analysis. Therefore, we used 2008; Wang et al., 2009a,b). Wang et al. (2009b) found 80 days
RMA for normalization that reflects more accurately the expres- after sound exposure that GlyR α subunit message was increased
sion level of genes and a statistical method sufficiently stringent in in the dorsal cochlear nucleus. However, the protein level was
assigning significance. We have observed differences in expression decreased. Mechanisms for this mismatch are still unknown, but
of some genes (e.g., Nptx2, Slc1a3, and Scn4b) using microarray studies demonstrate that information regarding post-translational
analysis whereas RT-qPCR did not confirm such a significant vari- modifications of the proteins and their translocation is not inher-
ation. This situation was also described after microarray analysis ently encoded in the gene sequences and cannot be derived from
in the IC by Holt et al. (2005), mainly in genes that are con- mRNA expression (see Benoit et al., 2011 review).
stitutively expressed at low levels. It should not be surprising that
when constitute expression of genes is extremely low the threshold THE IPSI- AND CONTRALATERAL IC 15 DAYS AFTER THE LESION TO THE
of microarrays are higher than qPCR arrays. CORTEX
Functional analysis of IC gene profile expression 15 days after
DISCUSSION the lesion shows an up-regulation in Serglycin, a proteoglycan
Unilateral auditory cortical deafferentation induces bilateral and expressed primarily by immune cells (Kolset and Pejler, 2011) with
asymmetric changes in gene expression profiling in the IC. In a key role in inflammatory processes (Kolset and Tveit, 2008),
adults, this plastic response is time-dependent, with more exten- suggesting that, in our model, cortical ablation induces in the
sive regulation at long-term post-lesion (90 days) than at short- short-term an inflammatory reaction in the ipsilateral IC which
term (15 days). Also, the nature of this reorganization is different undergoes a larger loss of cortical descending afferents. In agree-
for each time post-lesion, supporting the concept of a plastic ability ment with this finding, previous studies in the IC using a thiamine
in adults. deficiency model to analyze inflammation, cellular stress, metabo-
Auditory cortical ablation affects a large number of genes simul- lism, and structural damage after focal neuronal death (Vemuganti
taneously; at 15 days post-lesion we found 52 genes (33 in the et al., 2006) found an up-regulation in Podocalyxin, a cell adhe-
ipsilateral side and 19 in the contralateral side) whose expression sion regulator gene. More recently, other roles for this gene in
changes. Meanwhile, 90 days after the lesion this regulation affects neural development, neurite growth, branching, axonal fascicula-
640 genes in the IC (430 in the ipsilateral side and 210 in the tion, and synapse formation after neuronal death or inflammation
contralateral side). Unilateral cortical ablation strongly affects the have been shown (Vitureira et al., 2010). In our experimental
ipsilateral lesioned side at both times post-lesion (15 and 90 days), model, up-regulation of this gene in the ipsilateral IC suggests
due to the loss of a denser innervation. Ipsilateral regulation was that this adjustment correlates with the density of the lesioned
greater after long-term (90 days) than after short-term. This “late” pathway. Furthermore, Complement component 3 (C3), another
regulation suggests an important role for adult neural plastic- inflammation-related gene, was found to be up-regulated in the IC
ity events in this key auditory nucleus. After 15 days post-lesion, in thiamin deficiency animals (Vemuganti et al., 2006) and in our
gene expression regulation was mostly related to an inflamma- ablated animals; supporting the idea of an ongoing inflammatory
tory response, whereas after 90 days regulation was linked mainly process at 15 days post-lesion which is stronger in the ipsilateral IC,
to sprouting phenomena and synaptic transmission. We already accordingly with a heaviest preterminal fields degeneration after
know that the auditory system in adults is able to induce gene cortical ablation in the ipsilateral IC rather than the contralat-
modulation in the IC even after long periods of peripheral deaf- eral (Feliciano and Potashner, 1995). In addition, Stevens et al.
ferentation (Holt et al., 2005). Our approach has focused on a com- (2007) have demonstrated that C3 is a gene that tagged synapses
paratively minor contralateral IC deafferentation and a stronger to be eliminated during CNS development. These authors suggest
ipsilateral IC deafferentation resulting from unilateral cortical that complement-mediated synapse elimination (synaptic strip-
ablation. Our results show that this misbalance is enough to induce ping) may become aberrantly reactivated in neurological diseases.
a rearrangement in gene expression profiles in the IC, involving The process of synaptic elimination (pruning), even in adult ani-
the pathways necessary to trigger neuronal plasticity in adults. mals, is important for rewiring neural circuits. Butz et al. (2009),
Neurons convert environmental stimuli using a complex array using a model for analyzing cortical rewiring after deafferentation

showed that even small changes in homeostatic equilibrium imply (Saatman et al., 2010). In addition, we detected down-regulation of
formation of new synapses or pruning of existing ones. the Calsyntenin 3 (Clstn3), a gene which overexpression accelerates
On the other hand, in the ipsilateral IC at 15 days post-lesion neuronal death (Uchida et al., 2011). All this strongly suggests an
we also found down-regulated genes, like Latexin which is a car- ongoing cell death process in the ipsilateral IC at 90 days post-
boxypeptidase inhibitor which mediates inflammatory responses lesion. However we did not find changes in the expression of
(Aagaard et al., 2005), but it also known to be expressed by astro- initiator or effector caspases genes triggering the apoptotic process.
cytes, providing neuroprotective mechanisms (Yata et al., 2011). This could be related with limited microarray sensitivity. On the
However, our results suggest that a typical astrocytic reaction does other hand, deprivation of auditory nerve input in adult animals
not take place in the IC, because we did not find any regulation in does not result in significant neuronal loss in the cochlear nuclei
marker genes for reactive astrocytes like Vimentin or Glial fibrillary (Harris et al., 2005), supporting the idea that in the auditory system
acidic protein (GFAP) which in the case of a thiamine deficiency mature neurons are less sensitive to apoptotic cell death. It could
model were up-regulated (Vemuganti et al., 2006). be that the regulation of apoptotic cascade genes observed by us
Analyses of the contralateral IC 15 days after the cortical lesion may be related to other cellular pathways in which all these genes
show an up-regulation in the Spinster (Spns) gene, which is a nega- also performed a key role. Further specific proteomic experiments
tive synaptic growth regulator. The Drosophila Spns mutant shows of caspases cell cascades will be needed to elucidate this problem,
a 200% increase in the number of synaptic endings and a deficit mainly because the regulation of apoptotic cascades is complex
in presynaptic release (Sweeney and Davis, 2002). Spinster is also and involves transcriptional control as well as posttranscriptional
linked to a novel caspase-independent cell death pathway medi- protein modifications (Culmsee and Landshamer, 2006).
ated by autophagy (Yanagisawa et al., 2003). These data speak We also found in the ipsilateral IC 90 days after the lesion
in favor of a rearrangement process in the contralateral IC that down-regulation of Myelin-associated glycoprotein (Mag ), a gene
has lost a weaker corticofugal projection compared with the side able to inhibit axon regeneration after injury (Yiu and He, 2003).
ipsilateral to the lesion. The focus of this regulation may be in Studies in vivo demonstrate a modest but significant enhance-
synaptic pruning, looking for an efficient synaptic rewiring (Butz ment of axon regeneration in mice lacking Mag (Schnaar and
et al., 2009), or it may be related to changes in presynaptic release Lopez, 2009). Down-regulation of this gene in our material could
after lesion (Birthelmer et al., 2003), which affects activity in IC be an indication of axon elongation and indirectly of a sprout-
neurons (Nwabueze-Ogbo et al., 2002; Popelar et al., 2003). ing process subsequent to lesion in the ipsilateral IC. However,
In summary, microarray analyses at 15 days post-lesion show at this same post-lesion time an oligodendrocyte-specific pro-
that genes mainly related to inflammatory processes were up- tein, Claudin 11 (Cldn11), which encodes a molecular component
regulated in the ipsilateral IC. The relative increase in the activity present in tight junctions and which is also is involved in axon
of genes related to inflammation could be a consequence of exten- myelination, was down-regulated. This result probably indicates
sive brain injury (Block et al., 2005). However the asymmetry of an inefficient axon myelination during axon elongation. Cldn11
the corticofugal projection (Saldana et al., 1996; Bajo et al., 2007) down-regulation may affect biophysical properties of myelinated
geared toward the ipsilateral side, along with an ipsilateral predom- axons in the IC, because it is known that Cldn11-null mice present
inance of changes in gene expression suggest a local consequence of a 60% decrease in conduction velocity (Devaux and Gow, 2008). A
terminal degeneration in the IC. The biological functions of genes, low effective compartmentalization of the myelin sheet of sprout-
whose regulation is affected, indicate that beyond an inflamma- ing axon collaterals may be in accordance with previous results in
tory response an emergent plastic process, probably related with which after 90 days of cortical lesion, sound stimulation-induced
sprouting, and pruning on the adult collicular network, takes place c-Fos immunoreactivity in the IC was only partially recovered
bilaterally in the ICs after short-term post-lesion. (Clarkson et al., 2010a). In correspondence with this low activity
in the IC at this survival time after the cortical lesion, genes related
THE IPSI- AND CONTRALATERAL IC 90 DAYS AFTER THE LESION TO THE with synaptic activity also were affected with down-regulation in
CORTEX Synaptotagmin-11 (Syt11, a Ca2+ -sensor during vesicular traffick-
A much larger gene expression regulation pattern was found ing, Inoue et al., 2007), Calcium channel – voltage-dependent – γ3
in the IC 90 days after the cortical lesion. Microarray compar- (Cacng3, a gene with modulatory effects on the electrophysiologi-
isons between control and ipsilateral IC showed that changes cal characteristic of the CaV 2.1 channel, Rousset et al., 2001) and
affected remarkably genes involved in apoptosis. We found an also in Potassium inwardly rectifying channel, subfamily J, mem-
up-regulation in Programmed cell death 10 (Pdcd10) whose over- ber 16 (Kcnj16) which plays a physiological role in the potassium
expression is sufficient to induce neuronal apoptosis (Lin et al., buffering-action of brain astrocytes (Hibino et al., 2004). Holt et al.
2010). Fission 1 (Fis1) which participates in apoptotic mitochon- (2005) showed by RT-qPCR a rearrangement in synaptic transmis-
drial fission (Youle and Karbowski, 2005) was also up-regulated sion genes in the IC 90 days after cochlear ablation, with regulation
and so were Growth arrest-specific 2 (Gas2) gene, a substrate of affecting several glutamate and GABA receptors genes, supporting
Caspase-3 (Sgorbissa et al., 1999), and Cytochrome c oxidase sub- the hypothesis that, after long-term post-lesion, the adult auditory
unit VIIb (Cox7b) which is one of the last subunits that join the system is able to modify the gene neurotransmission machinery in
assembling cytochrome oxidase complex inducing apoptosis and the IC even after lack of auditory activity. Glial related genes such
affecting mitochondrial integrity (Fornuskova et al., 2010). We also as Connexin 30 and 43, which are specific gap junction proteins
found an up-regulation in Calpain 7 (Capn7), which is associated that mediate cell-to-cell communication (Gemel et al., 2008) and
after brain injury with neuron death and axonal degeneration Cadherin 13 involved in regulation of cell growth, survival, and

astrocyte proliferation (Huang et al., 2003) were down-regulated. et al., 2005). Also it was shown that Hpca overexpression dramat-
Recently studies from connexin 30 and 43 show that these gap ically elongated neurites (Oh et al., 2008). Similarly, Dopamine
junctions mediate astroglial networks scale synaptic activity, as receptor D1 interacting protein (Drd1ip) a known brain plastic-
they define the concentrations and dynamics of extracellular ions ity gene (Kruusmagi et al., 2007) that plays a specialized role in
and neurotransmitters during synaptic activity (Pannasch et al., removal of synaptic AMPA receptor (Davidson et al., 2009) was
2011). These glia gene regulations after ablation are important up-regulated. So it was N-myc downstream regulate gene 4 (Ndrg4)
for tripartite synapses model, in which brain function actually a contributor to neuronal differentiation, neurite formation, cell
arises from the coordinated activity of a network comprising both progression, and survival (Takahashi et al., 2005; Schilling et al.,
neurons and glia (Perea et al., 2009). 2009), Neural proliferation, differentiation, and control 1 (Npdc1)
The idea that in the ipsilateral IC 90 days after cortical ablation gene, which is able to down-regulate the proliferation of neural
plastic or adaptive reorganizations have been switched on is also precursors (Dupont et al., 1997), and Spinster that is a negative
reinforced by previous data showing that sound-induced c-Fos regulator of synaptic regrowth (Sweeney and Davis, 2002) were
immunoreactivity progressively increases from 90 to 180 days after also up-regulated. This balance between regrowth and growth
cortical ablation (Clarkson et al., 2010a), showing that neuronal suppression gene expression seems to be important to demon-
activity is still on its way to recovery. strate rewiring network processes in the contralateral IC 90 days
After 90 days of the lesion, the contralateral IC showed up- after lesion through activation of mechanisms involve in neuronal
regulation in genes involved in neurotransmission/signal propa- regulation.
gation/synaptic plasticity such as Sodium channel, type IV, beta In summary, up-regulation in genes related to axonal growth,
(Scn4b) whose overexpression induces neurite outgrowth, causes receptor expression, and trafficking as well in channels and synap-
thickening of dendrites and increases the post synaptic density of tic machinery may be compatible with plastic reorganization that
neuronal spines (Oyama et al., 2006). Also Potassium voltage chan- is more active in the contralateral IC. These differences in the tim-
nel, shaker related subfamily, member 6 (Kcna6 ) was up-regulated. ing for plasticity activation between both sides may be explained
This potassium channel regulates miniature inhibitory postsy- by the asymmetrical loss of excitation due to the asymmetrical
naptic currents (mIPSCs) by regulating glycine release from the density of the descending connections from the cortex.
endings (Shoudai et al., 2007). In astrocytes Kcna6 underlies part
of the delayed rectifier potassium current (Smart et al., 1997). COMPARING IPSI- AND CONTRALATERAL CHANGES AT 15 AND
Another family of potassium channels like KCNQ5 and KCNK15 90 DAYS POST-LESION
has been localized to neurons in the IC and are modulated by Comparison between ipsi- and contralateral IC after 15 days shows
hearing loss (Caminos et al., 2007; Dong et al., 2009). Further- a small number of altered genes (14), contrasting with the larger
more, up-regulation in opioid receptor, sigma 1 (Oprs1), found in number of regulated genes in the comparison (ipsi- vs. con-
our results, may be related to its role in neuronal plasticity, enhanc- tralateral) after 90 days subsequent to the cortical lesion (1659
ing growth factor-induced neurite outgrowth (Hashimoto, 2010; genes). Gene expression analysis between ipsi- and contralateral
Ruscher et al., 2011), and also regulating both Ca2+ entry and IC after 90 days following the cortical lesion showed up-regulation
Ca2+ mobilization from endoplasmic reticulum stores (Monnet, in potassium and chloride channels, and cholinergic, serotonin-
2005). Another opioid receptor involved in nociception, Opioid ergic, glycinergic receptors in the contralateral IC. Biological
receptor-like 1 (Oprl1) was also up-regulated, and is able to decrease function of up-regulated genes speaks in favor of a more active
acetylcholine release and dopamine release and elevates extracel- synaptic plasticity in the contralateral IC after long-term post-
lular glutamate and GABA levels (Schlicker and Morari, 2000). All lesion. In particular, the NMDA receptor subunit coded by Grin1
these changes were accompanied by an up-regulation in genes (glutamate receptor, ionotropic, N-methyl d-aspartate 1) and its
for neurotransmitter delivery at synaptic terminals. For exam- associated protein coded by Grina (glutamate receptor, ionotropic,
ple, in our material up-regulation of SV2a which exerts a main N-methyl d-aspartate-associated protein 1) were up-regulated in
role in neurotransmitter uptake, vesicle targeting, and membrane the contralateral IC. NMDA receptors are important for activity-
fusion (Elferink and Scheller, 1995), as well as Synaptogyrin 1 dependent synaptic plasticity (Kalev-Zylinska et al., 2009; Rebola
and 3 (Syngr1 and 3), both of which play an essential function et al., 2010). In vitro experiments demonstrated that activation
in synaptic plasticity without being required for neurotransmitter of NMDA receptors mediated a dramatic increase of both c-fos
release (Belizaire et al., 2004). These data agrees with the idea that expression and intracellular calcium (Lerea et al., 1992). In a pre-
in adult animals the contralateral IC that loses the weaker pro- vious work both Calretinin (an indirect marker of Ca2+ influx-
jection from the cortex is still able, after long-term post-lesion, to Clarkson et al., 2010b) and c-Fos in the IC contralateral to the
enhance its genetic machinery to try to compensate the imbalance lesion (Clarkson et al., 2010a) showed stronger immunoreactiv-
induced by lost cortical connections. This compensation could be ity relative to the ipsilateral side. Future experiments will be need
the result of fine plastic modulations, which may include sprout- to demonstrate in our in vivo model a direct correlation among
ing, pruning, and neurotransmission rearrangement. According NMDA receptor activation, c-Fos activation, and changes in the
to this idea, we also found an increase in Hippocalcin (Hpca) intracellular Ca2+ concentration.
which encodes a calcium-binding protein considered relevant An extensive group of genes potentially involved in neu-
for synaptic plasticity, acting as a molecular link between Ca2+ rotransmitter release, such as four members of the Synap-
entry through NMDA receptor and the subsequent endocytosis of totagmin family (Syt1, 2, 11, and 12; Chapman, 2008;
AMPA receptor subunits in long-term depression (LTD; Palmer Rizo and Rosenmund, 2008; Sudhof and Rothman, 2009), were

up-regulated in the contralateral side. In addition, genes involved adult animals it is larger after long-term post-lesion with specific
both in expression and trafficking of Synaptotagmin family mem- changes aimed at recovering activity loss in IC neurons.
bers, such as the SV2 family (SV2a, SV2c, and SV2 related protein;
Nowack et al., 2010) were also up-regulated in the contralateral AUTHOR CONTRIBUTION
IC. Not only were receptors and synaptic vesicle fusion genes up- Cheryl Clarkson Performed experiments, prepared figures and
regulated in the contralateral side after long-term lesion, but also wrote the manuscript. M. Javier Herrero-Turrión. Performed RT-
this rearrangement was accompanied by an up-regulation in key qPCRs experiments, analyzed statistically microarray data and
enzymes for GABA (4-aminobutyrate aminotransferase, Abat, and drafted the manuscript. Miguel A. Merchán. Designed, coordi-
Glutamic acid decarboxylase 1, Gad1), glycine (Glycyl-tRNA syn- nated, and supervised the study. All authors read and agreed the
thetase, Gars), glutamate (Glutaminyl-tRNA synthetase, Qars) and paper content.
endocannabinoid synthesis (Dyacylglycerol lipase alpha, Dagla),
all of them key components in short and long-term plastic synap- ACKNOWLEDGMENTS
tic changes. In addition, neurotransmitter transporters were up- The authors would like to thank Dr. José Juiz for his critical review
regulated in the contralateral IC after long-term cortical ablation and very helpful suggestions and Ignacio Plaza for his excellent
(for neuronal glutamate: Vesicular glutamate transporter 2, VGlut2, technical assistance. Financial support for this investigation was
or solute carrier family 17 member 6, Slc17a6 ; for proline: Slc6a7 ; provided by the grant BFU2009-13754-CO2 from the Spanish
for glycine: Slc6a9; for GABA: Slc6a11 and Slc32a1; and for glu- Ministerio de Investigación e Innovación (Minciin).
tamate: Slc1a3 or GLAST ). Up-regulation of both enzymes and
transporter genes clearly suggests a post-lesion shift in the synthe- SUPPLEMENTARY MATERIAL
sis and recycling of neurotransmitters. These findings support the The Supplementary Material for this article can be found
idea of a better recovery of neurotransmission in the contralateral online at http://www.frontiersin.org/Neural_Circuits/10.3389/
IC probably due to the comparatively lower loss of excitation in fncir.2012.00086/abstract
that side after cortical lesion. All these data also suggest activity-
Datasheet 1 | Naïve control vs. 15 days and 90 days post-lesion.
dependent compensatory mechanisms in the contralateral IC,
counteracting the most affected ipsilateral IC. In summary, these Datasheet 2 | Primers PCR arrays.
changes in gene expression are important due to the claim that
regulation depends on the extent of deafferentation, and even in Datasheet 3 | Funtional annotation of diferentially expresed genes.
REFERENCES Bledsoe, S. C. Jr., Nagase, S., Miller, Front. Comput. Neurosci. 3:10. Culmsee, C., and Landshamer, S. (2006).
Aagaard, A., Listwan, P., Cowieson, N., J. M., and Altschuler, R. A. (1995). doi:10.3389/neuro.10.010.2009 Molecular insights into mechanisms
Huber, T., Ravasi, T., Wells, C. A., Deafness-induced plasticity in the Caminos, E., Garcia-Pino, E., Martinez- of the cell death program: role in
et al. (2005). An inflammatory role mature central auditory system. Galan, J. R., and Juiz, J. M. the progression of neurodegenera-
for the mammalian carboxypepti- Neuroreport 7, 225–229. (2007). The potassium channel tive disorders. Curr. Alzheimer Res.
dase inhibitor latexin: relationship to Block, F., Dihne, M., and Loos, M. KCNQ5/Kv7.5 is localized in synap- 3, 269–283.
cystatins and the tumor suppressor (2005). Inflammation in areas of tic endings of auditory brainstem Davidson, H. T., Xiao, J., Dai, R., and
TIG1. Structure 13, 309–317. remote changes following focal nuclei of the rat. J. Comp. Neurol. Bergson, C. (2009). Calcyon is nec-
Bajo, V. M., Nodal, F. R., Bizley, J. K., brain lesion. Prog. Neurobiol. 75, 505, 363–378. essary for activity-dependent AMPA
Moore, D. R., and King, A. J. (2007). 342–365. Chapman, E. R. (2008). How does receptor internalization and LTD in
The ferret auditory cortex: descend- Bowen, G. P., Lin, D., Taylor, M. K., synaptotagmin trigger neurotrans- CA1 neurons of hippocampus. Eur.
ing projections to the inferior col- and Ison, J. R. (2003). Auditory cor- mitter release? Annu. Rev. Biochem. J. Neurosci. 29, 42–54.
liculus. Cereb. Cortex 17, 475–491. tex lesions in the rat impair both 77, 615–641. Dennis, G. Jr., Sherman, B. T., Hosack,
Belizaire, R., Komanduri, C., Wooten, temporal acuity and noise incre- Clarkson, C., Juíz, J. M., and Mer- D. A., Yang, J., Gao, W., Lane, H. C.,
K., Chen, M., Thaller, C., and Janz, R. ment thresholds, revealing a com- chán, M. A. (2010a). Long-term et al. (2003). DAVID: database for
(2004). Characterization of synap- mon neural substrate. Cereb. Cortex regulation in calretinin stain- annotation, visualization, and inte-
togyrin 3 as a new synaptic vesi- 13, 815–822. ing in the rat inferior colliculus grated discovery. Genome Biol. 4,
cle protein. J. Comp. Neurol. 470, Bullitt, E. (1995). Expression of c-fos- after unilateral auditory cortical P3.
266–281. like protein as a marker for neuronal ablation. J. Comp. Neurol. 518, Devaux, J., and Gow, A. (2008). Tight
Benoit, C. E., Rowe, W. B., Menards, C., activity following noxious stimula- 4261–4276. junctions potentiate the insulative
Sarret, P., and Quirion, R. (2011). tion in the rat. J. Comp. Neurol. 296, Clarkson, C., Juíz, J. M., and Merchán, properties of small CNS myelinated
Genomic and proteomic strategies 517–530. M. A. (2010b). Transient down- axons. J. Cell Biol. 183, 909–921.
to identify novel targets potentially Bush, A. L., and Hyson, R. L. (2008). regulation of sound-induced c-fos Dong, S., Mulders, W. H., Rodger, J.,
involved in learning and memory. Effects of lithium and deafferenta- protein expression in the inferior and Robertson, D. (2009). Changes
Trends Pharmacol. Sci. 32, 43–52. tion on expression of glycogen syn- colliculus after ablation of the audi- in neuronal activity and gene expres-
Birthelmer, A., Ehret, A., Amtage, F., thase kinase-3beta, NFkappaB, beta- tory cortex. Front. Neuroanat. 4:141. sion in guinea-pig auditory brain-
Forster, S., Lehmann, O., Jeltsch, catenin, and pCreb in the chick doi:10.3389/fnana.2010.00141 stem after unilateral partial hearing
H., et al. (2003). Neurotransmit- cochlear nucleus. Brain Res. 1203, Clarkson, C., López, D. E., and Merchán, loss. Neuroscience 159, 1164–1174.
ter release and its presynaptic mod- 18–25. M. A. (2010c). Long-term functional Druga, R., and Syka, J. (2001). Effect of
ulation in the rat hippocampus Butz, M., van Ooyen, A., and Wor- recovery in the rat auditory sys- auditory cortex lesions on NADPH-
after selective damage to cholinergic gotter, F. (2009). A model for tem after unilateral auditory cor- diaphorase staining in the inferior
or/and serotonergic afferents. Brain cortical rewiring following deaf- tex ablation. Acta Otolaryngol. 130, colliculus of rat. Neuroreport 12,
Res. Bull. 59, 371–381. ferentation and focal stroke. 326–332. 1555–1559.

Dupont, E., Sansal, I., Toru, D., Evrard, brainstem. J. Comp. Neurol. 493, Kalev-Zylinska, M. L., Symes,W.,Young, Monnet, F. P. (2005). Sigma-1 receptor
C., and Rouget, P. (1997). Identifi- 460–474. D., and During, M. J. (2009). as regulator of neuronal intracellular
cation of NPDC-1, gene involved in Hashimoto, K. (2010). Role of sigma- Knockdown and overexpression of Ca2+ : clinical and therapeutic rele-
the control of proliferation and dif- 1 receptors in neural plasticity NR1 modulates NMDA receptor vance. Biol. Cell 97, 873–883.
ferentiation of neural and glial pre- and in antipsychotic action. Nihon function. Mol. Cell. Neurosci. 41, Norena, A. J., and Eggermont, J. J.
cursors. C. R. Seances Soc. Biol. Fil. Shinkei Seishin Yakurigaku Zasshi 30, 383–396. (2006). Enriched acoustic environ-
191, 95–104. 123–127. Kandler, K. (2004). Activity-dependent ment after noise trauma abolishes
Edeline, J. M., and Weinberger, N. M. Hibino, H., Fujita, A., Iwai, K., Yamada, organization of inhibitory circuits: neural signs of tinnitus. Neuroreport
(1991a). Subcortical adaptive filter- M., and Kurachi, Y. (2004). Differ- lessons from the auditory sys- 17, 559–563.
ing in the auditory system: asso- ential assembly of inwardly rectify- tem. Curr. Opin. Neurobiol. 14, Nowack, A., Yao, J., Custer, K. L., and
ciative receptive field plasticity in ing K+ channel subunits, Kir4.1 and 96–104. Bajjalieh, S. M. (2010). SV2 regulates
the dorsal medial geniculate body. Kir5.1, in brain astrocytes. J. Biol. Keuroghlian, A. S., and Knudsen, E. I. neurotransmitter release via multi-
Behav. Neurosci. 105, 154–175. Chem. 279, 44065–44073. (2007). Adaptive auditory plastic- ple mechanisms. Am. J. Physiol. Cell
Edeline, J. M., and Weinberger, N. M. Holt, A. G., Asako, M., Duncan, R. ity in developing and adult animals. Physiol. 299, C960–C967.
(1991b). Thalamic short-term plas- K., Lomax, C. A., Juíz, J. M., and Prog. Neurobiol. 82, 109–121. Nwabueze-Ogbo, F. C., Popelar, J., and
ticity in the auditory system: associa- Altschuler, R. A. (2006). Deafness Kolset, S. O., and Pejler, G. (2011). Syka, J. (2002). Changes in the
tive returning of receptive fields in associated changes in expression of Serglycin: a structural and func- acoustically evoked activity in the
the ventral medial geniculate body. two-pore domain potassium chan- tional chameleon with wide impact inferior colliculus of the rat after
Behav. Neurosci. 105, 618–639. nels in the rat cochlear nucleus. Hear. on immune cells. J. Immunol. 187, functional ablation of the auditory
Edeline, J. M., and Weinberger, N. Res. 216–217, 146–153. 4927–4933. cortex. Physiol. Res. 51(Suppl. 1),
M. (1992). Associative retuning in Holt, A. G., Asako, M., Lomax, C. A., Kolset, S. O., and Tveit, H. (2008). Ser- S95–S104.
the thalamic source of input to MacDonald, J. W., Tong, L., Lomax, glycin – structure and biology. Cell. Oh, D. Y., Cho, J. H., Park, S. Y., Kim,
the amygdala and auditory cor- M. I., et al. (2005). Deafness-related Mol. Life Sci. 65, 1073–1085. Y. S., Yoon, Y. J., Yoon, S. H., et al.
tex: receptive field plasticity in the plasticity in the inferior colliculus: Kovacs, K. J. (2008). Measurement of (2008). A novel role of hippocalcin
medial division of the medial genic- gene expression profiling following immediate-early gene activation-c- in bFGF-induced neurite outgrowth
ulate body. Behav. Neurosci. 106, removal of peripheral activity. J. fos and beyond. J. Neuroendocrinol. of H19-7 cells. J. Neurosci. Res. 86,
81–105. Neurochem. 93, 1069–1086. 20, 665–672. 1557–1565.
Elferink, L. A., and Scheller, R. H. Howley, R., Kinsella, P., Buckley, P. Kruusmagi, M., Zelenin, S., Bris- Oyama, F., Miyazaki, H., Sakamoto, N.,
(1995). Synaptic vesicle proteins and G., Alcock, L., Jansen, M., Heffer- mar, H., and Scott, L. (2007). Becquet, C., Machida, Y., Kaneko,
regulated exocytosis. Prog. Brain Res. nan, J., et al. (2012). Comparative Intracellular dynamics of calcyon, K., et al. (2006). Sodium chan-
105, 79–85. genomic and proteomic analysis of a neuron-specific vesicular protein. nel beta4 subunit: down-regulation
Feliciano, M., and Potashner, S. J. high grade glioma primary cultures Neuroreport 18, 1547–1551. and possible involvement in neuritic
(1995). Evidence for a glutamatergic and matched tumor in situ. Exp. Cell Lerea, L. S., Butler, L. S., and McNa- degeneration in Huntington’s dis-
pathway from the guinea pig audi- Res. 318, 2245–2256. mara, J. O. (1992). NMDA and non- ease transgenic mice. J. Neurochem.
tory cortex to the inferior colliculus. Huang, D. W., Sherman, B. T., and NMDA receptor-mediated increase 98, 518–529.
J. Neurochem. 65, 1348–1357. Lempicki, R. A. (2009). Systematic of c-fos mRNA in dentate gyrus Palmer, C. L., Lim, W., Hastie, P. G.,
Fornuskova, D., Stiburek, L., Wenchich, and integrative analysis of large gene neurons involves calcium influx via Toward, M., Korolchuk, V. I., Bur-
L., Vinsova, K., Hansikova, H., lists using DAVID bioinformatics different routes. J. Neurosci. 12, bidge, S. A., et al. (2005). Hippocal-
and Zeman, J. (2010). Novel resources. Nat. Protoc. 4, 44–57. 2973–2981. cin functions as a calcium sensor
insights into the assembly and Huang, Z. Y., Wu, Y., Hedrick, N., Lin, C., Meng, S., Zhu, T., and Wang, X. in hippocampal LTD. Neuron 47,
function of human nuclear-encoded and Gutmann, D. H. (2003). T- (2010). PDCD10/CCM3 acts down- 487–494.
cytochrome c oxidase subunits 4, cadherin-mediated cell growth reg- stream of {gamma}-protocadherins Pannasch, U., Vargová, L., Reingruber,
5a, 6a, 7a, and 7b. Biochem. J. 428, ulation involves G2 phase arrest and to regulate neuronal survival. J. Biol. J., Ezan, P., Holcman, D., Giaume,
363–374. requires p21(CIP1/WAF1) expres- Chem. 285, 41675–41685. C., et al. (2011). Astroglial networks
Gao, E., and Suga, N. (1998). sion. Mol. Cell. Biol. 23, 566–578. Lyons, M. R., and West, A. E. scale synaptic activity and plastic-
Experience-dependent corti- Illing, R. B., Kraus, K. S., and Mei- (2011). Mechanisms of specificity ity. Proc. Natl. Acad. Sci. U.S.A. 108,
cofugal adjustment of midbrain dinger, M. A. (2005). Reconnect- in neuronal activity-regulated gene 8467–8472.
frequency map in bat auditory ing neuronal networks in the audi- transcription. Prog. Neurobiol. 94, Paxinos, G., and Watson, C. (2005).
system. Proc. Natl. Acad. Sci. U.S.A. tory brainstem following unilateral 259–295. The Rat Brain in Stereotaxic Coordi-
95, 12663–12670. deafening. Hear. Res. 206, 185–199. Ma, X., and Suga, N. (2005). Long-term nates, 5th Edn, San Diego: Elsevier
Gao, E., and Suga, N. (2000). Illing, R. B., and Reisch, A. (2006). Spe- cortical plasticity evoked by elec- Academic Press.
Experience-dependent plastic- cific plasticity responses to unilat- tric stimulation and acetylcholine Perea, G., Navarrete, M., and Araque,
ity in the auditory cortex and the erally decreased or increased hear- applied to the auditory cortex. A. (2009). Tripartite synapses: astro-
inferior colliculus of bats: role of ing intensity in the adult cochlear Proc. Natl. Acad. Sci. U.S.A. 102, cytes process and control synap-
the corticofugal system. Proc. Natl. nucleus and beyond. Hear. Res. 9335–9340. tic information. Trends Neurosci. 32,
Acad. Sci. U.S.A. 97, 8081–8086. 216–217, 189–197. Malmierca, M. S., and Merchán, M. 421–431.
Gemel, J., Lin, X., Collins, R., Veenstra, Inoue, S., Imamura, A., Okazaki, Y., (2004). “The auditory system”, in Perez-Otano, I., and Ehlers, M. D.
R. D., and Beyer, E. C. (2008). Cx30.2 Yokota, H., Arai, M., Hayashi, N., The Rat Nervous System, ed. G. Pax- (2005). Homeostatic plasticity and
can form heteromeric gap junction et al. (2007). Synaptotagmin XI inos (San Diego: Academic Press), NMDA receptor trafficking. Trends
channels with other cardiac connex- as a candidate gene for suscep- 995–1080. Neurosci. 28, 229–238.
ins. Biochem. Biophys. Res. Commun. tibility to schizophrenia. Am. J. McAlpine, D., Martin, R. L., Mossop, Popelar, J., Nwabueze-Ogbo, F. C., and
369, 388–394. Med. Genet. B Neuropsychiatr. Genet. J. E., and Moore, D. R. (1997). Syka, J. (2003). Changes in neuronal
Harris, J. A., Hardie, N. A., 144B, 332–340. Response properties of neu- activity of the inferior colliculus in
Bermingham-McDonogh, O., Jen, P. H., and Zhang, J. P. (1999). Corti- rons in the inferior colliculus of rat after temporal inactivation of
and Rubel, E. W. (2005). Gene cofugal regulation of excitatory and the monaurally deafened ferret the auditory cortex. Physiol. Res. 52,
expression differences over a critical inhibitory frequency tuning curves to acoustic stimulation of the 615–628.
period of afferent-dependent neu- of bat inferior collicular neurons. intact ear. J. Neurophysiol. 78, Rebola, N., Srikumar, B. N., and Mulle,
ron survival in the mouse auditory Brain Res. 841, 184–188. 767–779. C. (2010). Activity-dependent

synaptic plasticity of NMDA Schilling, S. H., Hjelmeland, A. B., rat auditory pathway. J. Comp. Neu- Wieloch, T., and Nikolich, K. (2006)
receptors. J. Physiol. 588(Pt 1), Radiloff, D. R., Liu, I. M., Wake- rol. 501, 509–525. Mechanisms of neural plasticity
93–99. man, T. P., Fielhauer, J. R., et al. Sweeney, S. T., and Davis, G. W. (2002). following brain injury. Curr. Opin.
Riquelme, R., Saldana, E., Osen, K. K., (2009). NDRG4 is required for cell Unrestricted synaptic growth in Neurobiol. 16, 258–264.
Ottersen, O. P., and Merchán, M. cycle progression and survival in spinster-a late endosomal protein Yanagisawa, H., Miyashita, T., Nakano,
A. (2001). Colocalization of GABA glioblastoma cells. J. Biol. Chem. 284, implicated in TGF-beta-mediated Y., and Yamamoto, D. (2003).
and glycine in the ventral nucleus 25160–25169. synaptic growth regulation. Neuron HSpin1, a transmembrane pro-
of the lateral lemniscus in rat: an Schlicker, E., and Morari, M. (2000). 36, 403–416. tein interacting with Bcl-2/Bcl-
in situ hybridization and semiquan- Nociceptin/orphanin FQ and neu- Takahashi, K., Yamada, M., Ohata, H., xL, induces a caspase-independent
titative immunocytochemical study. rotransmitter release in the cen- Honda, K., Yamada, M. (2005). autophagic cell death. Cell Death
J. Comp. Neurol. 432, 409–424. tral nervous system. Peptides 21, Ndrg2 promotes neurite outgrowth Differ. 10, 798–807.
Rizo, J., and Rosenmund, C. (2008). 1023–1029. of NGF-differentiated PC12 cells. Yata, K., Oikawa, S., Sasaki, R., Shindo,
Synaptic vesicle fusion. Nat. Struct. Schmittgen, T. D., and Livak, K. J. Neurosci. Lett. 388, 157–162. A.,Yang, R., Murata, M., et al. (2011).
Mol. Biol. 15, 665–674. (2008). Analyzing real-time PCR Turrigiano, G. (2012). Homeosta- Astrocytic neuroprotection through
Rousset, M., Cens, T., Restituito, data by the comparative C(T) tic synaptic plasticity: local and induction of cytoprotective mole-
S., Barrere, C., Black, J. L. III, method. Nat. Protoc. 3, 1101–1108. global mechanisms for stabiliz- cules; a proteomic analysis of mutant
McEnery, M. W., and Charnet, P. Schnaar, R. L., and Lopez, P. H. (2009). ing neuronal function. Cold Spring P301S tau-transgenic mouse. Brain
(2001). Functional roles of gamma2, Myelin-associated glycoprotein and Harb. Perspect. Biol. 4, a005736. Res. 1410, 12–23.
gamma3 and gamma4, three new its axonal receptors. J. Neurosci. Res. doi:10.1101/cshperspect.a005736 Yiu, G., and He, Z. (2003). Signaling
Ca2+ channel subunits, in P/Q-type 87, 3267–3276. Uchida, Y., Nakano, S., Gomi, F., and mechanisms of the myelin inhibitors
Ca2+ channel expressed in Xeno- Semple, M. N., and Kitzes, L. M. (1985). Takahashi, H. (2011). Up-regulation of axon regeneration. Curr. Opin.
pus oocytes. J. Physiol. (Lond.) 532, Single-unit responses in the inferior of calsyntenin-3 by beta-amyloid Neurobiol. 13, 545–551.
583–593. colliculus: different consequences of increases vulnerability of cortical Youle, R. J., and Karbowski, M. (2005).
Rubio, M. E. (2006). Redistribution of contralateral and ipsilateral audi- neurons. FEBS Lett. 585, 651–656. Mitochondrial fission in apoptosis.
synaptic AMPA receptors at glu- tory stimulation. J. Neurophysiol. 53, Vale, C., and Sanes, D. H. (2002). The Nat. Rev. Mol. Cell Biol. 6, 657–663.
tamatergic synapses in the dorsal 1467–1482. effect of bilateral deafness on excita- Zhang, Y., Suga, N., and Yan, J.
cochlear nucleus as an early response Sgorbissa, A., Benetti, R., Marzinotto, tory and inhibitory synaptic strength (1997). Corticofugal modulation
to cochlear ablation in rats. Hear. S., Schneider, C., and Brancol- in the inferior colliculus. Eur. J. of frequency processing in bat
Res. 216–217, 154–167. ini, C. (1999). Caspase-3 and Neurosci. 16, 2394–2404. auditory system. Nature 387,
Ruscher, K., Shamloo, M., Rickhag, M., caspase-7 but not caspase-6 cleave Vemuganti, R., Kalluri, H., Yi, J. H., 900–903.
Ladunga, I., Soriano, L., Gisselsson, Gas2 in vitro: implications for Bowen, K. K., and Hazell, A. S.
L., et al. (2011). The sigma-1 recep- microfilament reorganization dur- (2006). Gene expression changes
tor enhances brain plasticity and ing apoptosis. J. Cell. Sci. 112(Pt 23), in thalamus and inferior colliculus Conflict of Interest Statement: The
functional recovery after experimen- 4475–4482. associated with inflammation, cellu- authors declare that the research was
tal stroke. Brain 134, 732–746. Shoudai, K., Nonaka, K., Maeda, M., lar stress, metabolism and structural conducted in the absence of any com-
Rutkowski, R. G., and Weinberger, N. M. Wang, Z. M., Jeong, H. J., Higashi, damage in thiamine deficiency. Eur. mercial or financial relationships that
(2005). Encoding of learned impor- H., et al. (2007). Effects of vari- J. Neurosci. 23, 1172–1188. could be construed as a potential con-
tance of sound by magnitude of rep- ous K+ channel blockers on spon- Vitureira, N., Andres, R., Perez- flict of interest.
resentational area in primary audi- taneous glycine release at rat spinal Martinez, E., Martinez, A., Bribian,
tory cortex. Proc. Natl. Acad. Sci. neurons. Brain Res. 1157, 11–22. A., Blasi, J., et al. (2010). Podoca- Received: 22 May 2012; accepted: 29
U.S.A. 102, 13664–13669. Smart, S. L., Bosma, M. M., and Tem- lyxin is a novel polysialylated neural October 2012; published online: 26
Rybalko, N., Suta, D., Nwabueze-Ogbo, pel, B. L. (1997). Identification of the adhesion protein with multiple roles November 2012.
F., and Syka, J. (2006). Effect of audi- delayed rectifier potassium channel, in neural development and synapse Citation: Clarkson C, Herrero-Turrión
tory cortex lesions on the discrim- Kv1.6, in cultured astrocytes. Glia 20, formation. PLoS ONE 5, e12003. MJ and Merchán MA (2012) Cortical
ination of frequency-modulated 127–134. doi:10.1371/journal.pone.0012003 auditory deafferentation induces long-
tones in rats. Eur. J. Neurosci. 23, Stevens, B., Allen, N. J., Vazquez, L. E., Wang, H., Turner, J. G., Ling, L., Par- term plasticity in the inferior collicu-
1614–1622. Howell, G. R., Christopherson, K. S., rish, J. L., Hughes, L. F., and Cas- lus of adult rats: microarray and qPCR
Saatman, K. E., Creed, J., and Raghu- Nouri, N., et al. (2007). The clas- pary, D. M. (2009a). Age-related analysis. Front. Neural Circuits 6:86. doi:
pathi, R. (2010). Calpain as a ther- sical complement cascade mediates changes in glycine receptor subunit 10.3389/fncir.2012.00086
apeutic target in traumatic brain CNS synapse elimination. Cell 131, composition and binding in dorsal Copyright © 2012 Clarkson, Herrero-
injury. Neurotherapeutics 7, 31–42. 1164–1178. cochlear nucleus. Neuroscience 160, Turrión and Merchán. This is an open-
Saldana, E., Feliciano, M., and Mug- Sudhof, T. C., and Rothman, J. E. (2009). 227–239. access article distributed under the terms
naini, E. (1996). Distribution Membrane fusion: grappling with Wang, H., Brozoski, T. J., Turner, J. G., of the Creative Commons Attribution
of descending projections from SNARE and SM proteins. Science Ling, L., Parrish, J. L., Hughes, L. F. License, which permits use, distribution
primary auditory neocortex to 323, 474–477. et al. (2009b). Plasticity at glycin- and reproduction in other forums, pro-
inferior colliculus mimics the Sun, X., Xia, Q., Lai, C. H., Shum, D. ergic synapses in dorsal cochlear vided the original authors and source
topography of intracollicular pro- K., Chan, Y. S., and He, J. (2007). nucleus of rats with behavioral evi- are credited and subject to any copy-
jections. J. Comp. Neurol. 371, Corticofugal modulation of acousti- dence of tinnitus. Neuroscience 164, right notices concerning any third-party
15–40. cally induced Fos expression in the 747–759. graphics etc.

APPENDIX
FIGURE A1 | Sample quality control. Box whisker diagram showing the data of the samples [control, 15 and 90 days post-lesion; inferior colliculus right (R)
distribution of each of the samples. The diagram shows the dispersion of each and left (L)] and the degree of similarity between replicas.

Cortical Auditory Deafferentation Induces Long-Term Plasticity in The Inferior Colliculus of Adult Rats: Microarray and QPCR Analysis

Uploaded by

Copyright:

Available Formats

Cortical Auditory Deafferentation Induces Long-Term Plasticity in The Inferior Colliculus of Adult Rats: Microarray and QPCR Analysis

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Cortical Auditory Deafferentation Induces Long-Term Plasticity in The Inferior Colliculus of Adult Rats: Microarray and QPCR Analysis

Uploaded by

Copyright:

Available Formats

ORIGINAL RESEARCH ARTICLE

Cortical auditory deafferentation induces long-term

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 1

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 2

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 3

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 4

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 5

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 6

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 7

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 8

Microarray RT-qPCR Microarray RT-qPCR Microarray RT-qPCR

Frontiers in Neural Circuits

Neurotransmission: 10812879 5-hydroxytryptamine Htr1a NS NS NS NS 1.746 4,093 ± 1,23* Rn00561409_s1

November 2012 | Volume 6 | Article 86 | 9

Frontiers in Neural Circuits

Microarray RT-qPCR Microarray RT-qPCR Microarray RT-qPCR

(1)Change in intensity, expressed as fold change.

November 2012 | Volume 6 | Article 86 | 10

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 11

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 12

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 13

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 14

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 15

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 16

Frontiers in Neural Circuits www.frontiersin.org November 2012 | Volume 6 | Article 86 | 17

You might also like