Gene expression and plasticity in the rat auditory cortex after bilateral cochlear ablation

ORIGINAL ARTICLE

Gene expression and plasticity in the rat auditory cortex after bilateralcochlear ablation

SEUNG-HA OH1, CHONG-SUN KIM1 & JAE-JUN SONG2

1Department of Otolaryngology-Head and Neck Surgery, College of Medicine, Seoul National University, Seoul and2Department of Otorhinolaryngology � Head and Neck Surgery, Dongguk University International Hospital, Kyeonggi,

Korea

AbstractConclusion. The plastic changes in the auditory cortex after bilateral cochlear ablation are related to the immediate earlygenes as well as the neural plasticity-related genes. In addition, cross-modal plasticity may play an important role in the earlychanges in the auditory cortex after bilateral cochlear ablation. Objectives. The purpose of this study was to identifycandidate genes involved in the normal development of primary auditory cortex during the critical period as well as thosegenes specifically modulated under conditions of sensory deafferentation by bilateral cochlear ablation. Materials andmethods. We produced a bilaterally deaf rat model and used DNA microarray technology to analyze differential geneexpression in the primary auditory cortex of bilateral cochlear ablated and sham-operated age-matched control rats. Geneexpression in the auditory cortex was compared at 2, 4, and 12 weeks after surgery. For selected genes, the changes ingene expression were confirmed by real-time polymerase chain reaction (PCR). Results. In the cochlear ablation groups, theexpression of immediate early genes (Egr1 , 2 , 3 , 4 , c-fos , etc.) and neural plasticity-related genes (Arc , Syngr1 , Bdnf , etc.)was decreased at 2 weeks and increased at 4 weeks. The expression of neurotransmission-related genes (Gabra5 , Chrnb3,Chrne , etc.) was decreased at 12 weeks.

Keywords: Central auditory system, damage protection, genes, auditory cortex, critical period, microarray analysis, deafness

Introduction

Plasticity in the central nervous system is often

induced by changes in peripheral neuronal activity,

this response is called ‘activity-dependent plasticity’.

As the level of sensory stimulation can be easily

manipulated, the central auditory system provides an

excellent model for study of activity-dependent

plasticity. Hearing loss and noise over-stimulation

have been shown to lead to plastic changes in the

central auditory pathways [1]. These plastic changes

might rely on such mechanisms as the differences in

timing of expression of receptors that are important

for synaptic plasticity, differences in the molecular

factors that control the development of different

neural pathways, and differences in the degree of

early connectivity and activity-dependent shaping of

this connectivity [2]. The susceptibility of the central

sensory system to the change of the peripheral

activity opens for only a very short time in postnatal

development of the mammalian brain and we call it

the ‘critical period’.

In a previous study, we found hypometabolism in

the auditory cortex of prelingually deaf subjects, but

the area of hypometabolism decreased afterwards

without a new sensory input, which suggests the

existence of neural plasticity in the auditory cortex.

The presence of recovered metabolism may contri-

bute to the auditory-to-visual cross-modal plasticity

in prelingually deaf patients [3]. We found the same

phenomenon in animal studies. Four weeks after

cochlear ablation, rat auditory cortex demonstrated

the most severe hypometabolism and the metabolic

status recovered afterwards [4].

There have been a number of studies on the

plasticity of the auditory cortex [5,6]. However,

changes in the gene expression of auditory cortex

Correspondence: Jae-Jun Song, MD, Department of Otorhinolaryngology � Head and Neck Surgery, Dongguk University International Hospital, 814 Siksa-

dong, Ilsandong-gu, Goyang-si, Gyeonggi-do 410-773, Republic of Korea. Tel: �82 31 961 7436. Fax: �82 31 961 7427. E-mail: [email protected]

Acta Oto-Laryngologica, 2007; 127: 341�350

(Received 26 December 2006; accepted 12 February 2007)

ISSN 0001-6489 print/ISSN 1651-2551 online # 2007 Taylor & Francis

DOI: 10.1080/00016480701275246

Act

a O

tola

ryng

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

gary

on

03/1

4/13

For

pers

onal

use

onl

y.

after bilateral cochlear ablation have never been

assessed.

By characterizing the mechanisms of plastic

change in the auditory cortex after loss of hearing,

we might be able to determine the relationship

between intrinsic constraints and environmental

factors. Microarray technology now allows for a

holistic and high-throughput profiling of gene ex-

pression patterns. Our study is the first holistic gene

profiling investigation of the rat auditory cortex to be

undertaken. Our goal was to identify candidate

genes that are involved in the normal developmental

maturation of primary auditory cortex during the

critical period, as well as those genes that may be

specifically modulated under conditions of sensory

deafferentation by bilateral cochlear ablation.

Materials and methods

Experimental design

Forty-two Sprague�Dawley rats housed in cages in a

specific pathogen-free area were used for this study.

Institutional guidelines regarding animal experi-

ments were followed throughout. Rats were ran-

domly assigned to the groups that received bilateral

cochlear ablation (groups 1, 2, and 3) or to the

groups that received sham operation (groups 4, 5,

and 6). Rats aged 10�14 days were allocated to

group 0. Rats that underwent bilateral cochlear

ablation (groups 1, 2, and 3) or sham operation

(groups 4, 5, and 6) were sacrificed at 2, 4, and 12

weeks after the procedure.

The gene expression profile of the each group

(groups 1�6) was compared with group 0. There-

after, we compared gene expression of the cochlear

ablation groups with that of the sham-operated age-

matched control groups. Using this experimental

design, we could analyze differential gene expression

of the auditory cortex in the cochlear ablation group

and the sham-operated age-matched control group

at three different points in time, and determine the

change of the gene expression profiles over time in

each group.

Surgery for bilateral cochlear ablation

For the purpose of this study, the most appropriate

model was a prelingually deaf rat. As major devel-

opmental changes in the auditory pathway are

known to occur over about 2 weeks in rats, beginning

at around the 10th postnatal day, we ablated the

cochlea surgically at 10�14 days after birth [7]. The

surgical technique was reported previously [4].

Under anesthesia with ketamine, a posterior auricu-

lar incision was made and the bulla was identified.

The cochlea was found after sucking out the

mesodermal tissue in the middle ear. It was un-

capped using a pick and the fluid within the cochlea

was sucked out with a needle suction tip. The skin

was sutured after irrigating the middle ear cavity

with gentamicin. In the sham-operated age-matched

control group, the skin incision and the identification

of the bulla surface was performed and the skin was

then closed. The bulla was not opened, so as to

prevent unwanted conductive hearing loss.

Sample collection from auditory cortex

For the bilateral cochlear ablation group, the deaf

model was confirmed using auditory brainstem

evoked responses and the destruction of cochlea

was confirmed under otomicroscopic examination of

temporal bone. Normal hearing was confirmed by

auditory brainstem evoked responses in the sham-

operated age-matched control groups. The animals

in both the sham-operated age-matched control and

cochlear ablation groups were decapitated and their

brains were removed, slowly frozen on dry ice, and

stored at �208C. Serial coronal sections of brain of

100 m thick containing auditory cortex were col-

lected [8]. These slides were compared with the rat

brain atlas and the location of the auditory cortex

was confirmed. Auditory cortex tissue was collected

by punching the area of interest under the micro-

scope.

Preparation of fluorescent DNA probe and hybridization

Microarray experiments were performed by the

service provider (GenoCheck Co., Gyunggi, Korea).

Total RNA was extracted from the auditory cortex

tissues using the TRI Reagent† (Molecular Re-

search Center, Inc., OH, USA) according to the

manufacturer’s instructions. Fluorescence-labeled

cDNA probes were prepared from 5 ml of total

RNA by RT primer (Genisphere Inc. CA, USA)

primed polymerization using SuperScript reverse

transcriptase (Invitrogen, NY, USA) in a total

reaction volume of 10.5 ml. The reverse transcrip-

tion mixture included 100 U Superscript RNase

H-reverse transcriptase (Invitrogen), each 15 mM

dNTP. After reverse transcription, the sample RNA

was degraded by adding 1 ml of stop solution (0.5 M

NaOH/50 M EDTA) and incubating at 658C for 10

min. After two labeled cDNAs were mixed, the

mixture was denatured 958C for 2 min and then

incubated in a water chamber at 458C for 20 min.

The cDNA mixture was then placed on an Agilent

Rat Whole Genome 44K chip (G4131A, Agilent

Technologies, Inc., CA, USA) and covered by a

hybridization chamber. The slides were hybridized

for 12 h at 628C in a hybridization oven. The

342 S.-H. Oh et al.

Act

a O

tola

ryng

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

gary

on

03/1

4/13

For

pers

onal

use

onl

y.

hybridized slides were washed in 2�SSC, 0.1%

SDS for 5 min, 1�SSC for 10 min, and then 0.2�SSC for 5 min at room temperature. The slides were

centrifuged at 3000 rpm for 20 s to dry.

Microarray data analysis

Hybridized slides were scanned with the GenePix†

4000B scanner (Molecular Devices, Co., CA, USA)

and the scanned images were analyzed with the

software program GenePix Pro 5.1 (Molecular

Devices), GeneSpring 7.2 (Sillicongenetics, CA,

USA), and R package. To allow the algorithm to

eliminate all bad spots, points with no data were

eliminated by visual inspection from the initial

images. To determine the background signal inten-

sity, the negative control was spotted on each slide.

To filter out the unreliable data, spots with signal-

to-noise ratio below zero were not included in the

data. Data were normalized by global, lowness, and

scaled normalization for data reliability.

Gene expression of group 0 was used as the

control. The signal intensity of each of groups 1�6

was compared with that of group 0 and the ratio of

signal intensity was calculated. Thereafter, the ratio

of signal intensity for each gene was compared

between the bilateral cochlear ablation groups and

the sham-operated age-matched control groups at 2,

4, and 12 weeks after surgery. Genes of interest were

sorted by a difference of twofold change of signal

intensity in the cochlear ablation group compared to

the sham-operated age-matched control group at

each point in time. To understand the functions of

differentially expressed genes, we used the gene

ontology classification of genes, which assigns differ-

entially expressed genes to groups based on mole-

cular function and biological processes.

Real-time polymerase chain reaction (PCR)

The accuracy of microarray analysis in this study was

confirmed by real-time PCR analysis.

Templates for real-time PCR were oligo(dT)-

primed first-strand cDNA prepared from total

RNA isolated from auditory cortex tissue. Real-

time PCR was monitored by quantification of

SYBR Green I fluorescence using the ABI Prism†

7900 Sequence Detection System (Applied Biosys-

tems, CA, USA). Primers were designed using

Primer Express† (version 2.0, Applied Biosystems)

to have annealing temperatures of 59�608C and to

generate amplicons of 50�150 base pairs in length.

The specificity of all amplicons was further assessed

by using the dissociation curve protocol on the ABI

Prism† 7000 Sequence Detection System Software

(version 1.0, Applied Biosystems) and by agarose gel

electrophoresis of a reaction sample taken for each

primer pair. Relative quantification of transcript

expression was performed using the comparative

cycle threshold (CT) method. Briefly, real-time

PCR assays were carried out using parallel amplifi-

cation of the housekeeping gene glyceraldehyde-3-

dehydrogenase (GADPH) as a control and standard

curves were constructed to monitor the efficacy of

each assay. Measurements of the CT values were

obtained in the exponential phase of the PCR

reaction using the ABI Prism† 7000 Sequence

Detection System Software (version 1.0, Applied

Biosystems). The CT of a target gene was then

calibrated against that of GADPH amplified in

parallel from the same sample (CTtarget�CT

GAPDH�DCT). Relative amounts of the target gene in the

ablated group compared to the sham-operated age-

matched control group were then normalized

(CTdeaf�CT

normal�DDCT). The fold change of gene

expression was calculated using the formula (fold

change�2�DDCT).

The genes and primers used for real-time PCR

were as follows: egr (early growth response)-1 , c-fos

(FBJ murine osteosarcoma viral oncogene), arc

(activity-regulated cytoskeletal associated protein),

syt (synaptotagmin)12 , nt5 (5-nucleotidase), slc2a1

(solute carrier family 2 member1, GLUT1), slc2a3

(solute carrier family 2 member 3, GLUT3), slc2a4

(solute carrier family 2 member 4, GLUT4), syn-

gap1 (synaptic Ras GTPase activating protein 1)

(Table I).

Results

Identification of differentially expressed genes following

cochlear ablation

From the 41 372 probe sets used on the Agilent Rat

Whole Genome 44K chip, we found over 219 probe

sets that showed differential expression by more than

twofold at 2, 4, or 12 weeks after the bilateral

cochlear ablation. Of these, 32 genes showed in-

creased expression and 35 genes had decreased

expression at 2 weeks after the ablation (Table II).

By 4 weeks following ablation, 37 genes had

increased expression and 15 genes had decreased

expression (Table III). By 12 weeks following abla-

tion, 12 genes showed increased expression and 88

genes showed decreased expression (Table IV).

The expression pattern of the genes of interest was

verified by real-time PCR and was compared with

the results of the microarray experiments (Table V).

Immediate early genes

Characteristic gene expression patterns were ob-

served in many immediate early genes (IEGs).

Plasticity in auditory cortex after bilateral cochlear ablation 343

Act

a O

tola

ryng

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

gary

on

03/1

4/13

For

pers

onal

use

onl

y.

Egr1 and c-fos expression was further validated

by real-time PCR. The expression of Egr1 was

0.30-fold decreased at 2 weeks, 2.54-fold increased

at 4 weeks, and 0.67-fold decreased at 12 weeks

(Figure 1A, B). The expression of c-fos was 0.19-fold

decreased at 2 weeks, 3.71-fold increased at 4 weeks,

and 0.60-fold decreased at 12 weeks (Figure 1C, D).

Other IEGs showed a similar expression pattern

(Table V).

Neural plasticity- and synaptogenesis-related genes

Microarray analysis identified many neural plasti-

city- and synaptogenesis-related genes. Of these

genes, four were further assessed with real-time

PCR. The expression of Arc was 0.16-fold decreased

at 2 weeks, 4.47-fold increased at 4 weeks, and 0.72-

fold decreased at 12 weeks. The expression of Nt5

was 0.69-fold decreased at 2 weeks, and 1.21-fold

and 1.15-fold increased at 4 weeks and 12 weeks,

Table I. Sequences of primers of genes selected for real-time PCR.

Gene name Forward primer sequence Reverse primer sequence Size (bp)

Egr1 TTA TCC CAG CCA AAC TAC CC ACA AGG CCA CTG ACT AGG CT 126

c-Fos TAG CAA CAT GGA GCT GAA GG AAG GAA CCA GAC AGG TCC AC 117

Arc CCA TCA CAG AAC ACC TTT GG CGG AAG TAG AGG TTC CTT CG 172

Syt12 GGA CTT GGC TCC CTA TGG TA GAA ATC CTG ACC GAA GGT GT 94

Nt5 CAG CCA TCA AAG CAG ACA TT TGG GTG TCT GAG GTT GTT GT 176

Slc2a1 CTC CAT GCT GAT GAT GAA CC GGC CAC AGT ACA CTC CAA TG 122

Slc2a3 TGT CCT CCA GTT GTC TCA GC ATA GAT GGG CTC CTG GAC AC 97

Slc2a4 AGA GAG CGT CCA CTG TCC TT ACA GCA TTG ATG CCT GAG AG 113

Syngap1 TTC TGA AGG CCA GTT CCT CT GTG AGT GCC ATC CTG TCA TC 96

Table II. Genes with greater than twofold changes in expression at 2 weeks after cochlear ablation.

Gene title UniGene ID Deaf/normal

Increased genes

Ribosomal protein S5 Rn.2100 3.93

Phospholamban Rn.9740 2.97

Kynurenine aminotransferase III Rn.28263 2.81

Vitamin D receptor Rn.10911 2.63

Microtubule-associated protein 1 A Rn.11402 2.62

Karyopherin alpha 1 (importin alpha 5) Rn.6272 2.56

Nuclear receptor subfamily 5, group A, member 2 Rn.42941 2.42

Calcium channel, voltage-dependent, L type, alpha 1C subunit Rn.9827 2.37

2�4-Dienoyl-coenzyme A reductase 2, peroxisomal Rn.144598 2.31

Calcium-regulated heat stable protein 1 Rn.14584 2.29

Gap junction membrane channel protein alpha 4 Rn.23406 2.28

Regulator of G-protein signaling 1 Rn.14787 2.27

Dynein, axonemal, heavy polypeptide 1 Rn.92411 2.18

Iron-responsive element binding protein 2 Rn.10132 2.09

Insulin induced gene 2 Rn.16736 2.03

Decreased genes

Early growth response 2 Rn.89235 0.15

Activity-regulated cytoskeletal-associated protein Rn.10086 0.29



FBJ murine osteosarcoma viral oncogene homolog Rn.103750 0.39

Prostaglandin-endoperoxide synthase 2 Rn.44369 0.39

Angiotensin II receptor, type 2 Rn.138103 0.39



TGFB inducible early growth response Rn.2398 0.45

S100 protein, beta polypeptide Rn.8937 0.46

Synaptotagmin-like 4 (granuphilin-a) Rn.107417 0.46

Dyskeratosis congenita 1, dyskerin Rn.4223 0.48

Tyrosinase Rn.38928 0.48

Mucosal vascular addressin cell adhesion molecule 1 Rn.87175 0.49

344 S.-H. Oh et al.

Act

a O

tola

ryng

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

gary

on

03/1

4/13

For

pers

onal

use

onl

y.

respectively. Microarray analysis of brain-derived

neurotrophic factor (Bdnf) showed 0.54-fold de-

crease of expression at 2 weeks, 1.48-fold increase

at 4 weeks, and 0.56-fold decreased at 12 weeks.

The real-time PCR results for Syt12 and Syngap1

showed little change at each of the three points in

time (Table V).

Glucose metabolism-related genes

Real-time PCR results for solute carrier family 2

member1 (Slc2a1 , GLUT1) showed 0.65-fold

decrease in expression at 2 weeks, 1.21-fold increase

at 4 weeks, and 0.95-fold decrease at 12 weeks

(Figure 2A, B). Solute carrier family 2 member 4

(Slc2a4 , GLUT4) expression decreased 0.53-fold at

2 weeks and normalized at 4 weeks and 12 weeks

(Figure 2C, D). However, solute carrier family 2

member 3 (Slc2a3 , GLUT3) showed no change of

expression after bilateral cochlear ablation (Figure

2E, F) (Table V).

Neurotransmission-related genes

Microarray analysis identified that neurotransmis-

sion-related genes showed decreased expression at

12 weeks (Table V). The changes in expression of

these genes at 2 and 4 weeks were insignificant.

These are gamma aminobutyric acid A receptor

alpha 5 (Gabra5), nicotinic cholinergic receptor

beta 3 (Chrnb3), solute carrier family 1 member 2

(Slc1a2), nicotinic cholinergic receptor epsilon

(Chrne), and solute carrier family 6 member 9

(Slc6a9) (Table V).

Discussion

This is the first study to examine both normal

developmental and sensory-driven cortical changes

in gene expression of the auditory cortex using

microarray technology.

In the rat, thalamocortical axons arrive at their

target cortical layer four 3 days after birth and areas

of the cortex can be detected [9]. However, the

functional maturation of the auditory cortex occurs

Table III. Genes with greater than twofold change in expression at 4 weeks after cochlear ablation.


Increased genes

Wiskott-Aldrich syndrome protein Rn.48367 6.92

Transthyretin Rn.1404 5.74

Purinergic receptor P2X, ligand-gated ion channel, 1 Rn.91176 3.85


Interleukin 1 beta Rn.9869 3.56

Phosphatidylinositol 3 kinase, regulatory subunit, polypeptide 3 Rn.44448 2.65

Synaptogyrin 1 Rn.88235 2.54

Sodium channel, voltage-gated, type 1, alpha polypeptide Rn.32079 2.38

Lactate dehydrogenase A-like 6B Rn.52356 2.26

Synaptic Ras GTPase activating protein 1 homolog Rn.9908 2.22

Complement component 4 binding protein, alpha Rn.10408 2.22


Calcium-independent alpha-latrotoxin receptor homolog 3 Rn.17279 2.20


Solute carrier family 2 (facilitated glucose transporter), member 3 Rn.95055 2.17

Myogenic differentiation 1 Rn.9493 2.16

Activity-regulated cytoskeletal-associated protein Rn.10086 2.13

Protein phosphatase 1, regulatory (inhibitor) subunit 12A Rn.51134 2.05

Synaptic vesicle glycoprotein 2c Rn.24210 2.04

Decreased genes

Cyclin F Rn.15126 0.26

F-box and leucine-rich repeat protein 20 Rn.18281 0.27

Transmembrane 4 superfamily member 9 Rn.98240 0.41

Ceruloplasmin Rn.32777 0.45

Insulin receptor-related receptor Rn.44446 0.46

Myxovirus (influenza virus) resistance 2 Rn.10374 0.47

Bruton agammaglobulinemia tyrosine kinase Rn.26996 0.47

Growth hormone receptor Rn.2178 0.48

BH3 interacting (with BCL2 family) domain, apoptosis agonist Rn.89639 0.49

Sodium-dependent neutral amino acid transporter ASCT2 Rn.28965 0.50


Act

a O

tola

ryng

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

gary

on

03/1

4/13

For

pers

onal

use

onl

y.

during the second and third postnatal weeks [10].

Because the time of operation in this study was the

time when rats just begin to experience sound, the

animal model developed for this study is similar to

prelingual deafness in the human. As rats undergo

normal developmental processes during this period,

we designed our experiments to analyze the effect of

auditory deprivation and normal development as

well. Using this experimental design, we could

analyze the effect of bilateral cochlear ablation on

auditory cortex while excluding the effect of normal

development.

IEGs are activated by the diverse intracellular

messenger systems linking membrane events and the

nucleus. They are characterized by rapid activation

in response to external stimulation in the absence of

de novo protein synthesis [11].

Our data showed that many IEGs are significantly

affected by cochlear ablation (Table V).

c-fos represents the effects of exogenous factors on

gene transcription. In the auditory brainstem, the

expression of c-fos is induced by acoustic stimula-

tion, and is reduced by deprivation of acoustic

stimulation [12]. In the auditory cortex, frequency-

specific expression patterns of c-fos are induced by

band noise stimulation [13].

Egr1 codes for a transcription factor protein and

controls expression of other genes in the central

nervous system [14,15]. Egr1 plays an important

role in neural plasticity. Expression of Egr1 is

essential for the formation of the late phase of

long-term potentiation [16]. In the auditory brain-

stem, the expression of Egr1 is increased in a

stimulation-dependent manner [17].

In the visual cortex, increased expression of IEGs

is associated with onset of visual stimulation and

reaches peak values by postnatal third weeks [18]. In

sham-operated age-matched control groups, the

expression of the IEGs was increased at 2 weeks

after sham operation and gradually decreased after-

wards. This might be the result of the normal

development of auditory cortex. In the bilateral

cochlear ablation groups, peak expression of the

IEGs was at 4 weeks after ablation surgery and it is

postnatal 6th weeks. It is very interesting that IEGs

were increased in the auditory cortex with no

Table IV. Genes with greater than twofold change in expression at 12 weeks after cochlear ablation.


Increased genes

Procollagen, type I, alpha 2 Rn.107239 3.06

Tropomyosin 3, splice variant 1 Rn.37575 2.68

Bone morphogenic protein receptor, type II Rn.59276 2.31

Phosphoenolpyruvate carboxykinase 1 Rn.104376 2.27

Tumor necrosis factor receptor superfamily, member 12a Rn.105040 2.02

Endothelial cell-specific molecule 1 Rn.6376 2.02

Myotrophin Rn.3239 2.01

Decreased genes

Mitogen-activated protein kinase 9 Rn.9910 0.21

Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform Rn.44437 0.24

Interleukin 12 receptor, beta 2 Rn.118443 0.30


Gamma-aminobutyric acid A receptor, alpha 5 Rn.10368 0.34

Transglutaminase 2, C polypeptide Rn.10 0.35

Bone morphogenic protein receptor, type II (serine/threonine kinase) Rn.59276 0.35

Synaptonemal complex protein 3 Rn.34889 0.37

Phosphatidylinositol-4-phosphate 5-kinase, type II, beta Rn.30025 0.37

Cholinergic receptor, nicotinic, beta polypeptide 3 Rn.9694 0.38

Syntaxin 12 Rn.54570 0.40

Calcium/calmodulin-dependent protein kinase II alpha subunit Rn.107499 0.40

Mitogen-activated protein kinase 8 Rn.4090 0.41

Prostaglandin D2 synthase 2 Rn.10837 0.43


Na�-dependent glucose transporter 1 Rn.22741 0.43

Synaptic vesicle glycoprotein 2c Rn.24210 0.45

High mobility group nucleosomal binding domain 3 Rn.106652 0.45

Solute carrier family 18 (vesicular monoamine), member 2 Rn.9686 0.46

Solute carrier family 1 (glial high affinity glutamate transporter), member 2 Rn.10240 0.46

Myosin IXA Rn.88901 0.47

Solute carrier family 6 (neurotransmitter transporter, glycine), member 9 Rn.32110 0.48

Cholinergic receptor, nicotinic, epsilon polypeptide Rn.10301 0.48

346 S.-H. Oh et al.

Act

a O

tola

ryng

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

gary

on

03/1

4/13

For

pers

onal

use

onl

y.

Table V. Comparison of gene expression at each point in time.

Microarray Real-time PCR

Gene title UniGene ID 2 weeks 4 weeks 12 weeks 2 weeks 4 weeks 12 weeks

Immediate early genes (IEGs)

FBJ murine osteosarcoma viral oncogene Rn.103750 0.38 1.73 0.57 0.19 3.71 0.60

Early growth response 1 Rn.9096 0.33 1.75 0.51 0.30 2.54 0.67

Early growth response 2 Rn.89235 0.14 3.66 0.33



TGFB inducible early growth response Rn.2398 0.46 1.13 0.84

Neural plasticity- and synaptogenesis-related genes

Activity-regulated cytoskeletal associated protein Rn.10086 0.29 2.13 0.51 0.16 4.47 0.72

5-Nucleotidase Rn.40132 0.77 1.17 1.08 0.69 1.21 1.15

Synaptic Ras GTPase activating protein 1 Rn.9908 1.97 2.22 0.88 1.23 1.19 1.43

Synaptogyrin 1 Rn.88235 1.42 2.54 1.09

Synaptotagmin 12 Rn.10682 0.68 1.38 0.91 1.21 1.24 1.04

Brain-derived neurotropic factor Rn.11266 0.54 1.48 0.56

Glucose metabolism

Solute carrier family 2, member 1, (GLUT1) Rn.3205 0.83 1.03 0.89 0.65 1.21 0.95



Neurotransmission-related genes

GABA receptor alpha 5 Rn.10368 1.56 1.16 0.34

Nicotinic cholinergic receptor beta 3 Rn.9694 1.36 1.17 0.38

Solute carrier family 1, member 2 Rn.10240 1.15 1.47 0.46

Nicotinic cholinergic receptor epsilon Rn.10301 1.11 0.88 0.48

Solute carrier family 6, member 9 Rn.32110 1.16 1.08 0.48

Figure 1. The gene expression of the immediate early genes. (A, B) The expression pattern of Egr1 was similar in microarray (A) and real-

time PCR (B). It was decreased at 2 weeks, increased at 4 weeks, and decreased at 12 weeks. (C, D) The expression pattern of c-fos . (A)

Egr1 microarray. (B) Egr1 real-time PCR. (C) cfos microarray. (D) cfos real-time PCR.


Act

a O

tola

ryng

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

gary

on

03/1

4/13

For

pers

onal

use

onl

y.

auditory input. We think that this is the result of the

increased non-auditory input to auditory cortex by

the visual-auditory cross-modal plasticity.

During the critical period, the expression of IEGs

in the sensory cortex is sensitive to sensory deaf-

ferentation. Lachance et al. showed that IEGs are

particularly sensitive to monocular enucleation dur-

ing the critical period but are not significantly

modulated in adult rats in visual cortex [19].

However, because there is controversy about the

cross-modal reorganization of primary auditory cor-

tex after deafening, we need more direct evidence

of cross-modal plasticity and its relationship to

up-regulation of IEGs in future studies [2,20].

After postnatal auditory stimulation, the expres-

sion of Arc is increased in the brain cortex of the

chicken, which is analogous to the mammalian

auditory cortex [21]. Because Arc is usually induced

in an activity-dependent manner, we think that the

auditory cortex of the bilateral cochlear ablation

group at 4 weeks was activated by non-auditory

stimulation.

The expression pattern of the Bdnf was similar to

that of the IEGs. Bdnf is an important neuronal

survival factor in the central nervous system [22].

Pathways that prevent cell death can be triggered by

neuronal activity to initiate adaptations that increase

synaptic strength and promote neural plasticity [23].

In addition to a role in neuronal survival, Bdnf also

modulates synaptic activity [24].

Region-specific increased neuronal activity has

been shown to be associated with parallel increases

Figure 2. The expression pattern of glucose metabolism-related genes. (A, B) The expression pattern of slc2a1 . (C, D) The expression

pattern of slc2a4 . (E, F) The expression pattern of slc2a3 . (A) glut1 microarray. (B) glut1 real-time PCR. (C) glut4 microarray. (D) glut4

real-time PCR. (E) glut3 microarray. (F) glut3 real-time PCR.

348 S.-H. Oh et al.

Act

a O

tola

ryng

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

gary

on

03/1

4/13

For

pers

onal

use

onl

y.

in brain glucose uptake [25]. Increase of glucose

metabolism is associated with learning and memory

processing in the hippocampus and cortical brain

regions [26].

In the previous study, we reported that the

decreased glucose metabolism of auditory cortex

becomes prominent after a critical period after

bilateral cochlear ablation of the rat and decreases

as the duration of deafness increases [4]. However,

in this study, the changes in expression of the glucose

metabolism-related genes were not significant.

In this study, some neurotransmission-related

genes showed significantly decreased expression in

bilateral cochlear ablation group at 12 weeks. Many

neurotransmission-related genes showed significant

changes of expression in the inferior colliculus of

adult rat after bilateral cochlear ablation. Those

changes began early after the bilateral cochlear

ablation [27]. However, in this study, decreased

expression of the neurotransmission-related genes

occurred only at 12 weeks after bilateral cochlear

ablation. We think that during the critical period, the

IEGs and neural plasticity-related genes may play a

more important role than neurotransmission-related

genes.

Conclusion

In this study, we found that many genes play an

important role in early changes of the auditory

cortex after bilateral cochlear ablation during the

critical period. Our results provide insights into the

global changes in the gene expression of the auditory

cortex focusing on two primary conditions, such as

cortical development and disruption of sensory

input.

The peak expression of the IEGs in the sham-

operated age-matched control group was at 2 weeks

and the peak expression of IEGs was at 4 weeks in

the bilateral cochlear ablation groups. This time

delay seems to be related to the slow development of

the auditory cortex. However, this is the result in the

absence of auditory input and we think that this is

the result of the cross-modal plasticity after bilateral

cochlear ablation. This result implies that the devel-

opment of the auditory cortex can be modulated by

the peripheral activity.

We need more information about the age depen-

dency of this plastic change, auditory cortex speci-

ficity of the genes which showed change of

expression in this study, and visual-auditory cross-

modal plasticity after bilateral cochlear ablation.

Further studies are needed on these issues and

they will elucidate the roles of the molecules isolated

in this study in the context of cortical development

and plasticity.

References

[1] Syka J. Plastic changes in the central auditory system after

hearing loss, restoration of function, and during learning.

Physiol Rev 2002;/82:/601�36.

[2] Bavelier D, Neville HJ. Cross-modal plasticity: where and

how? Nat Rev Neurosci 2002;/3:/443�52.

[3] Lee DS, Lee JS, Oh SH, Kim SK, Kim JW, Chung JK, et al.

Cross-modal plasticity and cochlear implants. Nature 2001;/

409:/149�50.

[4] Ahn SH, Oh SH, Lee JS, Jeong JM, Lim D, Lee DS, et al.

Changes of 2-deoxyglucose uptake in the rat auditory path-

way after bilateral ablation of the cochlea. Hear Res 2004;/

196:/33�8.

[5] King AJ. Auditory plasticity: vocal output shapes auditory

cortex. Curr Biol 2005;/15:/R503�5.

[6] King AJ, Moore DR. Plasticity of auditory maps in the brain.

Trends Neurosci 1991;/14:/31�7.

[7] Clerici WJ, Coleman JR. Resting and high-frequency evoked

2-deoxyglucose uptake in the rat inferior colliculus: devel-

opmental changes and effects of short-term conduction

blockade. Brain Res 1986;/392:/127�37.

[8] Paxinos G, Watson C. The rat brain in stereotaxic coordi-

nates. New York: Academic Press; 1986.

[9] Pallas SL. Intrinsic and extrinsic factors that shape neocor-

tical specification. Trends Neurosci 2001;/24:/417�23.

[10] Metherate R, Cruikshank SJ. Thalamocortical inputs trigger

a propagating envelope of gamma-band activity in auditory

cortex in vitro. Exp Brain Res 1999;/126:/160�74.

[11] Herdegen T, Leah JD. Inducible and constitutive transcrip-

tion factors in the mammalian nervous system: control of

gene expression by jun, fos and krox, and creb/atf proteins.

Brain Res Brain Res Rev 1998;/28:/370�490.

[12] Keilmann A, Herdegen T. The c-fos transcription factor in

the auditory pathway of the juvenile rat: effects of acoustic

deprivation and repetitive stimulation. Brain Res 1997;/753:/

291�8.

[13] Friauf E. Tonotopic order in the adult and developing

auditory system of the rat as shown by c-fos immunocyto-

chemistry. Eur J Neurosci 1992;/4:/798�812.

[14] Cao XM, Koski RA, Gashler A, McKiernan M, Morris CF,

Gaffney R, et al. Identification and characterization of the

egr-1 gene product, a DNA-binding zinc finger protein

induced by differentiation and growth signals. Mol Cell

Biol 1990;/10:/1931�9.

[15] Knapska E, Kaczmarek L. A gene for neuronal plasticity in

the mammalian brain: Zif268/egr-1/ngfi-a/krox-24/tis8/zenk?

Prog Neurobiol 2004;/74:/183�211.

[16] Wei F, Xu ZC, Qu Z, Milbrandt J, Zhuo M. Role of egr1 in

hippocampal synaptic enhancement induced by tetanic

stimulation and amputation. J Cell Biol 2000;/149:/1325�34.

[17] Illing RB. Activity-dependent plasticity in the adult auditory

brainstem. Audiol Neurootol 2001;/6:/319�45.

[18] Kaminska B, Mosieniak G, Gierdalski M, Kossut M,

Kaczmarck L. Elevated ap-1 transcription factor DNA

binding activity at the onset of functional plasticity during

development of rat sensory cortical areas. Brain Res Mol

Brain Res 1995;/33:/295�304.

[19] Lachance PE, Chaudhuri A. Microarray analysis of devel-

opmental plasticity in monkey primary visual cortex. J

Neurochem 2004;/88:/1455�69.

[20] Kral A, Schroder JH, Klinke R, Engel AK. Absence of cross-

modal reorganization in the primary auditory cortex of

congenitally deaf cats. Exp Brain Res 2003;/153:/605�13.

[21] Bock J, Thode C, Hannemann O, Braun K, Darlison MG.

Early socio-emotional experience induces expression of the

immediate-early gene arc/arg3.1 (activity-regulated cytoske-


Act

a O

tola

ryng

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

gary

on

03/1

4/13

For

pers

onal

use

onl

y.

leton-associated protein/activity-regulated gene) in learning-

relevant brain regions of the newborn chick. Neuroscience

2005;/133:/625�33.

[22] Jones KR, Farinas I, Backus C, Reichardt LF. Targeted

disruption of the bdnf gene perturbs brain and sensory

neuron development but not motor neuron development.

Cell 1994;/76:/989�99.

[23] Girault JA, Costa A, Derkinderen P, Studler JM, Toutant M.

Fak and pyk2/cakbeta in the nervous system: a link between

neuronal activity, plasticity and survival? Trends Neurosci

1999;/22:/257�63.

[24] Poo MM. Neurotrophins as synaptic modulators. Nat Rev

Neurosci 2001;/2:/24�32.

[25] Barrett D, Shumake J, Jones D, Gonzalez-Lima F. Metabolic

mapping of mouse brain activity after extinction of a

conditioned emotional response. J Neurosci 2003;/23:/

5740�9.

[26] Friedman HR, Goldman-Rakic PS. Coactivation of pre-

frontal cortex and inferior parietal cortex in working memory

tasks revealed by 2dg functional mapping in the rhesus

monkey. J Neurosci 1994;/14:/2775�88.

[27] Holt AG, Asako M, Lomax CA, MacDonald JW, Tong L,

Lomax MI, et al. Deafness-related plasticity in the inferior

colliculus: gene expression profiling following removal of

peripheral activity. J Neurochem 2005;/93:/1069�86.

350 S.-H. Oh et al.

Act

a O

tola

ryng

ol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

gary

on

03/1

4/13

For

pers

onal

use

onl

y.

Documents

Gene expression and plasticity in the rat auditory cortex after bilateral cochlear ablation