Laterality, intensity, and frequency of electrical intracochlear stimulation are differentially...

Laterality, intensity, and frequency of electrical intracochlearstimulation are differentially mapped into specific patterns of geneexpression in the rat auditory brainstem

TILL JAKOB & ROBERT-BENJAMIN ILLING

Neurobiological Research Laboratory, Department of Otorhinolaryngology, University of Freiburg, Freiburg, Germany

AbstractSpecific patterns of sensory activity may invoke neuroplastic remodelling in the young and adult mammalian brain. Amongthe indicators for the initiation of neuronal remodelling is the expression of immediate early genes (IEGs). The IEG c-Fosencodes a monomer of the transcription factor dimer AP-1 that triggers cascades of activity-dependent protein synthesis. Inorder to investigate activity-dependent neuroplasticity in the central auditory system, we applied unilateral electricalintracochlear stimulation (EIS), adjusted to evoke amplitudes of the auditory brainstem response corresponding to about40 dB acoustic stimulation in adult urethane-anaesthetized rats for 2 h. The intracochlear electrode was baso-dorsallylocated and the stimulation was specified with respect to intensity (100 or 1500 impulses per min) and frequency (1.6, 50,100 or 400 Hz). Following EIS, we detected and counted neurons in cochlear nucleus, superior olive, inferior colliculus, andmedial geniculate body that initiate stimulation-dependent expression of the protein c-Fos. Unique patterns of geneexpression emerged across the major auditory brainstem regions with respect to laterality, intensity, and frequency of EIS.These data suggest a potential of neuroplastic construction to be exploited by running a cochlear implant in humans oneway rather than another.

Key words: c-Fos, cochlear implant, immediate early genes, plasticity

Introduction

Sensory stimulation of the ears triggers a cascade

of events affecting several major and minor brain-

stem nuclei that are interconnected by a complex

network of ascending, descending, and commissural

pathways. This network is designed to serve auditory

processing with respect to stimulus intensity, spectral

composition, and binaural balance. It is a funda-

mental result obtained by neuroscience research

over the past decades that processing and integration

of sensory stimuli by neuronal networks takes

place not only at the electrophysiological level of

fast and transient currents, but also on slower and,

under specific circumstances, lasting molecular and

morphological changes in specific populations

of neurons affecting their response properties to

subsequent input.

Such molecular changes consist in the modifica-

tion of enzymes, in the translocation of structural

elements, or in the activation of genes to supply the

affected neuron with functions it did not exert in the

time before the change took place. Stimulation-

dependent gene activation entails, in most cases

studied, the expression of immediate early gene

(IEG) products, many of which are known to act

as transcription factors. The emergence of the

IEG transcription factors (c-Fos (1) and Egr-1 (2)

(Zif-268 (3); NGFI-A (4); Krox 24(5)) is indicative

of synaptic plasticity in the adult mammalian brain

(6,7).

In the auditory brainstem, c-Fos expression is

massively increased in many types of neurons

following stimulation with specific sounds (8�10)

or after electrical stimulation of cochlea or eighth

cranial nerve (11�14). It is a monomer of the

activator protein 1 dimer (AP-1; (15,16)). Among

many potential consequences, emergence of AP-1

should contribute to transcription and translation of

the protein GAP-43 (17) and to the activation of

matrix metalloproteinases (18) via the AP-1 re-

sponse element in the promoter region of their

genes. Both molecules are involved in axonal growth

and synaptic plasticity (19�21).

The protein c-Fos, together with other IEGs

coding for transcription factors, is part of signalling

cascades crucial for the induction and/or the

Correspondence: R.-B. Illing, Neurobiological Research Laboratory, Universitats-HNO-Klinik, Killianstr. 5, D-79106 Freiburg, Germany. Tel: �49 761 270

4273. Fax: �49 761 270 4075. E-mail: [email protected]

Audiological Medicine. 2008; 6: 215�227

ISSN 1651-386X print/ISSN 1651-3835 online # 2008 Informa UK Ltd. (Informa Healthcare, Taylor & Francis As)

DOI: 10.1080/16513860701875521

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maintenance of long-term potentiation of synapses

effectively induced at stimulation frequencies around

100 Hz (22,23). In particular, up-regulation of

c-fos and egr-1 mRNA is related to stimulation-

dependent synaptic enhancement (6). Transcription

of c-fos mRNA requires a stronger stimulation to

be recruited in the processes of synaptic remodelling

(24). There is evidence that strong neuronal activity

triggers not only stronger responses, but also differ-

ent signalling cascades, than weak activity (25�28).

In contrast to what was originally thought, the rise

of c-Fos is not an indicator for electrophysiological

activity. The local and cellular mismatch between

stimulation dependence of c-Fos expression and

2-deoxyglucose uptake (29�32) indicates that these

markers monitor neuronal activity on different levels.

Instead of reflecting spiking intensity, the emergence

of c-Fos is suggestive of molecular, structural, and

eventually functional changes that are imposed onto

neurons by specific patterns of afferent activity

rather than by activity per se.

Based on these studies, we here attempted

to identify characteristic distributions of c-Fos

expression in the auditory brainstem as a function

of stimulus laterality, stimulus intensity, and stimu-

lus frequency. We chose to employ EIS rather than

acoustic stimulation to take advantage of a full

control of the spectral and temporal pattern of

electrophysiological activity in the system, an aspect

of central importance in auditory processing that

we seek to exploit in the future, ultimately to help

optimize the use of cochlear implants in human

patients.

Materials and methods

Animals

This study is based on the brains from 23 adult

Wistar rats aged 7 to 12 weeks (Table I). Care and

use of the animals as reported here was approved

by the appropriate agency. The rats were anaesthe-

tized with an intraperitoneally injected mixture

of ketamine (50 mg/kg, Ketanest, Parke-Davis,

Ann Arbor, MI) and xylazine (5 mg/kg, Rompun,

Bayer-Leverkusen, Germany) for ear bone removal.

Table I.

Raw number of sections and cellular nuclei (ipsilateral, contralateral) analysed for:

Exp. no.

Stim.

frequency AVCN DCN LSO CIC MGB

M1070 1Hz 7 (7, 12) 3 (177, 24) 4 (89, 117) 4 (326, 341) 2 (28, 42)

M1082 1Hz 11 (11, 15) 5 (429, 11) 3 (106, 118) 4 (1265, 672) 3 (34, 44)

M1086 1Hz 20 (9, 10) 6 (227, 14) 5 (187, 223) 4 (452, 259) 1 (3, 4)

M1106 1Hz 10 (2, 12) 4 (65, 26) 5 (129, 181) 4 (287, 550) 5 (38, 28)

M1114 1,6Hz 13 (3, 16) 5 (430, 129) 5 (70, 117) 4 (168, 225) 3 (22, 30)

M1131 1,6Hz 12 (4, 11) 7 (356, 35) 3 (91, 96) 4 (791, 819) 3 (78, 102)

M820 50Hz 20 (888, 36) 6 (411, 222) 5 (152, 211) 4 (1639, 2004) 3 (284, 245)

M986 50Hz 14 (57, 103) 3 (168, 87) 3 (169, 110) 4 (2183, 2529) �M1065 50Hz 11 (23, 18) 5 (340, 101) 5 (209, 144) 4 (2325, 1746) 3 (116, 198)

M1104 50Hz 18 (1148, 16) 6 (254, 134) 5 (128, 129) 4 (670, 1744) 3 (21, 24)

M1113 100Hz 15 (7, 26) 7 (975, 200) 6 (142, 147) 4 (891, 887) 5 (28, 30)

M1124 100Hz 11 (17, 27) 5 (228, 83) 3 (79, 97) 4 (250, 201) 3 (61, 81)

M1127 100Hz 9 (8, 3) 5 (158, 34) 2 (41, 92) 4 (795, 651) 5 (31, 28)

M1128 100Hz 18 (8, 39) 3 (380, 112) 4 (92, 78) 4 (494, 763) 3 (13, 13)

X14 100Hz 7 (29, 21) 4 (214, 92) 2 (146, 145) 4 (627, 607) 2 (22, 73)

M1056 400Hz 10 (20, 33) 3 (160, 63) 4 (142, 164) 4 (919, 825) 3 (105, 173)

M1061 400Hz 13 (44, 26) 4 (131, 46) 3 (121, 125) 4 (1771, 1645) 3 (194, 265)

M1062 400Hz 15 (308, 32) 4 (71, 24) 4 (154, 129) 4 (1322, 1243) 3 (176, 174)

M1091 400Hz 9 (10, 69) 5 (382, 77) 3 (169, 148) 4 (803, 883) 3 (19, 12)

M1085 Control 19 (1, 1) 8 (0, 6) 4 (0, 2) 4 (664, 882) �M1097 Control 10 (0, 1) 5 (0, 1) 4 (6, 3) 4 (1311, 888) 3 (15, 27)

M1098 Control 8 (0, 1) 4 (0, 1) 2 (0, 0) 4 (378, 365) 3 (10, 14)

M1090 acoustic

stim.

� � � � �

Experimental animals, type of stimulation, and number of quantitatively evaluated specimens.

216 T. Jakob & R.-B. Illing

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For EIS, anaesthesia was achieved with urethane

(1.5 g/kg i.p.; Fluka AG, Buchs, Switzerland).

Electrical intracochlear stimulation (EIS)

To reduce the level of hearing-dependent spiking

activity in the auditory system, the tympanic mem-

brane was disrupted and the malleus was removed

bilaterally three days before EIS. Judging from the

auditory brainstem response (ABR), loss of middle

ear transmission led to an immediate and sustained

rise of hearing threshold by 50dB (13). The cochlea

was exposed using a retroauricular surgical ap-

proach. A hole was made into the bony wall of the

basal cochlea, the site of high-frequency transduc-

tion, to insert the electrode carrier (33). In this series

of experiments, the position of the stimulation

electrode was strictly dorsal to the modiolus. For

stimulation, we applied bipolar stimuli, using a

cochlear implant (Model CI24M) run by the Nu-

cleus Implant Communicator, both kindly provided

by Cochlear AG (Basel, Switzerland, and Hannover,

Germany).

The electrically evoked brainstem potentials

(EABR) were recorded at the onset of stimulation

by placing steel needle electrodes subcutaneously at

vertex and mastoids to corroborate the correct

placement of the stimulation electrodes and to

determine an appropriate current level for stimula-

tion. The EABR was visualized using an averager

(Multiliner E; Evolution 1.70c; Toennies, Wurzburg,

Germany), calculating mean amplitudes over 100

sweeps. We aimed to obtain amplitudes in the range

of 3 to 6 mV, corresponding to acoustically evoked

amplitudes of 30 to 50dB (Figure 1). In conjunction

with the baso-dorsal position of the stimulation

electrode, the moderate current level served to

induce a local, or tonotopic, rather than a total, or

generalized, intracochlear stimulation.

We applied EIS unilaterally (left side) in acute

experiments for 2 h (Table I). Biphasic pulses with

50-ms phase width each and 20-ms phase gap

were applied with frequencies of either 1.6 Hz,

50 Hz, 100 Hz, or 400 Hz. Four experiments with

1 Hz stimulation frequency were performed in the

early stages of our study. Based on observations of

the frequency-dependency of synaptic plasticity in

brain slice preparations we consider results from

1 Hz experiments as indistinguishable from those of

1.6 Hz experiments and pooled both for statistical

analysis. The duty cycle was set to arrive at either

100 (1.6 and 100 Hz) or 1500 (50 and 400 Hz)

biphasic pulses per min. The current level was varied

between 260 and 950 mA between experiments to

compensate for unavoidable variations in the dis-

tance of the electrodes to the modiolus. The EABR

was recorded again at the end of the session to verify

unchanged electrode position and sustained effective

stimulation of the central auditory system.

Immunohistochemistry

Preceding transcardial perfusion, rats were given a

lethal dose of pentabarbital (0.6 ml/kg of Narco-

dorm-n, Alvetra GmbH, Neumunster, Germany,

i.p.). The fixative was the same as we used in previous

studies (34). Brains were cryo-cut into frontal

sections of 30-mm thickness. Immunocytochemistry

was performed using antibodies raised in rabbit

against c-Fos (0.5 mg/ml, Santa Cruz Biotechnology,

Santa Cruz, CA, No. Sc-253, lot no. A0603). After

incubation for 48 h, matching biotinylated secon-

dary antibodies and the avidin-biotin-technique

(Vector Laboratories, Burlingame, USA) were em-

ployed to visualize the binding sites of the primary

Figure 1. Auditory brainstem response following acoustic (upper lane) or electrical intracochlear (EIS, lower two lanes) stimulation. Arrows

point to time of stimulus onset (click or current). The two lanes labelled ‘EIS’ show two extremes of potential differentiation under

supposedly identical stimulation conditions. Time scale: 1 ms per unit.

Activity-induced gene expression in auditory brainstem 217

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antibodies with diaminobenzidine (DAB, 0.05 %,

Sigma-Aldrich, Germany), H2O2 (0.0015%) and

ammonium nickel sulphate (0.3%) for 2 min under

visual control. Stained cellular nuclei in the parabra-

chial region served as positive controls (13).

Counting and statistics

For the quantitative evaluation of our results,

photographs were taken from auditory regions on

both sides of the brainstem with a�10 objective

and a digital camera (Axicam, Zeiss, Germany) at an

8-bit grey tone scale. Whenever necessary, a mosaic

of photographs was taken and assembled to cover the

whole cross-sectional profile of the respective region

of the brain section. Photographs were then im-

ported into an image analysis program (analySIS,

Soft Imaging Systems GmbH, Germany). Detection

threshold of grey tone values was set between 115

and 125 to compensate for minor variations in

staining intensity of sections from various animals.

Following the definition of the region of interest

excluding large blood vessels, detection of stained

nuclei (Figure 2A, inset) was performed under

the optimized settings for aspect ratio (1�4), mean

diameter (2�15), elongation (1�5), area (2�90), grey

value minimum (140), and roundness (0.1�1).

Statistical analysis was performed with Prism

(GraphPad Software, Inc.). The number of c-Fos

positive nuclei was determined for each brainstem

region and each setting of stimulation parameters

across animals. These numbers were given as counts

per square millimetre. Mean and standard error of

mean were determined and used for statistical

testing. Significant differences of counts were sought

by applying the two-sided unpaired Students t-test,

with significance level set to pB0.05. Stereological

corrections for counting particles in the sectioned

material were not made as our conclusions are based

on numerical relationships rather than absolute

densities.

Results

Controls

As a consequence of the binding of antibodies

directed against c-Fos, nuclei turn black in perox-

idase-DAB-nickel-based immunocytochemistry in

cells that contain this protein (Figure 2A, upper

panel of inset). In normal hearing but not specifically

stimulated rats, c-Fos expression occurs in cells of all

auditory brainstem regions at a low level. While

some neuronal nuclei were moderately rich in c-Fos,

very few of them showed intense black staining. As a

consequence of a bilateral perforation of the tympa-

nic membrane and removal of the malleus, c-Fos

expression fell within two days below detection level

in ventral cochlear nucleus (VCN), lateral superior

olive (LSO), and medial geniculate body (MGB),

whereas some intensely c-Fos positive nuclei per-

sisted in dorsal cochlear nucleus (DCN) and central

nucleus of the inferior colliculus (CIC). Control

animals with unilateral or bilateral middle ear bone

dislocation did not show an increase of the number

of c-Fos positive cells anywhere in the auditory

brainstem. Even after two weeks of sensory depriva-

tion by bilateral middle ear bone dislocation, c-Fos

staining in the auditory brainstem remained de-

pressed. Animals with this kind of hearing loss

inflicted three days before analysis served as controls

in our study, against which a stimulation-dependent

rise of c-Fos positive nuclei after middle ear bone

removal could be readily detected.

VCN

We observed cases with a large number of stained

nuclei on the side of stimulation (Figure 2A) and

very few such nuclei contralaterally (Figure 2B).

However, substantial variations in c-Fos expression

even under supposedly identical set-up and stimula-

tion parameters occurred in VCN. The form of the

EABR appeared to have some predictive value for

this difference. Whenever we recorded an EABR

with three or more clearly distinguishable troughs

(Figure 1, lower lane), a strong c-Fos expression

occurred in the ipsilateral VCN. With fewer troughs,

c-Fos expression was sparser ipsilaterally, even to

the extent of being statistically indistinguishable

from the unstimulated side. A dependence of

EABR morphology on preoperative cochlear func-

tioning or malfunctioning was not noticed. Remark-

ably, variations in the number of stained nuclei in

VCN appeared to have few consequences for

amount and pattern of c-Fos expression beyond

VCN (see below). In cases with notable c-Fos

expression, stained nuclei were found dorsally in

VCN at a location corresponding tonotopically to

the intracochlear position of the stimulation elec-

trode (Figure 2A). The cross-sectional area of c-Fos

positive nuclei in VCN was on average 10.54 mm2.

Any kind of stimulation resulted in a significant

increase of c-Fos positive nuclei in the ipsilateral

VCN compared to the control, except at 400 Hz

(1.6 Hz: i: p�0.0118*; 50 Hz: i: p�0.0451*; 100

Hz: i: p�0.0474*; 400 Hz: i: p�0.1039; cp. Figure

2). Variations in stimulation frequency failed to result

in significant differences of c-Fos positive nuclei.

When the same number of impulses per minute

were given at 1.6 and 100 Hz (1.6 vs. 100 Hz: i:

p�0.1699; c: p�0.1179; Figure 2E), or at 50 and

400 Hz (50 vs. 400 Hz: i: p�0.2176; c: p�0.6918;


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Figure 2F), differences in c-Fos expression remained

insignificant. However, the number of impulses per

minute did make a difference, with more impulses

resulting in more stained nuclei (100 vs. 1500 stimuli

per min: i: p�0.0317; c: p�0.0043; Figure 2G) in

the VCN on both sides of the brainstem.

DCN

The average cross-sectional area of c-Fos positive

nuclei in DCN emerging after EIS was 11.35 mm2,

exceeding nuclear size in all areas investigated.

When compared to the control level, all stimulation

parameters led to a significant increase of c-Fos

expression in the DCN on both sides of the

brainstem (1.6 Hz: i: p�0.0006*; c: p�0.0378*;

50 Hz: i: pB0.0001* c: pB0.0001*; 100 Hz: i:

p�0.0032*; c: p�0.0011*; 400 Hz: i: p�0.0031*;

c: p�0.0002*). The increase was stronger on the

ipsilateral side than contralaterally in all cases

(Figure 3D), an asymmetry that was significant for

all but the highest frequency (1.6 Hz: p�0.0021*;

Figure 2. The pattern of c-Fos expression in VCN following unilateral EIS. (A) On the side of stimulation, c-Fos positive nuclei (black

dots) emerged in regions tonotopically corresponding to the intracochlear stimulation site. Scale bar for A to C: 200 mm. The inset shows

c-Fos positive profiles (upper part) and how they were identified by image analysis (lower part, scale bar 10 mm). (B) On the unstimulated

side, few c-Fos positive nuclei emerged. (C) In the deafened control, c-Fos positive profiles were absent. (D) Frequency dependence of

c-Fos expression. a: significant difference to stimulated animals at all frequencies, both ipsilateral and contralateral, except for 400 Hz

ipsilateral. b: significant difference between ipsilateral and contralateral side. (E) Comparison of the effect of stimulation with different

frequencies at equally low intensity (100 stimuli per min). (F) Comparison of the effect of stimulation with different frequencies at equally

high intensity (1500 stimuli per min). (G) Comparison of variable stimulation intensities independent of frequency. c: significant difference

to contralateral side at 100 and 1500 stimuli per min.


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50 Hz: p�0.0174*; 100 Hz: p�0.0415*; 400 Hz.

p�0.0616).

In the ipsilateral DCN, the number of c-Fos

positive nuclei rose with stimulation frequency

(Figure 3D) from 1.6 Hz (234.7 cells per mm2)

over 50 Hz (236.9) to 100 Hz (316.1), but fell off at

400 Hz (152.7). Contralaterally, there was a gradual

rise in number at 1.6 Hz (33.4) to 50 Hz (114.1),

but a subsequent decrease at 100 Hz (96.4) and

400 Hz (45.5).

When stimulating with 100 impulses per min,

100 Hz produced more positive nuclei in the

ipsilateral DCN than 1.6 Hz, but the difference

was not significant (p�0.4128). However, the

contralateral DCN showed a significant increase

of c-Fos expression with frequency (p�0.0343*;

Figure 3E). Stimulation with 1500 impulses per

min resulted in a frequency-dependent decrease of

c-Fos expression that was again insignificant ipsilat-

erally (p�0.1873) but significant contralaterally

(p�0.015*; Figure 3F).

Comparing animals stimulated with 100 impulses

per min with those stimulated with 1500 impulses

per min (Figure 3G), both showed indistinguishable

levels of c-Fos expression ipsilaterally (p�0.2231)

and contralaterally (p�0.4462), respectively. There

Figure 3. The pattern of c-Fos expression in DCN following unilateral EIS. (A) On the side of stimulation, c-Fos positive nuclei (black

dots) emerged throughout DCN. Scale bar for A to C: 200 mm. (B) On the unstimulated side, fewer cells expressed c-Fos, but most of these

corresponded tonotopically to the intracochlear stimulation site. (C) In the deafened control, c-Fos positive profiles were essentially absent.

(D) Frequency dependence of c-Fos expression. a: significant difference to all frequencies and both sides of the brainstem. b: significant

difference between ipsilateral and contralateral side. (E) Comparison of the effect of stimulation with different frequencies at equally low

intensity (100 stimuli per min). (F) Comparison of the effect of stimulation with different frequencies at equally high intensity (1500 stimuli

per min). (G) Comparison of variable stimulation intensities independent of frequency. c: significant difference to ipsilateral and

contralateral side at 100 and 1500 stimuli per min.


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was a correspondingly strong bias favouring c-Fos

expression in ipsilateral over contralateral DCN in

both groups (100 impulses: p�0.0004*; 1500 im-

pulses: p�0.005*).

The distribution of c-Fos positive nuclei was not

identical in the DCN ipsilateral and contralateral to

the side of stimulation. Ipsilaterally, c-Fos positive

nuclei were often distributed over the whole medio-

lateral extent of the DCN, mostly in the more

superficial layers. Some animals showed a more

restricted expression in ipsilateral DCN with more

cells stained mediodorsally than lateroventrally,

conforming to tonotopic representation of the in-

tracochlear stimulation side. By contrast, stained

nuclei on the contralateral side tended to be loca-

lized more dorsomedially and in the deeper layers of

DCN. These differences persisted across all stimula-

tion parameters applied.

LSO

Determining the average cross-sectional area of

c-Fos positive nuclei that emerged after EIS at

5.92 mm2 revealed that these were the smallest nuclei

across all regions analysed. Larger nuclei were seen

in periolivary regions (11.68 mm2 on average), but

these were not included in the quantitative evalua-

tion that follows.

Stimulation at all frequencies resulted in a highly

significant increase of c-Fos positive nuclei in

ipsilateral and contralateral LSO alike compared

to controls (1.6 Hz: i: pB0.0001*; c: pB0.0001*;

50 Hz: i: p�0.0009*; c: pB0.0001*; 100 Hz: i:

pB0.0001* c: pB0.0001*; 400 Hz: i: pB0.0001*;

c: pB0.0001*; Figure 4D). No significant differ-

ences were found between sides at any frequency

(1.6 Hz: p�0.0593; 50 Hz: p�0.7463; 100 Hz:

p�0.1000; 400 Hz: p�0.7236). Within the groups

run at either 100 or 1500 impulses per min,

no persistent differences were found (1.6 Hz vs.

100 Hz: i: p�0.9646; c: p�0.7162; 50 Hz vs.

400 Hz: i: p�0.9398; c: p�0.4454; Figure 4E, F).

However, there was a difference between groups

stimulated with 100 and 1500 impulses per min,

respectively, in that the stronger stimulation resulted

in significantly more nuclei on the stimulated side

(p�0.0025*), but no such difference was found

contralaterally (p�0.9928). One hundred impulses

per minute produced c-Fos expression in signifi-

cantly more nuclei contralaterally than ipsilaterally

(p�0.0082*; Figure 4G), a difference that did

not persist following 1500 impulses per min

(p�0.8679).

The distribution of c-Fos positive nuclei was

slightly different in ipsilateral and contralateral

LSO. Whereas most such nuclei were located

medially and fewer laterally on both sides of the

brainstem (conforming to the tonotopic representa-

tion of high frequencies), this polarization was

more obvious contralaterally than ipsilaterally.

CIC

For the quantitative evaluation only the central

inferior colliculus (CIC) was considered. Nuclei of

neurons in this region that turned c-Fos positive

upon EIS had an average cross-sectional area of

6.53 mm2, being rather small.

Compared to controls that already showed a fair

number of c-Fos positive nuclei, EIS did not

significantly increase their number except for ani-

mals stimulated at 50 Hz, where it was found to

rise bilaterally (1.6 Hz: i: p�0.4859; c: p�0.2542;

50 Hz: i: p�0.0314*; c: p�0.0043*; 100 Hz: i:

p�0.9375; c: p�0.9135; 400 Hz: i: p�0.1375; c:

p�0.2063; Figure 5D). No relationships appeared

to exist between high numbers of c-Fos positive cells

in VCN and high numbers of them in CIC. At no

frequency was there an ipsilateral-to-contralateral

difference (1.6 Hz: p�0.7534; 50 Hz: p�0.5970;

100 Hz: p�0.8465; 400 Hz: p�0.8434; Figure

5D). Again no differences were obtained between

groups with 100 impulses per min (i: p�0.5295; c:

p�0.2140; Figure 5E). Between the groups stimu-

lated with 1500 impulses per min, there was a

significant difference for the contralateral CIC,

with fewer stained cells at higher frequency (i:

p�0.2009; CICc: p�0.0460*; Figure 5F). Com-

paring the groups stimulated with 100 and 1500

impules per min, respectively, there was a bilateral

rise of c-Fos positive cells with stimulus intensity

(i: p�0.0056*; c: p�0.0020*; Figure 5G).

Bands of particularly dense c-Fos positive nuclei

running from dorsomedial to ventrolateral CIC

conformed tonotopically to the intracochlear posi-

tion of the stimulation electrode (cp. Illing et al.,

2002), but were more obvious in some animals than

in others.

MGB

The location of stimulation-dependent c-Fos expres-

sion within the MGB was always and exclusively

in its medial subdivision. The average size of

c-Fos positive nuclei in MGB was 7.54 mm2.

Following EIS, no significant rise in the number of

c-Fos expressing cells was seen against the control

except for the 400 Hz stimulation, resulting in a

bilateral increase (1.6 Hz, i: p�0.2795, c: p�0.209;

50 Hz, i: p�0.0962, c: p�0.0609; 100 Hz, i:

p�0.1662, c: p�0.1721; 400 Hz, i: p�0.0295*,

c: p�0.0296*; Figure 6D). Within each frequency,


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no ipsilateral-to-contralateral differences were seen

(1.6 Hz: p�0.5093; 50 Hz: p�0.9971; 100 Hz:

p�0.3568; 400 Hz: p�0.4637). Again no differ-

ences were observed comparing stimulations with

100 impulses per min (i: p�0.8096; c: p�0.5567)

or with 1500 impulses per min (i: p�0.6579; c:

p�0.8931; Figure 6E, F).

However, there was a clear effect of stimulus

intensity, raising the number of stained nuclei

on both sides with higher stimulus intensity (i:

p�0.0011*; c: p�0.0130*; Figure 6G). Against

controls, stimulation with 100 impulses per min had

no effect (i: p�0.1761; c: p�0.1632; Figure 6G),

but stimulation with 1500 impulses per min did

increase the number of positive nuclei on both sides

(i: p�0.0424*, c: p�0.0207*).

Discussion

Our set-up was designed to provide complete control

on as many stimulation parameters as possible.

By choosing EIS over acoustic stimulation, this

could be fully achieved with respect to the temporal

pattern of the stimulation, a major parameter of

Figure 4. The pattern of c-Fos expression in LSO following unilateral EIS. (A) On the side of stimulation, c-Fos positive nuclei (black dots)

emerged throughout LSO with a bias towards regions tonotopically corresponding to the intracochlear stimulation site. Scale bar for A to C:

100 mm. (B) On the unstimulated side, c-Fos positive nuclei emerged in comparable numbers. (C) In the deafened control, c-Fos positive

profiles were absent. (D) Frequency dependence of c-Fos expression. a: significant difference to all frequencies and both sides. (E)

Comparison of the effect of stimulation with different frequencies at equally low intensity (100 stimuli per min). (F) Comparison of the

effect of stimulation with different frequencies at equally high intensity (1500 stimuli per min). (G) Comparison of variable stimulation

intensities independent of frequency. c: significant difference to ipsilateral and contralateral side at 100 and 1500 stimuli per min. The

control level of c-Fos expression was significantly lower with respect to any stimulation.


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central auditory signal processing. By working on

anaesthetized animals, potential differences in arou-

sal states were largely levelled out. However, despite

reproducing electrode position with the greatest

possible precision, the EABR already indicated that

the effect of EIS varied considerably across animals.

A full control of stimulation patterns but limited

control in stimulation effectiveness reflects the

situation faced with human cochlear implant recei-

vers.

Unlike Saito et al. (35), we did not use the current

level of the stimulation as a variable. Instead, we

varied stimulation frequency and number of impulses

per time window. Performing statistical analysis on

the results obtained from three to six animals in each

group (Table I), we expected to invoke differences in

the pattern of stimulation-dependent gene expression

for laterality, frequency, and intensity of the stimula-

tion, but we were surprised to find a specific

fingerprint for each parameter across the major

auditory brainstem regions (Table II).

The choice of frequencies between 1 and 400 Hz

was motivated by experiments on stimulation-

dependent molecular and functional plasticity of

synapses in brain slice preparations, in which a

100 Hz burst of depolarizing pulses is considered a

Figure 5. The pattern of c-Fos expression in CIC following unilateral EIS. (A) On the side of stimulation, c-Fos positive nuclei (black dots)

were widely scattered. Scale bar for A to C: 200 mm. (B) On the unstimulated side, equal numbers of c-Fos positive nuclei were seen. (C)

Even in the deafened control, c-Fos positive profiles were numerous. (D) Frequency dependence of c-Fos expression. a: significant

difference to 50 Hz stimulation on both sides. (E) Comparison of the effect of stimulation with different frequencies at equally low intensity

(100 stimuli per min). (F) Comparison of the effect of stimulation with different frequencies at equally high intensity (1500 stimuli

per min). (G) Comparison of variable stimulation intensities independent of frequency. c: significant difference to contralateral staining

following 1500 stimuli per min.


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strong stimulation (23). Once these stimulation

frequencies prove effective in changing the molecular

profile of central auditory neurons, the frequency

range may be extended to cover and evaluate the

clinical settings of current cochlear implants in

humans.

VCN

The pattern of IEG expression in the neuronal

network of the VCN reflects the side on which

the stimulation occurred, with more affected cells

ipsilaterally. As this difference tended to exist but

failed to reach significance in the present study

(Figure 2F, G) due to a substantial scatter across

cases (Figure 2D), we base this claim on considera-

tion of previous experience that we and others

accumulated over the past years with a slightly

different set-up (13,35,36). It should be noted,

however, that Rouiller et al. (37) after acoustic

stimulation as well as Vischer et al. (11) and Zhang

et al. (38) after electrical stimulation also failed

to induce much c-Fos in VCN. We suggest that

an indicator for a substantial ipsilateral but not

Figure 6. The pattern of c-Fos expression in MGB following unilateral EIS. (A) Expression of c-Fos occurred in neuronal nuclei (black

dots) only in the medial part of the geniculate body. Scale bar for A to C: 200 mm. On the unstimulated side, equal numbers of c-Fos positive

cells were encountered (B). (C) In the deafened control, few c-Fos positive profiles were present. (D) Frequency dependence of c-Fos

expression. a: significant difference to effect of 400 Hz on both sides. (E) Comparison of the effect of stimulation with different frequencies

at equally low intensity (100 stimuli per min). (F) Comparison of the effect of stimulation with different frequencies at equally high intensity

(1500 stimuli per min). (G) Comparison of variable stimulation intensities independent of frequency. A stimulation-dependent effect on

c-Fos expression in MBG was only seen at high intensity stimulation. c: significant difference to effect of stimulation with 1500 stimuli per

min on both sides.


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contralateral induction of c-Fos expression in VCN

of adult rats is an intraoperative EABR that includes

at least three clearly identifiable troughs (Figure 1).

Apart from laterality, increasing intensity was

mapped onto the pattern of c-Fos expression in

VCN with an increasing number of c-Fos positive

neurons (Figure 2G). By contrast, a constant num-

ber of impulses presented at different frequencies

did not appear to matter for the network in VCN

with respect to the induction of c-Fos expression

(Figure 2E, F).

The fact that in some experiments very few

neurons in VCN but still numerous cells in other

auditory brainstem regions turned c-Fos positive

provides further evidence that c-Fos cannot be

mistaken as a marker for electrophysiological activ-

ity, as neurons in VCN must have been brought to

spiking in order to drive subordinated neuronal

populations and trigger stimulation-dependent mo-

lecular processes in them.

DCN

Expression of c-Fos has been induced in DCN with

acoustic (8,37) or electrical (11,39) stimulation. The

results obtained here largely conform to previous

findings. The tonotopic organization of DCN was

apparent following unilateral cochleotomy and sub-

sequent acoustic stimulation (40). Our results reflect

tonotopy in the contralateral but not in the ipsilateral

DCN, where c-Fos positive cells were often scattered

across the entire nucleus. An excitatory connection

between the DCN on both sides has been described

by several authors (Brown and Liu, 1995; Vischer

et al., 1994; Luo et al., 1999). The data presented

here point to an elaborated use of this connection

with respect to bilateral processing upon unilateral

stimulation.

Neurons of the DCN responded quite differently

to changing stimulation parameters with c-Fos

expression. There was a significant and non-linear

mapping of frequency on the side opposite to the

stimulation. Expression of c-Fos was significantly

stronger ipsilaterally than contralaterally at all but

the highest stimulation frequencies (Figure 3D).

This reflects a non-linear mapping of stimulation

frequency onto the DCN activity. When stimulating

at low intensity, there was an increase of c-Fos

expression in the contralateral DCN with frequency

(Figure 3E). Conversely, increased frequency re-

duced c-Fos expression in the contralateral DCN at

high stimulation intensity (Figure 3F). Averaging

these results cancels differences at varying intensity

(Figure 3G).

LSO

Earlier studies on the LSO have shown that binaural

acoustic stimulation causes no or little increase in

c-Fos expression (8,37,40). Following unilateral

cochleotomy, acoustic stimulation led to an increase

of c-Fos positive cells in the LSO ipsilateral to the

hearing ear (40). A rise of the number of c-Fos

positive cells was also seen on the side of a unilateral

electrical stimulation (38,39), with few effects on the

contralateral LSO.

In our set-up, c-Fos expression was induced in

neurons of the LSO on both sides of the brainstem.

This finding can be explained by projections to LSO

from both VCN. Whereas bushy cells of the ipsilat-

eral VCN project directly to LSO neurons (42�44),

neurons of the contralateral LSO are affected by

auditory stimulation through a relay in the medial

nucleus of the trapezoid body which affects LSO

neurons through glycinergic synapses (45�47)

As soon as stimulation was applied, c-Fos expres-

sion rose strongly over the control level in the LSO

bilaterally (Figure 4D). Changing stimulation fre-

quency at low (Figure 4E) or high (Figure 4F)

intensity did not make a difference to the number of

LSO neurons. The number of affected neurons in

the ipsilateral LSO caught up with the contralateral

level only when stimulation intensity increased

(Figure 4G).

CIC

The CIC harbours a considerable number of c-Fos

positive neurons without EIS stimulation and also

after middle ear bone dislocation (Figure 5C, D). A

unilateral EIS caused an increase of the number of

c-Fos positive neurons in the CIC on both sides

of the brainstem only at high stimulus intensity

(Figure 5G). High intensity stimulation appeared

to be more effective in driving CIC neurons to

express c-Fos (Figure 5G). Moreover, high intensity

Table II.

Laterality Intensity Frequency

VCN /�+i � 0

DCN �i 0 �LSO �c � 0

CIC 0 � �MGB 0 � 0

Regionally unique mapping of patterned sensory activity onto the

pattern of c-Fos expression in the auditory brainstem. Plus-signs

indicate change of expression under the specified parameter in the

respective region. All entries are based on the present study except

for the one marked by asterisk, which relies on previous work

(Saito et al., 1999; Illing et al., 2002). i: ipsilateral; c: contral-

ateral.


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(i.e. number of impulses per minute) was more

effective in driving gene expression at lower than at

high frequencies (Figure 5F).

With the stimulation parameters used here, few

c-Fos positive cells were encountered in the dorsal

inferior colliculus and almost none was seen in the

external inferior colliculus. Neither of these mid-

brain regions belongs to the lemniscal pathway,

and both of them appear to be involved, at least in

part, in non-auditory functions (48,49). In experi-

ments by other researchers using different set-ups

for unilateral electrical stimulation, plenty of c-Fos

positive cells emerged in dorsal and external inferior

colliculus, whereas few cells turned c-Fos positive in

CIC (11,39). The major difference from our experi-

mental set-up was that their stimulation affected the

entire cochlea or the entire cochlear nerve. Vischer

et al. (11) suspect that under generalized activation

distinctly tonotopic auditory brainstem regions such

as VCN, CIC, and ventral MGB failed to initiate a

stimulation-dependent gene expression.

MGB

Yet another pattern of stimulation-dependent gene

expression was encountered in the auditory thala-

mus (Table II). The MGB responded to EIS with

increased c-Fos expression only in its medial, non-

lemniscal subdivision (Figure 6A�C). As in CIC,

neurons of the medial MGB did not respond to

lower stimulation intensity with c-Fos expression as

the number of c-Fos positive nuclei did not differ at

100 impulses per min compared to the control.

There was no indication for a differential expression

pattern with changing frequency (Figure 6E, F).

However, there was a clear reflection of stimulus

intensity on both sides (Figure 6G).

Conclusions

Specific conditions of afferent activity prompted

specific populations of neurons in specific regions

of the auditory brainstem to prepare for a molecular,

structural, and functional remodelling within hours.

Laterality, frequency, and intensity of the stimulus

each affect different neurons in different regions.

Coding of laterality takes place below the midbrain,

but appears to be lost from the inferior colliculus

upwards. Each major brainstem region involved in

the analysis of auditory stimuli responded to the

stimulation parameters by a unique dynamic pattern

of c-Fos expression (Table II). Having chosen to

start our studies by reference to experiments on the

activity/molecular connection in synaptic plasticity,

we have made a first step. There is reason to consider

these data transferable to man. However, our study

is not comprehensive in the sense that it already

provides binding recommendations to CI program-

mers or users. Instead, it provides data showing for

the first time that the parameters of running a CI

bear a very real potential for neuroplastic remodel-

ling of the central auditory system.

Acknowledgements

We thank I. Hirschmuller-Ohmes, P. Pedersen, A.

Reisch, M. Fredrich, M. Meidinger, and H. Hildeb-

randt-Schoenfeld for valuable help, and R. Laszig

for continuous support. We gratefully received sup-

port from the Research Commission of Universi-

tatsklinikum Freiburg i. Br. and Cochlear GmbH,

Hannover.

References

1. Morgan JI, Curran T. Stimulus-transcription coupling in

neurons: role of cellular immediate-early genes. Trends

Neurosci. 1989;/12:/459�62.

2. Sukhatme VP, Cao XM, Chang LC, Tsai-Morris CH,

Stamenkovich D, Ferreira PC, et al. A zinc finger-encoding

gene coregulated with c-Fos during growth and differentia-

tion, and after cellular depolarization. Cell. 1988;/53:/37�43.

3. Christy BA, Lau LF, Nathans D. A gene activated in mouse

3T3 cells by serum growth factors encodes a protein with

‘zinc finger’ sequences. Proc Natl Acad Sci USA. 1988;/85:/

7857�61.

4. Milbrandt J. A nerve growth factor-induced gene encodes a

possible transcriptional regulatory factor. Science. 1987;/238:/

797�9.

5. Lemaire P, Revelant O, Bravo R, Charnay P. Two mouse

genes encoding potential transcription factors with identical

DNA-binding domains are activated by growth factors in

cultured cells. Proc Natl Acad Sci USA. 1988;/85:/4691�5.

6. Cole AJ, Saffen DW, Baraban JM, Worley PF. Rapid increase

of an immediate early gene messenger RNA in hippocampal

neurons by synaptic NMDA receptor activation. Nature.

1989;/340:/474�6.

7. Rampon C, Jiang CH, Don H, Tan YP, Lockhart D, Schultz

P, et al. Effects of environmental enrichment on gene

expression in the brain. Proc Natl Acad Sci USA. 2000;/97:/

12880�4.

8. Ehret G, Fischer R. Neuronal activity and tonotopy in the

auditory system visualized by c-Fos gene expression. Brain

Res. 1991;/567:/350�4.

9. Friauf E. c-Fos immunocytochemical evidence for acoustic

pathway mapping in rats. Behav Brain Res. 1995;/66:/217�24.

10. Hillman DE, Gordon CE, Troublefield Y, Stone E, Giacchi

RJ, Chen S. Effect of unilateral tympanotomy on auditory

induced c-Fos expression in cochlear nuclei. Brain Res. 1997;/

748:/77�84.

11. Vischer MW, Hausler R, Rouiller EM. Distribution of

Fos-like immunoreactivity in the auditory pathway of the

Sprague-Dawley rat elicited by cochlear electrical stimulation.

Neurosci Res. 1994;/19:/175�85.

12. Saito H, Miller JM, Altschuler RA. Cochleotopic fos im-

munoreactivity in cochlea and cochlear nuclei evoked by

bipolar cochlear electrical stimulation. Hear Res. 2000;/145:/

37�51.


Aud

iol M

ed D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Cal

ifor

nia

Irvi

ne o

n 10

/27/

14Fo

r pe

rson

al u

se o

nly.

13. Illing RB, Michler SA, Kraus KS, Laszig R. Transcription

factor modulation and expression in the rat auditory brain-

stem following electrical intracochlear stimulation. Exp Neu-

rol. 2002;/175:/226�44.

14. Nakamura M, Rosahl SK, Alkahlout E, Gharabaghi A, Walter

GF, Samii M. C-Fos immunoreactivity mapping of the

auditory system after electrical stimulation of the cochlear

nerve in rats. Hear Res. 2003;/184:/75�81.

15. 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 Rev. 1998;/28:/370�490.

16. Wisdom R. AP-1: one switch for many signals. Exp Cell Res.

1999;/253:/180�5.

17. De Groen PC, Eggen BJ, Gispen WH, Schotman P, Schrama

LH. Cloning and promoter analysis of the human B-50/GAP-

43 gene. J Mol Neurosci. 1995;/6:/109�19.

18. Rylski M, Kaczmarek L. AP-1 targets in the brain. Front

Biosci. 2004;/9:/8�23.

19. Benowitz LI, Routenberg A. GAP-43: an intrinsic determi-

nant of neuronal development and plasticity. Trends Neu-

rosci. 1997;/20:/84�91.

20. Kaczmarek L, Lapinska-Dzwonek J, Szymczak S. Matrix

metalloproteinases in the adult brain physiology: a link

between c-Fos, AP-1 and remodelling of neuronal connec-

tions? EMBO J. 2002;/21:/6643�8.

21. Meidinger MA, Hildebrandt-Schoenfeld H, Illing RB. Co-

chlear damage induces GAP-43 expression in cholinergic

synapses of the rat cochlear nucleus: a light and electron

microscopical study. Eur J Neurosci. 2006;/23:/3187�99.

22. Bliss TV, Lomo T. Long-lasting potentiation of synaptic

transmission in the dentate area of the anaesthetized rabbit

following stimulation of the perforant path. J Physiol. 1973;/

232:/331�56.

23. Hoelscher C, Richter-Levin G, editors. Neuronal Mechan-

isms of Memory Formation. Cambridge: Cambridge Univ.

Press; 2001.

24. Worley PF, Bhat RV, Baraban JM, Erickson CA, McNaugh-

ton BL, Barnes CA. Thresholds for synaptic activation of

transcription factors in hippocampus: correlation with long-

term enhancement. J Neurosci. 1993;/13:/4776�86.

25. Kaczmarek L. Expression of c-Fos and other genes encoding

transcription factors in long-term potentiation. Behav Neural

Biol. 1992;/57:/263�6.

26. Demmer J, Dragunow M, Lawlor PA, Mason SE, Leah JD,

Abraham WC, et al. Differential expression of immediate

early genes after hippocampal long-term potentiation in

awake rats. Mol Brain Res. 1993;/17:/279�86.

27. Jones MW, Errington ML, French PJ, Fine A, Bliss TV, Garel

S, et al. A requirement for the immediate early gene Zif268 in

the expression of late LTP and long-term memories. Nat

Neurosci. 2001;/4:/289�96.

28. Fleischmann A, Hvalby O, Jensen V, Strekalova T, Zacher C,

Layer LE, et al. Impaired long-term memory and NR2A-type

NMDA receptor-dependent synaptic plasticity in mice lack-

ing c-Fos in the CNS. J Neurosci. 2003;/23:/9116�22.

29. Duncan GE, Johnson KB, Breese GR. Topographic patterns

of brain activity in response to swim stress: assessment by

2-deoxyglucose uptake and expression of Fos-like immunor-

eactivity. J Neurosci. 1993;/13:/3932�43.

30. Reimer K. Simultaneous demonstration of Fos-like immu-

noreactivity and 2-deoxy-glucose uptake in the inferior

colliculus of the mouse. Brain Res. 1993;/616:/339�43.

31. White LE, Price JL. The functional anatomy of limbic status

epilepticus in the rat. I. Patterns of 14C-2-deoxyglucose

uptake and Fos immunocytochemistry. J Neurosci. 1993;/13:/

4787�809.

32. Kearney JA, Frey KA, Albin RL. Metabotropic glutamate

agonist-induced rotation: a pharmacological, FOS immuno-

histochemical, and [14C]-2-deoxyglucose autoradiographic

study. J Neurosci. 1997;/17:/4415�25.

33. Illing RB, Reisch A. Specific plasticity responses to unilat-

erally decreased or increased hearing intensity in the adult

cochlear nucleus and beyond. Hear Res. 2006;216/217,

189�97.

34. Illing RB, Horvath M, Laszig R. Plasticity of the auditory

brainstem: effects of cochlear lesions on GAP-43 immunor-

eactivity in the rat. J Comp Neurol. 1997;/382:/116�38.

35. Saito H, Miller JM, Pfingst BE, Atschuler RA. Fos-like

immunoreactivity in the auditory brainstem evoked by bipolar

intracochlear electrical stimulation: effects of current level

and pulse duration. Neuroscience. 1999;/91:/139�61.

36. Illing RB, Michler SA. Modulation of P-CREB and expres-

sion of c-Fos in cochlear nucleus and superior olive following

electrical intracochlear stimulation. Neuroreport. 2001;/12:/

875�8.

37. Rouiller EM, Wan XS, Moret V, Liang F. Mapping of c-Fos

expression elicited by pure tones stimulation in the auditory

pathways of the rat, with emphasis on the cochlear nucleus.

Neurosci Lett. 1992;/144:/19�24.

38. Zhang JS, Haenggeli CA, Tempini A, Vischer MW, Moret V,

Rouiller EM. Electrically induced Fos-like immunoreactivity

in the auditory pathway of the rat: effects of survival time,

duration, and intensity of stimulation. Brain Res Bull. 1996;/

39:/75�82.

39. Nakamura M, Rosahl SK, Alkahlout E, Walter GF, Samii

MM. Electrical stimulation of the cochlear nerve in rats:

analysis of c-Fos expression in auditory brainstem nuclei.

Brain Res. 2005;/1031:/39�55.

40. Luo L, Ryan AF, Saint Marie RL. Cochlear ablation alters

acoustically induced c-fos mRNA expression in the adult rat

auditory brainstem. J Comp Neurol. 1999;/404:/271�83.

41. Brown MC, Liu TS. Fos-like immunoreactivity in central

auditory neurons of the mouse. J Comp Neurol. 1995;/357:/

85�97.

42. Warr WB. Fibre degeneration following lesions in the anterior

ventral cochlear nucleus of the cat. Exp Neurol. 1966;/14:/

453�74.

43. Cant NB. The fine structure of the lateral superior olivary

nucleus of the cat. J Comp Neurol. 1984;/227:/63�77.

44. Glendenning KK, Hutson KA, Nudo RJ, Masterton RB.

Acoustic chiasm II: anatomical basis of binaurality in lateral

superior olive of cat. J Comp Neurol. 1985;/232:/261�85.

45. Moore MJ, Caspary DM. Strychnine blocks binaural inhibi-

tion in lateral superior olivary neurons. J Neurosci. 1983;/3:/

237�42.

46. Saint Marie RL, Luo L, Ryan AF. Effects of stimulus

frequency and intensity on c-fos mRNA expression in the

adult rat auditory brainstem. J Comp Neurol. 1999;/404:/258�70.

47. Adams JC, Mugnaini E. Immunocytochemical evidence for

inhibitory and disinhibitory circuits in the superior olive. Hear

Res. 1990;/49:/281�98.

48. Aitkin LM, Dickhaus H, Schult W, Zimmerman N. External

nucleus of the inferior colliculus: auditory and spinal soma-

tosensory afferents and their interactions. J Neurophysiol.

1978;/41:/421�40.

49. Oliver DL, Huerta MF. Inferior and superior colliculi. In:

Webster DB, Popper AN, Fay RR, editors. The Mammalian

Auditory Pathway: Neuroanatomy. New York: Springer;

1992. p. 168�221.


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