Electrical stimulation of the cochlear nerve in rats: analysis of c-Fos expression in auditory brainstem nuclei

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Brain Research 1031

Research report

Electrical stimulation of the cochlear nerve in rats: analysis of c-Fos

expression in auditory brainstem nuclei

Makoto Nakamuraa,*, Steffen K. Rosahlb, Eyad Alkahloutc,

Gerhard F. Walterd, Madjid M. Samiie

aDepartment of Neurosurgery, Klinikum Hannover Nordstadt, Hannover, GermanybDepartment of Neurosurgery, Albert Ludwigs University, Freiburg, Germany

cDepartment of Neurosurgery, Klinikum Duisburg, GermanydDepartment of Neuropathology, Medizinische Hochschule Hannover, Germany

eDepartment of Neurosurgery, International Neuroscience Institute, Hannover, Germany

Accepted 19 October 2004

Available online 28 November 2004

Abstract

We investigated functional activation of central auditory brainstem nuclei in response to direct electrical stimulation of the cochlear nerve

using c-Fos immunoreactivity as a marker for functional mapping. The cochlear nerve was stimulated in the cerebellopontine angle of Lewis

rats applying biphasic electrical pulses (120–250 AA, 5 Hz) for 30 min. In a control group, bilateral cochlectomy was performed in order to

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2004.10.024

Abbreviations: BI_DCN_L, left dorsal cochlear nucleus (control group); BI_DCN_R, right dorsal cochlear nucleus (control group); BI_DLL_L, left dorsa

nucleus of the lateral lemniscus (control group); BI_DLL_R, right dorsal nucleus of the lateral lemniscus (control group); BI_IC_L, left inferior colliculus

(control group); BI_IC_R, right inferior colliculus (control group); BI_ILL_L, left intermediate nucleus of the lateral lemniscus (control group); BI_ILL_R

right intermediate nucleus of the lateral lemniscus (control group); BI_LSO_L, left lateral superior olive (control group); BI_LSO_R, right lateral superior olive

(control group); BI_LTB_L, left lateral nucleus of the trapezoid body (control group); BI_LTB_R, right lateral nucleus of the trapezoid body (control group)

BI_MSO_L, left medial superior olive (control group); BI_MSO_R, right medial superior olive (control group); BI_MTB_L, left medial nucleus of the

trapezoid body (control group); BI_MTB_R, right medial nucleus of the trapezoid body (control group); BI_VCN_L, left ventral cochlear nucleus (contro

group); BI_VCN_R, right ventral cochlear nucleus (control group); BI_VLL_L, left ventral nucleus of the lateral lemniscus (control group); BI_VLL_R, righ

ventral nucleus of the lateral lemniscus (control group); BI_VTB_L, left ventral nucleus of the trapezoid body (control group); BI_VTB_R, right ventra

nucleus of the trapezoid body (control group); CIC, central nucleus of the inferior colliculus; DAB, 3-3Vdiaminobenzidine; DCIC, dorsal cortex of the inferio

colliculus; DCN, dorsal cochlear nucleus; DLL, dorsal nucleus of the lateral lemniscus; ECIC, external cortex of the inferior colliculus; EL_DCN_L, left dorsa

cochlear nucleus (group with electrical stimulation); EL_DCN_R, right dorsal cochlear nucleus (group with electrical stimulation); EL_DLL_L, left dorsa

nucleus of the lateral lemniscus (group with electrical stimulation); EL_DLL_R, right dorsal nucleus of the lateral lemniscus (group with electrical stimulation)

EL_IC_L, left inferior colliculus (group with electrical stimulation); EL_IC_R, right inferior colliculus (group with electrical stimulation); EL_ILL_L, lef

intermediate nucleus of the lateral lemniscus (group with electrical stimulation); EL_ILL_R, right intermediate nucleus of the lateral lemniscus (group with

electrical stimulation); EL_LSO_L, left lateral superior olive (group with electrical stimulation); EL_LSO_R, right lateral superior olive (group with electrica

stimulation); EL_LTB_L, left lateral nucleus of the trapezoid body (group with electrical stimulation); EL_LTB_R, right lateral nucleus of the trapezoid body

(group with electrical stimulation); EL_MSO_L, left medial superior olive (group with electrical stimulation); EL_MSO_R, right medial superior olive (group

with electrical stimulation); EL_MTB_L, left medial nucleus of the trapezoid body (group with electrical stimulation); EL_MTB_R, right medial nucleus of the

trapezoid body (group with electrical stimulation); EL_VCN_L, left ventral cochlear nucleus (group with electrical stimulation); EL_VCN_R, right ventra

cochlear nucleus (group with electrical stimulation); EL_VLL_L, left ventral nucleus of the lateral lemniscus (group with electrical stimulation); EL_VLL_R

right ventral nucleus of the lateral lemniscus (group with electrical stimulation); EL_VTB_L, left ventral nucleus of the trapezoid body (group with electrica

stimulation); EL_VTB_R, right ventral nucleus of the trapezoid body (group with electrical stimulation); IC, inferior colliculus; ILL, intermediate nucleus o

the lateral lemniscus; LL, lateral lemniscus; LSO, lateral superior olive; LTB, lateral nucleus of the trapezoid body; MSO, medial superior olive; MTB, media

nucleus of the trapezoid body; PB, phosphate buffer; PBST, phosphate buffer containing bovine serum albumine and Triton-X; SOC, superior olivary complex

TB, trapezoid body; VCN, ventral cochlear nucleus; VLL, ventral nucleus of the lateral lemniscus; VTB, ventral nucleus of the trapezoid body

* Corresponding author. Department of Neurosurgery, Nordstadt Hospital, Haltenhoffstr. 41, 30167 Hannover, Germany. Tel.: +49 511 9701534; fax: +49

511 9701606.

E-mail address: [email protected] (M. Nakamura).

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(2005) 39–55

M. Nakamura et al. / Brain Research 1031 (2005) 39–5540

assess the basal expression of c-Fos in the auditory brainstem nuclei. The completeness of cochlear ablations and the response of auditory

brainstem nuclei to electrical stimulation were electrophysiologically verified. C-Fos immunohistochemistry was performed using the free

floating method. In anaesthetized animals with unilateral electrical stimulation of the cochlear nerve, increased expression of c-Fos was

detected in the ipsilateral ventral cochlear nucleus (VCN), in the dorsal cochlear nucleus bilaterally (DCN), in the ipsilateral lateral superior

olive (LSO) and in the contralateral inferior colliculus (IC). A bilateral slight increase of c-Fos expression in all subdivisions of the lateral

lemniscus (LL) did not reach statistical significance. Contralateral inhibition of the nuclei of the trapezoid body (TB) was observed. Our data

show that unilateral electrical stimulation of the cochlear nerve leads to increased expression of c-Fos in most auditory brainstem nuclei,

similar to monaural auditory stimulation. They also confirm previous studies suggesting inhibitory connections between the cochlear nuclei.

C-Fos immunoreactivity mapping is an efficient tool to detect functional changes following direct electrical stimulation of the cochlear nerve

on the cellular level. This could be particularly helpful in studies of differential activation of the central auditory system by experimental

cochlear and brainstem implants.

D 2004 Elsevier B.V. All rights reserved.

Theme: Sensory Systems

Topic: Auditory systems: central anatomy

Keywords: c-Fos; Immediate early genes; Brainstem auditory system; Cochlear nerve; Electrical stimulation

1. Introduction

Immunohistochemical staining of the immediate early

gene product c-Fos has been widely used as an indicator of

neuronal activation induced by a variety of external stimuli

[18,19]. It may be considered as a functional marker with

cellular resolution [33], providing distinct advantages over

electrophysiological mapping which lacks spatial resolution,

and over time-consuming and costly autoradiographic

procedures.

Several studies in the past investigated c-Fos expression

within the auditory system following acoustic stimulation

[1,2,5,6,8,9,14,24,26,28,31]. Increased c-Fos immunoreac-

tivity was consistently found in neurons of the dorsal

cochlear nucleus (DCN) and inferior colliculus (IC). Effects

of cochlear ablation on c-Fos immunoreactivity in the

central auditory nuclei have been investigated in other

studies [16,25]. C-Fos expression in the auditory pathway

elicited by electrical stimulation of the cochlea was assessed

in an animal model of cochlear implantation. These studies

found c-Fos immunoreactivity mainly in the DCN and the

inferior colliculus with a pattern that resembled the results

after acoustic stimulation [12,20,29,35,37].

It has been shown that electrical stimulation of the central

auditory system by auditory brainstem implants evokes

useful hearing sensations in profoundly deaf patients even if

the connection between cochlea and cochlear nucleus, the

cochlear nerve, was interrupted bilaterally [7,11,17].

Recently, we have reported on the cellular basis of

activating central auditory structures by electrical stimula-

tion—an issue that had not been analyzed before. Our study

showed increased c-Fos immunoreactivity in the ipsilateral

VCN and bilateral DCN after unilateral electrical stimula-

tion of the cochlear nerve with a broad distribution of c-Fos

positive neurons in all tonotopic areas of the ventral and

dorsal cochlear nucleus [21].

The present study follows up on this topic and provides

an analysis of c-Fos expression in supra-cochlear auditory

brainstem nuclei following unilateral electrical stimulation

of the cochlear nerve in rats.

2. Materials and methods

2.1. Animals

Following contralateral cochlear ablation, the right

cochlear nerve was electrically stimulated in 10 adult female

Lewis rats. A control group (n=10) underwent bilateral

cochlear ablation in order to investigate the basal expression

of c-Fos in the cochlear nuclei. Cochlear ablations were

performed to eliminate any uncontrolled auditory input that

could lead to c-Fos expression in auditory brainstem nuclei.

Animals were kept in a sound shielded room in a holding

cage where they could freely move before the beginning of

the experiments. All surgical procedures were performed

under deep anesthesia with ketamine and medetomidine.

The depth of anesthesia was monitored by cutaneous

nociceptive and corneal reflexes, respectively.

2.2. Cochlear nerve stimulation

Stimulation of the cochlear nerve was performed 14 days

after contralateral cochlear ablation to ensure that no

contralateral auditory input could influence the activation

of c-Fos in auditory brainstem nuclei. Following a retro-

auricular incision, a lateral suboccipital craniectomy was

performed on the right side. After exposure of the

cerebellopontine angle, the cochlear nerve was identified.

A concentric Tungsten electrode was attached to the nerve

and stimulated with biphasic electrical pulses of 120–250

AA at a rate of 5 Hz for 30 min. The stimulus intensity was

M. Nakamura et al. / Brain Research 1031 (2005) 39–55 41

adjusted at 30% above the threshold of the electrically

evoked auditory brainstem response (EABR) which was

registered with a Viking IV electrophysiological amplifier

(Nicolet Biomedical). The frequency of 5 Hz for electrical

stimulation of the cochlear nerve was selected due to

previous reports, which showed successful c-Fos expression

in auditory brainstem nuclei after sound or pure tone

stimulation with a stimulus frequency of 5 Hz [1,9]. From

evoked potential studies, we also know that click stimuli

consisting of a wide range of acoustic frequencies produce a

very distinct evoked response in the rat brainstem at this

repetition rate.

2.3. Cochlear ablation

Bilateral cochlectomy was performed in 10 rats in order

to investigate basal expression of c-Fos in the auditory

brainstem nuclei. While genuine controls would have been

sound-deprived rats with intact cochleas, definite elimina-

tion of undesirable auditory input was practically not

achievable without cochlear ablation. Therefore, c-Fos

expression in bilaterally cochlectomized rats served as a

secondary baseline in this study.

In an aseptic surgical procedure, the bulla tympanica was

opened to expose the cochlea. The cochlea was destroyed

with a micro-drill and the completeness of cochlear

ablations was electrophysiologically verified. The incision

was closed on both sides and the animals were allowed to

survive for 14 days.

2.4. Perfusion and tissue preparation

After the experiments, the animals were perfused trans-

cardially with phosphate buffered saline (pH 7.4) followed

by a perfusion with buffered 4% paraformaldehyde. In all

animals undergoing cochlear stimulation, transcardial per-

fusion was performed 30 min after cessation of stimulation.

Brains were dissected and held in the fixative for 24 h. For

cryoprotection, they were subsequently infiltrated with 30%

sucrose dissolved in the fixative at 4 8C until they sank. The

brains were frozen (�40 8C) and transversely sectioned (60

Am) with a Cryostat. The auditory brainstem nuclei were

sectioned from the rostral to the caudal end.

2.5. Immunochemistry

The sections were transferred to wells containing 0.1 M

phosphate buffer (PB) and rinsed twice for 10 min.

Incubations in 0.3% hydrogen peroxide followed for 30

min. After washing in 0.1 M phosphate buffer twice, the

sections were pre-incubated for 1 h in phosphate buffer

containing 0.1% bovine serum albumine and 0.5% Triton-X

(PBST) to block non-specific binding sites. The sections

were then washed in PB twice, and incubation with the

primary anti-c-Fos antibody (Oncogene Research Products,

Cat #PC38, Cambridge, MA, USA; Anti-c-Fos rabbit

polyclonal antibody, 1:10000 dilution in PBST) at room

temperature followed for 24 h. After primary incubation, the

sections were washed twice in PB. Incubation with the

biotinylated secondary antibody (Vector Laboratories, goat

anti-rabbit IgG, diluted 1:200 in PBST) at room temperature

followed for 90 min. After rinsing in PB twice, the sections

were reacted with the ABC solution (Avidin–Biotin Com-

plex; Vectastain, Vector laboratories, diluted 1:800 in PBST)

for 120 min. After washing in PB twice, they were

incubated in a 3-3V diaminobenzidine (DAB) solution

(0.05% DAB, 0.001% H2O2 with metal intensification

using cobalt chloride and nickel ammonium sulfate) for 10

minF5 s. Finally, the sections were rinsed three times in PB,

mounted on slides, dehydrated and cover slipped. Every

second section underwent additional Nissl-counterstaining

for morphological orientation.

2.6. Data analysis and statistics

C-Fos labeled neurons in the auditory brainstem nuclei

were counted using a light microscope interfaced to a

computer system operated by image analysis software

(Castgrid, Olympus). C-Fos immunoreactivity was assessed

only in those sections that were processed for immunohis-

tochemistry without Nissl-counterstaining.

All available sections of each auditory brainstem nucleus

were included for data analysis in order to avoid an undue

bias towards special sections.

The region of interest within the section for counting

cells was generated with the image analysis program

(Castgrid, Olympus). The area under investigation was

delineated at a low magnification (40�) and the cells were

counted on the computer screen at magnification of 100�.

The counting frame was moved automatically by the

computer system and all cells within the counting frames

were manually counted by one person who did not know the

treatment history of the animal. Delineation of each auditory

brainstem nucleus was standardized using the same method

in all animals and sections.

In the control group (n=10), 70 sections were used for

assessment of the right ventral cochlear nucleus (BI_

VCN_R) and 69 for the left VCN (BI_VCN_L) with each

animal contributing 6–7 sections. In the same group, 38

sections were available for assessment of the right dorsal

cochlear nucleus (BI_DCN_R) and 36 for the left DCN

(BI_DCN_L) including 3–4 sections for each side per animal.

A total of 67 sections were used for assessment of the right

and left medial and lateral olive (BI_MSO_R, BI_MSO_L,

BI_LSO_R, BI_LSO_L) corresponding to 6–7 sections per

animal, 56 sections were assessed for the ventral, medial and

lateral nucleus of the trapezoid body (BI_VTB_R,

BI_VTB_L, BI_MTB_R, BI_MTB_L, BI_LTB_R,

BI_LTB_L) corresponding to 5–6 sections per animal, 42

sections were assessed for the ventral, intermediate and dorsal

nucleus of the lateral lemniscus (BI_VLL_R, BI_VLL_L,

BI_ILL_R, BI_ILL_L, VI_DLL_R, BI_DLL_L) correspond-


ing to 4–5 sections per animal and 76 sections were assessed

for the inferior colliculus (BI_IC_R, BI_IC_L) correspond-

ing to 7–8 sections per animal.

In the group with electrical stimulation (n=10), 58

sections were used for assessment of the right VCN

(EL_VCN_R) and 45 for the left VCN (EL_VCN_L)

corresponding to 5–6 sections per animal for the right side

and 4–5 sections for the left side. In the same group, 37

sections were assessed for quantitative analysis of c-Fos

immunoreactivity in the right DCN (EL_DCN_R) and 35

for the left DCN (EL_DCN_L). Three to four sections were

available for each side per animal. A total of 54 sections

were used for assessment of the right and left medial and

lateral olive (EL_MSO_R, EL_MSO_L, EL_LSO_R,

EL_LSO_L) corresponding to 5–6 sections per animal, 45

sections were assessed for the ventral, medial and lateral

nucleus of the trapezoid body (EL_VTB_R, EL_VTB_L,

EL_MTB_R, EL_MTB_L, EL_LTB_R, EL_LTB_L) corre-

sponding to 4–5 sections per animal, 44 sections were

assessed for the ventral, intermediate and dorsal nucleus of

the lateral lemniscus (EL_VLL_R, EL_VLL_L, EL_ILL_R,

EL_ILL_L, EL_DLL_R, EL_DLL_L) corresponding to 4–5

Fig. 1. (A) Average number of cell nuclei with c-Fos immunoreactivity (per 6

BI_VCN_L, left side) and DCN (BI_DCN_R, right side; BI_DCN_L, left side) of r

DCN in rats with bilateral cochlectomy. Low c-Fos immunoreactivity on both side

right side. (C) VCN (BI_VCN_L) and DCN (BI_DCN_L) of the left side.

sections per animal, 80 sections were assessed for the

inferior colliculus (EL_IC_R, EL_IC_L) corresponding to 8

sections per animal.

The slight difference of the number of sections per side in

the same animal is partly due to a minimal inclination of the

cleavage planes in the transverse orientation.

All immunostained cell nuclei in different focuses in all

sections (total thickness of 60 Am) except of the inner and

outer 10 Am of the section were counted. This was

performed due to possible tearing of cell nuclei during

cryotomy at the inner and outermost layer of each section

and to avoid potential bias among sections. The depth in

each section was calculated using the computer system

operated by image analysis software (Castgrid, Olympus).

Immunostained cell nuclei were counted and marked on the

computer screen to avoid double counting of the same cell

nucleus in different focuses.

The mean number of c-Fos positive cell nuclei per

section was calculated and averaged for each group.

To adjust for slight differences in staining intensity

across sections or series, a cell counting criterion was used

for quantitative assessment similar to the criterion reported

0-Am section)Fstandard error (SE) in the VCN (BI_VCN_R, right side;

ats with bilateral cochlectomy. (B+C) Coronal section through the VCN and

s. Scale bar 200 Am. (B) VCN (BI_VCN_R) and DCN (BI_DCN_R) of the


in a previous study [2]. Only uniformly dark-stained cell

nuclei well above the basal background staining of

surrounding tissue were taken into account. The cell nucleus

had to contain a concentration of grainy, speckled label

which may have varied in intensity among individual cells,

but was easily delimited as nuclear. A non-auditory region

(the central grey at the level of the ventral cochlear nucleus)

was used as a standard for darkness of staining. Neurons in

auditory brainstem nuclei were counted only if they were

stained as dark as or darker than neurons in the standard.

This was confirmed using densitometry measurements

with the image analysis program (Castgrid, Olympus).

Statistical comparisons were performed using the SPSS

statistical analysis software (SPSS). The mean numbers of c-

Fos positive cell nuclei per section were assessed on each side

(ipsilateral and contralateral) in each auditory brainstem

nucleus. Mean numbers in each auditory brainstem nucleus

were compared between each side or between the stimulated

and control group using the independent, two-tailed Student’s

t-test. Differences were considered significant at pb0.01.


EL_VCN_L, left side) and DCN (EL_DCN_R, right side; EL_DCN_L left side) o

sections through the VCN and DCN in rats with electrical stimulation of the right

Fos IR in VCN (EL_VCN_R) and DCN (EL_DCN_R). (C) Contralateral side with

immunoreactivity in the DCN (EL_DCN_L).

The experimental study was approved by the regional

committee for experiments on laboratory animals.

3. Results

The results concerning c-Fos reactive cell nuclei in the

VCN and in the DCN have been reported before [21]. In

animals with bilateral cochlear ablation, the number of c-

Fos reactive cell nuclei representing basal expression was

generally low in the VCN (BI_VCN_R, 7.94F1.05 and

BI_VCN_L, 6.06F0.78; no statistical difference between

both sides were emerged p=0.126) and DCN (BI_DCN_R,

9.05F1.42 and BI_DCN_L, 7.94F1.48; p=0.567) on both

sides (Fig. 1A–C).

In anaesthetized animals with unilateral electrical stim-

ulation of the right cochlear nerve, increased expression of

c-Fos was detected in the ipsilateral VCN (EL_VCN_R,

31.34F3.27) and bilateral DCN (EL_DCN_R, 46.16F6.02

and EL_DCN_L, 37.11F5.7). The ipsilateral VCN

0-Am section)Fstandard error (SE) in the VCN (EL_VCN_R, right side;

f rats with electrical stimulation of the right cochlear nerve. (B+C) Coronal

cochlear nerve. Scale bar 200 Am. (B) Side of stimulation with increased c-

only few labeled cell nuclei in the VCN (EL_VCN_L) and increased c-Fos


(EL_VCN_R) showed a statistically significant ( pb0.01),

11-fold increase in c-Fos expression compared to the

contralateral VCN (EL_VCN_L) [21]. C-Fos expression

was more abundant at the lateral and medial margins than in

the centre of the ventral cochlear nucleus. C-Fos immunor-

eactivity was also present within the granule cell regions of

the ventral cochlear nucleus. In counterstained sections,

some of these neurons were identified as granule cells by

their small size. Neurons showing c-Fos immunoreactivity

were small cells similar to those previously reported in the

cat [1].


BI_MSO_L, left side) and LSO (BI_LSO_R, right side; BI_LSO_L left side) of ra

LSO in rats with bilateral cochlectomy. Scale bar 200 Am. (B) MSO (BI_MSO_R

(BI_LSO_L) of the left side.

C-Fos expression was high in both DCN (Fig. 2A–C).

C-Fos immunoreactive neurons were found throughout all

layers, but labeling was more dense in the superficial

layers.

In animals with bilateral cochlear ablation, few c-Fos

reactive cell nuclei were found in the medial (MSO) and

lateral superior olive (LSO) with no significant side

difference. There were 1.01F0.347 immunoreactive cell

nuclei per section in the ipsilateral MSO (BI_MSO_R) and

0.64F0.255 cell nuclei in the contralateral MSO (BI_M-

SO_L) with no statistical difference ( p=0.387). In the

0-Am section)Fstandard error (SE) in the MSO (BI_MSO_R, right side;

ts with bilateral cochlectomy. (B+C) Coronal sections through the MSO and

) and LSO (BI_LSO_R) of the right side. (C) MSO (BI_MSO_L) and LSO


lateral olive (LSO), 1.27F0.303 immunoreactive cell

nuclei per section were counted on the ipsilateral side

(BI_LSO_R) compared to 0.6F0.185 cell nuclei per

section on the contralateral side (BI_LSO_L) ( p=0.06)

(Fig. 3A–C).

In animals with unilateral electrical stimulation of the

right cochlear nerve, c-Fos expression in the MSO was very

low on both sides with 0.2F0.104 immunoreactive cell

nuclei per section on the ipsilateral (EL_MSO_R) and 0 cell

nuclei on the contralateral side (EL_MSO_L) ( p=0.055).

Significant increase of c-Fos expression was observed in the


EL_MSO_L, left side) and LSO (EL_LSO_R, right side; EL_LSO_L left side) of

sections through the MSO and LSO in rats with electrical stimulation of the ri

(EL_LSO_R) of the right side. (C) MSO (EL_MSO_L) and LSO (EL_LSO_L) o

ipsilateral LSO (EL_LSO_R) with 12.93F0.862 immunor-

eactive cell nuclei per section, mostly distributed in the core

region of the nucleus, compared to 0.26F0.127 immunor-

eactive cell nuclei on the contralateral side (EL_LSO_L).

The difference between both sides was statistically signifi-

cant ( pb0.01) (Fig. 4A–C).

Analysis of c-Fos expression in the trapezoid bodies of

animals with bilateral cochlear ablation revealed

8.78F1.548 immunoreactive cell nuclei in the right VTB

(BI_VTB_R) compared to 6.93F1.299 cell nuclei per

section on the left side (BI_VTB_L). The difference was

0-Am section)Fstandard error (SE) in the MSO (EL_MSO_R, right side;

rats with electrical stimulation of the right cochlear nerve. (B+C) Coronal

ght cochlear nerve. Scale bar 200 Am. (B) MSO (EL_MSO_R) and LSO

f the left side.


not statistically significant ( p=0.257) (Fig. 5A–C).

11.22F1.422 c-Fos positive cell nuclei were counted per

section in the ipsilateral VTB (EL_VTB_R) of animals

undergoing unilateral electrical stimulation. 0.67F0.373 cell

nuclei per section showed c-Fos expression in the contrala-

teral VTB (EL_VTB_L). The difference was statistically

significant ( pb0.01) (Fig. 6A–C). There was no significant

increase of c-Fos expression in the ipsilateral VTB of

electrically stimulated animal compared to the basal

expression in the control group ( p=0.467).

In the MTB, there were 6.09F1.325 immunoreactive cell

nuclei on the right (BI_MTB_R) and 4.73F1.008 positive

cell nuclei on the left side (BI_MTB_L) in the control group

Fig. 5. (A) Average number of cell nuclei with c-Fos immunoreactivity (per 60-Amleft side), MTB (BI_MTB_R, right side; BI_MTB_L, left side) and VTB (BI_V

(B+C) Coronal sections through the MTB and VTB in rats with bilateral cochlectom

right side. (C) MTB (BI_MTB_L) and VTB (BI_VTB_L) of the left side.

( p=0.418) (Fig. 5A–C). In animals undergoing electrical

stimulation, 5.67F0.667 labeled cell nuclei were found in

the ipsilateral MTB (EL_MTB_R) compared to only

0.44F0.338 cell nuclei on the contralateral side

(EL_MTB_L) ( pb0.01) (Fig. 6A–C).

In the LTB of animals with bilateral cochlear ablation,

there was no significant side difference with 2.09F0.39

immunolabeled cell nuclei on the right side (BI_LTB_R)

compared to 2.13F0.438 cell nuclei on the left side

(BI_LTB_L) (Fig. 5A–C). After unilateral electrical stim-

ulation of the cochlear nerve, no significant increase of c-

Fos expression was found on the ipsilateral LTB

(EL_LTB_R) with 2.67F0.373 immunoreactive cell nuclei.

section)Fstandard error (SE) in the LTB (BI_LTB_R, right side; BI_LTB_L,

TB_R, right side; BI_VTB_L left side) of rats with bilateral cochlectomy.

y. Scale bar 200 Am. (B) MTB (BI_MTB_R) and VTB (BI_VTB_R) of the

Fig. 6. (A) Average number of cell nuclei with c-Fos immunoreactivity (per 60-Am section)Fstandard error (SE) in the LTB (EL_LTB_R, right side;

EL_LTB_L, left side), MTB (EL_MTB_R, right side; EL_MTB_L, left side) and VTB (EL_VTB_R, right side; EL_VTB_L left side) of rats with

electrical stimulation of the right cochlear nerve. (B+C) Coronal sections through the MTB and VTB in rats with electrical stimulation of the right

cochlear nerve. Scale bar 200 Am. (B) MTB (EL_MTB_R) and VTB (EL_VTB_R) of the right side. (C) MTB (EL_MTB_L) and VTB (EL_VTB_L) of

the left side.


C-Fos expression was low on the contralateral side with

0.22F0.147 cell nuclei (EL_LTB_L) ( pb0.01) (Fig. 6A–C).

In all subdivisions of the trapezoid body (VTB, MTB and

LTB), contralateral decrease of c-Fos expression was

observed after electrical stimulation. The decrease of c-Fos

labeling compared to basal expression in the control group

was significant ( pb0.01).

C-Fos immunoreactive neurons extended throughout all

subdivisions of the trapezoid body without any special

tonotopic distribution.

C-Fos expression in nuclei of the lateral lemniscus in

animals of the control group was similar on both sides.

13.62F1.829 immunoreactive cell nuclei per section were

counted on the ipsilateral DLL (BI_DLL_R) compared to

12.53F1.798 cell nuclei on the contralateral side (BI_

DLL_L) ( p=0.67). There were 12F1.611 immunoreactive

cell nuclei/section in the ipsilateral ILL (BI_ILL_R)

compared to 11.06F1.62 cell nuclei/section on the

contralateral side (BI_ILL_L) ( p=0.682). 8.59F1.129

immunoreactive cell nuclei were counted in the ipsilateral

VLL (BI_VLL_R) compared to 7.74F1.076 cell nuclei/

section on the contralateral side (BI_VLL_L) ( p=0.586)

(Fig. 7A–C).

Following unilateral electrical stimulation of the

cochlear nerve, the level of c-Fos expression in the

lateral lemniscus was generally higher with a slight

Fig. 7. (A) Average number of cell nuclei with c-Fos immunoreactivity (per 60-Am section)Fstandard error (SE) in the DLL (BI_DLL_R, right side;

BI_DLL_L, left side), ILL (BI_ILL_R, right side; BI_ILL_L, left side) and VLL (BI_VLL_R, right side; BI_VLL_L left side) of rats with bilateral cochlectomy.

(B+C) Coronal sections through the nuclei of the lateral lemniscus (DLL, ILL and VLL) in rats with bilateral cochlectomy. Scale bar 200 Am. (B) DLL

(BI_DLL_R), ILL (BI_ILL_R) and VLL (BI_VLL_R) of the right side. (C) DLL (BI_DLL_L), ILL (BI_ILL_L) and VLL (BI_VLL_L) of the left side.


increase on the ipsilateral side, although the side differ-

ence was not statistically significant. 21.3F1.994 immu-

noreactive cells/section were counted on the ipsilateral

DLL (EL_DLL_R) compared to 19.15F1.971 cell nuclei

on the contralateral side (EL_DLL_L) ( p=0.448).

17.15F1.652 c-Fos positive cells/section were registered

in the ipsilateral ILL (EL_ILL_R) compared to

15.45F1.629 cell nuclei/section on the contralateral side

(EL_ILL_L) ( p=0.468). 11.1F1.304 immunoreactive cell

nuclei/section were counted in the VLL (EL_VLL_R) of

the ipsilateral side compared to 10.35F1.597 cell nuclei/

section on the contralateral side (EL_VLL_L) ( p=0.506)

(Fig. 8A–C).

In all subdivisions of the lateral lemniscal nuclei, we

found c-Fos expression throughout the entire extent of each

nucleus, without any tonotopic distribution in both stimu-

lated and control animals.

Similar levels of c-Fos expression were observed in the

inferior colliculus (IC) on the right and left side in the

control group with 38.34F6.25 immunoreactive cell nuclei /

section on the right side (BI_IC_R) and 52.75F8.054 cell

nuclei/section on the left IC (BI_IC_L; p=0.16; Fig. 9A–C).

Fig. 8. (A) Average number of cell nuclei with c-Fos immunoreactivity (per 60-Am section)Fstandard error (SE) in the DLL (EL_DLL_R, right side;

EL_DLL_L, left side), ILL (EL_ILL_R, right side; EL_ILL_L, left side) and VLL (EL_VLL_R, right side; EL_VLL_L left side) of rats with electrical

stimulation of the right cochlear nerve. (B+C) Coronal sections through the nuclei of the lateral lemniscus (DLL, ILL and VLL) in rats with electrical

stimulation of the right cochlear nerve. Scale bar 200 Am. (B) DLL (EL_DLL_R), ILL (EL_ILL_R) and VLL (EL_VLL_R) of the right side. (C) DLL

(EL_DLL_L), ILL (EL_ILL_L) and VLL (EL_VLL_L) of the left side.


C-Fos expression increased significantly after electrical

stimulation of the cochlear nerve on the contralateral IC

(EL_IC_L) with 122.63F11.451 immunoreactive cell

nuclei/section, and was more pronounced in the external

(ECIC) and dorsal (DCIC) cortex of the inferior colliculus

than in the central (CIC) nucleus. In comparison, there were

only 43.61F6.201 labeled cell nuclei on the ipsilateral side

(EL_IC_R; pb0.01; Fig. 10A–C). No tonotopic distribution

was observed.

All results are summarized in Fig. 11.

4. Discussion

Previous studies have demonstrated that acoustic stim-

ulation leads to increased c-Fos immunoreactivity in

neurons in the cochlear nucleus, the superior olivary

complex and the inferior colliculus (IC) indicating neuronal

activation. C-Fos expression in the dorsal cochlear nucleus

(DCN) and IC was the most consistent finding in the

majority of studies [6,8,23,26,31]. Concerning c-Fos immu-

noreactivity in other brainstem auditory structures, there has

Fig. 9. (A) Average number of cell nuclei with c-Fos immunoreactivity (per 60-Am section)Fstandard error (SE) in the IC (BI_IC_R, right side; BI_IC_L, left

side) and of rats with bilateral cochlectomy. (B+C) Coronal sections through the IC in rats with bilateral cochlectomy. Scale bar 200 Am. (B) IC (BI_IC_R) of

the right side. (C) IC (BI_IC_L) of the left side.


been considerable variation between studies. In part, this

may be due to variations in the sensitivity and specificity of

the antibodies employed or may result from varying

experimental conditions (e.g. the type and duration of the

stimulus, the length of the post stimulus survival time or the

use of anesthetics). Although some recent studies have

shown a greater breadth of sound-induced expression

[1,2,9,13,15], it is generally agreed that sound-induced c-

Fos expression is tonotopically organized in the DCN and

IC but less consistent in other auditory brainstem. There are

few reports about tonotopically labeled bands of neurons in

the ventral cochlear nucleus (VCN) [1,9,26].

Studies with monaural acoustic stimulation showed clear

side differences of c-Fos expression in certain brainstem

nuclei [2,9]. C-Fos expression was higher on the ipsilateral

VCN and DCN compared to the contralateral side. In the

inferior colliculus (IC), most c-Fos immunoreactive cell

nuclei were on the contralateral side which receives the

main (crossing) projection from the ipsilateral cochlear

nucleus. Other auditory brainstem nuclei in which the

majority of c-Fos expression was located ipsilateral to the

auditory stimulation included the LSO and the LTB. In

contrast, the contralateral side dominated in the MTB, the

VTB, VNLL and IC. The INLL and DNLL contained about

Fig. 10. (A) Average number of cell nuclei with c-Fos immunoreactivity (per 60-Am section)Fstandard error (SE) in the IC (EL_IC_R, right side; EL_IC_L,

left side) and of rats with electrical stimulation of the right cochlear nerve. (B+C) Coronal sections through the IC in rats with electrical stimulation of the right

cochlear nerve. Scale bar 200 Am. (B) IC (EL_IC_R) of the right side. (C) IC (EL_IC_L) of the left side.


the equal numbers of c-Fos immunoreactive cell nuclei on

both sides [9].

The effect of cochlear ablation on c-Fos expression in

the auditory brainstem nuclei was investigated recently

[16,25]. A rapid and intense transient c-Fos expression was

found in the ipsilateral DCN which disappeared by 8 h

after cochlear ablation. C-Fos expression was inconsis-

tently found over cell nuclei of the contralateral medial

nucleus of the trapezoid body (MTB) after cochlear

ablation. No expression was observed elsewhere in the

VCN or in the superior olivary complex, nuclei of the

lateral lemniscus or inferior colliculus (IC) at any time up

to 6 months after ablation [16]. In our study, c-Fos

expression in auditory brainstem nuclei was analyzed 14

days after bilateral cochlear ablation, long after the

reported injury-induced increase was gone. In contrast to

the study by Luo et al. [16], we detected a low baseline

level of c-Fos immunoreactivity in most auditory brain-

stem nuclei with no significant side difference. The reason

for the discrepancy is unclear. It may be noteworthy, that

in the former study, expression of c-Fos mRNA was

analyzed as opposed to immunocytochemical labeling of c-

Fos protein in our study. We assume that collateral

projections may be sufficient to sustain an intrinsic low

Fig. 11. Summary of results presented in a scheme of the auditory system. The mean number of c-Fos immunoreactive cells is shownFstandard error (SE) in

each auditory brainstem nucleus of the right (ipsilateral) and left (contralateral) side. The average number of c-Fos positive cells in control animals is presented

in brackets (). Statistically significant increase or decrease of c-Fos immunoreactivity after electrical stimulation of the right cochlear nerve is shown in bold

numbers with arrows (zor A).


basal level of c-Fos expression in auditory brainstem

nuclei even after auditory deprivation due to bilateral

cochlear ablation.

All animals of the control group in our study were

subjected to bilateral cochlectomy. Therefore, they are not

controls in the true sense of the word. Genuine controls

would have been intact non-cochlectomized rats that were

merely anesthetized and treated and analyzed for basal c-Fos

expression identical to the experimental animals omitting

the stimulus. Bilateral cochlectomy in control animals and

unilateral cochlectomy in the stimulated group was per-

formed to eliminate any possible and uncontrolled auditory

stimulation which could lead to unwanted c-Fos expression

in auditory brainstem nuclei during the experiments.

Complete and definite elimination of auditory input is only

possible with cochlear ablation. However, it cannot be

excluded that bilateral ablation in the controls and also

unilateral ablation of the cochlea in stimulated animals

changed the natural basal expression and the functional

interrelationship between the components of the auditory

system of these rats.

C-Fos expression in the central auditory system after

electrical stimulation of the cochlea was investigated by

several authors [12,20,29,35,37] and was constantly found

in the VCN, DCN and IC. The location of c-Fos expression

in the auditory nuclei was consistent with expected


tonotopic areas in accordance with the site of electrical

stimulation in the cochlea.

After unilateral electrical stimulation of the cochlea,

dense c-Fos expression was found in the ipsilateral DCN,

and moderate expression in the contralateral DCN, respec-

tively [12,29,30,37]. C-Fos immunoreactive cell nuclei were

observed in the ipsilateral VCN [20,29,30,37] and contrala-

teral posteroventral cochlear nucleus [37]. We found

increased c-Fos immunoreactivity in the ipsilateral VCN

and bilateral DCN. Evidence of contralateral excitation of

DCN neurons after unilateral acoustic stimulation has also

been reported by several authors using c-Fos immunohis-

tochemistry in the past [2,16,35]. C-Fos expression in the

contralateral VCN after acoustic stimulation was less

pronounced compared to the contralateral DCN [2].

Increased c-Fos expression in contralateral cochlear

nuclei indicates that its neurons have connections to the

contralateral side. Earlier results employing anterograde and

retrograde axonal transport techniques [4,34] revealed direct

synaptic connections from one cochlear nucleus to the other.

Although the major target of the known commissural

connections were reported to be the contralateral VCN

[4,34]. It was previously reported that this pathway is likely

to be inhibitory because it contains the inhibitory transmitter

glycine [36].The reason for the discrepancy of the level of c-

Fos expression between the contralateral DCN and VCN in

our and previous studies remains unclear. Whether or not

the difference is associated with stronger contralateral

inhibition of the VCN remains to be elucidated. Because

the number of c-Fos positive neurons in the contralateral

VCN was slightly lower compared to the VCN of the

control group, this hypothesis is not unlikely. However, it

would not explain the significant increase of c-Fos

expression in the contralateral DCN after electrical stim-

ulation compared to the baseline expression in the control

group. Other possible pathways have been described, which

could finally result in contralateral excitation of the DCN.

For example, indirect projections to the DCN from

periolivary nuclei bilaterally and the ipsilateral inferior

colliculus have been considered [2,16]. It was reported that

the periolivary projection to the DCN could have a

significant non-inhibitory component [22]. However, the

principal, contralaterally driven excitatory input to the DCN

is considered to originate in the inferior colliculus [3,27].

This could be another explanation for contralateral c-Fos

expression in the DCN in stimulated animals.

Zhang et al. [37] reported sparse to moderate c-Fos

expression in the superior olivary complex, equally dis-

tributed on both sides, after unilateral electrical stimulation

of the cochlea. Saito et al. observed bilateral c-Fos

expression in the MTB within the SOC, with more

immunoreactive nuclei on the contralateral side [29] similar

to the results reported after unilateral acoustic stimulation

[9]. In the LTB, a few c-Fos positive cell nuclei were

observed on the ipsilateral side after unilateral electrical

cochlear stimulation [29] similar to the results after

monaural acoustic stimulation [9]. In the VTB, a few

immunoreactive cell nuclei were observed contralaterally at

high stimulus intensities [29] corresponding to results

reported by Friauf [9].

Our findings concerning c-Fos expression in nuclei of the

trapezoid body are somewhat different to the previously

published results. After electrical stimulation of the cochlear

nerve, c-Fos expression was more abundant in ipsilateral

nuclei of the trapezoid body (VTB, MTB and LTB).

However, c-Fos expression was not significantly higher on

the ipsilateral side compared to basal expression of c-Fos in

the trapezoid body after bilateral cochlear ablation. It must

be assumed that unilateral electrical stimulation of the

cochlear nerve led to suppression of the contralateral

trapezoid body rather than excitation on the ipsilateral side.

It remains unclear, whether this disparity compared to

previously published studies [9,29] is due to the different

stimulus condition used in our study.

In the LSO, unilateral cochlear electrical stimulation led

to increased c-Fos labelling on the ipsilateral side, immu-

noreactive cell nuclei were not observed in the contralateral

LSO and in the MSO of both sides [29]. Concerning the

MSO and LSO, our results were similar with almost no c-

Fos immunoreactive cell nuclei in the MSO on both sides

and significantly increased c-Fos expression in the ipsi-

lateral LSO. The MSO has been found to have many

neurons sensitive to low-frequency cues [10] while the LSO

showed a bias to high frequency stimuli. This may be the

reason for the almost total absence of c-Fos labeled cell

nuclei in the MSO. Other studies analyzing c-Fos expres-

sion after acoustic stimulation also failed to show c-Fos

immunoreactivity in the MSO [9,26] and it cannot be

excluded that neurons of the MSO need particular stim-

ulation requirements to express c-Fos.

In the lateral lemniscus, immunoreactivity was more

abundant in the DLL than in the VLL [37], and also more

prominent contralaterally after unilateral electrical stimula-

tion of the cochlea [29,37]. We have observed a similar

tendency with more pronounced c-Fos expression in the

DLL than VLL bilaterally in both electrically stimulated

animals and the control group. In both groups, however,

there was no significant side difference and the slight

bilateral increase of c-Fos expression after unilateral

electrical stimulation of the cochlear nerve was not

significant compared to the control group.

Reports about c-Fos expression in the lateral lemniscal

nuclei after acoustic stimulation are controversial. Some

expression in neurons of the VLL has been reported

[1,2,32], but expression elsewhere in the lateral lemniscal

nuclei has been described as either equivocal [1] or not

consistent [2]. Only two reports show c-Fos expression in

all three subdivisions of the lateral lemniscus after acoustic

stimulation [9,28]. There is clearly a need for further

investigations to clarify the response properties of neurons

in the lateral lemniscal nuclei after unilateral or bilateral

acoustic and electrical stimulation.


Expression of c-Fos in the IC is one of the most

consistently reported observations. C-Fos labelling was

shown to be more pronounced in the contralateral IC than

in the ipsilateral side with respect to the electrically

[12,20,29,37] or acoustically stimulated cochlea [2,9]. Our

results were consistent with previous reports with no

obvious increase or decrease of the ipsilateral IC from basal

c-Fos expression. These findings are consistent with

previous electrophysiological studies which showed excita-

tion of the majority of neurons in the IC by contralaterally

applied stimuli and the anatomical observation that the

majority of fibers from the ipsilateral cochlear nucleus

project to the contralateral IC.

There are previous studies reporting that only specific

types of neurons in auditory brainstem nuclei are capable to

express c-Fos [1,2,32]. Our study confirms these for the

cochlear nuclei where c-Fos expression was mainly limited

to the lateral and medial margins and to the granule cell

region of the ventral cochlear nucleus. Neurons showing c-

Fos immunoreactivity were small cells similar to those

previously reported in the cat [1]. It is unclear whether other

subtypes of neurons will express c-Fos when stimulation

parameters are varied. Previous investigations with acoustic

stimulation showed that the number of c-Fos labeled

neurons gradually increased with increasing sound pressure

levels [2,28]. Increasing stimulus intensity for electrical

stimulation of the cochlea [37] showed an expansion of c-

Fos expression into the deep layers of the DCN. It may be

assumed that different intensities of electrical stimulation of

the cochlear nerve would also influence the distribution and

number of c-Fos expression in auditory brainstem nuclei.

Electrical stimulation of the cochlear nerve produced a

broad distribution of c-Fos positive neurons throughout the

tonotopic organization of the ipsilateral VCN, bilateral

DCN, ipsilateral LSO and contralateral IC. The bilateral

slight increase of c-Fos expression in all subdivisions of the

lateral lemniscus was rather insignificant. It remains unclear

whether unilateral electrical stimulation led to contralateral

inhibition of the nuclei of the trapezoid body (VTB, MTB

and LTB).

Compared to localized electrical stimulation of the

cochlea, direct supra-threshold electrical stimulation of the

cochlear nerve in the cerebellopontine angle cannot be

expected to activate the auditory system in a tonotopic

manner.

Our data show that electrical stimulation of the cochlear

nerve leads to increased expression of c-Fos in auditory

brainstem nuclei that parallels electrophysiological

responses similar to results reported after acoustic stimula-

tion. The animal model may be employed to investigate the

effect of changes in the physical properties of the electrical

stimuli and the accessibility of tonotopic gradients in the

central auditory system by electrical stimulation. Compar-

ison of different stimulus intensities of electrical stimulation

and its effect on c-Fos expression may present an interesting

subject for further studies. Our study may contribute a

comparison with results obtained in cochlear electrical

stimulation with cochlear prostheses and provides basic

data of functional activation of the central auditory system

after electrical stimulation of the cochlear nerve. Our results

may be useful for further development of auditory nerve

prostheses, which are currently being re-considered for

clinical application.

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Electrical stimulation of the cochlear nerve in rats: analysis of c-Fos expression in auditory brainstem nuclei