auditory and vestibular defects in the circling (ci2) rat mutant

Auditory and vestibular defects in the circling (ci2) ratmutant

Alexander Kaiser,1 Maren Fedrowitz,2 Ulrich Ebert,2 Elke Zimmermann,1 Hans-JuÈrgen Hedrich,3 Dirk Wedekind3

and Wolfgang LoÈscher2

1Department of Zoology and2Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, 30559 Hannover, Germany3Institute for Laboratory Animal Science, Medical School, 30625 Hannover, Germany

Keywords: cochlea, cochlear nuclei, dopamine, hearing loss, rotational behaviour

Abstract

The circling rat is an autosomal recessive mutant (homozygous ci2/ci2) that displays lateralized circling behaviour, locomotorhyperactivity, ataxia and stereotypic head-movement. These abnormal behaviours occur in phases or bursts either spontaneously

or in response to stress. Heterozygous (ci2/+) littermates display normal spontaneous behaviours. We have previously found that

ci2/ci2 rats of both genders have a lower tissue content of dopamine in the striatum ipsilateral to the preferred direction ofrotation, indicating that the rats turn away from the brain hemisphere with higher striatal dopaminergic activity. In view of the

similarities of the motor syndrome of the ci2/ci2 mutant rat to that of mouse deafness mutants, the present study evaluated the

hearing ability of the circling rat mutant by recording brainstem auditory-evoked potentials. To test for vestibular dysfunction, aswimming test was conducted. Histological methods were used to examine the cochlear and vestibular parts of the inner ear and

the cochlear and vestibular brainstem nuclei for defects. The absence of auditory-evoked potentials demonstrated a complete

hearing loss in the adult ci2/ci2 mutant rat, whereas heterozygous littermates exhibited auditory-evoked potentials with thresholds

resembling those of other laboratory strains. Furthermore, the mutant rats were unable to swim. Histological analysis of the innerear of adult mutants revealed virtually complete loss of the cochlear neuroepithelium, while no such hair cell degeneration was

seen in the vestibular parts of the inner ear. However, part of the vestibular hair cells showed protrusions into the endolymphatic

space, suggesting alterations in the cytoskeletal architecture. The histological ®ndings in mutant circling rats strongly indicate thatthe hearing loss of the mutants is of the sensory neural type, the most prevalent type of hearing loss. In the cochlear nuclei of

the brain stem of mutant rats, neurons exhibited an abnormal shape, reduced size and increased density compared to controls.

In contrast, no abnormal neuronal morphology was seen in the vestibular nuclei, but a signi®cantly reduced neuronal density wasfound in the medial vestibular nucleus. Abnormal vestibular function would be a likely explanation for the disturbed balance of

mutant rats as exempli®ed by the ataxia and the inability to swim, whereas the previous data on these rats strongly indicate an

involvement of the basal ganglia in the abnormal circling behaviour. The genetic defect in the mutant rats, thus, results in a

clinical syndrome with features also seen in human genetic disorders with deafness and hyperkinesia, making the ci2/ci2 rat anexcellent model for investigating both cochlear/vestibular dysfunction and hyperkinetic movement disorders.

IntroductionWe have recently described a new rat mutant characterized by phases

of rapid lateralized circling, locomotor hyperactivity, hyperexcit-

ability, repetitive short-lasting arching of the neck (opisthotonus,

`stargazing'), and moderate ataxia (LoÈscher et al., 1996; Richter et al.,

1999; Fedrowitz et al., 2000). This phenotype, which was discovered

in a Lewis (LEW/Han) rat breeding colony, is caused by an

autosomal recessive mutation, which we have designated circling,

gene symbol ci. Because a mutant with the same behavioural

alterations (ci1) had been detected before in the same breeding colony

of LEW/Han rats (Deerberg et al., 1989), but was not further

characterized, the mutant described by us recently and used in the

present study was designated ci2 (Fedrowitz et al., 2000).

Neurochemical studies showed that ci2 rats have a lower tissue

content of dopamine in the striatum ipsilateral to the preferred

direction of rotation (LoÈscher et al., 1996; Richter et al., 1999),

indicating that the rats turn away from the brain hemisphere with

higher striatal dopaminergic activity, a common hypothesis of turning

behaviour in rodents (Pycock, 1980; Carlson & Glick, 1996). In

addition to lateralities in striatal dopaminergic function as a cause for

rotational behaviour in rats, circling is often observed in mouse

deafness mutants and is commonly suggested to be a consequence of

inner ear defects impairing vestibular functions (Kitamura et al.,

1991; Bloom & Hultcrantz, 1994; Alagramam et al., 1999; Rogers

et al., 1999). However, often the determination of inner ear defects in

such mutants is restricted to the cochlea. Similarly, in the deaf

stargazer (stg) rat mutant, abnormal circling behaviour was proposed

to be due to vestibular dysfunction, but hair cell degeneration was

only demonstrated for the cochlea (Truett et al., 1994). In another

more recently described rat mutant with circling (waltzing)

behaviour, the vestibular parts of the inner ear were examined and

Correspondence: Dr W. LoÈscher, 2Department of Pharmacology, Toxicologyand Pharmacy, as above.E-mail: [email protected]

Received 15 May 2001, revised 14 July 2001, accepted 31 July 2001

European Journal of Neuroscience, Vol. 14, pp. 1129±1142, 2001 ã Federation of European Neuroscience Societies

no abnormalities in vestibular hair cells were found (Rabbath et al.

2001). Previous examination of the inner ear in the ci2 mutant had not

revealed any gross abnormalities (LoÈscher et al., 1996), but speci®c

histological methods to demonstrate hair cell degeneration had not

been used yet in this rat mutant.

In view of the similarities of the motor syndrome of the ci2 mutant

rat to that of mouse deafness mutants and the deaf stargazer rat

mutant, the goal of the present study was to investigate the auditory

and vestibular system in mutant circling rats by an integrative

behavioural, neurophysiological and neuroanatomical approach.

Hearing ability was tested by auditory-evoked potentials and

vestibular functions by a swimming test. Results were related to the

morphology of the inner ear and cochlear and vestibular brainstem

nuclei.

Materials and methods

Animals

The mutant (ci2) rat described by us previously (LoÈscher et al., 1996;

Richter et al., 1999; Fedrowitz et al. 2000) and used in the present

study was isolated in F96 of LEW/Han rats and is maintained as a

segregated inbred strain LEW/Ztm-ci2. About 50% of the offspring

exhibit the mutation (ci2/ci2), which can be identi®ed by phenotype

behaviour (intense circling, hyperactivity, opisthotonus) as early as

10±14 days of age, while the remaining 50% of pups are unaffected

(ci2/+), showing normal behaviour. This makes them suitable

experimental controls for comparing behavioural and anatomical

differences. The abnormal motor behaviour of the mutants appears

either spontaneously in their home cage, or can be observed (and

ampli®ed) in a new environment, such as new cage or open-®eld

(LoÈscher et al., 1996). The abnormal behaviour of the mutant rats in

the home cage or a new environment appears in phases or bursts;

between these phases the rats behave normal, particularly when not

being disturbed. Most ci2 mutant rats of both genders exhibit a side-

preference of more than 70% in their rotational behaviour (LoÈscher

et al., 1996).

For the present study, adult, homozygous ci2/ci2 animals of both

sexes as well as age- and sex-matched, heterozygous ci2/+ rats were

used. All homozygous mutants differed by intense circling behaviour

and other behavioural abnormalities (locomotor hyperactivity,

opisthotonus, ataxia) from heterozygous (ci2/+) littermates. The

latter behaved normally like the genetic background strain (LEW/

Ztm). For determination of the side-preference of turning behaviour

in mutants, the spontaneous behavioural abnormalities of the mutant

rats were recorded by observation of the animals in a new, empty

plastic cage (one rat per cage). Following onset of circling in this new

environment, the rotations were counted for a period of 5 min. Only

complete (360°) left or right rotations were counted for quanti®cation

of circling behaviour. Recording of circling in a new cage was

repeated at least twice in each rat (the average number of recordings

was ®ve per rat, using separate days per recording) to study the

consistency of the rotational behaviour. The percentage directional

(side) preference of each rat's turning behaviour in each test was

determined by the following formula: 100 3 (full turns in preferred

direction/total full turns). Only rats with a side-preference of more

than 70% were used for the present experiments. Heterozygous (ci2/

+) rats (used as controls for comparison with the mutant ci2/ci2 rats)

showed no circling or other behavioural abnormalities in the home

cage or a new environment.

Rats were housed in groups under a 12-h light : 12-h dark cycle

(lights on at 07.00 h) and permitted food and water ad libitum. All

experiments were performed during the light period. All animal care

and handling was conducted in compliance with the German Animal

Welfare Act and was approved by the responsible governmental

agency in Hannover.

Testing of auditory function

Following testing for the presence of circling behaviour, rats of either

sex were classi®ed as homozygous (ci2/ci2) or heterozygous (ci2/+),

respectively (see above). We describe here recordings of brainstem

auditory-evoked potentials (BAEP) from six heterozygous and seven

homozygous adult ci2 rats (6±10 months of age). Individuals were

anaesthetized with an initial dose of chloral hydrate (360 mg/kg i.p.)

and placed on a controlled heating pad in a sound-attenuated (60 dB

measured at 1 kHz), double-walled anechoic chamber (G & H,

Hannover, Germany). Before and during the recording session, the

chamber was additionally heated by a 250-W heating bulb placed

about 80 cm above the animal. For maintaining anaesthesia,

supplemental doses of chloral hydrate were given when needed.

Rectal temperature was monitored and maintained at 37 °C. As a

measure of the physiological state and the depth of anaesthesia, a

combined electrocardiogram and muscle-potential recording was

monitored using two stainless steel insect pins (0.5 mm diameter),

which were inserted subcutaneously into the left extremities. At the

end of the experiment, the animals were allowed to recover from

anaesthesia in a moderately heated cage. For acoustic stimulation and

recording of BAEPs, we used hardware (System II) and software

(BioSig, SigGen) from Tucker-Davis Technologies (Gainesville,

Florida). Pure tones with a frequency of 5±60 kHz and sound pressure

levels (SPLs) from 20 to 120 dB (ref. 20 mPa) were presented in 5 dB

steps with a 10-ms duration (2 ms rise/fall-time) at a rate of 4 Hz.

Acoustic stimuli were generated using a digital signal processor,

which controlled a digital-to analogue-converter (Tucker-Davis

Technologies). The output of the converter was routed through an

antialiasing ®lter and digital attenuators which were either connected

to a low-frequency loudspeaker or an ampli®er connected to an

electrostatic high-frequency loudspeaker. Speakers were mounted in

front of the animal with a distance of 5 or 10 cm from the ear canal

openings and a 1/4¢ B & K microphone (BruÈel and Kjaer Denmark,

Type 4135, Spectris, Germany) was placed above the head for

recording of the sound stimuli. Calibration of the speaker was

performed using the same microphone without the animal at a

midline position at the same speaker distance as used in the recording

sessions.

For binaural recordings of BAEPs, four stainless steel insect pins

with a diameter of 0.5 mm were inserted subcutaneously with the

active recording electrodes (+) placed about 5 mm lateral from the

dorsal midline above the cerebellum. The reference electrodes (±)

were placed close to the ventral parts of the pinnae. In addition, a

ground electrode was placed subcutaneously at a mid position of the

lower jaw.

Signals from the electrodes were fed to a HS4 headstage, digitized

and routed to a digital control unit (DB4) via ®bre optic cables

(Tucker-Davies Technologies). The ampli®cation of the control unit

was set to 700 000 with band pass ®lter settings of 0.3 kHz and

3 kHz. Outgoing signals were fed to an analogue-to-digital-converter

(ADC; AD 1) with the sampling rate set to 40 ms. The ADC was

controlled via ®bre optic cables by a digital signal processor with

software (BioSig, Tucker-Davis Technologies) running on a personal

computer. Every stimulus was presented and averaged for 250 times,

stored on the computer hard disk and analysed off-line for the

presence of peaks. The minimum sound pressure level at a given

frequency that was necessary to evoke a peak within 8 ms of stimulus

1130 A. Kaiser et al.

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1129±1142

onset in the recorded trace was taken as a measure of threshold for the

audiograms presented in the result section.

Testing of vestibular function

Because vestibular-de®cient rodents are known to be unable to swim,

a swimming test was used (Porsolt et al., 1978) to test for vestibular

disruption as previously described for different rodent species with

inner ear defects (Gray et al., 1988; Sawada et al., 1994; Truett et al.,

1994; Sondag et al., 1998). The rats were individually forced to swim

in a rectangular box (50 3 50 3 50 cm), three quarters ®lled with

warm (30±35 °C) water, from which they could not escape. While in

the water the rats were observed to determine if they showed `normal'

swimming behaviour. Normal swimming behaviour was de®ned as

follows: after being placed in the water, normal rats quickly

resurfaced, managed to keep their noses above the surface of the

water and swam towards the wall of the cylinder.

A further test for vestibular dysfunction in rats with inner ear

defects is the air-righting re¯ex test (Ossenkopp et al., 1990). For this

test, rats are held supine and are dropped from a height of 30 cm onto

a foam cushion. While normal rats are able to right themselves in the

air and land on their feet, rats with vestibular dysfunction exhibit an

impairment of this normal righting re¯ex, thus, landing on their back

or side (Ossenkopp et al., 1990).

To test for postural changes associated with vestibular defects in

rats, the tail-hanging test was performed (Hunt et al., 1987). This test

consists of lifting the rats by the tail. Normal rats extend their

forelimbs toward the earth, thus, showing a `landing' response. In

contrast, rats with vestibular dysfunction tend to bend ventrally, thus,

tending towards an `occipital' landing (Hunt et al., 1987).

In all tests for vestibular function, mutant rats were compared with

age-matched, heterozygous (ci2/+) rats and rats of the background

strain (LEW/Ztm). In the air-righting re¯ex and tail-hanging tests,

each mutant or control rat was evaluated three times in each test.

Only rats showing a de®cit in each of the three tests were considered

to have a vestibular defect.

Cochlear and vestibular histology

Following the auditory-evoked potential testing, rats were reanaesthe-

tized and perfused transcardially with ringer solution followed by a

solution of 4% formaldehyde in 0.1 M phosphate buffer solution.

Immediately after the perfusion, the brains and the bullae including

the cochleae and vestibular organs were removed and stored for

several days in the same ®xative as above. Brains were processed for

cyto- and immunohistology of brainstem nuclei (see section below).

The cristae ampullaris, the macula utriculi and the macula sacculi

were removed from the bony labyrinth while the bone overlying the

cochleae was thinned with forceps and razor blades. To permit a

better penetration of the embedding medium, the bony and

membranous labyrinth were carefully opened on one side of the

cochleae. Following preparation, specimens were decalci®ed and

embedded in araldite (Durcupan, Fluka; Deisenhofen, Germany). For

a better visualization, some of the specimens were ®xed with osmium

tetroxide before the embedding procedure. Semi-thin sections (3±

5 mm) were cut using a Leica Microtom RM 2065 Supercut with a

diamond knife for thick sectioning (Diatome, Switzerland), mounted

on glass slides and coverslipped or counterstained with toluidine blue

to enhance visibility of cell types and stereocilia. Sections were

analysed with a Zeiss Axiophot microscope using bright light or

phase contrast illumination and documented with a cooled digital

camera with a 1315 pixels 3 1035 pixels CCD chip and a PC-based

software package (SPOT camera, Diagnostic Instruments;

MetaMorph, Universal Imaging Corporation; both Visitron

Systems, Puchheim, Germany). Digital processing of recorded

images was restricted to a shading correction, an unsharp mask ®lter

and brightness and contrast enhancement when necessary.

Histological examination of the cochlear and vestibular nucleiin the brainstem

The morphological studies of the cochlear and vestibular nuclei and

the cochlear neurons were performed in brain sections of nine

homozygous circling rats (ci2/ci2; six males, three females) and eight

heterozygous noncircling littermates (ci2/+ three males, ®ve

females). The age of the animals was 5±12 months. Only two rats

(one homozygous and one heterozygous animal) were younger

(5 weeks old). Because data from these younger rats did not differ

from the other animals, data were combined into group values. The

average side-preference of circling of the mutant rats used for these

experiments was 85% (mean of 2±7 tests per rat).

For histomorphological examination, the rats were deeply

anaesthetized with chloral hydrate and transcardially perfused with

0.1% phosphate-buffered saline (PBS; pH 7.4) followed by perfusion

with 4% formaldehyde/PBS or in four cases with 4% paraformalde-

hyde/PBS. Brains were removed and placed in 30% sucrose/PBS

overnight. Coronal sections were cut at 40 mm on a freezing

microtome and every second section was mounted on glass slides,

air-dried and Nissl-stained with thionine (Sigma, Deisenhofen,

Germany). All sections were dehydrated in graded ethanols, cleared

in xylene and coverslipped with Entellan (Merck, Darmstadt,

Germany).

The following cochlear and vestibular nuclei were examined

(based on the atlas of Paxinos & Watson, 1998): the anteroventral

cochlear nucleus (AVCN), the posteroventral cochlear nucleus

(PVCN), the dorsal cochlear nucleus (DCN), the superior vestibular

nucleus (SVN), the medial vestibular nucleus (MVN; parvicellular

and magnocellular part), the lateral vestibular nucleus (LVN; anterior

and posterior part), and the inferior (spinal) vestibular nucleus (IVN).

All examinations were made for both sides of the brain by an

investigator who was not aware of the side-preference in rotational

behaviour of the ci2/ci2 rats. After completion of the histological

examinations, data from mutant rats were grouped with respect to the

ipsilateral and contralateral side of the brain, referring to the side-

preference of rotational behaviour of the ci2/ci2 rats, and to the left

and right side of ci2/+rats.

For comparison of the size of cochlear and vestibular nuclei, the

areas of the subnuclei were measured in all coronal sections

containing the respective nuclei with an image analysis system (KS

300, Zeiss, Germany) at 25 3 magni®cation. The volume was

calculated according to the following equation: volume = (area

section 1 + area section 2¼ + area section n) 3 distance between

sections. For these measurements, the MVN was not divided into

magnocellular and parvicellular. For the IVN, the size was only

determined for its rostral part (±11.00 to ±11.48 mm from bregma)

and for the AVCN only for its caudal part (±9.88 to ±10.46 mm from

bregma). Because of partial damage during histological preparation,

it was not possible to determine the volume of the PVCN and DCN.

For these nuclei, the mean areas of two to three corresponding

sections were compared between mutants and controls.

As described in the Results section, the cell bodies of cochlear

neurons of the ci2/ci2 rats seemed to be morphologically different

from those of the ci2/+rats. They appeared to be of a rounder shape,

smaller, and with fewer primary dendrites. For quantitative evalu-

ation of this observation, the area, the perimeter, and the circularity

shape factor (4 3 p 3 area/perimeter2) of 25 neurons per side and

animal were measured in one of the cochlear nuclei, the AVCN, at

Deafness and vestibular defects in the circling rat 1131


400 3 magni®cation. To have comparable sections of the AVCN for

these comparisons in all rats, sections ±10.33 6 0.24 mm from

bregma were used. For the comparison between mutants and controls,

it was not possible to compare different types of AVCN neurons,

because no clear differentiation into different types of neurons was

visible in the AVCN of mutant rats. In order to avoid inclusion of

neurons which were cut at the edge of the sections, only cell bodies

with nucleolus were measured. In the vestibular nuclei, no morpho-

logical differences between the neurons of ci2/ci2 and ci2/+rats were

obvious, so that these measurements were restricted to the AVCN.

In another series of measurements, neurons were counted in the

different nuclei, using the optical disector method (Coggeshall &

Lekan, 1996). For these measurements, the MVN was divided into a

magnocellular and a parvicellular part. For each cochlear and

vestibular nucleus, neuronal density was determined in three

consecutive sections. At a magni®cation of 400 3, neurons were

counted in 30 square ®elds of 1434 mm2 each. Three counts per side

and animal were made and averaged. All cell counts were expressed

as neurons/mm3. In order to have comparable sections for the

comparisons between mutants and controls, sections at the following

coordinates were used for determination of neuronal densities (all

values in mm from bregma): AVCN, ±10.3 6 0.16; PVCN,

±10.76 6 0.26; DCN, ±10.76 6 0.26; SVN, ±10.38 6 0.24; MVN

parvicellular, ±10.22 6 0.2; MVN magnocellular, ±11.22 6 0.2;

LVN anterior, ±10.38 6 0.24; LVN posterior, ±10.98 6 0.28; IVN,

±11.22 6 0.28.

Statistics

Signi®cance of differences within groups were calculated by paired

tests (Wilcoxon signed rank test or Student's t-test), whereas,

differences between groups were calculated by nonpaired tests

(Mann±Whitney U-test or Student's t-test), the choice of test

depending on whether data were normally distributed or not.

Signi®cance of differences in vestibular tests was calculated by

Fisher's exact test. All tests used were two-sided and a P < 0.05 was

considered signi®cant. Because there was no obvious difference

between sexes in any measure, data from both sexes were combined.

FIG. 1. Examples of auditory brainstem potentials from a heterozygous (unaffected) ci2/+ rat (left column) and a homozygous (mutant) ci2/ci2 rat (rightcolumn). To the left of each trace the recorded side (L, left; R, right), the stimulation frequency (kHz) and the dB sound pressure level (SPL) values areindicated. Because the thresholds for unaffected ci2/+ rats were not different between left and right ears, recording examples are only shown for the left ear.An attenuation of 0 dB corresponds to 105 dB SPL. Whereas in ci2/+ animals the amplitude of BAEPs clearly increased with sound pressure, in ci2/ci2 ratseven with high sound intensities no potentials could be recorded at either side. Calibration for amplitude and time is given in the right part of the ®gure.



Results

Behavioural abnormalities

As previously reported (LoÈscher et al., 1996), homozygous (ci2/ci2)

rats showed several abnormal behaviours when observed after

transfer to a new environment (new clean cage) during the light

phase. These abnormal behaviours occurred in phases or bursts

(typically lasting about 5±10 s) and were characterized by circling

behaviour, locomotor hyperactivity, brief periods of opisthotonus

(`stargazing'), and moderate ataxia. Circling consisted of tight 360°rotations. Typically, when ci2/ci2 rats were placed in the new cage

they exhibited bursts of intense circling for about 2±4 min after

which they became inactive, so that observation in each trial was

restricted to 5 min. In a total of 88 mutant rats, the majority of rats

(85 out of 88 = 97%) exhibited a side-preference of at least 70% in

their rotational behaviour during a 5-min test trial. Repeated testing

of the same rats on separate days demonstrated that the side-

preference was consistent in 80% of the animals. Two subgroups

were observed, i.e. those showing right-sided and left-sided rotational

preferences. The average number of rotations of the 88 ci2/ci2 rats in

three subsequent 5-min trials was 18.8 6 1.4. Heterozygous (ci2/+)

littermates or rats from the background strain (LEW/Ztm) did not

show any circling or other behavioural abnormalities in the new cage.

Testing of auditory function

We initially observed that the mutant rats did not respond to intense

ultrasonics, which suggested that they have impaired hearing ability.

To quantify their hearing range and sensitivity, auditory-evoked

brainstem responses were recorded.

Comparison of hearing abilities of both groups of ci2 rats showed

unequivocally that the mutant circling (ci2/ci2) rats were completely

and binaurally deaf within the tested range of frequencies and sound

pressure levels (Figs 1 and 2). In contrast to an increasing BAEP

amplitude as a response to an increasing sound pressure level in

heterozygous ci2/+rats, no BAEP's could be recorded at any tested

stimulus for ci2/ci2 animals (Fig. 1). The overall course of the

averaged audiogram of the heterozygous group of rats resembles

those of other laboratory rats (cf. Fay, 1988). Maximum sensitivity

was in the frequency range of 10±30 kHz with individual thresholds

between 40 and 60 dB SPL (Fig. 2).

Testing of vestibular function

To test for vestibular function in the mutant rats, a swimming test was

conducted. A total of 30 adult ci2/+ rats and 30 adult ci2/ci2 mutant

rats were tested. Furthermore, some rats of the background strain

(LEW/Ztm) were examined. All the heterozygous ci2/+ and LEW/

Ztm rats displayed normal swimming behaviour resulting in proper

orientation of the rats with respect to the water surface. However, all

the ci2/ci2 mutant rats showed abnormal swimming behaviour

associated with lack of orientation: when placed in water, the

mutants spiralled underwater (in a corkscrew fashion) unable to

maintain their noses above the water surface. Mutants needed to be

rescued promptly to prevent drowning. Like all other behavioural

abnormalities (lateralized, circling, hyperactivity, ataxia, opisthoto-

nus), the swimming inability of the ci2/ci2 mutant rats did not change

with age as indicated by tests in rats of different age up to 1.5 years.

In the air-righting re¯ex and tail-hanging tests, 23 ci2/ci2,

nine ci2/+ and eight LEW/Ztm rats were examined. Whereas all 17

controls (ci2/+or LEW/Ztm) showed a normal air-righting re¯ex in

three subsequent tests, ci2/ci2 rats were less successful as the controls

to right themselves in the air and to land on their feet: data for the 23

ci2/ci2 rats in the three tests (number of rats with normal air-righting/

number of rats tested) were 3 out of 23, 8 out of 23, and 6 out of 23.

Twelve ci2/ci2 rats showed no normal air-righting in all three tests,

which was signi®cantly different from controls (P = 0.0003).

In the tail-hanging test, all controls (n = 17) extended their

forelimbs toward the ear, thus, showing a `landing' response in all

of the three subsequent tests. In contrast, most of the 23 ci2/ci2 rats

tended to bend ventrally and curl up their tail, thus, tending towards

an `occipital landing'. Some ci2/ci2 rats also showed a lateral body

bias during hanging, as described for rats with unilateral lesions of the

nigrostriatal pathway (Abrous et al., 1998), but there was no clear

correlation between this lateral bias and the preferred direction of

circling. In 13 ci2/ci2 rats, abnormalities in the tail-hanging test were

seen in all three tests, the difference from controls being highly

signi®cant (P = 0.0001).

Inner ear defects

A total of ®ve adult mutant circling (ci2/ci2) rats was examined for

inner ear defects. Five heterozygous (ci2/+) control rats served as

controls. The gross anatomic inspection of the bony and membranous

labyrinth of the circling ci2/ci2 rats did not reveal any differences

compared to heterozygous rats (Fig. 3a and b). Microscopical

examination of semi-thin sections of the inner ears from both sides,

however, showed clear defects in the cochlear hair cells of ci2/ci2 rats

(Fig. 3d and f). In the cochleae of homozygous ci2 rats, the basilar

membrane, the tectorial membrane and the Reissner's membrane

appeared normal, but the Organ of Corti, including inner and outer

hair cells, supporting cells and the Tunnel of Corti was completely

absent or occasionally reduced to a disarranged cell layer. There was

no longitudinal gradient in hair cell loss along the cochlea. As a result

of the hair cell degeneration, the cochleae of those animals are

functionally defective resulting in the observed complete deafness

FIG. 2. Audiogram of brainstem auditory-evoked potentials (BAEPs). Thelower curve shows the averaged audiogram from six ci2/+ rats as obtainedfrom BAEPs. Thresholds from individual animals (in dB sound pressurelevel, SPL) are indicated by different symbols above and below the curve.There was no difference in threshold for simultaneous recordings from theleft and right side of the brainstem (not shown). As there were no responsesdetected in ci2/ci2 rats at any combination of frequency and sound pressurelevel, the upper curve (open circles) represents the maximum soundpressure levels used for stimulation at the individual frequencies.



FIG. 3. Illustration of results from the study of inner ear morphology and cochlear histology of unaffected rats (ci2/+, left column, a, c and e) and mutantcircling rats (ci2/ci2, right column b, d and f). The anatomical (a and b) and histological (c±f) investigation of the hair cell organs of adult mutant ci2/ci2 ratsrevealed a normal gross anatomical appearance of the bony and membraneous labyrinth (b, d and f). Compared to heterozygous rats (a, c and e) the bony andmembraneous labyrinth (b, d and f) of the mutant circling rats showed a normal gross anatomical appearance of the turns of the cochlea. Highermagni®cation light-microscopical examination of the cochleae (d and f) of mutant circling rats, however, clearly showed the absence of hair cells andsupporting cells in the cochlea, while in the heterozygous animals those cell types were present. (c and d) Mid-modiolar section through a ci2/+ (c) and aci2/ci2-cochlea (d) showing different turns of the cochlea. In all turns of the ci2/ci2 rats both types of hair cells and supporting cells were absent. In rareexamples, remnants of the Organ of Corti were occasionally seen (not shown). Those cells did not form a Tunnel of Corti and had a disordered appearancecompared to a normal cochlea. Note that a rupture of the Reissner's membrane (for orientation see also e and f) as shown in the turn to the lower left side ind was due to preparation artifacts and was not speci®c for mutant rats. (e and f) Higher magni®cation of a section through the middle turn of the cochlea of aci2/+ (e) and a ci2/ci2 rat (f).Note that compared to the ci2/+ rats (e and c) there is an almost complete loss of ganglion cells in the ci2/ci2 mutant (f and d).SV, scala vestibuli; SM, scala media; ST, scala tympani; RM, Reissner's membrane; TM, tectorial membrane; BM, basilar membrane; St.V, Stria vascularis;IHC, inner hair cell; OHC, outer hair cell; GC, ganglion cochleare.

FIG. 4. Histology of vestibular organs in unaffected rats (ci2/+ left column, a, c, e and g) vs. circling (ci2/ci2) mutant rats (right column, b, d, f and h). Fromtop to bottom examples from the sacculus (a and b), utriculus (c and d), Crista ampullaris (e and f) and Scarpa's ganglion (g and h) are shown. In allepithelia, type I and type II hair cells and supporting cells were present. Supporting cells appear as a basal monolayer of cells beneath the hair cells.Compared to the unaffected ci2/+ animals (a, c and e) we found abnormal cytoplasmic protrusions (b, d and f) in some of the hair cells as indicated by openarrows. Furthermore in the ci2/+ rats, stereovilli appeared longer compared to the ci2/ci2 rats (®lled arrows). A qualitative comparison of the vestibularganglions of both phenotypes of rats revealed that there are less cell bodies and less thicker axons in the ci2/ci2 rats.



(see above). Furthermore, ganglion cell degeneration was clearly

visible in mutant rats (Fig. 3d and f).

In contrast to the lack of hair cells and ganglion cells in the cochlea

of ci2/ci2 mutants, the gross appearance of the epithelia of the

vestibular organs and Scarpa's ganglion appeared to be not different

from ci2/+ rats (Fig. 4). Both types of hair cells (type I, type II) were

present in the utriculus, sacculus and the cristae. Further inspection of

the epithelia, however, revealed that for some hair cells, the

cytoplasm is protruding into the endolymphatic space and that the

stereovilli appear shorter compared to controls (Fig. 4b and d and f).

Within Scarpa's ganglion, the cell bodies and ®bres had a normal

appearance (Fig. 4g and h), but the number of ganglion cells and the

number of thick axons tended to be reduced (Fig. 4h).

Morphology of the cochlear and vestibular nuclei in thebrainstem

In a ®rst series of experiments, the volume of the cochlear and

vestibular nuclei was determined separately for the left and right

hemisphere in unaffected (ci2/+) rats and ipsilateral and contralateral

in mutant rats, ipsilateral being the preferred direction of circling. As

shown in Table 1, no signi®cant volume asymmetries between the

ipsi- and contralateral hemisphere were found for the cochlear or

vestibular nuclei in mutant rats. Compared to heterozygous controls,

the volume of the MVN of mutants was signi®cantly smaller (by

about 40%), while no signi®cant differences were seen for the other

subregions of the vestibular or cochlear nuclei. However, there was a

clear tendency for cochlear nuclei of mutant rats to be smaller (by

about 25% on average) than respective nuclei in controls.

In a second series of experiments, the morphology of neurons in

vestibular and cochlear nuclei was compared between homozygous

ci2/ci2 rats and heterozygous littermates. The cell bodies of neurons

in all cochlear nuclei of mutant rats had an atypical round shape

which only rarely gave rise to primary dendrites, whereas normal

neuronal shape with emerging dendrites was seen in heterozygous

controls (Fig. 5). In order to quantify these differences between

mutant and ci2/+ control rats, the shape of neurons in the AVCN was

evaluated by image analysis. As shown in Fig. 6A, neurons of mutant

rats in the ipsi- and contralateral AVCN had a rounder shape than

respective neurons in controls (P < 0.0001). Furthermore, the

perimeter of ipsi- and contralateral AVCN neurons was signi®cantly

smaller (Fig. 6B) and the soma tended to be smaller (Fig. 6C)

compared to unaffected controls. In contrast to the cochlear nuclei of

mutant rats, neurons in the vestibular nuclei appeared normal and did

not exhibit any obvious morphological difference from heterozygous

control rats (not illustrated).

In a third series of experiments, neuronal density was determined

in the cochlear and vestibular nuclei of ci2/ci2 and ci2/+ rats using

the optical disector counting technique. As already indicated in

Fig. 5, neuronal density in the different cochlear nuclei was

signi®cantly higher in mutant rats compared to controls (Fig. 7).

No differences in neuronal density was obvious between ipsi- and

contralateral hemisphere in mutant or right and left hemisphere in

heterozygous control rats. In the vestibular nuclei of mutant and

control rats (Fig. 8), no signi®cant difference in neuronal density was

seen in the SVN, IVN and LVN, whereas, mutant rats had a

signi®cantly lower neuronal density than controls in the MVN. The

only regions in which we found a signi®cant difference between

ipsilateral and contralateral hemisphere in mutant rats was the

anterior LVN, in which neuronal density ipsilateral to the preferred

direction of circling was signi®cantly lower compared to the

contralateral side (Fig. 8).

Discussion

The auditory-evoked potential testing demonstrated a complete

hearing loss in the adult ci2/ci2 mutant rat, whereas, heterozygous

littermates exhibited a hearing range, which lay within the sensitiv-

ities reported from other laboratory strains (Fay, 1988). Histological

analysis of the inner ear revealed virtually complete loss of the

TABLE 1. Volume of subregions of cochlear and vestibular nuclei in mutant circling (ci2/ci2) rats and unaffected heterozygous (ci2/+) controls determined by

image analysis

Region

Volume

Control (ci2/+) Mutant (ci2/ci2)

Left Right Ipsilateral Contralateral

Cochlear nucleusAVCN (mm3) 0.419 6 0.041 0.390 6 0.054 0.284 6 0.029 0.302 6 0.027PVCN (mm2) 0.551 6 0.052 0.479 6 0.055 0.381 6 0.046 0.398 6 0.027DCN (mm2) 0.512 6 0.068 0.583 6 0.091 0.397 6 0.069 0.411 6 0.017

Vestibular nucleusSVN (mm3) 0.205 6 0.022 0.235 6 0.037 0.164 6 0.018 0.178 6 0.024MVN (mm3) 0.981 6 0.050 1.000 6 0.045 0.649 6 0.076* 0.603 6 0.060*LVN (mm3) 0.470 6 0.029 0.469 6 0.025 0.433 6 0.098 0.413 6 0.100IVN (mm3) 0.386 6 0.024 0.350 6 0.028 0.262 6 0.053 0.328 6 0.034

For the IVN, volume data refer to the rostral part of this subregion, for the AVCN volume data refer to the caudal part of this subregion. In the case of PVCN andDCN, partial damage during histological preparation did not allow us to determine the volume of these subregions; instead, the mean areas of two to threecorresponding sections were determined (in mm2) and used for comparison between mutants and controls. Data are means 6 SE of six to seven mutants and sevencontrols. All mutants exhibited a clear rotational side-preference, so that data from mutants are shown ipsilateral and contralateral to the preferred direction ofturning. Controls showed no turning or other behavioural abnormalities, so that data from controls are shown for the left and right hemisphere. Signi®cantdifferences between controls and mutants are indicated by asterisk (*P < 0.05). The MVN of mutants was signi®cantly smaller compared to controls, irrespectiveof whether the ipsi-/contralateral data or data from left and right hemisphere were used for comparison with controls (ANOVA: P < 0.0001). In mutants, data fromipsi- and contralateral sites did not differ signi®cantly. In controls, data from left and right hemisphere did not differ signi®cantly except for PVCN (P = 0.0313).AVCN, anteroventral cochlear nucleus; PVCN, posteroventral cochlear nucleus; DCN, dorsal cochlear nucleus; SVN, superior vestibular nucleus; MVN, medialvestibular nucleus; LVN, lateral vestibular nucleus; IVN, inferior vestibular nucleus.



cochlear neuroepithelium and ganglion cells in the adult mutants,

while no hair cell degeneration was seen in unaffected ci2/+ rats. The

histological ®ndings in mutant circling rats suggest that the hearing

loss of the mutants is of the sensory neural type, the most prevalent

type of hearing loss. Genetic deafness is a frequent sensory disorder

in the human population, affecting about 1 in 2000 births (Gasparini

et al., 1999; Skvorak Giersch & Morton, 1999). Many of these

patients show primary abnormalities of the sensory neuroepithelia of

the inner ear, as do several hearing-impaired mouse mutants (Fekete,

1999; Gasparini et al., 1999; Probst & Camper, 1999). In recent

years, mouse mutants have played an important role in the

identi®cation of human hereditary hearing loss genes (Steel, 1995;

Hughes, 1997; Fekete, 1999; Probst & Camper, 1999). To date,

numerous different genetic conditions associated with hearing

impairment have been described, and several of the genes involved

in these forms have been mapped and identi®ed, showing that myosin

and connexin mutations and mitochondrial dysfunction can lead to

deafness (Martini et al., 1997; Steel et al., 1997; Koehler et al., 1999;

Redowicz, 1999; Skvorak Giersch et al., 1999). Despite the

widespread use of rats in studies on auditory and vestibular functions,

in contrast to mice, only few mutants with hereditary deafness have

been described in the rat (Hedrich, 1990).

In the ci2/ci2 rat, in addition to the hair cell loss in the cochlea,

neurons in the cochlear nuclei of the brainstem exhibited morpho-

logical alterations, reduced size and increased density. A similar

change in morphological characteristics, i.e. smaller but densely

packed neurons, was found in deaf subjects of various species,

including mouse (Willott et al., 1994), guinea pig (Lesperance et al.,

1995), cat (Saada et al., 1996), dog (Niparko & Finger, 1997), and

man (Seldon & Clark, 1991). The shrinkage of cells in the cochlear

nuclei is thought to result from the lack of synapses formed by

incoming afferent ®bers from the cochlea to these primary auditory

neurons. The increased density of cochlear neurons was found to be

secondary to a reduced volume of these nuclei (Willott et al., 1994;

Saada et al., 1996).

In contrast to the marked abnormalities of the cochlear hair cells in

ci2/ci2 rats, no obvious hair cell degeneration was seen in the

different parts of the vestibular apparatus of the inner ear of adult

mutants by the methods employed. Thus, under light microscopy, the

epithelia of the cristae as well as those of the maculae utriculi and

sacculi had a normal gross appearance. However, examination of the

surface of the vestibular epithelia with light microscopy revealed a

possible cause for the difference of vestibular function in ci2/ci2

mutants. At least some hair cells of the epithelia of the utriculus,

sacculus and the cristae ampullaris showed a protrusion of the

FIG. 5. The micrographs show nissl-stained neurons from corresponding locations of the anteroventral cochlear nucleus of a heterozygous (nonaffected,control) ci2/+ (a and b) and a mutant ci2/ci2 rat (c and d). It can clearly be seen that in ci2/ci2 animals the cell bodies have a rounder shape with fewer cellprocesses. Furthermore, higher densities of cell bodies can be observed in ci2/ci2 rats. The same abnormalities were seen in all other parts of the cochlearnucleus, but not in the vestibular nuclei.



cytoplasm into the endolymphatic space. A similar defect has been

described for the shaker-2 mouse, in which, besides other

ultrastructural alterations, vestibular hair cells were observed pro-

truding into the endolymphatic space (Anniko et al., 1980). Such a

deformation of hair cells can be probably related to an alteration of

structural components of the cytoskeleton including the cuticular

plate and the stereovilli. Interestingly, recent studies in the shaker-2

mouse mutation, the homologue of human DFNB3, causing deafness

and circling behaviour, have discovered that mutation of the

unconventional myosin gene, Myo15, is responsible for the vestibular

defects, deafness and inner ear morphology of these mice (Probst

et al., 1998). We currently investigate whether mutations in

unconventional myosins may also be responsible for the phenotype

of the ci2/ci2 rat mutant.

The morphology of neurons in the vestibular nuclei did not differ

from controls, but the size and neuronal density of the MVN was

signi®cantly reduced. Abnormal vestibular function in ci2/ci2 rats

FIG. 6. Morphology of neurons in the cochlear nucleus as determined bymorphometric imaging analysis. Per rat and hemisphere, 25 neurons wererandomly chosen from the anteroventral cochlear nucleus (AVCN), usingsections at the same coordinates in mutants and controls. For each neuron,the circularity shape factor (A), the perimeter (B) and the area of the soma(C) were determined and averaged. These averaged values were then usedto calculate the means 6 SE of six to seven ci2/ci2 mutants and seven ci2/+ controls. All mutants exhibited a clear rotational side-preference, so thatdata from mutants are shown ipsilateral and contralateral to the preferreddirection of turning. Controls showed no turning or other behaviouralabnormalities, so that data from controls are shown for the left and righthemisphere. *P < 0.01, signi®cant differences between controls (left andright hemisphere) and mutants (ipsi- and contralateral hemispheres).

FIG. 7. Neuronal density determined by the optical disector countingtechnique in thionine-stained sections of nonaffected (heterozygous) controlsand mutant rats for the different cochlear nuclei. Data are means 6 SE ofsix to seven mutants and eight controls. *P < 0.05, signi®cant differencesbetween controls (left and right hemisphere) and mutants (ipsi- andcontralateral hemispheres). For further explanations see Fig. 5 legend.AVCN, anteroventral cochlear nucleus; PVCN, posteroventral cochlearnucleus; DCN, dorsal cochlear nucleus.



was indicated by the inability of these mutants to swim, because

swimming inability is a well-known consequence of vestibular

defects in rodents (Gray et al., 1988; Sawada et al., 1994; Truett et al.,

1994; Sondag et al., 1998). Interestingly, inability to swim and

deafness have also been reported for the circling stargazer rat mutant

(Truett et al., 1994), whereas, the recently described circling

(waltzing) rat mutant identi®ed in Pavia, Italy, could swim, but

exhibited other defects in vestibular function tests, including

abnormal behaviour in the tail-hanging test (Rabbath et al. 2001).

The ci2/ci2 rat also showed abnormal behaviour in this test and

righting problems in the air-righting re¯ex test, substantiating

vestibular dysfunction. Similar to the present ®ndings with ci2/ci2

rats, Rabbath et al. (2001) did not detect any gross alterations in

the vestibular sensory epithelium and concluded that the vestibular

de®cits may be due to other types of cellular impairment of the

labyrinth and/or abnormalities in the central processing of

vestibular information underlying gaze and postural control. We

have, however, found a more subtle deformation (see above) that

is a probable cause for the observed vestibular defects in the ci2/

ci2 rat mutant.

FIG. 8. Neuronal density determined by the optical disector counting technique in thionine-stained sections of nonaffected (ci2/+) controls and mutant rats forthe different vestibular nuclei. *P < 0.01, signi®cant differences between controls (left and right hemisphere) and mutants (ipsi- and contralateralhemispheres). For further explanations see Fig. 5 legend. SVN, superior vestibular nucleus; MVN, medial vestibular nucleus (separated into parvicellular andmagnocellular parts); LVN, lateral vestibular nucleus (separated into anterior and posterior parts); IVN, inferior vestibular nucleus.



In both the studies on the waltzing rat and the ci2/ci2 rat, adult

animals were used for histological examination of the inner ear. Thus,

it cannot be excluded that the morphology of cochlear and vestibular

hair cells was different during early development. For instance,

cochlear hair cells could be present or vestibular hair cells could be

reduced or absent during early stages of postnatal development.

Whereas, in mammals, cochlear hair cells do not have the ability to

regenerate, vestibular hair cells can regenerate (Forge et al., 1993;

Warchol et al., 1993; Rubel et al., 1995; Tanyeri et al., 1995; Lopez

et al., 1997). We have preliminary evidence for vestibular hair cell

loss in young (about 4 weeks of age) ci2 rat mutants (Kaiser et al.

2000) which would indicate that hair cell regeneration occurs during

further development of these animals, so that vestibular hair cells

appear morphologically normal in adult rat mutants examined in the

present study. In this respect, it is important to note that degeneration

of the cochlear and vestibular hair cells by administration of the

ototoxic drug, streptomycin to postnatal rats leads to deafness and

abnormal motor activity, including backward locomotion, circling,

repetitive head movements and hyperactivity, but that most of these

abnormalities, including the circling, are gradually diminished or

disappear during subsequent months, most likely as a result of

regeneration of vestibular hair cells (Alleva & Balazs, 1978). In

contrast, the lateralized circling behaviour and other abnormal motor

activities of the ci2/ci2 rats are present over the whole life-span of the

animals without any evidence for remission (LoÈscher et al., 1996).

One possible explanation for the circling and other motor

abnormalities in the ci2/ci2 rat is that peripheral vestibular defects

cause the circling, and that the central effects are simply a

consequence of the abnormal sensory input. Based on the present

data, it is not possible to determine whether circling, hyperactivity

and the other abnormalities exhibited by the ci2 rat mutant result from

abnormalities within the vestibular system, require a combination of

vestibular dysfunctions with other types of central defects, or occur

independently from the vestibular defects. An alternative possibility

to the vestibular system being the primary cause of the movement

abnormalities, is that the mutation affects susceptible tissues, such as

certain brain regions, in addition to the inner ear, resulting in

recognizable clinical syndromes as recently demonstrated for the

deafness dystonia syndrome as a result of a genetically induced

mitochondrial dysfunction (Jin et al., 1999; Koehler et al., 1999).

Based on our previous studies in ci2/ci2 rats and the fact that most

ci2/ci2 rats exhibit a consistent side-preference in their circling while

all alterations in the cochlear and vestibular system are bilateral, we

do not consider the abnormal circling behaviour of ci2/ci2 mutant rats

to be a direct consequence of vestibular dysfunction, but rather relate

this lateralized behaviour to the asymmetries in the striatal dopamine

system previously found in the mutants (LoÈscher et al., 1996; Richter

et al., 1999; Fedrowitz et al. 2000). As mentioned above, mutant rats

of both genders have a lower tissue content of dopamine in the

striatum ipsilateral to the preferred direction of rotation, indicating

that the rats turn away from the brain hemisphere with higher striatal

dopaminergic activity (LoÈscher et al., 1996; Richter et al., 1999).

Histological examination of the striatum and substantia nigra pars

compacta failed to disclose any morphological abnormality in the

mutant circling rat (Richter et al., 1999), indicating a biochemical,

rather than morphological, abnormality in the dopamine system.

Interestingly, whereas circling in the stargazer (stg) rat was initially

thought to be due to a vestibular defect (Truett et al., 1994),

subsequent studies led to the proposal that circling in this mutant is

due to genetically mediated dysfunction of the central dopaminergic

system (Brock & Ashby, 1996). These data in different circling rat

mutants suggest that the concept of circling behaviour in deaf rodent

mutants being simply a direct consequence of peripheral vestibular

defects needs to be reevaluated.

Whereas, abnormalities in dopaminergic function are likely

candidates to explain the circling behaviour and possibly also the

hyperactivity of the mutant ci2/ci2 rats, vestibular dysfunction could

cause the ataxia and opisthotonus observed in these animals.

Furthermore, as shown by the present experiments, the ci2/ci2

mutant rat is unable to swim, another balance disorder which is

probably a result of disturbed vestibular function. In view of the

various efferent connections of the vestibular nuclei (Rubertone et al.,

1995) and the ®nding that vestibular information is transmitted not

only to the cerebellum, cortex and spinal cord but also, via the

thalamus, to the striatum (Shiroyama et al., 1999), it is conceivable

that abnormalities in vestibular nuclei during brain development lead

to secondary changes in brain regions such as the striatum thought to

be critically involved in circling behaviour (Pycock, 1980; Carlson &

Glick, 1996). This view is in line with previous observations of

Alleva & Balazs (1978) using the ototoxic drug streptomycin in rats.

Whereas, this drug induced head tremor and dif®culty with the

righting re¯ex in adult rats, administration of streptomycin to

postnatal rats induced circling, hyperactivity, repetitive head move-

ments and backward locomotion. Alleva & Balazs (1978) concluded

from their experiments with streptomycin-induced auditory and

vestibular defects in rats, that the neurological syndrome developing

in postnatal rats involves both the vestibular apparatus and higher

motor centres and that particularly the hyperactivity and circling are

suggestive of a central site of action, presumably involving a

dopaminergic mechanism. In a subsequent study, this group found

increased dopamine D2 receptor binding in the striatum of dyskinetic

rats that had been treated subacutely with streptomycin, as neonates

(Seth et al., 1982). In ci2/ci2 mutant rats, we also recently found

increased dopamine receptor binding in the striatum (Richter et al.,

1999). Thus, the data from these different models seem to suggest

that vestibular defects during postnatal development, independent of

whether induced or inherited, lead to secondary changes in the

dopaminergic system within the basal ganglia, which would be a

likely explanation for the typical phenotype seen in these models.

In conclusion, the ci2 rat mutant provides interesting features as a

new model in the ®eld of deafness research and may be useful for

studies on cochlear and vestibular dysfunctions. Furthermore, the

behavioural phenotype of the ci2/ci2 rat mutant makes this animal an

interesting model for hyperkinetic movement disorders. Our previous

data on these rats suggest an involvement of the basal ganglia in the

abnormal circling behaviour of the ci2/ci2 mutant (LoÈscher et al.,

1996; Richter et al., 1999; Fedrowitz et al. 2000), whereas, the

present data suggest that abnormal vestibular function may be

responsible for the disturbed balance of the rats as exempli®ed by the

ataxia and the inability to swim. The genetic defect in the mutant rats,

thus, leads to a clinical syndrome with features also seen in human

genetic disorders with deafness and hyperkinesia, e.g. the human

deafness dystonia syndrome (Jin et al., 1996; Koehler et al., 1999).

The underlying genetic and neurochemical substrates of this

syndrome in circling mutant ci2/ci2 rats await further study.

Acknowledgements

We thank Dr Ivan Lopez (Division of Head and Neck Surgery, UCLA, LosAngeles, USA) for technical advise regarding the histological examination ofvestibular hair cells. Furthermore, we thank Dr GuÈnter Reuter and Dr SvenCords (Department of Otolaryngology, Medical School Hannover) for initialexamination of auditory-evoked potentials in mutant rats and Dr AngelikaRichter for advise and discussion. The experiments on the air-righting re¯ex



and tail-hanging were performed by Dr Sven Lindemann and Ms. AlinaLessenich. The technical assistance of Ms. E. Engelke, Ms. C. Bartling, Mr M.Weissing, and Mr M. Meyer is gratefully acknowledged. The study wassupported by a grant from the Deutsche Forschungsgemeinschaft (Lo 274/8±1).

Abbreviations

AVCN, anteroventral cochlear nucleus; BAEP, brainstem auditory-evokedpotential; DCN, dorsal cochlear nucleus; IVN, inferior (spinal) vestibularnucleus; LVN, lateral vestibular nucleus; MVN, medial vestibular nucleus;PBS, phosphate-buffered saline; PVCN, the posteroventral cochlear nucleus;SPL, sound pressure level; SVN, superior vestibular nucleus.

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auditory and vestibular defects in the circling (ci2) rat mutant

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