EARLY COMPENSATION OF VESTIBULO-OCULOMOTOR SYMPTOMSAFTER UNILATERAL VESTIBULAR LOSS IN RATS IS RELATED TO

GABAB RECEPTOR FUNCTION

A. K. MAGNUSSON,a* M. ULFENDAHLb and R. THAMc

aDepartment of Biomedicine and Surgery, Division of Cell Biology, Faculty of Health Sciences, SE-581 85 Linko«ping, SwedenbInstitute for Hearing and Communication Research and Department of Clinical Neuroscience, Karolinska Institutet,

SE-171 76 Stockholm, SwedencDepartment of Neuroscience and Locomotion, Faculty of Health Sciences, SE-581 85 Linko«ping, Sweden

Abstract6The horizontal vestibulo-oculomotor re£ex was studied in pigmented rats during the ¢rst 5 days after aunilateral chemical or surgical vestibular dea¡erentation. Spontaneous eye movements in darkness and slow phasevelocity gain of compensatory eye movements during horizontal sinusoidal rotation were evaluated. The most evidentvestibulo-oculomotor symptom immediately after a unilateral vestibular loss was a spontaneous nystagmus, whichgradually abated during the following days. Further, an asymmetry between ipsi- and contra-lesional gains was evidentduring sinusoidal vestibular stimulation. Single systemic doses of the GABAB receptor antagonist [3-[1-(S)-[[3-(cyclo-hexylmethyl)-hydroxyphosphinoyl]-2-(S)-hydroxypropyl]amino]ethyl]-benzoic acid (CGP 56433A), the agonist baclofen,or the GABAA receptor agonist (4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol (THIP) were given at di¡erent intervalsafter unilateral vestibular dea¡erentation. CGP 56433A highly aggravated the vestibulo-oculomotor symptoms, observedas an increase in spontaneous nystagmus and slow phase velocity gain asymmetry. This e¡ect was most pronouncedduring the ¢rst 2 days after unilateral vestibular loss, when CGP 56433A even decompensated the vestibular system to theextent that all vestibular responses were abolished. Baclofen caused no e¡ect during the ¢rst days after unilateralvestibular loss, but in parallel with the abatement of spontaneous nystagmus, the drug equilibrated or even reversedthe remaining spontaneous nystagmus with corresponding e¡ects on the slow-phase velocity gain asymmetry. The e¡ectsof baclofen were very similar after both chemical and surgical dea¡erentation. THIP caused a slight depression of allvestibular responses. All single dose e¡ects of the drugs were transient.Altogether these results reveal that endogenous stimulation of GABAB receptors in GABA-ergic vestibulo-oculomotor

circuits are important for reducing the vestibular asymmetry during the early period after unilateral vestibular deaf-ferentation. A possible role for GABAB receptors in the reciprocal inhibitory commissural pathways in the vestibularnuclei is suggested. D 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Key words: eye movement, baclofen, CGP 56433A, THIP.

Following an acute unilateral vestibular dea¡erentation(UVD), most vertebrates share an array of symptomssuch as spontaneous nystagmus with an ipsi-lesionalslow phase, eye deviation, head tilt, barrel rollingand circular walking (Dieringer, 1995; Curthoys andHalmagyi, 1995). These behavioural vestibulo-oculomo-tor and postural symptoms are reduced during a periodlasting from hours up to several days, depending on thespecies [Hamann et al., 1998; Magnusson et al., 2000(rat), Vibert et al., 1993 (guinea-pig), Baarsma andCollewijn, 1975 (rabbit), Maioli et al., 1983 (cat), Fetterand Zee, 1988 (monkey)]. After that period, a more or

less permanent partially compensated condition isreached. In the rat, the remaining de¢cits of the vesti-bulo-oculomotor system are characterised by a direc-tional preponderance observed as a slow ipsi-lesionaldrift of the eyes in darkness and asymmetrical compen-satory eye movements during horizontal rotational tests.Permanent postural de¢cits include an ipsi-lesional headtilt in the frontal plane and a twisting of the bodyaround its sagittal axis, when the animal is lifted bythe tail and the proprioceptive input from the legsexcluded (Hamann et al., 1998; Magnusson et al.,2000). The recovery of sensory-motor behaviour afterUVD is attributed to functional reorganisation of centralvestibular or parallel vestibular-related systems and notdue to regeneration of the vestibular sensory epitheliumand/or the vestibular primary a¡erents.The mechanisms behind the compensatory process

have been extensively studied in many species for a quar-ter of a century. However, there is yet no consensusregarding this multi-factorial process. One approach toinvestigate potential mechanisms, involved in the behav-ioural compensation, is to modulate neurotransmitter

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*Corresponding author. Tel. : +46-13-223929; fax: +46-13-223192.E-mail address: annma@mcb.liu.se (A. K. Magnusson).Abbreviations: CGP 56433A, [3-[1-(S)-[[3-(cyclohexylmethyl)-

hydroxyphosphinoyl]-2-(S)-hydroxypropyl]amino]ethyl]-benzoicacid; c.i., con¢dence interval; EDTA, ethylenediaminetetra-ace-tate; MVN, medial vestibular nucleus; SPV, slow phase velocity;THIP, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol; UVD,unilateral vestibular dea¡erentation.

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systems pharmacologically in vivo. It has been shownthat physiological stimulation of GABAB receptors con-tributes to normalisation of the vestibulo-oculomotorfunction in a partially compensated state, weeks ormonths after a UVD, by minimising the asymmetry inthe vestibular system (Magnusson et al., 1998, 2000).The aim of the present study was to investigate the

role of GABAB receptors during the initial period afterUVD, when an uncompensated condition of the vesti-bulo-oculomotor system, immediately after UVD, trans-forms to a partially compensated condition. Hence, weassessed the e¡ects of GABAB receptor inhibition orstimulation on vestibulo-oculomotor behaviour in alertrats during the ¢rst 5 days following a UVD. The UVDwas achieved either chemically, by an intra-tympanicinjection of sodium arsanilate, or surgically, by perform-ing a unilateral vestibular ganglionectomy/neurectomy.Also, the e¡ect of GABAA receptor stimulation wasassessed following a chemical UVD.

EXPERIMENTAL PROCEDURES

General preparation of animals

Sixty-six male DA-HAN rats (BK Universal, Sweden), weigh-ing 170^270 g, were used in 164 separate experiments. The ani-mals were housed in pairs and food and water was provided adlibitum. A normal daily awake/sleep cycle was maintained bymeans of a regular 12 h light/dark cycle. The number of animalsused was kept as low as possible and all e¡orts were made tominimise their su¡ering. All experiments were approved by theAnimal Care and Use Committee in Linko«ping in agreementwith Swedish law.The general preparations of the animals are described else-

where (Niklasson et al., 1990/91). In brief, all animals wereprepared for head ¢xation by attaching a metal nut to theskull utilising six stainless steel screws and dental acrylic duringpentobarbital sodium (50 mg/kg i.p.) anaesthesia. Following1 week of recovery, the animals were accustomed to beingrestrained in a padded plastic cage with their heads ¢xed bymeans of a screw inserted into the head nut. For recordings ofhorizontal eye movements, a magnetic search coil system wasused (Kasper et al., 1987). A small copper coil (external diam-eter 1.8 mm, 80 turns, weight 1 mg; IET, Marly, Switzerland)was glued to the temporal region of the cornea under topicalanaesthesia (Lidocaine 4%, Astra, Sweden). We made sure thatthe eye could move freely and that no signs of pain or stresswere present during the experiments. The head-¢xed rat wasplaced horizontally in the centre of a magnetic ¢eld which wasgenerated by two perpendicular pairs of single-turn coils. Theamplitude of the induction voltage generated in the eye coil waslinearly dependent on its horizontal angular position in the mag-netic ¢eld. Eye position signals due to eye movements and eyevelocity signals obtained by di¡erentiation of the eye positionsignals were recorded on-line (Linearcorder Mark VII, Graph-tec, Tokyo). Identical signals were also fed to a computer fordigital storage and o¡-line analysis. Slight variations in theshape of the pre-manufactured coils resulted in small di¡erencesin the ratio voltage output/angular orientation of the coils in themagnetic ¢eld. Hence, after an experiment, the search coil wasgently removed from the eye, attached to a plastic dummy andcalibrated within the accurate range of induction voltage. Thecorrelation factor was usually between 0.9 and 1.1.

Chemical UVD

Using a surgical microscope, 0.1 ml sodium^arsanilate (p-ami-nophenylarsonic acid, sodium salt; TCI, Japan) was injected

through the ventral part of the tympanic membrane on theright side of 50 animals during a short-lasting halothane (Fluo-thane) anaesthesia (Kaufman et al., 1993).

Vestibular ganglionectomy/neurectomy

Fourteen animals were anaesthetised with avertin (tribromo-ethanol, 300 mg/kg i.p.), and the site of surgery in¢ltrated withXylocain with adrenalin (2%, Astra, Sweden). The tympanicbulla was cleared with blunt dissection through a ventralapproach and opened by drilling until the pterygopalatineartery, running through the stapes, was clearly visible. Theartery was cauterised before the vestibulum was entered viathe region around the oval window with a drill. Care wastaken to spare the facial nerve during this procedure. Utriculus,sacculus and the visible ampullae (superior and lateral) werescraped out with hooks, needles and suction. The medial wallof the vestibulum was removed with a diamond drill-bit head(diameter 1 mm) until the dura was clearly visible but not dam-aged. With this approach both the superior and inferior divi-sions of the vestibular nerve and Scarpa’s ganglion should bedestroyed. Sham operations were performed when the bulla wasopened and the pterygopalatine artery cauterised in three ani-mals. Ringer’s solution with glucose was given by intraperito-neal and subcutaneous injections during the ¢rst post-operativedays.

Vestibular stimulation and analysis of eye movements

Spontaneous eye movements, without any vestibular stimu-lation, were recorded in darkness. The frequency of the sponta-neous nystagmus and slow phase velocity (SPV) were analysedo¡-line from chart strips.Whole body sinusoidal rotation of 0.2 Hz with a peak angular

velocity of 90‡/s was performed in darkness using a computer-controlled turntable. Signals were analysed o¡-line using Lab-VIEW 2 software (National Instruments, USA; for details seeAndersson et al., 1997). In brief, collected data were analysed in20 s blocks. Initially the saccades were removed, generatingcontinuous curves for both eye position and velocity. TotalSPV gain (SPV divided by turntable velocity) was estimatedby Fourier analysis. A separate ipsi-lesional SPV gain (duringrotation to the lesioned side) and a contra-lesional SPV gain(during rotation to the intact side) was estimated by comparisonof corresponding velocity curves with asymmetrical sinus signalswith di¡erent amplitudes, using a least-squares best-¢t curvealgorithm.

Time schedule of experiments and drug administration

All animals were weighed every day and postural symptomswere noted by observation of barrel rolling, circular walking,head tilt and twisting of the body around the sagittal axis,when the animal was lifted by the tail.Horizontal eye movements were recorded prior to application

of a drug according to the following test programme: (1) record-ing of spontaneous eye movements in darkness during 3^4 min,(2) recording of compensatory eye movements during 2 minspells of horizontal sinusoidal 0.2 Hz rotation in darkness, per-formed twice with a 5 min interval in between. The test drug,dissolved in saline, was injected intra-muscularly and recordingsof eye movements were usually obtained again after 20^30 min,according to the schedule above. After the ¢nal recording ses-sion, the animal was released from its ¢xation device and pos-tural symptoms and general behaviour were observed again. Thedrugs tested were the GABAB receptor antagonist [3-[1-(S)-[[3-(cyclohexylmethyl)-hydroxyphosphinoyl]-2-(S)-hydroxypropyl]-amino]ethyl]-benzoic acid (CGP 56433A; Novartis Pharma AG,Switzerland) at a dose of 2 Wmol/kg or 6 Wmol/kg, the GABABreceptor agonist baclofen (4-amino-3[p-chlorophenyl]-butyricacid, baclofen racemic mixture; Novartis Pharma AG, Switzer-land) at a dose of 14 Wmol/kg, and the GABAA agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol (THIP; Sigma-RBI,USA) at a dose of 57 Wmol/kg.

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The dose range for CGP 56433A was evaluated in pilotexperiments, both during the initial uncompensated as well asthe partially compensated state, before two e¡ective doses werechosen. Doses for baclofen and THIP were chosen on the basisof previous studies assessing dose-responsive characteristics ofboth drugs in partially compensated UVD rats (Magnusson etal., 1998). All three drugs were tested after chemical UVD whilstbaclofen was the only drug tested after surgical UVD.A majority of the animals participated more than once ac-

cording to a random pattern. At least 1 day elapsed betweenexperimental sessions. Following the ¢nal experimental sessionthe animals were killed with an overdose of anaesthetics.

Histology

The extent of the lesion was investigated in 7/14 animals aftersurgical UVD. After the experimental period, the animals weregiven an overdose of pentobarbital sodium before being per-fused transcardially with cold saline followed by phosphate-bu¡-ered (0.1 M) solution of glutaraldehyde (3%). The temporalbones were removed and the bulla opened on the intact sideto allow better penetration of glutaraldehyde during post-¢xa-tion. Following a standard procedure for decalci¢cation (0.1 MEDTA, approximately 3 weeks at room temperature), temporalbones were embedded in para⁄n. Serial sections (6 Wm) weremade on a microtome (LKB, Sweden), mounted on slides andstained with Toluidine Blue before being dried, cleared in xyleneand coverslipped.

Statistical analysis

Mean values of saccade frequency, and ipsi- and contra-lesional SPV gains during repeated sinusoidal rotation were cal-culated before and after application of drugs. Data presented in¢gures and tables display meanV 95% con¢dence intervals (c.i.).Saccade frequency was compared before and after drug admin-istration by means of Student’s paired t-test for equal variances.For comparison of SPV-gain asymmetry during sinusoidal stim-ulation before and after drug administration, the correspondingdi¡erence between contra- and ipsi-lesional gains was calculatedand Student’s paired t-test for equal variances applied. In alltests a di¡erence of P6 0.05 was considered statistically signi¢-cant (*P6 0.05; **P6 0.01; ***P6 0.001).

RESULTS

General behaviour and postural symptoms following UVD

Following unilateral arsanilate application, milderpostural symptoms were observed after 24 h, whichbecame maximal on the third day and included an ipsi-lesional head tilt in the frontal plane, ipsi-lesional circu-lar walking, barrel rolling and twisting of the bodyaround the sagittal axis when lifted by the tail. Thesepostural symptoms ameliorated within a couple of daysto subside into the characteristic partially compensatedsymptoms with a slight ipsi-lesional head tilt in the fron-tal plane and a twisting of the body along the sagittalaxis when lifted by the tail. Inter-individual variabilityregarding time-course and degree of recovery was verysmall for these symptoms. The animals showed no sig-ni¢cant changes in weight during the observation period.Following surgical UVD, an abnormal limb position

with an extension of the contra-lesional hindlimb and theipsi-lesional forelimb as well as ipsi-lesional barrel rollingwas observed as the anaesthetics wore o¡. These symp-toms ameliorated with some variability during the fol-

lowing days. A remaining ipsi-lesional head-tilt in thefrontal plane was present during the whole investigationperiod in 50% of the animals. When lifted by their tail,12/14 showed an ipsi-lesional sagittal twist of the body.The other two animals performed an unusual torsional£exion of their head and upper body towards their owntail with a clear ipsi-lesional twist. The surgical UVDanimals lost up to 20% of their weight during the ¢rstpost-operative days, in spite of receiving glucose ^ Ring-er’s solution supplement on a daily basis.

Vestibulo-oculomotor symptoms following UVD

Following chemical UVD, a spontaneous nystagmuswith an ipsi-lesional slow phase appeared after approx-imately 6 h. This nystagmus increased in frequency andSPV over the next 24 h up to values around 140 beats/min and 30‡/s, respectively. Within 1 week, the sponta-neous nystagmus had abated to a residual slow ipsi-lesional eye drift (6 5‡/s) in darkness (Fig. 1A).Approximately 1 h after surgical UVD, when the

anaesthetics wore o¡, spontaneous nystagmus wasrecorded. The saccade frequency and SPV increased upto 180 beats/min and 20‡/s, respectively, before abatingover the following 3 days (Fig. 1A). After 3 days, somerats showed a remaining ipsi-lesional slow eye driftwhilst other rats showed no or even a contra-lesionalslow eye drift in darkness.Both chemical and surgical UVD resulted in a severe

depression of the ipsi-lesional SPV gain as revealed dur-ing sinusoidal stimulation (Fig. 1B). After chemicalUVD, the contra-lesional gain remained relatively high,whilst a surgical UVD depressed it to a level only slightlybetter than the corresponding ipsi-lesional gain after thesurgical procedure (Fig. 1B). A larger individual varia-tion was observed after surgical than chemical UVD.Sham-operated animals showed no directional prepon-

derance, thus displaying no latent eye drift nor SPV-gainasymmetry during sinusoidal stimulation.

E¡ects of GABAB receptor mediated inhibition followingchemical UVD

Single doses of the GABAB receptor antagonist CGP56433A were given at di¡erent times after chemicalUVD. Two dose levels were investigated. A single doseof 2 Wmol/kg exaggerated the spontaneous nystagmus tomaximal values at 20^30 min after the injection (Fig. 2),probably re£ecting a steady-state or peak blood concen-tration at this time. When given during the ¢rst 2 daysafter UVD, a single dose of 6 Wmol/kg abolished allvestibular responses after 20^30 min (Fig. 2). This e¡ectwas preceded by a gradual exaggeration of the sponta-neous nystagmus up to around 200 beats/min with acorresponding SPV around 30‡/s already during the¢rst 5^10 min (Figs. 3 and 4). After this increase inspontaneous nystagmus, all vestibulo-oculomotor reac-tions disappeared abruptly (Figs. 3 and 4), a conditionwhich lasted about 2 h before the nystagmus reappearedagain (Fig. 4). When the higher dose was given on thethird and fourth day after UVD, it caused a signi¢cant

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GABAB receptor function during vestibular compensation 627

Fig. 2. Mean maximal saccade frequency directed to the intact side in darkness before (white columns) and 20^30 min aftersystemic CGP 56433A administration of 2 Wmol/kg (dark columns) or 6 Wmol/kg (patterned columns) versus time after achemical UVD. For explanation of the zero amplitude during the ¢rst 2 days see the Results section. Asterisks indicatesigni¢cant di¡erences of the frequency before and after drug administration evaluated by Student’s paired t-test. Error bars

indicate 95% c.i. and nv5 for each column. *P6 0.05; **P6 0.01; ***P6 0.001.

Fig. 1. (A) Mean saccade frequency in darkness versus time after UVD. Squares indicate chemical and triangles indicatesurgical UVD. (B) Mean SPV gain versus time following UVD. The contra-lesional gains are illustrated by ¢lled squares(chemical UVD) and triangles (surgical UVD), whilst ipsi-lesional gains are illustrated by un¢lled squares (chemical UVD)

and triangles (surgical UVD). Error bars indicate 95% c.i. and nv5 for each symbol at every time point.

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A. K. Magnusson et al.628

increase in spontaneous nystagmus without abolishingthe vestibular responses (Figs. 2 and 5). Furthermore,during this time period, a corresponding drug inducedincrease in SPV-gain asymmetry was evident duringsinusoidal stimulation (Fig. 5).In most of the experiments using CGP 56433A, eye

movement responses recovered to pre-injection valueswithin a couple of hours. The animals were alert andshowed no sign of drowsiness throughout the experi-ment.The postural symptoms were aggravated by CGP

56433A at both doses, given during the ¢rst 2 daysafter chemical UVD, with a more severe ipsi-lesionalhead tilt in the frontal plane and barrel rolling comparedto the pre-injection condition.

E¡ects of GABAB receptor mediated stimulation followingchemical or surgical UVD

Single doses of baclofen were given at di¡erent timesafter either chemical or surgical UVD. No e¡ects relatedto the drug were observed either during the ¢rst 2 daysafter arsanilate application or the ¢rst day after surgicalUVD (Figs. 6 and 7). However, in parallel with the grad-ual abatement of spontaneous nystagmus, e¡ects ofbaclofen emerged. Thus, from the third day and onwardsafter chemical (Fig. 6A) and the second day and onwardsafter surgical UVD (Fig. 6B), baclofen abolished theremaining nystagmus or even reversed it into a sponta-neous nystagmus with a contra-lesional slow phase direc-tion.

Fig. 4. Saccade frequency versus time after a systemic administration of CGP 56433A (6 Wmol/kg) from one rat 24 h after aright-sided chemical UVD. The response to the drug was registered every minute until all eye movements were abolished andfrom there on registered every ¢fth minute. During an interval between 110 and 130 min the saccade frequency was di⁄cultto estimate due to an irregular pattern, whilst the saccade frequency had recovered enough to give reliable values during the

remaining period of time.

Fig. 3. Horizontal spontaneous nystagmus from one rat 24 h after a right-sided chemical UVD. Eye movements wererecorded before as well as 7 and 20 min after systemic administration of CGP 56433A (6 Wmol/kg). (A) Eye-position signal ;(B) eye-velocity signal. Calibrations for eye-position and -velocity signals are indicated by vertical bars and the time scale by

the horizontal bar.

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GABAB receptor function during vestibular compensation 629

Also, parallel to the e¡ects on spontaneous nystagmusdescribed above, baclofen equilibrated or reversed theSPV-gain asymmetry during sinusoidal stimulation.This was observed as an increase in ipsi- and a decreasein contra-lesional SPV-gain responses in both chemical(Fig. 7A) and surgical (Fig. 7B) UVD animals.No changes in postural behaviour could be observed

following baclofen administration in any of the animals.

E¡ects of GABAA receptor mediated stimulation followingchemical UVD

Pharmacological GABAA stimulation with THIPcaused a small, but sometimes signi¢cant, decrease ofspontaneous nystagmus (Fig. 8) with correspondingdecreases in both ipsi- and contra-lesional SPV gains

during sinusoidal stimulation. Similar e¡ects of THIPwere observed during the whole observation time.

Histology after surgical UVD

The vestibular ganglion (Scarpa’s ganglion) has twosub-divisions, including a superior sub-division locatedclose to the superior vestibular and facial nerves andan inferior located in conjunction with the inferior ves-tibular and cochlear nerves. The border between the twosub-divisions is indistinct (Fig. 9A).Structural examination of temporal bones after surgi-

cal UVD showed a variation in the degree of destructionof Scarpa’s ganglion. In 4/7 rats, a complete destructionof both the superior and inferior vestibular ganglion wassuccessfully achieved (Fig. 9B). The remaining three ratshad morphological remnants of the inferior ganglion. Inall animals, the vestibular nerve was destroyed distally toScarpa’s ganglion.

DISCUSSION

GABA receptor antagonists and agonists

The newly developed GABAB receptor antagonistCGP 56433A used in this study has considerably higherreceptor a⁄nity (Froestl and Mickel, 1997) and moree⁄cient penetration of the blood^brain barrier (Froestlet al., 1996) than the antagonist CGP 36742, previouslyused in studying vestibular compensation (Magnusson etal., 2000). CGP 56433A as well as the GABAB receptoragonist baclofen are acting on both pre- and post-synap-tic GABAB receptors in the rat CNS (Deisz, 1997;Pozza et al., 1999). The GABAA receptor agonistTHIP is capable of penetrating the blood^brain barrier(Falch and Krogsgaard-Larsen, 1982) and has previouslybeen used in studying the vestibulo-oculomotor behav-iour in rats (Tham et al., 1990; Magnusson et al., 1998).There is morphological evidence for both pre- and post-

Fig. 5. Horizontal eye movements from one rat during a staticcondition and dynamic 0.2 Hz sinusoidal stimulation in darkness,4 days after a right-sided chemical UVD. (A) Table-position sig-nal ; (B) eye position signal before administration of drug; (C) eye-position signal 30 min after systemic administration of CGP56433A (6 Wmol/kg). Calibrations for table- and eye position sig-nals are indicated by vertical bars. The horizontal bar indicates

the time scale.

Fig. 6. Mean saccade frequency before (white columns) and 30 min after (patterned columns) systemic administration of14 Wmol/kg of baclofen, versus time after (A) chemical UVD and (B) surgical UVD. IS indicates saccades directed to theintact side and LS to the lesioned side. Asterisks indicate signi¢cant di¡erences of the frequency before and after drugadministration evaluated by Student’s paired t-test. Error bars indicate 95% c.i. and nv5 for each column. *P6 0.05;

**P6 0.01; ***P6 0.001.

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A. K. Magnusson et al.630

synaptic GABAB receptors (Holstein et al., 1992) andpost-synaptic GABAA receptors (Hironaka et al., 1990)in the rat vestibular nuclei. The pre-synaptic GABAB

receptors have a negative auto-regulatory function con-trolling the release of GABA (Kerr and Ong, 1995).

Role of GABAB receptor function after UVD

Our data reveal that endogenous stimulation ofGABAB receptors has a fundamental role in reducingthe asymmetrical function of the vestibular system dur-ing the early period after UVD, as deduced by adminis-tration of CGP 56433A.The drug caused severe aggravation of the spontane-

ous nystagmus and the higher dose (6 Wmol/kg) evendrove the vestibular system to overload, thus abolishing

all vestibular responses. The sensitivity to CGP 56433Adecrease on the third day after UVD, after which thedrug only cause a moderate but signi¢cant increase inthe frequency of spontaneous nystagmus. A similar e¡ectwas observed in long-term compensated rats whereGABAB receptor inhibition caused a moderate dose de-pendent increase in spontaneous nystagmus (Magnussonet al., 2000).Pharmacological stimulation of GABAB receptors by

administration of baclofen caused an opposite change inthe vestibulo-oculomotor behaviour compared to that ofinhibition. Both the remaining spontaneous nystagmusand SPV-gain asymmetry during sinusoidal stimulationwere abolished or reversed. However, these e¡ects couldnot be demonstrated until a couple of days after UVD,thus paralleling the abatement of the spontaneous nys-tagmus. Baclofen has the same e¡ects in long-term com-pensated rats (Magnusson et al., 1998, 2000).There is convincing evidence that spontaneous nystag-

mus, developing immediately after UVD, is associatedwith a bilateral imbalance in neuronal discharge activityin the medial vestibular nuclei (MVN). This is related toa loss of the primary a¡erent input and an excessivecommissural inhibition from the contra-lesional MVN,resulting in a silencing of the ipsi-lesional type I neurones(Shimazu and Precht, 1966; Smith and Curthoys, 1988;Ris et al., 1995). A highly important factor for compen-sation of the spontaneous nystagmus is a rebalancing ofneuronal discharge activity in the MVN (Precht et al.,1966; Yagi and Markham, 1984; Smith and Curthoys,1988; Newlands and Perachio, 1990). The commissuralinhibition is partly GABA-ergic (Precht et al., 1973). Ifthe GABA-ergic suppression could be relieved in someway, the resting discharge activity of ipsi-lesional type Ineurones would be partially restored, which would bemirrored by a decrease in spontaneous nystagmus.Recent ¢ndings in rat brain slice experiments indicate

that such an e¡ect is obtained by a down-regulation ofGABAB receptor sensitivity in the ipsi-lesional rostral

Fig. 7. Mean SPV gains during sinusoidal 0.2 Hz rotation before and after systemic administration of baclofen (14 Wmol/kg)versus time following (A) chemical UVD and (B) surgical UVD. Dotted and un¢lled columns illustrate ispi- and contra-lesional gains, respectively, before drug administration. Dashed and grey columns illustrate ipsi- and contra-lesional gains,respectively, 30 min after baclofen administration. Signi¢cant di¡erences of asymmetry, calculated as the di¡erence betweenipsi- and contra-lesional SPV gains (v), before and after baclofen administration, are given. Error bars indicate 95% c.i. and

nv5 for each column.

Fig. 8. Mean saccade frequency directed to the intact side before(white columns) and 30 min after systemic administration of 57Wmol/kg of THIP (patterned columns) versus time after chemicalUVD. Asterisks indicate signi¢cant di¡erences in the frequencybefore and after drug administration evaluated by Student’s pairedt-test. Error bars indicate 95% c.i. and nv5 for each column.

*P6 0.05; **P6 0.01.

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GABAB receptor function during vestibular compensation 631

MVN after UVD (Yamanaka et al., 2000; Johnston etal., 2001). Our behavioural results may re£ect these invitro ¢ndings. If post-synaptic GABAB receptors aredown-regulated ipsi-lesionally, administration of eitheran antagonist or agonist would mainly a¡ect contra-lesional MVN second-order neurones.The fact that GABAB receptor inhibition has its most

obvious e¡ect in the immediate period after UVD sug-gests another mechanism for relieving the ipsi-lesionalvestibular neurones from excessive GABA-ergic inhibi-tion. Pre-synaptic auto-regulatory GABAB receptorscould modulate the release of GABA from neurones inthe commissural system. An inhibition of these receptors,by CGP 56433A, would disturb such an inherent phys-iological compensatory mechanism after UVD. Othercompensatory changes, like a down-regulation ofGABA- and glycine receptor sensitivity in the MVN(Yamanaka et al., 2000; Vibert et al., 2000), changes inthe intrinsic properties of vestibular second-order neu-rones (Cameron and Dutia, 1997; Vibert et al., 1999),cerebellar inhibition of the hyperactive contra-lesionalneurones (Kitahara et al., 1998) or an increase in synap-tic input from the spinal cord (Dieringer, 1995), probablydevelop gradually over a short period of time followingUVD. Assuming a development of such compensatorymechanisms, the role of GABA-ergic auto-regulation,for diminishing the vestibular imbalance, would be lessimportant with time.

Role of GABAA receptor function after UVD

It is not possible to inhibit GABAA receptors by sys-temic application of an antagonist in vivo, due to severeside-e¡ects. Therefore, it is di⁄cult to properly elucidatethe role of GABAA receptors during the initial compen-satory period after UVD. However, the data generatedfrom administration of an agonist indicate that stimu-lation of GABAA receptors does not induce any com-pensatory e¡ects comparable to those induced bystimulation of GABAB receptors. For instance, the

drug did not reduce the SPV-gain asymmetry duringsinusoidal stimulation neither during the early periodafter UVD nor in long-term-compensated animals(Magnusson et al., 1998).

Surgical versus chemical UVD

Most researchers investigating the compensatorymechanisms following UVD in rats utilise a surgicalapproach (Cameron and Dutia, 1997; Balaban andRomero, 1998; Campos Torres et al., 1999; Fukushimaet al., 2001). This procedure inevitably involves anaesthe-sia and a post-traumatic period with disturbances of theanimals’ general condition, manifested by weight loss. Asurgical UVD also involves technical problems in rats. Itis possible to visualise the superior and lateral ampullae,but the posterior ampulla must be cleared out blindly. Inorder to get rid of the remaining vestibular epithelium, itis common to apply ethanol locally. Unfortunately, ahistological veri¢cation of the completeness of thisperipheral UVD is rarely reported. A destruction ofScarpa’s ganglion or the vestibular nerve has been per-formed by other investigators (Sirkin et al., 1984; Li etal., 1995; Hamann et al., 1998), some of which havedemonstrated the extent of the lesion by histology.Chemical UVD can be performed by intra-tympanic

application of a vestibulo-toxic substance, such assodium arsanilate, during general anaesthesia lastingless than 30 s, which would minimise the possible con-founding e¡ects on the compensatory mechanisms. Thegeneral condition of the animals is not disturbed duringthe following period, hence they show no weight loss.Histological investigations have shown a total loss ofthe vestibular sensory epithelium after systemic or localapplication of sodium arsanilate (Anniko and Wersa«ll,1977; Andersson et al., 1997).In the present investigation, a chemical UVD was per-

formed in most of the experiments. To eliminate thepossibility of a di¡erent modulation of GABAB receptorfunction after a chemical versus a surgical approach, we

Fig. 9. Photomicrographs showing Toluidine Blue-stained para⁄n sections of the temporal bone. (A) The vestibular ganglion(*) in the control animal has two sub-divisions, separated by a border indicated by the two arrows. (B) In the animal sub-jected to surgical UVD, the vestibular ganglion and the cochlear nerve are absent, leaving only remnants of the facial nerve.The section was mirror-reversed for comparison with the control. Abbreviations are: fn, facial nerve; cn, cochlear nerve; co,

basal turn of the cochlea. Scale bar = 500 Wm.

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A. K. Magnusson et al.632

investigated the e¡ects of baclofen on vestibulo-oculo-motor behaviour in surgical UVD animals as well. His-tological examination revealed remnants of the posteriorpart of Scarpa’s ganglion in some animals even thoughthe vestibular nerve was destroyed distally to the gan-glion. The inconsistency of the extent of the lesionmight explain the inter-individual variability observedin recovery of vestibulo-oculomotor and postural symp-toms later on.Our data demonstrate an obvious di¡erence in the

course of behavioural vestibulo-oculomotor recovery ofthe contra-lesional gains between chemical and surgicalUVD animals. The contra-lesional SPV gain is not farfrom normal after chemical UVD, whilst it is depressedduring the whole investigation period after surgicalUVD. Both types of ¢ndings have been reported previ-ously (Smith and Curthoys, 1989).Administration of baclofen caused identical changes to

the vestibulo-oculomotor pattern after both UVDapproaches, except that compensatory e¡ects of baclofenwere observed 1 day earlier in surgical UVD animals. Wetherefore conclude that the methods used for UVD inthis study are equal, in terms of the e¡ects of baclofen.

CONCLUSION

The results from this study provide evidence for a

fundamental role of GABAB receptors in the early com-pensation of the vestibular imbalance following UVD.This is manifested by speci¢c functional changes, relatedto the vestibulo-oculomotor system, under the in£uenceof GABAB receptor-related drugs. Accordingly, admin-istration of a GABAB receptor antagonist highly aggra-vated the asymmetry in the vestibulo-oculomotor systemand could even cause the system to overload during the¢rst 2 days after UVD. The reciprocal inhibitory com-missural system in the vestibular nuclei is suggested asthe major site for GABAB receptor modulation in thehorizontal vestibulo-oculomotor circuits.A potential clinical relevance exists since roughly 30%

of all patients compensate poorly from a unilateral ves-tibular loss (Curthoys and Halmagyi, 1999). If pharma-cological GABAB receptor stimulation could ‘boost’ thecompensation of the vestibular imbalance, some of thesepatients might be less incapacitated.

Acknowledgements+We would like to dedicate this work to thememory of our late friend and collaborator Birgitta Erikssonwho skilfully assisted us for more than 20 years. This projectwas supported by the Ansgarius Foundation, Lions Foundation,the Health Research Council in the South^East of Sweden, theSwedish Medical Research Council (Grant numbers 4767 and9888). We would like to thank Drs. Wolfgang Froestl, NovartisPharma AG, Switzerland, for the generous gift of CGP 56433Aand Sivert Lindstro«m for comments on the manuscript.

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(Accepted 13 December 2001)

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