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. 2000 Mar 1;523(Pt 2):413–424. doi: 10.1111/j.1469-7793.2000.t01-1-00413.x

Rapid compensatory changes in GABA receptor efficacy in rat vestibular neurones after unilateral labyrinthectomy

Toshiaki Yamanaka 1, Aydin Him 1, Susan A Cameron 1, Mayank B Dutia 1
PMCID: PMC2269812  PMID: 10699085

Abstract

  1. The inhibitory effects of the GABAA agonist muscimol and the GABAB agonist baclofen on tonically active medial vestibular nucleus (MVN) neurones were recorded in slices of the rat dorsal brainstem in vitro, to determine whether any changes occurred in the functional efficacy of GABAergic inhibition in these cells during the initial rapid stage of ‘vestibular compensation’, the behavioural recovery that takes place after unilateral labyrinthectomy (UL). These experiments were carried out in preparations where the midline was cut, severing all commissural connections between the two vestibular nuclei.

  2. Slices of the MVN were prepared from normal animals and animals that had been unilaterally labyrinthectomised 4 h earlier. The mean in vitro discharge rate of MVN neurones in the rostral region of the ipsi-lesional nucleus after UL was significantly higher than that in control slices, confirming our earlier reports of an increase in intrinsic excitability of these cells in the early stage of vestibular compensation. The in vitro discharge rates of caudal ipsi-lesional MVN cells, and rostral and caudal contra-lesional MVN cells, were not different from controls.

  3. Muscimol and baclofen caused reversible, dose-related inhibition of the tonic discharge rate of MVN cells in control slices. In slices prepared from UL animals, MVN cells in the rostral region of the ipsi-lesional nucleus showed a marked downregulation of their response to both muscimol and baclofen, seen as a rightward shift and a decrease in slope of the dose-response relationships for the two agonists. In the contra-lesional nucleus, there was a small but significant upregulation of the responsiveness of both rostral and caudal MVN cells to baclofen, and a marked upregulation of the responsiveness of caudal MVN cells to muscimol.

  4. In slices from animals that had undergone bilateral labyrinthectomy 4 h earlier, the downregulation of the functional efficacy of GABA receptors in the rostral MVN cells did not occur. The changes in GABA receptor efficacy after UL are therefore not due to the vestibular de-afferentation itself, but are instead due to the imbalance in excitability of the vestibular nuclei of the lesioned and intact sides, and the enhanced commissural inhibition of the ipsi-lesional MVN cells that follows UL.

  5. The downregulation of GABA receptor efficacy in the ipsi-lesional MVN neurones is functionally compensatory, in that their response to commissural and cerebellar inhibitory drive will be significantly reduced after UL. Their intrinsic membrane conductances, and their remaining excitatory synaptic inputs, will consequently be more effective in causing depolarisation and the restoration of resting activity. Simultaneously the upregulation of GABAergic efficacy in the contra-lesional MVN will tend to reduce the hyperactivity on the contralateral side. These adaptive changes therefore represent a plausible cellular mechanism for the recovery of resting discharge in the ipsi-lesional MVN neurones, and the ‘re-balancing’ of the excitability of the vestibular neurones of the lesioned and intact sides, as occurs after UL in vivo.

  6. We propose that the adaptive regulation of the functional efficacy of GABA receptors in the MVN neurones may be an important cellular mechanism for the ‘homeostasis of bilateral excitability’ of the vestibular nuclei of the two sides.

The process of ‘vestibular compensation’, the behavioural recovery that takes place following damage to one vestibular labyrinth or nerve, is an attractive experimental model of lesion-induced plasticity in the adult mammalian CNS. Ablation of the vestibular receptors of one inner ear (unilateral labyrinthectomy, UL), or unilateral vestibular neurectomy, results in severe oculomotor and postural symptoms including spontaneous ocular nystagmus, roll- and yaw-tilt of the head, contralateral limb extension and ipsilateral limb flexion, barrel rolling and circular walking (for reviews see Smith & Curthoys, 1989; Curthoys & Halmagyi, 1995; Dieringer, 1995). Remarkably, many of these initial symptoms ameliorate rapidly so that barrel rolling and circular walking cease within a few hours, and ocular nystagmus gradually subsides over 2–3 days, as vestibular compensation takes place. This initial behavioural recovery is followed by a much slower process in which dynamic oculomotor and postural performance recovers, though never completely. Since there is no regeneration of the lesioned labyrinth or nerve, the behavioural recovery after UL is attributed to lesion-induced plasticity in the central vestibular pathways (Curthoys & Halmagyi, 1995; Dieringer, 1995).

The neuronal mechanisms involved in vestibular compensation are largely unknown. Several recent studies have investigated the effects of UL on neurones in the medial vestibular nucleus (MVN), the largest of the four main cell groups in the brainstem vestibular complex. Immediately after UL the normally high resting activity of the majority of MVN cells (type I cells) on the side of the lesion (the ‘ipsi-lesional’ side) is largely abolished (rat: Hamann & Lannou, 1987; guinea-pig: Smith & Curthoys, 1988a,b; Ris et al. 1995; gerbil: Newlands & Perachio, 1990a,b). This is presumably due not only to the loss of excitatory drive from the lesioned ipsilateral primary vestibular afferents but also to an enhanced commissural inhibition from contralateral MVN cells, which become hyperactive due to the loss of inhibitory drive from the lesioned side (Smith & Curthoys, 1989; Curthoys & Halmagyi, 1995; Ris & Godaux, 1998). The profound imbalance in the resting activity of the MVN cells on the lesioned and intact sides is believed to cause the initial severe oculomotor and postural symptoms after UL. The abatement of these ‘static’ symptoms over 2–3 days is accompanied by a recovery of resting activity in the ipsi-lesional vestibular neurones, and a decrease in the hyperactivity of contra-lesional cells, so that the marked imbalance in neuronal activity on the two sides is reduced. However, there is not a strict correlation between the time course of neuronal recovery and the disappearance of the behavioural symptoms, implying that centres other than the vestibular nuclei are also involved in the initial behavioural compensation (Ris et al. 1997).

While many studies have sought evidence for changes in the neurochemical or synaptic organisation of the central vestibular pathways during vestibular compensation, the cellular mechanisms that bring about the recovery of resting discharge in the ipsi-lesional MVN cells after UL are unknown. We recently investigated changes in the intrinsic properties of MVN neurones in the early stages of recovery after UL in the rat. The MVN of the ipsi-lesional and contra-lesional sides were isolated in slice preparations in vitro, i.e. the midline was cut, severing all commissural connections between the two vestibular nuclei, after various periods of compensation following UL. The spontaneous in vitro discharge of the MVN cells, which is due largely to their intrinsic pacemaker-like membrane conductances (Serafin et al. 1991; Johnston et al. 1994; Sakai et al. 1996), was studied. Initial experiments showed that the ipsi-lesional MVN neurones, whose resting discharge is virtually abolished for many hours after UL in vivo, nevertheless continued to fire at their normal intrinsic in vitro firing rates when they were removed into a slice preparation within 2 h after UL (Cameron & Dutia, 1997). This contrast between the in vivo and in vitro situations demonstrates that the reason for the silencing of these cells after the lesion in vivo is the enhanced commissural inhibition from the contra-lesional (intact) side; removing the ipsi-lesional neurones into a slice preparation, in which the commissural pathways are cut, relieves these cells of the inhibitory drive that keeps them silent in vivo. In slices prepared after longer periods of compensation following UL, the intrinsic in vitro firing rates of rostral MVN cells in the ipsi-lesional nucleus were found to be significantly higher than normal, indicating that the intrinsic excitability of these cells had increased above normal between 2 and 4 h post-UL. This increase in intrinsic excitability of the ipsi-lesional cells was dependent on the activation of glucocorticoid receptors, which are presumably strongly activated in the course of the acute stress response that accompanies the severe initial symptoms after UL (Cameron & Dutia, 1999). We have proposed that the increased intrinsic excitability may be important in restoring the resting activity of the ipsi-lesional MVN cells after UL, by helping to overcome the initial disfacilitation and excessive commissural inhibition that silences them after UL in vivo (Cameron & Dutia, 1997, 1999). By contrast, Vibert et al. (1999) have recently reported that in the guinea-pig, an increase in the intrinsic excitability of the ipsi-lesional MVN cells occurs only after several days post-UL. This represents a species difference that may be related to the marked differences in the physiology of the stress axis in the guinea-pig (for a discussion see Cameron & Dutia, 1999).

In this study we have investigated the mechanisms underlying the rapid increase in the intrinsic excitability of rostral MVN cells after UL in the rat. As we have discussed earlier (Cameron & Dutia, 1997), a possible mechanism may involve the adaptive downregulation of the functional efficacy of GABA receptors in these cells, in response to the enhanced commissural inhibitory drive which they experience after UL in vivo. Here we have tested this possibility by determining the responsiveness of rostral and caudal MVN cells to the selective GABAA and GABAB receptor agonists muscimol and baclofen, in slices of the ipsi-lesional and contra-lesional MVN prepared from animals that had been unilaterally labyrinthectomised 4 h earlier. Previous studies have established that both GABAA and GABAB receptor subtypes are found postsynaptically on MVN cells, that almost all MVN cells are inhibited by GABA agonists, and that the inhibitory responses persist after the blockade of synaptic transmission either in Ca2+-modified medium or in TTX (Dutia et al. 1992; Holstein et al. 1992; Vibert et al. 1995; Hutchinson et al. 1995). Direct evidence indicating changes in GABAA receptor levels after UL in the rat has been provided by Calza et al. (1992), who demonstrated a decrease in benzodiazepine binding in the ipsi-lesional MVN within 3 h after UL, which then returned to normal within 3 days post-UL. MVN cells receive a substantial inhibitory projection from cerebellar Purkinje cells (De Zeeuw & Berrebi, 1995), and pharmacological evidence has shown that the mammalian commissural inhibitory pathway involves GABAergic neurones (Smith & Darlington, 1994; Furuya & Koizumi, 1998). Our results show that within 4 h after UL in the rat, marked compensatory changes take place in the functional efficacy of GABAergic inhibition in both ipsi-lesional and contra-lesional MVN neurones. These changes are in the appropriate direction to help restore resting activity in the ipsi-lesional MVN cells and lead to a re-balancing of the excitability of the MVN neurones on the lesioned and intact sides in vivo.

METHODS

Slices of the MVN were prepared from normal Sprague-Dawley rats (90–125 g) and animals that had undergone either a left labyrinthectomy or a bilateral labyrinthectomy under avertin (tribromoethanol) anaesthesia (300 mg kg−1, i.p.), 4 h previously. Labyrinthectomy was carried out by opening the horizontal semicircular canal duct in the temporal bone, drilling through the horizontal and anterior semicircular canal ampullae and aspirating the contents of the vestibule which was then rinsed with 100 % ethanol, as described in detail previously (Cameron & Dutia, 1997). The animals began to recover from avertin anaesthesia about 50–60 min following induction, typically 20–30 min after the labyrinthectomy was completed. The left labyrinthectomised animals showed all the characteristic symptoms of unilateral vestibular loss, including spontaneous nystagmus, barrel rolling, circular walking, loss of ipsilateral extensor tone, and head yaw- and roll-tilt. The bilaterally labyrinthectomised animals did not show any of these symptoms, but instead showed pronounced ataxia (Deliagina et al. 1997). The animals were allowed to recover in their home cages for 4 h after UL, at which point they were re-anaesthetised with 3–4 % halothane in oxygen, decapitated using a guillotine and the brain removed for the preparation of slices of the MVN for electrophysiological recording.

Slice preparation and recording

Extracellular recordings of the spontaneous tonic activity of MVN neurones were made in horizontal slices of the dorsal brainstem in vitro. Slices were prepared as described in detail previously (Dutia et al. 1992). The whole brain was removed into ice-cold artificial cerebrospinal fluid (aCSF; composition (mM): NaCl, 124; KCl, 5; KH2PO4, 1.2; MgSO4, 1.3; CaCl2, 2.4; NaHCO3, 26.0; and D-glucose, 10.0). The brainstem extending from the inferior colliculi to the obex was isolated, and the cerebellum removed to expose the MVN on the floor of the fourth ventricle. The brainstem was cemented with the fourth ventricle uppermost to the stage of a Vibroslice (Camden Instruments, London, UK), and slices of the dorsal brainstem (350–400 μm thickness) containing the MVN were prepared. Each slice was cut along the midline, to give two isolated medial vestibular nuclei. In experiments on unilaterally labyrinthectomised animals, care was taken to unambiguously identify the left (ipsilateral to the lesioned labyrinth) and right (contralateral) MVN. These were transferred to an interface-type incubation chamber that was continuously perfused with aCSF bubbled with 95 % O2-5 % CO2 (pH 7.4; flow, 1.5 ml min−1) and maintained at 33 ± 0.2°C. All slices were incubated for at least 1 h before recording. Single unit extracellular recordings were made from tonically active MVN neurones using conventional glass micropipettes filled with 2 M sodium gluconate (impedance, 10–30 MΩ) connected to the headstage of an Axoclamp-2A amplifier (Axon Instruments). The resting discharge of tonically active MVN neurones was displayed and analysed on-line using a CED 1401 plus laboratory interface (Cambridge Electronic Design, Cambridge, UK) linked to a microcomputer.

Drug application and data analysis

The GABAA agonist muscimol (5-aminomethyl-3-hydroxyisoxazole) and the GABAB agonist baclofen (4-amino-3-[4-chlorophenyl]butanoic acid) were obtained from Sigma. Small aliquots of stock solutions of the two drugs were made in distilled water and frozen until required. Test solutions of agonists at different concentrations were made up by diluting the stock solution in oxygenated aCSF immediately before use, and applied to the slices by switching the perfusion inlet tube to the appropriate reservoir by means of three-way taps. The inhibitory response of each cell to a 60 s test pulse of agonist was measured as the maximal decrease in discharge rate expressed as a percentage of the resting discharge rate (Dutia et al. 1992; Sulaiman & Dutia, 1998). Dose-response curves were fitted to the mean inhibitory responses for each concentration of agonist using a least-squares method. Although we assigned values of 100 % inhibition to all the responses where the tonic activity of the cells was completely inhibited by the GABA agonists, the parameters of the curve-fitting algorithm were not constrained to within this range. The dose-response relationships were compared using two-way ANOVA, and the parameters of the fitted dose-response curves (maximal response, half-maximal concentration (EC50) and the slope) were compared using Student's t test, significance being assumed when P < 0.05. Results are given as means ±s.e.m.

RESULTS

Increase in intrinsic excitability of ipsi-lesional MVN cells after UL

Extracellular recordings of the in vitro spontaneous activity of MVN cells were made in brainstem slices prepared from normal animals (28 animals), and animals which had undergone unilateral labyrinthectomy 4 h previously (UL group, 44 animals). In slices prepared from UL animals, the mean in vitro resting discharge rate of MVN cells in the rostral part of the ipsi-lesional nucleus was significantly higher than normal (21.5 ± 1.1 spikes s−1, n= 63 cells vs. 16.2 ± 1.3 spikes s−1, n= 40 cells in controls; P < 0.05, Student's t test). The mean discharge rates of MVN cells in the caudal region of the ipsi-lesional nucleus, and the rostral and caudal parts of the contra-lesional nucleus, were no different from those in the corresponding regions of normal control slices (ipsi-lesional caudal cells, 17.3 ± 1.0 spikes s−1, n= 45 cells vs. 17.0 ± 1.0 spikes s−1, n= 37 cells in controls; contra-lesional rostral cells, 16.9 ± 1.1 spikes s−1, n= 50 cells, and contra-lesional caudal cells, 16.7 ± 1.0 spikes s−1, n= 43 cells). These results confirm our earlier observation of a significant increase in the intrinsic excitability of the ipsi-lesional rostral but not caudal MVN cells within 4 h after UL in the rat (Cameron & Dutia, 1997, 1999).

Response of normal MVN cells to muscimol and baclofen in vitro

As illustrated in Fig. 1, in slices prepared from normal (labyrinth intact) animals bath application of the GABAA agonist muscimol and the GABAB agonist baclofen caused reversible, dose-related inhibition of the tonic discharge of all of the MVN cells tested. Table 1 shows the parameters of the first-order logistic curves fitted to the dose-response relationships (maximal inhibition, half-maximal agonist concentration (EC50) and slope) for all the data in these experiments. There was no significant difference between rostral and caudal MVN cells in their dose dependence for the two agonists in slices from normal animals (Table 1).

Figure 1. Examples of the inhibitory effects of muscimol (A) and baclofen (B) on the tonic in vitro firing rate of two representative normal MVN cells.

Figure 1

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The agonists were applied at the concentrations shown for 60 s, as indicated by the bars above the data. Arrowheads indicate the time of maximal inhibition; in cells where the tonic activity was silenced by the agonist, this was taken to be the mid-point between the silencing of the cell and the re-appearance of tonic activity.

Table 1.

Parameters of the dose-response relationships fitted to the mean inhibitory responses of MVN cells to muscimol and baclofen, in each of the experimental conditions

Muscimol Baclofen
Max inhibition (%) EC50 (μm) Slope Max inhibition (%) EC50 (μm) Slope
Normal MVN (controls)
 Rostral cells 107.9 ± 1.1 8.95 ± 0.21 1.14 ± 0.02 114.7 ± 2.3 5.02 ± 0.32 0.79 ± 0.02
 Caudal cells 132.5 ± 14.6 13.0 ± 3.42 0.94 ± 0.09 103.4 ± 5.3 2.92 ± 0.45 0.86 ± 0.09
Ipsi-lesional MVN
 Rostral cells 123.4 ± 7.7 45.3 ± 9.5* 0.78 ± 0.06* 114.2 ± 7.1 20.2 ± 2.2* 0.59 ± 0.02*
 Caudal cells 105.3 ± 10.2 10.5 ± 3.45 0.79 ± 0.12 97.3 ± 12.6 3.85 ± 1.89 0.69 ± 0.2
Contra-lesional MVN
 Rostral cells 112.9 ± 14.8 8.07 ± 2.9 0.93 ± 0.14 94.5 ± 4.6* 2.18 ± 0.33* 1.02 ± 0.12
 Caudal cells 101.4 ± 7.5 2.5 ± 0.51* 1.34 ± 0.29* 95.1 ± 1.5 1.54 ± 0.08* 0.92 ± 0.04
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*

Significant difference in parameter after labyrinthectomy compared to control (P < 0.05).

Changes in GABA receptor efficacy in ipsi-lesional MVN neurones 4 h after UL

Rostral cells

In slices prepared from animals that had been unilaterally labyrinthectomised 4 h previously, MVN cells in the rostral region of the ipsi-lesional nucleus showed a marked decrease in their responsiveness to both muscimol and baclofen. This was seen as a significant rightward shift of the dose-response relationships for both agonists compared to normal MVN cells (Fig. 2a and B, filled squares). The EC50 values of the fitted dose-response curves increased significantly for both muscimol and baclofen after UL, and this was accompanied by a significant decrease in slope in both cases (Table 1).

Figure 2. Effects of muscimol and baclofen on rostral ipsi-lesional MVN cells from control and 4 h post-UL animals.

Figure 2

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A and B, dose-response curves fitted to the mean (±s.e.m.) inhibitory effects of muscimol and baclofen, respectively, in rostral MVN cells in slices from normal animals (^) and in slices of the ipsi-lesional nucleus from animals unilaterally labyrinthectomised 4 h earlier (▪). The inhibitory response to each dose of agonist is expressed as a percentage decrease from the control (resting) firing rate of the cell. C and D, the times to maximal inhibition (Tinhib), measured from the time of the start of agonist application, for muscimol and baclofen, respectively. Asterisks indicate significant differences between control cells and cells 4 h after UL (P < 0.05, Student's t test).

This marked downregulation of the functional efficacy of GABAA and GABAB receptor-mediated inhibition in rostral MVN cells was accompanied by significant changes in the time course of their inhibitory response to muscimol, but not to baclofen. In these cells the inhibitory response to muscimol was slower to develop, and the inhibition of the tonic firing rate was longer in duration, than in normal MVN cells. We measured the time taken to reach the point of maximal inhibition (Tinhib) after the 60 s exposure to muscimol, in normal cells and in MVN cells 4 h post-UL. In cells whose tonic activity was silenced by the higher doses of muscimol, Tinhib was taken to be half-way between the silencing of the cell and the re-appearance of the tonic discharge (arrowheads in Fig. 1). As shown in Fig. 2C, the mean values for Tinhib in response to 10 and 30 μM muscimol in the ipsi-lesional rostral cells were significantly higher in MVN cells 4 h post-UL than in normal cells. By contrast there were no changes in the values of Tinhib for the response of the MVN cells to baclofen in post-UL slices (Fig. 2D).

The relationship between the in vitro resting firing rates of the ipsi-lesional rostral MVN cells and their inhibitory responses to the representative doses of 10 μM muscimol and 10 μM baclofen is illustrated in Fig. 3. In slices from 4 h post-UL animals (Fig. 3a and B, filled circles) more cells were found with resting firing rates above about 25 spikes s−1, reflecting the increase in the mean resting discharge rate of these cells after UL. In parallel with this the inhibitory effects of the two agonists were uniformly reduced, and there was no apparent dependence of the inhibitory response on the resting discharge rate of the cells.

Figure 3. Downregulation of GABA receptor efficacy in ipsi-lesional MVN cells is not related to their in vitro resting discharge rates.

Figure 3

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Distribution of the inhibitory responses of rostral MVN cells to a dose of 10 μM muscimol (A) and 10 μM baclofen (B), in control slices (□) and in slices of the ipsi-lesional nucleus 4 h after UL (•).

Caudal cells

For caudal MVN cells in the ipsi-lesional nucleus, the parameters of the fitted dose-response curves for the mean inhibitory effects of muscimol and baclofen were not significantly different 4 h post-UL compared to normal (Table 1, Fig. 4). However, the mean inhibitory responses evoked by the higher doses of muscimol (30 μM, Fig. 4a) and baclofen (30 μM, Fig. 4B) were significantly lower in 4 h post-UL slices than in normal slices (P < 0.05, Student's t test). In addition, the values for Tinhib in response to 3 and 10 μM doses of muscimol were not different from normal (Fig. 4C), but in response to 30 μM muscimol Tinhib increased from 136.0 ± 22.9 s in controls to 227.4 ± 62.0 s after UL (Fig. 4C). While this was reminiscent of the increase in Tinhib seen in the ipsi-lesional rostral MVN cells, it was not large enough to reach significance (P= 0.1 compared to control). As in the rostral cells, there were no changes in the time course of the inhibitory responses to baclofen after UL (Fig. 4D). These results suggest that within 4 h post-UL there may be some downregulation also of the functional efficacy of GABAA and GABAB receptors in the ipsi-lesional caudal MVN cells, though at this time after UL this is much less marked than the pronounced downregulation seen in the ipsi-lesional rostral cells.

Figure 4. Effects of muscimol and baclofen on caudal ipsi-lesional MVN cells.

Figure 4

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A and B, dose-response curves fitted to the mean (±s.e.m.) inhibitory effects of muscimol and baclofen, respectively, in caudal MVN cells in slices from normal animals (^) and in slices of the ipsi-lesional nucleus from animals unilaterally labyrinthectomised 4 h earlier (▪). C and D, Tinhib measured from the time of the start of agonist application, for muscimol and baclofen, respectively. Asterisks indicate significant differences between the inhibitory response to agonist in control cells and cells 4 h after UL (P < 0.05, Student's t test).

Changes in GABA receptor efficacy in contra-lesional MVN neurones after UL

In the contra-lesional nucleus, the dose-response relationship for the GABAA agonist muscimol in rostral MVN cells 4 h post-UL was not different from normal (Fig. 5a, filled squares; Table 1), and the inhibitory responses of these cells to muscimol did not show any changes in Tinhib. By contrast, there was a small but significant increase in the responsiveness to the GABAB agonist baclofen compared to normal, as indicated by a leftward shift of the dose- response curve and a significant decrease in the EC50 value 4 h post-UL, with no change in the slope (Fig. 5B, filled squares; Table 1).

Figure 5. Effects of muscimol and baclofen on contra-lesional MVN cells.

Figure 5

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A and B, dose-response curves fitted to the mean (±s.e.m.) inhibitory effects of muscimol and baclofen, respectively, in rostral MVN cells in slices from normal animals (^) and in slices of the contra-lesional nucleus from animals unilaterally labyrinthectomised 4 h earlier (▪). C and D, dose-response curves fitted to the mean (±s.e.m.) inhibitory effects of muscimol and baclofen, respectively, in caudal MVN cells in slices from normal animals (^) and in slices of the contra-lesional nucleus from animals unilaterally labyrinthectomised 4 h earlier (▪).

In the caudal region of the contra-lesional MVN, there was a marked increase in the responsiveness to muscimol of the MVN cells 4 h post-UL (Fig. 5C). This was seen as a significant leftward shift of the muscimol dose-response curve compared to normal, accompanied by a significant increase in slope (Fig. 5C, filled squares; Table 1). This marked upregulation of GABAA receptor efficacy was also accompanied by a smaller but significant increase of the responsiveness of the contra-lesional caudal cells to the GABAB agonist baclofen (Fig. 5D, filled squares; Table 1). There was a small but significant decrease in the EC50 value of the dose-response curve for baclofen compared to normal, similar to that seen in the contra-lesional rostral cells 4 h post-UL (Table 1).

Effects of bilateral labyrinthectomy on the intrinsic excitability and response to muscimol and baclofen of rostral MVN cells

To investigate whether the increased intrinsic excitability and the down-modulation of GABA receptor efficacy in the ipsi-lesional rostral MVN cells 4 h post-UL (Fig. 2) were due to the vestibular de-afferentation itself, or whether they were dependent instead on the imbalance in the resting activity of the MVN of the two sides after UL in vivo, further experiments were carried out in slices prepared from eight animals that had undergone bilateral labyrinthectomy (BL) 4 h earlier. In slices from BL animals, the in vitro discharge rates of rostral MVN cells were no different from normal (14.8 ± 1.0 spikes s−1, n= 72 cells vs. 15.2 ± 0.9 spikes s−1, n= 83 cells in sham-operated controls). By contrast, the mean firing rate of caudal MVN cells in slices from BL animals was significantly higher than that of caudal cells in slices from sham-operated controls (21.8 ± 1.2 spikes s−1, n= 82 cells vs. 12.9 ± 1.0 spikes s−1, n= 66 cells in controls, P < 0.001). There was no asymmetry in the in vitro firing rates of MVN cells in the left and right nuclei.

The responses to muscimol and baclofen of rostral MVN cells in both the left and right nuclei in slices prepared from BL animals were compared to those in normal slices, to determine whether the downregulation seen in the ipsi-lesional rostral cells after UL (Fig. 2) was also seen after BL. As shown in Fig. 6a and B, there were no significant differences in the mean inhibitory effects of muscimol and baclofen in the rostral MVN cells in slices prepared from 4 h post-BL animals compared to controls, and the downregulation seen in the UL animals did not occur after BL.

Figure 6. Downregulation of GABA receptor efficacy does not occur after bilateral labyrinthectomy.

Figure 6

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A and B, dose-response curves fitted to the mean (±s.e.m.) inhibitory effects of muscimol and baclofen, respectively, in rostral MVN cells in slices from normal animals (^) and in slices from animals bilaterally labyrinthectomised 4 h earlier (▪).

DISCUSSION

Cellular mechanisms underlying the recovery of resting discharge in ipsi-lesional MVN cells after UL

These results show that within 4 h after UL in the rat, significant changes take place in the functional efficacy of GABA receptor-mediated inhibition in MVN neurones on the lesioned and intact sides. These changes were observed when, after a 4 h period of vestibular compensation after UL in vivo, the MVN of the two sides were removed into slice preparations in vitro. Each MVN in vitro was isolated from its afferent inputs including those from the remaining vestibular nerve and from the commissural and cerebellar inhibitory pathways, so that changes in the intrinsic membrane properties and neurotransmitter responsiveness of the MVN cells could be specifically investigated. In particular the ipsi-lesional MVN cells were isolated from the strong commissural inhibition that silences them after UL in vivo; although at 4 h post-UL almost all ipsi-lesional MVN neurones are silent or nearly so in vivo, they are not silent in vitro (for a discussion see Cameron & Dutia, 1997). This experimental approach allowed investigation of the early adaptive changes in the properties of the MVN neurones that precede, and which may therefore be causally related to, the resumption of spike firing in ipsi-lesional cells in vivo. The present results show that the compensatory increase in intrinsic excitability of the ipsi-lesional MVN neurones that we have previously demonstrated (Cameron & Dutia, 1997, 1999), is accompanied by significant changes in the efficacy of GABA receptor function in the vestibular neurones.

In the ipsi-lesional MVN, there was a marked downregulation of the functional efficacy of both GABAA and GABAB receptors in rostral MVN cells after UL, seen as a significant rightward shift and a decrease in the slope of their dose-response relationships for both muscimol and baclofen. This demonstrates an adaptive downregulation of the affinity or efficacy of postsynaptic GABA receptors on the MVN neurones. As we have proposed earlier (Cameron & Dutia, 1997), this presumably occurs in response to the sustained excessive commissural inhibition of the ipsi-lesional MVN neurones over the 4 h period of recovery after UL in vivo. Such agonist-dependent regulation of receptor function has been studied in some detail for GABAA receptors (for a review see Barnes, 1996; Platt et al. 1996). The changes in Tinhib accompanying the downregulation of the responsiveness of the ipsi-lesional MVN cells to muscimol (Figs 2 and 4) are suggestive of a change in the binding or activation kinetics of the GABAA receptors, perhaps due to changes in their phosphorylation state or subunit composition (for a review see Smart, 1997). While GABAB receptors have also been reported to be up- or downregulated after long-term exposure to antagonists and agonists (Malcangio et al. 1993; Pratt & Bowery, 1993), rapid changes in GABAB receptor efficacy as seen here have not to our knowledge been previously reported. In the context of GABA receptor pharmacology, MVN neurones may prove to be a useful model where such dynamic modulation of GABAA and GABAB receptor function may be studied in vivo and in vitro.

The marked downregulation of GABA receptor efficacy in the ipsi-lesional MVN cells is functionally compensatory, in that it will significantly reduce the sensitivity of these cells to the excessive commissural inhibitory synaptic drive that silences them in vivo. Consequently their intrinsic, pacemaker-like membrane conductances (Serafin et al. 1991; Johnston et al. 1994; Sakai et al. 1996), as well as their remaining excitatory synaptic inputs in vivo, will be more effective in causing membrane depolarisation and the restoration of spike firing after UL. The rapid downregulation of GABA receptor efficacy in the ipsi-lesional MVN cells therefore represents an early-onset, adaptive cellular response that may lead to the recovery of resting discharge in these cells. Of the various mechanisms that have been proposed to explain the rapid recovery of resting activity in the ipsi-lesional cells after UL (for reviews see Smith & Curthoys, 1989; Curthoys & Halmagyi, 1995; Dieringer, 1995; Vibert et al. 1999), to our knowledge this represents the first such mechanistic explanation that is supported by direct experimental evidence.

In the longer term, it is conceivable that the adaptive changes in GABA receptor efficacy in the ipsi-lesional MVN cells persist indefinitely after UL. In this case the permanent loss of excitatory input from the lesioned primary vestibular afferents may be, in effect, compensated for by a decrease in responsiveness of the ipsi-lesional MVN cells to inhibitory synaptic inputs. The recent observation by Magnusson et al. (1998) that administration of baclofen, but not the GABAA agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridone), causes significant changes in vestibulo-ocular reflex function in long-term compensated rats, suggests that there might be persistent differences in GABAB receptor efficacy between the lesioned and intact sides, so that exogenous baclofen causes inhibition of one side of an otherwise re-balanced vestibular system. Further experiments to investigate the efficacy of GABAergic inhibition in the MVN cells at longer times after UL are necessary to examine the role of GABA receptor plasticity in the maintenance of vestibular compensation. The present results do not exclude the possibility that additional, presently unknown cellular mechanisms with various times of onset and duration after UL also contribute to compensation.

Changes in GABA receptor efficacy in contra-lesional MVN cells

In the contra-lesional MVN 4 h post-UL, there was a small but significant upregulation of GABAB receptor efficacy in both rostral and caudal MVN neurones, and a marked upregulation of GABAA receptor efficacy in the caudal MVN cells. This increase in inhibitory receptor efficacy will tend to counteract the hyperactivity on the contra-lesional side that follows UL in vivo, which may in turn reduce the inhibitory commissural drive to the ipsi-lesional side (Cameron & Dutia, 1997). It is possible that these changes are at least in part a consequence of the silencing of the ipsi-lesional MVN cells after UL, since this would cause a profound decrease in the commissural inhibitory drive from the ipsi-lesional cells and the dis-inhibition of contra-lesional MVN neurones. However, because largely homologous regions of the MVN are reciprocally connected by the brainstem commissures, it might be expected that the downregulation of GABA receptor efficacy in the ipsi-lesional rostral MVN cells would be mirrored in an upregulation of GABA receptor function in the contra-lesional rostral cells. Instead there was no change in GABAA receptor efficacy in the contra-lesional rostral cells, while there was an upregulation of GABAA receptor efficacy in contra-lesional caudal cells. Rather than being directly due to the brainstem commissures, therefore, it is possible that the changes in contra-lesional cells reflect alterations in the balance of cerebellar inhibitory drive after UL (Kitahara et al. 1997, 1998). The significance of the changes in GABA receptor function in contra-lesional cells remains to be further explored.

Relationship between intrinsic excitability and GABA receptor efficacy in MVN cells

Although in the ipsi-lesional rostral cells there was a significant increase in intrinsic excitability 4 h post-UL and a marked downregulation of GABA receptor efficacy, there was no apparent correlation between the resting in vitro discharge rates of these cells and their responsiveness to the GABA agonists (Fig. 3). In addition, the upregulation of GABAA receptor efficacy in the contra-lesional caudal cells is not accompanied by any significant change in their intrinsic excitability (Cameron & Dutia, 1997). These findings argue against our earlier suggestion that the increase in excitability of the ipsi-lesional rostral cells may be caused by a decreased sensitivity to endogenously released GABA within the slice (Cameron & Dutia, 1997). Instead, the increase in intrinsic excitability and the downregulation of GABA receptor efficacy appear to be independent, parallel cellular compensatory responses induced by UL. These two processes will act synergistically, possibly with other short-latency adaptive cellular responses that have yet to be identified, in bringing about the recovery of resting discharge in the ipsi-lesional MVN cells.

In our previous study (Cameron & Dutia, 1999), we showed that the development of the increase in intrinsic excitability in the rostral MVN cells after UL was dependent on the activation of glucocorticoid receptors, presumably in the course of the acute stress response that accompanies the initial vestibular symptoms. It is not presently known whether the changes in GABA receptor efficacy seen here are also dependent on glucocorticoid receptor activation. In the guinea-pig, Vibert et al. (1999) have recently reported that an increase in intrinsic excitability is seen in ipsi-lesional MVN cells only after several days post-UL. This species difference is likely to be accounted for by the fact that the guinea-pig normally has very high levels of circulating glucocorticoids, with consequential adaptations in glucocorticoid receptor-dependent physiological processes (see Cameron & Dutia, 1999, for a discussion). It remains to be determined whether the guinea-pig shows rapid adaptive changes in GABA receptor efficacy after UL similar to those seen here.

Role of the contra-lesional intact labyrinth in vestibular compensation

In bilaterally labyrinthectomised animals, in which there is an equal and simultaneous disfacilitation of the vestibular neurones of the two sides, neither the increase in intrinsic excitability of the rostral MVN cells nor the downregulation of GABA receptor efficacy occurred (Fig. 6). This indicates that the compensatory changes observed here are not due to the vestibular de-afferentation itself, since after the interruption of afferent activity in both vestibular nerves these changes would be expected to occur in the MVN of both sides. Instead the adaptive changes in intrinsic excitability and GABA receptor efficacy occur in response to the imbalance in excitability of the vestibular neurones of the lesioned and intact sides, and the enhanced commissural inhibition of the ipsi-lesional neurones that follows UL.

Implications for vestibular function and plasticity: the ‘homeostasis of bilateral excitability’

Our results show that the functional efficacy of GABAergic inhibition in MVN neurones undergoes adaptive up- or downregulation, over a period of hours, in response to a disparity in the afferent inputs to the vestibular nuclei of the two sides in vivo. The changes in GABA receptor efficacy are in the appropriate direction to help ‘re-balance’ the excitability of the vestibular neurones of the two sides. In relation to the normal function of the vestibular system, this dynamic regulation of GABAergic inhibitory efficacy is therefore a potential cellular mechanism for the ‘homeostasis of bilateral excitability’ of the vestibular nuclei. Such balanced excitability of the vestibular neurones of the two sides is believed to be a fundamental requirement for the normal bilateral control of eye and head movements by the vestibular system. The adaptive changes in GABAergic inhibitory efficacy in the MVN neurones are sufficiently rapid for them to be important in rectifying disparities in excitability of the vestibular nuclei that may arise from, for example, acute perturbations of the afferent input from an affected ear in vestibular neuritis or Meniere's disease, and age- or environmentally induced changes in peripheral afferent inputs from the two inner ears. In this light, the large compensatory changes in GABA receptor efficacy that occur after UL may be the maximal response of this normal homeostatic mechanism to the profound disparity in excitability between the two sides after the complete loss of afferent input from one ear. While the present results do not indicate whether the changes in GABA receptor function are synapse specific for a particular pathway, it is likely that the inhibitory vestibular commissures that link the vestibular nuclei of the two sides provide a major neural substrate for the homeostasis of bilateral excitability.

Acknowledgments

This work was supported by the Wellcome Trust (grants 043132, 051932). We are very grateful to Dr R. B. Barlow for his advice and help. A. Him is supported by a scholarship from the Turkish Ministry for Education.

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