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MS 0266 Received 25 October 1999; accepted after revision 3 March 2000.
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ABSTRACT |
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INTRODUCTION |
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Glycine and 
-amino-butyric acid (GABA) are the two major inhibitory neurotransmitters in the central nervous system (CNS). These neurotransmitters are known to act in separate pathways in the brain and spinal cord, but may also co-localize in the same presynaptic terminals, as demonstrated in the spinal cord (Todd et al. 1996; Jonas et al. 1998), cerebellum (Ottersen et al. 1988) and anteroventral cochlear nucleus (Juiz et al. 1996). These observations raise questions concerning the particular roles of the two transmitter systems. A co-operative action has been shown in a recent study by Jonas et al. (1998), demonstrating that glycine and GABA may be co-released from the same terminals to simultaneously activate postsynaptic ionotropic glycine and GABA receptors. Close association between GABA and glycine raises other possibilities, such as the modulation of glycinergic transmission by GABAergic activation of pre- or postsynaptic metabotropic receptors.
We have investigated the roles of glycine and GABA in a rat brainstem auditory nucleus, the anteroventral cochlear nucleus (AVCN). Previous immunohistochemical studies have demonstrated glycine or GABA within the AVCN of the rat (Altshuler et al. 1986) and other species (Wenthold et al. 1986; Glendenning & Baker, 1988; Juiz et al. 1989, 1996). Three types of inhibitory terminals have been identified in the AVCN: those containing glycine or GABA alone, and those containing both glycine and GABA (Oberdorfer et al. 1988; Glendenning & Baker, 1988; Juiz et al. 1996). Interestingly, although the majority of inhibitory terminals contain glycine, approximately half the glycinergic terminals also contain GABA (Juiz et al. 1996; see also Ostapoff et al. 1997). A role for both GABA and glycine has been suggested by previous physiological studies (Caspary et al. 1994; Backoff et al. 1997), although other studies indicate that postsynaptic inhibition in the AVCN is primarily mediated by glycine (Wu & Oertel, 1986; Wickesberg & Oertel, 1990). A variety of physiological and developmental roles have been suggested for glycine and GABA in the cochlear nucleus and other auditory brainstem nuclei, but the reason for the presence of both inhibitory neurotransmitters is not clear (Wu & Oertel, 1986; Wickesberg & Oertel, 1990; Caspary et al. 1994; Ostapoff et al. 1997; Backoff et al. 1997; Kotak et al. 1998).
In the present experiments, we have examined glycinergic and GABAergic transmission in AVCN bushy cells using immunohistochemistry and electrophysiological recordings of synaptic currents. Bushy cells exhibit a simple morphology, with a spherical or globular cell body and usually a short, tufted dendrite (Cant & Morest, 1979; Wu & Oertel, 1984). As the majority of synaptic contacts are made with the cell body of bushy cells, this provides a considerable advantage in the study of synaptic currents by avoiding the problems of dendritic electrotonic attenuation (Lim et al. 1999). Our recent study on inhibitory connections with bushy cells found a correlation between glycinergic quantal current amplitude and gephyrin immunoreactive cluster area (Lim et al. 1999). Gephyrin causes clustering of glycine receptors by linking the cytoplasmic loop of the 
-subunit of the glycine receptor to microtubules and microfilaments of the cytoskeleton (Kirsch et al. 1993; Meyer et al. 1995; Kirsch & Betz, 1998). Gephyrin may also associate with GABA receptors, specifically via an interaction with the 2 subunit of the GABAA receptor (Essrich et al. 1998). Therefore, it is possible that both glycine and GABA receptors are co-localized with gephyrin at a proportion of the inhibitory synapses on AVCN bushy cells.
In the present study, our immunohistochemical and electrophysiological results indicate that, despite a strong co-localization of glycine and GABA in the AVCN, glycine is the major inhibitory transmitter acting at postsynaptic ionotropic receptors on bushy cells. However, the results reveal a specific presynaptic role for GABA acting via presynaptic metabotropic GABAB receptors to depress release at glycinergic synapses on AVCN bushy cells.
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METHODS |
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Immunocytochemistry
Immunohistochemical results were obtained from rats in the early postnatal developmental period, since our electrophysiological recordings from rat AVCN slices are technically limited to the developmental period of less than 3 weeks postnatal (usually between 1 and 2 weeks). Three albino rats (3 weeks postnatal age) were injected (I.P.) with an overdose of sodium pentobarbital (> 80 mg kg-1) and perfused via the left ventricle with cold vascular rinse (0·01 M phosphate buffer with 137 mM NaCl, 3·4 mM KCl, and 6 mM NaHCO3) followed by fixative (4 % paraformaldehyde in 0·1 M phosphate buffer; pH 7·4) for 10-15 min. The brainstem and spinal cords were removed, not postfixed and stored at 4°C in 0·1 M phosphate buffer containing 15 % sucrose. This light fixation regime has proven adequate for recognition of epitopes in the extracellular N-terminal regions of the 
1 subunit of the glycine receptor and the 2/3 subunits of the GABAA receptor by monoclonal antibodies (mAb) 2b and bd-17, respectively (Alvarez et al. 1996, 1997). Light fixation also increases immunofluorescence for gephyrin using mAb 7a. All animal procedures were performed according to NIH guidelines and approved by the local animal welfare and ethics committee.
Transverse sections (30 µM thick) of the brainstem were obtained at the level of the cochlear nuclei on a freezing sliding microtome. The sections were immediately mounted on gelatin-coated slides, air-dried and frozen until use. Then, the sections were washed in phosphate-buffered saline with 0·1 % Triton X-100 (PBS-T, 0·01 M; pH 7·4) and incubated with one of the following primary antibodies: mouse mAb 7a diluted 1:100 (Boehringer Mannheim, Indianapolis, IN, USA), mouse mAb bd-17 diluted 1:10 (Boehringer Mannheim) or mouse mAb 2b diluted 1:50 (gift from Drs J. Kirsch and H. Betz, Max Plank Institute, Frankfurt, Germany). All dilutions were made in PBS-T. Cells were incubated overnight at 4°C. The sections were then washed in PBS-T and incubated for 3 h in FITC-conjugated goat anti-mouse IgG (1:50 in PBS-T; Jackson, West Grove, PA, USA). The preparations were coverslipped with anti-fading and fluorescence mounting medium (Vectashield, Vector, Burlingame, CA, USA).
Confocal microscopy
Immunofluorescence preparations were imaged using a laser scanning confocal microscope (Olympus Fluoview). Low magnification imaging was performed with a ×10 objective lens (NA 0·3; pixel size 1·60 µm2) and confocal conditions (confocal aperture, laser strength, scan velocity, photomultiplier tube sensitivity, gain and offset) were kept identical when imaging immunofluorescence for each of the three antibodies. Hence, grey intensity levels reflect the relative immunofluorescence intensity obtained with each antibody in Fig. 1. High magnification analysis was carried out with a ×60 oil immersion objective lens (NA 1·4) and digitally zoomed (×2; pixel size = 0·13 µm2). The optimal confocal conditions were set for each antibody and maintained while imaging different neurons in the same or serial sections. Differences in intensity of immunolabelling between cells for each particular antibody therefore parallel immunoreactivity differences between cells (Figs 2 and 3). For surface reconstruction of selected neurons a series of optical sections separated by either 0·5 or 1 µm in the z-axis were acquired throughout whole cells. For clarity, only half-cells (from a mid-cross section to the top or bottom surfaces) were reconstructed. Figure trimming, pasting and composition was done in ImagePro Plus (version 3.0.01; Media Cybernetics) and CorelDraw 3.0 (Corel, Ottawa, Canada).
Electrophysiology
Albino rats (Wistar, 12-16 days old) were anaesthetized with 20 mg kg-1 sodium pentobarbitone I.P. and decapitated. Parasagittal slices (150 µm) were made of the anteroventral cochlear nucleus (AVCN) as previously described (Isaacson & Walmsley, 1995; Lim et al. 1999). Whole-cell patch electrode recordings (at a membrane potential of -60 mV) were made from neurons visualized in the slices using infrared differential interference contrast optics. Afferent nerve fibres were stimulated (0·1 ms, 2-40 V pulses) using a glass microelectrode filled with artificial cerebrospinal fluid (ACSF) and placed close to the recorded cell. Experiments were performed at room temperature (22-25°C), and conducted on slices superfused with an ACSF solution containing (mM): 130 NaCl, 3·0 KCl, 1·3 Mg2SO4, 2·0 CaCl2, 1·25 NaH2PO4, 26·2 NaHCO3, 10 glucose, equilibrated with 95 % O2-5 % CO2. Patch electrodes (3-5 M
resistance) contained (mM): 120 CsCl, 4 NaCl, 4 MgCl2, 0·001 CaCl2, 10 Hepes, 3 Mg-ATP, 0·2 GTP-tris, and 0·2-10 EGTA (pH 7·2). In one series of experiments to examine postsynaptic GABAB responses, potassium methyl-sulphate was substituted for CsCl in the patch pipette solution, and recordings were made at membrane potentials of -20 to -60 mV. Series resistance, which was <10 M, was routinely compensated by >80 %. Synaptic currents were recorded and filtered at 10 kHz with an Axopatch-1D amplifier (Axon Instruments) before being digitized at 20 kHz. Data were also recorded on videotape with a Vetter videocassette recorder and digitized off line. Data acquisition and analysis was performed using Axograph software (Axon Instruments). The amplitudes of spontaneous IPSCs were measured using semi-automated detection procedures (Axograph 4.0), as previously described (Lim et al. 1999). Results are expressed as means ± standard error of the mean (S.E.M.). Significance of results was assessed using Student's paired t test. (In most cases, statistical tests on evoked IPSCs were carried out using relative changes, normalized with respect to the control IPSC, since the absolute control IPSC amplitude was arbitrarily set by the stimulus conditions for each cell.)
Drugs were added to the perfusate, as indicated, from stock H2O-based solutions (except where indicated otherwise): 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris; 20 mM stock, dissolved in DMSO), (±)-2-amino-5-phosphonopentanoic acid (APV; RBI; 50 mM stock), bicuculline methochloride (Tocris; 10 mM stock), bicuculline methiodide (Sigma; 10 mM stock), strychnine hydrochloride (Sigma; 1 mM stock), tetrodotoxin (TTX; Alamone; 1 mM stock). The GABAB antagonist, 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348; 10 mM stock) was a gift from Novartis.
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RESULTS |
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Immunohistochemistry
Immunolabelling experiments were carried out to provide a structural basis for subsequent electrophysiological experiments on the functional role of glycine and GABA in the AVCN. The distribution of immunolabelled glycine receptors (1 subunit), GABAA receptors (2/3 subunit) and gephyrin was examined over the surface of bushy cells in the rat AVCN. As a control for the AVCN immunolabelling, simultaneous observations were made of immunolabelled neurons from other adjacent brainstem nuclei (spinal vestibular nucleus, reticular formation), taken from the same histological sections. Identical imaging protocols were used to facilitate comparison between different regions and immunolabels (Methods).
Figure 1 illustrates immunolabelling results for the AVCN. GABAA receptor immunolabelling was found to be weak in most regions of the AVCN, except for a strong band of labelling in the granule cell layer (Fig. 1A). Labelling of this band is consistent with intense GABAA and GABAB binding studies (Juiz et al. 1994). In contrast, strong punctate immunolabelling of glycine receptors (Fig. 1B, E and H) and gephyrin (Fig. 1C, F and I) was found throughout the AVCN. High magnification images illustrate the punctate labelling of the receptor clusters of both the glycine receptors (Fig. 1E) and gephyrin (Fig. 1F). As reported previously (Wenthold et al. 1988), immunolabelling for gephyrin was more intense than for the 1 subunit of the glycine receptor, perhaps reflecting the better characteristics of gephyrin epitopes over 1 subunit epitopes for immunolocalization studies using these monoclonal antibodies (Kirsch & Betz, 1993; Alvarez et al. 1997). Labelling of GABAA receptors was generally diffuse with very few postsynaptic clusters (Fig. 1D). Surface reconstructions of individual cells shown by asterisks (Fig. 1D, E and F) exhibited strong punctate covering of bushy cell somas with gephyrin (Fig. 1I) and glycine receptor immunoreactivity (Fig. 1H). In contrast, GABAA receptor immunolabelling displayed only a few sparse weakly immunoreactive clusters and diffuse weak membrane labelling (Fig. 1G). This striking difference in labelling patterns suggests a lack of association between GABAA receptors and glycine receptor (or gephyrin) clusters in AVCN neurons, and indicates a predominance of glycine over GABA postsynaptic receptor clusters on AVCN neurons.
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Distribution of GABAA receptor | ||
Weak GABAA immunolabelling and low numbers of local surface intensities or 'hot-spots' on AVCN neurons were not due to technical difficulties with the immunocytochemistry. Uniform and robust immunolabelling was observed in adjacent areas of the same sections which contained weakly immunolabelled AVCN neurons. These neighbouring brainstem neurons frequently displayed 'hot-spots', in addition to continuous extrasynaptic immunolabelling (Fig. 2). Diffuse cytoplasmic GABAA receptor immunoreactivity and immunoreactive granules in the cytoplasm were also clearly visible in strongly immunolabelled brainstem neurons (Fig. 2B and C). These most probably correspond to immunoreactivity localized to the Golgi apparatus and to small transport vesicles, as demonstrated previously in spinal neurons with electron microscopy (Alvarez et al. 1996). Cytoplasmic immunolabelling was rare and weak on AVCN neurons, also indicating low levels of expression of GABAA receptor subunits. In contrast, immunofluorescence intensity for glycine receptors or gephyrin was no different in AVCN neurons than in other brainstem or spinal neurons and displayed identical patterns of surface clustering or 'hot-spots' (Fig. 3). In addition, some intracellular granules were seen immunolabelled for glycine receptor 1 subunits but not for gephyrin, perhaps reflecting differences in the trafficking mechanisms of both molecules.
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Comparison of immunofluorescence for the | ||
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Immunofluorescence for the | ||
In summary, GABAA receptor 2/3 subunits appear to be expressed at lower levels on AVCN neurons compared with other brainstem neurons. In contrast, glycine receptor 1 subunits and gephyrin are expressed at levels comparable to the highest expressing neurons in other brainstem nuclei, and appear concentrated in surface clusters known to represent postsynaptic receptor aggregates (Triller et al. 1985; Wenthold et al. 1988; Alvarez et al. 1997). Our results also suggest a closer association of gephyrin with postsynaptic glycine receptors than with GABAA receptors in AVCN neurons.
Electrophysiology
Whole-cell patch-clamp recordings were used to determine the relative contributions of glycine and GABAA receptor activation to inhibitory transmission in AVCN bushy cells. Figure 4A illustrates the postsynaptic currents recorded in a bushy cell in a thick slice preparation of the entire cochlear nucleus (approximately 300-400 µm thick), following stimulation of the auditory nerve stump with a bipolar stimulating electrode. The response demonstrates an initial large monosynaptic excitatory current (due to the endbulb of Held connections; Isaacson & Walmsley, 1995), followed by a later polysynaptic inhibitory current (Wu & Oertel, 1986). This response exemplifies the prominent but delayed nature of auditory nerve-mediated inhibitory input to bushy cells, generated within the cochlear nucleus itself (Wu & Oertel, 1986). As inhibitory inputs also originate outside the cochlear nucleus, and local inhibitory nerve fibres may be severed by the slice procedure, all subsequent experiments were carried out with IPSCs evoked using local stimulation close to the recorded cell. (Because similar results were obtained for a variety of stimulus conditions, it seems unlikely that the stimulation procedure restricted the inputs to an exclusive subset of input fibre type.) Bushy cells were visualized in thin AVCN slices (150 µm thick), identified initially on the basis of their morphology (with globular or spherical cell bodies) and, following whole-cell recording, by the presence of characteristically brief excitatory spontaneous synaptic currents, as shown in our previous studies (Isaacson & Walmsley, 1995). Following identification, AMPA and NMDA receptor-mediated excitatory currents were blocked using CNQX (10 µM) and APV (50 µM), respectively, in order to record IPSCs in isolation. Between 50 and 150 evoked IPSCs were averaged to obtain a reliable measure of the control inhibitory synaptic current. These IPSCs result from the activation of multiple fibres, as evidenced by the graded nature of IPSC amplitude with stimulus voltage. For each cell, the stimulation voltage was adjusted to obtain a stable IPSC, and the voltage was not altered during the recording period.
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A, stimulation of the auditory nerve (arrow) resulted in a monosynaptic excitatory postsynaptic current followed by a disynaptic inhibitory postsynaptic current in a bushy cell of the AVCN. B, whole-cell patch-clamp recordings from two bushy cells following local stimulation show that strychnine (1 µM) almost completely abolishes the IPSC, while the residual current is blocked by bicuculline (10 µM). Records are averages of 102 responses (a) and 141 responses (b). C, bicuculline (10 µM) has very little effect in blocking the IPSC, which is subsequently blocked by application of strychnine (1 µM). Records are averages of 173 responses (a) and 104 responses (b) from two different bushy cells. Arrows in all panels indicate times of stimulus artefact which have been removed from all traces. | ||
The relative glycine versus GABA contributions to evoked IPSCs were determined by addition of the pharmacological antagonists strychnine or bicuculline, respectively, to the perfusate. The concentrations of strychnine (1 µM) and bicuculline (10 µM) were chosen to provide a near-maximal effect, as determined by previous studies (e.g. Jonas et al. 1998). Although 10 µM bicuculline is likely to have very little effect on glycine receptors, there may be partial antagonism of GABA receptors by 1 µM strychnine (Jonas et al. 1998). Therefore, experiments were conducted by applying either strychnine or bicuculline first, followed by application of the other antagonist. Figure 4Ba and b illustrates the responses of two different bushy cells to application of the glycine receptor antagonist, strychnine (1 µM), followed by application of the GABAA receptor antagonist, bicuculline (10 µM). Figure 4Ca and b illustrates the responses of two other bushy cells in which bicuculline was added first, followed by strychnine. For 11 cells in which strychnine was applied first, the evoked IPSC was decreased by application of strychnine (1 µM) to a mean of 10 ± 3·4 % of the control peak amplitude (mean peak amplitude of control IPSC, 400·7 ± 78·8 pA; mean peak amplitude in presence of strychnine, 31·6 ± 14·5 pA). Application of bicuculline (10 µM) first reduced the evoked IPSC amplitude to 81 ± 6·1 % of the control amplitude (n = 5 cells; mean peak amplitude of control IPSC, 161·7 ± 49·2 pA; mean peak amplitude in presence of bicuculline, 137·6 ± 52·4 pA). The latter result provides a more reliable quantitative estimate of the contribution of GABA receptors to the evoked IPSC, although both results suggest that the majority of the evoked IPSC is mediated by glycine receptor activation.
We next recorded spontaneous miniature IPSCs (mIPCSs), in the presence of TTX (1 µM) to block presynaptic action potentials (in addition to 10 µm CNQX and 50 µM APV to block glutamatergic transmission). Figure 5 shows recordings of mIPSCs in two different AVCN bushy cells (Fig. 5Aa and Ba). Addition of strychnine (1 µM) to the perfusate eliminated nearly all of the mIPSCs in both cells (Fig. 5Ab and Bb). Similar results obtained in a total of five AVCN bushy cells demonstrated that, on average, 93 % of the detectable mIPSCs in these cells are glycinergic. We have shown previously that mean glycinergic mIPSC amplitude varies considerably between bushy cells (Lim et al. 1999). The small contribution of GABAA receptor- mediated spontaneous events did not appear to differ between bushy cells displaying either predominantly large (Fig. 5A) or small (Fig. 5B) glycinergic mIPSCs.
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A and B, continuous whole-cell recording from two different bushy cells with large (Aa) and small (Ba) spontaneous mIPSCs, in the presence of TTX (1 µM). Application of strychnine (1 µM) abolished the majority of detectable mIPSCs in both cells (Ab and Bb). | ||
These results suggest that glycine is the major inhibitory transmitter in the AVCN. However, a large complement of inhibitory terminals in the AVCN contain both glycine and GABA. It is therefore possible that GABA may mediate its effects through activation of metabotropic GABAB receptors. Activation of presynaptic GABAB receptors may depress transmitter release by reducing calcium currents in presynaptic terminals (Takahashi et al. 1998), while postsynaptic GABAB receptor activation may activate membrane conductances, such as a slow potassium conductance (Newberry & Nicoll, 1985). Preliminary experiments (n = 3), conducted using a potassium methylsulfate patch-electrode solution, membrane depolarization (-40 mV) and high frequency nerve stimulation (5 pulses, 50 Hz), failed to find evidence for the nerve-mediated activation of membrane currents (e.g. potassium currents) which may have resulted from postsynaptic GABAB receptor activation (data not shown).
We next investigated the possible involvement of presynaptic GABAB receptors. Evoked glycinergic IPSCs were recorded before and after the addition of baclofen. Figure 6A illustrates a marked reduction by baclofen (10 µM) of the evoked IPSC. A significant reduction was observed in all cells examined (mean, 16 ± 3·9 % of control amplitude; P < 0·01, n = 3 cells). We also found that the mean amplitude of mIPSCs was unchanged in the presence of baclofen (mean control mIPSC amplitude 94 ± 21·5 pA, mean mIPSC amplitude in baclofen 91 ± 18·7 pA, P > 0·76, n = 4 cells; see also Wall & Dale, 1993; Grudt & Henderson, 1998). The lack of a postsynaptic effect on glycinergic mIPSC amplitude indicates that the dramatic reduction in nerve-evoked IPSCs following application of baclofen is most likely via activation of presynaptic GABAB receptors. A presynaptic effect was supported by the action of baclofen to decrease the mean mIPSC frequency to 62 ± 37 % (n = 4 cells) of the control value (mean frequency of control mIPCSs, 37 ± 10 mIPCSs min-1; frequency in the presence of baclofen, 23 ± 6 mIPSCs min-1; n = 4 cells). We were therefore interested in determining whether presynaptic GABAB receptors could be activated physiologically through nerve stimulation.
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A, nerve-evoked glycinergic IPSC, recorded in the presence of CNQX (10 µM), APV (50 µM) and bicuculline (10 µM). Application of the GABAB receptor agonist baclofen (10 µM) significantly attentuated the evoked glycinergic IPSC (85 % reduction). Traces are averages of 98 records. B, glycinergic IPSCs during a pulse train (5 pulses at 50 Hz), followed by a single stimulus (delay 200 ms), before (continuous traces) and after (dashed lines) bath application of the GABAB antagonist CGP 35348 (500 µM). Traces are averages of 50 records. Stimulus artefacts have been removed (arrows). | ||
We examined the effect of the GABAB receptor antagonist CGP 35348 on the nerve-evoked glycinergic IPSC, during repetitive trains of stimuli. Figure 6B illustrates nerve-evoked glycinergic IPSCs isolated in the presence of CNQX, APV and bicuculline. The control trace (average of 5 pulse trains; continuous line) illustrates glycinergic IPSCs during a brief stimulus train (5 pulses, 50 Hz), followed by a single test stimulus 200 ms after the train. If GABA were co-released during the stimulus train, then it may act presynaptically to depress glycine release and thus reduce the evoked IPSCs. Application of the GABAB antagonist should therefore block this effect and produce an increase in the evoked glycinergic IPSCs. Figure 6B (dashed lines) shows that this is the case. An increase in the amplitude of the test IPSC can be seen, following bath application of CGP 35348 (500 µM; Isaacson et al. 1993). No significant change was observed in the amplitude of the first IPSC in the pulse train (P1), ruling out a possible non-specific effect of CGP 35348 (P > 0·05; mean control IPSC amplitude 713 ± 299 pA). In all six cells examined, an increase was observed in the ratio of the delayed test IPSC to the first (control) IPSC in the train, in the presence of CGP 35348 (mean increase, 26 ± 8·7 %; P < 0·01, n = 6 cells). This result demonstrates that presynaptic GABAB receptors can be activated by synaptically released GABA during repetitive nerve activation, resulting in a reduction of glycine release.
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DISCUSSION |
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Previous studies have reported a large proportion of inhibitory presynaptic terminals in the AVCN containing both GABA and glycine (Oberdorfer et al. 1988; Juiz et al. 1989, 1996; Ostapoff et al. 1997). The electrophysiological results presented here indicate that postsynaptic inhibition in bushy cells of the AVCN is mediated primarily by glycine, with only a minor contribution from GABAA receptors, as previously suggested in the mouse (Wu & Oertel, 1986). A major postsynaptic role for glycine in contrast to GABA is supported by our immunohistochemical results using monoclonal antibodies known to specifically immunolocalize GABAA receptor 2/3 subunits (mAb bd-17; Ewert et al. 1990), glycine receptor 1 subunits or gephyrin (mAbs 2b and 7a; Pfeiffer et al. 1984). Beta subunits are almost universal in functional brain GABAA receptors, since they are necessary for forming the native GABA/muscimol and picrotoxin binding sites and targeting of the receptor (Mehta & Ticku, 1999). In the AVCN, 2 and 3, but not 1, expression has been detected using in situ hybridization (Zhang et al. 1991). Therefore, it is likely that antibodies raised against a shared epitope of 2/3 subunits (Ewert et al. 1990) will detect a large proportion of (if not all) functional GABAA receptors expressed by AVCN neurons. In contrast to GABAA receptors, mature postsynaptic glycine receptors are generally formed by only two subunit types (Kuhse et al. 1993), although the presence of subunit homo-oligomers at postsynaptic sites has also been proposed (Legendre, 1997). Molecular variability in subunits has also been detected (reviewed in Betz, 1992) and AVCN neurons in animals younger than 5 weeks co-express all three subunits cloned in the rat (Sato et al. 1995). Alpha-1 subunits are upregulated after 4 days of postnatal development (Friauf et al. 1997), 2 subunits are downregulated after 5 weeks of postnatal development and 3 subunits are expressed throughout (Sato et al. 1995). A preponderance of 1 subunits is suggested, based on the relative intensity of the in situ hybridization signal (Sato et al. 1995). Hence, immunohistochemistry against the 1 subunit of the glycine receptor constitutes an adequate target to compare the distribution of glycine receptors to that of gephyrin or GABAA receptors. Finally, gephyrin is a protein associated with the inner leaf of the plasma membrane and specifically associated with postsynaptic sites at inhibitory synapses (Triller et al. 1985). Many studies have demonstrated an important role for gephyrin in the postsynaptic clustering of glycine receptors at spinal and brainstem synapses (Kirsch et al. 1993; Meyer et al. 1995; Kirsch & Betz, 1998). More recent studies provided evidence suggesting a possible interaction between gephyrin and GABAA receptors at certain synapses (Essrich et al. 1998), and it is therefore important to elucidate which receptors are preferentially expressed by neurons displaying prominent gephyrin clusters on their surfaces.
Glycine receptor and gephyrin immunolabelling showed many intense punctate clusters over the surface of AVCN bushy cells of very similar appearance, frequency and distribution, compared with weak, diffuse labelling of postsynaptic GABAA receptors. Weak GABAA receptor labelling and the fixation sensitivity of targeted epitopes may explain previous observations at the light microscopic level reporting undetectable labelling, using avidin-peroxidase techniques in more strongly fixed tissue with another monoclonal antibody of similar specificity to the one used here (Juiz et al. 1989). Weak 2/3 subunit immunolabelling is also consistent with low levels of GABAA 2/3 subunit expression detected using in situ hybridization (Zhang et al. 1991). We conclude that GABAA receptors are expressed at low levels in AVCN neurons and do not appear to greatly concentrate at postsynaptic membrane 'hot-spots'. Our results are consistent with GABA receptor 2/3 subunits located at both synaptic and extrasynaptic sites (Nusser et al. 1998). In contrast, both glycine receptor and gephyrin immunolabelling is concentrated in membrane clusters that ultrastructurally have been shown to correspond to postsynaptic sites (Triller et al. 1985; Wenthold et al. 1988; Alvarez et al. 1997).
Previous reports have shown a decrease in glutamatergic and GABAergic transmission via activation of presynaptic GABAB receptors by baclofen (e.g. Thompson & Gahwiler, 1992; Brenowitz et al. 1998). Our electrophysiological results demonstrate that the GABAB receptor agonist, baclofen, strongly attenuates the amplitude of the postsynaptic glycinergic IPSCs in AVCN bushy cells. Our results also show that baclofen does not change the mean amplitude of glycine mIPSCs, consistent with similar observations in Xenopus embryo motoneurons (Wall & Dale, 1993) and rat substantia gelatinosa neurons (Grudt & Henderson, 1998). Our results suggest that baclofen depresses transmitter release through activation of presynaptic GABAB receptors located on glycinergic terminals (Wall & Dale, 1993; Grudt & Henderson, 1998; Jonas et al. 1998), possibly due to a reduction in calcium entry into the presynaptic terminal (Wall & Dale, 1994; Takahashi et al. 1998). The existence of presynaptic GABAB receptors on nerve terminals does not provide direct evidence in support of a physiological role for these receptors. Importantly, such a role in the AVCN was supported by the further observation that nerve-mediated release of GABA following repetitive stimulation depresses glycinergic inhibitory transmission via activation of presynaptic GABAB receptors.
Nerve-mediated presynaptic inhibition may be due to a number of different mechanisms. The most direct mechanism is via presynaptic contacts, as demonstrated in the spinal cord (Walmsley et al. 1987). However, this is unlikely in the AVCN, since electron microscopy studies have not reported the existence of presynaptic boutons on nerve terminals contacting bushy cells (Wenthold, 1987; Ostapoff & Morest, 1991). Our observation of nerve-mediated activation of presynaptic GABAB receptors is therefore probably due to autoinhibition arising from co-release of GABA and glycine from the same terminals, or diffusion of GABA from nearby nerve terminals (Isaacson et al. 1993). Since the large excitatory auditory terminals (the endbulbs of Held) on the soma of bushy cells interdigitate with the inhibitory nerve terminals (Ostapoff & Morest, 1991; Lim et al. 1999), this raises the interesting possibility that GABA may spillover sufficiently to activate presynaptic receptors on the excitatory terminals. This possibility, supported by the observation that baclofen severely depresses transmission at the endbulb synapse (B. Walmsley, unpublished observations; Brenowitz et al. 1998), may provide an additional role for GABA in the AVCN.
Finally, it should be emphasized that the present results were obtained in immature rats (less than 3 weeks postnatal age), during a period when there are likely to be developmental changes in inhibitory transmission in the AVCN (Sato et al. 1995; Friauf et al. 1997). A developmental role for GABA in the brainstem auditory system has been suggested by Kotak et al. (1998), who have described a developmental transition from GABAergic to glycinergic transmission in the gerbil over the first 2 weeks postnatal. Whether there is a further shift in the relative roles of GABA and glycine in the adult rat AVCN remains to be determined.
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REFERENCES |
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We are very grateful to Drs Ian Forsythe and Sharon Oleskevich for comments on an earlier draft of the manuscript. This work was supported by the HFSP (F.J.A.), and NIH grants (33555 and 25547; F.J.A. and R. E. W. Fyffe).
Corresponding author
B. Walmsley: The Synaptic Structure and Function Group, Division of Neuroscience, The John Curtin School of Medical Research, The Australian National University, PO Box 334, Canberra, ACT 2601, Australia.
Email: bruce.walmsley{at}anu.edu.au
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