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. 1999 Apr 15;516(Pt 2):505–512. doi: 10.1111/j.1469-7793.1999.0505v.x

Quantal size is correlated with receptor cluster area at glycinergic synapses in the rat brainstem

Rebecca Lim *, Francisco J Alvarez *, Bruce Walmsley *
PMCID: PMC2269264  PMID: 10087348

Abstract

  1. Whole-cell patch electrode recordings of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were obtained in neurons of the rat anteroventral cochlear nucleus (AVCN). Mean mIPSC peak amplitude was found to vary considerably between AVCN neurons (range, -19·1 to -317·9 pA; mean ± s.d., -159·1 ± 100·7 pA; 14 cells).

  2. Immunolabelling of glycinergic receptor clusters in AVCN neurons was performed using antibodies against the glycine receptor clustering protein gephyrin. Measurements of the area of gephyrin immunoreactive clusters were obtained using confocal fluorescence microscopy. These measurements showed a large variability in cluster area, not only in the same cell (mean coefficient of variation, c.v., 0·66 ± 0·18; 16 cells), but also in mean cluster area between cells (range, 0·21-0·84 μm2; 16 cells).

  3. A possible relationship between mIPSC amplitude and receptor cluster area was investigated in a further series of experiments, in which mIPSCs recordings and immunolabelling of glycine receptor clusters were obtained for the same cells. In these experiments, AVCN neurons were identified using intracellular labelling with neurobiotin. Successful results using a combination of whole-cell recordings, neurobiotin identification and immunolabelling were obtained for a total of 10 AVCN neurons. Analysis of the results revealed a positive, statistically significant correlation between mean receptor cluster size and mean mIPSC amplitude (P < 0·05, 10 cells, Spearman's correlation test).

  4. These results provide direct experimental evidence supporting a hypothesis of central glycinergic transmission in which synaptic strength may be regulated by changes in the size of the postsynaptic receptor region.

Synaptic contacts between neurons in the central nervous system exhibit a highly specialized structural arrangement in which postsynaptic receptors are arranged in tight clusters, closely apposed to the presynaptic sites of quantal neurotransmitter release (Siekevitz, 1985; Walmsley, 1991; Kirsch et al. 1996; Nusser et al. 1997; Colledge et al. 1998; Walmsley et al. 1998). Considerable variability in the size and shape of these active zones has been observed, which may relate to differences in the functional parameters of individual synapses (Nusser et al. 1997; Schikorski & Stevens, 1997; Walmsley et al. 1998). A fundamental measure of synaptic function is provided by the postsynaptic current generated in response to the presynaptic release of a quantum of neurotransmitter. At an individual synapse, the amplitude of this quantal postsynaptic current depends theoretically on the concentration and time course of neurotransmitter in the synaptic cleft, and on the transmitter binding properties of the postsynaptic receptors (Clements, 1996). If the released neurotransmitter approaches a saturating concentration for the receptors, then the total number of postsynaptic receptors available to bind the neurotransmitter becomes an important limiting factor (Clements, 1996). The total number of available receptors is governed by structural factors such as receptor packing density and the size of the receptor cluster region. Previous studies at a variety of excitatory and inhibitory central synapses have demonstrated a wide range in both postsynaptic receptor cluster size and in the amplitude of quantal postsynaptic currents (Walmsley et al. 1998). Although these studies are suggestive, it has proven difficult to obtain experimental evidence on a potential relationship between these two parameters (Nusser et al. 1997; Walmsley et al. 1998).

At glycinergic inhibitory synapses in the central nervous system, recent experimental evidence has demonstrated that the size of receptor clusters may vary greatly, not only between synapses on the same postsynaptic neuron, but also in average size between different neuronal types (Alvarez et al. 1997). Interestingly, the size of glycine receptor clusters has also been shown to increase along the dendrites of particular neurons, leading to the suggestion that this provides compensation for the attenuation of synaptic potentials or currents travelling along the dendrites towards the cell soma (Triller et al. 1990; Alvarez et al. 1997). Synaptic activity has been shown to induce the focal membrane accumulation of gephyrin, a glycine receptor clustering protein which anchors the receptors to the subsynaptic cytoskeleton (Kirsch et al. 1993; Meyer et al. 1995; Kirsch & Betz, 1995, 1998). These studies suggest that receptor clustering may be related to function at glycinergic synapses. Very recent evidence, using gene targeting experiments, has demonstrated that gephyrin expression is an essential requirement for the clustering of glycine receptors (Feng et al. 1998). However, the question of whether or not receptor cluster size and/or receptor density are determining factors in synaptic strength is an important, unresolved issue, with obvious implications for our understanding of the mechanisms underlying synaptic plasticity during development and learning (Walmsley et al. 1998). In the present study we provide direct evidence on this relationship at a glycinergic synaptic connection in the anteroventral cochlear nucleus (AVCN) of the rat.

METHODS

Electrophysiology

Wistar rats (12-16 days old) were anaesthetized with 20 mg kg−1 sodium pentobarbitone i.p. and decapitated in accordance with Australian National University Ethics Committee guidelines (protocol no. JN7497). Parasagittal slices (150 μm thickness) were made of the anteroventral cochlear nucleus (AVCN), as previously described (Isaacson & Walmsley, 1995; Bellingham et al. 1998). Whole-cell patch electrode recordings were made from neurons visualized in the slices using infra-red differential interference contrast optics. All experiments were performed at room temperature (22-25°C), and conducted on slices superfused with a Ringer 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. Drugs (tetrodotoxin, CNQX, bicuculline, and strychnine) were added to the perfusion solution as indicated. Recordings of glycinergic currents were routinely carried out in the presence of TTX (1 μM), CNQX (1 μM) and bicuculline (10 μM). Control experiments were carried out in the absence of bicuculline. Patch electrodes (3-5 MΩ resistance) contained (mM): 120 CsCl, 4 NaCl, 4 MgCl2, 0·001 CaCl2, 10 Hepes, 3 magnesium ATP, 0·2 GTP-tris, and 0·2-10 EGTA (pH 7·2). For intracellular labelling, 0·2-2·0 mg ml−1 neurobiotin was added to the internal solution. Series resistance, which was < 10 MΩ, 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 VCR (Vetter) and digitized off-line. Data acquisition and analysis was performed using Axograph (Axon Instruments). The amplitudes of spontaneous IPSCs were measured using semi-automated detection procedures (Axograph 4.0).

Cell identification and immunohistochemistry

After recording, slices were immersion fixed for 5-30 min using 2-4 % paraformaldehyde in 0·1 M phosphate buffer (pH 7·4), dehydrated and rehydrated in alcohols to enhance penetration of antibodies and incubated for 3-4 days with mouse monoclonal antibodies against gephyrin (mAb7a: Boehringer-Mannheim, diluted 1 : 100) or glycine receptor α1 subunits (mAb2b: gift from Drs J. Kirsch and H. Betz, Max-Plank Institute, Frankfurt) both diluted 1 : 100 in 0·01 M phosphate-buffered saline (pH 7·2) with 0·3 % Triton X-100. Immunoreactive sites were visualized using secondary antibodies coupled to carboxymethylindocyanine (Cy3) (dilution 1 : 25; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Neurobiotin was revealed with fluorescein isothiocyanate (FITC) coupled avidin-D (dilution 1 : 10; Vector Laboratories Inc., Burlingame, CA, USA). Sections with neurobiotin-labelled cells were only processed with gephyrin antibodies. This combination allowed for longer fixation times and better structural analysis. The FITC-neurobiotin intracellular label and the Cy3-gephyrin immunofluorescence were imaged using a Leica confocal microscope. Optical sections of 0·5 μm in thickness were acquired through the whole cell body and dendritic arbor of the recorded cells. Full penetration of the immunostaining through the 150 μm section was confirmed under the confocal microscope. Gephyrin immunoreactive clusters were analysed using NIH Image (Bethesda, MD, USA). Measurements were made of area, immunostaining density, and the lengths of major and minor axes for each immunofluorescent cluster. For illustration, the top or bottom surfaces of half-cells were reconstructed from the stack of optical sections using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).

Unless otherwise stated, statistical analysis was performed using Spearman's non-parametric correlation test (Prism, Graphpad, San Diego, CA, USA).

RESULTS

The AVCN is a brainstem nucleus with an important role in sound localization (Oertel, 1991). Consistent with the precise timing required for sound localization, bushy cells of the AVCN receive powerful excitatory contacts, the endbulbs of Held, from individual auditory nerve fibres (Isaacson & Walmsley, 1995; Ryugo et al. 1996). Bushy cells also receive glycinergic inhibitory synaptic contacts which structurally resemble other inhibitory synapses in the brainstem and spinal cord (Wenthold & Hunter, 1990). Bushy cells are morphologically simple neurons with a large spherical or globular cell body, and usually a single short, tufted dendrite. Significantly, the lack of an extensive dendritic tree makes these cells ideal for the measurement of quantal synaptic currents, since the somatic location of synaptic contacts eliminates the electrotonic attenuation of these currents (Isaacson & Walmsley, 1995; Bellingham et al. 1998). In the present study, we have investigated the glycinergic inputs to AVCN neurons in slices of cochlear nuclei from rats 12-16 days old.

Immunolabelling of receptor clusters using antibodies to the glycine receptor clustering protein gephyrin reveals a profuse distribution of discrete clusters over the soma of AVCN neurons (Fig. 1), in agreement with previous studies using antibodies to glycine receptor α-subunits (Friauf et al. 1997). In some cells, a few proximal dendritic gephyrin immunoreactive (gephyrin-ir) clusters were observed, but these represented only a small percentage of the total number of clusters on these cells, and are therefore unlikely to influence the synaptic current recordings.

Figure 1. Glycine receptor cluster size varies between neurons in the AVCN.

Figure 1

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A and B, confocal fluorescence images of AVCN neurons labelled with monoclonal antibody 7a, directed against the glycine receptor clustering protein gephyrin. A illustrates a neuron exhibiting predominately large gephyrin immunoreactive (gephyrin-ir) clusters over the soma, in contrast to B which shows a neuron with small clusters. Arrows indicate cluster-free regions, the probable sites of large excitatory terminals, the endbulbs of Held, which are known to contact the soma of AVCN bushy cells. Scale bar (applies also to A), 10 μm. Insets show boxed regions at higher magnification (scale bar, 1 μm). C and D, histograms of gephyrin-ir cluster size (area, μm2) for the neurons shown in A and B, respectively (C, 122 clusters; D, 194 clusters). E, mean gephyrin-ir cluster size plotted against size variability (standard deviation), for sixteen AVCN neurons. Mean number of measurements = 137 clusters per cell (16 cells). F, comparison of the fluorescence intensity of gephyrin-ir clusters (hatched bars) for cells exhibiting small mean cluster size (0·27 μm2, 188 clusters, 3 cells) and large mean cluster size (0·75 μm2, 229 clusters, 3 cells). Error bars indicate s.e.m. Arbitrary intensity units.

Gephyrin associates with the β-subunit of the glycine receptor, and in situ hybridization of the rat AVCN has shown that during early development the β-subunit is expressed in association with α1, α2 and α3 glycine receptor subunits (Sato et al. 1995). In our AVCN slices, the patterns of labelling with antibodies against gephyrin and the α1 subunit are very similar. In addition, electron microscopy of the AVCN and other CNS neurons has shown that gephyrin immunolabelling is localized to the postsynaptic density (Alvarez et al. 1997; Wenthold & Hunter, 1990). Although the transmitter GABA may be present in some terminals in the AVCN (Wenthold & Hunter, 1990), there is little evidence for functional GABAergic transmission in bushy cells, as demonstrated by our control experiments carried out in the presence of TTX (1 μM) and CNQX (1 μM), showing that virtually all (> 99 %) of the spontaneous inhibitory postsynaptic currents are blocked by low concentrations of strychnine (< 1 μM). These data support previous proposals that glycine is the major inhibitory transmitter in the AVCN (Wenthold & Hunter, 1990), and demonstrates that the shape of the gephyrin-immunofluorescent patches accurately maps the structural features of postsynaptic glycine receptor clusters (see also Alvarez et al. 1997). In the present study, we have used gephyrin immunoreactivity as an excellent marker for glycine receptor clusters in preference to specific glycine receptor subunit antibodies, because of its robust fixation tolerence and greater visibility, particularly when used in conjunction with intracellular labels (see below).

High magnification analysis of gephyrin-ir clusters reveals a large variability of shapes and sizes. These clusters range in size from small immunofluorescent spots of around 0·2-0·5 μm in diameter to larger elongated patches, up to 3 μm in the longest axis, and often display complex structures including scalloped borders and perforations (Fig. 1A and B). Detailed measurements of the areas of > 2000 gephyrin-ir clusters from 16 cells demonstrate that cluster areas vary greatly, over two orders of magnitude, from 0·01 to 3·6 μm2. Histograms of cluster areas for individual cells show that there is considerable variability in cluster size over the surface of these cells (Fig. 1C and D). This variability is reflected in a large coefficient of variation (c.v.) of cluster areas (mean c.v., 0·66 ± 0·18, 16 cells). In addition, many of the histograms of cluster size exhibit a pronounced skew (mean skew, 0·83, 16 cells). However, a striking observation is that the mean cluster size varies greatly from cell to cell, as demonstrated by the plot of mean cluster size versus standard deviation for 16 cells (Fig. 1E). Mean cluster size was found to vary over a 4-fold range, from 0·21 to 0·84 μm2 (16 cells).

Although not a major aim of this study, we attempted to obtain an indication of receptor density using the method of Kirsch & Betz (1995), who demonstrated that gephyrin immunofluorescence intensity provides a quantitative measure of glycine receptor density. Our measurements were obtained from cells labelled under identical conditions, using the same confocal settings. Measurements were made of 188 clusters from three cells exhibiting small clusters (mean cluster size, 0·27 μm2), and 229 clusters from three cells with large clusters (mean cluster size, 0·75 μm2). These results show that, although there is variability in fluorescence intensity between clusters on individual cells, there is only a very small difference (8 %, Student's unpaired t test, P= 0·005) in mean fluorescence intensity between the clusters on small versus large cluster cells, despite an almost 3-fold difference in mean cluster size between these cells (Fig. 1F).

Following the observation that mean cluster size varies considerably from cell to cell, we next investigated whether or not the mean amplitude of glycinergic quantal currents also varies between AVCN neurons. In the presence of tetrodotoxin (1 μM) to block presynaptic action potentials, spontaneous inhibitory postsynaptic currents were measured using whole-cell patch clamp recordings in AVCN neurons (Fig. 2A-F). These miniature IPSCs (mIPSCs), recorded at a membrane potential of -60 mV, were isolated using CNQX (10 μM) to block AMPA receptor-mediated currents, and subsequently confirmed as glycinergic using the glycine receptor antagonist strychnine (1 μM). In an individual cell, a wide range of glycinergic mIPSC amplitudes is found, as illustrated for two cells shown in Fig. 2A-C and D-F. In parallel with our observations on receptor cluster sizes, the mean c.v. of mIPSC amplitudes for all cells is large (0·67 ± 0·15, 14 cells), and the mean amplitude of mIPSCs varies greatly between cells, ranging from -19·1 to -317·9 pA (mean, -159·2 ± 100·7 pA, 14 cells, Fig. 2G). One possible explanation for cell-to-cell differences in mean mIPSC amplitude could be that there are differences in the subunit composition of the heteromeric glycine receptors in each cell. As shown in studies of glycine receptor-mediated transmission in the spinal cord, a developmental switch in glycine receptor subunit composition from α2 to α1 is reflected by a major change in the mean channel open time of the receptor-channels, and in the decay time course of the synaptic currents (Takahashi et al. 1992). We examined this possibility and found that, although there is some cell-to-cell variability, there is no correlation between mean mIPSC amplitude and decay time course (half-width, P= 0·83, 14 cells). Thus, it is unlikely that a difference in receptor subunit composition is responsible for the large difference in mean mIPSC amplitude between cells. The mean 10-90 % rise time of mIPSCs was 0·42 ± 0·17 ms (14 cells). The decay time course was best fitted by the sum of two exponentials (mean τ1, 3·6 ± 1·5 ms; mean τ2, 28·4 ± 20·6 ms; 14 cells). These decay values are comparable to those found by Takahashi et al. (1992) for dorsal horn neurons in 16-day-old rats (mean τ1, 4·7 ms; mean τ2, 14·0 ms), although the rise times in our study are much briefer (see Fig. 5B in Takahashi et al. 1992), consistent with a somatic location of synaptic inputs on AVCN bushy cells.

Figure 2. Miniature glycinergic IPSCs in AVCN bushy cells exhibit large differences in mean amplitude between cells.

Figure 2

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A and D, continuous whole-cell recordings of mIPSCs from two different AVCN neurons in the presence of tetrodotoxin (1 μM), CNQX (10 μM), and bicuculline (10 μM). B and E, superimposed traces of mIPSCs, from the same neurons illustrated in A and D, respectively. C and F, histograms of mIPSC peak amplitudes from the same neurons illustrated in A and D, respectively (C, 582 mIPSCs; F, 282 mIPSCs). G, mean peak mIPSC amplitude plotted against standard deviation for 14 AVCN neurons. Mean number of mIPSC amplitude measurements, 506 per cell.

We next tested the hypothesis that mIPSC amplitude is directly related to the size of the receptor clusters. In this series of experiments, immunolabelling of gephyrin clusters was carried out on the same cells for which whole-cell recordings of mIPSCs were made, with each neuron identified using intracellular labelling with neurobiotin. Figure 3 illustrates two examples of neurobiotin-labelled neurons, together with their complement of gephyrin-ir clusters, the glycinergic mIPSCs recorded in each cell, and the histograms of cluster sizes and mIPSC amplitudes. The neuron shown in Fig. 3A and B exhibits large gephyrin-ir clusters and large mIPSCs (see histograms in Fig. 3C), whereas the neuron illustrated in Fig. 3D and E exhibits small clusters and small mIPSCs (see histograms in Fig. 3F). The success rate of these technically demanding combined experiments was very low. However, we were able to obtain successful results in which a combination of whole-cell recordings, neurobiotin identification and immunolabelling was achieved, for a total of 10 AVCN neurons. For each neuron, measurements were made of gephyrin cluster sizes and mIPSC amplitudes. Figure 4 illustrates a plot of mean cluster size versus mean mIPSC amplitude for all 10 neurons. Analysis of this plot demonstrated that there is a positive, statistically significant correlation between mean receptor cluster size and mean mIPSC amplitude (P < 0·05, 10 cells), supporting the proposal that synaptic strength may be influenced by receptor cluster size at central glycinergic synapses.

Figure 3. Combined immunolabelling of gephyrin clusters and whole-cell recordings of mIPSCs in the same neurons.

Figure 3

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A and D, recorded cells identified with neurobiotin fluorescence labelling. B and E, gephyrin-ir labelling of the cells shown in A and D, respectively. Scale bar (applies to A, B, D and E), 10 μm. Insets in B and E show boxed regions at higher magnification (scale bar, 1 μm). For the same cells shown in A and D, panels C and F show superimposed traces of mIPSCs (insets) and histograms of mIPSC amplitudes (top) and gephyrin-ir cluster sizes (bottom). Number of measurements for histograms in C, 1668 mIPSCs; 142 clusters; F, 228 mIPSCs; 263 clusters.

Figure 4. mIPSC amplitude is correlated with gephyrin-ir cluster size in AVCN neurons.

Figure 4

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Combined immunolabelling of gephyrin clusters, neurobiotin identification and whole-cell recordings of mIPSCs were obtained in 10 cells. Mean mIPSC amplitude is shown plotted against mean cluster size for all 10 cells (±s.e.m.). Mean number of measurements for cluster size, 127 per cell, and for mIPSC amplitudes, 137 per cell. Non-parametric analysis shows a statistically significant correlation between mean mIPSC amplitude and mean cluster size (Spearman's correlation test, P < 0·05).

DISCUSSION

Plasticity in the structure of synaptic contacts has been proposed to play a major role in regulating the efficacy of central synaptic transmission (Siekevitz, 1985; Calverley & Jones, 1990; Triller et al. 1990; Walmsley, 1991; Pierce & Mendell, 1993; Geinisman et al. 1996; Kirsch et al. 1996; Alvarez et al. 1997; Nusser et al. 1997; Schikorski & Stevens, 1997; Colledge et al. 1998; Walmsley et al. 1998). A change in synaptic efficacy may occur by an alteration in the total number of synapses, due to splitting and separation of individual synaptic contacts, and by a change in the efficacy of individual contacts. The strength of an individual synaptic contact is determined by the probability of quantal neurotransmitter release following the arrival of a nerve impulse, and by the amplitude of the postsynaptic response to transmitter release. An important underlying assumption of many structure-function hypotheses is that the amplitude of the postsynaptic response is related to total number of available receptors (Walmsley, 1995). Although receptor cluster size and receptor packing density may provide an indication of the total number of available postsynaptic receptors, the actual number of receptors activated by the release of neurotransmitter also depends on other factors, such as the duration and concentration of neurotransmitter in the synaptic cleft region and on the neurotransmitter binding affinity of the receptors (Clements, 1996). Thus, it is not obvious that a relationship should exist between postsynaptic receptor cluster morphology and the amplitude of the quantal postsynaptic current. Our results now provide direct experimental evidence supporting a relationship between postsynaptic receptor cluster size and synaptic strength, at central glycinergic synapses. However, this relationship is obviously not precise (see linear regression fit in Fig. 4), and may become asymptotic for very large cluster sizes, as proposed for GABAergic contacts by Nusser et al. (1997). Our results indicate that mean receptor packing density may be similar between cells, and therefore other factors such as receptor binding affinity and receptor subunit composition may contribute to the observed variability in synaptic efficacy between cells. In addition, there may be variability due to the rate of spontaneous release, which could differ between synapses and thus influence the distribution and mean value of mIPSC amplitudes (Walmsley, 1995).

The clustering of postsynaptic glycine receptors may be regulated by a variety of mechanisms, some involving specific clustering proteins such as gephyrin (Triller et al. 1990; Kirsch et al. 1993; Kirsch & Betz, 1995, 1998; Meyer et al. 1995; Alvarez et al. 1997; Feng et al. 1998). Recently, it has been demonstrated that the clustering of glycine receptors is regulated by presynaptic nerve activity, in a process involving the entry of calcium into the postsynaptic neuron (Kirsch & Betz, 1998; Craig, 1998). In previous studies, glycinergic synapses have been shown to exhibit activity-dependent long-term potentiation of synaptic strength (Oda et al. 1995). Alterations in synaptic strength may be due to either presynaptic or postsynaptic changes, or both. Our results suggest that activity-related changes in synaptic strength may be induced postsynaptically by changes in the size of receptor clusters. At central synapses, there is usually a close correspondence between the structural extent of the postsynaptic receptor region and the presynaptic release site or active zone (Walmsley et al. 1998). Therefore, changes in the size of the postsynaptic receptor region may result in a concomitant change in the size of the presynaptic active zone. If the probability of quantal transmitter release is related to the number of available vesicles and the size of the presynaptic active zone, then our present results also raise the interesting possibility that the properties of release may be indirectly influenced by changes in the size of the postsynaptic receptor region (Walmsley, 1991, 1995; Schikorski & Stevens, 1997; Walmsley et al. 1998).

Acknowledgments

We are grateful to Dr J. Clements for assistance with mIPSC acquisition and analysis software. This work was supported by the NHMRC of Australia (B. W.), the Human Frontiers in Science Program (F. J. A.) and NIH grants (33555 & 25547; F. J. A. and R. E. W. Fyffe).

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