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

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 microm2; 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.

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

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.

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

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.

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

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

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).