Gephyrin is critical for glycine receptor clustering but not for the formation of functional GABAergic synapses in hippocampal neurons - PubMed (original) (raw)

Gephyrin is critical for glycine receptor clustering but not for the formation of functional GABAergic synapses in hippocampal neurons

Sabine Lévi et al. J Neurosci. 2004.

Abstract

The role of the scaffolding protein gephyrin at hippocampal inhibitory synapses is not well understood. A previous study (Kneussel et al., 1999) reported a complete loss of synaptic clusters of the major GABA(A)R subunits alpha2 and gamma2 in hippocampal neurons lacking gephyrin. In contrast, we show here that GABA(A)R alpha2 and gamma2 subunits do cluster at pyramidal synapses in hippocampal cultures from gephyrin-/- mice, albeit at reduced levels compared with control neurons. Synaptic aggregation of GABA(A)R alpha1 on interneurons was identical between the culture types. Furthermore, we recorded miniature IPSCs (mIPSCs) from gephyrin-/- neurons. Although the mean mIPSC amplitude was reduced (by 23%) compared with control, the frequency of these events was unchanged. Cell surface labeling experiments indicated that gephyrin contributes, in part, to aggregation but not to insertion or stabilization of GABA(A)R alpha2 and gamma2 in the plasma membrane. Thus, a major gephyrin-independent component of hippocampal inhibitory synapse development must exist. We also report that glycine receptors cluster at GABAergic synapses in a subset of hippocampal interneurons and pyramidal neurons. Unlike GABA(A)Rs, synaptic clustering of glycine receptors was completely abolished in gephyrin-/- neurons. Finally, artificial extrasynaptic aggregation of GABA(A)R was able to redistribute and cocluster gephyrin by a mechanism requiring a neuron-specific modification or intermediary protein. We propose a model of hippocampal inhibitory synapse development in which some GABA(A)Rs cluster at synapses by a gephyrin-independent mechanism and recruit gephyrin. This clustered gephyrin may then recruit glycine receptors, additional GABA(A)Rs, and other signal-transducing components.

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Figures

Figure 1.

Figure 1.

Properties of GABAergic miniature synaptic currents in gephyrin mutant hippocampal neurons. mIPSCs were evident in gephyrin-/- and gephyrin+/+ neurons (A). The averaged events from each cell in A are shown in B, scaled at the right, with the gephyrin-/- trace in gray. The cumulative probability distributions of events pooled from all cells (n ≥ 350 events; ≤50 events per cell) showed a difference in amplitude distribution between gephyrin-/- and gephyrin+/+ groups (C; K-S; p < 0.001) but no difference in decay time constant (_p_ > 0.1).

Figure 2.

Figure 2.

Clustering of GABAARs in the absence of gephyrin. Similar morphology (A1-C1) and surface clusters of GABAAR γ2 (A2-C2) were seen in all three genotypes [gephyrin+/+ (A), gephyrin+/- (B), and gephyrin-/- (C)], despite the absence of detectable gephyrin (A3-C3) immunoreactivity in the mutants (C3). Boxed regions in the GABAAR images are shown enlarged below. Scale bar, 10 μm.

Figure 3.

Figure 3.

Synaptic localization of GABAAR subunits in gephyrin mutant neurons. Surface GABAAR γ2 (A-C, green), α2 (D-F, green), and α1 (G-I, green) subunits were clustered opposite GABAergic terminals labeled with GAD (red) in all genotypes [gephyrin+/+ (A, D, G), gephyrin+/- (B, E, H), and gephyrin-/- (C, F, J)]. Interneurons, identified by high levels of GAD in the soma, were analyzed for α1 (G-I), and pyramidal neurons for γ2 (A-C) and α2 (D-F). Boxed regions in the double-color images are shown as enlarged single-channel images below. These images represent some of the most highly clustered receptor distributions for all genotypes. Scale bar, 10 μm.

Figure 4.

Figure 4.

Levels of clustered GABAAR subunits in gephyrin mutant neurons. GAD-innervated dendrites were measured for surface or total cell GABAAR subunit immunofluorescence (see Materials and Methods). The percentage of receptor fluorescence present in aggregates was reduced in the gephyrin-/- neurons compared with gephyrin+/- controls for α2 and γ2 but not α1 (*p < 0.001; values represent integrated fluorescence in clusters as a percentage of integrated fluorescence of the whole dendrite region).

Figure 5.

Figure 5.

Surface localization of GABAARs in gephyrin mutant neurons. A, Neurons were labeled live with α2 Ab, fixed and permeabilized, incubated with secondary Ab (green), then incubated again with α2 Ab and new secondary Ab (red). Thus, surface receptor labels with both color secondary Ab (because all sites were not saturated), whereas internal receptor labels with red secondary Ab only. Internal aggregates of GABAAR were not observed in any genotype. B, Surface association of GABAAR was confirmed by confocal microscopy. Shown are a series of optical sections from a plane near the substrate following up the surface of a thick dendrite and soma. The receptor clusters are in the same optical sections as the GAD-labeled axons on the cell surface. Scale bars: A, 10 μm; B, 5 μm.

Figure 6.

Figure 6.

Clustering of glycine receptor at GABA synapses and dependence on gephyrin. Glycine receptor was detected in a subset of wild-type hippocampal neurons specifically aggregated at GABA synapses, colocalized with GABAAR and gephyrin opposite GAD and synapsin (A-C, arrowheads). Glycine receptors were not clustered at glutamatergic synapses identified by clusters of SynGAP (D, arrows). Glycine receptor clusters were detected on both interneurons (E) and pyramidal neurons (F). Whereas 44% of control gephyrin+/- neurons showed clusters of glycine receptor (G, arrowheads), glycine receptor clustering was essentially abolished in the absence of gephyrin (H, arrows; 2.5% of gephyrin-/- neurons showed any clusters). Scale bars: A, 10 μm; B-D, 5 μm; E, F, 10 μm; G, H, 10 μm.

Figure 7.

Figure 7.

Comparison of GABAR synaptic immunofluorescence and mIPSC amplitude distributions. Recordings of mIPSCs in wild-type hippocampal neurons were done with neurobiotin-filled electrodes, and cells were fixed, immunolabeled, and imaged in one batch for GABAAR α2 and GAD. Examples of regions of two cells recorded on the same day that exhibited larger (29F) or smaller (29C) synaptic GABAR clusters are shown (A). Compared with cell 29C, cell 29F exhibited a greater skew toward higher values in the distributions of integrated intensity of GABAAR α2 immunofluorescence per synapse (GABAR synaptic fluorescence) and of mIPSC peak amplitude (B). Comparison among the eight sister cells of mean cell intensity of GABAAR α2 per synapse versus mean cell mIPSC peak amplitude did not show a significant correlation (C; correlation coefficient, 0.54; Spearman correlation test; p > 0.1). Gray triangles in C represent cell 29C and 29F. The distributions of number of SDs of each value from the mean were not significantly difference for mIPSC amplitude values (red) and synaptic GABAR immunofluorescence values (blue) (D; data pooled from all cells; K-S; p > 0.1).

Figure 8.

Figure 8.

Coclustering of gephyrin by artifical aggregation of GABAR in neurons but not in cotransfected fibroblasts. Wild-type hippocampal neurons were fixed and then labeled with GABAAR α2 Ab, biotinylated secondary Ab, and FITC-streptavidin (A), or treated live with GABAAR α2 Ab, biotinylated secondary Ab, and FITC-streptavidin and then fixed (B; cross-linking T0), or treated live with GABAAR α2 Ab and biotinylated secondary Ab, incubated for 12 hr, and then treated with FITC-streptavidin and fixed (C; cross-linking T12). All neurons were immunolabeled for gephyrin (A2-C2; A4-C4, red) and synapsin (A3-C3; A5-C5, blue). Cross-linking at T0 resulted in formation of nonsynaptic aggregates of GABAAR α2 lacking gephyrin (B, arrows) as well as the usual synaptic coclusters of GABAAR α2, gephyrin, and synapsin (A, B, arrowheads). After another 12 hr, gephyrin joined the nonsynaptic aggregates of GABAAR α2 (C, arrows). The percentage of GABAAR α2 clusters localized to synapses was decreased by the cross-linking at T0 and T12, whereas the percentage of gephyrin clusters localized to synapses was decreased by the cross-linking at T12 but not T0, and the percentage of GABAAR α2 clusters associated with gephyrin decreased at T0 and increased again at T12 (D; *p < 0.001). A similar cross-linking experiment in fibroblasts transfected with GABAAR YFP-α2, β3, γ2, and gephyrin or CFP-gephyrin yielded no detectable coclustering of gephyrin with GABAR (E, F). Surface clustering was induced with biotinylated anti-GFP and AMCA-streptavidin (GABAR image), cells were incubated an additional 2 hr and then fixed and immunolabeled with gephyrin antibody (E; COS-7 cells) or imaged for CFP-gephyrin (F; CHO-K1 cells). Scale bars, 10 μm.

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