Gephyrin-independent GABA_AR mobility and clustering during plasticity

The activity-dependent modulation of GABA-A receptor (GABA_AR) clustering at synapses controls inhibitory synaptic transmission. Several lines of evidence suggest that gephyrin, an inhibitory synaptic scaffold protein, is a critical factor in the regulation of GABA_AR clustering during inhibitory synaptic plasticity induced by neuronal excitation. In this study, we tested this hypothesis by studying relative gephyrin dynamics and GABA_AR declustering during excitatory activity. Surprisingly, we found that gephyrin dispersal is not essential for GABA_AR declustering during excitatory activity. In cultured hippocampal neurons, quantitative immunocytochemistry showed that the dispersal of synaptic GABA_ARs accompanied with neuronal excitation evoked by 4-aminopyridine (4AP) or N-methyl-D-aspartic acid (NMDA) precedes that of gephyrin. Single-particle tracking of quantum dot labeled-GABA_ARs revealed that excitation-induced enhancement of GABA_AR lateral mobility also occurred before the shrinkage of gephyrin clusters. Physical inhibition of GABA_AR lateral diffusion on the cell surface and inhibition of a Ca²⁺ dependent phosphatase, calcineurin, completely eliminated the 4AP-induced decrease in gephyrin cluster size, but not the NMDA-induced decrease in cluster size, suggesting the existence of two different mechanisms of gephyrin declustering during activity-dependent plasticity, a GABA_AR-dependent regulatory mechanism and a GABA_AR-independent one. Our results also indicate that GABA_AR mobility and clustering after sustained excitatory activity is independent of gephyrin.