Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses

Super-resolution imaging has revealed that key synaptic proteins are dynamically organized within sub-synaptic domains (SSDs). To examine how different inhibitory receptors are regulated, we carried out dual-color direct stochastic optical reconstruction microscopy (dSTORM) of GlyRs and GABA_A Rs at mixed inhibitory synapses in spinal cord neurons. We show that endogenous GlyRs and GABA_A Rs as well as their common scaffold protein gephyrin form SSDs that align with pre-synaptic RIM1/2, thus creating trans-synaptic nanocolumns. Strikingly, GlyRs and GABA_A Rs occupy different sub-synaptic spaces, exhibiting only a partial overlap at mixed inhibitory synapses. When network activity is increased by 4-aminopyridine treatment, the GABA_A R copy numbers and the number of GABA_A R SSDs are reduced, while GlyRs remain largely unchanged. This differential regulation is likely the result of changes in gephyrin phosphorylation that preferentially occurs outside of SSDs. The activity-dependent regulation of GABA_A Rs versus GlyRs suggests that different signaling pathways control the receptors' sub-synaptic clustering. Taken together, our data reinforce the notion that the precise sub-synaptic organization of GlyRs, GABA_A Rs, and gephyrin has functional consequences for the plasticity of mixed inhibitory synapses.