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.