Gephyrin is a key scaffold protein mediating the anchoring of GABAA receptors at inhibitory synapses. Here we exploited super-resolution techniques combined with proximity-based clustering analysis and model simulations to investigate the single molecule gephyrin reorganization during plasticity of inhibitory synapses in mouse hippocampal cultured neurons. This approach revealed that during the expression of inhibitory long-term potentiation the increase of gephyrin density at postsynaptic sites is associated with the promoted formation of gephyrin nano-domains. We demonstrate that the gephyrin rearrangement in nano-domains stabilizes the amplitude of postsynaptic currents, thus indicating that, besides the number of synaptic GABAA receptors, the nanoscale distribution of GABAA receptors in the postsynaptic area is a crucial determinant for the expression of inhibitory synaptic plasticity. In addition, the methodology implemented here clears the way to the application of the graph-based theory to single molecule data for the description and quantification of the spatial organization of the synapse at the single molecule level.
The mechanisms of inhibitory synaptic plasticity are poorly understood, mainly because the size of synapse is below the diffraction limit, thus reducing the effectiveness of conventional optical and imaging techniques. Here we exploited super-resolution approaches combined with clustering analysis to study at unprecedented resolution the distribution of the inhibitory scaffold protein gephyrin in response to protocols inducing long-term potentiation of inhibitory synaptic responses (iLTP). We found that, during the expression of iLTP, the increase of synaptic gephyrin is associated with the fragmentation of gephyrin in sub-synaptic nano-domains. We demonstrate that such synaptic gephyrin nano-domains stabilize the amplitude of inhibitory postsynaptic responses, thus identifying the nanoscale gephyrin rearrangement as a key determinant for inhibitory synaptic plasticity.
The authors declare no competing financial interests.
This work has been supported by Telethon-Italy (GGP11043) to AB, Compagnia di San Paolo (ROL-4318) to AB and Compagnia di San Paolo (ROL-9687) to Marti Duocastella and AB.