Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

Abstract

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters whose precise alignment across the cleft in a trans-synaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses - those forming onto excitatory principal neurons (Ex[->]Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex[->]PV synapses). As in Ex[->]Ex synapses, we find that in Ex[->]PV synapses presynaptic Munc13-1 and postsynaptic PSD-95 both form nanoclusters that demonstrate alignment, underscoring synaptic nanostructure and the trans-synaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre-and postsynaptic Ex[->]PV nanostructure. Ex[->]PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex[->]Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell also had a retrograde impact on Munc13-1 organization, as Ex[->]PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of trans-synaptic alignment in these synapse types, revealing protein alignment in Ex[->]PV synapses over a distinct range of distances compared to Ex[->]Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses. Understanding the rules of synapse nanodomain assembly, which themselves are cell-type specific, will be essential for illuminating brain network dynamics.