A multi-faceted analysis of synapses reveals the role of neuroligin-1 cleavage in presynaptic vesicle accumulation in the lateral amygdala

Neuroligin-1 (NLGN1) is a cell adhesion molecule found at excitatory glutamatergic synapses in the brain which regulates synaptic function and maturation. Extracellular cleavage of NLGN1 by proteases has been shown to control vesicle release in cultured neurons, but nothing is known about the underlying changes to synapse structure that accompany this, or how synapse function is affected in brain tissue. We found that prevention of NLGN1 cleavage through mutation to the extracellular stalk domain increases synaptic vesicle docking and miniature excitatory post-synaptic current frequency at synapses of the lateral amygdala. Using a novel volume electron microscopy (vEM) analysis pipeline based on deep learning extraction of thousands of synapses and vesicles clouds and subsequent spatial analyses, we found that the total pool of synaptic vesicles shifts closer to the synapse in mutants. Furthermore, we observed an increased frequency of incomplete synapses that lack vesicle accumulation, pointing towards disruption of synaptic pruning and accumulation of putatively non-functioning synapses. Our study provides evidence of a structural and functional role of NLGN1 cleavage in native brain tissue, and establishes a foundation for vEM analysis of synapse-vesicle spatial relationships in other animal models of dysfunction and disease.