Quantitative Spatial Analysis of Neuroligin-3 mRNA Expression in the Enteric Nervous System Reveals a Potential Role in Neuronal-Glial Synapses and Reduced Expression in Mice.

Mutations in the Neuroligin-3 () gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and mutant mice. We measured mRNA neuronal and glial expression via quantitative three-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single-cell RNA sequencing (scRNASeq) data to assess for , , and their binding partners, , and , in enteric neural subsets. Most submucosal and myenteric neurons expressed mRNA. In contrast to other and binding partners, was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron-glia interactions. The autism-associated R451C mutation reduces mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, mRNA levels are reduced in calretinin, nNOS-labelled neurons and S100 β -labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.