| Summary: | Mutations in the Neuroligin-3 (<i>Nlgn3</i>) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular <i>Nlgn3</i> expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure <i>Nlgn3</i> mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and <i>Nlgn3<sup>R451C</sup></i> mutant mice. We measured <i>Nlgn3</i> 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 <i>Nlgn3</i>, <i>Nlgn1</i>, <i>Nlgn2</i> and their binding partners, <i>Nrxn1-3</i>, <i>MGDA1</i> and <i>MGDA2</i>, in enteric neural subsets. Most submucosal and myenteric neurons expressed <i>Nlgn3</i> mRNA. In contrast to other <i>Nlgns</i> and binding partners, <i>Nlgn3</i> was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron–glia interactions. The autism-associated R451C mutation reduces <i>Nlgn3</i> mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, <i>Nlgn3</i> 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 <i>Nlgn3,</i> which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.
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