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Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure Nlgn3 mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and Nlgn3R451C mutant mice. We measured Nlgn3 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 Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron–glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 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 Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.

To investigate the effects of microwave (MW) treatment on hays nutritive value, five types of hay (50 g) were treated with MW for 0 (control), 20, 40, 60, and 80 s (0, 440, 880, 1320, and 1760 kJ kg−1 of MW energy, respectively) and analyzed for nutritive value prior to scanning by an electron microscope to observe microstructure changes. The dry matter (DM) % of hays were increased with increasing treatment time (p < 0.001). The improved DM and digestible organic matter in the DM were recorded from MW treated lucerne (60 s), wheat (40 s), and canola (20 s) (p < 0.001), which might have been due to the MW ruptured cell wall. The MW energy required for maximal DM digestibility improvement was linearly related to control hay crude protein content (r2 = 0.79; p < 0.001). Overall, the study showed MW treatment can increase some hays’ digestibility. In addition, the crude protein content from control hay may be a proxy to indicate DM digestibility improvement.