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 Nlgn3R451C Mice

Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction but its cellular expression in the GI tract remains to be characterised. Localisation of NLGN3 protein is challenging in intestinal tissue due to the lack of target-specific antibodies. Here, we combined RNAScope in situ hybridization for Nlgn3 mRNA and immunofluorescence for markers of all enteric neurons, cholinergic submucosal neurons, non-cholinergic submucosal neurons, nitregic and calretinin-containing myenteric neurons as well as glial cells. We also developed a quantitative 3-dimensional image analysis method to measure Nlgn3 mRNA cellular expression levels in enteric neurons and glia. We show that Nlgn3 mRNA is expressed in most submucosal and myenteric neurons as well as in enteric glia. The R451C mutation reduces Nlgn3 mRNA expression levels in cholinergic, nitrergic and calretinin enteric neuronal subpopulations but does not affect Nlgn3 mRNA expression in VIPergic submucosal neurons. In summary, we show that the autism-associated R451C mutation in Nlgn3 reduces Nlgn3 mRNA expression in the mouse ENS. These findings could shed light on the pathophysiology of GI dysfunction in ASD.