The identification and chemical coding of cholinergic neurons in the small and large intestine of the mouse

BACKGROUND: The recent availability of antisera to the vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) that demonstrate peripheral cholinergic neurons has made possible the anatomical identification of cholinergic neurons in the enteric nervous system. In this study, we localised cholinergic neurons in the mouse small and large intestine and identified which substances are found colocalised in the cholinergic neurons. METHODS: Immunohistochemical single and double staining techniques were used on whole mount preparations and frozen sections to examine the localisation and chemical coding of cholinergic neurons in the small and large intestine of the mouse. Cholinergic neurons were identified using antisera to ChAT or VAChT. RESULTS: In both the small and large intestine, numerous ChAT-immunoreactive nerve cell bodies were present in the myenteric and submucous ganglia, and ChAT- and VAChT-immunoreactive nerve terminals were abundant in the myenteric and submucous plexuses and the external muscle. Previous studies have identified two major classes of myenteric neurons in the small intestine of the mouse--those containing calretinin plus substance P, and those containing nitric oxide synthase (NOS) plus vasoactive intestinal peptide (VIP). Double-label studies showed that the vast majority of the calretinin/substance P neurons were cholinergic neurons, whereas only a small proportion of the NOS/VIP cells were cholinergic; the noncholinergic NOS/VIP neurons were motor neurons or interneurons, whereas the cholinergic NOS/VIP neurons appeared to be exclusively interneurons. In the small intestine, all of the 5-HT-loaded neurons and a subpopulation of the calbindin neurons were also cholinergic. In the large intestine, there was a pattern of overlaps similar to that found in the small intestine, except that in the large intestine approximately 25% of the calretinin cells were not cholinergic. Only approximately one third of the GABA-loaded neurons in the large intestine were cholinergic. CONCLUSIONS: Large subpopulations of motor neurons and interneurons in the mouse small intestine are cholinergic neurons.