Identification of cholinergic neurons in enteric nervous system by antibodies against choline acetyltransferase

Several different monoclonal and polyclonal antibodies to choline acetyltransferase (ChAT) were screened to identify effective antibodies for immunocytochemical marking of cholinergic neurons in the enteric nervous system. Excellent immunohistochemical results were obtained with two of the antibodies in the myenteric plexus of the guinea pig stomach and small intestine. One was a mouse monoclonal antibody designated B3.9B3, and the second was a rabbit polyclonal antibody referred to as Peptide 3. Both antibodies clearly stained neuronal cell bodies as well as nerve fibers to the muscle layers and fibers encircling stained and unstained cell bodies. Cell counts indicated that ~64% (21.0 ± 8.6 cells/ganglion) of gastric myenteric neurons are ChAT positive. Pelvic ganglia and the inferior mesenteric ganglia were examined as controls. Strong labeling of the majority of neurons was found in the pelvic ganglia, whereas few immunoreactive cells were apparent in the predominantly noradrenergic inferior mesenteric ganglion. Lack of effective antibodies to enteric neuronal ChAT has hampered progress in the study of the neurophysiology of cholinergic neurons in the digestive tract. Application of the B3.9B3 and Peptide 3 antibodies now promises to facilitate investigation of this important subset of enteric neurons.