Abstract
The bowel is the only organ of the body in which neural reflexes can be elicited in the absence of input from the brain or spinal cord. This activity is mediated by the enteric nervous system (ENS), which contains primary afferent neurons. Experiments were carried out to locate the primary afferent neurons of the ENS. Two types of stimulation were used to activate neurons in the wall of the gut in vitro: exposure of the mucosa to cholera toxin or delivery of pressure to the mucosal surface with puffs of N2 from a micropipette. Neurons that became active in response to these stimuli were identified by demonstrating the intranuclear immunoreactivity of Fos, the product of the c-fos protooncogene. No Fos immunoreactivity could be detected in the absence of stimulation; however, application of cholera toxin and puffs of N2 each induced the appearance of Fos immunoreactivity in neurons in both the submucosal and myenteric plexuses. With either stimulus, the induction of Fos immunoreactivity was antagonized by TTX and therefore depended on neuronal activity. The appearance of Fos immunoreactivity could also be prevented by the 5-HT1P receptor antagonist N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide. In contrast, the stimulus-induced expression of Fos immunoreactivity was inhibited, but not abolished, by hexamethonium, which limited the spread of activation within the submucosal plexus and completely prevented expression of Fos immunoreactivity by myenteric neurons in response to mucosal puffs of N2. FluoroGold was injected into single ganglia of the myenteric plexus in order to identify submucosal neurons with myenteric projections. Submucosal neurons in which Fos immunoreactivity was induced by the stimuli were doubly labeled by FluoroGold. A subset of the submucosal, but not myenteric, neurons that expressed Fos immunoreactivity was doubly labeled by antibodies to calbindin. Submucosal calbindin-immunoreactive neurons were found to contain substance P immunoreactivity and could also be immunostained by anti-idiotypic antibodies that react with 5-HT1P receptors. A subset of dynorphin1–8-immunoreactive submucosal neurons (which are known to costore vasoactive intestinal peptide and to be secretomotor in function) expressed nuclear Fos immunoreactivity in response to cholera toxin, but not puffs of N2. These data suggest that intrinsic primary afferent neurons are located in the submucosal plexus, project to the myenteric plexus, and are activated by 5-HT acting on the 5-HT1P receptor subtype. These neurons are probably cholinergic and costore calbindin and substance P.