In situ identification and visualization of neurons that mediate enteric and enteropancreatic reflexes

To identify neurons participating in enteric and enteropancreatic reflexes, we validated the use of the activity-dependent markers FM1-43 and FM2-10 as "on-line" probes for the visualization of activated guinea pig enteric and pancreatic neurons. FM1-43 or FM2-10 labeling of neuronal perikarya and processes was induced by KCl (70 mM), veratridine (1.0 microM), intracellular injection of depolarizing current pulses, stimulation of afferent inputs, evoking reflexes (by inflating an intraluminal balloon, blowing puffs of N2 at, or applying glucose to, the villous surface of the duodenum), or injury; labeling was prevented by tetrodotoxin (0.5 microM). Intracellular recording and injection of Neurobiotin confirmed that FM1-43 labeled neurons that spike, but not those that exhibit only fast excitatory postsynaptic potentials. Perikarya did not label if axonal transport was blocked by colchicine. When pulses of N2 or glucose were directed at duodenal villi in vitro, labeling by FM1-43 or FM2-10 was observed in myenteric and pancreatic neurons, as well as in subsets of cells in pancreatic islets and intestinal crypts. Hexamethonium blocked the spread of label via nicotinic synapses and thus enabled primary afferent neurons to be located. Balloon distension elicited hexamethonium-resistant labeling of epithelial cells, interstitial cells, and Dogiel type II neurons in each plexus; however, in preparations stimulated with pulses of N2 or glucose, hexamethonium-resistant labeling of neurons occurred only in the submucosal plexus and not in myenteric ganglia. These observations suggest that primary afferent neurons responsible for mucosal pressure- or glucose-induced enteric and enteropancreatic reflexes are submucosal, whereas myenteric afferent neurons become activated only when the wall of the bowel is distended. The data are compatible with the possibility that primary afferent neurons are activated by a signaling molecule released from intestinal epithelial cells.