IN-SITU IDENTIFICATION AND VISUALIZATION OF NEURONS THAT MEDIATE ENTERIC AND ENTEROPANCREATIC REFLEXES

To identify neurons participating in enteric and enteropancreatic refl exes, we validated the use of the activity-dependent markers FM1-43 an d FM2-10 as ''on-line'' probes for the visualization of activated guin ea pig enteric and pancreatic neurons. FM1-43 or FM2-10 labeling of ne uronal perikarya and processes was induced by KCl (70 mM), veratridine (1.0 mu M), intracellular injection of depolarizing current pulses, s timulation of afferent inputs, evoking reflexes (by inflating an intra luminal balloon, blowing puffs of N-2 at, or applying glucose to, the villous surface of the duodenum), or injury; labeling was prevented by tetrodotoxin (0.5 mu M). Intracellular recording and injection of Neu robiotino(TM) confirmed that FM1-43 labeled neurons that spike, but no t those that exhibit only fast excitatory postsynaptic potentials. Per ikarya did not label if axonal transport was blocked by colchicine. Wh en pulses of N-2 or glucose were directed at duodenal villi in vitro, labeling by FM1-43 or FMB-10 was observed in myenteric and pancreatic neurons, as well as in subsets of cells in pancreatic islets and intes tinal crypts. Hexamethonium blocked the spread of label via nicotinic synapses and thus enabled primary afferent neurons to be located. Ball oon 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 N-2 or glucose, he xamethonium-resistant labeling of neurons occurred only in the submuco sal plexus and not in myenteric ganglia. These observations suggest th at primary afferent neurons responsible for mucosal pressure- or gluco se-induced enteric and enteropancreatic reflexes are submucosal, where as myenteric afferent neurons become activated only when the wall of t he bowel is distended. The data are compatible with the possibility th at primary afferent neurons are activated by a signaling molecule rele ased from intestinal epithelial cells. (C) 1996 Wiley-Liss, Inc.