Characterization of nitric oxidergic neurons in the alimentary tract of the snail Helix pomatia L:: Histochemical and physiological study

By using NADPH-diaphorase (NADPH-d) histochemistry, nitric oxide synthase (NOS) immunohistochemistry, Western blotting, and NO pharmacology, we investigated the distribution and possible function of NOS-containing neurons in different units of the alimentary tract of the snail, Helix pomatia. Discrete populations of neurons in the buccal ganglia displayed NADPH-d reactivity. NADPH-d-reactive and NOS-immunoreactive (NOS-IR) neurons were present in the caecum, and labeled fibers were found to innervate the circular muscles of the proesophagus and caecum and to form axosomatic connections with neurons of the myenteric and submucosal plexi of the caecum. A 65-kDa protein was found to be nNOS-IR in the caecum protein extract. The majority of the NADPH-d-reactive neurons also displayed FMRFamide immunoreactivity, whereas a mutual innervation by NADPH-diaphorase-reactive and catch-relaxing peptide (CARP)-IR neurons was observed in the caecum. Application of NO-donors [glyceryl trinitrate, S-nitroso-N-acetyl-DL-penicillamine, sodium nitroprusside (SNP)] evoked a dose-dependent increase in tension, frequency, and amplitude of the spontaneous muscle contractions of the proesophagus and caecum. Contractions could be blocked by applying the NO scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. FMRFamide evoked a response of the caecum similar to that with NO, and its simultaneous application was additive. Preincubation with CARP blocked the increase of tension evoked by SNP, whereas Mytilus inhibitory peptide (MIP) decreased the rhythmic contractions induced by the NO donor. Our findings indicate that NO is an important signal molecule in the feeding system of Helix, involved, partially in cooperation with different molluscan neuropeptides, in the regulation of both neuronal and muscular activities.