Grant: Discovery Grants Program - Individual

Background, Objective and Hypothesis Esophageal peristalsis is a vital motor reflex, but its neuronal mechanism is not well understood. Neurons in the central subnucleus of the tractus solitarius (NTSC) receive esophageal vago-afferent inputs via ionotropic glutamate receptors (iGluRs), and they abundantly express nitric oxide synthase (nNOS). This project seeks to study the role and mechanism of nNOS/NO signaling in the NTSC for the regulation of esophageal peristaltic pattern and motility in rats. Pilot studies showed that applying the NOS inhibitor L-NAME to the NTS reversibly decreased the distension-induced “distal inhibition”. Moreover, S-nitrosylated proteins were identified in the NTSC. We hypothesize that esophageal vago-afferent input increases NO production in the NTSC, which in turn decreases the activity of NTSC neurons by S-nitrosylation of iGluRs hence regulates esophageal peristaltic reflex. Proposed Studies I. nNOS/NO-signaling in the NTSC modulates esophagomotor reflex. Using esophageal manometry we will characterize effects of thoracic esophageal distension (TED) on the pattern and motility of esophageal peristalsis. We will then make micro/focal application of nNOS inhibitor(s) or NO-donor(s) to the NTS to investigate how manipulation of NO-signaling in the NTSC shifts the effects of TED on esophageal peristaltic reflex. II. nNOS/NO-signaling regulates esophageal vago-afferent evoked activity in the NTSC. We will record and typify the patterns and intensity of TED-induced unit discharges in the NTSC of sedated rats. We will then examine the effect of manipulating NO-signaling on the TED induced neuronal activity in the NTSC. We will also make patch-clamp recordings in single NTSC neurons of rat brainstem slices to portray excitatory postsynaptic potential/current (EPSP/C) evoked by stimulating the solitary tract. Then we will study the effects of manipulating NO-signaling on EPSP/C of NTSC neurons. III. Vago-afferent increases NO-production and S-nitrosylation of iGluRs in NTSC neurons. We will execute long-lasting TED after applying nNOS inhibitor to the NTS of sedated rats. The NO-production and S-nitrosylated proteins in the NTSC of test rats and sham-operated rats will be evaluated by fluorescent chemical, immunohistochemical and immunoblotting assays, respectively. We will also measure the level of S-nitrosylated NR2A/GluN2 and GluR1/GluA1 subunits in NTSC neurons using biotin-switch assay. Significances This is the first endeavour to study how nNOS/NO modulates NTSC neurons hence regulating esophageal motor reflex. Outcomes from the proposed studies will contribute new knowledge to the research field of vago-vagal motor reflex, ultimately improving the health of Canadians. Importantly, this research project consists of experiments that are associated with multiple advanced research techniques, providing an excellent opportunity for HQP training in Canada.