Preganglionic endings from nucleus of Edinger-Westphal in pigeon ciliary ganglion contain neuronal nitric oxide synthase

The avian ciliary ganglion (CG) controls choroidal blood flow by its choroi dal neurons, and pupil constriction and accommodation by its ciliary neuron s. It was previously reported that both choroidal and ciliary neurons label positively for NADPH diaphorase (NADPHd), a marker for nitric oxide syntha se (NOS), To assess if this labeling is preganglionic or postganglionic and to determine if it is attributable to neuronal NOS (nNOS), we studied pige on CG using NADPHd histochemistry and nNOS immunohistochemistry (IHC). Shor t-duration staining times by NADPHd histochemistry yielded intense labeling of structures that appeared to be the cap-like endings on ciliary neurons and the boutonal endings on choroidal neurons that arise from the nucleus o f Edinger-Westphal (EW), and light or nu postganglionic perikaryal staining . The light postganglionic staining that was observed tended to be localize d to ciliary neurons. Consistent with this, NADPHd+ nerve fibers were obser ved in the postganglionic ciliary nerves but rarely in the postganglionic c horoidal nerves. These same staining times yielded robust staining of neuro ns in the orbital pterygopalatine microganglia network, which are known to be nNOS+. Diffuse staining of CG perikarya was observed with longer stainin g durations, and this staining tended to mask the preganglionic labeling. P reganglionic NADPHd+ staining in CG with short staining times was blocked b y the NOS inhibitors iodonium diphenyl (IDP) and dichlorophenol-indophenol (DPIP), but the diffuse postganglionic staining observed with the longer st aining times was not completely blocked. Labeling of CG sections for substa nce P (SP) by IHC (which labels EW-originating preganglionic endings in CG) and subsequently for NADPHd confirmed that NADPHd was localized to pregang lionic endings on CG neurons. Immunohistochemical double labeling for nNOS and SP or enkephalin further confirmed that nNOS is found in boutonal and c ap-like endings in the CG. Two studies were then carried out to demonstrate that the nNOS + preganglionic endings in CG arise from EW. First, NADPHdand nNOS+ neurons were observed in EW in pigeons treated with colchicine to enhance perikaryal labeling. Second, NADPHd+ and nNOS+ preganglionic endin gs were eliminated from CG ipsilateral to an EW lesion. These various resul ts indicate that NOS is present in EW-arising preganglionic endings on chor oidal and ciliary neurons in avian CG. NOS also appears to be found in some ciliary neurons, but its presence in choroidal neurons is currently uncert ain.