Anatomical and functional evidence for progressive age-related decline in parasympathetic control of choroidal blood flow in pigeons.

The choroid receives extensive parasympathetic innervation, which in birds arises largely from the ciliary ganglion (CG). Since age-related changes in parasympathetic regulation of choroidal blood flow (ChBF) could contribute to age-related retinal decline, we used anatomical and functional methods to determine if ChBF control by the CG shows age-related decline in pigeons. The efficacy of the choroidal vasodilatory response to activation of the CG preganglionic input from the medial subdivision of the nucleus of Edinger-Westphal (EWM) was assessed using laser Doppler flowmetry (LDF). The EWM receives bisynaptic retinal input, and electrical stimulation of EWM or light stimulation of the retina in young animals produces dramatic choroidal vasodilation. Transcleral LDF was therefore used to measure both basal ChBF and the increases in ChBF elicited by electrical stimulation of EWM or by retinal illumination in 0.5-18 year old pigeons. Fixed cryostat sections of the eye from 0.5 to 22 year old pigeons were immunolabeled for the 3A10 neurofilament-associated antigen to determine if intrachoroidal nerve fibers arising from CG exhibited age-related loss. We focused on superior choroid, since it is the primary target for CG nerve fibers. There was a marked age-related loss in the ChBF vasodilatory response elicited by either EWM stimulation or retinal illumination, as was also true for basal ChBF. A progressive decrease in choroidal nerve fibers of CG origin, to 17% of youthful abundance by 22 years of age, was also observed. The evoked ChBF increase, and basal ChBF, achieved 50% of their age-related decline between the ages of 3 and 4 years, while half the loss in CG innervation of choroid was later, occurring by 10 years. Age-related loss of choroidal nerve fibers occurs in parallel with but more slowly than the reduction in basal ChBF and the choroidal vasodilation that can be elicited via natural (light) or electrical activation of the central neural input to CG choroidal neurons. The prominent age-related decline in parasympathetic control of ChBF early in the pigeon life span could contribute to the age-related retinal decline observed in pigeons.

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 choroidal neurons, and pupil constriction and accommodation by its ciliary neurons. It was previously reported that both choroidal and ciliary neurons label positively for NADPH diaphorase (NADPHd), a marker for nitric oxide synthase (NOS). To assess if this labeling is preganglionic or postganglionic and to determine if it is attributable to neuronal NOS (nNOS), we studied pigeon CG using NADPHd histochemistry and nNOS immunohistochemistry (IHC). Short-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 of Edinger-Westphal (EW), and light or no postganglionic perikaryal staining. The light postganglionic staining that was observed tended to be localized to ciliary neurons. Consistent with this, NADPHd+ nerve fibers were observed in the postganglionic ciliary nerves but rarely in the postganglionic choroidal nerves. These same staining times yielded robust staining of neurons in the orbital pterygopalatine microganglia network, which are known to be nNOS+. Diffuse staining of CG perikarya was observed with longer staining durations, and this staining tended to mask the preganglionic labeling. Preganglionic NADPHd+ staining in CG with short staining times was blocked by the NOS inhibitors iodonium diphenyl (IDP) and dichlorophenol-indophenol (DPIP), but the diffuse postganglionic staining observed with the longer staining times was not completely blocked. Labeling of CG sections for substance P (SP) by IHC (which labels EW-originating preganglionic endings in CG) and subsequently for NADPHd confirmed that NADPHd was localized to preganglionic endings on CG neurons. Immunohistochemical double labeling for nNOS and SP or enkephalin further confirmed that nNOS is found in boutonal and cap-like endings in the CG. Two studies were then carried out to demonstrate that the nNOS+ preganglionic endings in CG arise from EW. First, NADPHd+ and nNOS+ neurons were observed in EW in pigeons treated with colchicine to enhance perikaryal labeling. Second, NADPHd+ and nNOS+ preganglionic endings were eliminated from CG ipsilateral to an EW lesion. These various results indicate that NOS is present in EW-arising preganglionic endings on choroidal and ciliary neurons in avian CG. NOS also appears to be found in some ciliary neurons, but its presence in choroidal neurons is currently uncertain.

Neural nitric oxide mediates Edinger-Westphal nucleus evoked increase in choroidal blood flow in the pigeon.

PURPOSE: Nitric oxide (NO) has been identified as a putative neurotransmitter in choroidal perivascular nerve fibers originating parasympathetically. Although constitutively produced NO has been implicated in the regulation of the choroidal circulation, the specific role of neurally derived NO in choroidal vasodilation has not been determined. This study examined the role of neurally derived NO in the control of the choroidal blood flow (ChBF) in vivo. METHODS: Resting ChBF and a increase in ChBF elicited by electrical stimulation of the nucleus of Edinger-Westphal (EW) were measured transclerally by laser Doppler flowmetry in anesthetized pigeons before and after administration of a selective inhibitor of neural NO synthase, 7-Nitroindazole (7NI; 50 mg/kg given intraperitoneally); a nonselective NO synthase inhibitor, Ng-nitro-L-arginine methyl ester (L-NAME; 30 mg/kg given intravenously); L-arginine (300 mg/kg given intravenously) followed by 7NI (50 mg/kg given intraperitoneally); or vehicle. RESULTS: The 7NI and L-NAME, but not the vehicle, attenuated the EW-evoked response (maximally by 78% and 83%, respectively), and this effect lasted for at least 1 hour. Pretreatment with L-arginine abolished this effect of 7NI. Resting ChBF was reduced and systemic blood pressure was increased after L-NAME administration, but both were unchanged after 7NI or vehicle were administered. CONCLUSIONS: Neurally derived NO is responsible for a major component of the ChBF increase caused by EW stimulation in pigeons. This represents the first demonstration in vivo that neuronally produced NO is an important factor in the control of ChBF by the parasympathetic nervous system. In particular, neuronally produced NO appears to play a role in rapid upregulation of ChBF in the pigeon, whereas endothelially produced NO plays a major role in control of resting ChBF.