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.

Evidence that urocortin is absent from neurons of the Edinger-Westphal nucleus in pigeons.

The Edinger-Westphal nucleus (EWN) is a central preganglionic parasympathetic cell group that gives rise to cholinergic input to the ciliary ganglion, thereby regulating several neurovegetative ocular functions. Recently, the supposed presence of the neuropeptide urocortin (UCN) has been reported in EWN neurons in rodent brain. The purpose of the present study was to examine the distribution of UCN in avian brain and to investigate by immunohistochemical analysis the possible use of this substance as an EWN marker in a non-mammalian class of vertebrates. Brain tissue of pigeons was incubated with a specific antibody against UCN and the results showed labeling of many small neurons, forming a double wing in the dorsal mesodiencephalic transition area. Their size and shape, however, differed from those of EWN neurons, and they were preferentially located rostral to the EWN. Double-label experiments employing an antibody against the enzyme choline acetyltransferase (ChAT) showed that UCN is not localized to the cholinergic cells of the EWN and confirmed the rostral distributionof UCN never overlapping the ChAT+ EWN cells. Taken together, these results suggest that, at least in pigeons, the UCN+ population does not belong to the traditionally defined EWN.

Evidence that urocortin is absent from neurons of the Edinger-Westphal nucleus in pigeons

The Edinger-Westphal nucleus (EWN) is a central preganglionic parasympathetic cell group that gives rise to cholinergic input to the ciliary ganglion, thereby regulating several neurovegetative ocular functions. Recently, the supposed presence of the neuropeptide urocortin (UCN) has been reported in EWN neurons in rodent brain. The purpose of the present study was to examine the distribution of UCN in avian brain and to investigate by immunohistochemical analysis the possible use of this substance as an EWN marker in a non-mammalian class of vertebrates. Brain tissue of pigeons was incubated with a specific antibody against UCN and the results showed labeling of many small neurons, forming a double wing in the dorsal mesodiencephalic transition area. Their size and shape, however, differed from those of EWN neurons, and they were preferentially located rostral to the EWN. Double-label experiments employing an antibody against the enzyme choline acetyltransferase (ChAT) showed that UCN is not localized to the cholinergic cells of the EWN and confirmed the rostral distributionof UCN never overlapping the ChAT+ EWN cells. Taken together, these results suggest that, at least in pigeons, the UCN+ population does not belong to the traditionally defined EWN.

Influence of ophthalmic nerve fibers on choroidal blood flow and myopic eye growth in chicks.

Ophthalmic sensory nerve fibers containing substance P and calcitonin gene-related peptide' innervate the choroid in mammals and are known to vasodilate choroidal blood vessels. The avian choroid is also innervated by ophthalmic nerve fibers containing substance P and calcitonin gene-related peptide. The present studies were carried out to determine the influence of these sensory fibers on choroidal blood flow in birds and characterize their interaction with manipulations affecting eye growth. In these studies, ChBF was measured using laser Doppler flowmetry in both eyes in the following groups of birds: (1) normal chicks; (2) chicks with right optic nerve transected for 2 weeks; (3) chicks with right optic nerve transected and a goggle over the right eye for 2 weeks; and (4) chicks with right optic and ophthalmic nerves transected and a goggle over the right eye for 2 weeks. The eyes were refracted and various ocular dimensions measured after the blood-flow measurements. It was found that optic nerve transection reduced ChBF to 30% of normal. Placing a goggle (which increases ocular temperature by 4 degrees C) over an optic nerve transected eye nearly doubled choroidal blood flow over that in an optic nerve transected eye without a goggle. Additional transection of the ophthalmic nerve in a goggled optic nerve-transected eye, yielded choroidal blood flow that was indistinguishable from that in a nongoggled optic nerve-transected eye. Optic nerve transection had a slight stunting effect on axial growth of the eye. While myopic axial elongation was observed in goggled eyes with the optic nerve cut, the extent of myopia was less than in normal goggled eyes. Ophthalmic nerve transection further reduced the myopia induced by goggling in an optic nerve cut eye. These results suggest that ophthalmic nerve input to the choroid exerts a vasodilatory influence, which is activated in a goggled eye. This increased choroidal blood flow may be in response to elevated ocular temperatures caused by the goggling and this increase appears to be masked in goggled eyes with an intact optic nerve by the reduction in choroidal blood flow normally accompanying myopic eye growth. Our results thus show that the induction of myopic eye growth (as in our optic nerve cut eyes with a goggle) need not be accompanied by a decrease in choroidal blood flow from the baseline no-goggle condition (in this case, with the optic nerve cut).