revealed that NOS is found only at a low level in some ciliary neurons and is absent from choroidal neurons. The latter Þnding is consistent with the fact that NOS+ and NADPHd+ Þbers are much scarcer in choroid than are the cholinergic Þbers demonstrably arising from ciliary ganglion [62, 64], and those NOS+/NADPHd+ Þbers that are present are likely to arise from the PPG [63]. The prior report that NADPHd is abundant in choroidal neurons of the pigeon CG [351], thus, appears to have been based, regrettably, in a failure to distinguish between speciÞc and nonspeciÞc NADPHd staining. Consistent with the cholinergic projection of the ciliary ganglion to

the eye [152, 232, 285], Fischer et al. [84] found that M2, M3, and M4 receptors are present in chick choroid and ciliary body by Western blots and immunolabeling.

12.5.4.4 Function of vSCN-EWM-Ciliary Ganglion Circuit – Birds

Our anatomical data suggest that the vSCNEWM circuit participates in the light-regulated control of ChBF [284], and in a series of studies, we have shown that this is the case. For example, we showed that electrical stimulation of EWM increases ChBF in the ipsilateral eye, as measured by laser Doppler ßowmetry (LDF)

Fig. 12.22 Photomicrograph of a Þeld of view from a section immunolabeled according to immunoßuorescence procedures for the 3A10 neuroÞlament antigen that detects cholinergic ciliary ganglion input to the choroid. The image shows 3A10+ labeled Þbers in the choroid of the superior ocular quadrant of a normal pigeon eye. INL inner nuclear layer, IPL inner plexiform layer, RPE retinal pigment epithelium

(Fig. 12.23) [88]. The increases are driven by increases in choroidal volume (vasodilation) and are not accompanied by increases in systemic BP. In Fitzgerald et al. [90], we also showed that activation by electrical stimulation of what we now call vSCN yields increases in ChBF in the opposite eye, while retinal illumination yields ChBF increases in the illuminated eye, as would be predicted by the layout of the vSCN-EWM circuit (Fig. 12.23). Furthermore, the vSCNand lightelicited increases could be blocked reversibly by lidocaine injection into the EWM ipsilateral to

the recorded eye (Fig. 12.23). Control studies conÞrmed that the light-elicited increases were not artifactually generated by transocular illumination of the LDF probe. In a study on chicks, we showed that severing the ciliary nerves permanently dilates the pupil and causes increased ChBF [315]. We interpreted the ChBF increase to stem from the increased illumination falling on the retina due to the chronically dilated pupil. An opaque occluder that diminished light entry in chick eyes with severed ciliary nerves eliminated the ChBF increase [316]. These results conÞrmed that the vSCN-EWM-ciliary ganglion circuit regulates ChBF in response to retinal illumination and/or activity. Thus, a major natural stimulus activating this circuit is the pattern and intensity of retinal illumination [90], and perhaps ßicker may as well [317]. Such reßexive responses to increased retinal illumination or increased complexity of the patterns imaged on the retina may be adaptive, since such retinal activation alters the metabolic and/or thermal demands on the retina [35, 204, 325].

In a 1996 study [412], we evaluated the effects of two NOS inhibitors, 7NI and LNAME, on the increases in ChBF elicited by electrical stimulation of EWM in pigeons, as detected using transcleral LDF. We found that 7NI (which at the time we thought was nNOS selective but now know is not) and LNAME each attenuated the EWM-evoked response by about 80% (Fig. 12.24). In light of our Þndings that choroidal neurons do not contain NOS or make NO [64], the results of Zagvazdin et al. [412] indicate that endothelially derived NO must mediate EWM-elicited choroidal vasodilation. Since choroidal neurons of the avian ciliary ganglion do release acetylcholine [232], and since acetylcholine is known to stimulate endothelial NO release [242], we studied the role of muscarinic cholinergic mechanisms in ciliary ganglionmediated ChBF increases in pigeon [413]. Using LDF and atropine as well as selective blockers of the M3-type muscarinic receptor (4-diphenyl- acetoxy-N-methylpiperedine, 4DAMP) and the M2-type muscarinic receptor (himbacine), we found that atropine and the M3-type muscarinic receptor blockade greatly (by about 90%) inhib-

a

b

Nasal choroid 30 sec 200 µA pulse trains

Nasal choroid 30 sec 400 µA pulse trains

Superior choroid 30 sec 400 µA pulse trains

c

Choroidal blood flow Choroidal blood flow Choroidal blood flow Choroidal blood flow

Fig. 12.23 Chart records showing the effects SCN-EWM circuit activation on ChBF in pigeon, with ChBF measured using laser Doppler ßowmetry and expressed in relative units referred to as blood ßow units. The records in (a) show that stimulation of EWM yields blood ßow increases in superior and nasal choroid, with the increases being stimulus duration and stimulus amplitude dependent. The records in (b) show right eye ChBF responses to right EW activation or right retinal illumination. Note that both elicit ChBF increases and that the light-mediated

response is prevented by an EWM lesion, showing that it is mediated via EWM. The records in (c) show right eye ChBF responses to right EWM or left vSCN stimulation. Note both elicit clear ChBF increases that are stimulus duration dependent, and note that transiently inactivating EW with lidocaine reversibly diminishes both the ChBF increases obtained with vSCN or EW stimulation. These overall results are consistent with the interpretation that the vSCN-EWM circuit in birds is involved in retinal activity-dependent increases in ChBF

ited EWM-evoked increases in ChBF (Fig. 12.24), while M2-type receptor inhibition increased ChBF by about 100%. Based on our Þndings that the ciliary ganglion input to choroid does not synthesize NO but inhibitors of NO production do block EWM-evoked choroidal vasodilation, it seems likely that the M3 receptors acted on by 4DAMP are present on choroidal endothelial cells, and the ciliary ganglion thereby mediates choroidal vasodilation via M3 cholinergic stimulation of endothelial NO release. In contrast, M2 muscarinic receptors may play a presynaptic role in downregulating EWM-evoked parasympathetic cholinergic vasodilation in avian choroid. Inhibiting them thus would be a means to potentiate ChBF. Our Þnd-

ing that EW lesions signiÞcantly diminish ChBF in pigeons indicates that basal activity in the preganglionic input to the ciliary ganglion is needed to maintain basal choroidal tone [90], and our Þnding that severing the choroidal nerves from the choroidal neurons of the ciliary ganglion to the choroid in chickens vastly diminishes ChBF supports the view that ciliary ganglion input prominently controls basal choroidal tone [315].

12.5.4.5Studies of the Importance of ChBF Control by the vSCN-EWM-

Ciliary Ganglion Circuit – Birds

While large reductions in ChBF lead to severe photoreceptor loss [111, 226], even slight reduc-

a

Fig. 12.24 The chart records in (a) show examples of the effect of intravenous (1 mg/kg) and topical (4Ð5 drops of 5% solution) atropine, a nonselective muscarinic antagonist, on the increase in ChBF evoked by electrical stimulation of EWM and on systemic arterial blood pressure (BP) in pigeons. Eserine (100 mg/kg iv), an acetylcholine esterase inhibitor, was administered 30 min after atropine administration. CBF was measured by laser Doppler ßowmetry and is presented as relative blood ßow units (BFU). The electrical stimulation (Stim) was applied as threeÐfour 5Ð10 s trains with 5 s (iv atropine) and 40 s (topical atropine) intervals between trains. Note that atropine produced

an eserine-reversible decrease in the EWM-evoked response and an elevation in systemic BP. The chart records in (b) show examples of the effect of nitric oxide synthase inhibition by 7NI and LNAME on the increase in ChBF after 5 s electrical stimulation of EWM. The EWM stimulation was not associated with arterial BP changes and was diminished after the injection of 7NI (50 mg/kg administered ip) or LNAME (30 mg/kg). Note the reduction in baseline choroidal blood ßow and the increase in blood pressure after LNAME. Time (expressed as h/min/s) from the start of data acquisition is shown on the x-axis

tions in ChBF hinder the ability of oxygen and nutrients to reach the outer retina, and such ChBF reductions thereby have rapid and deleterious functional consequences for the outer retina [325, 399, 400]. ChBF is also vital for supporting the inner retina in those retinal regions poor in retinal vessels [37, 169]. Given that ChBF is likely to be regulated by the nervous system so as to match ChBF to retinal need, it is also likely that adaptive neural regulation of ChBF is important for the long-term health of the retina. We have speciÞcally addressed this issue in studies on the avian vSCN-EWM-ciliary ganglion system in which we destroyed EWM. Such lesions reduce basal ChBF to 50Ð75% of normal in the ipsilateral eye and block adaptive ChBF regulation by the vSCN-EWM-ciliary ganglion circuit (e.g., light-mediated ChBF increases) [90]. In pigeon eyes affected by EWM lesions, we have found evidence of retinal functional disturbance and pathology, including (1) increased glial Þbrillary acidic protein (GFAP) in retinal MŸller cells [87, 180] and (2) losses in behaviorally assessed visual acuity [145]. In the former studies, we examined the effects of EWM lesions on the health of the retina (as assessed by GFAP immunolabeling) in birds housed under normal circadian lighting conditions [174]. We found that the GFAP increases in MŸller cells following EWM destruction are progressive up to 24 weeks and occur preferentially in superior/temporal retina, which is heavily innervated by the ciliary ganglion in pigeons (Fig. 12.25). After 24 months, GFAP expression begins to diminish, but the GFAP upregulation is still evident 1 year after the lesion. In our behavioral studies, we examined the effects of EWM lesions on visual acuity in pigeons [145]. Bilateral lesions of EWM were made electrolytically, and visual acuity for highcontrast, square-wave gratings was determined behaviorally about 1 year later and compared to that in a group of pigeons that had received sham lesions of EW about 1 year prior to acuity testing (Fig. 12.26). Because lesions targeting EWM invariably result in damage to the adjoining EWL, two additional control groups were studied. In one control group, bilateral lesions in area pretectalis (AP), which innervates the pupillary control

part of EWL and thereby controls pupillary constriction [284], were made, and the effects on visual acuity determined about 1 year later. In the second additional control group, the effects of acute accommodative and pupillary dysfunction on acuity were studied in cyclopleged pigeons. The mean acuities of birds with AP lesions

different from normal. In contrast, pigeons with lesions that completely destroyed EW bilaterally showed visual acuity (2.7 ± 0.1 cycles/degree) that was well below the acuity of the shamand AP-lesion control groups. The acuity of the cycloplegic pigeons (4.8 ± 0.3 cycles/degree) and one pigeon with a nearly complete bilateral EWL but a unilateral EWM lesion (6.4 cycles/degree) indicated that only about half of the loss with a bilateral EW lesion could be attributed to accommodative dysfunction. Thus, bilateral destruction of EWM led to a loss in visual acuity, suggesting that disruption of adaptive neural regulation of ChBF causes retinal injury that impairs vision.

Our lesion studies, therefore, support the notion that interrupting neural control of ChBF by the vSCN-EWM-ciliary ganglion circuit is harmful for the retina. It seems likely therefore that retinopathy would also ensue from central or peripheral damage to facial, sensory, or sympathetic circuits controlling ChBF [277]. The precise nature of the retinal injury and the circumstances under which the impaired neural control of ChBF might be especially harmful, however, are uncertain and would depend on the precise role that the damaged circuit plays in supporting ocular health. It is possible that the retinal impairments observed with EWM lesions that disable parasympathetic control of ChBF stem from ChBF insufÞciency that renders the retina chronically hypoxic and ischemic (Fig. 12.27) [45, 87, 399, 400]. Impaired parasympathetic control of ChBF may also result in harmful accumulation of waste products in the outer retina or an inadequate nutrient supply for outer retina renewal (e.g., amino acids, sugars, and fats) [141, 199]. Regardless of the basis of the retinal damage that occurs with disturbed ciliary ganglionmediated control of ChBF in birds, it is likely that these same potentially damaging processes are

Fig.12.25 ImagesofrepresentativeGFAP-immunolabeled sections through the superior-central retina of normal pigeons (b, c), a pigeon with a left area pretectalis (AP) lesion (d, e), a pigeon that survived for 3 weeks with a complete right EW lesion (g), a pigeon that survived for 9 weeks with a complete right EW lesion (h), a pigeon that survived for 22 weeks with a complete right EW lesion (i), and a pigeon that survived for 40 weeks with a complete right EW lesion (j), all housed in a 12-h moderate light/12- h dark cycle. Images (a) and (f) show a 1-mm-thick toluidine blue-stained plastic section of pigeon retina, with the different retinal layers delimited by hash marks, and the hash mark between the outer nuclear layer and the inner segment layer located at the outer limiting membrane. Retinal sections from a normal pigeon never housed in an individual cage show no GFAP immunolabeling (b), while the retinal section from a normal pigeon housed for 3 weeks in an individual cage shows slight GFAP immunostaining of the nerve Þber layer (NFL) and ganglion cell layer (GCL). The lesion of left AP eliminated the pupil light reßex and chronically dilated the pupil of the right

eye. In the left eye, GFAP immunolabeling is weak and does not extend beyond the NFL, while GFAP immunolabeling in the right eye Þlls MŸller cell processes into the GCL. The view of the right eye (g) of a bird 3 weeks after a complete lesion of both the right EWM and EWL shows GFAP immunolabeling Þlls MŸller cell processes into the inner nuclear layer (INL). The image of the right eye (h) of a bird 9 weeks after a complete lesion of right EWM and EWL shows GFAP immunolabeling extends through the outer plexiform layer (OPL). The image of the right (i) eye of a bird 22 weeks after a complete lesion of EWM and EWL shows GFAP immunolabeling extends through the outer plexiform layer to the outer limiting membrane (OLM). Finally, the image of the right eye (j) of a pigeon 40 weeks after a complete lesion of EWM and EWL shows GFAP immunolabeling Þlled the MŸller cell processes through the INL. GCL ganglion cell layer, INL inner nuclear layer, IPL inner plexiform layer, IS inner segment layer, NFL nerve Þber layer, ONL outer nuclear layer, OS outer segment, RPE retinal pigment epithelium. MagniÞcation the same in all images

a

b

Acuity in cycles/degree

11

10

9

8

7

6

5

4

3

2

Fig. 12.26 Image (a) shows a schematic representation of the training chamber used to measure visual acuity in pigeons, using a discriminative behavioral task. Image (b) shows the mean acuity data (±SEM) for the complete EW-lesion birds, the partial EW-lesion bird, the AP-lesion birds, the sham-lesion birds, the cycloplegic birds treated with saline (Saline), and the same cycloplegic birds treated with vecuronium (Vec). Note that the mean data for the three birds with the complete bilateral destruction

(EWMbilat) are graphed separately from the data for the one bird with the bilateral EWL but unilateral EWM lesion (EWMunilat). The SEM is too small for the former birds to be evident, and no SEM can be calculated for the single unilateral EW bird. The visual acuity for the bilaterally EW-lesioned birds was signiÞcantly poorer than for the Þve other categories of birds, among which there were no statistically signiÞcant differences