Jeffrey H. Boatright

Stimulation of endogenous dopamine release and metabolism in amphibian retina by light- and K+-evoked depolarization.

The release and metabolism of dopamine (DA) in retina was assessed using an in vitro eye cup preparation of the African clawed frog. The concentration of DA in the incubation medium and of 3,4-dihydroxyphenylacetic acid (DOPAC) and DA in retinas was measured by high-performance liquid chromatography with electrochemical detection (HPLC-ED). K+-induced depolarization stimulated DA overflow from the eye cups into the incubation medium and increased tissue DOPAC levels in dark-adapted retinas. Basal and K+-stimulated DA overflow and DOPAC accumulation were Ca2+-dependent. Exposure of dark-adapted retinas to constant white light for 1 h also increased DA overflow and DOPAC levels, while 1 h of alternating 10 s periods of light and dark had no effect. The results indicate that DA release and metabolism may be stimulated as a function of light-adaptation.

The Role of Glia, Mitochondria, and the Immune System in Glaucoma

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group

AAV-Mediated Gene Therapy for Retinal Degeneration in the rd10 Mouse Containing a Recessive PDEβ Mutation

PURPOSE To test AAV-mediated gene therapy in the rd10 mouse, a natural model of recessive RP caused by mutation of the beta-subunit of rod photoreceptor cGMP phosphodiesterase. METHODS One eye of a cohort of rd10 mice kept in a dark environment was subretinally injected at postnatal day (P) 14 with 1 microL AAV5-smCBA-PDEbeta. The contralateral eye was not injected. The animals were then maintained for 2 weeks in the dark before they were moved to a normal 12-hour light/12-hour dark cycling light environment for visually guided behavioral training. Three weeks after injection, treated rd10 mice were examined by scotopic and photopic electroretinography and then killed for biochemical and morphologic examination. RESULTS Substantial scotopic ERG signals were maintained in treated rd10 eyes, whereas untreated eyes in the same animals showed minimal signals. Treated eyes showed photopic ERG b-wave amplitudes similar to those of the normal eyes; in untreated partner eyes, only half the normal amplitudes remained. Strong PDEbeta expression was observed in photoreceptor outer segments only in treated eyes. Light microscopy showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Electron microscopy showed good outer segment preservation only in treated eyes. A visually guided water maze behavioral test under dim light showed significantly improved performance in one eye-treated rd10 mice compared with untreated mice. CONCLUSIONS These data demonstrate that P14 administration of AAV5-smCBA-PDEbeta can prevent retinal degeneration in rd10 mice, as reflected by significant structural, biochemical, electrophysiological, and behavioral preservation/restoration. These results serve as a baseline for studying long-term retinal rescue in rd10 mice.

Regulation of endogenous dopamine release in amphibian retina by melatonin: the role of GABA

In the retina of the African clawed frog (Xenopus laevis), endogenous dopamine release increases in light and decreases in darkness. Exogenous melatonin and several chemical analogs of melatonin suppressed light-evoked dopamine release from frog retina in a concentration-dependent manner. The rank order of potency for inhibition of light-evoked dopamine release was melatonin >> 5-methoxytryptamine > or = N-acetylserotonin > 5-methoxytryptophol >>> serotonin. Melatonin did not suppress dopamine release below levels seen in darkness. The putative melatonin receptor antagonist luzindole inhibited the effect of melatonin. Luzindole enhanced dopamine release in darkness but had little effect in light. These data suggest a role for endogenous melatonin in dark-induced suppression of retinal dopamine. Picrotoxin and bicuculline, GABA-A receptor antagonists, blocked melatonin-induced suppression of dopamine release. In the presence of melatonin, bicuculline was significantly less potent in stimulating dopamine release. These results suggest that melatonin enhances GABAergic inhibition of light-evoked dopamine release. This mechanism may underlie the light/dark difference in dopamine release in vertebrate retina.

Dopamine mediates the light-evoked suppression of serotonin N-acetyltransferase activity in retina

The possible role of dopamine in the light-induced suppression of serotonin N-acetyltransferase (NAT) activity in retinas of the African clawed frog (Xenopus laevis) was investigated using an in vitro eye cup preparation. The nocturnal increase of retinal NAT activity was significantly inhibited by either light exposure or exogenous dopamine. Spiperone, a dopamine receptor blocker, antagonized this inhibitory effect of light on NAT activity, but had no effect in darkness. The effect of spiperone required the presence of cyclic nucleotide phosphodiesterase inhibitors, 3-isobutylmethylxanthine (IBMX), papaverine, or Ro 20-1724. Under the conditions employed in this study, neither spiperone nor the phosphodiesterase inhibitors significantly affected NAT activity when added alone. This observation suggests a synergistic interaction between the dopaminergic antagonists and the phosphodiesterase inhibitors. Other dopamine receptor blockers, including haloperidol, cis-flupenthixol, clozapine and metoclopramide, increased NAT activity of light-exposed retinas incubated in the presence of IBMX. SCH 23390, a D1-selective dopamine receptor antagonist, did not increase NAT activity, nor did the alpha- and beta-adrenergic receptor antagonists tested. The effect of spiperone and IBMX on NAT activity was blocked by apomorphine and by the D2-dopamine receptor agonist LY 171555, but not by the D1-receptor agonist SKF 38393-A. The concentration of 3,4-dihydroxyphenylacetic acid was higher in light-exposed retinas than in dark-adapted retinas, suggesting that light exposure increases dopamine metabolism in Xenopus retina. The results presented in this paper suggest that dopamine, released in response to light exposure and acting on D2-dopamine receptors, is partially responsible for the light-induced suppression of the nocturnal increase in retinal NAT activity.

Bile acids in treatment of ocular disease

Bear bile has been included in Asian pharmacopeias for thousands of years in treatment of several diseases, ranging from sore throat to hemorrhoids. The hydrophilic bile acids tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) are the major bile acids of bear bile. Both of these are available as synthetic formulations and are approved by the health administrations of several countries for treatment of cirrhosis and gallstones. This review briefly covers the use of bear bile in Traditional Chinese Medicine, bile acid physiology, approved use of UDCA and TUDCA in Western medicine, and recent research exploring their neuroprotective properties, including in models of ocular disease.

Aerobic Exercise Protects Retinal Function and Structure from Light-Induced Retinal Degeneration

Aerobic exercise is a common intervention for rehabilitation of motor, and more recently, cognitive function (Intlekofer and Cotman, 2013; Wood et al., 2012). While the underlying mechanisms are complex, BDNF may mediate much of the beneficial effects of exercise to these neurons (Ploughman et al., 2007; Griffin et al., 2011; Real et al., 2013). We studied the effects of aerobic exercise on retinal neurons undergoing degeneration. We exercised wild-type BALB/c mice on a treadmill (10 m/min for 1 h) for 5 d/week or placed control mice on static treadmills. After 2 weeks of exercise, mice were exposed to either toxic bright light (10,000 lux) for 4 h to induce photoreceptor degeneration or maintenance dim light (25 lux). Bright light caused 75% loss of both retinal function and photoreceptor numbers. However, exercised mice exposed to bright light had 2 times greater retinal function and photoreceptor nuclei than inactive mice exposed to bright light. In addition, exercise increased retinal BDNF protein levels by 20% compared with inactive mice. Systemic injections of a BDNF tropomyosin-receptor-kinase (TrkB) receptor antagonist reduced retinal function and photoreceptor nuclei counts in exercised mice to inactive levels, effectively blocking the protective effects seen with aerobic exercise. The data suggest that aerobic exercise is neuroprotective for retinal degeneration and that this effect is mediated by BDNF signaling.

Site-specific DNA hypomethylation permits expression of the IRBP gene.

Interphotoreceptor retinoid binding protein (IRBP), a putative component of the visual cycle, is expressed selectively in the retina and pineal gland. This study examined whether site-specific DNA hypomethylation plays a role in this expression regulation. Southern blotting of HpaII and MspI digests of DNA from various bovine and murine tissues (whole brain, retina, pineal gland, superior colliculus, cortex, thymus, habenular nucleus, cornea, liver, tail, and kidney) revealed that specific CpG dinucleotides in the IRBP gene promoter are hypomethylated in DNA from retinal photoreceptor cells and pineal gland compared to DNA from other tissues. These sites are methylated in DNA from non-photoreceptor retinal cells. Exogenous methylation of these sites diminished DNA:protein binding in electrophoretic mobility shift assays. HpaII methylation of chloramphenicol acetyltransferase reporter constructs suppressed IRBP but not SV40 promoter activity in transiently transfected primary cultures of embryonic chick retinal cells. These data indicate that specific cytosines in the bovine and murine IRBP promoters are unmethylated in photoreceptive cells but methylated in other tissues. This differential DNA methylation may modulate IRBP gene expression since exogenous methylation of the murine sites suppresses reporter gene transcription, apparently by inhibiting DNA:protein binding events.

Inhibition of endogenous dopamine release in amphibian retina byl-2-amino-4-phosphonobutyric acid (l-AP4) andtrans-2-aminocyclopentane-1,3-dicar☐ylate (ACPD)

The metabotropic glutamate receptor agonists 2-amino-4-phosphonobutyric acid (AP4) and trans-2-aminocyclopentane-1,3-dicarboxylate (ACPD) blocked light-stimulated dopamine release from Xenopus laevis retina. ACPD suppressed release in darkness but AP4 did not. AP4 blocked release stimulated in darkness by picrotoxin, a GABA-A receptor antagonist. The data suggest that regulation of dopamine release in Xenopus retina involves subpopulations of metabotropic glutamate receptors.

In Vivo Imaging of Retinal Oxidative Stress Using a Reactive Oxygen Species-Activated Fluorescent Probe.

PURPOSE In vivo methods for detecting oxidative stress in the eye would improve screening and monitoring of the leading causes of blindness: diabetic retinopathy, glaucoma, and age-related macular degeneration. METHODS To develop an in vivo biomarker for oxidative stress in the eye, we tested the efficacy of a reactive oxygen species (ROS)-activated, near-infrared hydrocyanine-800CW (H-800CW) fluorescent probe in light-induced retinal degeneration (LIRD) mouse models. After intravitreal delivery in LIRD rats, fluorescent microscopy was used to confirm that the oxidized H-800CW appeared in the same retinal layers as an established ROS marker (dichlorofluorescein). RESULTS Dose-response curves of increasing concentrations of intravenously injected H-800CW demonstrated linear increases in both intensity and total area of fundus hyperfluorescence in LIRD mice, as detected by scanning laser ophthalmoscopy. Fundus hyperfluorescence also correlated with the duration of light damage and functional deficits in vision after LIRD. In LIRD rats with intravitreal injections of H-800CW, fluorescent labeling was localized to photoreceptor inner segments, similar to dichlorofluorescein. CONCLUSIONS Hydrocyanine-800CW detects retinal ROS in vivo and shows potential as a novel biomarker for ROS levels in ophthalmic diseases.