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Ph.D. Program in Biology, The Graduate Center, CUNY, 365 Fifth Avenue, New York, NY 10016
1 Department of Biological Sciences, Hunter College, CUNY, 695 Park Avenue, New York, NY 10021
Zinc is an important trace element found in relatively high concentration in the pigment epithelium and visual cells of the vertebrate retina (1). Zinc deficiency has been associated with night blindness and age-related macular degeneration (AMD) (2). Dietary zinc supplementation is now commonly prescribed for AMD on the basis of the benefits recently reported from the Age-Related Eye Disease Study (3). Nevertheless, the role zinc plays in the processing of visual information or in retinal disease is not well understood (2). We believe the possible role of zinc in the processing of visual information is an important consideration in the diagnosis and treatment of retinal disorders. Abnormalities involving light or dark adaptation, contrast sensitivity, or glutamate toxicity in the outer retina should be investigated from this perspective.
Studies in the all-rod retina of the skate (Raja erinacea) support the notion of a role for endogenous zinc as a neuromodulator in the outer retina of vertebrates. Application of the zinc chelator histidine (100 µM) in the presence of the GABA-blocker picrotoxin (200 µM) doubled the amplitude of the skate electroretinogram (ERG) b-wave recorded in the skate eyecup preparation (4). Consistent with this observation, we found that application of histidine to the skate retinal slice preparation resulted in a reversible increase in inward current from identified horizontal cells recorded in voltage clamp mode (5). Here we report results from an ERG study that is similar to the one in skate but uses the in situ eyecup preparation of the zebrafish, a vertebrate whose retina has both rods and cones.
Adult zebrafish (Danio rerio) obtained from a colony maintained at the Marine Biological Laboratory, Woods Hole, Massachusetts, were anesthetized using 0.02% tricaine, immobilized with a 2-µl injection of 1% gallamine triethiodide (Flaxidil), and placed on their sides on a small sponge over a silver chloride pellet reference electrode. Their gills were irrigated with an oxygenated oral solution containing 2 g Instant Ocean per gallon distilled water (6). The cornea and lens were carefully removed to allow superfusion of the retina using a glass capillary manifold that could be switched from zebrafish Ringer (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2, and 5 mM HEPES) (6) to Ringer containing 200 µM picrotoxin alone or 200 µM picrotoxin plus 100 µM histidine. All solutions were adjusted to pH 7.2. A glass capillary agar bridge to another silver chloride pellet was placed in the eyecup as the recording electrode. Experiments were conducted in a darkened room in the presence of dim ambient illumination. ERG responses were recorded using a low-noise differential preamplifier (PAR 113) having a bandpass of 0.03 Hz to 1 kHz, and stored with a DigiData 1200 acquisition system and pClamp6 software. The intensity of the unattenuated beam (log I = 0) from a 100-W tungsten-halogen lamp operated at 8 amps from a dc-regulated power supply was 30 µW/cm2 at the retina. ERG responses recorded from the zebrafish eyecup preparation following removal of the cornea and lens were generally smaller than those recorded using a corneal electrode on the intact eye, possibly due to trauma caused during the microsurgery. Thereafter, responses remained stable for up to an hour.
Figure 1A shows ERG records obtained from one preparation in response to 1.1-s flashes of intensity log I = -0.7. The b-wave component of the response (arrow) recorded during superfusion with Ringer (upper trace) was not significantly changed after 10 min in picrotoxin (middle trace). This suggests that GABAergic mechanisms do not have a large impact on the magnitude of the ERG b-wave in the zebrafish retina. The amplitude of the b-wave nearly doubled, however, within 30 min after the superfusate was switched to Ringer containing both histidine and picrotoxin (Fig. 1A, lower trace). Note also the large apparent increase in the a-wave (photoreceptor potential), which is due probably to delayed onset of the b-wave as suggested by a 40% delay (50 ms ± 13, n = 5) in b-wave time to peak in the histidine solution.
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Intensity-response data (mean ± SEM) from the five preparations, normalized to the picrotoxin response at log I = -0.3, are shown in Figure 1D. The curves represent the best fit of each data set to the Naka-Rushton equation (7) obtained using the Origin analysis package. The maximum responses determined for the fitted curves were, respectively, Rmax = 0.96, 0.97, and 1.80 for Ringer, picrotoxin, and histidine plus picrotoxin. In the histidine solution, the value of 
, the intensity eliciting a response of 
Rmax (8), was shifted to the left by 0.4 log units. Thus, saturating responses (Rmax) were essentially the same for Ringer alone or with the addition of picrotoxin. However, when histidine was added to the superfusate, the maximum response increased by a factor of about 1.9, consistent with the near-doubling observed in responses at lower intensities.
On the basis of the results of this study, we conclude that the effects of the zinc chelator histidine on the zebrafish retina, which contains both rods and cones, is similar to that observed in the all-rod retina of the skate. It appears, therefore, that zinc may be acting as a neuromodulator in the outer retina. In addition to any direct effect zinc may have on the sensitivity of glutamate receptors on second-order cells or to other possible neuromodulatory effects of zinc, an interesting possibility of negative feedback of zinc co-released with glutamate from photoreceptor terminals to reduce calcium entry into these terminals has been proposed by Wu and his colleagues (9). Since calcium is a cofactor in synaptic vesicle release, they suggest that this feedback will reduce the release of photoreceptor transmitter, including zinc, until an equilibrium is reached. The zinc neuromodulatory hypothesis is also consistent with histological evidence of a band of free zinc in the region of photoreceptor terminals in salamander (9) and skate (10) retinas and with a recent report that the relative concentration of free zinc in that region changes with the state of light adaptation in a mammalian retina (1).
The authors express their appreciation to Dr. Alan Adolph and Kwoon Wong for their assistance concerning the zebrafish eyecup preparation. Supported by NIH Grant EY00777, PSC/CUNY Grants 62245-0031 and 64225-0033, as well as by an NIH/RISE (Research Institute for Scientific Enhancement) GM60665 award to Hunter College. Research Centers in Minority Institutions Award RR-03037 from the National Center for Research Resources of the NIH, which supports the infrastructure of the Biological Sciences Department at Hunter College, is also acknowledged. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR/NIH.
Literature Cited
This article has been cited by other articles:
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  R. L. Chappell, H. Qian, J. Zakevicius, and H. Ripps Histidine Suppresses Zinc Modulation of Connexin Hemichannels Biol. Bull., December 1, 2004; 207(3): 188 - 190. [Full Text] [PDF]
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S. Redenti and R. L. Chappell Zinc Chelation Enhances the Sensitivity of the ERG b-Wave in Dark-Adapted Skate Retina Biol. Bull., October 1, 2003; 205(2): 213 - 214. [Full Text] [PDF]
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