A dark decrement for enhanced dynamic sensitivity of retinal photoreceptors.

The skate retina provides a native all-rod retina suited for investigating a single type of photoreceptor regarding its properties and signaling to second order cells. Using the aspartate-induced isolated A-wave of the skate eyecup electroretinogram (ERG), it has been shown that adaptation in rods remains Weber-Fechner-like over a 6-log unit increase in background light intensity. Zinc, which can block calcium channels, has been found in the rod synaptic terminal and the synaptic cleft. Histidine is a zinc chelator. Voltage signals from neurons post-synaptic to rods indicate that histidine increases the dark release of glutamate and increases the horizontal cell light response. In histidine, the A-wave response to various light intensities in the dark-adapted retina increased more than fifty percent, corresponding to the effect on horizontal cells. In the presence of background light, although histidine-treated rod light responses remained Weber-Fechner-like, their increment threshold was raised significantly. This indicates that endogenous zinc feedback serves to increase rod sensitivity in a light-adapted retina, despite a corresponding reduction of threshold sensitivity in the dark. We propose that the increase in A-wave amplitude is a result of the increased conductance at the synaptic terminal and that the A-wave can be used to monitor changes in rod transmitter release. Furthermore, endogenous zinc may also provide the benefit of reducing metabolic stress and the risk of glutamate toxicity in the dark.

Review: Zinc's functional significance in the vertebrate retina.

This review covers a broad range of topics related to the actions of zinc on the cells of the vertebrate retina. Much of this review relies on studies in which zinc was applied exogenously, and therefore the results, albeit highly suggestive, lack physiologic significance. This view stems from the fact that the concentrations of zinc used in these studies may not be encountered under the normal circumstances of life. This caveat is due to the lack of a zinc-specific probe with which to measure the concentrations of Zn(2+) that may be released from neurons or act upon them. However, a great deal of relevant information has been garnered from studies in which Zn(2+) was chelated, and the effects of its removal compared with findings obtained in its presence. For a more complete discussion of the consequences of depletion or excess in the body's trace elements, the reader is referred to a recent review by Ugarte et al. in which they provide a detailed account of the interactions, toxicity, and metabolic activity of the essential trace elements iron, zinc, and copper in retinal physiology and disease. In addition, Smart et al. have published a splendid review on the modulation by zinc of inhibitory and excitatory amino acid receptor ion channels.

Cytoprotection by endogenous zinc in the vertebrate retina.

Our recent studies have shown that endogenous zinc, co-released with glutamate from the synaptic terminals of vertebrate retinal photoreceptors, provides a feedback mechanism that reduces calcium entry and the concomitant vesicular release of glutamate. We hypothesized that zinc feedback may serve to protect the retina from glutamate excitotoxicity, and conducted in vivo experiments on the retina of the skate (Raja erinacea) to determine the effects of removing endogenous zinc by chelation. These studies showed that removal of zinc by injecting the zinc chelator histidine results in inner retinal damage similar to that induced by the glutamate receptor agonist kainic acid. In contrast, when an equimolar quantity of zinc followed the injection of histidine, the retinal cells were unaffected. Our results are a good indication that zinc, co-released with glutamate by photoreceptors, provides an auto-feedback system that plays an important cytoprotective role in the retina.

Zinc modulation of calcium activity at the photoreceptor terminal: a calcium imaging study.

There is abundant experimental evidence that zinc ions (Zn(2+)) are present in the synaptic vesicles of vertebrate photoreceptors, and that they are co-released with glutamate. Here we show that increasing the concentration of extracellular zinc (2 µM-2 mM) suppresses the entry of calcium into the synaptic terminals of isolated salamander double cones. The resultant dose-dependent curve was fit by an inverse Hill equation having an IC50 of 38 µM, and Hill coefficient of 1.1. Because there is currently no reliable way to measure the concentration of extracellular zinc, it is not known whether the zinc released under normal circumstances is of physiological significance. In an attempt to circumvent this problem we used zinc chelators to reduce the available pool of endogenous zinc. This enabled us to determine how the absence of zinc affected calcium entry. We found that when intra- or extra-cellular zinc was chelated by 250 µM of membrane-permeable TPEN or 500 µM of membrane-impermeable histidine, there was a significant rise in the depolarization-induced intracellular calcium level within photoreceptor terminals. This increase in internal [Ca(2+)] will undoubtedly lead to a concomitant increase in glutamate release. In addition, we found that blocking the L-type calcium channels that are expressed on the synaptic terminals of photoreceptors with 50 µM nicardipine or 100 µM verapamil abolished the effects of zinc chelation. These findings are a good indication that, when released in vivo, the zinc concentration is sufficient to suppress voltage-gated calcium channels, and reduce the rate of glutamate release from photoreceptor terminals.

Zinc-mediated feedback at the synaptic terminals of vertebrate photoreceptors.

There is mounting evidence that zinc release from glutamatergic nerve terminals serves as a neuromodulator at synaptic sites within the retina and CNS. However, it has not been possible to reliably measure the concentration of zinc co-released with glutamate in the confines of the synaptic cleft. Thus, much of the evidence supporting this view derives from electrophysiological studies showing the modulatory effects of exogenous zinc on the membrane currents of ligand- and voltage-gated channels. In the present study, we took advantage of the unique properties of the glutamatergic photoreceptor terminal to demonstrate a feedback signal mediated by endogenous zinc at the synaptic sites from which it is discharged. Through its ability to block voltage-gated calcium channels in the photoreceptor terminal, zinc suppresses the radial dark current of the visual cell, and reduces its release of glutamate. It follows that chelation of extracellular zinc, e.g., with histidine, will lead to an increase both in the dark current and in the release of glutamate, changes that result in an enhancement of the light-evoked a-wave of the ERG and can account for the b-wave enhancement observed previously after zinc chelation when inner retinal responses were not blocked by aspartate.

Zinc release at the synaptic terminals of rod photoreceptors.

The presence of reactive zinc (Zn2+) within photoreceptor terminals, and evidence that exogenous zinc affects the electrophysiological activity of the distal retina, led to the suggestion that its co-release with glutamate could play an essential role in the modulation of information at the first synapse in the visual pathway. Although we had shown previously that zinc release could be visualized in the region of the outer synaptic layer of a retinal slice preparation, it could not be ascertained with certainty that the release sites were at the presynaptic terminal rather than from the mitochondria-rich inner segment or from zinc within the distal processes of photoreceptors and Müller cells. Using membrane permeant and membrane impermeant forms of a fluorescent zinc indicator (Newport green), we show both the intracellular distribution of Zn2+ and its depolarization-dependent discharge from the terminals of isolated zebrafish photoreceptors in culture. Zinc release could be detected in the dark-adapted preparation, and was further enhanced by brief exposures to black widow spider venom or high K+. Synaptically released zinc may significantly influence neural processing in the vertebrate retina by modulating the activity of excitatory and/or inhibitory receptors as well as intracellular signaling proteins.

Müller cell zinc transporter-3 labeling suggests a role in outer retina zinc homeostasis.

Labeling for zinc transporter protein-3 (ZnT-3), which can be found localized to glutamatergic vesicles elsewhere in the nervous system, has revealed an unexpectedly high concentration of this transporter protein in the outer limiting membrane region of the murine retina, a region that contains the mitochondria-rich portion of photoreceptor inner segments and is not involved with vesicle release. Having suggested the possibility that Müller cell apical villi forming the outer limiting membrane may be associated with the labeling observed, we used immunohistochemical techniques to look for ZnT-3 labeling of Müller cells isolated from rat and mouse retinas. With DAB labeling, rat Müller cell apical villi, soma, and endfeet exhibited ZnT-3 reactivity. FITC label and confocal analysis revealed that ZnT-3 protein appeared throughout the length of mouse Müller cells. We conclude from these observations that the dense labeling for ZnT-3 in the photoreceptor inner segment region of murine retinal slices is due to labeling of ZnT-3 protein associated with Müller cell apical villi. Based on these findings we suggest that Müller cells utilize ZnT-3 to regulate retinal zinc homeostasis and that this role is important to mitochondrial function in the photoreceptor inner segments.

Neuroimaging of zinc released by depolarization of rat retinal cells.

Zinc is associated with glutamatergic pathways in brain and retina, yet its role in neuromodulation remains unknown. High concentrations of reactive zinc in vertebrate photoreceptor terminals suggest a neuromodulatory role in the outer plexiform layer but zinc release has not been demonstrated. Using the membrane-impermeable form of the Zn(2+) sensitive fluorescent dye Newport Green, we have demonstrated increased release of Zn(2+) from the rat retina in response to potassium-induced depolarization of retinal cells. This increase was greatest in the outer retina with densest bands observed in the outer plexiform layer and photoreceptor inner segment regions of rat retinal slices.

Directional asymmetries in the optokinetic response of larval zebrafish (Danio rerio).

Photographic images of the optokinetic response (OKR) of larval zebrafish permitted the calculation of the amplitude and velocity of the response to gratings of various spatial frequencies rotating at different speeds. At low spatial frequencies, the amplitude of the OKR did not vary significantly for drum speeds ranging from 24 to 108 degrees/sec. Plotting the velocity of the OKR as a function of drum speed gave rise to a bell-shaped curve, with a maximum at about 48 degrees/sec. Interestingly, both eyes exhibited an asymmetric response to the rotating drum, that is, they were more responsive to temporal-to-nasal rotation than to nasal-to-temporal motion. Although this asymmetry persisted over the entire range of drum speeds tested, the situation was reversed when tested with gratings of higher spatial frequency (i.e., the eyes became more responsive to rotation in the nasal-to-temporal direction). The amplitude of the OKR for both eyes exhibited an inverse relation to increasing spatial frequency of the stimulus, whereas the velocity of the OKR showed a steep decline within the range of 0.08 to 0.14 cycles/degree. The data indicate that zebrafish are more responsive to objects with low spatial frequencies moving from behind the animal's head toward the frontal plane, and to high spatial frequencies of objects moving across the frontal plane (perpendicular to the anterior-posterior axis of the eye).

Localization of zinc transporter-3 (ZnT-3) in mouse retina.

Studies of the central nervous system have localized the zinc-transporter-3 (ZnT-3) protein to synaptic vesicles containing glutamate and zinc. We have examined the distribution of the ZnT-3 protein in the light-adapted mouse retina using immunohistochemical techniques. Light microscopic analysis of 15-30-microm retinal sections revealed a rich band of ZnT-3 protein in the region of the outer limiting membrane and photoreceptor inner segments. ZnT-3 reactivity was also present in the outer plexiform, inner nuclear, inner plexiform, and ganglion cell layers. The outer nuclear layer and photoreceptor outer segments did not exhibit ZnT-3 immunoreactivity. In the light-adapted murine retina, ZnT-3 appears localized in regions which have been found reactive for ionic zinc.

Endogenous zinc as a retinal neuromodulator: evidence from the skate (Raja erinacea).

The effects of zinc on skate (Raja erinacea) bipolar cell responses to glutamatergic agonists were examined using whole-cell voltage-clamp recording. Isolated ON bipolar cell currents mediated by the metabotropic agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (30 microM), L-2-amino-4-phosphonobutyrate (3-10 microM) and glutamate (0.3-10 microM) were blocked when zinc (1 microM) was added to the test solution. Similarly, isolated OFF bipolar cell responses to the ionotropic agonist kainate (300 microM) were blocked by zinc (1 microM). The effects of zinc were further studied using electroretinogram (ERG) recording. Skate eyecup preparations were superfused with picrotoxin (200 microM) to block GABAergic input. When histidine (100 microM), a zinc chelator, was added to the superfusate, ERG ON responses increased. This suggests that endogenous zinc plays a neuromodulatory role in the retina and is consistent with zinc's suppressive effect on isolated bipolar cells.

GABA(C) receptors modulate the rod-driven ERG b-wave of the skate retina.

Studies characterizing the types of GABA receptors present on cells isolated from the skate retina have allowed us to develop a working model of possible GABA interactions at the level of the outer plexiform layer (OPL). Earlier studies have shown an electrogenic GABA transport mechanism in horizontal cells presents a source of GABA in the OPL which could modulate feedback onto photoreceptors. GABA(A) receptors on Müller cells, or GABA(A) and/or GABA(C) receptors on bipolar cells. This model has been used for the interpretation of results of experiments in this study designed to test the role these interactions may exert on the electroretinogram (ERG). Simultaneous intracellular recording of the horizontal cell response (the S-potential) was used to monitor effects on photoreceptor transmitter release which would be altered if GABAergic photoreceptor feedback mechanisms were involved. Picorotoxin (50 microM), a chloride channel blocker which suppresses the responses of both GABA(A)Rs and GABA(C)Rs, reduced the ON (b-wave) component of the ERG substantially. Simultaneous intracellular horizontal cell recordings, however, showed no effect on their light-evoked response, suggesting that photoreceptor feedback is not involved in the picrotoxin effect on the ERG. On the other hand, even 100 microM bicuculline, a GABA(A)R antagonist produced no change in either the ERG or the horizontal cell response. This observation leads to the conclusion that the GABAARs on Miller cells and bipolar cells are not involved. Thus, there remains a distinct possibility that the ERG changes produced by picrotoxin are due to its ability to block the GABA(C)Rs on retinal bipolar cells.