Richard L. Chappell

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.

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.

Zinc Enhances Ionic Currents Induced in Skate Muller (Glial) Cells by the Inhibitory Neurotransmitter GABA

We describe here a novel effect of zinc on GABA receptors of glial cells in the skate retina. The GABA-induced currents of skate Mϋller cells, the radial glia of the retina, are mediated by activation of GABAA receptors (GABAARs). In other parts of the nervous system, GABAA-mediated currents are inhibited by zinc. However, in isolated, voltage-clamped Mϋller cells, coapplication of zinc (10 μM) and GABA (1 μM) resulted in an enhancement of the GABAAR current. Surprisingly, zinc alone induced a current similar in many respects to that elicited by GABA, i. e. the reversal potential was the same as for the GABA-induced current, the current was blocked by bicuculline and picrotoxin, and the current-voltage relation obtained in the presence of 10 μM zinc was virtually identical to that obtained with 1 μM GABA. Both bicuculline and picrotoxin suppressed a current that was present with cells bathed only in Ringer, suggesting that some of the GABA channels were spontaneously open in the absence of externally applied GABA. This possibility was supported by cell-attached patch recordings. Under conditions in which potassium and calcium currents were suppressed, spontaneous channel activity was observed. Moreover, the frequency of these channel openings was greater when zinc was included in the pipette solution, and reduced when bicuculline was added. These findings suggest that zinc acts directly to enhance the GABAA receptor activity of the Mϋller cells, and raise the possibility that the subunit composition of the GABAARs of skate Mϋller cells differs from that of GABAARs identified previously in other neuronal and glial preparations.

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 zincs suppressive effect on isolated bipolar cells.

Zinc Modulation of Hemichannel Currents in Xenopus Oocytes

Connexins are a multigene family of structural proteins comprising gap-junctional channels, the aqueous pores that link electrically coupled cells in tissues throughout the body. These narrow passages (d 16 A) allow the intercellular exchange of ions, second messengers, and other small molecules having a molecular mass 1 kDa. In the course of forming gap junctions, the connexins oligomerize into hexameric arrays known as “connexons” or “hemichannels” that assemble in the plasma membrane before docking with the connexons of adjacent cells (1). There is now abundant evidence that, at this penultimate stage of gap-junction formation, hemichannels can be activated both chemically and electrically (2, 3), that their properties often re fl ect those of fully formed gap junctions (4), and that the modulation of hemichannel activity may be of physiological signifi cance (5). Light-induced changes in the chemical environment of the vertebrate retina have profound effects on both neurotransmitter-gated and electrical synapses; and these effects can, in turn, alter the sensitivity, receptive fi eld organization, and signalling pathways that transmit the visual message to the CNS. One putative neuromodulator that has aroused a great deal of interest in recent years is zinc, which has been found in the synaptic vesicles of glutamatergic neurons in brain and retina (6). In the retinas of amphibia (7), fi sh (8), and mammals (6), zinc is present in photoreceptors, which signal second-order cells by regulating glutamate release in response to photic stimulation. Although the co-release of zinc with glutamate remains conjectural (9), the effects it exerts on retinal neurons have not been explored extensively (8, 10); and no studies have addressed the question of its effect on connexins or the channels they form. In the present study, we used the two-electrode voltage-clamp recording technique to examine the effects of zinc on the currents mediated by the connexons formed by the endogenous connexin (Cx38) of stage V-VI Xenopus oocytes, and those formed by perch Cx35, a connexin expressed in neurons of the vertebrate retina (11, 12). To study the behavior of Cx35, cells were tested 48 to 72 h after they were injected with 46 nl of a mixture of 10 ng/cell Cx35 cRNA and 10 ng/cell of an antisense oligonucleotide to Cx38. The recordings were made with the cells bathed in a Na-free medium to eliminate the large Na-dependent currents that are similar in time course to the hemichannel currents, but of opposite polarity (13); zinc chloride was added without substitution. Responses were elicited with a series of 10-s pulses from a holding potential of 40 mV to 60 mV (see protocol, Fig. 1B inset), and were recorded with low resistance electrodes (0.7–1.5 M) connected to a GeneClamp 500 amplifi er (Axon Instruments, Foster City, CA) and controlled by protocols generated in pClamp 8 (Axon).

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.

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 animals 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).

Endogenous Zinc as a Neuromodulator in Vertebrate Retina: Evidence From the Retinal Slice

Studies of the transretinal electroretinogram (ERG) of the skate (Raja erinacia) eyecup have provided evidence that endogenous zinc plays a role as a neuromodulator in vertebrate retina (1). With GABA receptor activity blocked by 200 /uM picrotoxin, superfusion of the zinc chelating agent histidine (100 /uM) increased by about 2-fold the ON (b-wave) and OFF (d-wave) components of the ERG. In addition, as shown first in the salamander retina (2) and more recently in mammalian retinas (3, 4), an accumulation of zinc has been localized to the base of the photoreceptors in skate (5). These observations support the suggestion that zinc, co-released with glutamate from photoreceptor terminals, may serve as

Zinc chelation enhances the sensitivity of the ERG b-wave in dark-adapted skate retina.

A decade ago, Wu et al. reported evidence of a dense band of ionic zinc in the region of the photoreceptor terminals of the salamander retina (1). They speculated that zinc may play a neuromodulatory role in the outer retina, including possible feedback onto photoreceptors to down-regulate transmitter release, as well as feedforward onto second order cells. Subsequently, a high ionic zinc concentration was identifi ed in a similar region near the base of the photoreceptors in the all-rod retina of the skate (2). In addition, Ugarte and Osborne (3) have reported recently that a dense band of ionic zinc, located in the photoreceptor region of the light-adapted rat retina, is redistributed under dark-adapted conditions. Zinc has been known to affect the response of receptors on a number of retinal cell types to various neurotransmitters (4). In the skate, zinc regulates both GABA (2) and glutamate (5) receptors of isolated cells. Furthermore, the zinc chelator histidine (an amino acid endogenous in the retina, where it may play various roles in cell metabolism and disease (6, 7, 8, 9, 10)) has been shown to enhance the size of the b-wave of the electroretinogram (ERG) of the skate (5) and zebrafi sh (11). Histidine can also increase the membrane currents recorded postsynaptically from horizontal cells during voltage-clamp in the skate retinal slice preparation (12). The effects of histidine support the notion that endogenous zinc may be playing a role in the physiological response of the retina to light. Recent studies of the effects of histidine on the zebra fi sh ERG have demonstrated an increase in sensitivity of its mixed rod-cone retina in the presence of a zinc chelator (11). Here, we report studies of the effect of histidine on the retina of the skate, Raja erinacea, indicating that application of this zinc chelator enhances the sensitivity of the b-wave of the ERG in an all-rod retina. Skates were obtained through the Marine Resources Center of the Marine Biological Laboratory (Woods Hole, MA). The animals were allowed to dark-adapt for at least one hour prior to an experiment. After approved euthanasia, the eyes were enucleated and dissected under dim red light. The anterior portion of the eye, including cornea and lens, was removed; and the remaining retinal eyecup preparation was used for ERG recordings. Eyecups were placed into a chamber over a silver chloride reference electrode within a Faraday cage. The active silver chloride electrode was connected to superfusion solutions in the eyecup via a glass capillary containing Ringer/agar. ERG responses to increasing intensities of illumination were recorded while the preparation was superfused (0.5 ml/min) with skate-modifi ed Ringer’s solution (2) alone, or to which 200 M picrotoxin (to block GABAergic receptors in the retina known to be zinc-sensitive (2)), and then 100 M histidine plus 200 M picrotoxin had been added. We had found that responses in picrotoxin did not increase after the fi rst 10

Responses of small- and large-field bipolar cells to GABA and glycine

Morphologically distinct subtypes of retinal bipolar cells transmit information along parallel pathways to convey different aspects of the visual scene, but the synaptic mechanisms that regulate signal transmission are largely unknown. The all-rod retina of skate provides a comparatively simple system in which to correlate bipolar cell morphology with responses to the inhibitory neurotransmitters GABA and glycine. Two subtypes of bipolar cells can be identified when isolated in culture: large-field bipolar cells with extensive dendritic arbors, and small-field bipolar cells with one or two dendritic branches. Under voltage-clamp, glycine elicited significant current responses from small-field cells, but not from large-field bipolar cells. Although all bipolar cells displayed GABA-activated chloride currents mediated by both GABA(A) and GABA(C) receptors, the small-field bipolar cells showed a significantly greater contribution from GABA(A) receptors. The results of the present study reveal for the first time that the relative expression of the two classes of GABA receptor on each bipolar cell type correlates with cell morphology and the presence of the glycine receptor.