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Hunter College, CUNY, 695 Park Ave., New York, New York 10021
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 µM picrotoxin, superfusion of the zinc chelating agent histidine (100 µM) 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 a neuromodulator in the outer plexiform layer of the vertebrate retina. By acting on the receptor terminal to reduce calcium entry, zinc could serve as a feedback signal to modulate transmitter release (2). If this is the case, one would expect to observe an effect of histidine application on the conductance of second-order cells in the retina of the skate.
We have tested this hypothesis by the use of whole-cell, patch-clamp recordings from horizontal cells in the skate retinal slice preparation. The slices (
200 µm thick) from the all-rod retina of the skate were positioned on a glass slide and visualized using a fixed-stage microscope equipped with a water-immersion objective and Nomarski differential interference contrast optics. Whole-cell patch recordings were obtained under conditions of steady ambient illumination from horizontal cells of the inner nuclear layer, located below the base of the photoreceptors (Fig. 1A). Glass capillary electrodes, pulled to a resistance of 2 to 4 megohms, were filled with a standard skate internal solution (5) and the fluorescent dye, Lucifer yellow (0.3%). In addition, cesium chloride (204 mM) was added to suppress potassium currents. Holding potentials of -40 mV were used, thus avoiding the transient outward currents seen in these cells when they are held at more negative potentials (6). This simplified the analysis of the relationship between membrane conductance and photoreceptor transmitter release. The preparation was superfused with a continuous flow of skate-modified Ringer solution (5) at approximately 1 ml per min. This could be rapidly exchanged with a Ringer solution to which histidine (100 or 500 µM) had been added. The higher concentration provided a faster increase in histidine concentration in the experimental chamber, but the ratios of the current increases measured were found to be independent of drug concentration.
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Data similar to that shown in Figure 1D were obtained from 6 horizontal cells, normalized to the current measured at VC = -120 mV in control Ringer solution, and averaged (Fig. 1E). The inward current increased 42% in histidine at VC = -120 mV; when returned to Ringer, the increment in current was reduced by 72%.
Since the skate horizontal cell has no ligand-gated GABA receptors (7), the well known effect of zinc on these receptors is not relevant, as it is for salamander horizontal cells (2). Glutamate receptors of skate horizontal cells have not been studied, but the possibility that zinc is acting directly on horizontal cells to reduce their permeability seems remote. Retinal horizontal cell glutamate receptors have been identified as AMPA/kainite receptors (8), although metabotropic glutamate receptors have been reported in one case (9). AMPA/kainite receptors studied on neurons elsewhere in the nervous system are generally enhanced by zinc at low concentrations (10,11). Similar observations have been reported for retinal horizontal cells (12), but most studies have shown no effect of zinc on these cells (2,13,14), with one exception, where currents were reduced (15). For example, a zinc concentration of 50 µM—while high enough to block glutamate release from salamander photoreceptors—showed no effect on horizontal cell responses to applied glutamate (2). Similarly, it is important to note that, as a chelating agent, histidine, which has a much higher affinity for Zn2+ than for Ca2+ and is not membrane-permeable, would be expected to reduce, not increase, the ERG response if it were acting directly to reduce calcium entry needed for photoreceptor transmitter release (1).
The skate horizontal cell can serve as a glutamate electrode, monitoring the amount of photoreceptor transmitter released; i.e., an increase in photoreceptor transmitter release will be reflected in an increase in horizontal cell conductance. With these considerations in mind, we interpret the increase in membrane conductance observed in the presence of histidine to represent an increase in photoreceptor transmitter release. We believe that this effect is due to the chelation by histidine of endogenous zinc. Thus, in the presence of histidine, the inhibitory feedback process is suppressed, calcium entry into the receptor terminals is increased, and transmitter release is enhanced.
This mechanism probably represents an important component of "neural" adaptation, comprising processes that are distinct from those governed directly by the bleaching and generation of rhodopsin (16,17). Moreover, it may well provide insight into mechanisms of response dynamics, such as the surround enhancement effects observed with dynamically-modulated spots of light (18,19), as well as phenomena referred to as suppressive rod-cone interaction in amphibians (20), cat (21,22), and man (23).
Supported by NIH Grant EY00777, PSC/CUNY Grants 622450031 and 632130032, as well as by an NIH/RISE (Research Institute for Scientific Enhancement) GM60665 award to Hunter College and by NIGMS grants 2T34 GM07823 (MARC) and R25 GM56945. Research Centers in Minority Institutions award RR-03037 from the National Center for Research Resources of the National Institutes of Health, 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.
Footnotes
1 Ph.D. Program in Biology, The Graduate School and University Center, CUNY, 365 Fifth Ave., New York, NY 10016. 
Literature Cited
This article has been cited by other articles:
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  J. Dufner-Beattie, B. P. Weaver, J. Geiser, M. Bilgen, M. Larson, W. Xu, and G. K. Andrews The mouse acrodermatitis enteropathica gene Slc39a4 (Zip4) is essential for early development and heterozygosity causes hypersensitivity to zinc deficiency Hum. Mol. Genet., June 15, 2007; 16(12): 1391 - 1399. [Abstract] [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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S. Redenti and R. L. Chappell Zinc Chelation Enhances the Zebrafish Retinal ERG b-Wave Biol. Bull., October 1, 2002; 203(2): 200 - 202. [Full Text] [PDF]
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 D.-Q. Zhang, C. Ribelayga, S. C. Mangel, and D. G. McMahon Suppression by Zinc of AMPA Receptor-Mediated Synaptic Transmission in the Retina J Neurophysiol, September 1, 2002; 88(3): 1245 - 1251. [Abstract] [Full Text] [PDF]
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