The Journal of Neurophysiology Vol. 78 No. 5 November 1997, pp. 2402-2412
Copyright ©1997 by the American Physiological Society

GABA Receptors of Bipolar Cells From the Skate Retina: Actions of Zinc on GABA-Mediated Membrane Currents

Haohua Qian^{1, 2}, Lihong Li^{1, 4}, Richard L. Chappell^{1, 4}, and Harris Ripps^{1, 3}

¹ The Marine Biological Laboratories, Woods Hole, Massachusetts 02543; ² Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138; ³ Department of Ophthalmology and Visual Sciences, University of Illinois College of Medicine, Chicago, Illinois 60612; and ⁴ Department of Biological Sciences, Hunter College and Graduate School, City University of New York, New York 10021

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Qian, Haohua, Lihong Li, Richard L. Chappell, and Harris Ripps. GABA receptors of bipolar cells from the skate retina: actions of zinc on GABA-mediated membrane currents. J. Neurophysiol. 78: 2402-2412, 1997. -Aminobutyric acid (GABA)-induced currents were recorded from isolated bipolar cells of the skate retina using perforated patch-clamp methodology. Pharmacological analysis of the responses, using selective agonists and antagonists of the major classes of GABA receptor, revealed the presence of both GABA_A and GABA_C receptors at both the dendrites and axon terminals of the bipolar cells. The two receptor types showed very different reactions to zinc, a divalent metallic cation that was detected in the synaptic terminal region of skate photoreceptors. Currents mediated by the activation of GABA_C receptors were down-regulated by zinc, a feature that is typical of the action of zinc on GABA_C receptors. On the other hand, the effects of zinc on GABA_A receptor-mediated activity was highly dependent on zinc concentration. Unlike theGABA_A receptors on other neurons, responses mediated by activation of the GABA_A receptor of skate bipolar cells were significantly enhanced by zinc concentrations in the range of 0.1-100 µM; at higher concentrations of zinc (>100 µM), response amplitudes were suppressed below control levels. The enhancement of GABA_A receptor activity on skate bipolar cells showed little voltage dependence, suggesting that zinc is acting on the extracellular domain of the GABA_A receptor. In the presence of 10 µM zinc, the dose-response curve for 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; a GABA_A agonist that suppresses GABA_C-activated currents) was shifted to the left of the curve obtained in the absence of zinc, but without a significant change in the response maximum. This finding indicates that the enhancing effect of zinc is due primarily to its ability to increase the sensitivity of the GABA_A receptor. The novel enhancement of neuronal GABA_A receptor activity by zinc, observed previously in the GABA_A-mediated responses of skate Müller (glial) cells, may reflect the presence of a unique subtype of GABA_A receptor on the bipolar and Müller cells of the skate retina.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The light-dependent release of an excitatory neurotransmitter from the synaptic terminals of vertebrate photoreceptors serves to transfer signals from the visual cells to bipolar cells in the distal retina. However, the message conveyed subsequently to more proximal neurons in the visual pathway can be profoundly influenced by the modulation of intercellular communication at pre- and postsynaptic sites (cf. Wu 1994). There is abundant evidence that -aminobutyric acid (GABA), the main inhibitory neurotransmitter in the nervous system, plays a role in this modulatory process (Kaneko and Tachibana 1986; Tachibana and Kaneko 1984, 1988; Wu 1986). Results obtained in a number of vertebrate species have shown that GABA and glutamic acid decarboxylase (GAD; the rate-limiting biosynthetic enzyme for GABA) are contained within various types of amacrine and horizontal cell (Agardh et al. 1987; Hendrickson et al. 1985; Mosinger et al. 1986; Sarthy and Fu 1989; Yazulla 1986), two classes of interneuron whose processes spread laterally within the plexiform layers of the retina. Both cell types make synaptic contacts with bipolar cells (Chun and Wässle 1989; Linberg and Fisher 1988; Marshak and Dowling 1987; Yang and Wu 1991), and there is evidence from recent studies (Feigenspan and Bormann 1994; Lukasiewicz et al. 1994; Matthews et al. 1994; Qian and Dowling 1995) that the GABA-induced responses of bipolar cells are governed by activation of more than one type of GABA receptor(GABAR).

There are at least three classes of membrane receptor mediating neuronal responses to GABA; they have been termed GABA_A, GABA_B, and GABA_C receptors, and are distinguishable by their channel properties and pharmacological profiles. Briefly summarized, GABA_A and GABA_C receptors (GABA_ARs and GABA_CRs) are ligand-gated chloride channels. They differ, however, in that the former are antagonized by bicuculline and allosterically modulated by barbiturates and benzodiazepines (Bormann 1988; Macdonald and Olsen 1994; Olsen 1982; Sivilotti and Nistri 1991), whereasGABA_CRs are bicuculline insensitive, and unresponsive to barbiturates or benzodiazepines (Arakawa and Okada 1988; Qian and Dowling 1993). In addition, responses mediated by GABA_ARs are typically transient in nature; those generated by activation of GABA_CRs are sustained and exhibit slower kinetics. GABA_BRs, on the other hand, are coupled to potassium and/or calcium channels through G-protein and intracellular second-messenger pathways (Bormann 1988; Sivilotti and Nistri 1991), are selectively activated by baclofen, and are antagonized by phaclofen and related compounds (Hill and Bowery 1981; Kerr et al. 1988). Clearly, the type and distribution of GABARs on bipolar cells will play an important role in determining their reactions to GABA, and the ways in which they integrate excitatory and inhibitory inputs.

In the present study, we have attempted to identify, and to characterize pharmacologically, the GABA receptors of bipolar cells isolated from the all-rod retina of the skate. Of particular concern were the effects of zinc, an endogenous heavy metal that has been shown to be an important modulator of GABA-induced currents in a broad range of neuronal cells (Smart et al. 1994). In an earlier study we found that the GABA_AR-mediated responses of skate Müller (glial) cells were greatly enhanced by low concentrations of zinc (Qian et al. 1996a). In this study we investigated whether similar mechanisms exist in retinal neurons, because this unusual finding had not been reported previously for neuronal preparations from retina or other regions of the vertebrate CNS. Our results provide evidence that the GABA_ARs of skate bipolar cells display a similar phenomenon. Moreover, we have found that both GABA_A and GABA_C receptors are present on skate bipolar cells, but zinc acts very differently on the GABA-mediated currents of the two receptor types. Some of these findings were presented at the annual meeting of the Association for Research in Vision and Ophthalmology (Chappell et al. 1995).

	METHODS

Abstract Introduction Methods Results Discussion References

Cell isolation

Isolated cells were enzymatically dissociated from the skate (Raja erinacea and R. ocellata) retina as described previously (Malchow and Ripps 1990). Briefly, eyes enucleated from anesthetized (0.02% 3-aminobenzoic acid ethyl ester, Tricaine), pithed animals, were rinsed in 70% ethanol, and hemisected. Pieces of neural retina were dissected free of the posterior eyecup, and incubated for 50 min in a modified (Malchow and Ripps 1990) culture medium (L-15, GIBCO, Grand Island, NY) containing 1.8 mg/ml papain (Calbiochem, La Jolla, CA) activated with 1 mg/mlL-cysteine. The tissue was thoroughly rinsed in an enzyme-free modified L-15 solution, triturated through a sterile pipette into conical tubes, and allowed a few minutes to sediment. Isolated cells in aliquots of the supernatant were plated on plastic culture dishes and stored (1-3 days) at 14°C. Before use, the medium was replaced with an elasmobranch Ringer solution that contained (in mM) 250 NaCl, 6 KCl, 1 MgCl₂, 4 CaCl₂, 360 urea, 10 glucose, a n d  5  N-2-h y d r o x y e t h y l p i p e r a z i n e-N-2-e t h a n e s u l f o n i c  a c i d(HEPES), titrated to pH 7.6 with NaOH. Because we have not explored fully the question of whether GABA_B receptors are present on skate bipolar cells, barium (10 mM) was added to the Ringer solution to suppress potassium and calcium currents; preliminary experiments in which barium was omitted from the bath solution gave similar results.

Bipolar cells were identified by their characteristic morphology (see RESULTS). It should be noted, however, that two immunochemically distinct types of bipolar cell have been identified in the intact skate retina (Schlemermeyer and Chappell 1996), and a greater number of morphologically different forms are seen in Golgi preparations (cf. Fig. 1A). We were unable to reliably distinguish one from the other when the cells were isolated, and no attempt was made to classify the type of bipolar cell from which recordings were obtained. In most respects, cells that were successfully patched gave qualitatively similar current responses for a wide range of experimental conditions; e.g., GABA invariably evoked outward currents in cells voltage clamped at 0 mV. However, the variability in response amplitude to identical concentrations of receptor agonist (cf. Figs. 2C and 8), may reflect the response characteristics of functionally different cell types, or simply the relative numbers of receptors retained after cell isolation.

View larger version (92K): [in this window] [in a new window]   FIG. 1. Cellular morphology of neurons and glia of the skate retina. A: camera lucida drawings of various bipolar cells seen in Golgi-impregnated retinal sections. Images were obtained from several slides, and markers indicate the approximate locations of the outer plexiform layer (OPL), inner nuclear layer (INL), and inner plexiform layer (IPL). B: examples of solitary bipolar cells enzymatically dissociated from skate retina; the cells had been in culture for 1 day. C and D: other cell types seen in the culture dish show the ease with which bipolar cells can be distinguished from Müller cells (C), and an internal horizontal cell with several photoreceptors (D). E: using the Timm's silver-sulfide method for zinc detection, a band of reaction product was localized near the terminals of the skate photoreceptors.

View larger version (19K): [in this window] [in a new window]   FIG. 2. -Aminobutyric acid (GABA)-mediated responses recorded from skate bipolar cells using the perforated-patch (amphotericin B) technique reveal the presence of GABAA and GABAC receptors. A: with the cell voltage clamped at 0 mV, GABA-induced outward currents grew in amplitude and became progressively more transient as the drug concentration was increased. B: coapplication of 100 µM bicuculline removed a transient component from the response to 100 µM GABA, leaving the more sustained, bicuculline-insensitive current mediated by the GABAC receptor. C: normalized responses to increasing concentrations of GABA () were fit by the Hill equation with an EC50 of 7.8 µM and a Hill coefficient of 1.2 (n = 5). Normalized responses to the same concentrations of GABA when coapplied with 500 µM bicuculline () produced a shift in the dose-response curve; i.e., the bicuculline-insensitive current was also well described by a Hill equation of similar slope (Hill coefficient = 1.3), but with an EC50 of 1.6 µM (n = 8). Error bars: ±SE.

View larger version (15K): [in this window] [in a new window]   FIG. 8. Effect of zinc on the dose-response curve of GABAAR -mediated responses elicited by increasing concentrations of THIP before and after enhancement by zinc. Normalized responses (from 6 cells) as a function of THIP concentration (control, ) were fit by the Hill equation with an EC50 at 230 µM, and a Hill coefficient of 1.14. The Hill equation also adequately described the THIP-induced responses (from another 6 cells) in the presence of 10 µM zinc (); response enhancement by zinc resulted in a lowering of the EC50 to 58 µM, but had no effect on the Hill coefficient (1.11). Error bars: SE.

Golgi (silver impregnation)

Pieces of eyecup were fixed for 1 h in 2.5% glutaraldehyde in 0.05 M sodium phosphate buffer, and then processed by the Golgi triple impregnation method as modified by Stell (1965). After silver impregnation, the tissue was embedded in collodion, sectioned at 90 µm, and mounted on glass slides. Individual, well-impregnated bipolar cells were viewed by light microscopy under a ×40 oil-immersion objective, and a two-dimensional pen-and-ink image of the cell was created with the aid of a camera lucida attachment. Silver did not always fill the cells fully, but it was possible in many instances to reconstruct the dendritic branches, and to visualize the bulbous axon terminal and identify the lamina to which it projected.

Zinc histochemistry

To visualize at the light-microscopic level the cellular distribution of zinc in retinal sections, we used a modified version of the sulfide silver method (neo-Timm) for cryosectioned tissue (cf. Danscher 1981). Pieces of skate eyecup were fixed by immersion for 3 min in 0.1% sodium sulfide (Na₂S) in 0.1 M phosphate buffer, pH 7.4, and then transferred to a solution containing 3.5% glutaraldehyde in 0.1 M phosphate buffer. After 3 min in the fixative, the tissue was placed in fresh sulfide solution for 10 min, and fixation was completed by submersion in the glutaraldehyde mixture for 1 h. The tissue was rinsed in 0.1 M phosphate buffer (3 × 15 min), cryoprotected overnight in a 30% sucrose solution at room temperature, coated with OCT (Miles, Elkhart, IN), and sectioned on a 20°C cryostat at 20 µm. The sections were thaw-mounted on gelatin-subbed slides, dried, coated with 0.5% gelatin, and allowed to dry for >1 h before physical development. The developer consisted of a colloid mixture of gum arabic, sodium citrate buffer containing hydroquinone (as reducing agent), and silver lactate (cf. Danscher 1981). The slides were developed in a staining jar in a 26°C light-tight water bath for 2.5 h, rinsed in running tap water at 40°C for at least 45 min, and briefly in distilled water. They were then dehydrated in graded ethanols, cleared in xylene, coverslipped in Permount (Fisher Scientific, Itaska, IL), and photographed under Nomarski optics.

Electrophysiology

Membrane currents were recorded under voltage clamp using the amphotericin perforated-patch technique devised by Rae et al. (1991). Despite a higher access resistance than achieved with conventional whole cell recording methods, the perforated patch severely restricts perfusion of the cell cytoplasm and thereby reduces "rundown" due to loss of multivalent ions and small molecules involved in intracellular signaling. Patch pipettes with tip diameters of ~1 µm were drawn on a two-stage electrode puller and used either with or without flame polishing. The electrodes were filled by capillary action to ~500 µm from the orifice with a solution containing (in mM) 191 CsAcetate, 13 KCl, 11 ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA), 1 CaCl₂, 1 mM MgCl₂, and 10 mM HEPES titrated to pH 7.6 with KOH; they were then backfilled with a similar solution that contained amphotericin B (240 µg/ml) prepared just before use. Although amphotericin has a low permeability to chloride, the chloride concentration in the pipette was made to approximate the intracellular concentration determined previously (Chappell et al. 1992); this served to reduce response degradation due to slow chloride permeation through the patch. With gentle suction, a small patch of membrane was drawn into the micropipette tip as it formed a gigaohm seal with the surrounding cell surface. Amphotericin partitioned across the membrane in <5 min, judging by the growth of GABA-induced currents, and subsequent response stability. The typical access resistance (uncompensated) was ~20 M; without compensation, a response of 200 pA would produce a 4-mV drop across the electrode, and result in a 6% error at a chloride driving force of 70 mV. Recordings of membrane currents were made with an Axopatch 200A integrating amplifier (Axon Instruments, Burlingame, CA) controlled by pClamp 6 software programs; the responses were stored on computer disks for subsequent analysis. For most pharmacological experiments, the cells were voltage clamped at 0 mV, considerably more positive than the typical resting membrane potential of bipolar cells, e.g., 21 to 50 mV (cf. Dowling 1987; Kaneko and Saito 1983; Saito et al. 1979; Shiells and Falk 1992); a holding potential of 0 mV was chosen primarily to elicit more robust GABA-mediated currents. Qualitatively similar results were obtained when bipolar cells were clamped to negative potentials, but there was a progressive decline in response amplitude as the chloride equilibrium potential was approached. Ascending and descending voltage ramps (250 mV/s) were used to obtain current-voltage relations, and for pairwise comparison of GABA-mediated responses with those obtained in the presence of a test drug.

Drug delivery

Studies were conducted at room temperature with the culture dish mounted on the stage of an inverted microscope. Cells were viewed at ×400 with Hoffman modulation-contrast optics, and superfused with solutions applied via a gravity-driven system controlled by a manifold of solenoid-activated valves. The system accommodated 20 solutions, and the switching time for solution exchange was 10-15 ms under either manual or computer control. Drugs were dissolved in the elasmobranch Ringer solution and delivered by a multibarrel pipette located 300-500 µm from the cell; the solution was removed by gentle suction. We usually allowed a 1-min interval between drug applications, and each experimental run (on a given cell) consisted of multiple sequential applications of test and control solutions to ensure the repeatibility of the drug effects.

Dose-response data were fit to the Hill equation where I_[C] is the cell membrane current elicited by a given drug concentration [C], I_max is the maximum current response of the cell, n is the Hill coefficient, and [EC₅₀] is the drug concentration that gives rise to a half-maximal response.

To identify the receptor type(s) at the distal and proximal ends of the bipolar cell, responses were recorded when drugs were delivered by pressure ejection (Picospritzer IID, General Valve, Fairfield, NJ) using small-bore pipettes placed at selected loci along the radial extent of the cell; the drug-delivery pipette and a suction-withdrawal pipette were aligned on opposite sides of the test location. This arrangement produced a laminar flow that could be directed either to the dendritic region or to the axon terminal (cf. Tachibana and Kaneko 1988) and effectively prevented the diffusion of test agents to the other end of the cell; drug localization was confirmed in preliminary experiments in which colored vegetable dye was added to the superfusate, and the fluid stream visualized with a video-based system. Except for 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) hydrochloride, bicuculline methchloride, and diazepam, obtained from Research Biochemicals International (Natick, MA), the chemicals used in the perfusates and pipettes were purchased from Sigma Chemical (St. Louis, MO).

	RESULTS

Abstract Introduction Methods Results Discussion References

Bipolar cell morphology

Although the bipolar cells of most vertebrate species display a number of distinguishing features, they assume a variety of shapes and sizes in situ (Kolb et al. 1992; Linberg et al. 1996; Shkolnik-Yarros and Podugolnikova 1978). This is readily apparent in the camera lucida drawings (Fig. 1A) of Golgi images of skate bipolar cells. The dendritic arborization that lies within the outer plexifiorm layer is of variable extent and complexity, and the axonal endings terminate either distally or proximally within the inner plexiform layer (IPL); the distal and proximal loci of the synaptic terminals probably correspond to the serotonin-accumulating and protein kinase C-reactive cells, respectively, described by Schlemermeyer and Chappell (1996).

Examples of some of the cell types that are identifiable after enzymatic dissociation of the skate retina are shown in Fig. 1, B-D. Isolated bipolar cells (Fig. 1B) exhibit features similar to those seen in the Golgi images, although many of the fine branches appear to have been lost in the dissociation procedure. Note also that the length of the bipolar cell, which projects only from the photoreceptor terminal to the inner plexiform (synaptic) layer, is often greater than that of the Müller cell (Fig. 1C), which extends radially from the innermost border of the retina to the photoreceptor nuclei in the distal retina. The disparity is due most likely to the loss of Müller cell processes as well as to the oblique course usually followed by the bipolar cell axons in situ. Several other forms of bipolar cell were seen in culture, but it was not possible to determine whether morphological differences represented different subtypes, or were artifacts of the isolation procedure. Solitary horizontal cells and rod photoreceptors (Fig. 1D) are also easy to identify, but amacrine and ganglion cells (not shown) cannot be distinguished except by dye marking or by electrophysiological procedures.

The localization of zinc within the skate retina was not grossly different from that shown previously in tiger salamander (Wu et al. 1993). With the use of a similar method (Danscher 1981), a band of reaction product was seen near the terminals of skate photoreceptors (Fig. 1E), but the level of resolution was not suffucient to determine whether the deposits extended to the synaptic junctions and whether they were contained in synaptic vesicles. Nevertheless, it is clear that zinc is present in the glutamatergic photoreceptors of the skate retina.

Membrane currents in response to GABA

Figure 2A shows a series of perforated-patch recordings of GABA-induced currents from a bipolar cell voltage clamped at 0 mV. With low concentrations of GABA (10 µM), the outward currents were sustained during drug application. In response to higher concentrations of GABA (>30 µM), the current traces appeared to fall exponentially toward an asymptotic level during the course of drug application. A graph of the normalized dose-response data for the peaks of the GABA-evoked current recordings from five cells (Fig. 2C, ) gives rise to a typical sigmoid curve with a Hill coefficient (n) of 1.2 and a half saturation value (EC₅₀) of 7.78 µM. Picrotoxin (100 µM) completely blocked the GABA-induced responses (data not shown), in agreement with earlier results indicating that the GABA currents of skate bipolar cells are mediated by chloride ions (Chappell et al. 1992; Malchow et al. 1991).

GABA activates GABA_A and GABA_C receptors

The currents induced by GABA concentrations of 30 and 100 µM shown in Fig. 2A appear to be comprised of two components: an initial transient response, followed by a more sustained response of lesser amplitude. The fact that there are two underlying components to the response can be better appreciated by observing the effects of bicuculline on the GABA-induced currents. As shown in Fig. 2B, a significant fraction of the current response to 100 µM GABA was suppressed by coapplication of 100 µM bicuculline, indicating the presence of both GABA_A and GABA_C receptors on skate bipolar cells. The normalized dose-response curve for the bicuculline-insensitive GABA_C-mediated currents recorded from eight cells (Fig. 2C, ) was well described by a sigmoidal function with a Hill coefficient of 1.35 and an EC₅₀ of ~1.6 µM.

A further indication of the nature of the receptors underlying the GABA-induced currents is illustrated by the effects of THIP, a potent GABA_AR agonist that suppressesGABA_CR activity (Kusama et al. 1993; Qian and Dowling 1994; Woodward et al. 1993). Application of 100 µM THIP elicited an outward current that was enhanced by 10 µM diazepam (Fig. 3A), and completely abolished by 100 µM bicuculline (Fig. 3B). On the other hand, the GABA_C-mediated currents elicited by the coapplication of GABA and bicuculline were not significantly affected by diazepam; the drug either had no effect, or produced a small decrease in response amplitude (Fig. 3C). These findings are a good indication that the GABA-mediated responses of skate bipolar cells involve activation of both GABA_A and GABA_C receptors.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Differential responses of GABAA and GABAC receptors on skate bipolar cells. A: the GABAA-mediated response induced by 200 µM 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) was enhanced by coapplication of 10 mM diazepam ( = +33 ± 5% SE, n = 5). Unless indicated otherwise, recordings shown here and in subsequent figures were obtained from cells voltage clamped at 0 mV. B: the response to 200 µM THIP is blocked by the addition of 500 µM bicuculline. C: the bicuculline-insensitive GABAC response, induced by coapplication of 10 µM GABA and 500 µM bicuculline, is not enhanced by the addition of 10 µM diazepam. Scale bars: vertical axis is 50 pA for A and B; 20 pA for C. Horizontal axis is 2 s. Duration of drug application is indicated by the horizontal bar under each response. The experiments shown in B and C were repeated on 5 cells with similar results.

Distribution of GABARs on skate bipolar cells

Owing to the different response properties of the GABA_A and GABA_C receptors (Qian and Dowling 1993), it is important to determine whether one or another type is restricted to either the dendritic or axonal region of the bipolar cell. As already mentioned, bicuculline blocks selectively the GABA_A receptors of skate bipolar cells, but has no apparent effect on the GABA_C receptor. Consequently, applying GABA should activate both GABA_A and GABA_C receptors, whereas application of GABA to cells bathed in a Ringer solution containing 100 µM bicuculline will elicit responses that are mediated predominantly by activation of the GABA_C receptor.

Figure 4 shows that current responses displaying both transient and sustained components were elicited when 100 µM GABA was puffed either at the dendrites or the axon terminals of the bipolar cell (the GABA responses in Ringer). At both sites, the transient components of the responses were blocked when GABA was delivered in the presence of 100 µM bicuculline, indicating that both GABA_A and GABA_C receptors are present on the dendrites and axon terminals of skate bipolar cells. Although the magnitudes of the currents were typically smaller at the cell terminals, the likelihood that there are changes in receptor composition and distribution, as well as the loss of dendritic and axonal processes due to the cell isolation procedure, precludes analysis of the proportion of receptor types at the two loci.

View larger version (12K): [in this window] [in a new window]   FIG. 4. GABAA and GABAC receptors are on the dendrites and axon terminals of skate bipolar cells. GABA (100 µM), applied alone or in the presence of 100 µM bicuculline, elicited outward currents both from the dendrites (top traces) and the axon terminal region (bottom traces). Recordings at the left represent the GABA-mediated responses that include contributions from both GABAA and GABAC receptors; those at the right reflect activation of the bicuculline-insensitive GABAC receptors. Comparable results were obtained from 7 bipolar cells.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Differential effects of zinc on GABAAR- and GABACR-mediated currents. A: the current evoked by 10 µM GABA is greatly enhanced by the addition of 10 µM zinc, but no response was elicited by the application of 10 µM zinc alone. Horizontal bar indicates duration of drug application. B: the GABACR response, isolated by adding 100 µM bicuculline to the test solution, was reduced by 10 µM zinc. C: the GABAAR current elicited by application of 100 µM THIP was enhanced by the addition of 10 µM zinc to the test solution. D: the dose-response relation shows the effect of various zinc concentrations on the relative amplitude of the current response to 100 µM THIP; zero on the scale of ordinates represents the response elicited by 100 µM THIP in the absence of zinc. In the range of 0.3-100 µM, responses increased almost linearly as a function of zinc concentration; above 100 µM, responses were depressed to below control level. Error bars: ±SE(n = 6).

Differential effects of zinc on GABA_A and GABA_C receptors

Perhaps the most significant finding that distinguishes the results obtained on the GABA_A receptors of skate bipolar cells from those reported for GABA_ARs of other neurons, relates to the effects of zinc on the GABA-induced currents. Although zinc alone had no effect on membrane current (see DISCUSSION), Fig. 5A shows that the current elicited by 10 µM GABA was greatly enhanced when 10 µM Zn²⁺ was coapplied with the GABA solution; a similar enhancement was observed when cells were bathed in zinc before coapplication of zinc and GABA.

The recordings in Fig. 5, B and C, provide evidence that the response enhancement by zinc was due solely to its effect on the GABA_A receptor. After adding 100 µM bicuculline to the GABA solution, thereby leaving the GABA_C response intact, the low concentration of zinc depressed the GABA-evoked current by ~50% (Fig. 5B). On the other hand, Fig. 5C shows that the current elicited by the GABA_A-selective agonist THIP (100 µM) was enhanced by the addition of zinc. Also noteworthy is the fact that responses elicited by low concentrations of GABA (Fig. 5A) or THIP (Fig. 5C) were relatively well sustained during drug application, but exhibited a large transient component (desensitization) when the GABA-mediated current was enhanced by zinc. This phenomenon can be attributed to the enhancement of GABA_A-receptor sensitivity by zinc (see below). The concentration dependence of the zinc effect on the THIP-induced currents, i.e., responses mediated by GABA_A receptors, is illustrated in Fig. 5D. With zinc concentrations up to ~100 µM, the responses to 100 µM THIP were enhanced in a dose-dependent fashion; at the maximum, the amplitude was ~45% greater than control. At higher concentrations of zinc, the THIP-induced currents were markedly reduced, with about an 80% loss of amplitude when superfused with 1 mm zinc.

Zinc alters the kinetics of the GABA-induced currents

The responses mediated by activation of GABA_A andGABA_C receptors exhibit very different kinetic properties. This is readily seen in the OFF phase of the drug responsesshown in Fig. 3, B and C, i.e., the responses to THIP(GABA_A) and GABA + bicuculline (GABA_C), respectively.The GABA-induced current mediated by GABA_C receptors returned to baseline at a much slower rate than that resulting from activation of GABA_A receptors. Although this feature has been noted previously (Amin and Weiss 1994; Qian and Dowling 1993, 1994), the results shown here represent the first demonstration of the differences in response dynamics of these classes of receptor on an individual neuron.

The observation that low concentrations of zinc exert opposite effects on the two types of GABA receptor raised the possibility that the action of zinc might affect the dynamics of the natural response to GABA, i.e., when both GABA_A and GABA_C receptors contribute to the response. To examine this possibility, we analyzed the time course of the falling phase of the responses at the termination of various drug applications. With the responses normalized to the amplitudes of the current recorded immediately before offset of the zinc/drug combinations, 10 µM zinc appeared to have no effect on the kinetics of either the GABA_C or GABA_A receptor-mediated response when tested individually (Fig. 6,A and B, respectively). Nevertheless, 10 µM zinc greatly accelerated the OFF response to 10 µM GABA (Fig. 6C).

View larger version (26K): [in this window] [in a new window]   FIG. 6. Zinc affects the decay kinetics of GABA-mediated responses. A: normalized current recordings during the decay phase of the GABACR responses (elicited by coapplication of 10 µM GABA plus 100 µM bicuculline) show no difference in time course before and after the addition of 10 µM zinc. Each response in this and subsequent recordings has been normalized to the current level reached immediately before the offset of drug application (indicated by arrowheads). B: GABAAR-mediated currents, recorded during the decay phase of the response to 100 µM THIP, also were not affected by the addition of 10 µM zinc. C: the decay phase of the response to 10 µM GABA (activating both GABAARs and GABACRs) fell to baseline at a much faster rate when 10 µM zinc was added to the test solution. D: normalizing the responses to THIP, GABA + bicuculline, and GABA (i.e., currents mediated by activation of the GABAAR, the GABACR, and the combined action of the 2 receptors, respectively) illustrates the fact that the rate of decay associated with the termination of the response to 10 µM GABA was intermediate between the slow response due to the GABACR and the faster response of the GABAAR. E: bar graphs summarizing the half-decay times for the conditions shown in A-D (error bars: SE; n = 5). See text for details.

Although seemingly paradoxical, the pharmacological basis for the zinc-induced acceleration in the response dynamics of the GABA-mediated current is seen in Fig. 6D.As already mentioned, both the rise and fall times of theGABA_A-induced currents are more rapid than those resulting from activation of GABA_C receptors. Because zinc enhances the GABA_A component of the total GABA response, while inhibiting the contribution from the GABA_C component, the proportion of the more rapid GABA_A response will be greatly increased in the presence of zinc, thereby accelerating the kinetics of the OFF response to GABA. The bar graphs in Fig. 6E show the half-decay times of responses to the various drug combinations, and the final pair illustrate quantitatively the significant reduction induced by zinc (>50%) in the t_1/2 of the GABA-induced response (P < 0.005).

Zinc enhancement of the GABA_A current is not voltage dependent

The enhancement of GABA_A receptor activity by zinc was not dependent on the membrane potential of the bipolar cell. Figure 7A shows voltage-ramp recordings of the current-voltage relation obtained in the presence of 200 µM THIP, and then after the addition of 10 µM zinc. For both conditions, the current reversed at about 60 mV and showed outward rectification owing to an imbalance in the chloride concentration across the cell membrane. However, the current was always greater in the presence of zinc, and as shown in Fig. 7B the response ratio, i.e., the relative enhancement by zinc, remained relatively constant with membrane voltages of 50 to +50 mV, the range over which reliable measurements could be obtained. Nevertheless, it is apparent that there is a gradual decline in the response ratio with increasing depolarization. This small degree of voltage dependence in the zinc effect was seen rather consistently and is due most likely to the nonlinear effect on response amplitude of the electrode access resistance (see METHODS). Specifically, the reduction in magnitude of the recorded current as a function of membrane voltage will be disproportionately greater at more depolarized membrane potentials; this will be even more pronounced for the larger currents elicited in the presence of zinc, and result in a small but consistent decline in the response ratio.

View larger version (17K): [in this window] [in a new window]   FIG. 7. Zinc enhancement of GABAAR-mediated currents is not voltage sensitive. A: currents recorded during voltage ramps with the bipolar cell in THIP (200 µM) and in THIP (200 µM) + zinc (10 µM). The reversal potential of the responses for this cell was 58 mV. B: the ratio of the current measured in THIP + zinc to that measured in THIP alone (from the ramp data in A) was relatively constant over the range from 50 to +50 mV. Similar results were obtained in 5 experimental runs.

Zinc shifts the GABA_A sensitivity curve

The normalized dose-response data for the GABA_A receptor obtained from measurements of the peak amplitude of the THIP-evoked currents (Fig. 8, ) were fit by a Hill equation with a coefficient n = 1.14 and an EC₅₀ of 230 µM. In the presence of 10 µM zinc, the THIP-induced responses from the same cells (normalized to the maximum of the THIP control) could be described by an almost identical function (Hill coefficient = 1.1), but with a pronounced shift in the dose-response curve toward the left on the scale of abscissae, i.e., a half saturation value of 58 µM (Fig. 8, ). These findings indicate that low concentrations of zinc increased by about fourfold the sensitivity of the GABA_A receptor, but had little effect on the response maximum or the Hill coefficient.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Zinc modulation of GABA-induced membrane currents

The present study was designed to test whether a GABA-sensitive retinal neuron, i.e., the bipolar cell, displays the unique pharmacology reported for GABA_A receptors of skate Müller (glial) cells, namely, a significant enhancement of GABA-mediated responses by low concentrations of zinc (Qian et al. 1996a). The results presented in Fig. 5 show that this is clearly the case; the currents induced both by GABA and the GABA_A agonist THIP were greatly enhanced by coapplication of 10 µM zinc. There were, however, notable differences in the two sets of data. Unlike the recordings obtained from Müller cells, zinc alone did not elicit a current response from skate bipolar cells (Fig. 5A). Moreover, only a single class of receptor (GABA_AR) appeared to be present on the Müller cell membrane (Malchow et al. 1989; Qian et al. 1996a), whereas the GABA-induced currents recorded from enzymatically isolated skate bipolar cells were mediated by activation of both GABA_A and GABA_C receptors (Figs. 2 and 3). In contrast to its effect on the GABA_AR, zinc reduced the GABA_C-mediated response (Fig. 5B), a finding similar to that reported by Qian and Dowling (1995) for the GABA_CRs of hybrid bass bipolar cells.

The enhancement by zinc of GABA_AR-mediated currents in retinal bipolar cells is perhaps the first unequivocal demonstration of this unusual phenomenon in a neuronal cell. Although there have been reports suggesting that zinc may enhance GABA-induced responses (Smart and Constanti 1983; Smart et al. 1994), interpretation of the data has been difficult, and it has not been possible to ensure that the effects resulted from a direct action on the GABAR complex. For example, relatively high concentrations of zinc have been shown to elicit periodic giant depolarizing potentials in pyramidal neurons of the rat hippocampus, but the effect was ascribed to a zinc-induced release of GABA (Xie and Smart 1993). Also noteworthy is the fact that 300-500 µM zinc enhanced the GABA-induced depolarizing voltage responses from rat olfactory cortex to iontophoretically applied GABA, but the enhancement appeared to be a consequence of a reduction in the cell's input conductance, rather than a direct effect on the GABA_AR (Smart and Constanti 1990). In other studies in which zinc modulation of neuronal GABA receptors has been studied, zinc either had no effect, or down-regulated the GABA-induced responses (Akaike et al. 1987; Celentano et al. 1991; Dong and Werblin 1995; Qian and Dowling 1995; Westbrook and Mayer 1987).

Similar to the results described previously for skate Müller cells (Qian et al. 1996b), an inhibitory action of zinc on the GABA_AR-mediated activity of bipolar cells could be revealed by using zinc concentrations in excess of ~100 µM (Fig. 5D). This observation, together with results indicating that the modulatory effects of zinc are relatively independent of voltage (Fig. 7B), suggests the possibility that zinc may interact with the GABA_AR at two external membrane binding sites, with very different affinities for zinc (Qian et al. 1996b). Although we have not explored this concept in the present study, the data obtained from skate Müller cells indicate that the high affinity site, activated at low concentrations of zinc, gives rise to enhancement of the GABA_A-induced current, whereas the low affinity site requires high concentrations of zinc to produce its inhibitory effect.

The zinc content of the retina is among the highest of the body tissues, and zinc deficiency has been suggested as a factor in a wide range of ocular disorders (cf. Newsome et al. 1988). Nevertheless, with regard to its putative modulatory role, it is important to stress that the question of whether zinc is released from the synaptic terminals of the visual cells has yet to be resolved. There is mounting evidence that zinc is associated with glutamatergic axon terminals in other regions of the CNS, and that discharge and uptake can be detected under appropriate experimental conditions (cf. Beaulieu et al. 1992; Harrison and Gibbons 1994). However, histochemical localization within photoreceptors (Fig. 1E) does not provide information relevent to the question of whether it undergoes release from this site, particularly in view of the fact that an endogenous source of zinc has been implicated in maintaining the structural and functional integrity of the rod photoreceptor (Karpen et al. 1993; Shuster et al. 1988, 1992). In this connection, it should be recalled that zinc is essential to the catalytic action of many enzymes, is involved in maintaining the stability and structure of proteins and subcellular organelles, and is indispensable for the transcription and translation of the genetic message (cf. Vallee and Auld 1990). Thus, in addition to studies on the modulatory effects of zinc, there is clearly the need to determine 1) the subcellular localization of zinc within photoreceptor terminals, and 2) whether release and uptake can be demonstrated under conditions that induce discharge and reformation of synaptic vesicles (cf. Ripps and Chappell 1991).

GABA_AR- and GABA_CR-mediated currents of skate biplar cells

The presence of more than one type of GABAR on the bipolar cell membrane is hardly surprising. Retinal bipolar cells in mammalian and other vertebrate species invariably express more than one class of GABAR (Feigenspan and Bormann 1994; Heidelberger and Matthews 1991; Lukasiewicz et al. 1994; Matthews et al. 1994; Qian and Dowling 1995). It becomes necessary, therefore, to isolate the currents mediated by each type to assess individually their pharmacological properties. In the case of GABA_A and GABA_C receptors, THIP and bicuculline are particularly useful agents. THIP is a selective agonist of GABA_ARs (Aprison and Lipkowitz 1989; Krogsgaard-Larsen and Johnston 1978) that exerts an antagonistic effect on GABA_C-gated currents (Kusama et al. 1993; Qian and Dowling 1994; Woodward et al. 1993). Bicuculline, on the other hand, selectively blocks the activity of the GABA_AR but has little affect on the GABA_CR (Qian and Dowling 1993; Wang et al. 1994). Both of these drugs, in various concentrations and combinations, were used extensively in the course of this study.

With regard to some of the pharmacological properties tested, the behavior of the two receptor types was consistent with results obtained in earlier studies. As with GABA_ARs on other neurons, the chloride-mediated currents gated by the GABA_ARs of skate bipolar cells are blocked by bicuculline and picrotoxin, and can be enhanced by benzodiazepines (Fig. 3, A and B). On the other hand, the GABA_CR of skate bipolar cells exhibits many of the same properties as the GABA_CRs of bipolar cells in rat (Bormann and Feigenspan 1995; Feigenspan et al. 1993), white perch (Qian and Dowling 1993), salamander (Lukasiewicz et al. 1994), and hybrid bass (Qian and Dowling 1994) retinae, notably, its insensitivity to bicuculline (Fig. 2B), the sustained nature of the GABA-induced current (Fig. 3C), its slow recovery at the termination of a GABA pulse (Fig. 6A), and the low EC₅₀ (1.6 µM) of the dose-response curve to coapplication of GABA and bicuculline (Fig. 2C, ).

It is difficult to assess accurately the relative sensitivities of the currents mediated by the two receptor types. The problem lies in determining the direct effect of GABA on the current response of the GABA_A receptor. Many of the available antagonists of the GABA_CR (e.g., I4AA), in concentrations adequate for block of the GABA_CR, also act as weak agonists of the GABA_AR (Bowery and Jones 1976; Kemp et al. 1986; Thomas and Prell 1995), whereas agonists specific for the GABA_AR such as THIP and related compounds have lower affinities than GABA for the GABA_AR-binding site (Aprison and Lipkowitz 1989; Krogsgaard-Larsen and Johnston 1978). Nevertheless, a rough indication of the relative sensitivities of the two receptor types can be obtained by comparing the dose-response relation of the bicuculline-insensitive GABA_C-mediated current (Fig. 2C, ) with that obtained in the presence of the bicuculline-sensitive GABA_AR current (Fig. 2C, ), i.e., the overall GABA response (cf. Feigenspan and Bormann 1994). Under the latter conditions, the dose-response curve yielded an EC₅₀ value of 7.8 µM and a Hill coefficient of 1.2, whereas the GABA_C current was described by a Hill equation with an EC₅₀ value of 1.6 µM and a coefficient of 1.35. Thus the presence of currents contributed by the GABA_AR to the GABA-mediated responses shifted the EC₅₀ of the dose-response relation to a significantly higher value, but had little effect on the Hill coefficient. These results provide a good indication that the sensitivity for GABA of theGABA_CR is at least five times greater than that of theGABA_AR, but that the two receptor types display a similar degree of cooperativity for agonist binding (Feigenspan and Bormann 1994).

Distribution of GABARs on skate bipolar cells

The finding that both the dendritic branches and axon terminals of skate bipolar cells contain GABA receptors (Fig. 4) is consistent with observations on the GABA-mediated responses of mouse (Suzuki et al. 1990), goldfish (Tachibana and Kaneko 1988), and hybrid bass bipolar cells (Qian and Dowling 1995), and suggests that the bipolar cells of these species can receive feed-foward inhibitory signals from horizontal cells and a feedback synapse from amacrine cells. Both of these GABAergic interneurons may participate in organizing the center-surround antagonistic receptive fields of bipolar and ganglion cells. However, the precise nature of the interplay between excitatory and inhibitory inputs that leads to this important functional property has yet to be established, and it is evident that signals from horizontal cells to photoreceptors, and from amacrine to ganglion cells may contribute to the process (cf. Tachibana and Kaneko 1984; Wu 1994).

Bipolar cells occupy a key position in the retinotectal pathway, linking the percipient elements with neurons that carry the visual message to the CNS. In skate, as well as in hybrid bass (Qian and Dowling 1995), GABA_ARs and GABA_CRs are present on the dendrites and axon terminals of the bipolar cells, but the significance of having at these loci two types of GABAR, both of which gate chloride conductances, has yet to be elucidated. This arrangement clearly presents the opportunity to selectively regulate the dynamics of GABA-mediated inhibition by virtue of the differences in the sensitivities of the GABA_A and GABA_C receptors (Fig. 2), in the effects of neuromodulators (e.g., zinc; Fig. 5), and in the characteristics of the responses mediated by the two receptor types (Fig. 3). Indeed, studies in the amphibian retina (Zhang and Slaughter 1995) indicate that activation of GABA_CRs suppress selectively the ON (depolarizing) pathway to amacrine and ganglion cells, presumably via a presynaptic mechanism, i.e., a reduction in bipolar cell input due to shunting at the synaptic terminal by the GABA_C-mediated current (cf. Feigenspan et al. 1993; Lukasiewicz and Werblin 1994), and a concomitant inhibition of calcium-induced transmitter release (Wellis and Werblin 1995). On the other hand, the differential effects of zinc suggest that its modulatory role in the distal retina may be to enhance the transient inhibitory response mediated by GABA_ARs, and suppress the sustained inhibitory effect on bipolar cell activity resulting from activation of GABA_CRs (Qian and Dowling 1995).

Molecular characterization of GABARs

Aside from the general features imparted by receptor type, it is important to recognize that the functional properties of membrane channel proteins are governed in large part by their subunit composition (Verdoorn 1994; Verdoorn et al. 1990). There is increasing evidence indicating that theGABA_C receptors in the vertebrate retina are formed by GABA -subunits (Bormann and Feigenspan 1995; Cutting et al. 1991; Qian and Dowling 1994; Zhang et al. 1995), and as shown recently, a single histidine residue of the GABA 1-subunit is essential for zinc inhibition of theGABA_C receptor (Wang et al. 1995). This residue is conserved among the GABA - (GABA_C) subunits cloned thus far, consistent with the available electrophysiological results, i.e., the GABA_CR-mediated currents of retinal neurons are inhibited by low concentrations of zinc.

In the case of the GABA_AR, on the other hand, molecular studies have revealed a remarkable diversity of receptor subtypes that arise by the association of different combinations of the various polypeptide subunits; a recent count (McKernan and Whiting 1996) indicates that there are already 13 known members of the mammalian family (6 -subunits, 3 -subunits, 3 -subunits, and 1 -subunit). Although the potential number of pentameric arrangements is staggering, the number of functional GABA_AR subtypes that are actually expressed throughout the nervous system is not known, and the question is being vigorously investigated with a variety of pharmacological, physiological, and molecular approaches (cf. Burt and Kamatchi 1991; Kaila 1994; Rabow et al. 1995). Of particular relevance to the present study is the action of zinc in revealing the response heterogeneity imposed by the subunit composition of GABA_ARs (Smart et al. 1994). For example, current recordings from cells transiently transfected with cDNAs encoding various subunit combinations showed that expressing the - and -subunits resulted in GABA_ARs that could be blocked by Zn²⁺, whereas the presence of the -subunit, irrespective of the other subunit components, was sufficient to produceGABA_ARs in which the GABA-activated currents were almost completely insensitive to Zn²⁺ (Draguhn et al. 1990; Smart et al. 1991). However, there is evidence that cells that express subunit combinations that include the -subunit, form GABA_ARs that are suppressed by zinc despite the presence of the -subunit (Saxena and Macdonald 1994). Interestingly, these authors found that a GABA_AR isoform consisting of the _{11 2L}-subunits resulted in cells whose GABA-mediated currents were enhanced an average of 17% in the presence of 10 µM zinc. Because there was no further characterization of the zinc-modulatable current, it would be premature to entertain the possibility that this subunit complex underlies the current recordings obtained in the present study. In any event, the nature of the zinc responses suggests that the subunit composition of GABA_ARs of skate bipolar and Müller cells represent a novel receptor configuration.

	ACKNOWLEDGEMENTS

  We are grateful to Dr. Robert Malchow for participation in a number of earlier experiments, to Dr. Laura Haugh-Scheidt for the design and construction of the perfusion system, and to J. Zakevicius for assistance throughout the course of the study. Golgi sections of the skate retina were very kindly provided by Dr. John Dowling; we thank him also for a critical reading of the manuscript.

  This research was funded by National Eye Institute Grants EY-06516, EY-00777, and EY-06603 and by awards from Research to Prevent Blindness, the Lions of Illinois, and the Illinois Eye Fund. H. Qian was supported by an Erik B. Fries Fellowship from the Marine Biological Laboratory.

	FOOTNOTES

  Address for reprint requests: H. Ripps, Dept. of Ophthalmology and Visual Sciences, University of Illinois College of Medicine, 1855 West Taylor St., Chicago, IL 60612.

  Received 18 February 1997; accepted in final form 2 July 1997.

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