Calcium Released From Intracellular Stores Inhibits GABAA-Mediated Currents in Ganglion Cells of the Turtle Retina

Abram Akopian1, Robert Gabriel1, 3, and Paul Witkovsky1, 2

1 Department of Ophthalmology and 2 Department of Physiology and Neuroscience, New York University School of Medicine, New York, New York 10016; and 3 Department of General Zoology and Neurobiology, Janus Pannonius University, H-7604 Pecs, Hungary

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Akopian, Abram, Robert Gabriel, and Paul Witkovsky. Calcium released from intracellular stores inhibits GABAA-mediated currents in ganglion cells of the turtle retina. J. Neurophysiol. 80: 1105-1115, 1998. We studied spiking neurons isolated from turtle retina by the whole cell version of the patch clamp. The studied cells had perikaryal diameters >15 µm and fired multiple spikes in response to depolarizing current steps, indicating they were ganglion cells. In symmetrical [Cl-], currents elicited by puffs of 100 µM gamma -aminobutyric acid (GABA) were inward at a holding potential of -80 mV. All of the GABA-evoked current was blocked by SR95331 (20 µM), indicating that it was mediated by a GABAA receptor. The GABA-evoked currents were unaltered by eliciting a transmembrane calcium current either just before or during the response to GABA. On the other hand caffeine (10 mM), which induces Ca2+ release from intracellular stores, inhibited the GABA-evoked current on average by 30%. The caffeine effect was blocked by introducing the calcium buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the cell but was unaffected by replacing [Ca2+]o with equimolar cobalt. Thapsigargin (10 µM), an inhibitor of intracellular calcium pumps, and ryanodine (20 µM), which depletes intracellular calcium stores, both markedly reduced a caffeine-induced inhibition of the GABA-evoked current. Another activator of intracellular calcium release, inositol trisphosphate (IP3; 50 µM), also progressively reduced the GABA-induced current when introduced into the cell. Dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP; 0.5 mM), a membrane-permeable analogue of cAMP, did not reduce GABA-evoked currents, suggesting that cAMP-dependent kinases are not involved in suppressing GABAA currents, whereas calmidazolium (30 µM) and cyclosporin A(20 µM), which inhibit Ca/calmodulin-dependent phosphatases, did reduce the caffeine-induced inhibition of the GABA-evoked current. Alkaline phosphatase (150 µg/ml) and calcineurin (300 µg/ml) had a similar action to caffeine or IP3. Antibodies directed against the ryanodine receptor or the IP3 receptor reacted with the great majority of neurons in the ganglion cell layer. We found that these two antibodies colocalized in large ganglion cells. In summary, intracellular calcium plays a role in reducing the currents elicited by GABA, acting through GABAA receptors. The modulatory action of calcium on GABA responses appears to work through one or more Ca-dependent phosphatases.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

In the vertebrate retina as in other portions of the CNS gamma -aminobutyric acid (GABA) is an inhibitory transmitter of primary importance (Massey and Redburn 1987). Its actions are mediated by three main types of GABA receptor, of which two (GABAA and GABAC) are ionotropic, whereas the GABAB receptor is metabotropic, i.e., linked to second messenger systems. All three GABA receptor classes have a wide distribution among retinal neurons (Lukasiewicz 1996; Slaughter 1995). The inner plexiform (synaptic) layer of the retina is particularly rich in GABA-containing neuronal processes (Yazulla 1986). Correspondingly, the trigger features of ganglion cells, which carry the retinal message to the brain, are strongly affected by GABAergic inputs. For example, the directional selectivity of ganglion cells is lost when GABAA receptors are blocked by selective antagonists (Massey et al. 1997; Wyatt and Daw 1976). The evident importance to ganglion cell function of GABA-dependent circuitry is the starting point for the current study, which is focused on the modulation of GABAA inputs to ganglion cells by calcium.

There is abundant evidence that GABA-mediated responses are subject to modulation. For example, Huidobro-Toro et al. (1996) reported that serotonin, acting through a G protein-dependent pathway linked to calcium, inhibited GABAA receptor function. Other studies showed that GABAA-dependent responses are modulated by intracellular calcium. Intracellular calcium level can be altered by calcium influx through voltage-gated (Inoue et al. 1986) or ligand-gated channels such as the NMDA receptor (Chen and Wong 1995). In addition, calcium can be released from neuronal intracellular stores through a caffeine-sensitive ryanodine receptor (Garaschuk et al. 1997) or by means of inositol trisphosphate (IP3) (Ross et al. 1989), which itself is generated through second messenger-linked pathways (Berridge 1993). Our data indicate that both caffeine and IP3 inhibit GABAA-dependent membrane currents in isolated ganglion cells of the turtle retina. On the other hand, calcium entry through voltage-gated channels does not appear to be involved in the regulation of the GABA-induced membrane currents we studied. Moreover, our experiments indicate that intracellular calcium interacts with the GABAA receptor through a calcium-dependent phosphatase. These data were reported in abstract form (Akopian and Witkovsky 1997).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cell isolation procedure

Turtles (red-eared sliders, Pseudemys scripta elegans) were obtained from a commercial supplier (Kons Scientific, Germantown, WI). The animal was rapidly decapitated, the eyes were enucleated and hemisected with a razor cut, and the retinas were stripped from the eyecups. Retinas were exposed to 20 U/mg papain (Worthington, Freehold, NJ) at room temperature (20-23°C) for 20 min and then washed several times with Ringer solution. The remaining procedure for cell isolation is identical to that reported previously (Akopian and Witkovsky 1996). Experiments were performed on cells within 1-2 h after isolation. We studied large cells (perikarya >15 µm diam), which after isolation often retained a portion of the axon and some short processes. On the basis of perikaryal dimensions (Kolb 1982) and the presence of an axon, the neurons we studied are ganglion cells. In current-clamp mode they fire multiple spikes in response to depolarizing current steps.

Immunocytochemistry

Eyecups were prepared as described previously, except that the retina was not separated from the back of the eye. The eyecup was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 12 h. The tissue was washed in phosphate buffer and cryoprotected in 30% sucrose dissolved in phosphate buffer, then embedded in Tissue-Tek compound (Miles Laboratories, Naperville, IL) and sectioned in a cryostat at 12 µm. Sections were placed in an antibody diluent solution for 1 h (Gabriel et al. 1992) supplemented with 5% normal goat serum. Primary antibodies (monoclonal mouse anti-ryanodine receptor, 1:200 dilution, and polyclonal rabbit anti-IP3 receptor, 1:250 dilution, both from Chemicon, Temecula, CA) were applied overnight, followed by the appropriate biotinylated secondary antibodies (Sigma, St. Louis, MO) in 1:100 dilution for 3 h and the streptavidin-biotinylated horseradish peroxidase complex (Vector, Burlingame, CA) in 1:200 dilution for 2-4 h. The reaction was developed in 0.05% diaminobenzidine and 0.01% H2O2 dissolved in 0.05 M tris(hydroxymethyl)aminomethane buffer (12-20 min). Sections were dehydrated in an ascending ethanol series and coverslipped in Permount.

For fluorescent double labeling, the same sections and primary antibodies were used as described previously. The secondary antibodies were horse anti-mouse coupled with Texas red (Vector) and goat anti-rabbit coupled with fluorescein isothiocyanate (Vector) at 1:50 dilution. Sections were coverslipped in Vectashield (Vector) mounting medium.

Omission of primary antibodies or their replacement with nonimmune sera resulted in no staining. We found no cross-reactivity between primaries and the noncorresponding secondaries. As positive controls for the specificity of antibody staining, rat retina and skeletal muscle were used for ryanodine receptor labeling; rat retina and cerebellum were employed to examine IP3 receptor immunochemistry. We obtained staining appropriate to the cellular location of the receptors, as described by others (McPherson and Campbell 1993; Ross et al. 1989).

Solutions

The Ringer solution contained (in mM) 100 NaCl, 3.3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH adjusted to 7.5 with NaOH. In experiments to study Ca2+ influx through voltage-gated Ca channels, the normal Ringer solution was modified by increasing CaCl2 to 20 mM and adding 1 µM tetrodotoxin (TTX), 5 mM 4-aminopyridine (4-AP), and 20 mM tetraethylammonium (TEA) to block sodium and potassium currents. The osmolarity was kept constant by removing an equimolar concentration of NaCl. The standard intracellular solution in the patch pipette contained (in mM) 100 CsCl, 2 MgCl2, 0.1 CaCl2, 0.5 ethylene glycol-bis (beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 2 ATP-Na salt at pH 7.2. GABA (50-100 µM), baclofen (500 µM), cis-4-aminocrotonic acid (CACA; 100 µM), caffeine (10-20 mM), bicuculline (30-100 µM), imidazole-4-acetic acid HCl (I4AA; 100 µM), and SR95331 (1-20 µM) (all from Research Biochemical International, Natick, MA) were applied extracellularly by pressure pulses with the use of a DAD-12 superfusion system (Adams and List Associates, Westbury, NY). GABA antagonists were applied before and during the agonist application, whereas caffeine was applied 10-20 s before a GABA application. Calmidazolium and cyclosporin A (20 µM, Research Biochemical International), bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA; 10 mM, Sigma), alkaline phosphatase (150 µg/ml, Boehringer Mannheim, Indianapolis, IN), and calcineurin (300 µg/ml, Sigma) were added to the standard patch pipette solution.

Recording procedure

Whole cell currents were recorded in a conventional way (Hamill et al. 1981) with the use of an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Recording pipettes were made from borosilicate glass tubing (1.2 mM OD, 0.6 mm ID). Electrode resistance was typically 3-5 MOmega in the bath solution. After seal rupture the series resistance (10-15 MOmega ) was compensated (70-80%) by a standard circuit. Whole cell currents induced by GABA (100 µM) were typically <1 nA (usually 300-700 pA); voltage errors resulting from inadequate compensation were estimated to be at most 3-4 mV. The average input resistance for ganglion cells, estimated from the steady-state current induced by a -10 mV voltage step from -60 mV, was 2.4 ± 0.4 (SE) GOmega (n = 8). Currents were filtered at 1 kHz by a low-pass Bessel filter and were typically sampled at 100 Hz. The pClamp software package (Axon Instruments) was used for data acquisition and analysis. Summary data are presented as means ± SE. Membrane potential usually was held at -60 mV and 1-2 s pressure pulses were applied to puff GABA onto the cell surface. The interval between puffs was >30 s.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Characteristics of GABA-induced current

It was shown in other preparations that a combination of Mg-ATP and low [Ca2+]i is critical for slowing a rundown of GABA-dependent responses (Chen et al. 1990; Stelzer and Wong 1987), which otherwise fall to ~10% initial value within 10 min. Accordingly, we routinely included MgCl2 (2 mM) and ATP (2 mM) in the intracellular solution and used a combination of 0.1 mM CaCl2 and 0.5 m M EGTA to clamp intracellular free Ca2+ level at ~50 nM (Hagiwara 1983). If the GABA-induced responses declined >10% from their initial value during the first 10 min, the experiment was terminated and the data from that cell were rejected.

Cells were held at -80 mV, and inward currents were elicited by 2-s puffs of 100 µM GABA. The mean inward current amplitude elicited by a GABA puff was 580 ± 40 pA (n = 25, range 300-900 pA). With symmetrical Cl- concentration inside the patch pipette and in the bath, an inward current was elicited at negative membrane potentials that reversed near 0 mV and became outward at positive potentials (Fig. 1A). When [Cl-] in the internal solution was reduced to 8 mM, by replacing it with gluconate the reversal potential was shifted to -60 mV, close to the value predicted by the Nernst equation for Cl- dependence (Fig. 1B). The current-voltage curves of GABA responses (Fig. 1B) were approximately linear in the membrane potential range of -60 to +30 mV. At low GABA concentrations (5-10 µM) the onset of currents was slow and the responses were sustained; however, at higher concentrations, desensitization of currents was observed in the continued presence of GABA (see Figs. 3 and 4).


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FIG. 1. gamma -Aminobutyric acid (GABA)-induced whole cell currents recorded from turtle retinal ganglion cells. A: GABA-elicited inward currents at negative holding potentials, reversing to outward currents at membrane potentials positive to +5 mV. The values of the holding potentials are indicated near each record. B: reversal potential of GABA-evoked current was near 0 mV with symmetrical [Cl-] inside and outside the cell (bullet ), but shifted to near -60 mV when [Cl-] in the patch pipette was reduced to 8 mM by an equimolar substitution of gluconate for Cl- salt (open circle ). C: bicuculline (100 µM) reversibly reduced GABA-evoked current by ~90%. D: SR-95531 (20 µM) completely blocked the GABA-evoked current.


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FIG. 3. Influx of Ca2+ through voltage-gated channels does not alter GABA-evoked current. A: GABA-induced currents recorded before and 100 ms after eliciting calcium current are superimposed. Ca current was evoked by a 500-ms depolarizing step from -60 to 0 mV. External solution contained 20 mM CaCl2 to enhance Ca current and 1 µM tetrodotoxin, 4 mM 4-aminopyridine (4-AP), and 20 mM tetraethylammonium to block Na and K currents. Patch pipette contained standard internal solution. The amplitude of the GABA-evoked current was not altered by the depolarizing step. B: cell different from A; Ca current elicited during a 4-s GABA puff (horizontal bar) altered neither the magnitude nor the kinetics of the GABA response, indicating that influx of Ca2+ through voltage-gated channels has no effect on GABA responses.


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FIG. 4. Effect of caffeine on GABA-induced current. A: caffeine reversibly reduced GABA-evoked current. GABA (100 µM) was puffed before and after 20-s preexposure of cells to 20 mM caffeine. B: reduction of the amplitude by caffeine was accompanied by a slowing of the onset (arrow) and decay of GABA-induced current. The decay components were fit by single exponentials (smooth lines) with time constants, tau , of 2.5 s (control) and 8.2 s (caffeine treated). C and D: histograms show the caffeine-induced changes in decay time constant (C) and the rise time to peak (D) of GABA-induced current. Bars indicate mean values ± SE from number of cells indicated.

As a further probe of the receptor subtype responsible for the generation of GABA-evoked responses, we applied known GABA receptor agonists and antagonists. The GABA pulses (1-2 s,100 µM) were applied at 30-50 s intervals to reduce desensitization. The GABA-induced responses were reversibly reduced on average by 90% (n = 6) in the presence of the GABAA receptor antagonist bicuculline (100 µM, Fig. 1C) and 100% by exposure to 20 µM SR95331 (Fig. 1D), a potent GABAA antagonist (Lukasiewicz and Werblin 1994). We examined the effect of the GABAC receptor agonist CACA as a test of whether the bicuculline-insensitive part of the GABAA response was mediated by an activation of GABAC receptors. In 4 of 11 cells studied, CACA (100 µM) induced a small sustained current (not shown). However, we found that currents elicited by CACA and GABA did not sum linearly. Moreover, 100 µM imidazole-4-acetic acid (I4AA), a known GABAC receptor antagonist (Qian and Dowling 1995), did not block the responses to CACA. Application of the GABAB agonist baclofen (500 µM) was without effect. We conclude that at least the great majority of receptors involved in the generation of the GABA-mediated currents examined were of the GABAA receptor subtype.

Effect of low intracellular Ca2+ perfusion on GABA-induced currents

In the first set of experiments we tested whether a reduction of [Ca2+]i affected GABA-induced responses. When the patch pipettes contained the standard internal solution (see METHODS) the GABA-induced currents usually were stable during the 15-20 min recording, but when they contained a Ca-free/BAPTA internal solution a progressive enhancement of the GABA-mediated responses was observed during the first 2-3 min recording. The responses increased by 28 ± 4% (n = 9) after 3-5 min perfusion with Ca-free internal solution compared with the initial value recorded within 20 s after the patch break. On the other hand, when the patch pipette contained a high Ca internal solution (5 Ca/1 EGTA, estimated free [Ca2+] ~1 µM), the GABA responses fell during the same time interval by 21 ± 8% (n = 5). Figure 2 summarizes these results, which indicate that the magnitude of the GABA-induced current is modulated by [Ca2+]i.


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FIG. 2. Effect of internal [Ca2+] on gamma -aminobutyric acid (GABA)-induced whole cell current. Histogram summarizes changes in the amplitude of GABA currents (IGABA) after 10-min recording in whole cell mode. Patch pipette contained standard internal solution (control, open bar); high Ca internal solution, which contained combination of 5 mM CaCl2/1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and Ca-free solution, which contained 10 mM BAPTA. Peak amplitude of GABA-evoked current recorded after 10 min was normalized to that obtained immediately after the patch break. Number of cells studied is indicated above each bar.

Effect of Ca2+ influx on GABA-induced responses

There are two obvious ways by which an elevation of [Ca]i might occur: a Ca2+ influx across the plasma membrane through voltage-gated Ca channels and/or its release from intracellular stores. As a test of the former possibility, GABA-induced currents were recorded first in normal Ringer solution and then in a bath solution containing 20 mM CaCl2 to enhance the Ca current and also in TTX, 4AP, and TEA to block sodium and potassium currents. GABA-dependent responses were not significantly affected by these changes in the composition of the external solution. To stimulate Ca2+ influx through voltage-gated channels the membrane potential was stepped from -80 to +10 mV for 500 ms (Fig. 3A), and the responses to GABA were recorded within 200 ms after the termination of the depolarizing steps. GABA-induced currents measured after the voltage steps did not differ from those recorded before them. Thus the mean GABA-evoked current for the control condition was 395 ± 55 pA, whereas it was 390 ± 68 pA after induction of a Ca current of 457 ± 81 pA, indicating that Ca influx through voltage-gated channels does not suppress GABA responses. We considered the possibility that, if the Ca influx were transient and fast, as reported for frog primary sensory neurons (Inoue et al. 1986), the interval between stimulation of Ca current and application of GABA in our experiment might be too long to observe the effect of Ca2+ influx. Although Ca currents in turtle retinal ganglion cells are reported to be sustained (Lasater and Witkovsky 1990) (see Fig. 3), we explored this possibility by activating Ca currents during a continuous application of GABA. If Ca2+ influx were to induce a transient reduction of GABA-mediated responses, then a relaxation of the GABA-induced inward current would be expected to occur immediately after the termination of the voltage step. No relaxation was seen in three cells examined (Fig. 3B).

Effect of Ca2+ released from intracellular stores

Experimental evidence supports the idea that Ca-induced Ca release (CICR) from intracellular stores, first described in muscle cells, also occurs in neurons (reviewed in Verkhratsky and Shmigol 1996). Caffeine is known to promote large increases of [Ca2+]i from intracellular stores by shifting the Ca2+ sensitivity of CICR to lower concentrations (Sitsapesan and Williams 1990). Superfusion of the cell surface with Ringer solution containing 10-20 mM caffeine did not evoke any current, but it reduced the amplitude of the GABA-evoked current elicited by a superimposed puff of GABA by 32 ± 3% (Fig. 4A; n = 40, range 20-60%). The caffeine effect was fully reversible by a wash with normal Ringer solution. The reduction of amplitude was accompanied by changes in the kinetics of activation and inactivation of the GABA-evoked current (Fig. 4B). The time course of desensitization of the (200 µM) GABA-induced current was fit by a single exponential; its time constant, tau , increased from 3.2 ± 0.15 s in control to 6.6 ± 0.9 s in the presence of caffeine (Fig. 4C). Caffeine also substantially slowed the onset of GABA-induced responses (Fig. 4B, arrow), increasing rise time from 120 ± 15 ms in control to 300 ± 30 ms (Fig. 4D).

A caffeine-induced reduction of GABA currents was observed over a wide range of GABA concentrations, from 5 µM to 1 mM. Figure 5 illustrates that caffeine reduced the maximum response without altering the concentration of GABA that elicited a half-maximal response. The finding that the normalized dose-response curves for GABA in the absence and in the presence of 10 mM caffeine are superimposed (Fig. 5, inset) indicates that caffeine has no effect on the affinity of GABA for the GABAA receptor. Moreover, the reversal potential of GABA-evoked responses was unaffected by caffeine (Fig. 6A). Inhibition of GABA-mediated responses by caffeine did not depend on membrane potential and was observed at holding potentials from -60 to +50 mV. This is illustrated in Fig. 6B, where the I-V curves of GABA-induced currents in control and in the presence of caffeine intersect near +10 mV.


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FIG. 5. Effect of caffeine on dose-response relation of the GABA-evoked current. Caffeine reduced peak amplitude of GABA currents over a wide range of GABA concentrations. Normalized dose-response curve (inset, bottom right) shows that affinity of GABA receptor was not affected by caffeine.


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FIG. 6. Voltage dependence of caffeine effect. A: 100 µM GABA-induced currents were recorded at different holding potentials in normal Ringer solution and after 20 s preexposure to 10 mM caffeine. Caffeine reduced GABA responses at different levels of holding membrane potential, whose values are indicated near the traces at left. B: current-voltage relationship of GABA-induced currents obtained from A. These results indicate that caffeine reduced GABA-evoked current amplitude without changing the reversal potential.

To explore the basis of the caffeine-induced inhibition of GABA-evoked current we tested for its possible dependence on extracellular Ca2+ by replacing extracellular Ca2+ (2 mM) with an equivalent amount of Co2+. In Co2+ medium caffeine still induced an inhibition of GABA-induced responses of 25 ± 5% (n = 7, not shown), a degree of inhibition that did not differ significantly from that observed in normal extracellular [Ca2+]. This result suggests that caffeine causes an elevation of [Ca2+]i by releasing Ca2+ from intracellular stores. If so, one would expect that chelation of internal Ca2+ by the high-affinity Ca buffer BAPTA should reduce a caffeine-induced inhibition of GABA-dependent responses. We found that, when the patch pipette contained Ca-free 10 mM BAPTA solution, the mean reduction of GABA responses by caffeine was reduced to 8 ± 3% (range 2-22%, n = 6, Fig. 7B).


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FIG. 7. Pharmacology of caffeine-induced inhibition of GABA-evoked current. A: caffeine (filled bar) induced a substantial reduction of GABA-evoked current in control Ringer solution (left panel). After 5-min exposure of cells to 10 µM thapsigargin (open bar), the inhibitory effect of caffeine was substantially reduced (right panel). B: caffeine-induced inhibition of GABA responses was abolished in the presence of 10 mM BAPTA in the patch pipette. C: calmidazolium (30 µM), an inhibitor of Ca/CaM phosphatase, applied intracellularly attenuated the caffeine-induced inhibition of GABA-evoked current, indicating an involvement of Ca/CaM-dependent phosphatases.

Because BAPTA will buffer intracellular calcium irrespective of its source, a more direct test of the putative participation of intracellular calcium stores was necessary. Therefore we explored the effect on GABA-mediated currents of the inhibition of Ca2+ uptake into the sarco(endo)plasmic reticulum; thapsigargin, a selective blocker of endoplasmic reticulum Ca2+-ATPase, was utilized for this test (Thastrup et al. 1990). Figure 7A illustrates a representative experiment in which the GABA-elicited responses first were recorded in normal Ringer solution and then in the presence of caffeine (left panel, filled bar). Thereafter the cells were exposed to 10 µM thapsigargin (open bar) for 5 min, and again GABA-evoked responses were recorded in the presence of caffeine (right panel). After exposing cells to thapsigargin, caffeine reduced GABA responses only by 7 ± 2% (n = 11).

Ryanodine was shown to bind to the ryanodine receptor channels and "lock" them in an subconducting state, leading to a depletion of the ryanodine-sensitive Ca stores (McPherson and Campbell 1993). In our experiments external application of ryanodine (2-20 µM) led to the reduction of GABA-induced responses in a concentration-dependent manner. The mean reductions of GABA-evoked currents after 5-min pretreatment with 2 and 20 µM ryanodine were 26 ± 3 and 32 ± 3%, respectively (n = 3-6). When the patch pipette contained a 10 mM BAPTA internal solution, the inhibitory effect of ryanodine (20 µM) on the GABA-evoked current was substantially reduced to 9% (n = 2). This result is consistent with the idea that ryanodine, acting through its receptor, causes a depletion of Ca stores and that released Ca subsequently induces an inhibition of the GABA-evoked current. Next we tested if ryanodine could block a caffeine-induced inhibition of the GABA-evoked current. In our experiments the inhibitory effect of caffeine was reduced from 32 ± 3% in the control solution to 14 ± 4% in the presence of ryanodine. Thus we did not observe a complete suppression of the caffeine effect in the presence of ryanodine.

Involvement of Ca-dependent enzymes

Ca-induced inhibition of GABA-evoked responses might be mediated by a direct interaction of Ca2+ with the GABA receptor or by an activation of Ca-dependent enzymatic processes that modify the GABA receptor. In relation to the latter possibility calmodulin is a ubiquitous intracellular protein that regulates the activity of various enzymes in a Ca2+-dependent manner (Cheung 1980). Ca/CaM was shown to stimulate adenylate cyclase activity in retinal preparations from different species (Gnegy et al. 1984; Sano 1985). Thus one possibility is that the effect of [Ca2+]i is mediated through an activation of adenylate cyclase, resulting in an elevation of adenosine 3',5'-cyclic monophosphate (cAMP). Moreover, caffeine, being a methylxanthine, has phosphodiesterase blocking activity (Verkhratsky and Shmigol 1996) and so might contribute to keeping the concentration of cAMP at a high level. If one assumes that caffeine acts primarily through cAMP, the expectation would be that GABA-dependent responses should be decreased by an elevation of [cAMP]i. We utilized a membrane-permeable analogue of cAMP, dBcAMP (500 µM), and found that in its presence GABA-elicited responses actually increased slightly (data not shown). These data suggest that activation of adenylate cyclase is not the primary mechanism by which caffeine modulates GABA-induced responses.

Another calcium-dependent enzyme is the calmodulin-dependent phosphatase calcineurin, which was found in chick retinal bipolar and ganglion cells (Cooper et al. 1985; Wood et al. 1980). Elevation of [Ca2+]i is known to trigger the activation of Ca/CaM-dependent phosphatases in the CNS (Klee et al. 1979). To test this possibility we used calmidazolium, which is a potent inhibitor of CaM-dependent phosphatase (Van Belle 1981). When calmidazolium (30 µM) was included in the pipette solution it substantially reduced the caffeine-induced inhibition of GABA-mediated responses (Fig. 7C). In these experiments recordings were performed 3-5 min after establishing the whole cell configuration to allow calmidazolium sufficient time to diffuse into the cell. The mean inhibition of GABA-dependent responses by caffeine in the presence of calmidazolium was 9 ± 2% (range 0-17%, n = 19). Additional support for an involvement of calcineurin was provided by experiments with cyclosporin A, a selective antagonist of calcineurin (Hashimoto et al. 1990). After 5-min incubation with 20 µM cyclosporin A, the inhibitory effect of caffeine on GABA-evoked current was reduced to 5 ± 2% (n = 9, range 0-12%, not shown). The effect of cyclosporin A was apparent within 1 min of establishing the whole cell configuration, indicating that the effective rates of endogenous phosphorylation and dephosphorylation are both high (Jones and Westbrook 1997).

As a further probe of the possible involvement of phosphatase activity in a downregulation of GABA-dependent responses, we recorded GABA-induced current in the presence of alkaline phosphatase (150 µg/ml) in the patch pipette. Alkaline phosphatases are nonspecific enzymes that hydrolyze phosphorus-containing compounds (McComb et al. 1979). In the presence of alkaline phosphatase we observed a dramatic rundown of the GABA-mediated currents, such that after ~5-min exposure, responses were reduced to 25-30% of their initial value (Fig. 8, A and E, open circle ). Finally, intracellular administration of calcineurin (300 µg/ml) caused a similar, but slower, rundown of GABA-evoked currents than did alkaline phosphatase. After 10-min incubation with calcineurin, responses to GABA application reduced to 40% of their initial value (Fig. 8B). In contrast there was no reduction of GABA-elicited currents over the same period in control cells tested with the normal pipette solution (Fig. 8E, bullet ). This difference suggests that the effect of [Ca2+]i on GABA-induced responses may be mediated through an activation of one or more Ca-dependent enzymes, of which the most probable is the Ca-dependent phosphatase calcineurin.


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FIG. 8. Effects of intracellularly applied alkaline phosphatase, calcineurin, and inositol trisphosphate (IP3) on GABA-induced current. Rundown of GABA-induced currents recorded with patch pipette containing 150 µg/ml alkaline phosphatase in the internal solution. Whole cell currents were recorded within 20 s after the patch break and after different time intervals of perfusion with alkaline phosphatase (A) and calcineurin (B). C: GABA-evoked current was substantially reduced after 8-min recording with a patch pipette containing 50 µM IP3 (1) compared with the current recorded immediately after the patch break (1). D: coapplication of heparin (50 µM) with IP3 in the pipette solution abolished an IP3-induced inhibition of GABA responses. E: time course of normalized peak GABA currents recorded with a patch pipette containing standard internal solution (bullet ) or in the presence of 150 µg/ml alkaline phosphatase (open circle ), calcineurin (triangle ), or 50 µM IP3 added to the standard internal solution (square ). Currents (It) were evoked at 1-min intervals and their peak amplitude normalized to the current, obtained within 20 s (Io) after the patch break. Each point represents the mean value ± SE for the indicated number of cells.

It is known that activation of glutamatergic receptors in CNS neurons induces an elevation of internal Ca2+ (Mayer et al. 1987), both by its influx through receptor-regulated channels and by release from intracellular stores. The latter can be brought about by 1) Ca-induced Ca release (Linn and Christensen 1992) and/or 2) by stimulation of Ca-dependent phospholipase C to produce IP3, which then mobilizes Ca2+ from internal stores (Wood et al. 1997).

We tested whether IP3 can modulate GABA-mediated responses. In the experiment illustrated in Fig. 8C, IP3 (50 µM) was added to the standard internal solution, and GABA-evoked responses were recorded within 20 s after the patch break (1) and after 8 min in whole cell mode (2). GABA-dependent currents progressively reduced in the presence of IP3. The mean inhibition of GABA-evoked current after 8-10 min perfusion with IP3 (Fig. 8E, square ) was 47 ± 5% (n = 7). The inhibitory action of IP3 was blocked when 10 mM BAPTA was added to the patch pipette, indicating an involvement of intracellular Ca2+. Heparin (50 µM), which blocks Ca release from IP3-sensitive stores (Frank and Fein 1991), applied together with IP3, substantially reduced the IP3-induced inhibition of GABA-induced responses (Fig. 8D). These data indicate that an IP3-induced reduction of GABA responses is most likely mediated through release of Ca2+ from IP3-sensitive stores. The histogram illustrated in Fig. 9 summarizes the results obtained in our study. It shows that caffeine-induced inhibition of GABA responses is reduced substantially in the presence of 1) ryanodine or 2) thapsigargin in the bath solution, 3) when the patch pipette contained Ca-free/BAPTA solution, or 4) when calmidazolium was present in the standard internal solution.


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FIG. 9. Summary of drug-induced inhibition of GABA-evoked current. Caffeine-induced inhibition of GABA responses in normal Ringer solution (open bar) is compared with that seen in the presence of ryanodine or thapsigargin in bath solution or when the pipette solution contained calmidazolium or BAPTA.

Immunocytochemical localization of ryanodine and IP3 receptors

In vertical sections of the turtle retina, most cells in the ganglion cell layer and a few cells in the inner nuclear layer were seen to be labeled with antibodies directed against either the ryanodine receptor or the IP3 receptor (Fig. 10). Most ganglion cells were immunoreactive for the ryanodine receptor (Fig. 10, A and B). Large ganglion cells invariably were immunoreactive for both ryanodine and IP3 receptors; occasionally dendritic processes extending into the inner plexiform layer also showed immunoreactivity (Fig. 10, C and D). In the perikaryon the stained areas were patchy, possibly indicating a local concentration of intracellular membranes. In double-labeling experiments both ryanodine and IP3 receptors were colocalized in numerous cells of the ganglion cell layer, including the large ganglion cells (Fig. 10, E and F), which are the subject of the physiological experiments reported previously. These data indicate that the large ganglion cells have both ryanodine and IP3 receptors.


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FIG. 10. Ryanodine receptor and inositol trisphosphate (IP3) receptor immunostaining. A: vertical retinal section showing ryanodine receptor immunoreactivity. Staining is seen in the ganglion cell layer (gcl), the inner nuclear layer (inl), and in the synaptic terminals of the photoreceptors (pl). Almost no immunoreactivity is seen in the inner plexiform layer (ipl). B: anti-IP3 receptor immunoreactivity. Similar staining pattern to A. C and D: ganglion cells at higher magnification: antiryanodine receptor in C and anti-IP3 receptor in D. Note the patchy staining in the perikarya. Marker bar in B applies to A and B; in F it applies to C through F. E and F: fluorescent double labeling of ganglion cells with antiryanodine receptor antibody (E) and anti-IP3 receptor (F). Arrow, large ganglion cell that colocalizes the 2 receptor types.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Ca-mediated inhibition of GABA-evoked current involves intracellular Ca stores

The current physiological results suggest that in turtle retinal ganglion cells an elevation of [Ca2+]i released from internal stores suppresses GABAA-induced responses through the intermediation of one or more calcium-dependent enzymes. Our immunoreactivity data indicate that the ganglion cells we studied have both ryanodine and IP3 receptors, which were implicated in the release of calcium from endoplasmic reticulum stores (Berridge 1993; Garaschuk et al. 1997).

Antiryanodine and IP3 receptor immunoreactivity

The IP3 receptor was localized to the endoplasmic reticulum of cerebellar Purkinje neurons (Ross et al. 1989), which are particularly abundant in the perikaryon. In contrast, in a variety of vertebrate retinas including that of turtle, Peng et al. (1991) found most IP3 receptor immunoreactivity in synaptic terminals rather than in perikarya. This staining difference may reflect the different antibodies used and/or the subcellular localization of different IP3 receptor isoforms. The latter possibility was advanced by Peng et al. (1995) to account for the absence of anti-IP3 receptor staining in retinal rods and horizontal cells in which IP3 turnover was reported (reviewed in Anderson and Brown 1988). However, in goldfish retina, Micci and Christensen (1996) found abundant IP3 receptor in ganglion cell perikarya.

Ryanodine receptors were localized by light- and electron microscopic immunocytochemistry to the endoplasmic reticulum of a variety of central neurons (Sah et al. 1993, 1994). In cultured hippocampal neurons, IP3 and ryanodine receptors colocalize and are most concentrated in the perikarya (Seymour-Laurent and Barish 1995). This is the same pattern we find for turtle retinal ganglion cells.

In many neurons, caffeine stimulates Ca release from ryanodine-sensitive stores by a mechanism known as CICR (Sitsapesan and Williams 1990; Verkhratsky and Shmigol 1996). In our experiments, caffeine reversibly reduced the membrane currents elicited by GABA and slowed the kinetics of activation and decay of the GABA-induced membrane current. Inhibition was observed over a wide range of GABA concentrations from 10 µM to 1 mM. The following results support our conclusion that the effect of caffeine is mediated through Ca2+ release from internal stores. 1) A caffeine-induced inhibition of GABA-dependent responses was not affected when extracellular Ca2+ was replaced by Co2+. 2) When cells were incubated with thapsigargin, a potent blocker of endoplasmatic Ca-ATPase, the effect of caffeine was substantially reduced. 3) Use of Ca-free solution to which 10 mM BAPTA was added in the patch pipette suppressed the inhibitory effect of caffeine on GABA-mediated responses. 4) Pretreatment of cells with ryanodine reduced the effect of caffeine.

The effect of caffeine can be blocked by thapsigargin, which prevents Ca2+ accumulation into stores through the inhibition of a Ca/ATPase pump, or by ryanodine, which greatly increases passive Ca exchange between the store and cytosol, thereby preventing a net Ca2+ accumulation by the store. In our experiments both these agents substantially reduced the caffeine-induced inhibition of the GABA. In addition, ryanodine by itself reduced the GABA-evoked current, but ryanodine was ineffective when the patch pipette solution contained 10 mM BAPTA, an indication that reduction of GABA-evoked current is most likely caused by a net cytosolic Ca2+ increase because of a depletion of Ca stores.

Ganglion cells in turtle retina possess high-voltage-activated Ca channels (Lasater and Witkovsky 1990). In our experimental conditions 500-ms depolarizing steps from -80 to 0 mV elicited a large Ca current (Fig. 3), but it had no effect on GABA-evoked responses, indicating that the Ca2+ that enters the cell through voltage-gated channels does not modulate GABA-induced current, whereas elevation of [Ca2+]i through release of internal stores does. Our finding is in contrast to results described in frog sensory neurons in which a relaxation of the GABA-activated Cl- current was observed after the termination of each depolarizing step (Behrends et al. 1988; Inoue et al. 1986). We did not observe such an effect in our experiments when the calcium current was activated during a sustained application of GABA (Fig. 3B). However, our finding that the source of calcium for modulation of GABA-elicited responses depends on intracellular stores rather than calcium influx through voltage-gated channels is consistent with results obtained in cultured porcine melanotrophs and rat central neurons (Mouginot et al. 1991; Mulle et al. 1992). The ineffectiveness of calcium influx through voltage-dependent calcium channels in modifying GABA-induced currents presumably reflects the compartmentalization of the cytoplasm. Studies of the calcium dependence of synaptic transmitter release (Llinás et al. 1995) indicate that calcium influx across the plasma membrane is confined to a microdomain. Possibly this calcium does not have access to a calcium-dependent component of the biochemical pathway that activates calcineurin, which is accessible to calcium released from intracellular stores.

Increases in [Ca2+]i were reported to reduce the open time of Cl- channels activated by GABAA receptors in pituitary cells (Taleb et al. 1987). In dentate granule cells elevation of [Ca2+]i caused depression of synaptic GABAA receptor-channels (De Koninck and Mody 1996). In contrast, maintenance of a low level of [Ca2+]i was required for full activation of GABAA current in guinea pig hippocampal neurons (Stelzer and Wong 1987). In retinal ganglion cells of tiger salamander Ca2+ released from internal stores is able to modulate metabotropic GABAB responses, possibly by altering the affinity of agonist binding to receptors (Shen and Slaughter 1997). However, in our study, caffeine reduced the maximum current induced by activation of GABAA receptors rather than by producing a shift in the dose-response curve along the concentration axis (Fig. 5).

Mechanisms of calcium-mediated modulation of GABA currents

The reversibility of the caffeine effect suggests that it is not likely to be mediated through proteolysis of GABA receptors by Ca-dependent proteases. Two alternative possibilities are that calcium is linked to a cAMP-dependent pathway or that it works through one or more calcium-dependent kinases and/or phosphatases. [Ca2+ ]i is known to regulate the activity of various enzymes through activation of calmodulin (Cheung 1980), and calmodulin-dependent adenylate cyclase was found in rat, rabbit, bovine, and fish retinas (Gnegy et al. 1984; Sano 1985). There is evidence from other systems that GABAA responses can be modulated by activation of a cAMP cascade. For example, activation of cAMP-dependent protein kinase A (PKA) is suggested to reduce GABAA receptor current in cultured mouse spinal neurons (Porter et al. 1990) and rat superior cervical ganglion cells (Moss et al. 1992), whereas in cerebellar Purkinje cells PKA activators increased a GABA-activated current (Kano and Konnerth 1992).

If one supposes that the effect of caffeine on GABA-induced responses in our experiments is mediated through an inhibition of phosphodiesterase or an activation of Ca/CaM-dependent adenylate cyclase by [Ca2+]i, then one would expect both cAMP and caffeine to produce an inhibition of GABA-dependent responses. However, exposure of cells to 500 µM dBcAMP failed to reduce GABA responses; in contrast we observed a slight enhancement of GABA-induced currents in three of four cells studied. This change suggests that activation of the cAMP cascade is probably not an intermediary in the caffeine-induced inhibition of GABA-mediated responses in turtle ganglion cells.

An alternative mechanism for caffeine-induced inhibition of GABA-evoked responses is through activation of Ca-dependent phosphatases, as described in hippocampal neurons (Chen et al. 1990; Chen and Wong 1995). Acceleration of rundown of GABA-induced currents in the presence of alkaline phosphatase and calcineurin (Fig. 8) supports this idea. In our experiments, calmidazolium and cyclosporin A, both potent inhibitors of Ca/CaM-dependent phosphatase (Van Belle 1981), substantially reduced the inhibitory effect of caffeine, indicating that dephosphorylation of the GABAA receptor or a related protein probably is responsible for the suppression of GABA responses induced by elevation of [Ca2+]i. Moreover, when experiments were performed with patch pipette containing Ca-free/BAPTA solution, we observed a progressive increase of GABA responses during the first 2-3 min recording in whole cell mode. A similar enhancement of GABA responses after the patch membrane break was observed in experiments in which calmidazolium was present in the recording pipette, suggesting that in intact cells the GABAA receptors partially are suppressed by active endogenous phosphatases.

Our data are consistent with the identification of a Ca-CaM-dependent phosphatase (calcineurin) in bipolar and ganglion cells of the chick retina (Cooper et al. 1985). In contrast, in rod bipolar cells of the rabbit retina, activation of protein kinase C (PKC) rather than PKA activation is involved in the modulation of GABA-evoked currents (Gillette and Dacheux 1996). Moreover, in rabbit rod bipolar cells, activation of a cAMP cascade is responsible for the modulation of GABA responses (Veruki and Yeh 1994). Evidently, multiple mechanisms exist for the regulation of GABAA induced currents that may be cell and species specific.

Interaction of glutamatergic and GABAergic pathways in the retina

In the retina, activation of ionotropic glutamate receptors can result in an elevation of [Ca2+]i either by influx through receptor-regulated channels or by release from internal stores (Duarte et al. 1996; Linn and Christensen 1992). In addition certain metabotropic glutamate receptors stimulate IP3 turnover, which in turn raises [Ca2+]i by releasing it from intracellular stores (Pin and Duvoisin 1995). GABA, in turn, affects glutamatergic transmission presynaptically by modulating calcium current and transmitter release from bipolar cell terminals (Heidelberger and Matthews 1991; Slaughter 1995). Metabotropic GABA receptors also modulate calcium current in ganglion cells (Zhang et al. 1997). The results of this study indicate that Ca-induced Ca2+ release (stimulated by caffeine) and IP3-induced Ca2+ release from internal stores down-modulate GABA-evoked current in turtle retinal ganglion cells. In summary, the major excitatory (glutamate) and inhibitory (GABA) transmitters of the vertebrate retina affect each others' actions. These data collectively point out the importance of the control of intracellular calcium for retinal function.

    ACKNOWLEDGEMENTS

  We thank Dr. Peter Lukasiewicz for helpful comments.

  This work was supported by National Eye Institute Grant EY-03570 to P. Witkovsky, an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology, and by the Hoffritz Foundation. P. Witkovsky is a Senior Scientific Investigator of Research to Prevent Blindness.

    FOOTNOTES

  Address for reprint requests: A. Akopian, Dept. of Ophthalmology, New York School of Medicine, 550 First Ave., New York, NY 10016.

  Received 26 November 1997; accepted in final form 11 May 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society