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Metabotropic and ionotropic glutamate receptors regulate calcium channel currents in salamander retinal ganglion cells

Wen Shen and Malcolm M. Slaughter

Received 3 December 1997; accepted after revision 27 April 1998.

	ABSTRACT

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1. Glutamate suppressed high-voltage-activated barium currents (I_Ba,HVA) in tiger salamander retinal ganglion cells. Both ionotropic (iGluR) and metabotropic (mGluR) receptors contributed to this calcium channel inhibition.

2. Trans-ACPD (1-aminocyclopentane-trans-1S,3R-dicarboxylic acid), a broad-spectrum metabotropic glutamate receptor agonist, suppressed a dihydropyridine-sensitive barium current. Kainate, an ionotropic glutamate receptor agonist, reduced an -conotoxin GVIA-sensitive current.

3. The relative effectiveness of selective agonists indicated that the predominant metabotropic receptor was the L-2-amino-4-phosphonobutyrate (L-AP4)-sensitive, group III receptor. This receptor reversed the action of forskolin, but this was not responsible for calcium channel suppression. l-AP4 raised internal calcium concentration. Antagonists of phospholipase C, inositol trisphosphate (IP₃) receptors and ryanodine receptors inhibited the action of metabotropic agonists, indicating that group III receptor transduction was linked to this pathway.

4. The action of kainate was partially suppressed by BAPTA, by calmodulin antagonists and by blockers of calmodulin-dependent phosphatase. Suppression by kainate of the calcium channel current was more rapid when calcium was the charge carrier, instead of barium. The results indicate that calcium influx through kainate-sensitive glutamate receptors can activate calmodulin, which stimulates phosphatases that may directly suppress voltage-sensitive calcium channels.

5. Thus, ionotropic and metabotropic glutamate receptors inhibit distinct calcium channels. They could act synergistically, since both increase internal calcium. These pathways provide negative feedback that can reduce calcium influx when ganglion cells are depolarized.

	INTRODUCTION

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Many neurotransmitters suppress voltage-activated calcium channels, particularly the high-voltage-activated L- and N-types (Hille, 1994). One function of this inhibition is to autoregulate transmitter release by presynaptic negative feedback. A conventional model is that transmitter release from the presynaptic terminal results in activation of postsynaptic ionotropic receptors and concomitant stimulation of presynaptic metabotropic receptors. However, there are many examples of postsynaptic metabotropic receptors. In fact, the first description of a metabotropic glutamate receptor was the retinal 'on' bipolar postsynaptic receptor (Slaughter & Miller, 1981; Nawy & Jahr, 1990; Shiells & Falk, 1990). There is also evidence that ionotropic glutamate receptors may suppress voltage-gated calcium channels (Nistri & Cherubini, 1991). Clearly, narrowly defined roles do not adequately detail the physiology of ionotropic and metabotropic glutamate receptors.

We chose to examine the glutamatergic regulation of calcium currents in amphibian retinal ganglion cells. These neurons possess a plethora of glutamate receptors, including the AMPA/kainate and NMDA ionotropic receptors (Slaughter & Miller, 1983) and at least one form of metabotropic receptor (Akopian & Witkovsky, 1996). Additionally, these neurons are thought to be purely postsynaptic in the retina and therefore ionotropic and metabotropic receptors may function in a unique manner to control postsynaptic integration rather than regulation of transmitter release.

Recordings from ganglion cells revealed sustained, high-voltage-activated calcium channel currents. These currents were suppressed by both ionotropic and metabotropic glutamate agonists. Ganglion cells possessed all three groups of metabotropic receptor, although group III agonists produced the largest effect on the calcium channel. The metabotropic receptors stimulated calcium release from internal stores, acting through both inositol trisphosphate (IP₃) and ryanodine receptors, leading to suppression of L-type calcium channels. The kainate-mediated inhibition of N-type calcium channels probably involves two mechanisms, one mediated by calcium-calmodulin and one by phosphatases.

	METHODS

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Experiments were performed in isolated neurons and a tissue slice of retina from the aquatic tiger salamander (Ambystoma tigrinum) in accordance with National Institutes of Health and University Animal Care guidelines. The methodology was described in detail previously (Tian & Slaughter, 1994; Pan & Slaughter, 1995). In brief, the animal was stunned, decapitated, double pithed, and the eye was enucleated. In retinal slice recordings, the retina was separated from the pigment epithelium and positioned, photoreceptor-side down, on filter paper. The retina and filter paper were sectioned into 200-300 µm-thick slices, which were placed in a perfusion chamber and observed with an upright microscope fitted with Hoffman modulation optics. For studies of isolated cells, the retina was placed in a papain-containing Ringer solution (12 U ml^-1 papain; Worthington Biochemicals) for 45-60 min at room temperature (22°C). Then the retina was dissociated in calcium-free Ringer solution. Cells were placed on coverslips treated with lectin to promote cell adhesion and stored in Ringer solution in a 17°C incubator. Neurons were studied within a few hours of dissociation.

In the retinal slice, recordings were obtained from neurons in the ganglion cell layer, a somatic layer that Lukasiewicz & Werblin (1988) found to be almost exclusively populated by ganglion cells. In the isolated cell preparation, neurons were identified as ganglion cells based on morphology, the presence of a large sodium current, and the absence of an inwardly rectifying current. These criteria were developed by characterizing properties of second- and third-order neurons in the slice preparation.

Whole-cell recordings were obtained with low-resistance electrodes (< 5 M) filled with pipette solution consisting of (mM): 106 potassium gluconate, 5 NaCl, 2 MgCl₂, 5 EGTA, and 5 Hepes, buffered to pH 7·4 with KOH, except where noted in the text. In addition, the solution contained an 'ATP regenerating cocktail' consisting of 4 mM ATP, 20 mM phosphocreatine, and 50 U ml^-1 creatine phosphokinase.

Control amphibian Ringer solution consisted of (mM): 111 NaCl, 2·5 KCl, 1·8 CaCl₂, 1 MgCl₂, 10 dextrose, and 5 Hepes, buffered to pH 7·8 and oxygenated. To isolate calcium currents, 10 mM BaCl₂ and 40 mM TEA-Cl replaced equimolar NaCl and CaCl₂ in the extracellular medium after whole-cell recordings were obtained. Tetrodotoxin (TTX, 1 µM) was employed to block sodium currents.

Because metabotropic agonists did not alter the membrane potential of neurons in the retinal slice preparation, we performed studies of metabotropic receptors in both the tissue slice and isolated cells. Studies involving ionotropic glutamate agonists were performed in isolated, single neurons to avoid trans-synaptic effects.

Electrophysiological data were collected with a List EPC-9 amplifier, HEKA Pulse software and a Macintosh computer, and analysed with Igor and Excel software. The analog signals were filtered at 5 kHz but were not leak subtracted since maintained ligand-gated currents were monitored. Access resistance was generally 6-12 M and was not compensated. Data are expressed as means ± standard error of the mean (S.E.M.). Student's t test was used for statistical comparisons between cells treated with different internal solutions.

Both voltage steps and ramps were used to monitor calcium channel currents and, since these channels inactivated slowly, both gave similar results, as illustrated in Fig. 1. This appears to justify using the two protocols interchangeably for the purposes of the experiments in this report. It was often more convenient to use voltage ramps to avoid calcium channel 'run-down'. The pipette solution contained high potassium, rather than caesium which is commonly employed to block potassium currents, since potassium might be important for enzyme action. This raised the question whether the calcium channel current was isolated under these experimental conditions. Figure 1C shows that, in external solution containing 10 mM barium and 40 mM TEA, almost all the inward current was blocked by 50 µM cadmium. At the most depolarized potentials there was an outward current, indicating that the potassium current was not completely blocked. However, for most of the voltage range (particularly the region below +10 mV), potassium contributed very little to the overall current. Therefore, this ion substitution was sufficient to evaluate the influence of glutamate analogues on calcium channel current. This figure also shows that kainate (in the presence of 50 µM cadmium) induced a current that was comparatively small and reversed near +10 mV. If cells were clamped to +10 mV in an external barium-TEA solution containing 100 µM cadmium, then kainate produced an outward current that averaged 16 ± 2 pA (n = 9). This indicates that ligand-gated glutamate receptor current will not appreciably distort measurements of barium current. In many of the following figures, a single step to +10 mV is illustrated for simplicity of presentation. This voltage evoked a near-maximal barium current, was very close to the reversal potential for glutamate-gated ionotropic currents (Fig. 1C), and produced little potassium current. Therefore, this potential was used to measure relative barium current suppression. However, full voltage ranges were examined for every drug tested.

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Figure 1. Protocols for measuring calcium channel current Inward currents were elicited by either 30 ms step pulses from -50 to +90 mV in 20 mV increments from a holding potential of -70 mV (A), or a 50 ms ramp from -50 to +90 mV (B). C, an inward current was evoked by a 50 ms voltage ramp (-70 to +50 mV). The ramp was repeated in the presence of 50 µM cadmium and again in the presence of both 50 µM cadmium and 100 µM kainate (KA) (dotted trace). Recordings in A and B were obtained from the same neuron in the ganglion cell layer of the slice preparation; the recording in C was obtained from an isolated neuron. The external solution contained 10 mM barium and 40 mM TEA.

To examine intracellular calcium concentration, cells were loaded with 3 µM fura-2 AM for 30 min in a modified Ringer solution (adding 1 mM EGTA and eliminating calcium). Then cells were bathed in normal Ringer solution for 1 h before they were placed on the stage of an inverted Nikon Diaphot, viewed with a × 40 oil-immersion objective, and stimulated with 340 and 380 nm light from a mercury lamp. Emitted fluorescence, passed through a 510 nm dichroic mirror and barrier filter, was detected with a Hamamatsu SIT camera and captured using Metafluor software (Universal Imaging Corp., West Chester, PA, USA). Cell bodies were marked by software as 'areas of interest' and fluorescence signals to 340 and 380 nm light were measured over the somata every 10 s, and the 340 nm/380 nm fluorescence ratio was used as an indicator of relative changes in internal calcium concentration.

All glutamate analogues were obtained from Tocris Cookson. Dantrolene, inositol trisphosphate, dihydropyridines, -conotoxin GVIA, trifluoperazine, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H-7), microcystin, okadaic acid, staurosporine, calmidazolium, Ruthenium Red and heparin were purchased from Research Biochemicals. All other chemicals were obtained from Sigma.

	RESULTS

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Glutamate and its analogues suppressed a sustained, high-voltage-activated barium current (IBa,HVA), as shown in Fig. 2. Application of 200 µM glutamate (which is not a saturating dose in the slice, presumably due to uptake mechanisms) produced an inward current that was apparent at the holding potential of -70 mV (Fig. 2A, arrow). It also produced a decrease in the inward barium current of slightly in excess of 50 %. When the same concentration of glutamate was applied in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an ionotropic glutamate receptor antagonist, no inward current was observed at -70 mV and there was a smaller reduction in barium current. This indicates that glutamate suppressed IBa,HVA through activation of both metabotropic and ionotropic receptors. This was confirmed by application of selective metabotropic (mGluR) and ionotropic (iGluR) receptor agonists. The general mGluR agonist 1-aminocyclopentane-trans-1S,3R-dicarboxylic acid (trans-ACPD) reduced the IBa,HVA evoked by a voltage ramp or a single step to +10 mV (Fig. 2B). IBa,HVA recovered after removing trans-ACPD. Trans-ACPD did not produce a standing current at the holding potential, similar to the effect of glutamate in the presence of CNQX. Kainate, an iGluR agonist, produced an inward current at the holding potential and caused a larger suppression of the barium current (Fig. 2C). The effect of kainate on IBa,HVA did not rapidly recover, even though the ionotropic current disappeared. On average, trans-ACPD reduced IBa,HVA by 16 ± 2 % (n = 50) while kainate reduced IBa,HVA by 35 ± 3 % (n = 18). After trans-ACPD suppressed a portion of IBa,HVA, kainate was still able to produce a large suppression of the remaining IBa,HVA. In six cells the combined effects of these agonists were tested. Trans-ACPD reduced the inward current by 15 ± 2 %, while trans-ACPD plus kainate reduced 44 ± 3 % of the current. The effect of kainate was completely suppressed by 25 µM CNQX, while the effect of trans-ACPD was unaffected by CNQX. These experiments indicate that ionotropic and metabotropic glutamate receptors independently suppress barium current.

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Figure 2. Ionotropic and metabotropic glutamate agonists reduced IBa,HVA A, the upper panel shows a barium inward current produced by a 30 ms step from -70 to +10 mV (continuous line) and the effect of 200 µM glutamate (Glu) on that inward current (dotted trace) and on the holding current (arrow). The lower panel shows the effect of the same concentration of glutamate in the presence of 25 µM CNQX (dotted trace). B, the effect of 100 µM trans-ACPD (ACPD) on the inward barium current is shown by the dotted traces (ramp and step protocols). The continuous lines show 2 traces: control and recovery after removing trans-ACPD. C, the change in the inward barium current and the holding current produced by 100 µM kainate (KA) is shown by the dotted trace, while the continuous traces show the currents before kainate application and after removal of kainate (Washout; arrows). The recording in A was obtained from a neuron in the ganglion cell layer of the slice preparation; recordings in B and C were from isolated cells.

Metabotropic glutamate receptor pharmacology

While trans-ACPD is a broad-spectrum mGluR agonist, there are selective agonists for each of the three subclasses of mGluR (reviewed by Pin & Duvoisin, 1995). Group I receptors (mGluR1 and 5) can be selectively activated by S-3,5-dihydrophenylglycine (DHPG; Schoepp et al. 1994); group II receptors (mGluR2 and 3) by (2S,1'R,2'R,3'R)-2-(2'-3'-dicarboxycyclopropyl)-glycine (DCG-IV; Hayashi et al. 1993); and group III receptors (mGluR4, 6, 7 and 8) by l-2-amino-4-phosphonobutyrate (l-AP4) (Tanabe et al. 1993). As shown in Fig. 3, selective agonists of group I and II produced very modest reductions in IBa,HVA. DHPG and DCG-IV reduced this current by approximately 5 and 4 %, respectively. In contrast, L-AP4 reduced IBa,HVA by approximately 10-12 %.

The small effect of DHPG was surprising because Akopian & Witkovsky (1996) reported that group I receptors were responsible for reducing approximately 50 % of the high-voltage-activated calcium current in Xenopus retinal ganglion cells. We therefore tested the effect of quisqualate, a potent activator of DHPG-sensitive mGluR1 and 5 as well as ionotropic glutamate receptors (Aramori & Nakanishi, 1992). Quisqualate blocked a large portion of the barium current (Fig. 3B); however, most of this effect was mediated by ionotropic receptors and blocked by CNQX. In the presence of this ionotropic antagonist, the effect of quisqualate was similar to that of DHPG.

The specificity of DHPG was examined in occlusion experiments with l-AP4 and (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I), an agonist of group II and III receptors when used at 30 µM. The action of DHPG was additive with both of the other agonists (Fig. 3E). Individual neurons were tested with each agonist alone and then in combination. In five cells, L-CCG-I alone reduced IBa,HVA by 13·5 ± 1·7 %, DHPG alone reduced the current by 4·4 ± 0·9 %, and in combination the decrease was 18·7 ± 2 %. The effect of DHPG was distinct from that of L-CCG-I (P < 0·01). The combined action of DHPG plus L-CCG-I was not statistically different from L-CCG-I alone, probably because of the small sample size. In another seven neurons, l-AP4 alone suppressed the current by 11·6 ± 1·2 %, DHPG again reduced the current by 4 ± 1 %, and together they reduced the current by 16·6 ± 1·7 %. In this case, the effect of DHPG plus l-AP4 was significantly greater (P < 0·05) than the effect of either agonist alone. Thus, the action of DHPG was distinct and did not overlap with agonists for group II or III mGluRs.

DCG-IV, a group II agonist, also produced a small effect, so L-CCG-I was also tested. At a concentration of 1 µM, L-CCG-I is a selective agonist of group II mGluRs, but at higher concentrations it activates other mGluRs (Nakanishi, 1992). L-CCG-I (1 µM) reduced IBa,HVA by 5 %, similar to the effect of DCG-IV. On the other hand, 30 µM L-CCG-I produced a much larger effect than DCG-IV, an effect that was even larger than that of L-AP4. Dose-response curves for L-CCG-I and l-AP4 revealed that both saturated near 30 µM and that at every dose the effect of L-CCG-I exceeded that of l-AP4 (Fig. 3C). L-CCG-I (30 µM) occluded the effects of DCG-IV (Fig. 3D) and l-AP4 (Fig. 3E). For example, in fifteen neurons, L-CCG-I alone decreased IBa,HVA by 14·9 ± 1·5 %, l-AP4 alone reduced IBa,HVA by 10 ± 1·2 %, and the combination produced a 13·5 ± 2·7 % suppression. Similarly, the effects of DCG-IV and L-CCG-I were compared in six cells (Fig. 3D). The action of both agonists in combination (14·1 ± 2·1 % suppression) was no greater than the action of L-CCG-I alone (14·2 ± 1·8 %). This indicates that 30 µM L-CCG-I stimulates both group II and III receptors. Overall, these experiments confirm that ganglion cells possess three distinct types of mGluRs and that group III receptors mediate the largest suppression of IBa,HVA.

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Figure 3. Effects of selective metabotropic glutamate agonists A, IBa,HVA is shown before (continuous traces) or during application of 30 µM L-CCG-I, 30 µM L-AP4 or 30 µM DCG-IV (dotted traces). B, control IBa,HVA (continuous traces) are compared with inward currents evoked during treatment with 30 µM DHPG, 50 µM quisqualate (QA), or 50 µM quisqualate plus 25 µM CNQX (dotted traces). C, the graph shows the concentration dependence of suppression of IBa,HVA for L-CCG-I () and L-AP4 (). D and E, the histograms compare suppression of IBa,HVA by mGluR agonists alone and in combination, using 30 µM of DHPG, L-CCG-I, DCG-IV and L-AP4. Recordings were obtained from neurons in the ganglion cell layer of the slice preparation.

The metabotropic receptor transduction pathway

The trans-ACPD-sensitive metabotropic receptors suppressed dihydropyridine-sensitive calcium channels. Nifedipine (50 µM) largely occluded the effect of 100 µM trans-ACPD, but not the effect of 100 µM kainate (Fig. 4A). In contrast, 800 nM -conotoxin GVIA, a blocker of N-type calcium channels, occluded the action of kainate. Thus, metabotropic and ionotropic glutamate receptors appear to be linked to discrete types of calcium channel. We generally found that -conotoxin GVIA, compared with dihydropyridines, suppressed much more of the barium current. This probably explains why kainate suppressed more of the total IBa,HVA than trans-ACPD did.

The effects of trans-ACPD, L-CCG-I and L-AP4 were all blocked when 1 mM GDPS was included in the pipette solution (data not shown), indicating the involvement of a G-protein. The action of trans-ACPD was not reversed by prepulse facilitation, although this protocol did reverse the inhibitory effect of GABA_B receptor activation (Zhang et al. 1997) (Fig. 4B). Prepulse facilitation is often used as a signature of direct G-protein inactivation of the calcium channel (Boland & Bean, 1993; Campbell et al. 1995).

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Figure 4. Metabotropic and ionotropic glutamate receptors regulate different high-voltage-activated calcium channels A, the inward current was evoked under control conditions (largest inward current in each panel), then in the presence of 50 µM nifedipine (Nif) or 800 nM -conotoxin GVIA (-CTX) (arrows), and then in the presence of glutamate analogues (100 µM trans-ACPD or 100 µM kainate) plus calcium channel antagonists (dotted traces). B, the effects of 100 µM trans-ACPD and 300 µM baclofen (Bac; a GABAB receptor agonist) on barium currents evoked by a test pulse (+10 mV) were compared (arrowheads and dashed lines) before and after a conditioning prepulse to +100 mV. Recordings were obtained from isolated neurons.

The prepulse experiments suggested that mGluRs activate a second messenger cascade. To explore the potential role of protein kinases, the actions of mGluR agonists were tested in the presence of broad-spectrum kinase inhibitors. However, trans-ACPD, L-CCG-I and L-AP4 continued to reduce barium current when a combination of 100 µM H-7, 1 µM staurosporine and 100 µM genistein was applied (Fig. 5A). This indicates that these metabotropic agonists do not require the activation of protein kinase A (PKA), protein kinase C (PKC) or tyrosine kinase, respectively (Fan & Rillema, 1992; Minami et al. 1994; Henry et al. 1996).

The ineffectiveness of kinase inhibitors was surprising because both l-AP4 and L-CCG-I are generally considered to act by reducing forskolin-stimulated cAMP accumulation (Pin & Duvoisin, 1995). This mechanism was tested directly. Forskolin produced a decrease in voltage-dependent barium current. Both l-AP4 and L-CCG-I reversed this effect, increasing the calcium current (Fig. 5B). 3-Isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, also decreased the barium current, but L-CCG-I and l-AP4 further decreased the barium current in the presence of IBMX (Fig. 5C). These experiments indicated that group III mGluR agonists reversed the effect of forskolin. However, this mechanism enhanced IBa,HVA, indicating that another mechanism accounted for the group III suppression of IBa,HVA.

When calcium release from internal stores was blocked, the effects of metabotropic glutamate agonists were suppressed (Fig. 5). Heparin, which inhibits IP₃ receptor activation (Vaca & Kunze, 1995; Lopez & Terzic, 1996), reduced the effect of metabotropic agonists. Heparin reduced the effect of trans-ACPD by 72 % (from 15·8 ± 1·8 to 4·4 ± 1 % reduction of barium current), of L-CCG-I by 70 % (from 14·5 ± 1·8 to 4·3 ± 0·9 %) and of l-AP4 by 69 % (from 10·1 ± 1·9 to 3·1 ± 0·7 %). Neomycin, a blocker of phospholipase C, was also able to suppress the effect of L-CCG-I and l-AP4. In the presence of neomycin, the action of L-CCG-I was reduced by 56 % (from 14·5 ± 1·8 to 6·6 ± 1·2 %), while the action of l-AP4 was reduced by 67 % (from 10·1 ± 1·9 to 3·3 ± 1·1 %). It is unclear why neomycin was less effective in counteracting the effect of l-CCG-I. However, the actions of heparin and neomycin indicate that the IP₃ system mediates at least part of the response to metabotropic agonists.

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Figure 5. The second messenger pathway of metabotropic glutamate agonists A, the histogram details the mean suppression of IBa,HVA produced by glutamate agonists in the presence of various antagonists of signal transduction. Agonists (trans-ACPD, L-CCG-I and L-AP4) were applied through a perfusion pipette near the cell. Antagonists were introduced by dialysis through the recording pipette, which contained the following concentrations: , kinase inhibitors: 100 µM H-7 + 1 µM staurosporine + 100 µM genistein; , 400 µg ml-1 heparin; , 20 µM Ruthenium Red; , 10 µM neomycin; , 100 µM ryanodine. , control. B, IBa,HVA was plotted (measured every 10 s, using a voltage step to +10 mV) during application of 25 µM forskolin alone or with 30 µM L-AP4 or 30 µM L-CCG-I. C, using a similar protocol, the actions of 1 mM IBMX with and without 30 µM L-CCG-I were compared.

Blockers of ryanodine receptors also suppressed the effect of metabotropic glutamate agonists. Internal dialysis with Ruthenium Red (Kuemmerle et al. 1994) had an effect that was similar in magnitude to that of heparin. The block of trans-ACPD action was 73 % (from 15·8 ± 1·8 to 4·3 ± 0·9 %), for L-CCG-I it was 76 % (from 14·5 ± 1·8 to 3·5 ± 0·8 %), and for l-AP4 it was 62 % (from 10·1 ± 1·9 to 3·8 ± 0·8 %). Dantrolene (50 µM), applied extracellularly, had a similar suppressive effect on these metabotropic agonists (data not shown). Including 100 µM ryanodine in the pipette solution reduced the response to l-AP4 by 79 % (from 10·1 ± 1·9 to 2·1 ± 1·7 %). This indicates that release of calcium from ryanodine-sensitive internal stores was involved in the action of L-CCG-I and l-AP4.

EGTA was the calcium buffer normally included in the pipette solution. Based on calculations using Maxchelator v6.81 software, internal bulk calcium was buffered at 9 nM. When 10 mM BAPTA was used to chelate internal calcium to a similar level (3 nM), the effect of trans-ACPD was significantly reduced (Fig. 6A). When BAPTA was in the pipette solution, trans-ACPD only reduced the barium current by 4·1 ± 1·5 % (n = 7), compared with a mean reduction of 15·8 ± 1·8 % (n = 50) when EGTA was the buffer. This supports the hypothesis that internal calcium is an important factor in mGluR modulation of voltage-gated calcium channels.

The influence of heparin and of Ruthenium Red were superadditive (each blocked about 70 % of the trans-ACPD effect), suggesting that metabotropic glutamate receptor transduction was mediated by an interaction between the IP₃ and ryanodine receptors. To examine this, the two pathways were regulated in combination. For example, in Fig. 6B (left panel) the cell was dialysed with heparin and then caffeine was applied. Caffeine suppressed IBa,HVA. Similar observations were made in three other neurons. In a related experiment, a cell was dialysed with IP₃ (Fig. 6B, middle panel), which caused a progressive decrease in IBa,HVA. In the presence of IP₃, caffeine application produced an additional, but smaller, suppression of the barium current. A straight line was drawn through the IP₃ data points, illustrating the progressive effect of IP₃ dialysis and the additional influence of caffeine application. Similar effects were seen in three cells. IBa,HVA did not decline when cells were dialysed with both heparin and IP₃ (not shown). While heparin did not block the action of caffeine, dantrolene did suppress the effect of IP₃. As shown in Fig. 6B (right panel), internal dialysis of a neuron with 100 µM IP₃ produced a progressive decrease in IBa,HVA. Application of dantrolene, a ryanodine receptor blocker, stopped the decline in the calcium current, and this decline resumed again when dantrolene was removed. Straight lines were drawn through the data to illustrate the rate of change of the calcium current under different conditions. These experiments indicate that IP₃ and ryanodine receptor activation can lead to a suppression of IBa,HVA. This suggests that the effect of IP₃ on barium current depends upon a functional ryanodine receptor pathway. In contrast, when the IP₃ system was blocked by heparin, caffeine was still able to suppress the barium current.

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Figure 6. Influence of internal calcium on IBa,HVA A, in the presence of 10 mM internal BAPTA, inward barium currents were measured before (continuous trace) and during application of 100 µM trans-ACPD (dotted trace). The histogram summarizes results from 50 control cells dialysed with control pipette solution (5 mM EGTA) and 7 neurons dialysed with pipette solution containing 10 mM BAPTA. B, these graphs show the influence of stimulators of internal calcium release. IBa,HVA was measured every 10 s during application of 10 mM caffeine or 50 µM dantrolene (filled bars) after the neuron was dialysed with either heparin (400 µg ml-1) or IP3 (100 µM). Dotted lines were fitted by eye. Recordings were obtained from isolated neurons.

These results suggest that activation of group III mGluRs caused an increase in internal calcium concentration. To investigate this directly, isolated neurons were loaded with fura-2 AM and internal calcium concentration was monitored. Cell bodies were marked as 'regions of interest' and their calcium fluorescence signals were monitored every 10 s. If the cells maintained a steady signal for several minutes, trans-ACPD or l-AP4 was bath applied. Both produced an increase in internal calcium concentration (Fig. 7). Since voltage-clamp apparatus was unavailable in the calcium-imaging station, NMDA was used to identify third-order neurons. Only amacrine cells and ganglion cells are stimulated by NMDA, which would be expected to increase internal calcium concentration either by permeation through NMDA channels or through activation of voltage-dependent calcium channels (Slaughter & Miller, 1983). The cells that were sensitive to trans-ACPD and l-AP4 were also sensitive to NMDA.

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Figure 7. Trans-ACPD and L-AP4 increased internal calcium concentration in third-order neurons The neuron was loaded with fura-2 AM. The effects of 100 µM trans-ACPD, 50 µM L-AP4 and 100 µM NMDA on the 340 nm/380 nm fluorescence ratio are shown.

The ionotropic receptor transduction pathway

Kainate modulation of IBa,HVA recruited a different second messenger pathway. Like the metabotropic glutamate receptor pathway, it was independent of protein kinase A, protein kinase C and tyrosine kinase. Staurosporine, H-7 and genistein did not interfere with suppression of the barium current by kainate, but calmodulin antagonists were effective blockers of the effect. For example, internal dialysis with 100 µM trifluoperazine (Vandonselaar et al. 1994) reduced the kainate-induced suppression of the barium current by 74 %, from a mean suppression of 35 ± 3 to 9 ± 3 % (n = 6; Fig. 8C and D). Trifluoperazine did not suppress the action of metabotropic agonists (Fig. 8A and B). Similarly, 100 µM calmidazolium (Van Belle, 1981) reduced the effect of kainate on IBa,HVA by 74 %, from a mean of 35 ± 3 to 9 ± 3 % (n = 6; Fig. 9). The reduced effect of kainate in the presence of trifluoperazine and calmidazolium was statistically significant (P < 0·01).

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Figure 8. Trifluoperazine, a calmodulin antagonist, suppressed the action of kainate, but not of trans-ACPD, on IBa,HVA A, the action of 100 µM trans-ACPD on IBa,HVA is compared in 1 neuron using control pipette solution (left trace) and in another neuron in which 100 µM trifluoperazine (TFP) was included in the pipette solution (right trace). B, summary of the percentage suppression produced by 100 µM trans-ACPD for 10 neurons with each pipette solution. C and D, experiments similar to those in A and B, testing the effect of 100 µM trifluoperazine on 100 µM kainate action. In A and C, dotted traces depict the responses in the presence of glutamate agonists; continuous traces indicate control currents. Arrowheads identify ionotropic glutamate currents in C. Recordings were obtained from isolated neurons.

Phosphatase inhibitors (Fig. 9) also suppressed the effect of kainate. Internal dialysis with 50 µM cyclosporin A (Wiederrecht et al. 1993) reduced the kainate suppression of I_Ba,HVA by 80 %, from 35 ± 3 to 7 ± 2 % (n = 10, P < 0·01). Dialysis with 3 µM okadaic acid reduced the kainate suppression by 63 %, from 35 ± 3 to 13 ± 3 % (n = 7, P < 0·01).

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Figure 9. Calmodulin phosphatase inhibitors suppress the response to kainate A, after cells were dialysed with various antagonists, inward currents were evoked in the presence (dotted traces) or absence (continuous traces) of 100 µM kainate. Arrowheads identify ionotropic glutamate currents. B, summary of results comparing the percentage suppression of IBa,HVA produced by kainate with normal pipette solution (Control) versus pipette solution containing calmodulin or phosphatase antagonists. Recordings were obtained from isolated neurons. CSA, cyclosporin A (50 µM); CMZ, calmidazolium (100 µM); OKA, okadaic acid (3 µM).

While these agents reduced the effect of kainate on the barium current, kainate was still able to produce a normal amplitude ionotropic current. For cells in which drugs were dialysed through the recording pipette, the mean ionotropic current (measured at -70 mV) was compared with the ionotropic current under control conditions. Under control conditions this kainate current was 192 ± 43 pA (n = 18), with cyclosporin A it was 187 ± 32 pA (n = 10), with okadaic acid it was 196 ± 46 pA (n = 7), with trifluoperazine it was 197 ± 49 pA (n = 6), and for calmodulin it was 181 ± 45 pA (n = 6). There was no indication that the inhibition of the action of kainate on I_Ba,HVA was due to a reduction in activity of the kainate receptor.

If calmodulin mediates the action of kainate, it suggests that influx of divalent ions through glutamate channels could be important in generating this response. Internal dialysis with BAPTA, a rapid chelator of divalent ions, was used to examine this pathway. When the recording pipette contained 10 mM BAPTA, kainate suppression of I_Ba,HVA was reduced by about half (from 35 ± 3 to 18 ± 2 %, n = 7) (data not shown).

A surprising observation was that the time required to reach a steady-state reduction of the barium current was longer for ionotropic agonists than for metabotropic agonists. The time course of agonist action was monitored by measuring I_Ba,HVA every 10 s during drug application (Fig. 10). Trans-ACPD produced a maximal suppression of the current measured at the first time point after drug application. In contrast, although a significant kainate effect was detected at the first time point, the influence of kainate continued to rise and did not reach a maximum until about 1 min. The slow response was not due to slow perfusion or receptor activation, since monitoring the ionotropic kainate current confirmed that kainate activated its receptors quickly. This suggests that kainate was producing a factor that slowly increased and progressively suppressed the high-voltage-activated calcium channels.

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Figure 10. The effect of kainate was faster when calcium, instead of barium, was the current carrier In A and B, the external solution contained 10 mM barium plus 40 mM TEA; in C, barium was replaced with 10 mM calcium. In each panel, the voltage-gated inward current (peak current during a step to +10 mV) was measured every 10 s and plotted (). In addition, the current measured at the holding potential (-70 mV) was plotted in the lower graphs (labelled ILigand). Kainate (100 µM) or trans-ACPD (100 µM) was applied as indicated by the filled bars. D and E, voltage-gated inward currents were measured in a calcium-free external solution containing TTX and 111 mM sodium. The effect of 100 µM kainate (D) or 30 µM L-AP4 (E) is shown (dotted traces). Arrowhead indicates presence of ionotropic current (D). Inward currents were evoked by steps from -70 to 0 mV. Recordings were obtained from isolated neurons.

The factor is probably divalent ions that cross the membrane to activate calmodulin. When calcium was used in place of external barium, the effect of kainate on I_Ca,HVA was rapid (Fig. 10C). This suggests that the slow action of kainate was due to the time course of events between barium entry and calmodulin activation. To explore this further, divalent ions were removed from the extracellular solution (111 mM NaCl, 2·5 mM KCl, 5·6 mM TEA-Cl, 5 mM Hepes and 1 mM EGTA), and potassium channels were blocked by the solution in the patch pipette (100 mM TEA-Cl, 5 mM NaCl, 1 mM MgCl₂, 5 mM Hepes and 5 mM EGTA, plus the ATP-regenerating components 4 mM ATP, 20 mM phosphocreatine and 50 U ml^-1 creatine phosphokinase). Under these conditions the calcium channel is very permeable to sodium. In this external medium a I_Na,HVA was observed that was blocked by 2 mM cadmium (this higher concentration of cadmium was probably required because of the presence of EGTA). Under these conditions kainate had comparatively little effect on voltage-activated inward current, while the effect of l-AP4 was similar to that observed previously (Fig. 10D and E). In eight cells, 100 µM kainate reduced the inward current by 7·5 ± 1·3 % while 30 µM l-AP4 reduced the current by 18·3 ± 1·9 %. The average kainate-induced receptor current was 189 ± 36 pA at a holding potential of -70 mV. This supports the conclusion that influx of divalent ions is important in mediating the action of kainate.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Glutamate reduced I_Ba,HVA in retinal ganglion cells through activation of iGluRs as well as mGluRs. The former inhibited -conotoxin GVIA-sensitive channels while the latter inhibited dihydropyridine-sensitive channels. The iGluRs produced a larger effect, in part because N-type calcium channels account for more current than L-type calcium channels. Both pathways increased intracellular calcium, although mGluR elevation resulted from release from internal stores, while iGluRs produced an influx from outside of the cell. Despite their common dependence on calcium, the second messenger pathways exhibited distinct differences, implying compartmentalization of the elevated calcium produced by the two receptor systems.

Metabotropic receptor pharmacology

Molecular studies have identified eight genes which encode mGluRs (reviewed by Pin & Duvoisin, 1995). The eight genes fall into three groups. The effects of selective agonists on retinal ganglion cells indicate that all three groups of mGluR exist in retinal ganglion cells. DHPG, DCG-IV and l-AP4 activated distinct receptors and appeared to be selective agonists for group I, II and III mGluRs, respectively. The group III receptors are activated by trans-ACPD and L-CCG-I in retinal ganglion cells, as are the group III receptors on retinal 'on' bipolar cells (Tian & Slaughter, 1994; Thoreson & Ulphani, 1995). Thus, the predominant mGluR in salamander ganglion cells is pharmacologically similar to that found in 'on' bipolar cells.

The group III receptor appears to mediate its response through activation of IP₃ receptors. This transduction mechanism is most often associated with group I receptors, while group II and III receptors are linked to inhibition of adenylyl cyclase. However, there is probably not a one-to-one correspondence between receptors and transduction mechanisms. Group III receptors can stimulate phosphotidylinositol hydrolysis in neurons (Schoepp & Johnson, 1988), and group II receptors (mGluR2) can increase inositol phosphate formation in expression systems (Tanabe et al. 1992). Group III receptors stimulate phosphodiesterase in retinal bipolar cells (Nawy & Jahr, 1990; Shiells & Falk, 1990) and guanylyl cyclase in retinal horizontal cells (Dixon & Copenhagen, 1997). Therefore, group III mGluRs can activate numerous transduction cascades. Studies have demonstrated a similar plethora of transduction mechanisms in the group I receptors (Aramori & Nakanishi, 1992).

Transduction pathways

The mGluR pathway was suppressed by GDPS, neomycin and heparin. This suggests that mGluRs stimulated G-proteins that activated phospholipase C, generating inositol trisphosphate and diacylglycerol. Diacylglycerol stimulation of protein kinase C was not required for mGluR action, since H-7 and staurosporine did not suppress the action of mGluR agonists. Direct application of IP₃ reduced I_Ba,HVA, indicating that this pathway could account for the effects of mGluR activation.

Antagonists of ryanodine receptors (dantrolene, ryanodine and Ruthenium Red) also suppressed the effect of mGluR agonists. Ryanodine and IP₃ receptor antagonists each blocked approximately 80 % of mGluR action, indicating that these two pathways were not independent. Furthermore, ryanodine receptor blockers suppressed the effect of exogenous IP₃, while IP₃ receptor inhibition did not block the effect of caffeine. This suggests that IP₃ receptor activation led to stimulation of ryanodine receptors. This sequence is incorporated in the tentative model shown in Fig. 11, where it is proposed that activation of group III mGluRs caused a G-protein stimulation of phospholipase C, leading to the formation of IP₃. Then, IP₃ activated release of calcium from internal stores that stimulated ryanodine receptors, leading to a further increase in internal calcium concentration. This suppressed I_Ba,HVA, although additional intervening steps may be required.

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Figure 11. A model of the transduction pathways for ionotropic and metabotropic glutamate receptors The boxed items indicate proposed transduction steps. The agents used to identify each step are listed adjacent to the processes they affect. The model proposes that metabotropic receptors activate phospholipase C (PLC) resulting in production of IP3 and increased internal calcium release, which stimulates further calcium release from ryanodine receptors. This leads to a suppression of L-type calcium channels. Ionotropic receptors produce a transmembrane increase in internal calcium that stimulates calmodulin and phosphatase, inhibiting N-type calcium channels, possibly by a direct dephosphorylation of the calcium channel.

The kainate inhibition of I_Ba,HVA was linked to calmodulin and phosphatases. Trifluoperazine and calmidazolium, calmodulin antagonists, each suppressed approximately 75 % of kainate action. Okadaic acid, a broad-spectrum phosphatase inhibitor, reduced the effect of kainate by 63 %, while cyclosporin A, a more specific inhibitor of calmodulin-dependent phosphatases, blocked 80 %. Antagonists of either calmodulin or phosphatase eliminated most of the kainate effect, again indicating that these pathways interacted. The simplest model of this transduction pathway is that kainate-sensitive glutamate receptor channels permitted influx of divalent ions, which activated calmodulin and a calcium-calmodulin-dependent phosphatase. The phosphatase may have directly dephosphorylated and suppressed I_Ca,HVA. The weak kainate effect in the absence of external divalent ions, and the difference in time course of the kainate action in the presence of external barium and external calcium support the importance of transmembrane movement of divalent ions in this process. A model of this transduction cascade is shown in Fig. 11.

A similar iGluR-mediated transduction pathway has been identified in other systems. Both NMDA and kainate have been shown to reduce a I_Ca,HVA in cultured rat hypothalamic neurons by a transduction mechanism that was completely blocked by trifluoperazine, calmidazolium and BAPTA (Zeilhofer et al. 1993). However, these neurons did not possess a parallel regulation by mGluRs and the effect of kainate was on both L- and N-type calcium channels. Another example is the NMDA receptor regulation of the Na⁺-K⁺-ATPase, which is mediated by calmodulin and calcineurin (Marcaida et al. 1996).

Calcium and possible synergy

BAPTA reduced the effects of both iGluRs and mGluRs, while EGTA did not. The primary difference between EGTA and BAPTA buffering was probably the buffering speed, and therefore the calcium diffusion distance (Naraghi & Neher, 1997). BAPTA was very effective at blocking mGluR effects, implying that internal calcium release sites were close to their effectors. BAPTA was less effective at blocking iGluR action. This may indicate that calmodulin had better access to divalent ion influx, either because it was very close to the mouth of the calcium channel or because of its relative affinity. The relative effectiveness of BAPTA versus EGTA indicates that the transduction pathways are spatially restricted in both receptor systems.

Metabotropic glutamate receptors in retinal ganglion cells

In situ hybridization and specific antibodies have been used to identify mGluRs in rat retina. Retinal ganglion cells express some members of all three groups: mGluR1, 2, 4, and 7 (Akazawa et al. 1994; Hartveit et al. 1995). This is consistent with our physiological studies in amphibian retina.

There have only been a few electrophysiological studies on mGluRs in retinal ganglion cells, with little convergence on general principles. Perhaps most surprising, isolated Xenopus ganglion cells have predominantly group I receptors, which inhibit almost half of the total high-voltage-activated calcium current (Akopian & Witkovsky, 1996). Interestingly, l-AP4 and L-CCG-I reduced 12 % and 14 % of the calcium current in Xenopus, respectively. This is similar to their effectiveness in salamander ganglion cells. Another similarity is that mGluRs inhibited dihydropyridine-sensitive calcium channels. The striking differences are that dihydropyridine-sensitive calcium channels represent a much smaller fraction of the high-voltage-activated channels in salamander ganglion cells, and that group I receptors modulate a modest proportion of these channels.

Studies in cultured postnatal mouse retinal ganglion cells yielded very different observations. Rothe et al. (1994) found that trans-ACPD, quisqualate (in the presence of iGluR blockers), and l-AP4 affected an -conotoxin-sensitive calcium current. Furthermore, these agonists did not alter internal calcium concentration. Again, this is surprising because quisqualate activates group I receptors, which generally activate the IP₃ system. Thus, while all the studies show that ganglion cells possess a variety of mGluRs, they also indicate that the relative balance of receptors may differ, as do the transduction pathways and the linkage to specific calcium channels.

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Acknowledgements

We thank Dr Michael Duffey for assistance with the calcium-imaging experiments. This work was supported by grant EY05725 from the National Eye Institute.

Corresponding author

W. Shen: SUNY, Department of Physiology and Biophysics, 124 Sherman Hall, Buffalo, NY 14214, USA.

Email: WENSHEN{at}ACSU.BUFFALO.EDU

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