Characterization of the mGluR1-Mediated Electrical and Calcium Signaling in Purkinje Cells of Mouse Cerebellar Slices

F. Tempia,1,2 M. E. Alojado,1 P. Strata,2 and T. Knöpfel1

 1Laboratory for Neuronal Circuit Dynamics, Brain Science Institute, RIKEN, Saitama 351-0198, Japan; and  2Department of Neuroscience and Rita Montalcini Centre for Brain Repair, University of Turin, I-10125 Turin, Italy


    ABSTRACT
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INTRODUCTION
METHODS
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DISCUSSION
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Tempia, F., M. E. Alojado, P. Strata, and T. Knöpfel. Characterization of the mGluR1-Mediated Electrical and Calcium Signaling in Purkinje Cells of Mouse Cerebellar Slices. J. Neurophysiol. 86: 1389-1397, 2001. The metabotropic glutamate receptor 1 (mGluR1) plays a fundamental role in postnatal development and plasticity of ionotropic glutamate receptor-mediated synaptic excitation of cerebellar Purkinje cells. Synaptic activation of mGluR1 by brief tetanic stimulation of parallel fibers evokes a slow excitatory postsynaptic current and an elevation of intracellular calcium concentration ([Ca2+]i) in Purkinje cells. The mechanism underlying these responses has not been identified yet. Here we investigated the responses to synaptic and direct activation of mGluR1 using whole cell patch-clamp recordings in combination with microfluorometric measurements of [Ca2+]i in mouse Purkinje cells. Following pharmacological block of ionotropic glutamate receptors, two to six stimuli applied to parallel fibers at 100 Hz evoked a slow inward current that was associated with an elevation of [Ca2+]i. Both the inward current and the rise in [Ca2+]i increased in size with increasing number of pulses albeit with no clear difference between the minimal number of pulses required to evoke these responses. Application of the mGluR1 agonist (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) by means of short-lasting (5-100 ms) pressure pulses delivered through an agonist-containing pipette positioned over the Purkinje cell dendrite, evoked responses resembling the synaptically induced inward current and elevation of [Ca2+]i. No increase in [Ca2+]i was observed with inward currents of comparable amplitudes induced by the ionotropic glutamate receptor agonist AMPA. The 3,5-DHPG-induced inward current but not the associated increase in [Ca2+]i was depressed when extracellular Na+ was replaced by choline, but, surprisingly, both responses were also depressed when bathing the tissue in a low calcium (0.125 mM) or calcium-free/EGTA solution. Thapsigargin (10 µM) and cyclopiazonic acid (30 µM), inhibitors of sarco-endoplasmic reticulum Ca2+-ATPase, had little effect on either the inward current or the elevation in [Ca2+]i induced by 3,5-DHPG. Furthermore, the inward current induced by 3,5-DHPG was neither blocked by 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy] ethyl-1H-imidazole, an inhibitor of store operated calcium influx, nor by nimodipine or omega-agatoxin, blockers of voltage-gated calcium channels. These electrophysiological and Ca2+-imaging experiments suggest that the mGluR1-mediated inward current, although mainly carried by Na+, involves influx of Ca2+ from the extracellular space.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Electrophysiological studies both in vitro and in vivo have demonstrated that activation of metabotropic glutamate receptors (mGluRs) induces an excitation of cerebellar Purkinje cells (Batchelor et al. 1997; Lingenhöhl et al. 1993; Staub et al. 1992; Tempia et al. 1998). Pharmacological and immunohistochemical studies have established that this response is mediated by the subtype mGluR1, which forms together with mGluR5, the group I within the family of mGluRs (Batchelor et al. 1997; Mateos et al. 2000; Nusser et al. 1994). In heterologous expression systems, group I mGluRs couple via Gq class alpha subunits of GTP-binding proteins (G proteins) and phospholipase C (PLC) to the inositol trisphosphate (IP3)/calcium and diacylglycerol/protein kinase C pathway (Knöpfel et al. 1995; Pin and Duvoisin 1995). In addition, at least the splice variant mGluR1a promotes cAMP accumulation (Aramori and Nakanishi 1992). In a variety of neurons, group I mGluRs (mGluR1 and mGluR5) are expressed at the somato-dendritic membrane and mediate excitatory responses by a multitude of mechanisms (Charpak et al. 1990; Chavis et al. 1996; Crepel et al. 1994; Guèrineau et al. 1995; Staub et al. 1992). Cerebellar Purkinje cells express high levels of mGluR1 in their dendritic spines where it is located perisynaptically (Mateos et al. 2000; Nusser et al. 1994). Work from several laboratories has established that in cerebellar Purkinje cells bath application of mGluR1 agonists or repetitive parallel fiber stimulation can induce an inward current and depolarization as well as an increase in [Ca2+]i (Batchelor et al. 1997; Finch and Augustine 1998; Staub et al. 1992; Takechi et al. 1998; Tempia et al. 1998; Vranesic et al. 1991; Wang et al. 2000). However, some of the previously reported observations are contradictory. For instance, it was reported that (±)-1-Aminocylopentano-trans-1,3-dicarboxylic acid (t-ACPD), a specific mGluR agonist, did not induce an elevation of [Ca2+]i, while repetitive parallel fiber stimulation could induce a mGluR-mediated calcium signal without accompanying membrane current (Finch and Augustine 1998; Llano et al. 1991; Takechi et al. 1998). Thus it is not clear whether there is a causal relationship between the mGluR1-mediated membrane current and the associated elevation of [Ca2+]i or whether these two signals are mediated by independent pathways. When changes in [Ca2+]i were suppressed by intracellular Ca2+ chelators, the mGluR1-induced current was either also depressed (Linden et al. 1994; Staub et al. 1992) or only slightly affected (Hirono et al. 1998; Tempia et al. 1998). PLC inhibitors were reported as either effective (Netzeband et al. 1997) or ineffective (Hirono et al. 1998; Tempia et al. 1998) in blocking the mGluR1-induced current. In cultured Purkinje cells the mGluR1-mediated current depended on the extracellular concentration of Na+ ([Na+]o) (Linden et al. 1994; Staub et al. 1992) and the mGluR1-mediated excitatory postsynaptic current (EPSC) and the (S)-3,5-dihydroxyphenylglycine (3,5-DHPG)-induced inward current were associated with an increase of the intracellular concentration of Na+ ([Na+]i) (Knöpfel et al. 2000). The present experiments were designed to further elucidate the relationship between calcium elevations and the mGluR1-mediated inward current in mouse cerebellar Purkinje cells.


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METHODS
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Electrophysiology

Parasagittal cerebellar slices were prepared from 18- to 28-day-old CD-1 (ICR) mice following previously established techniques (Batchelor et al. 1997; Knöpfel et al. 2000; Tempia et al. 1998). Briefly, the animals were anesthetized with ether and decapitated. The cerebellar vermis was removed and placed in ice-cold extracellular saline solution containing (in mM) 118 NaCl, 3 KCl, 1 MgCl2, 2 CaCl2, 1 NaH2PO4, 25 NaHCO3, and 10 D-glucose. The solution was gassed with 95% O2-5% CO2 resulting in a pH of 7.4. Parasagittal cerebellar slices (200 µm thick) were cut using a vibratome and then incubated at 35°C for the first hour and then at 25°C for up to 8 h. After at least 1 h of incubation, a single slice was transferred into a recording chamber and fully submerged in a continuously flowing extracellular saline solution (24-26°C, 2 ml/min) gassed with 95% O2-5% CO2.

Whole cell patch-clamp recordings were obtained from Purkinje cells visualized using a ×40 water-immersion objective of an upright fixed stage microscope. Pipettes were prepared from borosilicate glass and had a tip diameter of 2-3 µm. When filled with an intracellular solution their resistance was 1.7-3 MOmega . The internal pipette solution consisted of (in mM) 137 K gluconate, 10 HEPES, 4 MgCl2, 0.5 EGTA, 4 ATP dipotassium salt, 0.5 GTP sodium salt, and 0.2 mM Oregon Green 488 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA-1; Molecular Probes, Eugene, OR). The pH was adjusted to 7.3 with KOH. Recordings were performed in whole cell configuration voltage-clamp using an Axopatch amplifier (Axon Instruments, Foster City, CA). A glass pipette pulled from sodalime glass (tip diameter, 3-10 µm) and filled with extracellular solution was used for electrical stimulation. This stimulation electrode was placed gently into the slice with its tip above the border of the dye-filled dendrite of the Purkinje cell. Negative current pulses (200-300 µs) ranging from 5 to 40 µA were delivered for stimulation of parallel fibers. Direct activation of postsynaptic receptors was achieved by agonist application via a borosilicate glass pipette (tip diameter, 2-4 µm), which was positioned as described in the preceding text for electrical stimulation and which was connected to a pressure application system (Picospritzer, 20-30 psi, 5-100 ms). Calcium-free extracellular solution was prepared by omitting CaCl2, increasing MgCl2 to 3 mM and adding 1 mM EGTA. Solution containing 0.125 mM Ca2+ contained 2.875 mM MgCl2. Peptide toxins were directly dissolved in extracellular solution complemented with 0.1 mg/ml bovine serum albumin and the same solution was used as control.

Microfluorometry

Fluorescence of the calcium indicator Oregon Green 488 BAPTA-1 was excited by epi-illumination with light provided by a monochromator and detected by a cooled 12 bit CCD under control of Axon Imaging Workbench software (Axon Instruments). Excitation wavelength was 490 nm. Emission light passed a barrier filter (500 nm).

Fluorescence images were corrected for background fluorescence (measured from image regions free of dye). Changes of [Ca2+]i were expressed as relative fluorescence changes (Delta F/F values) as described earlier, and pseudo-color-coded maps of Delta F/F were masked with the fluorescence image of the cell (Muri and Knöpfel 1994). Analysis was done using Origin 5 software (Microcal). Values in the text are means ± SE. Student's t-test was used for statistical analysis.

Chemicals

6-Nitro-7-sulfamoylbenzo [f] quinoxaline-2,3-dione (NBQX), (S)-a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), thapsigargin, cyclopiazonic acid (CPA), 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF 96365), and D-2-amino-5-phosphono-pentanoic acid (DAPV), were obtained from Tocris Cookson (Bristol, UK); omega-agatoxin was purchased from Alomone Labs (Israel), and tetrodotoxin (TTX) and all other compounds were from Sigma.


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INTRODUCTION
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Repetitive stimulation of parallel fibers induces a slow inward current associated with an elevation of [Ca2+]i

Following the formation of the whole cell configuration, voltage-clamped Purkinje cells were loaded with the fluorescent calcium indicator in the patch pipette for 15-25 min. For the investigation of synaptically induced responses, Purkinje cells were voltage-clamped at -70 mV and recorded in the presence of bicuculline (20 µM) or picrotoxin (10 µM) to block gamma-aminobutyric acid-A (GABAA) receptors and NBQX (10 µM) and D-APV (50 µM) to block ionotropic glutamate receptors (Batchelor and Garthwaite 1997; Batchelor et al. 1997; Tempia et al. 1998). The stimulation electrode was placed over the dendritic tree of the dye loaded cell and, as previously established, two to six stimuli delivered at 100 Hz to parallel fibers induced a mGluR1-EPSC (Tempia et al. 1998). Figure 1 shows recordings of such a mGluR1-EPSC in combination with [Ca2+]i imaging. The mGluR1-EPSC was associated with a clear increase in [Ca2+]i that was restricted to a region of the dendrite close to the stimulation electrode (Fig. 1, A-D). We wondered whether the elevation of [Ca2+]i could be seen in isolation, that is, without an accompanying membrane current, if only a small number of pulses were applied. Such a different threshold for the calcium signals relative to the associated membrane current was recently suggested (Finch and Augustine 1998; Takechi et al. 1998). To test this possibility, we varied the number of stimulation pulses applied and plotted the membrane current and [Ca2+]i responses against the number of pulses. Under our experimental conditions, both the inward current and the rise in [Ca2+]i increased in size with increasing number of pulses with no clear difference in the minimal number of pulses required to evoke these responses (n = 4 cells; Fig. 1, E and F).



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Fig. 1. The synaptically induced metabotropic glutamate receptor 1-excitatory postsynaptic current (mGluR1-EPSC) is associated with a localized increase in [Ca2+]i. A: fluorescence image of a mouse Purkinje cell patch-clamped with a pipette containing 200 µM Oregon Green and voltage-clamped at VH = -70 mV. S, stimulation electrode placed over the dendrite. B: pseudo-color-coded map of changes of [Ca2+]i obtained during induction of a mGluR1-EPSC by 6 stimuli delivered at 100 Hz. C: magnification of responsive area indicated by red rectangle in A. D: mGluR1-EPSCs and changes of [Ca2+]i induced by 3, 4, and 6 stimuli applied at 100 Hz. Arrows indicate time of stimulation. The changes of [Ca2+]i. were monitored at the region indicated by green rectangle in C. E: plot of the amplitudes of the mGluR1-EPSC and the associated elevation of [Ca2+]i against the number of stimulation pulses delivered for the cell shown in A-D. F: plot as in E but means ± SE values from data obtained from 4 Purkinje cells. The extracellular solution contained 20 µM bicuculline to block GABAA receptors, 10 µM 6-nitro-7-sulfamoylbenzo [f] quinoxaline-2,3-dione (NBQX), and 50 µM D-2-amino-5-phosphono-pentanoic acid (D-APV) to block ionotropic glutamate receptors.

Isolation of postsynaptic mechanisms

To isolate the postsynaptic mechanism of the mGluR1-mediated inward current, we applied the specific group I mGluR agonist 3,5-DHPG (Schoepp et al. 1994) via a pipette positioned close to the cell body and subjected the pipette to short-lasting (5-100 ms) pressure pulses. In all experiments with application 3,5-DHPG, the extracellular solution contained, in addition to the blockers of ionotropic glutamate and GABA receptors described in the preceding text, 1 µM TTX. Pressure ejection of 3,5-DHPG induced a short-lasting inward current (ImGluR1). ImGluR1 was associated with an increase of [Ca2+]i that was restricted to a region of the dendrite close to the 3,5-DHPG-containing electrode (n >70 cells; Fig. 2A). In contrast, activation of voltage-gated calcium channels by a short lasting depolarization to -0 mV for 250 ms produced a much more widespread elevation in [Ca2+]i (n = 10 cells; Fig. 2A). It is known that fast dendritic synaptic currents are only under limited voltage-clamp control in Purkinje cells (Eilers et al. 1995). Therefore we needed to exclude the possibility that part of the observed elevation in [Ca2+]i seen with pressure application of 3,5-DHPG was due to a depolarization of the dendritic membrane and resultant activation of voltage-gated calcium channels. To test this possibility, we applied by the same protocol the ionotropic glutamate receptor agonist AMPA (in extracellular solution lacking the AMPA receptor antagonist NBQX). With pressure applications of AMPA (200 µM, 10-100 ms) inducing inward currents of comparable size, no change in [Ca2+]i was observed (n = 4 cells). Only with AMPA-induced currents exceeding 1 nA, obtained with longer-lasting drug ejection, failure of sufficient space clamp of the dendrite was indicated by a rise in [Ca2+]i (n = 2 cells). In all experiments with 3,5-DHPG, we therefore limited the amount of drug applied to levels such that ImGluR1 did not exceed 500 pA, excluding the possibility of significant activation of voltage-gated calcium channels.



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Fig. 2. Replication of the mGluR1-EPSC by pressure application of (S)-3,5-dihydroxyphenylglycine (3,5-DHPG, 100 µM) via a pipette. A: D and R, stimulation electrode placed over the dendritic field of the patch-clamped Purkinje cell and recording electrode, respectively. Following application of 3,5-DHPG (50-ms pressure pulse), the cell was subjected to a depolarizing voltage jump (from -70 to 0 mV). Middle: membrane current and changes of [Ca2+]i measured in the regions indicated by rectangles of corresponding colors superimposed on the fluorescence image of the cell. Right: pseudo-color-coded maps of changes of [Ca2+]i obtained during response to application of 3,5-DHPG (1) and during depolarizing voltage jump (2). B: experimental protocol as in A except of that the drug-application pipette contained the ionotropic glutamate receptor agonist AMPA (A). Note lack of elevation of [Ca2+]i during AMPA-induced membrane current. In A and all subsequent figures, the extracellular solution contained 20 µM bicuculline, 10 µM NBQX, 50 µM D-APV, and 1 µM TTX. The AMPA receptor blocker NBQX was omitted in B.

Effect of reducing the Na+ gradient on ImGluR1

In cultured Purkinje cells ImGluR1 depended on the extracellular Na+ concentration ([Na+]o) (Linden et al. 1994; Staub et al. 1992), and recently we have shown in acute cerebellar slices that the mGluR1-EPSC and the inward current induced by 3,5-DHPG is associated with an increase in [Na+]i (Knöpfel et al. 2000). The dependence on [Na+]o would be compatible with the proposal that ImGluR1 was generated by an electrogenic Na+/Ca2+ exchange or by the activity of nonselective cation (CAN) channels (Linden et al. 1994; Staub et al. 1992). If there was a significant contribution by electrogenic Na+/Ca2+ exchange, we would expect that the Ca2+ transient would increase with depression of exchanger activity. To test for this possibility, we investigated the effect of substitution of external sodium by choline (Fig. 3). Reduction of [Na+]o from 144 to 26 mM, that is, a reduction of [Na+]o by about 80%, depressed ImGluR1 to 37 ± 9% of control values (n = 8, P < 0.001), while the associated elevations of [Ca2+]i slightly increased by 17 ± 10% of control values (Fig. 3). The small increase of the [Ca2+]i elevation was not significant (n = 5, P > 0.1).



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Fig. 3. Effect of reducing the Na+ gradient on inward current and the associated elevation in [Ca2+]i induced by 3,5-DHPG. A: membrane current (top) and changes of [Ca2+]i (bottom) induced by 3,5-DHPG in control solution (144 mM Na+), with 26 mM Na+/118 mM choline (80% choline), and after return to control solution (wash). 3,5-DHPG was dissolved in 80% choline external solution and application produced a transient outward current (*). B: statistical analysis of responses obtained in 26 mM Na+/118 mM choline (80% choline) and 1 mM Na+/143 mM choline (99% choline). Lowering extracellular Na+ concentration significantly (**P < 0.001) reduced ImGluR1. The small increase of the [Ca2+]i elevation was not significant (P > 0.1).

Reducing [Na+]o to 1 mM virtually abolished ImGluR1 (Fig. 3, n = 7). Under this condition, spontaneous escapes from clamp control associated with large calcium transients occurred, and this limited our evaluation of 3,5-DHPG-induced calcium transients to the condition in which 80% of [Na+]o was substituted by choline.

ImGluR1 and the associated [Ca2+]i elevations require the presence of extracellular calcium

Direct activation of mGluR1 made it possible to test the requirement of extracellular calcium for the generation of ImGluR1. Responses induced by 3,5-DHPG were recorded first under control conditions and then after switching to a Ca2+-free extracellular solution. Influx of calcium during depolarization of the cell to -0 mV for 75 ms was used to monitor the wash out of extracellular calcium. About 5 min after switching to the Ca2+-free extracellular solution, depolarization of the membrane failed to elevate [Ca2+]i (Fig. 4B). At this time ImGluR1 and the elevation of [Ca2+]i were also strongly depressed (9.4 ± 5.2 and 11.2 ± 8.1% of control values; n = 9; P < 0.001). After returning to control medium, 3,5-DHPG-induced calcium elevations and inward currents recovered to control levels (Fig. 4B). Resting fluorescence of the calcium indicator did not fall significantly during these short-lasting perfusions with Ca2+-free medium, suggesting that a significant drop of [Ca2+]i and a consequent depletion of intracellular calcium stores was unlikely the reasons for the lack of 3,5-DHPG-induced elevations of [Ca2+]i in the absence of extracellular calcium. As an additional control for this possibility, we tested the effect of reducing [Ca2+]o from the control level of 2 to 0.125 mM. In three of six of those experiments, cells were pretreated with thapsigargin, that is, recordings were made under conditions where thapsigargin sensitive stores were already depleted. Lowering [Ca2+]o from 2 to 0.125 mM depressed ImGluR1 and the associated elevation of [Ca2+]i to 57.6 ± 8.6 and 48.2 ± 1.1% of control values (n = 6; P < 0.01; Fig. 4, C and D).



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Fig. 4. The 3,5-DHPG-induced current and the associated increase in [Ca2+]i depend on extracellular [Ca2+]. A: fluorescence image of a Purkinje cell with superimposed drawing indicating site of 3,5-DHPG application D and regions (rectangle) from which change in [Ca2+]i was monitored. B: recordings from the cell shown in A. 3,5-DHPG-induced inward current and elevation of [Ca2+]i in control solution, after switch to a Ca2+-free medium (0 [Ca2+]o) and after return to control solution. Wash out of Ca2+ was indicated by disappearance of depolarization-induced elevation of [Ca2+]i induced by depolarizing voltage jump to -0 mV for 250 ms. D: effect of reduction of [Ca2+]o from 2 mM (control) to 0.125 mM; statistical analysis of data obtained as illustrated in B and C. **, statistical significant effects (P < 0.01).

Inhibitors of sarcoplasmatic reticulum Ca2+-ATPase do not appreciably affect the mGluR1-mediated responses

The requirement of extracellular calcium for the mGluR1-induced elevation of [Ca2+]i suggested that release of Ca2+ from intracellular stores contributed less to the observed signal than previously assumed. We recently characterized a current with an initial appearance similar to the present ImGluR1 in dopamine cells. In this system, addition of 1 µM thapsigargin, an inhibitor of sarcoplasmatic reticulum Ca2+-ATPase, to the extracellular solution readily abolished 3,5-DHPG-induced calcium elevations (Guatteo et al. 1999). Surprisingly, 10 µM thapsigargin had no clear effect on the 3,5-DHPG-induced calcium elevations and ImGluR1 in Purkinje cells (Fig. 5; n = 10). After application of thapsigargin, ImGluR1 and the associated elevation of [Ca2+]i amounted 87.2 ± 7.5 and 82.5 ± 8.3% of control responses. Incubation with CPA (30 µM), another inhibitor of sarcoplasmic reticulum Ca2+-ATPase, had also only a small, albeit significant, effect on ImGluR1 or [Ca2+]i elevations induced by 3,5-DHPG (85.2 ± 2.8 and 75 ± 9.1% of control responses; Fig. 5; n = 6).



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Fig. 5. Effect of thapsigargin and cyclopiazonic acid (CPA), inhibitors of sarcoplasmic reticulum Ca2+-ATPase on the 3,5-DHPG-induced inward current and [Ca2+]i. A: D and R, stimulation electrode placed over the dendritic field of the patch-clamped Purkinje cell and recording electrode, respectively. Recording of 3,5-DHPG-induced membrane current and changes of [Ca2+]i before (control) and 9 and 19 min after wash in of 30 µm thapsigargin. B: 3,5-DHPG-induced responses before (control), during, and after application of CPA (30 µM, 7 min). D: statistical analysis of data obtained as illustrated in A and B. con, control responses; thap, responses after application of thapsigargin; and CPA, responses after CPA. The effect of thapsigargin was not significant (n = 10, P > 0.1); the small reduction of the responses by CPA was significant (n = 6, P < 0.05).

ImGluR1 and the associated [Ca2+]i elevations were not abolished by SKF 96365

The requirement of extracellular calcium for ImGluR1 and the associated elevation of Ca2+ opened the possibility that at least part of the calcium entry was mediated by store operated Ca2+ entry channels. To test this possibility, we tested SKF 96365 (Gibson et al. 1998) on the 3,5-DHPG-induced responses. In the presence of SKF 96365, ImGluR1 and the associated elevation of [Ca2+]i amounted 94.7 ± 10.7 and 84.6 ± 7.4% of control values (n = 7, Fig. 6).



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Fig. 6. The 3,5-DHPG-induced current and the associated increase in [Ca2+]i were not blocked by 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF 96365). A: 3,5-DHPG-induced inward current and elevation in [Ca2+]i in control solution and in the presence of 100 µM SKF 96365. B: statistical analysis of data obtained as illustrated in A.

[Ca2+]i elevation associated with ImGluR1 is not mediated by voltage-gated calcium channels

Group I mGluRs are known to modulate the activity of voltage-gated calcium channels (Chavis et al. 1996; Fagni et al. 2000). To exclude the possibility that ImGluR1 results from an upregulation of voltage-gated calcium channels or that the associated calcium elevation is mediated by this calcium entry pathway, we tested the effect of blockers of L- and P-type calcium channels. Neither 5 µM nimodipine (n = 6) nor 100 nM omega-AGA toxin (n = 4) had any significant effect on ImGluR1 (97.2 ± 2.9 and 89 ± 8.3% of control values; P > 0.1; Fig. 7). The blockade of L-type calcium channels is known to exhibit use dependency. To account for this, some of the preceding cells (n = 3) were bathed for more than 30 min in nimodipine (5 µM) and were repetitively depolarized (voltage pulses to +10 mV lasting 100-200 ms once per minute). Also the 3,5-DHPG-induced elevation of [Ca2+]i was resistant to both nimodipine (101.3 ± 8.3% of controls; P > 0.1) and omega-AGA toxin (95.3 ± 4.1% of controls; P > 0.1).



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Fig. 7. The 3,5-DHPG-induced current and the associated increase in [Ca2+]i were not blocked by nimodipine or omega-agatoxin. A: 3,5-DHPG-induced inward current and elevation in [Ca2+]i in control solution and in the presence of 5 µM nimodipine. B: responses from the same cell as shown in A after wash out of nimodipine (wash/control) and during superfusion with 100 nM omega AGA toxin. C: statistical analysis of data obtained as illustrated in A and B.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present experiments were designed to investigate, and in part to reinvestigate, the relation between the mGluR1-induced inward current and the associated elevation of [Ca2+]i in cerebellar Purkinje cells. We directly activated mGluR1 by short-lasting pressure applications of a specific agonist and showed that this mode of activation produced responses closely resembling those observed with synaptic activation of the receptors. We carefully considered the undesired possibility that the observed elevations of [Ca2+]i were due to an escape from voltage-clamp followed by an activation of voltage-gated calcium channels. This possibility could be excluded by showing that inward currents of similar amplitudes and time courses induced by AMPA were not associated with any measurable [Ca2+]i response. Furthermore, [Ca2+]i transients were not depressed by blockers of voltage-gated calcium channels known to be expressed in Purkinje cells. Moreover, the experiments in which we substituted [Na+]o by choline showed that the 3,5-DHPG-induced [Ca2+]i transients were not depressed even when the inward currents were strongly reduced. These later experiments also supported our previous conclusion, made in cultured Purkinje cells and in [Na+]i imaging experiments, that the main charge carrier of ImGluR1 is Na+ (Knöpfel et al. 2000; Staub et al. 1992).

As to the source of the mGluR1-mediated rise of [Ca2+]i, it is generally thought that the main effect mediated by mGluR1 in Purkinje cells is a production of IP3, which in turn liberates calcium from intracellular stores via IP3 receptors (Finch and Augustine 1998; Llano et al. 1991; Takechi et al. 1998), as it is the case for recombinant mGluR1 in heterologous expression systems (Knöpfel et al. 1995; Masu et al. 1991). The elevated [Ca2+]i might then modulate rather than cause the inward current (Batchelor and Garthwaite 1997).

The present data suggest a different picture. Most strikingly, omission or even just reduction of [Ca2+]o markedly depressed both the inward current and the associated elevation of [Ca2+]i. Depletion of intracellular Ca2+ stores by thapsigargin or CPA did not affect the observed responses to 3,5-DHPG, and therefore depletion of intracellular calcium stores was unlikely the reason for the lack of 3,5-DHPG-induced elevations of [Ca2+]i in the absence of extracellular calcium. Nevertheless it is difficult to exclude the possibility that intracellular Ca2+ stores were depleted during reduction of [Ca2+]o. We minimized the likelihood of this possibility by testing mGluR1 immediately after the cessation of depolarization-induced Ca2+ influx. Furthermore, we showed that reducing [Ca2+]o from the control level of 2-0.125 mM, a condition under which depletion of Ca2+ stores is even less likely, also caused a reduction of the responses to mGluR1 activation. Finally, lowering [Ca2+]o was also effective in reducing the responses to mGluR1 activation in cells that were pretreated with thapsigargin, that is, under conditions where thapsigargin sensitive Ca2+ stores were already depleted. Another issue related to the interpretation of our observation that reduction of [Ca2+]o caused a reduced response to 3,5-DHPG is the possibility that [Ca2+]o affects the responsiveness of mGluR1 to the glutamate analogue. It is known that mGluR1 is modulated by extracellular calcium, but this effect concerns to an upregulation of receptor activity by extracellular calcium (Kubo et al. 1998) and mGluR1 can readily be activated in the absence of extracellular calcium (Kawabata et al. 1998). We therefore conclude that the dependency of ImGluR1 on [Ca2+]o was not due to a modulation of the ability of 3,5-DHPG to activate mGluR1, but rather that a significant fraction of the observed 3,5-DHPG-induced elevation of [Ca2+]i is generated by calcium entering the cell from the extracellular space.

In the light of this scenario, the observation that application of thapsigargin or of CPA had no dramatic effect on the 3,5-DHPG-induced elevation of [Ca2+]i is not surprising. Consistent with our data, Wang et al. (2000) recently described that parallel-fiber and mGluR1-mediated elevation of [Ca2+]i was insensitive to thapsigargin in rat Purkinje cells. Only when these calcium signals were paired with climbing fiber activation, a thapsigargin-sensitive nonlinear interaction between climbing fiber and parallel fiber-mediated calcium signaling occurred (Wang et al. 2000). One possible explanation for the lack of a thapsigargin-sensitive component of the investigated mGluR1-mediated response is based on the fact that the type 1 IP3 receptor, which is expressed in Purkinje cells, has a particularly low sensitivity to IP3 (Finch and Augustine 1998). Therefore a significant contribution of IP3 receptor-mediated component to the [Ca2+]i signal will require a high density of activated mGluR1 receptors (Eilers et al. 1995; Finch and Augustine 1998; Takechi et al. 1998; Wang et al. 2000). The density of activated mGluR1, and hence the amount of possible accumulation of local IP3, most likely depends on the density of the stimulation current employed and hence on the type of stimulation electrodes (e.g., monopolar or bipolar; larger or smaller inner tip diameter) and on the choice of nominally constant current or constant voltage stimulation pulses. Notably, when using large bipolar electrodes (Batchelor et al. 1996), tetanic parallel fiber stimulation induced a large ImGluR1, but the associated calcium signals were much smaller than those described by Finch and Augustine (1998) and Takechi et al. (1998) in the absence of a detectable ImGluR1. The different type of responses obtained with "dense parallel fiber stimulation" as opposed to "sparse parallel fiber stimulation" as described by Wang et al. (2000) is another example underlining this issue.

Since our results indicate that a significant component of the ImGluR1-associated [Ca2+]i elevation is not due to Ca2+ released from internal stores, we considered the possibility that there is an influx of Ca2+ from the extracellular space. The possibility that Ca2+ entry is mediated by store operated plasmatic membrane channels is unlikely because SKF96365, a blocker of such channels, did not show any significant effect. However, we cannot exclude an involvement of SKF 96365-insensitive store operated channels. Another possible Ca2+ entry pathway is a rapid activation of voltage-gated Ca2+ channels by mGluR1. In fact, mGluR1 can modulate Ca2+ channels (Chavis et al. 1996; Choi and Lovinger 1996; Fagni et al. 2000; McCool et al. 1998; Swartz and Bean 1992), and in principle a shift of the activation curve to the left (Kammermeier and Ikeda 1999) could lead to openings even at the resting potential. Indeed, a mGluR1-mediated increase of L-type Ca2+ current has been shown in cerebellar granule cells, where it is due to a functional coupling between such channels and ryanodine receptors (Chavis et al. 1996). However, a significant influx of Ca2+ through voltage-gated Ca2+ channels is unlikely, since blockers of L- and P-type Ca2+ channels, which in Purkinje cells are responsible for more than 90% of Ca2+ currents (Mintz et al. 1992; Regan 1991), affected neither the ImGluR1 nor the associated [Ca2+]i elevation.

Thus the Ca2+ entry pathway is not through store-operated channels or via classical voltage-gated Ca2+ channels. Furthermore, the question arises, whether there is a causal relationship between ImGluR1 and the associated [Ca2+]i elevation. Such a causal relationship has been described in the CA1 region of the hippocampus, where a group I mGluR mediates a calcium-activated nonselective cationic current (ICAN) (Congar et al. 1997). Hippocampal ICAN is G-protein dependent (Congar et al. 1997), like the ImGluR1 in Purkinje cells (Tempia et al. 1998), but it is completely blocked by chelating [Ca2+]i by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) (Congar et al. 1997), while in Purkinje cells, ImGluR1 is only attenuated but not abolished by the same treatment (Hirono et al. 1998; Linden et al. 1994; Staub et al. 1992; Tempia et al. 1998). Moreover, this hypothesis requires another Ca2+ influx pathway responsible for ICAN activation.

On the basis of our results, we cannot exclude an involvement of an electrogenic Na+/Ca2+ exchanger. Such a mechanism has been established in basolateral amygdala neurons (Keele et al. 1997) and in rat ventromedial hypotalamic neurons (Lee and Boden 1997) and has been discussed in cultured cerebellar Purkinje cells (Linden et al. 1994; Staub et al. 1992). Involvement of Na+/Ca2+ exchange would be consistent with the intracellular Na+ accumulation associated with ImGluR1 (Knöpfel et al. 2000) and the dependency of this current on extracellular Na+. However 2-[2-[4-(4-nitrobenzyloxy)phenyl] ethyl]isothiourea methanesulphonate (KB-R7943), a blocker of at least some types and modes of Na+/Ca2+ exchangers, was shown to have little effect on ImGluR1 (Hirono et al. 1998). In any case, contribution of Na+/Ca2+ exchange for the generation of ImGluR1 would require an independent pathway of Ca2+ entry into the cytoplasmic space, like any of the other pathways discussed.

Finally, the possibility of an involvement of a nonselective cation channel, which is also permeable to Ca2+, but not activated by [Ca2+]i should be considered (Guèrineau et al. 1995). For example, Purkinje cells express some members of the TRP family of nonselective cationic channels recently described in mammals (Mori et al. 1998; Otsuka et al. 1998; for a review on TRP channels, see Harteneck et al. 2000). A Ca2+-permeable nonselective cation channel, belonging either to this or to another class of molecules, would provide the cell with a calcium entry pathway, which at the same time generates an inward current mainly carried by Na+, in agreement with the fact that ImGluR1 is associated with a significant intradendritic Na+ increase (Knöpfel et al. 2000).


    ACKNOWLEDGMENTS

We thank A. Takada for expert administrative and secretarial assistance.

This work was supported in part by an intramural grant from the RIKEN Brain Science Institute and grants of Italian Ministero dell' Università e della Ricerca Scientifica e Tecnologica and Consiglio Nazionale delle Ricerche.


    FOOTNOTES

Address for reprint requests: T. Knöpfel, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan (E-mail: knopfel{at}brain.riken.go.jp).

Received 30 January 2001; accepted in final form 16 May 2001.


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