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
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ABSTRACT |
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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.
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INTRODUCTION |
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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 M. 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 (F/F values) as
described earlier, and pseudo-color-coded maps of
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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RESULTS |
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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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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.
|
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).
|
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).
|
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).
|
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).
|
[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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DISCUSSION |
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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
).
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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