Medical Research Center for Regulation of Neuronal Cell Excitability and Department of Physiology, Sungkyunkwan University School of Medicine, 300 Chunchun-dong Jangan-ku, Suwon 440-746, Korea
* Author for correspondence (e-mail: mkpark{at}med.skku.ac.kr)
Accepted 14 March 2003
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Summary |
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Key words: Glutamate, AMPA receptor, NMDA receptor, Metabotropic glutamate receptor, Calcium, Dopamine neuron, Substantia nigra pars compacta
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Introduction |
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Dopamine neurons located at substantia nigra pars compacta (SNc) are known
to generate spontaneous firing and, glutamate, as a major excitatory
neurotransmitter, is reported to modulate the firing patterns of the SNc
dopamine neurons through many kinds of glutamate receptors
(Overton and Clark, 1997;
Meltzer et al., 1997
;
Kitai et al., 1999
).
Subthalamic and pedunculopontine nuclei and neurons in prefrontal cortex
provide glutamatergic inputs to the dopamine neurons and can regulate firing
patterns and increase their frequency of SAPs
(Cardozo, 1993
;
Meltzer et al., 1997
;
Kitai et al., 1999
;
Grillner and Mercuri, 2002
).
In addition to these inputs, dopamine neurons can be tonically exposed to
glutamate at variable concentrations owing to ambient glutamate levels, local
synaptic activities of glutamatergic neurons and pathologic conditions such as
seizures, injuries, and hypoxia (Benveniste
et al., 1984
; Lerma et al.,
1986
; Sah et al.,
1989
; Herrera-Marschitz et
al., 1996
; Obrenovitch and
Urenjak, 1997
). The increased SAP frequency could modulate target
cells by changing the dopamine release at the axon terminals as well as at the
somatodendritic trees, particularly in dopamine neurons
(Nedergaard et al., 1988
;
Jaffe et al., 1998
;
Chen and Rice, 2001
).
The VOCCs and Ca2+-dependent ion channels also appear to be
critical in the ionic mechanisms by which the cells spontaneously fire, and
the glutamate-mediated changes in [Ca2+]c could affect
the electrical activities (Hounsgaard et
al., 1992; Kang and Kitai,
1993
; Amini et al.,
1999
). In addition, Ca2+ signals in dopamine neurons
appear to play important roles in somatodendritic dopamine releases or
dendritic secretions (Nedergaard et al.,
1988
; Jaffe et al.,
1998
). Thus the glutamate-mediated Ca2+ signals appear
to be important in maintaining the functions of dopamine neurons. However, how
each glutamate receptor is activated and cooperatively contributes to the
[Ca2+]c dynamics is not clear in SNc dopamine neurons.
This is partly due to the specific situation of the neurons, which always
exist within the brain tissue and so are persistently influenced by networks
and nearby cells. Moreover, owing to the high ambient glutamate concentration
in cerebrospinal and interstitial fluids
(Herrera-Marschitz et al.,
1996
), it is difficult to observe the effect of glutamate on
[Ca2+]c dynamics in vivo or in brain slices, especially
at low glutamate concentrations.
Thus we have investigated how glutamate activates different glutamate receptors and raises [Ca2+]c at different glutamate concentrations, by using acutely isolated dopamine neurons from the rat SNc. By taking advantage of using freshly isolated cells, we were able to remove network interferences between neurons and/or glial cells and clearly clamp glutamate concentrations even at very low levels. Thus we show that the [Ca2+]c, depending on the glutamate concentration, can be differentially regulated by different glutamate receptors as well as by the rate of spontaneous firing. At low glutamate concentrations (0.3-3 µM), the level of [Ca2+]c was determined mainly by the activation of mGluR as well as the enhanced frequency of spontaneous firing. However, at high glutamate concentrations (>10 µM) [Ca2+]c was affected mainly by the activation of AMPA/kainate receptors.
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Materials and Methods |
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Solutions and chemicals
The normal bath solution contains (in mM): 140 NaCl, 5 KCl, 10 HEPES, 10
D-glucose, 1 CaCl2, 1 MgCl2. The pH and osmolarity were
adjusted to 7.4 and about 300 mOsm with NaOH and sucrose. When we applied
glutamate to stimulate NMDA receptors, we added 1 µM glycine in the bath
solution. Among the chemicals related to ionotropic/metabotropic glutamate
receptors and ion channels, (s)-3,5-dihydroxyphenylglycine (DHPG, group 1
mGluR agonist), 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, AMPA/kainate
receptor antagonist), D()-amino-5-phosphonopentanoic acid (AP-5, NMDA
receptor antagonist), tetrodotoxin (TTX, Na+ channel antagonist),
CPCCOEt (type1 mGluR antagonist), and (R,S)-AMPA were obtained from Tocris,
and nifedipine, -conotoxin GVIA, and
-agatoxin IVA were from
Alomone Laboratories (Jerusalem, Israel). All other materials were obtained
from Sigma.
Measuring cytosolic Ca2+ concentration
The isolated SNc cells were incubated with 2-5 µM fura-2-AM at room
temperature (20-24°C) for 20-35 minutes. After that, the cells were washed
with normal physiological salt solution twice. All cells were used within 3
hours of isolation. Single cell fluorescence intensity was measured using an
Olympus IX70 inverted microscope (40x objective or 60x water
immersion objective), attached with a charge coupled device (CCD) image
intensifier camera (Quantix) and Metafluor software (Universal Imaging). We
used 340/380 dual excitations with a 400 nm dichroic mirror and emitted light
was collected with a long pass filter of 450 nm. The details are described
previously (Park et al.,
2002).
The ratio (340/380) of fluorescence intensities measured at the cell bodies
was calibrated with the maximum and minimum ratio values obtained after
exposing to 15 µM ionomycin and 10 mM Ca2+ or 10 mM EGTA, by
using a dissociation constant of 150 nM for Ca2+-fura-2 at room
temperature (Neher and Augustine,
1992).
Measuring electrical activities
The patch clamp system (EPC-9, HEKA) was used to measure spontaneous firing
activities. Patch pipettes were made by a Sutter puller and pipette tips were
polished with Narishige Forge. The resistance of patch pipettes was 2-3 MW. We
made whole cell or cell-attached configurations in the current clamp mode. In
cell-attached patch experiments, the electrical signals were continuously
sampled at 2 kHz (1 kHz filter) and stored in an IBM-compatible computer for
further analysis. In this case, patch pipettes were filled with the bath
solution. The electrical signals were much the same in the extracellular
recordings (Grace and Bunney,
1983a; Grace and Bunney,
1983b
). Frequency conversion of SAPs was performed with Igor ver.
4 and some of data were analyzed with Origin ver. 6.0. When recorded in the
whole-cell configuration, patch pipettes were filled with the internal
solution whose compositions are (in mM): 125 K-gluconate; 5 KCl; 8 NaCl; 0.1
CaCl2; 1 MgCl2; 0.75 EGTA; 10 HEPES; 2 Mg-ATP; adjusted
to pH 7.3 with KOH.
Immunocytochemistry
The acutely isolated cells on glass coverslips were rinsed twice by
phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 40
minutes at room temperature. After fixation, the cells were washed with PBS
and then incubated in the PBS with 1% bovine serum albumin (BSA) and 0.1%
Triton X-100 for 60 minutes. After that, the cells were incubated for 2 hours
in PBS containing tyrosine hydroxylase antibodies (Pel-Freez, Rogers,
Arkansas, USA, diluted 1:100), 1% BSA, and 0.1% Triton X-100. Next, the cells
were rinsed three times with PBS and incubated with fluorescein isothiocyanate
(FITC)-conjugated goat anti-rabbit IgG (Molecular Probes) diluted by 1:200 in
PBS. After incubation for 1 hour at room temperature, the fluorescent
antibodies were removed by washing three times with PBS. The fluorescence
images were obtained in a Zeiss 510 confocal laser scanning microscope with a
488 nm excitation line and 505-545 nm emission filter.
Statistics
Paired student's t-test was used and P-values less than
0.05 were regarded as significantly different.
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Results |
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Spontaneous firing activities and cytosolic Ca2+
concentration
It is well known that dopamine neurons in substantia nigra generate
spontaneous firing in vivo, in vitro and even in isolated cells
(Grace and Bunney, 1983a;
Grace and Bunney, 1983b
;
Grace, 1988
;
Cardozo, 1993
;
Uchida et al., 2002
). Thus, in
order to test whether the neurons we isolated produce SAPs, we used
patch-clamp techniques. When we made the whole-cell configuration in a
current-clamp mode, we observed SAPs with an average frequency of 2-3 Hz
(n=10). The membrane potential fluctuated between -64 and -50 mV
(n=10, data not shown). Interestingly, this spontaneous firing was
also detected in a cell-attached configuration, whose shapes were similar to
those that Grace and Bunney previously reported in extracellular recording
conditions (Grace and Bunney,
1983b
) (Fig. 2). In
the cell-attached configuration we continuously recorded spontaneous firing
activities for a long time without much fluctuation. One more advantage of
this recording configuration is that it does not disturb natural
concentrations of Ca2+ buffers and soluble signalling molecules,
thereby we mainly recorded them in this condition.
|
To investigate the relationship between spontaneous firing activities and [Ca2+]c, we recorded them in the fura-2-loaded cells at the same time. As shown in Fig. 2, when we applied 0.5 µM TTX, the spontaneous firing was completely blocked (Fig. 2Aa,b) and [Ca2+]c decreased at the same time (Fig. 2Ac). After TTX washout, the SAPs and [Ca2+]c slowly restored to the previous level. Interestingly, in this particular cell there were some fluctuations of firing frequencies after washout of TTX, and the changes in [Ca2+]c were exactly mirrored by the frequency changes in SAPs. This is a good example showing that resting [Ca2+]c in spontaneously firing dopamine neurons is highly dynamic and easily affected by the frequency of SAPs. We observed a similar phenomenon in eight cells.
Since there was no neurotransmitter in the bath solution, we suspected that VOCCs were playing a major role in Ca2+ influx in the spontaneously firing cells. Thus we used a non-specific voltage-operated Ca2+ channel antagonist, Cd2+. As soon as 100 µM Cd2+ was applied, SAPs suddenly disappeared and then slowly restored to the previous level while the [Ca2+]c remained at the decreased level (Fig. 2Ba,b,c, n=5). This suggests that the SAPs at resting conditions could activate VOCCs to allow Ca2+ influx and help to keep [Ca2+]c elevated in SNc dopamine neurons.
Next we examined what kinds of VOCCs are expressed in SNc dopamine cells
(Fig. 3). To eliminate SAPs we
added 0.5 µM TTX to all the solutions and depolarized membrane potential
with a brief exposure (12 seconds) to 60 mM KCl. After the first stimulation
of cells with 60 mM KCl, we stimulated cells again with the same KCl solution
in the presence of various VOCC antagonists, such as nifedipine (L-type
Ca2+ channel antagonist, Fig.
3A), -conotoxin GVIA (N-type Ca2+ channel
antagonist, Fig. 3B), and
-agatoxin IVA, (P/Q-type Ca2+ channel antagonist,
Fig. 3C), a cocktail of the
three antagonists (Fig. 3D), or
100 µM Cd2+ (Fig.
3E). The average block effects were summarized in
Fig. 3F. In the SNc dopamine
neurons, despite the dominant contribution of L-type Ca2+ channels
to the rise in [Ca2+]c, other channels appear to
participate in the KCl-induced [Ca2+]c rises.
|
Glutamate-mediated [Ca2+]c rises and related
receptors
In the presence of 0.5 µM TTX, we applied glutamate at various
concentrations and measured [Ca2+]c. As shown in
Fig. 4, the
[Ca2+]c started to rise from submicromolar
concentrations (0.3 µM) and the peak value was obtained at near 100 µM
with a half activation dose of 3.9±0.1 µM.
|
To investigate what kinds of receptors or ion channels are involved, we used specific antagonists for various glutamate receptors and VOCCs. The representative traces are shown in Fig. 5A where the black curves are the [Ca2+]c rises in response to 100 µM glutamate and the red curves are those obtained in the presence of specific antagonists in the same cells. Interestingly the glutamate-induced [Ca2+]c rise was most significantly inhibited by the specific AMPA/kainate receptor antagonist, CNQX (30 µM, n=16). The NMDA receptor antagonist, AP-5, and VOCC antagonists slightly inhibited the glutamate-mediated [Ca2+]c rises (n=13, P<0.05 in paired student's t-test). In the case of nifedipine, this L-type Ca2+ channel antagonist did not inhibit as much as it did in the KCl-induced [Ca2+]c rise (Fig. 3F).
|
Therefore, to further clarify the Ca2+ influx pathways blocked
by CNQX, we directly stimulated AMPA/kainate receptors by adding AMPA to the
bath. In this case, AMPA/kainate receptors and VOCCs would be selectively
opened. As shown in Fig. 6,
nifedipine and other VOCC antagonists did not inhibit the glutamate-induced
[Ca2+]c rises as much as they did in the KCl-stimulated
cells (Fig. 3), suggesting that
the AMPA-mediated [Ca2+]c rise is not solely mediated by
VOCCs but involved with other pathways. When we added AMPA to a
Ca2+-free solution, there was no detectable change in
[Ca2+]c as shown in
Fig. 6A (n=4). Thus
AMPA did not appear to directly stimulate intracellular Ca2+
stores. Therefore, it is likely that, if cells were stimulated with high
concentrations of glutamate, the SNc dopamine neurons would raise
[Ca2+]c through mainly Ca2+-permeable
AMPA/kainate receptors
(Pellegrini-Giampietro et al.,
1997; Metzger et al.,
2000
) and some VOCCs.
|
Because the intracellular endoplasmic reticulum Ca2+ store is
the most important source of Ca2+ for many kind of cells, such as
cardiac, skeletal and pancreatic acinar cells
(Park et al., 2000;
Csordas et al., 2001
;
Bers, 2002
), we tested how this
store contributes to the [Ca2+]c rises in SNc dopamine
neurons. To this end, we used antagonists for ionotropic glutamate receptors
and stimulated cells with glutamate. In this case, the
[Ca2+]c rise reached 31.5±3.5% (n=6) of
the maximal [Ca2+]c increase that was obtained with 100
µM glutamate (Fig. 7A). In
the Ca2+-free solution, glutamate also increased
[Ca2+]c by 19.3±4.2% of control levels
(n=5, Fig. 7B,D). When
we directly stimulated cells with a group 1 mGluR agonist, DHPG, the
[Ca2+]c increased to a level similar to that shown in
the glutamate-stimulated cells in the Ca2+-free solution
(Fig. 7C,D).
|
Metabotropic glutamate receptor-mediated
[Ca2+]c rises
When we stimulated cells with DHPG in a long time scale, SNc showed a
characteristic shape of the [Ca2+]c rise as shown in
Fig. 8Aa. After the initial
rapid [Ca2+]c rise and drop, the sustained
[Ca2+]c elevation was observed in all cells tested
(n=9). The initial peak was not affected by removal of extracellular
Ca2+(Fig. 8Ab), but
only blocked by a specific type 1 mGluR antagonist, CPCCOEt (100 µM,
Fig. 9C). Moreover, the
sustained [Ca2+]c elevation was completely abolished by
removal of extracellular Ca2+
(Fig. 8Ab), suggesting two
kinds of Ca2+ rising mechanisms by mGluR; the initial
Ca2+ release from the intracellular Ca2+ stores and the
sustained Ca2+ influx out of the cell.
|
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Interestingly this characteristic [Ca2+]c rise was reproduced by the application of glutamate at low concentrations. As shown in Fig. 8Ba,b, 1 or 3 µM glutamate reproduced a [Ca2+]c rise similar to that shown in Fig. 8Aa. In these cells, 100 µM CPCCOEt not only blocked the first component of the [Ca2+]c rise (Fig. 9C) but also effectively inhibited the second sustained component (Fig. 8Ba,b). CNQX (30 µM) or AP-5 (50 µM) had a minimal effect (Fig. 8Ba,b), indicating that at low glutamate concentrations mGluR are dominantly operating in the SNc dopamine neurons.
Relative contributions of NMDA, AMPA and mGluR receptors
In the previous experiments, we showed that glutamate at low concentrations
mainly mimicked the DHPG-induced [Ca2+]c responses but
glutamate at high concentrations gave rise to [Ca2+]c
mainly through activation of the AMPA/kainate receptors
(Fig. 5). Thus, we thought that
glutamate could differentially raise [Ca2+]c according
to glutamate concentrations. Thus we examined how glutamate raises
[Ca2+]c at different glutamate concentrations in the
presence of specific antagonists for glutamate receptors such as CNQX, AP-5
and CPCCOEt. Fig. 9 shows the
results, where the phenomenon we observed was clearly disclosed. At low
glutamate concentrations (0.3-3 µM), CPCCOEt dominantly inhibited the
glutamate-elicited [Ca2+]c rises, whereas at high
glutamate concentrations CNQX effectively blocked the
[Ca2+]c rises. The blocking effect of AP-5 on the
glutamate-induced [Ca2+]c rise was not much
concentration-dependent but largest at near 3 µM glutamate. In
Fig. 9D, we summarized relative
contributions of each glutamate receptor to the glutamate-induced
[Ca2+]c rises at various concentrations by analyzing
antagonist effects in several cells.
Contribution of spontaneous firing to glutamate-mediated
[Ca2+]c rises
In Fig. 2, we show that SAPs
were important in maintaining the resting [Ca2+]c, and
changes in SAP frequency could affect the level of
[Ca2+]c. Thus we tried to investigate how SAP frequency
affects [Ca2+]c. To raise spontaneous firing frequency
without activating glutamate receptors, we gradually raised KCl concentration
from 5 mM until SAPs disappeared, and measured [Ca2+]c
at the same time. As shown in Fig.
10 (left), the elevation of KCl in a bath solution raised the
frequency of spontaneous firing as well as [Ca2+]c. The
frequency reached a peak at 10-12 mM KCl and the SAPs disappeared at 15 mM
KCl, probably as a result of too much depolarization of membrane potential. It
also suggests that SAPs can be generated between optimal ranges of the
membrane potential. One interesting finding in this figure is that the
[Ca2+]c was rising while the frequency was increasing.
However, as soon as SAPs disappeared, after addition of 15 mM KCl, the
[Ca2+]c decreased, despite the higher KCl concentration.
This suggests that SAP is more important in [Ca2+]c
rises than simple depolarization of membrane potential in our experimental
conditions. To test how the steady state depolarization (due to KCl) activate
VOCCs, we raised KCl concentration in the presence of 0.5 µM TTX. But in
this case, the [Ca2+]c rise was much smaller than that
observed in spontaneously firing cells (data not shown, n=5).
Therefore it is likely that the frequency of SAPs is an important factor in
the regulation of [Ca2+]c in SNc dopamine neurons.
|
Next, we tested how glutamate changes [Ca2+]c and
spontaneous firing frequency. Although glutamate is known to raise spontaneous
firing activity (Meltzer et al.,
1997), the concentration dependence of glutamate has not been
reported yet. On the right-hand side of
Fig. 10, we gradually raised
glutamate concentration in a bath, starting at 0.3 µM. Surprisingly
glutamate at very low concentrations strongly raised the SAP frequency as well
as [Ca2+]c (n=6). Glutamate very sensitively
and dramatically increased the SAP frequency compared with the increase caused
by KCl elevation (Figs 3,
10). Moreover, the
[Ca2+]c rise by glutamate in the presence of TTX was
much smaller (data not shown, n=5), suggesting that spontaneous
firing is also an important factor in contributing to the
[Ca2+]c rise in the SNc dopamine cells. Thus we could
conclude that glutamate at low concentrations not only elevates
[Ca2+]c by activation of mGluR but also gives rise to
[Ca2+]c by enhancing SAP frequency.
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Discussion |
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Cell types in substantia nigra pars compacta
The SNc dopamine neurons are well known to have multiple neurites and a
large cell body (Juraska et al.,
1977; Grace and Bunny,
1983a
; Hajos and Greenfield,
1993
; Cardozo and Bean,
1995
; Kitai et al.,
1999
). SNc also contains non-dopaminergic neurons, which are
different in shape and electrophysiological properties from the dopamine
neurons. When we isolated cells from the SNc area, which we deliberately tried
to confine, 79% of the isolated cells were large multipolar cells and among
them 91% were dopaminergic neurons (Fig.
1). If we include three TH+ cells among the atypical cells, the
total dopaminergic neurons in SNc would be 76% of the dissociated cells. This
suggests that the majority of cells within the SNc area are dopaminergic but a
substantial portion of the cells in SNc are not dopaminergic. However, in this
calculation we could not exclude the possibilities that some of cells may
originate from the nearby areas of the SNc during the isolation procedures and
some cells vulnerable to the dissociation procedures would be selectively
lost.
Contribution of VOCCs to [Ca2+]c rises in
dopamine neurons
In SNc dopamine neurons, VOCCs appear to participate in pacemaker-like
oscillations of membrane potential and many types of VOCCs are reported to be
present in SNc neurons (Cardozo and Bean,
1995; Kang and Kitai,
1993
; Takada et al.,
2001
). However it has not been reported what types of VOCCs are
important in [Ca2+]c homeostasis. According to the
whole-cell patch-clamp recordings in acutely isolated cells from 3-10-day old
rats (Cardozo and Bean, 1995
),
P/Q-type, N-type and L-type VOCCs are near equally activated by strong
depolarization pulses. However, the immunohistochemical data obtained from
brains from the adult Wistar rats (Takada
et al., 2001
) show the predominant presence of L-type
Ca2+ channels at dendrites and cell bodies of the dopamine neurons.
Our experimental data obtained from 7-16-day old rats indicate that L-type
VOCCs are a major contributor to [Ca2+]c changes in the
cell body. They are closer to the data obtained from the adult rats.
Roles of NMDA-, AMPA/kainate, and metabotropic glutamate receptors in
[Ca2+]c dynamics
Neurons in the brain can be exposed to highly variable concentrations of
glutamate and the glutamate concentration in the cerebrospinal fluid is as
high as several micromolar concentrations
(Lerma et al., 1986;
Sah et al., 1989
;
Herrera-Marschitz et al.,
1996
). Although resting neurons appear to be exposed more or less
to a micromolar concentration due to strong buffering activities of glial
cells, some glutamate receptors are reported to be tonically active at resting
glutamate concentrations (Sah et al.,
1989
). Since maintenance of basal Ca2+ concentrations
could be essential in neuronal functions, understanding of glutamate-mediated
Ca2+ influx pathways would be important. In SNc dopamine neurons,
modulation of firing patterns by glutamate is relatively well-studied (Metzler
et al., 1997; Kitai et al.,
1999
) but the Ca2+ signals relating to glutamate have
not been thoroughly studied yet.
In this experiment, we have dissected glutamate-mediated Ca2+
influx pathways at variable levels of glutamate concentrations. One
interesting finding is that the Ca2+ influx pathway, which is
closely linked with mGluR, is operating at low glutamate concentrations and
this pathway may be tonically active and contributes to resting
[Ca2+]c. In Fig.
8, we showed the two components of the mGluR-linked
[Ca2+]c rises. Among them the fast component is clearly
attributed to the intracellular Ca2+ stores and probably plays an
important role in acute responses. This is in agreement with the reports that
SNc neurons have the inositol 1,4,5-trisphophate mediated Ca2+
responses and it acts as an inhibitory signals
(Fiorillo and William, 1998;
Morikawa et al., 2000
;
Seutin et al., 2000
). By
contrast, the second component, persistent elevation of
[Ca2+]c, appears to be important in keeping
[Ca2+]c at some levels elevated in vivo, since
submicromolar concentrations of glutamate elicited the responses and
interstitial fluid contains glutamate at micromolar concentrations
(Lerma et al., 1986
;
Herrera-Marschitz et al.,
1996
). For this Ca2+ influx pathway, store-operated
Ca2+ channels or/and unidentified Ca2+ permeable
channels would be responsible (Guatteo et
al., 1999
; Fagni et al.,
2000
).
Regarding AMPA/kainate receptors in dopamine neurons, we have somewhat
different results from the general view that NMDA receptors are more important
than AMPA/kainate receptors in the glutamate-mediated Ca2+
influxes. In Fig. 5, we showed
that the contribution of NMDA receptors to the [Ca2+]c
rise, when stimulated with glutamate at high concentrations, was relatively
smaller than that of AMPA/kainate receptors. However, recently there have been
accumulating reports that [Ca2+]c can be increased
through AMPA/kainate receptors themselves and some types of AMPA/kainate
receptors are highly Ca2+-permeable and play a role in
AMPA-mediated neurotoxicity (Metzger et
al., 2000;
Pellegrini-Giampietro et al.,
1997
). Among them, it has been reported that joro spider toxin can
selectively inhibit Ca2+-permeable AMPA receptors
(Blaschke et al., 1993
). In
Fig. 5, we showed that
AMPA/kainate receptors are most important in the [Ca2+]c
rise in response to the high glutamate concentration. Thus we suspected the
presence of Ca2+-permeable AMPA receptors in SNc dopamine neurons.
However, when we tested joro spider toxin in our experimental conditions, it
did not inhibit the AMPA-mediated Ca2+ influx (data not shown,
n=7), suggesting the presence of joro spider toxin-resistant
Ca2+-permeable AMPA receptors
(Meucci et al., 1996
;
Meucci and Miller, 1998
) or
other unknown pathways.
Spontaneous firing and Ca2+ signals
Glutamate not only conveys electrical signals at synapses but also
regulates spontaneous firing patterns in SNc dopamine neurons. Thus the roles
of many kinds of glutamate receptors have long been studied. So far, all the
glutamate receptors such as NMDA, AMPA/kainate receptors, and mGluR are known
to be more or less involved in modulating spike patterns or shapes of spikes
(Pucak and Grace, 1994;
Metzler et al., 1997; Kitai et al.,
1999
). However, to our knowledge, it is not clear what
concentration of glutamate could modulate the spontaneous firing or change
spike patterns. Surprisingly we found that glutamate at submicromolar
concentrations strongly increases the firing rate of SNc neurons. Since firing
rate (Figs 2,
10) is directly related to the
level of [Ca2+]c, glutamate at low concentrations could
effectively maintain [Ca2+]c at slightly elevated
levels. Generally, in pancreatic acinar cells, immune cells, or cardiac
myocytes, the basal elevation or continuous oscillations of
[Ca2+]c are recently regarded as a wakeup signal in
which it activates subcellular organelles to boost metabolic processes or
trigger production of antibodies (Duchen,
1999
; Bers, 2002
;
Csordas et al., 2001
;
Lewis, 2001
;
Park et al., 2000
;
Park et al., 2001
). However,
in the SNc dopamine neurons the physiological role of the slightly elevated
[Ca2+]c at resting conditions, as a result of spontaneous firing,
remains elusive but it may play an important role in many biological
processes.
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Acknowledgments |
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