Download [Ca2+]c dynamics in spontaneously firing dopamine neurons of the

Research Article
2665
Glutamate-mediated [Ca2+]c dynamics in
spontaneously firing dopamine neurons of the rat
substantia nigra pars compacta
Yu Mi Choi, Shin Hye Kim, Dae Yong Uhm and Myoung Kyu Park*
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: [email protected])
Accepted 14 March 2003
Journal of Cell Science 116, 2665-2675 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00481
Summary
The mechanism by which glutamate regulates the cytosolic
free Ca2+ concentration ([Ca2+]c) in spontaneously firing
dopamine neurons is not clear. Thus we have investigated
the glutamate-mediated [Ca2+]c dynamics in the acutely
isolated dopamine neurons from the rat substantia nigra
pars compacta by measuring [Ca2+]c and spontaneously
occurring action potentials (SAPs). The freshly isolated
dopamine neurons showed tetrodotoxin (TTX)-sensitive
spontaneous firing of 2-3 Hz and the resting [Ca2+]c
decreased with abolition of the SAPs. The level of [Ca2+]c
was affected by the spontaneous firing rate. In the presence
of the Na+ channel antagonist, TTX (0.5 µM), glutamate
increased [Ca2+]c by activating different glutamate
receptors depending on the glutamate concentration used.
Addition of glutamate at low concentrations (<3 µM) raised
[Ca2+]c mainly by activating metabotropic glutamate
receptors (mGluR), whereas at high concentrations (>10
µM) it raised [Ca2+]c mainly by activating AMPA/kainate
Introduction
Glutamate-mediated rises in cytosolic free Ca2+ concentration
([Ca2+]c) are of the ultimate importance for neuronal
excitability (Nakanishi, 1992; Berridge, 1998); they also
play a crucial role in the outgrowth of dendrites and axons,
neuronal differentiation, synaptic remodeling and plasticity
(Collingridge and Singer, 1990), regulation of mitochondrial
metabolism (Duchen, 1999), gene expression (Gallin and
Greenberg, 1995; Ginty, 1997), and neurotoxicity (Sattler and
Tymianski, 2000). Since glutamate is involved in many such
functions and can activate various glutamate receptors, the
[Ca2+]c signals related to glutamate receptors in neuronal cells
have long been studied in a variety of neuronal cells (Mayer
and Miller, 1990; Gallin and Greenberg, 1995). Therefore, it is
well known that elevated glutamate, in addition to generating
excitatory synaptic potentials and changing membrane
potential, increases [Ca2+]c by the activation of AMPA/kainate
receptors, NMDA receptors, and group 1 metabotropic
glutamate receptors (mGluR), as well as by the secondary
opening of voltage-operated Ca2+ channels (VOCCs) (Mayer
and Miller, 1990; Nakanishi, 1992).
Dopamine neurons located at substantia nigra pars compacta
(SNc) are known to generate spontaneous firing and,
receptors. The contribution of NMDA receptors to the
glutamate-mediated [Ca2+]c rises was largest at
intermediate concentrations of glutamate. Activation of
mGluR elicited a Ca2+ release from intracellular Ca2+
stores and continuous Ca2+ influx out of the cell. The
spontaneous firing activities were highly enhanced by
submicromolar levels of glutamate and abolished at levels
above 10 µM. From these results, we conclude that at low
glutamate concentrations the [Ca2+]c in the dopamine
neurons is mainly governed by mGluR and the firing
activities, whose rate is regulated at submicromolar
glutamate concentrations, but at higher glutamate
concentrations [Ca2+]c is dominantly affected by
AMPA/kainate receptors.
Key words: Glutamate, AMPA receptor, NMDA receptor,
Metabotropic glutamate receptor, Calcium, Dopamine neuron,
Substantia nigra pars compacta
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; HerreraMarschitz 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
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Journal of Cell Science 116 (13)
[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.
Materials and Methods
Preparation of SNc dopamine neurons
We used the postnatal day 9 to 16 Sprague-Dawley rats. After
decapitation, whole brains were quickly removed and placed in icechilled oxygenated HEPES-buffered saline, which contains (in mM):
135 NaCl; 5 KCl; 1 CaCl2; 1 MaCl2; 25 D-glucose; 10 HEPES;
adjusted to pH 7.3 with NaOH. The brain was cut into a midbrain
block containing SNc and coronal slices of 300-400 µm thickness
were obtained with a vibratome (TPI, USA). Subsequently, the SNc
regions of the slices demarcated by dark color were dissected out with
a scalpel blade and digested with the fully oxygenated HEPESbuffered saline containing papain (4-10 U/ml, Worthington) for 20-60
minutes at 34-37°C. Next, the tissue segments were rinsed with
enzyme-free saline and then gently triturated with a graded series of
fire polished Pasteur pipettes (Chung et al., 2000). The isolated cells
were plated on poly-D-lysine-coated small glass coverslips that were
already fitted for a recording chamber mounted on an inverted
microscope equipped with a fluorescence measurement system.
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, ω-conotoxin GVIA, and
ω-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
(40× objective or 60× 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+-fura2 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 MΩ. We made whole cell or cellattached 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 Kgluconate; 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.
Glutamate-mediated [Ca2+]c changes in dopamine neurons
2667
Results
Identification of dopamine neurons
When we isolated cells from the selectively dissected SNc
brain slices, a majority of the cells showed a very large soma
having 3-6 neurites, which is characteristic of dopamine
neurons (Fig. 1Aa). But there were small cells having only two
bidirectional neurites (Fig. 1Ab, small bipolar cells) as well
as atypical cells such as a small body with multiple neurites
or a large body with two opposite neurites. Among the 104
cells isolated, 83 were large multipolar, 5 were small bipolar
and 16 were atypical cells. Although many papers have
already reported that dopaminergic neurons have a large body
and multiple neurites (Grace and Bunny, 1983a; Grace and
Bunny, 1983b; Grace, 1988; Cardozo, 1993; Chung et al.,
2000), in order to confirm whether the large multipolar cells
are dopaminergic, we stained cells with the antibody (PelFreez, USA) for tyrosine hydroxylase (TH), which is the main
enzyme for synthesizing dopamine. After incubation with
secondary antibodies tagged with FITC, we found that 76 out
of 83 large multipolar cells (91.5%) showed strong
fluorescence intensity as shown in Fig. 1, suggesting that most
of the large multipolar cells are dopaminergic neurons.
However, among the atypical cells, a few cells having a large
soma showed positive staining with TH antibodies (3/16),
but none of the small bipolar neurons was stained with
TH antibodies (0/5 cells). Fig. 1 shows two typical large
multipolar (Fig. 1Aa) and small bipolar cells (Fig. 1Ab) and
fluorescence images after staining with TH antibodies. Only
the large multipolar cells showed strong fluorescence. Thus
we have performed experiments only in these large multipolar
neurons.
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
Fig. 1. Identification of dopaminergic neurons. (A) Transmitted
images. Among the three neurons isolated, two large multipolor cells
(marked a), were dopaminergic, whereas a small bipolar cell (marked
b) was not (stained with tyrosine hydroxylase (TH) antibodies and
FITC-conjugated secondary antibodies). Bar, 20 µm.
(B) Classification of acutely isolated cells from the SNc according to
their shapes.
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
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Journal of Cell Science 116 (13)
Fig. 2. Electrical activities of acutely isolated SNc dopamine neurons. Spontaneous firing activities were recorded from the cell-attached
current-clamp cells (A,Ba). Their firing activities were converted into the frequency-time graph (A,Bb). The [Ca2+]c using fura-2 was measured
from the same cells (A,Bc). (A) The spontaneous firing activities were completely blocked by 500 nM TTX. Note that the change in [Ca2+]c
was mirrored by the firing rate. (B) The spontaneous firing was initially blocked by a nonspecific Ca2+ channel antagonist, 100 µM CdCl2. But
it recovered after some time even in the presence of CdCl2. Note the persistent decreased [Ca2+]c level after a full recovery of the spontaneous
electrical activities.
200
20
25
0
0
5
10
15
20
[Ca ]c (nM)
800
600
400
100 µM CdCl2
2+
10 µM Nif
1 µM ω-conoGVIA
1 µM ω-aga IVA
200
0
200
0
0
5
10
15
Time (min)
20
25
0
5
10
15
10
15
20
25
Time (min)
E
800
400
5
Time (min)
F
600
0
25
20
25
100
80
60
40
20
Ni f
15
KC l
10
Relative [Ca2+]c change (%)
5
Time (min)
[Ca2+]c (nM)
200
0
0
D
400
100 µM CdCl 2
0
400
600
L-,N-,P/Q-type
[Ca 2+ ] blocker
200
1 µM ω-cono GVIA
ω-aga IVA
400
600
ω-cono GVIA
10 µM Nif
1 µM ω-aga IVA
800
2+
600
C
800
[Ca ]c (nM)
B
800
[Ca2+]c (nM)
[Ca2+]c (nM)
A
0
Time (min)
Fig. 3. KCl-elicited [Ca2+]c rises through voltage-operated calcium channels. After application of 60 mM KCl for 12 seconds (䉱), the KCl
solution was reapplied under the presence of L-type Ca2+ channel (10 µM nifedipine, A), N-type Ca2+ channel (1 µM ω-conotoxin GVIA, B),
and P/Q-type Ca2+ channel (1 µM ω-agatoxin IVA, C) antagonists, a cocktail of the above three antagonists (D), or 100 µM CdCl2 (E).
(F) Summary of the inhibitions of the KCl-elicited [Ca2+]c rises by VOCC antagonists; nifedipine (36.0±3.0% of control, n=10), ω-conotoxin
GVIA (69.5±8.8%, n=5), ω-agatoxin IVA (92.3±6.6%, n=4), cocktail of the above three antagonists (23.2±4.1%, n=3), and CdCl2 (15.0±2.7%,
n=4). *Significant difference (paired student t-test, P<0.05). Nif, nifedipine; ω-cono GVIA, ω-conotoxin GVIA; ω-aga IVA, ω-agatoxin IVA.
Glutamate-mediated [Ca2+]c changes in dopamine neurons
A
Glutamate
2669
B
300 µM
100 µM
10 µM
3 µM
1 µM
c
1 min
30 µM
CNQX
50 µM
AP- 5
10 µM
Nif
f
Nif
1 µM
1 µM
ω-conoGVIA
ω-conoGVIA ω-agaIVA
ω-agaIVA
200 µM
CdCl2
CdCl2
ω-a ga IVA
ω-cono GVIA
CNQX
Nif+ω -cono GVIA
+ ω -aga IVA
0
Nifedipine
20
AP-5
40
Glutamate
Relative [Ca2+]c change (%)
100
60
1
10
100
1000
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
g
[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).
B
80
300
glutamate (µM)
e
d
600
0.1
200 nM
b
900
0
100 µM Glutamate
a
1200
30 s
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 (Ltype Ca2+ channel antagonist, Fig. 3A), ω-conotoxin GVIA (Ntype Ca2+ channel antagonist, Fig. 3B), and ω-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.
A
[Ca2+]c (nM)
Fig. 4. Dose dependence of glutamate-induced
[Ca2+]c rises. (A) The [Ca2+]c rises in response to
different glutamate concentrations in the same
cell. (B) The concentration-response curve of the
glutamate-induced [Ca2+]c rises. The
concentration of the half-maximum [Ca2+]c rise
(EC50) is 3.9±1.0 µM. All the solutions contain 1
µM glycine. Each point represents the average of
4-20 cells (mean ± s.e.m.).
200 nM
1500
Fig. 5. Glutamate-induced [Ca2+]c increases.
(A) Effects of antagonists for ionotropic
glutamate receptors (a,b) and VOCCs (c-g) on
the 100 µM glutamate-elicited [Ca2+]c rises.
The black curves are [Ca2+]c rises in response
to 100 µM glutamate and the red curves are
[Ca2+]c rises in the presence of specific
antagonists. (a) 30 µM CNQX; (b) 50 µM AP5; (c) 10 µM nifedipine; (d) 1 µM ω-conotoxin
GVIA; (e) 1 µM ω-agatoxin IVA; (f) cocktail of
the above VOCC antagonists; (g) 200 µM
CdCl2. (B) Summary of a-f experiments (means
± s.e.m., n). CNQX, 39.5±6.7% of control,
n=16; AP-5, 93.3±3.9%, n=13; nifedipine,
82.3±3.9%, n=7; ω-conotoxin GVIA,
93.9±0.3%, n=5; ω-agatoxin IVA, 100%, n=3;
cocktail of the above three antagonists,
67.5±2.4%, n=3; CdCl2, 27.7±4.8%, n=6. All
the solutions contain 1 µM glycine. *Significant
difference (paired student t-test, P<0.05).
2670
Journal of Cell Science 116 (13)
10 µM Nif
[Ca2+ ]c (nM)
A
200
calcium-free
0
0
[Ca2+ ]c (nM)
B
10
min
a
100 µM AMPA
1 µM
ω-cono GVIA
b
1 µM
ω-aga IVA
200
0
0
min 10
ω-aga IVA
ω-cono GVIA
Nifedipine
AMPA
C
Relative [Ca2+]c change (%)
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).
Fig. 6. AMPA-induced [Ca2+]c increases. 100 µM AMPA was
applied at each point indicated by an arrowhead for 12 seconds.
(A) Nifedipine (10 µM) inhibited the 100 µM AMPA-elicited [Ca2+]c
rises (n=4). In the Ca2+-free solution, AMPA did not rise [Ca2+]c at
all (n=3). (B) 1 µM ω-agatoxin IVA (n=2; b) but not 1 µM ωconotoxin GVIA (n=4; a) slightly inhibited the AMPA-induced
[Ca2+]c rises. (C) Summary of the above data. Nifedipine,
67.7±12.5%, n=4; ω-conotoxin GVIA, 95.0±0.5%, n=4; ω-agatoxin
IVA, 88.8±0.5%, n=2; *Significant difference (paired student t-test,
P<0.05).
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
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.
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-mediated [Ca2+]c changes in dopamine neurons
100 µM glutamate
100 µM glutamate
B
1200
1400
1000
1200
800
30 µM CNQX
+ 50 µM AP-5
600
400
200
[Ca2+]c (nM)
1000
800
600
0
15
20
25
0
Time (min)
1000
800
30 µM DHPG
400
200
0
5
10
15
20
25
Time (min)
Relative [Ca2+]c change (%)
D
1200
0
10
15
20
25
Time (min)
100 µM glutamate
600
5
100
80
60
40
DHPG
10
DHPG
Ca2+-free
5
Glutamate
2+
Ca -free
0
[Ca2+]c (nM)
Ca -free
200
0
C1400
2+
400
CNQX + AP5
[Ca2+]c (nM)
A
2671
20
0
Fig. 7. The [Ca2+]c increases by the activation of metabotropic glutamate receptors. (A) 100 µM glutamate-mediated [Ca2+]c rises in the
absence/presence of ionotropic glutamate receptor antagonists (30 µM CNQX + 50 µM AP-5, n=6). (B) 100 µM glutamate-mediated [Ca2+]c
rises with and without calcium in the bath solution (n=5). (C) 100 µM glutamate- and DHPG (mGluR agonist)-mediated [Ca2+]c increases
(n=10). (D) Summary of the above data. 100% (control) means the peak values of [Ca2+]c increases to 100 µM glutamate. CNQX and AP-5,
31.5±3.5% of control, n=6; glutamate in Ca2+-free solution, 23.7 ± 3.5%, n=5; DHPG in Ca2+-free solution, 19.3±4.2%, n=5; DHPG
(31.3±1.9%, n=10. All the solutions contain 1 µM glycine.
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 glutamateinduced [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 glutamatemediated [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
2672
A
10 µM DHPG
b
10 µM DHPG
Fig. 8. Two phases of the [Ca2+]c rise by
activation of the metabotropic glutamate
receptors. (A) Application of 10 µM DHPG (a)
elicited characteristic [Ca2+]c rises: the initial
transient Ca2+ rise and long-sustained [Ca2+]c
elevation (n=7). Disappearance of the later
persistent [Ca2+]c elevation (b) under the Ca2+free solution (n=7). (B) Effects of glutamate
receptor antagonists on the late phase of
glutamate-induced [Ca2+]c increases. All the
solutions contain 1 µM glycine. Note the
glutamate concentration.
50 nM
a
Journal of Cell Science 116 (13)
1 min
near 100 µM (Fig. 4). When cells are
stimulated
with
high
glutamate
2+ influx is
concentrations,
the
main
Ca
Ca2+ free
mediated by the activation of AMPA/kainate
receptors and VOCCs (Figs 5, 6). The
intracellular Ca2+ stores also contribute to
the glutamate-induced [Ca2+]c rise by about
30% of the maximal response (Fig. 7). The
3 µM glutamate
1 µM glutamate
activation of metabotropic glutamate
receptors leads to the initial rapid Ca2+ rise
and later sustained elevation of [Ca2+]c (Fig.
8). In Fig. 9, we clearly demonstrate that
different glutamate receptors differentially
100 µM CPCCOEt
participate in [Ca2+]c dynamics according to
glutamate
concentrations. At low glutamate
100 µM CPCCOEt
concentrations, the [Ca2+]c is mainly
30 µM CNQX
30 µM CNQX
regulated by activation of mGluR (Figs
8, 9), whereas at high glutamate
50 µM AP-5
50 µM AP-5
concentrations it mostly depends on the
activation of AMPA/kainate receptors (Figs
5, 6, 9). Moreover, the spontaneous firing
rate of the cell is another key factor
1 min
1 min
governing the [Ca2+]c level in the SNc
dopamine neurons, whose sensitivity is
conclude that glutamate at low concentrations not only elevates
modulated at the level of ambient glutamate concentrations
[Ca2+]c by activation of mGluR but also gives rise to [Ca2+]c
(Figs 2, 4, 10).
by enhancing SAP frequency.
B
a
200 nM
100 nM
b
Discussion
In this study, we have investigated the [Ca2+]c dynamics in
spontaneously firing single dopamine neurons and dissected
the Ca2+ influx pathways relating to glutamate receptors. The
freshly isolated dopamine neurons from SNc showed regular
SAPs of 2-3 Hz (Figs 2, 10) and their frequency was highly
affected by submicromolar concentrations of glutamate,
which is similar to the ambient glutamate concentration
reported in vivo (Lerma et al., 1986; Herrera-Marschit et al.,
1996). The frequency of SAPs is closely related to the level
of [Ca2+]c and the resting [Ca2+]c is slightly elevated due to
the resting level of spontaneous firing (Figs 2, 10). During
this firing, VOCCs are partially activated at resting condition
and allow Ca2+ influx out of the cells (Figs 2, 3). Among the
many VOCCs, L-type Ca2+ channels appear to contribute
mostly to the depolarization-induced Ca2+ rises in the SNc
dopamine neurons (Fig. 3). Glutamate, surprisingly, could
raise [Ca2+]c at submicromolar concentrations and peak at
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.
Glutamate-mediated [Ca2+]c changes in dopamine neurons
A
10 µM
glutamate
B
100 µM
glutamate
2673
100 µM
10 µM
3 µM
3 µM
200 nM
0.3 µM
1 µM
1 µM
0.3 µM
2 min
C
10 µM 100 µM
glutamate
3 µM
1 µM
0.3 µM
CPCCOEt
D
Relative Contribution(%)
AP-5
CNQX
mGluR
NMDAR
AMPAR
100
80
60
40
20
0
0.3 µM
1 µM
3 µM
10 µM 100 µM
Glutamate
Fig. 9. Inhibition of [Ca2+]c rises by antagonists for AMPA/kainate, NMDA, and metabotropic glutamate receptors at different glutamate
concentrations in SNc dopamine neurons. The black curves indicate the glutamate-induced [Ca2+]c rises at the indicated concentrations, and the
red curves indicate the glutamate-induced [Ca2+]c rises in the presence of 30 µM CNQX (A), 50 µM AP-5 (B), 100 µM CPCCOEt (C),
respectively. (D) Different contributions of AMPA/kainate, NMDA, and metabotropic glutamate receptors to the [Ca2+]c rises according to
glutamate concentrations. Each point was calculated by analysis of the data from 4-20 cells. All the solutions contain 1 µM glycine.
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 wholecell patch-clamp recordings in acutely isolated cells from 310-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; HerreraMarschitz 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
2674
Journal of Cell Science 116 (13)
A
glutamate ( µ M)
KCl (mM)
5
10
7
12
15
0.3
5
1
3
10
4
(mV)
0
100 sec
B
Frequency (Hz)
3
8
5
6
12
0.3
15
1
5
4
2
0
0
C
10
7
100
200
300
400
500
TIME (sec)
600
10
500
[Ca 2+ ]c (nM)
10
Fig. 10. Glutamate at submicromolar concentrations
increases the frequency of spontaneous firing and
[Ca2+]c in SNc dopamine neurons. (A) Spontaneous
firing activities of the dopamine cell were recorded in
the cell-attached current-clamp configuration. The
concentration of KCl was gradually increased and
thereafter the glutamate concentration increased too.
(B) Frequency plot of the data in A. When stimulated
with KCl, a maximum frequency was obtained at 1012 mM. Glutamate more dramatically raised the firing
frequency dose-dependently but the spontaneous firing
suddenly disappeared after exposure to 10 µM
glutamate. (C) The [Ca2+]c changes from the same cell.
The increased spontaneous firing reflects the increase
in [Ca2+]c. Glutamate has a much stronger effect on the
spontaneous firing and [Ca2+]c than KCl. The solution
contains 1 µM glycine.
3
400
1
300
5
200
10
7
12
15
5
0.3
100
0
0
100
200
300
400
500
TIME (sec)
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
600
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.
This work was supported by the Neurobiology Research Program
from the Korea Ministry of Science and Technology (M1-0108-000027) and by the Korea Science and Engineering Foundation
(KOSEF) grant through the Medical Research Center for Regulation
of Neuronal Cell Excitability at Sungkyunkwan University.
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