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The Journal of Neuroscience, September 1, 1998, 18(17):6693-6703
L-Type Calcium Channels Mediate a Slow Excitatory Synaptic
Transmission in Rat Midbrain Dopaminergic Neurons
Antonello
Bonci1,
Pernilla
Grillner1,
Nicola
B.
Mercuri1, 2, and
Giorgio
Bernardi1, 2
1 Ístituto Ricovero e Cura a Carattere Scientifico
Santa Lucia, 00179 Rome, Italy, and 2 Clinica
Neurologica, University of Tor Vergata, 00173 Rome, Italy
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ABSTRACT |
Patch pipettes were used to record whole-cell synaptic currents
under voltage-clamp in dopaminergic neurons in slices of rat substantia
nigra pars compacta and ventral tegmental area. We report that
dihydropyridines (DHPs), L-type Ca2+ channel
antagonists, depressed a slow EPSC (EPSCslow)
evoked by a train of focally delivered electrical stimuli. In fact, the amplitude of the EPSCslow was reduced by the DHP
antagonists nifedipine (1-100 µM), nimodipine (1-100
µM), and isradipine (30 nM-100
µM) in a concentration-dependent and reversible manner.
On the other hand, Bay-K 8644 (1 µM), an L-type
Ca2+ channel agonist, increased the
EPSCslow. The DHPs depressed the EPSCslow only
when the high-frequency stimulation that was used to evoke this
synaptic current lasted >70 msec. On the other hand, Bay-K 8644 increased the amplitude of the EPSCslow only when it was
evoked by a train <70 msec. Moreover, the DHPs did not affect the
EPSCfast, the IPSCfast, and the
IPSCslow. The inhibition of the EPSCslow caused
by the DHPs is attributed to presynaptic mechanisms because (1) the
inward current generated by exogenously administered glutamate was not
affected and (2) the EPSCslow was reduced to a similar
degree even when the activation state of postsynaptic L-type
Ca2+ channels was changed by holding the neurons at
 100, 60, and +30 mV. Finally, a DHP-sensitive component of the
EPSCslow could even be detected after the blockade of N-,
Q-, and P-type Ca2+ channels by the combination of
 -conotoxin GVIA, -agatoxin IVA, and -conotoxin
MVIIC. Taken together, these results indicate that under certain
patterns of synaptic activity, L-type Ca2+ channels
regulate the synaptic release of excitatory amino acids on the
dopaminergic neurons of the ventral mesencephalon.
Key words:
dopamine neurons; L-type calcium channels; dihydropyridines; excitatory postsynaptic currents; midbrain; electrophysiology
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INTRODUCTION |
The increase of calcium influx into
the presynaptic terminal is generally considered a fundamental event
that triggers neurotransmitter release (Katz and Miledi, 1969 ;
Llinàs et al., 1981; Augustine et al., 1987). Several types of
presynaptic calcium channels, particularly the N-type and P/Q-type,
have been thought to participate in calcium-mediated neurotransmitter
secretion (Bean, 1989; Seabrook and Adams, 1989; Mintz et al., 1992;
Luebke et al., 1993; Wheeler et al., 1994; Dunlap et al., 1995; Wright
and Angus, 1996). Despite observations that L-type calcium channels
play an important role in the release of neurotransmitters such as
catecholamines from chromaffin cells, dynorphin from the dendrites of
rat hippocampal granule cells, neuropeptides from the neurohypophysis,
and excitatory amino acids (EAAs) from the retina (Lemos and Nowycky,
1989; Takibana et al., 1993; Lopez et al., 1994; Simmons et al.,
1995; Von Gersdorff and Matthews, 1996), these channels are
thought to play a minor role in most neuronal excitation-secretion
events. In fact, it is generally thought that they are not active
during the generation of action potentials in the CNS and the
subsequent depolarization of the synaptic terminals (Miller, 1987;
Kullman et al., 1992; Dunlap et al., 1995; Reuter, 1996). Consistent
with these findings, the inhibition of L-type Ca2+
channels has little effect on synaptic transmission in areas such as
the frontal cortex, hippocampus, accumbens, cerebellum, and striatum
(Kamiya et al., 1988; Llinàs et al., 1989; Horne and Kemp, 1991;
Kullman et al., 1992; Mintz et al., 1992; Turner et al., 1992; Zhang et
al., 1993; Wheeler et al., 1994). In the present study, we report a
specific modulation by L-type Ca2+ channels of slow
EPSCs (EPSCsslow) evoked under voltage-clamp on
presumed dopamine-containing neurons of the rat midbrain, by using the
whole-cell patch-clamp technique (Wu et al., 1995; Bonci and Williams,
1997; Shen and Johnson, 1997). This slow synaptic excitatory event,
generated by the activation of NMDA and metabotropic receptors (Mercuri
et al., 1996; Shen and Johnson, 1997), is likely caused by the
release of aspartate/glutamate from inputs to the midbrain arising from
the cortex, subthalamus, and pedunculopontine nucleus (Christie et al.,
1985; Kita and Kitai, 1987; Sesack and Pickel, 1992; Lavoie and Parent,
1994). Given the considerable evidence that the dopaminergic neurons
influence various motor and behavioral states (Le Moal 1995), a
regulation of their excitatory afferents by L-type
Ca2+ channels is an important topic that might have
physiological and therapeutic implications (Seeman, 1995).
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MATERIALS AND METHODS |
Preparation of the tissue. Male albino Wistar rats
(150-300 gm) were killed under halothane anesthesia. The preparation
of the slices has been described previously (Mercuri et al., 1995). Briefly, a single horizontal slice (200-350 µm thick) containing the
substantia nigra and the ventral tegmental area was transferred to a
recording chamber and continuously perfused at a rate of 2.5 ml/min,
with a solution maintained at 35°C and oxygenated with a mixture of
95% CO2/5% O2. The standard solution
contained (in mM): NaCl 126, KCl 2.5, NaH2PO4 1.2, MgCl2 1.2, CaCl2 2.4, glucose 11, and NaHCO3 19, giving a
pH of 7.4.
Recording and stimulation. The slice was transferred to a
recording chamber on the stage of an upright microscope (Axioscope, Carl Zeiss) illuminated with infrared light. The presumed dopaminergic neurons were directly visualized and approached by positive pressure. Whole-cell recordings were made using patch pipettes having a resistance of 4-7 M and containing (in mM): potassium
gluconate 144, CaCl2 0.3, MgCl2 1.2, HEPES 10, EGTA 1, Mg-ATP 2, and GTP 0.25, pH 7.3. To record the
IPSCfast, 128 mM KCl and 20 mM NaCl were used instead of potassium gluconate. In the
experiments with the neurons clamped at 100 and +30 mV, the
intracellular solution contained 120 mM cesium gluconate
instead of potassium gluconate, whereas during the recordings at 30 mV
the internal solution also contained the sodium channel blocker QX-314
(10 mM). The series resistance compensation was usually set
at 80%, and the series resistance (Rs 5-16
M) was monitored during the experiments every time an EPSC was
evoked. The neurons were voltage-clamped using an Axopatch-1D amplifier
(Axon Instruments, Foster City, CA). The synaptic events were evoked
with bipolar tungsten-stimulating electrodes positioned in the ventral
mesencephalon (200-400 µm from the recording site). All of the
electrical stimuli were locally delivered by a Grass S88 stimulator.
The EPSCslow was generated in the dopaminergic neurons by a
repetitive electrical stimulation (100-300 Hz, 40-400 msec duration,
1-20 V, delivered at 45 sec intervals). To evoke the
EPSCfast and the GABAA IPSC, single square-wave (0.1-0.3 msec, 1-10 V,) pulses were applied every 20 sec. In
experiments examining the EPSCslow and the
EPSCfast, the superfusion medium contained
picrotoxin (100 µM) or bicuculline (30 µM),
saclofen (300 µM), and strychnine (1 µM),
to block GABAA, GABAB, and
glycine receptors, respectively. The GABAA IPSC was evoked
in the presence of DL-2-amino-5-phosphono-pentanoic acid
(APV) (30 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 µM) to block NMDA and AMPA/kainate receptors,
respectively. A stimulation protocol similar to that used to evoke the
EPSCslow was also used to elicit the
IPSCslow, but the superfusing solution contained
picrotoxin (100 µM) or bicuculline (30 µM),
APV (30 µM), and CNQX (10 µM). The synaptic
currents were captured and stored on a computer by using the Pclamp
software 6.0.3 (Axon Instruments) and the analog/digital Maclab Chart
software (AD Instruments, Castle Hill, Australia). The
EPSCslow amplitude was measured by averaging a period of 10 msec, 400 msec after the end of the train of stimuli. The data were
subsequently analyzed with an Axograph 3.0 (Axon Instruments). The
dose-response curves of the effects of the drugs and the statistical
analyses were performed by using Kaleidograph, Mac Draw, and Stat View
4.1 running on a MacIntosh computer.
Application of drugs. The drugs were bath-applied at a
defined concentration. Drug solutions entered the recording chamber no
later than 20 sec after a three-way tap was turned. Complete replacement of the medium in the chamber took 90 sec. In some experiments, glutamate (1 mM) was applied via a puffer
pipette (20-80 msec, 100-200 kPa) controlled by a Picrospritzer II
(General Valve Corporation, Fairfield, NJ) and placed ~50 µm above
the recorded cells. The following drugs were used: picrotoxin,
bicuculline methiodide, methionine enkephalin, dopamine, nifedipine,
and APV [all obtained from Sigma (St. Louis, MO)]. CNQX,
(±)- -methyl-4-carboxyphenylglycine (MCPG), and
2-hydroxysaclofen (saclofen) were obtained from Tocris Cookson.
Nimodipine and Bay-K 8644 were a gift from Bayer Italia. Isradipine was
obtained from Sandoz (Basel, Switzerland), and -conotoxin GVIA,
-agatoxin-IVA, and -conotoxin MVIIC were obtained from Alomone
Labs.
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RESULTS |
Properties of the dopaminergic neurons and characterization of the
slow synaptic currents
The "principal" or presumed dopaminergic neurons of the
midbrain were identified by their spontaneous firing, broad (>1.5 msec) action potentials (under current-clamp), a pronounced
hyperpolarization-activated inward rectification
(Ih), an outward current response to
dopamine (10-30 µM), and no outward current to the
superfusion of methionine enkephalin (10-30 µM) (Kita et
al., 1986; Grace and Onn, 1989; Lacey et al., 1989; Yung et al.,
1991; Johnson and North, 1992; Mercuri et al., 1995).
The local stimulation of the ventral mesencephalon by a train of
electrical stimuli determined an EPSC (EPSCslow) in
cells patch-clamped at 60 mV. Figure
1A shows that the size
of the EPSCslow was dependent on the duration of the train.
It is clear that 150-200 msec are necessary to fully generate the
EPSCslow for a given train of stimuli. The maximal
amplitude and the mean duration of the EPSCslow was
371 ± 16.2 pA (n = 60) and 2.12 ± 0.8 sec
(n = 20), respectively. The EPSCslow was
caused by the coactivation of NMDA and metabotropic EAA receptors
(Mercuri et al., 1996; Shen and Johnson, 1997). In fact, the
application of the non-NMDA receptor antagonist CNQX (10 µM) did not modify the amplitude of the
EPSCslow (n = 6) (Fig.
1Ba). In addition, the NMDA receptor antagonist APV
(30 µM) depressed this synaptic event by 59 ± 4.1%
(n = 11) (Fig. 1Ba), whereas the
nonspecific metabotropic receptors antagonist MCPG (300 µM) depressed the EPSCslow by 34.9 ± 5.9% (n = 9) (Fig. 1Bb).
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Figure 1.
Properties of the EPSCslow.
A, The graph indicates that the amplitude of the
EPSCslow is dependent on the duration of the train. The
amplitude of the synaptic current was measured after the stimulation.
Each point represents at least three experiments. Ba,
The NMDA antagonist APV depresses the EPSCslow in a
reversible manner, whereas the AMPA/kainate antagonist CNQX (10 µM) did not produce any effect on the
EPSCslow. The stimulus artifacts were blanked (records are
average of 4 sweeps). Bb, The metabotropic antagonist
(+)-MCPG (300 µM) also depresses the EPSCslow
in a reversible manner.
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L-type calcium channel antagonists reduce
the EPSCslow
Bath application of the L-type calcium channel antagonists (Janis
and Triggle, 1984) nifedipine (1-100 µM), nimodipine
(1-100 µM), and isradipine (30 nM-30
µM) decreased, in a dose-dependent manner, the amplitude
of the EPSCslow (n = 64) (Fig.
2). The EC50 values for the
effects of nimodipine, nifedipine, and isradipine were 5.3, 6.3, and
0.63 µM, respectively. The maximum degree of inhibition
was caused by 30 µM isradipine (48.6 ± 3.1%,
n = 5). Nifedipine (100 µM) and
nimodipine (100 µM) reduced the slow synaptic current by
42.2 ± 4.8% and 36.8 ± 4.8%, respectively. The depression of the EPSCslow induced by the DHPs reached a steady-state
in 5-12 min and was washed out in ~15-25 min.
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Figure 2.
Effects of dihydropyridines on the slow excitatory
synaptic transmission. The traces represent the EPSCslow in
control, during and after (wash) the effect of
nifedipine (3 µM), nimodipine (10 µM), and
isradipine (1 µM). The bottom traces in
Aa, Ab, and Ac are
superimposed (control vs the effect of the L-type
Ca2+ antagonists). B, Dose-response
plots of the inhibition of the EPSCslow caused by
nifedipine (left), nimodipine (middle),
and isradipine (right). Each point is an average of at
least four different experiments. Only one experiment per slice was
performed. To calculate the percentage inhibition of the
EPSCslow, each cell was taken as its own
control.
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Interestingly, there was a direct correlation between the degree of
inhibition of the slow synaptic current and the duration of the
stimulation necessary to produce the EPSCslow. In fact, the
reduction of DHP's EPSCslow was not observed when it was
generated by a train of stimuli having a duration <70 msec
(n = 11) (Fig. 3).
However, when the train of stimuli lasted >80 msec, the DHPs clearly
depressed the EPSCslow (n = 53) (Fig. 3).
In another series of experiments, a single stimulus and a train of
stimuli (200 msec duration) were delivered alternately on the
same presumed dopaminergic neuron to compare the effect of nifedipine
on the fast and slow EPSC (Fig. 3Aa,c). Nifedipine (10 µM) reduced the EPSCslow by 30.1 ± 2.1% (n = 4), whereas it did not affect the EPSCfast.
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Figure 3.
A, The percentage of inhibition of
the EPSCslow caused by nifedipine is dependent on the
duration of the electrical stimulation. Aa, Superimposed
EPSCfast in control condition and after the application of
nifedipine. Ab, Ac, A 50 msec and a 200 msec train of
stimuli were delivered to evoke EPSCsslow. In the presence
of nifedipine, the EPSCslow evoked by the longer train
(c) was depressed, but the EPSC evoked by the
short train (b) was not affected. The time and
current bars in c are also valid for b.
Note that the EPSCfast in a and the
EPSCslow in c were elicited
alternately in the same neuron. B, The graph
shows that the depression of the EPSCslow caused by
nifedipine (10 µM) is dependent on the duration of the
electrical stimulus.
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The superfusion of the slices with nifedipine (10-100
µM) changed neither the amplitude of the
IPSCfast (GABAA mediated) (Hausser and Yung,
1994) evoked by a single shock (n = 5) nor the
IPSCslow (GABAB mediated) evoked by a train of
stimuli of various durations, ranging from 50 to 400 msec
(n = 7) (Fig.
4Aa,b) (Wu et al.,
1995). It is also worth noting that the IPSCslow was evoked
(in the presence of APV and CNQX) by the same stimulation protocol that
evoked the EPSCslow (see Materials and Methods).
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Figure 4.
A, Nifedipine (10 µM)
had no effect on the amplitude of the GABAA
(a) and GABAB
(b) IPSCs. The GABAA current is
inward because the recording pipette was filled with a solution
containing potassium chloride (see Materials and Methods). Ba,
Bb, Superimposed traces showing that the L-type
Ca2+ channel agonist Bay-K 8644 (1 µM)
increased the EPSCslow evoked by a short train
(a) but did not augment the EPSCfast
(b). The EPSCfast and the
EPSCslow were elicited alternatively in the same
cell. c, Plot taken from the same dopamine neuron
showing the time course of the enhancing effect of Bay-K 8644 on the
EPSCslow.
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Bay-K 8644 enhances the EPSCslow
The application of the L-type Ca2+ channels
agonist Bay-K 8644 (1 µM) (Nowycky et al., 1985)
increased the amplitude of the EPSCslow evoked by a short
train of stimuli (50-70 msec duration) by 21.8 ± 2.1%
(n = 5) but did not affect the EPSCfast
(Fig. 4B). In four experiments, a single stimulus and
a train of stimuli (50 msec duration) were also delivered
alternately on the same dopaminergic neuron (Fig.
4B). However, when the train of stimuli lasted >200
msec, Bay-K 8644 did not produce any detectable effect on the
EPSCslow (n = 5) (data not shown). Thus,
when a strong stimulation activated maximally the presynaptic L-type
Ca2+ channels, no further increase of the slow
synaptic current was caused by this L-type channel agonist.
Furthermore, neither the IPSCfast nor IPSCslow
were affected by Bay-K 8644 (1 µM) (three cells for each
condition; data not shown).
Site of action
To exclude a possible involvement of postsynaptic L-type calcium
channels on the dihydropyridine-mediated depression of the EPSCslow, additional experiments were performed
by holding the potential of the dopaminergic neurons at +30
(n = 6) and 100 mV (n = 5).
Nifedipine (10 µM) reduced the EPSCslow at 30 mV, 60 mV, and 100 mV to a similar degree (Fig.
5). The percentage reduction was
27.6 ± 2.7% (n = 4), 29.4 ± 3.2%
(n = 4), and 31.8 ± 2.9% (n = 4)
at + 30 mV, 60 mV, and 100 mV, respectively
(p > 0.05) (Fig. 5b). Furthermore, a
train of stimuli lasting 200 msec and pressure-ejected glutamate (1 mM) were delivered alternately on the same
dopaminergic neuron. Nifedipine (10 µM) did not modify the inward current caused by locally applied glutamate; however, it did
reduce significantly the synaptic current (p < 0.05) (Fig. 6), consistent with a
presynaptic site of action.
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Figure 5.
Presynaptic effects of nifedipine.
a, Changes in the holding potential from 60 mV to 30 mV or 100 mV did not affect the depression of the EPSC produced by
nifedipine. Typical traces taken form experiments showing the similar
degree of inhibition produced by nifedipine (10 µM).
b, The horizontal columns show that the percentage of
inhibition caused by nifedipine (10 µM) at 30 mV, 60
mV, and 100 mV is not statistically different among the three groups
of neurons tested (p > 0.05). Each column
represents an average of at least five cells. To improve space-clamp
between proximal somatic and distal dendritic regions, the
Ih current was also reduced by extracellular
CsCl (1 mM) during the hyperpolarization.
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Figure 6.
a, Traces from an experiment in
which a 200 msec train was given alternately with a 50 msec
extracellular (puff) application of
glutamate (1 mM). Nifedipine (10 µM)
decreased the amplitude of the EPSCslow but not the inward
current evoked by the local application of glutamate. b,
The plot represents the time course of the experiment shown in
a.
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Effects of the blockade of calcium channels subtypes on
the EPSC
When the N- and P/Q-type Ca2+ channels were
inhibited by the application of -conotoxin GVIA (1 µM), -AGA-IVA (200 nM), and -conotoxin
MVIIC (5 µM) (Olivera et al., 1985; Williams et al., 1992; Takahashi and Momiyama, 1993; Boland et al., 1994; Randall and
Tsien, 1995), the EPSCfast was almost completely blocked
(n = 4; data not shown). The EPSCslow
evoked by a short train of stimuli (50 msec duration) was reduced by
88.2 ± 6.2% (n = 3) (Fig.
7A). The subsequent
application of nifedipine (30 µM) did not produce any
further effect (91.3 ± 2.7%; n = 3) (Fig.
7A). The next experiments examined the contribution of
L-type calcium channels to the EPSCslow elicited by a long
train of stimuli. The amplitude of the EPSCslow evoked by a
80-200 msec train of stimuli was reduced by the application of the
three toxins by 71.1 ± 2.3% (n = 4) (Fig.
7A). In contrast to the cells stimulated with a short train,
the addition of nifedipine (30 µM) produced a further
reduction to 89.2 ± 2.8%; n = 4) (Fig.
7A,B). Cd2+ (300 µM) had a
similar but more prominent effect on the residual EPSCslow
(n = 4). In three experiments, the concentration of
-conotoxin GVIA, -AGA IVA, and -conotoxin MVIIC was raised
from 1 to 1.5 µM, from 200 to 300 nM, and
from 5 to 7 µM, respectively. However, the depression of
the residual EPSCslow caused by nifedipine (30 µM) was similar (93.1 ± 2.9%; p > 0.05) to that observed with the lower concentrations of toxins.
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Figure 7.
A, The graph shows the depressant
effects of -conotoxin GVIA (1 µM), -AGA-IVA (200 nM), and -conotoxin MVIIC (5 µM) on the
EPSCslow evoked by a short train of stimuli. Note that
nifedipine (30 µM) added to the three toxins did not
depress the synaptic current. A, B, In the presence of
-conotoxin GVIA (1 µM), -AGA-IVA (200 nM), and -conotoxin MVIIC (5 µM),
nifedipine (30 µM) reversibly depressed the residual
EPSCslow. The residual synaptic current recorded after the
treatment with the toxins was blocked by cadmium (300 µM). Ba, Sample records of a cell obtained
at the times indicated by the numbers 1-6 in the graph.
Bb, Graph of the amplitude of the EPSC slow
during the application of the natural toxins nifedipine (30 µM) and cadmium (300 µM). Each point is an
average of four experiments performed on four different slices.
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DISCUSSION |
Our results indicate that high frequency stimulation of the
ventral mesencephalon reveals a specific role for L-type
Ca2+ channels in the release of excitatory amino
acids on dopaminergic neurons. The observation that the
EPSCslow was reduced but not blocked by -conotoxin-GVIA,
-agatoxin-IVA, and -conotoxin MVIIC not only demonstrates the
importance of the N- and P/Q-type Ca2+ channels in
the release of EAAs, but it also confirms previous biochemical and
electrophysiological findings showing that a part of the release of
glutamate in the brain is resistant to N- and P/Q-type
Ca2+ channel antagonists (Brown et al., 1986; Turner
et al., 1992; Huston et al., 1995). Thus, in contrast to a single or a
short train, a prolonged high-frequency stimulation may activate a
sufficient number of L-type Ca2+ channels by
producing a prolonged depolarization of the presynaptic terminals.
Consequently, the influx of Ca2+ ions through them
could significantly participate in the exocytosis of EAAs on the
dopaminergic cells. The fact that the DHPs do not reduce the excitatory
transmission mediated either by single electrical stimuli or by a
short-stimulus train supports a more specific function for L-type
calcium channels in controlling the excitatory synaptic events caused
by a long sequence of action potentials. In fact, as observed in other
parts of the CNS, the influx of calcium during a weak stimulation of
presynaptic terminals is certainly controlled by the cooperative
activation of N- and P/Q-type channels (Luebke et al., 1993; Mintz
et al., 1995) but not by L-type channels that require a stronger
depolarization to open.
It is conceivable that the naturally occurring release of
excitatory neurotransmitters on the dopaminergic neurons is not only
related to the invasion of the synaptic terminals by a single action
potential but also by a sequence of spikes. Indeed, the sustained
release of EAAs from cortical cells has an important role in regulating
the type of firing of the dopaminergic cells (bursting vs pacemaker)
(Grenhoff et al., 1988; Overton and Clark, 1992), the extracellular
level of dopamine (Taber and Fibiger, 1995a,b), and the activity of
dopaminergic neurons to salient stimuli (Schultz, 1992). The cortical
and subthalamic neurons that release EAAs on the dopaminergic cells are
certainly able to maintain a high rate of repetitive firing (Connors et
al., 1982; Stafstrom et al., 1985; Smith and Grace, 1992;
Overton and Greenfield, 1995; Kreiss et al., 1997) and to depolarize
their excitatory terminals for a longer time. The biophysical
properties of L-type calcium channels (time- and voltage-dependence)
allow them to be active under conditions of repetitive stimulation. Thus, the time-, voltage-, and use-dependent properties of DHPs on
L-type calcium channels (Sanguinetti and Kass, 1984) could account for
the depression of the EPSCslow. The DHP-sensitive Ca2+ channels might be localized on all the
different sources of EAAs that project to the ventral mesencephalon
(Christie et al., 1985; Kita and Kitai, 1987; Sesack and Pickel, 1992;
Lavoie and Parent, 1994). Alternatively, only selected nerve terminals
of a more heterogeneous population of afferents might bear L-type
channels that regulate EAA exocytosis. It is worth mentioning that
other neurons from various regions of the CNS are able to fire in a burst or in a sustained manner. For this reason, it could be postulated that the action of L-type Ca2+ channels on the slow
synaptic event observed on the dopaminergic neurons might occur in
other areas of the brain.
All three DHP antagonists used in this study depressed in a
reversible manner the EPSCslow in a micromolar range. This
is a rather peculiar effect, because micromolar doses of DHP
antagonists do not usually change fast synaptic transmission in the
dopaminergic cells and in various central neurons in in
vitro conditions (Takayashi and Momiyama, 1993; Wheeler et al.,
1994; Dunlap et al., 1995; Sim and Griffith, 1996; Poncer, 1997). It is
also interesting to note that, as observed in other areas of the brain,
the fast and slow inhibitory synaptic currents were not affected by the DHP antagonists. This suggests that the exocytosis of
glutamate/aspartate but not that of GABA is controlled by L-type
Ca2+ channels during a sustained stimulation.
Because the DHPs reduce only the long EPSCslow but leave
unaffected the amplitude and duration of the short EPSCslow
and the IPSCslow, it is unlikely that a nonspecific
depression of excitability of presynaptic fibers could account for the
reduced release of transmitter after a train of stimuli. Thus, L-type
calcium channels could, during high frequency stimulation, modulate
excitatory transmission to dopaminergic cells. The manipulation of the
holding potential of the dopaminergic cells from 100 to +30 mV
indicates that postsynaptic voltage-dependent dihydropyridine-sensitive
calcium channels are not required for nifedipine action on the
EPSCslow. Although at 100 mV the L-type calcium channels
should be closed, at 30 mV the contribution of postsynaptic L-type
channels to the synaptic event should be minimal (Cardozo and Bean,
1995). The reversal of the EPSCslow indicates that adequate
voltage-clamp was maintained. These sets of experiments together with
those in which glutamate was locally applied imply that L-type
presynaptic calcium channels are involved.
Conclusions
In conclusion, the experiments described in the present paper
demonstrate that L-type calcium channels play an important role in
triggering the release of excitatory neurotransmitters on the dopaminergic neurons of the ventral mesencephalon. Thus, the activation of presynaptic boutons by a relatively long burst of action potentials could open not only the N- and P/Q-type but also the L-type
Ca2+channels that participate in controlling the
strength of excitation of the dopaminergic neurons.
Our data also suggest a rationale for the pharmacological manipulation
of the EAA inputs to the dopaminergic neurons by drugs that modulate
presynaptic L-type calcium channels in neurological and psychiatric
disorders involving the dopamine system.
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FOOTNOTES |
Received April 24, 1998; revised June 12, 1998; accepted June 16, 1998.
We thank Robert C. Malenka, Thomas Knopfel, and Bruce P. Bean for their
helpful comments and M. Federici for the technical assistance.
Correspondence should be addressed to Nicola B. Mercuri, Experimental
Neurology Laboratory, Ístituto Ricovero e Cura a Carattere Scientifico Santa Lucia, Via Ardeatina 306, 00179 Roma,
Italy.
Dr. Bonci's present address: Department of Psychiatry and Physiology,
School of Medicine, University of California, 401 Parnassus Avenue, San
Francisco, CA 94143.
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REFERENCES |
-
Augustine GJ,
Charlton MP,
Smith SJ
(1987)
Calcium action in synaptic transmitter release.
Annu Rev Neurosci
10:633-693[ISI][Medline].
-
Bean BP
(1989)
Classes of calcium channels in vertebrate cells.
Annu Rev Physiol
51:367-384[ISI][Medline].
-
Boland LM,
Morrill JA,
Bean BP
(1994)
Omega-conotoxin block of N-type calcium channels in frog and rat sympathetic neurons.
J Neurosci
14:5011-5027[Abstract].
-
Bonci A,
Williams JT
(1997)
Increased probability of GABA release after chronic morphine treatment.
J Neurosci
17:678-689.
-
Brown AM,
Kunze DL,
Yatani A
(1986)
Dual effects of dihydropyridines on whole cell and unitary calcium currents in single ventricular cells of guinea-pig.
J Physiol (Lond)
379:495-514[Abstract/Free Full Text].
-
Cardozo DL,
Bean BP
(1995)
Voltage-dependent calcium channels in a rat midbrain dopamine neurons: modulation by dopamine and GABAB receptors.
J Neurophysiol
74:1137-1148[Abstract/Free Full Text].
-
Christie MJ,
Bridge S,
James LB,
Beart PM
(1985)
Excitotoxins lesion suggest an aspartatergic projection from rat medial prefrontal cortex to ventral tegmental area.
Brain Res
333:169-172[ISI][Medline].
-
Connors BW,
Gutnick MJ,
Prince DA
(1982)
Electrophysiological properties of neocortical neurons in vitro.
J Neurophysiol
48:1302-1320[Abstract/Free Full Text].
-
Dunlap K,
Luebke JI,
Turner TJ
(1995)
exocitotic Ca++ channels in mammalian central neurons.
Trends Neurosci
18:89-98[ISI][Medline].
-
Grace AA,
Onn SP
(1989)
Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro.
J Neurosci
9:3463-3481[Abstract].
-
Grenhoff J,
Ugedo L,
Svennson TH
(1988)
Firing patterns of midbrain dopamine neurons: diversity in intrinsic differences between A9 and A10.
Acta Physiol Scand
134:271-284[ISI][Medline].
-
Hausser MA,
Yung WH
(1994)
Inhibitory synaptic potentials in guinea pig substantia nigra dopamine neurones in vitro.
J Physiol (Lond)
479:401-422[ISI][Medline].
-
Horne AL,
Kemp JA
(1991)
The effect of -conotoxin GVIA on synaptic transmission within the nucleus accumbens and hippocampus of the rat in vitro.
Br J Pharmacol
103:1733-1739[ISI][Medline].
-
Huston E,
Cullen GP,
Burley JR,
Dolphin AC
(1995)
The involvement of multiple calcium channel sub-types in glutamate release from cerebellar granule cells and its modulation by GABAB receptor activation.
Neuroscience
68:465-478[ISI][Medline].
-
Janis D,
Triggle DJ
(1984)
1,4-dihydropyridine calcium channel antagonists and activators: a comparison of binding characteristics with pharmacology.
Drug Dev Res
4:257-274[ISI].
-
Johnson SW,
North RA
(1992)
Two types of neurones in the rat ventral tegmental area and their synaptic inputs.
J Physiol (Lond)
450:455-468[Abstract/Free Full Text].
-
Kamiya H,
Sawada S,
Yamamoto C
(1988)
Synthetic -conotoxin blocks synaptic transmission in the hippocampus in vitro.
Neurosci Lett
91:84-88[ISI][Medline].
-
Katz B,
Miledi R
(1969)
Tetrodotoxin-resistant electric activity in presynaptic terminal.
J Physiol (Lond)
203:459-487[Abstract/Free Full Text].
-
Kita H,
Kitai ST
(1987)
Efferent projection of the subthalamic nucleus in the rat: light and electron microscopic analysis with the PHA-L method.
J Comp Neurol
260:435-452[ISI][Medline].
-
Kita T,
Kita H,
Kitai ST
(1986)
Electrical membrane properties of rat substantia nigra compacta neurons in an in vitro slice preparation.
Brain Res
372:21-30[ISI][Medline].
-
Kreiss DS,
Mastropietro CW,
Rawji SS,
Walters JR
(1997)
The response of subthalamic nucleus neurons to dopamine receptor stimulation in a rodent model of Parkinson's disease.
J Neurosci
17:6807-6819[Abstract/Free Full Text].
-
Kullman DM,
Perkell DJ,
Manabe T,
Nicoll RA
(1992)
Ca++ entry via post-synaptic voltage-sensitive Ca++ channels can transiently potentiate excitatory synaptic transmission in the hippocampus.
Neuron
9:1175-1183[ISI][Medline].
-
Lacey MG,
Mercuri NB,
North RA
(1989)
Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids.
J Neurosci
9:1233-1241[Abstract].
-
Lavoie B,
Parent A
(1994)
Pedunculopontine nucleus in the squirrel monkey: cholinergic and glutamatergic projections to the substantia nigra.
J Comp Neurol
344:232-241[ISI][Medline].
-
Le Moal M
(1995)
Mesocorticolimbic dopaminergic neurons.
In: Psychopharmacology: the fourth generation of progress (Bloom FE,
Kupfer DJ,
eds), pp 283-294. New York: Raven.
-
Lemos JR,
Nowycky MC
(1989)
Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals.
Neuron
2:1419-1426[ISI][Medline].
-
Llinàs R,
Steinberg IZ,
Walton K
(1981)
Relationship between presynaptic calcium current and postsynaptic potential in squid giant synapse.
Biophys J
33:323-352[Abstract/Free Full Text].
-
Llinàs R,
Sugimori M,
Lin JW,
Cherskey B
(1989)
Blocking and isolation of a Ca++ channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison.
Proc Natl Acad Sci USA
86:1689-1693[Abstract/Free Full Text].
-
Lopez MG,
Villaroya M,
Lara B,
Martinez Sierra R,
Albillos A,
Garcia AG,
Gandia L
(1994)
Q- and L-type Ca++ channels dominate the control of secretion in bovine chromaffin cells.
FEBS Lett
349:331-337[ISI][Medline].
-
Luebke JI,
Dunlap K,
Turner TJ
(1993)
Multiple calcium channel types control glutamatergic synaptic transmission in the hippocampus.
Neuron
11:895-902[ISI][Medline].
-
Mercuri NB,
Bonci A,
Calabresi P,
Stratta F,
Bernardi G
(1995)
Properties of the hyperpolarization-activated cation current Ih in rat midbrain dopaminergic neurons.
Eur J Neurosci
7:462-469[ISI][Medline].
-
Mercuri NB,
Grillner P,
Bernardi G
(1996)
N-methyl-D-aspartate receptors mediate a slow excitatory post-synaptic potential in the rat midbrain dopaminergic neurons.
Neuroscience
74:785-792[ISI][Medline].
-
Miller RJ
(1987)
Multiple calcium channels and neuronal function.
Science
235:46-52[Abstract/Free Full Text].
-
Mintz IM,
Venema VJ,
Swiderek KM,
Lee TD,
Bean BP
(1992)
P-type calcium channels blocked by the spider toxin -Aga-IVA.
Nature
355:827-829[Medline].
-
Mintz IM,
Sabatini BL,
Regehr WG
(1995)
Calcium control of transmitter release at a cerebellar synapse.
Neuron
15:675-688[ISI][Medline].
-
Nowycky MC,
Fox AP,
Tsien RW
(1985)
Long-opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644.
Proc Natl Acad Sci USA
82:2178-2182[Abstract/Free Full Text].
-
Olivera BM,
Gray WR,
Zeikus R,
McIntosh JM,
Varga J,
Rivier J,
de Santos V,
Cruz LJ
(1985)
Peptide neurotoxins from fish-hunting cone-snails.
Science
230:1338-1343[Abstract/Free Full Text].
-
Overton P,
Clark D
(1992)
Iontophoretically administered drugs acting at the N-methyl-D-aspartate receptor modulate burst firing in A9 dopamine neurons in the rat.
Synapse
10:131-140[ISI][Medline].
-
Overton PG,
Greenfield SA
(1995)
Determinants of neuronal firing pattern in the guinea-pig subthalamic nucleus: an in vivo and in vitro comparison.
J Neural Transm Park Dis Dement Sect
10:41-54[ISI][Medline].
-
Poncer JC
(1997)
Either N- or P-type calcium channels mediate GABA release at distinct hippocampal inhibitory synapses.
Neuron
18:463-472[ISI][Medline].
-
Randall A,
Tsien RW
(1995)
Pharmacological dissection of multiple types of calcium channel currents in rat cerebellar granule cells.
J Neurosci
15:2995-3012[Abstract].
-
Reuter H
(1996)
Diversity and function of presynaptic calcium channels in the brain.
Curr Opin Neurobiol
6:331-337[ISI][Medline].
-
Sanguinetti MC,
Kass RS
(1984)
Voltage-dependent block of calcium channels current in the calf cardiac Purkinje fiber by dihydropyridine calcium channel antagonists.
Circ Res
55:336-348[Abstract/Free Full Text].
-
Schultz W
(1992)
Activity of dopamine neurons in the behaving primate.
Semin Neurosci
4:129-138.
-
Seabrook GR,
Adams DJ
(1989)
Inhibition of neurally-evoked transmitter release by calcium channel antagonists in rat parasympathetic ganglia.
Br J Pharmacol
97:1125-1136[ISI][Medline].
-
Seeman P
(1995)
Dopamine receptors: clinical correlates.
In: Psychopharmacology: the fourth generation of progress (Bloom FE,
Kupfer DJ,
eds), pp 295-302. New York: Raven.
-
Sesack SR,
Pickel VM
(1992)
Prefrontal cortical efferents in the rat synapse on unlabeled neuronal target of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area.
J Comp Neurol
320:145-160[ISI][Medline].
-
Shen KZ,
Johnson SW
(1997)
A slow excitatory postsynaptic current mediated by G-protein-coupled metabotropic glutamate receptors in a rat ventral tegmental dopamine neurons.
Eur J Neurosci
9:48-57[ISI][Medline].
-
Sim JA,
Griffith WH
(1996)
Muscarinic inhibition of glutamatergic transmission onto rat magnocellular basal forebrain neurons in a thin slice preparation.
Eur J Neurosci
8:880-891[ISI][Medline].
-
Simmons ML,
Terman GW,
Gibbs SM,
Chavkin C
(1995)
L-type calcium channels mediate dynorphin neuropeptide release from dendrites but not axons of hippocampal granule cells.
Neuron
14:1265-1272[ISI][Medline].
-
Smith ID,
Grace AA
(1992)
Role of subthalamic nucleus in the regulation of nigral dopamine neuron activity.
Synapse
12:287-303[ISI][Medline].
-
Stafstrom CE,
Schwindt PC,
Chubb MC,
Crill WE
(1985)
Properties of persistent sodium conductance and calcium conductance of layer V neurons from cat sensory-motor cortex in vitro.
J Neurophysiol
53:153-170[Abstract/Free Full Text].
-
Taber MT,
Fibiger HC
(1995a)
Electrical stimulation of the prefrontal cortex increases dopamine release in the nucleus accumbens of the rat: modulation by metabotropic glutamate receptors.
J Neurosci
15:3896-3904[Abstract].
-
Taber MT,
Fibiger HC
(1995b)
Cortical regulation of subcortical dopamine release: mediation via the ventral tegmental area.
J Neurochem
65:1407-1410[ISI][Medline].
-
Takahashi T,
Momiyama A
(1993)
Different types of calcium channels mediate central synaptic transmission.
Nature
366:156-158[Medline].
-
Takibana M,
Okada T,
Arimura T,
Kobayashi K,
Piccolino M
(1993)
dihydropyridine-sensitive calcium current mediates neurotransmitter release from bipolar cells of the goldfish retina.
J Neurosci
13:2898-2909[Abstract].
-
Turner TJ,
Adams ME,
Dunlap K
(1992)
Multiple calcium channel types coexist to regulate synaptosomal neurotransmitter release.
Proc Natl Acad Sci USA
90:9518-9522[Abstract/Free Full Text].
-
Von Gersdorff H,
Matthews G
(1996)
Calcium-dependent inactivation of calcium current in synaptic terminals of retinal bipolar neurons.
J Neurosci
16:115-122[Abstract/Free Full Text].
-
Wheeler DB,
Randall A,
Tsien RW
(1994)
Roles of N-type and Q-type calcium channels in supporting hippocampal synaptic transmission.
Science
264:107-111[Abstract/Free Full Text].
-
Williams ME,
Brust PF,
Feldman DH,
Patthi S,
Simerson S,
Maroufi A,
Mc Crue AF,
Velicelebi G,
Ellis SB,
Harpold MM
(1992)
Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel.
Science
257:389-395[Abstract/Free Full Text].
-
Wright CE,
Angus JA
(1996)
Effects of N-, P- and Q- type neuronal calcium channels antagonists on mammalian peripheral neurotransmission.
Br J Pharmacol
119:49-56[ISI][Medline].
-
Wu Y,
Mercuri NB,
Johnson SW
(1995)
Presynaptic inhibition of
 -amino-butyric acid B-mediated synaptic currents by adenosine recorded in vitro in midbrain dopamine neurons.
J Pharmacol Exp Ther
273:576-581[Abstract/Free Full Text]. -
Yung WH,
Hausser MA,
Jack JJB
(1991)
Electrophysiology of dopaminergic and non-dopaminergic neurons of the guinea-pig substantia nigra pars compacta in vitro.
J Physiol (Lond)
436:643-667[Abstract/Free Full Text].
-
Zhang JF,
Randall AD,
Ellinor PT,
Horne WA,
Sather WA,
Tanabe T,
Schwartz TL,
Tsien RW
(1993)
Distinctive pharmacology and kinetics of cloned neuronal calcium channels and their possible counterparts in mammalian CNS neurons.
Neuropharmacology
32:1075-1088[ISI][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18176693-11$05.00/0
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Abstract
Introduction
