Laboratory of Pharmacology, University of Liège, Tour de Pathologie (B23), B-4000 Sart Tilman/Liège 1, Belgium
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Seutin, Vincent, Fatiha Mkahli, Laurent Massotte, and Albert Dresse. Calcium Release From Internal Stores Is Required for the Generation of Spontaneous Hyperpolarizations in Dopaminergic Neurons of Neonatal Rats. J. Neurophysiol. 83: 192-197, 2000. We recently have demonstrated the existence of spontaneous hyperpolarizations in midbrain dopaminergic neurons of neonatal but not adult rats. These events are mediated by the opening of apamin-sensitive K+ channels after a rise in the intracellular concentration of Ca2+. They are resistant to tetrodotoxin in most cases and are probably endogenous (i.e., not synaptically activated). Here their mechanism was investigated. Cyclopiazonic acid (10 µM), a specific inhibitor of endoplasmic reticulum Ca2+ ATPases, reversibly abolished the events. Caffeine, which promotes Ca2+ release from intracellular stores, had concentration-dependent effects. At 1 mM, it markedly and steadily increased the frequency and the amplitude of the hyperpolarizations. At 10 mM, it induced a transient increase in their frequency followed by their cessation. All these effects were quickly reversible. Ryanodine (10 µM), which decreases the conductance of Ca2+ release channels, irreversibly blocked the spontaneous hyperpolarizations. Dantrolene (100 µM), a blocker of Ca2+ release from sarcoplasmic reticulum of striated muscle, did not affect the events. On the other hand, Cd2+ (100-300 µM), a broad antagonist of membrane voltage-gated Ca2+ channels, significantly reduced the amplitude and the frequency of the hyperpolarizations. However, when the frequency of the events was increased by 1 mM caffeine, Cd2+ affected them to a smaller extent, whereas cyclopiazonic acid still abolished them. We conclude that internal stores are the major source of Ca2+ ions that induce the K+ channel openings underlying the spontaneous hyperpolarizations of these neurons.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Increases in the intracellular concentration of
Ca2+ play a critical role in the control of
neuronal excitability by modulating the opening of various types of ion
channels (Hille 1992) and by altering the strength of
synaptic inputs (Malenka 1994
). Depending on the amount
of increase in [Ca]i, long-term depression or
potentiation can be induced at many, but not all (McBain et al.
1999
), synapses in the CNS.
Cai is clearly important in the regulation of the
excitability of midbrain dopaminergic neurons. Each action potential of these spontaneously firing neurons is followed by a large slow afterhyperpolarization (sAHP), which is mediated by apamin-sensitive K+ channels (Shepard and Bunney
1991). These channels belong to the small conductance
(SK) class of Ca2+-activated K+ channels
(Köhler et al. 1996
). Blockade of the sAHP has
been shown in vitro to modulate the spontaneous firing of DA neurons (Shepard and Bunney 1991
) and to enhance the ability of
bath-applied N-methyl-D-aspartate (NMDA) to
induce burst firing in these cells (Seutin et al. 1993
).
More recently, apamin-sensitive channels also have been shown to
underlie a metabotropic receptor-induced slow inhibitory postsynaptic
potential (IPSP) (Fiorillo and Williams 1998
). This IPSP
appears to be due to second-messenger-mediated mobilization of
Ca2+ from caffeine-sensitive intracellular stores.
In the course of a developmental study on the electrophysiological
properties of DA neurons, we found that SK channels can activate under
yet another circumstance. Thus using intracellular recordings in brain
slices, we showed that DA neurons from neonatal (PD 8-15) but not
adult rats undergo irregular, spontaneous hyperpolarizations (frequency: 0.2-1.5 Hz) that can be observed clearly when neurons are
hyperpolarized by current injection to about 60 mV (Seutin et
al. 1998
). These events had an amplitude and a duration of 2-8
mV and 100-400 ms, respectively. They were blocked specifically by
apamin and by quaternary salts of bicuculline, which also block SK
channels (Seutin and Johnson 1999
), but not by a more
specific GABAA antagonist. Finally, they were attenuated
strongly or abolished when the recording electrode contained the
Ca2+ chelator
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid (BAPTA), suggesting that they were due to a rise in the
intracellular concentration of this ion in the recorded neuron.
In the present series of experiments, we sought to identify the
source(s) of this rise. In particular, we investigated whether the
cellular mechanism of these spontaneous hyperpolarizations would be
similar to the one described for the metabotropic receptor-induced slow
IPSP. A major difference between this IPSP and the spontaneous hyperpolarizations is that the latter were unaffected by an antagonist of group I metabotropic receptors (Seutin et al. 1998).
They also were unaffected by antagonists of other receptors coupled to
the phosphoinositide pathway, namely muscarinic, 5HT2 and
1 receptors (Seutin, unpublished observations).
Some of the results of this study have been published previously in
abstract form (Seutin et al. 1999).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods were similar to the ones used in a previous paper
(Seutin et al. 1998). Briefly, neonatal (9-16 days)
Wistar and Sprague Dawley rats were used. They were housed and handled
in accordance with the Guide for the Care and Use of Laboratory
Animals, National Institutes of Health Publication 85-23, revised 1985. They were anesthetized with halothane and decapitated.
Horizontal slices were prepared as described (Seutin et al.
1998). After a 30 min-recovery period, slices were transferred into a recording chamber in which they were submerged completely in a
solution of the following composition (in mM): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.4 CaCl2, 11 glucose, and 18 NaHCO3, saturated with 95%
O2-5% CO2 (pH 7.4). Flow
rate and temperature were set at 2.5 ml/min. and 34.5 ± 0.5°C,
respectively (mean ± SE).
Intracellular recordings were made using microelectrodes (resistance:
60-150 M) filled with 2 M KCl. Recordings were obtained from 60 neurons exhibiting characteristic features of DA neurons, as described
previously (Seutin et al. 1998
). These neurons had a
spontaneous firing rate of 1.9 ± 0.2 spikes/s. Their input
resistance was 283 ± 15 M
. All traces shown in the figures
were obtained in the bridge balance mode around
60 mV. Because most
DA neurons were spontaneously active, a constant negative current
injection (
40 to
200 pA) was applied. For a given experiment, the
spontaneous hyperpolarizations were observed at the same mean membrane
potential in the control condition and in the presence of a drug. When
a drug induced a steady change in the mean membrane potential (see RESULTS), its effect on this parameter was offset by
changing the amplitude of the constant current injection. Only
recordings from neurons having an input resistance >100 M
were
analyzed. Off-line analysis was performed using Flukeview software.
Several parameters were measured to assess possible nonspecific effects
of the drugs that were applied: they included the amplitude of the
spike (from its threshold, which was close to 40 mV), the width of
the spike at the half of its maximal amplitude, the amplitude and time
to peak of the sAHP. The amplitude of the sAHP was estimated by
measuring the difference between the voltage reached at the end of the
fast AHP and at the peak of the sAHP. The time to peak was estimated as
the delay between the peak of the spike and the peak of the sAHP. Care
was taken to measure all these parameters at the same firing rate in
the control condition and in the presence of the drug. The input
resistance was measured by injecting small pulses (
20 to
30 pA) of
negative current while the cell was hyperpolarized to
60 mV by a
constant current injection. Some active currents were examined
qualitatively by observing voltage deflexions induced by pulses of
negative current of increasing amplitude (range:
60 to
200 pA).
They included the Ih current (Mercuri
et al. 1995
), which activates during such pulses, as well as an
IA type and a low-threshold Ca2+
current, which activate in some cells after the end of the current injection.
The spontaneous hyperpolarizations usually were seen easily at 60 mV.
However, in a few cells (n = 3), GABAA
IPSPs interfered with their visualization. In these cases, the
GABAA antagonist 2-[carboxy-3'-propyl]-3-amino-6-paramethoxy-phenyl-pyridazinium bromide (SR95531, 10 µM) (Heaulme et al. 1987
) was
superfused throughout the experiment.
Quantification of the spontaneous hyperpolarizations was done as
follows. Both their frequency and their amplitude were measured. At
least 20 events were considered for the amplitude measurements in one
condition except when their frequency was markedly decreased by a drug.
The frequency was measured over a time period of 30 s. Only events
2 mV were considered.
Drugs used and their supplier were as follows: apamin, baclofen, bicuculline methochloride (BMC), Cd2+, caffeine, cyclopiazonic acid (CPA), muscimol, ryanodine (Sigma, St Louis, MO), tetrodotoxin (TTX; ICN Biomedicals, Aurora, OH), SR95531 (gift from Sanofi, Paris), and theophylline (gift from SMB, Brussels).
A phosphate-free solution had to be used for the Cd2+ experiments. In this case, this solution was superfused first and served as the control condition before Cd2+ was applied. CPA was dissolved in DMSO and subsequently diluted 1000 times. Equivalent final concentrations of DMSO had no detectable effect. All drugs were applied by superfusion. Approximately 30 s were required for the drug solution to enter the recording chamber.
All data are expressed as means ± SE. Statistical differences were assessed using Student's t-test. Differences were considered significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dopaminergic neurons undergo two types of spontaneous hyperpolarizations
We suggested previously that spontaneous hyperpolarizations may be
heterogeneous in these cells (Seutin et al. 1998). This hypothesis was confirmed in the present study. Figure
1 shows an example of a DA neuron in
which both types of hyperpolarizations were observed. Some of these
events were preceded by a spike-like depolarization, whereas others
(Fig. 1,
) were not. Events of the first type were abolished by TTX,
but the others were not [their amplitude and/or frequency, however,
were reduced by 25-50% in a majority of cells (Fig. 3 of
Seutin et al. 1998
)]. The TTX-sensitive events were
only observed in three neurons (5%), and their mechanism was not
investigated further. These hyperpolarizations also are observed rarely
in neurons from adult animals (Seutin, unpublished observations). On
the other hand, the TTX-resistant events were found in most neurons
(n = 54; 90%). They were seen with the same frequency
in neurons from Wistar and Sprague-Dawley rats. We therefore focus on
these hyperpolarizations in this paper. The kinetics of these events
was significantly slower than the one of the sAHP. Indeed, their time
to peak was 67 ± 2 ms (n = 55 events from 6 cells), as compared with 51 ± 3 ms for the sAHP
(n = 20; P < 0.001).
|
Effect of agents interfering with Ca2+ release from internal stores
CPA is a selective inhibitor of endoplasmic reticulum
Ca2+ ATPases (Seidler et al.
1989). We used it instead of thapsigargin because it had been
shown that the latter agent also may block membrane
Ca2+ channels at the concentrations that have to
be used in brain slices (see Taylor and Broad 1998
). At
10 µM, CPA abolished the spontaneous hyperpolarizations in all
neurons tested (n = 14), as shown in Fig.
2. This effect occurred after 5-8 min.
It was very slowly reversible after removal of the drug: a partial
recovery was observed after 45-60 min. The effect of CPA was similar
in the absence (n = 8) and in the presence
(n = 6) of TTX. CPA did not have any effect on other
electrophysiological parameters of DA neurons. It did not affect the
baseline membrane potential, and it altered neither the shape of the
action potential and of the sAHP (Table
1) nor the input resistance of the
neurons. Moreover, active currents did not seem to be significantly
affected by CPA. Finally, CPA did not have a significant effect on the
spontaneous firing rate of the cells (2 ± 0.5 vs. 1.8 ± 0.4 spikes/s in the control condition, n = 6, NS).
|
|
Caffeine is known to activate Ca2+ release from
ryanodine-sensitive intracellular stores at high concentrations
(Rousseau and Meissner 1989). Effects of caffeine on the
hyperpolarizations were concentration dependent. It had no significant
effect at 100 µM (n = 5). At 1 mM, it induced a
steady increase in the frequency and the amplitude of the events (Fig.
3). The frequency rose from 0.67 ± 0.07 to 1.37 ± 0.17 Hz (n = 11, P < 0.01), whereas the mean amplitude increased from 3.15 ± 0.18 to 4.51 ± 0.3 mV (n = 11, P < 0.001). The duration of the events also probably increased, but this
was difficult to assess because they often overlapped with each other
in the presence of caffeine. These effects occurred within 2-3 min and
were rapidly reversible (after 3-4 min) on wash-out of the drug.
Caffeine had no significant effect on the baseline membrane potential
(
0.4 ± 0.7 mV, n = 11, NS) at this concentration. As shown in Table 1, its effect on other
electrophysiological parameters were negligible. Another pattern was
observed when caffeine was applied at a concentration of 10 mM (Fig.
4). After a transient increase in the
frequency of the hyperpolarizations (duration: ± 1 min), a complete
cessation was seen after ~3 min (n = 8 neurons). On
the other hand, this concentration of caffeine also induced a steady
hyperpolarization of the baseline membrane potential (
6.6 ± 0.9 mV, n = 10 applications) as well as a decrease in input
resistance of 15 ± 5% (n = 8). However, the
effect of caffeine on the spontaneous events persisted when the
membrane potential was brought back to
60 mV by changing the
amplitude of the injected current (see METHODS). Moreover,
control experiments showed that agents that induce a similar decrease
in input resistance (1 µM muscimol and 300 nM baclofen, n
total = 6) did not abolish the spontaneous hyperpolarizations (not
shown). The effects of 10 mM caffeine were partially reversible after
5-10 min. The steady hyperpolarization induced by 10 mM caffeine was
antagonized partly (from 9 ± 0.6 to 3.3 ± 0.7 mV) by the SK
channel blockers BMC (300 µM) or apamin (300 nM; n
total = 3). TTX did not modify any of these actions of caffeine.
Caffeine also induced nonspecific effects at this concentration (Table
1). It prolonged the action potential and the time to peak of the sAHP
while reducing their amplitude. As a control experiment, we tested the
effect of 100 µM theophylline. This compound had no effect on the
spontaneous hyperpolarizations (n = 3) although it
blocks adenosine receptors at this concentration in the hippocampus
(Mitchell et al. 1993
; Seutin, unpublished
observations). This confirms that the effects of caffeine on the
spontaneous events are due to its interaction with
Ca2+ release channels.
|
|
Ryanodine (10 µM), which locks release channels in a low conductance
state (Hille 1992), abolished the spontaneous
hyperpolarizations after 5-7 min in all tested cells
(n = 4; not shown). This effect was irreversible.
Ryanodine did not induce significant nonspecific effects, except for a
small (5-30%) reduction in the amplitude of the sAHP.
Finally, dantrolene (100 µM; applications lasting 40 min), which is
a blocker of Ca2+ release from sarcoplasmic
reticulum (Ohta et al. 1990
), had no effect on the
spontaneous hyperpolarizations (n = 6; not shown). It
also did not affect them when their frequency was enhanced by 1 mM
caffeine (n = 2).
Effect of cadmium
The effect of Cd2+ (100-300 µM), a broad
antagonist of voltage-dependent Ca2+ channels
(VDCCs), was examined in 10 cells [both in the absence (n = 5) and in the presence (n = 5) of
TTX, which again did not modulate the effects of the blocker].
Cd2+ markedly reduced the frequency of the
hyperpolarizations (n = 5) or abolished them
(n = 5; Fig. 5). The
effect of Cd2+ appeared rapidly (2-3 min) and
was partially reversible 10-30 min after removal of the drug. The
overall reduction of frequency produced by Cd2+
was 81 ± 9% (n = 10). It also reduced the mean
amplitude of the events in the five cells in which it had a partial
effect (from 3.1 ± 0.3 to 2.1 ± 0.1 mV). Interestingly, it
induced a small steady hyperpolarization (2.8 ± 0.6 mV) in 5 of
10 experiments. On the other hand, application of
Cd2+ suppressed the spontaneous firing (which is
Ca2+-dependent) (Kang and Kitai
1993), as well as the sAHP (Table 1), suggesting that it
effectively blocks Ca2+ channels at these
concentrations. It also markedly reduced the amplitude and the width of
the spike (Table 1). The input resistance was not affected by
Cd2+ at the time when measurements
were made (3-7 min.). We noticed, however, that longer-lasting
applications of the blocker could induce a decrease in input resistance
and a depolarization that were sometimes irreversible.
|
Effect of cyclopiazonic acid and of cadmium in the presence of 1 mM caffeine
We took advantage of the frequency-augmenting effect of 1 mM caffeine to examine effects of drugs under conditions in which the phenomenon was enhanced (Figs. 6 and 7). In the presence of this concentration of caffeine, CPA still abolished the hyperpolarizations in all tested cells (Fig. 6, n = 5). The effect of Cd2+ was overall significant but much less marked (Fig. 7, n = 5, P < 0.001 vs. CPA). Indeed, the events were never abolished in these conditions. Their frequency decreased by 48 ± 5% (from 1.34 ± 0.25 to 0.72 ± 0.18 Hz). This effect was significantly smaller than the one observed with Cd2+ in the absence of caffeine (P < 0.05). The amplitude of the events was reduced as well. The time course of the effects of both CPA and Cd2+ was the same as in the absence of caffeine.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results show that three compounds known to interfere with Ca2+ signaling originating in intracellular stores (CPA, caffeine, and ryanodine) have a major effect on spontaneous hyperpolarizations of neonatal DA neurons. Another compound that blocks release from Ca2+ stores of the sarcoplasmic reticulum (dantrolene) has no effect. On the other hand, a blocker of VDCCs (Cd2+) has a smaller and less consistent inhibitory effect than the one of CPA.
The fact that CPA, caffeine, and ryanodine markedly affect the
spontaneous hyperpolarizations is strong evidence that release of
Ca2+ from ryanodine-sensitive stores is a
critical step in their generation. Our results are similar in this
respect to those of Fiorillo and Williams (1998), who
showed that 10 µM CPA, 10 mM caffeine, and 10 µM ryanodine block
the occurrence of the IPSP mediated by SK channels in DA neurons of
adult rats (see INTRODUCTION). Therefore it is likely that
the spontaneous release of Ca2+ that we
demonstrate here originates from stores that are very similar or
identical to those that are activated after synaptic stimulation of
metabotropic glutamate receptors in adult DA neurons.
In addition, we show that a lower concentration of caffeine (1 mM) has
a facilitating effect on the hyperpolarizations. In our view, this
apparently paradoxical result has a simple explanation. At 1 mM,
caffeine moderately stimulates the release of
Ca2+ from the stores, whereas its effect at 10 mM
is so potent that it completely depletes the stores, thereby
inactivating the process. A similar interpretation for this dual effect
of caffeine was proposed in a recent Ca2+ imaging
study of CA1 pyramidal neurons (Sandler and Barbara
1999). An observation that is in favor of this interpretation
in our study is the fact that the higher but not the lower
concentration of caffeine induces a steady hyperpolarization of the
neurons. Furthermore this steady hyperpolarization is attenuated by SK channel blockers. This suggests that, in these conditions, the cytoplasmic concentration of Ca2+ undergoes a
continuous increase that steadily activates SK channels as well as
other (unidentified) channels, possibly BK-type
Ca2+-dependent K+ channels
(Hille 1992
).
CPA and 1 mM caffeine had a major effect on the spontaneous hyperpolarizations without affecting the sAHP. Moreover, the kinetics of the two types of events were different. Taken together, these results strongly suggest that the sAHP and the spontaneous hyperpolarizations are regulated differentially in DA neurons. It is likely that the sAHP is induced mainly by Ca2+ entering the cell through VDCCs (see the effects of Cd2+ on the sAHP).
One interesting observation is that a high concentration of dantrolene
(100 µM) did not affect the process that we describe (dantrolene was
not tested by Fiorillo and Williams). This suggests that the
pharmacology of the Ca2+ stores in DA neurons may
be special. Indeed, there is strong evidence in skeletal muscle that
caffeine and dantrolene have opposite effects on
Ca2+ release from ryanodine sensitive stores
(e.g., see Ohta et al. 1990). Moreover, similar evidence
has been obtained in neurons of the rat suprachiasmatic nucleus
(Ding et al. 1998
; the effect of dantrolene was observed
at 20 µM in a slice preparation), suggesting that the pharmacology of
Ca2+ stores may be heterogeneous in the CNS.
CPA appears to be a valuable tool to examine the contribution of
intracellular Ca2+ stores to cell physiology.
Indeed, this compound appeared to act very selectively on this target
without having any major unspecific effect. It is interesting to note
that CPA did not induce a steady hyperpolarization of the cells,
whereas 10 mM caffeine did. The most likely explanation of this
observation is that the kinetics of the increase in cytoplasmic
Ca2+ brought about by both drugs is very
different. Thus this rise is presumably slow with CPA, allowing
cytoplasmic buffering systems and/or membrane
Ca2+ transporters to counteract it. The situation
is very different with 10 mM caffeine, which probably induces a brutal
increase in the cytoplasmic concentration of
Ca2+. It should be noted that CPA did not affect
the firing rate of neonatal DA neurons. Assuming that its effect on the
spontaneous hyperpolarizations is very specific, this finding suggests
that these events do not have a significant influence on the firing rate of these neurons, although we had suggested this as a possibility (Seutin et al. 1998).
The Cd2+ experiments suggest that membrane VDCCs contribute to the spontaneous hyperpolarizations, albeit to a lesser extent than intracellular stores. Indeed, the effect of the divalent cation was smaller than the one of CPA, especially in the presence of 1 mM caffeine. Membrane VDCCs might play several roles: induce the phenomenon known as Ca2+-induced Ca2+ release, be involved in the replenishment of the stores, and modulate the phenomenon indirectly. The fact that Cd2+ was less potent in the presence of caffeine than in its absence is inconsistent with the second hypothesis. Because caffeine enhances the release and therefore tends to deplete the stores, Cd2+ should be more potent in the presence of caffeine. Our data do not allow to discriminate with certainty between the two other hypotheses.
Our results strongly suggest that repetitive elevations of the
cytoplasmic concentration of Ca2+ occur in
microdomains of DA neurons in neonatal rats. In view of the role of
such events in synaptic plasticity, it will be interesting in future
experiments to determine whether such plasticity exists in neonatal DA
neurons and whether it is modulated by the phenomenon described here.
It should be noted in this respect that a NMDA-receptor-dependent
long-term potentiation of glutamatergic synapses has been recently
demonstrated in these cells in slices taken from slightly older animals
(Bonci and Malenka 1999).
Finally, our study suggests that release of Ca2+
from intracellular stores in central neurons may be even more common
than was thought previously. Indeed, it shows that, besides their
activation after synaptic stimulation (Finch and Augustine
1998; Fiorillo and Williams 1998
; Takechi
et al. 1998
), these stores also can release
Ca2+ spontaneously, at least at a well-defined
stage of development.
![]() |
ACKNOWLEDGMENTS |
---|
We thank Dr. Elizabeth Thomas for helpful discussions.
V. Seutin and A. Dresse were supported in part by a grant from the National Fund for Scientific Research (F.N.R.S., Belgium).
![]() |
FOOTNOTES |
---|
Address reprint requests to V. Seutin.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 June 1999; accepted in final form 23 September 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|