Calcium Release From Internal Stores Is Required for the Generation of Spontaneous Hyperpolarizations in Dopaminergic Neurons of Neonatal Rats

Vincent Seutin, Fatiha Mkahli, Laurent Massotte, and Albert Dresse

Laboratory of Pharmacology, University of Liège, Tour de Pathologie (B23), B-4000 Sart Tilman/Liège 1, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1 receptors (Seutin, unpublished observations).

Some of the results of this study have been published previously in abstract form (Seutin et al. 1999).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ) 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 MOmega . 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 MOmega 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, up-arrow ) 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).



View larger version (4K):
[in this window]
[in a new window]
 
Fig. 1. Two types of spontaneous hyperpolarizations in dopaminergic neurons of neonatal rats. Some events were preceded by a spike-like depolarization, whereas others were not (up-arrow ).

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).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Cyclopiazonic acid abolishes the TTX-resistant spontaneous hyperpolarizations. A constant current injection of -130 pA was applied throughout the experiment.


                              
View this table:
[in this window]
[in a new window]
 
Table 1. Effect of drugs affecting Ca2+ signaling on various electrophysiological properties of dopaminergic neurons from neonatal rats

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.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. Caffeine (1 mM) steadily increases the frequency and the amplitude of the events. A constant current injection of -100 pA was applied throughout the experiment. 2-[carboxy-3'-propyl]-3-amino-6-paramethoxy-phenyl-pyridazinium bromide (SR95531; 10 µM) was superfused continuously.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 4. Caffeine (10 mM) transiently enhances the frequency and the amplitude of the events then abolishes them. Note that the effect at 1 min is similar to the one observed in Fig. 3. Constant current injection was -95 pA except at 3 min in caffeine, at which time it was -55 pA; 0.5 µM TTX was superfused throughout the experiment.

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.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5. An experiment in which Cd2+ abolished the spontaneous hyperpolarizations. Constant current injections were -60 (control), -45 (Cd2+), and -80 (wash) pA. TTX (0.5 µM) was superfused throughout the experiment.

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.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6. Cyclopiazonic acid abolishes the events when they are enhanced by 1 mM caffeine. Note that the hyperpolarizations were very broad in this cell in the presence of caffeine. Constant current injections were -95 (control and cyclopiazonic acid) and -120 (wash) pA. TTX (0.5 µM) was superfused throughout the experiment.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7. Inhibitory effect of Cd2+ is less marked in the presence of caffeine. Note that the frequency of the events was only decreased by ~50%. Constant current injections were -110 (control and Cd2+) and -130 (wash) pA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society