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The Journal of Neuroscience, 2000, 20:RC103:1-5

RAPID COMMUNICATION
Inositol 1,4,5-Triphosphate-Evoked Responses in Midbrain Dopamine Neurons

Hitoshi Morikawa¹, Farzin Imani², Kamran Khodakhah², and John T. Williams¹

¹ Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, and ² Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver, Colorado 80262

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Synaptically released glutamate evokes slow IPSPs mediated by metabotropic glutamate receptors (mGluRs) in midbrain dopamine neurons. These mGluR IPSPs are caused by release of Ca²⁺ from intracellular stores and subsequent activation of small-conductance Ca²⁺-activated K⁺ channels (SK channels). To further investigate the intracellular mechanisms involved, the effect of photolyzing intracellular caged inositol 1,4,5-triphosphate (InsP₃) on membrane conductance and intracellular Ca²⁺ concentration ([Ca²⁺]_i) was examined in rat midbrain slices. Photolytic release of InsP₃ elicited a transient outward current and a sharp rise in [Ca²⁺]_i that lasted for ~5 sec. Apamin, a blocker of SK channels, abolished the InsP₃-induced outward current without affecting the rise in [Ca²⁺]_i. Depleting intracellular Ca²⁺ stores with cyclopiazonic acid completely blocked both the outward current and the Ca²⁺ transient elicited by InsP₃. InsP₃-evoked Ca²⁺ mobilization was not affected by blockade of ryanodine receptors with ruthenium red, whereas depleting ryanodine-sensitive Ca²⁺ stores with ryanodine almost eliminated InsP₃-induced Ca²⁺ release. Increasing the size of intracellular Ca²⁺ stores by means of prolonged depolarization added a late component to the outward current and a slow component to the rising phase of [Ca²⁺]_i. These effects of depolarization were blocked by ruthenium red. These results show that InsP₃ activates SK channels by releasing Ca²⁺ from InsP₃-sensitive stores that also contain ryanodine receptors. Increasing intracellular Ca²⁺ stores boosts InsP₃-evoked responses by invoking Ca²⁺-induced Ca²⁺ release through ryanodine receptors. This intracellular signaling pathway may play a significant role in regulating the excitability of midbrain dopamine neurons.

Key words: intracellular Ca²⁺; inositol 1,4,5-triphosphate; inositol 1,4,5-triphosphate receptors; ryanodine receptors; SK channels; midbrain dopamine neurons; flash photolysis

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Intracellular Ca²⁺ plays a pivotal role in controlling the excitability of neurons by activating various Ca²⁺-sensitive ion channels on the plasma membrane (Vergara et al., 1998). One major pathway to elevate intracellular Ca²⁺ concentration ([Ca²⁺]_i) is mobilization of Ca²⁺ from intracellular stores. This is achieved by activating inositol 1,4,5-triphosphate (InsP₃) receptors or ryanodine receptors located on the membranes of these stores. Firing of presynaptic fibers is known to evoke a rise in [Ca²⁺]_i in postsynaptic neurons, mainly via Ca²⁺ influx (Denk et al., 1996). Recent evidence has demonstrated that synaptically released glutamate, acting at metabotropic glutamate receptors (mGluRs), results in the generation of InsP₃ and subsequent mobilization of Ca²⁺ in cerebellar Purkinje neurons (Finch and Augustine, 1998; Takechi et al., 1998) and hippocampal pyramidal neurons (Nakamura et al., 1999).

Dopaminergic neurons in the ventral midbrain (ventral tegmental area and substantia nigra pars compacta) are involved in the perception of reward, motivational behavior, and the reinforcing actions of addictive drugs. In addition, impaired functioning of dopamine neurons is associated with the etiology of human disorders such as Parkinson's disease and schizophrenia (for review, see Schultz, 1998). It has recently been shown that glutamate released from presynaptic terminals elicits slow IPSPs mediated by activation of mGluRs in midbrain dopamine neurons (Fiorillo and Williams, 1998). This mGluR-mediated hyperpolarization results from mobilization of Ca²⁺ from intracellular stores, probably through production of InsP₃, and subsequent activation of small-conductance Ca²⁺-activated K⁺ channels (SK channels). Furthermore, spontaneous release of Ca²⁺ from internal stores has been implicated in the generation of spontaneous hyperpolarizations observed in dopamine neurons from neonatal rats (Seutin et al., 2000). The involvement of ryanodine-sensitive Ca²⁺ stores has been suggested in both cases on the basis of sensitivity to caffeine and ryanodine.

In the present study, the intracellular signaling pathway ensuing from the generation of InsP₃ was investigated in midbrain dopamine neurons using flash photolysis of caged InsP₃ loaded into the cell (Walker et al., 1989). The results demonstrate that InsP₃ elicits activation of SK channels through mobilization of Ca²⁺ from intracellular stores and that these InsP₃-sensitive stores are invariably equipped with ryanodine receptors. In addition, Ca²⁺-induced Ca²⁺ release (CICR) through ryanodine receptors was found to boost InsP₃-evoked release of Ca²⁺ when internal Ca²⁺ stores were filled by depolarization-induced Ca²⁺ influx.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Whole-cell recordings were made from dopamine neurons in horizontal midbrain slices (250 µm) from Wistar rats (10-21 d). Preparation of slices has been described previously (Cameron and Williams, 1994). Slices were placed in a recording chamber and superfused with warmed (35°C) physiological saline containing (in mM): 125 NaCl, 2.5 KCl, 1 MgCl₂, 26 NaHCO₃, 1.25 NaH₂PO₄, 11 glucose, 2.4 CaCl₂, bubbled with 95% O₂/5% CO₂; pH 7.4, 295 mOsm/kg. Cells were visualized using an upright microscope with infrared optics, and recordings were made with patch pipettes containing (in mM): 145 KMeSO₄, 8 KCl, 10 HEPES, 2 MgATP, and 0.2 GTP; pH 7.2, 285 mOsm/kg. The pipette solution also contained Fura-6F (500 µM; Molecular Probes, Eugene, OR) and caged InsP₃ [100 µM; made in house; Womack et al. (2000)]. Ruthenium red (20 µM; Calbiochem, San Diego, CA) or ryanodine (10 µM; Sigma, St. Louis, MO) was added to the pipette solution when indicated. The membrane potential was clamped at 60 mV unless stated otherwise. Recordings were started at least 15 min after whole-cell access was gained to ensure equilibrium of the pipette solution with the cytosol. Dopamine cells were identified by the presence of a large I_H current (>200 pA at 120 mV) (Johnson and North, 1992).

In experiments in which mGluR-mediated IPSCs were measured, EGTA (100 µM) was added to the pipette solution instead of Fura-6F and caged InsP₃. The superfusion medium contained 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]-quinoxaline (5 µM), picrotoxin (100 µM), and CGP35348 (100 µM) to block AMPA-, GABA_A-, and GABA _B-mediated synaptic currents, and slices were pretreated with MK-801 (50 µM) to block the NMDA-mediated synaptic current. A train of 10 stimuli (500 µsec at 70 Hz) was delivered every 60 sec, using a bipolar tungsten stimulating electrode placed close (30-100 µm) to the soma. The stimulus intensity was adjusted to obtain a maximal IPSC in each cell. The slow IPSC thus recorded was inhibited by the mGluR antagonist (S)--methyl-4-carboxyphenylglycine (1 mM).

Fluorescence measurements were made from an area just covering the soma, defined by a rectangular diaphragm in a conjugate image plane of the microscope. The Ca²⁺ indicator dye Fura-6F (K_d = 5.3 µM), introduced into the cell via the whole-cell patch pipette, was excited at a single wavelength of 425 ± 15 nm, and the emitted light was collected at 510 ± 15 nm. Fura indicators, when fully saturated with Ca²⁺, emit negligible fluorescence when excited at ~420 nm (Ogden et al., 1995). [Ca²⁺]_i was calculated from the formula [Ca²⁺] = K_d(F  F_min)/(F_max  F), where F is the background-corrected fluorescence, F_min is fluorescence of the indicator at 0 [Ca²⁺], and F_max is fluorescence of the indicator at saturating [Ca²⁺], which was assumed to be zero as stated above. F_min was obtained in each cell by assuming that the resting fluorescence emitted by the indicator, after background correction, reflects fluorescence at 50 nM [Ca²⁺]_i.

A xenon arc lamp (Carin Research, Faversham, UK) produced UV pulses of ~1 msec in duration to photolyze a known fraction of caged InsP₃ loaded into the cytosol. The interval between two successive pulses was >4 min to allow reequilibration of caged InsP₃ with the cytosol. The extent of photolysis was calibrated using a fluorescent pH indicator, taking advantage of the stoichiometric release of a proton with ATP during photolysis of caged MgATP, which has the same photolytic efficiency as caged InsP₃ (Walker et al., 1988).

Data are expressed as means ± SEM. Statistical significance was determined with Student's t test (unpaired or paired) or ANOVA. The difference was considered significant if p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Intracellular InsP₃ induces SK channel activation in dopamine neurons

Whole-cell recordings (holding potential, 60 mV) were made from dopamine neurons dialyzed with the calcium indicator Fura-6F (500 µM) and caged InsP₃ (100 µM) in ventral midbrain slices. Pulses of UV light (1 msec) were applied to rapidly release (~3 msec) (Walker et al., 1989) a known concentration of InsP₃, and the resulting changes in membrane current and fluorescence were measured. Photolytic release of InsP₃ (30 µM) in the cytosol elicited a transient outward current and a rapid rise in [Ca²⁺]_i, which decayed over a period of ~5 sec (Fig. 1A). The outward current invariably decayed faster than [Ca²⁺]_i, which may reflect the actual subplasmalemmal [Ca²⁺]_i and also the steep Ca²⁺ concentration-dependence of apamin-sensitive SK channels (Köhler et al., 1996). On average, the amplitude of the peak outward current and the magnitude of the increase in [Ca²⁺]_i produced by InsP₃ (30 µM) was 104 ± 18 pA and 1.1 ± 0.1 µM, respectively (n = 18). These InsP₃-evoked responses were reproducible throughout the duration of recordings (~3 hr).

View larger version (17K): [in this window] [in a new window]   Figure 1.   Photolytic release of InsP3 induced apamin-sensitive SK channel activation in dopamine neurons. A, A pulse of UV light (1 msec) was applied at the time indicated by the arrow to rapidly release InsP3 (30 µM) in the cytosol. The resulting changes in membrane current (top traces) and [Ca2+]i (bottom traces) are shown before (left) and after (right) bath application of apamin (100 nM). B, Pooled data from four cells tested for the effect of apamin (100 nM). *p < 0.05. C, Scatter plot of the amplitude of outward current versus the peak [Ca2+]i evoked by photolytic release of InsP3 (0.75-30 µM). The data are from 18 cells. Up to five different concentrations of InsP3 were tested in each cell.

Extracellular application of apamin (100 nM), a blocker of SK channels (Köhler et al., 1996), irreversibly blocked the InsP₃-induced outward current (10 ± 4% of control, n = 4) without significantly affecting the [Ca²⁺]_i elevation (94 ± 5% of control, n = 4) (Fig. 1A,B). The amplitude of the outward current produced by various concentrations of InsP₃ (0.75-30 µM) correlated with the peak [Ca²⁺]_i (r = 0.70) (Fig. 1C).

The effects of cyclopiazonic acid (CPA) on the InsP₃-evoked responses were examined next. CPA depletes intracellular Ca²⁺ stores by blocking the endoplasmic reticulum Ca²⁺-ATPase (Seidler et al., 1989). The experiment in Figure 2A illustrates that bath application of CPA (10 µM) completely abolished both the outward current and the rise in [Ca²⁺]_i evoked by InsP₃. In five cells tested, CPA (10 µM) reduced the InsP₃-induced increase in [Ca²⁺]_i to 9 ± 4% of control and completely inhibited the outward current (Fig. 2B). In contrast, CPA had no effect on the rise in [Ca²⁺]_i caused by a depolarizing step to 0 mV (1 sec), although it slowed the decay time course of [Ca²⁺]_i after depolarization (Fig. 2A).

View larger version (22K): [in this window] [in a new window]   Figure 2.   Depletion of intracellular Ca2+ stores abolished InsP3-evoked responses. A, A depolarizing step to 0 mV (1 sec; voltage protocol illustrated below) followed 20 sec later by photolytic release of InsP3 (30 µM; arrow) was applied before (left) and after (right) perfusion of CPA (10 µM). The resulting changes in membrane current (top traces) and fluorescence (bottom traces) are shown. In this cell, a high-affinity Ca2+ indicator Fura-2 (200 µM; kd = 140 nM) was used to enhance detection of small changes in [Ca2+]i. The fluorescence was therefore not converted to [Ca2+]i. B, Pooled data from five cells tested for the effect of CPA (10 µM). *p < 0.05.

Taken together, these results indicate that InsP₃ released in the cytosol mobilizes Ca²⁺ from intracellular stores and activates apamin-sensitive SK channels.

InsP₃ receptors and ryanodine receptors are colocalized on the same intracellular Ca²⁺ stores

It has been suggested that activation of mGluRs triggers CICR through ryanodine receptors in midbrain dopamine neurons (Fiorillo and Williams, 1998). To investigate whether photolytic release of InsP₃ evokes CICR, the contribution of ryanodine receptors was assessed with ruthenium red and ryanodine. Ruthenium red is a blocker of ryanodine receptors, whereas ryanodine locks ryanodine receptor channels in a subconductance open state and depletes ryanodine-sensitive Ca²⁺ stores (Smith et al., 1988). Ruthenium red (20 µM in the patch pipette) had no significant effect on the outward current or the rise in [Ca²⁺]_i caused by release of InsP₃ (30 µM) (Fig. 3A), indicating that CICR through ryanodine receptors does not play a major role in the InsP₃-induced mobilization of Ca²⁺. Furthermore, the amplitude of mGluR IPSCs elicited by extracellular stimuli was not significantly different between control and ruthenium red-containing internal solutions (121 ± 26 pA, n = 5 vs 106 ± 25 pA, n = 6, p > 0.6). In contrast, ryanodine (10 µM in the patch pipette) almost completely blocked InsP₃-induced responses (Fig. 3A). However, after a depolarizing step (5 sec) to 0 mV, InsP₃ elicited a clear increase in [Ca²⁺]_i and a small outward current even in the presence of ryanodine. The effect of depolarization lasted for at least 30 sec (n = 3). This observation suggests that Ca²⁺ entry during depolarization partially refilled the InsP₃-sensitive stores that had been depleted by ryanodine. The inhibitory action of ryanodine (10 µM) was fully blocked by co-addition of ruthenium red (20 µM) to the internal solution (Fig. 3A).

View larger version (36K): [in this window] [in a new window]   Figure 3.   The effects of ruthenium red and ryanodine on InsP3-induced responses. A, Summary histogram showing InsP3-evoked responses in cells dialyzed with control pipette solution, ruthenium red (20 µM), ryanodine (10 µM), and both ruthenium red (20 µM) and ryanodine (10 µM). The number of cells tested is indicated in parentheses. *p < 0.05. B, InsP3 (30 µM) was released at the time indicated by the arrow without (left) or with (right) a preceding depolarizing step to 0 mV (5 sec) in a cell dialyzed with ryanodine (10 µM). The voltage protocol is shown below. The interval between the depolarizing step and release of InsP3 was 5 sec. Top traces and bottom traces depict membrane current and [Ca2+]i, respectively.

Taken together, these data strongly suggest that InsP₃ elicits Ca²⁺ mobilization from the InsP₃-sensitive stores that also express ryanodine receptors but does not trigger CICR through these ryanodine receptors.

Increasing Ca²⁺ stores invokes CICR

We next asked under what conditions ryanodine receptors could be activated in dopamine neurons. Repetitive firing of dopamine neurons for 20-50 sec has been shown to induce transient facilitation of mGluR IPSPs that lasts for several minutes, an effect that may be caused by loading of intracellular Ca²⁺ stores (Fiorillo and Williams, 1998). Thus, filling internal Ca²⁺ stores by prolonged depolarization may invoke a component of the InsP₃-evoked Ca²⁺ release that is attributable to CICR. A prolonged depolarization (20 sec) was applied 1 min before photolytic release of InsP₃ to examine this possibility. This depolarization induced a slow component in the rising phase of [Ca²⁺]_i, a marked prolongation of the overall duration of the Ca²⁺ transient, and a late component in the outward current (Fig. 4A). These effects of depolarization persisted for >5 min (n = 3). When cells were dialyzed with ruthenium red (20 µM), depolarization produced no significant change in the profile of the InsP₃-elicited Ca²⁺ transient, and no late component was observed in the outward current (n = 3) (Fig. 4B). These results suggest that CICR through ryanodine receptors augments the InsP₃-evoked Ca²⁺ release when intracellular stores are enlarged by prolonged depolarization. A transient reduction in the peak amplitude of the InsP₃-induced current was also observed after the prolonged depolarization with both control and ruthenium red-containing internal solutions (Fig. 4A,B). The reason for this decrease in InsP₃-induced current is not known.

View larger version (24K): [in this window] [in a new window]   Figure 4.   Prolonged depolarization added a CICR-mediated component to InsP3-induced responses. A, InsP3 (30 µM)-evoked responses before (left traces) and after (middle and right traces) application of a prolonged depolarizing step to 0 mV (20 sec; open arrow), which caused a large increase in [Ca2+]i (>2 µM). The interval between depolarization and release of InsP3, which is indicated below, was at least 1 min to allow the membrane current and [Ca2+]i to recover to control levels after depolarization. The late component in the outward current and the slow component in the rising phase of [Ca2+]i are marked with open and closed circles, respectively, which were observed when InsP3 was released 1 min after depolarization. B, Same experiment as in A with ruthenium red (20 µM) included in the pipette solution. C, Intracellular signaling cascade elicited by InsP3 in midbrain dopamine neurons. Solid arrows represent the pathway in control conditions, and dashed arrows represent the possible pathways invoked when intracellular Ca2+ stores are enlarged with prolonged depolarization. IP3R, InsP3 receptor; RyR, ryanodine receptor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study demonstrates that intracellularly released InsP₃ induces activation of apamin-sensitive SK channels through mobilization of Ca²⁺ from intracellular stores in midbrain dopamine neurons. It is shown that InsP₃-sensitive stores are functionally connected to ryanodine-sensitive stores. Furthermore, evidence is provided suggesting that increasing Ca²⁺ stores facilitates InsP₃-induced mobilization of Ca²⁺ by bringing ryanodine receptors into play.

The amplitude of the outward current was correlated with the peak [Ca²⁺]_i after release of InsP₃. Thus, the increase in membrane conductance can be a reasonable measure of the increase in free cytosolic Ca²⁺ concentration. Furthermore, the relationship between the peak [Ca²⁺]_i and the current amplitude illustrated in Figure 1C is in good agreement with the known EC₅₀ of Ca²⁺ for activation of cloned SK channels determined from inside-out patches (630-700 nM) (Köhler et al., 1996).

Depleting intracellular Ca²⁺ stores with CPA almost completely blocked the InsP₃-evoked Ca²⁺ transient, even when the high-affinity Ca²⁺ indicator Fura-2 was used to enhance detection of a small change in [Ca²⁺]_i. Furthermore, InsP₃ elicited no change in the holding current after treatment with CPA, indicating that the outward current results entirely from mobilization of Ca²⁺ from intracellular stores. The time course of decay of [Ca²⁺]_i after Ca²⁺ influx attributable to membrane depolarization was slowed in the presence of CPA, possibly reflecting the blockade of Ca²⁺ sequestration into intracellular stores. Thus, Ca²⁺ entering the cell during depolarization can indeed be pumped into intracellular stores and charge them with Ca²⁺. It should be noted, however, that internal Ca²⁺ stores remained stable in dopamine cells clamped at 60 mV, because release of InsP₃ could elicit reproducible responses without membrane depolarization throughout the duration of the recording.

Ruthenium red, which blocks ryanodine receptors, failed to affect the InsP₃-induced release of Ca²⁺ and the amplitude of mGluR IPSCs. Hence, ryanodine receptors do not appear to make a significant contribution to the InsP₃-mediated responses. On the other hand, depletion of ryanodine-sensitive Ca²⁺ stores with ryanodine abolished InsP₃-induced Ca²⁺ mobilization as well as the outward current. Ryanodine also blocked mGluR IPSPs (Fiorillo and Williams, 1998) and an outward current induced by iontophoresis of acetylcholine (Fiorillo and Williams, 2000) in dopamine neurons. Ruthenium red reversed the inhibitory effects of ryanodine on the InsP₃-evoked responses (Fig. 3A) and the acetylcholine-induced outward current (Fiorillo and Williams, 2000), providing a positive control for the effectiveness of ruthenium red. These data, together with the lack of effect of ruthenium red on InsP₃-induced Ca²⁺ mobilization, suggest that InsP₃-sensitive stores also possess ryanodine receptors and can thus be depleted by ryanodine. Interestingly, the InsP₃-evoked responses abolished by ryanodine partially recovered after depolarization of the cells. This observation is consistent with the idea that depolarization-induced Ca²⁺ influx refilled the InsP₃-sensitive stores that had been depleted by ryanodine. This effect of depolarization could not be ascribed to CICR triggered by an increase in [Ca²⁺]_i after depolarization, because CICR is blocked in the presence of ryanodine. Furthermore, it is unlikely that this could be caused by Ca²⁺-induced activation of InsP₃ receptors, because when InsP₃ was released 5 sec after the depolarizing step, [Ca²⁺]_i was still considerably high, i.e., ~800 nM (Fig. 3B), which is in a concentration range where Ca²⁺ inhibits InsP₃ receptors (Bezprozvanny et al., 1991). It has also been reported that InsP₃-sensitive stores are equipped with ryanodine receptors in cerebellar Purkinje neurons (Khodakhah and Armstrong, 1997) and hippocampal pyramidal neurons (Nakamura et al., 1999). However, the possibility remains that there are separate stores, in addition to these InsP₃-sensitive stores equipped with ryanodine receptors, that are regulated exclusively by ryanodine receptors.

Increasing the size of intracellular Ca²⁺ stores with prolonged depolarization potentiated InsP₃-evoked responses by inducing CICR through ryanodine receptors. Regenerative release of Ca²⁺ via ryanodine receptors produced a slow component in the rising phase of [Ca²⁺]_i and an overall prolongation of the duration of Ca²⁺ transient, which was reflected in a late component of the outward current. It should be noted that increasing the size of internal stores with prolonged depolarization did not cause an increase in the magnitude of [Ca²⁺]_i elevation when ryanodine receptors were blocked with ruthenium red. It is possible that filling the InsP₃-sensitive stores did not result in an elevated peak [Ca²⁺]_i attained by Ca²⁺ mobilization through InsP₃ receptors themselves, because enhanced initial release of Ca²⁺ can in turn cause Ca²⁺-induced inactivation of InsP₃ receptors to terminate InsP₃ receptor-mediated Ca²⁺ release (Ogden and Capiod, 1997). Alternatively, Ca²⁺ entering the cell during depolarization may have preferentially filled separate ryanodine-sensitive stores that are devoid of InsP₃ receptors.

The schematic illustration depicted in Figure 4C summarizes how generation of InsP₃ inside the cell leads to activation of SK channels on the plasma membrane in midbrain dopamine neurons. Intracellular InsP₃ activates InsP₃ receptors on the internal stores that also contain ryanodine receptors. Under control conditions in which the membrane potential is clamped at 60 mV, InsP₃ receptor-mediated release of Ca²⁺ leads to activation of SK channels with no significant contribution from ryanodine receptors. When intracellular stores are enlarged by depolarization-induced Ca²⁺ influx, Ca²⁺ released from the InsP₃-sensitive stores can now elicit ryanodine receptor-mediated release of Ca²⁺, i.e., CICR, from the same stores and/or from separate stores that possess only ryanodine receptors. This will result in an augmentation of Ca²⁺ mobilization and a prolongation of SK channel activation. Dopamine neurons are spontaneously active and fire continuously both in vivo and in vitro (Sanghera et al., 1984). It is therefore possible that this spontaneous firing keeps internal Ca²⁺ stores filled and thus enables InsP₃ to induce CICR under physiological conditions.

SK channels are known to participate in controlling the firing pattern of midbrain dopamine neurons (Shepard and Bunney, 1991). Thus, it is very likely that mobilization of Ca²⁺ from intracellular stores plays an important role in regulating the activity of dopamine neurons. Activation of dopamine neurons is critically involved in the reinforcing actions of drugs of abuse, such as opioids and psychostimulants (Koob et al., 1998). Future investigation on how these drugs interact with the intracellular signaling pathway described here may lead to a better understanding of the cellular mechanisms underlying drug addiction.

	FOOTNOTES

Received June 28, 2000; revised Aug. 1, 2000; accepted Aug. 1, 2000.

This work was supported by National Institutes of Health Grant DA08163, a National Ataxia Foundation grant, and a Uehara Memorial Foundation fellowship. We thank Drs. James M. Brundege, Carlos Paladini, and Laura S. Stone for helpful comments on this manuscript.

Correspondence should be addressed to John T. Williams, Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201. E-mail: williamj{at}ohsu.edu.

This article is published in The Journal of Neuroscience, Rapid Communications Section, which publishes brief, peer-reviewed papers online, not in print. Rapid Communications are posted online approximately one month earlier than they would appear if printed. They are listed in the Table of Contents of the next open issue of JNeurosci. Cite this article as: JNeurosci, 2000, 20:RC103 (1-5). The publication date is the date of posting online at www.jneurosci.org.

	REFERENCES

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