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Dopamine D₂ Receptor-Activated Ca²⁺ Signaling Modulates Voltage-Sensitive Sodium Currents in Rat Nucleus Accumbens Neurons

Department of Cellular and Molecular Pharmacology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois

Submitted 29 July 2004; accepted in final form 3 November 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Receptor-mediated dopamine (DA) modulation of neuronal excitability in the nucleus accumbens (NAc) has been shown to be critically involved in drug addiction and a variety of brain diseases. However, the mechanisms underlying the physiological or pathological molecular process of DA modulation remain largely elusive. Here, we demonstrate that stimulation of DA D₂ class receptors (D₂R) enhanced voltage-sensitive sodium currents (VSSCs, I_Na) in freshly dissociated NAc neurons via suppressing tonic activity of the cyclic AMP/PKA cascade and facilitating intracellular Ca²⁺ signaling. D₂R-mediated I_Na enhancement depended on activation of G_i/o proteins and was mimicked by direct inhibition of PKA. Furthermore, increasing free [Ca²⁺]_in by activating inositol 1,4,5-triphosphate receptors (IP₃Rs), blocking Ca²⁺ reuptake, or adding buffered Ca²⁺, all enhanced I_Na. Under these circumstances, D₂R-mediated I_Na enhancement was occluded. In contrast, D₂R-mediated I_Na enhancement was blocked by inhibition of IP₃Rs, chelation of free Ca²⁺, or inhibition of Ca²⁺/calmodulin-activated calcineurin (CaN), but not by inhibition of phospholipase C (PLC). Although stimulation of muscarinic cholinergic receptors (mAChRs) also increased I_Na, this action was blocked by PLC inhibitors. Our findings indicate that D₂Rs mediate an enhancement of VSSCs in NAc neurons, in which cytosolic free Ca²⁺ plays a crucial role. Our results also suggest that D₂R-mediated reduction in tonic PKA activity may increase free [Ca²⁺]_in, primarily via disinhibition of IP₃Rs. IP₃R activation then facilitates Ca²⁺ signaling and subsequently enhances VSSCs via decreasing PKA-induced phosphorylation and increasing CaN-induced dephosphorylation of Na⁺ channels. This study provides insight into the complex and dynamic role of D₂Rs in the NAc.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Dysfunction within the mesolimbic dopamine (DA) system has been implicated in a variety of neuropsychiatric disorders, including schizophrenia, attention deficit hyperactive disorder, and drug addiction. However, the mechanism underlying D₁/D₂ class receptor (D₁R/D₂R) modulation of the activity of DA-targeted nucleus accumbens (NAc) neurons has not been fully elucidated. Recent studies reveal that DA regulation of ion channel function and Ca²⁺ signaling plays an important role in modulating the excitability of striatal neurons (see Greengard et al. 1999 for review). For instance, while D₁R stimulation reduces voltage-sensitive Ca²⁺ currents (VSCCs, I_Ca) via a protein phosphatase 1 (PP1)-mediated mechanism (Fienberg et al. 1998; Hernandez-Lopez et al. 1997; Surmeier et al. 1995; Zhang et al. 2002), D₂R stimulation increases the cytosolic levels of free Ca²⁺ ([Ca²⁺]_in) (Chang and Shin 1997; Greengard et al. 1999; Nishi et al. 1997; Parikh et al. 1996) even though it may occur concurrently with a reduction in L-type I_Ca (Hernandez-Lopez et al. 2000). The mechanism of the D₂R-mediated increase in [Ca²⁺]_in has not been identified (Greengard et al. 1999). In addition, although the D₁R-mediated I_Na reduction involves PKA-induced phosphorylation of Na⁺ channels (Cantrell et al. 1997; Catterall 1997; Schiffmann et al. 1995, 1998; Zhang et al. 1998), the effects of D₂R mediation on I_Na seem to be equivocal (White et al. 1997). However, preserving cytosolic free Ca²⁺ abolishes D₂R-mediated I_Na reduction and enhances I_Na in the majority of NA neurons (Zhang and White 1997), suggesting that D₂R-mediated increase in free [Ca²⁺]_in is also vital to DA regulation of VSSCs.

D₂R-mediated increase in [Ca²⁺]_in, which facilitates Ca²⁺ signaling, is significant for intracellular signal transduction, including the cAMP/PKA cascade in the mesolimbic DA systems (Sunahara et al. 1996). Because adenylyl cyclase (AC) is subclassfied as Ca²⁺-stimulated, Ca²⁺-inactivated, and protein kinase C (PKC)-activated isotypes (Sunahara et al. 1996), PKA activity can be facilitated or attenuated by increased [Ca²⁺]_in. Indeed, because Ca²⁺-inactivated AC_9,5 is predominant in the NAc (Sunahara et al. 1996), PKA activity is inhibited by increased [Ca²⁺]_in (Antoni et al. 1998a). In contrast, [Ca²⁺]_in may also be regulated by PKA because phosphorylation of inositol 1,4,5-triphosphate receptors (IP₃Rs) by this kinase decreases Ca²⁺ flux (Cameron et al. 1995; Ferris et al. 1991; Quinton and Dean 1992; Supattapone et al. 1988; Tertyshnikova and Fein 1998; but also see Tang et al. 2003). In addition, depending on its levels, free Ca²⁺ can regulate calcium release via a positive or negative feedback mechanism, in which increased [Ca²⁺]_in acts as either a physiological activator or inhibitor of Ca²⁺ release (Bezprozvanny et al. 1991; Ehrlich et al. 1994; Hagar et al. 1998).

Facilitated Ca²⁺ signaling may modulate the D₂R-mediated increase in I_Na via activating CaN, a protein phosphatase (PP2B) that regulates ion channel activity and signaling (Yakel 1997). Activated CaN dephosphorylates Na⁺ channels (Chen et al. 1995; Murphy et al. 1993) and DA and adenosine 3,5,-monophosphate-regulated phosphoprotein (32 kDa) phosphorylated by PKA at threonine 34 (p-Thr.34-DARPP-32) (Nishi et al. 1997, 1999; Schiffmann et al. 1998), thereby increasing I_Na and inhibiting p-Thr.34-DARPP-32-induced stabilization of the phosphorylation state of Na⁺ channels, respectively. In contrast, suppression of CaN not only disinhibits Ca²⁺-inactivated AC_9,5 and p-Thr.34-DARPP-32 (Antoni et al. 1998a; Greengard 2001; Nishi et al. 1997), thereby increasing PKA-induced phosphorylation of Na⁺ channels (Chen et al. 1995), but also abolishes dephosphorylation and expression of IP₃Rs (Genazzani et al. 1999); this may subsequently decrease Ca²⁺-modulated I_Na. Based on these findings, which suggest that Ca²⁺/CaN signaling plays an integrative role in regulating Na⁺ channel activity, we hypothesized that D₂R-mediated I_Na enhancement is modulated via facilitated Ca²⁺/CaN signaling. The present study was performed to determine whether the D₂R-mediated increase in Ca²⁺/CaN signaling enhances I_Na and to elucidate the possible molecular mechanisms underlying the D₂R modulation. Our results suggest that the D₂R-mediated I_Na enhancement in rat NAc neurons should be attributed to increased Ca²⁺/CaN signaling, primarily via disinhibition of IP₃Rs.

	METHODS

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Animals

Young adult (age of 4–5 wk) male Sprague-Dawley rats were housed in groups in a temperature- and humidity-controlled vivarium under a 12-h light/dark cycle. Food and water were freely available. After 3 day acclimation to the vivarium, rats were used for acute experiments regarding modulation of voltage-sensitive sodium currents.

Preparation of brain slices

All procedures were performed in strict compliance with the National Research Council Guide for the Care and Use of Laboratory Animals (1996) and were approved by our Institutional Animal Care and Use Committee. Rats were decapitated under halothane anesthesia, and brain tissues containing the NAc were rapidly excised and dissected into blocks. The thickness of brain blocks for cell dissociation was 3–4 mm before slicing.

Whole cell voltage-clamp recordings

Medium spiny neurons (MSNs) in the NAc were freshly dissociated from slice preparations obtained from rats as described in the preceding text. Brain slices (350 µM) in coronal section were cut while bathed in an ice-cold high-sucrose solution [which contained (in mM) 180 sucrose, 2.5 KCl, 26 NaHCO₃, 1.2 Na₂HPO₄, 25 glucose, 0.1 CaCl₂, 1 MgCl₂, and 19 MgSO₄, pH 7.35, 300–305 mosM/l]. Slices were then incubated for 1–5 h at room temperature (20–22°C) in a NaHCO₃-buffered saline (Earle's balanced salts solution, EBSS), bubbled with mixed gas of 95% O₂-5% CO₂. Slices were then moved into a low-Ca²⁺ (100 µM), HEPES-buffered salt solution [which contained (in mM) 140 sodium isethionic acid, 23 glucose, 15 HEPES, 2 KCl, 4 MgCl₂, and 0.1 CaCl₂, pH 7.4, 300–305 mosM/l]. With the aid of a dissecting microscope, the NAc (including both core and shell regions) was dissected and placed in an oxygenated stir chamber containing protease (Type XIV; 1–1.5 mg/ml) in HEPES-buffered HBSS at 35°C. After 30 min of digestion, the tissue was rinsed three times in the low-Ca²⁺ HEPES-buffered saline and mechanically dissociated with a graded series of fire-polished Pasture pipettes in normal external solution (see following text). The cell suspension was then plated into a petri dish mounted on the stage of an inverted microscope containing external bath solution. The dissociated cells were allowed to settle before recording.

Standard whole cell voltage-clamp recording techniques were used as described in our previous study (Zhang et al. 1998). Electrodes were pulled from Corning 7056 glass capillaries (1.65 mm OD, 1.1 mm ID) and fire-polished with a microforge before use. I_Na was isolated by different internal (with or without the Ca²⁺ chelator EGTA) and external (background) solutions—internal (in mM): 120 CsF, 10 NaCl, 2 Na₂ATP, 10 HEPES, and 10 EGTA (or replaced by glucose in the cases of EGTA absence), pH 7.3 (with 1 M CsOH), 280–285 mosM/l; and external (in mM): 110 choline chloride, 30 NaCl, 5 CsCl, 1 MgCl₂, 1 CaCl₂, 0.4 CdCl₂, 10 glucose, and 10 HEPES, pH 7.3–7.4, 300–305 mosM/l. To determine the effects of Ca²⁺ signaling on modulation of VSSCs, cytosolic free Ca²⁺ was not chelated in NAc neurons with the absence of Ca²⁺ chelators in the internal solution (also see following text). In addition, the relatively low concentration of NaCl (30 mM) in the external solution was used to minimize voltage-clamp error. Electrodes filled with internal solution had a resistance of 2–3 M. The junction potential of –5 mV was measured between the electrode and bath solution and was not compensated. Recordings were obtained with an Axon Instruments 200A patch-clamp amplifier and controlled/monitored with a PC running pCLAMP7 with a 2-kHz filter. The membrane potential was held at –70 mV and was depolarized to –20 mV for activation of VSSCs. Step depolarizing pulses (20 ms) were applied at intervals of 5 or 10 s to allow enough time for voltage-gated Na⁺ channels to recover from inactivation. After seal rupture, series resistance (<10 M) was compensated (70–80%) and periodically monitored. All currents were leak-subtracted. Adequate voltage control was determined by standard methods (Colatsky and Tsien 1979). Recordings were made from only medium-sized NAc neurons (8- to 14-µm somal diameter) that had none or a few short proximal dendrites. All experiments were performed at room temperature (20–22°C). The initial control levels of I_Na were recorded and compared between two groups of NAc neurons, either with or without internal EGTA. A time-I_Na response course (6 min) was recorded and compared between NAc neurons with cytosolic free Ca²⁺ completely chelated by EGTA and cells with free Ca²⁺ buffered by EGTA at different concentrations (see following text).

Drug application

Separate subgroups of neurons were recorded with application of different drugs in different experiments. Drugs acting on D₂Rs, pertussis toxin (PTX)-sensitive G_i/o proteins, PKA, PLC, mAChRs, IP₃Rs, Ca²⁺-ATPase, CaN (PP2B), and free Ca²⁺ buffered by EGTA at different concentrations were used to manipulate the levels of cytosolic free Ca²⁺ to identify the possible pathway(s) that may be involved in D₂R-mediated I_Na enhancement. For some neurons, drugs were applied in bath solution, either controlled with a DAD-12 superfusion system (ALA Scientific Instruments, Westbury, NY) or controlled manually with a micropipette. For other cells, drugs were applied via internal dialyzing from recording pipettes to the cytosol. After the whole cell configuration was formed, a brief period of time (1–1.5 min) was given to stabilize the basal levels of VSSCs prior to data acquisition. To determine the role of D₂Rs in Ca²⁺ modulation of I_Na, the selective D₂R class agonist quinpirole (1–2 µM) and antagonist eticlopride (10 µM) were used. PTX (2.5 µM; 5- to 8-h pretreatment by incubation), a selective inhibitor for G_i/o proteins, was used to determine whether the increase in VSSCs after D₂R stimulation was coupled to activation of G_i/o proteins. Selective inhibitors for PKA (Rp-cAMPs, 100 µM) and PLC [U-73122, 10 µM and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholin (ET-18-OCH₃), 100 µM], as well as a selective agonist (5-methylfurmethide, 5-MFT, 10 µM) and antagonist (atropine, 10–40 µM) for mAChRs, were used to determine whether D₂R-mediated I_Na enhancement is associated with and modulated via G_q/PLC coupling. Internally applied IP₃ (90 nM) was used to initiate intracellular Ca²⁺ release from the endoplasmic reticulum (ER) and to increase the cytosolic levels of free Ca²⁺. Selective inhibitors for IP₃Rs (xestospongin C, 0.5–1 µM, and heparin, 10 µM) were also used to block the effects of IP₃ on I_Na. Thapsigargin (275 nM), a selective and irreversible inhibitor for Ca²⁺ ATPase, was used to block reuptake of free Ca²⁺ to the ER. The effects of cytosolic free Ca²⁺, which was buffered by EGTA at different concentrations, on I_Na were studied via direct dialysis from recording pipettes to cytosol. A software platform (WINMAXC) was used to calculate the final free [Ca²⁺]_in buffered by EGTA (Bers et al. 1994). EGTA (10 mM) was used to chelate the resting levels of cytosolic free Ca²⁺ in some control neurons, while concentrations of free Ca²⁺ in those neurons were expressed as 0 nM. EGTA (10 mM) was also used to buffer different concentrations of applied Ca²⁺ in the internal pipette solution to obtain a final free [Ca²⁺]_in at different levels (100 µM). Higher concentrations of free Ca²⁺ buffered by EGTA at µM levels were also tested in some cells. In addition, [1,2-bis(o-aminophenoxyethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester] (BAPTA-AM, 50 µM), another Ca²⁺ chelator, was used in some cases for further determining the effects of free Ca²⁺ and D₂R stimulation on I_Na. Finally, the CaN inhibitor cyclosporin A (50 µM) and CaN autoinhibitory peptide (CAP, 200 nM) were used to determine the possible effects of CaN in D₂R and Ca²⁺ modulation of VSSCs.

Statistical analysis

Comparisons of the I_Na density (pA/pF) and their percent changes obtained from different experimental groups were made with either paired or unpaired t-test (*P < 0.05 and **P < 0.01). ² test was used to analyze the different responses of I_Na with or without EGTA-induced Ca²⁺ chelation (*P < 0.05 and **P < 0.01). Other comparisons between the time-I_Na curves were made with a two-way ANOVA with repeated measures on one variable (I_Na densities).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Stimulation of D₂Rs enhances I_Na with cytosolic free Ca²⁺ in NAc neurons

Whole cell voltage-clamp techniques were used for recording of I_Na in freshly dissociated NAc neurons. These neurons (with capacitance <10 pF) exhibited DA receptor modulation of VSSCs (Zhang et al. 1998) that was identical to that observed in the dorsal striatum (Cepeda et al. 1995; Schiffmann et al. 1995; Surmeier et al. 1992). Although EGTA was commonly used as a cytosolic free Ca²⁺ chelator in previous investigations elsewhere, it was not always used in our experiments. To determine the physiological effects of the cytosolic free Ca²⁺ on I_Na after D₂R stimulation, different groups of NAc neurons were recorded in the presence or absence of EGTA. First, we evaluated the effects of quinpirole-induced D₂R stimulation on whole cell I_Na, either with (n = 31 cells) or without (n = 27 cells) EGTA in the internal pipette solution. With chelation of Ca²⁺ by EGTA in cytosol, quinpirole suppressed VSSCs in 18 of 31 neurons (58%), but enhanced I_Na in 8 of 31 cells (26%). In contrast, removal of EGTA from cytosol not only markedly decreased the number of cells showing suppressed VSSCs to D₂R stimulation [n = 2/27 cells, 7.4%; EGTA(+) vs. EGTA(–): 58 vs. 7.4%, ² = 16.39, **P < 0.01] but also significantly increased the number of cells showing I_Na enhancement to quinpirole [n = 22/27, 81.5%; EGTA(+) vs. EGTA(–): 26 vs. 81.5%, ² = 17.91, **P < 0.01] (Fig. 1A1). The remaining cells showed no change in I_Na to quinpirole [EGTA(+) and EGTA(–): n = 5/31, 16%, and n = 3/27, 11.1%, respectively]. Presence of an effect on VSSCs was defined as 10% in the change of I_Na density. Moreover, the initial (control) levels of I_Na were not significantly affected by removal of EGTA as compared with cells recorded with EGTA [EGTA(+) vs. EGTA(–): 310.3 ± 48.4 vs. 312.3 ± 31.4 pA/pF, P > 0.05; Fig. 1A2]. The percent enhancement in I_Na density in response to quinpirole was 27.7 ± 10.9% as compared with controls (327.6 ± 38.7% versus 274.2 ± 78.1 pA/pF, paired t-test, t = 5.6552, **P < 0.01; Fig. 1B). This effect of quinpirole on I_Na was washed out or blocked by application of the D₂R class antagonist reticlopride (1 µM). In addition, this D₂R-mediated I_Na enhancement was apparently coupled to activation of G_i/o proteins because it was prevented by inactivation of G_i/o proteins after incubation of slices with PTX (2.5 µl/ml, 5–8 h; Fig. 1C1). Thus there was no significant difference in peak I_Na between PTX-treated NAc neurons with or without quinpirole (322.6 ± 49 vs. 351.4 ± 57 pA/pF or 100 vs. 107 ± 3%, n = 6/6 cells, paired t-test, P > 0.05) (Fig. 1, C₂). Because exclusion of EGTA from the cytosol did not significantly affect the initial control levels of I_Na, the following experiments were generally performed without the Ca²⁺ chelator.

View larger version (41K): [in this window] [in a new window]   FIG. 1. The modulatory effects of cytosolic free Ca2+ on INa with D2 class receptors (D2R) stimulation in nucleus accumbens (NAc) neurons. A1: with the presence of EGTA, quinpirole (1–2 µM) suppressed voltage-sensitive sodium currents (VSSCs) in the majority of NAc neurons (18 of 31, 58%), but enhanced INa in 8 of 31 cells (26%). Removal of EGTA abolished quinpirole-induced suppression in VSSCs (2 test, **P < 0.01), while INa was enhanced in the majority of NAc cells (2 test, **P < 0.01). The remaining cells showed no changes in INa in response to quinpirole. A2: removing EGTA from cytosol did not significantly affect the control levels of VSSCs in NAc neurons [EGTA(+) vs. EGTA(–), P > 0.05]. B1: representative traces indicating that when the cytosolic resting levels of free Ca2+ were not interrupted by chelation, stimulation of D2Rs by quinpirole enhanced INa in a NAc neuron recorded. This effect of quinpirole on INa was washed out or blocked by eticlopride. B2: the bar graphs showing a significant increase of INa in response to quinpirole as compared with the initial control levels of VSSCs (paired t-test, **P < 0.01). C1: inactivation of D2R-coupled Gi/o by pertussis toxin (PTX, 2.5 µg/l) pretreatment blocked quinpirole-induced INa enhancement. C2: there was no significant difference in the INa between PTX-treated NAc neurons with or without quinpirole (paired t-test, P > 0.05).

It is well known that stimulation of D₂Rs is negatively coupled to the cAMP/PKA cascade. Thus if the enhanced I_Na in response to D₂R stimulation is mediated via the cAMP/PKA cascade, blockade of PKA activity should resemble the effects of quinpirole on I_Na. In fact, direct inhibition of PKA by internally applied Rp-cAMPs (100 µM) significantly enhanced I_Na (initial control vs. peak: 325.5 ± 42.2 vs. 405.9 ± 43 pA/pF or 100 vs. 129.6 ± 9.2%, n = 8/8, paired t-test; t = 2.5308, *P = 0.0392; Fig. 2, A and B). Under these circumstances, the ability of quinpirole to increase I_Na appeared to be occluded and was no longer observed (Rp-cAMPs/peak vs. Rp-cAMPs ± quinpirole: 405.9 ± 43 vs. 401.9 ± 42.1 pA/pF or 129.6 ± 9.2 vs. 130.1 ± 10.5%, n = 8/8 cells, paired t-test, P > 0.05; Fig. 2, A and B). In addition, bath applied Rp-cAMPs (100 µM) also increased I_Na (n = 2/2 cells; Fig. 2C), further proving the effects of inhibition of PKA on VSSCs. To clarify whether stimulation of D₂Rs also affected the activity of PLC (thereby influencing intracellular IP₃ formation and Ca²⁺ signaling), a variety of agonists and antagonists for PLC were used. In contrast to inhibition of PKA, internally dialyzed U-73122 (10 µM), a PLC inhibitor, did not produce any significant changes in the control levels of I_Na (control vs. U-73122: 295.4 ± 70.3 vs. 254.5 ± 38.5 pA/pF, t-test, P > 0.05) and failed to block D₂R stimulation-induced I_Na enhancement (Fig. 2D). Under this condition, stimulation of D₂Rs by quinpirole was still able to increase I_Na (U-73122 control vs. U-73122 + quinpirole: 287.7 ± 45.1 vs. 254.5 ± 38.5 pA/pF or percent increase in I_Na: +12.9 ± 3.7%; n = 6/6, paired t-test: t = 3.4912, *P < 0.05; Fig. 2E). In addition, ET-18-OCH₃ (ET), another PLC inhibitor, was used to confirm the lack of effects of inhibition of PLC on I_Na. Internally applied ET (50 µM) failed to block the quinpirole-induced increase of I_Na (ET control vs. ET + quinpirole: 242.3 ± 61.4 vs. 290.5 ± 69.2 pA/pF or percent increase in I_Na: +22.7 ± 6.5%; n = 6/6 cells, paired t-test: t = 3.6932, *P < 0.02; Fig. 2F). Bath-applied ET (100 µM) also failed to block the effects of quinpirole on I_Na (n = 3/4 cells; Fig. 2G).

View larger version (34K): [in this window] [in a new window]   FIG. 2. Inhibition of PKA, but not phospholipase C (PLC), increases INa and occludes the effects of quinpirole on VSSCs. A: representative traces showing that, with free Ca2+, inhibition of PKA by internally applied Rp-cAMPs (100 µM) markedly increased INa in NAc neurons. Under this condition, quinpirole failed to cause further increase in INa. B: INa was significantly increased from its initial levels after application of Rp-cAMPs (paired t-test, *P < 0.05). However, there was no significant difference in the percentage increase in INa between cells treated with Rp-cAMPs and cells treated with combined Rp-cAMPs + quinpirole (P > 0.05). C: representative traces showing that bath-applied Rp-cAMPs (100 µM) was also able to increase INa, and subsequently, to occlude the effects of quinpirole on enhancing INa. D: in contrast to inhibition of PKA, inhibition of PLC by U-73122 had no significant effects on INa and failed to affect quinpirole-induced INa enhancement. Internal application of U-73122 (10 µM) failed to either induce any significant changes in the control levels of INa or to block quinpirole-enhanced INa. E: there was a significant increase in INa after concurrent application of U-73122 and quinpirole as compared with U-73122 alone (paired t-test, *P < 0.05). F: inhibition of PLC by internal application of ET-18-OCH3 (ET, 50 µM) failed to block quinpirole-induced INa enhancement. Inset: quinpirole was still able to induce a significant increase in INa (paired t-test, *P < 0.05). G: externally applied ET (100 µM) also failed to block quinpirole-increased INa after D2R stimulation (n = 3/4 cells).

View larger version (40K): [in this window] [in a new window]   FIG. 3. Inhibition of PLC blocks INa enhancement induced by stimulation of mAChRs. A: representative traces showing that activation of PLC-coupled mAChRs by 5-methylfurmethide (5-MFT, 10 µM) markedly increased VSSCs. This effect of 5-MFT was completely blocked by concurrent application of the mAChR antagonist atropine (10–40 µM). B: peak INa was significantly increased in the majority of NAc neurons in response to application of 5-MFT (paired t-test, **P < 0.01). C: the PLC inhibitor U-73122, which produced no significant changes in VSSCs, completely blocked the effects of 5-MFT on INa enhancement. D: there was no significant difference in INa between the 2 groups of NAc neurons treated with U-73122 and with combined U73122 [GenBank] plus 5-MFT (t-test, P > 0.05).

Previous investigations have shown that phosphorylation of IP₃Rs by PKA interrupts function of Ca²⁺-releasing channels and suppresses Ca²⁺ flux (see INTRODUCTION). Therefore D₂R-mediated reduction of PKA activity may result in disinhibition of IP₃Rs from a tonic inhibition of PKA and an increase in intracellular Ca²⁺ release. This may be particularly important for IP₃-induced Ca²⁺ flux after D₂R stimulation in NAc neurons because activation of PLC appears not to be associated with D₂R stimulation (see preceding text). On the other hand, low nanomolar concentrations of IP₃ have been found to induce substantial Ca²⁺ release (Parekh et al. 1997). To assess whether IP₃-induced Ca²⁺ mobilization modulated I_Na in NAc neurons, IP₃ (90 nM) was internally dialyzed into the cytosol from the recording pipette. IP₃ promptly enhanced I_Na in all NAc neurons recorded (n = 10; Fig. 4, A and B). Compared with the initial control levels, the peak I_Na was significantly increased after IP₃ application (318 ± 69 vs. 476 ± 101 pA/pF or 100 vs. 149.4 ± 7.3%, paired t-test: t = 4.3562, **P < 0.01; Fig. 4C). When co-applied with IP₃, the enhancing effects of D₂R stimulation by quinpirole on I_Na were occluded (n = 4/4; Fig. 4A). There was no significant difference in the percent increase in I_Na between cells treated with IP₃ and those treated with combined IP₃ plus quinpirole (P > 0.05; Fig. 4C). Prolonging the perfusion time of IP₃ did not produce a further increase in I_Na but usually reduced I_Na from its peak levels (data not shown), suggesting a negative feedback mechanism might be initiated (see DISCUSSION). IP₃-induced I_Na enhancement was completely blocked by xestospongin C (0.5–1 µM), a selective IP₃R inhibitor (Gafni et al. 1997) [IP₃ (peak) vs. IP₃ + xestospongin C: 476 ± 101 vs. 242 ± 47 pA/pF or 149.4 ± 7.3% vs. 106.4 ± 10%, n = 5/5, paired t-test: t = 4.6767, *P < 0.02; Fig. 4, B and C]. There was no significant difference in I_Na among NAc neurons recorded at the control levels (IP₃ initial) and that with combined IP₃ plus xestospongin C (318 + 69 vs. 242 ± 47 pA/pF, 100 vs. 106 ± 10%, P > 0.05; Fig. 4C). In addition, internally dialyzed heparin (2.5 mg/ml, 5–8 min), another IP₃R inhibitor, also prevented quinpirole-induced I_Na enhancement (n = 6/6 cells; Fig. 4D).

View larger version (35K): [in this window] [in a new window]   FIG. 4. IP3 also increases INa and occludes quinpirole-induced INa enhancement. A: representative traces showing that, without chelation of free Ca2+, cytosolic application of IP3 (90 nM) markedly increased INa (n = 10 cells) and occluded the facilitatory effects of quinpirole on INa. B: application of the IP3R inhibitor xestospongin C (500 nM–1 µM) completely reversed IP3-induced INa increase to the control levels (n = 4/4 cells). C: the bar graph indicating that the peak INa was significantly increased with IP3 application as compared with the initial control levels (paired t-test, **P < 0.01). There was no significant difference in INa recorded from NAc neurons with IP3 treatment and neurons with combined IP3 plus quinpirole (n = 4, P > 0.05). In addition, the IP3-produced increase in INa was completely blocked by the IP3R inhibitor xestospongin C (paired t-test, #P < 0.02). D: moreover, internally dialyzed heparin (2.5 mg/ml), another selective IP3R inhibitor, also prevented quinpirole-induced INa enhancement (n = 6/6 cells).

Ca²⁺ ATPase plays a very important role in regulating intracellular free Ca²⁺ levels by "pumping" released free Ca²⁺ back into the ER. Inhibition of Ca²⁺ ATPase blocks reuptake of free Ca²⁺, depletes stored Ca²⁺, and increases cytosolic free [Ca²⁺]_in. Application of thapsigargin (275 nM), an irreversible Ca²⁺ ATPase blocker, produced a significant increase in I_Na as compared with the initial control levels (control vs. thapsigargin: 332.7 ± 35.9 vs. 387.4 ± 47.6 pA/pF or 100 vs. 115.8 ± 5.6%, n = 7/7, paired t-test, t = 2.5215, *P < 0.05; Fig. 5, A and B). In addition, the effects of D₂R stimulation by quinpirole on enhancing VSSCs were occluded after thapsigargin application (Fig. 5C). There was no significant difference in I_Na between neurons treated with thapsigargin alone and cells treated with combined thapsigargin and quinpirole (346.9 ± 101 vs. 339.1 ± 111 pA/pF, n = 6 cells, paired t-test, P > 0.05).

View larger version (29K): [in this window] [in a new window]   FIG. 5. Inhibition of Ca2+ ATPase enhances INa and occludes quinpirole-induced INa enhancement. A: inhibition of Ca2+-ATPase by bath-applied thapsigargin (275 nM) increased INa in NAc neurons with cytosolic free Ca2+. B: bar graph exhibiting a significant increase in INa after thapsigargin application (paired t-test, *P < 0.05). C: blockade of Ca2+ reuptake by thapsigargin also occluded quinpirole-induced increase of INa in NAc neurons (n = 6/6).

It has been proposed that free Ca²⁺, even in the presence of resting levels of IP₃, is the actual messenger that opens the IP₃R (Taylor and Marshall 1992). In addition, Ca²⁺ is also the only known physiological inhibitor of IP₃Rs. Excessive increases in [Ca²⁺]_in (>300 nM) decrease the binding of IP₃, suppress activity of IP₃R-gated channels, and inhibit Ca²⁺ release (Ehrlich et al. 1994). Although stimulation of D₂Rs leads to an increase in [Ca²⁺]_in, whether the increased Ca²⁺ signaling is able to modulate VSSCs is unknown. In this study, different concentrations of cytosolic free Ca²⁺ (12 nM to 100 µM) were buffered with EGTA (see METHODS) and were dialyzed into the cytosol of NAc neurons. The I_Na recorded with complete chelation of cytosolic free Ca²⁺ (0 nM) by EGTA (10 mM) during a 6-min period of recording time was used as control. A 6- to 7-min recording time period was chosen to avoid possible current deterioration, which could occur in some whole cell patched cells with application of high concentration of free Ca²⁺ and longer time periods of recording. There was no significant difference in the I_Na recorded during this period of time [n = 7 cells, 1-way ANOVA, F(7,30) = 0.7517, P > 0.05; Fig. 6C, ]. However, when the free [Ca²⁺]_in was buffered with EGTA to 12 nM, a rapid and robust increase in I_Na was observed (Fig. 6, A and C, ). The enhanced I_Na achieved its peak levels within 2–3 min followed by a plateau that lasted 2–3 min. In the presence of free Ca²⁺ (12 nM), the peak I_Na levels were significantly greater than its initial controls (n = 4, initial vs. peak: 390.8 ± 68.4 vs. 929.5 ± 124.1 pA/pF or 100 ± 17.5 vs. 244.4 ± 21.3%, paired t-test, t = 6.1543, **P < 0.01; Fig. 6B). Under these circumstances, quinpirole-induced I_Na enhancement was occluded (Fig. 6A). There was no significant difference in the peak I_Na recorded from NAc neurons with application of 12 nM free Ca²⁺ and that with combined free Ca²⁺ and quinpirole (n = 4/4, 929.5 ± 124.1 pA/pF vs. 986.7 ± 127.4 pA/pF or 244.4 ± 21.3 vs. 257.4 ± 23.6%, paired t-test, P > 0.05; Fig. 6B).

View larger version (23K): [in this window] [in a new window]   FIG. 6. Increase of cytosolic free [Ca2+]in buffered by EGTA enhances INa and occludes quinpirole-induced INa enhancement. A: representative traces showing that increasing the levels of free Ca2+ by EGTA-buffered Ca2+ (12 nM) rapidly and robustly increased INa in NAc neurons. Under these circumstances, stimulation of D2Rs by quinpirole was unable to produce further increase in INa. B: in response to the increased free [Ca2+]in, peak INa was significantly enhanced as compared with its initial control level (paired t-test, **P < 0.01). In contrast, there was no significant difference in the percent increase of peak INa between neurons recorded with or without combined quinpirole (paired t-test, P > 0.05). C: the time-INa response curves showing that cytosolic application of free Ca2+ buffered by EGTA at different concentrations alters INa. In control neurons with free Ca2+ completely chelated (0 nM) by EGTA, there was no significant change in INa during a 6-min recording session (, 1-way ANOVA, P > 0.05). However, when free [Ca2+]in was buffered with EGTA at 12 nM, peak INa was potently enhanced. There was a significant difference in the time-INa response curves between cells recorded with 0 nM of Ca2+ and that with 12 nM of Ca2+ ( vs. , ** P < 0.01). Cytosolic application of free Ca2+ buffered by EGTA at a concentration of 164 nM also produced significant increase in peak INa ( vs. , **P < 0.01). However, elevating EGTA-buffered [Ca2+]in to 1–100 µM caused a remarkable reduction in INa as compared with that induced by either 12 nM ( vs. , P < 0.01) or 164 nM ( vs. , P < 0.01; Fig. 6C). Under this condition, there was no significant difference in the recorded INa between the 0 nM [Ca2+]in-treated and 1–100 µM [Ca2+]in-treated cells ( vs. , P > 0.05).

Inhibition of CaN eliminates the effects of both free Ca²⁺ and D₂R stimulation on I_Na enhancement

Ca²⁺/calmadulin-dependent CaN has been reported to increase I_Na via dephosphorylation of Na⁺ channels (see INTRODUCTION). In our study, experiments were performed to determine whether D₂R-mediated I_Na enhancement was modulated by increase of [Ca²⁺] _in, and subsequently a facilitated Ca²⁺/CaN signaling. The CaN inhibitors cyclosporin A and CaN autoinhibitory peptide (CAP) were used to determine whether higher concentration of free Ca²⁺ and D₂R stimulation-induced I_Na enhancement could be blocked via inhibition of CaN activity. Increase in I_Na induced by free Ca²⁺ (buffered by EGTA at 12 nM) was abolished after application of cyclosporin A (50 µM, n = 7 cells; Fig. 7A ). When I_Na was suppressed and returned to the control levels, quinpirole also lost its ability to enhance I_Na (n = 4/4 cells; Fig. 7A). This effect of cyclosporin A on free Ca²⁺-modulated I_Na enhancement was reversible and washed out with fresh medium. Under this condition, the ability of free Ca²⁺ (12 nM) to increase I_Na was completely restored (Fig. 7A). The bar graph shows that the significant increase of I_Na after application of free Ca²⁺ (12 nM) was blocked by cyclosporin A [n = 7/7 cells, MANOVA, F(1,6) = 12.29, P < 0.01, post hoc Newman-Keuls test: *P < 0.05, **P < 0.01; Fig. 7B]. In addition, cyclosporin A also produced a moderate decrease in the control levels of I_Na (n = 3/4 cells; Fig. 7C), unmasking a tonic activity of endogenous CaN in modulating the function of Na⁺ channels. Similar to cyclosporin A, internally dialyzed CAP (200 nM, 6–8 min) abolished the ability of quinpirole to enhance I_Na (n = 3/3 cells; Fig. 7D). A summary of the possible mechanism underlying D₂R-mediated facilitation of Ca²⁺/CaN signaling that modulates I_Na enhancement in NAc neurons is illustrated in Fig. 8.

View larger version (39K): [in this window] [in a new window]   FIG. 7. Inhibition of CaN blocks the ability of cytosolic free Ca2+ and D2R stimulation to enhance INa. A: the INa enhancement induced by EGTA-buffered free Ca2+ was blocked by the CaN inhibitor cyclosporin A. Under these circumstances, the ability of D2R stimulation by quinpirole (1 µM) to enhance INa was abolished. The facilitatory effects of EGTA-buffered free Ca2+ on INa were then restored after washout of this CaN inhibitor. B: EGTA-buffered free Ca2+ produced a significant increase in whole cell INa, that was completely blocked by cyclosporin A (*P < 0.05, **P < 0.01). C: cyclosporin A decreased the control levels of INa in NAc neurons with cytosolic free Ca2+. D: internal application of CaN autoinhibitory peptide (CAP), another inhibitor of CaN, also abolished the ability of quinpirole to enhance INa.

View larger version (36K): [in this window] [in a new window]   FIG. 8. The D2R/Gi,o/AC/cAMP/PKA/IP3R/Ca2+/CaN pathway for modulation of INa in rat NAc neurons. Based on the results from the present study and the previous findings from other investigations, we propose here that D2R-mediated INa enhancement in medium spiny NAc neurons of rats is modulated via inhibition of PKA and disinhibition of the Ca2+/CaN signaling. Increased cytosolic free [Ca2+]in and activated CaN play a critical role in the facilitated Ca2+ signaling after D2R stimulation. These findings also suggest that the D2R-mediated increase in INa should be primarily attributed to disinhibition of IP3Rs. Because stimulation of D1Rs leads to activation of PKA, phosphorylation of Na+ channels and reduction in VSSCs, the D2R/Ca2+-mediated INa enhancement would play an important role in balancing the physiological effects of DA on ion channel function and the membrane excitability of NAc neurons. Increased INa should also contribute to excitatory responsiveness through which the output of information from the NAc to other brain regions would be enhanced. The numbers indicate the experimental steps that have been taken in determining the D2R/Ca2+-modulated INa enhancement in NAc neurons. The upward "open" arrows in the NAc neuron indicate an increase in free Ca2+ levels or INa, and the downward arrows indicate a reduction in INa. The sign of (–) indicates inhibition and (+) indicates stimulation/activation.

	DISCUSSION

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Another important and novel finding in this study is that, similar to D₂R stimulation, elevating free [Ca²⁺]_in via Ca²⁺ mobilization without activation of VSCCs also enhances I_Na in NAc neurons. First of all, intracellularly applied IP₃ rapidly enhanced I_Na, whereas this effect of IP₃ on I_Na was reversed by xestospongin C and heparin through inhibition of IP₃Rs. Second, the facilitatory effects of IP₃ on I_Na were mimicked by thapsigargin, which irreversibly blocks free Ca²⁺ reuptake and thereby increasing free [Ca²⁺]_in (Inesi and Sagara 1992, 1994; Sabala et al. 1993). Third, elevating free [Ca²⁺]_in buffered by EGTA also induced robust enhancement in I_Na. More impor-tantly, under these circumstances, the effects of D₂R stimulation on I_Na enhancement were occluded, suggesting that the facilitatory action of D₂R stimulation on I_Na was replaced by enhanced Ca²⁺ signaling. In addition, our findings reveal that the I_Na enhancement, which was associated with increased [Ca²⁺]_in, was concentration-dependent and probably regulated by a negative feedback mechanism. As mentioned in the preceding text, to clarify the physiological effects of free Ca²⁺ on modulation of I_Na, Ca²⁺ chelators were excluded in some experiments. This "unconventional" way of voltage-clamp recording did not affect the basal levels of VSSCs in NAc neurons because no significant difference in the initial control levels of I_Na were observed between cells recorded with or without chelation of Ca²⁺ by EGTA. In contrast, elevating [Ca²⁺]_in significantly increased I_Na, indicating that free Ca²⁺ functionally modulated enhancement of I_Na. These results are consistent with previous findings that suggest that the physiological resting levels of free Ca²⁺ may be below the nanomolar (nM) levels, and elevating [Ca²⁺]_in increases the activity of IP₃R and Ca²⁺ flux (Ehrlich et al. 1994). It is unclear why Ca²⁺-induced I_Na enhancement at higher [Ca²⁺]_in (164 nM) appeared to be less potent than lower [Ca²⁺]_in (12 nM) because neither of them apparently exceeds a threshold level (300 nM) (Ehrlich et al. 1994). It is possible, however, that the actual [Ca²⁺]_in might be significantly higher than the applied Ca²⁺ concentrations because Ca²⁺ release could be facilitated by increased cytosolic free Ca²⁺.

The failure to produce greater elevations in I_Na in response to high concentration (micromolar levels) of free [Ca²⁺]_in suggests that a negative feedback mechanism that suppressed Ca²⁺ release might have been initiated as a result of excessive increases in [Ca²⁺]_in (>300 nM). Under these circumstances, many IP₃Rs are inactivated though others may still stay open (Bezprozvanny et al. 1991; Ehrlich et al. 1994; Hagar et al. 1998). The "up-down" activity of IP₃Rs in response to increased [Ca²⁺]_in has been displayed as a normal distribution in a bell-shaped Ca²⁺-dependence activation curve (Hagar et al. 1998), indicating that excessive increases in [Ca²⁺]_in have turned free Ca²⁺ from a physiological activator to an inhibitor of Ca²⁺ release. Our results are in agreement with these findings, suggesting that the reduced I_Na after application of high [Ca²⁺]_in was functionally modulated by such negative feedback mechanism. On the other hand, excessive free Ca²⁺ would also disrupt the activity of Na⁺ channels, causing a significant reduction in the amplitude of single-channel Na⁺ currents by directly binding within the pore and occluding the conductance pathway of Na⁺ channels (Zamponi and French 1995).

Our findings also suggest that facilitated Ca²⁺ signaling may modulate I_Na via inhibition of Ca²⁺-inactivited AC and activation of CaN. It is well established that AC can be subclassified into nine different isotypes, and the type IX and V Ca²⁺-inactivated ACs (AC_9,5) are predominant in the caudate putamen and NAc (Antoni et al. 1998a; Glatt and Snyder 1993; Paterson et al. 1995; Premont et al. 1996; Xia et al. 1992). Therefore elevating [Ca²⁺]_in may diminish the tonic action of AC_{9, 5} and PKA, particularly PKA-induced phosphorylation, on Na⁺ channels and IP₃Rs. On the other hand, increased [Ca²⁺]_in would activate CaN (Snyder-Keller and Keller 1998), thereby enhancing dephosphorylation of Na⁺ channels and IP₃Rs. Importantly, activated CaN also diminishes the activity of Ca²⁺-inactivated AC (Antoni et al. 1998a, b) and inhibits PKA-activated p-Thr.34-DARPP-32 (Greengard et al. 1999). All these actions contribute to an increase in whole cell VSSCs. Moreover, we found that inhibition of CaN not only eliminated both free Ca²⁺- and quinpirole-induced I_Na enhancement but also reduced the basal levels of VSSCs. These findings suggest that CaN functions dynamically in a final common path in which the D₂R-mediated I_Na enhancement is modulated by facilitated Ca²⁺ signaling.

Although previous (see INTRODUCTION) and the present findings suggest that the D₂R-mediated increase in [Ca²⁺]_in is likely modulated via disinhibition of IP₃Rs from a tonic phosphorylation of PKA, a recent study reports that IP₃ formation can be promoted through a D₂R/G_q/PLC₁ coupling in striatal neurons (Hernandez-Lopez et al. 2000). This finding seems to contradict others showing that D₂Rs are either not coupled to IP₃ formation (Gupta and Mishra 1990; Kelly et al. 1988; Rubinstein and Hitzemann 1990), or actually function in an inhibitory manner (Pizzi et al. 1987, 1988), in the rat striatum. These findings give rise to a question: should the D₂R-mediated increase in [Ca²⁺]_in (and subsequently enhanced I_Na) be primarily attributed to an increase in IP₃ formation via activation of the D₂R/G_q/PLC coupling or to disinhibition of IP₃Rs from tonic phosphorylation of PKA in NAc neurons? Our results support the latter prospect. Unlike inhibition of PKA, which led to increased I_Na, inhibition of PLC produced no significant changes, and failed to block quinpirole-induced I_Na enhancement, in I_Na of NAc neurons. However, these results do not rule out the possibility that activated PLC may still be functional to form IP₃ and facilitate Ca²⁺ signaling in NAc neurons. In fact, we demonstrate that stimulation of mAChRs, which could activate PLC and increase IP₃ formation and Ca²⁺ signaling, effectively increased VSSCs in NAc neurons. The increased I_Na after mAChR stimulation was reversed by blockade of mAChRs and prevented by inhibition of PLC. Taken together, these findings suggest that activation of PLC may not be critical for D₂R-mediated I_Na enhancement, whereas the D₂R-activated Ca²⁺/CaN signaling, likely via dephosphorylation of IP₃Rs and Na⁺ channels, modulates I_Na enhancement in NAc neurons. The proposed mechanism underlying the D₂R-activated Ca²⁺/CaN signaling, which modulates the enhancement of I_Na, is illustrated in Fig. 8.

A recent finding indicates that fluoride (F^–), a common component of the internal solution for patch-clamp recording, inhibits the activity of PKA (Vargas et al. 1999). Thus it seems to be possible that the increased whole cell VSSCs observed in our study might also be affected by F^–. However, although I_Na appeared to be slightly increased (<10%) after a 6-min recording period with internal CsF, this change in VSSCs was not significant. In contrast, under the same experimental condition, I_Na was significantly increased after stimulation of D₂Rs or inhibition of PKA (by Rp-cAMPs), whereas the increased I_Na was abolished by inhibition of D₂R-coupled G_i/o proteins. Importantly, D₂R/Ca²⁺-modulated I_Na was increased during a similar, or even shorter (2–4 min), recording period as compared with the unchanged VSSCs in control cells. These findings are apparently in agreement with the study of Vargas et al. (1999), in which PKA-induced phosphorylation is slightly decreased by <10% with internal F^– during an initial 5-min period of action time but is inhibited at much greater levels after a longer period (20 min) of F^– treatment. Taken together, these results suggest that, during a relative short period of recording time, fluoride-induced inhibition in PKA activity may not have a significant impact in D₂R/Ca²⁺-modulated I_Na enhancement.

The NAc is a structure in which functionally distinct ensembles of neurons are recruited by convergent DAergic and other inputs to coordinate patterns of movement and affective behavior (Pennartz et al. 1994). Through modulating the function of membrane ion channels, DA participates in control and regulation of the excitability of NAc neurons. Given that DA dynamically modulates the activity of NAc neurons via regulating the function of membrane ion channels, including but not limited to suppression of I_Na and I_Ca though D₁R-mediated signaling (Fienberg et al. 1998; Hu et al. 2004; Schiffmann et al. 1995, 1998; Zhang et al. 1998, 2002), the D₂R-mediated increase in Ca²⁺ signaling and I_Na would provide a physiological balance in modulation of the membrane excitability. An increase in I_Na also contributes to regulation of excitatory responsiveness through which the output of information from the NAc to other brain regions is enhanced. Determination of this function of D₂Rs is significant and will be helpful for further understanding of the mechanisms related to not only the normal physiological processing but also neurodegenerative diseases, sensitization, self-administration, and withdrawal effects of psychostimulants after chronic exposure.

In summary, our study has demonstrated that D₂R-stimulation enhances whole cell VSSCs in freshly dissociated medium spiny NAc neurons. The incased I_Na is modulated by facilitated Ca²⁺/CaN signaling, most likely via disinhibition of IP₃Rs. Our findings also suggest that CaN plays an critical role in integrating intracellular signals initiated by D₂R stimulation and that activation of the D₂R/G_i/o/AC/PKA/IP₃R/Ca²⁺/CaN pathway may decrease phosphorylation and increase dephosphorylation of both IP₃Rs and voltage-sensitive Na⁺ channels, thereby increasing VSSCs in NAc neurons.

	GRANTS

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This study was supported by National Institute of Drug Abuse Grant DA-04093 and Research Scientist Development Award DA-00456 to F. J. White.

	ACKNOWLEDGMENTS

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The authors acknowledge K. Ford, C. Grevers, and L. Baker for excellent technical assistance. We also thank Drs. Donald C. Cooper and Anthony West for helpful comments regarding this manuscript.

Present addresses: Y. Dong, Nancy Friend Pritzker Laboratory, Dept. of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 1201 Welch Rd., Room P152, Palo Alto, CA 94304–5485; and X.-F. Zhang, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064.

	FOOTNOTES

 
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.

Address for reprint requests and other correspondence: X.-T. Hu, Dept. of Cellular and Molecular Pharmacology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Rd., North Chicago, IL 60064-3095 (E-mail: Xiu-Ti.Hu{at}rosalindfranklin.edu)

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