Repeated Cocaine Exposure Decreases Dopamine D₂-Like Receptor Modulation of Ca²⁺ Homeostasis in Rat Nucleus Accumbens Neurons

Abstract

The nucleus accumbens (NAc) is a limbic structure in the forebrain that plays a critical role in cognitive function and addiction. Dopamine modulates activity of medium spiny neurons (MSNs) in the NAc. Both dopamine D₁-like and D₂-like receptors (including D1R or D_1,5R and D2R or D_2,3,4R, respectively) are thought to play critical roles in cocaine addiction. Our previous studies demonstrated that repeated cocaine exposure (which alters dopamine transmission) decreases excitability of NAc MSNs in cocaine-sensitized, withdrawn rats. This decrease is characterized by a reduction in voltage-sensitive Na⁺ currents and high voltage-activated Ca²⁺ currents, along with increased voltage-gated K⁺ currents. These changes are associated with enhanced activity in the D1R/cAMP/PKA/protein phosphatase 1 pathway and diminished calcineurin function. Although D1R-mediated signaling is enhanced by repeated cocaine exposure, little is known whether and how the D2R is implicated in the cocaine-induced NAc dysfunction. Here, we performed a combined electrophysiological, biochemical, and neuroimaging study that reveals the cocaine-induced dysregulation of Ca²⁺ homeostasis with involvement of D2R. Our novel findings reveal that D2R stimulation reduced Ca²⁺ influx preferentially via the L-type Ca²⁺ channels and evoked intracellular Ca²⁺ release, likely via inhibiting the cAMP/PKA cascade, in the NAc MSNs of drug-free rats. However, repeated cocaine exposure abolished the D₂R effects on modulating Ca²⁺ homeostasis with enhanced PKA activity and led to a decrease in whole-cell Ca²⁺ influx. These adaptations, which persisted for 21 days during cocaine abstinence, may contribute to the mechanism of cocaine withdrawal.

INTRODUCTION

The nucleus accumbens (NAc) is a forebrain structure that regulates cognitive function and drug-motivated behaviors in humans and animals (Hyman et al., 2006; Robbins and Everitt, 2002). Dopamine innervation from the midbrain mediates function of medium spiny neurons (MSNs) in the NAc by activating the D1R (D_1,5R) and D2R (D_2,3,4R). D2R modulates cellular activity via numerous signaling pathways (Beaulieu et al., 2007; Beom et al., 2004; Pedrosa et al., 2004; Senogles, 2000; Zhang et al., 2004). In striatal MSNs, D2R stimulation facilitates G_βγ/phospholypase C_β1 coupling (Hernandez-Lopez et al., 2000) and phosphorylation of inositol-1,4,5-trisphosphate receptors (IP₃R) by PKA (Hu et al., 2005a). Such actions increase intracellular Ca²⁺ release and decrease activity of L-type Ca²⁺ channels, respectively (Bonci and Hopf, 2005; Greengard, 2001). Both the D2R effects activate calcineurin, which dephosphorylates L-channels and IP₃Rs, thereby decreasing Ca²⁺ influx (Day et al., 2002; Hernandez-Lopez et al., 2000) but facilitating intracellular Ca²⁺ release (Bultynck et al., 2003; Groth et al., 2003).

Repeated cocaine exposure disturbs Ca²⁺ signaling in MSNs by enhancing and prolonging D1R/D2R stimulation. These changes result in part from reduced Ca²⁺ influx and calcineurin function (Hu et al., 2004, 2005b; Zhang et al., 2002), leading to a reduction in NAc excitability (see Hu, 2007 for review). Alterations in Ca²⁺ channel activity vary, depending upon subtypes of the channel. For instance, D1R stimulation increases L-channel activity with reinstatement of cocaine-seeking behavior in rats (Anderson et al., 2008; Self et al., 1998), whereas repeated cocaine exposure reduces Ca²⁺ currents (I_Ca) via N- and R-type Ca²⁺ channels in NAc MSNs (Zhang et al., 2002). Such differences can be attributed in part to enhanced phosphorylation of L-channels by PKA (Hernandez-Lopez et al., 1997) and dephosphorylation of N-/R-channels by protein phosphatase 1, respectively (Surmeier et al., 1995; Zhang et al., 2002). However, it is unknown whether and how D2R-mediated Ca²⁺ homeostasis in NAc MSNs is altered after repeated cocaine exposure. Repeated D2R stimulation reduces D2R-coupled G_i/G_o protein levels in the NAc (Nestler, 2004; Nestler et al., 1990) and induces desensitization and internalization of D2R after phosphorylation (Gainetdinov et al., 2004). These findings strongly suggest that dysregulated Ca²⁺ homeostasis and signaling are associated with D2R downregulation in striatal MSNs.

Cocaine-induced D2R dysfunction plays a critical role in neuroadaptation of Ca²⁺ influx and related signaling. Thus, chronic cocaine exposure reduces the a-subunits of D2R-coupled G_i/o protein (Nestler et al., 1990), Ca²⁺ influx (Hu et al., 2004; Zhang et al., 2002), and activity of the G_i/o/adenylate cyclase/cAMP/PKA/Ca²⁺/ calcineurin pathway in NAc MSNs (Hu et al., 2005b). Cocaine abuse also decreases D2R availability in the striatum of abstinent humans (Volkow et al., 1999). On the basis of these findings, we hypothesized that D2R modulation of Ca²⁺ homeostasis and signaling is decreased in NAc MSNs in cocaine-sensitized, withdrawn rats. This study was performed to determine whether (1) D2R modulates Ca²⁺ channel function and intracellular Ca²⁺ release in NAc MSNs of drug-free rats, and (2) repeated cocaine exposure decreases D2R modulation of Ca²⁺ channel activity and Ca²⁺ release via the D2R-coupled Ca²⁺/calcineurin pathways.

MATERIALS AND METHODS

Animals and pretreatments

Adolescent male Sprague-Dawley rats at 4–5 weeks of age (Spear, 2000), which were more vulnerable to the development of drug addiction (Badanich et al., 2006, 2008; Schramm-Sapyta et al., 2006), were used in this study. They were group housed in a temperature and humidity-controlled vivarium under a 12-h light/dark cycle. Food and water were freely available. After 3 days acclimation to the vivarium, rats were randomly assigned to two groups and received daily repeated intraperitoneal injections of saline (0.9% NaCl) or cocaine HCl (15 mg/kg) for 5 consecutive days. All rats used in this study received repeated injections of saline or cocaine in their home cage. All experiments were performed after a short-(3-day) or long-term (21-day) withdrawal from pre-treatments.

Whole-cell recordings in brain slices

All procedures were in strict accordance with the National Research Council Guide for the Care and use of Laboratory Animals (NIH Publication No. 85-23, 1996) and were approved by our Institutional Animal Care and Use Committee. Rats were decapitated under halothane anesthesia. Brain was immediately excised and immersed in ice-cold artificial cerebrospinal fluid containing (in mM): NaCl 124, KCl 2.5, NaHCO₃ 26, MgCl₂ 2, CaCl₂ 2, and glucose 10; pH 7.4; 310 mosM/L. Coronal slices (300 μm) containing the NAc were cut with a vibratome (Leica VT1000S, Bannockburn, IL) and incubated in oxygenated (95% O₂/5% CO₂) artificial cerebrospinal fluid for 1 h at room temperature before recording. Slices were anchored in a recording chamber and perfused with gravity-fed oxygenated artificial cerebrospinal fluid (34°C) at a flow rate of 2–3 ml/min. Patch recording pipettes (3–5 MΩ) were pulled from Corning 7056 glass capillaries (Corning, NY) with a horizontal pipette puller (Flaming/Brawn P-97, Sutter Instruments, Novato, CA) and filled with internal recording solution (in mM): CsOH 130, HEPES 10, MgCl₂ 2, Na₂-phosphocreatine 10, Na₂ATP 3, and NaGTP 0.3, pH 7.3 with gluconic acid (50%), 280 mosM/l. To avoid influence of Ca²⁺ chelation that alters dynamic D2R modulation of ion channel activity, Ca²⁺ chelators were not included in the pipette solution (Hu et al., 2005a,b). Recordings were initiated in visually identified MSNs within the core region of the NAc using differential interference contrast microscopy and an amplifier (Axopatch 200B, Axon Instruments, Union City, CA).

After whole-cell configuration was formed, voltage-clamp mode was converted to current-clamp mode. Voltage signals were recorded, amplified in a bridge mode, and digitized by an interface (DigiData 1322A Series) into a computer running analysis software (pCLAMP 9) (Axon Instruments). To prevent influences of synaptic activities on the membrane potential (V_m), glutamate receptors and GABA_A receptors were blocked during recording. Na⁺ and K⁺ channel were also blocked to separate the voltage-gated Ca²⁺ channels (see Drug Application below for detail). High voltage-activated Ca²⁺ plateau potentials were generated by injecting step depolarizing current pulses starting from 0 nA with 0.05 nA increments and 40 ms duration, which were delivered at 10 s intervals. The recording period in each episode was 3 s. Stabilized Ca²⁺ potentials were recorded before application of agonists, antagonists, or blockers as control. Characteristics of Ca²⁺ potentials were obtained from the initial Ca²⁺ spike evoked by the minimal depolarizing current (rheobase). MSNs with stable resting membrane potential were recorded and used for analysis. Resting membrane potential was held at –80 mV (near the mean of –78 mV) during drug application and recording. This gave each NAc MSN the same basal potential level; thus, the results obtained from different cells would be comparable (Hu et al., 2004). The amplitude of Ca²⁺ potentials was measured from the spike threshold to its peak. The half-amplitude duration of Ca²⁺ potentials was measured at the amplitude level at which one-half of the spike peak was reached. The integrated area (size) of Ca²⁺ potentials was defined and measured under the curve, which initiated at the rising part of the spike from resting membrane potential and ended with the recording period.

Separate subgroups of NAc MSNs were recorded with application of different drugs and ion channel blockers that were added in artificial cerebrospinal fluid immediately before use. Selective blockers/inhibitors for Na⁺ channels (tetrodotoxin, TTX, 1 μM), K⁺ channels (tetraethylammonium, TEA, 20 mM), gluta-mate receptors (kynurenic acid, 2.5 mM), and GABA_A receptors [SR-95531 or 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide, 4 mM] were applied in bath in all experiments. Quinpirole, a selective D2R agonist (1–10 μM), was used to assess D2R modulation of Ca²⁺ spikes and release. The selective D2R antagonist eticlopride (10 μM) was used to block the effects of quinpirole. Selective Ca²⁺ channel blockers for L-type (nifedipine, 5 μM), N- (ω-conotoxin GVIA, 1 μM), and P/Q-type (ω-conotoxin MVIIC, 2 μM) channels were also applied in the bath to assess the role of non-L-type Ca²⁺ channels. Active calcineurin (100 U) was added in the internal solution and applied directly in cytosol. Cyclosporin A (20 μM), a selective inhibitor for calcineurin, was applied externally.

PKA assays

The NAc and motor cortex from saline- or cocaine-pretreated rats, with a short- or long-term withdrawal, were dissected and lysed in hypotonic buffer [10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 1 mM DTT, 25 μM (–)-p-bromotetramisole oxalate, 5 μM cantharidin, 5 μM microcystin-LF, and 5 μM cyclosporin A] and were supplemented with Complete Protease Inhibitor tablets (Roche Diagnostics, Indianapolis, IN). PKA activity in 2 μg of each sample was determined by the PepTag PKA assay (Promega, Madison, WI) (Dong et al., 2005). Positive controls contained 10 ng of purified PKA catalytic subunit (Promega), whereas the negative controls contained no PKA. The PepTag assay used the Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) peptide substrate tagged with a UV-fluorescent dye. The PKA activity was detected by the amount of phosphorylated substrate migrating toward the anode. Quantification was performed by calculating luminence intensity using TotalLab software (Nonlinear Dynamics, Durham, NC).

Two-photon calcium imaging

Live-cell Ca²⁺ imaging of individual neurons in thick brain slice preparations was performed using a custom-made video-rate two-photon imaging system based on an Olympus BX51 microscope frame (Nguyen et al., 2001), which is the gold standard technique currently available. Relative changes in [Ca²⁺]_in were readily measured and compared within and between samples. Although the absolute [Ca²⁺]_in could not be measured, a relative change from this baseline and estimated concentrations based on established baseline values were made. The resting Ca²⁺ levels were interpreted to be similar based on similar fluorescence intensity values at the same laser intensity and power levels, and similar depths within the tissue slice. In addition, other Ca²⁺-dependent effects that would affect passive and active membrane properties were also not different between groups, which were consistent with the observed fluorescent levels. Individual MSNs were filled with the Ca²⁺ indicator fura-2 (50 μM) via a patch pipette as described (Stutzmann et al., 2003). Excitation was provided by trains (80 MHz) of ~100 fs pulses at 780 nm from a Ti:sapphire laser (Mai Tai Broadband, Spectra-Physics, Mountain View, CA). The laser beam was scanned at 30 fps using a custom-built scanner and focused through a 40× water-immersion objective (NA = 0.8). Emitted fluorescence light was detected by a wide-field photomultiplier (Electron Tubes, Rockaway, NJ) to derive a video signal that was captured and analyzed by Video Savant 5.0 software (IO Industries, ON, Canada). Further analysis of background-corrected images was performed using Metamorph software. For clarity, images and traces of fura-2 fluorescence are expressed as inverse pseudo-ratios: F₀/ΔF (F₀ is the average resting fluorescence before stimulation, and ΔF is the decrease of fura-2 fluorescence resulting from increased [Ca²⁺] when excited at 780 nM), so that increases in [Ca²⁺] correspond to increasing ratios.

Statistical analysis

Student's t-test was used for comparison of drug effects on the membrane properties, characteristics of Ca²⁺ plateau potentials, PKA activity, and intracellular Ca²⁺ release between control and drug-treated NAc cells in saline- or cocaine-pretreated rats. Repeated-measures ANOVA was used for comparison of the quinpirole-induced changes in the dose-response curves between saline- and cocaine-withdrawn groups. Newman-Keuls test was carried out for post hoc comparisons.

RESULTS

D2R stimulation reduces the size of evoked Ca²⁺ plateau potentials in NAc MSNs

All medium spiny NAc cells were recorded in the core region. Blockade of K⁺ channels (TEA in the bath and cesium in cytosol) depolarized resting membrane potential in NAc MSNs (SAL group = –61.1 ± 2.2, COC group = –60.7 ± 2.0 mV) when compared with that without such blockade (~–79 mV) (Zhang et al., 1998), which effectively stabilized generation of Ca²⁺ potentials (Hu et al., 2004; Nasif et al., 2005a). This membrane depolarization was attributed mainly to blockade of the outflowing K⁺ currents that were activated at the resting status of cells to maintain resting membrane potential at hyperpolarized levels. This change in resting membrane potential was not significantly affected by D2R stimulation (data not shown). Ca²⁺ plateau potentials were evoked by depolarizing current pulses with blockade of Na⁺ channels and K⁺ channels (Fig. 1A). Bath-applied quinpirole (1–10 μM) decreased Ca²⁺ channel activity in a dose-dependent manner (Figs. 1B and 1C). Because 10 μM of quinpirole was effective in producing a significant reduction in the size of evoked Ca²⁺ plateau potentials (n = 40 cells, one-way ANOVA, F_(4,39) = 5.07, *P < 0.03), it was used in the rest of experiments of this study. D2R-mediated decrease in Ca²⁺ influx was reflected by a reduction in the size of evoked Ca²⁺ spikes (control vs. quinpirole: 57,196 ± 8353 vs. 39,055 ± 5629 mV × ms; n = 15 cells, paired t-test, t_(1,14) = 5.61, *P < 0.05), and the duration of Ca²⁺ potentials (measured at the half-amplitude level (control vs. quinpirole: 336.3 ± 23.8 vs. 213 ± 23.4 ms and 47.5 ± 2.7 vs. 38.5 ± 2.9 mV, respectively; n = 15 cells/group, paired t-test, t = 5.20 and t = 5.21, all *P < 0.05) (Fig. 2A–D). Rheobase and firing threshold of evoked Ca²⁺ spikes were not significantly affected by quinpirole (SAL vs. COC: 0.7 ± 0.05 vs. 0.7 ± 0.05 nA, and 213.6 ± 1.7 vs. 212.4 ± 1.5 mV, respectively). The effects of D2R stimulation on Ca²⁺ influx were washed out by fresh medium (n = 15, paired t-test, P > 0.05) (Figs. 2A–2D) or blocked by the selective D2R antagonist eticlopride (10 μM) (control vs. eticlopride + quinpirole: area, 34,479 ± 1969 vs. 35,450 ± 2709 mV × ms; amplitude: 38.3 ± 4.1 vs. 41.2 ± 2.3 mV; duration: 235.1 ± 15.8 vs. 255.3 ± 24.5 ms; n = 10 cells, paired t-test, P > 0.05) (Fig. 2E–H).

Fig. 1.

D2R stimulation reduced the size of evoked Ca²⁺ plateau potentials in NAc MSNs of control rats. A: Representative trace shows an evoked Ca²⁺ potential. The amplitude of Ca²⁺ spikes was measured from the spike threshold to its peak. The half-amplitude duration of Ca²⁺ potentials was measured at the amplitude level at which one-half of the spike peak was reached. The integrated area of Ca²⁺ potentials was defined and measured under the trace which initiates at the beginning of the evoked potential from the resting membrane potential (held at –80 mV; the horizontal dash line) and ends at the end of the recording period (3 s). B: D2R stimulation by quinpirole (2, 4, 6, and 10 μM) reduced the duration of Ca²⁺ spikes in a concentration-dependent fashion. C: Bar graphs show a signifi-cant decrease in the size of Ca²⁺ spikes with 10 μM quinpirole (means ± S.E, n = 10 cells; paired t-test, *P < 0.05).

Fig. 2.

D2R-modulated inhibition in Ca²⁺ potentials was reversible and prevented by D2R antagonist. A: Representative recording traces showing that D2R-modulated inhibition of Ca²⁺ spikes was reversible. B–D: Bar graphs indicate that the significant reductions in the size, amplitude, and duration of Ca²⁺ spikes induced by quinpirole (10 μM) were reversed following washout (n = 10 NAc cells; paired t-test, *P < 0.05). Short bars represent means ± S.E. E: Concurrent application of the selective D2R antagonist eticlopride (10 μM) blocked the quinpirole-induced reduction in the size of Ca²⁺ potentials. F–H: There was no significant difference in the area, amplitude, and duration of Ca²⁺ spikes between control and eticlopride + quinpirole-treated cells. Bath-applied eticlopride produced no significant change in Ca²⁺ potentials (means ± S.E., n = 10 cells; paired t-test, all P > 0.05).

Selective blockade of L-type, but not N- and P/Q-type Ca²⁺ channel, mimics and occludes D2R-mediated reduction in Ca²⁺ spikes

Given the fact that generation of Ca²⁺ spikes in striatal cells of young rats depends primarily on activation of high voltage-activated Ca²⁺ channels (Hu et al., 2004; Surmeier et al., 1995; Zhang et al., 2002), we evaluated whether and how the activity and which subtype of high voltage-activated Ca²⁺ channels was modulated by the D2R. Bath application of nifedipine (5 μM), a specific L-channel blocker, significantly reduced the amplitude, duration, and size of evoked Ca²⁺ spikes (control vs. nifedipine: 51.0 ± 2.9 vs. 42.5 ± 4.4 mV; 319.6 ± 32.6 vs. 251.7 ± 35.7 ms, and 50,816 ± 5800 vs. 39,811 ± 4425 mV × ms, respectively; n = 10 cells, paired t-test, t = 3.51, t = 4.22, and t = 3.07, all *P < 0.05) (Fig. 3A). With blockade of L-channels, quinpirole (10 μM) was no longer able to suppress Ca²⁺ potentials (nifedipine vs. nifedipine + quinpirole: 39,811 ± 4425 vs. 37,802 ± 4539 mV × ms, 42.5 ± 4.4 vs. 39.8 ± 4.7 mV, and 251.7 ± 35.7 vs. 248 ± 37.9 ms, respectively; n = 10 cells, paired t-test, all P > 0.05) (Fig. 3A). Because the size of Ca²⁺ spikes was affected by the duration and amplitude, it was measured and used as the primary result and was compared among all experimental groups throughout this study.

Fig. 3.

Blockade of L-, but not N- or P/Q-type Ca²⁺ channels, mimicked and occluded D2R-modulated inhibition of Ca²⁺ spikes. A₁: Representative traces showing that selective blockade of L-channel by nifedipine (5 μM) reduced the duration and amplitude of Ca²⁺ spikes, whereas the D2R-modulated inhibition of Ca²⁺ potentials was occluded by blockade of L-channel. A₂: Bar graphs indicate a significant decrease in the Ca²⁺ spike area with L-channel blockade (n = 10 cells, respectively; means ± S.E., paired t-test, both *P < 0.05). There was no significant difference in the Ca²⁺ spike between nifedipine-treated and nifedipine plus quinpirole-treated cells (n = 10 cells; paired t-test, ^#P > 0.05). B₁: Selective blockade of N-type Ca²⁺ channels with ω-conotoxin GVIA (1 μM) reduced the duration of Ca²⁺ potentials, and this effect was further enhanced by concurrent application of quinpirole. B₂: There was a significant decrease in the Ca²⁺ spike area between control cells and that with application of ω-conotoxin GVIA or ω-conotoxin GVIA plus quinpirole and between cells treated with ω-conotoxin GVIA and ω-conotoxin GVIA + quinpirole (n = 10 cells; paired t-test, all *P < 0.05). C₁: Selective blockade of P/Q-type channels with ω-conotoxin MVIIC (2 μM) reduced the Ca²⁺ spike area, which was also enhanced by quinpirole. C₂: There was a significant difference in the spike area between control cells and that with ω-conotoxin MVIIC or ω-conotoxin MVIIC + quinpirole and between cells treated with ω-conotoxin MVIIC and ω-conotoxin MVIIC + quinpirole (n = 10 cells; paired t-test, all *P < 0.05).

In contrast, blockade of other types of high voltage-activated Ca²⁺ channels did not eliminate D2R-mediated inhibition in Ca²⁺ spikes. Bath-applied ω-conotoxin GVIA (5 μM), a specific N-type Ca²⁺ channel blocker, significantly reduced the area of Ca²⁺ spikes (control vs. ω-conotoxin GVIA: 58,290 ± 1117 vs. 48,208 ± 10079 mV × ms, n = 10 cells, paired t-test, t = 3.55, *P < 0.05) (Fig. 3B). The N-channel blocker-induced reduction in Ca²⁺ spikes was enhanced by concurrent application of quinpirole, which produced an additive decrease in the size of Ca²⁺ spikes (ω-conotoxin GVIA vs. ω-conotoxin GVIA + quinpirole: 43,393 ± 8605 vs. 27,773 ± 5891 mV × ms, n = 10 cells, paired t-test; t = 4.42, *P < 0.05) (Fig. 3B). ω-Conotoxin MVIIC (2 μM), a specific blocker for P/Q-type Ca²⁺ channels (Brown and Randall, 2005; Phillips and Stamford, 2000), also significantly diminished the area of Ca²⁺ spikes (control vs. ω-conotoxin MVIIC: 43,245 ± 4962 vs. 33,042 ± 2309 mV × ms, n = 10 cells, paired t-test, t = 2.77, *P < 0.05) (Fig. 3C). Application of quinpirole with the P/Q-channel blocker also produced an additional reduction in the size of Ca²⁺ spike (ω-conotoxin MVIIC vs. ω-conotoxin MVIIC + quinpirole: 33,042 ± 2309 vs. 21,383 ± 5609 mV × ms, n = 10 cells, paired t-test, t = 4.45, *P < 0.05) (Fig. 3C).

Calcineurin suppresses Ca²⁺ plateau potentials and occludes D2R-mediated inhibition of Ca²⁺ spikes

To determine whether and how Ca²⁺ channel activity is modulated by the D2R-coupled Ca²⁺/calcineurin pathway, we evaluated the interaction of calcineurin and D2R stimulation. We found that cytosolic application of active calcineurin (100 U) mimicked the D2R-mediated reduction in Ca²⁺ spikes (control vs. calcineurin: 54,460 ± 3059 vs. 32,722 ± 2141 mV × ms, n = 10 cells, t-test, t = 5.84, *P < 0.05). However, concurrent application of quinpirole with calcineurin produced no further reduction; and the calcineurin-induced decrease in the size of Ca²⁺ potentials was not significantly affected by quinpirole (calcineurin vs. calcineurin + quinpirole: 32,722 ± 2141 vs. 33,823 ± 2397 mV × ms, n = 10 cells, paired t-test; P > 0.05; control vs. calcineurin + quinpirole: 54,460 ± 3059 vs. 33,823 ± 2397 mV × ms; paired t-test, t = 5.06, *P < 0.05) (Fig. 4A–B).

Fig. 4.

D2R/calcineurin-modulated inhibition of Ca²⁺ spikes. A: Representative traces showing that cytosolic application of exogenous calcineurin reduced the duration of Ca²⁺ potentials that occluded the effects of quinpirole on reducing Ca²⁺ spikes. B: Calcineurin (100 U) significantly decreased the Ca²⁺ spike area, whereas concurrent application of quinpirole produced no further effects on reducing Ca²⁺ potentials (n = 10 cells; paired t-test, *P < 0.05 and P > 0.05, respectively). Bars represent means ± S.E. C: Representative traces show that inhibition of calcineurin activity by cyclosporin A (20 μM) increased the area of Ca²⁺ potentials and prevented the inhibitory effects of quinpirole on reducing the area of Ca²⁺ spikes. D: There was a significant difference in the area of Ca²⁺ potentials between control cells and that treated with cyclosporin A or cyclosporin A + quinpirole (n = 10 cells; paired t-test, both *P < 0.05). Coapplication of quinpirole did not produce any significant change in the area of Ca²⁺ spikes in NAc neurons when compared with cyclosporin A group (n = 10 cells; paired t-test, P > 0.05).

To further investigate the calcineurin effects on Ca²⁺ channel activity, we assessed if inhibition of calcineurin activity could induce an opposite responsiveness in Ca²⁺ spikes. In contrast to calcineurin, bath application of cyclosporin A (20 μM) (Hu et al., 2005a), a selective inhibitor for calcineurin, significantly increased the duration and size of Ca²⁺ potentials in NAc MSNs (control vs. cyclosporin A: 367.9 ± 24 vs. 404.9 ± 33.1 ms and 48,980 ± 2686 vs. 55,988 ± 3773 mV × ms, n = 10 cells, respectively; paired t-test, t = 3.28 and t = 5.43, respectively, both *P < 0.05) (Fig. 4C and D). This effect of cyclosporin A on Ca²⁺ potentials was not affected by concurrent application of quinpirole (cyclosporin A + vs. cyclosporin A quinpirole: 55,988 ± 3773 vs. 54,596 ± 3874 mV × ms, n = 10 cells, paired t-test, P > 0.05; control vs. cyclosporin A + quinpirole: 48,980 ± 2686 vs. 54,596 ± 3874 mV × ms, paired t-test, t = 3.55, *P < 0.05) (Fig. 4D).

Repeated cocaine exposure and withdrawal decreases Ca²⁺ channel function and abolishes D2R-mediated inhibition of Ca²⁺ spikes

In this study, we extended our earlier research regarding the decreased activity of high voltage-activated Ca²⁺ channels in medium spiny NAc neurons of rats after a 3-day withdrawal (Hu et al., 2004) by investigating two additional questions: (1) does the decreased Ca²⁺ channel activity persist after longer withdrawal? And (2) are the effects of D2R on modulating high voltage-activated Ca²⁺ channel activity in NAc MSNs of cocaine-pretreated rats altered after cocaine withdrawal? To study the first question, we compared the integrated area of Ca²⁺ spikes in NAc MSN from saline- and cocaine-pretreated rats. The size of Ca²⁺ potentials was significantly reduced in cocaine-pretreated NAc neurons either after a 3-day or a 21-day abstinence (3-day/withdrawl: SAL vs. COC = 53,138 ± 7742 vs. 35,285 ± 1707 mV × ms; 21-day/withdrawl: SAL vs. COC = 46,062 ± 4595 vs. 32,380 ± 2808 mV × ms; n = 14 cells/each group, paired t-test, t = 2.99 and t = 2.63, respectively, both *P < 0.05) (Fig. 5A–D). This change resulted from reduced duration (3-day/withdrawl: SAL vs. COC = 323.5 ± 18.5 vs. 262.4 ± 13.5 ms; 21-day/withdrawl: SAL vs. COC = 233.1 ± 16.6 vs. 174.6 ± 11.2 ms; t = 2.16 and t = 2.33; all *P < 0.05). Rheobase was also increased in cocaine-pretreated cells (3-day/withdrawl: SAL vs. COC = 0.66 ± 0.04 vs. 1.0 ± 0.06 nA; 21-day/withdrawl: SAL vs. COC = 0.7 ± 0.08 vs. 1.1 ± 0.11 nA; t-test, t = 3.5 and t = 2.4, respectively; all *P < 0.05) (Fig. 5E). These changes indicate that the intrinsic excitability of medium spiny NAc neurons was remarkably reduced by downregulating high voltage-activated Ca²⁺ channel function in cocaine-sensitized, withdrawn rats.

Fig. 5.

Repeated cocaine exposure attenuated both Ca²⁺ channel function and D2R-modulated inhibition of Ca²⁺ spikes in NAc MSNs after a short- or long-term withdrawal. A, B: Representative traces show that the duration of Ca²⁺ potentials was reduced in NAc MSNs after a 3-day (3-day/withdrawl) or a 21-day (21-day/withdrawl) cocaine withdrawal. A greater rheobase was also needed in generating Ca²⁺ spikes in NAc MSNs of cocaine-withdrawn rats. C, D: Bar graphs indicate a significant reduction in the Ca²⁺ spike size in cocaine (COC)-pretreated MSNs when compared with saline (SAL)-pretreated controls after a 3-day or a 21-day withdrawal (n = 14 cells/each group, unpaired t-test, both *P < 0.05). The bars represent mean ± S.E. E: D2R stimulation with quinpirole (10 μM) reduced the Ca²⁺ spike area in SAL-pretreated NAc neurons after a 3-day withdrawal. F: Repeated cocaine administration reduced the Ca²⁺ spike duration in NAc MSNs after a 3-day withdrawal (comparing the two top traces in 5E and 5F). Quinpirole failed to induce any significant change in the size of Ca²⁺ potentials in cocaine-pretreated MSNs. G, H: Bar graphs show that quinpirole induced a significant reduction in the size of Ca²⁺ spikes in saline-pretreated rats after either a 3-day withdrawal or a 21-day withdrawal (SAL 3-day/withdrawl and SAL 21-day/withdrawl, n = 14 and 12 cells, respectively, paired t-test, *P < 0.05). However, this effect was abolished in cocaine-pretreated rats after a 3-day or a 21-day withdrawal (COC 3-day/withdrawl and 21-day/withdrawl, n = 13 and11 cells, respectively; two-way ANOVA with repeated measures, both *P < 0.05). Bars represent the means and the vertical lines indicate ± S.E.

The second question was studied by comparing the spike area of medium spiny NAc neurons between saline- and cocaine-pretreated rats. Associated with cocaine-induced decrease in Ca²⁺ influx, the D2R effects on reducing the duration of evoked Ca²⁺ spikes were diminished in cocaine-pretreated NAc neurons after withdrawal. Under this circumstance, quinpirole was no longer able to suppress Ca²⁺ spikes in cocaine-withdrawn NAc neurons, even at a higher concentration (3-day/withdrawl group: SAL vs. COC: n = 14 vs. 13 cells, and 21-day/withdrawl group: SAL vs. COC, n = 12 vs. 11 cells; two-way ANOVA with repeated measures, F_(2,46) = 4.16, *P < 0.05, and F_(2,40) = 14.2, *P < 0.05, respectively, both compared to quinpirole 0 μM; control) (Fig. 5E–H).

Repeated cocaine administration persistently increases PKA activity in the NAc after a 3-day or a 21-day withdrawal

Previous findings indicated that repeated cocaine treatment increases PKA activity in various brain regions, including NAc (Edwards et al., 2007; Hope et al., 2005; Scheggi et al., 2007). To determine if the persistent dysregulation of Ca²⁺ channels in NAc cells (see above) was associated with this change, we assessed PKA activity in the NAc using a highly specific fluorescent peptide substrate (kemptide) after a 3-day or a 21-day withdrawal. The net levels of this peptide were changed from positive to negative, depending upon the extent of phosphorylation of the peptide by PKA, which allowed electrophoretic separation and quantification of the phosphorylated substrate (Dong et al., 2005; Ford et al., 2009). We found that PKA activity was significantly increased in NAc cells after a 3-day (SAL vs. COC: 100% ± 11.8% vs. 137.2% ± 13.8%, n = 20/22 rats; t-test, t = 2.026, *P < 0.05) (Fig. 6A) or a 21-day cocaine withdrawal (SAL vs. COC: 100% ± 9.0% vs. 134.5% ± 11.6%, n = 22/22 rats; t = 2.370, *P < 0.03). However, there was no significant change of PKA activity in the motor cortex (3-day/withdrawl: 100% ± 6.2% vs. 102.2% ± 7.6%, n = 17/19 rats; 21-day/withdrawl: 100% ± 6.4% vs. 97.5% ± 5.7%, n = 27/27 rats; both P > 0.05) (Fig. 6B), indicating that the cocaine-induced changes in PKA activity were region specific.

Fig. 6.

Repeated cocaine exposure enhanced PKA activity in the NAc but not motor cortex. A: Examples of gels show phosphorylation of peptide substrates resulting from PKA activation in the tissues from the NAc obtained from SAL- and COC-pretreated rats. PKA activity was increased after the short- or long-term cocaine withdrawal (3-day/withdrawl or 21-day/withdrawl, respectively). There was a significant difference in PKA activity in the NAc between SAL- and COC-pretreated rats after both short- and long-term withdrawal (SAL 3-day/withdrawl vs. COC 3-day/withdrawl, n = 20 vs. 22 rats, unpaired t-test, *P < 0.05; SAL 21-day/withdrawl vs. COC 21-day/withdrawl, n = 14 vs. 14 rats, unpaired t-test, *P < 0.05). The bars and vertical lines represent the mean ± S.E. B: There was no significant change in PKA activity in the motor cortex of SAL-pretreated rats when compared with COC-pretreated rats.

Repeated exposure to cocaine decreases D2R modulation of cytosolic Ca²⁺ release in NAc MSNs after a 3-day or a 21-day withdrawal

It has been suggested that D2R stimulation increases Ca²⁺ release in striatal cells (Greengard, 2001). Stimulation of D2R also elevates [Ca²⁺]_in in cortical astrocytes (Khan et al., 2001). However, little is known whether and how D2R could dynamically modulate this activity in medium spiny NAc neurons. Here, we used a two-photon laser scanning Ca²⁺ imaging technology to evaluate cytosolic Ca²⁺ release in medium spiny NAc neurons in slice preparations from saline- or cocaine-withdrawn rats. The basal levels of resting Ca²⁺ level, as indicated by relative fluorescent intensity of the calcium indicator fura-2, were imaged for 3 min in both saline- and cocaine-pretreated rats. There was no significant difference in the relative baseline levels of fluorescent intensity between NAc neurons in saline- and cocaine-pretreated rats after a 3-day or a 21-day withdrawal (both P > 0.05) (Fig. 7A). D2Rs localized on the cell membrane of medium spiny NAc neurons were stimulated with bath application of quinpirole (10 μM) for 5 min, and then the changes in [Ca²⁺]_in after D2R stimulation were recorded and compared to the baseline. D2R stimulation by quinpirole evoked a marked relative increase in the somatic levels of free Ca²⁺ in NAc MSNs in saline-pretreated rats (F₀/ΔF = 0.62% ± 0.21% or 162% ± 20.8% of the predrug baseline measurement, n = 8 cells in four rats) (Fig. 7A and C). This D2R effect lasted ~5 min and was washed out completely with fresh artificial cerebrospinal fluid, as indicated in the raw two-photon images (Fig. 7A). The relative changes in Ca²⁺ release in saline- or cocaine-pretreated NAc cells in response to D2R stimulation are presented with pseudocolored images (Fig. 7B).

Fig. 7.

D2R modulation of intracellular Ca²⁺ release was abolished in NAc MSNs in cocaine-pretreated rats after a short- or long-term withdrawal. A: Background-subtracted two-photon Ca²⁺ images of quinpirole-stimulated Ca²⁺ release in saline (top)- and cocaine-pretreated (bottom) NAc MSNs after a 3-day withdrawal. Representative images show averaged fluorescent intensity before application of quinpirole (left), with peak Ca²⁺ response during quinpirole (middle), and after quinpirole was washed out (right). Similar responses were observed in NAc MSNs from 21-day withdrawn rats (not shown). Note that with the Ca²⁺ indicator fura-2 at 780 nm two-photon excitation (which corresponds to greater fluorescence emission with calcium in the unbound state), an increase in Ca²⁺ results in a decrease in fluorescence. Therefore, a dimmer image indicates greater Ca²⁺ release. B: Psuedocolored images with color scale indicate the F₀/ΔF levels during the maximal Ca²⁺ response induced by D2R stimulation with quinpirole in NAc MSNs from saline (top)- vs. cocaine (bottom)-withdrawn rats after a 3-day withdrawal. C: Bar graphs indicate the relative changes in the Ca²⁺ levels (F₀/ΔF) evoked by quinpirole in NAc MSNs recorded in saline-(open) and cocaine-(filled) pretreated rats with a 3-day or a 21-day withdrawal (left and right, respectively). Asterisks indicates a significant difference between saline- and cocaine-pretreated cells (unpaired t-test, *P < 0.05, **P < 0.01).

Repeated cocaine exposure abolished the D2R-mediated intracellular Ca²⁺ release in medium spiny NAc neurons. There was no detected Ca²⁺ release in the presence of quinpirole in cocaine-pretreated NAc cells when compared with that in saline-pretreated cells after either a 3-day withdrawal (F₀/ΔF = –0.02% ± 0.1% or 98.3% ± 7.1% of baseline predrug response; n = 9 cells, t_(1,16) = 3.02; **P < 0.001) (Fig. 7A and C) or a 21-day withdrawal (SAL- vs. COC-pretreated: n = 10/9 cells, four rats in each group; F₀/ΔF = 0.37 ± 0.16 vs. –0.02 ± 0.02, respectively, t_(1,17) = 2.29; *P < 0.05) (Fig. 7C, right panel). Given the fact that the quinpirole-induced increase in [Ca²⁺]_in was observed without activation of voltage-gated Ca²⁺ channels and with blockade of ionotropic glutamate receptors, these findings indicate that D2R-mediated increase in [Ca²⁺]_in resulted from intracellular stores.

Protein levels of L-type Ca²⁺ channels and IP₃R are not significantly altered in the rat NAc after chronic cocaine treatment and withdrawal

Ca²⁺ influx via high voltage-activated Ca²⁺ channels and Ca²⁺ release from intracellular stores depend not only on the activity but also on the number of these Ca²⁺ channels and IP₃ receptors, respectively. Thus, we also evaluated if the decrease of Ca²⁺ influx and Ca²⁺ release in cocaine-pretreated NAc cells was affected by reduced protein levels (reflecting a decrease in the number) of the L-channels and/or IP₃Rs. Specific antibodies were used to measure the levels of L-channels and IP₃Rs in the rat NAc. The total protein levels of α1 subunit (pore-forming and ligand-binding protein) of L-channel and IP₃R were not significantly affected in the rat NAc after repeated cocaine treatment and withdrawal (n = 18–31/group, all P > 0.05; data not shown). Whether the surface expression (a.k.a. trafficking) of the L-channels and IP₃Rs was altered by repeated cocaine exposure and withdrawal remains to be determined in future investigations.

DISCUSSION

This study determined that D2R (D_2,3,4R) stimulation modulates Ca²⁺ homeostasis and related signaling in rat NAc MSNs in the core region by decreasing Ca²⁺ influx preferentially via the L-channels and increasing intracellular Ca²⁺ release. Repeated cocaine exposure increased PKA activity and abolished the D2R modulation. These changes are found after a 3-day or a 21-day cocaine abstinence, indicating enduring neuroadaptations of NAc function in cocaine-sensitized, withdrawn rats.

D2R stimulation decreases Ca²⁺ influx by preferentially reducing L-channel activity: implications of Ca²⁺ release and calcineurin activation

D2R-mediated decrease in Ca²⁺ influx was reflected by reduced “size” of Ca²⁺ potentials, which was reversible, receptor specific, and dose dependent. This decrease was mimicked and occluded by blockade of the L-, but not N- and P/Q-type Ca²⁺ channels, suggesting that D2R modulation preferentially reduced L-channel activity in NAc MSNs. Intracellular Ca²⁺ release and downstream calcineurin activation were implicated in the mechanisms, which may underlie the D2R-modulated decrease of L-channel activity. We previously revealed that D2R stimulation facilitates Na⁺ channel activity, most likely via elevating intracellular [Ca²⁺]_in in rat NAc MSNs (Hu et al., 2005a). Nevertheless, such Ca²⁺ release has never been proven by real-time Ca²⁺ imaging study. This study demonstrates that selective D2R stimulation increased [Ca²⁺]_in in the absence of membrane depolarization with blockade of ionotropic glutamate receptors, indicating a dynamic intracellular Ca²⁺ release in rat NAc MSNs. Such increased Ca²⁺ release could activate calcineurin and dephosphorylate L-channels, thereby subsequently reducing Ca²⁺ influx via the L-channel (Day et al., 2002; Groth et al., 2003). Moreover, we also found that calcineurin mimicked and occluded D2R-mediated suppression of Ca²⁺ spikes, and inhibition of calcineurin activity by cyclosporin A not only prolonged the duration but also prevented D2R suppression of Ca²⁺ spikes. These findings suggest that via a D2R-coupled, cAMP/PKA/IP₃R/Ca²⁺-mediated pathway (Hu et al., 2005a), and likely the others (Hernandez-Lopez et al., 2000), D2R stimulation activates calcineurin in a converged common path that may facilitate dephosphorylation of L-channels and therefore reduce Ca²⁺ influx in NAc MSNs (Fig. 8A).

Fig. 8.

D2R modulation of L-type Ca²⁺ channel activity is reduced after chronic exposure to cocaine: alterations in the D2R/AC/PKA/IP₃R/Ca²⁺/calcineurin pathway. On the basis of the findings of this study and previous results, we propose here a working model that will help us to better understand the mechanisms that may underlie D2R modulation of Ca²⁺ homeostasis in rat NAc MSNs, either under physiological condition or after repeated cocaine exposure and withdrawal. A: D2R modulation of Ca²⁺ homeostasis in control NAc MSNs. D2R stimulation with quinpirole inhibits the cAMP/PKA cascade (by decreasing PKA activity) that reduces phosphorylation of IP₃Rs localized on endoplasmic reticulum. This D2R action triggers intracellular Ca²⁺ release, which subsequently elevates [Ca²⁺]_in and activates Ca²⁺/calmodulin-dependent calcineurin (CaN). Calcineurin-induced dephosphorylation of L-channels diminishes I_Ca via the L-channels and therefore suppresses evoked Ca²⁺ spikes. These changes lead to a decrease in the intrinsic excitability of NAs neurons. The D2R effects on suppressing Ca²⁺ spikes are blocked by selective inhibition of D2R (eticlopride), mimicked (and occluded) by cytosolic application of calcineurin, eliminated by inhibition of calcineurin activity (cyclosporin A), and abolished by specific blockade of L-channel (nifedipine). However, inhibition of D2R did not significantly affect function of non-L-type high voltage-activated Ca²⁺ channels. B: Repeated cocaine exposure and withdrawal dysregulates D2R-modulated Ca²⁺ homeostasis in NAc MSNs, leading to a significant decrease in D2R function with increased PKA activity. Such changes facilitate IP₃R phosphorylation, reduce Ca²⁺ release, decrease [Ca²⁺]_in, and diminish calcineurin function in NAc MSNs. Although attenuated dephosphorylation of L-channels by calcineurin intends to increase I_Ca via the channel, it is overcome by a significant reduction in I_Ca via N- and R-type Ca²⁺ channels. As described above, activity of N- and R-type Ca²⁺ channels is decreased in cocaine-pretreated NAc MSNs in drug-withdrawn rats, likely via enhanced dephosphorylation of these channels by protein phosphatase 1, which is activated by PKA. Together, these findings indicate that neuroadaptation of D2R-mediated Ca²⁺ homeostasis by repeated cocaine exposure dysregulates NAc MSNs, causing a persistent decrease in the intrinsic excitability of these cells.

Repeated cocaine exposure decreases Ca²⁺ influx with enhanced PKA activity

Another major finding of this study is that the cocaine-induced decrease in Ca²⁺ influx was associated with enhanced PKA activity in the NAc. Given the fact that the MSNs constitute 95% cell population in this brain region (Pasik, 1979), we could reasonably assume that the cocaine-induced increase of PKA activity in the NAc occurred mainly in MSNs. The mechanism underlying the decreased Ca²⁺ influx has been related to enhanced D1R signaling and reduced D2R function with increased phosphorylation and diminished dephosphorylation of Ca²⁺ channels, respectively (see Hu, 2007; Nestler, 2004 for review). Compelling evidence shows that cocaine-induced neuroadaptation in ion channels is attributable, at least in part, to facilitation of the cAMP/PKA cascade. For instance, upregulated D1R signaling (e.g., increase of G_s-coupled cAMP formation and PKA activity) is found in NAc cells after repeated cocaine treatment (Hope et al., 2005; Self et al., 1995; Terwilliger et al., 1991). With enhanced PKA/DARPP-32 activity and reduced calcineurin function (Hu et al., 2005b), both the Ca²⁺ influx via high voltage-activated Ca²⁺ channels and the evoked Ca²⁺ spikes are significantly diminished in cocaine-pretreated NAc MSNs (Hu et al., 2004; Zhang et al., 2002).

Cocaine-induced decrease in Ca²⁺ influx in NAc MSNs could result from enhanced PKA activity via a direct and indirect manner. Although direct phosphorylation of L-channels by PKA can facilitate activity of the channel, whole-cell Ca²⁺ influx is actually reduced because of decreased I_Ca through N- and R-type Ca²⁺ channels in cocaine-withdrawn NAc MSNs (Zhang et al., 2002). This decrease in I_Ca is most likely related to an indirect PKA action by which protein phosphatase 1 is activated and non-L-type high voltage-activated Ca²⁺ channels are dephosphorylated (Surmeier et al., 1995; Zhang et al., 2002). It is worth noting that I_Ca via the L-channels contribute to only ~30% of whole-cell Ca²⁺ conductance, whereas the combined N- and R-type Ca²⁺ currents consist of about 50% of Ca²⁺ influx in control, drug-free NAc MSNs (Zhang et al., 2002). Even though repeated cocaine exposure tended to increase Ca²⁺ influx via L-channels, such change was not statistically significant (at least not in the soma). Thus, we suggest that the reduced amount of I_Ca across N- and R-type Ca²⁺ channels was greater than the probably increased Ca²⁺ currents via the L-channels and therefore led to suppression of Ca²⁺ potentials in cocaine-preexposed NAc MSNs (Fig. 8B).

Repeated cocaine treatment abolishes D2R-modulated Ca²⁺ release and inhibition of L-channel activity

This study also reveals an intracellular Ca²⁺ release induced by D2R stimulation. This novel finding provides the first evidence for dynamic D2R modulation of Ca²⁺ mobilization in rat NAc MSNs. However, repeated cocaine exposure decreased the D2R effect. This decrease could be attributed in part to a reduction in D2R/G_i/o coupling and IP₃R activity, but not to a decrease in IP₃R levels. Such conclusion results from the following facts: First, PKA phosphorylation inhibits IP₃R activity and diminishes Ca²⁺ release from endoplasmic reticulum (Cameron et al., 1995; Ferris et al., 1991; Quinton and Dean, 1992; Tertyshnikova and Fein, 1998). Second, D2R-coupled Ca²⁺ modulation of ion channel activity involves inhibition of PKA activity and disinhibition of IP₃R (Hu et al., 2005b). Third, D2R-mediated suppression of Ca²⁺ spikes relies on reduced L-channel activity via dephosphorylation of the channel by calcineurin (Day et al., 2002; Hernandez-Lopez et al., 2000). Fourth, repeated cocaine exposure increases D1R/G_s coupling (Terwilliger et al., 1991) and decreases D2R/G_i coupling (Nestler et al., 1990) as well as calcineurin levels and efficacy in NAc MSNs (Hu et al., 2005b). Fifth, the IP₃R protein levels are not changed by repeated cocaine exposure. Thus, reduced D2R modulation of intracellular Ca²⁺ release by cocaine provides an informative knowledge for us to better understand the mechanism of cocaine withdrawal. Although the exact subtypes of D2R implicated in the cocaine-induced changes remain unknown (all D_2,3,4R are found in the NAc, but may or may not be in the same cells), these findings suggest that diminished D2R signaling plays an important role in dysregulating Ca²⁺ homeostasis in NAc MSNs of cocaine-preexposed, abstinent rats.

Cocaine-induced adaptations in NAc MSNs persist during withdrawal

The reduced Ca²⁺ influx, enhanced PKA activity, decreased intracellular Ca²⁺ release, and abolished inhibition of L-channel activity mediated by D2R persisted for at least 21 days after cocaine withdrawal. These findings are consistent with earlier in vivo studies, showing that repeated cocaine exposure enduringly increases D1R-modulated inhibition of NAc activity in cocaine-withdrawn rats (Henry and White, 1991; Henry et al., 1989). They are also correlated with cocaine-induced behavioral sensitization and neuroadaptations in synaptic activity of NAc cells, which may increase cocaine craving and/or relapse in response to cocaine and cocaine-related cues (Conrad et al., 2008; Kourrich et al., 2007). These findings are in agreement with the perspective that cocaine-induced neuroadaptations in dopamine signaling pathways and ion channel function decrease the intrinsic excitability (Dong et al., 2006; Zhang et al., 1998) and activity of NAc MSNs (Hu, 2007; Kalivas and Hu, 2006). Such decrease in NAc activity provides a support for the reduced basal activity in the reward circuitry revealed by brain imaging study in cocaine-abstinent human (Kufahl et al., 2005; Volkow et al., 2003b), which may contribute to the neuropathophysiology of cocaine addiction.

It is worth noting that cocaine-induced changes in the L-channels, voltage-gated K+ channels, and PKA activity also occur in the medial prefrontal cortex (mPFC) (Dong et al., 2005; Ford et al., 2009; Nasif et al., 2005a,b), suggesting that cocaine-induced neuroadaptation preferentially affects function of the mesocorticolimbic dopamine system (a.k.a. the reward pathway). Given that both NAc and mPFC play critical roles in regulating cognitive function and addiction, these findings suggest that cocaine-induced neuroadaptation in neuronal activity may primarily or initially occur in the two brain regions.

Functional implications

Reduced D2R modulation of Ca²⁺ homeostasis in MSNs reveals dysregulation of the NAc after withdrawal from repeated cocaine exposure. These results are in agreement with previous findings, showing involvement of D2R dysfunction in the mechanisms of cocaine withdrawal. For instance, brain imaging studies in cocaine-abstinent humans indicate decreased D2R availability in striatal cells (Volkow et al., 2003b). This decrease (likely via phosphorylation-induced internalization) is correlated with reduced glucose metabolism and oxygen consumption in the orbital and medial PFC during cocaine abstinence (known as PFC hypoactivity) (Volkow et al., 2003a, 2007). Such changes reflect a decreased basal activity in the orbital-mPFC and reduced excitatory outputs from these cortical regions to the NAc (Kalivas and Hu, 2006) and therefore may contribute to the neuro-pathogenesis of cocaine withdrawal symptoms, including but not limited to depression, apathy, anhedonia, and drug seeking (Hu, 2007). Our novel findings provide support for the perspective that cocaine-induced behavioral changes are fundamentally based upon neuroadaptations in ion channel function and dopamine/Ca²⁺ signaling in the NAc and mPFC, in which D2R dysregulation, along with D1R dysfunction, plays a crucial role. Determining the altered D2R function in the NAc of cocaine-preexposed rats extends our knowledge to better understand the mechanisms of cocaine addiction, which may eventually help us to develop more effective therapeutic treatments for drug addiction.

Abbreviations

cAMP

cyclic adenosine monophosphate

D1R

dopamine D₁-like receptors

D2R

dopamine D₂-like receptors

IP₃R

inositol-1,4,5-trisphosphate receptor

MSNs

medium spiny neurons

NAc

nucleus accumbens

PKA

protein kinase A