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Repeated Cocaine Administration Suppresses HVA-Ca²⁺ Potentials and Enhances Activity of K⁺ Channels in Rat Nucleus Accumbens Neurons

Neuropsychopharmacology Laboratory, Department of Cellular and Molecular Pharmacology, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064-3095

Submitted 4 March 2004; accepted in final form 3 May 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The nucleus accumbens (NAc) is an important forebrain area involved in sensitization, withdrawal effects, and self-administration of cocaine. However, little is known about cocaine-induced alterations in the neuronal excitability and whole cell neuroplasticity in this region that may affect behaviors. Our recent investigations have demonstrated that repeated cocaine administration decreases voltage-sensitive sodium and calcium currents (VSSCs and VSCCs, respectively) in freshly dissociated NAc neurons of rats. In this study, current-clamp recordings were performed in slice preparations to determine the effects of chronic cocaine on evoked Ca²⁺ potentials and voltage-sensitive K⁺ currents in NAc neurons. Repeated cocaine administration with 3–4 days of withdrawal caused significant alterations in Ca²⁺ potentials, including suppression of Ca²⁺-mediated spikes, increase in the intracellular injected current intensity required for generation of Ca²⁺ potentials (rheobase), reduced duration of Ca²⁺ plateau potentials, and abolishment of secondary Ca²⁺ potentials associated with the primary Ca²⁺ plateau potential. Application of nickel (Ni²⁺), which blocks low-voltage activated T-type Ca²⁺ channels, had no impact on evoked Ca²⁺ plateau potentials in NAc neurons, indicating that these Ca²⁺ potentials are high-voltage activated (HVA). In addition, repeated cocaine pretreatment also hyperpolarized the resting membrane potential, increased the amplitude of afterhyperpolarization in Ca²⁺ spikes, and enhanced the outward rectification observed during membrane depolarization. These findings indicate that repeated cocaine administration not only suppressed HVA-Ca²⁺ potentials but also significantly enhanced the activity of various K⁺ channels in NAc neurons. They also demonstrate an integrative role of whole cell neuroplasticity during cocaine withdrawal, by which the subthreshold membrane excitability of NAc neurons is significantly decreased.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Repeated cocaine administration causes various behavioral changes in both humans and experimental animals. In cocaine addicts, drug dependence is accompanied by withdrawal symptoms, including anergia, anhedonia, depression, anxiety, and cocaine craving (Gawin 1991). Many of these changes are also found in cocaine-sensitized animal models (Carroll and Lac 1987; Kokkinidis and McCarter 1990; Markou and Koob 1991; Mutschler and Miczek 1998). In addition, the changes in dopamine receptor (DA-R) modulation within the mesoaccumbens and mesocortical DA system have been attributed to induction and expression of cocaine-induced sensitization and withdrawal effects (for reviews, see Kalivas et al. 1998; Pierce and Kalivas 1997; White and Kalivas 1998; White et al. 1995a, 1997), suggesting a functional involvement of the nucleus accumbens (NAc) and the prefrontal cortex in cocaine addiction.

Our previous studies have demonstrated that the behavioral changes in cocaine-sensitized animals are correlated with neuroadaptation in both pre- and postsynaptic aspects of DA and glutamate transmission, leading to alterations in the basal activity and neuronal responses to dopaminergic and glutamatergic agonists (Henry and White 1991, 1995; Henry et al. 1989, 1998; Li et al. 1999; White et al. 1995a, b; Zhang et al. 1997). Increased inhibitory responses to DA D1 class receptor (D1R) agonists (Henry and White 1991, 1995; Henry et al. 1989) and decreased excitatory responses to glutamate receptor agonists (White et al. 1995b) strongly suggest that the membrane excitability of medium spiny neurons (MSNs) is reduced in the cocaine-sensitized NAc. Although the cellular and molecular mechanisms underlying chronic cocaine-induced changes in neuronal excitability are not fully understood, various alterations in membrane ion channel function and intracellular signaling pathways have been observed in NAc neurons (Hu et al. 2003; Zhang et al. 1998, 2002).

The membrane excitability of NAc neurons is predominantly regulated and controlled by Na⁺, Ca²⁺, and different K⁺ channels (Higashi et al. 1989; Nicola and Malenka 1997; North and Uchimura 1989; O'Donnell and Grace 1993; Uchimura et al. 1990). Our recent studies have demonstrated that repeated cocaine administration renders NAc neurons much less excitable in response to depolarizing influences via depressing whole cell voltage-sensitive sodium and calcium currents (VSSCs and VSCCs) (White et al. 1995b; Zhang et al. 1998, 2002). More importantly, these changes in Na⁺ and Ca²⁺ channel function are found to be associated with increased activity of the D1R/G_s/cAMP/protein kinase A (PKA) signaling pathway (Hu et al. 2003; Nestler 1997; Self and Nestler 1995; Zhang et al. 2002) and decreased function of calcineurin (protein phosphatase 2B, PP-2B), suggesting that an increased phosphorylation and decreased dephosphorylation may both contribute to the mechanism that underlies cocaine-induced whole cell neuroplasticity in the NAc (Hu et al. 2003). However, it is unknown how the reduced VSCCs dynamically affect the subthreshold membrane excitability and generation of Ca²⁺ potentials in NAc neurons. In addition, it is also unknown whether potassium channels in NAc neurons are functionally involved in the altered membrane excitability observed after repeated cocaine pretreatment. Based on our previous findings, we hypothesized that chronic cocaine-induced I_Ca reduction would result in a marked decrease in the subthreshold excitability and suppress generation of Ca²⁺ potentials in NAc neurons. To further investigate the effects of repeated cocaine administration on the subthreshold membrane excitability, which is primarily modulated by membrane Na⁺, Ca²⁺ and K⁺ channels, the present study was performed to determine how the voltage-sensitive Ca²⁺ potentials and whether K⁺ channel activity were affected, respectively, in cocaine-withdrawn NAc neurons.

	METHODS

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Animals and pretreatments

Male Sprague-Dawley rats (Harlan, Indianapolis, IN) initially weighing 175–200 g were housed in groups of two to three in a temperature and humidity-controlled vivarium under a 12-h light/dark cycle. Food and water were freely available. The animals 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 recording experiments were conducted on the third or fourth day after cessation of pretreatment.

Preparation of brain slices

All procedures were in strict accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23) and were approved by the Institutional Animal Care and Use Committee of our school. Rats were decapitated under halothane anesthesia and brains were rapidly excised and dissected into blocks (3–4 mm) containing the NAc. The blocks of tissue were immersed in ice-cold artificial cerebrospinal fluid [ACSF, containing (in mM) 124 NaCl, 5 KCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 2 MgSO₄, 2.4 CaCl₂, and 10 D-glucose, pH 7.4 ± 0.05] and sectioned in the coronal plane (400 µm) with a motorized vibrating microtome. Brain slices were transferred to a holding chamber and incubated at room temperature in oxygenated (95% O₂-5% CO₂) ACSF for 60 min. Slices were then transferred to an interface-type recording chamber where they rested on nylon mesh and were superfused continuously for 60 min with warm (34–35°C) oxygenated ACSF (flow rates of 1–2 ml/min) before recording.

Intracellular current-clamp recordings

Both "sharp" electrodes and patch electrodes were used in this study. The patch electrodes were used specifically for internal dialysis of K⁺ channel blockers. Sharp electrodes were made with borosilicate glass capillaries (1.0/0.58 mm OD/ID), pulled with a tip diameter <1 µm and filled with 3 M potassium acetate (resistance: 70–150 M). Current-voltage (I-V) relationships were studied by injecting step constant current pulses through recording electrode using an active bridge circuit of an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). Electrical activity was first amplified by a head stage located near the slice before being sent to the amplifier, which was connected to an oscilloscope and to a computer running pCLAMP software (Version 9.0) through an interface (Axon Instruments). In this study, all recordings were made from the core region of the NAc (and the effects of chronic cocaine on the shell region of NAc neurons are currently under investigation in a separate experiment). The resting membrane potential (RMP) was originally measured in the absence of current injection prior to the initiation of other experiments. Sodium action potentials (APs) were generated by injecting step depolarizing current pulses of 0.1-nA increments into NAc neurons. Calcium potentials were evoked by depolarizing current pulses with external and/or internal application of different K⁺ channel blockers (see following text). The characteristics of Ca²⁺ potential were obtained from the initial spike evoked by the minimal depolarizing current pulse (rheobase) in each NAc neuron. The amplitude of Ca²⁺ potential was measured from the threshold of spike to its peak. The half-amplitude duration of Ca²⁺ plateau potentials was measured at the amplitude level that one-half of the peak of plateau potential was reached. The amplitude (deepness) of afterhyperpolarization (AHP) was measured from the equipotential point of the spike threshold to the maximum deflection of the hyperpolarization following the end of Ca²⁺ potentials. In addition, to determine the possible differences in the I-V relationship between saline- and cocaine-pretreated NAc neurons, RMP was held at –80 mV for each NAc neuron by injecting a small constant current through manual control.

To detect the possible influence of K⁺ channels, particularly the voltage-gated K⁺ channels (delayed rectifiers), in generation of Ca²⁺ potentials, patch electrodes were used in some NAc neurons. They were pulled from Corning 7056 glass capillary tubes using a micropipette puller (Sutter Instrument, Novato, CA; resistance: 3–5 M) and filled with cesium (Cs⁺)-based internal pipette solution to block all K⁺ channels from the inside of membrane [which contained (in mM) 140 Cs gluconate, 10 HEPES, 2 MgCl₂, 3 Na₂ATP, and 0.3 Na₂GTP, pH 7.3, 280–285 mosM/l]. The patch electrode, which has larger tip opening as compared with sharp electrodes, allowed Cs⁺ to be dialyzed into cytosol. Both NAc neurons and electrodes were visualized using an infra-red upright microscope (Nikon, Melville, NY) fitted with a CCD camera (Hamamatsu) feeding into a monitor. Whole cell patch clamps were formed first by lowering the electrodes onto neurons using a micromanipulator (Sutter Instrument). Once whole cell configuration was formed, recording was switched to current clamp. RMP was then held at –80 mV for study of the I-V relationship as described in the preceding text. With concurrent application of Na⁺ and K⁺ channel blockers in the bath (see following text), Ca²⁺ potentials were evoked by injection of step constant current pulses (20- to 40-ms duration, 0 to +2.0 nA with 0.05-nA increments) through the recording electrode (SEC-05L/H single-electrode amplifier, npi Electronic Systems, Tamm, Germany). Electrical activity was also collected using Clampex 9.0 software (pCLAMP, Axon Instruments). The same measurements were used for characterization of Ca²⁺ plateau potentials as described above. In addition, the whole "area" of the Ca²⁺ plateau potential was also measured by integrating the area between the peak of Ca²⁺ potential and the end of the Ca²⁺ potential while keeping RMP as the baseline (mV x ms; Origin 7.0).

Drug application

The effects of different ion channel blockers on the membrane properties and evoked action potentials were studied in NAc neurons of saline- or cocaine-pretreated rats. Drugs used in this study were applied externally via bath solution and/or internally dialyzed from the pipette solution. Bath solutions with ion channel blockers were made with ACSF immediately before use and were applied via a pump. Such ion channel blockers included the selective Na⁺ channel blocker tetrodotoxin (TTX, 1 µM), the Ca²⁺ channel blocker cadmium (Cd²⁺, 200 µM), the relatively selective A-type K⁺ channel blocker 4-aminopyridine (4-AP) at a low concentration (500 µM), and the K⁺ channel blocker tetraethylammonium chloride (TEA, 20 mM). Termination of the effects of these drugs was achieved by washout using drug-free ACSF. In some cases, another K⁺ channel blocker Cs⁺ was internally applied into NAc neurons via dialyzing from recording pipette (see preceding text). Under this circumstance, some NAc neurons were also studied with application of nickel (Ni²⁺) at a relatively low concentration (100 µm), which usually blocks the low-voltage-activated (LVA-) Ca²⁺ channels in neurons. However, the evoked Ca²⁺ plateau potentials were not significantly affected by such Ni²⁺ treatment (see the results in the following text), indicating that the Ca²⁺ potentials evoked by depolarizing currents in NAc neurons were high-voltage activated (HVA).

Statistical analysis

Comparisons between the I-V curves were made with a two-way ANOVA (ANOVA) with repeated measures on one variable (depolarizing currents). Other comparisons were made with either Student's t-test or ² tests.

	RESULTS

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Characterization of different forms of HVA-Ca²⁺ potentials in NAc neurons

All NAc neurons were recorded within the core region (O'Donnell and Grace 1993; Zahm and Brog 1992) with stabilized RMP at the levels of –85 to –60 mV. There was no spontaneous activity in NAc neurons under the stabilized RMP. Various ion channel blockers were applied externally and/or internally to determine their effects on evoked Ca²⁺ potentials and other membrane properties. Injection of depolarizing current pulses evoked a Na⁺ action potential (AP), which was blocked by TTX (Fig. 1A). With blockade of Na⁺ currents and 4-AP-sensitive A-type K⁺ currents (I_A) (Nakajima 1966; Thompson 1977) by TTX and 4-AP (500 mM) respectively, depolarizing currents evoked a slow-developing, small Ca²⁺ spike (n = 8/8), which was blocked by Cd²⁺ (400 µM, n = 4/4; Figs. 1B and 3A). With application of TEA (20 mM), depolarizing currents evoked a Na⁺ AP immediately followed by a Ca²⁺ plateau potential (n = 12/12 cells; Figs. 1C, 2A, and 3B), which were blocked by combined application of TTX and Cd²⁺ (n = 4/4; Fig. 1C). With concurrent blockade of K⁺ and Na⁺ currents by TEA plus TTX, a typical Ca²⁺ plateau potential was evoked (Fig. 1D). In addition, additional blockade of TEA-insensitive I_A by internally applied Cs⁺ (Fig. 3C) markedly prolonged the duration of Ca²⁺ plateau potentials (comparing the saline-pretreated cells in Figs. 1D and 3C and note the difference in the time scales). Nevertheless, the afterhyperpolarization, which was produced by activation of the Ca²⁺-activated K⁺ currents [I_K(Ca)] and generally found in the slow-developing Ca²⁺ spike without blockade of I_K(Ca) (Fig. 3C), was never observed in Ca²⁺ plateau potentials with complete blockade of all types of K⁺ currents from the inside and outside of the membrane (comparing saline-pretreated NAc cells in Fig. 3, A and C).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Representative traces illustrating various action potentials generated by injection of depolarizing current pulses in the absence or presence of different ion channel blockers. A: Na+-dependent action potential was evoked by injection of a depolarizing current pulse in the absence of any ion channel blockers. This Na+ spike was blocked by bath application of TTX. B: with concurrent application of TTX (1 µM) and 4-aminopyridine (4-AP; 500 µM), a slow-developing Ca2+ spike was evoked by a depolarizing current pulse and was then blocked by application of Cd2+. C: with application of TEA (20 mM), an evoked Na+ action potential was immediately followed by a Ca2+ plateau potential. The combined Na+ and Ca2+ potentials were blocked by concurrent application of TTX and Cd2+ (400 µM). D: with concurrent application of TTX and TEA, a separate Ca2+ plateau potential was evoked without a preceding Na+ action potential. This potential was then completely blocked by bath application of Cd2+.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Repeated cocaine administration suppresses evoked Ca2+ potentials in NAc neurons. A: the rheobase and the amplitude of afterhyperpolarizations were markedly increased and deepened, respectively, in a slow-developing Ca2+ spike generated from a cocaine-pretreated NAc neuron as compared with that from a saline-pretreated cell. B: repeated cocaine administration not only markedly reduced the duration of primary Ca2+ plateau potentials but also abolished the secondary Ca2+ potential in NAc neurons. Note that the rheobase for cocaine-pretreated NAc neurons was also increased as compared with saline-pretreated cells. C: completed blockade of K+ channels (by external TEA and internal Cs+) prolonged the duration of Ca2+ plateau potentials in both saline- and cocaine-pretreated NAc neurons. This manipulation also made measurement of the threshold of Ca2+ plateau potentials possible (Comparing the durations and scales in B and C). Under these circumstances, the duration of Ca2+ plateau potential recorded from cocaine-pretreated NAc neurons was still remarkably shorter than that observed in saline-pretreated cells. - - -, the interrupted (B) and actual (C) threshold of Ca2+ plateau potentials in NAc neurons.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Secondary Ca2+ potentials modulate the generation of Na+ spike and Ca2+ plateau potentials. A: after blockade of TEA-sensitive K+ channels, a small membrane depolarization (the secondary Ca2+ potential) was observed at the end of an initially evoked Ca2+ plateau potential in saline-pretreated nucleus accumbens (NAc) neurons (n = 12/12). B: this secondary Ca2+ potential was capable to occasionally evoke additional Na+ spikes (n = 2 cells). C and D: blockade of Na+ currents by TTX did not eliminate the secondary Ca2+ potential. In some cases, concurrent blockade of voltage-sensitive sodiumcurrents (VSSCs) and TEA-sensitive as well as TEA-insensitive K+ currents (TTX + 4-AP + TEA), resulted in small Ca2+ potentials that were occasionally enlarged and converted to a secondary Ca2+ plateau, from which multiple Ca2+ plateau potentials could be generated (n = 3 cells). Arrows indicate the locations of the secondary Ca2+ potential and its effects.

Repeated cocaine administration inhibits HVA-Ca²⁺ potentials in NAc neurons

Repeated cocaine administration not only affected generation of both forms of Ca²⁺ potentials but also significantly altered their characteristics in NAc neurons. Concurrent blockade of VSSCs and 4-AP-sensitive I_A appeared to be necessary for evoking the slow-developing Ca²⁺ spikes in saline-pretreated NAc neurons. However, the slow-developing Ca²⁺ spikes were much less frequently evoked in cocaine-pretreated NAc neurons even with higher depolarizing currents (saline vs. cocaine group: 100% or n = 8/8 cells vs. 45.5% or n = 5/11 cells, ² = 6.3949, *P < 0.05; Figs. 3A and 4A). Meanwhile, the rheobase for evoking the slow-developing Ca²⁺ spikes was significantly increased in cocaine-pretreated neurons (saline vs. cocaine group: 0.9 ± 0.2 vs. 1.7 ± 0.3 nA, t = 2.6525, *P < 0.05; Figs. 3A and 4A). Moreover, the amplitude of the AHP observed in the slow-developing Ca²⁺ spike was significantly greater in these cocaine-withdrawn cells (saline vs. cocaine group: 22.3 ± 2.6 vs. 34.6 ± 3.2 mV, t = 2.9643, **P < 0.01; Figs. 3A and 4A). These changes were accompanied with a significantly hyperpolarized RMP (saline vs. cocaine group: –79.0 ± 1.0 vs. –84.0 ± 1.0 mV, t = 4.7412, **P < 0.001; Table 1). In addition, a tendency toward a decrease of the input resistance, increase of the threshold, and reduction in the amplitude and the duration of this spike were also observed in cocaine-withdrawn neurons although they were not statistically significant (Table 1).

View larger version (48K): [in this window] [in a new window]   FIG. 4. Effects of repeated cocaine administration on the characteristics of Ca2+ potentials. A: repeated cocaine administration significantly increased the rheobase (SAL-pretreated vs. COC-pretreated: n = 8 vs. 11 cells, 0.9 ± 0.2 vs. 1.7 ± 0.3 nA, t = 2.6525, *P < 0.05), deepened the amplitude of afterhyperpolarization (AHP; SAL-pretreated vs. COC-pretreated: 22.3 ± 2.6 vs. 34.6 ± 3.2 mV, t = 2.9643, *P < 0.02), and decreased the number of cells showing an evoked slow-developing Ca2+ spike (SAL-pretreated vs. COC-pretreated: n = 8/8 cells or 100% vs. n = 5/11 cells or 45.5%, 2 = 6.3949, *P < 0.05) as compared with saline-pretreated controls. These NAc neurons were recorded with blockade of Na+ and some TEA-insensitive A-type K+ currents by concurrent application of TTX and 4-AP. B: repeated cocaine administration significantly increased the rheobase (SAL-pretreated vs. COC-pretreated: n = 12 vs. 10 cells, 0.6 ± 0.1 vs. 1.0 ± 0.2 nA, t = 2.2378, *P < 0.05), reduced the duration of Ca2+ plateau potential measured at 1/2 of the amplitude (SAL-pretreated vs. COC-pretreated: 145.7 ± 24.7 vs. 55.5 ± 11.8 ms, t = 3.0844, **P < 0.01), and decreased the number of cells showing the small membrane depolarization (also known as the secondary Ca2+ potential, SAL-pretreated vs. COC-pretreated: n = 12/12 cells or 100% vs. n = 4/10 cells or 40%, 2 = 10.0768, **P < 0.01) as compared with saline-pretreated controls. These NAc neurons were recorded with blockade of TEA-sensitive K+ currents by TEA. C: repeated cocaine administration also significantly increased the rheobase (SAL-pretreated vs. COC-pretreated: n = 16 vs. 15 cells, 0.9 ± 0.1 vs. 1.2 ± 0.1 nA, t = 2.422, *P < 0.03), reduced the duration of Ca2+ plateau potential measured at 1/2 of the amplitude (SAL-pretreated vs. COC-pretreated: 315.4 ± 24.3 vs. 210.1 ± 16.3 ms, t = 3.674, **P < 0.01), and decreased the whole area of evoked Ca2+ plateau potential (SAL-pretreated vs. COC-pretreated: 38,016 ± 2,889 vs. 25,502 ± 2,042 unit, t = 3.612, **P < 0.001) as compared with saline-pretreated controls. These NAc neurons were recorded with blockade of Na+ currents and all TEA-sensitive as well as TEA-insensitive K+ currents by concurrent application of TTX, TEA and Cs+. All bars represent means ± SE.

Similar alterations in the membrane properties and characteristics of Ca²⁺ plateau potentials were observed in cocaine-pretreated NAc neurons with concurrent block of VSSCs and all types of K⁺ currents. With combined application of external TTX, TEA and internal Cs⁺ (Figs. 3C and 4C), a significant increase in the rheobase, decrease in the duration of Ca²⁺ plateau potential measured at the one-half-amplitude, and reduction in the total area of the Ca²⁺ plateau potential were observed in cocaine-withdrawn NAc neurons (saline vs. cocaine group: 0.9 ± 0.1 vs. 1.2 ± 0.1 nA, t = 2.422, *P < 0.05; 315.4 ± 24.3 vs. 210.1 ± 16.3 ms, t = 3.674, **P < 0.01; and 38,016 ± 2,889 vs. 25,502 ± 2,042 U, t = 3.612, **P < 0.01, respectively; Figs. 3C and 4C). Moreover, internally applied Cs⁺ eliminated the hyperpolarizing effects of chronic cocaine on RMP. Therefore there was no significant difference in the RMP between saline- and cocaine-pretreated NAc cells with internal presence of Cs⁺ (–68.7 ± 1.2 vs. 70.1 ± 0.7 mV, P > 0.05; Table 1). In addition, Cs⁺-induced depolarization in RMP was also observed in saline-pretreated NAc neurons (Table 1). These results indicate that Cs⁺-induced blockade of TEA-insensitive K⁺ currents, including I_Kir and I_A in this case, plays an important role in modulating RMP and Ca²⁺ plateau potentials, respectively. The cocaine-induced alterations in the characteristics of Ca²⁺ plateau potentials are summarized in Fig. 4 and Table 1.

Although HVA-Ca²⁺ currents are thought to make a major contribution for generation of the plateau potential in rat striatal neurons (Bargas et al. 1991b, 1994), a recent report suggests that some LVA T-type Ca²⁺ channels are expressed in the striatum of newborn and juvenile rats (McRory et al. 2001). To determine whether these LVA-Ca²⁺ channels are present and functionally involved in generation of Ca²⁺ plateau potentials in NAc neurons of adult rats, a relatively low concentration of Ni²⁺ (100 µM) was used in this study to block any possible effects of LVA T-type Ca²⁺ channels. However, application of Ni²⁺ affected neither the duration of Ca²⁺ plateau potentials (n = 5/5, control vs. Ni²⁺: 276.77 ± 55.65 vs. 307.35 ± 53.47 ms, P > 0.05) nor other characteristics in saline-pretreated NAc neurons (Fig. 5), indicating that the evoked Ca²⁺ plateau potential was generated primarily by HVA-Ca²⁺ currents in NAc neurons of these young adult rats.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Application of Ni2+ did not affect evoked Ca2+ plateau potentials in saline-pretreated NAc neurons. Although the concentration of Ni2+ (100 µM) utilized in the current study usually blocked the low-voltage-activated (LVA) T-type Ca2+ channels in other types of neurons, representative Ca2+ plateau potentials recorded from a saline-pretreated NAc neuron exhibited overlapping traces either with or without Ni2+. All neurons recorded under these circumstances were also treated with internally dialyzed Cs+ and externally applied TEA plus TTX. There was no significant difference either in the duration of Ca2+ plateau potential measured at a half-amplitude (insertion, n = 5/5 cells; control vs. Ni2+: 276.77 ± 55.65 vs. 307.35 ± 53.47 ms, P > 0.05) or in the threshold and amplitude of Ca2+ plateau potentials (not shown) between control and nickel-treated NAc neurons, indicating that the Ca2+ plateau potential evoked in NAc neurons is high voltage activated (HVA).

As described in the preceding text in saline-pretreated NAc cells, depolarizing current pulses induced a slowly developed, subthreshold membrane depolarization that gradually reached the threshold and eventually evoked a Na⁺ AP (Fig. 6A1). TTX inhibited the Na⁺-dependent action potential by suppressing the subthreshold depolarization, which therefore revealed an outward rectification represented by a downward deflection of membrane potential traces in response to increment of depolarizing current pulses (Fig. 6A2). This outward rectification was initiated near the membrane potential levels around –60 mV (Fig. 6C), at which I_K channels (including the somatic I_A) began to be activated (Nisenbaum and Wilson 1995a, b). There was a significant difference in the I-V curves of saline-pretreated NAc neurons during membrane depolarization before and after TTX application [SAL vs. SAL/TTX group, n = 10 cells, F(1,18) = 24.936, P < 0.001; Fig. 6C].

View larger version (38K): [in this window] [in a new window]   FIG. 6. Repeated cocaine administration enhanced outward rectification in NAc neurons during membrane depolarization: blockade of VSSCs. A: in a saline-pretreated NAc neuron, TTX suppressed the increase in subthreshold membrane excitability (the "ramp" potentials) induced by depolarizing current pulses and the generation of a Na+-dependent action potentials (A1). These changes revealed an outward rectification indicated by a downward reflection of membrane potential traces during membrane depolarization (A2). B: repeated cocaine administration caused a similar inhibition in the subthreshold membrane excitability and suppression of Na+ action potentials during membrane depolarization (B1). Note that higher depolarizing current was needed for generation of Na+ action potentials in cocaine-pretreated NAc neurons as compared with that in saline-pretreated cells. TTX did not produce further downward reflection of membrane potentials during membrane depolarization (B2). C: the current-voltage (I-V) curves indicate alterations in the I-V relationship of NAc neurons following repeated cocaine administration. In saline-pretreated NAc neurons, membrane depolarization initially induced an inward rectification ( SAL, n = 10), which was represented by an upward reflection in the I-V curve. This inward rectification was suppressed by application of TTX ( SAL/TTX, n = 10 cells). There was a significant difference in the I-V curve between the SAL/control and SAL/TTX-treated NAc neurons[ SAL vs. SAL/TTX, F(1,18) = 24.936, P < 0.001]. Repeated cocaine administration mimicked the effects of TTX on the I-V relationship. There was a significant difference in the I-V curve between saline- and cocaine-pretreated NAc neurons [ SAL vs. COC, F(1,18) = 31.266, P < 0.001]. However, this effect of cocaine on the I-V curve was not significantly affected by TTX ( COC vs. COC/TTX, n = 10/10 cells, P > 0.05). In addition, it is noted that in either SAL/TTX-treated NAc neurons () or COC-withdrawn cells ( or ), the inward rectification was converted to an outward rectification represented by a downward deflection in the I-V curve. This outward rectification was initiated at the subthreshold potential levels (approximately –70 to –60 mV) during membrane depolarization, in which the voltage-gated delayed rectifiers (IK) were activated. , the time point during the depolarizing current pulses where the membrane potentials were measured.

An enhanced outward rectification during membrane depolarization was also observed in cocaine-pretreated NAc neurons with blockade of both VSSCs and HVA-Ca²⁺ currents. Cocaine-pretreated neurons showed a marked increase in outward rectification in their I-V curves in response to increment of depolarizing current pulses with application of both TTX and Cd²⁺ compared with saline-pretreated NAc neurons (Fig. 7, A and B). Again, there was a significant difference in the I-V curves regarding the outward rectification between saline- and cocaine-pretreated neurons during membrane depolarization with application of TTX and Cd²⁺ [SAL vs. COC, n = 14/16 cells, F(1,28) = 9.84, *P < 0.05; Fig. 7C]. The integrated effects of repeated cocaine administration on the activity of VSSCs, HVA-Ca²⁺ currents and a variety of K⁺ currents including I_Kir(rest), I_K, and presumably I_K(Ca) (see DISCUSSION), which leads to decrease of membrane excitability of NAc neurons, are summarized in Fig. 8.

View larger version (38K): [in this window] [in a new window]   FIG. 7. Repeated cocaine administration enhanced the outward rectification of NAc neurons during membrane depolarization: blockade of both VSSCs and volstage-sensitive calcium currents (VSCCs). Representative traces of membrane potential showing the responses of NAc neurons to injection of depolarizing current pulses with application of TTX and Cd2+ in saline- (A) or cocaine-pretreated (B) NAc neurons. B: repeated cocaine pretreatment enhanced the outward rectification (downward deflection of membrane potential traces) of NAc neurons. C: the I-V curve of cocaine-pretreated NAc neurons shows a downward (more hyperpolarized) shift as compared with saline-pretreated cells, indicating a significant enhancement in the outward rectification during membrane depolarization [SAL-pretreated vs. COC-pretreated: n = 14/16; F(1,28) = 9.84, *P < 0.05]. , the time points where a steady-state Vm was measured.

View larger version (34K): [in this window] [in a new window]   FIG. 8. The mechanism underlying suppressed HVA-Ca2+ potentials and enhanced activity of K+ channels in NAc neurons of cocaine-withdrawn rats. This model incorporates some well-established findings from previous investigations and our present results. Repeated cocaine administration upregulates the D1R/Gs/AC/cAMP/protein kinase A (PKA) signaling pathway. Consequentially, enhanced PKA activity alters the function of voltage-sensitive Na+, Ca2+ and K+ channels in cocaine-pretreated NAc neurons in several ways, including the blockade of whole cell VSSCs and VSCCs. The decreased VSSCs are attributed to increased PKA phosphorylation and decreased calcineurin dephosphorylation of Na+ channels. The reduced activity of VSCCs may result from increased dephosphorylation of N- and R-type Ca2+ channels resulting from PKA-mediated activation of protein phosphatase 1 (PP1). Moreover, the reduced duration of HVA-Ca2+ plateau potentials along with abolishment of the secondary Ca2+ potential indicates that repeated cocaine administration not only decreases VSCCs but also prevents the dendritic propagation of Ca2+/Na+ action potentials, thereby decreasing somatic as well as dendritic membrane excitability in NAc neurons. In addition, repeated cocaine administration also increases activity of a variety of K+ channels, including the inwardly rectifiers [IKir(rest)], the voltage-gated slow delayed rectifiers (IKs), TEA-insensitive IA, and the Ca2+-activated BK channels. These functional changes in K+ channels have also been associated with increased PKA phosphorylation with the exception of the enhanced outward Kir(rest), which requires further investigation. Taken together, the integrated alterations in the function of membrane ion channels and the D1R/Gs/AC/cAMP/PKA signaling pathway result in decrease of membrane excitability in NAc neurons during cocaine withdrawal. Open arrows, cocaine-induced alterations in the ion channel functions and the membrane excitability.

	DISCUSSION

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Repeated cocaine administration inhibits HVA-Ca²⁺ potentials in NAc neurons

The major finding in the present study is that repeated cocaine administration inhibited HVA-Ca²⁺ potentials in NAc neurons. Significant alterations in the characteristics of HVA-Ca²⁺ potentials include the suppression of Ca²⁺ spikes, increased rheobase, and most remarkably, reduced duration of Ca²⁺ plateau potentials in cocaine-pretreated cells. These effects of chronic cocaine on Ca²⁺ potentials are expected and primarily attributed to reduced N- and R-type Ca²⁺ currents (Zhang et al. 2002). In addition, the increased rheobase should also be attributed to the reduced subthreshold excitability in which the activity of voltage-sensitive Na⁺ (Zhang et al. 1998) and K⁺ currents (see following text) were decreased and enhanced, respectively. It is well known that in rat striatal MSNs, plateau potentials are generated by VSCCs (Bargas et al. 1994, 1999; Cherubini and Lanfumey 1987; Dunia et al. 1996; Hernandez-Lopez et al. 1997; Higashi et al. 1988; Surmeier et al. 1995) after primarily activation of HVA-Ca²⁺ channels (Bargas et al. 1994; Hoehn et al. 1993; Surmeier et al. 1995). Given that stimulation of DA D1Rs indirectly decreases VSCCs via dephosphorylation of N-, R-, and P/Q-type channels by protein phosphatase 1 (PP-1) (Surmeier et al. 1995; Zhang et al. 2002) and that repeated cocaine administration upregulates the D1R/G_s/AC/cAMP/PKA pathway (Nestler 1997; Self and Nestler 1995), our findings indicate that suppression of HVA-Ca²⁺ potentials in cocaine-withdrawn NAc neurons is mediated by the upregulated D1R/G_s/AC/cAMP/PKA/PP1 pathway.

Another interesting finding in this study is that repeated cocaine administration abolished the secondary Ca²⁺ potential that is usually associated with the primary Ca²⁺ plateau potentials. "Active" propagation of APs from the axosomatic region to dendrites (known as backpropagation) as well as that from the dendrites to soma (known as "forward" propagation) has been demonstrated in different types of neurons (Hanson et al. 2004; Jaffe et al. 1992; Magee 2000; Magee and Carruth 1999; Stuart and Sakmann 1994). In these processes, voltage-gated dendritic Na⁺ and Ca²⁺ channels are activated by back- or forward-propagation of spikes (Debarbieux et al. 2003; Kavalali et al. 1997; Reuveni et al. 1993; Yuste et al. 1994), thereby eliciting VSSC and VSCC activation in dendrites (Markram and Sakmann 1994; Schiller et al. 1995; Spruston et al. 1995). Such alterations in the activity of dendritic Na⁺ and Ca²⁺ channels provide a prolonged inward current that is capable of generating multiple APs in the soma (Magee and Carruth 1999). Similar to these findings, backpropagation of APs is also found in striatal neurons (Bargas et al. 1991a, 1994; Hernandez-Lopez et al. 1997). Although the present recording methods do not allow us to make conclusions about dendritic events, our studies examining the secondary Ca²⁺ potential, as well as additional Na⁺ spikes, secondary Ca²⁺ plateau, and multiple Ca²⁺ plateau potentials evoked by it, suggest that both backpropagation and forward propagation of Na⁺/Ca²⁺ potentials may be present in NAc neurons. However, this secondary Ca²⁺ potential was abolished after repeated cocaine administration, suggesting that the assumed dendritic Na⁺/Ca²⁺ influx were also reduced in cocaine-withdrawn NAc neurons.

Repeated cocaine administration enhances multiple K⁺ currents in NAc neurons

Repeated cocaine administration also apparently alters the activity of somatic K⁺ channels in NAc neurons, evidenced by the hyperpolarized RMP, enhanced outward rectification during membrane depolarization, and deepened amplitude of afterhyperpolarization. Previous studies have determined the existence of various K⁺ currents in dorsal striatal and NAc MSNs, including but not limited to the inward rectifiers (I_Kir) (Galarraga et al. 1994; Nisenbaum and Wilson 1995a, b; Pacheco-Cano et al. 1996; Uchimura et al. 1989), delayed rectifiers (I_K) (Nisenbaum et al. 1994; Surmeier and Kitai 1993, 1997), and Ca²⁺-activated K⁺ currents [I_K(Ca)], which flow through the large (BK) and small (SK) conductance K⁺ channels, respectively (Bargas et al. 1999; Köhler et al. 1996). Given that one of the usual functions of I_Kir is to maintain RMP by carrying outward K⁺ current at the membrane potential levels slightly above the E_K (Hille 2001), it is not surprising that blockade of K_ir(rest) by internal Cs⁺ depolarized the RMP of NAc cells. In contrast, the RMP of NAc neurons was hyperpolarized after repeated cocaine treatment, suggesting that K_ir(rest) was enhanced in cocaine-withdrawn NAc neurons.

Associated with a hyperpolarized RMP, there was an enhanced outward rectification during membrane depolarization. Previous studies have demonstrated that there are at least three types of I_K in striatal MSNs involved in the regulation of membrane excitability (Nisenbaum and Wilson 1995a, b; Nisenbaum et al. 1994; Surmeier et al. 1991, 1992). Among them, the slowly inactivating A current (I_As) and noninactivating K⁺ current (persistent K⁺ current, I_Kper) are activated at the subthreshold levels (–60 –75 mV, respectively) to restrict the amplitude of APs and to dampen repetitive firing. In contrast, the fast-inactivating A current (I_Af) is activated near suprathreshold levels (approximately –40 mV) to assist membrane repolarization. Given that all NAc neurons in the I-V relationship study were initially held at the RMP level of –80 mV and that the outward rectification during membrane depolarization was activated at the subthreshold levels and was significantly enhanced in cocaine-withdrawn NAc neurons, our results indicate that repeated cocaine administration enhanced the voltage-gated I_K in NAc neurons. This finding is consistent with previous studies that have demonstrated that repeated cocaine administration upregulates PKA activity (Nestler 1997; Self and Nestler 1995), while PKA-induced phosphorylation increases not only I_Ks (Hille 2001) but also the open probability of I_A (Koh et al. 1996). Therefore we propose that the enhanced outward rectification in cocaine-pretreated NAc neurons during membrane depolarization is caused most likely by increased I_Ks (I_As and/or I_Kper), but not I_Af because the later is downregulated by PKA-induced phosphorylation (Yuan et al. 2002). Moreover, the deepened AHP observed with blockade of I_A also implies an enhanced I_K(Ca) in cocaine-sensitized NAc neurons. Although the subtype of I_K(Ca) responsible for this enhancement is currently unknown, previous investigations suggest involvement of Ca²⁺-activated BK rather than SK because PKA-induced phosphorylation increases BK but shuts down SK in different types of cells (Bringmann et al. 1997; Köhler et al. 1996).

In addition to the somatic K⁺ channels mentioned in the preceding text, repeated cocaine administration, which eliminates the secondary Ca²⁺ potentials, may also alter the activity of dendritic K⁺ currents because dendritic signal propagation is also modulated by I_A. For instance, activation of a high density of I_A channels prevents initiation of APs, limits backpropagation of APs, and therefore reduces excitatory synaptic events in dendrites (Hoffman et al. 1997). In contrast, downregulation of dendritic I_A increases the amplitude of backpropagating APs (Yuan et al. 2002). Combined with these previous findings, our results, particularly the abolished secondary Ca²⁺ potentials, suggest that repeated cocaine administration not only reduces dendritic Na⁺/Ca²⁺ influx but might also enhance I_A during dendritic membrane depolarization. Taken together, it is likely that chronic cocaine treatment disrupts the function of multiple ion channels critically involved in regulating the backpropagation and forward propagation of APs in NAc neurons.

Repeated cocaine administration decreases membrane excitability of NAc neurons via disruption of normal signal transduction

We have recently demonstrated the remarkable alterations in the activity of membrane ion channels of NAc neurons occurred after repeated cocaine administration (Zhang et al. 1998, 2002; the present findings), however, the exact molecular mechanisms underlying the reduced VSCCs and VSSCs, as well as enhanced K⁺ currents, remain to be thoroughly elucidated. Because the results regarding DA-R proliferation in the NAc and other DA-targeted areas after repeated cocaine administration are inconsistent (Farfel et al. 1992; Kleven et al. 1990; Laurier et al. 1994; Lim et al. 1990; Peris et al. 1990), functional alterations beyond the DA-R have to be considered. In fact, recent investigations have suggested that repeated cocaine administration increases D1R-stimulated activity in cAMP/PKA cascade (Nestler 1997; Self and Nestler 1995), decreases D2R-coupled G_i/G_o protein subunits (Nestler 1997; Nestler et al. 1990; Striplin and Kalivas 1993; Terwilliger et al. 1991), elevates the mRNA levels of D1Rs and D2Rs (Laurier et al. 1994), and attenuates the function of calcineurin but enhances the activity of DARPP-32 (Hu et al. 2003) in NAc neurons. These findings indicate that chronic cocaine-induced alterations in the function of membrane ion channels in NAc neurons are based on the integrated changes in different signaling pathways and gene expression.

In summary, the present study demonstrates that repeated cocaine administration not only suppresses somatic HVA-Ca²⁺ potentials but also enhances the activity of a variety of K⁺ currents [e.g., I_Kir(rest), I_A and I_K(Ca)] in rat NAc MSNs. It also suggests that dendritic propagation of Na⁺/Ca²⁺ potentials is suppressed by repeated cocaine administration. The integrated changes in the membrane ion channel function leads to decreased membrane excitability during cocaine withdrawal (see Fig. 8 for summarization). Given that most addictive substances affect DA neurotransmission in the mesoaccumbens and mesocortical DA system, this study provides a useful model for further investigation of whole cell intrinsic neuroplasticity in addictive drug-induced sensitization and withdrawal effects.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

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

	ACKNOWLEDGMENTS

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The authors acknowledge K. Ford and L. Baker for excellent technical assistance. We also thank Dr. Donald Cooper for helpful comments regarding this manuscript.

	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 reprint requests and other correspondence to: X.-T. Hu (E-mail: Xiu-Ti.Hu{at}rosalindfranklin.edu).

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