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Altered cocaine effects in mice lacking Cav2.3 (α1E) calcium channel
BBRC Biochemical and Biophysical Research Communications 299 (2002) 299–304 www.academicpress.com
Altered cocaine effects in mice lacking Cav2:3 (a1E) calcium channel Wenhua Han, Hironao Saegusa, Shuqin Zong, and Tsutomu Tanabe* Department of Pharmacology and Neurobiology, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan CREST, Japan Science and Technology Corporation, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan Received 7 October 2002
Abstract Much evidence indicates that calcium channel plays a role in cocaine-induced behavioral responses. We assessed the contributions of Cav 2:3 (a1E ) calcium channel to cocaine effects using Cav 2:3 knockout mice (Cav 2:3)/)). Acute administration of cocaine enhanced the locomotor activity in wild-type mice (Cav 2:3+/+), but failed to produce any response in Cav 2:3)/) mice. Repeated exposure to cocaine induced the behavioral sensitization and conditioned place preference in both genotypes. Pretreatment with a D1-receptor antagonist, SCH23390, blocked the cocaine-induced place preference in Cav 2:3+/+ mice; however, it had no significant effect in Cav 2:3)/) mice. Microdialysis and RT-PCR analysis revealed that the levels of extracellular dopamine and dopamine D1 and D2 receptor mRNAs were not altered in Cav 2:3)/) mice. These data indicate that Cav 2:3 channel contributes to the locomotorstimulating effect of cocaine, and the deletion of Cav 2:3 channel reveals the presence of a novel pathway leading to cocaine rewarding which is insensitive to D1 receptor antagonist. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Cav 2:3; Knockout mouse; R-type; Calcium channel; Cocaine; Locomotor activity; Sensitization; D1 receptor; Rewarding; Place preference
Cocaine is an abused psychomotor stimulant that can cause a range of psychiatric and other medical disorders in humans. Absence of effective medications for cocaine abuse has led to intense research efforts on understanding the neural mechanisms of cocaine abuse [1]. Much of recent progress in understanding the mechanisms of cocaine addiction is attributed to the development of animal models of addiction. In mice, acute administration of cocaine induces locomotor hyperactivity. Furthermore, repeated exposure to cocaine leads to behavioral sensitization, which is characterized by a progressive enhancement of locomotor response [2,3]. On the other hand, cocaine also produces rewarding effect. Conditioned place preference (CPP) test provides a technically tractable measure of this property [4,5]. Cocaine elevates the extracellular dopamine in the striatum by blocking the dopamine transporter (DAT). This increase in dopamine has been thought to contribute to cocaine behavioral responses [6–8]. * Corresponding author. Fax: +81-3-5803-0122. E-mail address: [email protected] (T. Tanabe).
Recently, a growing body of evidence indicates that Ca2þ influx through voltage-dependent calcium channels (VDCCs) plays an important role in behavioral effects of cocaine [9,10]. VDCCs are classified into L-, N-, P/Q-, R-, and T-types based on their electrophysiological and pharmacological properties. VDCCs are protein complexes composed of several subunits (a1 , a2 –d, b, and c), among which a1 subunit is the most important for determining the pharmacological and electrophysiological subtypes of VDCCs. Ten different genes encoding a1 subunit have been identified. According to the sequence similarities, these genes are classified into three groups (Cav 1, Cav 2, and Cav 3) [11]. The Cav 2 family includes Cav 2:1, Cav 2:2, and Cav 2:3 (also referred to as a1A , a1B , and a1E , respectively) channels. The Cav 2:3 channel is thought to contribute to the R-type calcium channel [12]. It has been indicated that microinjection of N- and L-type calcium channel antagonists into the nucleus accumbens, a brain region essential for cocaine addiction, attenuated cocaine-induced locomotor activity and behavioral sensitization [9]. The whole-cell recordings revealed that not only L- and N-type channels contrib-
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 6 3 2 - 3
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ute to the Ca2þ current, but also approximately 20% of the Ca2þ current are carried by R-type channel in the nucleus accumbens [13]. Until very recently, absence of specific toxins or drugs makes it difficult to delineate the functional contribution of R-type calcium channel to the cocaine-induced behavioral responses. To assess the role of Cav 2:3 channel, which supports the R-type current, in cocaine addiction, we examined the mice lacking Cav 2:3 channel that we generated previously [14]. By analyzing this knockout mouse, we have reported that Cav 2:3 calcium channel played a role in controlling pain behaviors [14], participated in the formation of accurate spatial memory [15], and played a protective role in ischemic neuronal injury [16]. In the present study, we analyzed locomotor activity and behavioral sensitization induced by cocaine both in Cav 2:3 knockout mice and their wild-type littermates. We also assessed cocaine-induced CPP in both genotypes. In order to elucidate the mechanisms underlying the cocaine behavioral responses in Cav 2:3)/) mice, we examined the effect of D1 receptor antagonist on these behavioral effects. Further, we investigated the extracellular dopamine by microdialysis and measured the expression levels for dopamine D1 and D2 receptor mRNAs by reverse transcription (RT)-PCR. Materials and methods Animals. Cav 2:3+/+ and Cav 2:3)/) mice were obtained as previously described [14]. Drugs. Cocaine–HCl was purchased from Sigma. SCH23390, ketanserin, and sulpiride were purchased from TOCRIS. All drugs except sulpiride were dissolved in saline solution (0.9% NaCl). Sulpiride was dissolved in distilled water with the aid of 0.1 N HCl and then pH was adjusted to 6.5–7.0 with NaHCO3 . Locomotor activity. Locomotor activity was evaluated in a polyvinyl chloride open field chamber (50 50 40 cm). Locomotion pathways were recorded with a video camera and the distances were measured using a software from OÕHara, Japan [15]. To determine the development and the maintenance of the sensitization to cocaine, cocaine (20 mg/kg) was administered once a day, by intraperitoneal (i.p.) injection, for 5 consecutive days. After a 9-day drug free period, cocaine (20 mg/kg) was once again injected on day 15 [17]. To examine the effect of D1 receptor antagonist on cocaine-induced locomotion, SCH23390 (0.05 mg/kg, i.p.) was injected 20 min before each treatment of cocaine. Locomotor activity was recorded on day 1, day 5, and day 15. On the test day, animals were habituated to the test chamber for 60 min before cocaine injection. The spontaneous locomotion was recorded for 3 min in every 10 min. After cocaine injection, locomotor activity was then recorded for another 9 min. Conditioned place preference. Tests were performed in a Plexiglas chamber composed of three distinct compartments [18]. Two larger compartments (15 15 15 cm) were separated by a smaller one (15 15 9 cm). One of the larger compartments had a smooth floor with black walls; the other had a wire mesh floor with white walls. The central area provided access to the larger compartments. The CPP schedule consisted of three phases: (1) preconditioning phase, during which mice were placed in the central area and allowed free access to each compartment. Locations of mice were recorded for 20 min. (2) Conditioning phase, during which mice were trained with saline and cocaine (5 or 20 mg/kg) once a day for 3 days. In details, animals were
restricted to the black compartment for 20 min after injection of saline, removed to their home cages for 4 h, and then placed to the white compartment for another 20 min after the injection of cocaine. To determine the involvement of D1, D2, and serotonin 2A/2C (5HT-2A/ 2C) receptors in cocaine CPP, the antagonists against these receptors, SCH23390 (0.05 mg/kg), sulpiride (50 mg/kg), or ketanserin (1 mg/kg) were administered (i.p.) before every treatment with cocaine (20 mg/kg, i.p.). (3) Testing phase, during which mice were again placed in the central area 24 h after the final conditioning session, with free access to each compartment, for 20 min. The time they spent in the cocainepaired chamber during the testing phase minus that during the preconditioning phase provides a measure of CPP. In vivo microdialysis. Mice were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). Microdialysis probes (with 2 mm active membrane) were implanted vertically into the dorsal striatum (anterior, 0.0; lateral, 1.8 mm; and vertical, 4 mm from the bregma) [19]. After the surgery, animals were allowed a recovery period of 24 h. On the test day, the probe was connected to a syringe pump (Eicom, Japan) and continuously perfused with RingerÕs solution (in mM: 147 NaCl, 4 KCl, and 2.2 CaCl2 ) at a rate of 2 ll/min. After a 60 min equilibration period, samples were collected for 30 min to determine the basal level of dopamine, and then cocaine (20 mg/kg, i.p.) was administered. Samples were collected for another 2 h. All samples were automatically injected to the HTEC-500 microdialysis analysis system (Eicom, Japan) every 6 min using a fully automatic online system. Dopamine was determined by HPLC and electrochemical detection. Chromatographic data were acquired and processed using Powerchrom software (Eicom, Japan). At the end of the experiment, mice were anesthetized and then were perfusion-fixed with 4% formaldehyde. The brains were sectioned at 30-lm thickness in serial coronal slices. The place of the probe was ascertained according to the atlas. Results from mice implanted incorrectly were discarded. RT-PCR amplification of D1 and D2 receptors. Mice were treated with cocaine (20 mg/kg, i.p.) or saline for 5 days and sacrificed 18–24 h after the last injection. Striatal tissues were isolated by gross dissection [18]. Total RNA from the striatum was extracted using Trizol Reagent (Gibco-BRL). The first-strand DNA synthesis was performed with random hexamers and Superscript II (Gibco-BRL). The primer pairs for D1 and D2 receptors were used as described elsewhere [20,21]. The primer pair for b-actin was designed as follows: b-actin-F, 50 -GTG GGC CGC TCT AGG CAC CAA-30 ; b-actin-R, 50 -CTC TTT GAT GTC ACG CAC GAT TTC-30 . The sizes of the PCR products of D1 and D2 receptors and b-actin were 225, 241, and 540 bp, respectively. PCR was performed using serial dilutions of 1:16, 1:32, and 1:64 of the original cDNA. Results from the dilution of 1:64 were used, because at this dilution amplifications of all of the three kinds of cDNA products were not saturated. D1 or D2 receptor cDNA was amplified together with b-actin cDNA in the same reaction tube. PCR products were run on 1.5% agarose gels. The gels were stained with ethidium bromide and the analysis of the band intensity was accomplished using IMAGE QUANT software (Molecular Dynamics). Data were expressed as the ratio of D1 (or D2) receptor to b-actin. Statistical analysis. Data are presented as means SEM. All data were analyzed by two-way ANOVA (genotype and treatment) and StudentÕs t test. Less than 5% level of probability was considered significant. All the experiments were performed in a blind manner.
Results Effects of cocaine on locomotor activity Cav 2:3)/) mice showed a significantly reduced spontaneous locomotor activity when they were first introduced to an open field test chamber, as previously reported
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Fig. 1. Cocaine effects on locomotor activity in Cav 2:3+/+ and Cav 2:3)/) mice. (A) Locomotor activities in the open field of Cav 2:3+/+ (n ¼ 14) and Cav 2:3)/) (n ¼ 13) mice. **, Statistically significant difference between Cav 2:3+/+ and Cav 2:3)/) mice (p < 0:01). (B) Locomotor responses to cocaine on day 1, 5, and 15 in Cav 2:3+/+ (n ¼ 14) and Cav 2:3)/) (n ¼ 13) mice. *p < 0:05 and ***p < 0:01, versus the saline-treated group in the same genotype. #p < 0:05 versus Cav 2:3+/+ mice receiving the same treatment. (C) Effect of SCH23390 on cocaine-induced locomotor responses of Cav 2:3+/+ and Cav 2:3)/) mice. **p < 0:01 and ***p < 0:001 versus the SCH23390 + cocaine group in the same genotype (n ¼ 8). °p < 0:05 versus the day 1 of the SCH23390 + cocaine group in the same genotype.
[14]. After 1 h habituation, the locomotor activities in Cav 2:3)/) and Cav 2:3+/+ mice reached the same level (Fig. 1A). On day 1, cocaine significantly increased locomotor activity in wild-type mice compared with saline (p < 0:001), but it had no effect in Cav 2:3)/) mice. On day 5, locomotor activities of both genotypes after the cocaine administration were markedly enhanced. However the locomotor response to cocaine in Cav 2:3)/) mice was still significantly lower than that in the wild-type group (p < 0:05). On day 15, the locomotor activities of the two genotypes reached the same level (Fig. 1B). It has been demonstrated that D1-receptor plays an important role in the cocaine-induced locomotor response [22], hence we tested the effect of a D1 receptor antagonist, SCH23390. In both genotypes, pretreatment of SCH23390 reduced the cocaine-induced locomotor responses. But SCH23390 did not completely eliminate the development of locomotor sensitization, because the locomotor activities on day 15 were still significantly enhanced compared with those on day 1 (p < 0:05). Furthermore, SCH23390 pretreatment abolished the differences between the wild-type and Cav 2:3)/) mice (Fig. 1C). Cocaine-induced conditioned place preference (cocaine CPP) We tested animals in a conditioned place preference protocol to assess the rewarding properties of cocaine.
During the preconditioning phase, Cav 2:3+/+ and Cav 2:3)/) mice showed no particular preference for one test chamber over the other (data not shown). Like their wild-type littermates, Cav 2:3)/) mice displayed dosedependent increase in the time spent in the chamber paired with cocaine (p < 0:05) (Fig. 2A). No significant difference was observed between the genotypes. To
Fig. 2. Cocaine CPP in Cav 2:3+/+ and Cav 2:3)/) mice. (A) Scores are shown as the differences (in seconds) between post- and pre-conditioning time spent in the compartment associated with cocaine. Cocaine (5 and 20 mg/kg) dose-dependently increased the time spent in the drug-paired compartment in Cav 2:3+/+ (n ¼ 7–9) and Cav 2:3)/) (n ¼ 9) mice. *p < 0:05, **p < 0:01, and ***p < 0:001 versus the saline-treated mice in the same genotype. (B) Effects of SCH23390, sulpiride, and ketanserin on cocaine CPP (n ¼ 8). *p < 0:05 versus cocaine (20 mg/kg)-treated wild-type mice.
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Cocaine-induced dopamine release Numerous studies have reported that cocaine can increase the dopamine levels in the striatum, and the behavioral responses to cocaine are closely correlated with the increased dopamine levels [7,19,23]. We investigated the effect of cocaine on striatal dopamine release in freely moving mice of both genotypes by means of in vivo microdialysis. Wild-type and mutant mice showed similar basal dopamine levels (10:3 0:15 and 10:4 0:06 pg/sample, respectively). Cocaine increased striatal dopamine levels in both genotypes, and the maximal increase of dopamine elicited by cocaine in Cav 2:3)/) mice did not differ significantly from that in wild-type mice (Fig. 3). Fig. 3. Cocaine-induced dopamine release in the striatum of Cav 2:3+/+ and Cav 2:3)/) mice by in vivo microdialysis. Samples were collected every 6 min from Cav 2:3+/+ (n ¼ 5) and Cav 2:3)/) (n ¼ 6) mice. *p < 0:01, **p < 0:05, and ***p < 0:001 versus the basal values of the same genotype.
search for possible differences of mechanisms underlying cocaine CPP in Cav 2:3)/) mice, we examined the effects of antagonists against dopamine D1 and D2-receptors and 5HT-2A/2C receptors. In wild-type mice, pretreatment with SCH23390 almost completely inhibited cocaine CPP, as the mice receiving both SCH23390 and cocaine spent significantly less time on cocaine-paired side than mice given cocaine alone (p < 0:05). But in Cav 2:3)/) mutant mice, SCH23390 did not significantly affect the cocaine CPP. Sulpiride, a D2 receptor antagonist, and ketanserin, a 5HT-2A/2C receptors antagonist, did not significantly affect cocaine CPP in both genotypes (Fig. 2B).
D1 and D2 receptor mRNA levels in the striatum Next we examined the D1 and D2 receptor mRNA levels in the striatum of saline and cocaine-treated mice using semi-quantitative RT-PCR. The relative amounts of D1 and D2 receptor mRNA were calculated as a ratio to b-actin mRNA levels. Comparisons of the levels of D1and D2 receptor mRNAs between Cav 2:3+/+ and Cav 2:3)/) mice after the 5 day treatment of saline or cocaine showed no significant difference (Fig. 4).
Discussion We have shown that Cav 2:3 mutant mice exhibited no response to acute cocaine administration. This absence of acute response in Cav 2:3)/) mice is not due to a nonspecific locomotor deficit, because the locomotor
Fig. 4. RT-PCR analysis of D1 and D2 receptor mRNA expressions in the striatum. Representative data on D1 (A) and D2 (B) are shown. Sal, treated with saline; Coc, treated with cocaine. Gels stained with ethidium bromide were scanned with FluorImager 595 (Molecular Dynamics). (C,D) Fluorescence intensities were analyzed with IMAGE QUANT software. No significant differences were observed between wild-type and Cav 2:3)/) mice (n ¼ 5).
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activities in response to repeated cocaine exposure in the Cav 2:3)/) mice finally amounted to the same level as those in Cav 2:3+/+ mice. Cocaine increased the extracellular dopamine in the striatum by inhibiting DAT. The increased dopamine is thought to evoke the cocaine-induced locomotor hyperactivity [7,19]. Thus, the dopamine level in the striatum may be lowered in Cav 2:3)/) mice. To test this possibility, we measured the dopamine release in the striatum by in vivo microdialysis study. We found, however, that the basal dopamine level, as well as the level of cocaine-induced increase of extracellular dopamine, was not significantly different between Cav 2:3+/+ and Cav 2:3)/) mice. These data clearly indicate that the enhanced dopamine release in the striatum dose not necessarily cause the increase of locomotor activity. These data also indicate that Cav 2:3 channel is not significantly involved in controlling dopamine release in the striatum. On the other hand, the sensitization caused by chronic cocaine administration in the Cav 2:3)/) mice was still preserved, even though the locomotor response to acute cocaine was abolished. It has been demonstrated that D1 receptor antagonist attenuates the increase in locomotor activity evoked by cocaine, without preventing the development of locomotor sensitization to cocaine in rats [24]. Our results in the wild-type mice are consistent with this study: SCH23390, a D1 receptor antagonist, blocked not only the hyperactivity induced by acute cocaine injection, but also the sensitization induced by chronic cocaine administration. SCH23390 inhibited the cocaine-induced sensitization in the Cav 2:3)/) mice as well, reducing the locomotor activities of the two genotypes to the same level. However, the inhibition of locomotor sensitization by SCH23390 was not complete, there still remained a component of sensitization that was not blocked by SCH23390. These data suggest that the locomotor sensitization to cocaine in both genotypes is attributed to both D1-dependent and D1-independent mechanisms. Because SCH23390 abolished the difference between the two genotypes, D1-dependent mechanism might be involved in the reduced locomotor response to cocaine in the Cav 2:3)/) mice. A possible explanation for the absence of acute response and the preservation of locomotor sensitization to cocaine in Cav 2:3)/) mice might be as follows: cocaine-induced increase of dopamine in the striatum, which is intact in Cav 2:3)/) mice, is the initial step for the locomotor hyperactivity. However, the downstream system after dopamine releases might be defective in Cav 2:3)/) mice and this defect results in the absence of acute locomotor response to cocaine in Cav 2:3)/) mice. On the other hand, repeated exposure to cocaine may overcome the defect, possibly by the up or down regulation of certain gene(s) expression in the downstream system and eliminate the differences of the efferent sys-
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tems between the two genotypes. Thus the sensitization to locomotor response was observed in Cav 2:3)/) mice. We further used CPP to assess the rewarding properties of cocaine in Cav 2:3)/) mice. The CPP procedure provides a measure of detecting the rewarding effects of a number of drugs [25]. Our results showed that Cav 2:3)/) mice exhibited similar cocaine CPP as wildtype mice. It has been considered that mesolimbic dopaminergic system mediates the rewarding property of cocaine [26]. D1 receptor antagonist, SCH23390, completely blocked the establishment of cocaine CPP in Cav 2:3+/+ mice, as observed in rats [27]. However, SCH23390 did not inhibit the cocaine CPP in Cav 2:3)/) mice significantly. The inability of D1 receptor antagonist to block the cocaine CPP in Cav 2:3)/) mice can not be explained by the altered expressions of dopamine receptors, because the expression levels of D1 and D2 receptor mRNAs were not changed in Cav 2:3)/) mice. Involvement of serotonin pathways in cocaine CPP has been suggested recently [28]. Among the 14 kinds of serotonin receptors, 5HT-2A/2C receptors are expressed in the striatum and are thought to play a role in mediating cocaine-induced behavioral responses [29]. Therefore we examined the effect of a 5HT-2A/2C receptor antagonist in cocaine CPP in both genotypes. We also tested the effect of dopamine D2 receptor antagonist, because D2 receptor is thought to play a role in opiate rewarding effects [30]. However, both D2 and 5HT-2A/ 2C receptor antagonists had no effects on the establishment of cocaine CPP. These results indicate the presence of other unknown pathways responsible for cocaine rewarding in Cav 2:3)/) mice. Further rigorous studies are necessary to delineate the mechanism underlying cocaine CPP in Cav 2:3)/) mice. In conclusion, we have demonstrated that Cav 2:3 channel is involved in cocaine-induced acute hyperactivity. However, it is not essential for the acquisition of the sensitization to locomotor hyperactivity. Deletion of Cav 2:3 calcium channel results in the appearance of novel pathways leading to cocaine rewarding responses which are not sensitive to D1 receptor antagonist. Clarifying this pathway may be useful and/or important for the treatment of patients suffering from cocaine addiction.
Acknowledgments We thank Dr. Takashi Kurihara for advice and encouragement throughout the work; Mutsumi Kondoh and Junyang Wang for technical assistance.
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Altered cocaine effects in mice lacking Cav2:3 (a1E) calcium channel Wenhua Han, Hironao Saegusa, Shuqin Zong, and Tsutomu Tanabe* Department of Pharmacology and Neurobiology, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan CREST, Japan Science and Technology Corporation, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan Received 7 October 2002
Abstract Much evidence indicates that calcium channel plays a role in cocaine-induced behavioral responses. We assessed the contributions of Cav 2:3 (a1E ) calcium channel to cocaine effects using Cav 2:3 knockout mice (Cav 2:3)/)). Acute administration of cocaine enhanced the locomotor activity in wild-type mice (Cav 2:3+/+), but failed to produce any response in Cav 2:3)/) mice. Repeated exposure to cocaine induced the behavioral sensitization and conditioned place preference in both genotypes. Pretreatment with a D1-receptor antagonist, SCH23390, blocked the cocaine-induced place preference in Cav 2:3+/+ mice; however, it had no significant effect in Cav 2:3)/) mice. Microdialysis and RT-PCR analysis revealed that the levels of extracellular dopamine and dopamine D1 and D2 receptor mRNAs were not altered in Cav 2:3)/) mice. These data indicate that Cav 2:3 channel contributes to the locomotorstimulating effect of cocaine, and the deletion of Cav 2:3 channel reveals the presence of a novel pathway leading to cocaine rewarding which is insensitive to D1 receptor antagonist. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Cav 2:3; Knockout mouse; R-type; Calcium channel; Cocaine; Locomotor activity; Sensitization; D1 receptor; Rewarding; Place preference
Cocaine is an abused psychomotor stimulant that can cause a range of psychiatric and other medical disorders in humans. Absence of effective medications for cocaine abuse has led to intense research efforts on understanding the neural mechanisms of cocaine abuse [1]. Much of recent progress in understanding the mechanisms of cocaine addiction is attributed to the development of animal models of addiction. In mice, acute administration of cocaine induces locomotor hyperactivity. Furthermore, repeated exposure to cocaine leads to behavioral sensitization, which is characterized by a progressive enhancement of locomotor response [2,3]. On the other hand, cocaine also produces rewarding effect. Conditioned place preference (CPP) test provides a technically tractable measure of this property [4,5]. Cocaine elevates the extracellular dopamine in the striatum by blocking the dopamine transporter (DAT). This increase in dopamine has been thought to contribute to cocaine behavioral responses [6–8]. * Corresponding author. Fax: +81-3-5803-0122. E-mail address: [email protected] (T. Tanabe).
Recently, a growing body of evidence indicates that Ca2þ influx through voltage-dependent calcium channels (VDCCs) plays an important role in behavioral effects of cocaine [9,10]. VDCCs are classified into L-, N-, P/Q-, R-, and T-types based on their electrophysiological and pharmacological properties. VDCCs are protein complexes composed of several subunits (a1 , a2 –d, b, and c), among which a1 subunit is the most important for determining the pharmacological and electrophysiological subtypes of VDCCs. Ten different genes encoding a1 subunit have been identified. According to the sequence similarities, these genes are classified into three groups (Cav 1, Cav 2, and Cav 3) [11]. The Cav 2 family includes Cav 2:1, Cav 2:2, and Cav 2:3 (also referred to as a1A , a1B , and a1E , respectively) channels. The Cav 2:3 channel is thought to contribute to the R-type calcium channel [12]. It has been indicated that microinjection of N- and L-type calcium channel antagonists into the nucleus accumbens, a brain region essential for cocaine addiction, attenuated cocaine-induced locomotor activity and behavioral sensitization [9]. The whole-cell recordings revealed that not only L- and N-type channels contrib-
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ute to the Ca2þ current, but also approximately 20% of the Ca2þ current are carried by R-type channel in the nucleus accumbens [13]. Until very recently, absence of specific toxins or drugs makes it difficult to delineate the functional contribution of R-type calcium channel to the cocaine-induced behavioral responses. To assess the role of Cav 2:3 channel, which supports the R-type current, in cocaine addiction, we examined the mice lacking Cav 2:3 channel that we generated previously [14]. By analyzing this knockout mouse, we have reported that Cav 2:3 calcium channel played a role in controlling pain behaviors [14], participated in the formation of accurate spatial memory [15], and played a protective role in ischemic neuronal injury [16]. In the present study, we analyzed locomotor activity and behavioral sensitization induced by cocaine both in Cav 2:3 knockout mice and their wild-type littermates. We also assessed cocaine-induced CPP in both genotypes. In order to elucidate the mechanisms underlying the cocaine behavioral responses in Cav 2:3)/) mice, we examined the effect of D1 receptor antagonist on these behavioral effects. Further, we investigated the extracellular dopamine by microdialysis and measured the expression levels for dopamine D1 and D2 receptor mRNAs by reverse transcription (RT)-PCR. Materials and methods Animals. Cav 2:3+/+ and Cav 2:3)/) mice were obtained as previously described [14]. Drugs. Cocaine–HCl was purchased from Sigma. SCH23390, ketanserin, and sulpiride were purchased from TOCRIS. All drugs except sulpiride were dissolved in saline solution (0.9% NaCl). Sulpiride was dissolved in distilled water with the aid of 0.1 N HCl and then pH was adjusted to 6.5–7.0 with NaHCO3 . Locomotor activity. Locomotor activity was evaluated in a polyvinyl chloride open field chamber (50 50 40 cm). Locomotion pathways were recorded with a video camera and the distances were measured using a software from OÕHara, Japan [15]. To determine the development and the maintenance of the sensitization to cocaine, cocaine (20 mg/kg) was administered once a day, by intraperitoneal (i.p.) injection, for 5 consecutive days. After a 9-day drug free period, cocaine (20 mg/kg) was once again injected on day 15 [17]. To examine the effect of D1 receptor antagonist on cocaine-induced locomotion, SCH23390 (0.05 mg/kg, i.p.) was injected 20 min before each treatment of cocaine. Locomotor activity was recorded on day 1, day 5, and day 15. On the test day, animals were habituated to the test chamber for 60 min before cocaine injection. The spontaneous locomotion was recorded for 3 min in every 10 min. After cocaine injection, locomotor activity was then recorded for another 9 min. Conditioned place preference. Tests were performed in a Plexiglas chamber composed of three distinct compartments [18]. Two larger compartments (15 15 15 cm) were separated by a smaller one (15 15 9 cm). One of the larger compartments had a smooth floor with black walls; the other had a wire mesh floor with white walls. The central area provided access to the larger compartments. The CPP schedule consisted of three phases: (1) preconditioning phase, during which mice were placed in the central area and allowed free access to each compartment. Locations of mice were recorded for 20 min. (2) Conditioning phase, during which mice were trained with saline and cocaine (5 or 20 mg/kg) once a day for 3 days. In details, animals were
restricted to the black compartment for 20 min after injection of saline, removed to their home cages for 4 h, and then placed to the white compartment for another 20 min after the injection of cocaine. To determine the involvement of D1, D2, and serotonin 2A/2C (5HT-2A/ 2C) receptors in cocaine CPP, the antagonists against these receptors, SCH23390 (0.05 mg/kg), sulpiride (50 mg/kg), or ketanserin (1 mg/kg) were administered (i.p.) before every treatment with cocaine (20 mg/kg, i.p.). (3) Testing phase, during which mice were again placed in the central area 24 h after the final conditioning session, with free access to each compartment, for 20 min. The time they spent in the cocainepaired chamber during the testing phase minus that during the preconditioning phase provides a measure of CPP. In vivo microdialysis. Mice were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). Microdialysis probes (with 2 mm active membrane) were implanted vertically into the dorsal striatum (anterior, 0.0; lateral, 1.8 mm; and vertical, 4 mm from the bregma) [19]. After the surgery, animals were allowed a recovery period of 24 h. On the test day, the probe was connected to a syringe pump (Eicom, Japan) and continuously perfused with RingerÕs solution (in mM: 147 NaCl, 4 KCl, and 2.2 CaCl2 ) at a rate of 2 ll/min. After a 60 min equilibration period, samples were collected for 30 min to determine the basal level of dopamine, and then cocaine (20 mg/kg, i.p.) was administered. Samples were collected for another 2 h. All samples were automatically injected to the HTEC-500 microdialysis analysis system (Eicom, Japan) every 6 min using a fully automatic online system. Dopamine was determined by HPLC and electrochemical detection. Chromatographic data were acquired and processed using Powerchrom software (Eicom, Japan). At the end of the experiment, mice were anesthetized and then were perfusion-fixed with 4% formaldehyde. The brains were sectioned at 30-lm thickness in serial coronal slices. The place of the probe was ascertained according to the atlas. Results from mice implanted incorrectly were discarded. RT-PCR amplification of D1 and D2 receptors. Mice were treated with cocaine (20 mg/kg, i.p.) or saline for 5 days and sacrificed 18–24 h after the last injection. Striatal tissues were isolated by gross dissection [18]. Total RNA from the striatum was extracted using Trizol Reagent (Gibco-BRL). The first-strand DNA synthesis was performed with random hexamers and Superscript II (Gibco-BRL). The primer pairs for D1 and D2 receptors were used as described elsewhere [20,21]. The primer pair for b-actin was designed as follows: b-actin-F, 50 -GTG GGC CGC TCT AGG CAC CAA-30 ; b-actin-R, 50 -CTC TTT GAT GTC ACG CAC GAT TTC-30 . The sizes of the PCR products of D1 and D2 receptors and b-actin were 225, 241, and 540 bp, respectively. PCR was performed using serial dilutions of 1:16, 1:32, and 1:64 of the original cDNA. Results from the dilution of 1:64 were used, because at this dilution amplifications of all of the three kinds of cDNA products were not saturated. D1 or D2 receptor cDNA was amplified together with b-actin cDNA in the same reaction tube. PCR products were run on 1.5% agarose gels. The gels were stained with ethidium bromide and the analysis of the band intensity was accomplished using IMAGE QUANT software (Molecular Dynamics). Data were expressed as the ratio of D1 (or D2) receptor to b-actin. Statistical analysis. Data are presented as means SEM. All data were analyzed by two-way ANOVA (genotype and treatment) and StudentÕs t test. Less than 5% level of probability was considered significant. All the experiments were performed in a blind manner.
Results Effects of cocaine on locomotor activity Cav 2:3)/) mice showed a significantly reduced spontaneous locomotor activity when they were first introduced to an open field test chamber, as previously reported
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Fig. 1. Cocaine effects on locomotor activity in Cav 2:3+/+ and Cav 2:3)/) mice. (A) Locomotor activities in the open field of Cav 2:3+/+ (n ¼ 14) and Cav 2:3)/) (n ¼ 13) mice. **, Statistically significant difference between Cav 2:3+/+ and Cav 2:3)/) mice (p < 0:01). (B) Locomotor responses to cocaine on day 1, 5, and 15 in Cav 2:3+/+ (n ¼ 14) and Cav 2:3)/) (n ¼ 13) mice. *p < 0:05 and ***p < 0:01, versus the saline-treated group in the same genotype. #p < 0:05 versus Cav 2:3+/+ mice receiving the same treatment. (C) Effect of SCH23390 on cocaine-induced locomotor responses of Cav 2:3+/+ and Cav 2:3)/) mice. **p < 0:01 and ***p < 0:001 versus the SCH23390 + cocaine group in the same genotype (n ¼ 8). °p < 0:05 versus the day 1 of the SCH23390 + cocaine group in the same genotype.
[14]. After 1 h habituation, the locomotor activities in Cav 2:3)/) and Cav 2:3+/+ mice reached the same level (Fig. 1A). On day 1, cocaine significantly increased locomotor activity in wild-type mice compared with saline (p < 0:001), but it had no effect in Cav 2:3)/) mice. On day 5, locomotor activities of both genotypes after the cocaine administration were markedly enhanced. However the locomotor response to cocaine in Cav 2:3)/) mice was still significantly lower than that in the wild-type group (p < 0:05). On day 15, the locomotor activities of the two genotypes reached the same level (Fig. 1B). It has been demonstrated that D1-receptor plays an important role in the cocaine-induced locomotor response [22], hence we tested the effect of a D1 receptor antagonist, SCH23390. In both genotypes, pretreatment of SCH23390 reduced the cocaine-induced locomotor responses. But SCH23390 did not completely eliminate the development of locomotor sensitization, because the locomotor activities on day 15 were still significantly enhanced compared with those on day 1 (p < 0:05). Furthermore, SCH23390 pretreatment abolished the differences between the wild-type and Cav 2:3)/) mice (Fig. 1C). Cocaine-induced conditioned place preference (cocaine CPP) We tested animals in a conditioned place preference protocol to assess the rewarding properties of cocaine.
During the preconditioning phase, Cav 2:3+/+ and Cav 2:3)/) mice showed no particular preference for one test chamber over the other (data not shown). Like their wild-type littermates, Cav 2:3)/) mice displayed dosedependent increase in the time spent in the chamber paired with cocaine (p < 0:05) (Fig. 2A). No significant difference was observed between the genotypes. To
Fig. 2. Cocaine CPP in Cav 2:3+/+ and Cav 2:3)/) mice. (A) Scores are shown as the differences (in seconds) between post- and pre-conditioning time spent in the compartment associated with cocaine. Cocaine (5 and 20 mg/kg) dose-dependently increased the time spent in the drug-paired compartment in Cav 2:3+/+ (n ¼ 7–9) and Cav 2:3)/) (n ¼ 9) mice. *p < 0:05, **p < 0:01, and ***p < 0:001 versus the saline-treated mice in the same genotype. (B) Effects of SCH23390, sulpiride, and ketanserin on cocaine CPP (n ¼ 8). *p < 0:05 versus cocaine (20 mg/kg)-treated wild-type mice.
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Cocaine-induced dopamine release Numerous studies have reported that cocaine can increase the dopamine levels in the striatum, and the behavioral responses to cocaine are closely correlated with the increased dopamine levels [7,19,23]. We investigated the effect of cocaine on striatal dopamine release in freely moving mice of both genotypes by means of in vivo microdialysis. Wild-type and mutant mice showed similar basal dopamine levels (10:3 0:15 and 10:4 0:06 pg/sample, respectively). Cocaine increased striatal dopamine levels in both genotypes, and the maximal increase of dopamine elicited by cocaine in Cav 2:3)/) mice did not differ significantly from that in wild-type mice (Fig. 3). Fig. 3. Cocaine-induced dopamine release in the striatum of Cav 2:3+/+ and Cav 2:3)/) mice by in vivo microdialysis. Samples were collected every 6 min from Cav 2:3+/+ (n ¼ 5) and Cav 2:3)/) (n ¼ 6) mice. *p < 0:01, **p < 0:05, and ***p < 0:001 versus the basal values of the same genotype.
search for possible differences of mechanisms underlying cocaine CPP in Cav 2:3)/) mice, we examined the effects of antagonists against dopamine D1 and D2-receptors and 5HT-2A/2C receptors. In wild-type mice, pretreatment with SCH23390 almost completely inhibited cocaine CPP, as the mice receiving both SCH23390 and cocaine spent significantly less time on cocaine-paired side than mice given cocaine alone (p < 0:05). But in Cav 2:3)/) mutant mice, SCH23390 did not significantly affect the cocaine CPP. Sulpiride, a D2 receptor antagonist, and ketanserin, a 5HT-2A/2C receptors antagonist, did not significantly affect cocaine CPP in both genotypes (Fig. 2B).
D1 and D2 receptor mRNA levels in the striatum Next we examined the D1 and D2 receptor mRNA levels in the striatum of saline and cocaine-treated mice using semi-quantitative RT-PCR. The relative amounts of D1 and D2 receptor mRNA were calculated as a ratio to b-actin mRNA levels. Comparisons of the levels of D1and D2 receptor mRNAs between Cav 2:3+/+ and Cav 2:3)/) mice after the 5 day treatment of saline or cocaine showed no significant difference (Fig. 4).
Discussion We have shown that Cav 2:3 mutant mice exhibited no response to acute cocaine administration. This absence of acute response in Cav 2:3)/) mice is not due to a nonspecific locomotor deficit, because the locomotor
Fig. 4. RT-PCR analysis of D1 and D2 receptor mRNA expressions in the striatum. Representative data on D1 (A) and D2 (B) are shown. Sal, treated with saline; Coc, treated with cocaine. Gels stained with ethidium bromide were scanned with FluorImager 595 (Molecular Dynamics). (C,D) Fluorescence intensities were analyzed with IMAGE QUANT software. No significant differences were observed between wild-type and Cav 2:3)/) mice (n ¼ 5).
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activities in response to repeated cocaine exposure in the Cav 2:3)/) mice finally amounted to the same level as those in Cav 2:3+/+ mice. Cocaine increased the extracellular dopamine in the striatum by inhibiting DAT. The increased dopamine is thought to evoke the cocaine-induced locomotor hyperactivity [7,19]. Thus, the dopamine level in the striatum may be lowered in Cav 2:3)/) mice. To test this possibility, we measured the dopamine release in the striatum by in vivo microdialysis study. We found, however, that the basal dopamine level, as well as the level of cocaine-induced increase of extracellular dopamine, was not significantly different between Cav 2:3+/+ and Cav 2:3)/) mice. These data clearly indicate that the enhanced dopamine release in the striatum dose not necessarily cause the increase of locomotor activity. These data also indicate that Cav 2:3 channel is not significantly involved in controlling dopamine release in the striatum. On the other hand, the sensitization caused by chronic cocaine administration in the Cav 2:3)/) mice was still preserved, even though the locomotor response to acute cocaine was abolished. It has been demonstrated that D1 receptor antagonist attenuates the increase in locomotor activity evoked by cocaine, without preventing the development of locomotor sensitization to cocaine in rats [24]. Our results in the wild-type mice are consistent with this study: SCH23390, a D1 receptor antagonist, blocked not only the hyperactivity induced by acute cocaine injection, but also the sensitization induced by chronic cocaine administration. SCH23390 inhibited the cocaine-induced sensitization in the Cav 2:3)/) mice as well, reducing the locomotor activities of the two genotypes to the same level. However, the inhibition of locomotor sensitization by SCH23390 was not complete, there still remained a component of sensitization that was not blocked by SCH23390. These data suggest that the locomotor sensitization to cocaine in both genotypes is attributed to both D1-dependent and D1-independent mechanisms. Because SCH23390 abolished the difference between the two genotypes, D1-dependent mechanism might be involved in the reduced locomotor response to cocaine in the Cav 2:3)/) mice. A possible explanation for the absence of acute response and the preservation of locomotor sensitization to cocaine in Cav 2:3)/) mice might be as follows: cocaine-induced increase of dopamine in the striatum, which is intact in Cav 2:3)/) mice, is the initial step for the locomotor hyperactivity. However, the downstream system after dopamine releases might be defective in Cav 2:3)/) mice and this defect results in the absence of acute locomotor response to cocaine in Cav 2:3)/) mice. On the other hand, repeated exposure to cocaine may overcome the defect, possibly by the up or down regulation of certain gene(s) expression in the downstream system and eliminate the differences of the efferent sys-
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tems between the two genotypes. Thus the sensitization to locomotor response was observed in Cav 2:3)/) mice. We further used CPP to assess the rewarding properties of cocaine in Cav 2:3)/) mice. The CPP procedure provides a measure of detecting the rewarding effects of a number of drugs [25]. Our results showed that Cav 2:3)/) mice exhibited similar cocaine CPP as wildtype mice. It has been considered that mesolimbic dopaminergic system mediates the rewarding property of cocaine [26]. D1 receptor antagonist, SCH23390, completely blocked the establishment of cocaine CPP in Cav 2:3+/+ mice, as observed in rats [27]. However, SCH23390 did not inhibit the cocaine CPP in Cav 2:3)/) mice significantly. The inability of D1 receptor antagonist to block the cocaine CPP in Cav 2:3)/) mice can not be explained by the altered expressions of dopamine receptors, because the expression levels of D1 and D2 receptor mRNAs were not changed in Cav 2:3)/) mice. Involvement of serotonin pathways in cocaine CPP has been suggested recently [28]. Among the 14 kinds of serotonin receptors, 5HT-2A/2C receptors are expressed in the striatum and are thought to play a role in mediating cocaine-induced behavioral responses [29]. Therefore we examined the effect of a 5HT-2A/2C receptor antagonist in cocaine CPP in both genotypes. We also tested the effect of dopamine D2 receptor antagonist, because D2 receptor is thought to play a role in opiate rewarding effects [30]. However, both D2 and 5HT-2A/ 2C receptor antagonists had no effects on the establishment of cocaine CPP. These results indicate the presence of other unknown pathways responsible for cocaine rewarding in Cav 2:3)/) mice. Further rigorous studies are necessary to delineate the mechanism underlying cocaine CPP in Cav 2:3)/) mice. In conclusion, we have demonstrated that Cav 2:3 channel is involved in cocaine-induced acute hyperactivity. However, it is not essential for the acquisition of the sensitization to locomotor hyperactivity. Deletion of Cav 2:3 calcium channel results in the appearance of novel pathways leading to cocaine rewarding responses which are not sensitive to D1 receptor antagonist. Clarifying this pathway may be useful and/or important for the treatment of patients suffering from cocaine addiction.
Acknowledgments We thank Dr. Takashi Kurihara for advice and encouragement throughout the work; Mutsumi Kondoh and Junyang Wang for technical assistance.
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