Psychopharmacology (2002) 159:407–414 DOI 10.1007/s00213-001-0936-7
O R I G I N A L I N V E S T I G AT I O N
Patrick E. Karper · Herminia De La Rosa Eva R. Newman · Catherine M. Krall · Arbi Nazarian Sanders A. McDougall · Cynthia A. Crawford
Role of D1-like receptors in amphetamine-induced behavioral sensitization: a study using D1A receptor knockout mice Received: 30 April 2001 / Accepted: 10 September 2001 / Published online: 20 November 2001 © Springer-Verlag 2001
Abstract Rationale: The role played by D1-like receptors in amphetamine-induced behavioral sensitization has been examined using both the D1-like receptor antagonist, SCH 23390, and the D1A receptor knockout mouse (i.e. D1A-deficient mice). Studies using these two approaches have provided conflicting evidence about the importance of D1-like receptors for amphetamineinduced behavioral sensitization. Objective: The purpose of the present study was to determine: (a) whether D1A-deficient mice exhibit amphetamine-induced locomotor sensitization after 3 and 17 drug abstinence days, and (b) whether SCH 23390, which binds to both D1A and D1B receptor subtypes, blocks development of amphetamine sensitization in wild-type and D1A-deficient mice. Methods: In the first experiment, adult wild-type and D1A-deficient mice were injected with amphetamine (0, 1, 2, 4, or 8 mg/kg, IP) for 7 consecutive days. In the second experiment, wild-type and D1A-deficient mice were pretreated with SCH 23390 (0, 0.15, or 0.5 mg/kg, IP) 30 min prior to being injected with amphetamine (0 or 8 mg/kg, IP). After each daily amphetamine injection, mice were placed in activity chambers where distance traveled (i.e. horizontal locomotor activity) was measured for 60 min. On the test days, which occurred after 3 or 17 drug abstinence days, mice were injected with 1 mg/kg amphetamine and locomotion was measured for 120 min. Results: Both wild-type and D1A-deficient mice exhibited amphetamine-induced locomotor sensitization. Pretreatment with 0.5 mg/kg SCH 23390 P.E. Karper · H. De La Rosa · E.R. Newman · C.M. Krall A. Nazarian · S.A. McDougall · C.A. Crawford (✉) Department of Psychology, California State University, San Bernardino, CA 92407, USA e-mail:
[email protected] Fax: +1-909-880-7003 Present addresses: P.E. Karper, Department of Psychology, 125 Nightingale Hall, Northeastern University, Boston, MA 02115, USA A. Nazarian, Department of Psychology, Hunter College of the City University of New York, 695 Park Avenue, New York, NY 10021, USA
blocked the development of locomotor sensitization in wild-type mice, but did not alter the sensitized responding of D1A-deficient mice. Conclusions: It appears that D1-like receptors are necessary for the development of amphetamine sensitization in wild-type mice, while neither the D1A nor D1B receptor subtypes are necessary for the amphetamine-induced locomotor sensitization of D1A-deficient mice. A possible explanation for these conflicting results is that D1A-deficient mice may have a compensatory mechanism (not involving D1B receptors) that allows them to exhibit amphetamine-induced behavioral sensitization in the absence of the D1A receptor. Keywords Behavioral sensitization · D1A receptor knockout mice · Amphetamine · SCH 23390
Introduction Repeated exposure to various indirect dopamine agonist drugs (e.g. amphetamine, methamphetamine, cocaine, and methylphenidate) results in an augmented locomotor response called behavioral sensitization (Robinson and Becker 1986; Kalivas and Stewart 1991). Although the neural mechanisms responsible for behavioral sensitization have not been fully determined, many researchers have postulated that the D1-like family of receptors, which includes the D1A and D1B receptor subtypes, is important for psychostimulant-induced behavioral sensitization (Henry and White 1991, 1995; Kalivas 1995; Bjijou et al. 1996; Vezina 1996). As examples, Vezina (1996) suggests that stimulation of D1-like receptors in the ventral tegmental area is required for the development of behavioral sensitization; whereas Henry and White (1991, 1995) have postulated that a supersensitivity of D1-like receptors in the nucleus accumbens is involved in the long-term persistence and expression of the sensitized response. Even though D1-like receptors are often credited with playing a prominent role in behavioral sensitization, data linking D1-like receptor functioning to sensitization are
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complex. For example, the necessity of having functional D1-like receptors appears to vary depending on the type of psychostimulant administered. More specifically, there is abundant evidence showing that the D1-like receptor antagonist, SCH 23390, blocks both the development and expression of amphetamine- and methamphetamine-induced behavioral sensitization (Vezina and Stewart 1989; Hamamura et al. 1991; Kuribara and Uchihashi 1994; Kuribara 1995a; Vezina 1996). In contrast, D1-like receptor antagonism blocks the expression, but not the development of cocaine-induced behavioral sensitization (Kuribara and Uchihashi 1993; McCreary and Marsden 1993; Mattingly et al. 1994; White et al. 1998). Thus, pharmacological manipulation of D1-like receptors has produced an unexpected pattern of results, because the development of amphetamine-, but not cocaine-, induced behavioral sensitization is blocked by SCH 23390. In addition to these receptor antagonist studies, the role of D1-like receptors in behavioral sensitization has been examined using D1A receptor knockout mice. These knockout mice (also called D1A-deficient mice) completely lack the D1A receptor subtype, while having a full complement of D1B (homologous with D5 receptors in humans) and D2-like receptors (Drago et al. 1994; Montague et al. 2001). Not surprisingly, repeated treatment with cocaine does not produce locomotor sensitization in D1A-deficient mice (Xu et al. 2000), presumably because D1A receptors are necessary for the expression of the sensitized response. The latter interpretation is consistent with receptor antagonist studies showing that SCH 23390 blocks the expression, but not the development, of cocaine-induced behavioral sensitization (Kuribara and Uchihashi 1993; McCreary and Marsden 1993; Mattingly et al. 1994; White et al. 1998). In contrast, D1A-deficient mice pretreated with amphetamine show an augmented locomotor response when given a challenge injection of amphetamine after 4 drug abstinence days (Crawford et al. 1997; see also Xu et al. 2000). The ability of D1A-deficient mice to exhibit amphetamine-induced locomotor sensitization is surprising, considering the number of studies which report that SCH 23390 blocks the development and expression of amphetamine- and methamphetamine-induced behavioral sensitization (Vezina and Stewart 1989; Hamamura et al. 1991; Kuribara and Uchihashi 1994; Kuribara 1995a; Vezina 1996). In summary, evidence gained using receptor antagonist and homologous recombination research strategies supports different conclusions, with only the antagonist (i.e. SCH 23390) studies indicating that the D1-like receptor is necessary for amphetamine sensitization. The purpose of the present study was to assess more fully the importance of the D1-like receptor for amphetamine-induced locomotor sensitization. In the initial experiment, different groups of wild-type and D1A-deficient mice received daily pretreatment injections of amphetamine (1–8 mg/kg), with locomotor sensitization being assessed after 3 and 17 drug abstinence days. In a
subsequent experiment, wild-type and D1A-deficient mice were pretreated with both amphetamine and SCH 23390 (0.15 or 0.5 mg/kg) and locomotor responding to a challenge injection of amphetamine was assessed after 3 drug abstinence days.
Materials and methods Subjects Subjects were 177 (n=6–10 per group) F2 generation wild-type (+/+) and D1A-deficient (–/–) mice derived from a colony originally established at the National Institute of Health (NIH). D1Adeficient mice were generated as described in Drago et al. (1994) from embryonic stem cells where one of the D1A receptor alleles was targeted in vitro by homologous recombination. Briefly, a targeting construct containing a neomycin phosphotransferase gene was inserted into a region of the D1A receptor gene encoding the fifth transmembrane domain. As part of the targeting construct, a gene sequence (0.75 kb) downstream from the insertion site was also excised. This excised gene sequence codes for the third intracytoplasmic loop. The insertion of the neomycin phosphotransferase gene and the removal of the gene sequence generated an inactive gene product. The targeting construct was then transfected into a J1 line of 129/Sv stem cells. Positive clones were used to create chimeric mice. Chimeric males were then mated to female C57BL/6 mice to create heterozygotes (+/–). Heterozygous mice from the original breeding colony at NIH were used to create a colony at California State University, San Bernardino. Wild-type and D1A-deficient mice were obtained by mating identified heterozygotes at the university vivarium at California State University, San Bernardino. Mouse pups were housed with both parents in clear polycarbonate cages (21×23×21 cm). At weaning, mice were transferred to identical polycarbonate cages and housed with same sex litter mates (three or four per cage). Mice were not segregated by genotype. Wild-type and D1A-deficient mice began behavioral testing at 90–120 days of age, with an approximately equal number of male and female mice being assigned to each treatment group. Food and water were available ad lib. The colony room was maintained at 22–24°C and kept under a 12 L:12 D cycle. Subjects were cared for according to the guidelines of the “Guide for the Care and Use of Laboratory Animals” (1996) under a research protocol approved by the Institutional Animal Care and Use Committee of California State University, San Bernardino. Genotyping Mice were genotyped using polymerase chain reaction (PCR) as described previously (Miner et al. 1995; Bender et al. 1997). The genomic DNA for the assays was obtained from tail biopsies and extracted using the PureGene DNA isolation kit (Gentra Systems, Minneapolis, Minn., USA). Two independent PCR reactions were performed for genotyping. The first reaction determined the presence of the neomycin phosphotransferase gene. This reaction used a forward primer (D1.5; 5′-ctgattagcgtagcatggactttgtc-3′) and a reverse primer (PGK1; 5′-tggatgtggaatgtgtgcgag-3′). PCR conditions were 35 cycles of 94°C (20 min), 58°C (20 min), 72°C (1 min), followed by 72°C (6 min). PCR products were separated on a 1.5% agarose gel, with a 330 bp band indicating the presence of at least one transgenic allele. The second reaction determined the presence of the normal D1A gene. It used a forward primer (JD.27; 5′aaagttccttaagatgtcct-3′) and a reverse primer (JD.26; 5′-tggtggctggaaaacatcaga-3′). PCR conditions were the same as in the first reaction, with the exception that the annealing temperature was 55°C instead of 58°C. The presence of a 350 bp band indicated at least one wild-type allele.
409 Apparatus Behavioral testing was done in commercially available (Coulbourn Instruments, Allentown, Penn., USA) activity monitoring chambers (25.5×25.5×41 cm), consisting of Plexiglas walls, a plastic floor, and an open top. Each chamber included an X–Y photobeam array, with 16 photocells and detectors, which was used to determine distance traveled (horizontal locomotor activity). Drugs d-Amphetamine sulfate and SCH 23390 (Sigma, St Louis, Mo., USA) were dissolved in saline and injected intraperitoneally (IP) at a volume of 5 ml/kg.
17 drug abstinence days. This result is not consistent with studies showing that D1-like receptor antagonists (e.g. SCH 23390) block both the development and expression of amphetamine-induced behavioral sensitization (Vezina and Stewart 1989; Hamamura et al. 1991; Kuribara and Uchihashi 1994; Kuribara 1995a; Vezina 1996). One obvious possibility is that the D1B receptor, rather than the D1A receptor, is necessary for behavioral sensitization (i.e. SCH 23390 antagonizes both D1A and D1B receptor subtypes, while D1A-deficient mice lack only the D1A receptor). In the second experiment, therefore, wild-type and D1A-deficient mice were injected with SCH 23390 or saline prior to daily amphetamine treatment. The occurrence of amphetamine-induced behavioral sensitization was assessed after 3 abstinence days. Procedure
Experiment I: amphetamine-induced behavioral sensitization Procedure Wild-type and D1A-deficient mice were randomly assigned to one of five drug pretreatment groups. Specifically, mice from both genotypes were transported to the testing room and given a single daily injection of saline or amphetamine (1, 2, 4, or 8 mg/kg, IP). After being injected, mice were immediately placed in activity chambers where distance traveled was measured for 60 min. On completion of behavioral assessment, mice were returned to their home cage. The pretreatment phase lasted 7 consecutive days, with individual mice being given the identical drug pretreatment each day. The occurrence of behavioral sensitization was determined on 2 separate test days. Specifically, mice received a challenge injection of amphetamine (1 mg/kg, IP) after 3 drug abstinence days, and then again after 14 additional drug abstinence days (i.e. 17 days after the end of the pretreatment phase). On both test days, distance traveled was measured for 120 min. In summary, a total of 45 wild-type mice were placed into one of the following five pretreatment groups [the numbers of males (M) and females (F) in each group are indicated in parentheses]: saline (6 M; 4 F), 1 mg/kg (6 M; 4 F), 2 mg/kg (5 M; 3 F), 4 mg/kg (6 M; 3 F), and 8 mg/kg amphetamine (5 M; 3 F). Likewise, a total of 32 D1A-deficient mice were placed into the same pretreatment groups: saline (3 M; 3 F), 1 mg/kg (3 M; 4 F), 2 mg/kg (3 M; 4 F), 4 mg/kg (3 M; 3 F), and 8 mg/kg amphetamine (3 M; 3 F). Separate groups of wild-type (5 M; 3 F) and D1A-deficient (3 M; 4 F) mice were pretreated with saline and then given a challenge injection of saline after 3 abstinence days. Thus, their first exposure to amphetamine was on the second test day. Statistical analysis Distance traveled data from the pretreatment phase were analyzed using a 2×5×7 [Genotype×Drug×Day] repeated measures analysis of variance (ANOVA), whereas data from the two test days were analyzed using 2×5×12 [Genotype×Drug×Time block] repeated measures ANOVAs. Preliminary analyses showed that interactions involving gender did not reach statistical significance, so gender was not included as a variable in the final statistical analyses. The lack of statistically reliable gender effects may have been due to the small number of male and female mice assigned to each group (as few as three in some cases), because it has often been reported that rats and mice show different intensities of sensitized responding depending on the gender of the animal tested (Sershen et al. 1998; Becker 1999; Cailhol and Mormède 1999). When required, additional lower-order ANOVAs were used to analyze distance traveled data of wild-type and D1A-deficient mice. Post hoc analysis of distance traveled data was made using Tukey tests (P
