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NEUROPHARMACOLOGY
Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina
Received July 11, 2007; accepted August 9, 2007.
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Abstract |
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q-coupled receptors, including metabotropic glutamate receptor (mGluR) subtype 5 in neuronal and non-neuronal cell lines. In the brain, both RGS4 and mGluR5 receptors are enriched in the striatum, and their functions have been linked to psychostimulant-induced behavior and synaptic plasticity. However, it is not known whether RGS4 and mGluR5 interactions occur in rat striatum and whether chronic amphetamine (AMPH) treatment produces changes in RGS4 levels that are correlated with mGluR5 receptor activity. Using coimmunoprecipitation, the present study demonstrated that endogenous RGS4 binds mGluR5 receptors as well as key mGluR5-associated proteins, Gq/11, and phospholipase C-
1 (PLC1) in preparations from rat striatum. In the next experiment, rats were treated with AMPH (5 mg/kg i.p. daily) for 5 days followed by 3 weeks of abstinence. At this time point, animals pretreated with AMPH displayed sensitized behavioral responses to AMPH challenge and decreased RGS4 protein in dorsal striatum and nucleus accumbens. Behavioral sensitization to AMPH was also accompanied by an increase in Gq/11 and PLC1 in dorsal striatum. In contrast, total levels of mGluR5 receptors in the striatum were not altered by any AMPH treatment. In conclusion, the present study demonstrates that RGS4 protein is an integral part of the mGluR5 protein complex in the striatum. This study further suggests that AMPH-induced changes in mGluR5-associated protein levels (RGS4, Gq/11, and PLC1) may be related to altered coupling of striatal mGluR5 receptors in animals sensitized to AMPH.
i- and/or Gq-coupled receptors (Hepler, 1999
). Many RGS proteins display a regionspecific pattern of distribution in the brain (Gold et al., 1997), indicating that certain RGS subtypes could be associated with specific receptor transduction pathways. In fact, RGS proteins seem to have some degree of functional selectivity for particular receptors (Abramow-Newerly et al., 2006). RGS4, an RGS subtype enriched in the neocortex and striatum, is one of the most studied RGS proteins at the biochemical and cellular level. RGS4 is known to regulate the signaling of several Gi- and Gq-coupled receptors, including group I metabotropic glutamate receptors (mGluR; Saugstad et al., 1998), µ-opioid receptors (Georgoussi et al., 2006), M1–4 muscarinic receptors (Bernstein et al., 2004; Ding et al., 2006), and 5-hydroxytryptamine1A/2A (serotonin) receptors (Ghavami et al., 2004). However, most of this evidence on RGS4-receptor interactions stems from heterologous overexpression systems rather than native systems or in vivo conditions. Therefore, it is not clear whether this selectivity occurs in the brain and whether it involves a physical interaction between RGS4 and receptor complexes.
Because RGS proteins negatively regulate G-protein-coupled receptor (GPCR) signaling, changes in RGS protein levels in the brain are thought to modulate the intensity and duration of signaling of cognate receptors (Abramow-Newerly et al., 2006). Interestingly, expression of several RGS proteins in the brain is rapidly altered in response to psychostimulants (Burchett, 2005). Previous reports from our laboratory have documented a decrease of RGS4 mRNA in the striatum lasting from 1 to 6 h after acute amphetamine (AMPH) (Gonzalez-Nicolini and McGinty, 2002; Schwendt et al., 2006a). However, RGS4 protein changes are less robust and more transient than mRNA changes (Schwendt et al., 2006a). Regulation of forebrain RGS4 mRNA levels by repeated AMPH or cocaine has been primarily analyzed shortly (30 min–24 h) after the last injection of the drug (Burchett et al., 1999; Yuferov et al., 2003; Zhang et al., 2005). Whether alterations of RGS4 levels in the forebrain correlate with enduring behavior induced by chronic exposure psychostimulants is not known. The effects of psychostimulants on RGS4 gene and protein expression are differentially modulated via D1 and D2 dopamine receptors: D1 receptor blockade increases, whereas D2 receptor blockade decreases, RGS4 mRNA and protein levels in the striatum (Taymans et al., 2003; Schwendt et al., 2006a). Even though RGS4 expression is under the tight control of dopamine receptors, it does not seem to exercise direct feedback inhibition of Gi-coupled D2 receptors (Ghavami et al., 2004; Ding et al., 2006). Therefore, AMPH-induced changes in RGS4 levels are likely to affect the signaling of other receptors that are involved in modulating AMPH actions in the brain.
The striatum, relative to other parts of the brain, contains high concentrations of both RGS4 and mGluR5 receptors. Several lines of evidence suggest that group I mGluRs (mGluR1 and -5) play a role in drug-induced behavior and gene expression (Chiamulera et al., 2001; McGeehan et al., 2004; Parelkar and Wang, 2004; Herzig et al., 2005). These effects are likely to be tied to activity of dopamine receptors, because mGluR5 antagonists attenuate locomotor activity and cellular signaling induced by stimulation of dopamine D1 receptors (David and Abraini, 2001; Voulalas et al., 2005). It is thought that the functional convergence of dopamine- and glutamate-induced signaling in the striatum is essential for persistent neuroplasticity and behavior induced by chronic psychostimulant administration (Vanderschuren and Kalivas, 2000). However, the complex cellular mechanisms of dopamine and glutamate interactions and their changes after chronic psychostimulant treatment are far from being understood.
Because RGS4 regulates signaling of mGluR5 receptors in neuronal cell lines and hippocampal pyramidal neurons (Saugstad et al., 1998), we hypothesized that amphetamine-induced and dopamine receptor-driven changes of RGS4 expression in the striatum are related to altered function of striatal mGluR5 receptors. Furthermore, most of the mGluR5 receptors in the brain are coupled via Gq and G11 to phospholipase C-1 (PLC1) and phosphoinositide signaling cascades (Conn and Pin, 1997). Hence, manipulation of either Gq/11 or PLC1 protein dramatically affects mGluR5-induced postsynaptic currents and lasting synaptic plasticity (Chuang et al., 2001; Atkinson et al., 2006). Therefore, in the present study, we investigated whether endogenous RGS4 coimmunoprecipitates with mGluR5 receptors and other mGluR5-associated proteins in rat striatum. Furthermore, using a well established animal model of lasting drug-induced neuroplasticity (AMPH-induced behavioral sensitization), we investigated changes of RGS4 and individual RGS4-interacting proteins in the dorsal striatum (dSTR) and nucleus accumbens (NAc). To induce behavioral sensitization, we applied a dosing regimen of AMPH (5 mg/kg daily for 5 days), known to produce robust and lasting sensitization, especially when coupled with a low-dose AMPH challenge (Wolf and Jeziorski, 1993; Wang and McGinty, 1995). It has been shown also that repeated AMPH treatment produces robust sensitization to subsequent stimulant challenge only after prolonged periods of abstinence (2–3 weeks) but not at all or weakly during the first week (Robinson and Becker, 1986). In agreement, 5-day AMPH treatment (identical to the present study) produced changes in mRNA or protein levels of ionotropic glutamate receptors in NAc after 2 weeks but not after 3 days of abstinence (Lu and Wolf, 1999; Lu et al., 1999). Therefore, in the present study, we investigated AMPH-induced mRNA and protein changes in the striatum after extended (3-week) abstinence from repeated AMPH treatment using this well established regimen. At this time point, molecular adaptations in the striatum are most likely to be related to enduring psychostimulant-induced behaviors rather than to acute withdrawal symptoms (Vanderschuren and Kalivas, 2000).
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Materials and Methods |
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Coimmunoprecipitation. For immunoprecipitation, protein preparations from the whole striatum of nontreated animals were prepared under weakly denaturing conditions to permit the proteinprotein interactions, as described previously (Takagi et al., 2000) with some modifications. Briefly, tissue samples were homogenized and solubilized in ice-cold lysis buffer containing: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% SDS and protease, phosphate, and proteasome inhibitors (Complete Mini protease inhibitor; Roche Diagnostics, Indianapolis, IN), Halt phosphatase inhibitor (Pierce Chemical, Rockford, IL), 5 mM MG-132, and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO). The solubilized preparation was then centrifuged at 700g for 10 min to remove the nuclear fraction (P1). The supernatant was again centrifuged at 15,000g for 30 min to obtain cytosolic (S2) and membrane (P2) fractions. The P2 pellet was solubilized in 1% sodium deoxycholate, and its activity was subsequently quenched by 0.1% Triton X-100. Insoluble proteins were sedimented at 15,000g for 20 min, and supernatants (500 µg of protein at 1 µg/µl concentration) were used for immunoprecipitation with rabbit affinity-purified antibodies against mGluR5 (Upstate Biotechnology, Charlottesville, VA), Gq/11, or anti-PLC1 (Chemicon International, Temecula, CA). Immunocomplexes were captured using TrueBlot anti-rabbit IgG IP beads (eBioscience, San Diego, CA). After a series of washes, immunocomplexes were dissociated by adding denaturing Laemmli buffer (containing 50 mM dithiothreitol) and heating to 100°C for 5 to 10 min. Proteins were then resolved by SDS-PAGE and analyzed by immunoblotting as described below. Cross-reactivity with rabbit IgG fragments was prevented by using rabbit IgG Trueblot horseradish peroxidase-conjugated secondary antibodies or primary antibodies from different host species than rabbit (chicken RGS4 antibody; at 1:2000; Abcam Inc., Cambridge, MA). All coimmunoprecipitation experiments included a negative control (mock immunoprecipitate).
Amphetamine Administration. All animals were habituated to their home cage and test environment (locomotor activity chamber) for 3 days before any drug treatment to minimize any stress-induced behaviors. On the test day, rats were randomly assigned to two groups (n = 16 per group), and they were injected with physiological saline (SAL) or 5 mg/kg i.p. AMPH (D-amphetamine sulfate; Sigma-Aldrich) in the test environment, and locomotor activity was recorded for 3 h (as described below). Rats were injected daily for 5 days followed by 3 weeks of abstinence (in their home cages). On day 21, SAL- and AMPH-treated rats were randomly divided into two subgroups, and then they were transferred into the test environment, injected with SAL or AMPH (1 mg/kg i.p.), and locomotor activity was recorded as described below. Three hours after the injection, all groups of rats were anesthetized with equithesin (10 ml/kg i.p.), and the rats were decapitated. One hemisphere was flash frozen in isopentane at –40°C for in situ hybridization, and the dSTR and NAc were dissected from the contralateral hemisphere for Western blotting. All brain tissues were stored at –80°C until they were processed.
Behavioral Measurements. Behavioral activity was measured in the test environment by using automated photocell beam activity chambers (Accuscan Instruments, Columbus, OH). Horizontal (total distance traveled) as well as vertical activity (rearing) was recorded by Accuscan/Digimax software (Accuscan Instruments). All rats were adapted to the environment and injected with SAL (i.p.) for 3 days before the start of the experiment. On each day of the experiment, rats were placed in the chambers 1 h preceding SAL or AMPH injection. After the injection, horizontal and vertical activity was recorded in 5-min bins for 3 h.
In Situ Hybridization Histochemistry. Quantitative in situ hybridization histochemistry was performed as described previously (Schwendt et al., 2006a). Briefly, 12-µm brain sections mounted onto gelatin-coated slides were initially pretreated in a series of steps that fixed and defatted the tissue and blocked nonspecific hybridization. Synthetic cDNA oligodeoxynucleotide probes complementary to rat mGluR5 (bases 637–682, D_10891) and RGS4 (bases 109–156, AF_117211) sequences were end-labeled with [-35S]ATP (1250 Ci/mmol; GE Healthcare, Piscataway, NJ) using terminal deoxynucleotidyl transferase (Roche Diagnostics). Slides were incubated with 5 x 105 cpm/25 µl of hybridization buffer/section overnight at 37°C. After incubation, slides were washed and air-dried before being placed into a film cassette, along with 14C standards (American Radiolabeled Chemicals, St. Louis, MO) and with Biomax film (Eastman Kodak, Rochester, NY) for 5 days (RGS4) or 2 weeks (mGluR5). Quantitation of each mRNA hybridization signal in the dSTR and NAc was performed using NIH Image 1.62 software (http://rsb.info.nih.gov/nih-image). Gene expression values, represented by mean integrated density, were measured in selected brain areas from three adjacent brain sections.
Immunoblotting. Tissues from dSTR and NAc were hand-dissected from 2-mm-thick coronal slabs on the day of the experiment, quickly frozen on dry ice, and stored at –80°C until processed. Samples were then solubilized in 1% SDS/phosphate-buffered saline buffer containing protease, phosphatase, and proteasome inhibitors (Complete Mini protease inhibitor; Roche Diagnostics), Halt phosphatase inhibitor (Pierce Chemical), 5 mM MG-132, and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). Protein concentration in the samples was measured with BCA assay (Pierce Chemical). Subsequently, equal amounts of total protein were resolved using SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% milk/phosphate-buffered saline containing 0.05% Tween 20, and it was probed with rabbit antibodies against the following proteins: RGS4 (U1079) characterized by Krumins et al. (2004) and Schwendt et al. (2006a) at 1:2000; mGluR5 (Upstate Biotechnology) at 1:5000; Homer1bc at 1:1200; Gq/11 at 1:1000,; and phospholipase C-1 at 1:500 (all Chemicon International). After the incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibodies at 1:5000 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA), immunoreactive bands on the membranes were detected by ECL+ chemiluminescence reagents on Hyperfilm ECL (GE Healthcare). Integrated density of the bands was measured with Gel-Pro 3.1 software (Media Cybernetics, Inc., Silver Spring, MD). Equal loading and transfer of proteins were confirmed by Ponceau S staining and by immunolabeling of the same membranes with a rabbit anti-calnexin antibody (Nventa Biopharmaceuticals, San Diego, CA), because calnexin is an independent protein that was not altered by AMPH.
Statistical Analysis. Behavioral data were analyzed by calculating the area under the curve (AUC) for the activity counts plotted against time. Area under the curve values were subjected to a two-way ANOVA followed by Tukey's honestly significant difference test to determine specific differences between the groups. Because the integrated density values of the in situ hybridization data (collected from three adjacent sections of the same brain) are strongly correlated and cannot be treated independently, a nested ANOVA with repeated measures was applied to analyze the data (mixed model SAS 9.1; SAS Institute, Cary, NC). Immunoblotting data, represented by band density values, were normalized for the density of calnexin immunoreactivity within the same sample, and they were analyzed by a two-way ANOVA followed by Tukey's honestly significant difference test to determine specific differences between the groups. Both mRNA and protein data were then expressed as the percentage of the values from SAL-treated rats within the same time point or treatment. Unless otherwise noted, SigmaStat (SPSS Inc., Chicago, IL) software was used for statistical analysis.
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Results |
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130- and 250-kDa bands corresponding to mGluR5 monomer and dimer forms. In contrast, no bands were detected in "mock" samples (X) where precipitating antibody was not present, suggesting specific and effective pull-down of endogenous mGluR5 receptors under these experimental conditions. In support, probing the mGluR5-immunoprecipitated fraction with antibodies against proteins known to interact with mGluR5 receptors yielded bands corresponding to Gq/11, PLC1, and the mGluR5-scaffolding protein Homer1b/c (Fig. 1A, top). Furthermore, the presence of RGS4 protein in mGluR5-immunoprecipitates was revealed for the first time. Two different antibodies, rabbit anti-RGS4 (U1079) and chicken anti-RGS4 (Abcam Inc.), which recognize distinctive epitopes of RGS4 protein, detected an identical 28-kDa band corresponding to rat endogenous RGS4 found in total striatal lysates (Fig. 1A, bottom). Another band migrating at 35 kDa is likely to be of nonspecific origin, as discussed in Schwendt et al. (2006a).
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q/11 (Fig. 1B) or anti-PLC1 (Fig. 1C) antibodies yielded protein fractions in which Gq/11 or PLC1 were enriched. Both tested proteins were detected in the striatal preparations used as input for coimmunoprecipitation experiments (TOT lanes) but not in mock samples where precipitating antibody was omitted (X lanes). As expected, mGluR5 protein was present in both Gq/11- and PLC1-immunoprecipitated fractions. Again, endogenous RGS4 protein was detected in both Gq/11 and PLC1 immunoprecipitates with a two-antibody approach (Fig. 1, B and C, respectively). Unfortunately, reverse immunoprecipitation experiments for RGS4 could not be performed because both RGS4 antibodies used in this study did not have a high enough titer for immunoprecipitation. These results suggest that endogenous RGS4 in the striatum physically interacts with mGluR5 receptors and with other proteins of the mGluR5-signaling complex under our experimental conditions.
Regulation of RGS4, mGluR5, and mGluR5-Associated Proteins in Rat Striatum by Repeated Amphetamine and/or Amphetamine Challenge. The coimmunoprecipitation experiment indicated that RGS4 is part of the mGluR5 receptor signaling complex in the striatum. Knowing the involvement of RGS4 and mGluR5 in the actions of psychostimulants in the brain, the next set of experiments was designed to test the hypothesis that levels of RGS4, mGluR5, Gq/11, and PLC1 are altered by chronic AMPH treatment.
Rats injected daily with AMPH (5 mg/kg i.p.) progressively developed behavioral sensitization over the course of 5 days, as evidenced by augmentation of initial stereotyped behaviors and increased poststereotypy locomotion (data not shown), as described previously by Wolf and Jeziorski (1993). Furthermore, a challenge injection of AMPH (1 mg/kg i.p.) 3 weeks after the end of AMPH treatment, elicited significantly greater horizontal and vertical activity in AMPH-pretreated rats than in SAL-pretreated rats (Fig. 2, A and B, respectively). In our experimental setting, a conditioned effect of the drug-paired environment was not observed, because AMPH-pretreated/SAL-challenged rats displayed the same behavioral activity as their SAL-treated/SAL-challenged counterparts (Fig. 2).
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). Because dSTR is the region with higher concentrations of basal RGS4 levels and more prominent AMPH-induced changes in RGS4 levels, the investigation of AMPH-induced changes of RGS4-associated proteins in mGluR5-signaling complex was limited to this region.
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In contrast to RGS4, the levels of mGluR5 mRNA or protein (monomer and dimer) in dSTR were not altered in any treatment group (Fig. 4, A and B, respectively). However, semiquantitative immunoblot analysis of the cognate G-protein of the mGluR5 receptor revealed a significant increase of Gq/11 protein in dSTR of AMPH-pretreated and AMPH-challenged rats (AA group) compared with their SAL counterparts (p < 0.05; Fig. 5A, SA group). Total levels of another mGluR5/RGS4-associated protein, PLC1, were also elevated in dSTR of all animals pretreated with repeated AMPH (AS and AA groups), regardless of the challenge injection (Fig. 5B).
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Discussion |
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q and RGS4-PLC1 interactions. Despite the fact that these proteins are likely to form a functional signaling complex in vivo, this study also demonstrated that they are differentially regulated by repeated AMPH. Although repeated AMPH treatment produces lasting behavioral sensitization in response to a challenge, this behavioral state was not accompanied by changes in total levels of mGluR5 receptors in dSTR. However, a sustained decrease of RGS4 and an increase of the facilitatory signaling factors Gq/11 and PLC1 could be translated into enhanced mGluR5 postsynaptic coupling in animals sensitized to AMPH.
In the present report, we demonstrated in vivo proteinprotein interactions between RGS4, mGluR5 receptors, Gq/11, and PLC1. Physical and functional interactions between RGS4 and these proteins have been reported previously in recombinant and non-neuronal native systems, but not in the brain (Saugstad et al., 1998; Dowal et al., 2001; Huang et al., 2007). To reinforce the validity of the present findings, RGS4 protein in mGluR5-, Gq-, and PLC1-immunoprecipitated fractions, using RGS4 antisera raised in two different species (rabbit and chicken) and directed toward two different epitopes, was detected. The fact that both Gq/11 and PLC1 also associate with native mGluR5 receptors (Farr et al., 2004; current study) further suggests that RGS4 physically contributes to the formation of an mGluR5 signaling complex interacting with GPCRs coupled to Gq/11 and PLC1. Our results are in agreement with the concept that RGS proteins are an integral part of multiprotein postsynaptic signaling complexes of G-protein-coupled receptors (Abramow-Newerly et al., 2006).
Little is known about whether and how psychostimulant-induced changes are related to altered signaling of particular striatal receptors. Therefore, we examined whether behavioral sensitization elicited by an AMPH challenge in rats after repeated AMPH exposure induces changes in the mRNA and/or protein expression of RGS4, mGluR5 receptors, Gq/11, and PLC1 in the striatum. All proteins were analyzed at the single time point after a low dose of AMPH as a challenge. Although the time of analysis (3 h post-AMPH challenge) was based on maximum response of RGS4 mRNA and protein to acute AMPH (Schwendt et al., 2006a), the low dose of AMPH (1 mg/kg i.p.) is required to reveal augmented behavioral response to AMPH challenge in sensitized rats, as observed in the present study and several previous reports (Wolf and Jeziorski, 1993; Wang and McGinty, 1995). Repeated AMPH treatment followed by an AMPH challenge resulted in a decrease of RGS4 protein in both the dSTR and NAc and an increase in Gq/11 and PLC1 proteins in the dSTR, suggesting an increase in the activity of GPCRs coupled to Gq/11 and PLC1 in AMPH- but not SAL-pretreated rats. Because the expression of mGluR5 was not altered, the GPCR(s) involved in this augmented response was not identified with certainty. Thus, even though mGluR5, RGS4, Gq/11, and PLC1 have a close physical (present study) and functional (Saugstad et al., 1998) relationship in the brain, these data suggest that AMPH sensitization causes changes in the post-GPCR RGS4-Gq/11-PLC1 complex that may affect the duration of GPCR signaling. Compared with RGS4 protein, RGS4 mRNA in dSTR was decreased by an AMPH challenge (1 mg/kg i.p.), regardless of the previous exposure to SAL or AMPH. This sensitivity of RGS4 mRNA to a low dose of AMPH and the discrepancy between the RGS4 mRNA and protein response has been previously documented after acute AMPH exposure (Schwendt et al., 2006a), suggesting that RGS4 protein stability can withstand transient, low-intensity challenges that reduce transcription. In contrast to the dSTR, a decrease in RGS4 mRNA in NAc was only detected in AMPH-sensitized rats, possibly because basal levels of RGS4 mRNA in NAc and the responses to AMPH in general were lower than in the dSTR. These data support a report by Bishop et al. (2002) who observed that AMPH challenge decreased RGS4 mRNA levels in the NAc in rats pretreated with repeated AMPH 15 days earlier. However, they did not investigate RGS4 mRNA changes in the dSTR or the effect of abstinence alone (e.g., no drug challenge).
Three weeks of abstinence after repeated AMPH treatment without re-exposure to the drug resulted in a decrease of both RGS4 mRNA and protein in dSTR, implying that a chronic reduction in transcription was responsible for the decrease in RGS4 protein levels. Such an enduring decrease in RGS4 suggests that the duration of Gq-coupled receptor activity in the striatum may be augmented during abstinence. Abstinence from repeated AMPH also produced an enduring increase of PLC1, but not Gq/11, protein in dSTR, reinforcing that PLC1 is downstream of RGS4 and that abstinence from repeated AMPH induces a prolonged disturbance in RGS4-PLC1 coupling. Whether such a disturbance is critical to the altered response to an AMPH challenge in rats with a AMPH history must be investigated in future studies.
RGS4 mRNA and protein levels are under tight control by dopamine D1 and D2 receptors (Taymans et al., 2003; Schwendt et al., 2006a). It is the prevailing D1 influence that drives down-regulation of RGS4 after acute AMPH administration (Schwendt et al., 2006a). Thus, a lasting decrease of RGS4 mRNA and protein by repeated AMPH and augmented regulation of RGS4 protein by AMPH challenge may be due to dominant D1 or diminished D2 receptor tone controlling RGS4 gene expression in the dSTR. In agreement, augmented D1 receptor function together with long-term reductions in D2 receptor availability has been documented in the striatum after prolonged treatment with psychostimulants (Henry and White, 1991; Unterwald et al., 1996; Chen et al., 1999; Ginovart et al., 1999).
There is some inconsistency in the literature about the effects of repeated psychostimulant treatment on mGluR5 receptor expression. In this study, the expression of striatal mGluR5 receptors was not altered by AMPH sensitization or abstinence. In contrast, repeated cocaine treatment increased mGluR5 mRNA in dSTR and NAc (Ghasemzadeh et al., 1999), whereas a lasting decrease of mGluR5 mRNA signal throughout the dSTR and NAc after repeated AMPH was reported by Mao and Wang (2001). The discrepancies in the findings of the latter study and the present study could stem from the different dosing regimens of AMPH (4 versus 5 mg/kg i.p.) or differences in mRNA signal detection and analysis. Furthermore, Swanson et al. (2001) reported a decrease in NAc mGluR5 protein after repeated cocaine; however, authors measured only the levels of mGluR5 monomer and overlooked the fact that functional mGluR5 receptors in vivo predominantly exist in a dimer form (Romano et al., 2001). Analyzing both monomer and dimer forms of mGluR5 receptor (present study) provides more accurate information about mGluR5 receptor levels.
Recent data from our laboratory indicate that the sensitization paradigm used in the current study is accompanied by augmented phosphorylation of extracellular signal-regulated kinase (ERK) in dSTR (Shi and McGinty, 2007). Activation of the striatal ERK cascade by AMPH depends on both mGluR5 (stimulation) and RGS4 (inhibition) (Choe et al., 2002; Schwendt et al., 2006b). Thus, it is possible that augmented ERK activation in animals sensitized to AMPH is related to enhanced mGluR5 coupling (increased levels of Gq/11 and PLC1) and diminished mGluR5 inhibition (decrease of RGS4). Because the function of mGluR5 receptors depends on the coupled postsynaptic signaling assembly, AMPH-induced changes in mGluR5-associated proteins could alter mGluR5 receptor function in the dSTR, even without changes in total protein levels of the receptor.
In conclusion, the present results demonstrate that endogenous RGS4 protein is an integral part of the mGluR5 signaling complex in the striatum. Furthermore, these results demonstrate that behavioral sensitization to AMPH is not necessarily accompanied by changes in the expression of mGluR5 receptors in the striatum but rather by more complex, fine-tuned adaptations in the levels of receptor-associated proteins, such as RGS4, Gq/11, and PLC1. Whether these changes are reflected in the actual dynamic of mGluR5-postsynaptic complex composition remains to be determined in future studies. Because dopamine D1/D2 receptors exert tight control over RGS4 expression, RGS4 could belong to the growing family of factors regulating convergence of dopamine and glutamate signaling in the striatum (Girault et al., 2007). These properties, which are thought to underlie addiction, would predetermine the role of RGS4 in persistent alterations elicited by drugs of abuse.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: RGS, regulator of G-protein signaling; mGluR, metabotropic glutamate receptor(s); GPCR, G-protein-coupled receptor; AMPH, amphetamine; PLC1, phospholipase C-1; dSTR, dorsal striatum; NAc, nucleus accumbens; MG-132, MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; PAGE, polyacrylamide gel electrophoresis; SAL, saline; ANOVA, analysis of variance; IP, immunoprecipitate; SS, repeated SAL and challenged with SAL; SA, repeated SAL and challenged with AMPH; AS, repeated AMPH and challenged with SAL; AA, repeated AMPH and challenged with AMPH ERK, extracellular signal-regulated kinase.
Address correspondence to: Dr. Marek Schwendt, Department of Neurosciences, Medical University of South Carolina, 173 Ashley Ave., BSB 403, Charleston, SC 29425. E-mail: schwendt{at}musc.edu
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