ORIGINAL INVESTIGATION

RGS4 overexpression in the rat dorsal striatum modulatesmGluR5- and amphetamine-mediated behavior and signaling

Marek Schwendt & Stacey A. Sigmon & Jacqueline F. McGinty

Received: 25 October 2011 /Accepted: 30 November 2011 /Published online: 23 December 2011# Springer-Verlag 2011

AbstractRationale Regulator of G-protein signaling 4 (RGS4) is abrain-enriched negative modulator of G-protein-coupled re-ceptor signaling. Decreased availability of RGS4 in thefrontal cortex and striatum has been described in animalmodels of schizophrenia and drug addiction. However, cel-lular and behavioral consequences of dysregulated RGS4-dependent receptor signaling in the brain remain poorlyunderstood.Objective This study aims to investigate whether RGS4,through inhibiting the function of mGluR5 receptors in thedorsal striatum (dSTR), regulates cellular and behavioralresponses to acute amphetamine.Methods After herpes simplex virus-RGS4 was infused intothe dSTR, RGS4 overexpression as well as binding ofrecombinant RGS4 to mGluR5 was assessed. The effect ofRGS4 overexpression on behavioral activity induced by theintrastriatal mGluR5 agonist, DHPG, or amphetamine wasrecorded. Activation of extracellular signal-regulated kinase(ERK) and Akt (protein kinase B) was measured in thedSTR tissue at the end of each behavioral experiment.Results RGS4 overexpressed in the dSTR coimmunoprecipi-tated with mGluR5 receptors and suppressed both behavioralactivity and phospho-ERK levels induced by DHPG. RGS4overexpression or the mGluR5 antagonist, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP), attenuated amphetamine-induced phospho-ERK (but not phospho-Akt) levels. RGS4suppressed amphetamine-induced vertical activity and aug-mented horizontal activity over 90 min. Similarly, MTEP

augmented amphetamine-induced horizontal activity, but didnot affect vertical activity.Conclusions The present data demonstrate that RGS4 in thedSTR attenuates amphetamine-induced ERK signaling anddecreases the behavioral efficacy of acute amphetaminelikely by limiting mGluR5 function.

Keywords Akt . Amphetamine . Dorsal striatum . ERK .

Locomotor activity . mGluR5 . Overexpression . RGS4

Introduction

Regulator of G-protein signaling 4 (RGS4) is a member ofthe large family of RGS proteins that function as negativemodulators of G-protein-coupled receptor (GPCR)-mediatedsignaling pathways (Siderovski and Willard 2005). AllRGS proteins bind directly to the GTP-bound Gαi orGαq subunit of activated heterotrimeric G-proteins andincrease the rate of GTP hydrolysis. This accelerated“turn-off” of activated G-proteins provides a cellularmechanism for limiting temporal and spatial resolutionof GPCR-signaling and GPCR-mediated synaptic plastic-ity by RGS proteins (Abramow-Newerly et al. 2006;Kimple et al. 2011). A growing body of evidence indi-cates that disruption of this regulatory mechanism is acritical component of pathophysiologies underlying vari-ous human diseases (Emilsson et al. 2006; Nishiguchi etal. 2004; Talkowski et al. 2006; Tekumalla et al. 2001).

RGS4 is a small RGS protein enriched in the brain, withhighest levels of RGS4 protein present in the frontal cortex,dorsal striatum (dSTR), amygdala, and thalamus (Gold et al.1997). As such, dysregulation of RGS4 has been linked toseveral neuropsychiatric disorders including schizophrenia(Ding and Hegde 2009; Mirnics et al. 2001), Parkinson’s

M. Schwendt : S. A. Sigmon : J. F. McGinty (*)Department of Neurosciences,Medical University of South Carolina,173 Ashley Avenue, BSB 403, MSC 510,Charleston, SC 29425-5100, USAe-mail: mcginty@musc.edu

Psychopharmacology (2012) 221:621–635DOI 10.1007/s00213-011-2606-8

disease (Ding et al. 2006; Zhang et al. 2005), Alzheimer’sdisease (Emilsson et al. 2006), and drug addiction (Hooks etal. 2008). With regard to addiction, our laboratory as well asothers has documented that acute exposure to psychostimu-lants or opiates results in rapid downregulation of RGS4mRNA and protein levels, predominately in the striatum(Chase et al. 2010; Gonzalez-Nicolini and McGinty 2002;Schwendt et al. 2006; Yuferov et al. 2003). Furthermore,chronic exposure to both experimenter- and self-administeredcocaine resulted in a lasting RGS4 decrease in the prefrontalcortex and striatum, followed by a rapid upregulation of RGS4levels as a result of cue-induced drug-seeking (Schwendt et al.2007). This suggests that RGS4 can play a role in cellularadaptations underlying acute dopaminergic activity (hyperlo-comotion) as well as enduring drug-induced behaviors (such asrelapse to drug-seeking). However, the identity of GPCRs andsignaling pathways affected by the fluctuation of RGS4 levelsremains obscure.

Although a number of receptors regulated by RGS4 havebeen identified in heterologous cell lines (as reviewed byBansal et al. 2007), few studies have addressed the receptorspecificity and the regulatory function of RGS4 in nativeneuronal cultures or directly in the brain. In one of thosestudies (Saugstad et al. 1998), it was demonstrated thatRGS4 is a potent inhibitor of mGluR1/5-induced signalingin hippocampal neurons. In the striatum, which containshigh concentrations of both RGS4 and mGluR5 (but notmGluR1) receptors, this functional interaction is likely tooccur via direct physical association of RGS4 with themGluR5 receptor signaling complex (Schwendt andMcGinty 2007). mGluR5 receptors in the brain are coupledto downstream signaling pathways either through a conven-tional Gαq-to-phospholipase C, subtype β1 (PLCβ1) path-way or through a pathway utilizing Homer scaffoldingproteins (Conn and Pin 1997; Ribeiro et al. 2010). Whilethe conventional pathway leads to the release of calciumfrom intracellular stores and activation of mitogen-activatedprotein kinase (MAPK) signaling pathways, the Homer-dependent pathway is calcium-independent and leads tothe activation of both MAPK and PI3K-Akt-mTOR path-ways (Mao et al. 2005; Ronesi and Huber 2008). In agree-ment, local administration of the mGluR1/5 agonist, DHPG,into the dSTR dose-dependently increased the phosphoryla-tion of extracellular signal-regulated kinase (ERK) 1/2MAPK that was blocked by pretreatment with an mGluR1/5antagonist (Choe and Wang 2001). Activation of mGluR5receptors in the striatum is also necessary for the inductionof ERK1/2 signaling by D-amphetamine sulfate (Amph)(Choe et al. 2002), and it is likely that this signaling mecha-nism is also involved in regulating mGluR5-dependent syn-aptic plasticity after exposure to psychostimulants (Fourgeaudet al. 2004; Grueter et al. 2006). On the other hand, regulationof PI3K-Akt-mTOR signaling in the STR by mGluR5

receptors has not been investigated. In addition to cellularsignaling, striatal mGluR5s can regulate behavior in experi-mental animals, as demonstrated by the finding that intra-striatal administration of DHPG induced complex locomotorand stereotypical behaviors (Wang and Mao 2000). However,despite a number of studies investigating the cellular andbehavioral significance of striatal mGluR5 receptors, thereare considerable gaps in our understanding of their role inpsychostimulant addiction.

Therefore, in the present study, we hypothesized thatRGS4 limits mGluR1/5 signaling in the dSTR and, further,that the mGluR5–RGS4 functional relationship is mani-fested by altered behavioral responses to an mGluR5 agonistand/or to Amph. Since psychostimulants decrease the levelsof RGS4 in the dSTR, we employed a technique of viral-mediated gene transfer to overexpress RGS4 within thedSTR in order to study the effects of elevated RGS4 proteinlevels on cellular signaling and behavior.

Materials and methods

Animals

Adult male Sprague–Dawley rats (275–300 g) (CharlesRiver Laboratories, Wilmington, MA, USA) were single-housed in clear plastic cages and maintained on a 12-h light/dark cycle with food and water available ad libitum. Allanimals were acclimated to their home cage environmentfor a minimum of 3 days prior to surgery and were handledfor an additional 5 days prior to drug administration tominimize the effects of stress on behavioral and neuro-chemical parameters. Every animal procedure in this studywas approved by the Institutional Animal Care and UseCommittee and was performed in strict accordance with the“Guide for the Care and Use of Laboratory Animals”(Institute of Laboratory Animal Resources, National AcademyPress 1996).

Drugs

Amph was purchased from Sigma-Aldrich (St. Louis, MO)or acquired from the NIDA Controlled Substances Program(Research Triangle Institute, NC). The mGluR1/5 agonist,(RS)-3,5-DHPG, was purchased from Tocris Bioscience(Ellisville, MO); and the selective mGluR5 antagonist, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP) hydrochlo-ride, was purchased from Ascent Scientific (Bristol, UK).All drugs were freshly prepared on the day of the experi-ment. Amph was dissolved in physiological saline (Sal)solution and injected intraperitoneally (i.p.) in a volume of1 ml/kg. For intrastriatal infusions, DHPG was first dis-solved in 25% dimethylsulfoxide (DMSO) and then diluted

622 Psychopharmacology (2012) 221:621–635

in artificial cerebrospinal fluid (aCSF) (in mM: NaCl123, CaCl2 0.86, KCl 3.0, MgCl2 0.89, NaH2PO4 0.50,and Na2HPO4 0.25, pH 7.4) to a final concentration of125 or 250 nmol/μl. MTEP was dissolved in 1% Tween80 in aCSF to a final concentration of 5 μg/μl. DMSO incombination with aCSF or Tween 80 in combinationwith aCSF was therefore used as vehicle (Veh) controlfor the respective agents. Solutions of all drugs wereneutralized to pH 7.2–7.4 with 1 N NaOH, if necessary.The concentrations of the drugs used were determinedbased on the results of previously published studies (Choe andWang 2001; Gass and Olive 2009; Gonzalez-Nicolini andMcGinty 2002; Molina-Hernandez et al. 2006), as well asour preliminary data.

Stereotaxic surgery

On the day of surgery, rats were anesthetized with ketamine/xylazine (66 mg/kg and 1.33 mg/kg, i.p.), followed byEquithesin (0.5 ml/kg, i.p.) and ketorolac (2.0 mg/kg, i.p.).Rats were mounted onto a stereotaxic device (Stoelting,Wood Dale, IL), and bilateral stainless steel guide cannulaeprecut (24-gauge, Plastics One, Roanoke, VA, USA) wereimplanted 2 mm above the dSTR infusion target (+1.2 mmanteroposterior, ±3.4 mm mediolateral, and −3.4 mm dorso-ventral relative to bregma according to Paxinos and Watson2007). Animals were allowed to recover for 5 days prior tothe beginning of behavioral experiments.

HSV-mediated gene transfer

Recombinant herpes simplex virus (HSV)-LacZ andHSV-RGS4 constructs were gifts from Drs. StephenGold and David Self (UT Southwestern, Dallas, TX).They were generated in the laboratory of Dr. RachelNeve (McGovern Institute for Brain Research, MIT,Cambridge, MA) and were previously described (Neveet al. 1997; Rahman et al. 2003). The average titer ofthe purified virus stocks was >108 infectious units/μl.Two microliters of HSV vectors was bilaterally infusedinto the dSTR at a rate of 0.2 μl/min using a 33-gaugeinjector cannula inserted to a depth of 2 mm below thetip of the guide cannula. The injector was connected viapolyethylene tubing to a 10-μl gastight Hamilton syringemounted in an infusion pump (PHD 2000, Harvard Apparatus,Holliston, MA). The injector remained in place for 5 min afterthe infusion to prevent backflow along the cannula track.HSV-mediated transgene expression was confirmed by β-galactosidase stain for HSV-LacZ or by immunohisto-chemistry and immunoblot for HSV-RGS4. All behavior-al experiments were performed on day 3 after HSVinjection, at the peak of HSV-driven transgene expression(Rahman et al. 2003).

Drug administration and locomotor activity

DHPG (125 and 250 nmol), MTEP (5 μg), or the appropri-ate vehicle was bilaterally infused into the dSTR (1 μl/side)through a 33-gauge injector over a period of 4 min using aninfusion pump (as described in detail for HSV microinfu-sions). After the infusions, the injectors were left in place foran additional 1 min. Amph (2.5–3 mg/kg, i.p.) or Sal wasadministered to rats 15 min later. This dose of Amph hasbeen shown to reliably stimulate horizontal and verticalactivities in rats (Wang and McGinty 1995). To measurebaseline and drug-induced behavioral activity, animals wereplaced in automated photocell beam activity chambers(Accuscan Instruments, Columbus, OH, USA), and horizon-tal activity (total distance traveled) as well as vertical activ-ity (rearing) were recorded for 15 min–3 h as describedpreviously (Schwendt et al. 2006). At the end of eachbehavioral experiment, whole brains or selected brain tis-sues were harvested for protein analysis as described below.

Immunohistochemistry

For the immunohistochemical analysis of HSV-driven LacZor RGS4 expression, rats were anesthetized and perfusedwith 4% paraformaldehyde in phosphate-buffered saline(PBS) solution (in mM: NaCl 137, KCl 2.7, Na2HPO4 4.3,and KH2PO4 1.47, pH 7.4). Brains were removed and post-fixed for 4 h and saturated in 30% sucrose/PBS solution.After embedding and freezing, 30 μm sections were cut on afreezing microtome and used for histology (cannulae place-ments) or for confirmation of transgene expression. Detec-tion of β-galactosidase expression (LacZ staining) wasperformed as described previously (Neve et al. 1997). Viraloverexpression of RGS4 was detected via immunohisto-chemistry using a specific antibody against FLAG-taggedRGS4, anti-FLAG M2 HRP-conjugated antibody (1:1,000,Sigma-Aldrich). The staining was visualized using VIPsubstrate (Vector Labs, Burlingame, CA). The sections wereviewed and photographed with an Olympus microscopeequipped with a digital camera.

Coimmunoprecipitation

For immunoprecipitation of FLAG-tagged RGS4, dSTRtissue was homogenized by a brief sonication followed bysolubilization of striatal membrane proteins in a mild Triton-based lysis buffer (50 mM Tris–HCl, pH 7.4, with 150 mMNaCl, 1 mM EDTA, and 1% Triton X-100) containing thefollowing inhibitors: complete mini protease inhibitor(Roche Diagnostics, Indianapolis, IN, USA), Halt phospha-tase inhibitor (Pierce Chemical, Rockford, IL, USA), and5 mM MG-132 proteasome inhibitor (Tocris). Insolubleproteins were sedimented at 15,000×g for 10 min, and

Psychopharmacology (2012) 221:621–635 623

supernatants (500 μg of protein at 1 μg/μl concentration)were used for immunoprecipitation of FLAG-tagged RGS4with anti-FLAG M2 agarose resin according to the manu-facturer’s instructions (Sigma-Aldrich). After a series ofwashes, immunocomplexes were dissociated from the beadsunder native conditions by a competition with a 3×FLAGpeptide. Proteins were then resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)and analyzed by immunoblotting as described below. Co-immunoprecipitation experiments included a positive con-trol (immunoprecipitation of FLAG-BAP fusion protein,Sigma-Aldrich) as well as negative controls (omission oflysate, FLAG M2 beads, or no overexpression of FLAG-tagged RGS4).

Immunoblotting

For immunoblotting analysis, 5-mm-thick forebrain coronalslices containing dSTR were collected using a precisionbrain slicer (Braintree Scientific, Braintree, MA) and rapidlyfrozen in isopentane on dry ice. Frozen coronal slices werethen trimmed in the cryostat to a 2-mm thicknesscorresponding to ~2.7–0.7 mm anterior to bregma (Paxinosand Watson 2007), and the dSTR was bilaterally dissectedusing a 2-mm tissue micropuncher and stored at −80°C untilprocessed. This approach allowed for monitoring of cannu-lae placement and potential tissue damage. Total tissueprotein was extracted from dSTR by sonication in 1%SDS/PBS buffer with inhibitors (as described above), dena-tured for10 min at 85°C, and centrifuged for 10 min at12,000×g to pellet insoluble proteins. Protein concentra-tions were measured with the micro-bicinchoninic acid as-say kit (Pierce Chemical, Rockford, IL, USA). Equal proteinamounts (15 μg) were separated by SDS-PAGE (4–15%polyacrylamide) and transferred onto polyvinylidenedifluoride membranes. Membranes were blocked for 1 h in5% milk/tris-buffered saline solution and probed overnightat 4°C with a primary antibody diluted in 3% or 5% milk/tris-buffered saline solution with 0.1% Tween 20. The fol-lowing primary antisera were used: rabbit phospho-ERK(1:2,500), rabbit tERK (1:15,000), rabbit phospho-Akt-Thr308 (1:1,000), rabbit phospho-Akt-Ser473 (1:2,000),and rabbit tAkt (1:7,500), all from Cell Signaling Technol-ogy (Danvers, MA); rabbit RGS4 (1:5,000) and rabbitmGluR5 (1:5,000; Millipore, Billerica, MA); mouse FLAGM2 (1:1,000, Sigma-Aldrich); mouse β-galactosidase(1:2,500; Promega, Madison, WI); mouse D1 receptor(1:250; Millipore); rabbit D2 receptor (1:1,000; Abcam,Cambridge, MA); and calnexin (1:10,000; Enzo Life Sciences,Farmingdale, NY). After the incubation with an appropriateHRP-conjugated secondary antiserum (Jackson ImmunoResearch, West Grove, PA), immunoreactive bands on themembranes were detected by ECL+ chemiluminescence

reagents on an X-ray film (GE Healthcare, Piscataway, NJ).Equal loading and transfer of proteins were confirmed bystripping and reprobing of the same membranes either forproteins independent of their phosphorylation state (tERK,tAkt) or for calnexin, an intracellular protein that was notaltered by any experimental treatment. The integrated banddensity of each protein sample was measured using ImageJsoftware (U.S. National Institutes of Health, Bethesda, MD).

Statistical analysis

Results are expressed as mean ± standard error of the mean(S.E.M.). Behavioral data were analyzed by calculating thearea under the curve (AUC) for the total distance traveledand vertical activity plotted against time starting at timezero. For the immunoblotting data, the ratios of phospho-to-total protein levels (ERK and Akt) or ratios of protein-to-calnexin levels are presented. Because of the less abundantlevels of phospho-ERK1 in the dSTR and because phospho-ERK2 has been identified as the isoform that mediates theneurochemical and behavioral effects of psychostimulants(Girault et al. 2007), only the phospho-ERK2/tERK2 ratiowas analyzed. Behavioral and immunoblotting data wereevaluated using a one-way ANOVA followed by Student–Newman–Keuls (SNK) multiple comparison tests. Sigma-Stat (Systat Software, Chicago, IL, USA) software was usedfor all statistical analyses.

Results

Infusion of HSV-RGS4 into the dSTR resulted in a robustoverexpression of recombinant RGS4 protein and bindingto mGluR5 receptors

In order to study the role of RGS4 in the dSTR in mGluR5-and psychostimulant-induced behaviors and signaling, weoverexpressed the protein specifically in the dSTR usingHSV vectors previously demonstrated to be effective for invivo gene transfer (Carlezon et al. 2000; Han et al. 2010;Rahman et al. 2003). A single bilateral microinfusion ofHSV-RGS4 or HSV-LacZ directed into dSTR (as depictedin Fig. 1a, top panel) resulted in the expression of FLAG-tagged RGS4 or β-galactosidase restricted to an area of ~1–1.5 mm in diameter as detected 3 days postinfection byimmunohistochemical staining (Fig. 1a, middle panels).Microinfusion of HSV vectors was associated with minimaltissue damage, comparable to the damage seen after HSVvehicle (10% sucrose) as demonstrated by a Nissl stain ofthe dSTR sections collected at the site of infusion (Fig. 1a,lower panel). Overexpression of RGS4 was also confirmedby immunoblotting, again at 3 days postinfection (Fig. 1b).Microinfusion of HSV-RGS4 (but not HSV-LacZ) resulted

624 Psychopharmacology (2012) 221:621–635

in a robust expression of FLAG-tagged RGS4 as detected bythe antibody against the FLAG tag as well as the antibodyagainst the endogenous RGS4 protein (Fig. 1b). Anti-RGS4antibody recognized both endogenous RGS4 and overex-pressed FLAG-tagged RGS4 which has a higher molecularweight due to the presence of the FLAG tag.

We have previously shown that endogenous RGS4 pro-tein coimmunoprecipitates with mGluR5 receptors in ratdSTR (Schwendt and McGinty 2007). In order to assessthe behavioral and neurochemical significance of this inter-action, we first had to examine whether FLAG-tagged RGS4overexpressed in the dSTR also interacts with mGluR5receptors. To that effect, a specific anti-FLAG antibody

was used to immunoprecipitate the RGS4-FLAG proteinfraction from dSTR lysates obtained 3 days after HSV-RGS4-FLAG microinfusion. As illustrated in Fig. 1c (toppanel), probing the immunoprecipitated sample (+++) andtotal (T) striatal lysates with anti-mGluR5 antibody revealed130- and 250-kDa bands corresponding to monomer anddimer isoforms of mGluR5 receptors. In contrast, no bandswere detected in negative control samples in which lysate orFLAG-IP beads were omitted as well as in immunoprecipi-tates from the dSTR with no RGS4-FLAG overexpression.To confirm the efficiency and specificity of immunoprecip-itation, samples were also probed with the antibody againstthe FLAG tag itself. As demonstrated in the middle panel

B

Flag (29 kDa)

β-Gal (110 kDa)

RGS4 (28kDa)

Calnexin (90 kDa)

HSVLacZ

HSV

-

-

--

-

RGS4-Flag (29 kDa)

RGS4-Flag

IP: Flag

IB: mGluR5

IB: Flag

- dimer (250kDa)

- monomer (130 kDa)

- Flag (29kDa)

lysateFlag IP beadsHSV-RGS4-Flag

M T - + + ++ + +-+ + +-

- D1R (~48 kDa)

- D2R (~50kDa)

lysateFlag IP beadsHSV-RGS4-Flag

T - + + ++ + +-+ + +-

IB: D1R

IB: D2R

IP: Flag

C

A + 1.20 mm

Nissl stain

HSV-RGS4-Flag

HSV-LacZ

10x

D

Fig. 1 Infusion of HSV-RGS4 into the dSTR results in overexpressionof RGS4 protein as well as binding of recombinant RGS4 to mGluR5receptors. Animals received bilateral intra-dSTR infusion of HSV-RGS4-FLAG or a control vector HSV-LacZ (2 μl/side), and brainswere analyzed 3 days later. a Upper panel: Outline of the rat braincoronal section depicting a representative placement of HSV infusions,adapted from Paxinos and Watson 2005. Lower panels: Immunohisto-chemical detection of RGS4 and β-galactosidase overexpression in thedSTR near the infusion site (black box represents a tip of the injector).Representative image of Nissl-stained striatal section showing no

necrosis or abnormal cytoarchitecture in the proximity of the infusionsite. b Protein levels of β-galactosidase, RGS4, RGS4-FLAG, andcalnexin as measured by immunoblotting in tissue lysates preparedfrom the dSTR. c FLAG-tagged RGS4 coimmunoprecipitates withmGluR5, but not with dopamine D1 or D2 receptors in the dSTR.(d). Immunoprecipitates (IP) were analyzed by immunoblotting (IB)using antibodies against FLAG tag, mGluR5, or D1 and D2 receptors.T indicates total lysate used as an immunoprecipitation input. co-IPanalysis included negative controls in which lysate, FLAG-IP beads, orRGS4-FLAG overexpression was omitted

Psychopharmacology (2012) 221:621–635 625

(Fig. 1c), FLAG signal was only detected in the total dSTRlysate (T) and complete FLAG tag immunoprecipitates (+++),but not in a series of negative controls. In addition to mGluR5,other Gαi- and Gαq-coupled GPCRs have been shown tointeract with RGS4 protein when coexpressed in various celllines (Jaen and Doupnik 2006; Ruiz de Azua et al. 2010;Wang et al. 2009). In this study, we tested the ability ofFLAG-tagged RGS4 to interact with D1 and D2 dopaminereceptors, which are both abundant in the STR and critical forpsychostimulant-induced behaviors (Berke and Hyman2000). However, coimmunoprecipitation experimentsrevealed no interaction of RGS4-FLAG with D1 or D2 recep-tors in dSTR lysates (Fig. 1d). These results suggest a specificinteraction of overexpressed RGS4 with native mGluR5 (butnot dopamine) receptors in the rat dSTR under these experi-mental conditions.

Overexpression of RGS4 in the dSTR attenuatedmGluR5-dependent behavior and phospho-ERK signaling

In the next series of experiments, we sought to determinewhether increasing levels of RGS4 in the dSTR interferewith mGluR5 function, assessed as a suppression ofmGluR5-dependent behavioral activation and phosphoproteinsignaling.

In agreement with previous observations (Choe andWang 2001), intrastriatal infusion of DHPG (125 and250 nmol) dose-dependently increased phospho-ERK2 lev-els 30 min postinfusion (Fig. 2a, left panel). A one-wayANOVA (F(2,13)04.92, p<0.001) followed by SNK multiplecomparisons revealed not only significantly higherphospho-ERK2 levels in both DHPG-treated groups vs.the vehicle-treated group (p<0.01) but also a significantdose-dependent effect (DHPG250 vs. DHPG125, p<0.05).Similarly, there was a dose-dependent effect of DHPG onphospho-Akt-Thr308 levels in the dSTR (Fig. 2a, right

panel). However, in the latter case, DHPG induced adose-dependent dephosphorylation of Akt-Thr308 as dem-onstrated by one-way ANOVA (F(2,13)012.85, p00.001)followed by SNK multiple comparison test (DHPG250 vs.DHPG125, p<0.05). Because phosphorylation of bothThr308 and Ser473 is thought to be required for fullkinase activity (Alessi et al. 1996; Manning and Cantley2007), the level of Akt phosphorylation at Ser473 wasalso examined throughout this study. However, nochanges in phospho-Akt-Ser473 were found amonggroups in all the experiments (data not shown). It shouldbe noted that the two doses of DHPG used in this studydid not alter the total levels of ERK2 or Akt in the dSTR.In addition, microinfusion of 125 or 250 nmol DHPG hadno effect on protein levels of endogenous RGS4 in thedSTR as measured 2 h postinfusion (Fig. 2b). This wasnot due to the inability of DHPG to induce mGluR5-dependent protein expression because 250 nmol signifi-cantly induced Arc protein levels in the dSTR (data notshown).

In a subsequent study, the effect of RGS4 overexpressionon DHPG-induced behavior and signaling was investigated.Rats received bilateral microinfusions of HSV-RGS4 intodSTR in order to overexpress RGS4, or they were infusedwith HSV-LacZ. Three days later, 250 nmol DHPG (orvehicle) was delivered into the dSTR, and behavior wasanalyzed for the first 30 min after the infusion. As shownin Fig. 3a, DHPG-infused rats demonstrated a robust in-crease in locomotor activity (total distance traveled) whencompared to vehicle-infused rats 20–30 min postinfusion.Quantitative analysis of the total distance traveled over thelast 10 min of the test (20–30 min postinfusion) revealed asignificant difference in AUC between treatment groups asdetected by ANOVA (F(3,14)019.60, p<0.01). Pairwisecomparisons showed that in control animals overexpressingβ-galactosidase in the dSTR, DHPG microinfusion induced

* *

^* *

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

pER

K2

/ tE

RK

2

0

0.5

1

1.5

2

2.5

3

3.5

pAkt

(30

8) /

tAkt

A

* *^

*

BVehDHPG 125DHPG 250

0

0.5

1

1.5

2

2.5

RG

S4

/ cal

nexi

n

Fig. 2 Intrastriatal infusion of mGluR5 agonist DHPG dose-dependentlyregulates phospho-ERK and phospho-Akt-Thr308, but not RGS4 proteinlevels in the dSTR. Animals received bilateral infusion of DHPG (125 or250 nmol), and the brain was analyzed 30 min or 2 h postinfusion. aLevels of phospho-ERK and phospho-Akt-Thr308 in the dSTR as ana-lyzed by immunoblotting 30 min after intrastriatal infusion of DHPG or

vehicle. b RGS4 protein levels in the dSTR as analyzed by immunoblot-ting 2 h after intrastriatal infusion of DHPG or vehicle. Data are expressedas mean±S.E.M. of phospho-ERK2/tERK2, phospho Akt/tAkt, andRGS4/calnexin integrated density ratio (n04–6 samples/group). *p<0.05vs. Veh, **p<0.01 vs. Veh, ^p<0.05 vs. DHPG 125 nmol

626 Psychopharmacology (2012) 221:621–635

a significant increase of the total distance traveled (LacZ-DHPG vs. LacZ-Veh, p<0.01). Interestingly, behavioralactivation by DHPG was blunted in animals overexpressingRGS4 in the dSTR (RGS4-DHPG vs. LacZ-DHPG, p<0.01).The effects of DHPG on vertical activity (rearing) were notsignificantly different in any treatment group (data notshown).

At the end of the experiment (30 min post-Veh/DHPGmicroinfusion), the dSTR was harvested for phosphoproteinanalysis (Fig. 3b, c). In accordance with the previous experi-ment, DHPG induced ERK phosphorylation but to a differentdegree across treatment groups (Fig. 3b;F(3,14)07.84, p<0.01).SNK pairwise comparison tests revealed a significantly greaterincrease in phospho-ERK2 levels in the dSTR of LacZ-DHPGanimals than in LacZ-Veh rats (p<0.01). In addition, RGS4overexpression suppressed DHPG-induced phospho-ERK2levels (RGS4-DHPG vs. LacZ-DHPG, p<0.01). On the otherhand, DHPG-induced dephosphorylation of Akt-Thr308 wasnot prevented by RGS4 overexpression (Fig. 3c). One-wayANOVA (F(3,14)010.29, p<0.01) followed by SNK pairwisecomparisons revealed that phospho-Akt-Thr308 levels weredownregulated by DHPG regardless of β-galactosidase orRGS4 overexpression (LacZ-DHPG vs. LacZ-Veh, p<0.01and RGS4-DHPG vs. RGS4-LacZ, p<0.05).

The effects of RGS4 overexpression in the dSTRon early-onset Amph-induced locomotion and phosphoproteinsignaling: comparison to intra-dSTR mGluR5 receptorblockade

The preceding experiments suggested that RGS4 regulatesmGluR5-mediated locomotor behavior and ERK phos-phorylation in the dSTR. Follow-up experiments soughtto determine whether RGS4–mGluR5 functional interac-tions play a role in psychostimulant-induced behaviorsand protein phosphorylation in the dSTR. Therefore, theeffects of RGS4 overexpression were compared to asite-specific blockade of mGluR5s in the dSTR afteracute Amph exposure.

Animals received microinfusion of HSV-LacZ or HSV-RGS4 into the dSTR, and 3 days postinfection, locomotor

0

500

1000

1500

2000

2500

5 10 15 20 25 30 35 40 45 50 55 60 5 10 15 20 25 30

LacZ-VehLacZ-DHPG **RGS4-VehRGS4-DHPG ^^

tota

l dis

tan

ce (

cm /

5 m

in)

time (min)

A

B

* *

^

0

0.2

0.4

0.6

0.8

1

1.2

1.4

pE

RK

2/tE

RK

2

LacZ-VehLacZ-DHPGRGS4-VehRGS4-DHPG

- pERK1

- pERK2

- tERK1

- tERK2

Veh DHPG Veh DHPG

LacZ RGS4

* *#

0

0.2

0.4

0.6

0.8

1

1.2

1.4 LacZ-VehLacZ-DHPGRGS4-VehRGS4-DHPG

pA

kt (

308)

/tA

kt

- pAkt (308)

- tAkt

Veh/DHPG

Veh DHPG Veh DHPG

LacZ RGS4

C

Fig. 3 Overexpression of RGS4 in the dSTR attenuates DHPG-inducedbehavior and ERK phosphorylation. Animals received bilateral infusionof HSV-RGS4-FLAG or a control vector HSV-LacZ (2 μl/side) followedby the bilateral infusion of either DHPG (250 nmol) or vehicle into thedorsolateral STR 3 days later. a Locomotor activity (total distance trav-eled) as recorded for 60 min preinfusion and 30 min postinfusion ofDHPG. Mean total distance traveled (per 5 min)±S.E.M. plotted overtime and analyzed as AUC. *p<0.05 vs. LacZ-Vehicle, ^p<0.05 RGS4-DHPG vs. LacZ-DHPG. Immunoblotting analysis of phospho-ERK (b)and phospho-Akt-Thr308 (c) levels in the dSTR 30 min after the infusionof DHPG or Veh. Mean integrated density ratio±S.E.M. of phospho-ERK2/tERK2 and phospho-Akt-Thr308/tAkt (n04–5 samples/group).*p<0.05 vs. LacZ-Vehicle, **p<0.01 vs. LacZ-Vehicle, ^p<0.05 vs.LacZ-DHPG, #p<0.05 vs. RGS4-Vehicle

R

Psychopharmacology (2012) 221:621–635 627

and phosphorylation responses to Amph were measured(Fig. 4). A single injection of Amph (2.5 mg/kg, i.p.) in-duced significant locomotor hyperactivity (F(3,19)04.57, p<0.05) in both LacZ-Amph and RGS4-Amph groups whencompared to their saline counterparts (Fig. 4a; LacZ-Amphvs. LacZ-Sal, p<0.05 and RGS4-AMPH vs. RGS4-Sal, p<0.05). Phosphoprotein analysis revealed that Amph in-creased ERK2 phosphorylation as previously reported (Shiand McGinty 2007), although to a different degree acrosstreatment groups (Fig. 4b; F(3,20)06.64, p<0.05). There wasa significant increase in phospho-ERK2 levels in the LacZ-Amph group (p<0.01, vs. LacZ-Sal), which was attenuatedin animals overexpressing RGS4 (RGS4-Amph vs. LacZ-Amph, p <0.05; Fig. 4b). Amph treatment also inducedphospho-Akt-Thr308 levels (F(3,21)04.24, p<0.05) as pre-viously reported (Shi and McGinty 2007). SNK testsrevealed that phospho-Akt-Thr308 levels were significantlygreater in the LacZ-Amph group than in the LacZ-Sal group(p<0.05). RGS4 overexpression did not attenuate Amph-induced phosphorylation of Akt-Thr308 when compared toLacZ-Sal animals (p00.4); nevertheless, it prevented theAmph-induced rise in phospho-Akt308 levels (RGS4-Amphvs. RGS4-Sal, p00.21; Fig. 4c).

In the next experiment, the selective mGluR5 antagonist,MTEP, was bilaterally infused into dSTR 15 min beforeAmph (2.5 mg/kg i.p.) or Sal injection. Amph inducedsignificant locomotor hyperactivity (total distance traveled)when compared to Sal rats (t(21)02.77, p<0.05) during the15-min period following i.p. injections (Fig. 4d). However,no group-specific effects of Veh or MTEP pretreatment onAmph-induced locomotor activity emerged during this shorttime interval. As expected, acute Amph induced an increasein phospho-ERK2 levels (Fig. 4e; F(3,19)010.96, p<0.01).SNK multiple comparison tests showed that elevatedphospho-ERK2 levels were present in the Veh-Amph groupwhen compared to the Veh-Sal group (p<0.01), whereasphospho-ERK2 was significantly suppressed in the Amphgroup pretreated with MTEP (MTEP-Amph vs. Veh-Amph,p<0.01). Amph also induced phospho-Akt-Thr308 levels(F(3,19)03.44, p<0.05), specifically in Veh-pretreated ani-mals (SNK test: p<0.05, Veh-Sal vs. Veh-Amph). MTEPmicroinfusion showed a trend toward partial inhibition ofAmph-induced phosphorylation of Akt-Thr308 (p00.06,Fig. 4f).

The effects of RGS4 overexpression in the dSTRor intra-dSTR mGluR5 receptor blockade on extendedbehavioral activity after acute Amph

The previous experiments suggested that RGS4 and mGluR5may share a commonmechanism in regulating Amph-inducedphosphoprotein signaling in the dSTR. However, due to theshort time period of analysis, it remained unclear whether

local manipulation of RGS4 (and/or mGluR5s) in the dSTRalters the overall pattern of post-Amph behavioral activation.Therefore, in the following experiment, the effects of RGS4overexpression or mGluR5 inhibition in the dSTR on Amph-induced behaviors were measured over a 2-h period.

First, HSV-LacZ or HSV-RGS4 was microinfused into thedSTR as illustrated by the distribution of cannulae placementsites in Fig. 5a. Three days later, rats were injected i.p. with Salor Amph (2.5 mg/kg), and behavioral activity was recordedfor the following 2 h. Quantitative analysis of the total dis-tance traveled (over the period of peak activity, 15–90 minpostinjection) revealed significant differences between AUCvalues among the treatment groups as detected by ANOVA(F(3,13)014.06, p<0.01; Fig. 5b). Pairwise SNK comparisonsshowed that in LacZ animals, Amph induced a significantincrease in the total distance traveled (LacZ-Amph vs. LacZ-Sal, p<0.01). Interestingly, Amph-induced distance traveledwas augmented in animals overexpressing RGS4 in the dSTR(RGS4-Amph vs. LacZ-Amph, p<0.05). Similarly, Amphsignificantly increased the peak vertical activity of animals(F(3,13)017.93, p<0.01; Fig. 5c). Further, pairwise compari-sons revealed that Amph induced a significant increase invertical activity of LacZ animals (LacZ-Amph vs. LacZ-Sal,p<0.01). In contrast to horizontal activity, Amph-inducedvertical activity was blunted in animals overexpressingRGS4 in the dSTR (RGS4-Amph vs. LacZ-Amph, p<0.05;Fig. 5c).

In the next experiment, MTEP was microinfused intodSTR 15 min before an i.p. Sal or Amph (2.5 mg/kg)

Fig. 4 The effects of RGS4 (or blockade of mGluR5 receptors) onearly onset Amph-induced behavior and phosphoprotein levels in thedSTR. a–c Left column: Animals received bilateral infusion of HSV-RGS4-FLAG or a control vector HSV-LacZ (2 μl/side). Three dayslater, animals received Amph (2.5 mg/kg, i.p) or saline injection, andbehavioral activity and phosphoprotein levels in the dSTR were ana-lyzed. a Total distance traveled as recorded for 60 min pre- and 15 minpost-Sal or Amph administration. Mean total distance traveled (per5 min)±S.E.M. plotted over time and analyzed as AUC. *p<0.05 vs.LacZ-Sal, ^p<0.05 RGS4-Amph vs. LacZ-Amph. b, c Upper panels:representative immunoblot images for each treatment group. Lowerpanels: immunoblotting analysis of phospho-ERK2/tERK2 (b) andphospho-Akt-Thr308/tAkt (c) levels in the dSTR 20 min after theinjection of Sal or Amph. Mean integrated density ratio±S.E.M. ofphospho-ERK2/tERK2 and phospho-Akt-Thr308/tAkt (n05–7 sam-ples/group). *p<0.05, **p<0.01 vs. LacZ-Sal, ^p<0.01 vs. LacZ-Amph. d–f Right column: Animals received bilateral infusion of Vehor MTEP (5 μg/side). Fifteen minutes later, animals received Amph(2.5 mg/kg, i.p) or saline injection, and behavioral activity and phos-phoprotein levels in the dSTR were analyzed. d Total distance traveledas recorded for 60-min pre- and 15-min post-Sal or Amph administra-tion. Mean total distance traveled (per 5 min)±S.E.M. plotted overtime and analyzed as AUC. e, f Upper panels: Representative immu-noblot images for each treatment group. Lower panels: Immunoblot-ting analysis of phospho-ERK2/tERK2 (e) and pAkt-Thr308/tAkt (f)levels in the dSTR 20 min after the injection of Sal or Amph. Meanintegrated density ratio±S.E.M. of phospho-ERK2/tERK2 and pAkt-Thr308/tAkt (n05–7 samples/group). *p<0.05, *p<0.01 vs. Veh-Sal,^^p<0.01 vs. Veh-Amph

b

628 Psychopharmacology (2012) 221:621–635

B E

C F

* *

^ ^

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

pE

RK

2/tE

RK

2

Veh-Sal

Veh-Amph

MTEP-Sal

MTEP-Amph

*

0

0.2

0.4

0.6

0.8

1

1.2

1.4

pA

kt (

308)

/tA

kt

Veh-Sal

Veh-Amph

MTEP-Sal

MTEP-Amph

A D

0

500

1000

1500

2000

0 10 20 30 40 50 60 5 10 15

time (mins)

tota

l dis

tan

ce (

cm /

5min

)Sal/Amph

0

500

1000

1500

0 10 20 30 40 50 60 0 5 10 15

tota

l dis

tan

ce (

cm /

5min

)

time (mins)

Sal/AmphVeh/MTEP

Sal Amph Sal Amph

LacZ RGS4

*

0

0.2

0.4

0.6

0.8

1

pA

kt (

308)

/tA

kt

LacZ-Sal

LacZ-Amph

RGS4-Sal

RGS4-Amph

* *

^

0

0.2

0.4

0.6

0.8

1

1.2

1.4

pE

RK

2/tE

RK

2

LacZ-Sal

LacZ-Amph

RGS4-Sal

RGS4-Amph

-pERK1

-pERK2

- tERK1

- tERK2

Sal Amph Sal Amph

LacZ RGS4

- pAkt (308)

- tAkt

Sal Amph Sal Amph

Veh MTEP

-pERK1

-pERK2

- tERK1

- tERK2

Sal Amph Sal Amph

Veh MTEP

- pAkt (308)

- tAkt

LacZ-SalLacZ-AmphRGS4-SalRGS4-Amph

Veh-SalVeh-AmphMTEP-SalMTEP-Amph

Psychopharmacology (2012) 221:621–635 629

D

0

750

1500

2250

3000

3750

0 15 30 45 60 15 30 45 60 75 90 105 120

A

time (min)

0

100

200

300

400

500

600

700

0 15 30 45 60 15 30 45 60 75 90 105 120

time (min)

bea

m b

reak

s / 5

min

tota

l dis

tan

ce (

cm/5

min

)

FC

B E

+0.84 mm

+1.20 mm

+1.68 mm

Sal/Amph

LacZ-Sal (AUC=8723)

RGS4-Sal (AUC=7894)RGS4-Amph (AUC=149704)^

LacZ-Amph (AUC=105711)**

LacZ-Sal (AUC=678)

RGS4-Sal (AUC=591)RGS4-Amph (AUC=16429)^

LacZ-Amph (AUC=23888)**

Sal/Amph

tota

l dis

tan

ce (

cm/5

min

)

0

1000

2000

3000

4000

5000

6000

0 15 30 45 60 15 30 45 60 75 90 105 120

time (min)

Sal/AmphVeh/MTEP

0

100

200

300

400

500

600

0 15 30 45 60 15 30 45 60 75 90 105 120

Veh/MTEP

Sal/Amph

time (min)

bea

m b

reak

s / 5

min

Veh-Sal (AUC=366)

MTEP-Sal (AUC=1664)MTEP-Amph (AUC=269849)^

Veh-Amph (AUC=173507)**

Veh-Sal (AUC=0)

MTEP-Sal (AUC=122)MTEP-Amph (AUC=17132)

Veh-Amph (AUC=23880)**

+1.08 mm

+1.20 mm

+1.80 mm

+0.36 mm +0.48 mm

630 Psychopharmacology (2012) 221:621–635

injection, and behavior was again monitored for 2 h. Distri-bution of individual cannulae placement sites is shown inFig. 5d. Quantitative analysis of the total distance traveled(over the period of the peak Amph-induced activity, 15–90 min postinjection) revealed significant differences be-tween AUC values among the treatment groups as detectedby ANOVA (F(3,13)018.48, p<0.01; Fig. 5e). Pairwise SNKcomparisons showed that in Veh-treated rats, Amph induceda significant increase in the total distance traveled (Veh-Amph vs. Veh-Sal, p<0.01, Fig. 5e). In a striking similarityto the effect of RGS4 overexpression, Amph-induced dis-tance traveled was augmented in animals that received intra-striatal MTEP infusion (MTEP-Amph vs. Veh-Amph, p<0.05). As expected, Amph significantly increased the peakvertical activity of rats (F(3,13)011.55, p<0.01; Fig. 5f).Pairwise comparisons revealed that Amph induced a signif-icant increase in the vertical activity of Veh-treated rats(Veh-Amph vs. Veh-Sal, p<0.01). In contrast to RGS4 over-expression, Amph-induced vertical activity was not bluntedby intrastriatal administration of MTEP (MTEP-Amph vs.Veh-Amph, p00.15).

Discussion

The present study investigated the role of striatal RGS4protein in regulating mGluR5- and Amph-dependent cellu-lar signaling and behavioral output. First, infusion of HSV-RGS4 into the dSTR resulted in a robust overexpression ofRGS4 protein, which coimmunoprecipitated with mGluR5receptors. Second, overexpression of RGS4 in the dSTRsuppressed early locomotor activation as well as increased

phospho-ERK2 levels induced by local administration of themGluR5 agonist, DHPG. However, RGS4 overexpressiondid not reverse DHPG-induced dephosphorylation of Akt-Thr308 in the dSTR. Third, HSV-driven RGS4 overexpres-sion attenuated Amph-induced phospho-ERK2 levels in amanner similar to the mGluR5 antagonist, MTEP. In con-trast, RGS4 (and MTEP) only partially inhibited phospho-Akt-Thr308 upregulation by Amph. Fourth, although nei-ther RGS4 nor MTEP showed an effect on early onsetAmph-induced behaviors, analysis of the extended 3-h behavioral profile of acute Amph revealed that RGS4overexpression caused a bidirectional regulation of behav-ior, augmenting peak Amph-induced horizontal activitywhile suppressing peak vertical activity. Similarly, micro-infusion of MTEP into dSTR also augmented peak Amph-induced horizontal locomotion.

A growing body of evidence suggests that group I mGluRsplay an important role in the behavioral and neurochemicalactions of Amph as well as other abused drugs (for review, seeBird and Lawrence 2009). As such, systemic administration ofmGluR5 antagonists inhibited Amph-induced hyperactivity(Gormley and Rompre 2011;McGeehan et al. 2004; Pietraszeket al. 2004) and expression of Amph-conditioned place prefer-ence (Herzig et al. 2005). However, some studies providedcontradictory evidence regarding the role of mGluR5 in theregulating, rewarding, and locomotor-stimulating effects ofpsychostimulants (Gormley and Rompre 2011; McGeehanand Olive 2003; Veeneman et al. 2011). Discrepancies couldstem from the fact that whereas different drugs exert region-specific effects on the brain, a population of mGluR1/5 recep-tors in the whole brain was typically blocked in these studies.Furthermore, there is only limited information regardingbrain region-specific effects of mGluR1/5 antagonists onpsychostimulant-induced behavior and cellular signaling.In one study, intra-dSTR blockade of group I mGluRssuppressed Amph-induced ERK and CREB phosphoryla-tion within this brain region (Choe et al. 2002). Thisfinding was not surprising considering that (1) STRreceives major excitatory inputs from all regions of thecortex and from the thalamus (McGeorge and Faull1989; Mengual et al. 1999; Smith et al. 2004), (2) striatalmedium spiny neurons are densely populated with gluta-mate receptors, including group I mGluRs, and (3)Amph-induced glutamate release plays an important rolein controlling Amph-induced behaviors and activation ofimmediate early genes (Ferguson and Robinson 2004;Gray et al. 1999; Mao and Wang 1999). The presentstudy was the first one to investigate the effects ofintra-dSTR delivery of an mGluR5 antagonist on bothcellular signaling and behavior after acute Amph. Thesuppressive effect of MTEP on ERK phosphorylation isin agreement with the previous findings (Choe and Wang2001) and is likely mediated by a suppression of both

Fig. 5 The effects of RGS4 overexpression (or a blockade of mGluR5receptors) in the dSTR on extended Amph-induced behavioral activity.a–c Left column: Animals received bilateral infusion of HSV-RGS4-FLAG or a control vector HSV-LacZ (2 μl/side) into dSTR. Three dayslater, animals received Amph (2.5 mg/kg, i.p) or saline injection, andbehavioral activity was recorded for 2 h. a Outline of the rat braincoronal section depicting distribution of cannulae placements adaptedfrom Paxinos and Watson 2005. b Total distance traveled as recordedfor 60 min predrug and 120 min postdrug administration. Mean totaldistance traveled (per 5 min)±S.E.M. plotted over time and analyzed asAUC. **p<0.01 vs. LacZ-Sal, ^p<0.05 RGS4-Amph vs. LacZ-Amph.c Vertical activity (rearing) is shown as mean number of beam breaks(per 5 min)±S.E.M. plotted over time and analyzed as area under thecurve. **p<0.01 vs. LacZ-Sal, ^p<0.05 RGS4-Amph vs. LacZ-Amph.d–f Right column: Animals received bilateral infusion of Veh or MTEP(5 μg/side). Fifteen minutes later, animals received Amph (2.5 mg/kg,i.p) or saline injection, and behavioral activity was recorded for 2 h. dOutline of the rat brain coronal section depicting distribution of cannulaeplacements adapted from Paxinos and Watson 2005. e Total distancetraveled as recorded for 60 min pre- and 120 min post-Sal or Amphadministration. Mean total distance traveled (per 5 min)±S.E.M. plottedover time and analyzed as AUC. **p<0.01 vs. Veh-Sal, ^p<0.05MTEP-Amph vs. Veh-Amph. f Vertical activity (rearing) is shown as meannumber of beam breaks (per 5 min)±S.E.M. plotted over time andanalyzed as area under the curve. **p<0.01 vs. Veh-Sal

R

Psychopharmacology (2012) 221:621–635 631

conventional Gαq/PLCβ1- and Homer-dependent path-ways (Mao et al. 2005). The precise mechanism ofmGluR5-mediated regulation of Akt activity is not clear,but it seems to be of a transient character. In the hippo-campus, administration of DHPG resulted in a rapidincrease in phospho-Akt308 at 5 min, returning back tobaseline levels within 20 min postadministration (Houand Klann 2004). Further, the current study suggests thatDHPG treatment actually produces delayed dephosphor-ylation of Akt-Thr308 in the dSTR 30 min after thetreatment. Surprisingly, manipulating mGluR5 function(by RGS4 or MTEP) in combination with DHPG orAmph treatment revealed none or very limited controlof this receptor over the phosphorylation/dephosphoryla-tion equilibrium of Akt-Thr308 in the dSTR. In agree-ment, delayed dephosphorylation of phospho-Akt308 inthe STR after Amph treatment has been found to dependon the activation of D2 receptors (Beaulieu et al. 2007;Shi and McGinty 2011), the GPCRs not likely regulatedby RGS4 (Ghavami et al. 2004; Ding et al. 2006).

A unique observation of the current study was the strikingresemblance between the effects of an mGluR5 antagonistand those of RGS4 overexpression, supporting the hypothe-sis that RGS4 negatively regulates mGluR5 signaling in thedSTR. Indeed, overexpression of RGS4 in nonneuronal celllines inhibited Gαq-coupled GPCRs and attenuated MAPKactivation (Yan et al. 1997). It is also significant that both thelong-form Homer protein and RGS4 are necessary to fine-tune calcium oscillations downstream of Gαq-coupledreceptors (Shin et al. 2003). Taken together, these findingssuggest that aberrant regulation of RGS4 levels has directconsequences for the cellular signaling downstream ofmGluR5 receptors. Since abused drugs (for the most part)downregulate RGS4 levels in the striatum, it could be hy-pothesized that this leads to hypersensitivity of mGluR5receptors manifested as augmented ERK phosphorylationduring reexposure to the drug or drug-paired cues (Valjentet al. 2006; Schwendt et al. 2010).

Besides cellular signaling, overexpression of RGS4and mGluR5 inhibition altered Amph-induced behavioralprofiles in a similar, but complex, manner. It is possiblethat both the mGluR5 antagonist and RGS4 decrease thebehavioral efficacy of Amph since increased time spentin horizontal activity at the expense of vertical activitycorresponds to a behavioral repertoire characteristic oflower doses of Amph (Antoniou and Kafetzopoulos1991; Kuczenski and Segal 1989; Wang and McGinty1995). It is likely that cellular mechanisms of this com-plex behavioral shift are not limited to a single signalingpathway or exclusive to inhibition of mGluR5 receptorsin the dSTR. For example, direct inhibition of ERKactivity results in suppression of both horizontal andvertical post-Amph hyperactivities (Shi and McGinty

2006; Sutton et al. 2000), whereas selective inhibitionof PI3K/Akt activity in the dSTR results in a behavioralprofile similar to the one observed in animals with RGS4overexpression (Shi et al. 2009). However, we observedthat both MTEP administration and RGS4 overexpressionresult in greater inhibition of ERK than Akt activity inthe STR. It is possible that in our study, manipulating themGluR5 function has different outcomes depending onthe cell-type-specific localization of the receptor. Al-though mGluR5s are abundantly expressed in both D1-and D2-positive populations of striatal medium spinyneurons (Tallaksen-Greene et al. 1998), evidence sug-gests that mGluR5 receptors are colocalized and closelyinteract with D1 receptors to regulate striatal neurotrans-mission (Paolillo et al. 1998; Schotanus and Chergui2008; Voulalas et al. 2005) and many of the long-termeffects of addictive drugs (Novak et al. 2010). On theother hand, mGluR5s can dimerize with D2 receptorsresulting in suppressed function and cellular signalingof D2 receptors present in this heterodimer (Fuxe et al.2009). Thus, inhibition of mGluR5 function (by MTEPor RGS4) could also alter Amph-induced activation ofD2 receptors and behaviors mediated by the indirect(striatopallidal) pathway. Due to the complex characterof signaling and behavioral consequences of mGluR5(and RGS4) manipulations in the dSTR, future studieswill have to examine the functional mGluR5/RGS4 rela-tionship in a cell-type-specific manner.

In conclusion, the data presented in this study shownovel evidence for the significance of mGluR5 receptorsand mGluR5–RGS4 interactions in the dSTR in modu-lating psychostimulant-induced behaviors and signaling.Genetic disruption of either mGluR5 (Chiamulera et al.2001) or mGluR5-interacting proteins (Atkinson et al.2006; Chuang et al. 2001; Kammermeier et al. 2000;Swanson et al. 2001) produces profound alterations inthe responses to psychomotor stimulants, similar to directpharmacological inhibition of group I mGluRs (Herzigand Schmidt 2004; McGeehan and Olive 2003). Sinceour data suggest that RGS4 tightly regulates mGluR5signaling, dynamic regulation of striatal RGS4 levels bypsychostimulants could have a dramatic effect on thebehavioral and neural functions of mGluR5 receptors.Therefore, modulating RGS4–mGluR5 interactions mayrepresent a novel mechanism suitable for therapeuticinterventions for psychostimulant addictions as well asfor other psychiatric disorders (Sjogren et al. 2010).

Acknowledgments The authors would like to thank Amena Smith,Adrian Gomez, and John Yang for their excellent technical assistance.This work was supported by the National Institutes of Health grantsR01 DA03982 (JFM), R21 DA025846 (MS), and CO6 RR01 5155from the Extramural Research Facilities Program of the NationalCenter for Research Resources.

632 Psychopharmacology (2012) 221:621–635

References

Abramow-Newerly M, Roy AA, Nunn C, Chidiac P (2006) RGSproteins have a signalling complex: interactions between RGSproteins and GPCRs, effectors, and auxiliary proteins. Cell Signal18:579–591

Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P,Hemmings BA (1996) Mechanism of activation of protein kinaseB by insulin and IGF-1. EMBO J 15:6541–6551

Antoniou K, Kafetzopoulos E (1991) A comparative study of thebehavioral effects of D-amphetamine and apomorphine in therat. Pharmacol Biochem Behav 39:61–70

Atkinson PJ, Young KW, Ennion SJ, Kew JN, Nahorski SR, ChallissRA (2006) Altered expression of G(q/11alpha) protein shapesmGlu1 and mGlu5 receptor-mediated single cell inositol 1,4,5-trisphosphate and Ca(2+) signaling. Mol Pharmacol 69:174–184

Bansal G, Druey KM, Xie Z (2007) R4 RGS proteins: regulation of G-protein signaling and beyond. Pharmacol Ther 116:473–495

Beaulieu JM, Tirotta E, Sotnikova TD,Masri B, Salahpour A, GainetdinovRR, Borrelli E, CaronMG (2007) Regulation of Akt signaling by D2and D3 dopamine receptors in vivo. J Neurosci 27:881–885

Berke JD, Hyman SE (2000) Addiction, dopamine, and the molecularmechanisms of memory. Neuron 25:515–532

Bird MK, Lawrence AJ (2009) Group I metabotropic glutamate recep-tors: involvement in drug-seeking and drug-induced plasticity.Curr Mol Pharmacol 2:83–94

Carlezon WA Jr, Nestler EJ, Neve RL (2000) Herpes simplex virus-mediated gene transfer as a tool for neuropsychiatric research. CritRev Neurobiol 14:47–67

Chase SL, Taussig RL, Neve DW, Self DW (2010) The role of RGS4in cocaine and D2 sensitization. (Neuroscience Meeting Planner.Online.). Society for Neuroscience, San Diego, CA, USA, pp770.25

Chiamulera C, Epping-Jordan MP, Zocchi A, Marcon C, Cottiny C,Tacconi S, Corsi M, Orzi F, Conquet F (2001) Reinforcing andlocomotor stimulant effects of cocaine are absent in mGluR5 nullmutant mice. Nat Neurosci 4:873–874

Choe ES, Wang JQ (2001) Group I metabotropic glutamate receptoractivation increases phosphorylation of cAMP response element-binding protein, Elk-1, and extracellular signal-regulated kinasesin rat dorsal striatum. Brain Res Mol Brain Res 94:75–84

Choe ES, Chung KT, Mao L, Wang JQ (2002) Amphetamine increasesphosphorylation of extracellular signal-regulated kinase and tran-scription factors in the rat striatum via group I metabotropicglutamate receptors. Neuropsychopharmacology 27:565–575

Chuang SC, Bianchi R, Kim D, Shin HS, Wong RK (2001) Group Imetabotropic glutamate receptors elicit epileptiform discharges inthe hippocampus through PLCbeta1 signaling. J Neurosci 21:6387–6394

Conn PJ, Pin JP (1997) Pharmacology and functions of metabotropicglutamate receptors. Annu Rev Pharmacol Toxicol 37:205–237

Ding L, Hegde AN (2009) Expression of RGS4 splice variants indorsolateral prefrontal cortex of schizophrenic and bipolar disor-der patients. Biol Psychiatry 65:541–545

Ding J, Guzman JN, Tkatch T, Chen S, Goldberg JA, Ebert PJ, Levitt P,Wilson CJ, Hamm HE, Surmeier DJ (2006) RGS4-dependent atten-uation of M4 autoreceptor function in striatal cholinergic interneur-ons following dopamine depletion. Nat Neurosci 9:832–842

Emilsson L, Saetre P, Jazin E (2006) Low mRNA levels of RGS4splice variants in Alzheimer’s disease: association between a rarehaplotype and decreased mRNA expression. Synapse 59:173–176

Ferguson SM, Robinson TE (2004) Amphetamine-evoked gene ex-pression in striatopallidal neurons: regulation by corticostriatalafferents and the ERK/MAPK signaling cascade. J Neurochem91:337–348

Fourgeaud L, Mato S, Bouchet D, Hemar A, Worley PF, Manzoni OJ(2004) A single in vivo exposure to cocaine abolishesendocannabinoid-mediated long-term depression in the nucleusaccumbens. J Neurosci 24:6939–6945

Fuxe K, Marcellino D, Woods AS, Giuseppina L, Antonelli T, FerraroL, Tanganelli S, Agnati LF (2009) Integrated signaling in hetero-dimers and receptor mosaics of different types of GPCRs of theforebrain: relevance for schizophrenia. J Neural Transm 116:923–939

Gass JT, Olive MF (2009) Role of protein kinase C epsilon (PKCvar-epsilon) in the reduction of ethanol reinforcement due to mGluR5antagonism in the nucleus accumbens shell. Psychopharmacology(Berl) 204:587–597

Ghavami A, Hunt RA, Olsen MA, Zhang J, Smith DL, Kalgaonkar S,Rahman Z, Young KH (2004) Differential effects of regulator ofG protein signaling (RGS) proteins on serotonin 5-HT1A, 5-HT2A, and dopamine D2 receptor-mediated signaling andadenylyl cyclase activity. Cell Signal 16:711–721

Girault JA, Valjent E, Caboche J, Herve D (2007) ERK2: a logicalAND gate critical for drug-induced plasticity? Curr Opin Pharma-col 7:77–85

Gold SJ, Ni YG, Dohlman HG, Nestler EJ (1997) Regulators of G-protein signaling (RGS) proteins: region-specific expression ofnine subtypes in rat brain. J Neurosci 17:8024–8037

Gonzalez-Nicolini V, McGinty JF (2002) Gene expression profile fromthe striatum of amphetamine-treated rats: a cDNA array and insitu hybridization histochemical study. Brain Res Gene ExprPatterns 1:193–198

Gormley S, Rompre PP (2011) Blockade of mGLUR5 receptors dif-ferentially alters amphetamine-induced enhancement of locomo-tor activity and of brain stimulation reward. J Psychopharmacol25:393–401

Gray AM, Rawls SM, Shippenberg TS, McGinty JF (1999) The kappa-opioid agonist, U-69593, decreases acute amphetamine-evokedbehaviors and calcium-dependent dialysate levels of dopamineand glutamate in the ventral striatum. J Neurochem 73:1066–1074

Grueter BA, Gosnell HB, Olsen CM, Schramm-Sapyta NL, NekrasovaT, Landreth GE, Winder DG (2006) Extracellular-signal regulatedkinase 1-dependent metabotropic glutamate receptor 5-inducedlong-term depression in the bed nucleus of the stria terminalis isdisrupted by cocaine administration. J Neurosci 26:3210–3219

Han MH, Renthal W, Ring RH, Rahman Z, Psifogeorgou K, HowlandD, Birnbaum S, Young K, Neve R, Nestler EJ, Zachariou V(2010) Brain region specific actions of regulator of G proteinsignaling 4 oppose morphine reward and dependence but promoteanalgesia. Biol Psychiatry 67:761–769

Herzig V, Schmidt WJ (2004) Effects of MPEP on locomotion, sensi-tization and conditioned reward induced by cocaine or morphine.Neuropharmacology 47:973–984

Herzig V, Capuani EM, Kovar KA, Schmidt WJ (2005) Effects ofMPEP on expression of food-, MDMA- or amphetamine-conditioned place preference in rats. Addict Biol 10:243–249

Hooks SB, Martemyanov K, Zachariou V (2008) A role of RGSproteins in drug addiction. Biochem Pharmacol 75:76–84

Hou L, Klann E (2004) Activation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway is re-quired for metabotropic glutamate receptor-dependent long-termdepression. J Neurosci 24:6352–6361

Jaen C, Doupnik CA (2006) RGS3 and RGS4 differentially associatewith G protein-coupled receptor-Kir3 channel signaling com-plexes revealing two modes of RGS modulation. Precouplingand collision coupling. J Biol Chem 281:34549–34560

Kammermeier PJ, Xiao B, Tu JC, Worley PF, Ikeda SR (2000) Homerproteins regulate coupling of group I metabotropic glutamatereceptors to N-type calcium and M-type potassium channels. JNeurosci 20:7238–7245

Psychopharmacology (2012) 221:621–635 633

Kimple AJ, Bosch DE, Giguere PM, Siderovski DP (2011) Regulatorsof G-protein signaling and their Galpha substrates: promises andchallenges in their use as drug discovery targets. Pharmacol Rev63:728–749

Kuczenski R, Segal D (1989) Concomitant characterization of behav-ioral and striatal neurotransmitter response to amphetamine usingin vivo microdialysis. J Neurosci 9:2051–2065

Manning BD, Cantley LC (2007) AKT/PKB signaling: navigatingdownstream. Cell 129:1261–1274

Mao L, Wang JQ (1999) Protection against acute amphetamine-induced behavior by microinjection of a group II metabotropicglutamate receptor agonist into the dorsal striatum of rats. Neuro-sci Lett 270:103–106

Mao L, Yang L, Tang Q, Samdani S, Zhang G, Wang JQ (2005) Thescaffold protein Homer1b/c links metabotropic glutamate receptor5 to extracellular signal-regulated protein kinase cascades in neu-rons. J Neurosci 25:2741–2752

McGeehan AJ, Olive MF (2003) The mGluR5 antagonist MPEPreduces the conditioned rewarding effects of cocaine but not otherdrugs of abuse. Synapse 47:240–242

McGeehan AJ, Janak PH, Olive MF (2004) Effect of the mGluR5antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP) on theacute locomotor stimulant properties of cocaine, D-amphetamine,and the dopamine reuptake inhibitor GBR12909 in mice. Psycho-pharmacology (Berl) 174:266–273

McGeorge AJ, Faull RL (1989) The organization of the projectionfrom the cerebral cortex to the striatum in the rat. Neuroscience29:503–537

Mengual E, de las Heras S, Erro E, Lanciego JL, Gimenez-Amaya JM(1999) Thalamic interaction between the input and the outputsystems of the basal ganglia. J Chem Neuroanat 16:187–200

Mirnics K, Middleton FA, Stanwood GD, Lewis DA, Levitt P (2001)Disease-specific changes in regulator of G-protein signaling 4(RGS4) expression in schizophrenia. Mol Psychiatry 6:293–301

Molina-HernandezM, Tellez-Alcantara NP, Perez-Garcia J, Olivera-LopezJI, Jaramillo MT (2006) Antidepressant-like and anxiolytic-likeactions of the mGlu5 receptor antagonist MTEP, microinjected intolateral septal nuclei of maleWistar rats. Prog NeuropsychopharmacolBiol Psychiatry 30:1129–1135

Neve RL, Howe JR, Hong S, Kalb RG (1997) Introduction of theglutamate receptor subunit 1 into motor neurons in vitro and invivo using a recombinant herpes simplex virus. Neuroscience79:435–447

Nishiguchi KM, Sandberg MA, Kooijman AC, Martemyanov KA, PottJW, Hagstrom SA, Arshavsky VY, Berson EL, Dryja TP (2004)Defects in RGS9 or its anchor protein R9AP in patients with slowphotoreceptor deactivation. Nature 427:75–78

Novak M, Halbout B, O’Connor EC, Rodriguez Parkitna J, Su T,Chai M, Crombag HS, Bilbao A, Spanagel R, Stephens DN,Schutz G, Engblom D (2010) Incentive learning underlyingcocaine-seeking requires mGluR5 receptors located on dopa-mine D1 receptor-expressing neurons. J Neurosci 30:11973–11982

Paolillo M, Montecucco A, Zanassi P, Schinelli S (1998) Potentiationof dopamine-induced cAMP formation by group I metabotropicglutamate receptors via protein kinase C in cultured striatal neu-rons. Eur J Neurosci 10:1937–1945

Paxinos G, Watson C (2007) The rat brain in sterotaxic coordinates, 6thedn. Academic Press, San Diego

Pietraszek M, Rogoz Z, Wolfarth S, Ossowska K (2004) Oppositeinfluence of MPEP, an mGluR5 antagonist, on the locomotorhyperactivity induced by PCP and amphetamine. J Physiol Phar-macol 55:587–593

Rahman Z, Schwarz J, Gold SJ, Zachariou V,WeinMN, Choi KH, KovoorA, Chen CK, DiLeone RJ, Schwarz SC, Selley DE, Sim-Selley LJ,Barrot M, Luedtke RR, Self D, Neve RL, Lester HA, Simon MI,

Nestler EJ (2003) RGS9 modulates dopamine signaling in the basalganglia. Neuron 38:941–952

Ribeiro FM, Paquet M, Cregan SP, Ferguson SS (2010) Group Imetabotropic glutamate receptor signalling and its implication inneurological disease. CNS Neurol Disord Drug Targets 9:574–595

Ronesi JA, Huber KM (2008) Homer interactions are necessary formetabotropic glutamate receptor-induced long-term depressionand translational activation. J Neurosci 28:543–547

Ruiz de Azua I, Scarselli M, Rosemond E, Gautam D, Jou W, GavrilovaO, Ebert PJ, Levitt P, Wess J (2010) RGS4 is a negative regulator ofinsulin release from pancreatic beta-cells in vitro and in vivo. ProcNatl Acad Sci USA 107:7999–8004

Saugstad JA, Marino MJ, Folk JA, Hepler JR, Conn PJ (1998) RGS4inhibits signaling by group I metabotropic glutamate receptors. JNeurosci 18:905–913

Schotanus SM, Chergui K (2008) Dopamine D1 receptors and group Imetabotropic glutamate receptors contribute to the induction oflong-term potentiation in the nucleus accumbens. Neuropharma-cology 54:837–844

Schwendt M, McGinty JF (2007) Regulator of G-protein signaling 4interacts with metabotropic glutamate receptor subtype 5 in ratstriatum: relevance to amphetamine behavioral sensitization. JPharmacol Exp Ther 323:650–657

Schwendt M, Gold SJ, McGinty JF (2006) Acute amphetamine down-regulates RGS4 mRNA and protein expression in rat forebrain:distinct roles of D1 and D2 dopamine receptors. J Neurochem96:1606–1615

Schwendt M, Hearing MC, See RE, McGinty JF (2007) Chroniccocaine reduces RGS4 mRNA in rat prefrontal cortex and dorsalstriatum. Neuroreport 18:1261–1265

Schwendt M, Madell RL, McGinty JF (2010) Relapse to cocaine-seeking after abstinence is associated with increased ERK1/2phosphorylation in the dorsolateral striatum: the role of mGluR5receptors. (Neuroscience Meeting Planner Online). Society forNeuroscience, Chicago, IL, USA, pp 770.19

Shi X, McGinty JF (2006) Extracellular signal-regulated mitogen-activated protein kinase inhibitors decrease amphetamine-inducedbehavior and neuropeptide gene expression in the striatum. Neuro-science 138:1289–1298

Shi X, McGinty JF (2007) Repeated amphetamine treatment increasesphosphorylation of extracellular signal-regulated kinase, proteinkinase B, and cyclase response element-binding protein in the ratstriatum. J Neurochem 103:706–713

Shi X, McGinty JF (2011) D1 and D2 dopamine receptors differ-entially mediate the activation of phosphoproteins in thestriatum of amphetamine-sensitized rats. Psychopharmacology(Berl) 214:653–663

Shi X, Gomez A, McGinty JF (2009) Inhibition of phosphoinositide 3-kinase decreases the activation of protein kinase B in rat striatumwithout altering the phosphorylation of extracellular signal-regulated kinase induced by amphetamine. (Neuroscience MeetingPlanner. Online.). Society for Neuroscience, Chicago, IL, USA, pp66.25

Shin DM, Dehoff M, Luo X, Kang SH, Tu J, Nayak SK, Ross EM,Worley PF, Muallem S (2003) Homer 2 tunes G protein-coupledreceptors stimulus intensity by regulating RGS proteins andPLCbeta GAP activities. J Cell Biol 162:293–303

Siderovski DP, Willard FS (2005) The GAPs, GEFs, and GDIs ofheterotrimeric G-protein alpha subunits. Int J Biol Sci 1:51–66

Sjogren B, Blazer LL, Neubig RR (2010) Regulators of G proteinsignaling proteins as targets for drug discovery. Prog Mol BiolTransl Sci 91:81–119

Smith Y, Raju DV, Pare JF, Sidibe M (2004) The thalamostriatalsystem: a highly specific network of the basal ganglia circuitry.Trends Neurosci 27:520–527

634 Psychopharmacology (2012) 221:621–635

Sutton MA, McGibney K, Beninger RJ (2000) Conditioned locomo-tion in rats following amphetamine infusion into the nucleusaccumbens: blockade by coincident inhibition of protein kinaseA. Behav Pharmacol 11:365–376

Swanson CJ, Baker DA, Carson D, Worley PF, Kalivas PW (2001)Repeated cocaine administration attenuates group I metabo-tropic glutamate receptor-mediated glutamate release and be-havioral activation: a potential role for Homer. J Neurosci21:9043–9052

Talkowski ME, Chowdari K, Lewis DA, Nimgaonkar VL (2006) CanRGS4 polymorphisms be viewed as credible risk factors forschizophrenia? A critical review of the evidence. Schizophr Bull32:203–208

Tallaksen-Greene SJ, Kaatz KW, Romano C, Albin RL (1998) Local-ization of mGluR1a-like immunoreactivity and mGluR5-like im-munoreactivity in identified populations of striatal neurons. BrainRes 780:210–217

Tekumalla PK, Calon F, Rahman Z, Birdi S, Rajput AH, HornykiewiczO, Di Paolo T, Bedard PJ, Nestler EJ (2001) Elevated levels ofDeltaFosB and RGS9 in striatum in Parkinson’s disease. BiolPsychiatry 50:813–816

Valjent E, Corbillé AG, Bertran-Gonzalez J, Hervé D, Girault JA(2006) Inhibition of ERK pathway or protein synthesis duringreexposure to drugs of abuse erases previously learned placepreference. Proc Natl Acad Sci USA 103(8):2932–2937

Veeneman MM, Boleij H, Broekhoven MH, Snoeren EM, GuitartMasip M, Cousijn J, Spooren W, Vanderschuren LJ (2011) Dis-sociable roles of mGlu5 and dopamine receptors in the rewarding

and sensitizing properties of morphine and cocaine. Psychophar-macology (Berl) 214:863–876

Voulalas PJ, Holtzclaw L, Wolstenholme J, Russell JT, Hyman SE(2005) Metabotropic glutamate receptors and dopamine receptorscooperate to enhance extracellular signal-regulated kinase phos-phorylation in striatal neurons. J Neurosci 25:3763–3773

Wang JQ, Mao L (2000) Sustained behavioral stimulation followingselective activation of group I metabotropic glutamate receptors inrat striatum. Pharmacol Biochem Behav 65:439–447

Wang JQ, McGinty JF (1995) Dose-dependent alteration in zif/268 andpreprodynorphin mRNA expression induced by amphetamine ormethamphetamine in rat forebrain. J Pharmacol Exp Ther 273:909–917

Wang Q, Liu-Chen LY, Traynor JR (2009) Differential modulation ofmu- and delta-opioid receptor agonists by endogenous RGS4protein in SH-SY5Y cells. J Biol Chem 284:18357–18367

Yan Y, Chi PP, Bourne HR (1997) RGS4 inhibits Gq-mediated activa-tion of mitogen-activated protein kinase and phosphoinositidesynthesis. J Biol Chem 272:11924–11927

Yuferov V, Kroslak T, Laforge KS, Zhou Y, Ho A, Kreek MJ (2003)Differential gene expression in the rat caudate putamen after“binge” cocaine administration: advantage of triplicate microarrayanalysis. Synapse 48:157–169

Zhang Y, James M, Middleton FA, Davis RL (2005) Transcriptionalanalysis of multiple brain regions in Parkinson’s disease supportsthe involvement of specific protein processing, energy metabo-lism, and signaling pathways, and suggests novel disease mech-anisms. Am J Med Genet B Neuropsychiatr Genet 137B:5–16

Psychopharmacology (2012) 221:621–635 635