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.2018 Feb;32(2):191-203.
doi: 10.1177/0269881117742100. Epub 2018 Jan 24.

Regulator of G protein signaling-12 modulates the dopamine transporter in ventral striatum and locomotor responses to psychostimulants

Affiliations

Regulator of G protein signaling-12 modulates the dopamine transporter in ventral striatum and locomotor responses to psychostimulants

Joshua D Gross et al. J Psychopharmacol.2018 Feb.

Abstract

Regulators of G protein signaling are proteins that accelerate the termination of effector stimulation after G protein-coupled receptor activation. Many regulators of G protein signaling proteins are highly expressed in the brain and therefore considered potential drug discovery targets for central nervous system pathologies; for example, here we show that RGS12 is highly expressed in microdissected mouse ventral striatum. Given a role for the ventral striatum in psychostimulant-induced locomotor activity, we tested whether Rgs12 genetic ablation affected behavioral responses to amphetamine and cocaine. RGS12 loss significantly decreased hyperlocomotion to lower doses of both amphetamine and cocaine; however, other outcomes of administration (sensitization and conditioned place preference) were unaffected, suggesting that RGS12 does not function in support of the rewarding properties of these psychostimulants. To test whether observed response changes upon RGS12 loss were caused by changes to dopamine transporter expression and/or function, we prepared crude membranes from the brains of wild-type and RGS12-null mice and measured dopamine transporter-selective [3H]WIN 35428 binding, revealing an increase in dopamine transporter levels in the ventral-but not dorsal-striatum of RGS12-null mice. To address dopamine transporter function, we prepared striatal synaptosomes and measured [3H]dopamine uptake. Consistent with increased [3H]WIN 35428 binding, dopamine transporter-specific [3H]dopamine uptake in RGS12-null ventral striatal synaptosomes was found to be increased. Decreased amphetamine-induced locomotor activity and increased [3H]WIN 35428 binding were recapitulated with an independent RGS12-null mouse strain. Thus, we propose that RGS12 regulates dopamine transporter expression and function in the ventral striatum, affecting amphetamine- and cocaine-induced increases in dopamine levels that specifically elicit acute hyperlocomotor responses.

Keywords: Amphetamine; cocaine; dopamine transporter; regulators of G protein signaling.

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Conflict of interest statement

Declaration of conflicting interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1
Figure 1
Independent targeting of two separate ablations to the mouseRgs12 locus. (a) Multiple, protein-protein interaction domain architecture of the encoded regulators of G protein signaling (RGS)12 protein, including a PSD-95/Dlg/ZO-1 (PDZ;orange) domain capable of binding the C-termini of the mitogen-activated protein kinase (MAPK) middle-tier kinase MAPK-kinase (MEK2) and the interleukin-8 receptor CXCR2 (Snow et al., 1998; Willard et al., 2007), a phosphotyrosine-binding (PTB;pink) domain thought to bind the NGF receptor TrkA (Willard et al., 2007), an RGS domain (yellow) which binds and inactivates heterotrimeric G protein Ggdα subunits of the Gi/o subfamily (Snow et al., 1998), a tandem repeat of Ras-binding domains (RBDs;cyan) that bind indicated members of the Ras guanine nucleotide triphosphate (GTP) ase and MAPK first-tier (Raf) kinases (ref. Willard et al., 2007), a GoLoco motif which binds inactive Gα subunits of the Gi subfamily (ref. Kimple et al., 2002), and regions of predicted low complexity (rose). Vertical lines indicate exon-exon junctions within the open-reading frame encoded by theRgs12 mRNA. (b) and (c) Mice lacking functional RGS12 (Rgs12Δ5-8/Δ5-8) were obtained from the Texas A and M Institute of Genomic Medicine (TIGM) in a 129/Sv×C57BL/6 mixed background. Because congenic C57BL/6 mice are standard for neuropsychopharmacological behavioral assays, we back-crossed the TIGMRgs12Δ5-8/Δ5-8 mice with C57BL/6J mice for more than ten generations. Henceforth,Rgs12Δ5-8/Δ5-8 mice will refer to congenicRgs12Δ5-8/Δ5-8 mice on the C57BL/6J background. Exons 5–8, encoding the entire polypeptide sequence of the RGS domain of RGS12 (yellow; panel (a)), were targeted for replacement with a neomycin resistance, drug-selectable marker. (d) Coronal in situ hybridization (Allen Brain Atlas) reveals markedRgs12 mRNA labeling in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc), beds of the mesolimbic and nigrostriatal dopaminergic soma. Analyses also reveal hippocampus (HPC) Rgs12 mRNA, particularly in the dentate gyrus. (e) Coronal in situ hybridization from our group shows sparse Rgs12 mRNA in dorsal striatum (dSTR) and ventral striatum (vSTR) and cortex (CTX). Marked labeling was observed in the claustrum (CLA). (f) Immunoblotting for RGS12 protein levels in microdissected frontal cortex, dSTR, vSTR, and midbrain in wild-type (WT) and RGS12-null mice. â-Actin immunoreactivity served as a loading control. (g) Densitometric analysis demonstrates that RGS12 in vSTR is higher than frontal CTX, dSTR, and midbrain and, specifically is 157% more abundant than in dSTR. Data are means±standard error of the mean (SEM) (n=6 mice per group) relative to dSTR set to100%. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons: dSTR vs frontal CTXp=0.018 (*p<0.05), dSTR vs vSTRp=0.0014 (**p<0.01), dSTR vs midbrainp=0.724 (not significant (ns),p>0.05).
Figure 2
Figure 2
Acute psychostimulant-induced locomotion inRgs12-null mice. (a) Locomotor activity inRgs12 Δ5–6/Δ5–6 mice (i.e. conditionalRgs12fl/fl mice cross-bred with an ubiquitousCMV::Cre transgene) following 30 min acclimation to activity chambers and then saline (at a final volume of 10 mL/kg) administration. Data are means±standard error of the mean (SEM) (n=7–8 per group). (b) (+)-amphetamine (AMPH) (3 mg/kg)-induced hyperlocomotion inRgs12 Δ5–6/Δ5–6 mice for 80 min following 30 min acclimation to activity chambers. Data are means±SEM (n=7–8 per group). (c) AMPH (3 mg/kg)-induced hyperlocomotion in constitutiveRgs12 Δ5–8/Δ5–8 mice for 80 min following 30 min acclimation to activity chambers. Data are means±SEM (n=9–23 per group). (d) Cocaine (10 mg/kg)-induced hyperlocomotion in constitutiveRgs12 Δ5–8/Δ5–8 mice for 60 min following 30 min acclimation to activity chambers. Data are means±SEM (n=9–10 per group). (e) Total locomotion by constitutiveRgs12 Δ5–8/Δ5–8 mice over 80 min following intraperitoneal (ip) injection of saline or various doses of AMPH after 30 min acclimation to activity chambers. Data are means±SEM (n=8–23 per group). (f) Total locomotion by constitutiveRgs12 Δ5–8/Δ5–8 mice over 60 min following ip injection of saline or various doses of cocaine after 30 min acclimation to activity chambers. Data are means±SEM (n=7–20 per group). (g) Total locomotion by constitutiveRgs12 Δ5–8/Δ5–8 mice over 60 min following ip injection of saline or indicated doses of SKF 81927 after 30 min acclimation to activity chambers. Data are means±SEM (n=8 per group).
Figure 3
Figure 3
Psychostimulant-induced locomotor sensitization and conditioned place preference in regulators of G protein signaling (RGS)12-null mice. Total locomotion over 80 min following (+)-amphetamine (AMPH) (2 mg/kg; (a) and (b)) or 60 min following cocaine (20 mg/kg; (d) and (e)). Total locomotion normalized to Day 1 total locomotion plotted as fold change across drug treatment days ((c) and (f)). Data are means±standard error of the mean (SEM) (n=6–9 per group). Psychostimulant-induced conditioned place preference inRgs12-null mice. Data shown are the difference in the time spent between AMPH (2 mg/kg) (panel (g))- or cocaine (10 mg/kg) (panel (h))-paired and saline-paired chamber for pre-conditioning (Day 2) and post-conditioning (Day 9). Data are means±SEM (n=7–8 per group).
Figure 4
Figure 4
(+)-Amphetamine (AMPH)-stimulated c-Fos induction in regulators of G protein signaling (RGS)12-null mice. (a) Immunohistochemical analysis of c-Fos in the nucleus accumbens (NAc) of RGS12-null and wild-type (WT) mice injected with AMPH (3 mg/kg). (b) Quantification of the number of c-Fos-positive neurons per field in RGS12-null vs WT dorsal striatum (dSTR) and ventral striatum (vSTR) (3–4 matched sections per genotype). Data are means±standard error of the mean (SEM) (n=3–4 mice per group).
Figure 5
Figure 5
Analysis of dopamine transporter (DAT) function and expression in regulators of G protein signaling (RGS)12-null mice. [3H]Dopamine uptake in ventral (a) and dorsal (b) striatal synaptosomes prepared from RGS12-null and wild-type mice. ((b, inset) Uptake rates (Vmax) of [3H] dopamine in ventral and dorsal striatal synaptosomes derived from data in panels (a) and (b); non-specific uptake determined in the presence of 10 μM cocaine. Data are means±standard error of the mean (SEM) (n=9–12 per group). (c) Uptake rates (Vmax) by DAT of [3H]dopamine in ventral striatal synaptosomes derived from data in Supplementary Material, Figure S4; non-specific uptake determined in the presence of 10 μM GBR12935 (a selective dopamine reuptake inhibitor). Data are means±SEM (n=6 per group). (d) and (e) Levels of DAT determined from [3H]WIN 35428 saturation binding analysis of ventral (d) and dorsal (e) striatal crude membrane fractions. Non-specific binding was determined in the presence of 10 μM GBR12935. ((e) inset) Maximal DAT binding sites at 52.5 nM [3H]WIN 35428 in ventral and dorsal striatal membranes derived from data in (d) and (e). Data are means±SEM (n=6–9 per group). (f) Maximal DAT binding sites at 52.5 nM [3H]WIN 35428 in ventral striatal membranes derived from data in Supplementary Material, Figure S4. Data are means±SEM (n=6 per group). dSTR: dorsal striatum; vSTR: ventral striatum.
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