A. Targeting vector design for generating GASP-1 KO mice. A cassette expressing the G418 resistance gene flanked by lox P sites was inserted into the intron upstream of the GASP-1 open reading frame (ORF) (intron 4) and a third loxP site was inserted in the intron downstream of the GASP ORF (intron 5). ES cells from C57/Bl6 mice were transfected with this vector. Properly targeted clones (see B, C) were transfected with Cre-recombinase and blastocysts from clones in which the GASP-1 ORF was disrupted were implanted into C57/BL6 females. B, C. Southern blotting analysis identified homologous recombination and single insertion using B. 5′ and C. Neo probes. D. Immuno-detection of GASP-1 in KO mice. Whole brain lysates from WT, HET and KO mice were prepared, separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted for GASP-1 using a GASP specific antibody (upper panel) or β-Actin (lower panel) to control for protein loading.

A-D. Cocaine sensitization in WT and GASP-1 KO mice. Mice were injected daily (IP) with saline (WT n = 27, GASP-1 KO n = 25) or cocaine (15 mg/kg, WT n = 29, GASP-1 KO n = 30) and the distance traveled reported for A. 15 min or B. 60 min post-injection. WT mice exhibited a sensitized locomotor response to cocaine by day 3 (WT cocaine, day 3 versus day 1, ##p<0.01 as analyzed by one-way ANOVA followed by Bonferroni multiple comparison tests between corresponding days), while GASP-1 KOs developed a significantly lower sensitized response within this first 15 min by day 4 (GASP-KO cocaine, day 4 versus day 1 ##p<0.01 as analyzed by one-way ANOVA followed by Bonferroni multiple comparison tests between corresponding days). GASP-1 KO mice showed significantly reduced sensitized locomotor response to cocaine compared to WT mice on all days (GASP-1 KO cocaine versus WT cocaine where *p<0.05, **p<0.01 and ***p<0.001 as analyzed by two-way ANOVA followed by Bonferroni multiple comparison tests between corresponding days). GASP-1 KO mice showed reduced locomotor responsiveness to cocaine after repeated treatment both 15 min (C. ***p<0.001) and 60 min post-injection (D. *p<0.05). E, F. Stereotypic behaviors of WT and GASP-1 KO mice after 1 (E) and 5 (F) days of cocaine exposure. On day 5 there were significant differences in stereotypy at 5, 10 and 15 min (GASP-1 KO cocaine versus WT cocaine where ***p<0.001, **p<0.01 and *p<0.05 respectively as analyzed by two-way ANOVA followed by Bonferroni multiple comparison tests between corresponding days) post-injection between cocaine treated genotypes. Data is presented as mean ± S.E.M.

A. D1R number in striatal membranes was not affected by cocaine treatment in either genotype when assessed by saturation binding using [³H]-SCH23390. B. Cocaine treatment reduced D2R number in WT mice (**p<0.01 WT cocaine versus WT saline). Saline treated GASP-1 KO showed reduced D2R number (GASP-1 KO saline versus WT saline where *p<0.05) and D2R number was increased in cocaine treated GASP-1 KO mice (#p<0.05 GASP-1 KO cocaine versus GASP-1 KO saline). D2R number was assessed by saturation binding using [³H]-raclopride. Data shown are normalized to saline treated groups and are from three independent radioligand binding experiments, performed in duplicate, with at least eight mice per group.

A, Dose response of quinpirole pretreatment. WT C57/BL6 mice were injected daily (IP) with saline or one of three doses of quinirole (0.5, 1 or 5 mg/kg n = 8 per group) for 7 days. On day 8 all mice were injected (IP) with 20 mg/kg cocaine and their locomotor activity recorded. B. Mice become tolerant to the locomotor-suppressing effects of quinpirole. WT C57/BL6 mice were injected daily (IP) with saline (n = 24) or quinpirole (5 mg/kg, n = 24) and their locomotor activity recorded once per day for 7 days. On days 1 and 2, quinpirole reduced locomotor activity when compared to saline treated animals (***p<0.001 and *p<0.05 respectively). WT mice became tolerant to the locomotor inhibitory effects of quinpirole on day 4 and remained so for days 6 and 7 compared to day 1 (#p<0.05 and ###p<0.001 respectively). C, D. Repeated quinpirole pre-treatment augments locomotor response to acute cocaine. On day 8, mice from (B) were injected with cocaine (IP, 20 mg/kg) and their locomotor activity (D) and stereotypy (E) recorded. Mice pretreated with repeated quinpirole showed enhanced locomotor and stereotypic responses to acute cocaine compared to saline treated mice (***p<0.001, **p<0.01 and *p<0.05). Data was analyzed by either one or two-way ANOVA with Bonferroni multiple comparison tests and presented as mean ± S.E.M.

A. Aripiprazole does not induce endocytosis of D2Rs. HEK 293 cells stably expressing N-terminal FLAG-tagged D2Rs were incubated with M1 antibody to the extracellular tag for 20 mins then stimulated with quinpirole or aripiprazole (10 µM), or left untreated for 30 mins. Cells were fixed, blocked, permeabilised and receptors visualized with fluorescent secondary antibody Alexa 488. Quinpirole but not aripiprazole induced endocytosis of the D2R. B. Acute aripiprazole inhibits locomotion. WT C57/BL6 mice were injected daily (IP) with vehicle (n = 24) or aripiprazole (5 mg/kg, n = 24) and their locomotor activity recorded once per day for 7 days. Aripiprazole reduced locomotor activity when compared to vehicle treated animals across all 7 days (*p<0.05, ***p<0.001) and there was no significant change in the effect of aripiprazole and any day compared to day 1. C, D. Repeated aripiprazole pretreatment augments locomotor response to acute cocaine. On day 8, mice from (B) were injected with cocaine (IP, 20 mg/kg) and their locomotor activity (D) and stereotypy (E) recorded. Mice pretreated with repeated aripiprazole showed enhanced locomotor and stereotypic responses to acute cocaine compared to saline treated mice (***p<0.001, **p<0.01 and *p<0.05). Data was analyzed by either one or two-way ANOVA with Bonferroni multiple comparison tests and presented as mean ± S.E.M.