A Novel Molecule “Shati” Is Involved in Methamphetamine-Induced Hyperlocomotion, Sensitization, and Conditioned Place Preference

Expression of shati mRNA in the various organs of mice. A, RT-PCR analysis of shati in the various organs in mice. Mice were decapitated without any treatment, and the brains were quickly removed. The sets of primers used for PCR are listed in Table 1. B, Increase in the production of the three sets of target sequences of shati induced by repeated METH treatment in the NAc of mice. Mice were administered METH (2 mg/kg, s.c.) for 6 d and decapitated 2 h after the last METH treatment. Values are means ± SE (n = 5). *p < 0.05 versus saline-treated mice. The sets of primers used for PCR are listed in Table 1. To standardize the PCR products, we used primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control.

METH induced expression of shati mRNA in the brain. A, Dose-dependent effect of repeated METH treatment on shati mRNA expression in the NAc. Mice were administered METH (0.3, 1, and 2 mg/kg, s.c.) for 3 d. Mice were decapitated 2 h after the last METH treatment. Values are means ± SE (n = 8). *p < 0.05 versus saline-treated mice. B, Time course changes in the expression of shati mRNA after repeated METH treatment in the NAc. Mice were administered METH (2 mg/kg, s.c.) for 6 d and decapitated 1, 2, 6, 24, and 48 h and 1 week after the last METH treatment. Values are means ± SE (n = 6–7). *p < 0.05 versus saline-treated mice. C, Changes in the expression of shati mRNA in the various brain regions (Fc, NAc, CPu, and Hip) of the mice after single and repeated METH treatment. Mice were administered METH (2 mg/kg, s.c.) for 5 d and challenged with METH (2 mg/kg, s.c.) or saline on day 6. Mice were decapitated 2 h after last treatment of METH (2 mg/kg, s.c.) or saline challenge. Values are means ± SE (n = 8–10). *p < 0.05 versus saline-treated mice. D, The effects of the DA D₁-like receptor antagonist R(+)-SCH23390 or D₂-like receptor antagonist raclopride on METH-induced expression of shati mRNA in the NAc. Mice were treated with R(+)-SCH23390 (0.1 mg/kg, s.c.) or raclopride (2 mg/kg, s.c.) 30 min before daily METH (2 mg/kg, s.c. for 6 d) treatment. Mice were decapitated 2 h after the last METH treatment. Values are means ± SE (n = 6–8). *p < 0.05 versus vehicle/saline-treated mice. ^#p < 0.05 versus vehicle/METH-treated mice.

Immunostaining of shati in the NAc after repeated treatment with METH. Mice were administered METH (2 mg/kg, s.c.) for 6 d and decapitated 24 h after the last treatment. A, Double-labeling fluorescence photomicrographs for shati and NeuN or GFAP. The shati-immunopositive cells (green) were colocalized with NeuN-immonopositive cells (red). Double immunostaining for S-3 or S-4 and NeuN in the NAc reveals shati expression in neuronal cells. Scale bars, 20 μm. B, Effect of shati-AS on METH-induced increase in shati expression. METH-induced increase in shati expression in the NAc was inhibited by shati-AS. Scale bar, 20 μm.

Roles of shati in METH-induced hyperlocomotion and sensitization. An osmotic minipump was used to deliver a continuous infusion of shati-AS (1.8 nmol/6 μl per day), shati-SC (1.8 nmol/6 μl per day), or CSF into the right ventricle (AP −0.5 mm, ML +1.0 mm from bregma, and DV −2.0 mm from the skull). A, Experimental schedule for the real-time RT-PCR using shati-AS. B, Effect of shati-AS on shati mRNA expression. Mice were administered METH (1 mg/kg, s.c.) for 3 d and decapitated 2 h after METH treatment on the day 3. Values are means ± SE (n = 8). *p < 0.05 versus saline-treated mice. ^#p < 0.05 versus shati-SC-treated mice. C, Experimental schedule for measurement of locomotor activity using shati-AS. D, Effect of shati-AS on repeated METH-induced behavioral sensitization. Mice were administered METH (1 or 2 mg/kg, s.c.) or saline for 5 d and challenged with METH (0.3 mg/kg, s.c.) on day 10. Locomotor activity was measured for 2 h on the days 1, 3, 5, and 10. Values are means ± SE (n = 5–7). ANOVA with repeated measures revealed significant differences in METH-induced sensitization (group, F_(8,47) = 51.238, p < 0.01; day, F_(2,94) = 68.423, p < 0.01; group × day, F_(16,94) = 4.412, p < 0.01). *p < 0.05 versus METH plus CSF-treated mice. ^#p < 0.05 versus METH plus shati-SC-treated mice. ^$p < 0.05 versus the locomotor activity on day 1 in the same group.

Effects of shati-AS on METH-induced dopaminergic responses. A, Experimental schedule for the measurement of overflow of DA using in vivo microdialysis using shati-AS. B, Effect of shati-AS on METH-induced increase in overflow of DA in the NAc. Mice were administered METH (1 mg/kg, s.c.) for 3 d. On day 3, levels of DA were measured in the NAc (AP +1.7 mm, ML −0.8 mm from bregma, and DV −4.0 mm from the skull) for 220 min after METH treatment by in vivo microdialysis. Basal levels of DA were 0.30 ± 0.08, 0.31 ± 0.05, and 0.30 ± 0.04 nm for the CSF-treated, shati-SC-treated, and shati-AS-treated mice, respectively. ANOVA with repeated measures revealed significant differences in METH-induced increase in overflow of DA (group, F_(2,14) = 5.662, p < 0.05; time, F_(10,140) = 35.646, p < 0.01; group × time, F_(20,140) = 1.927, p < 0.05). Values are means ± SE (n = 5–6). *p < 0.05 versus CSF-treated mice. ^#p < 0.05 versus shati-SC-treated mice. C, Experimental schedule for the [³H]DA uptake assay using shati-AS. D, Effect of shati-AS on METH-induced decrease of synaptosomal [³H]DA uptake. Mice were administered METH (1 mg/kg, s.c.) for 3 d and decapitated 1 h after the last injection. The synaptosomal [³H]DA uptake was 0.32 ± 0.04, 0.29 ± 0.03, 0.20 ± 0.02, 0.18 ± 0.01, 0.20 ± 0.01, and 0.09 ± 0.01 pmol/mg protein per 4 min for the saline plus CSF-treated, saline plus shati-SC-treated, saline plus shati-AS-treated, METH plus CSF-treated, METH plus shati-SC-treated, and METH plus shati-AS-treated mice, respectively. The final concentration of [³H]DA was 5 nm. Values are means ± SE (n = 7–8). *p < 0.05 versus saline-treated mice. ^#p < 0.05 versus shati-SC-treated mice. E, Effect of shati-AS on METH-induced decrease of vesicular [³H]DA uptake. Mice were administered METH (1 mg/kg, s.c.) for 3 d and decapitated 1 h after the last injection. The vesicular [³H]DA uptake was 3.76 ± 0.25, 4.05 ± 0.29, 2.80 ± 0.20, 1.74 ± 0.21, 1.85 ± 0.14, and 0.90 ± 0.14 pmol/mg protein per 4 min for the saline plus CSF-treated, saline plus shati-SC-treated, saline plus shati-AS-treated, METH plus CSF-treated, METH plus shati-SC-treated, and METH plus shati-AS-treated mice, respectively. The final concentration of [³H]DA was 30 nm. Values are means ± SE (n = 8). *p < 0.05 versus saline-treated mice. ^#p < 0.05 versus shati-SC-treated mice.