Differential effects of methamphetamine and SCH23390 on the expression of members of IEG families of transcription factors in the rat striatum

Abstract

Methamphetamine (METH) is a psychostimulant that can cause long-lasting neurodegenerative effects in humans and animals. These toxic effects appear to occur, in part, via activation of dopamine (DA) D1 receptors. This paper assessed the possibility that the DA D1 receptor antagonist, SCH23390, might inhibit METH-induced changes in the expression of several members of immediate early genes (IEGs) which are known to control more delayed expression of other genes. We found that injections of METH (4x10 mg/kg, given at 2 h intervals) caused significant increases in c-fos and fra-2 expression which lasted from 30 min to 4 h. Pre-treatment with SCH23390, given 30 min before each METH injection, completely blocked METH-induced expression of c-fos, but only partially inhibited fra-2 mRNA expression. These results were confirmed by Western blot analysis which showed METH-induced changes in c-Fos protein expression that were blocked by pretreatment with SCH23390. There were also delayed METH-induced DA D1 receptor-dependent effects on fosB mRNA expression. Even though fra-1 expression was not affected by pretreatment with METH alone, the repeated injections of SCH23390 caused substantial decreases in fra-1 mRNA expression in both the presence and absence of METH. The repeated injections of METH caused no changes in the mRNAs for c-jun, junB or junD. However, there were significant increases in the phosphorylation of c-Jun protein (ser63). Phosphorylation of c-Jun occurred in a delayed fashion (16 and 24 h after the last METH injections) and was attenuated by SCH23390 pretreatment. Interestingly, SCH23390 given alone caused significant decreases in phospho-c-Jun at all time-points. The METH injections also caused delayed induction in the expression of members of the Egr family of transcription factors in a DA D1 receptor-dependent fashion. Repeated injections of SCH23390 caused substantial suppression of basal striatal egr-1 and egr-2 mRNA expression but not of that of egr-3. Both crem and arc mRNA levels were induced by METH in a SCH23390-sensitive fashion. Moreover, multiple injections of SCH23390 given alone caused marked inhibition of basal arc expression. These results show that multiple injections of METH can differentially affect the expression of several IEGs, some of which occurred in a DA D1 receptor dependent fashion. The SCH23390-mediated suppression of basal fra-1, egr-1, and egr-2 mRNA levels suggests that their basal expression in the striatum might be dependent on tonic stimulation of the DA D1 receptor.

Fig. 1. Effects of METH and SCH23390 on the expression of the fos family genes

METH administration caused induction of (A) c-fos, (C) fosB and (E) fra-2, but did not influence fra-1 expression (D). Amplification curve for c-fos gene expression for the 30 min time-point is shown in fig. 1B and was reproducible. The levels of mRNA were normalized to clathrin mRNA levels. Data were obtained from RNA isolated from six animals per group and quantitation determined individually. Statistical significance was determined by ANOVA followed by protected least-squares difference (PLSD). Key to statistics: *, **, *** = p < 0.05, 0.01, 0.001, respectively in comparison to the control (C) group. #, ##, ### = p < 0.05, 0.01, 0.001, respectively in comparison to the METH (M) group.

Fig. 2. Effects of METH and SCH23390 on c-Fos protein level

(A) Representative photomicrograph of results showing the effects of METH and SCH23390 pre-treatment on striatal c-Fos protein levels at different time-points. (B) The quantitative data of the western blots represent means ± SEM (n = 3). The experiments were repeated three times for each protein. For quantification, the signal intensity was normalized over the signal intensity of α-tubulin. (B) METH caused significant DA D1 receptor-dependent increases in c-Fos protein. Statistics are as described in Fig. 1. Key to statistics: *, **,*** = p < 0.05, 0.01, 0.001, respectively in comparison to the control (C) group. #, ##, ### = p < 0.05, 0.01, 0.001, respectively in comparison to the METH (M) group.

Fig. 3. Effects of METH and SCH23390 on the levels of jun transcripts

The expression of (A) c-jun, (B) junB and (C) junD mRNA expression was not affected by METH treatment although SCH23390 pretreatment caused significant decreases in junD at 4 hr post drug treatment (C). Normalization, quantification, and statistics are as described in Fig. 1. Key to statistics: #, ## = p < 0.05, 0.01, respectively in comparison to the METH (M) group.

Fig. 4. Effects of METH and SCH23390 on c-Jun and phospho-c-Jun protein levels

(A and C) show representative photomicrographs of results showing the effects of METH and SCH23390 pre-treatment on the levels of c-Jun and phospho-c-Jun in rat striatal tissues et different time-points. (B and D) show the quantitative data of the western blots represent means ± SEM (n = 3). The experiments were repeated three times for each protein. For quantification, the signal intensity was normalized over the signal intensity of α-tubulin. (C) METH-induced changes in c-Jun were seen only at the 2-hr time point and this was blocked by SCH23390 pre-treatment. (D) Phospho-c-Jun showed significant increases at 16 hr and 24 hr after METH treatment. Pretreatment of SCH23390 blocked METH-induced changes in phospho-c-Jun. Normalization, quantification, and statistics are as described in Fig. 1. Key to statistics: *, **,*** = p < 0.05, 0.01, 0.001, respectively in comparison to the control (C) group. #, ##, ### = p < 0.05, 0.01, 0.001, respectively in comparison to the METH (M) group.

Fig. 5. METH caused induction of striatal egr mRNA levels

(A, B, C) Time-dependent induction of egr-1, egr-2 and egr-3 was observed after METH injections. SCH23390 blocked METH-induces changes in these genes. Normalization, quantification, and statistics are as described in Fig. 1. Key to statistics *, **, *** = p < 0.05, 0.01, 0.001, respectively in comparison to the control (C) group. #, ##, ### = p < 0.05, 0.01, 0.001, respectively in comparison to the METH (M) group.

Fig. 6. METH caused rapid induction of crem and arc mRNA levels

(A) METH induced rapid increases in the crem transcript levels. (B) arc expression was also induced by METH. Normalization, quantification, and statistics are as described in Fig. 1. Key to statistics: *, **, *** = p < 0.05, 0.01, 0.001, respectively in comparison to the control (C) group. #, ##, ### = p < 0.05, 0.01, 0.001, respectively in comparison to the METH (M) group.