METH induced behavioral sensitization
The model of behavioral sensitization was established by 7 days of METH (5.0 mg/kg, s.c.) administration followed by 7 days of withdrawal and challenged by single METH (1.0 mg/kg, s.c.) on day 15. One-way ANOVA showed the difference between groups (F(2, 21) = 14.58, P<0.001). Rats in the METH sensitization group (METH-METH group) showed significantly higher locomotor activity than either the saline control (NS-NS) or METH acute treatment group (NS-METH) (Tukey’s post hoc test, P<0.001, vs NS-NS group; P<0.01, vs NS-METH group, Figure 1B), indicating the enhanced locomotor response of sensitization. Compared with the NS-NS group, rats in the NS-METH group showed longer distances (P<0.05, Figure 1B).
Changes of mRNA in the PFC of METH-induced behavioral sensitization rat
An mRNA microarray was performed to determine a genome-wide profile of METH-induced behavioral sensitization. The cut-off value was set at a fold change of ≤0.5 or ≥2.0 (P<0.01). Figure 2 shows the clustering and a heat map of the mRNA expression among the NS-NS group, NS-METH group, and METH-METH group. A large number of genes were changed by METH exposure (P<0.01) (Figure 2B). Among these, 275 genes were differentially expressed in the METH-METH group compared with the NS-METH group (97 genes were upregulated and 178 genes were downregulated), and 232 genes were changed in the NS-METH group compared with the NS-NS group (122 genes were upregulated and 110 genes were downregulated) (Table S2).
Ontological and pathway analysis of differentially expressed mRNAs in the PFC of METH-induced sensitization rats
In order to investigate whether the clustering of differentially expressed genes induced by METH exposure correlates with functional groupings, we performed GO analysis and categorized the differentially expressed genes. Figure 3A and Table S3 show the top 10 ranked GO terms and the number of genes in a category. Compared with METH acute treatment (NS-METH), the biological process in the METH-induced behavioral sensitization (METH-METH) group was markedly constructed of biological regulation, cellular process and organism process. For molecular function, binding and protein binding were enriched in the METH-METH group.
To assess the functional features of METH-induced behavioral sensitization-mediated gene sets, we further performed KEGG pathway enrichment analysis. The top 10 significantly ranked pathways are shown in Figure 3B and Table S4. Many signal transduction pathways were enriched in the METH-induced behavioral sensitization group, including cell adhesion, PI3K-AKT signaling pathway, olfactory transduction, and cell cycle (METH-METH group vs. NS-METH, P<0.0001). For the METH acute treatment (NS-METH) group, the genes categorize by KEGG analysis were enriched in the pathways including the cell adhesion, the pathway in cancer, and dopaminergic pathway.
Changes in histone acetylation in the PFC of METH-induced sensitization rats
ChIP coupled with DNA microarray analysis revealed that METH resulted in increased histone acetylation modification (H3 or H4) on a large number of gene promoters (Figure 4), in accordance with mRNA activation. Compared with the NS-METH group, the METH-METH group induced much more acetylation modifications of H3 than H4 on the promoters; specifically, 821 genes presented H3 hyperacetylation and 10 genes showed H4 hyperacetylation (Table S5). The NS-METH group itself also induced hyperacetylation modifications on the gene promoters, including 947 genes at H3 and 4902 genes at H4. There was very little H3/H4 hyperacetylation that overlapped between the METH-METH and NS-METH groups.
Real-time PCR confirmation of the candidate genes
Based on the mRNA microarray, GO and KEGG enrichment analyses, the genes listed in Table S6 were likely to associate with METH-induced behavioral sensitization. Of these genes, Avp, Bcl2l1, Egr1, E2f3, Lnx2, Shoc2, Stx2 and Zfp36 clustered into the top 10 GO and KEGG classifications, and Anp32a, Eml2, Exog, Hira, Metrn, Pou3f2, Stk32, Syt8, and Trim17 were included in the GO category. According to the GO category analysis, Lnx2, Shoc2 and Stx2 were changed by METH single injection, and the other genes above were affected by METH-induced behavioral sensitization (METH-METH) compared with METH acute treatment (NS-METH). We then used qPCR to confirm some genes of interest. The results showed that METH challenge in the behavioral sensitization model caused a significant increase in the mRNA levels of acidic nuclear phosphoprotein 32 family member A (Anp32a), calcium/calmodulin-dependent protein kinase II inhibitor 1 (Camk2n1), echinoderm microtubule associated protein like 2 (Eml2) and POU class 3 homeobox 2 (Pou3f2), and a decrease in syntaxin 2 (Stx2), tripartite motif-containing 17 (Trim17),and zinc finger protein 36 (Zfp36) (P<0.05, one-way ANOVA followed by Turkey’s post hoc test, Figures 5, 6).
Alterations in ANP32A and POU3F2 in the development, withdrawal and challenge periods of METH-induced sensitization
Then, we selected ANP32A and POU3F2 and further measured the expression of mRNA and histone modification of the two genes. In addition to the challenge of behavioral sensitization, the development and withdrawal phases were also involved to investigate the changes in ANP32A and POU3F2 throughout the whole process of METH-induced behavioral sensitization. After METH (5 mg/kg, s.c.) chronic treatment for 7 days (development phase), the expression of ANP32A mRNA and H4 acetylation was markedly increased compared with the NS group (P<0.05, t-test, Figures 6A, 6B). However, ANP32A mRNA returned to the normal level, while H4 acetylation remained at the high level after 7 days of withdrawal (P<0.05, t-test). Then, injection of METH (1 mg/kg, s.c.) was used for challenge on day 15. One-way ANOVA showed a significant difference in mRNA (F(2, 18) = 8.824, P<0.01) and H4 acetylation (F(2, 26) = 6.072, P<0.01) between groups, and Tukey’s post hoc test showed that challenge induced the expression of ANP32A mRNA (P<0.01, METH-METH vs. NS-NS) and H4 acetylation (P<0.01, METH-METH vs. NS- METH, P<0.05, METH-METH vs. NS-NS) in the METH pretreatment group (METH-METH). The injection of METH alone also caused the elevation of ANP32A mRNA (P<0.05, NS-METH vs. NS-NS) but did not affect histone acetylation.
We next measured the change in POU3F2 mRNA and histone modification. POU3F2 mRNA was significantly elevated by chronic METH treatment for 7 days and decreased after 7 days of withdrawal (P<0.05, t-test), while H3 or H4 acetylation was not affected by the treatment (Figures 6C, 6D). In the challenge phase, one-way ANOVA showed that challenge with a low dose of METH caused a significant difference in mRNA (F(2, 45) = 4.01, P<0.05) and H3 and H4 hyperacetylation (H3: F(2, 36) = 4.17, P<0.05; H4: F(2, 38) = 3.47, P<0.05) between groups. Tukey’s post hoc test showed that the behavioral sensitization group produced significant elevation in mRNA (P<0.05, METH-METH vs. NS-NS), as well as H3 and H4 hyperacetylation (H3: P<0.05, METH-METH vs. NS-NS, METH-METH vs. NS-METH; H4: P<0.05, METH-METH vs. NS-NS). Notably, the single METH injection group (NS-METH) itself also increased the expression of POU3F2 mRNA (P<0.05, vs. NS-NS) but not H3/H4 acetylation.