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 d15. One-way ANOVA showed the difference between groups (F(2, 18) = 18.93, P < 0.001). Rats in METH sensitization group (METH-METH group) showed significant higher locomotor activity than either saline control (NS-NS) or METH acute treatment group (NS-METH) (P < 0.001, vs NS-NS group; P < 0.01, vs NS-METH group, Fig. 1B), indicating the enhanced locomotor response of sensitization. Comparing with NS-NS group, rats in NS-METH group showed longer distance (P < 0.05, Fig. 1B).
The change of mRNA and histone modification in METH-induced behavioral sensitization
mRNA and ChIP microarray were performed to find out a genome-wide profile in METH-induced behavioral sensitization. The cut-off value was set at ≤ 0.5 or ≥ 2.0 of fold change (P < 0.01). Figure 2 shows clustering and a heat-map of the mRNA expression among NS-NS group, NS-METH group, and METH-METH group. A large number of genes was changed by the exposure of METH (P < 0.01) (Fig. 2B). Among these, 275 genes were differentially expressed in METH-METH group compared with NS-METH group (97 genes was upregulated and 178 genes was downregulated), and 232 genes were changed in NS-METH group compared with NS-NS group (122 genes was upregulated and 110 genes downregulated).
Ontological and pathway analysis of mRNAs expression in METH-induced behavioral sensitization
In order to investigate whether the clustering of differential expression genes induced by the exposure of METH correlates with functional groupings, we performed GO analysis and categorized the differential expression genes. Figure 3A and table S1 show the top 10 ranked GO terms and the number of genes in a category. Comparing with METH acute treatment (NS-METH), the biological process in the group of METH-induced behavioral sensitization (METH-METH) was markedly constructed by biological regulation, cellular process and organism process. For the molecular function, binding and protein binding were enriched in group of METH-METH.
To assess the functional features of METH-induced behavioral sensitization mediated gene sets, we further performed the KEGG pathway enrichment analysis. The top 10 significant ranked pathways were shown in Fig. 3B and table S2. Many signal transduction pathways were enriched in the group of METH-induced behavioral sensitization, including cell adhesion, PI3K-AKT signalling pathway, olfactory transduction, cell cycle, et.al (METH-METH group vs. NS-METH, P < 0.0001), which were different from the KEGG pathway enrichment after METH acute treatment (NS-METH group vs. NS-NS).
The change of histone acetylation in METH-induced behavioral sensitization
ChIP coupled with DNA microarray analysis revealed that METH resulted in the increased histone acetylation modification (H3 or H4) on a large number of genes’ promoters (Fig. 4), in accordance with the mRNA activation. Comparing with NS-METH group, METH-METH group induced much more acetylation modification in H3 than H4 on the promoters, in which 821 genes presented H3 hyperacetylation and 10 genes showed H4 hyperacetylation. Group of NS-METH itself also induced hyperacetylation modifications on the gene promotes, including 947 genes in H3 and 4902 genes in H4. There was very few H3/H4 hyperacetylation that was overlapped between METH-METH and NS-METH group.
The alteration of ANP32A and POU3F2 in the development, withdrawal and challenge period of METH-induced behavioral sensitization
We further analysed the genes screened out by mRNA and ChIP microarray in the present study, and listed the genes that were likely to be participated in METH-induced behavioral sensitization (see table S3) by taking into account the biological functions associated with neuropsychological diseases. Then we selected ANP32A (Acidic leucine-rich nuclear phosphoprotein-32A) and POU3F2 (The POU domain, class 3, transcription factor 2) and further measured the expression of mRNA and histone modification of the two genes.
Besides the challenge of behavioral sensitization, development and withdrawal phases were also be involved to investigate the change of ANP32A and POU3F2 in 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 NS group (P < 0.05, Fig. 5A, 5B). However, ANP32A mRNA returned to the normal level while H4 acetylation still remained in the high level after 7 days of withdrawal (P < 0.05). Then, injection of METH (1 mg/kg, s.c.) was used for challenge on d15, which induced the increased expression of ANP32A mRNA (P < 0.01, METH-METH vs. NS-NS) and H4 acetylation (P < 0.01, METH-METH vs. NS-NS, P < 0.05, NS-METH vs. NS-NS) in METH pre-treatment group (METH-METH) (P < 0.01, METH-METH vs. NS-NS). 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 of POU3F2 mRNA and histone modification. mRNA of POU3F2 was significantly elevated by METH chronic treatment for 7 days and decreased after 7 days of withdrawal (P < 0.05, Fig. 5C, 5D), while the H3 or H4 acetylation was not affected by the treatment. In the challenge phase, challenge by low dose of METH caused the pronounced elevation of POU3F2 mRNA in the behavioural sensitization group (P < 0.05, METH-METH vs. NS-NS), as well as H3 and H4 hyperacetylation. Notably, the single METH injection itself also increased the expression of POU3F2 mRNA, but not H3/H4 acetylation in NS-METH group (P < 0.05, vs. NS-NS).