Behaviors

Figure 1a shows the timeline of the behavioral experiments. METH-trained rats increased their drug intake and control rats decreased lever pressing for saline during the SA training phase (Fig. 1b). The repeated measures ANOVA for earned infusions included the between-subject factor reward type (saline, METH) and the within-subject factor of SA day (training days 1–20). The analysis showed a significant effect of training day x reward type [F(19,456) = 25.31, p < 0.0001]. The significant interaction reflects the fact that METH-trained rats continued to increase their drug intake over the training days whereas the saline rats decreased and stabilized their intake to very low values (Fig. 1b).

Figure 1 Extended access to methamphetamine and footshock punishment lead to compulsive methamphetamine taking or abstinence in rats. (a) Timeline of behavioral experiments. (b) Rats with long access to METH escalate drug self-administration. The groups were separated post-facto based on their responses to footshocks. (c) Increasing footshock intensity leads to reduction of METH intake in SS but not in SR animals. (d) Mean drug intake on the last 3 days of METH SA compared to last 3 days of shocks. (e) Rats show incubation of METH craving at withdrawal day (WD) 30 in comparison to WD2. SR rats showed higher lever pressing at WD30 in comparison to the SS rats. Full size image

During the training plus punishment phase, footshock intensity was increased from 0.18 to 0.30 A over a period of 5 days (Fig. 1c). The increased shock intensity caused reinforced responding to significantly decrease in the shock-sensitive (SS) but not in the shock-resistant (SR) rats (Fig. 1c). The statistical analysis of METH infusions earned included the between-subject factor of groups [(SR) and (SS)] and the within-subject factor of shockday (shockdays 1–5). There was a significant effect of shockday x group [F(4,56) = 16.95, p < 0.0001], with the highest intensity (0.30 mA) showing almost total suppression of METH intake in the SS rats (Fig. 1c). Figure 1d shows the effects of the highest intensity (0.30 mA, 3 days of footshocks) on METH intake in comparison to the last 3 days of training before application of foot-shocks. Rats that were yoked to SR rats to receive non-contingent footshocks (YSR) received significant more footshocks than rats yoked (YSS) to the SS group [(291 ± 39 vs 94 ± 13, respectively), p < 0.001].

We also measured cue-induced reinstatement at withdrawal days 2 and 30 (Fig. 1e) and found that both SR and SS animals increased lever pressing at WD 30 in comparison to WD2. In addition, the SS rats showed less lever pressing than the SR rats at both time points.

Microarray Analyses

We used the Affymetrix array platform that contain a total of 68,842 probes to measure transcriptional changes in the NAc and dorsal striatum of animals euthanized one day after the second extinction test. These probes consist of 24,753 protein coding, 28,724 noncoding, and unassigned pseudogenes. The analysis of raw array data revealed that 9044 genes were expressed in the NAc and 9754 genes were expressed in the dorsal striatum. This was determined by comparing the average signal values of transcripts in the control animals to the average signal value of the bioB gene in the control animals. Gene with average signals above the average signal value of the bioB gene were considered as expressed in the specific brain region. The expressed genes were included in further analyses of differential gene expression between the experimental groups. There were 181 genes differentially expressed in the NAc and 304 genes were differentially expressed in the dorsal striatum.

The results of the microarray analyses in the NAc are shown in Figs 2a and 3a, Table 1, and in supplementary Tables S1 and S1. There were multiple pairwise comparisons that included: shock-resistant versus control (SRvsCT), shock-sensitive versus CT (SSvsCT), and SR versus SS (SRvsSS) groups, yoked SR versus CT (YSRvsCT), yoked SS versus CT (YSRvsCT), and YSRvsYSS (see Figs 2a and 3a and Table 1, and in Supplementary Tables S1 and S1). The differences in gene expression between YSR and YSS are shown in the Supplementary Table S2. In the NAc, there were 13 (6 up- and 7 downregulated) differentially expressed transcripts in the SRvsCT comparison (±1.7-fold change, p < 0.05) (Fig. 2a). The SSvsCT comparison contained only 3 (1 up and 2 down) differentially expressed genes while the SRvsSS comparison had 9 (1 up-and 8 downregulated) differentially expressed transcripts. We used DAVID and literature searches to generate functional annotation and classification analyses for significantly expressed transcripts. The names of these genes and their classification are shown in Table 1. The list includes genes that participate in metabolism and signal transduction as well as several microRNA precursor transcripts. Genes of interest in the SRvsCT comparison include oxytocin that showed 6.19-fold increases in the SR group. OXT was also increased in the SR in comparison to the SS group (5.71-fold) (Table 1). The yoked animals also showed some changes in gene expression in comparison to control (Fig. 3a). Rats yoked to the SR group showed changes in 6 transcripts (3 up and 3 up) in comparison to control. Animals yoked to the SS group showed 9 (4 up and 5 down) differentially expressed genes. The list of genes also included genes involved in metabolism, phosphorylation cascades, and signal transduction (Supplementary Table S1).

Figure 2 Microarray analysis of gene expression in the NAc and dorsal striatum of rats after one month of withdrawal from methamphetamine SA. Comparison of gene expression in pairwise comparisons between SR, SS, and control rats in NAc (a) in dorsal striatum (b) [n = 6 in each group]. The number of up- and downregulated genes in each comparison is shown in red and green, respectively. The list of genes and their classification are shown in Tables 1 and 2. Full size image

Figure 3 Microarray analysis of gene expression in the NAc and striatum of rats after one month of withdrawal from methamphetamine SA. Comparison of gene expression in pairwise comparisons between YSR (n = 3), YSS (n = 3), and control (n = 6) rats in NAc (a) in dorsal striatum (b). The number of up- and downregulated genes in each comparison is shown in red and green, respectively. The list of genes and their classification are shown in Tables 3 and 4. Full size image

Table 1 Classification of NAc genes with altered expression after METH SA and footshocks. Full size table

The results of the striatal microarray data are shown in Figs 2b and 3b, Table 2, and in supplementary Tables S3 and S4. Figure 2b shows that there were 17 (16 up and 1 down) differentially expressed transcripts in the SRvsCT comparison, 4 (3 up and 1 down) transcripts in the SSvsCT comparison, and 9 (6 up and 3 down) transcripts in the SRvsSS comparison. Table 2 shows a list of genes that participate cell adhesion, metabolism, signal transduction, and transcription regulation in the dorsal striatum. Figure 3b shows the results of the comparison between the yoked animals and the control group. There were 13 (6 up and 7 down) differentially expressed transcripts in the YSRvsCT comparison, 60 (42 up and 18 down) in the YSSvsCT comparison and 29 (22 up and 7 down) differentially expressed genes in the YSRvs YSS comparison. The genes are listed in supplementary Table S3. The differences in gene expression between YSR and YSS in the dorsal striatum are shown in Supplementary Table S4.

Table 2 Classification of striatal genes with differential expression after METH SA and footshocks. Full size table

Validation of array-identified genes by quantitative PCR

Compulsive METH taking-related genes

To validate the changes in oxytocin observed in the microarray data, we ran quantitative PCR using RNAs from the various groups of rats. Figure 4 presents the effects of METH SA and footshocks on the expression of oxytocin (OXT) in the nucleus accumbens and dorsal striatum. NAc OXT mRNA levels displayed significant changes [F(4,19) = 5.44, p = 0.0043] in expression. Post-hoc analyses demonstrated that the SR group showed substantial increases in OXT mRNA expression in comparison to the CT, SS, and yoked animals (Fig. 4a). YSR and YSS groups showed small decreases that were not significant in comparison to the control group (Fig. 4a). OXT receptor (OXTR) mRNA levels also exhibited significant changes [F(4,19) = 7.19, p = 0.0011] after withdrawal from METH SA, with post-hoc analyses indicating that all groups showed small increases in comparison to the control group (Fig. 4b). In contrast, there were no significant changes in OXT [F(4,24) = 1.643, p = 0.1961] or OXTR [F(4,27) = 1.715, p = 0.1767] mRNA levels in the dorsal striatum (Fig. 4b and d, respectively).

Figure 4 PCR validation of changes in oxytocin mRNA levels in the rat NAc after a month withdrawal from methamphetamine SA and footshocks. We conducted quantitative PCR using individual RNA from the NAc (a,c) and dorsal striatum (b,d) of rats from the various conditions (CT, n = 6–8; SR, n = 6–8; SS, n = 6–9; YSR, n = 3; YSS, n = 3–6). The shock-resistant rats showed significant increases in oxytocin (a) and oxytocin receptor (c) in the NAc but not in the dorsal striatum (b and d, respectively). Values are means ± SEM fold changes relative to the control group. Key to statistics: **p < 0.01, ***p < 0.001, in comparison to the control group; ##p < 0.01, in comparison to YSR rats; ^^p < 0.01, in comparison to the YSS group; $p < 0.05, $$p < 0.01 in comparison to the SR group. Full size image

We also validated the expression of striatal CARTpt that showed increased expression between SR and SS in the microarray data (Table 2). Figure 5a showed confirmation of the significant [F(4,28) = 4.220, p = 0.0085] increases in CARTpt in the striatum of SR rats. There were, however, no significant changes in the NAc [F(4,26) = 1.059, p = 0.3966] (Fig. 5b).

Figure 5 PCR validation of changes in CARTpt mRNA expression in the dorsal striatum after a month withdrawal from methamphetamine SA and footshocks. We conducted quantitative PCR using individual RNA from the dorsal striatum (a) and NAc (b) of rats. The shock-resistant rats showed significant increases in CARTpt in the dorsal striatum (a) but not in the NAc (b). Values are means ± SEM fold changes relative to the control group. Key to statistics: *p < 0.05, in comparison to the control group; ##p < 0.01, in comparison to YSR rats; ^^p < 0.01, in comparison to the YSS group; $$p < 0.01 in comparison to the SR group. Full size image

Footshock-responsive genes

We also validated the expression of some genes that showed changes in rats that were yoked to the METH SA to receive similar number of footshocks as the METH SA animals (Fig. 6). Figure 6a and b show that animals that received footshocks experienced no significant changes in FMO2 (flavin-containing monooxygenase) in the NAc but showed significant [F(4,19) = 6.849, p = 0.0014] changes in the dorsal striatum. Post-hoc analyses showed that the YSR group (291 ± 39 footshocks) exhibited significantly higher mRNA levels in the dorsal striatum than the other groups (Fig. 6b). Figure 6c and d show that there were significant changes in the expression of PDK4 (pyruvate dehydrogenase kinase 4) in the NAc [F(4,19) = 6.388, p = 0.0020] and dorsal striatum [F(4,28) = 6.497, p = 0.0008]. In addition to the YSR group showing higher levels than the other groups in the NAc, post-hoc analyses showed that both SR and SS groups [but not the YSS (94 ± 13 footshocks) rats] also showed small increases in comparison to the control animals. The results suggest that METH might have partially attenuated the transcriptional effects of the large number of footshocks that the SR rats had received (compare YSR to SR results in Fig. 6c). In the dorsal striatum (Fig. 6d), YSR rats also showed higher expression than the other groups whereas the SR animals showed lower expression than the control group, suggesting the large amount of METH taken by the SR rats might have significantly suppressed the effects of footshocks alone (YSR) in this brain region. Smaller number of footshocks and lower amount of METH (SS) did not significantly influence the expression of PDK4 in the dorsal striatum (Fig. 6d). These data support the notion that the NAc and dorsal striatum may respond differentially to a diversity of exogenous stimuli.

Figure 6 PCR measures of changes in FMO2, PDK4, and PTPRO mRNA levels in the rat NAc and dorsal striatum after a month withdrawal from methamphetamine SA and footshocks. We conducted quantitative PCR using individual RNA from the NAc (a,c,e) and dorsal striatum (b,d,f) of rats from the 5 groups of rats. The YSR that received many non-contingent footshocks (291 ± 39) showed significant increases in FMO2 in the dorsal striatum (b) but not in the NAc (a). YSR rats also experienced increases in PDK4 in both the NAc (c) and dorsal striatum (d). PTPRO mRNA levels were also increased in the NAc (e) and dorsal striatum (f) of YSR rats. Values are means ± SEM fold changes relative to the control group. Key to statistics: *p < 0.05, **p < 0.01, ***p < 0.001, in comparison to the control group; #p < 0.01, ##p < 0.01, ###p < 0.001, in comparison to the YSR rats. Full size image

Figure 6e and f show the effects of METH and footshocks on the expression of PTPRO (protein tyrosine phosphatase, receptor type O) in the NAc and dorsal striatum, respectively. PTPRO mRNA expression was significantly [F(4.19) = 4.890, p = 0.0070] affected in the NAc. Post-hoc analyses showed that the YSR and SS groups exhibited higher mRNA expression than controls (Fig. 6e). In addition, SR rats showed lower expression than YSR animals, again suggesting that large amount of METH might have inhibited the effects of footshocks alone on PTPRO mRNA levels in the NAc. In the dorsal striatum, there were also significant [F(4,28) = 3.982, p = 0.0111] increases in the levels of PTPRO mRNA in the YSR group when compared to the other groups (Fig. 6f). Post-hoc tests indicated that PTPRO mRNA levels in the other groups were comparable to those of controls (Fig. 6f).