Biochemical Neuroadaptations in the Rat Striatal Dopaminergic System after Prolonged Exposure to Methamphetamine Self-Administration

Abstract

Perturbations in striatal dopamine (DA) homeostasis might underlie the behavioral and pathobiological consequences of METH use disorder in humans. To identify potential consequences of long-term METH exposure, we modeled the adverse consequence DSM criterion of substance use disorders by giving footshocks to rats that had escalated their intake of METH during a drug self-administration procedure. Next, DA D1 receptor antagonist, SCH23390 was injected. Thereafter, rats were euthanized to measure several indices of the striatal dopaminergic system. Footshocks split the METH rats into two phenotypes: (i) shock-sensitive that decreased their METH-intake and (ii) shock-resistant that continued their METH intake. SCH23390 caused substantial dose-dependent reduction of METH taking in both groups. Stopping SCH23390 caused re-emergence of compulsive METH taking in shock-resistant rats. Compulsive METH takers also exhibited greater incubation of METH seeking than non-compulsive rats during withdrawal from METH SA. Analyses of DA metabolism revealed non-significant decreases (about 35%) in DA levels in resistant and sensitive rats. However, striatal contents of the deaminated metabolites, DOPAL and DOPAC, were significantly increased in sensitive rats. VMAT2 and DAT protein levels were decreased in both phenotypes. Moreover, protein expression levels of the D1-like DA receptor, D5R, and D2-like DA receptors, D3R and D4R, were significantly decreased in the compulsive METH takers. Our results parallel findings in post-mortem striatal tissues of human METH users who develop Parkinsonism after long-term METH intake and support the use of this model to investigate potential therapeutic interventions for METH use disorder.

Keywords: DOPAL; dopamine; dopamine metabolites; dopamine receptors; dorsal striatum; methamphetamine use disorder; parkinsonism.

Figure 1

(a) Experimental timeline for METH SA, contingent footshocks, and administration of the D1 antagonist, SCH23390. (b) Patterns of METH (n = 16) or saline (CT, n = 8) SA in male Sprague–Dawley rats. Infusion patterns during 20 days of SA training include escalation (1–14 days), maintenance (15–20 days) and footshock (21–31 days) phases. During the shock phase, current intensity was increased gradually from 0.18 to 0.42 mA over 11 days. METH SA rats were segregated into two distinct phenotypes based on their response to contingent footshocks. The shock-resistant rats (SR, n = 7) showed less than 20% decreases in the average number of METH infusions from pre-shock levels despite the footshocks. The shock-sensitive rats (SS, n = 9) significantly reduced their METH intake. On the days of SCH23390 administration, rats were given the drug 30 min prior to the METH SA sessions. SCH23390 reduced the number of METH infusions (p < 0.0001) in both phenotypes. After stopping SCH23390 administration, SR, but not SS, rats returned to compulsive METH-taking behaviors. Data represent the number of daily infusions during 9 h of access to METH or saline (0.1 mg/kg/infusion). (c) Total METH intake by SR (n = 7) and SS (n = 9) rats during the 20 days of METH SA training followed by footshock (0.18–0.42 mA). The values represent means ± SEM. Key to statistics: ##, ### = p < 0.01, 0.001, comparison between SR and SS METH groups.

Figure 2

The DA antagonist, SCH23390, suppresses METH-taking behavior. (a) METH intake during footshocks and the effects of SCH23390 treatment and cessation of SCH23390 treatment. Increased intensity of footshocks reduced METH intake in SS but not in SR rats (# p < 0.05, ### p < 0.001, SR vs. SS). Administration of SCH23390 (35–37 d/days) before METH SA sessions significantly decreased METH intake in SR rats in comparison to days 26–28 (&&& p < 0.001) and days 29–31 (^^^ p < 0.001) of training plus contingent shocks. Stopping SCH23390 treatment led to re-emergence (days 40–42) of compulsive METH SA in comparison to SCH23390 treatment phase (!!! p < 0.001). Two-way repeated measures analysis for METH consumed included the between-subject factors (treatment groups, SR vs. SS) and within-subject factor (four intervals of training days, 26–28 d; 29–31 d; 35–37 d and 40–42 d), and their interactions. We found significant effects of treatment groups (F (1,14) = 134.40, p < 0.0001), training days (F (3,42) = 36.58, p < 0.0001) and the interaction of the two (F (3,42) = 19.04, p < 0.0001). Key to statistics: # p < 0.05; ### p < 0.001, comparison to respective SR group; &&& p < 0.001, comparison of SR rats between treatment phase (35–37 d) and 26–28 d of footshock; ^^^ p < 0.001, comparison of SR rats between treatment phase (35–37 d) and 29–31 d of footshock; $$ p < 0.01, comparison of SR rats between re-emergence phase (40–42 d) and 26–28 d of footshock; !!! p < 0.001, comparison of SR rats between re-emergence phase (40–42 d) and treatment phase (35–37 d). (b) Dose-related effects of SCH23390 on METH infusions. Intraperitoneal injections of increasing doses of SCH23390 caused dose-dependent decreases in the number of METH infusions (F (11,84) = 33.43, p < 0.0001), analysis of variance followed by Sidak’s post hoc test. The values represent means ± SEM. Key to statistics: *** p < 0.001 vs. control; ### p < 0.001 vs. SR. (c) Time-dependent cue-induced METH seeking in SR and SS rats. The figure shows lever presses on drug-associated (active) lever on withdrawal days 2 (WD2), 7 (WD7) and 30 (WD30) of METH withdrawal. SR and SS phenotypes show minimal cue-induced drug seeking behavior on WD2. However, SR, but not SS, rats showed significant cue-induced METH seeking at WD7 and WD30. Two-way ANOVA analysis included groups (SR, SS), withdrawal days (WD2, WD7 and WD30). There were significant effects of group (F (1, 14) = 6.36, p = 0.0244), withdrawal days (F (2, 28) = 9.52, p = 0.0007) and interactions (F (2, 28) = 3.583, p = 0.0412), indicating incubation of METH seeking over the withdrawal period. Post hoc tests (Sidak’s) showed greater responses at active levers by SR rats for both WD7 (p < 0.001) and WD30 (p < 0.01) compared to WD2, whereas SS rats showed no incubation. There were also greater responses in SR rats on WD7 (p < 0.05) and WD30 (p < 0.05) in comparison to SS rats. Data are presented as means ± SEM of number of active lever presses. Key to statistics: && p < 0.01, &&& p < 0.001, significantly different from WD2; # p < 0.05, significantly different from SR rats.

Figure 3

Levels of dopamine and its metabolites in the dorsal striatum. (a) DA, (b) cys-DA (c) DOPAL, and (d) DOPAC in SR, SS, and control rats. Key to statistics: *** p < 0.001, comparison control vs. SR and SS groups; # p < 0.05, ### p < 0.001, comparison between SR and SS groups. All values represent means ± SEM. CT (n = 8), control saline rats; SR (n = 7), shock-resistant, vulnerable, compulsive METH takers; SS (n = 9), shock-sensitive, non-vulnerable group.

Figure 4

Protein levels of DA metabolizing enzymes. (a) Immunoblot analysis of MAO following withdrawal from METH self-administration. The anti-MAO antibody detected both MAO-A (59 kDa) and MAO-B (58 kDa). (b) Shock-sensitive rats exhibited higher levels of the MAO-A protein. (c) There were no differences in MAO-B levels between the three groups. (d) Western blot analysis of the ALDH enzyme in SR, SS, and control rats. ALDH protein levels were higher in the SS rats in comparison to SR rats. For quantification, the protein levels of MAO-A, MAO-B and ALDH were normalized to α-tubulin and then analyzed. Key to statistics: *** p < 0.001, comparison SR and SS vs. CT group; ## p < 0.01, ### p < 0.001, comparison between SR and SS groups. All values represent means ± SEM. CT (n = 8), control saline rats; SR (n = 7), shock-resistant, vulnerable, compulsive METH takers; SS (n = 9), shock-sensitive, non-vulnerable group.

Figure 5

METH SA is associated with decreased VMAT2 and DAT protein levels in the dorsal striatum of both SR and SS rats. Protein levels of TH (a), VMAT2 (b) and DAT (c) were determined by Western blot analyses. The left panel shows the immunoblot images, and the right panel shows the quantitative measures. For quantification, the protein levels of TH, VMAT2 and DAT were normalized to cyclophilin and then analyzed. All values represent means ± SEM (n = 6–7 rats per group). Key to statistics: * p < 0.05, ** p < 0.01, *** p < 0.001, comparison to control saline group. The statistical analysis was performed using one-way ANOVA and Tukey’s post hoc tests.

Figure 6

Protein levels of DA receptors are differentially impacted by METH SA in the dorsal striatum of rats. In the dorsal striatum, SS, but not SR, rats showed decreased D1R protein levels in comparison to the CT group (a). SR rats exhibited decreased D5R protein expression in comparison to CT and SS groups (b). There were no significant changes in D2R protein levels (c). Significant decreases in D3R and D4R protein levels were observed in both SR and SS rats (d,e). For quantification, the protein levels of D1R, D2R, D3R, D4R and D5R were normalized to cyclophilin and then analyzed. All values represent means ± SEM (n = 6–7 rats per group). Key to statistics: ** p < 0.01, *** p < 0.001, comparison to control saline group; ## p < 0.01, comparison to SR groups. The statistical analysis was performed using one-way ANOVA and Tukey’s post hoc tests.

Figure 7

Schema illustrating potential mechanisms for the observed changes in the striatal dopaminergic system of METH self-administering rats. METH SA is accompanied by increased release of DA in dopaminergic terminals followed by DA release into the synaptic cleft where DA interacts with DA receptors. Increased cytosolic DA release is associated with enzymatic and non-enzymatic formation of reactive oxygen species known to cause significant decreases in VMAT2 and DAT protein levels as observed in both SR and SS rats herein. The differential expression of post-synaptic DA D1-like receptors (D1R and D5R) supports the notion that these genes might be differentially regulated. The decreased expression of the D2-like receptors, D3R and D4R, indicates that they might have undergone similar molecular adaptations in the presence of high levels of DA released in the dorsal striatum during METH SA.