Parkin regulates drug-taking behavior in rat model of methamphetamine use disorder

Abstract

There is no FDA-approved medication for methamphetamine (METH) use disorder. New therapeutic approaches are needed, especially for people who use METH heavily and are at high risk for overdose. This study used genetically engineered rats to evaluate PARKIN as a potential target for METH use disorder. PARKIN knockout, PARKIN-overexpressing, and wild-type young adult male Long Evans rats were trained to self-administer high doses of METH using an extended-access METH self-administration paradigm. Reinforcing/rewarding properties of METH were assessed by quantifying drug-taking behavior and time spent in a METH-paired environment. PARKIN knockout rats self-administered more METH and spent more time in the METH-paired environment than wild-type rats. Wild-type rats overexpressing PARKIN self-administered less METH and spent less time in the METH-paired environment. PARKIN knockout rats overexpressing PARKIN self-administered less METH during the first half of drug self-administration days than PARKIN-deficient rats. The results indicate that rats with PARKIN excess or PARKIN deficit are useful models for studying neural substrates underlying "resilience" or vulnerability to METH use disorder and identify PARKIN as a novel potential drug target to treat heavy use of METH.

Fig. 1. Validation of PARKIN overexpression.

A Timeline for experiments with PARKIN overexpression. B, C Validation of the overexpression site (nucleus accumbens, 0.7–2.7 mm from bregma) and spread (medial-lateral, dorsal-ventral, and anterior-posterior). D Dose–response of PARKIN overexpression (0, 0.7, 2, or 6.5 × 10⁷ TUs/side) in the nucleus accumbens (n = 3 or 4/group). The data followed a linear regression curve (R² = 0.993, p < 0.0001). E Time course of PARKIN overexpression (2 × 10⁷ TUs/side) showing that PARKIN overexpression reached the maximum at around 3.5 weeks after the gene transfer vector microinjection and excess of PARKIN persisted at least for the duration of the study (~4 weeks) (D). Parkin band migrated to ~52 kDa (relate to the marked molecular weight of 55 kDa). Parkin band was the only band detected; a representative full blot is presented in Fig. 4.

Fig. 2. Noncoding-AAV and Sham-microinjection control for behavioral experiments.

Neither noncoding AAV2/6 (NC-AAV) nor sham microinjection (SM, microinjection of phosphate-buffered saline) had an effect on methamphetamine (METH) self-administration or preference for METH-paired compartment in the conditions place preference test in wild type (WT) rats. A Lever presses in NC-AAV and SM group. B METH intake in NC-AAV and SM group. C Conditioned place preference test results for NC-AAV and SM group. D METH intake in WT vs. NC-AAV and SM rats. E Preference for METH-paired compartment in WT vs. NC-AAV and SM rats. Data are expressed as mean ± SEM, n = 4/group. Abbreviations: CPP conditioned place preference, NAc nucleus accumbens, L left, R right.

Fig. 3. PARKIN knockout increases extended-access methamphetamine self-administration (EA METH SA) and METH reward.

A Timeline of EA METH SA experiment. B, C Escalation of METH taking in Parkin gene knockout (PKO) rats as compared to wild-type (WT) rats during an increasing ratio schedule of reinforcement: FR1, FR2, and FR5. The PKO rats pressed for METH at a significantly higher rate than the WT rats (B) with a consequent higher consumption of METH (C). There was no significant difference between PKO and WT rats in respect of the number of presses for saline (B inset, n = 4/group). *p < 0.05, **p < 0.01, ***p < 0.001, n = 11/group. D Total amount of METH consumed by PKO rats was significantly higher than that consumed by WT rats (+39% **p < 0.001). E Timeline of conditioned place preference experiment. F The PKO rats displayed a significantly higher preference for METH (4 mg/kg) than the WT rats. ***p < 0.001 as compared to the respective saline (SAL) controls, ^#p < 0.05 WT vs. PKO, n = 6/group. Data are expressed as mean ± SEM. Abbreviations: CPP conditioned place preference, FR fixed ratio.

Fig. 4. Extended-access methamphetamine self-administration (EA METH SA) decreases PARKIN protein levels in the nucleus accumbens.

A Representative PARKIN bands (~52 kDa) assessed in nucleus accumbens (NAc) of wild type (WT) rats, with β-ACTIN serving as a loading control: SAL (black) represents saline-yoked WT rats while METH (red) represents WT rats that underwent EA METH SA; both group of rats were killed 10 days after the last operant session. B Quantification of PARKIN bands. As compared to saline (SAL) administration, EA METH SA led to a deficit (−24%) in PARKIN levels in the nucleus accumbens of WT rats at 10 days after the last operant session. **p < 0.01, n = 8 or 9/group. Data are expressed as mean ± SEM.

Fig. 5. PARKIN overexpression in the nucleus accumbens of WT rats attenuates extended-access methamphetamine self-administration (EA METH SA) and METH reward.

Escalation of METH taking in rats overexpressing PARKIN in the nucleus accumbens as compared to non-expressing wild-type (WT) rats during an increasing ratio schedule of reinforcement: FR1, FR2, and FR5. A At the low PARKIN overexpression (4–6-fold), the PARKIN overexpressing (PO) rats did not press for METH at a significantly lower rate than the WT rats and, consequently, B they did not consume less METH than the WT rats. C At the high PARKIN overexpression (15–20-fold), the PO rats did press for METH at a significantly lower rate than the WT rats and D consumed less METH than the WT rats. *p < 0.05, **p < 0.01, ***p < 0.001, WT vs. PO-H, n = 6/group. E Quantification of total METH intake in PO-L and PO-H rats as compared to their respective WT control groups. There was no significant difference in total METH intake between the PO-L group and WT controls whereas the PO-H group consumed significantly less METH than the WT group (−33%, ***p < 0.001). F Correlation analysis of total METH intake with PARKIN levels in PO-H group (red dots: PO-H group; green dots: WT group). G The 15–20-fold excess of PARKIN was sufficient to significantly attenuate preference for METH (4 mg/kg) in WT rats in the conditioned place preference test. ***p < 0.001 as compared to the respective saline controls, ^#p < 0.05 WT vs. PO-H, n = 6/group. Data are expressed as mean ± SEM. Abbreviations: NAc nucleus accumbens, FR fixed ratio, PO-L lower PAKIN overexpression, PO-H higher PARKIN overexpression, SAL saline.

Fig. 6. The effect of PARKIN overexpression in the nucleus accumbens of Parkin gene knockout (PKO) rats.

High PARKIN overexpression (15–20-fold) in the nucleus accumbens of Parkin knockout PKO rats significantly attenuated methamphetamine (METH) taking in the first half of the extended-access METH self-administration as compared to non-overexpressing PKO rats. A Lever presses for METH, B METH intake. C Total amount of METH consumed by PKO PO rats was significantly lower than the total amount of METH consumed by PKO rats (−18%). D Correlation analysis of total METH intake with PARKIN levels in PKO PO group (red dots: PKO PO group; green dots: PKO group). *p < 0.05, **p < 0.01, n = 5 or 6/group. Data are expressed as mean ± SEM.