Role of Dorsomedial Striatum Neuronal Ensembles in Incubation of Methamphetamine Craving after Voluntary Abstinence

Abstract

We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we studied the role of dorsolateral striatum (DLS) and dorsomedial striatum (DMS) in this incubation. We trained rats to self-administer palatable food pellets (6 d, 6 h/d) and methamphetamine (12 d, 6 h/d). We then assessed relapse to methamphetamine seeking under extinction conditions after 1 and 21 abstinence days. Between tests, the rats underwent voluntary abstinence (using a discrete choice procedure between methamphetamine and food; 20 trials/d) for 19 d. We used in situ hybridization to measure the colabeling of the activity marker Fos with Drd1 and Drd2 in DMS and DLS after the tests. Based on the in situ hybridization colabeling results, we tested the causal role of DMS D₁ and D₂ family receptors, and DMS neuronal ensembles in "incubated" methamphetamine seeking, using selective dopamine receptor antagonists (SCH39166 or raclopride) and the Daun02 chemogenetic inactivation procedure, respectively. Methamphetamine seeking was higher after 21 d of voluntary abstinence than after 1 d (incubation of methamphetamine craving). The incubated response was associated with increased Fos expression in DMS but not in DLS; Fos was colabeled with both Drd1 and Drd2 DMS injections of SCH39166 or raclopride selectively decreased methamphetamine seeking after 21 abstinence days. In Fos-lacZ transgenic rats, selective inactivation of relapse test-activated Fos neurons in DMS on abstinence day 18 decreased incubated methamphetamine seeking on day 21. Results demonstrate a role of DMS dopamine D₁ and D₂ receptors in the incubation of methamphetamine craving after voluntary abstinence and that DMS neuronal ensembles mediate this incubation.

Significance statement: In human addicts, abstinence is often self-imposed and relapse can be triggered by exposure to drug-associated cues that induce drug craving. We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we used classical pharmacology, in situ hybridization, immunohistochemistry, and the Daun02 inactivation procedure to demonstrate a critical role of dorsomedial striatum neuronal ensembles in this new form of incubation of drug craving.

Keywords: Daun02 inactivation; incubation of drug craving; neuronal ensembles; relapse; self-administration; voluntary abstinence.

Figure 1.

Incubation of methamphetamine (Meth) craving is associated with the activation of DMS, but not of DLS (behavioral data). A, Timeline of the experiment. B, Food and methamphetamine self-administration (mean ± SEM number of food rewards; 5 pellets/lever press) and methamphetamine infusions during the 6 h sessions. C, Discrete choice sessions during training: mean ± SEM food rewards and methamphetamine infusions earned during the three discrete choice sessions during training. D, Voluntary abstinence: mean ± SEM number of food rewards and methamphetamine infusions earned during the 19 discrete choice sessions. E, Relapse tests: mean ± SEM number of nonreinforced lever presses during the 60 min test sessions. *p < 0.05 different from day 1. n = 5/group. Circles represent individual data. SA, self-administration.

Figure 2.

Incubation of methamphetamine craving is associated with the activation of DMS, but not of DLS: RNAscope data. A, Fos expression in Drd1- and Drd2-positive cells: DMS. Left, Fos-positive cells in the DMS in rats that were tested for relapse to methamphetamine seeking on abstinence days 1 and 21 (*p < 0.05, different from day 1). Black dotted line indicates Fos expression level in the no-test control group. Circles represent individual data. Right, Percentages of Fos-positive cells that coexpress Drd1 mRNA (Drd1 plus Fos), Drd2 mRNA (Drd2 plus fos), or neither (Fos alone) in DMS. Bottom panels, Representative images of DMS region (left) and Fos labeling in the no test and relapse test groups (middle), and Drd1 or Drd2 labeling in the relapse test group (right; Fos, white; Drd1, green; Drd2, red; DAPI, blue). Arrows indicate representative cells. B, Fos expression in Drd1- and Drd2-positive cells. Left, DLS Fos-positive cells in the DLS in rats that were not tested or tested for methamphetamine seeking in an relapse test on abstinence days 1 and 21. Black dotted line indicates Fos expression level in the No-test controls. Circles represent individual data. Right, Percentages of Fos-positive cells that coexpress Drd1 (Drd1 plus Fos), Drd2 mRNA (Drd2 plus Fos), or neither (Fos alone) in DLS. Bottom panels, Representative images of DLS region (left; scale bar, 1000 μm) and Fos labeling in both the no-test and relapse test groups (middle; scale bar, 20 μm), and Drd1 or Drd2 labeling in the relapse test group (right; scale bar, 20 μm; Fos, white; Drd1, green; Drd2, red; DAPI, blue). Arrows indicate representative cells. n = 5/group for day 1 and day 21 test groups, and n = 6 for the no-test group (black dashed line).

Figure 3.

Blockade of DMS D₁ and D₂ family receptors decreased incubated methamphetamine seeking after voluntary abstinence. A, Timeline of the experiment. B, Food and methamphetamine (Meth) self-administration (SA): mean ± SEM number of food rewards and methamphetamine infusions during the 6 h sessions. C, Discrete choice sessions during training: mean ± SEM number of food rewards and methamphetamine infusions earned during the three discrete choice sessions during training. D, Voluntary abstinence: mean ± SEM number of food rewards and methamphetamine infusions earned during the 19 discrete choice sessions. E, Relapse tests: mean ± SEM number of nonreinforced lever presses during the 60 min relapse tests on abstinence days 1 and 21. *Different from day 1, p < 0.05. F, Lever presses on the previously active lever during the 60 min relapse session on day 21. Circles represent individual data. *Different from vehicle, p < 0.05. G, Representative photomicrograph of the cannula placement in DMS (scale bar, 800 μm). n = 9 for day 1 groups and n = 10–11 for day 21 groups.

Figure 4.

Selective inhibition of methamphetamine (Meth) incubation-related activated DMS neurons with Daun02 decreased incubation of drug craving. A, Timeline of the experiment. B, Food and methamphetamine self-administration (SA): mean ± SEM number of food rewards and methamphetamine infusions during the 6 h sessions. C, Discrete choice sessions during training: mean ± SEM number of food rewards and methamphetamine infusions earned during the three discrete choice sessions during training. D, Voluntary abstinence: mean ± SEM number of food rewards and methamphetamine infusions earned during the 14 discrete choice sessions. E, Relapse tests: mean ± SEM number of nonreinforced presses on the methamphetamine-associated active lever during the 90 min relapse tests on abstinence day 21. Circles represent individual data. F, Time course of the methamphetamine-associated active lever presses during testing. G, Representative photomicrograph of cannula placements in DMS (scale bar, 800 μm). H, β-Gal-positive cells in the DMS (top) and DLS (bottom) in rats that were tested for relapse to methamphetamine seeking on abstinence day 21. Circles represent individual data. I, Representative photomicrograph of β-gal staining (top, DMS; bottom, DLS). *Different from vehicle, p < 0.05. n = 11–14/group. J, K, Fos plus β-gal and NeuN plus Fos double labeling: representative photomicrographs (20×) of Fos plus β-gal (J) or NeuN plus Fos (K) in DMS of vehicle-treated Fos-LacZ rats (n = 4) that were tested for relapse to methamphetamine seeking on abstinence 21. SA, self-administration.

Figure 5.

Selective inhibition of methamphetamine (Meth) relapse-encoding DMS neuronal ensembles with Daun02 had no effect on food seeking. A, Timeline of the experiment. B, Food and methamphetamine self-administration (SA): mean ± SEM number of food rewards and methamphetamine infusions during the 6 h sessions. C, Discrete choice sessions during training: mean ± SEM number of food rewards and methamphetamine infusions earned during the three discrete choice sessions during training. D, Voluntary abstinence: mean ± SEM number of food rewards and methamphetamine infusions earned during the 14 discrete choice sessions. E, Food relapse tests: mean ± SEM number of nonreinforced presses on the food-associated active lever during the 90 min test session on abstinence day 21. Circles represent individual data. F, Time course of the food-associated active lever presses. G, Representative photomicrograph of cannula placements in DMS (scale bar, 800 μm). H, β-Gal-positive cells in the DMS in rats that were tested for food seeking on abstinence 21. Circles represent individual data. I, Representative photomicrograph of β-gal staining. n = 13/group. SA, self-administration.