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

Role of nucleus accumbens core but not shell in incubation of methamphetamine craving after voluntary abstinence

We recently introduced an animal model to study incubation of drug craving after prolonged voluntary abstinence, mimicking the human condition of relapse after successful contingency management treatment. Here we studied the role of the nucleus accumbens (NAc) in this model. We trained rats to self-administer a palatable solution (sucrose+maltodextrin 1%, 6 h/day, 6 days) and methamphetamine (6 h/day, 12 days). We then evaluated relapse to methamphetamine seeking after 1 and 15 days of voluntary abstinence, achieved via a discrete choice procedure between the palatable solution and methamphetamine (14 days). We used RNAscope in-situ hybridization to quantify the co-labeling of the neuronal activity marker Fos, and dopamine Drd1- and Drd2-expressing medium spiny neurons (MSNs) in NAc core and shell during the incubation tests. Next, we determined the effect of pharmacological inactivation of NAc core and shell by either GABAA and GABAB agonists (muscimol+baclofen, 50+50 ng/side), Drd1-Drd2 antagonist (flupenthixol, 10 µg/side) or the selective Drd1 or Drd2 antagonists (SCH39166 1.0 µg/side or raclopride 1.0 µg/side) during the relapse tests. Incubated methamphetamine seeking after voluntary abstinence was associated with a selective increase of Fos expression in the NAc core, but not shell, and Fos was co-labeled with both Drd1- and Drd2-MSNs. NAc core, but not shell, injections of muscimol+baclofen, flupenthixol, SCH39166, and raclopride reduced methamphetamine seeking after 15 days of abstinence. Together, our results suggest that dopamine transmission through Drd1 and Drd2 in NAc core is critical to the incubation of methamphetamine craving after voluntary abstinence

Neural mechanisms of relapse to methamphetamine seeking after voluntary abstinence in a rat model

Relapse to drug use after prolonged abstinence is the key impediment to successful treatment of drug addiction (Wikler, 1973; Jaffe, 1990). It has been known for many years that during abstinence exposure to cues and contexts previously associated with the rewarding effects of drugs can provoke relapse to drug use (Wikler, 1973; O'Brien et al., 1992). Relapse to drug use during abstinence has been the focus of preclinical studies for over 30 years (Stewart and de Wit, 1987). The abstinence period preceding relapse testing in studies using animal models is typically experimenter-imposed or forced; this is achieved either by removal of the laboratory animal from the drug self-administration environment or through extinction training (Shalev et al., 2002; Shaham et al., 2003). However, in humans abstinence is often voluntary due to either the negative consequences of chronic drug use or the presence of alternative non-drug rewards (Katz and Higgins, 2003; Epstein et al., 2006) Based on these considerations, the aim of my thesis was twofold: (1) to develop an animal model of relapse to drug seeking, which incorporates some aspects of the voluntary abstinence, and (2) to explore the neuronal mechanisms underlying this form of relapse. Based on recent studies of Ahmed and colleagues (Lenoir et al., 2007; Cantin et al., 2010; Ahmed et al., 2013b), we first investigated how to achieve voluntary abstinence in rats. We found that by using a mutually exclusive discrete choice procedure, rats voluntarily abstain from methamphetamine because of the presence of a non-drug reward (high carbohydrate palatable food pellets). Indeed, independently of the daily drug access conditions and the withdrawal period, the rats strongly preferred palatable food over methamphetamine, even when they were given free access to the palatable food in the home cage (Caprioli et al., 2015a). Subsequently, we used this choice procedure to develop a rat model of incubation of drug craving in which abstinence is achieved voluntarily (Caprioli et al., 2015b). In this model, we first train rats with free access to \u2018animal facility\u2019 nutritionally-balanced food to self-administer palatable food pellets and then to self-administer intravenous methamphetamine. Next, we assess cue-induced methamphetamine seeking in extinctions test during early (day 1) and late (day 14 or 21) abstinence. Between tests, the rats undergo voluntary abstinence in their drug environment; this is achieved via a discrete choice procedure between methamphetamine and palatable food. In this model, cue-induced methamphetamine seeking is higher after 21 abstinence days than after 1 day (incubation of methamphetamine craving). Using this animal model we showed a robust incubation of methamphetamine craving after prolonged periods of choice-based voluntary abstinence (Caprioli et al., 2015b). This effect was observed under two different self-administration procedures that are widely used to model drug addiction: extended daily access drug self-administration procedure (Ahmed and Koob, 1998; Ahmed, 2011) and a long-term training procedure used to identify addicted rats based on DSM-IV criteria (Deroche-Gamonet et al., 2004; Piazza and Deroche-Gamonet, 2013) A question that derives from our study is whether similar or different neurobiological mechanisms control incubation of methamphetamine craving after voluntary versus forced abstinence. For this purpose, we used AZD8529, a new and highly selective positive allosteric modulator (PAM) of metabotropic glutamate receptor 2 (mGluR2) (Justinova et al., 2015). AZD8529 had a similar effect on incubated cue-induced methamphetamine seeking after voluntary and forced abstinence, suggesting mechanistic similarities (Caprioli et al., 2015b). An important question moving forward was which neural mechanisms are critical for the relapse triggered by drug-associated cues after voluntary abstinence. To address this question, we used the neuronal activity marker Fos (Curran and Morgan, 1995) and local dopamine receptor blockade and demonstrated a critical role of central amygdala (CeA) in cue-induced methamphetamine seeking after voluntary abstinence. Moreover, in our follow-up tracing study we used the retrograde tracer cholera toxin subunit b (CTb) in combination with Fos and identified a selective activation of the anterior insula (AI) to CeA projection in this form of relapse. These results are consistent with human imaging studies showing that cue-induced cocaine craving is associated with insula and amygdala activation (Grant et al., 1996; Garavan et al., 2000) and that cue-induced methamphetamine craving is associated with insula activation (Yin et al., 2012).Relapse to drug use after prolonged abstinence is the key impediment to successful treatment of drug addiction (Wikler, 1973; Jaffe, 1990). It has been known for many years that during abstinence exposure to cues and contexts previously associated with the rewarding effects of drugs can provoke relapse to drug use (Wikler, 1973; O'Brien et al., 1992). Relapse to drug use during abstinence has been the focus of preclinical studies for over 30 years (Stewart and de Wit, 1987). The abstinence period preceding relapse testing in studies using animal models is typically experimenter-imposed or forced; this is achieved either by removal of the laboratory animal from the drug self-administration environment or through extinction training (Shalev et al., 2002; Shaham et al., 2003). However, in humans abstinence is often voluntary due to either the negative consequences of chronic drug use or the presence of alternative non-drug rewards (Katz and Higgins, 2003; Epstein et al., 2006) Based on these considerations, the aim of my thesis was twofold: (1) to develop an animal model of relapse to drug seeking, which incorporates some aspects of the voluntary abstinence, and (2) to explore the neuronal mechanisms underlying this form of relapse. Based on recent studies of Ahmed and colleagues (Lenoir et al., 2007; Cantin et al., 2010; Ahmed et al., 2013b), we first investigated how to achieve voluntary abstinence in rats. We found that by using a mutually exclusive discrete choice procedure, rats voluntarily abstain from methamphetamine because of the presence of a non-drug reward (high carbohydrate palatable food pellets). Indeed, independently of the daily drug access conditions and the withdrawal period, the rats strongly preferred palatable food over methamphetamine, even when they were given free access to the palatable food in the home cage (Caprioli et al., 2015a). Subsequently, we used this choice procedure to develop a rat model of incubation of drug craving in which abstinence is achieved voluntarily (Caprioli et al., 2015b). In this model, we first train rats with free access to \u2018animal facility\u2019 nutritionally-balanced food to self-administer palatable food pellets and then to self-administer intravenous methamphetamine. Next, we assess cue-induced methamphetamine seeking in extinctions test during early (day 1) and late (day 14 or 21) abstinence. Between tests, the rats undergo voluntary abstinence in their drug environment; this is achieved via a discrete choice procedure between methamphetamine and palatable food. In this model, cue-induced methamphetamine seeking is higher after 21 abstinence days than after 1 day (incubation of methamphetamine craving). Using this animal model we showed a robust incubation of methamphetamine craving after prolonged periods of choice-based voluntary abstinence (Caprioli et al., 2015b). This effect was observed under two different self-administration procedures that are widely used to model drug addiction: extended daily access drug self-administration procedure (Ahmed and Koob, 1998; Ahmed, 2011) and a long-term training procedure used to identify addicted rats based on DSM-IV criteria (Deroche-Gamonet et al., 2004; Piazza and Deroche-Gamonet, 2013) A question that derives from our study is whether similar or different neurobiological mechanisms control incubation of methamphetamine craving after voluntary versus forced abstinence. For this purpose, we used AZD8529, a new and highly selective positive allosteric modulator (PAM) of metabotropic glutamate receptor 2 (mGluR2) (Justinova et al., 2015). AZD8529 had a similar effect on incubated cue-induced methamphetamine seeking after voluntary and forced abstinence, suggesting mechanistic similarities (Caprioli et al., 2015b). An important question moving forward was which neural mechanisms are critical for the relapse triggered by drug-associated cues after voluntary abstinence. To address this question, we used the neuronal activity marker Fos (Curran and Morgan, 1995) and local dopamine receptor blockade and demonstrated a critical role of central amygdala (CeA) in cue-induced methamphetamine seeking after voluntary abstinence. Moreover, in our follow-up tracing study we used the retrograde tracer cholera toxin subunit b (CTb) in combination with Fos and identified a selective activation of the anterior insula (AI) to CeA projection in this form of relapse. These results are consistent with human imaging studies showing that cue-induced cocaine craving is associated with insula and amygdala activation (Grant et al., 1996; Garavan et al., 2000) and that cue-induced methamphetamine craving is associated with insula activation (Yin et al., 2012)

Novel models of drug relapse and craving after voluntary abstinence

n/aWe introduced two novel models of choice-based voluntary abstinence and demonstrated the profound protective effects of positive social interaction on drug addiction and relapse in rat models. Our findings support wider implementation of social-based behavioral treatments, which include not only the established community reinforcement approach, but also social-based psychotherapies and family-based social support systems to provide social support before and during drug-seeking episodes