Dopamine D3 Receptor Modulates Akt/mTOR and ERK1/2 Pathways Differently during the Reinstatement of Cocaine-Seeking Behavior Induced by Psychological versus Physiological Stress.

Stress triggers relapses in cocaine use that engage the activity of memory-related nuclei, such as the basolateral amygdala (BLA) and dentate gyrus (DG). Preclinical research suggests that D3 receptor (D3R) antagonists may be a promising means to attenuate cocaine reward and relapse. As D3R regulates the activity of the Akt/mTOR and MEK/ERK1/2 pathways, we assessed the effects of SB-277011-A, a D3R antagonist, on the activity of these kinases during the reinstatement of cocaine-induced conditioned place preference (CPP) induced by psychological (restraint) and physiological (tail pinch) stress. Both stimuli reactivated an extinguished cocaine-CPP, but only restrained animals decreased their locomotor activity during reinstatement. Cocaine-seeking behavior reactivation was correlated with decreased p-Akt, p-mTOR, and p-ERK1/2 activation in both nuclei of restrained animals. While a D3R blockade prevented stress-induced CPP reinstatement and plasma corticosterone enhancement, SB-277011-A distinctly modulated Akt, mTOR, and ERK1/2 activation depending on the stressor and the dose used. Our data support the involvement of corticosterone in the SB-277011-A effects in restrained animals. Additionally, the ratios p-mTOR/mTOR and/or p-ERK1/2 /ERK1/2 in the BLA during stress-induced relapse seem to be related to the locomotor activity of animals receiving 48 mg/kg of the antagonist. Hence, our study indicates the D3R antagonist's efficacy to prevent stress-induced relapses in drug use through distinct modulation of Akt/mTOR and MEK/ERK1/2 pathways in memory-processing nuclei.

Morphine-withdrawal aversive memories and their extinction modulate H4K5 acetylation and Brd4 activation in the rat hippocampus and basolateral amygdala.

Chromatin modification is a crucial mechanism in several important phenomena in the brain, including drug addiction. Persistence of drug craving and risk of relapse could be attributed to drug-induced epigenetic mechanisms that seem to be candidates explaining long-lasting drug-induced behaviour and molecular alterations. Histone acetylation has been proposed to regulate drug-seeking behaviours and the extinction of rewarding memory of drug taking. In this work, we studied the epigenetic regulation during conditioned place aversion and after extinction of aversive memory of opiate withdrawal. Through immunofluorescence assays, we assessed some epigenetic marks (H4K5ac and p-Brd4) in crucial areas related to memory retrieval -basolateral amygdala (BLA) and hippocampus-. Additionally, to test the degree of transcriptional activation, we evaluated the immediate early genes (IEGs) response (Arc, Bdnf, Creb, Egr-1, Fos and Nfkb) and Smarcc1 (chromatin remodeler) through RT-qPCR in these nuclei. Our results showed increased p-Brd4 and H4K5ac levels during aversive memory retrieval, suggesting a more open chromatin state. However, transcriptional activation of these IEGs was not found, therefore suggesting that other secondary response may already be happening. Additionally, Smarcc1 levels were reduced due to morphine chronic administration in BLA and dentate gyrus. The activation markers returned to control levels after the retrieval of aversive memories, revealing a more repressed chromatin state. Taken together, our results show a major role of the tandem H4K5ac/p-Brd4 during the retrieval of aversive memories. These results might be useful to elucidate new molecular targets to improve and develop pharmacological treatments to address addiction and to avoid drug relapse.

Molecular Mechanisms Underlying the Retrieval and Extinction of Morphine Withdrawal-Associated Memories in the Basolateral Amygdala and Dentate Gyrus.

Despite their indisputable efficacy for pain management, opiate prescriptions remain highly controversial partially due to their elevated addictive potential. Relapse in drug use is one of the principal problems for addiction treatment, with drug-associated memories being among its main triggers. Consequently, the extinction of these memories has been proposed as a useful therapeutic tool. Hence, by using the conditioned place aversion (CPA) paradigm in rats, we investigated some of the molecular mechanisms that occurr during the retrieval and extinction of morphine withdrawal memories in the basolateral amygdala (BLA) and the hippocampal dentate gyrus (DG), which control emotional and episodic memories, respectively. The retrieval of aversive memories associated with the abstinence syndrome paralleled with decreased mTOR activity and increased Arc and GluN1 expressions in the DG. Additionally, Arc mRNA levels in this nucleus very strongly correlated with the CPA score exhibited by the opiate-treated rats. On the other hand, despite the unaltered mTOR phosphorylation, Arc levels augmented in the BLA. After the extinction test, Arc and GluN1 expressions were raised in both the DG and BLA of the control and morphine-treated animals. Remarkably, Homer1 expression in both areas correlated almost perfectly with the extinction showed by morphine-dependent animals. Moreover, Arc expression in the DG correlated strongly with the extinction of the CPA manifested by the group treated with the opiate. Finally, our results support the coordinated activity of some of these neuroplastic proteins for the extinction of morphine withdrawal memories in a regional-dependent manner. Present data provide evidence of differential expression and activity of synaptic molecules during the retrieval and extinction of aversive memories of opiate withdrawal in the amygdalar and hippocampal regions that will likely permit the development of therapeutic strategies able to minimize relapses induced by morphine withdrawal-associated aversive memories.

Distinct Regulation of Dopamine D3 Receptor in the Basolateral Amygdala and Dentate Gyrus during the Reinstatement of Cocaine CPP Induced by Drug Priming and Social Stress.

Relapse in the seeking and intake of cocaine is one of the main challenges when treating its addiction. Among the triggering factors for the recurrence of cocaine use are the re-exposure to the drug and stressful events. Cocaine relapse engages the activity of memory-related nuclei, such as the basolateral amygdala (BLA) and the hippocampal dentate gyrus (DG), which are responsible for emotional and episodic memories. Moreover, D3 receptor (D3R) antagonists have recently arisen as a potential treatment for preventing drug relapse. Thus, we have assessed the impact of D3R blockade in the expression of some dopaminergic markers and the activity of the mTOR pathway, which is modulated by D3R, in the BLA and DG during the reinstatement of cocaine-induced conditioned place preference (CPP) evoked by drug priming and social stress. Reinstatement of cocaine CPP paralleled an increasing trend in D3R and dopamine transporter (DAT) levels in the BLA. Social stress, but not drug-induced reactivation of cocaine memories, was prevented by systemic administration of SB-277011-A (a selective D3R antagonist), which was able, however, to impede D3R and DAT up-regulation in the BLA during CPP reinstatement evoked by both stress and cocaine. Concomitant with cocaine CPP reactivation, a diminution in mTOR phosphorylation (activation) in the BLA and DG occurred, which was inhibited by D3R blockade in both nuclei before the social stress episode and only in the BLA when CPP reinstatement was provoked by a cocaine prime. Our data, while supporting a main role for D3R signalling in the BLA in the reactivation of cocaine memories evoked by social stress, indicate that different neural circuits and signalling mechanisms might mediate in the reinstatement of cocaine-seeking behaviours depending upon the triggering stimuli.

Blockade of D3 receptor prevents changes in DAT and D3R expression in the mesolimbic dopaminergic circuit produced by social stress- and cocaine prime-induced reinstatement of cocaine-CPP.

BACKGROUND: Cocaine may cause persistent changes in the brain, which are more apparent in DA transporter (DAT) and DA receptor availability within the nucleus accumbens (NAc). On the other hand, the DA D3 receptor (D3R) has emerged as a promising pharmacotherapeutic target for substance use disorders. AIMS: This study aims to assess the impact of selective D3R antagonism on DAT and D3R after reinstatement of cocaine preference (CPP) induced by an acute session of social defeat stress (SDS) and a cocaine prime in mice after a period of abstinence. METHODS: Male mice were conditioned with 25 mg/kg of cocaine for 4 days. After 60 days of extinction training mice were pretreated with the selective D3R antagonist SB-277011A before the re-exposure to a priming dose of cocaine or to a single SDS session. CPP scores were determined and levels of DAT, D3R, phospho Akt (pAkt) and phospho mTOR (pmTOR) were assessed in the NAc shell. RESULTS: An increase in DAT and D3R expression was seen in the NAc after both a cocaine prime- and SDS-induced reinstatement of CPP. Pretreatment with SB-277011A blocked elevated DAT and D3R expression as well as SDS-induced reinstatement. By contrast, the blockade of D3R did not modified the cocaine prime-induced CPP. Changes in DAT and D3R expression do not seem to occur via the canonic pathway involving Akt/mTOR. CONCLUSIONS: Our results suggest that the selective D3R antagonist ability to inhibit DAT and D3R up-regulation could represent a possible mechanism for its behavioral effects in cocaine-memories reinstatement induced by social stress.