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There are currently no pharmacological treatments for cocaine use disorder. Recently there has been a great deal of interest in the potential of psychedelic drugs such as psilocybin to treat psychiatric disorders. Human studies have indicated that a single administration of psilocybin can have long-lasting effects. Few preclinical studies have examined a role for psilocybin in addiction models. The goal of the current study was to determine whether psilocybin would enhance extinction following cocaine self-administration in male and female mice and rats and thus result in an attenuation of cue-induced drug-seeking. In experiments in mice, 16 female and 19 male mice underwent 8d of cocaine self-administration (0.5 mg/kg/infusion) and extinction training. Immediately following extinction trials, mice were injected with vehicle or 1.0 mg/kg psilocybin. Following the conclusion of extinction training, mice were tested for cue-induced reinstatement. In experiments in rats, 24 female and 23 male rats underwent 15d of cocaine self-administration (0.8 mg/kg/infusion) and extinction training. Immediately following extinction trials, rats were injected with vehicle, 1.0 mg/kg psilocybin, or 2.5 mg/kg psilocybin. Following the conclusion of extinction training, rats were tested for cue-induced reinstatement. Psilocybin administered following extinction trials had no effect, as both female and male mice and rats demonstrated significant cue-induced reinstatement. These data suggest that psilocybin is ineffective at altering cocaine-seeking behavior in the paradigm and doses used in the current study. It remains to be seen whether treatment with psilocybin under different conditions may be useful in the long-standing goal of finding pharmacotherapies to treat CUD.

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Dopamine signaling in the amygdala is known to play a role in associative learning and memory, including the process of learning to associate environmental cues with the reinforcing properties of drugs like cocaine. Evidence suggests that the ventral tegmental area (VTA) dopamine (DA) projection specifically to the basolateral amygdala (BLA) participates in establishing cocaine-cue associations that can promote later craving- and relapse-like responses to the cue alone. In order to further investigate the specific role of VTA-BLA projections in cocaine-reinforced learning, we used chemogenetics to manipulate VTA DA inputs to the BLA during cocaine self-administration, cue- and cocaine-primed reinstatement, and conditioned place preference. We found inhibiting DA input to the BLA during cocaine self-administration inhibited acquisition and weakened the ability of the previously cocaine-paired cue to elicit cocaine-seeking, while acutely inhibiting the pathway on the day of cue-induced reinstatement testing had no effect. Conversely, exciting the projection during self-administration boosted the salience of the cocaine-paired cue as indicated by enhanced responding during cue-induced reinstatement. Importantly, interfering with DA input to the BLA had no impact on the ability of cocaine to elicit a place preference or induce reinstatement in response to a priming cocaine injection. Overall, we show that manipulation of projections underlying DA signaling in the BLA may be useful for developing therapeutic interventions for substance use disorders.

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Environmental enrichment combined with the glycine transporter-1 inhibitor Org24598 (EE+ORG) during cocaine-cue extinction (EXT) inhibited reacquisition of 1.0 mg/kg cocaine self-administration in male but not female rats in a previous investigation. In this investigation, we determined if this treatment benefit in males required EXT training and ascertained the molecular basis for the observed sex difference in treatment efficacy. Nine groups of male rats trained to self-administer 1.0 mg/kg cocaine or receiving yoked-saline underwent EXT or NoEXT with or without EE and/or ORG. Next, they underwent reacquisition of cocaine self-administration or were sacrificed for molecular analysis of 9 protein targets indicative of neuroplasticity in four brain regions. Two groups of female rats trained to self-administer 1.0 mg/kg cocaine also underwent EXT with or without EE + ORG and were sacrificed for molecular analysis, as above. EE + ORG facilitated the rate of EXT learning in both sexes, and importantly, the therapeutic benefit of EE + ORG for inhibiting cocaine relapse required EXT training. Males were more sensitive than females to neuroplasticity-inducing effects of EE + ORG, which prevented reductions in total GluA1 and PSD95 proteins selectively in basolateral amygdala of male rats trained to self-administer cocaine and receiving EXT. Females were deficient in expression of multiple protein targets, especially after EE + ORG. These included total GluA1 and PSD95 proteins in basolateral amygdala, and total TrkB protein in basolateral amygdala, dorsal hippocampus, and ventromedial prefrontal cortex. Together, these results support the clinical view that sex-specific pharmacological and behavioral treatment approaches may be needed during cue exposure therapy to inhibit cocaine relapse.

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The increasing rates of drug misuse highlight the urgency of identifying improved therapeutics for treatment. Most drug-seeking behaviours that can be modelled in rodents utilize the repeated intravenous self-administration (SA) of drugs. Recent studies examining the mesolimbic pathway suggest that Kv7/KCNQ channels may contribute to the transition from recreational to chronic drug use. However, to date, all such studies used noncontingent, experimenter-delivered drug model systems, and the extent to which this effect generalizes to rats trained to self-administer drugs is not known. Here, we tested the ability of retigabine (ezogabine), a Kv7 channel opener, to regulate instrumental behaviour in male Sprague Dawley rats. We first validated the ability of retigabine to target experimenter-delivered cocaine in a conditioned place preference (CPP) assay and found that retigabine reduced the acquisition of place preference. Next, we trained rats for cocaine-SA under a fixed-ratio or progressive-ratio reinforcement schedule and found that retigabine pretreatment attenuated the SA of low to moderate doses of cocaine. This was not observed in parallel experiments, with rats self-administering sucrose, a natural reward. Compared with sucrose-SA, cocaine-SA was associated with reductions in the expression of the Kv7.5 subunit in the nucleus accumbens, without alterations in Kv7.2 and Kv7.3. Therefore, these studies reveal a reward-specific reduction in SA behaviour and support the notion that Kv7 is a potential therapeutic target for human psychiatric diseases with dysfunctional reward circuitry.

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RATIONALE: When people with drug addiction encounter cues associated with drug use, this can trigger cravings and relapse. These cues can include conditioned stimuli (CSs) signaling drug delivery and discriminative stimuli (DSs) signaling drug availability. Compared to CS effects, DS effects are less explored in preclinical studies on cue-induced relapse. OBJECTIVE: We compared CS and DS effects on reward seeking following abstinence from intermittent-access cocaine (or sucrose) self-administration. METHODS: During 15-20 intermittent-access sessions, rats self-administered i.v. cocaine or sucrose pellets paired with a light-tone CS. Cocaine/sucrose was available for 5-min (signalled by a light; DS+) and unavailable for 25 min (signalled by different lighting conditions; DS-), and this cycled for 4 h/session. Following abstinence, we measured cocaine/sucrose seeking under extinction triggered by CS and DS presentation, and instrumental responding reinforced by these cues. RESULTS: Across intermittent-access sessions, rats increased lever pressing for cocaine or sucrose during DS+ periods and decreased responding during DS- periods. On days 2 and 21 of abstinence, only presentation of the DS+ or DS+ and CS combined elicited increased cocaine/sucrose-seeking behaviour (i.e., increased active lever presses). Presenting the DS- alongside the DS+ suppressed the increased cocaine-seeking behaviour otherwise produced by the DS+ . Finally, on day 21 of abstinence, rats showed equivalent levels of lever pressing reinforced by the DS+ , CS and by the DS+ and CS combined, suggesting comparable conditioned reinforcing value. CONCLUSIONS: After intermittent self-administration, cocaine-associated DSs and CSs acquire similar conditioned reinforcing properties, but DSs more effectively trigger increases in drug seeking.

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Vaccination active, promising alternative immunological strategy to treat of CUD. Various models of cocaine vaccines have been evaluated in animals and humans with relative success. In this sense, it is necessary to improve or optimize the cocaine vaccines already evaluated. Our laboratory previously reported the efficacy of the tetanus toxoid-conjugated morphine vaccine (M6-TT). The M6-TT vaccine can generate high titers of antibodies and reduce heroin-induced behavioral effects in rodents. So, it would be plausible to assume that if we modify the M6-TT vaccine by changing the hapten and maintaining the rest of the structural elements of the vaccine, we will maintain the properties of the M6-TT vaccine (high antibody titers). The objective of this study was to determine whether the antibodies generated by a tetanus toxoid-conjugated cocaine vaccine (COC-TT) can recognize and capture cocaine and decrease the cocaine-induced reinforcing effects. Male Wistar rats were immunized with the COC-TT. A solid-phase antibody-capture ELISA was used to monitor antibody titer responses after each booster dose in vaccinated animals. The study used cocaine self-administration and place-preference testing to evaluate the cocaine-reinforcing effects. The COC-TT vaccine could generate high levels of anti-cocaine antibodies. The antibodies reduced the cocaine self-administration and cocaine place preference. In addition, they decreased the cocaine-induced Fos protein expression. These findings suggest that the COC-TT vaccine generates a robust immunogenic response capable of reducing the reinforcing effects of cocaine, which supports its possible future use in clinical trials in patients with CUD.

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Background: Exercise has shown promise as a treatment for cocaine use disorder; however, the mechanism underlying its efficacy has remained elusive. Methods: We used a rat model of relapse (cue-induced reinstatement) and exercise (wheel running, 2 hours/day) coupled with RNA sequencing to establish transcriptional profiles associated with the protective effects of exercise (during early withdrawal [days 1-7] or throughout withdrawal [days 1-14]) versus noneffective exercise (during late withdrawal [days 8-14]) against cocaine-seeking and sedentary conditions. Results: As expected, cue-induced cocaine seeking was highest in the sedentary and late-withdrawal exercise groups; both groups also showed upregulation of a Grin1-associated transcript and enrichment of Drd1-Nmdar1 complex and glutamate receptor complex terms. Surprisingly, these glutamate markers were also enriched in the early- and throughout-withdrawal exercise groups, despite lower levels of cocaine seeking. However, a closer examination of the Grin1-associated transcript revealed a robust loss of transcripts spanning exons 9 and 10 in the sedentary condition relative to saline controls that was normalized by early- and throughout-withdrawal exercise, but not late-withdrawal exercise, indicating that these exercise conditions may normalize RNA mis-splicing induced by cocaine seeking. Our findings also revealed novel mechanisms by which exercise initiated during early withdrawal may modulate glutamatergic signaling in dorsomedial prefrontal cortex (e.g., via transcripts associated with non-NMDA glutamate receptors or those affecting signaling downstream of NMDA receptors), along with mechanisms outside of glutamatergic signaling such as circadian rhythm regulation and neuronal survival. Conclusions: These findings provide a rich resource for future studies aimed at manipulating these molecular networks to better understand how exercise decreases cocaine seeking.

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RATIONALE: The adverse consequences of human addictive drug use could be the result of either addictive drug consumption resulting in punishment (e.g., incarceration) or failure to engage in negative-reinforced behaviors that might compete with drug-maintained behaviors (e.g., contingency management strategies that reset payment amounts for drug free urines). OBJECTIVE: The goal of the present study was to establish a discrete-trial cocaine-vs-negative reinforcer (SNR) choice procedure where rats were presented with a simplified model of this conflict: choose negative reinforcement (i.e., escape or avoid foot shock) or choose an intravenous (IV) cocaine infusion followed by an inescapable shock. METHODS: Responding was maintained in male and female rats by IV cocaine infusions (0.32-1.8 mg/kg/inf) and a SNR (0.1-0.7 mA shock) under a discrete-trial concurrent "choice" schedule during daily sessions. Following parametric reinforcer magnitude and response requirement experiments, the effects of 12 h extended access cocaine self-administration and acute diazepam (0.32-10 mg/kg, IP) pretreatment were determined on cocaine-vs-SNR choice. RESULTS: Negative reinforcement was chosen over all cocaine doses. Lowering shock magnitude or increasing SNR response requirement failed to promote behavioral reallocation towards cocaine. Extended access cocaine self-administration sessions resulted in high daily cocaine intakes but failed to significantly increase cocaine choice in all (19) but one rat. Acute diazepam pretreatment also did not alter choice behavior up to doses that produced behavioral depression. CONCLUSIONS: These results suggest that SNRs may be a source of reinforcement that effectively compete with and mitigate maladaptive addictive drug-maintained behaviors in the general population.

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BACKGROUND: Epidemiological data indicate a high rate of comorbidity of depression and cocaine use disorder (CUD). The role of serotonin 2C (5-HT2C) receptors in the mechanisms responsible for the coexistence of depression and CUD was not investigated. METHODS: We combined bilateral olfactory bulbectomy (OBX), an animal model of depression, with intravenous cocaine self-administration and extinction/reinstatement in male rats to investigate two 5-HT2C receptor agonists (Ro 60-0175 (RO) and WAY 161503 (WAY)) and the 5-HT2C-receptor preferring antagonist mirtazapine (MIR; an antidepressant), with the goal of determining whether these drugs alter cocaine-induced reinforcement and seeking behaviors. Additionally, neurochemical analyses were performed following cocaine self-administration and its abstinence period in the brain structures in OBX rats and SHAM-operated controls. RESULTS: Acute administration of RO reduced, while WAY non-significantly attenuated cocaine reinforcement in both rat phenotypes. Moreover, RO or WAY protected against cocaine-seeking behavior after acute or after repeated drug administration during extinction training in OBX and SHAM rats. By contrast, acutely administered MIR did not alter cocaine reinforcement in both rat phenotypes, while it's acute (but not repeated) pretreatment reduced cocaine-seeking in OBX and SHAM rats. In neurochemical analyses, cocaine reinforcement increased 5-HT2C receptor levels in the ventral hippocampus; a preexisting depression-like phenotype enhanced this effect. The 10-daily cocaine abstinence reduced 5-HT2C receptor expression in the dorsolateral striatum, while the coexistence of depression and CUD enhanced local receptor expression. CONCLUSION: The results support a key role of 5-HT2C receptors for treating CUD and comorbid depression and CUD. They may be backs the further research of pharmacological strategies with drug targeting receptors.

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Recent years have provided more and more evidence confirming the important role of Wnt/ß-catenin signaling in the pathophysiology of mental illnesses, including cocaine use disorder. High relapse rates, which is a hallmark of drug addiction, prompt the study of changes in Wnt signaling elements (Wnt5a, Wnt7b, and Ctnnb1) in the motivational aspects of cocaine use and early drug-free period (3 days after the last exposure to cocaine). For this purpose, an animal model of intravenous cocaine self-administration and two types of drug-free period (extinction training and abstinence in the home cage) were used. The studies showed that chronic cocaine self-administration mainly disturbs the expression of Wnt5a and Ctnnb1 (the gene encoding ß-catenin) in the examined brain structures (striatum and hippocampus), and the examined types of early abstinence are characterized by a different pattern of changes in the expression of these genes. At the same time, in cocaine self-administrated animals, there were no changes in the level of Wnt5a and ß-catenin proteins at the tested time points. Moreover, exposure to cocaine induces a significant reduction in the striatal and hippocampal expression of miR-374 and miR-544, which can regulate Wnt5a levels post-transcriptionally. In summary, previous observations from experimenter-administered cocaine have not been fully validated in the cocaine self-administration model. Yoked cocaine administration appears to disrupt Wnt signaling more than cocaine self-administration. The condition of the cocaine-free period, the routes of drug administration, and the motivational aspect of drug administration play an important role in the type of drug-induced molecular changes observed. Furthermore, in-depth research involving additional brain regions is needed to determine the exact role of Wnt signaling in short-term and long-lasting plasticity as well as in the motivational aspects of cocaine use, and thus to assess its potential as a target for new drug therapy for cocaine use disorder.

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Relapse to drug use is one of the major challenges in treating substance use disorders. Exposure to drug-related cues and contexts triggers drug craving, which drives cocaine seeking, and increases the probability of relapse. Clinical and animal studies have shown a progressive intensification of cocaine seeking and craving that develops over the course of abstinence, a phenomenon commonly referred to as incubation of cocaine craving. Although the neurobiology underlying incubation of cocaine craving has been examined - particularly within the context of glutamate plasticity- the extent to which increased cocaine craving engenders mesolimbic dopamine (DA) changes has received relatively little attention. To assess whether incubation of cocaine craving is associated with alterations in DA terminal neurotransmission in the nucleus accumbens core (NAc), we used ex vivo fast scan cyclic voltammetry in female and male rats to assess DA dynamics following short access, long access, or intermittent access to cocaine self-administration followed by 28 days of abstinence. Results indicated that both long access and intermittent access to cocaine produced robust incubation of cocaine craving, which was associated with increases in cocaine potency. In addition, intermittent access self-administration also produced a robust increase in DA uptake rate at baseline. In contrast, short access to cocaine did not engender incubation of cocaine craving, nor produce changes in DA neurotransmission. Together these observations indicate that incubation of cocaine craving coincides with changes in DA transmission, suggesting that underlying changes in mesolimbic DA signaling may contribute to the progressive intensification of drug craving that occurs across periods of abstinence.

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Neurochemical studies were previously performed on the effects of a 10 day extinction learning from cocaine self-administration on D2R and A2AR recognition and D2R Gi/o coupling in the ventral striatum. In the present study biochemical receptor binding and proximity ligation assay were used to study possible changes in the allosteric receptor-receptor interactions and the density of the A2AR-D2R heterocomplexes in the ventral striatum (nucleus accumbens shell) in extinction from cocaine self-administration including cue induced reinstatement of cocaine seeking. A significant and clear-cut reduction of active lever pressing was observed in extinction on day 10 from cocaine use. In cue induced reinstatement of cocaine self-administration a significant return in active lever presses developed. In extinction, significant increases in the density of A2AR-D2R and D2R-Sigma1R heterocomplexes were observed in nucleus accumbens shell. In contrast, cue-induced reinstatement of cocaine seeking produced no significant changes in these heteroreceptor complexes of the nucleus accumbens shell. In the 3H raclopride/quinpirole competition binding experiments, the extinction led to a significant increase in the D2R Ki, High dissociation constant in the ventral striatum upon ex vivo exposure to CGS 21680 (100 nM), compared to the same exposure performed in membrane preparations from yoked saline rats. No significant changes in D2R Ki, High values were observed in membrane preparations from rats after cue-induced reinstatement of cocaine-seeking undergoing the same exposure ex vivo to CGS 21680 when compared with membrane preparations from yoked saline rats undergoing the same procedures. It seems likely that increased formation of A2AR-D2R and putative A2AR-D2R-Sigma1R heterocomplexes in the nucleus accumbens shell is part of the mechanism for the enhanced antagonistic allosteric A2AR-D2R interactions developed in extinction learning from cocaine. It reduces cocaine reward through reduced D2R function, and these inhibitory mechanisms are no longer in operation in cue induced reinstatement of cocaine seeking.

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BACKGROUND AND PURPOSE: It has been well established that glutamate in the nucleus accumbens (NAc) plays a critical role in the motivation to take drugs of abuse. We have previously demonstrated that rats with ablation of the serotonin transporter (SERT-/- rats) show increased cocaine intake reminiscent of compulsivity. EXPERIMENTAL APPROACH: By comparing SERT-/- to SERT+/+ rats, we investigated whether SERT deletion influences glutamate homeostasis under control conditions as well as after short access (ShA: 1 h per session) or long access (LgA: 6 h per session) to cocaine self-administration. Rats were killed at 24 h after the last self-administration session for ex vivo molecular analyses of the main determinants of the glutamate system, including transporters (vesicular and glial), receptors (main post-synaptic subunits of NMDA and AMPA receptors together with the metabotropic subunit mGLUR5), and scaffolding proteins (SAP102, SAP97, and GRIP) in the NAc shell (sNAc) KEY RESULTS: In cocaine-naive animals, SERT deletion was associated with changes indicative for a reduction in glutamate signalling. ShA and LgA exposure led to a further dysregulation of the glutamatergic synapse. CONCLUSION: SERT deletion may render the glutamatergic synapses of the NAc shell more responsive to both ShA and LgA intake of cocaine.

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Drug addiction is characterized by relapse when addicts are re-exposed to drug-associated environmental cues, but the neural mechanisms underlying cue-induced relapse are unclear. In the present study we investigated the role of a specific dopaminergic (DA) pathway from ventral tegmental area (VTA) to nucleus accumbens core (NAcore) in mouse cue-induced relapse. Optical intracranial self-stimulation (oICSS) was established in DAT-Cre transgenic mice. We showed that optogenetic excitation of DA neurons in the VTA or their projection terminals in NAcore, NAshell or infralimbic prefrontal cortex (PFC-IL) was rewarding. Furthermore, activation of the VTA-NAcore pathway alone was sufficient and necessary to induce reinstatement of oICSS. In cocaine self-administration model, cocaine-associated cues activated VTA DA neurons as assessed by intracellular GCaMP signals. Cue-induced reinstatement of cocaine-seeking was triggered by optogenetic stimulation of the VTA-NAcore pathway, and inhibited by chemogenetic inhibition of this pathway. Together, these results demonstrate that cue-induced reinstatement of reward seeking is in part mediated by activation of the VTA-NAcore DA pathway.

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BACKGROUND AND PURPOSE: It is well established that the nucleus accumbens and glutamate play a critical role in the motivation to take drugs of abuse. We have previously demonstrated that rats with ablation of the serotonin (5-HT) transporter (SERT-/- rats) show increased cocaine intake reminiscent of compulsivity. EXPERIMENTAL APPROACH: By comparing SERT-/- to SERT+/+ rats, we set out to explore whether SERT deletion influences glutamate neurotransmission under control conditions as well as after short access (1 h/session) or long access (6 h/session) to cocaine self-administration. KEY RESULTS: Rats were killed at 24 h after the final self-administration session for ex vivo molecular analyses of the glutamate system (vesicular and glial transporters, post-synaptic subunits of NMDA and AMPA receptors and their related scaffolding proteins). Such analyses were undertaken in the nucleus accumbens core. In cocaine-naïve animals, SERT deletion evoked widespread abnormalities in markers of glutamatergic neurotransmission that, overall, indicate a reduction of glutamate signalling. These results suggest that 5-HT is pivotal for the maintenance of accumbal glutamate homeostasis. We also found that SERT deletion altered glutamate homeostasis mainly after long access, but not short access, to cocaine. CONCLUSION AND IMPLICATIONS: Our findings reveal that SERT deletion may sensitize the glutamatergic synapses of the nucleus accumbens core to the long access but not short access, intake of cocaine. LINKED ARTICLES: This article is part of a themed issue on New discoveries and perspectives in mental and pain disorders. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.17/issuetoc.

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Infralimbic cortical (IL) manipulations indicate that this region mediates extinction learning and suppresses cocaine seeking following cocaine self-administration. However, little work has recorded IL activity during the inhibition of cocaine seeking due to the difficulty of determining precisely when cocaine-seeking behaviour is inhibited within a cocaine-seeking session. The present study used in vivo electrophysiology to examine IL activity across extinction as well as during cocaine self-administration and reinstatement. Sprague-Dawley rats underwent 6-h access cocaine self-administration in which the response lever was available during discrete signalled trials, a procedure which allowed for the comparison between epochs of cocaine seeking versus the inhibition thereof. Subsequently, rats underwent extinction and cocaine-primed reinstatement using the same procedure. Results indicate that theta rhythms (4-10 Hz) dominated IL local-field potential (LFP) activity during all experimental stages. During extinction, theta power fluctuated significantly surrounding the lever press and was lower when rats engaged in cocaine seeking versus when they withheld from doing so. These patterns of oscillatory activity differed from self-administration and reinstatement stages. Single-unit analyses indicate heterogeneity of IL unit responses, supporting the idea that multiple neuronal subpopulations exist within the IL and promote the expression of different and even opposing cocaine-seeking behaviours. Together, these results are consistent with the idea that aggregate synaptic and single-unit activity in the IL represent the engagement of the IL in action monitoring to promote adaptive behaviour in accordance with task contingencies and reveal critical insights into the relationship between IL activity and the inhibition of cocaine seeking.

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Cocaine use disorder is a serious, chronic and relapsing disease of the nervous system, for which effective treatments do not yet exist. Recently, the role of the N-methyl-d-aspartate (NMDA) receptor subunit GluN2B has been highlighted in cocaine abstinence followed by extinction training. Since the GluN2B subunit is stabilized at synaptic level by the interaction with its scaffolding protein PSD95, in this study we aimed at investigating efficacy of Tat-NR2B9c peptide, a PSD95 inhibitor, which disrupts the interaction of PSD95 with GluN2B, in the attenuation of cocaine seeking-behavior or cue-induced reinstatement. We found that Tat-NR2B9c, administered intravenously, attenuated the reinstatement of active lever presses induced by a priming dose of cocaine or by drug-associated conditioned stimuli. At the same time, the GluN2B/PSD95 complex levels were decreased in the ventral hippocampus of rats that previously self-administered cocaine injected with Tat-NR2B9c during cocaine- or cue-induced reinstatement. In conclusion, we here provide the first evidence showing that the disruption of the GluN2B/PSD95 complexes during cocaine abstinence followed by extinction training may represent a useful strategy to reduce reinstatement of cocaine-seeking behavior.

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The dopamine (DA) transporter (DAT) is part of a presynaptic multiprotein network involving interactions with scaffold proteins via its C-terminal PDZ domain-binding sequence. Using a mouse model expressing DAT with mutated PDZ-binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here, we show by application of direct stochastic reconstruction microscopy not only that the striatal level of transporter is reduced in DAT-AAA mice but also that the nanoscale distribution of this transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal DA adaptations and changes in DA-related behaviors distinct from those seen in other genetic DAT mouse models. DA levels in the striatum are reduced to ∼45% of that of WT, accompanied by elevated DA turnover. Nonetheless, fast-scan cyclic voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared with WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels, whereas immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed ratio of one and progressive ratio schedule of reinforcement but a reduction compared with WT when using cocaine as reinforcer. In summary, our data demonstrate how disruption of PDZ domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics and related behaviors.

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The basolateral amygdala (BLA) is a critical brain region for cocaine-memory reconsolidation. Corticotropin-releasing factor receptor type 1 (CRFR1) is densely expressed in the BLA, and CRFR1 stimulation can activate intra-cellular signaling cascades that mediate memory reconsolidation. Hence, we tested the hypothesis that BLA CRFR1 stimulation is necessary and sufficient for cocaine-memory reconsolidation. Using an instrumental model of drug relapse, male and female Sprague-Dawley rats received cocaine self-administration training in a distinct environmental context over 10 days followed by extinction training in a different context over 7 days. Next, rats were re-exposed to the cocaine-paired context for 15 min to initiate cocaine-memory retrieval and destabilization. Immediately or 6 h after this session, the rats received bilateral vehicle, antalarmin (CRFR1 antagonist; 500 ng/hemisphere), or corticotropin-releasing factor (CRF; 0.2, 30 or 500 ng/hemisphere) infusions into the BLA. Resulting changes in drug context-induced cocaine seeking (index of context-cocaine memory strength) were assessed three days later. Female rats self-administered more cocaine infusions and exhibited more extinction responding than males. Intra-BLA antalarmin treatment immediately after memory retrieval (i.e., when cocaine memories were labile), but not 6 h later (i.e., after memory reconsolidation), attenuated drug context-induced cocaine seeking at test independent of sex, relative to vehicle. Conversely, intra-BLA CRF treatment increased this behavior selectively in females, in a U-shaped dose-dependent fashion. In control experiments, a high (behaviorally ineffective) dose of CRF treatment did not reduce BLA CRFR1 cell-surface expression in females. Thus, BLA CRFR1 signaling is necessary and sufficient, in a sex-dependent manner, for regulating cocaine-memory strength.

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