CB1 Receptor Signaling Modulates Amygdalar Plasticity during Context-Cocaine Memory Reconsolidation to Promote Subsequent Cocaine Seeking.

Contextual drug-associated memories precipitate craving and relapse in cocaine users. Such associative memories can be weakened through interference with memory reconsolidation, a process by which memories are maintained following memory retrieval-induced destabilization. We hypothesized that cocaine-memory reconsolidation requires cannabinoid type 1 receptor (CB1R) signaling based on the fundamental role of the endocannabinoid system in synaptic plasticity and emotional memory processing. Using an instrumental model of cocaine relapse, we evaluated whether systemic CB1R antagonism (AM251; 3 mg/kg, i.p.) during memory reconsolidation altered (1) subsequent drug context-induced cocaine-seeking behavior as well as (2) cellular adaptations and (3) excitatory synaptic physiology in the basolateral amygdala (BLA) in male Sprague Dawley rats. Systemic CB1R antagonism, during, but not after, cocaine-memory reconsolidation reduced drug context-induced cocaine-seeking behavior 3 d, but not three weeks, later. CB1R antagonism also inhibited memory retrieval-associated increases in BLA zinc finger 268 (zif268) and activity regulated cytoskeletal-associated protein (Arc) immediate-early gene (IEG) expression and changes in BLA AMPA receptor (AMPAR) and NMDA receptor (NMDAR) subunit phosphorylation that likely contribute to increased receptor membrane trafficking and synaptic plasticity during memory reconsolidation. Furthermore, CB1R antagonism increased memory reconsolidation-associated spontaneous EPSC (sEPSC) frequency in BLA principal neurons during memory reconsolidation. Together, these findings suggest that CB1R signaling modulates cellular and synaptic mechanisms in the BLA that may facilitate cocaine-memory strength by enhancing reconsolidation or synaptic reentry reinforcement, or by inhibiting extinction-memory consolidation. These findings identify the CB1R as a potential therapeutic target for relapse prevention.SIGNIFICANCE STATEMENT Drug relapse can be triggered by the retrieval of context-drug memories on re-exposure to a drug-associated environment. Context-drug associative memories become destabilized on retrieval and must be reconsolidated into long-term memory stores to persist. Hence, targeted interference with memory reconsolidation can weaken maladaptive context-drug memories and reduce the propensity for drug relapse. Our findings indicate that cannabinoid type 1 receptor (CB1R) signaling is critical for context-cocaine memory reconsolidation and subsequent drug context-induced reinstatement of cocaine-seeking behavior. Furthermore, cocaine-memory reconsolidation is associated with CB1R-dependent immediate-early gene (IEG) expression and changes in excitatory synaptic proteins and physiology in the basolateral amygdala (BLA). Together, our findings provide initial support for CB1R as a potential therapeutic target for relapse prevention.

Vaporized Cannabis Extracts Have Reinforcing Properties and Support Conditioned Drug-Seeking Behavior in Rats.

Recent trends in cannabis legalization have increased the necessity to better understand the effects of cannabis use. Animal models involving traditional cannabinoid self-administration approaches have been notoriously difficult to establish and differences in the drug used and its route of administration have limited the translational value of preclinical studies. To address this challenge in the field, we have developed a novel method of cannabis self-administration using response-contingent delivery of vaporized Δ9-tetrahydrocannabinol-rich (CANTHC) or cannabidiol-rich (CANCBD) whole-plant cannabis extracts. Male Sprague-Dawley rats were trained to nose-poke for discrete puffs of CANTHC, CANCBD, or vehicle (VEH) in daily 1 h sessions. Cannabis vapor reinforcement resulted in strong discrimination between active and inactive operanda. CANTHC maintained higher response rates under fixed ratio schedules and higher break points under progressive ratio schedules compared with CANCBD or VEH, and the number of vapor deliveries positively correlated with plasma THC concentrations. Moreover, metabolic phenotyping studies revealed alterations in locomotor activity, energy expenditure, and daily food intake that are consistent with effects in human cannabis users. Furthermore, both cannabis regimens produced ecologically relevant brain concentrations of THC and CBD and CANTHC administration decreased hippocampal CB1 receptor binding. Removal of CANTHC reinforcement (but not CANCBD) resulted in a robust extinction burst and an increase in cue-induced cannabis-seeking behavior relative to VEH. These data indicate that volitional exposure to THC-rich cannabis vapor has bona fide reinforcing properties and collectively support the utility of the vapor self-administration model for the preclinical assessment of volitional cannabis intake and cannabis-seeking behaviors.SIGNIFICANCE STATEMENT The evolving legal landscape concerning recreational cannabis use has increased urgency to better understand its effects on the brain and behavior. Animal models are advantageous in this respect; however, current approaches typically used forced injections of synthetic cannabinoids or isolated cannabis constituents that may not capture the complex effects of volitional cannabis consumption. We have developed a novel model of cannabis self-administration using response-contingent delivery of vaporized cannabis extracts containing high concentrations of Δ9 tetrahydrocannabinol (THC) or cannabidiol. Our data indicate that THC-rich cannabis vapor has reinforcing properties that support stable rates of responding and conditioned drug-seeking behavior. This approach will be valuable for interrogating effects of cannabis and delineating neural mechanisms that give rise to aberrant cannabis-seeking behavior.

Expression of a heroin contextually conditioned immune effect in male rats requires CaMKIIα-expressing neurons in dorsal, but not ventral, subiculum and hippocampal CA1.

The physiological and motivational effects of heroin and other abused drugs become associated with environmental (contextual) stimuli during repeated drug use. As a result, these contextual stimuli gain the ability to elicit drug-like conditioned effects. For example, after context-heroin pairings, exposure to the heroin-paired context alone produces similar effects on peripheral immune function as heroin itself. Conditioned immune effects can significantly exacerbate the adverse health consequences of heroin use. Our laboratory has shown that exposure to a heroin-paired context suppresses lipopolysaccharide (LPS)-induced splenic nitric oxide (NO) production in male rats, and this effect is mediated in part by the dorsal hippocampus (dHpc). However, specific dHpc output regions, whose efferents might mediate conditioned immune effects, have not been identified, nor has the contribution of ventral hippocampus (vHpc) been investigated. Here, we evaluated the role of CaMKIIα-expressing neurons in the dHpc and vHpc main output regions by expressing Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) under a CaMKIIα promoter in the dorsal subiculum and CA1 (dSub, dCA1) or ventral subiculum and CA1 (vSub, vCA1). After context-heroin conditioning, clozapine-N-oxide (CNO, DREADD agonist) or vehicle was administered systemically prior to heroin-paired context (or home-cage control) exposure and LPS immune challenge. Chemogenetic inhibition of CaMKIIα-expressing neurons in dHpc, but not vHpc, output regions attenuated the expression of conditioned splenic NO suppression. These results establish that the main dHpc output regions, the dSub and dCA1, are critical for this context-heroin conditioned immune effect.

Dorsal hippocampal neural immune signaling regulates heroin-conditioned immunomodulation but not heroin-conditioned place preference.

Repeated pairings of heroin and a context results in Pavlovian associations which manifest as heroin-conditioned appetitive responses and peripheral immunomodulation upon re-exposure to heroin-paired conditioned stimuli (CS). The dorsal hippocampus (DH) plays a key role in the neurocircuitry governing these context-heroin associations. Within the DH, expression of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) is required for heroin-conditioned peripheral immunomodulation to occur. However, the role of signaling via IL-1 receptor type 1 (IL-1R1) has not been examined. Furthermore, it has not been evaluated whether the involvement of IL-1 in associative learning extends to classically conditioned appetitive behaviors, such as conditioned place preference (CPP). The first set of experiments investigated whether DH IL-1R1 signaling during CS re-exposure modulates heroin-conditioned immunomodulation and heroin-CPP. The second set of experiments employed chemogenetic techniques to examine whether DH astroglial signaling during CS re-exposure alters the same Pavlovian responses. This line of investigation is based on previous research indicating that astrocytes support hippocampal-dependent learning and memory through the expression of IL-1ß protein and IL-1R1. Interestingly, IL-1R1 antagonism disrupted heroin-conditioned suppression of peripheral immune parameters but failed to alter heroin-CPP. Similarly, chemogenetic stimulation of Gi-signaling in DH astrocytes attenuated heroin-conditioned peripheral immunomodulation but failed to alter heroin-CPP. Collectively our data show that both IL-1R1 stimulation and astrocyte signaling in the DH are critically involved in the expression of heroin-conditioned immunomodulation but not heroin-CPP. As such these findings strongly suggest hippocampal neuroimmune signaling differentially regulates Pavlovian immunomodulatory and appetitive behaviors.

Interleukin-1 signaling in the basolateral amygdala is necessary for heroin-conditioned immunosuppression.

Heroin administration suppresses the production of inducible nitric oxide (NO), as indicated by changes in splenic inducible nitric oxide synthase (iNOS) and plasma nitrate/nitrite. Since NO is a measure of host defense against infection and disease, this provides evidence that heroin can increase susceptibility to pathogens by directly interacting with the immune system. Previous research in our laboratory has demonstrated that these immunosuppressive effects of heroin can also be conditioned to environmental stimuli by repeatedly pairing heroin administration with a unique environmental context. Re-exposure to a previously drug-paired context elicits immunosuppressive effects similar to heroin administration alone. In addition, our laboratory has reported that the basolateral amygdala (BLA) and medial nucleus accumbens shell (mNAcS) are critical neural substrates that mediate this conditioned effect. However, our understanding of the contributing mechanisms within these brain regions is limited. It is known that the cytokine interleukin-1 (IL-1) plays an important role in learning and memory. In fact, our laboratory has demonstrated that inhibition of IL-1ß expression in the dorsal hippocampus (DH) prior to re-exposure to a heroin-paired context prevents the suppression of measures of NO production. Therefore, the present studies sought to further investigate the role of IL-1 in heroin-conditioned immunosuppression. Blockade of IL-1 signaling in the BLA, but not in the caudate putamen or mNAcS, using IL-1 receptor antagonist (IL-1Ra) attenuated heroin-conditioned immunosuppression of NO production as measured by plasma nitrate/nitrite and iNOS mRNA expression in spleen tissue. Taken together, these findings suggest that IL-1 signaling in the BLA is necessary for the expression of heroin-conditioned immunosuppression of NO production and may be a target for interventions that normalize immune function in heroin users and patient populations exposed to opiate regimens.

Requisite Role of Basolateral Amygdala Glucocorticoid Receptor Stimulation in Drug Context-Induced Cocaine-Seeking Behavior.

BACKGROUND: Exposure to cocaine-associated stimuli triggers a robust rise in circulating glucocorticoid levels. Glucocorticoid receptors are richly expressed in the basolateral amygdala, a brain region that controls the reinstatement of cocaine-seeking behavior upon exposure to a previously cocaine-paired environmental context. In the present study, we investigated whether glucocorticoid receptor stimulation in the basolateral amygdala is integral to drug context-induced motivation to seek cocaine in a rat model of drug relapse. METHODS: Rats were trained to lever press for cocaine reinforcement in a distinct environmental context and were then given daily extinction training sessions in a different context. At test, the rats received bilateral glucocorticoid receptor antagonist (mifepristone; 3 or 10ng/hemisphere) or vehicle microinfusions into either the basolateral amygdala or the overlying posterior caudate-putamen (anatomical control region). Immediately thereafter, drug-seeking behavior (i.e., nonreinforced lever presses) was assessed in the previously cocaine-paired context and locomotor activity was assessed in a novel context. RESULTS: Intra-basolateral amygdala, but not intra-posterior caudate-putamen, mifepristone dose-dependently attenuated drug context-induced cocaine-seeking behavior relative to vehicle, such that responding was similar to that observed in the extinction context. In contrast, mifepristone treatment did not alter locomotor activity. CONCLUSIONS: These findings suggest that basolateral amygdala glucocorticoid receptor stimulation is necessary for drug context-induced motivation to seek cocaine.

Acquisition of heroin conditioned immunosuppression requires IL-1 signaling in the dorsal hippocampus.

Opioid users experience increased incidence of infection, which may be partially attributable to both direct opiate-immune interactions and conditioned immune responses. Previous studies have investigated the neural circuitry governing opioid conditioned immune responses, but work remains to elucidate the mechanisms mediating this effect. Our laboratory has previously shown that hippocampal IL-1 signaling, specifically, is required for the expression of heroin conditioned immunosuppression following learning. The current studies were designed to further characterize the role of hippocampal IL-1 in this phenomenon by manipulating IL-1 during learning. Experiment 1 tested whether hippocampal IL-1 is also required for the acquisition of heroin conditioned immunosuppression, while Experiment 2 tested whether hippocampal IL-1 is required for the expression of unconditioned heroin immunosuppression. We found that blocking IL-1 signaling in the dorsal hippocampus with IL-1RA during each conditioning session, but not on interspersed non-conditioning days, significantly attenuated the acquisition of heroin conditioned immunosuppression. Strikingly, we found that the same IL-1RA treatment did not alter unconditioned immunosuppression to a single dose of heroin. Thus, IL-1 signaling is not a critical component of the response to heroin but rather may play a role in the formation of the association between heroin and the context. Collectively, these studies suggest that IL-1 signaling, in addition to being involved in the expression of a heroin conditioned immune response, is also involved in the acquisition of this effect. Importantly, this effect is likely not due to blocking the response to the unconditioned stimulus since IL-1RA did not affect heroin's immunosuppressive effects.

Cocaine seeking and taking: role of hippocampal dopamine D1-like receptors.

Despite the well-documented involvement of dopamine D1-like receptor stimulation in cocaine-induced goal-directed behaviours, little is known about the specific contribution of D1-like receptor populations in the dorsal hippocampus (DH) to drug context-induced cocaine-seeking or drug-reinforced instrumental behaviours. To investigate this question, rats were trained to lever press for un-signalled cocaine infusions in a distinct context followed by extinction training in a different context. Cocaine-seeking behaviour (non-reinforced lever responding) was then assessed in the previously cocaine-paired and extinction contexts. SCH23390-induced D1-like receptor antagonism in the DH, but not the overlying trunk region of the somatosensory cortex, dose-dependently inhibited drug context-induced cocaine-seeking behaviour, without altering cocaine-reinforced instrumental responding, cocaine intake, food-reinforced instrumental responding, or general motor activity, relative to vehicle treatment. These findings suggest that D1-like receptor stimulation in the DH is critical for the incentive motivational effects and/or memory of cocaine-paired contextual stimuli that contribute to drug-seeking behaviour.

Region-specific contribution of the ventral tegmental area to heroin-induced conditioned immunomodulation.

Dopamine receptor stimulation is critical for heroin-conditioned immunomodulation; however, it is unclear whether the ventral tegmental area (VTA) contributes to this phenomenon. Hence, rats received repeated pairings of heroin with placement into a distinct environmental context. At test, they were re-exposed to the previously heroin-paired environment followed by systemic lipopolysaccharide treatment to induce an immune response. Bilateral GABA agonist-induced neural inactivation of the anterior, but not the posterior VTA, prior to context re-exposure inhibited the ability of the heroin-paired environment to suppress peripheral nitric oxide and tumor necrosis factor-α expression, suggesting a role for the anterior VTA in heroin-conditioned immunomodulation.

Dorsal Raphe to Basolateral Amygdala Corticotropin-Releasing Factor Circuit Regulates Cocaine-Memory Reconsolidation.

Environmental stimuli elicit drug craving and relapse in cocaine users by triggering the retrieval of strong cocainerelated contextual memories. Retrieval can also destabilize drug memories, requiring reconsolidation, a protein synthesis-dependent storage process, to maintain memory strength. Corticotropin-releasing factor (CRF) signaling in the basolateral amygdala (BLA) is necessary for cocainememory reconsolidation. We have hypothesized that a critical source of CRF in the BLA is the dorsal raphe nucleus (DR) based on its neurochemistry, anatomical connectivity, and requisite involvement in cocaine-memory reconsolidation. To test this hypothesis, male and female Sprague-Dawley rats received adeno-associated viruses to express Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) selectively in CRF neurons of the DR and injection cannulae directed at the BLA. The rats were trained to self-administer cocaine in a distinct environmental context then received extinction training in a different context. They were then briefly reexposed to the cocaine-predictive context to destabilize (reactivate) cocaine memories. Intra-BLA infusions of the DREADD agonist deschloroclozapine (DCZ; 0.1 mM, 0.5 µL/hemisphere) after memory reactivation attenuated cocaine-memory strength, relative to vehicle infusion. This was indicated by a selective, DCZ-induced and memory reactivation-dependent decrease in drug-seeking behavior in the cocaine-predictive context in DREADD-expressing males and females at test compared to respective controls. Notably, BLA-projecting DR CRF neurons that exhibited increased c-Fos expression during memory reconsolidation co-expressed glutamatergic and serotonergic neuronal markers. Together, these findings suggest that the DRCRF → BLA circuit is engaged to maintain cocaine-memory strength after memory destabilization, and this phenomenon may be mediated by DR CRF, glutamate, and/or serotonin release in the BLA.

Dorsal raphe to basolateral amygdala corticotropin-releasing factor circuit regulates cocaine-memory reconsolidation.

Environmental stimuli elicit drug craving and relapse in cocaine users by triggering the retrieval of strong cocaine-related contextual memories. Retrieval can also destabilize drug memories, requiring reconsolidation, a protein synthesis-dependent storage process, to maintain memory strength. Corticotropin-releasing factor (CRF) signaling in the basolateral amygdala (BLA) is necessary for cocaine-memory reconsolidation. We have hypothesized that a critical source of CRF in the BLA is the dorsal raphe nucleus (DR) based on its neurochemistry, anatomical connectivity, and requisite involvement in cocaine-memory reconsolidation. To test this hypothesis, male and female Sprague-Dawley rats received adeno-associated viruses to express Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) selectively in CRF neurons of the DR and injection cannulae directed at the BLA. The rats were trained to self-administer cocaine in a distinct environmental context then received extinction training in a different context. Next, they were briefly re-exposed to the cocaine-predictive context to destabilize (reactivate) cocaine memories. Intra-BLA infusions of the DREADD agonist deschloroclozapine (DCZ; 0.1 mM, 0.5 µL/hemisphere) immediately after memory reactivation attenuated cocaine-memory strength, relative to vehicle infusion. This was indicated by a selective, DCZ-induced and memory reactivation-dependent decrease in drug-seeking behavior in the cocaine-predictive context in DREADD-expressing males and females at test compared to respective controls. Notably, BLA-projecting DR CRF neurons that exhibited increased c-Fos expression during memory reconsolidation co-expressed the glutamatergic neuronal marker, vesicular glutamate transporter 3. Together, these findings suggest that the DRCRF → BLA circuit is engaged to maintain cocaine-memory strength after memory destabilization, and this phenomenon may be mediated by DR CRF and/or glutamate release in the BLA.

Heroin-induced conditioned immunomodulation requires expression of IL-1ß in the dorsal hippocampus.

Opioid-associated environmental stimuli elicit robust immune-altering effects via stimulation of a neural circuitry that includes the basolateral amygdala and nucleus accumbens. These brain regions are known to have both direct and indirect connections with the hippocampus. Thus, the present study evaluated whether the dorsal hippocampus (DH), and more specifically interleukin-1 beta (IL-1ß) within the DH, is necessary for the expression of heroin-induced conditioned immunomodulation. Rats received five Pavlovian pairings of systemic heroin administration (1.0mg/kg, SC) with placement into a distinct environment (conditioned stimulus, CS). Six days after conditioning, a GABAA/B agonist cocktail or IL-1ß small interfering RNA (siRNA) was microinfused into the DH to inhibit neuronal activity or IL-1ß gene expression prior to CS or home cage exposure. Control animals received saline or negative control siRNA microinfusions. Furthermore, all rats received systemic administration of lipopolysaccharide (LPS) to stimulate proinflammatory nitric oxide production. CS exposure suppressed LPS-induced nitric oxide production relative to home cage exposure. Inactivation of, or IL-1ß silencing in, the DH disrupted the CS-induced suppression of nitric oxide production relative to vehicle or negative control siRNA treatment. These results are the first to show a role for DH IL-1ß expression in heroin-conditioned suppression of a proinflammatory immune response.

Subregion-specific role of glutamate receptors in the nucleus accumbens on drug context-induced reinstatement of cocaine-seeking behavior in rats.

The functional integrity of the nucleus accumbens (NAC) core and shell is necessary for contextual cocaine-seeking behavior in the reinstatement animal model of drug relapse; however, the neuropharmacological mechanisms underlying this phenomenon are poorly understood. The present study evaluated the contribution of metabotropic glutamate receptor subtype 1 (mGluR1) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor populations to drug context-induced reinstatement of cocaine-seeking behavior. Rats were trained to lever press for un-signaled cocaine infusions in a distinct context followed by extinction training in a different context. Cocaine-seeking behavior (non-reinforced active lever pressing) was then assessed in the previously cocaine-paired and extinction contexts after JNJ16259685 (mGluR1 antagonist: 0.0, 0.6, or 30 pg/0.3 µl/hemisphere) or CNQX (AMPA/kainate receptor antagonist: 0.0, 0.03, or 0.3 µg/0.3 µl /hemisphere) administration into the NAC core, medial or lateral NAC shell, or the ventral caudate-putamen (vCPu, anatomical control). JNJ16259685 or CNQX in the NAC core dose-dependently impaired contextual cocaine-seeking behavior relative to vehicle. Conversely, CNQX, but not JNJ16259685, in the lateral or medial NAC shell attenuated, whereas CNQX or JNJ16259685 in vCPu failed to inhibit, this behavior. The manipulations failed to alter instrumental behavior in the extinction context, general motor activity or food-reinforced instrumental behavior in control experiments. Thus, glutamate-mediated changes in drug context-induced motivation for cocaine involve distinct neuropharmacological mechanisms within the core and shell subregions of the NAC, with the stimulation of mGlu1 and AMPA/kainate receptors in the NAC core and the stimulation of AMPA/kainate, but not mGlu1, receptors in the NAC shell being necessary for this phenomenon.

Interaction between the basolateral amygdala and dorsal hippocampus is critical for cocaine memory reconsolidation and subsequent drug context-induced cocaine-seeking behavior in rats.

Contextual stimulus control over instrumental drug-seeking behavior relies on the reconsolidation of context-response-drug associative memories into long-term memory storage following retrieval-induced destabilization. According to previous studies, the basolateral amygdala (BLA) and dorsal hippocampus (DH) regulate cocaine-related memory reconsolidation; however, it is not known whether these brain regions interact or independently control this phenomenon. To investigate this question, rats were trained to lever press for cocaine reinforcement in a distinct environmental context followed by extinction training in a different context. Rats were then briefly re-exposed to the cocaine-paired context to destabilize cocaine-related memories, or they were exposed to an unpaired context. Immediately thereafter, the rats received unilateral microinfusions of anisomycin (ANI) into the BLA plus baclofen/muscimol (B/M) into the contralateral (BLA/DH disconnection) or ipsilateral DH, or they received contralateral or ipsilateral microinfusions of vehicle. They then remained in their home cages overnight or for 21 d, followed by additional extinction training and a test of cocaine-seeking behavior (nonreinforced active lever responding). BLA/DH disconnection following re-exposure to the cocaine-paired context, but not the unpaired context, impaired subsequent drug context-induced cocaine-seeking behavior relative to vehicle or ipsilateral ANI + B/M treatment. Prolonged home cage stay elicited a time-dependent increase, or incubation, of drug-context-induced cocaine-seeking behavior, and BLA/DH disconnection inhibited this incubation effect despite some recovery of cocaine-seeking behavior. Thus, the BLA and DH interact to regulate the reconsolidation of cocaine-related associative memories, thereby facilitating the ability of drug-paired contexts to trigger cocaine-seeking behavior and contributing to the incubation of cocaine-seeking behavior.

Optogenetic inhibition of the dorsal hippocampus CA3 region during early-stage cocaine-memory reconsolidation disrupts subsequent context-induced cocaine seeking in rats.

The dorsal hippocampus (DH) is key to the maintenance of cocaine memories through reconsolidation into long-term memory stores after retrieval-induced memory destabilization. Here, we examined the time-dependent role of the cornu ammonis 3 DH subregion (dCA3) in cocaine-memory reconsolidation by utilizing the temporal and spatial specificity of optogenetics. eNpHR3.0-eYFP- or eYFP-expressing male Sprague-Dawley rats were trained to lever press for cocaine infusions in a distinct context and received extinction training in a different context. Rats were then re-exposed to the cocaine-paired context for 15 min to destabilize cocaine memories (memory reactivation) or remained in their home cages (no-reactivation). Optogenetic dCA3 inhibition for one hour immediately after memory reactivation reduced c-Fos expression (index of neuronal activation) in dCA3 stratum pyramidale (SP) glutamatergic and GABAergic neurons and in stratum lucidum (SL) GABAergic neurons during reconsolidation. Furthermore, dCA3 inhibition attenuated drug-seeking behavior (non-reinforced lever presses) selectively in the cocaine-paired context three days later (recall test), relative to no photoinhibition. This behavioral effect was eNpHR3.0-, memory-reactivation, and time-dependent, indicating a memory-reconsolidation deficit. Based on this observation and our previous finding that protein synthesis in the DH is not necessary for cocaine-memory reconsolidation, we postulate that recurrent pyramidal neuronal activity in the dCA3 may maintain labile cocaine memories prior to protein synthesis-dependent reconsolidation elsewhere, and SL/SP interneurons may facilitate this process by limiting extraneous neuronal activity. Interestingly, SL c-Fos expression was reduced at recall concomitant with impairment in cocaine-seeking behavior, suggesting that SL neurons may also facilitate cocaine-memory retrieval by inhibiting non-engram neuronal activity.

Neural Substrates and Circuits of Drug Addiction.

Drug addiction is a chronic relapsing disorder, and a significant amount of research has been devoted to understand the factors that contribute to the development, loss of control, and persistence of compulsive addictive behaviors. In this review, we provide an overview of various theories of addiction to drugs of abuse and the neurobiology involved in elements of the addiction cycle. Specific focus is devoted to the role of the mesolimbic pathway in acute drug reinforcement and occasional drug use, the role of the mesocortical pathway and associated areas (e.g., the dorsal striatum) in escalation/dependence, and the contribution of these pathways and associated circuits to conditioned responses, drug craving, and loss of behavioral control that may underlie drug relapse. By enhancing the understanding of the neurobiological factors that mediate drug addiction, continued preclinical and clinical research will aid in the development of novel therapeutic interventions that can serve as effective long-term treatment strategies for drug-dependent individuals.

Basolateral amygdala CB1 receptors gate HPA axis activation and context-cocaine memory strength during reconsolidation.

Re-exposure to a cocaine-associated context triggers craving and relapse through the retrieval of salient context-drug memories. Upon retrieval, context-drug memories become labile and temporarily sensitive to modification before they are reconsolidated into long-term memory stores. The effects of systemic cannabinoid type 1 receptor (CB1R) antagonism indicate that CB1R signaling is necessary for cocaine-memory reconsolidation and associated glutamatergic plasticity in the basolateral amygdala (BLA); however, the contribution of BLA CB1R signaling to cocaine-memory reconsolidation is unknown. Here, we assessed whether intra-BLA CB1R manipulations immediately after cocaine-memory retrieval alter cocaine-memory strength indexed by subsequent drug context-induced cocaine-seeking behavior in an instrumental rodent model of drug relapse. Administration of the CB1R antagonist, AM251 (0.3 µg/hemisphere) into the BLA increased subsequent drug context-induced cocaine-seeking behavior in a memory retrieval-dependent and anatomically selective manner. Conversely, the CB1R agonist, WIN55,212-2 (0.5 or 5 µg/hemisphere) failed to alter this behavior. In follow-up experiments, cocaine-memory retrieval elicited robust hypothalamic-pituitary-adrenal axis activation, as indicated by a rise in serum corticosterone concentrations. Intra-BLA AM251 administration during memory reconsolidation selectively increased this cocaine-memory retrieval-induced corticosterone response. Intra-BLA corticosterone administration (3 or 10 ng/hemisphere) during memory reconsolidation did not augment subsequent cocaine-seeking behavior, suggesting that CB1R-dependent effects of corticosterone on memory strength, if any, are mediated outside of the BLA. Together, these findings suggest that CB1R signaling in the BLA gates cocaine-memory strength, possibly by diminishing the impact of cue-induced arousal on the integrity of the reconsolidating memory trace or on the efficacy of the memory reconsolidation process.

Basolateral amygdala corticotropin-releasing factor receptor type 1 regulates context-cocaine memory strength during reconsolidation in a sex-dependent manner.

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.

Involvement of the lateral orbitofrontal cortex in drug context-induced reinstatement of cocaine-seeking behavior in rats.

Orbitofrontal cortex (OFC) damage produces impaired decision-making, impulsivity and perseveration and potentially contributes to compulsive drug seeking in cocaine users. To further explore this phenomenon, we assessed the role of the lateral OFC (lOFC) in drug context-induced cocaine-seeking behavior in the reinstatement model of drug relapse. Rats were trained to lever press for intravenous cocaine infusions in a distinct environmental context (cocaine-paired context) followed by extinction training in a different context (extinction-paired context). Reinstatement of cocaine seeking (non-reinforced lever presses) was assessed in the cocaine context in the absence of response-contingent stimuli. In Experiment 1, we evaluated whether acute inhibition of lOFC output alters context-induced cocaine-seeking behavior by infusing the GABA(B + A) agonists (baclofen + muscimol) or vehicle into the lOFC immediately before exposure to the cocaine-paired context. In Experiments 2 and 3, we assessed how prolonged loss of lOFC output affects drug context-induced cocaine seeking by administering bilateral N-methyl-d-aspartic acid or sham lesions of the lOFC either before or after self-administration and extinction training. Remarkably, IOFC functional inactivation attenuated, post-training lesions failed to alter and pre-training lesions potentiated drug context-induced cocaine seeking without altering responding in the extinction context. These results suggest that neural activity in the lOFC promotes context-induced cocaine-seeking behavior. However, prolonged loss of lOFC output enhances the motivational salience of cocaine-paired contextual stimuli probably by eliciting compensatory neuroadaptations, with the effects of post-training lOFC lesions reflecting an intermediate state of compensatory neuroplasticity. Overall, these findings support the idea that OFC dysfunction may promote cue reactivity and enhance relapse propensity in cocaine users.

Dorsal hippocampal regulation of memory reconsolidation processes that facilitate drug context-induced cocaine-seeking behavior in rats.

Exposure to a cocaine-paired context increases the propensity for relapse in cocaine users and prompts cocaine-seeking behavior in rats. According to the reconsolidation hypothesis, upon context re-exposure, established cocaine-related associations are retrieved and can become labile. These associations must undergo reconsolidation into long-term memory to effect enduring stimulus control. The dorsal hippocampus (DH), dorsolateral caudate-putamen and dorsomedial prefrontal cortex are critical for the expression of context-induced cocaine seeking, and these brain regions may also play a role in the reconsolidation of cocaine-related memories that promote this behavior. To test this hypothesis, rats were trained to press a lever for unsignaled cocaine infusions (0.2 mg/infusion, i.v.) in a distinct environmental context (cocaine-paired context), followed by extinction training in a different context (extinction context). Rats were then re-exposed to the cocaine-paired context for 15 min in order to reactivate cocaine-related memories or received comparable exposure to a novel unpaired context. Immediately thereafter, rats received bilateral microinfusions of the protein synthesis inhibitor anisomycin, the sodium channel blocker tetrodotoxin or vehicle into one of the above brain regions. After additional extinction training in the extinction context, reinstatement of cocaine-seeking behavior (i.e., non-reinforced lever presses) was assessed in the cocaine-paired context. Tetrodotoxin, but not anisomycin, administered into the DH inhibited drug context-induced cocaine-seeking behavior in a memory reactivation-dependent manner. Other manipulations failed to alter this behavior. These findings suggest that the DH facilitates the reconsolidation of associative memories that maintain context-induced cocaine-seeking behavior, but it is not the site of anisomycin-sensitive memory restabilization per se.