Extracellular Signal-Regulated Kinase in the Basolateral Amygdala,but not the Nucleus Accumbens Core,is Critical for Context-Response-Cocaine Memory Reconsolidation in Rats

The reconsolidation of cocaine memories following retrieval is necessary for the sustained ability of a cocaine-paired environmental context to elicit cocaine seeking. Extracellular signal-regulated kinase (ERK) is an intracellular signaling molecule involved in nucleus accumbens core (NACc)-mediated reconsolidation of Pavlovian cocaine memories. Here, we used a rodent model of drug context-elicited relapse to test the hypothesis that ERK would be similarly required for the reconsolidation of context-response-cocaine memories that underlie drug context-induced reinstatement of instrumental cocaine-seeking behavior, with a focus on the NACc and on the basolateral amygdala (BLA), another important locus for the reconsolidation of cocaine memories. We show that the mitogen-activated protein kinase (MEK)/ERK1/2 inhibitor, U0126 (1.0 μg/0.5 μl/hemisphere), microinfused bilaterally into the BLA—but not the NACc—immediately after brief re-exposure to a previously cocaine-paired context (that is, cocaine-memory reactivation), significantly attenuated subsequent drug context-induced cocaine seeking relative to vehicle (VEH). This effect in the BLA was associated with a transient inhibition of ERK1/2 phosphorylation, and it depended on memory reactivation given that U0126 administered following exposure to a novel context did not alter subsequent cocaine seeking. Furthermore, similar to U0126, baclofen+muscimol-induced (B+M; 106.8/5.7 ng/0.5 μl/hemisphere) neural inactivation of the NACc, following cocaine-memory reactivation, failed to alter subsequent cocaine seeking. These findings demonstrate that ERK activation in the BLA, but not the NACc, is required for the reconsolidation of context-response-cocaine associative memories. Together with prior research, these results suggest that contextual drug-memory reconsolidation in Pavlovian and instrumental settings involves distinct neuroanatomical mechanisms.     

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.Subject terms: Consolidation, Addiction, Motivation     

Role of a Hippocampal Src-Family Kinase-Mediated Glutamatergic Mechanism in Drug Context-Induced Cocaine Seeking

Glutamatergic neurotransmission in the dorsal hippocampus (DH) is necessary for drug context-induced reinstatement of cocaine-seeking behavior in an animal model of drug relapse. Furthermore, in vitro studies suggest that the Src family of tyrosine kinases critically regulates glutamatergic cellular functions within the DH. Thus, Src-family kinases in the DH may similarly control contextual cocaine-seeking behavior. To test this hypothesis, 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 AP5 (N-methyl-D-aspartate glutamate (NMDA) receptor (NMDAR) antagonist; 0.25 or 2.5 μg/0.5 μl/hemisphere), PP2 (Src-family kinase inhibitor; 6.25 or 62.5 ng/0.5 μl/hemisphere), Ro25-6981 (NR2B subunit-containing NMDAR antagonist; 0.2 or 2 μg/0.5 μl/hemisphere), or vehicle administration into the DH. Administration of AP5, PP2, or Ro25-6981 into the DH dose-dependently impaired drug context-induced reinstatement of cocaine-seeking behavior relative to vehicle, without altering instrumental behavior in the extinction context or food-reinforced instrumental responding and general motor activity in control experiments. Cocaine-seeking behavior during the first 20 min of the test session in the cocaine-paired context was associated with an increase in NR2B subunit activation, and intra-DH PP2 pretreatment disrupted this relationship. Together, these findings suggest that Src-family kinase activation, NMDAR stimulation, and likely Src-family kinase-mediated NR2B subunit-containing NMDAR activation in the DH are necessary for incentive motivational and/or memory processes that promote contextual cocaine-seeking behavior.     

Involvement of amygdalar protein kinase A, but not calcium/calmodulin-dependent protein kinase II, in the reconsolidation of cocaine-related contextual memories in rats

Rationale

Contextual control over drug relapse depends on the successful reconsolidation and retention of context-response–cocaine associations in long-term memory stores. The basolateral amygdala (BLA) plays a critical role in cocaine memory reconsolidation and subsequent drug context-induced cocaine-seeking behavior; however, less is known about the cellular mechanisms of this phenomenon.

Objectives

The present study evaluated the hypothesis that protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) activation in the BLA is necessary for the reconsolidation of context-response–cocaine memories that promote subsequent drug context-induced cocaine-seeking behavior.

Methods

Rats were trained to lever-press for cocaine infusions in a distinct context, followed by extinction training in a different context. Rats were then briefly re-exposed to the previously cocaine-paired context or an unpaired context in order to reactivate cocaine-related contextual memories and initiate their reconsolidation or to provide a similar behavioral experience without explicit cocaine-related memory reactivation, respectively. Immediately after this session, rats received bilateral microinfusions of vehicle, the PKA inhibitor, Rp-adenosine 3’,5’-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS), or the CaMKII inhibitor, KN-93, into the BLA or the posterior caudate putamen (anatomical control region). Rats were then tested for cocaine-seeking behavior (responses on the previously cocaine-paired lever) in the cocaine-paired context and the extinction context.

Results

Intra-BLA infusion of Rp-cAMPS, but not KN-93, following cocaine memory reconsolidation impaired subsequent cocaine-seeking behavior in a dose-dependent, site-specific, and memory reactivation-dependent fashion.

Conclusions

PKA, but not CaMKII, activation in the BLA is critical for cocaine memory re-stabilization processes that facilitate subsequent drug context-induced instrumental cocaine-seeking behavior.     

β-Endorphin via the Delta Opioid Receptor is a Major Factor in the Incubation of Cocaine Craving

Cue-induced cocaine craving intensifies, or ‘incubates'', during the first few weeks of abstinence and persists over extended periods of time. One important factor implicated in cocaine addiction is the endogenous opioid β-endorphin. In the present study, we examined the possible involvement of β-endorphin in the incubation of cocaine craving. Rats were trained to self-administer cocaine (0.75 mg/kg, 10 days, 6 h/day), followed by either a 1-day or a 30-day period of forced abstinence. Subsequent testing for cue-induced cocaine-seeking behavior (without cocaine reinforcement) was performed. Rats exposed to the drug-associated cue on day 1 of forced abstinence demonstrated minimal cue-induced cocaine-seeking behavior concurrently with a significant increase in β-endorphin release in the nucleus accumbens (NAc). Conversely, exposure to the cue on day 30 increased cocaine seeking, while β-endorphin levels remained unchanged. Intra-NAc infusion of an anti-β-endorphin antibody (4 μg) on day 1 increased cue-induced cocaine seeking, whereas infusion of a synthetic β-endorphin peptide (100 ng) on day 30 significantly decreased cue response. Both intra-NAc infusions of the δ opioid receptor antagonist naltrindole (1 μg) on day 1 and naltrindole together with β-endorphin on day 30 increased cue-induced cocaine-seeking behavior. Intra-NAc infusion of the μ opioid receptor antagonist CTAP (30 ng and 3 μg) had no behavioral effect. Altogether, these results demonstrate a novel role for β-endorphin and the δ opioid receptor in the development of the incubation of cocaine craving.     

Post-Retrieval Extinction Attenuates Cocaine Memories

Recent studies have shown that post-retrieval extinction training attenuates fear and reward-related memories in both humans and rodents. This noninvasive, behavioral approach has the potential to be used in clinical settings to treat maladaptive memories that underlie several psychiatric disorders, including drug addiction. However, few studies to date have used a post-retrieval extinction approach to attenuate addiction-related memories. In the current study, we attempted to disrupt cocaine-related memories by using the post-retrieval extinction paradigm in male Sprague Dawley rats. Results revealed that starting extinction training 1 h after cocaine contextual memory was retrieved significantly attenuated cocaine-primed reinstatement of conditioned place preference (CPP) and relapse of cocaine CPP (drug-free and cocaine-primed) following 30 days of abstinence. However, animals that did not retrieve the contextual cocaine memory before extinction training, or animals that began extinction training 24 h after retrieval (outside of the reconsolidation window), demonstrated normal cocaine CPP. Conversely, animals that received additional CPP conditioning, rather than extinction training, 1 h after reactivation of cocaine memory showed enhanced cocaine CPP compared with animals that did not reactivate the cocaine memory before conditioning. These results reveal that a behavioral manipulation that takes advantage of reconsolidation and extinction of drug memories may be useful in decreasing preference for, and abuse of, cocaine.     

Stimulation of the Noradrenergic System during Memory Formation Impairs Extinction Learning but not the Disruption of Reconsolidation

The noradrenergic system plays a critical role in the ‘consolidation'' of emotional memory. If we are to target ‘reconsolidation'' in patients with anxiety disorders, the noradrenergic strengthening of fear memory should not impair the disruption of reconsolidation. In Experiment I, we addressed this issue using a differential fear conditioning procedure allowing selective reactivation of one of two fear associations. First, we strengthened fear memory by administering an α₂-adrenergic receptor antagonist (ie, yohimbine HCl; double-blind placebo-controlled study) 30 min before acquisition (time for peak value yohimbine HCl <1 h). Next, the reconsolidation of one of the fear associations was manipulated by administering a β-adrenergic receptor antagonist (ie, propranolol HCl) 90 min before its selective reactivation (time for peak value propranolol HCl <2 h). In Experiment II, we administered propranolol HCl after reactivation of the memory to rule out a possible effect of the pharmacological manipulation on the memory retrieval itself. The excessive release of noradrenaline during memory formation not only delayed the process of extinction 48 h later, but also triggered broader fear generalization. Yet, the β-adrenergic receptor blocker during reconsolidation selectively ‘neutralized'' the fear-arousing aspects of the noradrenergic-strengthened memory and undermined the generalization of fear. We observed a similar reduction in fear responding when propranolol HCl was administered after reactivation of the memory. The present findings demonstrate the involvement of noradrenergic modulation in the formation as well as generalization of human fear memory. Given that the noradrenergic strengthening of fear memory impaired extinction learning but not the disruption of reconsolidation, our findings may have implications for the treatment of anxiety disorders.     

On Disruption of Fear Memory by Reconsolidation Blockade: Evidence from Cannabidiol Treatment

The search for reconsolidation blockers may uncover clinically relevant drugs for disrupting memories of significant stressful life experiences, such as those underlying the posttraumatic stress disorder. Considering the safety of systemically administered cannabidiol (CBD), the major non-psychotomimetic component of Cannabis sativa, to animals and humans, the present study sought to investigate whether and how this phytocannabinoid (3–30 mg/kg intraperitoneally; i.p.) could mitigate an established memory, by blockade of its reconsolidation, evaluated in a contextual fear-conditioning paradigm in rats. We report that CBD is able to disrupt 1- and 7-days-old memories when administered immediately, but not 6 h, after their retrieval for 3 min, with the dose of 10 mg/kg being the most effective. This effect persists in either case for at least 1 week, but is prevented when memory reactivation was omitted, or when the cannabinoid type-1 receptors were antagonized selectively with AM251 (1.0 mg/kg). Pretreatment with the serotonin type-1A receptor antagonist WAY100635, however, failed to block CBD effects. These results highlight that recent and older fear memories are equally vulnerable to disruption induced by CBD through reconsolidation blockade, with a consequent long-lasting relief in contextual fear-induced freezing. Importantly, this CBD effect is dependent on memory reactivation, restricted to time window of <6 h, and is possibly dependent on cannabinoid type-1 receptor-mediated signaling mechanisms. We also observed that the fear memories disrupted by CBD treatment do not show reinstatement or spontaneous recovery over 22 days. These findings support the view that reconsolidation blockade, rather than facilitated extinction, accounts for the aforementioned CBD results in our experimental conditions.     

Stimulation of Medial Prefrontal Cortex Serotonin 2C (5-HT2C) Receptors Attenuates Cocaine-Seeking Behavior

Serotonin 2C receptor (5-HT_2CR) agonists administered systemically attenuate both cocaine-primed and cue-elicited reinstatement of extinguished cocaine-seeking behavior. To further elucidate the function of these receptors in addiction-like processes, this study examined the effects of microinfusing the 5-HT_2CR agonist MK212 (0, 10, 30, 100 ng/side/0.2 μl) into the medial prefrontal cortex (mPFC) on cocaine self-administration and reinstatement of extinguished cocaine-seeking behavior. Male Sprague–Dawley rats were trained to self-administer cocaine (0.75 mg/kg, i.v.) paired with light and tone cues. Once responding stabilized, rats received MK212 microinfusions before tests for maintenance of cocaine self-administration. Next, extinction training to reduce cocaine-seeking behavior, defined as responses performed without cocaine reinforcement available, occurred until low extinction baselines were achieved. Rats then received MK212 microinfusions before tests for reinstatement of extinguished cocaine-seeking behavior elicited by cocaine-priming injections (10 mg/kg, i.p.) or response-contingent presentations of the cocaine-associated cues; operant responses during cocaine-primed reinstatement tests produced no consequences. MK212 microinfusions into the prelimbic and infralimbic, but not anterior cingulate, regions of the mPFC dose-dependently attenuated both cocaine-primed and cue-elicited reinstatement of extinguished cocaine-seeking behavior, but did not reliably affect cocaine self-administration. A subsequent experiment showed that the effects of MK212 (100 ng/side/0.2 μl) on reinstatement of extinguished cocaine-seeking behavior were blocked by co-administration of the 5-HT_2CR antagonist SB242084 (200 ng/side/0.2 μl). MK212 administered alone into the mPFC as a drug prime produced no discernable effects on cocaine-seeking behavior. These findings suggest that stimulation of 5-HT_2CRs in the mPFC attenuates the incentive motivational effects produced by sampling cocaine or exposure to drug-paired cues.     

Bidirectional Modulation of Alcohol-Associated Memory Reconsolidation through Manipulation of Adrenergic Signaling

Alcohol addiction is a problem of great societal concern, for which there is scope to improve current treatments. One potential new treatment for alcohol addiction is based on disrupting the reconsolidation of the maladaptive Pavlovian memories that can precipitate relapse to drug-seeking behavior. In alcohol self-administering rats, we investigated the effects of bidirectionally modulating adrenergic signaling on the strength of a Pavlovian cue-alcohol memory, using a behavioral procedure that isolates the specific contribution of one maladaptive Pavlovian memory to relapse, the acquisition of a new alcohol-seeking response for an alcohol-associated conditioned reinforcer. The β-adrenergic receptor antagonist propranolol, administered in conjunction with memory reactivation, persistently disrupted the memory that underlies the capacity of a previously alcohol-associated cue to act as a conditioned reinforcer. By contrast, enhancement of adrenergic signaling by administration of the adrenergic prodrug dipivefrin at reactivation increased the strength of the cue-alcohol memory and potentiated alcohol seeking. These data demonstrate the importance of adrenergic signaling in alcohol-associated memory reconsolidation, and suggest a pharmacological target for treatments aiming to prevent relapse through the disruption of maladaptive memories.     

The CB1 Receptor Antagonist AM251 Impairs Reconsolidation of Pavlovian Fear Memory in the Rat Basolateral Amygdala

We have investigated the requirement for signaling at CB1 receptors in the reconsolidation of a previously consolidated auditory fear memory, by infusing the CB1 receptor antagonist AM251, or the FAAH inhibitor URB597, directly into the basolateral amygdala (BLA) in conjunction with memory reactivation. AM251 disrupted memory restabilization, but only when administered after reactivation. URB597 produced a small, transient enhancement of memory restabilization when administered after reactivation. The amnestic effect of AM251 was rescued by coadministration of the GABA_A receptor antagonist bicuculline at reactivation, indicating that the disruption of reconsolidation was mediated by altered GABAergic transmission in the BLA. These data show that the endocannabinoid system in the BLA is an important modulator of fear memory reconsolidation and that its effects on memory are mediated by an interaction with the GABAergic system. Thus, targeting the endocannabinoid system may have therapeutic potential to reduce the impact of maladaptive memories in neuropsychiatric disorders such as posttraumatic stress disorder.     

Interaction of the Basolateral Amygdala and Orbitofrontal Cortex is Critical for Drug Context-Induced Reinstatement of Cocaine-Seeking Behavior in Rats

The basolateral amygdala (BLA) and lateral orbitofrontal cortex (OFC) are critical elements of the neural circuitry that regulates drug context-induced reinstatement of cocaine-seeking behavior. Given the existence of dense reciprocal anatomical connections between these brain regions, this study tested the hypothesis that serial information processing by the BLA and OFC is necessary for drug context-induced cocaine-seeking behavior. Male Sprague–Dawley rats were trained to lever press for un-signaled cocaine infusions (0.15 mg/infusion, i.v.) in a distinct environment (cocaine-paired context) then underwent extinction training in a different environment (extinction context). During four subsequent test sessions, rats were re-exposed to the cocaine-paired and extinction contexts in order to assess cocaine-seeking behavior (non-reinforced active lever responding). Immediately before each test session, rats received microinfusions of the GABA_A/GABA_B agonist cocktail, baclofen+muscimol (BM: 1.0/.01 mM), or vehicle unilaterally into the BLA plus the contralateral or ipsilateral OFC, or unilaterally into the OFC alone. Exposure to the previously cocaine-paired context, but not the extinction context, reinstated extinguished cocaine-seeking behavior. BM-induced unilateral OFC inactivation failed to alter this behavior, similar to the effect of unilateral BLA inactivation in our previous study (Fuchs et al, 2007). Conversely, neural inactivation of the BLA plus the contralateral or ipsilateral OFC equally attenuated drug context-induced cocaine seeking without altering food-reinforced instrumental responding, relative to vehicle pretreatment. These findings suggest that the BLA and OFC co-regulate drug context-induced motivation for cocaine either through sequential information processing via intra- and interhemispheric connections or by providing converging input to a downstream brain region.     

Systems Reconsolidation Reveals a Selective Role for the Anterior Cingulate Cortex in Generalized Contextual Fear Memory Expression

After acquisition, hippocampus-dependent memories undergo a systems consolidation process, during which they become independent of the hippocampus and dependent on the anterior cingulate cortex (ACC) for memory expression. However, consolidated remote memories can become transiently hippocampus-dependent again following memory reactivation. How this systems reconsolidation affects the role of the ACC in remote memory expression is not known. Using contextual fear conditioning, we show that the expression of 30-day-old remote memory can transiently be supported by either the ACC or the dorsal hippocampus following memory reactivation, and that the ACC specifically mediates expression of remote generalized contextual fear memory. We found that suppression of neural activity in the ACC with the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) impaired the expression of remote, but not recent, contextual fear memory. Fear expression was not affected by this treatment if preceded by memory reactivation 6 h earlier, nor was it affected by suppression of neural activity in the dorsal hippocampus with the GABA-receptor agonist muscimol. However, simultaneous targeting of both the ACC and the dorsal hippocampus 6 h after memory reactivation disrupted contextual fear memory expression. Second, we observed that expression of a 30-day-old generalized contextual fear memory in a novel context was not affected by memory reactivation 6 h earlier. However, intra-ACC CNQX infusion before testing impaired contextual fear expression in the novel context, but not the original training context. Together, these data suggest that although the dorsal hippocampus may be recruited during systems reconsolidation, the ACC remains necessary for the expression of generalized contextual fear memory.     

Preclinical Evaluation of Reconsolidation Blockade by Clonidine as a Potential Novel Treatment for Posttraumatic Stress Disorder

Exposure to traumatic events can lead to posttraumatic stress disorder (PTSD). Current PTSD treatments typically only produce partial improvement. Hence, there is a need for preclinical research to identify new candidate drugs and to develop novel therapeutic approaches. Animal studies have indicated that fear memories can be weakened by blocking restabilization after retrieval, a process known as reconsolidation. Furthermore, evidence suggests that there are important alterations of the noradrenergic system in PTSD, and hence it may be of interest to study drugs that target this pathway. Here, we investigated the efficacy of clonidine, an α₂-adrenoreceptor agonist, to block reconsolidation in an animal model of persistent traumatic memories. Using an auditory fear conditioning paradigm in rats, we tested the efficacy of clonidine to weaken fear memory retention when administered systemically after retrieval. We evaluated dosage, number of treatments, and specificity in reconsolidation blockade. We found that postretrieval administration of clonidine disrupts fear-related memories in a dose-dependent manner and that two treatments are sufficient for maximal memory impairment. Furthermore, we determined that this effect is long lasting and specific to reconsolidation processes as shown by the selectivity to affect reactivated memories and the absence of spontaneous recovery and of postreactivation short-term memory impairment. Our results demonstrate the efficacy of systemic administration of clonidine following retrieval to persistently disrupt fear memory retention through reconsolidation blockade. This study provides important preclinical parameters for future therapeutic strategies involving clonidine to block reconsolidation as a novel treatment for PTSD symptoms.     

Inhibition of Hippocampal β-Adrenergic Receptors Impairs Retrieval But Not Reconsolidation of Cocaine-Associated Memory and Prevents Subsequent Reinstatement

Retrieval of drug-associated memories is critical for maintaining addictive behaviors, as presentation of drug-associated cues can elicit drug seeking and relapse. Recently, we and others have demonstrated that β-adrenergic receptor (β-AR) activation is necessary for retrieval using both rat and human memory models. Importantly, blocking retrieval with β-AR antagonists persistently impairs retrieval and provides protection against subsequent reinstatement. However, the neural locus at which β-ARs are required for maintaining retrieval and subsequent reinstatement is unclear. Here, we investigated the necessity of dorsal hippocampus (dHipp) β-ARs for drug-associated memory retrieval. Using a cocaine conditioned place preference (CPP) model, we demonstrate that local dHipp β-AR blockade before a CPP test prevents CPP expression shortly and long after treatment, indicating that dHipp β-AR blockade induces a memory retrieval disruption. Furthermore, this retrieval disruption provides long-lasting protection against cocaine-induced reinstatement. The effects of β-AR blockade were dependent on memory reactivation and were not attributable to reconsolidation disruption as blockade of β-ARs immediately after a CPP test had little effect on subsequent CPP expression. Thus, cocaine-associated memory retrieval is mediated by β-AR activity within the dHipp, and disruption of this activity could prevent cue-induced drug seeking and relapse long after treatment.     

Potentiation of GluN2C/D NMDA Receptor Subtypes in the Amygdala Facilitates the Retention of Fear and Extinction Learning in Mice

NMDA receptors are glutamate receptor ion channels that contribute to synaptic plasticity and are important for many forms of learning and memory. In the amygdala, NMDA receptors are critical for the acquisition, retention, and extinction of classically conditioned fear responses. Although the GluN2B subunit has been implicated in both the acquisition and extinction of conditioned fear, GluN2C-knockout mice show reduced conditioned fear responses. Moreover, D-cycloserine (DCS), which facilitates fear extinction, selectively enhances the activity of GluN2C-containing NMDA receptors. To further define the contribution of GluN2C receptors to fear learning, we infused the GluN2C/GluN2D-selective potentiator CIQ bilaterally into the basolateral amygdala (3, 10, or 30 μg/side) following either fear conditioning or fear extinction training. CIQ both increased the expression of conditioned fear 24 h later and enhanced the extinction of the previously conditioned fear response. These results support a critical role for GluN2C receptors in the amygdala in the consolidation of learned fear responses and suggest that increased activity of GluN2C receptors may underlie the therapeutic actions of DCS.     

Effects of Genetic Deletion of Endogenous Opioid System Components on the Reinstatement of Cocaine-Seeking Behavior in Mice

The repeated cycles of cessation of consumption and relapse remain the major clinical concern in treating drug addiction. The endogenous opioid system is a crucial component of the reward circuit that participates in the adaptive changes leading to relapse in the addictive processes. We have used genetically modified mice to evaluate the involvement of μ-opioid receptor (MOR) and δ-opioid receptor (DOR) and their main endogenous ligands, the enkephalins derived from proenkephalin (PENK) and prodynorphin (PDYN), in the reinstatement of cocaine-seeking behavior. Constitutive knockout mice of MOR, DOR, PENK, and PDYN, and their wild-type littermates were trained to self-administer cocaine or to seek for palatable food, followed by a period of extinction and finally tested on a cue-induced reinstatement of seeking behavior. The four lines of knockout mice acquired operant cocaine self-administration behavior, although DOR and PENK knockout mice showed less motivation for cocaine than wild-type littermates. Moreover, cue-induced relapse was significantly decreased in MOR and DOR knockout mice. In contrast, PDYN knockout mice showed a slower extinction and increased relapse than wild-type littermates. C-Fos expression analysis revealed differential activation in brain areas related with memory and reward in these knockout mice. No differences were found in any of the four genotypes in operant responding to obtain palatable food, indicating that the changes revealed in knockout mice were not due to unspecific deficit in operant performance. Our results indicate that MOR, DOR, and PDYN have a differential role in cue-induced reinstatement of cocaine-seeking behavior.     

PTSD-Like Memory Generated Through Enhanced Noradrenergic Activity is Mitigated by a Dual Step Pharmacological Intervention Targeting its Reconsolidation

Background:

Traumatic memories have been resilient to therapeutic approaches targeting their permanent attenuation. One of the potentially promising pharmacological strategies under investigation is the search for safe reconsolidation blockers. However, preclinical studies focusing on this matter have scarcely addressed abnormal aversive memories and related outcomes.

Methods:

By mimicking the enhanced noradrenergic activity reported after traumatic events in humans, here we sought to generate a suitable condition to establish whether some clinically approved drugs able to disrupt the reconsolidation of conditioned fear memories in rodents would still be effective.

Results:

We report that the α₂-adrenoceptor antagonist yohimbine was able to induce an inability to restrict behavioral (fear) and cardiovascular (increased systolic blood pressure) responses to the paired context when administered immediately after acquisition, but not 6h later, indicating the formation of a generalized fear memory, which endured for over 29 days and was less susceptible to suppression by extinction. It was also resistant to reconsolidation disruption by the α₂-adrenoceptor agonist clonidine or cannabidiol, the major non-psychotomimetic component of Cannabis sativa. Since signaling at N-methyl-D-aspartate (NMDA) receptors is important for memory labilization and because a dysfunctional memory may be less labile than is necessary to trigger reconsolidation on its brief retrieval and reactivation, we then investigated and demonstrated that pre-retrieval administration of the partial NMDA agonist D-cycloserine allowed the disrupting effects of clonidine and cannabidiol on reconsolidation.

Conclusions:

These findings highlight the effectiveness of a dual-step pharmacological intervention to mitigate an aberrant and enduring aversive memory similar to that underlying the post-traumatic stress disorder.     

Previous Stress Attenuates the Susceptibility to Midazolam's Disruptive Effect on Fear Memory Reconsolidation: Influence of Pre-Reactivation D-Cycloserine Administration

It is well known that, under certain boundary conditions, the retrieval of a stable consolidated memory results into a labile one. During this unstable phase, memory can be vulnerable to interference by a number of pharmacological agents, including benzodiazepines. One of the goals of this study was to evaluate the vulnerability to midazolam (MDZ) after reactivation of recent and remote contextual fear memories in animals that experienced a stressful situation before learning. Animals were subjected to a restraint session and trained in a contextual fear paradigm the following day; consolidated memories were reactivated at different times after learning and different MDZ doses (1.5, 3.0 mg/kg) were administered to rats after reactivation. Our results show that MDZ did not affect memory reconsolidation in older-than-one-day memories of stressed animals, even after the administration of a higher MDZ dose and a longer reactivation session (5 min). In contrast, MDZ was effective in blocking reconsolidation at all memory ages in unstressed animals. In addition, the current research investigated whether activating NMDA sites before reactivation promotes the destabilization of resistant memories such as those of stressed animals. We tested the influence of pre-reactivation D-cycloserine (DCS), a partial NMDA agonist, on MDZ''s effect on fear memory reconsolidation in stressed animals. Our findings indicate that DCS before reactivation promotes retrieval-induced lability in resistant memory traces, as MDZ-induced memory impairment in stressed rats became evident with pre-reactivation DCS but not after pre-reactivation sterile isotonic saline.