Activation of Epac in the BLA disrupts reconsolidation and attenuates heroin‐seeking behaviour

The susceptibility to drug cravings evoked by stimuli poses a formidable hurdle in the treatment of addiction and the prevention of relapse. Pharmacological interventions targeting drug‐associated memories hold promise for curbing relapse by impeding the process of memory reconsolidation, predominantly governed by cAMP signalling. Exchange Protein Activated by cAMP (Epac) serves as a distinctive mediator of cAMP signalling, which has been implicated in reinforcing the effects of cocaine and facilitating the acquisition. Nonetheless, the role of Epac in heroin‐related memory and the subsequent seeking behaviour remains enigmatic. In this study, we explored the impact of Epac activation on the reconsolidation process of drug‐related memories associated with heroin self‐administration. Over the course of 10 consecutive days, rats underwent training, wherein they acquired the behaviour of nose poking to obtain heroin accompanied by a tone + light cue. This nose‐poking behaviour was subsequently extinguished when heroin infusion and cue presentation were discontinued. Subsequently, we administered 8‐pCPT‐cAMP (8‐CPT), an Epac‐specific activator, into the basolateral amygdala at various time points, either in the presence or absence of a conditioned stimulus. Our findings demonstrate that administering 8‐CPT immediately after memory retrieval effectively reduces cue‐ and heroin‐induced reinstatement, with the observed effects persisting significantly for a minimum of 28 days. However, infusion of 8‐CPT for a duration of 6 h following the memory retrieval trial, or without it altogether, had no discernible impact. Thus, these findings strongly suggest that Epac activation can disrupt the reconsolidation of heroin‐associated memory, thereby diminishing the reinstatement of heroin‐seeking behaviour.

Opioid use disorder represents a persistent and recurring affliction of the brain, characterized by maladaptive patterns of learning and memory.These patterns give rise to intense cravings and compulsions to engage in opioid use, which are triggered by cues associated with drug consumption and memories tied to the rewarding effects of opioids. 1,2cumulating evidence shows that animal models of addiction, when exposed to drug self-administration, display heightened activity levels and a preference for environments associated with drug use.These behavioural changes are influenced by the brain's response to the cues and memories linked to drug-related experiences. 3,4The enduring nature of drug-related memories, reinforced by underlying neurobiological mechanisms, presents a formidable challenge in their eradication, ultimately culminating in compulsive drug-seeking behaviour and relapses, even following prolonged periods of abstinence.
Exposure therapy, relying on the process of extinction to diminish the association between drug cues and the rewarding effects of drugs, exhibits certain limitations, as the effects of extinction are highly context dependent and transient. 5In contrast, targeting the reconsolidation of memories following their retrieval represents a potentially effective strategy in the treatment of drug addiction.This approach aims to weaken the strength of drug-related memories and decrease the likelihood of relapse. 6,7Disrupting the reconsolidation process can lead to enduring effects that are not reliant on specific contexts, setting it apart from extinction-based interventions.Therefore, the exploration of methods to disrupt the reconsolidation of memories that link drug use with environmental stimuli holds great promise as a means of enhancing addiction therapy.
Exchange Protein Activated by cAMP (Epac) was originally identified as a novel target protein for cAMP in 1998.The discovery of Epac, comprising Epac1 and Epac2, revolutionized the prevailing understanding of cAMP signalling, which had hitherto been primarily associated with protein kinase A (PKA). 8 Investigations into neuronal signalling mediated by cAMP-Epac have shed light on its crucial role in neural development, synaptic remodelling, neurotransmitter release and learning and memory. 9,10Notably, the intracellular mechanism of memory reconsolidation mainly involves cAMP signalling and several studies suggested that Epac may contribute to long-term potentiation, 11 for which, be involved in the reconsolidation process.Prior research has revealed that the down-regulation of Epac impairs the retrieval of fear memory, 12 while the inhibition of PKA, another cAMP sensor, disrupts the reconsolidation of drug-related memories. 13Furthermore, activation of Epac has been shown to impede the reconsolidation of memories associated with cocaine cues and disrupt the association between drug cues and the rewarding effects of drug use. 14It is worth noting that discrepancies have been observed between psychostimulants and opiates. 15,16However, the precise impact of Epac activity on the reconsolidation process, particularly concerning memories linked to heroin use, remains inadequately understood.
The basolateral amygdala (BLA), a critical subregion of the amygdala, plays a pivotal role in associative learning, wherein a neutral stimulus is repeatedly paired with either a positive or negative event. 17evious studies have demonstrated that inactivation of the BLA impairs cue-induced recovery of heroin-seeking behaviour in animal models. 18Similarly, heroin-related cues elicit robust activation in the amygdala among individuals with a history of heroin use, triggering drug cravings. 19,20Pharmacological interventions targeting the BLA during memory reactivation or immediately thereafter have shown promising results in preventing the reinstatement of drug-seeking behaviour.These interventions include the use of DNA methyltransferase inhibitors (such as 5-AZA), 21,22 histone deacetylase inhibitors (such as TSA), 23 NMDA receptor antagonists (such as MK801 and D-APV), 24,25 β-adrenergic receptor antagonists (such as propranolol) 26,27 and extracellular signal-regulated kinase inhibitors (such as U0126). 28,29These findings underscore the pivotal involvement of the BLA in the reconsolidation of memories associated with drug use.
Moreover, studies have demonstrated the requirement of the cAMPdependent protein kinase (PKA) in the reconsolidation of fear, cocaine and heroin-associated memories in the BLA. 13,30Nonetheless, the specific role of Epac, another sensor of cAMP, in the reconsolidation of heroin-associated memories remains largely unexplored.Consequently, our study aimed to investigate the effects of Epac activation through 8-CPT on the reconsolidation of heroin-associated memories and its subsequent impact on drug-seeking behaviour triggered by heroinrelated cues or the passage of time.

| Animals
Male Sprague Dawley rats, initially weighing between 260-280 g, were divided into groups of five and housed in a temperaturecontrolled environment with a reversed 12-h light/dark cycle.They had ad libitum access to food and water.Prior to the commencement of the experiment, the rats underwent 3 min of daily handling for five consecutive days to acclimate them to the experimental procedures.
All animal care and experimental protocols adhered to the guidelines outlined in the National Research Council's Guide for the Care and Use of Laboratory Animals.The climate-controlled facility maintained a constant temperature of 23 ± 2 C, with humidity levels approximately at 60%.The lights were turned off at 8:00 AM and turned on at 8:00 PM.A total of 69 rats were used for experiments, and eight rats were excluded from the experiments due to catheter patency failure or failure to acquire heroin self-administration.

| Intravenous surgery
The rats, weighing between 300-320 g at the time of surgery, were anaesthetized using isoflurane (4-5% for induction and 1.5-2% for maintenance) as described previously. 6,31,32A cannula was inserted into the right jugular vein, with the tip positioned near the right atrium.To ensure stability, the catheter was secured to a curved connector made of 22-gauge stainless steel.Additionally, four stainless steel screws and dental acrylic were used to firmly anchor the catheter to the rat's cranial structure.The rats received intravenous infusions of 0.1 ml heparinized saline solution (containing 30 USP heparin per saline; Hospira) every 48 h to maintain the patency of the intravenous line.A recovery period of 5-7 days was provided before the commencement of the experiment.

| Drug infusions
The Epac-specific agonist, 8-(4-chlorophenylthio)-2 0 -O-methyladenosine-3 0 ,5 0 -cyclic monophosphate (8-CPT), was obtained from Sigma-Aldrich (St. Louis, MO, United States).The infusion volume of 8-CPT in the BLA (dorsoventral: À8.5 mm, mediolateral: ±5.0 mm and rostrocaudal: À2.8 mm) was 10 nmol per side, following the parameters established in previous studies. 21Hamilton syringes were connected to 30-gauge injectors (Plastics One) to facilitate the administration of the drug, which was delivered to a depth of 1 mm below the guide cannula within a duration of 1 min.The drug was allowed to diffuse for an additional 1 min without removing the injection needles.In all experimental procedures, rats received intracranial injections of the Epac-specific agonist or vehicle (0.5 μl), with or without exposure to conditioned stimulus (CS).The dose of 8-CPT was chosen based on previous studies. 14Subsequently, the rats were returned to their home cages and kept there until the subsequent testing.Control groups underwent an equivalent volume infusion of 1 Â PBS, with the same procedure being applied across all administered drugs.All infusion sites in the BLA are shown in Figure 5.

| Intravenous heroin self-administration training
The training protocol and conditions for heroin self-administration were conducted in accordance with our previous studies. 21,32An operant chamber equipped with two nosepoke operandi (AniLab Software and Instruments, China) positioned 5 cm above the ground and equipped with light stimulation was utilized.Rats underwent training to receive intravenous heroin (0.05 mg/kg/infusion) over a period of 10 days, with three sessions per day.Each training session consisted of a 1-h session followed by a 5-min break.A continuous reinforcement schedule, specifically a fixed ratio 1 (FR1) schedule, was employed, allowing a 40-s rest period after each heroin infusion.To outline the procedure briefly, rats were connected to a polyethylene tubing linked to a 10 ml syringe pump via a fluid swivel connected to a metal tether covered with metal mesh.When a session commenced, the chamber's light was turned on for the entire duration.Intravenous heroin was administered upon nosepokes into the active operandum, accompanied by the presentation of a 5-s tone-light cue.

| CS exposure
Twenty-four hours after the final nosepoke extinction session, a 15-min session was conducted to reactivate the memories associated with heroin.The CS exposure conditions were identical to those used during the training for heroin self-administration, except that active nosepokes resulted in reinforcement from drug cues alone, without the administration of heroin.

| Cue extinction
During the 3-h cue extinction phase, the conditions in the chamber were similar to those during the daily heroin self-administration training, with the exception that no heroin injection was administered after the presentation of the tone-light cue.

| Cue-induced reinstatement test
Twenty-four hours after receiving either an Epac or vehicle treatment, rats underwent a reinstatement test triggered by drug cues.The experimental conditions during the reinstatement test closely resembled those of the heroin self-administration training.
The only distinction was that active nosepokes resulted in the presentation of a tone-light cue that had previously been associated with heroin, but without the actual administration of heroin as reinforcement.The duration of the reinstatement test was 1 h.

| Heroin-induced reinstatement test
Five minutes prior to entering the self-administration context, the rats were administered heroin (0.25 mg/kg, s.c.).The test procedures during the reinstatement test were identical to those of the drug selfadministration training, with the exception that active nosepokes were reinforced with drug cues instead of heroin.The duration of the reinstatement test remained 1 h.The experimental procedure for Experiment 4 was identical to that of Experiment 1, except that the rats received an intra-BLA 8-CPT injection 6 h after a reactivation session of 15 min.

| Statistical analysis
The analysis of the experimental outcomes was performed using GraphPad, v.8.0 presented as mean ± SEM.For each experiment, repeated measure ANOVAs were conducted with a between-subjects treatment condition factor and a within-subjects test condition factor, followed by Tukey's post hoc test.Statistical significance was defined as p<0.05.

| Experiment 1: Immediate post-CS 8-CPT treatment in BLA decreases following cue-and heroininduced reinstatement heroin seeking
To investigate the impact of the post-reactivation 8-CPT infusion in BLA between cue-and heroin-induced reinstatement of drug seeking, rats were separated to two groups (Figure 1A).There was no difference in the total number of infusions between the groups that would   2D).In the spontaneous recovery test, the Epac group significantly attenuated the drug-seeking behaviour ( p < 0.01) (Figure 2E).These findings imply that heroin craving is inhibited immediately following CS Epac activation in BLA and that this effect lasts for up to 28 days.

| Experiment 3: Without CS reactivation, intra-BLA infusion of 8-CPT has no impact on later reinstatement heroin seeking
For the considerable effect of CS Epac strategy on heroin craving, we investigated whether CS reactivation affects the therapeutic benefits  3E).These findings showed that the ameliorative effect of Epac on heroin seeking was owing to the activation of drug-related memories synergistically; the selectively activation of Epac had no effect on the reinstatement of heroin seeking that occurs after cue-induced or heroin-primed reinstatement.= 0.06665, p = 0.7998; interaction of test day Â 8-CPT dose: F [1, 15] = 0.3148, p = 0.5831; Figure 4E).The findings of this study suggest that Epac has a temporally specific impact on the reconsolidation of heroin-associated memory.

| DISCUSSION
In this study, we investigated the influence of Epac in BLA on the reconsolidation of instrumental-learned memory.Specifically, we examined the effects of intra-BLA infusion of 8-CPT, a selective Epac agonist, immediately or 6 h after memory reactivation, on heroinassociated memory and subsequent heroin-seeking behaviour in rats.
Our results revealed that the intra-BLA infusion of 8-CPT during Importantly, the inhibitory effects of 8-CPT on heroin-seeking behaviour were observed for at least 28 days.However, when 8-CPT was administered with a delay of 6 h or without a retrieval trial, it did not exhibit any beneficial effect on the reinstatement of heroin-seeking behaviour induced by cues or priming with heroin.These findings suggest that the inhibitory impact of 8-CPT on heroin-seeking behaviour is dependent on the timing of administration and specifically targets the retrieval of the memory.
In the present work, we employed a heroin instrumental learning model to examine the impact of Epac activity in the BLA on the reconsolidation of heroin-associated memory.Our experimental manipulations targeted the reconsolidation process, which occurs after memory retrieval. 7,33,34Rats were trained to respond for heroin in the presence of cue stimuli, and this behaviour was subsequently extinguished by removing the heroin pump and the cue.During a retrieval trial, the tone and light cues were presented without the delivery of heroin. 13We found that immediate activation of Epac following memory retrieval resulted in a reduction of cue-induced drug-seeking behaviour on the following day.Importantly, no effect was observed when the intra-BLA infusion of 8-CPT occurred 6 h after retrieval or without a retrieval session, indicating that the inhibitory effect of 8-CPT was specific to the reconsolidation of the heroin-associated memory rather than a general suppression of operant responding.
Numerous studies have demonstrated that the process of memory reconsolidation is dependent on the synthesis of new proteins and can occur within a timeframe of 6 h following memory reactivation. 35The effectiveness of interventions aimed at disrupting memory reconsolidation is influenced by two key factors: the temporal proximity to the initial memory reactivation and the presence of a memory reactivation procedure.Our findings align with the characteristics of memory reconsolidation.The activation of Epac exhibits a memory recall-dependent and time-sensitive effect on the erasure of heroinassociated memories.Therefore, we propose that Epac activity in the BLA plays a role in mediating the reconsolidation of heroin-associated memories.
In this study, we focused on investigating the role of Epac, a cAMP-activated Ras-like GTPases guanine nucleotide exchange factor, in the reconsolidation of heroin-associated memories.Epac is a cAMP receptor that, along with PKA, regulates intracellular signal transduction. 36Both PKA and Epac are crucial cAMP effectors that have downstream signalling targets, enabling cells to respond and adapt to behavioural changes driven by cAMP fluctuations. 37,38The cAMP-PKA signalling pathway has been extensively studied in the context of drug addiction, encompassing various classes of drugs. 39,40evious research has highlighted the significance of PKA in the reconsolidation of drug-related memories. 13Given the close relationship between cAMP and these two sensors, we hypothesized that Epac might modulate PKA signalling and exhibit anti-seeking effects.
Indeed, a prior study demonstrated that activation of Epac following memory reactivation reduced cue-induced reinstatement of cocaineseeking behaviour, and this effect was found to be dependent on PKA activity. 14In our study, we observed that intra-BLA infusion of 8-CPT during the reconsolidation process disrupts the heroin-associated memories in rats.These findings suggest that the two cAMP-activated signalling pathways, PKA and Epac, may exert opposing effects on the reconsolidation of drug-related memories.Exploring the precise relationship between these two cAMP effectors in the context of reconsolidation is an important area for future research.
While our study showed that activation of Epac in the BLA disrupted the reconsolidation of heroin-associated memory, we were unable to identify the direct targets of Epac due to its two isoforms.
The Epac agonist we used, 8-CPT, is a general activator of Epac but has been found to be more effective in activating Epac1 than Epac2. 41This suggests that Epac1 may be involved in the reconsolidation process.Epac1 is expressed in various tissues, whereas Epac2 is primarily found in the brain, adrenal gland, pancreas and liver. 41Interestingly, a recent study by Liu et al. 42 demonstrated the contribution of Epac2 to cocaine reinforcement, suggesting a potential role of Epac2 in drug reward effects.They found that downregulating Epac2 genetically or pharmacologically impaired the acquisition of cocaine self-administration, which appears to conflict with our findings where Epac activation reduced heroin-seeking behaviour.To explain this phenomenon, we speculate that both Epac1 and Epac2 may be involved in extinction memory, leading to a bidirectional effect on drug-seeking behaviour.In our study, we included an additional extinction process, which could have influenced the outcome.Although no studies have directly investigated the role of Epac in extinction, several studies have highlighted the importance of cAMP-PKA signalling in this process. 43,44Therefore, it is plausible that the activity of Epac is required for extinction, and further research is needed to explore the specific role of Epac in extinction processes.

2. 4 . 2 |
Nosepoke extinction Following the drug self-administration phase, rats underwent extinction training.During the daily 3-h nosepoke extinction sessions, nosepokes into either of the operandi did not lead to any predetermined outcomes.Extinction training continued until the frequency of active nosepoke responses decreased to less than 20% of the average responding observed during the previous three heroin selfadministration sessions, and this criterion had to be met for at least two consecutive days.

2. 4 . 7 |
Spontaneous recovery testAfter a period of 28 days of prolonged withdrawal, nosepokes were recorded for 1 h during the cue-induced reinstatement test.The testing conditions remained identical to those used during the initial cue-induced reinstatement test.2.5 | Specific experiments 2.5.1 | Experiment 1: The effect of immediate post-CS reactivation Epac treatment in BLA on subsequent cue-induced and drug-primed reinstatement of heroin seeking In the same operant chambers, the rats were trained in heroin selfadministration for 10 days, followed by nosepoke extinction for 10 consecutive days.Twenty-four hours after the last nosepoke extinction, rats were re-exposed to the heroin training context for 15 min to induce CS reactivation on Day 22.To find out whether administering 8-CPT immediately after memory reactivation disrupt the expression of the heroin-associated memory, a cue-induced reinstatement test was carried out 24 h after the post-CS 8-CPT injected in the BLA.And cue extinction sessions were then performed for 2 days (Days 25 and 26).Saline and heroin priming-induced reinstatement were carried out on Days 27 and 28, respectively.2.5.2 | Experiment 2: The effect of acute treatment with 8-CPT in BLA on cue-induced reinstatement and spontaneous recovery after 28 days In this study, after a stable training of heroin self-administration acquisition and extinction, rats then had a 15-min CS reactivation trial before beginning the 8-CPT administration.A cue-induced reinstatement test was conducted 24 h later, followed by 28 days of abstention and the test for the spontaneous recovery of heroin-associated memory.F I G U R E 1 Immediate postconditioned stimulus 8-CPT treatment in BLA decreases following Cue-and heroininduced reinstatement heroin seeking.(A) Schematic diagram of the experimental procedure.(B) Total number of infusions across heroin selfadministration acquisition.(C) Total quantity of active nosepoke responses during all extinction treatments.(D) Active nosepoke responses during the last extinction treatment and the cueinduced reinstatement test.(E) Active nosepoke responses during the saline-or heroin-primed reinstatement test.Data were shown as means ± SEM (n ≥ 8 per group).*P < 0.05 Epac versus control group.Rein test refers to reinstatement test.Last ext day refers to last extinction day.Cue ext day refers to cue extinction day.

2. 5 . 3 |
Experiments 3: The effect of 8-CPT treatment in BLA without CS reactivation on subsequent cue-and drug-induced reinstatement of heroin seeking Except that rats got an intracranial injection of 8-CPT in BLA just after a 15-min no-reactivation session, the experimental protocol was the same as that of Experiment 1.2.5.4 | Experiment 4:The effect of delayed post-CS reactivation 8-CPT treatment in BLA on subsequent cue-and heroin-primed reinstatement of drug seeking

F I G U R E 2
Immediate 8-CPT treatment in BLA following conditioned stimulus reactivation has long-term inhibition effects on heroin seeking.(A) Schematic representation of the experimental procedure.(B) Total number of infusions across heroin selfadministration sessions.(C) Total number of active nosepoke responses across extinction sessions.(D) Active nosepokes during the last extinction session and cueinduced reinstatement test.(E) Active nosepokes during the last extinction session and spontaneous recovery of heroin-associated memory (after 28 days of abstinence).Data were shown as means ± SEM (n ≥ 8 per group).*P < 0.05 Epac versus control group.Rein test refers to reinstatement test.Last ext day refers to last extinction day.Cue ext day refers to cue extinction day.SR refers to spontaneous recovery.simultaneous retrieval of heroin-related memories significantly suppressed drug-seeking behaviour triggered by cues or the drug itself.

F I G U R E 3
Without conditioned stimulus reactivation, intra-BLA infusion of 8-CPT has no impact on later reinstatement heroin seeking.(A) Schematic representation of the experimental procedure.(B) Total number of infusions across acquisition of heroin self-administration. (C) Total number of active nosepoke responses across extinction sessions.(D) Active nosepoke responses during the last extinction session and cue-induced reinstatement test.(E) Active nosepoke responses during the saline-or heroin-primed reinstatement test.Data were shown as means ± SEM (n ≥ 8 per group).Rein test refers to reinstatement test.Last ext day refers to last extinction day.Cue ext day refers to cue extinction day.

F I G U R E 4
After conditioned stimulus reactivation, a 6 h-delayed intra-BLA 8-CPT infusion had little impact on the subsequent reinstatement heroin seeking.Schematic representation of the experimental procedure.(B) Total number of infusions across heroin selfadministration sessions.(C) Nosepoke responses during the reactivation trial.(D) Active nosepoke responses during the last extinction session and the cueinduced reinstatement test.(E) Active nosepokes during the saline-or heroinprimed reinstatement test.Data were shown as means ± SEM (n ≥ 8 per group).Rein test refers to reinstatement test.Last ext day refers to last extinction day.Cue ext day refers to cue extinction day.F I G U R E 5 Diagram showing the region where infusions were located.The numbers indicate the anterior-posterior position relative to bregma (mm).