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High relapse rate is a key feature of opioid addiction. In humans, abstinence is often voluntary due to negative consequences of opioid seeking. To mimic this human condition, we recently introduced a rat model of incubation of oxycodone craving after electric barrier-induced voluntary abstinence. Incubation of drug craving refers to time-dependent increases in drug seeking after cessation of drug self-administration. Here, we used the activity marker Fos, muscimol-baclofen (GABAa + GABAb receptor agonists) global inactivation, Daun020-selective inactivation of putative relapse-associated neuronal ensembles, and fluorescence-activated cell sorting of Fos-positive cells and quantitative polymerase chain reaction to demonstrate a key role of vSub neuronal ensembles in incubation of oxycodone craving after voluntary abstinence, but not homecage forced abstinence. We also used a longitudinal functional magnetic resonance imaging method and showed that functional connectivity changes in vSub-related circuits predict opioid relapse after abstinence induced by adverse consequences of opioid seeking. Funding Agencies|Intramural Research Program of the NIDA-NIH [ZIA-DA000434-22]; Swedish Research Council International Postdoc grant [2019-00658]

We recently developed a rat model of relapse to drug seeking after food choice-induced voluntary abstinence. Here, we used this model to study the role of the orbitofrontal cortex (OFC) and its afferent projections in relapse to fentanyl seeking. We trained male and female rats to self-administer palatable food pellets for 6 d (6 h/d) and intravenous fentanyl (2.5 μg/kg/infusion) for 12 d (6 h/d). We assessed relapse to fentanyl seeking after 13–14 voluntary abstinence days, achieved through a discrete choice procedure between fentanyl infusions and palatable food (20 trials/d). In both sexes, relapse after food choice-induced abstinence was associated with increased expression of the activity marker Fos in the OFC. Pharmacological inactivation of the OFC with muscimol plus baclofen (50 + 50 ng/side) decreased relapse to fentanyl seeking. We then determined projection-specific activation of OFC afferents during the relapse test by using Fos plus the retrograde tracer cholera toxin B (injected into the OFC). Relapse to fentanyl seeking was associated with increased Fos expression in the piriform cortex (Pir) neurons projecting to the OFC, but not in projections from the basolateral amygdala and thalamus. Pharmacological inactivation of the Pir with muscimol plus baclofen decreased relapse to fentanyl seeking after voluntary abstinence. Next, we used an anatomical disconnection procedure to determine whether projections between the Pir and OFC are critical for relapse to fentanyl seeking. Unilateral muscimol plus baclofen injections into the Pir in one hemisphere plus unilateral muscimol plus baclofen injections into the OFC in the contralateral, but not ipsilateral, hemisphere decreased relapse. Our results identify Pir–OFC projections as a new motivation-related pathway critical to relapse to opioid seeking after voluntary abstinence.SIGNIFICANCE STATEMENTThere are few preclinical studies of fentanyl relapse, and these studies have used experimenter-imposed extinction or forced abstinence procedures. In humans, however, abstinence is often voluntary, with drug available in the drug environment but forgone in favor of nondrug alternative reinforcers. We recently developed a rat model of drug relapse after palatable food choice-induced voluntary abstinence. Here, we used classical pharmacology, immunohistochemistry, and retrograde tracing to demonstrate a critical role of the piriform and orbitofrontal cortices in relapse to opioid seeking after voluntary abstinence.

Neuronal ensembles in ventromedial prefrontal cortex (vmPFC) play a role in both cocaine and palatable food seeking. However, it is unknown whether similar or different vmPFC neuronal ensembles mediate food and cocaine seeking. Here, we used the Daun02 inactivation procedure to assess whether the neuronal ensembles mediating food seeking and cocaine seeking can be functionally distinguished. We trained male and female Fos-LacZ rats to self-administer palatable food pellets and cocaine on alternating days for 18 days. We then exposed the rats to a brief non-reinforced food- or cocaine-seeking test to induce Fos and β-gal in neuronal ensembles associated with food or cocaine seeking, respectively, and infused Daun02 into vmPFC to ablate the β-gal-expressing ensembles. Two days later, we tested the rats for food or cocaine seeking under extinction conditions. Although inactivation of the food-seeking ensemble did not influence food or cocaine seeking, inactivation of the cocaine-seeking ensemble reduced cocaine seeking, but not food seeking. Results indicate that the neuronal ensemble activated by cocaine seeking in vmPFC is functionally separate from the ensemble activated by food seeking.

In abstinent drug addicts, cues formerly associated with drug-taking experiences gain relapse-inducing potency (‘incubate’) over time. Animal models of incubation may help develop treatments to prevent relapse, but these models have ubiquitously focused on the role of conditioned stimuli (CSs) signaling drug delivery. Discriminative stimuli (DSs) are unique in that they exert stimulus-control over both drug taking and drug seeking behavior and are difficult to extinguish. For this reason, incubation of the excitatory effects of DSs that signal drug availability, not yet examined in preclinical studies, could be relevant to relapse prevention. We trained rats to self-administer cocaine (or palatable food) under DS control, then investigated DS-controlled incubation of craving, in the absence of drug-paired CSs. DS-controlled cocaine (but not palatable food) seeking incubated over 60 days of abstinence and persisted up to 300 days. Understanding the neural mechanisms of this DS-controlled incubation holds promise for drug relapse treatments., eLife digest More than 85% of people who give up an addictive drug begin using it again within a year. Relapse rates have changed little over the past five decades. Situations, places and objects associated with drug-taking can trigger relapse long after a person’s last exposure to a drug. We can study relapse by training animals to self-administer drugs such as cocaine. For example, rats can learn to press a lever to receive an infusion of a drug paired with a cue (a conditioned stimulus), such as a tone or a light. After training, the rats continue to press the lever to seek the drug, even if this behavior no longer delivers it. In addition, their lever pressing in response to cues increases with time for several months after their last drug exposure. This phenomenon, known as incubation of drug craving, mirrors the increase in cravings reported by abstinent drug users. In drug users, cues such as the crack pipe or syringe used to take the drug can later contribute to drug relapse during abstinence. Most studies modeling this phenomenon have focused on how the rats respond to a conditioned stimulus that signaled the delivery of a drug during training. However, a second type of signal, known as the discriminative stimulus, can also influence relapse. Discriminative stimuli are sets of cues that signal whether drugs are about to become available or not; for example, the presence of people selling drugs on a street corner as opposed to the presence of police. Madangopal et al. now show that discriminative stimuli – in the absence of conditioned stimuli – can also control the incubation of drug craving. Rats learned to press a lever in response to a light signaling the availability of cocaine (the positive discriminative stimulus), and to avoid responding to a different light indicating that cocaine was unavailable (the negative discriminative stimulus). When tested during abstinence, the rats only increased their lever pressing to the first light over time, i.e., they showed an incubation of drug craving controlled by the positive discriminative stimulus. Lever pressing peaked after 60 days of abstinence and persisted for up to 300 days (almost half the rats' lifespan). By contrast, the same discriminative stimuli did not trigger increased lever pressing when used to signal the availability of a palatable food. Discriminative stimuli are thus powerful and persistent triggers of craving for addictive drugs. They signal the availability of a drug prior to both drug-taking and relapse, making them a critical target for intervention strategies. Understanding the mechanisms by which discriminative stimuli promote drug craving could lead to new treatments to prevent relapse.

Results from clinical studies suggest that drug relapse and craving are often provoked by exposure to drug-associated contexts. Since 2002, this phenomenon has been modeled in laboratory animals using the ABA renewal model. In the classical version of this model, rats with a history of drug self-administration in one context (A) undergo extinction in a different context (B) and reinstate (or relapse to) drug seeking after exposure to the original drug-associated context (A). In a more recent version of the model introduced in 2013, the experimental conditions in context A are identical to those used in the classical model, but drug-reinforced responding in context B is suppressed by probabilistic punishment. The punishment-based ABA renewal model is proposed to resemble abstinence in humans, which is often initiated by the desire to avoid the negative consequences of drug use. The goal of our review is to discuss similarities and differences in mechanisms that play a role in suppression of drug seeking in context B and context-induced relapse to drug seeking in context A in the two models. We first describe psychological mechanisms that mediate extinction and punishment of drug-reinforced responding in context B. We then summarize recent findings on brain mechanisms of context-induced relapse of drug seeking after extinction, or punishment-imposed abstinence. These findings demonstrate both similarities and differences in brain mechanisms underlying relapse in the two variations of the ABA renewal model. We conclude by briefly discussing clinical implications of the preclinical studies.

In abstinent drug addicts, cues formerly associated with drug-taking experiences gain relapse-inducing potency ('incubate') over time. Animal models of incubation may help develop treatments to prevent relapse, but these models have ubiquitously focused on the role of conditioned stimuli (CSs) signaling drug delivery. From a translational perspective this is problematic because people encounter these stimuli only during or after relapse. For this reason, incubation in response to discriminative stimuli (DSs) that signal drug availability before relapse, not yet examined in preclinical studies, could be more relevant to relapse prevention. We trained rats to self-administer cocaine (or palatable food) under DS control, then investigated DS-controlled incubation of craving, in the absence of drug-paired CSs. DS-controlled cocaine (but not palatable food) seeking incubated over 60 days of abstinence and persisted up to 300 days. Understanding the neural mechanisms of this DS-controlled incubation holds significant promise for drug relapse treatments.

Many preclinical studies examined cue-induced relapse to heroin and cocaine seeking in animal models, but most of these studies examined only one drug at a time. In human addicts, however, polydrug use of cocaine and heroin is common. We used a polydrug self-administration relapse model in rats to determine similarities and differences in brain areas activated during cue-induced reinstatement of heroin and cocaine seeking. We trained rats to lever press for cocaine (1.0 mg/kg/infusion, 3-h/d, 18 d) or heroin (0.03 mg/kg/infusion) on alternating days (9 d for each drug); drug infusions were paired with either intermittent or continuous light cue. Next, the rats underwent extinction training followed by tests for cue-induced reinstatement where they were exposed to either heroin- or cocaine-associated cues. We observed cue-selective reinstatement of drug seeking: the heroin cue selectively reinstated heroin seeking and the cocaine cue selectively reinstated cocaine seeking. We used Fos immunohistochemistry to assess cue-induced neuronal activation in different subregions of the medial prefrontal cortex (mPFC), dorsal striatum (DS), nucleus accumbens (NAc), and amygdala. Fos expression results indicated that only the prelimbic cortex (PL) was activated by both heroin and cocaine cues; in contrast, no significant cue-induced neuronal activation was observed in other brain areas. RNA in situ hybridization indicated that the proportion of glutamatergic and GABAergic markers in PL Fos-expressing cells were similar for the heroin and cocaine cue-activated neurons. Overall the results indicate that PL may be a common brain area involved in both heroin and cocaine seeking during polydrug use.

In a rat model of drug craving and relapse, cue-induced drug seeking progressively increases after withdrawal from methamphetamine and other drugs, a phenomenon termed 'incubation of drug craving'. However, current experimental procedures used to study incubation of drug craving do not incorporate negative consequences of drug use, which is a common factor promoting abstinence in humans. Here, we studied whether incubation of methamphetamine craving is observed after suppression of drug seeking by adverse consequences (punishment). We trained rats to self-administer methamphetamine or palatable food for 9 h per day for 14 days; reward delivery was paired with a tone-light cue. Subsequently, for one group within each reward type, 50% of the lever-presses were punished by mild footshock for 9-10 days, whereas for the other group lever-presses were not punished. Shock intensity was gradually increased over time. Next, we assessed cue-induced reward seeking in 1-h extinction sessions on withdrawal days 2 and 21. Response-contingent punishment suppressed extended-access methamphetamine or food self-administration; surprisingly, food-trained rats showed greater resistance to punishment than methamphetamine-trained rats. During the relapse tests, both punished and unpunished methamphetamine- and food-trained rats showed significantly higher cue-induced reward seeking on withdrawal day 21 than on day 2. These results demonstrate that incubation of both methamphetamine and food craving occur after punishment-induced suppression of methamphetamine or palatable food self-administration. Our procedure can be used to investigate mechanisms of relapse to drug and palatable food seeking under conditions that more closely approximate the human condition.

Background and rationale: Results from many clinical studies suggest that drug relapse and craving are often provoked by acute exposure to the self-administered drug or related drugs, drug-associated cues or contexts, or certain stressors. During the last two decades, this clinical scenario has been studied in laboratory animals by using the reinstatement model. In this model, reinstatement of drug seeking by drug priming, drug cues or contexts, or certain stressors is assessed following drug self-administration training and subsequent extinction of the drug-reinforced responding. Objective: In this review, we first summarize recent (2009-present) neurobiological findings from studies using the reinstatement model. We then discuss emerging research topics, including the impact of interfering with putative reconsolidation processes on cue- and context-induced reinstatement of drug seeking, and similarities and differences in mechanisms of reinstatement across drug classes. We conclude by discussing results from recent human studies that were inspired by results from rat studies using the reinstatement model. Conclusions: Main conclusions from the studies reviewed highlight: (1) the ventral subiculum and lateral hypothalamus as emerging brain areas important for reinstatement of drug seeking, (2) the existence of differences in brain mechanisms controlling reinstatement of drug seeking across drug classes, (3) the utility of the reinstatement model for assessing the effect of reconsolidation-related manipulations on cue-induced drug seeking, and (4) the encouraging pharmacological concordance between results from rat studies using the reinstatement model and human laboratory studies on cue- and stress-induced drug craving.

We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we studied the role of dorsolateral striatum (DLS) and dorsomedial striatum (DMS) in this incubation. We trained rats to self-administer palatable food pellets (6 d, 6 h/d) and methamphetamine (12 d, 6 h/d). We then assessed relapse to methamphetamine seeking under extinction conditions after 1 and 21 abstinence days. Between tests, the rats underwent voluntary abstinence (using a discrete choice procedure between methamphetamine and food; 20 trials/d) for 19 d. We usedin situhybridization to measure the colabeling of the activity markerFoswithDrd1andDrd2in DMS and DLS after the tests. Based on thein situhybridization colabeling results, we tested the causal role of DMS D1and D2family receptors, and DMS neuronal ensembles in “incubated” methamphetamine seeking, using selective dopamine receptor antagonists (SCH39166 or raclopride) and the Daun02 chemogenetic inactivation procedure, respectively. Methamphetamine seeking was higher after 21 d of voluntary abstinence than after 1 d (incubation of methamphetamine craving). The incubated response was associated with increasedFosexpression in DMS but not in DLS;Foswas colabeled with bothDrd1andDrd2. DMS injections of SCH39166 or raclopride selectively decreased methamphetamine seeking after 21 abstinence days. InFos-lacZtransgenic rats, selective inactivation of relapse test-activatedFosneurons in DMS on abstinence day 18 decreased incubated methamphetamine seeking on day 21. Results demonstrate a role of DMS dopamine D1and D2receptors in the incubation of methamphetamine craving after voluntary abstinence and that DMS neuronal ensembles mediate this incubation.SIGNIFICANCE STATEMENTIn human addicts, abstinence is often self-imposed and relapse can be triggered by exposure to drug-associated cues that induce drug craving. We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we used classical pharmacology,in situhybridization, immunohistochemistry, and the Daun02 inactivation procedure to demonstrate a critical role of dorsomedial striatum neuronal ensembles in this new form of incubation of drug craving.

Despite decades of research on neurobiological mechanisms of psychostimulant addiction, the only effective treatment for many addicts is contingency management, a behavioral treatment that uses alternative non-drug reward to maintain abstinence. However, when contingency management is discontinued, most addicts relapse to drug use. The brain mechanisms underlying relapse after cessation of contingency management are largely unknown, and, until recently, an animal model of this human condition did not exist. Here we used a novel rat model, in which the availability of a mutually exclusive palatable food maintains prolonged voluntary abstinence from intravenous methamphetamine self-administration, to demonstrate that the activation of monosynaptic glutamatergic projections from anterior insular cortex to central amygdala is critical to relapse after the cessation of contingency management. We identified the anterior insular cortex-to-central amygdala projection as a new addiction- and motivation-related projection and a potential target for relapse prevention.

Rationale and objective: In humans, exposure to contexts previously associated with heroin use can provoke relapse. In rats, exposure to heroin-paired contexts after extinction of drug-reinforced responding in different contexts reinstates heroin seeking. We previously demonstrated that the projections from ventral medial prefrontal cortex (vmPFC) to nucleus accumbens (NAc) shell play a role in this reinstatement. The ventral subiculum (vSub) sends glutamate projections to NAc shell and vmPFC. Here, we determined whether these projections contribute to context-induced reinstatement. Methods: We trained rats to self-administer heroin (0.05–0.1 mg/kg/infusion) for 3 h per day for 12 days; drug infusions were paired with a discrete tone–light cue. Lever pressing in the presence of the discrete cue was subsequently extinguished in a different context. We then tested the rats for reinstatement in the heroin- and extinction-associated contexts under extinction conditions. We combined Fos with the retrograde tracer Fluoro-Gold (FG) to determine projection-specific activation during the context-induced reinstatement tests. We also used anatomical disconnection procedures to determine whether the vSub → NAc shell and vSub → vmPFC projections are functionally involved in this reinstatement. Results: Exposure to the heroin but not the extinction context reinstated lever pressing. Context-induced reinstatement of heroin seeking was associated with increased Fos expression in vSub neurons, including those projecting to NAc shell and vmPFC. Anatomical disconnection of the vSub → NAc shell projection, but not the vSub → vmPFC projection, decreased this reinstatement. Conclusions: Our data indicate that the vSub → NAc shell glutamatergic projection, but not the vSub → vmPFC projection, contributes to context-induced reinstatement of heroin seeking.

In many human alcoholics, abstinence is self-imposed because of the negative consequences of excessive alcohol use, and relapse is often triggered by exposure to environmental contexts associated with prior alcohol drinking. We recently developed a rat model of this human condition in which we train alcohol-preferring P rats to self-administer alcohol in one context (A), punish the alcohol-reinforced responding in a different context (B), and then test for relapse to alcohol seeking in Contexts A and B without alcohol or shock. Here, we studied the role of projections to nucleus accumbens (NAc) shell from ventral subiculum (vSub), basolateral amygdala, paraventricular thalamus, and ventral medial prefrontal cortex in context-induced relapse after punishment-imposed abstinence. First, we measured double-labeling of the neuronal activity marker Fos with the retrograde tracer cholera toxin subunit B (injected in NAc shell) and demonstrated that context-induced relapse is associated with selective activation of the vSub→NAc shell projection. Next, we reversibly inactivated the vSub with GABA receptor agonists (muscimol+baclofen) before the context-induced relapse tests and provided evidence for a causal role of vSub in this relapse. Finally, we used a dual-virus approach to restrict expression of the inhibitory κ opioid-receptor based DREADD (KORD) in vSub→NAc shell projection neurons. We found that systemic injections of the KORD agonist salvinorin B, which selectively inhibits KORD-expressing neurons, decreased context-induced relapse to alcohol seeking. Our results demonstrate a critical role of vSub in context-induced relapse after punishment-imposed abstinence and further suggest a role of the vSub→NAc projection in this relapse.SIGNIFICANCE STATEMENTIn many human alcoholics, abstinence is self-imposed because of the negative consequences of excessive use, and relapse is often triggered by exposure to environmental contexts associated with prior alcohol use. Until recently, an animal model of this human condition did not exist. We developed a rat model of this human condition in which we train alcohol-preferring P rats to self-administer alcohol in one context (A), punish the alcohol-reinforced responding in a different context (B), and test for relapse to alcohol seeking in Contexts A and B. Here, we used neuroanatomical, neuropharmacological, and chemogenetic methods to demonstrate a role of ventral subiculum and potentially its projections to nucleus accumbens in context-induced relapse after punishment-imposed abstinence.

In humans, exposure to contexts previously associated with heroin use can provoke relapse. In rats, exposure to heroin-paired contexts after extinction of drug-reinforced responding in different contexts reinstates heroin seeking. This effect is attenuated by inhibition of glutamate or dopamine transmission in nucleus accumbens shell, or inactivation of ventral medial prefrontal cortex (mPFC). Here, we used an anatomical asymmetrical disconnection procedure to demonstrate that an interaction between glutamatergic projections from ventral mPFC to accumbens shell and local dopamine D1postsynaptic receptors contributes to context-induced reinstatement of heroin seeking. We also combined the marker of neuronal activity, Fos, with the retrograde tracer Fluoro-Gold to assess activation in this pathway during context-induced reinstatement. Rats were trained to self-administer heroin for 12 d; drug infusions were paired with a discrete tone–light cue. Lever pressing was subsequently extinguished in a nondrug-associated context in the presence of the discrete cue. Rats were then tested in the heroin- or extinction-associated contexts under extinction conditions. Injections of muscimol + baclofen into ventral mPFC in one hemisphere and D1-family receptor antagonist SCH 23390 into the contralateral or ipsilateral accumbens shell decreased context-induced reinstatement. Unilateral injections of muscimol + baclofen into ventral mPFC or SCH 23390 into the accumbens shell had no effect. Context-induced reinstatement was associated with increased Fos expression in ventral mPFC neurons, including those projecting to accumbens shell, with higher double-labeling in the ipsilateral projection than in the contralateral projection. Our results demonstrate that activation of glutamatergic projections from ventral mPFC to accumbens shell, previously implicated in inhibition of cocaine relapse, promotes heroin relapse.

Learned associations between effects of abused drugs and the drug administration environment are important in drug addiction. Histochemical and electrophysiological studies suggest that these associations are encoded in sparsely distributed nucleus accumbens neurons that are selectively activated by drugs and drug-associated cues. Although correlations have been observed between nucleus accumbens neuronal activity and responsivity to drugs and drug cues, no technique exists for selectively manipulating these activated neurons and establishing their causal role in behavioral effects of drugs and drug cues. Here we describe a new approach, which we term the 'Daun02 inactivation method', that selectively inactivates a minority of neurons previously activated by cocaine in an environment repeatedly paired with cocaine to demonstrate a causal role for these activated neurons in context-specific cocaine-induced psychomotor sensitization in rats. This method provides a new tool for studying the causal roles of selectively activated neurons in behavioral effects of drugs and drug cues and in other learned behaviors.

In operant learning, initial reward-associated memories are thought to be distinct from subsequent extinction-associated memories. Memories formed during operant learning are thought to be stored in “neuronal ensembles.” Thus, we hypothesize that different neuronal ensembles encode reward- and extinction-associated memories. Here, we examined prefrontal cortex neuronal ensembles involved in the recall of reward and extinction memories of food self-administration. We first trained rats to lever press for palatable food pellets for 7 d (1 h/d) and then exposed them to 0, 2, or 7 daily extinction sessions in which lever presses were not reinforced. Twenty-four hours after the last training or extinction session, we exposed the rats to either a short 15 min extinction test session or left them in their homecage (a control condition). We found maximal Fos (a neuronal activity marker) immunoreactivity in the ventral medial prefrontal cortex of rats that previously received 2 extinction sessions, suggesting that neuronal ensembles in this area encode extinction memories. We then used the Daun02 inactivation procedure to selectively disrupt ventral medial prefrontal cortex neuronal ensembles that were activated during the 15 min extinction session following 0 (no extinction) or 2 prior extinction sessions to determine the effects of inactivating the putative food reward and extinction ensembles, respectively, on subsequent nonreinforced food seeking 2 d later. Inactivation of the food reward ensembles decreased food seeking, whereas inactivation of the extinction ensembles increased food seeking. Our results indicate that distinct neuronal ensembles encoding operant reward and extinction memories intermingle within the same cortical area. SIGNIFICANCE STATEMENT A current popular hypothesis is that neuronal ensembles in different prefrontal cortex areas control reward-associated versus extinction-associated memories: the dorsal medial prefrontal cortex (mPFC) promotes reward seeking, whereas the ventral mPFC inhibits reward seeking. In this paper, we use the Daun02 chemogenetic inactivation procedure to demonstrate that Fos-expressing neuronal ensembles mediating both food reward and extinction memories intermingle within the same ventral mPFC area.

In the past decade, novel methods using engineered receptors have enabled researchers to manipulate neuronal activity with increased spatial and temporal specificity. One widely used chemogenetic method in mice and rats is the DREADD (designer receptors exclusively activated by designer drugs) system in which a mutated muscarinic G protein-coupled receptor is activated by an otherwise inert synthetic ligand, clozapine-N-oxide (CNO). Recently, the Roth laboratory developed a novel inhibitory DREADD in which a mutated kappa-opioid receptor (KORD) is activated by the pharmacologically inert drug salvinorin B (SalB; Vardy et al, 2015). They demonstrated the feasibility of using KORD to study brain circuits involved in motivated behavior in mice. Here, we used behavioral, electrophysiological, and neuroanatomical methods to demonstrate the feasibility of using the novel KORD to study brain circuits involved in motivated behavior in rats. In Exp. 1, we show that SalB dose-dependently decreased spontaneous and cocaine-induced locomotor activity in rats expressing KORD to midbrain (ventral tegmental area/substantia nigra). In Exp. 2, we show that SalB completely inhibited tonic firing in KORD-expressing putative dopamine neurons in midbrain. In Exp. 3, we used a 'retro-DREADD' dual-virus approach to restrict expression of KORD in ventral subiculum neurons that project to nucleus accumbens shell. We show that KORD activation selectively decreased novel context-induced Fos expression in this projection. Our results indicate that the novel KORD is a promising tool to selectively inactivate brain areas and neural circuits in rat studies of motivated behavior.

Cue-induced methamphetamine seeking progressively increases after withdrawal (incubation of methamphetamine craving), but the underlying mechanisms are largely unknown. We determined whether this incubation is associated with alterations in candidate genes in dorsal striatum (DS), a brain area implicated in cue- and context-induced drug relapse. We first measured mRNA expression of 24 candidate genes in whole DS extracts after short (2 d) or prolonged (1 month) withdrawal in rats following extended-access methamphetamine or saline (control condition) self-administration (9 h/d, 10 d). We found minimal changes. Next, using fluorescence-activated cell sorting, we compared gene expression in Fos-positive dorsal striatal neurons, which were activated during “incubated” cue-induced drug-seeking tests after prolonged withdrawal, with nonactivated Fos-negative neurons. We found significant increases in mRNA expression of immediate early genes (Arc, Egr1),Bdnfand its receptor (Trkb), glutamate receptor subunits (Gria1, Gria3, Grm1),and epigenetic enzymes (Hdac3, Hdac4, Hdac5, GLP, Dnmt3a, Kdm1a) in the Fos-positive neurons only. Using RNAscope to determine striatal subregion and cell-type specificity of the activated neurons, we measured colabeling ofFoswithDrd1andDrd2in three DS subregions.Fosexpression was neither subregion nor cell-type specific (52.5 and 39.2% ofFosexpression colabeled withDrd1andDrd2, respectively). Finally, we found that DS injections of SCH23390 (C17H18ClNO), a D1-family receptor antagonist known to block cue-induced Fos induction, decreased incubated cue-induced methamphetamine seeking after prolonged withdrawal. Results demonstrate a critical role of DS in incubation of methamphetamine craving and that this incubation is associated with selective gene-expression alterations in cue-activated D1- and D2-expressing DS neurons.

Background Cue-induced methamphetamine craving increases after prolonged forced (experimenter-imposed) abstinence from the drug (incubation of methamphetamine craving). Here, we determined whether this incubation phenomenon would occur under conditions that promote voluntary (self-imposed) abstinence. We also determined the effect of the novel metabotropic glutamate receptor 2 positive allosteric modulator, AZD8529, on incubation of methamphetamine craving after forced or voluntary abstinence. Methods We trained rats to self-administer palatable food (6 sessions) and then to self-administer methamphetamine under two conditions: 12 sessions (9 hours/day) or 50 sessions (3 hours/day). We then assessed cue-induced methamphetamine seeking in extinction tests after 1 or 21 abstinence days. Between tests, the rats underwent either forced abstinence (no access to the food- or drug-paired levers) or voluntary abstinence (achieved via a discrete choice procedure between methamphetamine and palatable food; 20 trials per day) for 19 days. We also determined the effect of subcutaneous injections of AZD8529 (20 and 40 mg/kg) on cue-induced methamphetamine seeking 1 day or 21 days after forced or voluntary abstinence. Results Under both training and abstinence conditions, cue-induced methamphetamine seeking in the extinction tests was higher after 21 abstinence days than after 1 day (incubation of methamphetamine craving). AZD8529 decreased cue-induced methamphetamine seeking on day 21 but not day 1 of forced or voluntary abstinence. Conclusions We introduce a novel animal model to study incubation of drug craving and cue-induced drug seeking after prolonged voluntary abstinence, mimicking the human condition of relapse after successful contingency management treatment. Our data suggest that positive allosteric modulators of metabotropic glutamate receptor 2 should be considered for relapse prevention.

Drug addiction is characterized by persistent relapse vulnerability during abstinence. In abstinent drug users, relapse is often precipitated by re-exposure to environmental contexts that were previously associated with drug use. This clinical scenario is modeled in preclinical studies using the context-induced reinstatement procedure, which is based on the ABA renewal procedure. In these studies, context-induced reinstatement of drug seeking is reliably observed in laboratory animals that were trained to self-administer drugs abused by humans. In this review, we summarize neurobiological findings from preclinical studies that have focused on the role of corticostriatal circuits in context-induced reinstatement of heroin, cocaine, and alcohol seeking. We also discuss neurobiological similarities and differences in the corticostriatal mechanisms of context-induced reinstatement across these drug classes. We conclude by briefly discussing future directions in the study of context-induced relapse to drug seeking in rat models. Our main conclusion from the studies reviewed is that there are both similarities (accumbens shell, ventral hippocampus, and basolateral amygdala) and differences (medial prefrontal cortex and its projections to accumbens) in the neural mechanisms of context-induced reinstatement of cocaine, heroin, and alcohol seeking. This article is part of a Special Issue entitled SI:Addiction circuits.

We developed recently a binge-eating model in which female rats with a history of intermittent food restriction show binge-like palatable food consumption after 15 min exposure to the sight of the palatable food. This “frustration stress” manipulation also activates the hypothalamic–pituitary–adrenal stress axis. Here, we determined the role of the stress neurohormone corticotropin-releasing factor (CRF) in stress-induced binge eating in our model. We also assessed the role of CRF receptors in the bed nucleus of the stria terminalis (BNST), a brain region implicated in stress responses and stress-induced drug seeking, in stress-induced binge eating. We used four groups that were first exposed or not exposed to repeated intermittent cycles of regular chow food restriction during which they were also given intermittent access to high-caloric palatable food. On the test day, we either exposed or did not expose the rats to the sight of the palatable food for 15 min (frustration stress) before assessing food consumption for 2 h. We found that systemic injections of the CRF1 receptor antagonist R121919 (2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7 dipropylamino pyrazolo[1,5-a]pyrimidine) (10–20 mg/kg) and BNST (25–50 ng/side) or ventricular (1000 ng) injections of the nonselective CRF receptor antagonistd-Phe-CRF(12–41)decreased frustration stress-induced binge eating in rats with a history of food restriction. Frustration stress also increased Fos (a neuronal activity marker) expression in ventral and dorsal BNST. Results demonstrate a critical role of CRF receptors in BNST in stress-induced binge eating in our rat model. CRF1 receptor antagonists may represent a novel pharmacological treatment for bingeing-related eating disorders.

Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We previously developed a fluorescence-activated cell sorting (FACS)-based method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. However, this method requires pooling samples from many rats. We now describe a modified FACS-based method to characterize molecular alterations in Fos-expressing dorsal striatal neurons from a single rat using a multiplex pre-amplification strategy. Fos and NeuN (a neuronal marker) immunohistochemistry indicate that 5-6% of dorsal striatum neurons were activated 90 min after acute methamphetamine injections (5 mg/kg, i.p.) while less than 0.5% of neurons were activated by saline injections. We used FACS to separate NeuN-labeled neurons into Fos-positive and Fos-negative neurons and assessed mRNA expression using RT-qPCR from as little as five Fos-positive neurons. Methamphetamine induced 3-20-fold increases of immediate early genes arc, homer-2, c-fos, fosB, and its isoforms (ΔfosB and a novel isoform ΔfosB-2) in Fos-positive but not Fos-negative neurons. Immediate early gene mRNA induction was 10-fold lower or absent when assessed in unsorted samples from single dorsal striatum homogenates. Our modified method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models. Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We here report an improved method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. We used FACS along with targeted PCR pre-amplification to assess acute methamphetamine-induced gene expression from as few as 5 Fos-expressing neurons from a single rat dorsal striatum. Methamphetamine induced 3-20-fold increases of immediate early genes (IEGs) in Fos-positive but not Fos-negative neurons. Targeted PCR pre-amplification makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models. Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We here report an improved method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. We used FACS along with targeted PCR pre-amplification to assess acute methamphetamine- induced gene expression from as few as 5 Fos-expressing neurons from a single rat dorsal striatum. Methamphetamine induced 3-20-fold increases of immediate early genes (IEGs) in Fos-positive but not Fos-negative neurons. Targeted PCR pre-amplification makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models.

Relapse to maladaptive eating habits during dieting is often provoked by stress. Recently, we identified a role of dorsal medial prefrontal cortex (mPFC) neurons in stress-induced reinstatement of palatable food seeking in male rats. It is unknown whether endogenous neural activity in dorsal mPFC drives stress-induced reinstatement in female rats. Here, we used an optogenetic approach, in which female rats received bilateral dorsal mPFC microinjections of viral constructs coding light-sensitive eNpHR3.0–eYFP or control eYFP protein and intracranial fiber optic implants. Rats were food restricted and trained to lever press for palatable food pellets. Subsequently, pellets were removed, and lever pressing was extinguished; then the effect of bilateral dorsal mPFC light delivery on reinstatement of food seeking was assessed after injections of the pharmacological stressor yohimbine (an α-2 andrenoceptor antagonist) or pellet priming, a manipulation known to provoke food seeking in hungry rats. Dorsal mPFC light delivery attenuated yohimbine-induced reinstatement of food seeking in eNpHR3.0-injected but not eYFP-injected rats. This optical manipulation had no effect on pellet-priming-induced reinstatement or ongoing food-reinforced responding. Dorsal mPFC light delivery attenuated yohimbine-induced Fos immunoreactivity and disrupted neural activity duringin vivoelectrophysiological recording in awake rats. Optical stimulation caused significant outward currents and blocked electrically evoked action potentials in eNpHR3.0-injected but not eYFP-injected mPFC hemispheres. Light delivery alone caused no significant inflammatory response in mPFC. These findings indicate that intracranial light delivery in eNpHR3.0 rats disrupts endogenous dorsal mPFC neural activity that plays a role in stress-induced relapse to food seeking in female rats.

In humans, exposure to environmental contexts previously associated with drug intake often provokes relapse to drug use, but the mechanisms mediating this relapse are unknown. Based on early studies by Bouton & Bolles on context-induced ‘renewal’ of learned behaviours, we developed a procedure to study context-induced relapse to drug seeking. In this procedure, rats are first trained to self-administer drug in one context. Next, drug-reinforced lever responding is extinguished in a different (non-drug) context. Subsequently, context-induced reinstatement of drug seeking is assessed by re-exposing rats to the drug-associated context. Using variations of this procedure, we and others reported reliable context-induced reinstatement in rats with a history of heroin, cocaine, heroin–cocaine combination, alcohol and nicotine self-administration. Here, we first discuss potential psychological mechanisms of context-induced reinstatement, including excitatory and inhibitory Pavlovian conditioning, and occasion setting. We then summarize results from pharmacological and neuroanatomical studies on the role of several neurotransmitter systems (dopamine, glutamate, serotonin and opioids) and brain areas (ventral tegmental area, accumbens shell, dorsal striatum, basolateral amygdala, prefrontal cortex, dorsal hippocampus and lateral hypothalamus) in context-induced reinstatement. We conclude by discussing the clinical implications of rat studies on context-induced reinstatement of drug seeking.