Different Effects of Peer Sex on Operant Responding for Social Interaction and Striatal Dopamine Activity.

When rats are given discrete choices between social interactions with a peer and opioid or psychostimulant drugs, they choose social interaction, even after extensive drug self-administration experience. Studies show that like drug and nondrug food reinforcers, social interaction is an operant reinforcer and induces dopamine release. However, these studies were conducted with same-sex peers. We examined if peer sex influences operant social interaction and the role of estrous cycle and striatal dopamine in same- versus opposite-sex social interaction. We trained male and female rats (n = 13 responders/12 peers) to lever-press (fixed-ratio 1 [FR1] schedule) for 15 s access to a same- or opposite-sex peer for 16 d (8 d/sex) while tracking females' estrous cycle. Next, we transfected GRAB-DA2m and implanted optic fibers into nucleus accumbens (NAc) core and dorsomedial striatum (DMS). We then retrained the rats for 15 s social interaction (FR1 schedule) for 16 d (8 d/sex) and recorded striatal dopamine during operant responding for a peer for 8 d (4 d/sex). Finally, we assessed economic demand by manipulating FR requirements for a peer (10 d/sex). In male, but not female rats, operant responding was higher for the opposite-sex peer. Female's estrous cycle fluctuations had no effect on operant social interaction. Striatal dopamine signals for operant social interaction were dependent on the peer's sex and striatal region (NAc core vs DMS). Results indicate that estrous cycle fluctuations did not influence operant social interaction and that NAc core and DMS dopamine activity reflect sex-dependent features of volitional social interaction.

Improving translation of animal models of addiction and relapse by reverse translation.

Critical features of human addiction are increasingly being incorporated into complementary animal models, including escalation of drug intake, punished drug seeking and taking, intermittent drug access, choice between drug and non-drug rewards, and assessment of individual differences based on criteria in the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). Combined with new technologies, these models advanced our understanding of brain mechanisms of drug self-administration and relapse, but these mechanistic gains have not led to improvements in addiction treatment. This problem is not unique to addiction neuroscience, but it is an increasing source of disappointment and calls to regroup. Here we first summarize behavioural and neurobiological results from the animal models mentioned above. We then propose a reverse translational approach, whose goal is to develop models that mimic successful treatments: opioid agonist maintenance, contingency management and the community-reinforcement approach. These reverse-translated 'treatments' may provide an ecologically relevant platform from which to discover new circuits, test new medications and improve translation.

Incubation of palatable food craving is associated with brain-wide neuronal activation in mice.

Studies using rodent models have shown that relapse to drug or food seeking increases progressively during abstinence, a behavioral phenomenon termed "incubation of craving." Mechanistic studies of incubation of craving have focused on specific neurobiological targets within preselected brain areas. Recent methodological advances in whole-brain immunohistochemistry, clearing, and imaging now allow unbiased brain-wide cellular resolution mapping of regions and circuits engaged during learned behaviors. However, these whole-brain imaging approaches were developed for mouse brains, while incubation of drug craving has primarily been studied in rats, and incubation of food craving has not been demonstrated in mice. Here, we established a mouse model of incubation of palatable food craving and examined food reward seeking after 1, 15, and 60 abstinence days. We then used the neuronal activity marker Fos with intact-brain mapping procedures to identify corresponding patterns of brain-wide activation. Relapse to food seeking was significantly higher after 60 abstinence days than after 1 or 15 days. Using unbiased ClearMap analysis, we identified increased activation of multiple brain regions, particularly corticostriatal structures, following 60 but not 1 or 15 abstinence days. We used orthogonal SMART2 analysis to confirm these findings within corticostriatal and thalamocortical subvolumes and applied expert-guided registration to investigate subdivision and layer-specific activation patterns. Overall, we 1) identified brain-wide activity patterns during incubation of food seeking using complementary analytical approaches and 2) provide a single-cell resolution whole-brain atlas that can be used to identify functional networks and global architecture underlying the incubation of food craving.

Sex Differences in Opioid and Psychostimulant Craving and Relapse: A Critical Review.

A widely held dogma in the preclinical addiction field is that females are more vulnerable than males to drug craving and relapse. Here, we first review clinical studies on sex differences in psychostimulant and opioid craving and relapse. Next, we review preclinical studies on sex differences in psychostimulant and opioid reinstatement of drug seeking after extinction of drug self-administration, and incubation of drug craving (time-dependent increase in drug seeking during abstinence). We also discuss ovarian hormones' role in relapse and craving in humans and animal models and speculate on brain mechanisms underlying their role in cocaine craving and relapse in rodent models. Finally, we discuss imaging studies on brain responses to cocaine cues and stress in men and women.The results of the clinical studies reviewed do not appear to support the notion that women are more vulnerable to psychostimulant and opioid craving and relapse. However, this conclusion is tentative because most of the studies reviewed were correlational, not sufficiently powered, and not a priori designed to detect sex differences. Additionally, imaging studies suggest sex differences in brain responses to cocaine cues and stress. The results of the preclinical studies reviewed provide evidence for sex differences in stress-induced reinstatement and incubation of cocaine craving but not cue- or cocaine-induced reinstatement of cocaine seeking. These sex differences are modulated in part by ovarian hormones. In contrast, the available data do not support the notion of sex differences in craving and relapse/reinstatement for methamphetamine or opioids in rodent models. SIGNIFICANCE STATEMENT: This systematic review summarizes clinical and preclinical studies on sex differences in psychostimulant and opioid craving and relapse. Results of the clinical studies reviewed do not appear to support the notion that women are more vulnerable to psychostimulant and opioid craving and relapse. Results of preclinical studies reviewed provide evidence for sex differences in reinstatement and incubation of cocaine seeking but not for reinstatement or incubation of methamphetamine or opioid seeking.

Role of Piriform Cortex and Its Afferent Projections in Relapse to Fentanyl Seeking after Food Choice-Induced Voluntary Abstinence.

We previously demonstrated a role of piriform cortex (Pir) in relapse to fentanyl seeking after food choice-induced voluntary abstinence. Here, we used this model to further study the role of Pir and its afferent projections in fentanyl relapse. We trained male and female rats to self-administer palatable food pellets for 6 d (6 h/day) and fentanyl (2.5 µg/kg/infusion, i.v.) for 12 d (6 h/day). We assessed relapse to fentanyl seeking after 12 voluntary abstinence sessions, achieved through a discrete choice procedure between fentanyl and palatable food (20 trials/session). We determined projection-specific activation of Pir afferents during fentanyl relapse with Fos plus the retrograde tracer cholera toxin B (injected into Pir). Fentanyl relapse was associated with increased Fos expression in anterior insular cortex (AI) and prelimbic cortex (PL) neurons projecting to Pir. We next used an anatomical disconnection procedure to determine the causal role of these two projections (AI→Pir and PL→Pir) in fentanyl relapse. Contralateral but not ipsilateral disconnection of AI→Pir projections decreased fentanyl relapse but not reacquisition of fentanyl self-administration. In contrast, contralateral but not ipsilateral disconnection of PL→Pir projections modestly decreased reacquisition but not relapse. Fluorescence-activated cell sorting and quantitative PCR data showed molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse. Finally, we found minimal or no sex differences in fentanyl self-administration, fentanyl versus food choice, and fentanyl relapse. Our results indicate that AI→Pir and PL→Pir projections play dissociable roles in nonreinforced relapse to fentanyl seeking versus reacquisition of fentanyl self-administration after food choice-induced voluntary abstinence.SIGNIFICANCE STATEMENT We previously showed a role of Pir in fentanyl relapse after food choice-induced voluntary abstinence in rats, a procedure mimicking human abstinence or a significant reduction in drug self-administration because of the availability of alternative nondrug rewards. Here, we aimed to further characterize the role of Pir in fentanyl relapse by investigating the role of Pir afferent projections and analyzing molecular changes in relapse-activated Pir neurons. We identified dissociable roles of two Pir afferent projections (AI→Pir and PL→Pir) in relapse to fentanyl seeking versus reacquisition of fentanyl self-administration after voluntary abstinence. We also characterized molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse.

Effect of Selective Lesions of Nucleus Accumbens µ-Opioid Receptor-Expressing Cells on Heroin Self-Administration in Male and Female Rats: A Study with Novel Oprm1-Cre Knock-in Rats.

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

Orbitofrontal cortex and dorsal striatum functional connectivity predicts incubation of opioid craving after voluntary abstinence.

We recently introduced a rat model of incubation of opioid craving after voluntary abstinence induced by negative consequences of drug seeking. Here, we used resting-state functional MRI to determine whether longitudinal functional connectivity changes in orbitofrontal cortex (OFC) circuits predict incubation of opioid craving after voluntary abstinence. We trained rats to self-administer for 14 d either intravenous oxycodone or palatable food. After 3 d, we introduced an electric barrier for 12 d that caused cessation of reward self-administration. We tested the rats for oxycodone or food seeking under extinction conditions immediately after self-administration training (early abstinence) and after electric barrier exposure (late abstinence). We imaged their brains before self-administration and during early and late abstinence. We analyzed changes in OFC functional connectivity induced by reward self-administration and electric barrier-induced abstinence. Oxycodone seeking was greater during late than early abstinence (incubation of oxycodone craving). Oxycodone self-administration experience increased OFC functional connectivity with dorsal striatum and related circuits that was positively correlated with incubated oxycodone seeking. In contrast, electric barrier-induced abstinence decreased OFC functional connectivity with dorsal striatum and related circuits that was negatively correlated with incubated oxycodone seeking. Food seeking was greater during early than late abstinence (abatement of food craving). Food self-administration experience and electric barrier-induced abstinence decreased or maintained functional connectivity in these circuits that were not correlated with abated food seeking. Opposing functional connectivity changes in OFC with dorsal striatum and related circuits induced by opioid self-administration versus voluntary abstinence predicted individual differences in incubation of opioid craving.

Animal Models of Drug Relapse and Craving after Voluntary Abstinence: A Review.

Relapse to drug use during abstinence is a defining feature of addiction. During the last several decades, this clinical scenario has been studied at the preclinical level using classic relapse/reinstatement models in which drug seeking is assessed after experimenter-imposed home-cage forced abstinence or extinction of the drug-reinforced responding in the self-administration chambers. To date, however, results from studies using rat relapse/reinstatement models have yet to result in Food and Drug Administration-approved medications for relapse prevention. The reasons for this state of affairs are complex and multifaceted, but one potential reason is that, in humans, abstinence is often self-imposed or voluntary and occurs either because the negative consequences of drug use outweigh the drug's rewarding effects or because of the availability of nondrug alternative rewards that are chosen over the drug. Based on these considerations, we and others have recently developed rat models of relapse after voluntary abstinence, achieved either by introducing adverse consequences to drug taking (punishment) or seeking (electric barrier) or by providing mutually exclusive choices between the self-administered drug and nondrug rewards (palatable food or social interaction). In this review, we provide an overview of these translationally relevant relapse models and discuss recent neuropharmacological findings from studies using these models. We also discuss sex as a biological variable, future directions, and clinical implications of results from relapse studies using voluntary abstinence models. Our main conclusion is that the neuropharmacological mechanisms controlling relapse to drug seeking after voluntary abstinence are often different from the mechanisms controlling relapse after home-cage forced abstinence or reinstatement after extinction. SIGNIFICANCE STATEMENT: This review describes recently developed rat models of relapse after voluntary abstinence, achieved either by introducing adverse consequences to drug taking or seeking or by providing mutually exclusive choices between the self-administered drug and nondrug rewards. This review discusses recent neuropharmacological findings from studies using these models and discusses future directions and clinical implications.

The Protective Effect of Social Reward on Opioid and Psychostimulant Reward and Relapse: Behavior, Pharmacology, and Brain Regions.

Until recently, most modern neuroscience research on addiction using animal models did not incorporate manipulations of social factors. Social factors play a critical role in human addiction: social isolation and exclusion can promote drug use and relapse, while social connections and inclusion tend to be protective. Here, we discuss the state of the literature on social factors in animal models of opioid and psychostimulant preference, self-administration, and relapse. We first summarize results from rodent studies on behavioral, pharmacological, and circuit mechanisms of the protective effect of traditional experimenter-controlled social interaction procedures on opioid and psychostimulant conditioned place preference, self-administration, and relapse. Next, we summarize behavioral and brain-mechanism results from studies using newer operant social-interaction procedures that inhibit opioid and psychostimulant self-administration and relapse. We conclude by discussing how the reviewed studies point to future directions for the addiction field and other neuroscience and psychiatric fields, and their implications for mechanistic understanding of addiction and development of new treatments.SIGNIFICANCE STATEMENT In this review, we propose that incorporating social factors into modern neuroscience research on addiction could improve mechanistic accounts of addiction and help close gaps in translating discovery to treatment. We first summarize rodent studies on behavioral, pharmacological, and circuit mechanisms of the protective effect of both traditional experimenter-controlled and newer operant social-interaction procedures. We then discuss potential future directions and clinical implications.

Abstinence-dependent dissociable central amygdala microcircuits control drug craving.

We recently reported that social choice-induced voluntary abstinence prevents incubation of methamphetamine craving in rats. This inhibitory effect was associated with activation of protein kinase-Cδ (PKCδ)-expressing neurons in central amygdala lateral division (CeL). In contrast, incubation of craving after forced abstinence was associated with activation of CeL-expressing somatostatin (SOM) neurons. Here we determined the causal role of CeL PKCδ and SOM in incubation using short-hairpin RNAs against PKCδ or SOM that we developed and validated. We injected two groups with shPKCδ or shCtrlPKCδ into CeL and trained them to lever press for social interaction (6 d) and then for methamphetamine infusions (12 d). We injected two other groups with shSOM or shCtrlSOM into CeL and trained them to lever press for methamphetamine infusions (12 d). We then assessed relapse to methamphetamine seeking after 1 and 15 abstinence days. Between tests, the rats underwent either social choice-induced abstinence (shPKCδ groups) or homecage forced abstinence (shSOM groups). After test day 15, we assessed PKCδ and SOM, Fos, and double-labeled expression in CeL and central amygdala medial division (CeM). shPKCδ CeL injections decreased Fos in CeL PKCδ-expressing neurons, increased Fos in CeM output neurons, and reversed the inhibitory effect of social choice-induced abstinence on incubated drug seeking on day 15. In contrast, shSOM CeL injections decreased Fos in CeL SOM-expressing neurons, decreased Fos in CeM output neurons, and decreased incubated drug seeking after 15 forced abstinence days. Our results identify dissociable central amygdala mechanisms of abstinence-dependent expression or inhibition of incubation of craving.

Pharmacological and behavioral divergence of ketamine enantiomers: implications for abuse liability.

Ketamine, a racemic mixture of (S)-ketamine and (R)-ketamine enantiomers, has been used as an anesthetic, analgesic and more recently, as an antidepressant. However, ketamine has known abuse liability (the tendency of a drug to be used in non-medical situations due to its psychoactive effects), which raises concerns for its therapeutic use. (S)-ketamine was recently approved by the United States' FDA for treatment-resistant depression. Recent studies showed that (R)-ketamine has greater efficacy than (S)-ketamine in preclinical models of depression, but its clinical antidepressant efficacy has not been established. The behavioral effects of racemic ketamine have been studied extensively in preclinical models predictive of abuse liability in humans (self-administration and conditioned place preference [CPP]). In contrast, the behavioral effects of each enantiomer in these models are unknown. We show here that in the intravenous drug self-administration model, the gold standard procedure to assess potential abuse liability of drugs in humans, rats self-administered (S)-ketamine but not (R)-ketamine. Subanesthetic, antidepressant-like doses of (S)-ketamine, but not of (R)-ketamine, induced locomotor activity (in an opioid receptor-dependent manner), induced psychomotor sensitization, induced CPP in mice, and selectively increased metabolic activity and dopamine tone in medial prefrontal cortex (mPFC) of rats. Pharmacological screening across thousands of human proteins and at biological targets known to interact with ketamine yielded divergent binding and functional enantiomer profiles, including selective mu and kappa opioid receptor activation by (S)-ketamine in mPFC. Our results demonstrate divergence in the pharmacological, functional, and behavioral effects of ketamine enantiomers, and suggest that racemic ketamine's abuse liability in humans is primarily due to the pharmacological effects of its (S)-enantiomer.

Role of Projections between Piriform Cortex and Orbitofrontal Cortex in Relapse to Fentanyl Seeking after Palatable Food Choice-Induced Voluntary Abstinence.

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 STATEMENT There 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.

Time to connect: bringing social context into addiction neuroscience.

Research on the neural substrates of drug reward, withdrawal and relapse has yet to be translated into significant advances in the treatment of addiction. One potential reason is that this research has not captured a common feature of human addiction: progressive social exclusion and marginalization. We propose that research aimed at understanding the neural mechanisms that link these processes to drug seeking and drug taking would help to make addiction neuroscience research more clinically relevant.

Fos-expressing neuronal ensemble in rat ventromedial prefrontal cortex encodes cocaine seeking but not food seeking in rats.

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 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 nonreinforced food- or cocaine-seeking test to induce Fos and ß-gal in neuronal ensembles associated with food or cocaine seeking, respectively and infused Daun02 into vmPFC to ablate the ß-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.

Animal Models of (or for) Aggression Reward, Addiction, and Relapse: Behavior and Circuits.

Inappropriate and pathological aggression plays a leading role in the suffering and death of millions of people, and further places an untenable strain on the caregivers and families of those afflicted. In some cases, such as addictive drugs, aggression can be highly rewarding (appetitive) and continually pursued despite short- and long-term negative consequences. Similarly, recidivism (relapse) rates for repeat violent offenders are as high as relapse rates for drug addicts. Appetitive aggression and relapse to aggression seeking can be modeled in mice studies using conditioned place preference and self-administration procedures followed by a period of abstinence and subsequent tests for relapse to aggression preference and aggression seeking. These procedures allow for the study of the mechanisms that control the appetitive versus the consummatory (attack) phases of aggressive behavior. In this review, we first discuss the behavioral procedures developed to probe appetitive aggression in mouse models, spanning from Pavlovian to operant tasks, and we also describe the recently proposed phenomenon of "aggression addiction." Next, we discuss the pharmacological and circuit mechanisms of aggression conditioned place preference and aggression self-administration, seeking, and relapse, highlighting mechanistic congruence and divergence between appetitive and consummatory phases of aggression. We conclude by discussing clinical implications of the studies reviewed.

Nucleus Accumbens Drd1-Expressing Neurons Control Aggression Self-Administration and Aggression Seeking in Mice.

We recently developed a mouse model of appetitive operant aggression and reported that adult male outbred CD-1 mice lever-press for the opportunity to attack subordinate male mice and relapse to aggression seeking during abstinence. Here we studied the role of nucleus accumbens (NAc) dopamine receptor (Drd)1- and Drd2-expressing neurons in aggression self-administration and aggression seeking. We trained CD-1 mice to self-administer intruders (9 d, 12 trials/d) and tested them for aggression self-administration and aggression seeking on abstinence Day 1. We used immunohistochemistry and in situ hybridization to measure the neuronal activity marker Fos in the NAc, and cell-type-specific colocalization of Fos with Drd1- and Drd2-expressing neurons. To test the causal role of Drd1- and Drd2-expressing neurons, we validated a transgenic hybrid breeding strategy crossing inbred Drd1-Cre and Drd2-Cre transgenic mice with outbred CD-1 mice and used cell-type-specific Cre-DREADD (hM4Di) to inhibit NAc Drd1- and Drd2-expressing neuron activity. We found that aggression self-administration and aggression seeking induced higher Fos expression in NAc shell than in core, that Fos colocalized with Drd1 and Drd2 in both subregions, and that chemogenetic inhibition of Drd1-, but not Drd2-, expressing neurons decreased aggression self-administration and aggression seeking. Results indicate a cell-type-specific role of Drd1-expressing neurons that is critical for both aggression self-administration and aggression seeking. Our study also validates a simple breeding strategy between outbred CD-1 mice and inbred C57-based Cre lines that can be used to study cell-type and circuit mechanisms of aggression reward and relapse.SIGNIFICANCE STATEMENT Aggression is often comorbid with neuropsychiatric diseases, including drug addiction. One form, appetitive aggression, exhibits symptomatology that mimics that of drug addiction and is hypothesized to be due to dysregulation of addiction-related reward circuits. However, our mechanistic understanding of the circuitry modulating appetitive operant aggression is limited. Here we used a novel mouse model of aggression self-administration and relapse, in combination with immunohistochemistry, in situ hybridization, and chemogenetic manipulations to examine how cell types in the nucleus accumbens are recruited for, and control, operant aggression self-administration and aggression seeking on abstinence Day 1. We found that one population, dopamine receptor 1-expressing neurons, act as a critical modulator of operant aggression reward and aggression seeking.

Separate vmPFC Ensembles Control Cocaine Self-Administration Versus Extinction in Rats.

Recent studies suggest that the ventral medial prefrontal cortex (vmPFC) encodes both operant drug self-administration and extinction memories. Here, we examined whether these opposing memories are encoded by distinct neuronal ensembles within the vmPFC with different outputs to the nucleus accumbens (NAc) in male and female rats. Using cocaine self-administration (3 h/d for 14 d) and extinction procedures, we demonstrated that vmPFC was similarly activated (indexed by Fos) during cocaine-seeking tests after 0 (no-extinction) or 7 extinction sessions. Selective Daun02 lesioning of the self-administration ensemble (no-extinction) decreased cocaine seeking, whereas Daun02 lesioning of the extinction ensemble increased cocaine seeking. Retrograde tracing with fluorescent cholera toxin subunit B injected into NAc combined with Fos colabeling in vmPFC indicated that vmPFC self-administration ensembles project to NAc core while extinction ensembles project to NAc shell. Functional disconnection experiments (Daun02 lesioning of vmPFC and acute dopamine D1-receptor blockade with SCH39166 in NAc core or shell) confirm that vmPFC ensembles interact with NAc core versus shell to play dissociable roles in cocaine self-administration versus extinction, respectively. Our results demonstrate that neuronal ensembles mediating cocaine self-administration and extinction comingle in vmPFC but have distinct outputs to the NAc core and shell that promote or inhibit cocaine seeking.SIGNIFICANCE STATEMENT Neuronal ensembles within the vmPFC have recently been shown to play a role in self-administration and extinction of food seeking. Here, we used the Daun02 chemogenetic inactivation procedure, which allows selective inhibition of neuronal ensembles identified by the activity marker Fos, to demonstrate that different ensembles for cocaine self-administration and extinction memories coexist in the ventral mPFC and interact with distinct subregions of the nucleus accumbens.

Opposite Effects of Basolateral Amygdala Inactivation on Context-Induced Relapse to Cocaine Seeking after Extinction versus Punishment.

Studies using the renewal procedure showed that basolateral amygdala (BLA) inactivation inhibits context-induced relapse to cocaine-seeking after extinction. Here, we determined whether BLA inactivation would also inhibit context-induced relapse after drug-reinforced responding is suppressed by punishment, an animal model of human relapse after self-imposed abstinence due to adverse consequences of drug use. We also determined the effect of central amygdala (CeA) inactivation on context-induced relapse.We trained rats to self-administer cocaine for 12 d (6 h/d) in Context A and then exposed them to either extinction or punishment training for 8 d in Context B. During punishment, 50% of cocaine-reinforced lever-presses produced an aversive footshock of increasing intensity (0.1-0.5 or 0.7 mA). We then tested the rats for relapse to cocaine seeking in the absence of cocaine or shock in Contexts A and B after BLA or CeA injections of vehicle or GABA agonists (muscimol-baclofen). We then retrained the rats for cocaine self-administration in Context A, repunished or re-extinguished lever pressing in Context B, and retested for relapse after BLA or CeA inactivation.BLA or CeA inactivation decreased context-induced relapse in Context A after extinction in Context B. BLA, but not CeA, inactivation increased context-induced relapse in Context A after punishment in Context B. BLA or CeA inactivation provoked relapse in Context B after punishment but not extinction. Results demonstrate that amygdala's role in relapse depends on the method used to achieve abstinence and highlights the importance of studying relapse under abstinence conditions that more closely mimic the human condition.SIGNIFICANCE STATEMENT Relapse to drug use during abstinence is often provoked by re-exposure to the drug self-administration environment or context. Studies using the established extinction-reinstatement rodent model of drug relapse have shown that inactivation of the basolateral amygdala inhibits context-induced drug relapse after extinction of the drug-reinforced responding. Here, we determined whether basolateral amygdala inactivation would also inhibit relapse after drug-reinforced responding is suppressed by punishment, a model of human relapse after self-imposed abstinence. Unexpectedly, we found that basolateral amygdala inactivation had opposite effects on relapse provoked by re-exposure to the drug self-administration environment after extinction versus punishment. Our results demonstrate that depending on the historical conditions that lead to abstinence, amygdala activity can either promote or inhibit relapse.

Role of Anterior Intralaminar Nuclei of Thalamus Projections to Dorsomedial Striatum in Incubation of Methamphetamine Craving.

Relapse to methamphetamine (Meth) seeking progressively increases after withdrawal from drug self-administration (incubation of Meth craving). We previously demonstrated a role of dorsomedial striatum (DMS) dopamine D1 receptors (D1Rs) in this incubation. Here, we studied the role of afferent glutamatergic projections into the DMS and local D1R-glutamate interaction in this incubation in male rats. We first measured projection-specific activation on day 30 relapse test by using cholera toxin b (retrograde tracer) + Fos (activity marker) double-labeling in projection areas. Next, we determined the effect of pharmacological reversible inactivation of lateral or medial anterior intralaminar nuclei of thalamus (AIT-L or AIT-M) on incubated Meth seeking on withdrawal day 30. We then used an anatomical asymmetrical disconnection procedure to determine whether an interaction between AIT-L→DMS glutamatergic projections and postsynaptic DMS D1Rs contributes to incubated Meth seeking. We also determined the effect of unilateral inactivation of AIT-L and D1R blockade of DMS on incubated Meth seeking, and the effect of contralateral disconnection of AIT-L→DMS projections on nonincubated Meth seeking on withdrawal day 1. Incubated Meth seeking was associated with selective activation of AIT→DMS projections; other glutamatergic projections to DMS were not activated. AIT-L (but not AIT-M) inactivation or anatomical disconnection of AIT-L→DMS projections decreased incubated Meth seeking. Unilateral inactivation of AIT-L or D1R blockade of the DMS had no effect on incubated Meth craving, and contralateral disconnection of AIT-L→DMS projections had no effect on nonincubated Meth seeking. Our results identify a novel role of AIT-L and AIT-L→DMS glutamatergic projections in incubation of drug craving and drug seeking.SIGNIFICANCE STATEMENT Methamphetamine seeking progressively increases after withdrawal from drug self-administration, a phenomenon termed incubation of methamphetamine craving. We previously found that D1R-mediated dopamine transmission in the dorsomedial striatum plays a critical role in this incubation phenomenon. Here, we used neuroanatomical and neuropharmacological methods in rats to demonstrate that an interaction between the glutamatergic projection from the lateral anterior intralaminar nuclei of the thalamus to the dorsomedial striatum and local dopamine D1 receptors plays a critical role in relapse to methamphetamine seeking after prolonged withdrawal. Our study identified a novel motivation-related thalamostriatal projection critical to relapse to drug seeking.

Role of mu, but not delta or kappa, opioid receptors in context-induced reinstatement of oxycodone seeking.

Relapse to non-medical use of prescription opioids often occurs after exposure to places previously associated with drug use. Here, we describe a rat model of context-induced reinstatement of oxycodone seeking after repeated cycles of drug self-administration and extinction-induced abstinence. We also determined the role of mu, delta and kappa opioid receptors (MOR, DOR, KOR) in this reinstatement. We trained rats to self-administer oxycodone for 6 h/day in context A; lever pressing was paired with a discrete cue. Next, we extinguished the lever pressing in the presence of the discrete cue in context B and then tested the rats for reinstatement of oxycodone seeking in both contexts. We retrained rats to self-administer oxycodone in context A, re-extinguished their lever pressing in context B and retested them for reinstatement in both contexts. Prior to testing, we injected the rats with vehicle or antagonists of MOR (naltrexone; 0.5 or 1.0 mg/kg), DOR (naltrindole; 7.5 or 15 mg/kg) or KOR (LY2456302; 5 or 10 mg/kg). We also tested the effect of naltrexone, naltrindole and LY2456302 on oxycodone self-administration under fixed-ratio-1 (FR1) and progressive ratio (PR) reinforcement schedules. We observed context-induced reinstatement of oxycodone seeking after repeated cycles of drug self-administration and extinction. Naltrexone, but not naltrindole or LY2456302, injections decreased this reinstatement. Additionally, naltrexone increased oxycodone self-administration under the FR1 schedule and decreased oxycodone self-administration under the PR schedule; naltrindole and LY2456302 were ineffective. Results demonstrate a critical role of MOR, but not DOR or KOR, in context-induced reinstatement of oxycodone seeking and oxycodone self-administration.