Epigenetic function during heroin self-administration controls future relapse-associated behavior in a cell type-specific manner.

Opioid use produces enduring associations between drug reinforcement/euphoria and discreet or diffuse cues in the drug-taking environment. These powerful associations can trigger relapse in individuals recovering from opioid use disorder (OUD). Here, we sought to determine whether the epigenetic enzyme, histone deacetylase 5 (HDAC5), regulates relapse-associated behavior in an animal model of OUD. We examined the effects of nucleus accumbens (NAc) HDAC5 on both heroin- and sucrose-seeking behaviors using operant self-administration paradigms. We utilized cre-dependent viral-mediated approaches to investigate the cell-type-specific effects of HDAC5 on heroin-seeking behavior, gene expression, and medium spiny neuron (MSN) cell and synaptic physiology. We found that NAc HDAC5 functions during the acquisition phase of heroin self-administration to limit future relapse-associated behavior. Moreover, overexpressing HDAC5 in the NAc suppressed context-associated and reinstated heroin-seeking behaviors, but it did not alter sucrose seeking. We also found that HDAC5 functions within dopamine D1 receptor-expressing MSNs to suppress cue-induced heroin seeking, and within dopamine D2 receptor-expressing MSNs to suppress drug-primed heroin seeking. Assessing cell-type-specific transcriptomics, we found that HDAC5 reduced expression of multiple ion transport genes in both D1- and D2-MSNs. Consistent with this observation, HDAC5 also produced firing rate depression in both MSN classes. These findings revealed roles for HDAC5 during active heroin use in both D1- and D2-MSNs to limit distinct triggers of drug-seeking behavior. Together, our results suggest that HDAC5 might limit relapse vulnerability through regulation of ion channel gene expression and suppression of MSN firing rates during active heroin use.

Serotonin Signaling in Hippocampus during Initial Cocaine Abstinence Drives Persistent Drug Seeking.

The initiation of abstinence after chronic drug self-administration is stressful. Cocaine-seeking behavior on the first day of the absence of the expected drug (Extinction Day 1, ED1) is reduced by blocking 5-HT signaling in dorsal hippocampal cornu ammonis 1 (CA1) in both male and female rats. We hypothesized that the experience of ED1 can substantially influence later relapse behavior and that dorsal raphe (DR) serotonin (5-HT) input to CA1 may be involved. We inhibited 5-HT1A/1B receptors (WAY-100635 plus GR-127935), or DR input (chemogenetics), in CA1 on ED1 to test the role of this pathway on cocaine-seeking persistence 2 weeks later. We also inhibited 5-HT1A or 5-HT1B receptors in CA1 during conditioned place preference (CPP) for cocaine, to examine mechanisms involved in the persistent effects of ED1 manipulations. Inhibition of DR inputs, or 5-HT1A/1B signaling, in CA1 decreased drug seeking on ED1 and decreased cocaine seeking 2 weeks later revealing that 5-HT signaling in CA1 during ED1 contributes to persistent drug seeking during abstinence. In addition, 5-HT1B antagonism alone transiently decreased drug-associated memory performance when given prior to a CPP test, whereas similar antagonism of 5-HT1A alone had no such effect but blocked CPP retrieval on a test 24 h later. These CPP findings are consistent with prior work showing that DR inputs to CA1 augment recall of the drug-associated context and drug seeking via 5-HT1B receptors and prevent consolidation of the updated nondrug context via 5-HT1A receptors. Thus, treatments that modulate 5-HT-dependent memory mechanisms in CA1 during initial abstinence may facilitate later maintenance of abstinence.

Vagus Nerve Stimulation (VNS) Modulates Synaptic Plasticity in the Infralimbic Cortex via Trk-B Receptor Activation to Reduce Drug-Seeking in Male Rats.

Drugs of abuse cause changes in the prefrontal cortex (PFC) and associated regions that impair inhibitory control over drug-seeking. Breaking the contingencies between drug-associated cues and the delivery of the reward during extinction learning reduces relapse. Vagus nerve stimulation (VNS) has previously been shown to enhance extinction learning and reduce drug-seeking. Here we determined the effects of VNS-mediated release of brain-derived neurotrophic factor (BDNF) on extinction and cue-induced reinstatement in male rats trained to self-administer cocaine. Pairing 10 d of extinction training with VNS facilitated extinction and reduced drug-seeking behavior during reinstatement. Rats that received a single extinction session with VNS showed elevated BDNF levels in the medial PFC as determined via an enzyme-linked immunosorbent assay. Systemic blockade of tropomyosin receptor kinase B (TrkB) receptors during extinction, via the TrkB antagonist ANA-12, decreased the effects of VNS on extinction and reinstatement. Whole-cell recordings in brain slices showed that cocaine self-administration induced alterations in the ratio of AMPA and NMDA receptor-mediated currents in Layer 5 pyramidal neurons of the infralimbic cortex (IL). Pairing extinction with VNS reversed cocaine-induced changes in glutamatergic transmission by enhancing AMPAR currents, and this effect was blocked by ANA-12. Our study suggests that VNS consolidates the extinction of drug-seeking behavior by reversing drug-induced changes in synaptic AMPA receptors in the IL, and this effect is abolished by blocking TrkB receptors during extinction, highlighting a potential mechanism for the therapeutic effects of VNS in addiction.

Incubation of methamphetamine craving in punishment-resistant individuals is associated with activation of specific gene networks in the rat dorsal striatum.

Methamphetamine use disorder (MUD) is characterized by loss of control over compulsive drug use. Here, we used a self-administration (SA) model to investigate transcriptional changes associated with the development of early and late compulsivity during contingent footshocks. Punishment initially separated methamphetamine taking rats into always shock-resistant (ASR) rats that continued active lever pressing and shock-sensitive (SS) rats that reduced their lever pressing. At the end of the punishment phase, rats underwent 15 days of forced abstinence at the end of which they were re-introduced to the SA paradigm followed by SA plus contingent shocks. Interestingly, 36 percent of the initial SS rats developed delayed shock-resistance (DSR). Of translational relevance, ASR rats showed more incubation of methamphetamine craving than DSR and always sensitive (AS) rats. RNA sequencing revealed increased striatal Rab37 and Dipk2b mRNA levels that correlated with incubation of methamphetamine craving. Interestingly, Bdnf mRNA levels showed HDAC2-dependent decreased expression in the AS rats. The present SA paradigm should help to elucidate the molecular substrates of early and late addiction-like behaviors.

A disinhibitory microcircuit of the orbitofrontal cortex mediates cocaine preference in mice.

Both clinical and animal studies showed that the impaired functions of the orbitofrontal cortex (OFC) underlie the compulsive drug-seeking behavior of drug addiction. However, the functional changes of the microcircuit in the OFC and the underlying molecular mechanisms in drug addiction remain elusive, and little is known for whether microcircuits in the OFC contributed to drug addiction-related behaviors. Utilizing the cocaine-induced conditioned-place preference model, we found that the malfunction of the microcircuit led to disinhibition in the OFC after cocaine withdrawal. We further showed that enhanced Somatostatin-Parvalbumin (SST-PV) inhibitory synapse strength changed microcircuit function, and SST and PV inhibitory neurons showed opposite contributions to the drug addiction-related behavior of mice. Brevican of the perineuronal nets of PV neurons regulated SST-PV synapse strength, and the knockdown of Brevican alleviated cocaine preference. These results reveal a novel molecular mechanism of the regulation of microcircuit function and a novel circuit mechanism of the OFC in gating cocaine preference.

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.

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.

Prelimbic Cortex Activity during a Distress Tolerance Task Predicts Cocaine-Seeking Behavior in Male, But Not Female Rats.

Distress tolerance (DT) is defined as the ability to persist in challenging goal-directed behavior in the face of stress, and individuals with low DT exhibit heightened drug-seeking behavior. However, no preclinical studies have examined the neurobiology underlying this phenomenon. To assess this, in vivo electrophysiology was used in Long Evans male and female rats during a DT task to record neural activity in the prelimbic cortex (PrL), a brain region implicated in drug-seeking. Rats were first assessed for DT, defined as the amount of time elapsed before rats quit seeking reward in an increasingly difficult operant task. Subsequently, rats underwent 2 weeks of self-administration for either water/saline or cocaine for 6 h/day. Animals then began a 1 month period of experimenter-imposed abstinence to induce heightened drug-seeking behavior. On day 28 of abstinence, DT and neural activity were reassessed; and on day 30, cocaine-seeking behavior was examined under extinction. Males had significantly higher DT than females and exhibited significantly more phasic PrL activity during the DT task. Furthermore, in male rats with a history of cocaine, PrL activity shifted to track DT; and this change in activity significantly correlated with the change in DT. Additionally, male (but not female) rats with low DT after 28 d of abstinence had significantly heightened drug-seeking behavior. Finally, PrL activity during the DT task predicted cocaine-seeking behavior. Collectively, these data demonstrate an important role for the PrL in DT in males, and link this neural activity and behavior to drug-seeking, particularly in males.SIGNIFICANCE STATEMENT Distress tolerance (DT) is defined as the ability to persist in challenging goal-directed behavior in the face of stress, and individuals with low DT exhibit heightened drug-seeking. Here, we investigated the role of the prelimbic cortex (PrL) in DT and its relationship to cocaine-seeking in male and female rats. We found that males had significantly higher DT than females and exhibited significantly more PrL activity during the DT task. Furthermore, male (but not female) rats with low DT after 28 d of abstinence had significantly heightened drug-seeking behavior. Finally, PrL activity during the DT task predicted cocaine-seeking. These data demonstrate an important role for the PrL in DT and link this neural activity and behavior to drug-seeking in males.

Pan-striatal reduction in the expression of the astrocytic dopamine transporter precedes the development of dorsolateral striatum dopamine-dependent incentive heroin seeking habits.

The emergence of compulsive drug-seeking habits, a hallmark feature of substance use disorder, has been shown to be predicated on the engagement of dorsolateral striatal control over behaviour. This process involves the dopamine-dependent functional coupling of the anterior dorsolateral striatum (aDLS) with the nucleus accumbens core, but the mechanisms by which this coupling occurs have not been fully elucidated. The striatum is tiled by a syncytium of astrocytes that express the dopamine transporter (DAT), the level of which is altered in individuals with heroin use disorder. Astrocytes are therefore uniquely placed functionally to bridge dopamine-dependent mechanisms across the striatum. Here we tested the hypothesis that exposure to heroin influences the expression of DAT in striatal astrocytes across the striatum before the development of DLS-dependent incentive heroin seeking habits. Using Western-blot, qPCR, and RNAscope™, we measured DAT protein and mRNA levels in whole tissue, culture and in situ astrocytes from striatal territories of rats with a well-established cue-controlled heroin seeking habit and rats trained to respond for heroin or food under continuous reinforcement. Incentive heroin seeking habits were associated with a reduction in DAT protein levels in the anterior aDLS that was preceded by a heroin-induced reduction in DAT mRNA and protein in astrocytes across the striatum. Striatal astrocytes were also shown to be susceptible to direct dopamine- and opioid-induced downregulation of DAT expression. These results suggest that astrocytes may critically regulate the striatal dopaminergic adaptations that lead to the development of incentive heroin seeking habits.

Random interval schedule of reinforcement influences punishment resistance for cocaine in rats.

In an animal model of compulsive drug use, a subset of rats continues to self-administer cocaine despite footshock consequences and is considered punishment resistant. We recently found that punishment resistance is associated with habits that persist under conditions that typically encourage a transition to goal-directed control. Given that random ratio (RR) and random interval (RI) schedules of reinforcement influence whether responding is goal-directed or habitual, we investigated the influence of these schedules on punishment resistance for cocaine or food. Male and female Sprague Dawley rats were trained to self-administer either intravenous cocaine or food pellets on a seeking-taking chained schedule of reinforcement, with the seeking lever requiring completion of either an RR20 or RI60 schedule. Rats were then given four days of punishment testing with footshock administered at the completion of seeking on a random one-third of trials. For cocaine-trained rats, the RI60 schedule led to greater punishment resistance (i.e., more trials completed) than the RR20 schedule in males and females. For food-trained rats, the RI60 schedule led to greater punishment resistance (i.e., higher reward rates) than the RR20 schedule in female rats, although male rats showed punishment resistance on both RR20 and RI60 schedules. For both cocaine and food, we found that seeking responses were suppressed to a greater degree than reward rate with the RI60 schedule, whereas response rate and reward rate were equally suppressed with the RR20 schedule. This dissociation between punishment effects on reward rate and response rate with the RI60 schedule can be explained by the nonlinear relation between these variables on RI schedules, but it does not account for the enhanced resistance to punishment. Overall, the results show greater punishment resistance with the RI60 schedule as compared to the RR20 schedule, indicating that schedules of reinforcement are an influencing factor on resistance to negative consequences.

Working Memory Performance Predicts, but Does Not Reduce, Cocaine and Cannabinoid Seeking in Adult Male Rats.

BACKGROUND: Cognitive deficits reflecting impaired executive function are commonly associated with psychiatric disorders, including substance use. Cognitive training is proposed to improve treatment outcomes for these disorders by promoting neuroplasticity within the prefrontal cortex, enhancing executive control, and mitigating cognitive decline due to drug use. Additionally, brain derived neurotrophic factor (BDNF) can facilitate plasticity in the prefrontal cortex and reduce drug-seeking behaviors. We investigated whether working memory training could elevate BDNF levels in the prefrontal cortex and if this training would predict or protect against cocaine or cannabinoid seeking. METHODS: Adult male rats were trained to perform a "simple" or "complex" version of a delayed-match-to-sample working memory task. Rats then self-administered cocaine or the synthetic cannabinoid WIN55,212-2 and were tested for cued drug seeking during abstinence. Tissue from the prefrontal cortex and dorsal hippocampus was analyzed for BDNF protein expression. RESULTS: Training on the working memory task enhanced endogenous BDNF protein levels in the prelimbic prefrontal cortex but not the dorsal hippocampus. Working memory training did not impact self-administration of either drug but predicted the extent of WIN self-administration and cocaine seeking during abstinence. CONCLUSIONS: These results suggest that working memory training promotes endogenous BDNF but does not alter drug-seeking or drug-taking behavior. However, individual differences in cognitive performance before drug exposure may predict vulnerability to future drug use.

Microglia contribute to methamphetamine reinforcement and reflect persistent transcriptional and morphological adaptations to the drug.

Methamphetamine use disorder (MUD) is a chronic, relapsing disease that is characterized by repeated drug use despite negative consequences and for which there are currently no FDA-approved cessation therapeutics. Repeated methamphetamine (METH) use induces long-term gene expression changes in brain regions associated with reward processing and drug-seeking behavior, and recent evidence suggests that methamphetamine-induced neuroinflammation may also shape behavioral and molecular responses to the drug. Microglia, the resident immune cells in the brain, are principal drivers of neuroinflammatory responses and contribute to the pathophysiology of substance use disorders. Here, we investigated transcriptional and morphological changes in dorsal striatal microglia in response to methamphetamine-taking and during methamphetamine abstinence, as well as their functional contribution to drug-taking behavior. We show that methamphetamine self-administration induces transcriptional changes associated with protein folding, mRNA processing, immune signaling, and neurotransmission in dorsal striatal microglia. Importantly, many of these transcriptional changes persist through abstinence, a finding supported by morphological analyses. Functionally, we report that microglial ablation increases methamphetamine-taking, possibly involving neuroimmune and neurotransmitter regulation. In contrast, microglial depletion during abstinence does not alter methamphetamine-seeking. Taken together, these results suggest that methamphetamine induces both short and long-term changes in dorsal striatal microglia that contribute to altered drug-taking behavior and may provide valuable insights into the pathophysiology of MUD.

Persistent increase of accumbens cocaine ensemble excitability induced by IRK downregulation after withdrawal mediates the incubation of cocaine craving.

The incubation phenomenon, cue-induced drug craving progressively increasing over prolonged withdrawal, accounts for persistent relapse, leading to a dilemma in the treatment of cocaine addiction. The role of neuronal ensembles activated by initial cocaine experience in the incubation phenomenon was unclear. In this study, with cocaine self-administration (SA) models, we found that neuronal ensembles in the nucleus accumbens shell (NAcSh) showed increasing activation induced by cue-induced drug-seeking after 30-day withdrawal. Inhibition or activation of NAcSh cocaine-ensembles suppressed or promoted craving for cocaine, demonstrating a critical role of NAcSh cocaine-ensembles in incubation for cocaine craving. NAcSh cocaine-ensembles showed a specific increase of membrane excitability and a decrease of inward rectifying channels Kir2.1 currents after 30-day withdrawal. Overexpression of Kir2.1 in NAcSh cocaine-ensembles restored neuronal membrane excitability and suppressed cue-induced drug-seeking after 30-day withdrawal. Expression of dominant-negative Kir2.1 in NAcSh cocaine-ensembles enhanced neuronal membrane excitability and accelerated incubation of cocaine craving. Our results provide a cellular mechanism that the downregulation of Kir2.1 functions in NAcSh cocaine-ensembles induced by prolonged withdrawal mediates the enhancement of ensemble membrane excitability, leading to incubation of cocaine craving.

Sex-Specific Impacts of Early Life Sleep Disruption: Ethanol Seeking, Social Interaction, and Anxiety Are Differentially Altered in Adolescent Prairie Voles.

Early life sleep is important for neuronal development. Using the highly social prairie vole rodent model, we have previously reported that early life sleep disruption (ELSD) during the preweaning period results in interference with social bonding and increases ethanol consumption following a stressor in adulthood. Furthermore, ELSD increases parvalbumin expression and reduces glutamatergic neurotransmission in cortical regions in adult prairie voles. To understand the impact of ELSD on the lifespan, an examination of an earlier time in life is necessary. The aim of the present study was to examine behavioral outcomes of ELSD on adolescent prairie voles. Given the known effects of ELSD on development of neuronal systems involved in mood and social motivation, we hypothesized that anxiety, risk, and reward-related behaviors would be impacted by ELSD in adolescent prairie voles. We report that both male and female adolescent prairie voles that experienced ELSD showed heightened anxiety-like behavior compared to age-matched controls (CONs) as measured by a light-dark box. Additionally, both male and female ELSD voles showed reductions in both ethanol preference and consumption, and affiliative behavior compared to CONs. These results suggest that adolescent prairie voles of both sexes experience heightened anxiety-like behavior and reduced reward-seeking behaviors after ELSD. These results further suggest that early life sleep is critically important for neurotypical behaviors in adolescence.

A Conditioned Place Preference for Heroin Is Signaled by Increased Dopamine and Direct Pathway Activity and Decreased Indirect Pathway Activity in the Nucleus Accumbens.

Repeated pairing of a drug with a neutral stimulus, such as a cue or context, leads to the attribution of the drug's reinforcing properties to that stimulus, and exposure to that stimulus in the absence of the drug can elicit drug-seeking. A principal role for the NAc in the response to drug-associated stimuli has been well documented. Direct and indirect pathway medium spiny neurons (dMSNs and iMSNs) have been shown to bidirectionally regulate cue-induced heroin-seeking in rats expressing addiction-like phenotypes, and a shift in NAc activity toward the direct pathway has been shown in mice following cocaine conditioned place preference (CPP). However, how NAc signaling guides heroin CPP, and whether heroin alters the balance of signaling between dMSNs and iMSNs, remains unknown. Moreover, the role of NAc dopamine signaling in heroin reinforcement is unclear. Here, we integrate fiber photometry for in vivo monitoring of dopamine and dMSN/iMSN calcium activity with a heroin CPP procedure in rats to begin to address these questions. We identify a sensitization-like response to heroin in the NAc, with prominent iMSN activity during initial heroin exposure and prominent dMSN activity following repeated heroin exposure. We demonstrate a ramp in dopamine activity, dMSN activation, and iMSN inactivation preceding entry into a heroin-paired context, and a decrease in dopamine activity, dMSN inactivation, and iMSN activation preceding exit from a heroin-paired context. Finally, we show that buprenorphine is sufficient to prevent the development of heroin CPP and reduce Fos activation in the NAc after conditioning. Together, these data support the hypothesis that an imbalance in NAc activity contributes to the development of drug-cue associations that can drive addiction processes.SIGNIFICANCE STATEMENT The attribution of the reinforcing effects of drugs to neutral stimuli (e.g., cues and contexts) contributes to the long-standing nature of addiction, as re-exposure to drug-associated stimuli can reinstate drug-seeking and -taking even after long periods of abstinence. The NAc has an established role in encoding the value of drug-associated stimuli, and dopamine release into the NAc is known to modulate the reinforcing effects of drugs, including heroin. Using fiber photometry, we show that entering a heroin-paired context is driven by dopamine signaling and NAc direct pathway activation, whereas exiting a heroin-paired context is driven by NAc indirect pathway activation. This study provides further insight into the role of NAc microcircuitry in encoding the reinforcing properties of heroin.

IGF-1 Microinjection in the Prefrontal Cortex Attenuates Fentanyl-Seeking Behavior in Mice.

BACKGROUND: Opioid use disorder (OUD) is a chronic relapsing psychiatric disorder with an enormous socioeconomic burden. Opioid overdose deaths have reached an epidemic level, especially for fentanyl. One of the biggest challenges to treat OUD is the relapse to drug seeking after prolonged abstinence. Abnormalities in insulin-like growth factor-1 (IGF-1) have been reported in various neurological and psychiatric disorders, including OUD. However, whether IGF-1 and its downstream signaling pathways are associated with relapse to fentanyl seeking remains unclear. METHODS: Mice were subjected to daily 2-hour fentanyl (10 µg/mL, 27 µL/infusion) oral self-administration training for 14 days, followed by 14-day fentanyl cessation. Expression levels of IGF-1/IGF-1 receptor and downstream signaling pathways in the dorsomedial prefrontal cortex (dmPFC) were detected. Then, IGF-1 was bilaterally microinjected into the dmPFC from fentanyl cessation day 9 to day 13. Fentanyl-seeking behavior and excitatory synaptic transmission of pyramidal neurons in PFC were evaluated. RESULTS: We found that 14-day cessation from fentanyl oral self-administration caused significant downregulation of IGF-1 and IGF-1 receptor phosphorylation in the dmPFC. These changes were accompanied by inhibition of the downstream Akt and S6 signaling pathway. In addition, local administration of IGF-1 in the dmPFC attenuated context-induced fentanyl-seeking behavior. Furthermore, electrophysiology and immunohistochemistry analyses showed that IGF-1 blocked fentanyl-induced reduction of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors-mediated excitatory synaptic transmission as well as synaptic expression of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and N-methyl-D-aspartate receptor subunits. CONCLUSIONS: These results suggest that IGF-1 in the PFC plays a pivotal role in regulating fentanyl seeking after prolonged cessation from fentanyl oral self-administration.

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.

Neuroadaptations in the dorsal hippocampus underlie cocaine seeking during prolonged abstinence.

Relapse vulnerability in substance use disorder is attributed to persistent cue-induced drug seeking that intensifies (or "incubates") during drug abstinence. Incubated cocaine seeking has been observed in both humans with cocaine use disorder and in preclinical relapse models. This persistent relapse vulnerability is mediated by neuroadaptations in brain regions involved in reward and motivation. The dorsal hippocampus (DH) is involved in context-induced reinstatement of cocaine seeking but the role of the DH in cocaine seeking during prolonged abstinence has not been investigated. Here we found that transforming growth factor-ß (TGF-ß) superfamily member activin A is increased in the DH on abstinence day (AD) 30 but not AD1 following extended-access cocaine self-administration compared to saline controls. Moreover, activin A does not affect cocaine seeking on AD1 but regulates cocaine seeking on AD30 in a bidirectional manner. Next, we found that activin A regulates phosphorylation of NMDA receptor (NMDAR) subunit GluN2B and that GluN2B-containing NMDARs also regulate expression of cocaine seeking on AD30. Activin A and GluN2B-containing NMDARs have both previously been implicated in hippocampal synaptic plasticity. Therefore, we examined synaptic strength in the DH during prolonged abstinence and observed an increase in moderate long-term potentiation (LTP) in cocaine-treated rats compared to saline controls. Lastly, we examined the role of DH projections to the lateral septum (LS), a brain region implicated in cocaine seeking and found that DH projections to the LS govern cocaine seeking on AD30. Taken together, this study demonstrates a role for the DH in relapse behavior following prolonged abstinence from cocaine self-administration.

Estradiol Regulation of the Prelimbic Cortex and the Reinstatement of Cocaine Seeking in Female Rats.

Relapse susceptibility in women with substance use disorders (SUDs) has been linked to the estrogen, 17ß-estradiol (E2). Our previous findings in female rats suggest that the influence of E2 on cocaine seeking can be localized to the prelimbic prefrontal cortex (PrL-PFC). Here, we investigated the receptor mechanisms through which E2 regulates the reinstatement of extinguished cocaine seeking. Sexually mature female rats underwent intravenous cocaine self-administration (0.5 mg/inf; 14 × 2 h daily) and extinction, and then were ovariectomized before reinstatement testing. E2 (10 µg/kg, i.p.) alone did not reinstate cocaine seeking, but it potentiated reinstatement when combined with an otherwise subthreshold priming dose of cocaine. A similar effect was observed following intra-PrL-PFC microinfusions of E2 and by systemic or intra-PrL-PFC administration of the estrogen receptor (ER)ß agonist, DPN, but not agonists at ERα or the G-protein-coupled ER1 (GPER1). By contrast, E2-potentiated reinstatement was prevented by intra-PrL-PFC microinfusions of the ERß antagonist, MPP, or the GPER1 antagonist, G15, but not an ERα antagonist. Whole-cell recordings in PrL-PFC layer (L)5/6 pyramidal neurons revealed that E2 decreases the frequency, but not amplitude, of GABAA-dependent miniature IPSCs (mIPSC). As was the case with E2-potentiated reinstatement, E2 reductions in mIPSC frequency were prevented by ERß and GPER1, but not ERα, antagonists and mimicked by ERß, but not GPER1, agonists. Altogether, the findings suggest that E2 activates ERß and GPER1 in the PrL-PFC to attenuate the GABA-mediated constraint of key outputs that mediate cocaine seeking.

Synaptic Adaptations at the Rostromedial Tegmental Nucleus Underlie Individual Differences in Cocaine Avoidance Behavior.

Although cocaine is powerfully rewarding, not all individuals are equally prone to abusing this drug. We postulate that these differences arise in part because some individuals exhibit stronger aversive responses to cocaine that protect them from cocaine seeking. Indeed, using conditioned place preference (CPP) and a runway operant cocaine self-administration task, we demonstrate that avoidance responses to cocaine vary greatly between individual high cocaine-avoider and low cocaine-avoider rats. These behavioral differences correlated with cocaine-induced activation of the rostromedial tegmental nucleus (RMTg), measured using both in vivo firing and c-fos, whereas slice electrophysiological recordings from ventral tegmental area (VTA)-projecting RMTg neurons showed that relative to low avoiders, high avoiders exhibited greater intrinsic excitability, greater transmission via calcium-permeable AMPA receptors (CP-AMPARs), and higher presynaptic glutamate release. In behaving animals, blocking CP-AMPARs in the RMTg with NASPM reduced cocaine avoidance. Hence, cocaine addiction vulnerability may be linked to multiple coordinated synaptic differences in VTA-projecting RMTg neurons.SIGNIFICANCE STATEMENT Although cocaine is highly addictive, not all individuals exposed to cocaine progress to chronic use for reasons that remain unclear. We find that cocaine's aversive effects, although less widely studied than its rewarding effects, show more individual variability, are predictive of subsequent propensity to seek cocaine, and are driven by variations in RMTg in response to cocaine that arise from distinct alterations in intrinsic excitability and glutamate transmission onto VTA-projecting RMTg neurons.