Astrocytes in the ventral pallidum extinguish heroin seeking through GAT-3 upregulation and morphological plasticity at D1-MSN terminals.

GABAergic projections from the nucleus accumbens core to the dorsolateral ventral pallidum are necessary for drug-conditioned cues to initiate relapse-like drug seeking. Astrocytes in the ventral pallidum are situated perisynaptically and regulate GABA transmission through expression of GABA uptake transporters, but whether they are involved in regulating drug seeking is unknown. To determine the contribution of ventral pallidal astrocytes to heroin seeking, we labeled astrocytes in male and female rats with a membrane-bound fluorescent tag and used confocal microscopy to quantify astroglial expression of the GABA transporter GAT-3 and astrocyte synaptic proximity after withdrawal from heroin self-administration and during 15 min of cued heroin seeking. We found that GAT-3 was upregulated in rats that had extinguished heroin seeking, but not in animals that were withdrawn from heroin without extinction training or in rats that extinguished sucrose seeking. When GAT-3 upregulation was reversed using a vivo-morpholino oligo, heroin seeking was restored in the extinguished context and extinction of cued heroin seeking was disrupted compared to control animals. Although astrocyte synaptic proximity was not altered overall after heroin withdrawal, examination of astrocyte proximity to accumbens D1- or D2-expressing afferents revealed a selective increase in astrocyte proximity with D1-expressing terminals during extinction of heroin self-administration. Experimentally-induced reduction of astrocyte synaptic proximity through knockdown of the astrocyte-selective actin-binding protein ezrin also markedly disrupted extinction of heroin seeking. Notably, GAT-3 or ezrin knockdown had no impact on context- or cue-induced seeking in sucrose-trained animals. These data show that astrocytes in the ventral pallidum undergo plasticity after extinction of heroin use that reduces seeking and highlight the importance of astrocyte-neuron interactions in shaping behaviors associated with opioid use disorder.

Matrix metalloproteinase activity during methamphetamine cued relapse.

Relapse to drug seeking involves transient synaptic remodelling that occurs in response to drug-associated cues. This remodelling includes activation of matrix metalloproteinases (MMPs) to initiate catalytic signalling in the extracellular matrix in the nucleus accumbens core (NAcore). We hypothesized that MMP activity would be increased in the NAcore during cue-induced methamphetamine (meth) seeking in a rat model of meth use and relapse. Male and female rats had indwelling jugular catheters and bilateral intracranial cannula targeting the NAcore surgically implanted. Following recovery, rats underwent meth or saline self-administration (6 h/day for 15 days) in which active lever responding was paired with a light + tone stimulus complex, followed by home cage abstinence. Testing occurred after 7 or 30 days of abstinence. On test day, rats were microinjected with a fluorescein isothiocyanate (FITC)-quenched gelatin substrate that fluoresces following cleavage by MMP-2,9, allowing for the quantification of gelatinase activity during cued-relapse testing. MMP-2,9 activity was significantly increased in the NAcore by meth cues presentation after 7 and 30 days of abstinence, indicating that remodelling by MMPs occurs during presentation of meth associated cues. Surprisingly, although cue-induced seeking increased between Days 7 and 30, MMP-2,9 activity did not increase. These findings indicate that although MMP activation is elicited during meth cue-induced seeking, MMP activation did not parallel the meth seeking that occurs during extended drug abstinence.

Accumbens D2-MSN hyperactivity drives antipsychotic-induced behavioral supersensitivity.

Antipsychotic-induced dopamine supersensitivity, or behavioral supersensitivity, is a problematic consequence of long-term antipsychotic treatment characterized by the emergence of motor abnormalities, refractory symptoms, and rebound psychosis. The underlying mechanisms are unclear and no approaches exist to prevent or reverse these unwanted effects of antipsychotic treatment. Here we demonstrate that behavioral supersensitivity stems from long-lasting pre, post and perisynaptic plasticity, including insertion of Ca2+-permeable AMPA receptors and loss of D2 receptor-dependent inhibitory postsynaptic currents (IPSCs) in D2 receptor-expressing medium spiny neurons (D2-MSNs) in the nucleus accumbens core (NAcore). The resulting hyperexcitability, prominent in a subpopulation of D2-MSNs (21%), caused locomotor sensitization to cocaine and was associated with behavioral endophenotypes of antipsychotic treatment resistance and substance use disorder, including disrupted extinction learning and augmented cue-induced cocaine-seeking behavior. Chemogenetic restoration of IPSCs in D2-MSNs in the NAcore was sufficient to prevent antipsychotic-induced supersensitivity, pointing to an entirely novel therapeutic direction for overcoming this condition.

Assessing combined effects of varenicline and N-acetylcysteine on reducing nicotine seeking in rats.

Nicotine addiction is a chronic relapsing brain disorder, and cigarette smoking is the leading cause of preventable death in the United States. Currently, the most effective pharmacotherapy for smoking cessation is Varenicline (VRN), which reduces both positive and negative reinforcement by nicotine. Clinically, VRN attenuates withdrawal symptoms and promotes abstinence, but >50% of smokers relapse within 3 months following a quit attempt. This may indicate that VRN fails to ameliorate components of nicotine-induced neuroplasticity that promote relapse vulnerability. Animal models reveal that glutamate dysregulation in the nucleus accumbens is associated with nicotine relapse. N-acetylcysteine (NAC) normalizes glutamate transmission and prolongs cocaine abstinence. Thus, combining VRN and NAC may promote and maintain, respectively, nicotine abstinence. In rats, we found that VRN effectively reduced nicotine self-administration and seeking in early abstinence, but not seeking later in abstinence. In contrast, NAC reduced seeking only later in abstinence. Because VRN and NAC are sometimes associated with mild adverse effects, we also evaluated a sequential approach combining subthreshold doses of VRN during self-administration and early abstinence with subthreshold doses of NAC during late abstinence. As expected, subthreshold VRN did not reduce nicotine intake. However, subthreshold VRN and NAC reduced seeking in late abstinence, suggesting a combined effect. Overall, our results suggest that combining subthreshold VRN and NAC is a viable and drug-specific approach to promote abstinence and reduce relapse while minimizing adverse effects. Our data also suggest that different components and time points in addiction engage the different neurocircuits targeted by VRN and NAC.

Relapse-Associated Transient Synaptic Potentiation Requires Integrin-Mediated Activation of Focal Adhesion Kinase and Cofilin in D1-Expressing Neurons.

Relapse to drug use can be initiated by drug-associated cues. The intensity of cue-induced drug seeking in rodent models correlates with the induction of transient synaptic potentiation (t-SP) at glutamatergic synapses in the nucleus accumbens core (NAcore). Matrix metalloproteinases (MMPs) are inducible endopeptidases that degrade extracellular matrix (ECM) proteins, and reveal tripeptide Arginine-Glycine-Aspartate (RGD) domains that bind and signal through integrins. Integrins are heterodimeric receptors composed of αß subunits, and a primary signaling kinase is focal adhesion kinase (FAK). We previously showed that MMP activation is necessary for and potentiates cued reinstatement of cocaine seeking, and MMP-induced catalysis stimulates ß3-integrins to induce t-SP. Here, we determined whether ß3-integrin signaling through FAK and cofilin (actin depolymerization factor) is necessary to promote synaptic growth during t-SP. Using a small molecule inhibitor to prevent FAK activation, we blocked cued-induced cocaine reinstatement and increased spine head diameter (dh). Immunohistochemistry on NAcore labeled spines with ChR2-EYFP virus, showed increased immunoreactivity of phosphorylation of FAK (p-FAK) and p-cofilin in dendrites of reinstated animals compared with extinguished and yoked saline, and the p-FAK and cofilin depended on ß3-integrin signaling. Next, male and female transgenic rats were used to selectively label D1 or D2 neurons with ChR2-mCherry. We found that p-FAK was increased during drug seeking in both D1 and D2-medium spiny neurons (MSNs), but increased p-cofilin was observed only in D1-MSNs. These data indicate that ß3-integrin, FAK and cofilin constitute a signaling pathway downstream of MMP activation that is involved in promoting the transient synaptic enlargement in D1-MSNs induced during reinstated cocaine by drug-paired cues.SIGNIFICANCE STATEMENT Drug-associated cues precipitate relapse, which is correlated with transient synaptic enlargement in the accumbens core. We showed that cocaine cue-induced synaptic enlargement depends on matrix metalloprotease signaling in the extracellular matrix (ECM) through ß3-integrin to activate focal adhesion kinase (FAK) and phosphorylate the actin binding protein cofilin. The nucleus accumbens core (NAcore) contains two predominate neuronal subtypes selectively expressing either D1-dopamine or D2-dopamine receptors. We used transgenic rats to study each cell type and found that cue-induced signaling through cofilin phosphorylation occurred only in D1-expressing neurons. Thus, cocaine-paired cues initiate cocaine reinstatement and synaptic enlargement through a signaling cascade selectively in D1-expressing neurons requiring ECM stimulation of ß3-integrin-mediated phosphorylation of FAK (p-FAK) and cofilin.

Circuit selectivity in drug versus natural reward seeking behaviors.

Substance use disorder (SUD) is characterized, in part by behavior biased toward drug use and away from natural sources of reward (e.g., social interaction, food, sex). The neurobiological underpinnings of SUDs reveal distinct brain regions where neuronal activity is necessary for the manifestation of SUD-characteristic behaviors. Studies that specifically examine how these regions are involved in behaviors motivated by drug versus natural reward allow determinations of which regions are necessary for regulating seeking of both reward types, and appraisals of novel SUD therapies for off-target effects on behaviors motivated by natural reward. Here, we evaluate studies directly comparing regulatory roles for specific brain regions in drug versus natural reward. While it is clear that many regions drive behaviors motivated by all reward types, based on the literature reviewed we propose a set of interconnected regions that become necessary for behaviors motivated by drug, but not natural rewards. The circuitry is selectively necessary for drug seeking includes an Action/Reward subcircuit, comprising nucleus accumbens, ventral pallidum, and ventral tegmental area, a Prefrontal subcircuit comprising prelimbic, infralimbic, and insular cortices, a Stress subcircuit comprising the central nucleus of the amygdala and the bed nucleus of the stria terminalis, and a Diencephalon circuit including lateral hypothalamus. Evidence was mixed for nucleus accumbens shell, insular cortex, and ventral pallidum. Studies for all other brain nuclei reviewed supported a necessary role in regulating both drug and natural reward seeking. Finally, we discuss emerging strategies to further disambiguate the necessity of brain regions in drug- versus natural reward-associated behaviors.

Cocaine and sucrose rewards recruit different seeking ensembles in the nucleus accumbens core.

Poorly regulated reward seeking is a central feature of substance use disorder. Recent research shows that rewarding drug-related experiences induce synchronous activation of a discrete number of neurons in the nucleus accumbens that are causally linked to reward-related contexts. Here we comprehensively characterize the specific ensemble of neurons built through experience that are linked to seeking behavior. We additionally address the question of whether or not addictive drugs usurp the neuronal networks recruited by natural rewards by evaluating cocaine- and sucrose-associated ensembles within the same mouse. We used FosCreERT2/+/Ai14 transgenic mice to tag cells activated by and potentially encoding cocaine and sucrose seeking. We tagged ~1% of neurons in the core subregion of the accumbens (NAcore) activated during cue-induced seeking for cocaine or sucrose. The majority of tagged cells in the seeking ensembles were D1-MSNs, and specifically activated during seeking, not during extinction or when mice remained in the home cage. To compare different reward-specific ensembles within the same mouse, we used a dual cocaine and sucrose self-administration protocol allowing reward-specific seeking. Using this model, we found ~70% distinction between the cells constituting the cocaine- compared to the sucrose-seeking ensemble. Establishing that cocaine recruits an ensemble of NAcore neurons largely distinct from neurons recruited into an ensemble coding for sucrose seeking suggest a finely tuned specificity of ensembles. The findings allow further exploration of the mechanisms that transform reward-based positive reinforcement into maladaptive drug seeking.

Ventral Pallidum Is the Primary Target for Accumbens D1 Projections Driving Cocaine Seeking.

Outputs from the nucleus accumbens (NAc) include projections to the ventral pallidum and the ventral tegmental area and subtantia nigra in the ventral mesencephalon. The medium spiny neurons (MSN) that give rise to these pathways are GABAergic and consist of two populations of equal number that are segregated by differentially expressed proteins, including D1- and D2-dopamine receptors. Afferents to the ventral pallidum arise from both D1- and D2-MSNs, whereas the ventral mesencephalon is selectively innervated by D1-MSN. To determine the extent of collateralization of D1-MSN to these axon terminal fields we used retrograde labeling in transgenic mice expressing tdTomato selectively in D1-MSN, and found that a large majority of D1-MSN in either the shell or core subcompartments of the accumbens collateralized to both output structures. Approximately 70% of D1-MSNs projecting to the ventral pallidum collateralized to the ventral mesencephalon, whereas >90% of mesencephalic D1-MSN afferents collateralized to the ventral pallidum. In contrast, <10% of dorsal striatal D1-MSNs collateralized to both the globus pallidus and ventral mesencephalon. D1-MSN activation is required for conditioned cues to induce cocaine seeking. To determine which D1-MSN projection mediates cued cocaine seeking, we selectively transfected D1-MSNs in transgenic rats with an inhibitory Gi-coupled DREADD. Activation of the transfected Gi-DREADD with clozapine-N-oxide administered into the ventral pallidum, but not into the ventral mesencephalon, blocked cue-induced cocaine seeking. These data show that, although accumbens D1-MSNs largely collateralize to both the ventral pallidum and ventral mesencephalon, only D1-MSN innervation of the ventral pallidum is necessary for cue-induced cocaine seeking.SIGNIFICANCE STATEMENT Activity in D1 dopamine receptor-expressing neurons in the NAc is required for rodents to respond to cocaine-conditioned cues and relapse to drug seeking behaviors. The D1-expressing neurons project to both the ventral pallidum and ventral mesencephalon, and we found that a majority of the neurons that innervate the ventral pallidum also collateralize to the ventral mesencephalon. However, despite innervating both structures, only D1 innervation of the ventral pallidum mediates cue-induced cocaine seeking.

N-Acetylcysteine treatment during acute stress prevents stress-induced augmentation of addictive drug use and relapse.

Converging epidemiological studies show that a life-threatening event increases the incidence of posttraumatic stress disorder (PTSD), which carries 30% to 50% comorbidity with substance use disorders (SUDs). Such comorbidity results in greater drug use and poorer treatment outcomes. There is overlap between the enduring synaptic neuroadaptations produced in nucleus accumbens core (NAcore) by acute restraint stress and cocaine self-administration. Because of these coincident neuroadaptations, we hypothesized that an odor paired with acute restraint stress would reinstate drug seeking and chose two mechanistically distinct drugs of abuse to test this hypothesis: alcohol and cocaine. Rats were trained to self-administer either drug beginning 3 weeks after odor pairing with acute stress or sham, and acute restraint stress increased alcohol consumption. Following context extinction training, the stress-paired odor reinstated both alcohol and cocaine seeking, while an unpaired odor had no effect. N-Acetylcysteine (NAC) restores drug and stress-induced reductions in glial glutamate transporter-1 and has proven effective at reducing cue-induced reinstatement of drug seeking. We administered NAC for 5 days prior to reinstatement testing and abolished the capacity of the stress-paired odor to increase alcohol and cocaine seeking. Importantly, daily NAC given during or just following experiencing acute restraint stress also prevented the capacity of stress-paired odors to reinstate alcohol and cocaine seeking and prevented stress-induced deficits in behavioral flexibility. These data support using daily NAC treatment during or immediately after experiencing a strong acute stress to prevent subsequent conditioned stress responding, in particular relapse and cognitive deficits induced by stress-conditioned stimuli.

The loss of NMDAR-dependent LTD following cannabinoid self-administration is restored by positive allosteric modulation of CB1 receptors.

Glutamatergic plasticity in the nucleus accumbens core (NAcore) is a key neuronal process in appetitive learning and contributes to pathologies such as drug addiction. Understanding how this plasticity factors into cannabis addiction and relapse has been hampered by the lack of a rodent model of cannabis self-administration. We used intravenous self-administration of two constituents of cannabis, Δ9 -tetrahydrocannabinol (THC) and cannabidiol (CBD) to examine how contingent cannabis use and cue-induced cannabinoid-seeking alters glutamatergic neurotransmission and synaptic plasticity in NAcore. NMDA receptor (NMDAR)-dependent long-term depression (LTD) in the NAcore was lost after cannabinoid, but not sucrose self-administration. Surprisingly, when rats underwent cue-induced cannabinoid seeking, LTD was restored. Loss of LTD was accompanied by desensitization of cannabinoid receptor 1 (CB1R). CB1R are positioned to regulate synaptic plasticity by being expressed on glutamatergic terminals and negatively regulating presynaptic excitability and glutamate release. Supporting this possibility, LTD was restored by promoting CB1R signaling with the CB1 positive allosteric modulator GAT211. These data implicate NAcore CB1R as critical regulators of metaplasticity induced by cannabis self-administration and the cues predicting cannabis availability.

Transient synaptic potentiation in nucleus accumbens shell during refraining from cocaine seeking.

Repeated exposure to drug-associated cues without reward (extinction) leads to refraining from drug seeking in rodents. We determined if refraining is associated with transient synaptic plasticity (t-SP) in nucleus accumbens shell (NAshell), akin to the t-SP measured in the NAcore during cue-induced reinstatement of drug seeking. Using whole cell patch electrophysiology, we found that medium spiny neurons (MSNs) in NAshell expressed increased ratio of AMPA to NMDA glutamate receptor currents during refraining, which normalized to baseline levels by the end of the 2-hour extinction session. Unlike t-SP observed in NAcore during reinstated drug seeking, neither dendrite spine head enlargement nor activation of matrix metalloproteases (MMP2/9) accompanied the increased AMPA:NMDA in NAshell during refraining. Refraining was also not associated with changes in paired pulse ratio, NMDA receptor current decay time, or AMPA receptor rectification index in NAshell MSNs. Our preliminary data in transgenic mice suggest that t-SP may increase D2-MSN inputs relative to D1-MSN inputs.

Drug Refraining and Seeking Potentiate Synapses on Distinct Populations of Accumbens Medium Spiny Neurons.

Cocaine-associated cues and contexts can precipitate drug seeking in humans and in experimental animals. Glutamatergic synapses in the core subcompartment of the nucleus accumbens (NAcore) undergo transient potentiation in response to presenting drug-associated cues. The NAcore contains two populations of medium spiny neurons (MSNs) that differentially express D1 or D2 dopamine receptors. By recording the ratio of AMPA and NMDA glutamate receptor currents (AMPA/NMDA ratio) from MSNs in NAcore tissue slices, we endeavored to understand which subpopulation of MSNs was undergoing transient potentiation. Transgenic female and male mice differentially expressing fluorescent reporters in D1 or D2 MSNs were withdrawn for 2-3 weeks after being trained to self-administer cocaine. In some mice, discrete cocaine-conditioned cues were isolated from the drug-associated context via extinction training, which causes rodents to refrain from drug seeking in the extinguished context. By measuring AMPA/NMDA ratios in the drug context with or without contextual or discrete cues, and with or without extinction training, we made the following three discoveries: (1) mice refraining from cocaine seeking in the extinguished context showed selective elevation in AMPA/NMDA ratios in D2 MSNs; (2) without extinction training, the drug-associated context selectively increased AMPA/NMDA ratios in D1 MSNs; (3) mice undergoing cue-induced cocaine seeking after extinction training in the drug-associated context showed AMPA/NMDA ratio increases in both D1 and D2 MSNs. These findings reveal that the NAcore codes drug seeking through transient potentiation of D1 MSNs, and that refraining from cocaine seeking in an extinguished context is coded through transient potentiation of D2 MSNs.SIGNIFICANCE STATEMENT Relapse is a primary symptom of addiction that can involve competition between the desire to use drugs and the desire to refrain from using drugs. Drug-associated cues induce relapse, which is correlated with transiently potentiated glutamatergic synapses in the nucleus accumbens core. We determined which of two cell populations in the accumbens core, D1-expressing or D2-expressing neurons, undergo transient synaptic potentiation. After being trained to self-administer cocaine, mice underwent withdrawal, some with and others without extinguishing responding in the drug-associated context. Extinguished mice showed transient potentiation in D2-expressing neurons in the extinguished environment, and all mice engaged in context-induced or cue-induced drug seeking showed transient potentiation of D1-expressing neurons. A simple binary engram in accumbens for seeking drugs and refraining from drugs offers opportunities for cell-specific therapies.

Targeting redox regulation to treat substance use disorder using N­acetylcysteine

Substance use disorder (SUD) is a chronic relapsing disorder characterized by transitioning from acute drug reward to compulsive drug use. Despite the heavy personal and societal burden of SUDs, current treatments are limited and unsatisfactory. For this reason, a deeper understanding of the mechanisms underlying addiction is required. Altered redox status, primarily due to drug-induced increases in dopamine metabolism, is a unifying feature of abused substances. In recent years, knowledge of the effects of oxidative stress in the nervous system has evolved from strictly neurotoxic to include a more nuanced role in redox-sensitive signaling. More specifically, S-glutathionylation, a redox-sensitive post-translational modification, has been suggested to influence the response to drugs of abuse. In this review we will examine the evidence for redox-mediating drugs as therapeutic tools focusing on N-acetylcysteine as a treatment for cocaine addiction. We will conclude by suggesting future research directions that may further advance this field.

Accumbens brain-derived neurotrophic factor (BDNF) transmission inhibits cocaine seeking.

Brain-derived neurotrophic factor (BDNF) regulates a variety of physiological processes, and several studies have explored the role of BDNF in addiction-related brain regions like the nucleus accumbens core (NAcore). We sought to understand the rapid effects of endogenous BDNF on cocaine seeking. Rats were trained to self-administer cocaine and extinguished. We then microinjected two inhibitors of BDNF stimulation of tropomyosin receptor kinase B (TrkB), the non-competitive receptor antagonist ANA-12 and TrkB/Fc, a fusion protein that binds BDNF and prevents TrkB stimulation. Blocking TrkB or inactivating BDNF in NAcore potentiated active lever pressing, showing that endogenous BDNF tone was present and supplying inhibitory tone on cue-induced reinstatement. To determine if exogenous BDNF also negatively regulated reinstatement, BDNF was microinjected into NAcore 15 minutes before cue-induced reinstatement. BDNF decreased cocaine seeking through TrkB receptor binding, but had no effect on inactive lever pressing, spontaneous or cocaine-induced locomotion, or on reinstated sucrose seeking. BDNF-infusion potentiated within trial extinction when microinjected in the NAcore during cue- and context + cue induced reinstatement, and the inhibition of lever pressing lasted at least 3 days post injection. Although decreased reinstatement endured for 3 days when BDNF was administered prior to a reinstatement session, when microinjected before an extinction session or in the home cage, BDNF did not alter subsequent cued-reinstatement. Together, these data show that endogenous BDNF acts on TrKB to provide inhibitory tone on reinstated cocaine seeking, and this effect was recapitulated by exogenous BDNF.

Loss of Plasticity in the D2-Accumbens Pallidal Pathway Promotes Cocaine Seeking.

Distinct populations of D1- and D2-dopamine receptor-expressing medium spiny neurons (D1-/D2-MSNs) comprise the nucleus accumbens, and activity in D1-MSNs promotes, whereas activity in D2-MSNs inhibits, motivated behaviors. We used chemogenetics to extend D1-/D2-MSN cell specific regulation to cue-reinstated cocaine seeking in a mouse model of self-administration and relapse, and found that either increasing activity in D1-MSNs or decreasing activity in D2-MSNs augmented cue-induced reinstatement. Both D1- and D2-MSNs provide substantial GABAergic innervation to the ventral pallidum, and chemogenetic inhibition of ventral pallidal neurons blocked the augmented reinstatement elicited by chemogenetic regulation of either D1- or D2-MSNs. Because D1- and D2-MSNs innervate overlapping populations of ventral pallidal neurons, we next used optogenetics to examine whether changes in synaptic plasticity in D1- versus D2-MSN GABAergic synapses in the ventral pallidum could explain the differential regulation of VP activity. In mice trained to self-administer cocaine, GABAergic LTD was abolished in D2-, but not in D1-MSN synapses. A µ opioid receptor antagonist restored GABA currents in D2-, but not D1-MSN synapses of cocaine-trained mice, indicating that increased enkephalin tone on presynaptic µ opioid receptors was responsible for occluding the LTD. These results identify a behavioral function for D1-MSN innervation of the ventral pallidum, and suggest that losing LTDGABA in D2-MSN, but not D1-MSN input to ventral pallidum may promote cue-induced reinstatement of cocaine-seeking. SIGNIFICANCE STATEMENT: More than 90% of ventral striatum is composed of two cell types, those expressing dopamine D1 or D2 receptors, which exert opposing roles on motivated behavior. Both cell types send GABAergic projections to the ventral pallidum and were found to differentially promote cue-induced reinstatement of cocaine seeking via the ventral pallidum. Furthermore, after cocaine self-administration, synaptic plasticity was selectively lost in D2, but not D1 inputs to the ventral pallidum. The selective impairment in D2 afferents may promote the influence of D1 inputs to drive relapse to cocaine seeking.

Accumbens nNOS Interneurons Regulate Cocaine Relapse.

Relapse to drug use can be initiated by drug-associated cues. The intensity of cue-induced relapse is correlated with the induction of transient synaptic potentiation (t-SP) at glutamatergic synapses on medium spiny neurons (MSNs) in the nucleus accumbens core (NAcore) and requires spillover of glutamate from prefrontal cortical afferents. We used a rodent self-administration/reinstatement model of relapse to show that cue-induced t-SP and reinstated cocaine seeking result from glutamate spillover, initiating a metabotropic glutamate receptor 5 (mGluR5)-dependent increase in nitric oxide (NO) production. Pharmacological stimulation of mGluR5 in NAcore recapitulated cue-induced reinstatement in the absence of drug-associated cues. Using NO-sensitive electrodes, mGluR5 activation by glutamate was shown to stimulate NO production that depended on activation of neuronal nitric oxide synthase (nNOS). nNOS is expressed in ∼1% of NAcore neurons. Using a transgene strategy to express and stimulate designer receptors that mimicked mGluR5 signaling through Gq in nNOS interneurons, we recapitulated cue-induced reinstatement in the absence of cues. Conversely, using a transgenic caspase strategy, the intensity of cue-induced reinstatement was correlated with the extent of selective elimination of nNOS interneurons. The induction of t-SP during cued reinstatement depends on activating matrix metalloproteinases (MMPs) and selective chemogenetic stimulation of nNOS interneurons recapitulated MMP activation and t-SP induction (increase in AMPA currents in MSNs). These data demonstrate critical involvement of a sparse population of nNOS-expressing interneurons in cue-induced cocaine seeking, revealing a bottleneck in brain processing of drug-associated cues where therapeutic interventions could be effective in treating drug addiction. SIGNIFICANCE STATEMENT: Relapse to cocaine use in a rat model is associated with transient increases in synaptic strength at prefrontal cortex synapses in the nucleus accumbens. We demonstrate the sequence of events that mediates synaptic potentiation and reinstated cocaine seeking induced by cocaine-conditioned cues. Activation of prefrontal inputs to the accumbens by cues initiates spillover of synaptic glutamate, which stimulates metabotropic glutamate receptor 5 (mGluR5) on a small population of interneurons (∼1%) expressing neuronal nitric oxide synthase. Stimulating these glutamate receptors increases nitric oxide (NO) production, which stimulates matrix metalloprotease-2 (MMP-2) and MMP-9 activity in the extracellular space. Manipulating the interaction between mGluR5, NO production, or MMP-2 and MMP-9 pharmacologically or genetically is sufficient to recapitulate transient synaptic potentiation and reinstate cocaine seeking.

Chemogenetic Activation of an Extinction Neural Circuit Reduces Cue-Induced Reinstatement of Cocaine Seeking.

UNLABELLED: The ventromedial prefrontal cortex (vmPFC) has been shown to negatively regulate cocaine-seeking behavior, but the precise conditions by which vmPFC activity can be exploited to reduce cocaine relapse are currently unknown. We used viral-mediated gene transfer of designer receptors (DREADDs) to activate vmPFC neurons and examine the consequences on cocaine seeking in a rat self-administration model of relapse. Activation of vmPFC neurons with the Gq-DREADD reduced reinstatement of cocaine seeking elicited by cocaine-associated cues, but not by cocaine itself. We used a retro-DREADD approach to confine the Gq-DREADD to vmPFC neurons that project to the medial nucleus accumbens shell, confirming that these neurons are responsible for the decreased cue-induced reinstatement of cocaine seeking. The effects of vmPFC activation on cue-induced reinstatement depended on prior extinction training, consistent with the reported role of this structure in extinction memory. These data help define the conditions under which chemogenetic activation of extinction neural circuits can be exploited to reduce relapse triggered by reminder cues. SIGNIFICANCE STATEMENT: The ventromedial prefrontal cortex (vmPFC) projection to the nucleus accumbens shell is important for extinction of cocaine seeking, but its anatomical proximity to the relapse-promoting projection from the dorsomedial prefrontal cortex to the nucleus accumbens core makes it difficult to selectively enhance neuronal activity in one pathway or the other using traditional pharmacotherapy (e.g., systemically administered drugs). Viral-mediated gene delivery of an activating Gq-DREADD to vmPFC and/or vmPFC projections to the nucleus accumbens shell allows the chemogenetic exploitation of this extinction neural circuit to reduce cocaine seeking and was particularly effective against relapse triggered by cocaine reminder cues.

Glutamate Transport: A New Bench to Bedside Mechanism for Treating Drug Abuse.

Drug addiction has often been described as a "hijacking" of the brain circuits involved in learning and memory. Glutamate is the principal excitatory neurotransmitter in the brain, and its contribution to synaptic plasticity and learning processes is well established in animal models. Likewise, over the past 20 years the addiction field has ascribed a critical role for glutamatergic transmission in the development of addiction. Chronic drug use produces enduring neuroadaptations in corticostriatal projections that are believed to contribute to a maladaptive deficit in inhibitory control over behavior. Much of this research focuses on the role played by ionotropic glutamate receptors directly involved in long-term potentiation and depression or metabotropic receptors indirectly modulating synaptic plasticity. Importantly, the balance between glutamate release and clearance tightly regulates the patterned activation of these glutamate receptors, emphasizing an important role for glutamate transporters in maintaining extracellular glutamate levels. Five excitatory amino acid transporters participate in active glutamate reuptake. Recent evidence suggests that these glutamate transporters can be modulated by chronic drug use at a variety of levels. In this review, we synopsize the evidence and mechanisms associated with drug-induced dysregulation of glutamate transport. We then summarize the preclinical and clinical data suggesting that glutamate transporters offer an effective target for the treatment of drug addiction. In particular, we focus on the role that altered glutamate transporters have in causing drug cues and contexts to develop an intrusive quality that guides maladaptive drug seeking behaviors.

Phenotype-dependent inhibition of glutamatergic transmission on nucleus accumbens medium spiny neurons by the abused inhalant toluene.

Abused inhalants are voluntarily inhaled at high concentrations to produce intoxicating effects. Results from animal studies show that the abused inhalant toluene triggers behaviors, such as self-administration and conditioned place preference, which are commonly associated with addictive drugs. However, little is known about how toluene affects neurons within the nucleus accumbens (NAc), a brain region within the basal ganglia that mediates goal-directed behaviors and is implicated in the development and maintenance of addictive behaviors. Here we report that toluene inhibits a component of the after-hyperpolarization potential, and dose-dependently inhibits N-methyl-D-aspartate (NMDA)-mediated currents in rat NAc medium spiny neurons (MSN). Moreover, using the multivariate statistical technique, partial least squares discriminative analysis to analyze electrophysiological measures from rat NAc MSNs, we show that toluene induces a persistent depression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated currents in one subtype of NAc MSNs, and that the electrophysiological features of MSN neurons predicts their sensitivity to toluene. The CB1 receptor antagonist AM281 blocked the toluene-induced long-term depression of AMPA currents, indicating that this process is dependent on endocannabinoid signaling. The neuronal identity of recorded cells was examined using dual histochemistry and shows that toluene-sensitive NAc neurons are dopamine D2 MSNs that express preproenkephalin mRNA. Overall, the results from these studies indicate that physiological characteristics obtained from NAc MSNs during whole-cell patch-clamp recordings reliably predict neuronal phenotype, and that the abused inhalant toluene differentially depresses excitatory neurotransmission in NAc neuronal subtypes.

Reinstatement of nicotine seeking is mediated by glutamatergic plasticity.

Nicotine abuse and addiction is a major health liability. Nicotine, an active alkaloid in tobacco, is self-administered by animals and produces cellular adaptations in brain regions associated with drug reward, such as the nucleus accumbens. However, it is unknown whether, akin to illicit drugs of abuse such as cocaine or heroin, the adaptations endure and contribute to the propensity to relapse after discontinuing nicotine use. Using a rat model of cue-induced relapse, we made morphological and electrophysiological measures of synaptic plasticity, as well as quantified glutamate overflow, in the accumbens after 2 wk of withdrawal with extinction training. We found an enduring basal increase in dendritic spine head diameter and in the ratio of AMPA to NMDA currents in accumbens spiny neurons compared with yoked saline animals at 2 wk after the last nicotine self-administration session. This synaptic potentiation was associated with an increase in both AMPA (GluA1) and NMDA (GluN2A and GluN2B) receptor subunits, and a reduction in the glutamate transporter-1 (GLT-1). When nicotine seeking was reinstated by presentation of conditioned cues, there were parallel increases in behavioral responding, extracellular glutamate, and further increases in dendritic spine head diameter and ratio of AMPA to NMDA currents within 15 min. These findings suggest that targeting glutamate transmission might inhibit cue-induced nicotine seeking. In support of this hypothesis, we found that pharmacological inhibition of GluN2A with 3-Chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulfonamide (TCN-201) or GluN2B with ifenprodil abolished reinstated nicotine seeking. These results indicate that up-regulated GluN2A, GluN2B, and rapid synaptic potentiation in the accumbens contribute to cue-induced relapse to nicotine use.