Cocaine Conditioned Place Preference Research Articles

A role for BD2‐selective BET inhibitors in cocaine‐seeking behaviors

Epigenetic mechanisms play important roles in the maladaptive transcriptional and behavioral responses to drugs of abuse. Inhibition of bromodomain and extra‐terminal domain (BET) proteins, a class of histone acetylation reader proteins, has been shown to reduce cocaine‐seeking behaviors in animal models of addiction. To date, the role of BET protein in animal models of substance use disorder (SUD) has only been evaluated using pan‐BET inhibitors, small molecules that bind to both bromodomains (BD1 and BD2) in all BET proteins. Although these initial studies are encouraging, side effects associated with pan‐BET inhibitors may limit their clinical utility as a SUD treatment. In order to evaluate safer and more selective approaches, we utilized a clinically tested BD2 selective BET inhibitor, RVX‐208. We show that RVX‐208 dose‐dependently reduced cocaine conditioned place preference in male mice, similar to the pan‐BET inhibitor JQ1. Additionally, RVX‐208 did not alter other behaviors, such as: locomotor activity, elevated zero maze, or novel object recognition memory. At the transcriptional level, RVX‐208 attenuated the expression of multiple cocaine‐induced genes in the nucleus accumbens. RVX‐208 also produced a distinct transcriptional response in stimulated primary neurons compared to JQ1 but had little effect on gene expression in non‐stimulated neurons. Together, these data indicate that targeting domain‐specific BET mechanisms may be an effective and safer strategy to reduce cocaine‐induced neurobehavioral adaptations.

Expression of stable and reliable preference and aversion phenotypes following place conditioning with psychostimulants.

Drug-seeking behavior occurs more readily in some individuals than others. This phenomenon is considered in studies of drug self-administration in which high drug-seeking/taking individuals can be identified. In contrast, studies of conditioned place preference (CPP) often involve a random sample of drug-naïve rodents that includes phenotypes not considered relevant to addiction. The main objective of the current studies was to determine if a priori identification of different conditioning phenotypes could improve the validity and sensitivity of CPP expression as a preclinical test for vulnerability to addiction. Analysis of cocaine place conditioning data from 443 Swiss-Webster mice revealed a trimodal distribution with peaks corresponding to means of k = 3 clusters. The cluster means occurred at high, low, or negative preference scores, the latter suggesting a phenotype acquiring conditioned place aversion (CPA). The same clusters were identified in mice conditioned with methamphetamine, MDPV, or amphetamine, and these clusters remained stable and reliable during three additional expression tests spaced at 24h. A meta-analysis of effect sizes obtained from CPP literature revealed a positively skewed distribution affected by sample size, consistent with the existence of a CPA phenotype within the populations tested. A dopamine receptor antagonist, flupentixol, blocked cocaine CPP expression in a group containing all phenotypes, but sensitivity improved markedly when CPA phenotypes were excluded from the dataset. These studies suggest that taking phenotype into consideration when designing place conditioning studies will improve their application as a preclinical tool in addiction biology and drug discovery.

Effects of ketosis on cocaine-induced reinstatement in male mice

In recent years, the benefits of the ketogenic diet (KD) on different psychiatric disorders have been gaining attention, but the substance abuse field is still unexplored. Some studies have reported that palatable food can modulate the rewarding effects of cocaine, but the negative metabolic consequences rule out the recommendation of using it as a complementary treatment. Thus, the main aim of this study was to evaluate the effects of the KD on cocaine conditioned place preference (CPP) during acquisition, extinction, and reinstatement. 41 OF1 male mice were employed to assess the effects of the KD on a 10 mg/kg cocaine-induced CPP. Animals were divided into three groups: SD, KD, and KD after the Post-Conditioning test. The results revealed that, while access to the KD did not block CPP acquisition, it did significantly reduce the number of sessions required to extinguish the drug-associated memories and it blocked the priming-induced reinstatement.

Domain-selective BET inhibition attenuates transcriptional and behavioral responses to cocaine

Epigenetic pharmacotherapies have emerged as a promising treatment option for substance use disorder (SUD) due to their ability to reverse maladaptive transcriptional and behavioral responses to drugs of abuse. In particular, inhibitors of bromodomain and extra terminal domain (BET) reader proteins have been shown to reduce cocaine- and opioid-seeking behaviors in rodents. However, only pan-BET inhibitors, small molecules that bind to both bromodomains (BD1 and BD2) with all BET proteins, have been investigated in animal models of SUD. Given the potential side effects associated with pan-BET inhibitors, safer and more selective strategies are needed to advance BET therapeutics as a potential treatment for SUD. Here, we show that RVX-208, a clinically tested, BD2-selective BET inhibitor, dose-dependently reduced cocaine conditioned place preference in male and female mice, similar to the pan-BET inhibitor JQ1. In other behavioral experiments, RVX-208 treatment did not alter distance traveled, anxiety-like behavior, or novel object recognition memory. At the transcriptional level, RVX-208 attenuated the expression of multiple cocaine-induced genes in the nucleus accumbens in a sex-dependent manner. RVX-208 produced a distinct transcriptional response in stimulated primary neurons compared to JQ1 but had little effect on gene expression in non-stimulated neurons. Together, these data indicate that targeting domain-specific BET mechanisms may be an effective and safer strategy to reduce cocaine-induced neurobehavioral adaptations.

Brief Maternal Separation Inoculates Against the Effects of Social Stress on Depression-Like Behavior and Cocaine Reward in Mice

Exposure to intermittent repeated social defeat (IRSD) increases the vulnerability of mice to the rewarding effects of cocaine in the conditioned place preference (CPP) paradigm. According to the “inoculation of stress” hypothesis, a brief period of maternal separation (MS) can provide protection against the negative effects of IRSD. The aim of the present study was to assess whether exposure to a brief episode of MS prevents the subsequent short-term effects of IRSD on depression- and anxiety-like behaviors and to explore its long-term effects on cocaine CPP in mice. Four groups of male C57BL/6 mice were employed; two groups were separated from their mother [6 h on postnatal day (PND) 9], while the other two groups were not (controls). On PND 47, 50, 53 and 56, mice that had experienced MS were exposed to social defeat in the cage of an aggressive resident mouse (MS + IRSD group) or were allowed to explore an empty cage (MS + EXPL group). The same procedure was performed with control mice that had not experienced MS (CONTROL + IRSD and CONTROL + EXPL groups). On PND57-58, all the mice performed the elevated plus maze and the hole-board, social interaction and splash tests. Three weeks after the last episode of defeat, all the mice underwent the CPP procedure with cocaine (1 mg/kg). Irrespective of whether or not MS had taken place, a reduction in open arms measures, dips, and social interaction was observed in mice that experienced IRSD. A higher latency of grooming and acquisition of cocaine-induced CPP were observed only in mice exposed to IRSD alone (CONTROL + IRSD). These results suggest that exposure to a brief episode of stress early in life increases the subsequent resilience of animals to the effects of social stress on vulnerability to cocaine.

Cocaine induces input and cell-type-specific synaptic plasticity in ventral pallidum-projecting nucleus accumbens medium spiny neurons.

Cocaine use and abstinence induce long-term synaptic alterations in the excitatory input to nucleus accumbens (NAc) medium spiny neurons (MSNs). The NAc regulates reward-related behaviors through two parallel projections to the ventral pallidum (VP)-originating in D1 or D2-expressing MSNs (D1-MSNs→VP; D2-MSNs→VP). The activity of these projections depends on their excitatory synaptic inputs, but it is not known whether and how abstinence from cocaine affects the excitatory transmission to D1-MSNs→VP and D2-MSNs→VP. Here we examined different forms of cocaine-induced synaptic plasticity in the inputs from the basolateral amygdala (BLA) and medial prefrontal cortex (mPFC) to NAc D1-MSNs→VP and putative D2-MSNs→VP (pD2-MSNs→VP) in the core and shell subcompartments of the NAc. We used the whole-cell patch-clamp technique to record excitatory postsynaptic currents from D1-tdTomato mice injected with ChR2 in either the BLA or the mPFC and retrograde tracer (RetroBeads) in the VP. We found that cocaine conditioned place preference (CPP) followed by abstinence potentiated the excitatory input from the BLA and mPFC to both D1-MSNs→VP and pD2-MSNs→VP. Interestingly, while the strengthening of the inputs to D1-MSNs→VP was of postsynaptic origin and manifested as increased AMPA to NMDA ratio, in pD2-MSNs→VP plasticity was predominantly presynaptic and was detected as changes in the paired-pulse ratio and coefficient of variation. Lastly, some of the changes were sex-specific. Overall our data show that abstinence from cocaine changes the excitatory inputs to both D1-MSNs→VP and pD2-MSNs→VP but with different mechanisms. This may help understand how circuits converging into the VP change after cocaine exposure.

The Src-Kinase Fyn is Required for Cocaine-Associated Memory Through Regulation of Tau.

Drug-associated context-induced relapse of cocaine-seeking behaviour requires the retrieval of drug-associated memory. Studies exploring the underlying neurobiological mechanism of drug memory formation will likely contribute to the development of treatments for drug addiction and the prevention of relapse. In our study, we applied a cocaine-conditioned place preference (CPP) paradigm and a self-administration paradigm (two drug-associated memory formation model) to confirm the hypothesis that the Src kinase Fyn critically regulates cocaine-associated memory formation in the hippocampus. For this experiment, we administered the Src kinase inhibitor PP2 into the bilateral hippocampus before cocaine-CPP and self-administration training, and the results showed that pharmacological manipulation of the Src kinase Fyn activity significantly attenuated the response to cocaine-paired cues in the cocaine-CPP and self-administration paradigms, indicating that hippocampal Fyn activity contributes to cocaine-associated memory formation. In addition, the regulation of cocaine-associated memory formation by Fyn depends on Tau expression, as restoring Tau to normal levels disrupted cocaine memory formation. Together, these results indicate that hippocampal Fyn activity plays a key role in the formation of cocaine-associated memory, which underlies cocaine-associated contextual stimulus-mediated regulation of cocaine-seeking behaviour, suggesting that Fyn represents a promising therapeutic target for weakening cocaine-related memory and treating cocaine addiction.

Optogenetic stimulation of dynorphinergic neurons within the dorsal raphe activate kappa opioid receptors in the ventral tegmental area and ablation of dorsal raphe prodynorphin or kappa receptors in dopamine neurons blocks stress potentiation of cocaine reward

Behavioral stress exposure increases the risk of drug-taking in individuals with substance use disorders by mechanisms involving the dynorphins, which are the endogenous neuropeptides for the kappa opioid receptor (KOR). KOR agonists have been shown to encode dysphoria, aversion, and changes in reward valuation, and kappa opioid antagonists are in clinical development for treating substance use disorders. In this study, we confirmed that KORs were expressed in dopaminergic neurons in the ventral tegmental area (VTA) of male C57BL6/J mice. Genetic ablation of KORs from dopamine neurons blocked the potentiating effects of repeated forced swim stress on cocaine conditioned place preference (CPP). KOR activation inhibited dopamine neuron GCaMP6m calcium activity in VTA during swim stress and caused a rebound enhancement during the period after stress exposure. Transient optogenetic inhibition of VTA dopamine neurons with AAV5-DIO-SwiChR was acutely aversive in a real time place preference assay and blunted cocaine CPP when inhibition was administered concurrently with cocaine conditioning. However, when inhibition preceded cocaine conditioning by 30 min, cocaine CPP was enhanced. Retrograde tracing with CAV2-DIO-ZsGreen identified a population of prodynorphinCre neurons in the dorsal raphe nucleus (DRN) projecting to the VTA. Optogenetic stimulation of dynorphinergic neurons within the DRN by Channelrhodopsin2 activated KOR in VTA and ablation of prodynorphin blocked stress potentiation of cocaine CPP. Together, these studies demonstrate the presence of a dynorphin/KOR midbrain circuit that projects from the DRN to VTA and is involved in altering the dynamic response of dopamine neuron activity to enhance drug reward learning.

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.

The GLT-1 enhancer clavulanic acid suppresses cocaine place preference behavior and reduces GCPII activity and protein levels in the rat nucleus accumbens

The β-lactam antibiotic ceftriaxone (CTX) is a glutamate transporter subtype 1 (GLT-1) enhancer that reduces cocaine reinforcing efficacy and relapse in rats, but pharmacokinetic liabilities limit translational utility. An attractive alternative is clavulanic acid (CLAV), a structurally related β-lactamase inhibitor and component of FDA-approved Augmentin. CLAV retains the GLT-1 enhancing effects of CTX but displays greater oral bioavailability, brain penetrability and negligible antibacterial activity. CLAV reduces morphine conditioned place preference (CPP) and ethanol consumption in rats, but knowledge about the efficacy of CLAV in preclinical models of drug addiction remains sparse. Here, we investigated effects of CLAV (10 mg/kg, IP) on the acquisition, expression, and maintenance of cocaine CPP in rats, and on two glutamate biomarkers associated with cocaine dependence, GLT-1 and glutamate carboxypeptidase II (GCPII). CLAV administered during cocaine conditioning (10 mg/kg, IP x 4 d) did not affect the development of cocaine CPP. However, a single CLAV injection, administered after the conditioning phase, reduced the expression of cocaine CPP. In rats with established cocaine preference, repeated CLAV administration facilitated extinction of cocaine CPP. In the nucleus accumbens, acute CLAV exposure reduced GCPII protein levels and activity, and a 10-d CLAV treatment regimen enhanced GLT-1 levels. These results suggest that CLAV reduces expression and maintenance of cocaine CPP but lacks effect against development of CPP. Moreover, the ability of a single injection of CLAV to reduce both GCPII activity and protein levels, as well as expression of cocaine CPP, points toward studying GCPII as a therapeutic target of CLAV.

Rewarding Social Interaction in Rats Increases CaMKII in the Nucleus Accumbens.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is known to be involved in the sensitized locomotor responses and drug-seeking behavior to psychostimulants. However, little is known about the contribution of CaMKII signaling in the nucleus accumbens (NAc) in natural rewards such as social interaction. The present experiments explored the implication of CaMKII signaling in drug versus natural reward. In the NAc of rats expressing cocaine or social interaction conditioned place preference (CPP), αCaMKII activation was induced in those expressing social interaction but not cocaine CPP. In order to investigate the role of NAc CaMKII in the expression of reward-related learning of drug versus non-drug stimuli, we inhibited CaMKII through an infusion of KN-93, a CaMKII inhibitor, directly into the NAc shell or core, before the CPP test in a concurrent paradigm in which social interaction was made available in the compartment alternative to the one associated with cocaine during conditioning. Whereas vehicle infusions led to equal preference to both stimuli, inhibition of CaMKII by a KN-93 infusion before the CPP test in the shell but not the core of the NAc shifted the rats’ preference toward the cocaine-associated compartment. Altogether, these results suggest that social interaction reward engages CaMKII in the NAc.

Synaptic Zn2+ potentiates the effects of cocaine on striatal dopamine neurotransmission and behavior

Cocaine binds to the dopamine (DA) transporter (DAT) to regulate cocaine reward and seeking behavior. Zinc (Zn2+) also binds to the DAT, but the in vivo relevance of this interaction is unknown. We found that Zn2+ concentrations in postmortem brain (caudate) tissue from humans who died of cocaine overdose were significantly lower than in control subjects. Moreover, the level of striatal Zn2+ content in these subjects negatively correlated with plasma levels of benzoylecgonine, a cocaine metabolite indicative of recent use. In mice, repeated cocaine exposure increased synaptic Zn2+ concentrations in the caudate putamen (CPu) and nucleus accumbens (NAc). Cocaine-induced increases in Zn2+ were dependent on the Zn2+ transporter 3 (ZnT3), a neuronal Zn2+ transporter localized to synaptic vesicle membranes, as ZnT3 knockout (KO) mice were insensitive to cocaine-induced increases in striatal Zn2+. ZnT3 KO mice showed significantly lower electrically evoked DA release and greater DA clearance when exposed to cocaine compared to controls. ZnT3 KO mice also displayed significant reductions in cocaine locomotor sensitization, conditioned place preference (CPP), self-administration, and reinstatement compared to control mice and were insensitive to cocaine-induced increases in striatal DAT binding. Finally, dietary Zn2+ deficiency in mice resulted in decreased striatal Zn2+ content, cocaine locomotor sensitization, CPP, and striatal DAT binding. These results indicate that cocaine increases synaptic Zn2+ release and turnover/metabolism in the striatum, and that synaptically released Zn2+ potentiates the effects of cocaine on striatal DA neurotransmission and behavior and is required for cocaine-primed reinstatement. In sum, these findings reveal new insights into cocaine’s pharmacological mechanism of action and suggest that Zn2+ may serve as an environmentally derived regulator of DA neurotransmission, cocaine pharmacodynamics, and vulnerability to cocaine use disorders.

Distinct Role of Dopamine in the PFC and NAc During Exposure to Cocaine-Associated Cues.

BackgroundDopamine neurotransmission plays a critical role in reward in drug abuse and drug addiction. However, the role of dopamine in the recognition of drug-associated environmental stimuli, retrieval of drug-associated memory, and drug-seeking behaviors is not fully understood. MethodsRoles of dopamine neurotransmission in the prefrontal cortex (PFC) and nucleus accumbens (NAc) in the cocaine-conditioned place preference (CPP) paradigm were evaluated using in vivo microdialysis. ResultsIn mice that had acquired cocaine CPP, dopamine levels in the PFC, but not in the NAc, increased in response to cocaine-associated cues when mice were placed in the cocaine chamber of an apparatus with 2 separated chambers. The induction of the dopamine response and the development of cocaine CPP were mediated through activation of glutamate NMDA (N-methyl-D-aspartate)/AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor signaling in the PFC during conditioning. Activation of dopamine D1 or D2 receptor signaling in the PFC was required for cocaine-induced locomotion, but not for the induction of the dopamine response or the development of cocaine CPP. Interestingly, dopamine levels in the NAc increased in response to cocaine-associated cues when mice were placed at the center of an apparatus with 2 connected chambers, which requires motivated exploration associated with cocaine reward. ConclusionsDopamine neurotransmission in the PFC is activated by the exposure to the cocaine-associated cues, whereas dopamine neurotransmission in the NAc is activated in a process of motivated exploration of cues associated with cocaine reward. Furthermore, the glutamate signaling cascade in the PFC is suggested to be a potential therapeutic target to prevent the progression of drug addiction.

Social Interaction With Relapsed Partner Facilitates Cocaine Relapse in Rats.

Social factors strongly contribute to drug use and relapse, and epidemiological studies have found that members of peer groups influence each other to use drugs. However, previous animal models mostly failed to incorporate social factors and demonstrate the effects of social partners on drug addiction and relapse. In the present study, we investigated the transfer of relapse to cocaine seeking between drug-addicted partners in rats. Male Sprague–Dawley rats were pair-housed and subjected to training and extinction of cocaine self-administration and conditioned place preference (CPP). 24 h after extinction test, the targeted rats interacted with a cocaine-primed (relapsed) partner or stranger, or saline-injected (unrelapsed) partner for 30 min, after which the targeted rats were tested for drug seeking behavior. We found that social interaction with a relapsed partner increased drug seeking behavior in cocaine self-administration and CPP models in rats, while social interaction with an unrelapsed partner or relapsed stranger had no effect on cocaine seeking. Moreover, the effect of social interaction on cocaine seeking could last for at least 1 day. Our findings demonstrate a facilitation effect of relapsed social partners on drug relapse in rats and provide a novel animal model for social transfer of drug relapse.

Stress decreases serotonin tone in the nucleus accumbens in male mice to promote aversion and potentiate cocaine preference via decreased stimulation of 5-HT1B receptors.

Stress-induced release of dynorphins (Dyn) activates kappa opioid receptors (KOR) in serotonergic neurons to produce dysphoria and potentiate drug reward; however, the circuit mechanisms responsible for this effect are not known. In male mice, we found that conditional deletion of KOR from Slc6a4 (SERT)-expressing neurons blocked stress-induced potentiation of cocaine conditioned place preference (CPP). Within the dorsal raphe nucleus (DRN), two overlapping populations of KOR-expressing neurons: Slc17a8 (VGluT3) and SERT, were distinguished functionally and anatomically. Optogenetic inhibition of these SERT+ neurons potentiated subsequent cocaine CPP, whereas optical inhibition of the VGluT3+ neurons blocked subsequent cocaine CPP. SERT+/VGluT3- expressing neurons were concentrated in the lateral aspect of the DRN. SERT projections from the DRN were observed in the medial nucleus accumbens (mNAc), but VGluT3 projections were not. Optical inhibition of SERT+ neurons produced place aversion, whereas optical stimulation of SERT+ terminals in the mNAc attenuated stress-induced increases in forced swim immobility and subsequent cocaine CPP. KOR neurons projecting to mNAc were confined to the lateral aspect of the DRN, and the principal source of dynorphinergic (Pdyn) afferents in the mNAc was from local neurons. Excision of Pdyn from the mNAc blocked stress-potentiation of cocaine CPP. Prior studies suggested that stress-induced dynorphin release within the mNAc activates KOR to potentiate cocaine preference by a reduction in 5-HT tone. Consistent with this hypothesis, a transient pharmacological blockade of mNAc 5-HT1B receptors potentiated subsequent cocaine CPP. 5-HT1B is known to be expressed on 5-HT terminals in NAc, and 5-HT1B transcript was also detected in Pdyn+, Adora2a+ and ChAT+ (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT1B transcript was selectively elevated in Pdyn+ cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward.

The association between risky decision making and cocaine conditioned place preference is moderated by sex

BackgroundExcessive risk taking is a characteristic trait of several psychiatric conditions, including substance use disorders. High risk-taking (HiR) rats self-administer more cocaine compared to low risk-taking (LoR) rats. However, research has not determined if risk taking is associated with enhanced cocaine conditioned place preference (CPP). MethodsMale and female Sprague Dawley rats (n = 48 each sex) were first tested in the risky decision task (RDT), in which a response on one lever resulted in safe delivery of one food pellet, and a response on a different lever resulted in delivery of two pellets and probabilistic delivery of foot shock. Following RDT training, rats were tested for cocaine CPP. The first session was a pretest that measured rats’ preference for three compartments that provided different visual and tactile cues. Rats then learned to associate one compartment with cocaine (either 10.0 mg/kg or 20.0 mg/kg; i.p.) and one compartment with saline (1.0 ml/kg; i.p.) across eight conditioning sessions. Finally, rats explored all three compartments in a drug-free state. ResultsSex significantly moderated the association between risky decision making and cocaine CPP. While increased risk aversion was somewhat positively associated with cocaine CPP in males, increased risk taking was positively correlated with cocaine CPP in females. ConclusionsThese results highlight the moderating role of sex on the relationship between risky decision making and cocaine reward.

HuD Regulates mRNA-circRNA-miRNA Networks in the Mouse Striatum Linked to Neuronal Development and Drug Addiction.

Simple SummaryGene expression controls all aspects of life, including that of humans. Genes are expressed by copying the information stored in the DNA into RNA molecules, and this process is regulated in part by multiple RNA-binding proteins (RBPs). One such protein, HuD, plays a critical role in the development of neurons and has been implicated in childhood brain tumors, neurodegenerative disorders (Parkinson’s, Alzheimer’s, and ALS), and drug abuse. In addition, HuD participates in neuronal remodeling mechanisms in the mature brain and promotes regeneration of peripheral nerves. HuD primarily binds to transcribed messenger RNAs, which are then stabilized for translation into proteins. However, recent studies demonstrate that HuD also regulates the expression of non-coding RNAs, such as circular RNAs (circRNAs) and microRNAs (miRNAs). In this study, we examined the role of HuD in the control of non-coding RNA expression in the mouse striatum, a brain region associated both with normal behaviors and pathological conditions such as drug abuse. Our results show that HuD regulates mRNA-circRNA-miRNA networks involved in the expression of genes associated with brain development and remodeling of neuronal connections. These findings suggest the possibility of new mechanisms controlling brain development, neurodegenerative diseases, and substance use disorders.The RNA-binding protein HuD (a.k.a., ELAVL4) is involved in neuronal development and synaptic plasticity mechanisms, including addiction-related processes such as cocaine conditioned-place preference (CPP) and food reward. The most studied function of this protein is mRNA stabilization; however, we have recently shown that HuD also regulates the levels of circular RNAs (circRNAs) in neurons. To examine the role of HuD in the control of coding and non-coding RNA networks associated with substance use, we identified sets of differentially expressed mRNAs, circRNAs and miRNAs in the striatum of HuD knockout (KO) mice. Our findings indicate that significantly downregulated mRNAs are enriched in biological pathways related to cell morphology and behavior. Furthermore, deletion of HuD altered the levels of 15 miRNAs associated with drug seeking. Using these sets of data, we predicted that a large number of upregulated miRNAs form competing endogenous RNA (ceRNA) networks with circRNAs and mRNAs associated with the neuronal development and synaptic plasticity proteins LSAMP and MARK3. Additionally, several downregulated miRNAs form ceRNA networks with mRNAs and circRNAs from MEF2D, PIK3R3, PTRPM and other neuronal proteins. Together, our results indicate that HuD regulates ceRNA networks controlling the levels of mRNAs associated with neuronal differentiation and synaptic physiology.

Lateral hypothalamic LEPR neurons drive appetitive but not consummatory behaviors.

SUMMARYAssigning behavioral roles to genetically defined neurons within the lateral hypothalamus (LH) is an ongoing challenge. We demonstrate that a subpopulation of LH GABAergic neurons expressing leptin receptors (LHLEPR) specifically drives appetitive behaviors in mice. Ablation of LH GABAergic neurons (LHVGAT) decreases weight gain and food intake, whereas LHLEPR ablation does not. Appetitive learning in a Pavlovian conditioning paradigm is delayed in LHVGAT-ablated mice but prevented entirely in LHLEPR-ablated mice. Both LHVGAT and LHLEPR neurons bidirectionally modulate reward-related behaviors, but only LHVGAT neurons affect feeding. In the Pavlovian paradigm, only LHLEPR activity discriminates between conditioned cues. Optogenetic activation or inhibition of either population in this task disrupts discrimination. However, manipulations of LHLEPR→VTA projections evoke divergent effects on responding. Unlike food-oriented learning, chemogenetic inhibition of LHLEPR neurons does not alter cocaine-conditioned place preference but attenuates cocaine sensitization. Thus, LHLEPR neurons may specifically regulate appetitive behaviors toward non-drug reinforcers.

The Buprenorphine Analogue BU10119 Attenuates Drug-Primed and Stress-Induced Cocaine Reinstatement in Mice.

There are no Food and Drug Administration-approved medications for cocaine use disorder, including relapse. The μ-opioid receptor (MOPr) partial agonist buprenorphine alone or in combination with naltrexone has been shown to reduce cocaine-positive urine tests and cocaine seeking in rodents. However, there are concerns over the abuse liability of buprenorphine. Buprenorphine's partial agonist and antagonist activity at the nociception receptor (NOPr) and κ-opioid receptor (KOPr), respectively, may contribute to its ability to inhibit cocaine seeking. Thus, we hypothesized that a buprenorphine derivative that exhibits antagonist activity at MOPr and KOPr with enhanced agonist activity at the NOPr could provide a more effective treatment. Here we compare the pharmacology of buprenorphine and two analogs, BU10119 and BU12004, in assays for antinociception and for cocaine- and stress-primed reinstatement in the conditioned place preference paradigm. In vitro and in vivo assays showed that BU10119 acts as an antagonist at MOPr, KOPr, and δ-opioid receptor (DOPr) and a partial agonist at NOPr, whereas BU12004 showed MOPr partial agonist activity and DOPr, KOPr, and NOPr antagonism. BU10119 and buprenorphine but not BU12004 lessened cocaine-primed reinstatement. In contrast, BU10119, BU12004, and buprenorphine blocked stress-primed reinstatement. The selective NOPr agonist SCH221510 but not naloxone decreased cocaine-primed reinstatement. Together, these findings are consistent with the concept that NOPr agonism contributes to the ability of BU10119 and buprenorphine to attenuate reinstatement of cocaine-conditioned place preference in mice. The findings support the development of buprenorphine analogs lacking MOPr agonism with increased NOPr agonism for relapse prevention to cocaine addiction. SIGNIFICANCE STATEMENT: There are no Food and Drug Administration-approved medications for cocaine use disorder. Buprenorphine has shown promise as a treatment for cocaine relapse prevention; however, there are concerns over the abuse liability of buprenorphine. Here we show a buprenorphine analogue, BU10119, which lacks μ-opioid receptor agonism and inhibits cocaine-primed and stress-primed reinstatement in a conditioned place-preference paradigm. The results suggest the development of BU10119 for the management of relapse to cocaine seeking.

CB1R activation in nucleus accumbens core promotes stress-induced reinstatement of cocaine seeking by elevating extracellular glutamate in a drug-paired context

Preclinical models of stress-induced relapse to drug use have shown that the dysregulation of glutamatergic transmission within the nucleus accumbens (NA) contributes notably to the reinstatement of cocaine-seeking behavior in rodents. In this sense, there has been increasing interest in the cannabinoid type-1 receptor (CB1R), due to its crucial role in modulating glutamatergic neurotransmission within brain areas involved in drug-related behaviors. This study explored the involvement of CB1R within the NA subregions in the restraint stress-induced reinstatement of cocaine-conditioned place preference (CPP), as well as in the regulation of glutamatergic transmission, by using a pharmacological approach and the in vivo microdialysis sampling technique in freely moving rats. CB1R blockade by the antagonist/inverse agonist AM251 (5 nmol/0.5 μl/side) or CB1R activation by the agonist ACEA (0.01 fmol/0.5 μl/side), prevented or potentiated restraint stress-induced reinstatement of cocaine-CPP, respectively, after local administration into NAcore, but not NAshell. In addition, microdialysis experiments demonstrated that restraint stress elicited a significant increase in extracellular glutamate in NAcore under reinstatement conditions, with the local administration of AM251 or ACEA inhibiting or potentiating this, respectively. Interestingly, this rise specifically corresponded to the cocaine-associated CPP compartment. We also showed that this context-dependent change in glutamate paralleled the expression of cocaine-CPP, and disappeared after the extinction of this response. Taken together, these findings demonstrated the key role played by CB1R in mediating reinstatement of cocaine-CPP after restraint stress, through modulation of the context-specific glutamate release within NAcore. Additionally, CB1R regulation of basal extracellular glutamate was demonstrated and proposed as the underlying mechanism.