Synthetic contraceptive hormones occlude the ability of nicotine to reduce ethanol consumption in ovary-intact female rats.

Rates of tobacco and alcohol use in women are rising, and women are more vulnerable than men to escalating tobacco and alcohol use. Many women use hormonal birth control, with the oral contraceptive pill being the most prevalent. Oral contraceptives contain both a progestin (synthetic progesterone) and a synthetic estrogen (ethinyl estradiol; EE) and are contraindicated for women over 35 years who smoke. Despite this, no studies have examined how synthetic contraceptive hormones impact this pattern of polysubstance use in females. To address this critical gap in the field, we treated ovary-intact female rats with either sesame oil (vehicle), the progestin levonorgestrel (LEVO; contained in formulations such as Alesse®), or the combination of EE+LEVO in addition to either undergoing single (nicotine or saline) or polydrug (nicotine and ethanol; EtOH) self-administration (SA) in a sequential use model. Rats preferred EtOH over water following extended EtOH drinking experience as well as after nicotine or saline SA experience, and rats undergoing only nicotine SA (water controls) consumed more nicotine as compared to rats co-using EtOH and nicotine. Importantly, this effect was occluded in groups treated with contraceptive hormones. In the sequential use group, both LEVO alone and the EE+LEVO combination occluded the ability of nicotine to decrease EtOH consumption. Interestingly, demand experiments suggest an economic substitute effect between nicotine and EtOH. Together, we show that chronic synthetic hormone exposure impacts nicotine and EtOH sequential use, demonstrating the crucial need to understand how chronic use of different contraceptive formulations alter patterns of polydrug use in women.

Oxytocin Reduces Sensitized Stress-Induced Alcohol Relapse in a Model of Posttraumatic Stress Disorder and Alcohol Use Disorder Comorbidity.

BACKGROUND: There is high comorbidity of posttraumatic stress disorder (PTSD) and alcohol use disorder with few effective treatment options. Animal models of PTSD have shown increases in alcohol drinking, but effects of stress history on subsequent vulnerability to alcohol relapse have not been examined. Here we present a mouse model of PTSD involving chronic multimodal stress exposure that resulted in long-lasting sensitization to stress-induced alcohol relapse, and this sensitized stress response was blocked by oxytocin (OT) administration. METHODS: Male and female mice trained to self-administer alcohol were exposed to predator odor (TMT) + yohimbine over 5 consecutive days or left undisturbed. After reestablishing stable alcohol responding/intake, mice were tested under extinction conditions, and then all mice were exposed to TMT or context cues previously associated with TMT before a reinstatement test session. Separate studies examined messenger RNA expression of Oxt and Oxtr in hypothalamus following chronic stress exposure. A final study examined the effects of systemic administration of OT on stress-induced alcohol relapse in mice with and without a history of chronic stress experience. RESULTS: Chronic stress exposure produced long-lasting sensitization to subsequent stress-induced alcohol relapse that also generalized to stress-related context cues and transcriptional changes in hypothalamic OT system. OT injected before the reinstatement test session completely blocked the sensitized stress-induced alcohol relapse effect. CONCLUSIONS: Collectively, these results provide support for the therapeutic potential of OT, along with highlighting the value of utilizing this model in evaluating other pharmacological interventions for treatment of PTSD/alcohol use disorder comorbidity.

Alcohol dependence and the ventral hippocampal influence on alcohol drinking in male mice.

Examining neural circuits underlying persistent, heavy drinking provides insight into the neurobiological mechanisms driving alcohol use disorder. Facilitated by its connectivity with other parts of the brain such as the nucleus accumbens (NAc), the ventral hippocampus (vHC) supports many behaviors, including those related to reward seeking and addiction. These studies used a well-established mouse model of alcohol (ethanol) dependence. After surgery to infuse DREADD-expressing viruses (hM4Di, hM3Dq, or mCherry-only) into the vHC and position guide cannula above the NAc, male C57BL/6J mice were treated in the CIE drinking model that involved repeated cycles of chronic intermittent alcohol (CIE) vapor or air (CTL) exposure alternating with weekly test drinking cycles in which mice were offered alcohol (15% v/v) 2 h/day. Additionally, smaller groups of mice were evaluated for either cFos expression or glutamate release using microdialysis procedures. In CIE mice expressing inhibitory (hM4Di) DREADDs in the vHC, drinking increased as expected, but CNO (3 mg/kg intraperitoneally [i.p.]) given 30 min before testing did not alter alcohol intake. However, in CTL mice expressing hM4Di, CNO significantly increased alcohol drinking (∼30%; p < 0.05) to levels similar to the CIE mice. The vHC-NAc pathway was targeted by infusing CNO into the NAc (3 or 10 µM/side) 30 min before testing. CNO activation of the pathway in mice expressing excitatory (hM3Dq) DREADDs selectively reduced consumption in CIE mice back to CTL levels (∼35-45%; p < 0.05) without affecting CTL alcohol intake. Lastly, activating the vHC-NAc pathway increased cFos expression and evoked significant glutamate release from the vHC terminals in the NAc. These data indicate that reduced activity of the vHC increases alcohol consumption and that targeted, increased activity of the vHC-NAc pathway attenuates excessive drinking associated with alcohol dependence. Thus, these findings indicate that the vHC and its glutamatergic projections to the NAc are involved in excessive alcohol drinking.

Dynorphin/Kappa Opioid Receptor Activity Within the Extended Amygdala Contributes to Stress-Enhanced Alcohol Drinking in Mice.

BACKGROUND: While there is high comorbidity of stress-related disorders and alcohol use disorder, few effective treatments are available and elucidating underlying neurobiological mechanisms has been hampered by a general lack of reliable animal models. Here, we use a novel mouse model demonstrating robust and reproducible stress-enhanced alcohol drinking to examine the role of dynorphin/kappa opioid receptor (DYN/KOR) activity within the extended amygdala in mediating this stress-alcohol interaction. METHODS: Mice received repeated weekly cycles of chronic intermittent ethanol exposure alternating with weekly drinking sessions ± forced swim stress exposure. Pdyn messenger RNA expression was measured in the central amygdala (CeA), and DYN-expressing CeA neurons were then targeted for chemogenetic inhibition. Finally, a KOR antagonist was microinjected into the CeA or bed nucleus of the stria terminalis to examine the role of KOR signaling in promoting stress-enhanced drinking. RESULTS: Stress (forced swim stress) selectively increased alcohol drinking in mice with a history of chronic intermittent ethanol exposure, and this was accompanied by elevated Pdyn messenger RNA levels in the CeA. Targeted chemogenetic silencing of DYN-expressing CeA neurons blocked stress-enhanced drinking, and KOR antagonism in the CeA or bed nucleus of the stria terminalis significantly reduced stress-induced elevated alcohol consumption without altering moderate intake in control mice. CONCLUSIONS: Using a novel and robust model of stress-enhanced alcohol drinking, a significant role for DYN/KOR activity within extended amygdala circuitry in mediating this effect was demonstrated, thereby providing further evidence that the DYN/KOR system may be a valuable target in the development of more effective treatments for individuals presenting with comorbidity of stress-related disorders and alcohol use disorder.

Activation of hypothalamic oxytocin neurons reduces binge-like alcohol drinking through signaling at central oxytocin receptors.

Preclinical and clinical evidence suggests that exogenous administration of oxytocin (OT) may hold promise as a therapeutic strategy for reducing heavy alcohol drinking. However, it remains unknown whether these effects are mediated by stimulation of endogenous sources of OT and signaling at oxytocin receptors (OTR) in brain or in the periphery. To address this question, we employed a targeted chemogenetic approach to examine whether selective activation of OT-containing neurons in the paraventricular nucleus of the hypothalamus (PVN) alters alcohol consumption in a binge-like drinking ("Drinking-in-the-Dark"; DID) model. Adult male Oxt-IRES-Cre mice received bilateral infusion of a Cre-dependent virus containing an excitatory DREADD (AAV8-hSyn-DIO-hM3Dq-mCherry) or control virus (AAV8-hSyn-DIO-mCherry) into the PVN. Chemogenetic activation of PVNOT+ neurons following clozapine-N-oxide injection reduced binge-like alcohol drinking in a similar manner as systemic administration of the neuropeptide. Pretreatment with a brain-penetrant OTR antagonist (L-368,899) reversed this effect while systemic administration of a peripherally restricted OTR antagonist (Atosiban) did not alter reduced alcohol drinking following chemogenetic activation of PVNOT+ neurons. Altogether, these data are the first to demonstrate that targeted activation of hypothalamic (endogenous) OT reduces alcohol consumption, providing further evidence that this neuropeptide plays a role in regulation of alcohol self-administration behavior. Further, results indicate that the ability OT to reduce alcohol drinking is mediated by signaling at OTR in the brain.

Effect of caffeine on alcohol drinking in mice.

Mixing alcohol (ethanol) with caffeinated beverages continues to be a common and risky practice. Energy drinks are one type of caffeinated beverage that may be especially problematic when used as mixers, due to their relatively high caffeine content in combination with their highly sweetened flavor profile. The present study used a mouse model of limited-access drinking and lickometer circuitry to examine the effects of an energy drink anid its caffeine content on ethanol consumption. Predictably, the highly sweetened energy drink significantly increased ethanol intake compared to a plain ethanol solution (6.34 ± 0.2 vs. 5.01 ± 0.3 g/kg; Cohen's d = 1.79). Interestingly, adulterating a plain ethanol solution with the same concentration of caffeine (without sweetener) found in the energy drink also increased ethanol intake (5.47 ± 0.3 vs. 4.11 ± 0.3 g/kg; Cohen's d = 1.4). A lower concentration of caffeine was without effect on ethanol drinking. Interestingly, plain caffeine solutions at both tested concentrations provoked high numbers of bottle contacts, indicating that the mice found the solution palatable. These findings suggest that altering the bitterness profile of an ethanol solution with the addition of caffeine can increase intake in a similar manner as sweetening the solution. Further, the findings underscore the importance of taste in motivating ethanol consumption and the potential role that caffeine can have in this process.

Effects of ceftriaxone on ethanol drinking and GLT-1 expression in ethanol dependence and relapse drinking.

Repeated cycles of chronic intermittent ethanol (CIE) exposure increase voluntary consumption of alcohol (ethanol) in mice. Previous reports from our laboratory show that CIE increases extracellular glutamate in the nucleus accumbens (NAc) and that manipulating accumbal glutamate concentrations will alter ethanol drinking, indicating that glutamate homeostasis plays a crucial role in ethanol drinking in this model. A number of studies have shown that ceftriaxone increases GLT-1 expression, the major glutamate transporter, and that treatment with this antibiotic reduces ethanol drinking. The present studies examined the effects of ceftriaxone on ethanol drinking and GLT-1 in a mouse model of ethanol dependence and relapse drinking. The results show that ceftriaxone did not influence drinking at any dose in either ethanol-dependent or non-dependent mice. Further, ceftriaxone did not increase GLT-1 expression in the accumbens core or shell, with the exception of the ethanol-dependent mice receiving the highest dose of ceftriaxone. Interestingly, ethanol-dependent mice treated with only vehicle displayed reduced expression of GLT-1 in the accumbens shell and of the presynaptic mGlu2 receptor in the accumbens core. The reduced expression of the major glutamate transporter (GLT-1), as well as a receptor that regulates glutamate release (mGlu2), may help explain, at least in part, increased glutamatergic transmission in this model of ethanol dependence and relapse drinking.

Kappa opioid receptors in the bed nucleus of the stria terminalis regulate binge-like alcohol consumption in male and female mice.

Binge drinking is the most common pattern of excessive alcohol consumption and is a significant contributor to the development of Alcohol Use Disorder and dependence. Previous studies demonstrated involvement of kappa opioid receptors (KOR) in binge-like drinking in mice using the Drinking-in-the-Dark model. The current studies examined the role of KOR specifically in the bed nucleus of the stria terminals (BNST) in binge-like alcohol consumption in male and female mice. Direct administration of the long lasting KOR antagonist, nor-BNI, into the BNST decreased binge-like alcohol consumption and blood alcohol concentrations in male and female C57BL/6J mice. Similarly, direct nor-BNI administration into the BNST modestly reduced sucrose consumption and the suppression of fluid intake was not related to reduced locomotor activity. To further determine the role of KOR within the BNST on binge-like alcohol consumption, the KOR agonist U50,488 was administered systemically which resulted in a robust increase in alcohol intake. Microinjection of nor-BNI into the BNST blocked the high level of alcohol intake after systemic U50,488 challenge reducing intake and resultant blood alcohol concentrations. Together, these data suggest that KOR activity in the BNST contributes to binge-like alcohol consumption in both male and female mice. This article is part of the special issue on 'Neuropeptides'.

Dynamic c-Fos changes in mouse brain during acute and protracted withdrawal from chronic intermittent ethanol exposure and relapse drinking.

Alcohol dependence promotes neuroadaptations in numerous brain areas, leading to escalated drinking and enhanced relapse vulnerability. We previously developed a mouse model of ethanol dependence and relapse drinking in which repeated cycles of chronic intermittent ethanol (CIE) vapor exposure drive a significant escalation of voluntary ethanol drinking. In the current study, we used this model to evaluate changes in neuronal activity (as indexed by c-Fos expression) throughout acute and protracted withdrawal from CIE (combined with or without a history of ethanol drinking). We analyzed c-Fos protein expression in 29 brain regions in mice sacrificed 2, 10, 26, and 74 hours or 7 days after withdrawal from 5 cycles of CIE. Results revealed dynamic time- and brain region-dependent changes in c-Fos activity over the time course of withdrawal from CIE exposure, as compared with nondependent air-exposed control mice, beginning with markedly low expression levels upon removal from the ethanol vapor chambers (2 hours), reflecting intoxication. c-Fos expression was enhanced during acute CIE withdrawal (10 and 26 hours), followed by widespread reductions at the beginning of protracted withdrawal (74 hours) in several brain areas. Persistent reductions in c-Fos expression were observed during prolonged withdrawal (7 days) in prelimbic cortex, nucleus accumbens shell, dorsomedial striatum, paraventricular nucleus of thalamus, and ventral subiculum. A history of ethanol drinking altered acute CIE withdrawal effects and caused widespread reductions in c-Fos that persisted during extended abstinence even without CIE exposure. These data indicate that ethanol dependence and relapse drinking drive long-lasting neuroadaptations in several brain regions.

Brain Regional and Temporal Changes in BDNF mRNA and microRNA-206 Expression in Mice Exposed to Repeated Cycles of Chronic Intermittent Ethanol and Forced Swim Stress.

Brain-derived neurotrophic factor (BDNF) expression and signaling activity in brain are influenced by chronic ethanol and stress. We previously demonstrated reduced Bdnf mRNA levels in the medial prefrontal cortex (mPFC) following chronic ethanol treatment and forced swim stress (FSS) enhanced escalated drinking associated with chronic ethanol exposure. The present study examined the effects of chronic ethanol and FSS exposure, alone and in combination, on Bdnf mRNA expression in different brain regions, including mPFC, central amygdala (CeA), and hippocampus (HPC). Additionally, since microRNA-206 has been shown to negatively regulate BDNF expression, the effects of chronic ethanol and FSS on its expression in the target brain regions were examined. Mice received four weekly cycles of chronic intermittent ethanol (CIE) vapor or air exposure and then starting 72-h later, the mice received either a single or 5 daily 10-min FSS sessions (or left undisturbed). Brain tissue samples were collected 4-h following final FSS testing and Bdnf mRNA and miR-206 levels were determined by qPCR assay. Results indicated dynamic brain regional and time-dependent changes in Bdnf mRNA and miR-206 expression. In general, CIE and FSS exposure reduced Bdnf mRNA expression while miR-206 levels were increased in the mPFC, CeA, and HPC. Further, in many instances, these effects were more robust in mice that experienced both CIE and FSS treatments. These results have important implications for the potential link between BDNF signaling in the brain and ethanol consumption related to stress interactions with chronic ethanol experience.

Dynorphin-kappa opioid receptor activity in the central amygdala modulates binge-like alcohol drinking in mice.

Although previous research has demonstrated a role for kappa opioid receptor-mediated signaling in escalated alcohol consumption associated with dependence and stress exposure, involvement of the dynorphin/kappa opioid receptor (DYN/KOR) system in binge-like drinking has not been fully explored. Here we used pharmacological and chemogenetic approaches to examine the influence of DYN/KOR signaling on alcohol consumption in the drinking-in-the-dark (DID) model of binge-like drinking. Systemic administration of the KOR agonist U50,488 increased binge-like drinking (Experiment 1) while, conversely, systemic administration of the KOR antagonist nor-BNI reduced drinking in the DID model (Experiment 2). These effects of systemic KOR manipulation were selective for alcohol as neither drug influenced consumption of sucrose in the DID paradigm (Experiment 3). In Experiment 4, administration of the long-acting KOR antagonist nor-BNI into the central nucleus of the amygdala (CeA) decreased alcohol intake. Next, targeted "silencing" of DYN+ neurons in the CeA was accomplished using a chemogenetic strategy. Cre-dependent viral expression in DYN+ neurons was confirmed in CeA of Pdyn-IRES-Cre mice and functionality of an inhibitory (hM4Di) DREADD was validated (Experiment 5). Activating the inhibitory DREADD by CNO injection reduced binge-like alcohol drinking, but CNO injection did not alter alcohol intake in mice that were treated with control virus (Experiment 6). Collectively, these results demonstrate that DYN/KOR signaling in the CeA contributes to excessive alcohol consumption in a binge-drinking model.

Increasing Brain-Derived Neurotrophic Factor (BDNF) in medial prefrontal cortex selectively reduces excessive drinking in ethanol dependent mice.

The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) has been implicated in a number of neuropsychiatric disorders, including alcohol use disorder. Studies have shown that BDNF activity in cortical regions, such as the medial prefrontal cortex (mPFC) mediates various ethanol-related behaviors. We previously reported a significant down-regulation in Bdnf mRNA in mPFC following chronic ethanol exposure compared to control mice. The present study was conducted to extend these findings by examining whether chronic ethanol treatment reduces BDNF protein expression in mPFC and whether reversing this deficit via direct injection of BDNF or viral-mediated overexpression of BDNF in mPFC alters voluntary ethanol consumption in dependent and nondependent mice. Repeated cycles of chronic intermittent ethanol (CIE) exposure was employed to model ethanol dependence, which produces robust escalation of ethanol intake. Results indicated that CIE treatment significantly increased ethanol intake and this was accompanied by a significant decrease in BDNF protein in mPFC that lasted at least 72 h after CIE exposure. In a separate study, once dependence-related increased drinking was established, bilateral infusion of BDNF (0, 0.25, 0.50 µg) into mPFC significantly decreased ethanol intake in a dose-related manner in dependent mice but did not affect moderate drinking in nondependent mice. In a third study, viral-mediated overexpression of BDNF in mPFC prevented escalation of drinking in dependent mice but did not alter intake in nondependent mice. Collectively, these results provide evidence that adaptations in cortical (mPFC) BDNF activity resulting from chronic ethanol exposure play a role in mediating excessive ethanol drinking associated with dependence.

Corticostriatal plasticity, neuronal ensembles, and regulation of drug-seeking behavior.

The idea that interconnected neuronal ensembles code for specific behaviors has been around for decades; however, recent technical improvements allow studying these networks and their causal role in initiating and maintaining behavior. In particular, the role of ensembles in drug-seeking behaviors in the context of addiction is being actively investigated. Concurrent with breakthroughs in quantifying ensembles, research has identified a role for synaptic glutamate spillover during relapse. In particular, the transient relapse-associated changes in glutamatergic synapses on accumbens neurons, as well as in adjacent astroglia and extracellular matrix, are key elements of the synaptic plasticity encoded by drug use and the metaplasticity induced by drug-associated cues that precipitate drug-seeking behaviors. Here, we briefly review the recent discoveries related to ensembles in the addiction field and then endeavor to link these discoveries with drug-induced striatal plasticity and cue-induced metaplasticity toward deeper neurobiological understandings of drug seeking.

Prosapip1-Dependent Synaptic Adaptations in the Nucleus Accumbens Drive Alcohol Intake, Seeking, and Reward.

The mammalian target of rapamycin complex 1 (mTORC1), a transducer of local dendritic translation, participates in learning and memory processes as well as in mechanisms underlying alcohol-drinking behaviors. Using an unbiased RNA-seq approach, we identified Prosapip1 as a novel downstream target of mTORC1 whose translation and consequent synaptic protein expression are increased in the nucleus accumbens (NAc) of mice excessively consuming alcohol. We demonstrate that alcohol-dependent increases in Prosapip1 levels promote the formation of actin filaments, leading to changes in dendritic spine morphology of NAc medium spiny neurons (MSNs). We further demonstrate that Prosapip1 is required for alcohol-dependent synaptic localization of GluA2 lacking AMPA receptors in NAc shell MSNs. Finally, we present data implicating Prosapip1 in mechanisms underlying alcohol self-administration and reward. Together, these data suggest that Prosapip1 in the NAc is a molecular transducer of structural and synaptic alterations that drive and/or maintain excessive alcohol use.

Oxytocin Reduces Ethanol Self-Administration in Mice.

BACKGROUND: Excessive ethanol (EtOH) consumption remains an important health concern and effective treatments are lacking. The central oxytocin system has emerged as a potentially important therapeutic target for alcohol and drug addiction. These studies tested the hypothesis that oxytocin reduces EtOH consumption. METHODS: Male C57BL/6J mice were given access to EtOH (20% v/v) using a model of binge-like drinking ("drinking in the dark") that also included the use of lickometer circuits to evaluate the temporal pattern of intake as well as 2-bottle choice drinking in the home cage. In addition, EtOH (12% v/v) and sucrose (5% w/v) self-administration on fixed- and progressive-ratio schedules were also evaluated. A wide range of systemically administered oxytocin doses were tested (0 to 10 mg/kg) in these models. RESULTS: Oxytocin (0, 0.3, 1, 3, or 10 mg/kg) dose dependently reduced EtOH consumption (maximal 45% reduction) in the binge drinking model, with lower effective doses having minimal effects on general locomotor activity. Oxytocin's effect was blocked by pretreatment with an oxytocin receptor antagonist, and the pattern of contacts (licks) at the EtOH bottle suggested a reduction in motivation to drink EtOH. Oxytocin decreased 2-bottle choice drinking without altering general fluid intake. Oxytocin also reduced operant responding for EtOH and sucrose in a dose-related manner. However, oxytocin decreased responding and motivation (breakpoint values) for EtOH at doses that did not alter responding for sucrose. CONCLUSIONS: These results indicate that oxytocin reduces EtOH consumption in different models of self-administration. The effects are not likely due to a general sedative effect of the neuropeptide. Further, oxytocin reduces motivation for EtOH at doses that do not alter responding for a natural reward (sucrose). While some evidence supports a role for oxytocin receptors in mediating these effects, additional studies are needed to further elucidate underlying mechanisms. Nevertheless, these results support the therapeutic potential of oxytocin as a treatment for alcohol use disorder.

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.

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.

Chronic intermittent ethanol exposure and withdrawal leads to adaptations in nucleus accumbens core postsynaptic density proteome and dendritic spines.

Alcohol use disorder is a chronic relapsing brain disease characterized by the loss of ability to control alcohol (ethanol) intake despite knowledge of detrimental health or personal consequences. Clinical and pre-clinical models provide strong evidence for chronic ethanol-associated alterations in glutamatergic signaling and impaired synaptic plasticity in the nucleus accumbens (NAc). However, the neural mechanisms that contribute to aberrant glutamatergic signaling in ethanol-dependent individuals in this critical brain structure remain unknown. Using an unbiased proteomic approach, we investigated the effects of chronic intermittent ethanol (CIE) exposure on neuroadaptations in postsynaptic density (PSD)-enriched proteins in the NAc of ethanol-dependent mice. Compared with controls, CIE exposure significantly changed expression levels of 50 proteins in the PSD-enriched fraction. Systems biology and functional annotation analyses demonstrated that the dysregulated proteins are expressed at tetrapartite synapses and critically regulate cellular morphology. To confirm this latter finding, the density and morphology of dendritic spines were examined in the NAc core of ethanol-dependent mice. We found that CIE exposure and withdrawal differentially altered dendrite diameter and dendritic spine density and morphology. Through the use of quantitative proteomics and functional annotation, these series of experiments demonstrate that ethanol dependence produces neuroadaptations in proteins that modify dendritic spine morphology. In addition, these studies identified novel PSD-related proteins that contribute to the neurobiological mechanisms of ethanol dependence that drive maladaptive structural plasticity of NAc neurons.

Kv7 channels in the nucleus accumbens are altered by chronic drinking and are targets for reducing alcohol consumption.

Alcohol use disorders (AUDs) are a major public health issue and produce enormous societal and economic burdens. Current Food and Drug Administration (FDA)-approved pharmacotherapies for treating AUDs suffer from deleterious side effects and are only effective in a subset of individuals. It is therefore essential to find improved medications for the management of AUDs. Emerging evidence suggests that anticonvulsants are a promising class of drugs for treating individuals with AUDs. In these studies, we used integrative functional genomics to demonstrate that genes that encode Kv7 channels (i.e. Kcnq2/3) are related to alcohol (ethanol) consumption, preference and acceptance in rodents. We then tested the ability of the FDA-approved anticonvulsant retigabine, a Kv7 channel opener, to reduce voluntary ethanol consumption of Wistar rats in a two-bottle choice intermittent alcohol access paradigm. Systemic administration and microinjections of retigabine into the nucleus accumbens significantly reduced alcohol drinking, and retigabine was more effective at reducing intake in high- versus low-drinking populations of Wistar rats. Prolonged voluntary drinking increased the sensitivity to the proconvulsant effects of pharmacological blockade of Kv7 channels and altered surface trafficking and SUMOylation patterns of Kv7.2 channels in the nucleus accumbens. These data implicate Kcnq2/3 in the regulation of ethanol drinking and demonstrate that long-term drinking produces neuroadaptations in Kv7 channels. In addition, these results have identified retigabine as a potential pharmacotherapy for treating AUDs and Kv7 channels as a novel therapeutic target for reducing heavy drinking.

Sensitization and Tolerance Following Repeated Exposure to Caffeine and Alcohol in Mice.

BACKGROUND: Energy drinks are popular mixers with alcohol. While energy drinks contain many ingredients, caffeine is an important pharmacologically active component and is generally present in larger amounts than in other caffeinated beverages. In these studies, we investigated the hypothesis that caffeine would influence the effects of alcohol (ethanol [EtOH]) on conditioned taste aversion (CTA), ataxia, and locomotor activity (LA) after repeated exposure. METHODS: Four groups of mice were exposed by oral gavage twice daily to vehicle, EtOH (4 g/kg), caffeine (15 mg/kg), or the EtOH/caffeine combination. CTA to saccharin and ataxia in the parallel rod task was evaluated after 8 or 16 gavages, respectively, using EtOH (1 to 3 g/kg) or EtOH/caffeine (3 mg/kg + 2 g/kg) challenges. In addition, LA was evaluated initially and after repeated exposure to oral gavage of these drugs and doses. RESULTS: Repeated oral gavage of EtOH produced significant locomotor sensitization, with those mice increasing total distance traveled by 2-fold. The locomotor response to caffeine, while significantly greater than vehicle gavage, did not change with repeated exposure. On the other hand, repeated gavage of caffeine/EtOH combination produced a substantial increase in total distance traveled after repeated exposure (~4-fold increase). After repeated EtOH exposure, there was significant tolerance to EtOH in the CTA and parallel rod tests. However, neither a history of caffeine exposure nor including caffeine influenced EtOH-induced CTA. Interestingly, a history of caffeine exposure increased the ataxic response to the caffeine/EtOH combination and appeared to reduce the ataxic response to high doses of EtOH. CONCLUSIONS: The data support the general hypothesis that repeated exposure to caffeine influences the response to EtOH. Together with previously published work, these data indicate that caffeine influences some EtOH-related behaviors, notably locomotion and ataxia, but appears not to influence the expression of conditioned behaviors.