NK1 receptors: Previous work in the lab found that L822429, a rat-specific NK1 antagonist, dose-dependently suppressed 1) stress-induced reinstatement of alcohol seeking in Wistar rats, and 2) self-administration in alcohol preferring P-rats (Schank et al., 2011; Schank et al., 2013). Furthermore, an upregulation of NK1R expression was found in the central amygdata (CeA) of P-rats. Fos-mapping showed that stress-induced reinstatement of alcohol seeking is associated with neuronal activation in the amygdala (amg), NAc, dorsal raphe nucleus (DR), medial prefrontal cortex (mPFC) and bed nucleus of the stria terminalis. NK1R antagonism suppressed the stress-induced increase in Fos in a subset of these regions, particularly the DR and NAc shell, suggesting that these regions mediate the effect of NK1R antagonism to block stress-induced relapse (Schank et al., 2015). DNA methylation: Substantial evidence supports a role of epigenetic mechanisms in the regulation of alcohol-related behaviors. We found that i.c.v. infusion of RG-108, a specific DNA methyltransferase inhibitor, decreased operant alcohol self-administration in post-dependent (PD) rats, and reversed long-term expression changes encoding synaptic proteins involved in neurotransmitter release in the mPFC induced by a history of dependence. The effects of RG-108 were reproduced with local infusion into the mPFC over 7-10 days and were behaviorally selective. Infusion of RG108 prevented both escalation of alcohol consumption and dependence-induced down-regulation of 4 out of the 7 transcripts modified in PD rats. Specifically, RG108 treatment directly reversed the down-regulation of synaptotagmin 2 (syt2), which was caused by alcohol-induced hypermethylation. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in alcohol seeking. (Barbier et al., 2015). microRNA regulation: Following up on previous work (Tapocik et al., 2012), we qPCR confirmed that alcohol dependence results in persistent up-regulation of miR-206 in the mPFC. Overexpression of miR-206 in the mPFC of non-dependent rats reproduced the escalation of alcohol self-administration. Accordingly, BDNF was downregulated in PD rats on microarray analysis, and this was confirmed by qPCR. In vitro, BDNF expression was repressed by miR-206 in a 3'UTR reporter assay, confirming BDNF as a functional target of miR-206. Inhibition of miR-206 expression in differentiated rat cortical primary neurons significantly increased secreted levels of BDNF. Therefore, recruitment of miR-206 in the mPFC contributes to escalated alcohol consumption and BDNF regulation (Tapocik et al, 2014). We have recently confirmed this finding in a mouse model of alcohol dependence where BDNF expression is down and mir-206 expression is upregulated. Furthermore, miR-206 KO mice have a blunted ethanol response to alcohol vapor (Tapocik et al., in prep.). Melanin Concentrating Hormone (MCH): MCH is a hypothalamic appetite regulating peptide that is also involved in reward, motivation and anxiety-like behaviors mediated by the MCH-1 receptor (MCH-1R) in rodents. MCH1-R blockade suppresses alcohol self-administration (Cippitelli et al., 2010). Because alcohol is also a caloric nutrient, self-administration results could be confounded by appetite effects. We therefore evaluated the role of the MCH system for the rewarding properties of alcohol using conditioned place preference (CPP). Alcohol-induced CPP was markedly decreased in mice with a genetic deletion of the MCH1-R as well as after pharmacological treatment with an MCH1-R antagonist, GW803430. In contrast, an isocaloric dose of dextrose did not produce CPP. Furthermore, wild-type (WT) mice treated with alcohol showed increased levels of DARPP-32 in the NAcSh, suggesting that MCH1-Rs regulate the rewarding properties of alcohol through interactions with signaling cascades downstream of DA receptors in the NAcSh (Karlsson et al. in prep.). Proinflammatory activity: Innate immunity might be involved in stress responses and behaviors related to addiction. We investigated the role of the IL-1 receptor (IL-1R) in alcohol related behaviors. Mice lacking IL-1Rs consumed less alcohol in a two bottle choice test, but CPP experiments showed that the decreased alcohol consumption in the IL-1R mice is not due to effects on alcohol reward. IL1-R KO mice had significantly longer sleep time when evaluated for LORR, indicating higher sensitivity to sedative/ataxic properties of alcohol than controls. In a model of stress-induced drinking, social defeat stress (SDS), both genotypes potently increased their consumption acutely, but no genotype differences were seen. Because IL-1 beta and TNF-a share overlapping pathways and mayact in parallel, we next used a double KO mouse model lacking both IL-1 and TNF-1 receptors. The double KOs consumed less alcohol, and did not escalate their consumption with increasing alcohol concentrations. This is not likely to be due to differences in the rewarding properties of alcohol, as double KO mice showed a robust expression of alcohol induced CPP (Karlsson et al., in prep.). Post-Traumatic Stress Disorder (PTSD): High rates of comorbidity exist between alcoholism and PTSD and it is unclear how exposure to traumatic experience increases the risk for alcoholism in adulthood. We developed a novel rat model of PTSD whereby rats were exposed to a snake (live predator exposure; LPE). LPE caused a robust activation of c-Fos expression in the lateral subregion of the lateral habenula (LHb). Furthermore, LPE rats spent more time in the closed arms of the elevated plus maze indicative of a classic anxiety-like phenotype. During fear conditioning, LPE animals displayed more freezing during contextual and cue expression tests than control animals. No baseline drinking differences were observed, however, context exposure significantly increased drinking in LPE animals. Exposure to a live predator induces PTSD-like symptoms and significantly escalates context induced consumption offering a novel comorbidity model to study the link between PTSD and alcoholism (Tapocik et al., in prep.). Gamma-aminobutyric acid (GABAB) receptor: GABAB and GABAB receptors plays an important role in the neurobiology of addiction. GABAB is involved in emotional processing, decision-making and anxious behavior. In clinical studies, GABAB agonists such as baclofen affect alcohol drinking and other drugs of abuse. We recently investigated the role of another drug from this group, ADX71441, testing whether different concentrations (1, 3 10 and 30 mg/kg) would reduce lever pressing for ethanol in rats. All concentrations resulted in a significant reduction in lever pressing during self-administration sessions, as well as reductions in active lever pressing during cue- and stress-induced reinstatement (Augier et al., in prep.). Self-administration of 20 % ethanol: Operant self-administration (SA) is an important model of motivation to consume ethanol (EtOH), but low rates of voluntary consumption in rats are thought to necessitate water deprivation and saccharin/sucrose fading for acquisition of responding. We sought to devise a model of SA that does not use water deprivation or saccharin/sucrose fading. Specifically, we tested if Wistar rats would acquire and maintain SA behavior of 20 % EtOH under two conditions, water deprivation (WD) and non-water deprivation (NWD). We found that Wistar rats acquire and maintain stable SA of 20%EtOH without the use of WD, extended access training, or saccharin/sucrose fading. These findings provide a method for voluntary oral EtOH SA in rats that is convenient for experimenters and eliminates the potential confound of sweeteners in EtOH-operant SA studies (Augier et al., 2014).
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