• Energy Research

Recent animal studies have shown that the level of stress-responsive arginine vasopressin (AVP) gene expression in the amygdala is increased during early withdrawal from long-term heroin or cocaine administration. The selective AVP V1b receptor antagonist SSR149415 (capable of exerting antidepressant-like and anxiolytic effects in animal models) also blocked stress-induced reinstatement of drug-seeking behavior. This study was undertaken to investigate the effects of alcohol and to determine whether (i) there are genetically determined differences in basal AVP mRNA levels in the medial/central amygdala (Me/CeA) and medial hypothalamus (MH) between selectively bred Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rats; (ii) the AVP mRNA levels are altered by long-term alcohol drinking in sP rats; and (iii) the V1b receptor antagonist SSR149415 alters alcohol drinking in sP rats.In Experiment 1, AVP mRNA levels were measured in the Me/CeA and MH of alcohol-naïve sP and sNP rats, and sP rats exposed to the standard, homecage 2-bottle "alcohol versus water" choice regimen 24 h/d for 17 days. In Experiment 2, SSR149415 (0, 3, 10, or 30 mg/kg; intraperitoneal) was acutely administered 30 minutes before lights off to alcohol-experienced sP rats. Alcohol, water, and food intake were monitored 6 and 24 hours later.We found higher basal AVP mRNA levels in both Me/CeA and MH of alcohol-naïve sP than sNP rats; alcohol consumption decreased AVP mRNA levels in both brain regions of sP rats, suggesting genetically determined differences between the 2 rat lines and in the effects of alcohol drinking in sP rats. Acute treatment with SSR149415 significantly reduced alcohol intake of sP rats.The stress-responsive AVP/V1b receptor system is 1 component of the neural circuitry underlying high alcohol drinking in sP rats.

BackgroundEvidence obtained in humans and rodents indicates that beta‐endorphin (encoded by the proopiomelanocortin [POMC] gene) is critical in the regulation of alcohol drinking behavior. However, the alcohol effect on POMC gene expression has not been studied in rodent mesolimbic regions, such as the nucleus accumbens (NAc).MethodsIn this study, we first utilized POMC‐enhanced green fluorescent protein (EGFP) transgenic mice to visualize POMC neurons and found that POMC‐EGFP cells were modestly distributed throughout the NAc shell and core, in addition to the hypothalamic arcuate nucleus. POMC mRNA expression in the NAc of mice and rats was confirmed using reverse transcriptase‐polymerase chain reaction and solution hybridization assays. We then investigated whether there are genetically determined differences in basal mRNA levels of POMC and mu opioid receptor (MOP‐r) between selectively bred Sardinian alcohol‐preferring (sP) and nonpreferring (sNP) rats, and whether these mRNA levels are altered in sP rats after alcohol drinking (10%, unlimited access) for 17 days.ResultsAlcohol‐naïve sP rats had higher basal POMC mRNA levels than sNP rats only in hypothalamus. Alcohol drinking increased POMC mRNA levels in both the NAc shell (by 100%) and the hypothalamus (by 50%) of sP rats. Although sP rats had lower basal levels of MOP‐r mRNA and GTPγS binding in NAc shell than sNP rats, voluntary alcohol consumption had no effect on MOP‐r mRNA levels in the NAc shell.ConclusionsOur results define the distribution of POMC‐expressing neurons in the NAc of mice and rats. Higher POMC expression at basal levels in sP rats (genetically determined), along with increases after drinking (alcohol‐induced) in the NAc shell and hypothalamus, suggests that the POMC systems play a role in high alcohol preference and consumption.

Addictive diseases, including addiction to alcohol, opiates or cocaine, pose massive public health costs. Addictions are chronic relapsing brain diseases, caused by drug-induced direct effects and persistent neuroadaptations at the molecular, cellular and behavioral levels. These drug-type specific neuroadapations are mainly contributed by three factors: environment, including stress, the direct reinforcing effects of the drug on the CNS, and genetics. Results from animal models and basic clinical research (including human genetic study) have shown important interactions between the stress responsive systems and alcohol abuse. In this review we will discuss the involvement of the dysregulation of the stress responsive hypothalamic-pituitary-adrenal (HPA) axis in alcohol addiction (Section I). Addictions to specific drugs such as alcohol, psychostimulants and opiates (e.g., heroin) have some common direct or downstream effects on several brain stress-responsive systems, including vasopressin and its receptor system (Section II), POMC and mu opioid receptor system (Section III) and dynorphin and kappa opioid receptor systems (Section IV). Further understanding of these systems, through laboratory-based and translational studies, have the potential to optimize early interventions and to discover new treatment targets for the therapy of alcoholism. This article is part of the Special Issue entitled 'CNS Stimulants'.

Metabolic dysfunction, mood disorders, anxiety disorders, and substance abuse disorders are associated with disruptions in circadian rhythm and circadian clock gene machinery. While the effects of alcohol on several core components of the clock genes have been described in rodent models, pharmacological activation or inhibition of clock gene functions has not been studied on alcohol drinking behaviors.We investigated whether cryptochrome (CRY1/2) activator KL001 altered alcohol intake in mice in excessive and relapse-like alcohol drinking models.Mice, subjected to 3 weeks of chronic intermittent alcohol drinking (IAD) (two-bottle choice, 24-h access every other day) developed excessive alcohol intake and high preference. We evaluated the pharmacological effects of KL001 after either 1-day acute withdrawal from IAD or 1-week chronic withdrawal using the alcohol deprivation effect (ADE) model.Single pretreatment with KL001 at 1-4 mg kg-1 reduced alcohol intake and preference after acute withdrawal in a dose-related manner. The effect of KL001 on reducing excessive alcohol consumption seems alcohol specific, as the compound does not alter sucrose (caloric reinforcer) or saccharin (noncaloric reinforcer) consumption in mice. Both single- and multiple-dosing regimens with an effective dose of KL001 (4 mg kg-1) prevented the ADE after chronic withdrawal, with no tolerance development after the multi-dosing regimen.Pretreatment with KL001 (a CRY1/2 activator) reduces excessive and "relapse" alcohol drinking in mice. Our in vivo results with a CRY activator suggest a possible novel target for alcohol treatment intervention.

AbstractPersistent alterations of proopiomelanocortin (Pomc) and mu‐opioid receptor (Oprm1) activity and stress responses after alcohol are critically involved in vulnerability to alcohol dependency. Gene transcriptional regulation altered by alcohol may play important roles. Mice with genome‐wide deletion of neuronal Pomc enhancer1 (nPE1−/−), had hypothalamic‐specific partial reductions of beta‐endorphin and displayed lower alcohol consumption, compared to wildtype littermates (nPE1+/+). We used RNA‐Seq to measure steady‐state nuclear mRNA transcripts of opioid and stress genes in hypothalamus of nPE1+/+ and nPE1−/− mice after 1‐day acute withdrawal from chronic excessive alcohol drinking or after water. nPE1−/− had lower basal Pomc and Pdyn (prodynorphin) levels compared to nPE1+/+, coupled with increased basal Oprm1 and Oprk1 (kappa‐opioid receptor) levels, and low alcohol drinking increased Pomc and Pdyn to the basal levels of nPE1+/+ in the water group, without significant effects on Oprm1 and Oprk1. In nPE1+/+, excessive alcohol intake increased Pomc and Oprm1, with no effect on Pdyn or Oprk1. For stress genes, nPE1−/− had lowered basal Oxt (oxytocin) and Avp (arginine vasopressin) that were restored by low alcohol intake to basal levels of nPE1+/+. In nPE1+/+, excessive alcohol intake decreased Oxt and Avpi1 (AVP‐induced protein1). Functionally examining the effect of pharmacological blockade of mu‐opioid receptor, we found that naltrexone reduced excessive alcohol intake in nPE1+/+, but not nPE1−/−. Our results provide evidence relevant to the transcriptional profiling of the critical genes in mouse hypothalamus: enhanced opioid and reduced stress gene transcripts after acute withdrawal from excessive alcohol may contribute to altered reward and stress responses.

Alcohol relapse is a treatment goal for alcohol dependence and the target for medications' development. Clinically utilized nalfurafine (NFF) is a potent and selective kappa- opioid receptor (KOP-r) agonist, with fewer side effects (e.g., sedation or anhedonia) than classic KOP-r full agonists. We have recently found that NFF reduces excessive alcohol drinking in mice via a KOP-r-mediated mechanism. Here, we further investigated whether NFF alone (1-10 μg/kg) or in combination with naltrexone (NTX, mu-opioid receptor [MOP-r] antagonist) altered alcohol relapse-like drinking using a mouse alcohol deprivation effect (ADE) paradigm to mimic the relapse episodes in human alcoholics. Nalmefene (NMF, clinically utilized KOP-r partial agonist with MOP-r antagonism) was used as a reference compound for the effects on mouse ADE of new NFF + NTX combination. After exposed to 3-week intermittent- access alcohol drinking (two-bottle choice, 24-h access every other day), both male and female mice displayed excessive alcohol intake and then pronounced ADE after 1-week abstinence. NFF prevented the ADE in a dose-dependent manner in both male and female mice. A combination of NFF with NTX reduced the ADE without sex differences at doses lower than those individual effective ones, suggesting synergistic effects between the two compounds. NMF prevented the ADE in both sexes, while selective KOP-r antagonist nor-BNI had no effect. Our new study suggests that a combination of clinically-utilized, potent KOP-r agonist NFF with low-dose NTX has therapeutic potential in alcohol "relapse" treatment.

In humans, alcoholism is associated with a decrease in basal ACTH and cortisol levels, and blunted pituitary ACTH responses to administered corticotropin-releasing hormone (CRH) during active drinking and after long-term abstinence. Preclinical studies indicate that a persistent decrease in pituitary activation after chronic exposure to ethanol is due to a direct effect of ethanol on the corticotrope of the anterior pituitary. The present studies were undertaken to determine if ethanol has effects on proopiomelanocortin (POMC) gene transcription activity in mouse anterior pituitary corticotrope tumor cell AtT20. We measured the levels of the POMC primary nuclear RNA transcript (PT), processing intermediate, and mature mRNA in the nucleus and the levels of the POMC mRNA in the cytoplasm after treatment of AtT20 cells with 5-15 mM concentrations of ethanol. After 15 mM ethanol for 60 to 120 min, the POMC PT levels were significantly decreased. This decreased POMC gene transcription activity was coupled with a significant reduction of the POMC cytoplasmic mRNA levels. After ethanol for 4h, however, both the decreases were no longer observed. After 8h, a decrease in the ACTH secretion in the medium was found. We further investigated if CRH or glutamate modulates the effects of ethanol on the POMC gene transcription activity. CRH at 10nM after 60 min increased the POMC PT levels, and 15mM ethanol attenuated the effect of CRH on the nuclear transcription activity. Glutamate receptor proteins, including NMDA receptor subtype NR1 (but not NR2A or NR2B) and GluR2, were identified by Western immunoblot analysis in AtT20 cells, with similar sizes to those in mouse hypothalamus. The inhibitory effect of 60 min ethanol at 5 to 15 mM on the POMC PT levels was attenuated by 50 μM L-glutamate. Together, our data showed that: (1) ethanol treatment in intoxicate doses significantly inhibited POMC gene transcription activity in a dose- and time-dependent manner in AtT20 cells, and (2) the POMC gene transcription activity in response to CRH or glutamate was altered by ethanol. Our results suggest that ethanol has an inhibitory effect on the POMC gene transcription activity in the anterior pituitary corticotrope, which may contribute to the persistent decrease in pituitary activation after chronic ethanol exposure.

Though it is well known that G protein-coupled receptor kinase 2 [GRK2] is involved in regulation of mu opioid receptor [MOR] desensitization and morphine-related behaviors, the potential role of GRK2 in regulation of kappa opioid receptor [KOR] functions in vivo has not been established yet. A couple of recent studies have found that GRK2 activity desensitizes KOR functions via decreasing G protein-coupled signaling with sensitizing arrestin-coupled signaling. Nalfurafine, a G protein-biased KOR full agonist, produces an inhibitory effect on alcohol intake in mice, with fewer side effects (sedation, aversion, or anxiety/depression-like behaviors). Using RNA sequencing (RNA-seq) analysis, we first identified that nuclear transcript level of grk2 [adrbk1] (but not other grks) was significantly up-regulated in mouse nucleus accumbens shell (NAcs) after chronic excessive alcohol drinking, suggesting alcohol specifically increased NAcs grk2 expression. We then tested whether selective GRK2/3 inhibitor CMPD101 could alter alcohol intake and found that CMPD101 alone had no effect on alcohol drinking. Therefore, we hypothesized that the grk2 increase in the NAcs could modulate the nalfurafine effect on alcohol intake via interacting with the G protein-mediated KOR signaling. Nalfurafine decreased alcohol drinking in a dose-related manner, and pretreatment with CMPD101 enhanced the reduction in alcohol intake induced by nalfurafine, indicating an involvement of GRK2/3 blockade in modulating G protein-biased KOR agonism of nalfurafine. Together, our study provides initial evidence relevant to the transcriptional change of grk2 gene in the NAc shell after excessive alcohol drinking. Pharmacological GRK2/3 blockade enhanced nalfurafine's efficacy, suggesting a GRK2/3-mediated mechanism, probably through the G protein-mediated KOR signaling.