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Excessive ethanol drinking is a major problem within the United States, causing alterations in brain plasticity and neurocognitive function. Astrocytes are glial cells that regulate neurosynaptic plasticity, modulate neurochemicals, and monitor other homeostatic roles. Astrocytes have been found to play a part in modulating excessive ethanol consumption. The basolateral amygdala (BLA), central amygdala (CeA), and bed nucleus of the stria terminalis (BNST) are brain regions that process stress, anxiety, and reward; they are also implicated in modulating ethanol intake. Little is understood, however, about how astrocyte expression in each region is modulated by chronic and binge-like ethanol drinking patterns. In the present report, we utilized two separate animal models of excessive drinking: chronic intermittent ethanol (CIE) and "Drinking-in-the-dark" (DID). Following these paradigms, animal brains were processed through immunohistochemistry (IHC) and stained for glial fibrillary acidic protein (GFAP). Collected data illustrated a sex-dependent relationship between ethanol intake and GFAP immunoreactivity (IR) in the BLA and BNST, but not in the CeA. Specifically, CIE and DID ethanol drinking resulted in blunted GFAP-IR (specifically via GFAP-positive cell count) in the BLA and BNST, particularly in males. These findings may implicate sex-dependent ethanol-induced changes in BLA and BNST astrocytes, providing a potential therapeutic target for anxiety and stress disorders.

Repetitive bouts of binge drinking can lead to neuroplastic events that alter ethanol's pharmacologic effects and perpetuate excessive consumption. The corticotropin-releasing factor (CRF) system is an example of ethanol-induced neuroadaptations that drive excessive ethanol consumption. Our laboratory has previously shown that CRF antagonist, when infused into the central amygdala (CeA), reduces binge-like ethanol consumption. The present study extends this research by assessing the effects of silencing CRF-producing neurons in CeA on binge-like ethanol drinking stemming from "Drinking in the Dark" (DID) procedures. CRF-ires-Cre mice underwent surgery to infuse G

Alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD) are highly comorbid, yet there is a lack of preclinical research investigating how prior ethanol (EtOH) dependence influences the development of a PTSD-like phenotype. Furthermore, the neuroimmune system has been implicated in the development of both AUD and PTSD, but the extent of glial involvement in this context remains unclear. A rodent model was developed to address this gap in the literature.We used a 15-day exposure to the 5% w/v EtOH low-fat Lieber-DeCarli liquid diet in combination with the stress-enhanced fear learning (SEFL) paradigm to investigate the effects of chronic EtOH consumption on the development of a PTSD-like phenotype. Next, we used a reverse transcription quantitative real-time polymerase chain reaction to quantify mRNA expression of glial cell markers GFAP (astrocytes) and CD68 (microglia) following severe footshock stress in EtOH-withdrawn rats. Finally, we tested the functional contribution of dorsal hippocampal (DH) astrocytes in the development of SEFL in EtOH-dependent rats using astrocyte-specific GResults demonstrate that chronic EtOH consumption and withdrawal exacerbate future SEFL. Additionally, we found significantly increased GFAP mRNA expression in the dorsal and ventral hippocampus and amygdalar complex following the severe stressor in EtOH-withdrawn animals. Finally, the stimulation of the astroglial GCollectively, results indicate that prior EtOH dependence and withdrawal combined with a severe stressor potentiate future enhanced fear learning. Furthermore, DH astrocytes significantly contribute to this change in behavior. Overall, these studies provide insight into the comorbidity of AUD and PTSD and the potential neurobiological mechanisms behind increased susceptibility to a PTSD-like phenotype in individuals with AUD.

Bed nucleus of the stria terminalis (BNST) neurons that synthesize corticotropin-releasing factor (CRF) drive binge alcohol drinking and anxiety. Here, we found that female C57BL/6J mice binge drink more than males and have greater basal BNSTCRF neuron excitability and synaptic excitation. We identified a dense VGLUT2 + synaptic input from the paraventricular thalamus (PVT) that releases glutamate directly onto BNSTCRF neurons but also engages a large BNST interneuron population to ultimately inhibit BNSTCRF neurons, and this polysynaptic PVTVGLUT2-BNSTCRF circuit is more robust in females than males. Chemogenetic inhibition of the PVTBNST projection promoted binge alcohol drinking only in female mice, while activation reduced avoidance behavior in both sexes. Lastly, repeated binge drinking produced a female-like phenotype in the male PVT-BNSTCRF excitatory synapse without altering the function of PVTBNST neurons per se. Our data describe a complex, feedforward inhibitory PVTVGLUT2-BNSTCRF circuit that is sex-dependent in its function, behavioral roles, and alcohol-induced plasticity., Bed nucleus of the stria terminalis (BNST) neurons that synthesize and release the stress neuropeptide corticotropin-releasing factor drive binge alcohol drinking and anxiety. The authors describe a complex feedforward inhibitory PVTVGLUT2-BNSTCRF circuit in mice that plays sex-dependent roles in alcohol drinking and avoidance behavior.

Neuropeptide Y (NPY) signaling via limbic NPY1 and 2 receptors (NPY1R and NPY2R, respectively) is known to modulate binge-like ethanol consumption in rodents. However, the role of NPY signaling in the medial prefrontal cortex (mPFC), which provides top-down modulation of the limbic system, is unknown. Here, we used "drinking-in-the-dark" (DID) procedures in C57BL/6J mice to address this gap in the literature. First, the impact of DID on NPY immunoreactivity (IR) was assessed in the mPFC. Next, the role of NPY1R and NPY2R signaling in the mPFC on ethanol consumption was evaluated through site-directed pharmacology. Chemogenetic inhibition of NPY1R+ neurons in the mPFC was performed to further evaluate the role of this population. To determine the potential role of NPY1R+ neurons projecting from the mPFC to the basolateral amygdala (BLA) this efferent population was selectively silenced. Three, 4-day cycles of DID reduced NPY IR in the mPFC. Intra-mPFC activation of NPY1R and antagonism of NPY2R resulted in decreased binge-like ethanol intake. Silencing of mPFC NPY1R+ neurons overall, and specifically NPY1R+ neurons projecting to the BLA, significantly reduced binge-like ethanol intake. We provide novel evidence that (1) binge-like ethanol intake reduces NPY levels in the mPFC; (2) activation of NPY1R or blockade of NPY2R reduces binge-like ethanol intake; and (3) chemogenetic inhibition of NPY1R+ neurons in the mPFC and NPY1R+ mPFC neurons projecting to the BLA blunts binge-like drinking. These observations provide the first direct evidence that NPY signaling in the mPFC modulates binge-like ethanol consumption.

Objectives: Alcohol dependence leads to dysregulation of the neuroimmune system, but the effects of excessive alcohol consumption on key players of the neuroimmune response after episodic binge drinking in nondependence has not been readily assessed. These studies seek to determine how the neuroimmune system within the hippocampus responds to binge-like consumption prior to dependence or evidence of brain damage. Methods: C57BL/6J mice underwent the drinking in the dark (DID) paradigm to recapitulate binge consumption. Immunohistochemical techniques were employed to determine the effects of ethanol on cytokine and astrocyte responses within the hippocampus. Astrocyte activation was also assessed using qRT-PCR. Results: Our results indicated that binge-like ethanol consumption resulted in a 3.6-fold increase in the proinflammatory cytokine interleukin (IL)-1β immunoreactivity in various regions of the hippocampus. The opposite effect was seen in the anti-inflammatory cytokine IL-10. Binge-like consumption resulted in a 67% decrease in IL-10 immunoreactivity but had no effect on IL-4 or IL-6 compared with the water-drinking control group. Moreover, astrocyte activation occurred following ethanol exposure as GFAP immunoreactivity was increased over 120% in mice that experienced 3 cycles of ethanol binges. PCR analyses indicated that the mRNA increased by almost 4-fold after one cycle of DID, but this effect did not persist in abstinence. Conclusions: Altogether, these findings suggest that binge-like ethanol drinking prior to dependence causes dysregulation to the neuroimmune system. This altered neuroimmune state may have an impact on behavior but could also result in a heightened neuroimmune response that is exacerbated from further ethanol exposure or other immune-modulating events.

The inbred high drinking in the dark (iHDID1 and iHDID2) strains are two replicate lines bred from the parent HS/Npt (HS) line for achieving binge levels of blood ethanol concentration (&ge, 80 mg/dL BEC) in a four-hour period. In this work, we sought to evaluate differences in baseline and ethanol-induced c-Fos activation between the HS, iHDID1, and iHDID2 genetic lines in brain regions known to process the aversive properties of ethanol. Methods: Male and female HS, iHDID1, and iHDID2 mice underwent an IP saline 2 3 g/kg ethanol injection. Brain sections were then stained for c-Fos expression in the basolateral/central amygdala (BLA/CeA), bed nucleus of the stria terminals (BNST), A2, locus coeruleus (LC), parabrachial nucleus (PBN), lateral/medial habenula (LHb/MHb), paraventricular nucleus of the thalamus (PVT), periaqueductal gray (PAG), Edinger&ndash, Westphal nuclei (EW), and rostromedial tegmental nucleus (RMTg). Results: The iHDID1 and iHDID2 lines showed similar and distinct patterns of regional c-Fos, however, in no region did the two both significantly differ from the HS line together. Conclusions: These data lend further support to altered baseline or ethanol-induced activation in brain regions associated with processing the aversive properties of ethanol in the iHDID1 and iHDID2 genetic lines.

Bed nucleus of the stria terminalis (BNST) neurons that synthesize and release the stress neuropeptide corticotropin-releasing factor (CRF) drive binge alcohol drinking and anxiety, behaviors that are primary risk factors for alcohol use disorder (AUD) and comorbid neuropsychiatric diseases more common in women than men. Here, we show that female C57BL/6J mice binge drink more than males and have greater basal BNSTCRF neuron excitability and synaptic excitation. We identified a dense VGLUT2+ glutamatergic synaptic input from the paraventricular thalamus (PVT) that is anatomically similar in males and females. These PVTBNST neurons release glutamate directly onto BNSTCRF neurons but also engage a large BNST interneuron population to ultimately provide a net inhibition of BNSTCRF neurons, and both components of this polysynaptic PVTVGLUT2-BNSTCRF circuit are more robust in females than males. Chemogenetic inhibition of the PVTBNST projection promoted binge alcohol drinking in females without affecting males, and chemogenetic activation of the pathway was sufficient to reduce avoidance behavior in both sexes in anxiogenic contexts. Lastly, we show that withdrawal from repeated binge drinking produces a female-like phenotype in the male PVT-BNSTCRF excitatory synapse without altering the function of PVTBNST neurons per se. Our data describe a complex feedforward inhibitory PVTVGLUT2-BNSTCRF glutamatergic circuit that is more robust in females, plays sex-dependent roles in alcohol drinking and avoidance behavior, and undergoes sex-dependent alcohol-induced plasticity.

Corticotropin-releasing factor (CRF) circuitry is a key component in plasticity underlying the transition to ethanol (EtOH) dependence. We have previously shown that chemogenetic silencing of CRF neurons stemming from the dorsolateral bed nucleus of the stria terminalis (dlBNST) and projecting to the ventral tegmental area (VTA) significantly blunts binge-like EtOH consumption. While CRF neurons in the BNST are thought to entail primarily a GABA phenotype, glutamatergic neurons within the BNST also innervate the VTA and influence consummatory behaviors. Here, we combined the well-validated Vgat-ires-Cre transgenic mice with chemogenetic tools to extend our previous findings and corroborate the contribution of the VTA-projecting dlBNST GABAergic circuitry in modulating binge-like EtOH consumption using “drinking-in-the-dark” procedures. Mice were given bilateral injection of Gi-coupled chemogenetic viral vector (or control virus) into the dlBNST and bilateral cannulae into the VTA. On test day, clozapine-N-oxide (CNO; or vehicle) was infused directly into the VTA to silence VTA-projecting dlBNST neurons and subsequent binge-like EtOH consumption was assessed. We then used immunohistochemistry (IHC) to determine the co-expression of CRF and viral vector. Our results showed that relative to vehicle treatment or CNO treatment in mice expressing the control virus, silencing VTA-projecting dlBNST GABAergic neurons by CNO treatment in mice expressing Gi-coupled chemogenetic virus significantly reduced binge-like EtOH intake. This effect was not seen with sucrose consumption. Our IHC results confirm a population of CRF-expressing GABAergic neurons within the dlBNST. This study directly establishes that VTA-projecting GABAergic neurons of the dlBNST modulate binge-like EtOH consumption.

There is strong evidence that ethanol entails aversive effects that can act as a deterrent to overconsumption. We have found that in doses that support the development of a conditioned taste aversion ethanol increases the activity of tyrosine hydroxylase (TH) positive neurons in the locus coeruleus (LC), a primary source of norepinephrine (NE). Using cre-inducible AAV8-ChR2 viruses in TH-ires-cre mice we found that the LC provides NE projections that innervate the rostromedial tegmental nucleus (RMTg), a brain region that has been implicated in the aversive properties of drugs. Because the neurocircuitry underlying the aversive effects of ethanol is poorly understood, we characterized the role of the LC to RMTg circuit in modulating aversive unconditioned responses and binge-like ethanol intake. Here, both male and female TH-ires-cre mice were cannulated in the RMTg and injected in the LC with rAVV viruses that encode for a Gq-expressing designer receptor exclusively activated by designer drugs (DREADDs) virus, or its control virus, to directly control the activity of NE neurons. A Latin Square paradigm was used to analyze both 20% ethanol and 3% sucrose consumption using the “drinking-in-the-dark” (DID) paradigm. Chemogenetic activation of the LC to RMTg pathway significantly blunted the binge-ethanol drinking, with no effect on the sucrose consumption, increased the emission of mid-frequency vocalizations and induced malaise-like behaviors in mice. The present findings indicate an important involvement of the LC to RMTg pathway in reducing ethanol consumption, and characterize unconditioned aversive reactions induced by activation of this noradrenergic pathway.

A growing body of literature implicates noradrenergic (NE) signaling in the modulation of ethanol consumption. However, relatively few studies have detailed specific brain pathways that mediate NE-associated binge-like ethanol consumption. To begin to fill this gap in the literature, male and female C57BL6/J and TH-ires-cre mice underwent pharmacological and chemogenetic testing, respectively, in combination with “drinking in the dark” procedures to model binge-like consumption of ethanol or sucrose solutions. First, we showed that intraperitoneal administration of the NE reuptake inhibitor, reboxetine, blunted binge-like ethanol intake in C57BL6/J mice. Chemogenetic activation of locus coeruleus (LC) tyrosine hydroxylase (TH)-expressing neurons blunted binge-like ethanol intake regardless of sex. Chemogenetic activation of LC projections to the lateral hypothalamus (LH), a region implicated in ethanol consumption, blunted binge-like ethanol drinking without altering sucrose intake in ethanol-experienced or ethanol-naive mice. In C57BL/6 J mice, LH-targeted microinfusion of an α1-adrenergic receptor (AR) agonist blunted binge-like ethanol intake across both sexes, while LH infusion of a β-AR agonist blunted binge-like ethanol intake in females exclusively. Finally, in mice with high baseline ethanol intake both an α1- AR agonist and an α-2 AR antagonist blunted binge-like ethanol intake. The present results provide novel evidence that increased NE tone in a circuit arising from the LC and projecting to the LH reduces binge-like ethanol drinking in mice, and may represent a novel approach to treating binge or heavy drinking prior to the development of dependence. This article is part of the special Issue on “Neurocircuitry Modulating Drug and Alcohol Abuse”.

The assessment of binge ethanol-induced neuronal activation, using c-Fos immunoreactivity (IR) as a marker of neuronal activity, is typically accomplished via forced ethanol exposure, such as intraperitoneal injection or gavage. Neuronal activity using a voluntary binge-like drinking model, such as “drinking-in-the-dark” (DID), has not been thoroughly explored. Additionally, studies assessing ethanol-elicited neuronal activation may or may not involve stereotaxic surgery, which could impact c-Fos IR. The experiments detailed herein aimed to assess the effects of voluntary binge-like ethanol consumption on c-Fos IR in brain regions implicated in ethanol intake in animals with and without surgery experience. Age-matched male C57BL/6J mice underwent either stereotaxic surgery (Study 1) or no surgery (Study 2). Then, mice experienced one 4-day DID cycle, tail blood samples were collected immediately after test conclusion on day 4, and mice were subsequently sacrificed. In each study, mice that drink ethanol were sorted into those that achieved binge-equivalent blood ethanol concentrations (BECs ≥ 80 mg/dl) versus those that did not. Relative to water-consuming controls, mice with BECs ≥ 80 mg/dl showed significantly elevated c-Fos IR in several brain regions implicated in neurobiological responses to ethanol. In general, the brain regions exhibiting binge-induced c-Fos IR were the same between studies, though differences were noted, highlighting the need for caution when interpreting ethanol-induced c-Fos IR when subjects have a prior history of surgery. Altogether, these results provide insight into the brain regions that modulate binge-like ethanol intake stemming from DID procedures among animals with and without surgery experience.

Background Corticotropin-releasing factor (CRF) signaling at the CRF 1 receptor (CRF 1 R) in the ventral tegmental area (VTA) can modulate ethanol consumption in rodents. However, the effects of binge-like ethanol drinking on this system have not been thoroughly characterized, and little is known about the role of CRF 2 R or the CRF neurocircuitry involved. Methods The effects of binge-like ethanol consumption on the VTA CRF system were assessed following drinking-in-the-dark procedures. Intra-VTA infusions of selective CRF 1 R and/or CRF 2 R compounds were employed to assess the contributions of these receptors in modulating binge-like ethanol consumption ( n = 89). To determine the potential role of CRF projections from the bed nucleus of the stria terminalis (BNST) to the VTA, CRF neurons in this circuit were chemogenetically inhibited ( n = 32). Binge-induced changes in VTA CRF system protein and messenger RNA were also assessed ( n = 58). Results Intra-VTA antagonism of CRF 1 R and activation of CRF 2 R resulted in decreased ethanol intake, which was eliminated by simultaneous blockade of both receptors. Chemogenetic inhibition of local CRF neurons in the VTA did not alter binge-like ethanol drinking, but inhibition of VTA-projecting CRF neurons from the BNST significantly reduced intake. Conclusions We provide novel evidence that 1) blunted binge-like ethanol consumption stemming from CRF 1 R blockade requires intact CRF 2 R signaling, and CRF 2 R activation reduces binge-like drinking; 2) inhibiting VTA-projecting BNST CRF neurons attenuates binge-like drinking; and 3) binge-like ethanol drinking alters protein and messenger RNA associated with the VTA-CRF system. These data suggest that ethanol-induced activation of BNST-to-VTA CRF projections is critical in driving binge-like ethanol intake.

Aims The combination of bupropion and naltrexone has shown efficacy in reducing binge drinking in animal models. This study assessed the tolerability and potential utility of combined naltrexone and bupropion in reducing binge drinking in human subjects. Methods This preliminary study employed an open-label, single-arm, 12-week, prospective design. Twelve men and women who exhibited a minimum of five (men) or three (women) binge drinking episodes per month over the past 3 months were recruited. All subjects received both bupropion-extended release 300 mg/day and naltrexone 50 mg/day and were monitored throughout the 3-month treatment period. Binge drinking was assessed using the timeline follow-back method. Results Treatment with combined naltrexone and bupropion reduced the average number of drinks per binge drinking day from 7.8 drinks to 6.4 drinks and reduced the average percentage of binge drinking days per month from 19% (5.7 days/month) to 5% (1.5 days/month). Naltrexone and bupropion were generally well tolerated, with insomnia, headache and nausea/diarrhea being the most common side effects. Six subjects elected to stay on medication after the trial. Conclusions This study suggests that combined naltrexone and bupropion therapy should be further investigated for tolerability and efficacy in reducing binge drinking in humans.

In recent years, neuropeptides which display potent regulatory control of stress-related behaviors have been extensively demonstrated to play a critical role in regulating behaviors associated with substance abuse and affective disorders. Somatostatin (SST) is one neuropeptide known to significantly contribute to emotionality and stress behaviors. However, the role of SST in regulating behavior has received relatively little attention relative to other stress-involved peptides, such as neuropeptide Y or corticotrophin releasing factor. This review characterizes our current understanding of the role of SST and SST-expressing cells in general in modulating several behaviors intrinsically linked to substance abuse and affective disorders, specifically: anxiety and fear; stress and depression; feeding and drinking; and circadian rhythms. We further summarize evidence of a direct role for the SST system, and specifically somatostatin receptors 2 and 4, in substance abuse disorders. This article is part of the special issue on 'Neuropeptides'.

The melanocortin (MC) system consists of neuropeptides that are cleaved from the polypeptide precursor pro-opiomelanocortin (POMC). In the brain, MC neuropeptides signal primarily through the MC-3 and MC-4 receptors, which are widely expressed throughout the brain. While the MC system has been largely studied for its role in food intake and body weight regulation, converging evidence has emerged over approximately the last 20-years showing that alcohol (ethanol), and other drugs of abuse influence the central MC system, and that manipulating MC receptor signalling modulates ethanol intake. Although there is divergent evidence, the wealth of data appears to suggest that activating MC signalling, primarily through the MC-4 receptor, is protective against excessive ethanol consumption. In the present review, we first describe the MC system and then detail how ethanol exposure and consumption alters central MC and MC-receptor expression and levels. This is followed by a review of the data, from pharmacological and genetic studies, which show that manipulations of MC receptor activity alter ethanol intake. We then briefly highlight studies implicating a role for the MC system in modulating neurobiological responses and intake of other drugs of abuse, including amphetamine, cocaine and opioids. Finally, we introduce relatively new observations that the drug, bupropion (BUP), a drug that activates central MC activity, significantly reduces ethanol intake in rodent models when administered alone and in combination with the non-selective opioid receptor antagonist, naltrexone. Phase II clinical trials are currently underway to assess the efficacy of BUP as a treatment for alcohol use disorders.

Psychological depression is frequently linked to alcohol abuse and even serves as key indicators of an alcohol use disorder (AUD). This relationship is supported by preclinical findings in which depression-like phenotypes develop in animals exposed to chronic intermittent ethanol vapor, a common preclinical model of alcohol dependence. However, the emergence of these maladaptive phenotypes following repeated binge-like ethanol drinking remains relatively unexplored. The purpose of this study was to evaluate depression-like behaviors associated with binge-like consumption in mice. Using the drinking-in-the-dark (DID) paradigm, we examined the impact of multiple binge-like cycles (1, 3, or 6) on depression-like behaviors in the forced swim test (FST) and sucrose preference as a test for anhedonia. We also assessed the effect of repeated binge cycles on the consumption of bitter and sweet tastants over a range of concentrations. Results indicated that binge-like ethanol drinking did not lead to depression-like behavior as repeated cycles of DID did not alter sucrose consumption or preference nor did it impact time spent immobile during the FST. Animals that experienced six cycles of DID showed increased quinine consumption and increased quinine preference, which may be indicative of an escalated preference for tastants that resemble the gustatory aspects of ethanol. Interestingly, an unexpected ~20% increase in hypermobility was observed after three cycles of binge-like ethanol drinking. Although the FST is most frequently used to model depression-like behavior, emerging evidence suggests that increased hypermobility during the FST could be indicative of an inability to cope in a stressful situation, suggesting that repeated ethanol exposure in the present experiment transiently enhances stress reactivity.

Background In recent years, much attention has been given to the lack of reproducibility in biomedical research, particularly in preclinical animal studies. This is a problem that also plagues the alcohol research field, particularly in consistent consumption in animal models of alcohol use disorders. One often overlooked factor that could affect reproducibility is the maintenance diet used in preclinical studies. Methods Herein, 2 well-established models of alcohol consumption, the "drinking in the dark" (DID) procedure and the continuous 2-bottle choice (C2BC) paradigm, were employed to determine the effects of diet on ethanol (EtOH) consumption. Male C57BL/6J mice were given 1 of 6 standard rodent chow diets obtained from Purina LabDiet(®) , Inc. (Prolab(®) RMH 3000) or Harlan(®) Laboratories, Inc. (Teklad Diets T.2916, T.2918, T.2920X, T.7912, or T.8940). A separate group of animals were used to test dietary effects on EtOH pharmacokinetics and behavioral measures following intraperitoneal (IP) injections of various doses of EtOH. Results Mice eating Harlan diets T.2916 (H2916) and T.2920X (H2920) consumed significantly less EtOH and exhibited lower blood EtOH concentrations (BECs) during DID; however, during C2BC, animals maintained on Harlan T.7912 (H7912) consumed more EtOH and had a higher EtOH preference than the other diet groups. EtOH consumption levels did not stem from changes in alcohol pharmacokinetics, as a separate group of animals administered EtOH IP showed no difference in BECs. However, animals on Harlan diet T.2920X (H2920) were more sensitive to alcohol-induced locomotor activity in an open-field task. No diet-dependent differences were seen in alcohol-induced sedation as measured with loss of righting reflex. Conclusions Although these data do not identify a specific mechanism, together, they clearly show that the maintenance diet impacts EtOH consumption. It is incumbent upon the research community to consider the importance of describing nutritional information in methods, which may help decrease interlaboratory reproducibility issues.

Summary paragraph Binge alcohol drinking is a tremendous public health problem because it leads to the development of numerous pathologies including alcohol abuse, and anxiety1–4. It is thought to do so by hijacking brain systems that regulate stress and reward, including neuropeptide Y (NPY) and corticotropin–releasing factor (CRF). The central actions of NPY and CRF play opposing functional roles in the regulation of emotional and reward–seeking behaviors; therefore, dysfunctional interactions between these peptidergic systems could play a role in the development of these pathologies. Here, we used converging physiological, pharmacological, and chemogenetic approaches to identify a precise neural mechanism in the bed nucleus of the stria terminalis (BNST), a limbic brain region involved in pathological reward and anxiety behaviors, underlying the interactions between NPY and CRF in the regulation of binge alcohol drinking in both mice and monkeys. We found that NPY Y1 receptor (Y1R) activation in the BNST suppressed binge alcohol drinking by enhancing inhibitory synaptic transmission specifically in CRF neurons via a novel, Gi-mediated, PKA-dependent postsynaptic mechanism. Further, chronic alcohol drinking led to persistent alterations in Y1R function in the BNST of both mice and monkeys, highlighting the enduring, conserved nature of this effect across mammalian species. Together, these data provide both a cellular locus and signaling framework for the development of novel therapeutics for treatment of neuropsychiatric diseases, including alcohol use disorders.

Neuropeptide Y (NPY) is a neuromodulator that is widely expressed throughout the central nervous system (CNS) and which is cosecreted with classic neurotransmitters including GABA and glutamate. There is a long history of research implicating a role for NPY in modulating neurobiological responses to alcohol (ethanol) as well as other drugs of abuse. Both ethanol exposure and withdrawal from chronic ethanol have been shown to produce changes in NPY and NPY receptor protein levels and mRNA expression in the CNS. Importantly, manipulations of NPY Y1 and Y2 receptor signaling have been shown to alter ethanol consumption and self-administration in a brain region-specific manner, with Y1 receptor activation and Y2 receptor blockade in regions of the extended amygdala promoting robust reductions of ethanol intake. Similar observations have been made in studies examining neurobiological responses to nicotine, psychostimulants, and opioids. When taken together with observations of potential genetic linkage between the NPY system and the human alcohol abuse disorders, NPY represents a promising target for treating problematic alcohol and drug use, and in protecting individuals from relapse during abstinence.

The melanocortin (MC) system regulates feeding and ethanol consumption. Recent evidence shows that melanocortin 4 receptor (MC4-R) stimulation within the nucleus accumbens (NAc) elicits anorectic responses and reduces ethanol consumption and ethanol palatability in adult rats. Ethanol exposure during adolescence causes long-lasting changes in neural pathways critically involved in neurobehavioral responses to ethanol. In this regard, binge-like ethanol exposure during adolescence reduces basal alpha-melanocyte-stimulating hormone (α-MSH) and alters the levels of agouti-related peptide (AgRP) in hypothalamic and limbic areas. Given the protective role of MC against excessive ethanol consumption, disturbances in the MC system induced by binge-like ethanol exposure during adolescence might contribute to excessive ethanol consumption during adulthood. In the present study, we evaluated whether binge-like ethanol exposure during adolescence leads to elevated ethanol intake and/or eating disturbance during adulthood. Toward that aim, Sprague-Dawley rats were treated with ethanol (3 g/kg i.p.; BEP group) or saline (SP group) for 14 days (PND 25 to PND 38). On PND73, all the groups were given access to 20% ethanol on an intermittent schedule. Our results showed that adult rats given intermittent access (IAE) to 20% ethanol achieved high spontaneous ethanol intake that was not significantly enhanced by binge-like ethanol pretreatment during adolescence. However, BEP group exhibited an increase in food intake without a parallel increase in body weight (BW) relative to SP group suggesting caloric efficiency disturbance. Additionally, we evaluated whether binge-like ethanol exposure during adolescence alters the expected reduction in feeding and ethanol consumption following NAc shell administration of a selective MC4-R agonist in adult rats showing high rates of ethanol consumption. For that, animals in each pretreatment condition (SP and BEP) were divided into three subgroups and given bilateral NAc infusions of the selective MC4-R agonist cyclo(NH-CH2-CH2-CO-His-D-Phe-Arg-Trp-Glu)-NH2 (0, 0.75 or 1.5 μg). Results revealed that MC4-R stimulation within the NAc reduced feeding and ethanol intake in high ethanol-drinking adult rats, regardless of previous binge-like ethanol exposure during adolescence, which adds new evidence regarding the dual ability of MC compounds to control excessive ethanol and food intake.

Neuropeptide Y (NPY) is a neuromodulator that is widely expressed throughout the central nervous system (CNS) and which is cosecreted with classic neurotransmitters including GABA and glutamate. There is a long history of research implicating a role for NPY in modulating neurobiological responses to alcohol (ethanol) as well as other drugs of abuse. Both ethanol exposure and withdrawal from chronic ethanol have been shown to produce changes in NPY and NPY receptor protein levels and mRNA expression in the CNS. Importantly, manipulations of NPY Y1 and Y2 receptor signaling have been shown to alter ethanol consumption and self-administration in a brain region-specific manner, with Y1 receptor activation and Y2 receptor blockade in regions of the extended amygdala promoting robust reductions of ethanol intake. Similar observations have been made in studies examining neurobiological responses to nicotine, psychostimulants, and opioids. When taken together with observations of potential genetic linkage between the NPY system and the human alcohol abuse disorders, NPY represents a promising target for treating problematic alcohol and drug use, and in protecting individuals from relapse during abstinence.

Neuropeptides are short sequences of amino acids that are coexpressed with neurotransmitters and which are widely expressed throughout the central nervous system. There is a large database of data pointing to critical roles for neuropeptides in modulating neurobiological responses to alcohol and drugs of abuse. Continued alcohol and drug use promote allostatic alterations in neuropeptide systems, and these changes contribute to excessive and uncontrolled intake that emerges with dependence. The neuropeptides that are reviewed in this chapter represent some of the most well-studied targets in the current drug and alcohol abuse literature. The goal of this chapter is to convey the significant roles that neuropeptides play in neurobiological responses to alcohol and drugs of abuse, and reinforce the idea that targeting neuropeptides and their receptors continue to be attractive avenues for treating drug and alcohol use disorders, as well as eating disorders.

Background A growing body of literature suggests that epigenetic mechanisms, including histone acetylation, may play key roles in drug abuse and the development of addiction. Experiments in this study were designed to investigate the role of histone acetylation in ethanol (EtOH)-induced locomotor sensitization. Methods Immunohistochemical, Western blotting, and site-directed pharmacological techniques were used to explore the roles of histone acetylation at histone H3 (acH3K9) in both the expression of and acquisition of EtOH-induced locomotor sensitization. A commonly used sensitization protocol, in which animals were exposed to repeated injections of a low dose of EtOH while in their home cage, was used to examine this behavioral phenomenon. Additionally, site-directed administration of the histone deacetylase inhibitor (HDACi) Trichostatin A (TSA), in the absence of repeated EtOH injections, was used to examine the role of hyperacetylation in the nucleus accumbens (NAC) shell in EtOH-induced locomotor sensitization. Results Sensitized mice displayed elevated acH3K9 immunoreactivity (IR) localized to the shell of the NAC. This augmentation in acH3K9 IR was confirmed, in a separate experiment, using Western blot analyses. Next, repeated intra-accumbal infusions of TSA, in the absence of repeated EtOH injections, were sufficient to induce an augmented locomotor response to a later injection of a low dose (2.0 g/kg, intraperitoneally) of EtOH, indicative of cross-sensitization to this locomotor stimulation between TSA and EtOH. Finally, a local infusion of TSA into the shell of the accumbens was also associated with a significant increase in acH3K9 IR within this region. Conclusions Together, the present observations suggest that histone acetylation, particularly within the shell of the NAC, is important for the development and expression of EtOH-induced locomotor sensitization.

Recent data have implicated the melanocortin (MC) system in modulating voluntary ethanol consumption. Administration of melanotan-II (MTII), a nonselective melanocortin receptor (MCR) agonist, reduces voluntary ethanol consumption in C57BL/6J mice. Previous studies have demonstrated that central infusion of MTII effectively reduced voluntary ethanol drinking in mutant mice lacking normal expression of MC3R (MC3R −/− mice) but failed to alter ethanol drinking in mice lacking expression of MC4R, demonstrating that central MTII administration reduces voluntary ethanol drinking by signaling through the MC4R. However, evidence shows that the neurocircuitry recruited during excessive binge-like ethanol drinking versus moderate ethanol drinking are not identical. Thus the present study sought to investigate the potential role of the MC3R in binge-like ethanol intake. To this end, the "drinking in the dark" (DID) procedure, a commonly used animal model of binge-like ethanol drinking, was employed. Wild-type MC3R +/+ and MC3R −/− mice were given intracerebroventricular (i.c.v.) infusion of MTII (0.0, 0.25, 0.50, or 1.0μg) prior to the onset of a 4-h testing period in which mice were given access to 20% (v/v) ethanol. Immediately after the 4-h testing period, tail blood samples were collected from each animal in order to assess blood ethanol concentrations (BECs). Consistent with previous findings, central administration of MTII blunted binge-like ethanol drinking in both MC3R +/+ and MC3R −/− mice. Interestingly, all doses of MTII blunted binge-like ethanol drinking in MC3R −/− mice during the first hour of testing, while only the 1.0μg dose reduced binge-like drinking in MC3R +/+ mice. Thus, MC3R −/− mice were more sensitive to the protective effects of MTII. These data suggest that MC3Rs oppose the protective effects of MTII against binge-like ethanol drinking, and thus selective MC3R antagonists may have potential therapeutic roles in treating excessive ethanol drinking.

Repeated cycles of binge alcohol drinking and abstinence are key components in the development of dependence. However, the precise behavioral mechanisms underlying binge-like drinking and its consequences on striatal synaptic physiology remain unclear. In the present study, ethanol and water drinking patterns were recorded with high temporal resolution over 6 weeks of binge-like ethanol drinking using the ‘drinking in the dark' (DID) protocol. The bottle exchange occurring at the beginning of each session prompted a transient increase in the drinking rate that might facilitate the acquisition of ethanol binge-like drinking. Ethanol drinking mice also displayed a ‘front-loading' behavior, in which the highest rate of drinking was recorded during the first 15 min. This rate increased over weeks and paralleled the mild escalation of blood ethanol concentrations. GABAergic and glutamatergic transmission in the dorsal striatum were examined following DID. Spontaneous glutamatergic transmission and the density of dendritic spines were unchanged after ethanol drinking. However, the frequency of GABAA receptor-mediated inhibitory postsynaptic currents was depressed in medium spiny neurons of ethanol drinking mice. A history of ethanol drinking also increased ethanol preference and altered the acute ethanol effects on GABAergic transmission differentially in dorsolateral and dorsomedial striatum. Together, the study shows that the bottle exchange during DID promotes fast, voluntary ethanol drinking and that this intermittent pattern of ethanol drinking causes a depression of GABAergic transmission in the dorsal striatum.

Background Recent reports have demonstrated that binge-like ethanol drinking leads to an increase in hypothalamic orexin (OX) signaling and that suppressing this signaling via systemic administration of an orexin receptor (OXR) antagonist blocks this behavior; however, the specific OX pathways that modulate this behavior remain unknown. The goal of the present study was to further elucidate the role of the OX system in binge-like ethanol drinking using behavioral, molecular, and pharmacological techniques. Methods The drinking-in-the-dark (DID) paradigm was used to model binge-like drinking behavior in male C57BL/6J mice. Experiment 1 examined changes in the OX precursor, prepro-orexin, within the hypothalamus following multiple cycle ethanol or sucrose DID using polymerase chain reaction (PCR) analysis. In experiments 2a & 2b, we used site-directed infusion of an OXR antagonist to examine the individual contribution of each OXR subtype within the ventral tegmental area (VTA) and central amygdala (CeA), repectively, in binge-like ethanol or sucrose drinking. Results Findings from our PCR study revealed that multiple cycles of binge-like ethanol drinking did not lead to changes in prepro-orexin mRNA as a function of binge-like ethanol drinking. However, data from site-directed pharmacology studies indicate that the OX1R is the predominate receptor subtype within the VTA and CeA that regulates binge-like ethanol drinking. Interestingly, inhibition of OX1Rs did not affect binge-like sucrose intake, which suggests that these OX circuits are specific for ethanol consumption. Conclusions As a whole, these data suggest that the VTA and CeA are important regions in which OX regulates binge-like ethanol drinking behavior. Moreover, these findings identify OXR antagonists as a potential treatment option that may be used to ameliorate problematic drinking behavior while leaving responding to natural rewards relatively intact. This article is protected by copyright. All rights reserved.

Excessive ethanol consumption alters the neuroimmune system and particularly impacts the cytokine milieu of the CNS. Cytokine dysregulation has been shown to underlie addictive-like behaviors including alcohol abuse; however, many studies focus primarily on the proinflammatory cytokine profile during alcohol dependence. The current study furthers this research by determining the impact of excessive ethanol consumption on interleukin-10 (IL-10) and interleukin-4 (IL-4) activity in a model of non-dependent binge consumption called the “drinking in the dark” (DID) paradigm. Furthermore, the ability of IL-10 to modulate ethanol consumption was tested using site-directed pharmacology. Immunohistochemistry analyses determined that ethanol decreased IL-10 by 50 % in the basolateral amygdala (BLA) but had no effect on IL-4. Neither IL-10 nor IL-4, however, were altered in the central amygdala (CEA). Enzyme linked immunosorbent assays confirmed that IL-10 was decreased in the amygdala but not in the serum, suggesting changes of this cytokine with the DID paradigm are restricted to the central nervous system. Finally, bilateral infusions of IL-10 into the BLA, but not CeA, reduced binge-like drinking and corresponding blood ethanol concentrations without impacting either locomotor activity or anxiety-like behavioral correlates. Together, these data support the idea that alcohol abuse dysregulates specific anti-inflammatory cytokines; however, ameliorating alcohol-induced effects on cytokines, like IL-10, may prove to be an effective therapy in curbing excessive consumption.

Contains fulltext : 168138.pdf (Publisher’s version ) (Closed access) Serotonin (also known as 5-hydroxytryptamine (5-HT)) is a neurotransmitter that has an essential role in the regulation of emotion. However, the precise circuits have not yet been defined through which aversive states are orchestrated by 5-HT. Here we show that 5-HT from the dorsal raphe nucleus (5-HTDRN) enhances fear and anxiety and activates a subpopulation of corticotropin-releasing factor (CRF) neurons in the bed nucleus of the stria terminalis (CRFBNST) in mice. Specifically, 5-HTDRN projections to the BNST, via actions at 5-HT2C receptors (5-HT2CRs), engage a CRFBNST inhibitory microcircuit that silences anxiolytic BNST outputs to the ventral tegmental area and lateral hypothalamus. Furthermore, we demonstrate that this CRFBNST inhibitory circuit underlies aversive behaviour following acute exposure to selective serotonin reuptake inhibitors (SSRIs). This early aversive effect is mediated via the corticotrophin-releasing factor type 1 receptor (CRF1R, also known as CRHR1), given that CRF1R antagonism is sufficient to prevent acute SSRI-induced enhancements in aversive learning. These results reveal an essential 5-HTDRN-->CRFBNST circuit governing fear and anxiety, and provide a potential mechanistic explanation for the clinical observation of early adverse events to SSRI treatment in some patients with anxiety disorders.

Background The melanocortin (MC) peptides and opioid peptide β-endorphin are cleaved from the polypeptide precursor pro-opiomelanocortin (POMC). POMC-derived peptides are generated by extensive posttranslational processing that involves several enzymes including prohormone convertase 1/3 and 2 (PC1/3 and PC2). Because ethanol (EtOH) decreases POMC mRNA levels, we determined whether the exposure to an EtOH-containing diet (ED) would significantly reduce central immunoreactivity (IR) of POMC, PC1/3, PC2, and β-endorphin. Methods Male Sprague-Dawley rats were given 18 days of access to a normal rodent chow or a control diet (CD), or short-term (4 days) or long-term (18 days) access to an ED. At the end of the study, rats were perfused with 4% paraformaldehyde, and their brains were sectioned into sets for processing with POMC, PC1/3, PC2, and β-endorphin IR. Results Rats exposed to an ED for 18 days (ED18) exhibited significant reductions of POMC and PC1/3 IR in the arcuate nucleus of the hypothalamus (Arc) relative to rats pair-fed a CD. On the other hand, rats exposed to an ED did not show any changes of central β-endorphin or PC2 IR relative to rats pair-fed a CD, regardless of length of exposure. Because there were no differences in body weights or caloric intake between the CD and ED groups, reductions of POMC and PC1/3 IR in ED-treated rats are best explained by EtOH exposure rather than altered energy balance. Conclusions This study shows that EtOH site-specifically reduces POMC and PC1/3 IR in rat brain. These observations are consistent with EtOH-induced reductions of α-melanocyte-stimulating hormone (α-MSH) and POMC IR that were previously reported. As MC agonists have been shown to blunt EtOH intake in rodents, exogenous MC receptor agonists, as well as targets that may increase the synthesis of endogenous α-MSH (e.g., PC1/3), may have therapeutic value for treating alcohol abuse disorders and alcoholism.

Binge alcohol (ethanol) drinking is a destructive pattern of ethanol consumption that may precipitate ethanol dependence, a chronic, debilitating, and prevalent health problem. While an abundance of research has focused on the neurochemical underpinnings of ethanol dependence, relatively little is known about the mechanisms underlying the heavy consumption characteristic of binge ethanol drinking. Recently, a simple preclinical model termed “drinking in the dark” (DID) was developed to examine binge-like ethanol consumption in a rodent population. This assay capitalizes on the predisposition of C57BL/6J mice to voluntarily consume substantial quantities of a high concentration (20% v/v) ethanol solution, resulting in pharmacologically relevant blood ethanol concentrations (BECs). This review provides a comprehensive overview of recent literature utilizing this model to investigate the neuromodulatory systems that may influence binge ethanol drinking. Studies examining the glutamatergic and opioidergic systems not only provide evidence for these systems in the modulation of binge-like ethanol consumption, but also suggest this preclinical model has predictive validity and may be an appropriate tool for screening novel pharmacological compounds aimed at treating binge ethanol drinking in the human population. Additionally, this review presents evidence for the involvement of the GABAergic, dopaminergic, nicotinic, and endocannabinoid systems in modulating binge-like ethanol consumption. Finally, recent evidence shows that corticotropin-releasing factor (CRF), agouti-related protein (AgRP), neuropeptide Y (NPY), and ghrelin are also implicated as impacting this pattern of ethanol consumption.

It is widely accepted that stress, anxiety, depression and alcohol abuse-related disorders are in large part controlled by corticotropin-releasing factor (CRF) receptors. However, evidence is accumulating that some of the actions on these receptors are mediated not by CRF, but by a family of related Urocortin (Ucn) peptides Ucn1, Ucn2 and Ucn3. The initial narrow focus on CRF as the potential main player acting on CRF receptors appears outdated. Instead it is suggested that CRF and the individual Ucns act in a complementary and brain region-specific fashion to regulate anxiety-related behaviors and alcohol consumption. This review, based on a symposium held in 2011 at the research meeting on “Alcoholism and Stress” in Volterra, Italy, highlights recent evidence for regulation of these behaviors by Ucns. In studies on stress and anxiety, the roles of Ucns, and in particular Ucn1, appear more visible in experiments analyzing adaptation to stressors rather than testing basal anxiety states. Based on these studies, we propose that the contribution of Ucn1 to regulating mood follows a U-like pattern with both high and low activity of Ucn1 contributing to high anxiety states. In studies on alcohol use disorders, the CRF system appears to regulate not only dependence-induced drinking, but also binge drinking and even basal consumption of alcohol. While dependence-induced and binge drinking rely on the actions of CRF on CRFR1 receptors, alcohol consumption in models of these behaviors is inhibited by actions of Ucns on CRFR2. In contrast, alcohol preference is positively influenced by actions of Ucn1, which is capable of acting on both CRFR1 and CRFR2. Because of complex distribution of Ucns in the nervous system, advances in this field will critically depend on development of new tools allowing site-specific analyses of the roles of Ucns and CRF.

A well established body of work indicates a crucial role for corticotropin-releasing factor (CRF) in neurobiological responses associated with excessive dependence-like ethanol drinking in ethanol-vapor-exposed rodents. Recent evidence demonstrates a role for CRF in the modulation of binge-like ethanol consumption by nondependent mice, a behavior that can precede ethanol dependence. The CRF circuitry that is engaged by binge-like ethanol exposure, however, is unknown. Using converging approaches, we provide evidence that, similar to ethanol-vapor-induced increases in ethanol intake, CRF signaling in the central nucleus of the amygdala (CeA) is engaged during binge-like ethanol consumption by C57BL/6J mice. Specifically, we found that binge-like consumption of an ethanol solution (20% ethanol v/v) was attenuated by pretreatment with the CRF1R antagonists antalarmin, 4-ethyl-[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]amino-1-butanol, and NBI-27914 at doses (30 mg/kg, i.p.) that did not alter nonbinge-like ethanol consumption. Binge-like ethanol consumption resulted in significant increases of CRF immunoreactivity in the CeA immediately following ethanol drinking and 18–24 h following ethanol removal and also blocked the ability of CRF to enhance GABAergic transmission in the CeA 18–24 h following ethanol removal. Pretreatment with bilateral injections of antalarmin (1 μg/0.5 μl per side) into the CeA, but not the adjacent basolateral amygdala, significantly attenuated binge-like ethanol consumption. These findings suggest that CRF signaling in the CeA is recruited during excessive ethanol intake, before the development of dependence. We hypothesize that plastic changes in CRF signaling develop with repeated binge-like drinking episodes, contributing to the transition to dependence.

Alcohol (ethanol) dependence and relapse in abstinent alcoholics are major health problems in the United States and neurochemical pathways that modulate these disorders are currently under investigation. However, heavy alcohol use and binge alcohol drinking patterns, which can emerge prior to the onset of dependence, have received far less attention. A ‘binge’ is defined by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) as a pattern of drinking that produces blood ethanol concentrations (BECs) greater than 0.08% (80 mg/dl) (NIAAA, 2004). The pattern of alcohol drinking required to produce these BECs is about 5 and 4 drinks in 2-hours for the average adult male and female, respectively. Interestingly, while about 90% of the ethanol consumed by individuals under the age of 21 in the United States is in the form of binge drinking, 70% of binge drinking episodes in the US involve adults age 26 years and older (Naimi et al., 2003). Thus, binge drinking is not restricted to the young but is a risky behavior prevalent in adults. As with all abusive patterns of alcohol drinking, frequent binge drinking is associated with numerous negative short- and long-term consequences. Binge drinking increases the risk of mood disorders (Okoro et al., 2004), increases aggressive and violent behavior (Shepherd et al., 2006), and impairs decision making and judgment (Goudriaan et al., 2007). Furthermore, heavy binge drinking has been linked to long-term health consequences including heart disease, high blood pressure, and type 2 diabetes (Fan et al., 2008). Perhaps most alarming is the finding of increased risk for developing alcohol dependence in individuals that binge drink early in life (Hingson et al., 2005; Miller et al., 2007). Thus, it is of paramount importance to identify neurochemical pathways in the brain that modulate binge drinking as such knowledge may provide insight into novel pharmaceutical treatments that could protect against this dangerous behavior. The manuscript by Kaur and colleagues (Kaur et al., in press) in the current volume of Alcoholism: Clinical and Experimental Research is an exciting example of recent work aimed at identifying the neurobiology of binge alcohol drinking.

Neuropeptide Y (NPY) and Protein Kinase A (PKA) have been implicated in neurobiological responses to ethanol. We have previously reported that mutant mice lacking normal production of the RIIβ subunit of PKA (RIIβ−/− mice) show enhanced sensitivity to the locomotor stimulant effects of ethanol and increased behavioral sensitization relative to littermate wild-type RIIβ+/+ mice. We now report that RIIβ−/− mice also show increased NPY immunoreactivity in the nucleus accumbens (NAc) core and the ventral striatum relative to RIIβ+/+ mice. These observations suggest that elevated NPY signaling in the NAc and/or striatum may contribute to the increased sensitivity to ethanol-induced behavioral sensitization that is characteristic of RIIβ−/− mice. Consistently, NPY−/− mice failed to display ethanol-induced behavioral sensitization that was evident in littermate NPY+/+ mice. To more directly examine the role of NPY in the locomotor stimulant effects of ethanol, we infused a recombinant adeno-associated virus (rAAV) into the region of the NAc core of DBA/2J mice. The rAAV-FIB-NPY13-36 vector expresses and constitutively secretes the NPY fragment NPY13-36 (a selective Y2 receptor agonist) from infected cells in vivo. Mice treated with the rAAV-FIB-NPY13-36 vector exhibited reduced expression of ethanol-induced behavioral sensitization compared to mice treated with a control vector. Taken together, the current data provide the first evidence that NPY signaling in the NAc core and the Y2 receptor modulate ethanol-induced behavioral sensitization.

Neuropeptide Y (NPY) is a 36-amino-acid neuromodulator that is distributed throughout the central nervous system and has been implicated in a wide range of neurobiological responses including the integration of emotional behavior. The anxiolytic properties of NPY are modulated by NPY signaling in the hippocampus and in the central (CeA) and basolateral (BLA) nuclei of the amygdala. Recently, the neurotoxin saporin, when conjugated to NPY (NPY–SAP), was shown to selectively kill NPY receptor-expressing neurons and has been used as a tool to study the central NPY neurocircuitry involved with feeding behaviors. Here we determined if NPY–SAP can be used as a tool to study the central NPY neurocircuitry that modulates anxiety-like behaviors. BALB/cJ mice were given injection of either NPY–SAP or a control blank saporin (B-SAP) into the CeA or the basomedial hypothalamus (BMH) as a control injection site. The elevated zero maze test was used to assess anxiety-like behavior and NPY–SAP-induced lesions were verified using NPY Y1 receptor (Y1R) immunoreactivity (IR). Results showed that injection of NPY–SAP into the CeA site-specifically blunted Y1R IR in the CeA which was associated with a significant increase in anxiety-like behavior. Injection of NPY–SAP into the BMH, while locally blunting Y1R IR, promoted a compensatory increase of Y1R IR in the BLA and the CA3 region of the hippocampus which was associated with a significant reduction of anxiety-like behavior. The present set of experiments suggest that the NPY–SAP neurotoxin may be a useful tool for studying the NPY neurocircuitry that modulates anxiety-like behaviors.

Alcoholism is a chronic disorder characterized by cycling periods of excessive ethanol consumption, withdrawal, abstinence and relapse, which is associated with progressive changes in central corticotropin-releasing factor (CRF) receptor signaling. CRF and urocortin (Ucn) peptides act by binding to the CRF type 1 (CRF1R) or the CRF type 2 (CRF2R) receptors, both of which have been implicated in the regulation of neurobiological responses to ethanol. The current review provides a comprehensive overview of preclinical evidence from studies involving rodents that when viewed together, suggest a promising role for CRF receptor (CRFR) antagonists in the treatment of alcohol abuse disorders. CRFR antagonists have been shown to protect against excessive ethanol intake resulting from ethanol dependence without influencing ethanol intake in non-dependent animals. Similarly, CRFR antagonists block excessive binge-like ethanol drinking in non-dependent mice but do not alter ethanol intake in mice drinking moderate amounts of ethanol. CRFR antagonists protect against increased ethanol intake and relapse-like behaviors precipitated by exposure to a stressful event. Additionally, CRFR antagonists attenuate the negative emotional responses associated with ethanol withdrawal. The protective effects of CRFR antagonists are modulated by the CRF1R. Finally, recent evidence has emerged suggesting that CRF2R agonists may also be useful for treating alcohol abuse disorders.

Recent evidence suggests that corticotropin-releasing factor (CRF) receptor (CRFR) signaling is involved in modulating binge-like ethanol consumption in C57BL/6J mice. In this report, a series of experiments were performed to further characterize the role of CRFR signaling in binge-like ethanol consumption. The role of central CRFR signaling was assessed with intracerebroventricular (i.c.v.) infusion of the nonselective CRFR antagonist, alpha-helical CRF(9-41) (0, 1, 5, 10 microg/1 microl). The contribution of central CRF type 2 receptor (CRF(2)R) signaling was assessed with i.c.v. infusion of the selective CRF(2)R agonist, urocortin (Ucn) 3 (0, 0.05, 0.1, or 0.5 microg/1 microl). The role of the hypothalamic-pituitary-adrenal (HPA) axis was assessed by pretreating mice with intraperitoneal (i.p.) injection of (1) the corticosterone synthesis inhibitor, metyrapone (0, 50, 100, 150 mg/kg) or (2) the glucocorticoid receptor antagonist, mifepristone (0, 25, 50 mg/kg), and (3) by using radioimmunoassay to determine whether binge-like ethanol intake influenced plasma corticosterone levels. Finally, we determined whether the ability of the CRF(1)R antagonist, CP-154,526 (CP; 0, 10, 15 mg/kg, i.p.), to blunt binge-like drinking required normal HPA axis signaling by comparing the effectiveness of CP in adrenalectomized (ADX) and normal mice. Results showed that i.c.v. infusion of a 1 microg dose of alpha-helical CRF(9-41) significantly attenuated binge-like ethanol consumption relative to vehicle treatment, and i.c.v. infusion of Ucn 3 dose-dependently blunted binge-like drinking. On the other hand, metyrapone nonselectively reduced both ethanol and sucrose consumption, mifepristone did not alter ethanol drinking, and binge-like drinking did not correlate with plasma corticosterone levels. Finally, i.p. injection of CP significantly attenuated binge-like ethanol intake in both ADX and normal mice. Together, these results suggest that binge-like ethanol intake in C57BL/6J mice is modulated by CRF(1)R and CRF(2)R signaling, such that blockade of CRF(1)R or activation of CRF(2)R effectively reduces excessive ethanol intake. Furthermore, normal HPA axis signaling is not necessary to achieve binge-like drinking behavior.

Background: The alcohol deprivation effect (ADE) is characterized by transient excessive alcohol consumption upon reinstatement of ethanol following a period of ethanol deprivation. While this phenomenon has been observed in rats using both bottle drinking (consummatory behavior) and operant self-administration (consummatory and appetitive “ethanol-seeking” behavior) procedures, ADE studies in mice have primarily relied on bottle drinking measures. Furthermore, the neurochemical pathways that modulate the ADE are not well understood. Therefore, we determined whether the ADE can be observed in C57BL/6J mice using operant self-administration procedures and if expression of the ADE is modulated by the corticotropin releasing factor-1 (CRF-1) receptor. Methods: C57BL/6J mice were trained in a 2-hour operant self-administration paradigm to lever press for 10% ethanol or water on separate response keys. Between operant sessions, mice had access to ethanol in their homecage. Once stable responding occurred, mice were deprived of ethanol for 4 days and were then retested with ethanol in the operant paradigm for 3 consecutive days. Next, to assess the role of the CRF-1 receptor, mice were given intraperitoneal (i.p.) injection (0, 10, or 20 mg/kg) of the CRF-1 receptor antagonist CP-154,526 30 minutes before ADE testing. Additional experiments assessed (i) ADE responding in which the alternate response lever was inactive, (ii) the effects of CP-154,526 on self-administration of a 1% sucrose solution following 4 days of deprivation, and (iii) ADE responding in which mice did not received i.p. injections throughout the experiment. Results: Mice exhibited a significant increase in postdeprivation lever responding for ethanol with either a water reinforced or inactive alternate lever. Interestingly, i.p. injection of a 10 mg/kg dose of CP-154,526 protected against the ADE while not affecting lever responding for a sucrose solution. Finally, baseline and deprivation-induced increases of ethanol reinforced lever responding were greater in mice not given i.p. injections. Conclusions: The ADE in C57BL/6J mice can be modeled using the operant self-administration paradigm and increased ethanol self-administration associated with the ADE is modulated by CRF-1 receptor signaling.

Stress may be a key contributor to the development of ethanol dependence and relapse (Breese et al., 2005; Koob, 2003). Stressful life events, such as those underlying post-traumatic stress disorder, are comorbid with ethanol abuse disorders and human laboratory studies show that stress increases the self-report of craving in abstinent alcoholics (Back et al., 2006; Breslau et al., 2003; Fox et al., 2007). Clinical research implicates stress in the relapse to pathological ethanol use in formerly abstinent alcoholics, perhaps as a means to self-medicate heightened anxiety and negative affect associated with withdrawal and abstinence from alcohol (Brady and Sonne, 1999; Breese et al., 2005; Kushner et al., 1994; Sinha, 2001). Recent investigations show that stress can also impact ethanol consumption in animal models (Chester et al., 2004; Croft et al., 2005; Le et al., 2000; Little et al., 1999; Liu and Weiss, 2002; Sillaber et al., 2002). Various stress paradigms reliably elicit stress-induced increases in ethanol consumption, especially among low ethanol consuming animals (Chester et al., 2004; Croft et al., 2005; Little et al., 1999). For example, selectively bred ethanol non-preferring NP rats exposed to 10 days of restraint stress showed significant and enduring increases in ethanol consumption beginning approximately 2 weeks following the stress procedure, while ethanol preferring P rats showed only transient stress-induced increases in ethanol drinking immediately after the stress procedure (Chester et al., 2004). Additionally, 3 weeks of stress induced by daily saline injections (Little et al., 1999) or 5 consecutive days of social defeat stress (Croft et al., 2005), significantly increased ethanol consumption approximately 2 weeks after the stress procedure among C57BL/10 mice displaying initially low preference for ethanol. An interesting commonality among many animal studies that assess the effects of stress on ethanol intake is that the effects of stress on ethanol drinking are delayed, typically occurring weeks after stress exposure (Chester et al., 2004; Croft et al., 2005; Little et al., 1999). Both ethanol and stress activate the hypothalamic-pituitary-adrenal (HPA) axis by inducing the release of corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and glucocorticoids (Brady and Sonne, 1999). The relationship between ethanol and the HPA-axis appears to be bidirectional, as exogenous administration of CRF, ACTH, and glucocorticoids alter ethanol consumption (Bell et al., 1998; O’Callaghan et al., 2002; Thorsell et al., 2005). Given that neurobiological responses to both stress and ethanol exposure involve HPA-axis signaling, it is possible that the neurochemicals and hormones associated with the HPA-axis modulate stress-induced increases of ethanol consumption. One such candidate is CRF, a 41 amino acid polypeptide that integrates both neuroendocrine and behavioral responses to stress (Smith et al., 1998). CRF-containing neurons are expressed throughout the brain, including in regions implicated in neurobiological responses to ethanol such as the bed nucleus of the stria terminalis, the amygdala, and the lateral hypothalamus (Koob, 2003). Of the two G protein-coupled receptors, the CRF1 receptor (CRF1R) appears to be involved with the integrate emotional behavior while the CRF2 receptor (CRF2R) may modulate ingestive behaviors (Koob, 2003; Zorrilla and Koob, 2004; Zorrilla et al., 2004). Corticotropin-releasing factor receptor signaling has been implicated in a variety of neurobiological responses to ethanol. For example, CRF receptor antagonists attenuate the anxiogenic effect of ethanol withdrawal (Breese et al., 2004; Knapp et al., 2004; Overstreet et al., 2004; Rassnick et al., 1993), prevent excessive ethanol self-administration in dependent animals (Funk et al., 2007; Valdez et al., 2002), and block foot shock-induced reinstatement of ethanol-seeking behavior (Liu and Weiss, 2002). The CRF1R also appears to be involved in stress-induced increases in ethanol consumption. Mutant mice lacking normal production of the CRF1R displayed significantly greater ethanol consumption beginning approximately 2 weeks after a social defeat stress procedure, an effect that was not evident in normal wild-type mice. Subsequent exposure to forced swim stress further augmented ethanol consumption in CRF1R knockout mice (Sillaber et al., 2002). While the Sillaber et al. (2002) study provides genetic evidence suggesting a role for the CRF1R in modulating stress-induced increases in ethanol consumption, the goal of the present experiment was to use a pharmacological approach to determine if pretreatment with the selective CRF1R antagonist, CP-154,526, would buffer the effects of stress and thus attenuate the development of stress-induced increases in ethanol intake in BALB/cJ mice. Therefore, we predicted that (1) ethanol consumption would increase among animals with a history of stress exposure and (2) pretreatment with CP-154,526 would attenuate stress-induced increases in ethanol consumption among animals with a history of stress. BALB/cJ mice were chosen because this strain has been shown to have high sensitivity to the effects of stress on both behavioral and neurobiological measures (Crawley et al., 1997) and drinks low levels of ethanol (Belknap et al., 1993). We also assessed the effects of stress exposure on ethanol consumption by C57BL/6N mice, a strain that voluntarily consumes high amounts of ethanol (Belknap et al., 1993). Here we show that 5 consecutive days of exposure to a 5-minute forced swim stress procedure caused significant and delayed increases in voluntary ethanol consumption in BALB/cJ mice, an effect which was attenuated by pretreatments with the CRF1R antagonist before each stress session. On the other hand, stress exposure did not alter ethanol intake by C57BL/6N mice.

Rodent models of alcoholism, including inbred and selectively bred strains have been useful tools for identifying the genetic and neurobiological factors that underlie this disease. However, in many cases, rodents do not consume enough alcohol to reach the point of behavioral and/or pharmacological intoxication (Spanagel, 2000). Recently, “drinking in the dark” (DID) procedures have been developed to induce excessive ethanol drinking in C57BL/6J mice, which result in blood ethanol concentrations (BECs) reaching levels that have measurable effects on physiology and/or behavior (Rhodes et al., 2005, 2007). With these procedures, C57BL/6J mice are given access to a 20% ethanol solution for 2 to 4 hours, starting with 3 hours into their dark cycle. C57BL/6J can achieve BECs of >100 mg% and exhibit signs of behavioral intoxication as measured by motor deficits on the rotarod and balance beam (Rhodes et al., 2005, 2007). It has been argued that the DID model has predictive validity for testing potential pharmacological targets aimed at treating alcohol abuse disorders as naltrexone, an opioid receptor antagonist which is currently used to treat alcoholism, dose dependently attenuates high levels of ethanol drinking induced by DID procedures (Kamdar et al., 2007). Corticotropin releasing factor (CRF) is a 41 amino acid neuromodulator that is widely expressed throughout the central nervous system (Bloom et al., 1982; Merchenthaler et al., 1982). CRF has been shown to modulate diverse biological functions including food intake, stress and anxiety-like behaviors, and neurobiological responses to ethanol [for reviews, see (Heilig and Koob, 2007; Valdez, 2006; Zorrilla and Koob, 2004; Zorrilla et al., 2003)]. Increases in CRF immunoreactivity (Olive et al., 2002; Zorrilla et al., 2001) and levels of extracellular CRF (Funk et al., 2006) are seen in the amygdala following ethanol withdrawal. Exposure to ethanol causes robust activation of the hypothalamic-pituitary-adrenal (HPA)-axis (Rivier, 1996; Rivier et al., 1990), which is initiated by ethanol-induced increases of CRF activity within the hypothalamus (Li et al., 2005; Rivier and Lee, 1996). Recent pharmacological and genetic evidence support the hypothesis that CRF exerts its effects on ethanol consumption through activation of the CRF1 receptor (CRF1R). Blockade of the CRF1R attenuates ethanol intake in dependent, but not nondependent rodents (Funk et al., 2007; Gehlert et al., 2007). Consistently, CRF1R deficient mice failed to show increased ethanol consumption following the acquisition of ethanol dependence and a period of abstinence that was observed in wild-type mice (Chu et al., 2007). Interestingly, a genetic polymorphism at the Crhr1 locus, which encodes the CRF1R was found to be significantly linked to alcoholism (Treutlein et al., 2006). Because CRF receptor signaling has been implicated in a wide range of neurobiological responses to ethanol, the goal of the present set of experiments was to determine whether the increased consumption of ethanol associated with DID procedures can be modulated by pretreatment with CP-154,526, a CRF1R antagonist. Specifically, because ethanol triggers HPA-axis signaling which is initiated by ethanol-induced increases of CRF activity within the hypothalamus (Li et al., 2005; Rivier and Lee, 1996), and because levels of corticosterone, a HPA-axis-associated hormone, have been shown to positively correlated with ethanol intake (Fahlke et al., 1994, 1995, 1996), we predicted that CRF1R blockade would attenuate increased ethanol drinking promoted by DID procedures.