Alcohol-seeking Behavior Research Articles

Cue-alcohol associative learning in female rats.

The ability of environmental cues to trigger alcohol-seeking behaviors is believed to facilitate problematic alcohol use. We previously showed that the development of this cue-evoked alcohol approach reflects cue-alcohol learning and memory in the adult male rat; however, we do not know whether the same is true for similarly aged female rats. Consequently, adult Long-Evans female rats were allowed to drink unsweetened alcohol in the home cage (Monday, Wednesday, Friday; 24-h two-bottle choice; 5 weeks) and were subsequently split into two experimental groups: Paired and Unpaired. Groups were matched for ingested doses and alcohol bottle preference across the pre-conditioning home cage period. Both groups were trained in conditioning chambers using a Pavlovian procedure. For the Paired group, the chamber houselight was illuminated to signal access to an alcohol sipper. Houselight onset was yoked for the Unpaired group, but access to the alcohol sipper was scheduled to occur only during the intervening periods (in the absence of light). We found that in the Paired, but not Unpaired group, an alcohol approach reaction was conditioned to houselight illumination, and the level of cue-conditioned reactivity predicted drinking behavior within trials. Groups experienced equivalently low but non-negligible blood alcohol concentrations over the course of conditioning sessions. We conclude that cue-triggered alcohol-seeking behavior in adult female rats reflects associative learning about the relationship between alcohol availability and houselight illumination.

Baclofen but Not Diazepam Alleviates Alcohol-Seeking Behavior and Hypothalamic-Pituitary-Adrenal Axis Dysfunction in Stressed Withdrawn Mice.

This study compares the impact of repeated injections of baclofen (an agonist of GABAB receptors) or diazepam (a benzodiazepine having an agonist action on GABAA receptors) given during the alcohol-withdrawal period on the stress-induced restoration of alcohol-seeking behavior and hypothalamic–pituitary–adrenal (HPA) axis dysfunction after a long (4 weeks) abstinence. Thus, C57BL/6 mice were submitted to a 6-month alcohol consumption [12% volume/volume (v/v)] and were progressively withdrawn to water before testing. Diazepam (Valium®, Roche) and baclofen (Baclofen®, Mylan) were administered intraperitoneally for 15 consecutive days (1 injection/day) during the withdrawal period at decreasing doses ranging from 1.0 mg/kg (Day 15) to 0.25 mg/kg (Day 1) for diazepam and from 1.5 mg/kg (Day 15) to 0.37 mg/kg (Day 1) for baclofen. Alcohol-seeking behavior was evaluated by alcohol-place preference in an odor recognition task. In the stress condition, mice received three electric footshocks 45 min before behavioral testing. Blood was sampled immediately after behavioral testing, and plasma corticosterone concentrations were measured by commercial enzyme immunoassay kits. Results showed that non-stressed withdrawn mice did not exhibit alcohol-place preference or alteration of plasma corticosterone concentrations relative to water controls. After stress, however, withdrawn mice exhibited a significant alcohol-place preference and higher circulating corticosterone concentrations as compared to stressed water controls. Interestingly, repeated administration during the withdrawal phase of baclofen but not diazepam suppressed both the alcohol-place preference and normalized corticosterone levels in stressed withdrawn animals. In conclusion, this study evidences that a pre-treatment with baclofen but not with diazepam during the withdrawal phase normalized, even after a long period of abstinence, the HPA axis response to stress, which contributes to the long-term preventing effects of this compound on alcohol-seeking behavior.

Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope® revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting Ih and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.

Ghrelin: From a Gut Hormone to a Potential Therapeutic Target for Alcohol Addiction

Alcohol use disorder (AUD) is a chronic relapsing disease with significant medical, psychosocial, and economic burden. Pharmacological treatments for AUD are, however, limited in number and efficacy. Beyond classic neurotransmitter systems, there has been a growing interest in understanding the role of peripheral pathways (e.g., gut‐brain communications) in AUD, with the ultimate goal of identifying novel therapeutic targets. Ghrelin is an orexigenic peptide synthesized by enteroendocrine cells primarily located in the gastric mucosa. Ghrelin is known as the ‘hunger hormone’, given its role in stimulating both homeostatic and hedonic food intake. Ghrelin has also been shown to modulate alcohol seeking and consummatory behaviors, possibly through interactions with reward and stress pathways. Here we investigated whether and how pharmacological manipulation of the ghrelin system may affect biobehavioral correlates of alcohol use in heavy‐drinking alcohol‐dependent individuals. First, in a randomized, double‐blind, placebo‐controlled, human laboratory study, intravenous (IV) ghrelin/placebo was administered and two experiments were conducted: progressive‐ratio IV alcohol self‐administration (IV‐ASA) and brain functional magnetic resonance imaging (fMRI). Results showed that IV ghrelin, compared to placebo, significantly increased the number of alcohol infusions self‐administered (p=0.04) and reduced the time to initiate alcohol self‐administration (p=0.03). In addition, ghrelin increased the alcohol‐related signal in the amygdala (p=0.01) and modulated the food‐related signal in the medial orbitofrontal cortex (p=0.01) and nucleus accumbens (p=0.08). In the second study, we examined the effects of a novel ghrelin receptor inverse agonist (PF‐5190457) co‐administered with alcohol in both rodents and humans. No pharmacological interactions between alcohol and PF‐5190457 were found. PF‐5190457 did not interact with the effects of alcohol on locomotor activity or loss‐of‐righting reflex. In humans, PF‐5190457 was well tolerated, did not cause any significant side effect, and did not alter alcohol pharmacokinetics or alcohol‐induced subjective effects. As a secondary outcome, a cue‐reactivity procedure was also conducted in a bar‐like laboratory setting. Results showed that PF‐5190457, compared to placebo, reduced cue‐elicited craving for alcohol (p=0.05), as well as attention to alcohol cues (p=0.02). In conclusion, the ghrelin system seems to regulate both behavioral and neurobiological correlates of alcohol consumption and may represent a potential target for developing novel medications to treat AUD.Support or Funding InformationThis work was supported by A) NIH intramural funding ZIA‐AA000218 jointly supported by NIAAA and NIDA, and B) NCATS grant UH2/UH3‐TR000963. Development of the Computerized Alcohol Infusion System software was supported by the NIAAA‐funded Indiana Alcohol Research Center (AA007611). Pfizer provided the PF‐5190457 drug under the NCATS grant UH2/UH3‐TR000963 grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oxytocin attenuates stress-induced reinstatement of alcohol seeking behavior in male and female mice.

The neuropeptide oxytocin (OXT) has emerged as a potential therapeutic intervention in the treatment of both alcohol use disorder (AUD) and stress-related psychiatric illnesses. The present study evaluates the effects of systemically administered (intraperitoneal (i.p.)) OXT treatment on alcohol relapse-like behavior in male and female mice. Adult male and female C57BL/6J mice were trained to lever respond in operant conditioning chambers for alcohol in daily self-administration sessions. Once lever responding and alcohol intake stabilized, mice were tested under extinction conditions for 14days before reinstatement testing. All mice underwent stress-induced reinstatement testing using either predator odor (2,3,5-trimethyl-3-thiazoline (TMT)) or the α-2 adrenergic receptor agonist yohimbine. In study 1, mice were exposed to TMT for 15min and then immediately placed into operant conditioning chambers to examine alcohol-seeking behavior under extinction conditions. At 30min prior to test session, separate groups of mice were injected with vehicle or OXT (0.1, 0.5, 1mg/kg). In study 2, mice were injected with yohimbine (0.3, 0.625mg/kg) 1h prior to reinstatement testing. At 30min post-yohimbine injection, mice are injected (i.p.) with vehicle or OXT (1mg/kg). OXT attenuated alcohol-seeking behavior in a dose-related manner in male and female mice in response to acute challenge with a predator odor. Additionally, OXT administration produced a similar decrease in alcohol relapse-like behavior triggered by the pharmacological stressor yohimbine in both sexes. Systemic oxytocin administration attenuates stress-induced reinstatement of alcohol seeking in male and female mice.

Endocannabinoids Promote Ethanol Drinking Via Cb <sub>1</sub> Receptor-Mediated Increase in Ghrelin Acylation and Signaling in the Stomach

The dynamic interplay between energy homeostasis and motivational behavior is exemplified by mediators involved in regulating both. Endocannabinoids acting via CB1 receptors (CB1R) or the stomach-derived hormone ghrelin acting via ghrelin receptors (GHS-R1A) both promote food intake and support alcohol seeking behavior. Here we report that the peripheral CB1R antagonist JD5037 reduces ethanol drinking in mice by inhibiting the formation of biologically active octanoyl-ghrelin without affecting its inactive precursor desacyl-ghrelin. In ghrelin-producing stomach cells, JD5037 reduced the level of the substrate octanoyl-carnitine generated from palmitoyl-carnitine due to increased fatty acid β-oxidation. Blocking gastric vagal afferents abrogated the ability of either CB1R or GHS-R1A blockade to reduce ethanol drinking. We conclude that blockade of CB1R in ghrelin-producing cells inhibits the formation of biologically active ghrelin and its signaling via gastric vagal afferents to facilitate voluntary ethanol intake. Thus, peripheral CB1R blockade affecting the gut-brain axis may have therapeutic potential in alcoholism.

α5GABAA subunit-containing receptors and sweetened alcohol cue-induced reinstatement and active sweetened alcohol self-administration in male rats.

GABAA receptors containing the α5 subunit (i.e., α5GABAA receptors) appear to be critically involved in the reinforcing and subjective effects of alcohol. Their role in alcohol relapse remains unknown. Pharmacological approaches were used to probe the role of α5GABAA receptors in alcohol seeking induced by re-exposure to a sweetened alcohol-paired cue, as well as in alcohol + sucrose vs. sucrose self-administration. For reinstatement studies, rats were trained to self-administer alcohol under a fixed-ratio schedule in which responding was maintained by alcohol + sucrose deliveries and an alcohol-paired stimulus. Sweetened alcohol seeking was extinguished by eliminating solution deliveries and the sweetened alcohol-paired stimulus. During reinstatement tests, animals received pretreatments of an α5GABAA inverse agonist (L-655,708) or an agonist (QH-ii-066) prior to sessions in which presentation of the sweetened alcohol-paired stimulus was restored, but no solution was delivered. For self-administration studies, rats were trained to self-administer alcohol + sucrose or sucrose under a fixed-ratio schedule. Once stable, animals received pretreatments of QH-ii-066, L-655,708, the inverse agonist RY-023, or naltrexone. L-655,708 attenuated reinstatement of sweetened alcohol seeking by alcohol + sucrose-paired cues; whereas sweetened alcohol-seeking behavior was augmented by QH-ii-066, albeit at different doses in different rats. Both L-655,708 and RY-023 selectively reduced alcohol + sucrose vs. sucrose self-administration. In contrast, naltrexone reduced both alcohol + sucrose and sucrose self-administration; whereas QH-ii-066 enhanced sucrose self-administration only. α5GABAA receptors play a key role in the modulation of sweetened alcohol cue-induced reinstatement, as well as in alcohol + sucrose but not sucrose self-administration. Inverse agonist activity at α5GABAA receptors may offer a novel strategy for both the reduction of problematic drinking and the prevention of relapse.

Cue-elicited craving and human Pavlovian-to-instrumental transfer

Background: Drug cue-reactivity can be measured by the well-established cue-elicited craving model, or by the more recently developed Pavlovian to instrumental transfer (PIT) procedure, which quantifies the impact of drug cues on drug-seeking behaviour. It remains unclear whether these two models produce similar cue reactive effects.Method: To test this, 38 young adult beer drinkers completed an alcohol cue-elicited craving procedure followed by a specific PIT procedure with alcohol cues.Results: There was a significant effect of alcohol cues on craving (p = .007) and on alcohol-seeking behaviour in the PIT procedure (p < .001). Contrary to expectations, these two indices of cue-reactivity were not correlated (r = −.08, p = .66). However, analysis indicated that the alcohol PIT effect was correlated with the self-reported belief that alcohol cues signalled greater effectiveness of the alcohol-seeking response (r = .44, p = .008).Conclusions: These findings suggest that different measures of cue-reactivity might tap into different responses within an individual. Future research is necessary to consider whether this variance is due to which aspect of cue reactivity is being assessed and whether different types of cue-reactivity are differentially influenced by variables such as outcome expectancy.

Oleoylethanolamide, Neuroinflammation, and Alcohol Abuse.

Neuroinflammation is a complex process involved in the physiopathology of many central nervous system diseases, including addiction. Alcohol abuse is characterized by induction of peripheral inflammation and neuroinflammation, which hallmark is the activation of innate immunity toll-like receptors 4 (TLR4). In the last years, lipid transmitters have generated attention as modulators of parts of the addictive process. Specifically, the bioactive lipid oleoylethanolamide (OEA), which is an endogenous acylethanolamide, has shown a beneficial profile for alcohol abuse. Preclinical studies have shown that OEA is a potent anti-inflammatory and antioxidant compound that exerts neuroprotective effects in alcohol abuse. Exogenous administration of OEA blocks the alcohol-induced TLR4-mediated pro-inflammatory cascade, reducing the release of proinflammatory cytokines and chemokines, oxidative and nitrosative stress, and ultimately, preventing the neural damage in frontal cortex of rodents. The mechanisms of action of OEA are discussed in this review, including a protective action in the intestinal barrier. Additionally, OEA blocks cue-induced reinstatement of alcohol-seeking behavior and reduces the severity of withdrawal symptoms in animals, together with the modulation of alcohol-induced depression-like behavior and other negative motivational states associated with the abstinence, such as the anhedonia. Finally, exposure to alcohol induces OEA release in blood and brain of rodents. Clinical evidences will be highlighted, including the OEA release and the correlation of plasma OEA levels with TLR4-dependent peripheral inflammatory markers in alcohol abusers. In base of these evidences we hypothesize that the endogenous release of OEA could be a homeostatic signal to counteract the toxic action of alcohol and we propose the exploration of OEA-based pharmacotherapies to treat alcohol-use disorders.

Compulsive Alcohol Seeking Results from a Failure to Disengage Dorsolateral Striatal Control over Behavior.

The acquisition of drug, including alcohol, use is associated with activation of the mesolimbic dopamine system. However, over the course of drug exposure the control over drug seeking progressively devolves to anterior dorsal striatum (aDLS) dopamine-dependent mechanisms. The causal importance of this functional recruitment of aDLS in the switch from controlled to compulsive drug use in vulnerable individuals remains to be established. Here we tested the hypothesis that individual differences in the susceptibility to aDLS dopamine-dependent control over alcohol seeking predicts and underlies the development of compulsive alcohol seeking. Male alcohol-preferring rats, the alcohol-preferring phenotype of which was confirmed in an intermittent two-bottle choice procedure, were implanted bilaterally with cannulae above the aDLS and trained instrumentally on a seeking-taking chained schedule of alcohol reinforcement until some individuals developed compulsive seeking behavior. The susceptibility to aDLS dopamine control over behavior was investigated before and after the development of compulsivity by measuring the extent to which bilateral aDLS infusions of the dopamine receptor antagonist α-flupenthixol (0, 5, 10, and 15 μg/side) decreased alcohol seeking at different stages of training, as follows: (1) after acquisition of instrumental taking responses for alcohol; (2) after alcohol-seeking behavior was well established; and (3) after the development of punishment-resistant alcohol seeking. Only alcohol-seeking, not alcohol-taking, responses became dependent on aDLS dopamine. Further, marked individual differences in the susceptibility of alcohol seeking to aDLS dopamine receptor blockade actually predicted the vulnerability to develop compulsive alcohol seeking, but only in subjects dependent on aDLS dopamine-dependent control.SIGNIFICANCE STATEMENT Over the course of addictive drug exposure, there is a transition in the control over drug seeking from ventral to anterior dorsal striatum (aDLS) dopamine-dependent mechanisms, but it is unclear whether this is causally involved in the development of compulsive drug seeking. We tested the hypothesis that individual differences in the reliance of alcohol seeking on aDLS dopamine predicts and underlies the emergence of compulsive alcohol seeking. We identified individual differences in the reliance of well established alcohol seeking, but not taking behavior, on aDLS mechanisms and also showed that this predicted the subsequent development of compulsive alcohol-seeking behavior. Thus, those individuals in whom alcohol seeking depended on aDLS mechanisms were vulnerable subsequently to display compulsivity.

Anterior Insular Cortex is Critical for the Propensity to Relapse Following Punishment-Imposed Abstinence of Alcohol Seeking.

Humans with alcohol use disorder typically abstain because of the negative consequences associated with excessive drinking, and exposure to contexts previously associated with alcohol use can trigger relapse. We used a rat model that captures a characteristic of this human condition: namely voluntary abstinence from alcohol use because of contingent punishment. There is substantial variability in the propensity to relapse following extended periods of abstinence, and this is a critical feature preventing the successful treatment of alcohol use disorder. Here we examined relapse following acute or prolonged abstinence. In male alcohol preferring P rats, we found an increased propensity to relapse in Context B, the punishment context after prolonged abstinence. Next, we found that neither alcohol intake history nor the motivational strength of alcohol predicted the propensity to relapse. We next examined the putative circuitry of context-induced relapse to alcohol seeking following prolonged abstinence using Fos as a marker of neuronal activation. The anterior insular cortex (AI) was the only brain region examined where Fos expression correlated with alcohol seeking behavior in Context B after prolonged abstinence. Finally, we used local infusion of GABAA and GABAB receptor agonists (muscimol + baclofen) to show a causal role of the AI in context-induced relapse in Context B, the punishment context after prolonged abstinence. Our results show that there is substantial individual variability in the propensity to relapse in the punishment-associated context after prolonged abstinence, and this is mediated by activity in the AI.SIGNIFICANCE STATEMENT A key feature of alcohol use disorder is that sufferers show an enduring propensity to relapse throughout their lifetime. Relapse typically occurs despite the knowledge of adverse consequences including health complications or relationship breakdowns. Here we use a recently developed rodent model that recapitulates this behavior. After an extended period of abstinence, relapse propensity is markedly increased in the "adverse consequence" environment, akin to humans with alcohol use disorder relapsing in the face of adversity. From a circuitry perspective, we demonstrate a causal role of the anterior insular cortex in relapse to alcohol seeking after extended abstinence following punishment imposed voluntary cessation of alcohol use.

Brief Exposure to Pictures Depicting Poor Environments Leads to Increased Consumption of Beer in Adult Social Drinkers

Previous studies have suggested a trait-like association between neighborhood deprivation and alcohol consumption. However, it is not known whether temporarily manipulating poverty and affluence states by exposure to stimuli signifying resource-scarcity or resource-wealth would influence alcohol-seeking behavior. Here, we aimed to investigate whether implicit exposure to affluence and poverty-related pictures would influence beer consumption. Participants in a “poverty” group viewed pictures depicting impoverished environments, and participants in an “affluence” group viewed images of wealthy environments. After priming, participants were provided with nonalcoholic beer (which they were told was alcohol-containing beer) and orange juice under the guise of a bogus taste test, to measure their alcohol-seeking behavior. Results showed that priming participants with a resource-scarce environment led to an increase in beer consumption (as a percentage of total fluid consumed), compared to priming with a resource-rich environment. The same pattern of results was obtained in both a Western European sample (Experiment 1) and a West Indian sample (Experiment 2). In Experiment 2, we also tested whether risk-taking behavior, measured by the Balloon Analogue Risk Task, was influenced by the environmental priming; no differences between groups were observed. These results provide the first experimental evidence that manipulation of poverty-affluence state, by brief exposure to pictures of impoverished or wealthy neighborhoods, can influence alcohol-seeking behavior in adult social drinkers.

Alcohol-associated antecedent stimuli elicit alcohol seeking in non-dependent rats and may activate the insula.

Alcohol self-administration produces brain and behavior adaptations that facilitate a progressive loss of control over drinking and contribute to relapse. One possible adaptation is the ability of antecedent environmental stimuli that are consistently paired with alcohol to trigger alcohol-seeking behaviors. We previously modeled this adaptation in rats using a Pavlovian conditioning procedure in which illumination of a houselight preceded the presentation of a sipper tube that produced unsweetened alcohol when licked. However, in our previous work we did not demonstrate whether this adaptation represented a consequence of repeated exposure to alcohol or the houselight, or whether it was the consequence of associative learning and memory. Thus, in the present study, we tested the associative basis of alcohol seeking in response to houselight illumination in our task using adult male rats that were not food- or water-deprived and were not dependent on alcohol. Separate groups of rats received houselight illumination that was explicitly paired or unpaired with presentation of the retractable sipper that provided access to unsweetened alcohol. Our primary dependent variable was appetitive alcohol-directed behavior: the frequency of movement toward and interaction with the hole in the wall of the chamber through which the sipper was presented during the period of houselight illumination trial before each sipper presentation. However, we also analyzed consummatory sipper licking behavior and blood ethanol concentration in the same rats. Finally, we explored the brain basis of cue-elicited alcohol seeking using c-Fos immunohistochemistry. Our findings confirmed the associative basis of cue-elicited alcohol seeking in our paradigm and mapped these onto the insular cortex, suggesting a role for this brain region in early stages of brain and behavior adaptation to regular alcohol use.

Effect of caffeine on alcohol consumption and alcohol-induced conditioned place preference in rodents

Background The aim of the study was to determine the effect of caffeine on alcohol consumption with or without deprivation and alcohol-induced conditioned place preference. Methods In the present study, we examined the effects of caffeine (2.5, 5 and 10 mg/kg) on alcohol consumption in Wistar rats with or without periods of deprivation in an unlimited-access, two-bottle, free choice drinking procedure after a stable baseline alcohol consumption was established. Conditioned place preference (CPP) was established by intraperitoneal injections of alcohol (2 g/kg) in a 12-day conditioning schedule in mice. The effect of caffeine (3 mg/kg) on CPP expression was determined by a final post-conditioning test following 12 conditioning sessions with alcohol. The effect of caffeine (3 mg/kg) on the reinstatement of alcohol-induced CPP was determined in a final post-conditioning test following 12 conditioning sessions with alcohol and the extinction of alcohol-induced CPP. Results Alcohol deprivation for 3 days did not result in alcohol deprivation effect (ADE). While caffeine (10 mg/kg) caused a significant (p<0.05) reduction in alcohol consumption compared with the baseline following a period of alcohol deprivation, it did not cause a change in alcohol consumption compared with the baseline in the study without alcohol deprivation phase. Caffeine significantly (p<0.05) reduced the expression of alcohol-induced CPP compared to saline and blocked the reinstatement of alcohol-induced CPP following the injection of a priming dose (0.4 g/kg) of alcohol. Conclusions Given that caffeine is an adenosine receptor antagonist, our findings suggest a role for adenosine receptors in the alcohol reward and alcohol-seeking behaviour.

Mammalian target of rapamycin complex 1 and its downstream effector collapsin response mediator protein-2 drive reinstatement of alcohol reward seeking.

Alcohol use disorder is a chronic relapsing disease. Maintaining abstinence represents a major challenge for alcohol-dependent patients. Yet the molecular underpinnings of alcohol relapse remain poorly understood. In the present study, we investigated the potential role of the mammalian target of rapamycin complex 1 (mTORC1) in relapse to alcohol-seeking behavior by using the reinstatement of a previously extinguished alcohol conditioned place preference (CPP) response as a surrogate relapse paradigm. We found that mTORC1 is activated in the nucleus accumbens shell following alcohol priming-induced reinstatement of alcohol place preference. We further report that the selective mTORC1 inhibitor, rapamycin, abolishes reinstatement of alcohol place preference. Activation of mTORC1 initiates the translation of synaptic proteins, and we observed that reinstatement of alcohol CPP is associated with increased protein levels of one of mTORC1's downstream targets, collapsin response mediator protein-2 (CRMP2), in the nucleus accumbens. Importantly, the level of mTORC1 activation and CRMP2 expression positively correlate with the CPP score during reinstatement. Finally, we found that systemic administration of the CRMP2 inhibitor, lacosamide, attenuates alcohol priming-induced reinstatement of CPP. Together, our results reveal that mTORC1 and its downstream target, CRMP2, contribute to mechanisms underlying reinstatement of alcohol reward seeking. Our results could have important implications for the treatment of relapse to alcohol use and position the Food and Drug Administration approved drugs, rapamycin and lacosamide, for the treatment of alcohol use disorder.

GAL3 receptor knockout mice exhibit an alcohol-preferring phenotype.

Galanin is a neuropeptide which mediates its effects via three G-protein coupled receptors (GAL1-3 ). Administration of a GAL3 antagonist reduces alcohol self-administration in animal models while allelic variation in the GAL3 gene has been associated with an increased risk of alcohol use disorders in diverse human populations. Based on the association of GAL3 with alcoholism, we sought to characterize drug-seeking behavior in GAL3 -deficient mice for the first time. In the two-bottle free choice paradigm, GAL3 -KO mice consistently showed a significantly increased preference for ethanol over water when compared to wildtype littermates. Furthermore, male GAL3 -KO mice displayed significantly increased responding for ethanol under operant conditions. These differences in alcohol seeking behavior in GAL3 -KO mice did not result from altered ethanol metabolism. In contrast to ethanol, GAL3 -KO mice exhibited similar preference for saccharin and sucrose over water, and a similar preference for a high fat diet over a low fat diet as wildtype littermates. No differences in cognitive and locomotor behaviors were observed in GAL3 -KO mice to account for increased alcohol seeking behavior. Overall, these findings suggest genetic ablation of GAL3 in mice increases alcohol consumption.

The galanin receptor-3 antagonist, SNAP 37889, inhibits cue-induced reinstatement of alcohol-seeking and increases c-Fos expression in the nucleus accumbens shell of alcohol-preferring rats.

This study aimed to investigate the effects of the galanin-3 receptor antagonist, SNAP 37889, on c-Fos protein expression after cue-induced reinstatement of alcohol-seeking in the brains of alcohol-preferring rats. Eighteen alcohol-preferring rats were trained to self-administer 10% v/v ethanol in the presence of response-contingent cues, which was followed by extinction. Rats were then treated with SNAP 37889 (30 mg/kg, i.p.) or vehicle, before being tested for cue-induced reinstatement. Administration of SNAP 37889 reduced cue-induced reinstatement of ethanol-seeking behaviour. To examine the effect of SNAP 37889 and cue-induced reinstatement on neuronal activation, c-Fos expression was measured in subregions of the medial prefrontal cortex and nucleus accumbens. SNAP 37889 administration increased c-Fos immunoreactivity in the nucleus accumbens shell, but was without effect in the nucleus accumbens core and the medial prefrontal cortex. Dual-label Fos/tyrosine hydroxylase immunohistochemistry was used to examine the effects of SNAP 37889 on dopamine neurons in the ventral tegmental area; however, no differences between SNAP 37889 and vehicle-treated rats were found. These data support previous findings of galanin-3 receptor involvement in cue-induced reinstatement of alcohol-seeking behaviour, and provide novel evidence that the ability of galanin-3 receptor antagonism to attenuate cue-induced reinstatement relates to activation of the nucleus accumbens shell.

Stress, Motivation, and the Gut-Brain Axis: A Focus on the Ghrelin System and Alcohol Use Disorder.

Since its discovery, the gut hormone, ghrelin, has been implicated in diverse functional roles in the central nervous system. Central and peripheral interactions between ghrelin and other hormones, including the stress-response hormone cortisol, govern complex behavioral responses to external cues and internal states. By acting at ventral tegmental area dopaminergic projections and other areas involved in reward processing, ghrelin can induce both general and directed motivation for rewards, including craving for alcohol and other alcohol-seeking behaviors. Stress-induced increases in cortisol seem to increase ghrelin in the periphery, suggesting a pathway by which ghrelin influences how stressful life events trigger motivation for rewards. However, in some states, ghrelin may be protective against the anxiogenic effects of stressors. This critical review brings together a dynamic and growing literature, that is, at times inconsistent, on the relationships between ghrelin, central reward-motivation pathways, and central and peripheral stress responses, with a special focus on its emerging role in the context of alcohol use disorder.

Opposing actions of CRF-R1 and CB1 receptors on VTA-GABAergic plasticity following chronic exposure to ethanol.

Dopamine neurons in the ventral tegmental area (VTA) influence learned behaviors and neuropsychiatric diseases including addiction. The stress peptide corticotrophin-releasing factor (CRF) contributes to relapse to drug and alcohol seeking following withdrawal, although the cellular actions are poorly understood. In this study, we show that presynaptic CRF type 1 receptors (CRF-R1) potentiate GABA release onto mouse VTA dopamine neurons via a PKC-Ca2+ signaling mechanism. In naive animals, activation of CRF-R1 by bath application of CRF or ethanol enhanced GABAA inhibitory postsynaptic currents (IPSCs). Following 3 days of withdrawal from four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure, spontaneous IPSC frequency was enhanced while CRF and ethanol potentiation of IPSCs was intact. However, withdrawal for 3 weeks or more was associated with reduced spontaneous IPSC frequency and diminished CRF and ethanol responses. Long-term withdrawal was also accompanied by decreased sensitivity to the CB1 receptor agonist WIN55212 as well as greatly enhanced sensitivity to the CB1 antagonist AM251. Inclusion of BAPTA in the internal recording solution restored the responsiveness to CRF or ethanol and reduced the potentiating actions of AM251. Together, these data suggest that GABAA inhibition of VTA dopamine neurons is regulated by presynaptic actions of CRF and endocannabinoids and that long-term withdrawal from CIE treatment enhances endocannabinoid-mediated inhibition, thereby suppressing CRF facilitation of GABA release. Such findings have implications for understanding the impact of chronic alcohol on stress-related, dopamine-mediated alcohol-seeking behaviors.

Synaptic Plasticity in the Bed Nucleus of the Stria Terminalis: Underlying Mechanisms and Potential Ramifications for Reinstatement of Drug- and Alcohol-Seeking Behaviors.

The bed nucleus of the stria terminalis (BNST) is a component of the extended amygdala that shows significant changes in activity and plasticity through chronic exposure to drugs and stress. The region is critical for stress- and cue-induced reinstatement of drug-seeking behaviors and is thus a candidate region for the plastic changes that occur in abstinence that prime addicted patients for reinstatement behaviors. Here, we discuss the various forms of long-term potentiation (LTP) and long-term depression (LTD) in the rodent BNST and highlight the way that these changes in excitatory transmission interact with exposure to alcohol and other drugs of abuse, as well as other stressors. In addition, we highlight potential areas for future research in this area, including investigating input- and cell-specific bidirectional changes in activity. As we continue to accrue foundational knowledge in the mechanisms and effects of plasticity in the BNST, molecular targets and treatment strategies that are relevant to reinstatement behaviors will also begin to emerge. Here, we briefly discuss the effects of catecholamine receptor modulators on synaptic plasticity in the BNST due to the role of norepinephrine in LTD and dopamine on the short-term component of LTP as well as the role that signaling at these receptors plays in reinstatement of drug- and alcohol-seeking behaviors. We hope that insights gained on the specific changes in plasticity that occur within the BNST during abstinence from alcohol and other drugs of abuse will provide insight into the biological underpinnings of relapse behavior in human addicts and inform future treatment modalities for addiction that tackle this complex biological problem.