Voluntary Alcohol Intake Research Articles

Activation of Melanocortin-4 Receptor Inhibits Both Neuroinflammation Induced by Early Exposure to Ethanol and Subsequent Voluntary Alcohol Intake in Adulthood in Animal Models: Is BDNF the Key Mediator?

The concept that neuroinflammation induced by excessive alcohol intake in adolescence triggers brain mechanisms that perpetuate consumption has strengthened in recent years. The melanocortin system, composed of the melanocortin 4 receptor (MC4R) and its ligand α-melanocyte-stimulating hormone (α-MSH), has been implicated both in modulation of alcohol consumption and in ethanol-induced neuroinflammation decrease. Chronic alcohol consumption in adolescent rats causes a decrease in an α-MSH release by the hypothalamus, while the administration of synthetic agonists of MC4R causes a decrease in neuroinflammation and a decrease in voluntary alcohol consumption. However, the mechanism that connects the activation of MC4R with the decrease of both neuroinflammation and voluntary alcohol consumption has not been elucidated. Brain-derived neurotrophic factor (BDNF) has been implicated in alcohol drinking motivation, dependence and withdrawal, and its levels are reduced in alcoholics. Deficiencies in BDNF levels increased ethanol self-administration in rats. Further, BDNF triggers important anti-inflammatory effects in the brain, and this could be one of the mechanisms by which BDNF reduces chronic alcohol intake. Interestingly, MC4R signaling induces BDNF expression through the activation of the cAMP-responsive element-binding protein (CREB). We hypothesize that ethanol exposure during adolescence decreases the expression of α-MSH and hence MC4R signaling in the hippocampus, leading to a lower BDNF activity that causes dramatic changes in the brain (e.g., neuroinflammation and decreased neurogenesis) that predispose to maintain alcohol abuse until adulthood. The activation of MC4R either by α-MSH or by synthetic agonist peptides can induce the expression of BDNF, which would trigger several processes that lead to lower alcohol consumption.

Effects of pair housing on voluntary alcohol intake in male and female Wistar rats.

A number of different voluntary alcohol intake paradigms are available for home cage drinking studies. Traditionally, these paradigms involve single housing in order for individual intake to be measured. This study aimed at investigating the effects of pair housing on voluntary alcohol intake. Male and female Wistar rats were housed in pairs or individually for studies of voluntary alcohol intake using the modified intermittent access paradigm with alcohol access during three consecutive days per week followed by four days of water only. Individual intake of 20% alcohol solution and water was measured during 12 sessions, i.e., 4 weeks. Pair-housed animals could interact freely with their cage mate for four consecutive days each week and were then separated by an inserted mesh divider for three consecutive days each week during alcohol intake sessions. Alcohol intake and preference were compared between pair-housed and individually housed rats. The results revealed higher alcohol intake in females than in males. Pair-housed males had a higher alcohol intake and preference during the first 3 weeks, but not during the fourth week, compared to individually housed males No effect of housing condition was observed in female rats. The alcohol intake was higher on the first day of access relative to the two consecutive days in pair-housed males and higher on the first two days relative to the third day in female rats. Social rank or female estrus cycle had no effect on alcohol intake or preference. Taken together, the use of a divider during alcohol intake sessions had no impact on alcohol intake in female rats and may not exert long-term influences in male rats. Future studies are needed in order to elucidate whether the use of a divider can constitute an experimental refinement as an alternative to individual housing in studies of voluntary alcohol intake using the limited access and/or intermittent access paradigms.

Knock-in Mice Expressing an Ethanol-Resistant GluN2A NMDA Receptor Subunit Show Altered Responses to Ethanol.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-activated, heterotetrameric ligand-gated ion channels critically important in virtually all aspects of glutamatergic signaling. Ethanol (EtOH) inhibition of NMDARs is thought to mediate specific actions of EtOH during acute and chronic exposure. Studies from our laboratory, and others, identified EtOH-sensitive sites within specific transmembrane (TM) domains involved in channel gating as well as those in subdomains of extracellular and intracellular regions of GluN1 and GluN2 subunits that affect channel function. In this study, we characterize for the first time the physiological and behavioral effects of EtOH on knock-in mice expressing a GluN2A subunit that shows reduced sensitivity to EtOH. A battery of tests evaluating locomotion, anxiety, sedation, motor coordination, and voluntary alcohol intake were performed in wild-type mice and those expressing the GluN2A A825W knock-in mutation. Whole-cell patch-clamp electrophysiological recordings were used to confirm reduced EtOH sensitivity of NMDAR-mediated currents in 2 separate brain regions (mPFC and the cerebellum) where the GluN2A subunit is known to contribute to NMDAR-mediated responses. Male and female mice homozygous for the GluN2A(A825W) knock-in mutation showed reduced EtOH inhibition of NMDAR-mediated synaptic currents in mPFC and cerebellar neurons as compared to their wild-type counterparts. GluN2A(A825W) male but not female mice were less sensitive to the sedative and motor-incoordinating effects of EtOH and showed a rightward shift in locomotor-stimulating effects of EtOH. There was no effect of the mutation on EtOH-induced anxiolysis or voluntary EtOH consumption in either male or female mice. These findings show that expression of EtOH-resistant GluN2A NMDARs results in selective and sex-specific changes in the behavioral sensitivity to EtOH.

Development of a novel alcohol and nicotine concurrent access (ANCA) self-administration procedure in baboons

BackgroundSelf-administration of either alcohol or nicotine under single-access conditions has been studied extensively in laboratory animals. Relatively few studies have examined the co-use of these substances, even though alcohol and nicotine use and abuse commonly co-occur in humans. The objectives of this study were to develop a baboon model of concurrent alcohol and nicotine self-administration, and examine effects of varenicline on alcohol and nicotine co-use. MethodsIn Experiment 1, five male baboons were trained to self-administer drinks of alcohol (4% w/v) and injections of nicotine (0.032–0.1 mg/kg) under single-access and then concurrent-access conditions, and intake of alcohol (g/kg) and nicotine (mg/kg) was compared under single- and concurrent-access conditions. In Experiment 2, three male baboons self-administered drinks of alcohol (4% w/v) and injections of nicotine (0.056 mg/kg) under concurrent-access conditions. Pretreatment with varenicline (0.32–1.0 mg/kg, s.c.) or an equal volume of its vehicle before concurrent-access sessions was repeated for 5 consecutive days. ResultsSelf-administration of nicotine and alcohol was successfully established under both single- and concurrent-access conditions that produced reliable levels of voluntary alcohol and nicotine intake. Co-self-administration of both drugs produced levels of intake similar to that produced by each drug alone. Varenicline significantly reduced intake of both alcohol and nicotine when compared to the vehicle condition. ConclusionsThis baboon model provides a valuable tool for further investigation of the behavioral and pharmacological mechanisms involved in co-use of nicotine and alcohol. A single pharmacotherapeutic agent (e.g., varenicline) may be useful in treating nicotine and alcohol co-use.

Caffeine modulates voluntary alcohol intake in mice depending on the access conditions: Involvement of adenosine receptors and the role of individual differences

Caffeine is the most consumed psychoactive stimulant and the main active ingredient of energy drinks. Epidemiology studies have shown a positive correlation between the consumption of energy drinks and that of ethanol. The popular belief is that caffeine antagonizes the intoxicating effects of alcohol. Both drugs act on the adenosine system but have opposite effects. Caffeine is a methylxanthine that acts as a nonselective adenosine receptor antagonist, binding to A1 and A2A receptor subtypes. In contrast, ethanol increases extracellular adenosinergic tone. The purpose of this study was to examine the impact of a broad range of doses of caffeine and of selective adenosine A1 and A2A receptor antagonists on voluntary ethanol intake under different ethanol access conditions. C57BL/6 J male mice had access to ethanol (10% w/v) under different conditions: restricted (2 h in the dark), unrestricted (24 h access), or after 4 days of alcohol removal following several periods of unrestricted access. Mice reduced ethanol intake in the restricted access condition after receiving caffeine (20.0 mg/kg), or theophylline (20.0 mg/kg), another methylxanthine. Selective A1 and A2A adenosine receptor antagonists, or their combination, did not have any effect. However, under unrestricted access conditions caffeine and the adenosine A2A receptor antagonist increased ethanol intake. After splitting animals into high, moderate and low ethanol consumers, caffeine (2.5–20.0 mg/kg) significantly increased ethanol consumption in moderate consumers with no effect on low or high consumers. In addition, after reintroducing ethanol access, caffeine (5.0 mg/kg) decreased ethanol consumption among moderate consumers. Thus, caffeine produced different effects on ethanol intake depending on the access condition and the baseline consumption of ethanol.

Repeated maternal separation: Alcohol consumption, anxious behavior and corticosterone were reversed by a non-pharmacological treatment

Adverse events in early life have been related to a maladaptive stress response during adulthood, which could predispose individuals to psychiatric and physiological disorders. The purpose of this work was to study the implications of repeated maternal separation (RMS) plus a physical stressor (cold stress), voluntary ethanol consumption and plasmatic levels of corticosterone (Cor) via conflict behavior tests. To this aim, pups were separated daily from their mothers for one hour and subjected to cold stress (4 °C) between postnatal days (PD) 2 and 20. Control groups were left undisturbed with their mothers. Afterwards, all groups were exposed to voluntary ethanol (6%) or dextrose (1%) intake for 7 days. After a 30-day period of environmental enrichment (EE), the animals were again exposed to the voluntary intake protocol for 7 days. At 66 days, they were subjected to different conflict tests. Thereafter, rats were sacrificed by decapitation and blood trunk was collected to determine plasma corticosterone levels. We demonstrated that early RMS increased both voluntary alcohol intake and Cor levels. Moreover, young adult animals showed excessive activity in conflict tests. Whereas in animals exposed to a non-pharmacological treatment, known as environmental enrichment (EE), the effects previously obtained were reversed and/or prevented. In summary, we can conclude that the combination of maternal separation in early life plus cold stress increase both the voluntary exposure to alcohol and disruptive behaviors. This is a risk factor for the development of chronic diseases such as alcoholism and long-term depression. However, we found that an enriched environment may have a beneficial effect with respect to alcohol intake and aggressive behaviors.

Predisposition to Alcohol Drinking and Alcohol Consumption Alter Expression of Calcitonin Gene-Related Peptide, Neuropeptide Y, and Microglia in Bed Nucleus of Stria Terminalis in a Subnucleus-Specific Manner.

Excessive alcohol consumption is often linked to anxiety states and has a major relay center in the anterior part of bed nucleus of stria terminalis (BNST). We analyzed the impact of (i) genetic predisposition to high alcohol preference and consumption, and (ii) alcohol intake on anterior BNST, namely anterolateral (AL), anteromedial (AM), and anteroventral (lateral + medial subdivisions: AVl, AVm) subnuclei. We used two rat lines selectively bred for low- and high-alcohol preference and consumption, named Sardinian alcohol-non preferring (sNP) and -preferring (sP), respectively, the latter showing also inherent anxiety-related behaviors. We analyzed the modulation of calcitonin gene-related peptide (CGRP; exerting anxiogenic effects in BNST), neuropeptide Y (NPY; exerting mainly anxiolytic effects), and microglia activation (neuroinflammation marker, thought to increase anxiety). Calcitonin gene-related peptide-immunofluorescent fibers/terminals did not differ between alcohol-naive sP and sNP rats. Fiber/terminal NPY-immunofluorescent intensity was lower in BNST-AM and BNST-AVm of alcohol-naive sP rats. Activation of microglia (revealed by morphological analysis) was decreased in BNST-AM and increased in BNST-AVm of alcohol-naive sP rats. Prolonged (30 consecutive days), voluntary alcohol intake under the homecage 2-bottle “alcohol vs. water” regimen strongly increased CGRP intensity in BNST of sP rats in a subnucleus-specific manner: in BNST-AL, BNST-AVm, and BNST-AM. CGRP area sum, however, decreased in BNST-AM, without changes in other subnuclei. Alcohol consumption increased NPY expression, in a subnucleus-specific manner, in BNST-AL, BNST-AVl, and BNST-AVm. Alcohol consumption increased many size/shapes parameters in microglial cells, indicative of microglia de-activation. Finally, microglia density was increased in ventral anterior BNST (BNST-AVl, BNST-AVm) by alcohol consumption. In conclusion, genetic predisposition of sP rats to high alcohol intake could be in part mediated by anterior BNST subnuclei showing lower NPY expression and differential microglia activation. Alcohol intake in sP rats produced complex subnucleus-specific changes in BNST, affecting CGRP/NPY expression and microglia and leading to hypothesize that these changes might contribute to the anxiolytic effects of voluntarily consumed alcohol repeatedly observed in sP rats.

Fenofibrate -a PPARα agonist- increases alcohol dehydrogenase levels in the liver: implications for its possible use as an ethanol-aversive drug.

After ethanol consumption, disulfiram increases blood-acetaldehyde levels, generating an aversive reaction that deters alcohol drinking. Given the major secondary effects of disulfiram, finding other effective drugs to reduce alcohol consumption in individuals with alcohol-use-disorder is highly desirable. It has been reported that administering fenofibrate to high-drinking rats increases hepatic catalase levels and blood acetaldehyde after administering ethanol and a 60-70% inhibition of voluntary alcohol intake. This work evaluated whether fenofibrate has an additional effect on the activity of other ethanol-metabolizing enzymes, which could contribute to the high acetaldehyde levels generated upon administering ethanol. Male high-drinker rats were allowed to voluntary drink 10% ethanol or water for 2 months. Subsequently, fenofibrate (100 mg/kg/day) or vehicle was administered orally for 14 days. Then, alcohol dehydrogenase (ADH1) and aldehyde dehydrogenase (ALDH2) protein levels and enzymatic activities in the livers were quantified. Fenofibrate treatment produced a marked increase in ADH1 protein levels (396% ± 18%, p < 0.001) and enzymatic activity (425% ± 25%, p < 0.001). Fenofibrate did not result in differences in ALDH2 activity or in ALDH2 protein levels. The studies show that treatment with fenofibrate not only increased the activity of catalase in the liver of alcohol-drinking rats, as reported earlier, but also increased the levels and enzymatic activity of ADH1, while ALDH2 remained unchanged. The increases in ADH1 contribute to explaining the remarkable effect of fenofibrate in raising blood levels of acetaldehyde in ethanol-consuming animals, in which a marked reduction of alcohol intake is recorded.

Dopaminergic-GABAergic interplay and alcohol binge drinking

The dopamine D3 receptor (D3R), in the nucleus accumbens (NAc), plays an important role in alcohol reward mechanisms. The major neuronal type within the NAc is the GABAergic medium spiny neuron (MSN), whose activity is regulated by dopaminergic inputs. We previously reported that genetic deletion or pharmacological blockade of D3R increases GABAA α6 subunit in the ventral striatum. Here we tested the hypothesis that D3R-dependent changes in GABAA α6 subunit in the NAc affect voluntary alcohol intake, by influencing the inhibitory transmission of MSNs.We performed in vivo and ex vivo experiments in D3R knockout (D3R −/−) mice and wild type littermates (D3R +/+). Ro 15-4513, a high affinity α6-GABAA ligand was used to study α6 activity.At baseline, NAc α6 expression was negligible in D3R+/+, whereas it was robust in D3R−/−; other relevant GABAA subunits were not changed. In situ hybridization and qPCR confirmed α6 subunit mRNA expression especially in the NAc. In the drinking-in-the-dark paradigm, systemic administration of Ro 15-4513 inhibited alcohol intake in D3R+/+, but increased it in D3R−/−; this was confirmed by intra-NAc administration of Ro 15-4513 and furosemide, a selective α6-GABAA antagonist. Whole-cell patch-clamp showed peak amplitudes of miniature inhibitory postsynaptic currents in NAc medium spiny neurons higher in D3R−/− compared to D3R+/+; Ro 15-4513 reduced the peak amplitude in the NAc of D3R−/−, but not in D3R+/+.We conclude that D3R-dependent enhanced expression of α6 GABAA subunit inhibits voluntary alcohol intake by increasing GABA inhibition in the NAc.

Persistent Neuroadaptations in the Expression of Genes Involved in Cholesterol Homeostasis Induced by Chronic, Voluntary Alcohol Intake in Rats.

Alcohol use disorder (AUD) is associated with persistent adaptations in the brain that are believed to participate in the long-lasting vulnerability to relapse after abstinence. Cholesterol, the major sterol compound found in the central nervous system (CNS), plays a major role in maintenance of neuronal morphology, synaptogenesis and synaptic communication and may be involved in alcohol-induced neuroadaptations. In this study, we investigated whether alcohol consumption in a two-bottle choice paradigm followed by 3 weeks of abstinence could alter the expression of genes encoding proteins involved in cholesterol homeostasis in brain regions involved in addiction and relapse, namely the prefrontal cortex (PFC), the nucleus accumbens (NAc), the mesencephalon and the amygdala. We found that voluntary alcohol intake followed by 3 weeks of forced abstinence produces changes in the transcription of several genes encoding proteins directly involved in cholesterol synthesis such as 3-hydroxyl-3-methylglutaryl-coenzyme A (HMGCoA) reductase, farnesyl-diphosphate farnesyltransferase 1 (FDFT1) and farnesyl diphosphate synthase (FDPS) and in its regulation such as sterol regulatory element-binding factor-2 (SREBF2), in cholesterol transport such as ATP-binding cassette subfamily A member 1 (ABCA1) and in cholesterol degradation such as CYP46A1. Interestingly, these changes appeared to be region-specific and suggest that previous chronic exposure to alcohol might durably increase cholesterol metabolism in the PFC, the NAc and the mesencephalon and decrease cholesterol metabolism in the amygdala. Altogether, these results suggest that alcohol consumption leads to durable deregulations in cholesterol metabolism in key areas involved in loss of control over drug use and addiction. These long-term neuroadaptations may participate in the changes in brain structure and functioning that are responsible for the long-lasting risks of relapse to alcohol.

No effect of sex on ethanol intake and preference after dopamine transporter (DAT) knockdown in adult mice.

Dopamine levels are controlled in part by transport across the cell membrane by the dopamine transporter (DAT), and recent evidence showed that a polymorphism in the gene encoding DAT is associated with alcoholism. However, research in animal models using DAT knockout mice has yielded conflicting results. The present study was planned to evaluate the effects of DAT knockdown in the nucleus accumbens (Nacc) on voluntary ethanol consumption and preference in male and female C57BL/6J mice. For this purpose, animals were stereotaxically injected with DAT siRNA-expressing lentiviral vectors in the Nacc, and using a voluntary, continuous access two-bottle choice model of alcohol, we investigated the importance of accumbal DAT expression in voluntary alcohol intake and preference. We also investigated the effects of DAT knockdown on saccharin and quinine consumption and ethanol metabolism. We show that females consumed more alcohol than males. Interestingly, DAT knockdown in the Nacc significantly decreased alcohol intake and preference in both groups, but no significant sex by group interaction was observed. Also, DAT knockdown did not alter total fluid consumption, saccharin or quinine consumption, or blood ethanol concentrations. Using Pearson correlation, results indicated a strong positive relationship between DAT mRNA expression and ethanol consumption and preference. Taken together, these data provide further evidence that DAT plays an important role in controlling ethanol intake and that accumbal DAT contributes in the modulation of the reinforcing effects of ethanol. Overall, the results suggest that DAT inhibitors may be valuable in the pharmacotherapy of alcoholism.

Toll-like receptor 3 activation increases voluntary alcohol intake in C57BL/6J male mice

Many genes differentially expressed in brain tissue from human alcoholics and animals that have consumed large amounts of alcohol are components of the innate immune toll-like receptor (TLR) pathway. TLRs initiate inflammatory responses via two branches: (1) MyD88-dependent or (2) TRIF-dependent. All TLRs signal through MyD88 except TLR3. Prior work demonstrated a direct role for MyD88-dependent signaling in regulation of alcohol consumption. However, the role of TLR3 as a potential regulator of excessive alcohol drinking has not previously been investigated. To test the possibility TLR3 activation regulates alcohol consumption, we injected mice with the TLR3 agonist polyinosinic:polycytidylic acid (poly(I:C)) and tested alcohol consumption in an every-other-day two-bottle choice test. Poly(I:C) produced a persistent increase in alcohol intake that developed over several days. Repeated poly(I:C) and ethanol exposure altered innate immune transcript abundance; increased levels of TRIF-dependent pathway components correlated with increased alcohol consumption. Administration of poly(I:C) before exposure to alcohol did not alter alcohol intake, suggesting that poly(I:C) and ethanol must be present together to change drinking behavior. To determine which branch of TLR signaling mediates poly(I:C)-induced changes in drinking behavior, we tested either mice lacking MyD88 or mice administered a TLR3/dsRNA complex inhibitor. MyD88 null mutants showed poly(I:C)-induced increases in alcohol intake. In contrast, mice pretreated with a TLR3/dsRNA complex inhibitor reduced their alcohol intake, suggesting poly(I:C)-induced escalations in alcohol intake are, at least partially, dependent on TLR3. Together, these results strongly suggest that TLR3-dependent signaling drives excessive alcohol drinking behavior.

Genetic Contribution to Initial and Progressive Alcohol Intake Among Recombinant Inbred Strains of Mice.

We profiled individual differences in alcohol consumption upon initial exposure and during 5 weeks of voluntary alcohol intake in female mice from 39 BXD recombinant inbred strains and parents using the drinking in the dark (DID) method. In this paradigm, a single bottle of 20% (v/v) alcohol was presented as the sole liquid source for 2 or 4 h starting 3 h into the dark cycle. For 3 consecutive days mice had access to alcohol for 2 h followed by a 4th day of 4 h access and 3 intervening days where alcohol was not offered. We followed this regime for 5 weeks. For most strains, 2 or 4 h alcohol intake increased over the 5-week period, with some strains demonstrating greatly increased intake. There was considerable and heritable genetic variation in alcohol consumption upon initial early and sustained weekly exposure. Two different mapping algorithms were used to identify QTLs associated with alcohol intake and only QTLs detected by both methods were considered further. Multiple suggestive QTLs for alcohol intake on chromosomes (Chrs) 2, 6, and 12 were identified for the first 4 h exposure. Suggestive QTLs for sustained intake during later weeks were identified on Chrs 4 and 8. Thirty high priority candidate genes, including Entpd2, Per3, and Fto were nominated for early and sustained alcohol intake QTLs. In addition, a suggestive QTL on Chr 15 was detected for change in 2 h alcohol intake over the duration of the study and Adcy8 was identified as a strong candidate gene. Bioinformatic analyses revealed that early and sustained alcohol intake is likely driven by genes and pathways involved in signaling, and/or immune and metabolic function, while a combination of epigenetic factors related to alcohol experience and genetic factors likely drives progressive alcohol intake.

Interneuronal δ-GABAA receptors regulate binge drinking and are necessary for the behavioral effects of early withdrawal.

Extensive evidence points to a role for GABAergic signaling in the amygdala in mediating the effects of alcohol, including presynaptic changes in GABA release, suggesting effects on GABAergic neurons. However, the majority of studies focus solely on the effects of alcohol on principal neurons. Here we demonstrate that δ-GABAARs, which have been suggested to confer ethanol sensitivity, are expressed at a high density on parvalbumin (PV) interneurons in the basolateral amygdala (BLA). Thus, we hypothesized that δ-GABAARs on PV interneurons may represent both an initial pharmacological target for alcohol and a site for plasticity associated with the expression of various behavioral maladaptations during withdrawal from binge drinking. To investigate this, we used a mouse model of voluntary alcohol intake (Drinking-in-the-Dark-Multiple Scheduled Access) to induce escalating heavy binge drinking and anxiety-like behavior in mice. This pattern of intake was associated with increased δ protein expression on parvalbumin positive interneurons in both the BLA and hippocampus. Loss of δ-GABAARs specifically in PV interneurons (PV:δ-/-) increased binge drinking behavior, reduced sensitivity to alcohol-induced motor incoordination, enhanced sensitivity to alcohol-induced hyperlocomotion and blocked the expression of withdrawal from binge drinking. This study is the first to demonstrate a role for δGABAARs specifically in PV-expressing interneurons in modulating binge alcohol intake and withdrawal-induced anxiety.

Protective influences of N-acetylcysteine against alcohol abstinence-induced depression by regulating biochemical and GRIN2A, GRIN2B gene expression of NMDA receptor signaling pathway in rats

Evidences have indicated a high degree of comorbidity of alcoholism and depression. N-acetylcysteine (NAC) has shown its clinical efficiency in the treatment of several psychiatric disorders and is identified as a multi-target acting drug. The ability of NAC to prevent alcohol abstinence-induced depression-like effects and underlying mechanism(s) have not been adequately addressed. This study was aimed to investigate the beneficial effects of NAC in the alcohol abstinence-induced depression developed following long-term voluntary alcohol intake. For evaluation of the effects of NAC, Sprague–Dawley rats were enabled to voluntary drinking of 4.5%, 7.5% and 9% v/v alcohol for fifteen days. NAC (25, 50, and 100 mg/kg) and fluoxetine (5 mg/kg) were injected intraperitoneally for three consecutive days during the alcohol abstinence period on the days 16, 17, 18. The behavioral studies were conducted employing forced swim test (FST), and tail suspension test (TST) on day 18 to determine the effects of N-acetylcysteine and fluoxetine in the ethanol withdrawal induced-depression. Blood alcohol concentration, alcohol biomarkers like SGPT, SGOT, ALP, GGT, and MCV were estimated by using commercially available kits. Serotonin concentrations were measured in the plasma, hippocampus and pre-frontal cortex using the rat ELISA kit. The expression of GRIN1, GRIN2A, GRIN2B genes for the N-methyl d-aspartate receptors (NMDAR) subunits in the hippocampus and the prefrontal cortex were also examined by reverse-transcription quantitative polymerase chain reaction. The results revealed that alcohol abstinence group depicted increased immobility time in FST and TST. Further, NAC exerted significant protective effect at the doses 50 mg/kg and 100 mg/kg, but 25 mg/kg showed insignificant protection against alcohol abstinence-induced depression. The increased level of biochemical parameters following ethanol abstinence were also reversed by NAC at the dose of 100 mg/kg. The significant reversal effect of NAC on the serotonin level following alcohol abstinence was greater in the hippocampus as compared to the third-day alcohol withdrawal group. The increased expression levels of GRIN2A and GRIN2B following ethanol abstinence were reversed with a higher dose of NAC (100 mg/kg) treatment. In conclusion, the results of the study reveal that NAC has remarkable protective effects in the alcohol abstinence-induced depression by modulating alcohol markers, serotonin levels and GRIN2A, GRIN2B gene expression of NMDAR signaling pathway in rats.

Critical Role for Gi/o-Protein Activity in the Dorsal Striatum in the Reduction of Voluntary Alcohol Intake in C57Bl/6 Mice.

The transition from non-dependent alcohol use to alcohol dependence involves increased activity of the dorsal striatum. Interestingly, the dorsal striatum expresses a large number of inhibitory G-protein-coupled receptors (GPCRs), which when activated may inhibit alcohol-induced increased activity and can decrease alcohol consumption. Here, we explore the hypothesis that dorsal striatal Gi/o-protein activation is sufficient to reduce voluntary alcohol intake. Using a voluntary, limited-access, two-bottle choice, drink-in-the-dark model of alcohol (10%) consumption, we validated the importance of Gi/o signaling in this region by locally expressing neuron-specific, adeno-associated-virus encoded Gi/o-coupled muscarinic M4 designer receptors exclusively activated by designer drugs (DREADD) in the dorsal striatum and observed a decrease in alcohol intake upon DREADD activation. We validated our findings by activating Gi/o-coupled delta-opioid receptors (DORs), which are natively expressed in the dorsal striatum, using either a G-protein biased agonist or a β-arrestin-biased agonist. Local infusion of TAN-67, an in vitro-determined Gi/o-protein biased DOR agonist, decreased voluntary alcohol intake in wild-type and β-arrestin-2 knockout (KO) mice. SNC80, a β-arrestin-2 biased DOR agonist, increased alcohol intake in wild-type mice; however, SNC80 decreased alcohol intake in β-arrestin-2 KO mice, thus resulting in a behavioral outcome generally observed for Gi/o-biased agonists and suggesting that β-arrestin recruitment is required for SNC80-increased alcohol intake. Overall, these results suggest that activation Gi/o-coupled GPCRs expressed in the dorsal striatum, such as the DOR, by G-protein biased agonists may be a potential strategy to decrease voluntary alcohol consumption and β-arrestin recruitment is to be avoided.

P.2.021 - Functional changes in the dorsolateral striatum in loss of control over reward seeking

Substance addiction inflicts an enormous burden on patients with formidable costs to society. Loss of control over substance use is a hallmark of addiction. It marks the stage where substance use has progressed from casual to compulsive and persists despite negative consequences. It is thought that this progression arises after lengthy and excessive substance use, as a result of neurobiological dysfunctions. One of the neural mechanisms that has been proposed to contribute to loss of control use is exaggerated involvement of the dorsolateral striatum (DLS), which mediates habitual behaviour [1,2]. This study examines how changes in DLS functioning contribute to loss of control over reward seeking. First, we assessed the effect of long-term exposure to alcohol on synaptic plasticity in a cortical projection onto neurons in the DLS. To map cortical inputs towards the DLS, rats were bilaterally injected with a retrograde rabies virus (Rabies-SadDG-GFP) in the DLS. Using immunohistochemistry, we found GFP expression in different cortical areas, including the primary and secondary motor cortex. Next, voluntary home-cage alcohol consumption was monitored in outbred Lister-Hooded rats. We used a two-month intermittent alcohol access paradigm and found substantial individual variation in alcohol intake, consistent with our previous findings. Based on a rank score of their voluntary alcohol intake, low and high consuming animals were then selected and bilaterally injected with an adeno-associated Channelrhodopsin virus (AAV5-CamKII-Chr2-eYFP) targeted at the identified motor cortices that project onto neurons in the DLS. Whole-cell patch clamp recordings were performed to measure optically induced excitatory post-synaptic currents in neurons in the DLS. Our preliminary results indicate increased facilitation of paired-pulse responses in the DLS in rats that show a high voluntary alcohol consumption. This increase is caused by reduced amplitude of the first post-synaptic response, indicative of a reduced presynaptic release probability. In parallel, we assessed whether activation of the DLS is sufficient to drive habitual behaviour and loss of control over sucrose seeking. To investigate habitual behavior, changes in sensitivity for outcome devaluation were measured after pre-feeding animals with either sucrose or chow before operant training. Loss of control over sucrose seeking was examined in a task in which we measure continued responding during a conditioned cue that predicts probabilistic footshock punishment. To enhance neuronal activity of the DLS, rats received bilateral injections of an adeno-associated DREADD virus (AAV5-hSyn-hMD3q-mCherry). Systemic clozapine N-oxide (CNO) injections did not change the sensitivity to outcome devaluation. Furthermore, we show that the presentation of the footshock-predictive cue profoundly suppresses responding for sucrose, but this is unaffected by CNO injections. In sum, our data indicate that long-term ingestion of large quantities of alcohol modulates short-term synaptic plasticity in glutamatergic cortical inputs into the DLS. However, chemogenetic activation of the DLS does not seem sufficient to evoke habitual or compulsive behaviour.

Co-Administration of Low-Dose Naltrexone and Bupropion Reduces Alcohol Drinking in Alcohol-Preferring (P) Rats.

This study examined whether combining naltrexone (NTX) with bupropion (BUP) is more effective in reducing alcohol drinking in alcohol-preferring (P) rats with a genetic predisposition toward high voluntary alcohol intake than either drug alone. Alcohol-experienced, adult, male, P rats were fed NTX alone in a dose of 10.0mg/kg BW, BUP alone in a dose of 10.0mg/kg BW, BUP alone in a dose of 20.0mg/kg BW, NTX (10.0mg/kg BW)+BUP (10.0mg/kg BW), or vehicle (VEH) at 1hour prior to onset of a daily 2-hour alcohol access period for 5 consecutive days. When administered alone, neither NTX (10.0mg/kg BW) nor BUP, in either of 2 doses (10.0mg/kg BW or 20.0mg/kg BW), reduced voluntary alcohol intake in P rats. However, NTX combined with BUP (10.0mg/kg NTX+10.0mg/kg BUP) and given as a single medication significantly reduced alcohol consumption throughout prolonged treatment. Combining low doses of NTX and BUP, each of which is ineffective when given alone, increases the efficacy of the medication. Low drug doses circumvent the problem of negative side effects that can occur with higher doses of either drug. A reduction in side effects can facilitate patient compliance and improve clinical outcomes for alcoholics and heavy drinkers who want to reduce their alcohol intake. The results, together with those from our prior studies, demonstrate the strength of a combinatorial pharmacotherapeutic approach to the treatment of alcohol use disorder.

Acute stress induces voluntary alcohol intake in mice through anxiety mitigated by toll-like receptor 4 antagonist

Event Abstract Back to Event Acute stress induces voluntary alcohol intake in mice through anxiety mitigated by toll-like receptor 4 antagonist Huei G. Chuang1, Sangu Muthuraju1*, Nur Naznee H. Abd Aziz2, Mustapha Muzaimi2, Jafri M. Abdullah1, 2 and Zamzuri Idris1, 2 1 Hospital Universiti Sains Malaysia, Health Campus, Universiti Sains Malaysia, Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, Malaysia 2 Universiti Sains Malaysia Health Campus, Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, Malaysia Background Evidences previously supporting the functional involvement of toll-like receptor 4 (TLR4) in the mediation of neuroimmune system, further promoting the emergence of alcohol drinking behaviour following stress exposure. Thus, this study focused to investigate the effect of TLR4 antagonist Lipopolysaccharide-Rhodobacter Sphaeroides(LPS-RS) on the stress-induced voluntary alcohol drinking behaviour, neuronal component, and genes expression associating with stress and addiction. Methods This study involved the exposure of restraint and social isolation stress using Swiss Albino mice. Two-bottle choice ethanol exposure method was used in the evaluation of voluntary ethanol drinking behaviour. Several behavioural assessments including elevated plus-maze, light-dark box exploration, open field maze, beam walking test, and wire hanging test were carried out to assess the fear and anxiety-like behaviour, locomotion, motor coordination, and neuromuscular ability. Morphological and immunoreactivity analysis and genes expression analysis were done after the completion of behavioural assessments. Results TLR4 antagonist LPS-RS treated stressed-mice showed a significant decrease in the ethanol intake compared to stressed mice. Behaviourally, acute stress did not cause any significant deficits on the motor coordination, neuromuscular ability, locomotion, exploratory behaviour and risk-assessment behaviour. Behavioural results proved that acute stress exposure causing the emergence of fear and anxiety-like behaviour in the stressed mice. Morphological analysis showed no significant changes in prefrontal cortex and hippocampus among all groups, while in immunoreactivity analysis, stressed-mice showed a significant increase immunoreactivity of c-FOS in both prefrontal cortex and hippocampus, significant increase immunoreactivity of TLR4 in prefrontal cortex and GFAP in hippocampus. Stressed-mice too showed significant increase in the TLR4, NF-Kappa, iNOS, DRD2, CREB-1, and OPRM-1 genes expression compared to control and LPS-RS treated mice. Conclusion TLR4 suppression using antagonist LPS-RS could be effective in reducing the ethanol intake among stress-exposed mice suggested that TLR4 suppression might provide a therapeutic value in the treatment of stress-induced alcohol addiction. Acknowledgements We thank the School of Medical Sciences, Pusat Pengajian Sains Perubatan (PPSP), Universiti Sains Malaysia. This work has been supported by project number 304/PPSP/61313158 from the Short-term Grant received by Dr. Sangu Muthuraju. Keywords: Toll-like recepter 4 (TLR4), acute stress, Addiction, Alcohol drinking behavior, Lipopolysaccharide-Rhodobacter Sphaeroides(LPS-RS) Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Oral Presentation Topic: Miscellaneous Citation: Chuang HG, Muthuraju S, Abd Aziz NH, Muzaimi M, Abdullah JM and Idris Z (2019). Acute stress induces voluntary alcohol intake in mice through anxiety mitigated by toll-like receptor 4 antagonist. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00073 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Sangu Muthuraju, Hospital Universiti Sains Malaysia, Health Campus, Universiti Sains Malaysia, Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Kelantan Darul Naim, 16150, Malaysia, muthuraju67@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Huei G Chuang Sangu Muthuraju Nur Naznee H Abd Aziz Mustapha Muzaimi Jafri M Abdullah Zamzuri Idris Google Huei G Chuang Sangu Muthuraju Nur Naznee H Abd Aziz Mustapha Muzaimi Jafri M Abdullah Zamzuri Idris Google Scholar Huei G Chuang Sangu Muthuraju Nur Naznee H Abd Aziz Mustapha Muzaimi Jafri M Abdullah Zamzuri Idris PubMed Huei G Chuang Sangu Muthuraju Nur Naznee H Abd Aziz Mustapha Muzaimi Jafri M Abdullah Zamzuri Idris Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Few long-term consequences after prolonged maternal separation in female Wistar rats.

Environmental factors during the early-life period are known to have long-term consequences for the adult phenotype. An intimate interplay between genes and environment shape the individual and may affect vulnerability for psychopathology in a sex-dependent manner. A rodent maternal separation model was here used to study the long-term effects of different early-life rearing conditions on adult behavior, HPA axis activity and long-term voluntary alcohol intake in female rats. Litters were subjected to 15 min (MS15) or 360 min (MS360) of daily maternal separation during postnatal day 1–21. In adulthood, the behavioral profiles were investigated using the multivariate concentric square field™ (MCSF) test or examined for HPA axis reactivity by cat-odor exposure with subsequent characterization of voluntary alcohol intake and associated changes in HPA axis activity. Adult female MS360 offspring showed mostly no, or only minor, effects on behavior, HPA axis reactivity and long-term alcohol intake relative to MS15. Instead, more pronounced effects were found dependent on changes in the natural hormonal cycle or by the choice of animal supplier. However, changes were revealed in corticosterone load after long-term alcohol access, as females subjected to MS360 had higher concentrations of fecal corticosterone. The present findings are in line with and expand on previous studies on the long-term effects of maternal separation in female rats with regard to behavior, HPA axis activity and voluntary alcohol intake. It can also be a window into further studies detailing how early-life experiences interact with other risk and protective factors to impact the adult phenotype and how possible sex differences play a role.