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Abstract: Brain regional γ-aminobutyric acid type A (GABAA) receptor subunit mRNA expression was studied in ethanol-preferring AA (Alko, Alcohol) rats after moderate ethanol drinking for up to 2 years of age. In situ hybridization with oligonucleotide probes specific for 13 different subunits was used with coronal cryostat sections of the brains. Selective alterations were observed by ethanol exposure and/or aging in signals for several subunits. Most interestingly, the putative highly ethanol-sensitive α4 and β3 subunit mRNAs were significantly decreased in several brain regions. The age-related alterations in α4 subunit expression were parallel to those caused by lifelong ethanol drinking, whereas aging had no significant effect on β3 subunit expression. The results suggest that prolonged ethanol consumption leading to blood concentrations of about 10mM may downregulate the mRNA expression of selected GABAA receptor subunits and that aging might have partly similar effects. [Copyright &y& Elsevier]

Abstract: Glutamatergic neurotransmission has been suggested to modulate cue-induced drug-seeking behavior. Here we examined the effects of metabotropic glutamate receptor agonists on alcohol self-administration and cue-induced reinstatement. Rats were trained to self-administer 10% w/v ethanol under an FR1 schedule of reinforcement during 30-min sessions. In the reinstatement experiments, ethanol and a non-rewarding quinine solution (available on alternating days) were paired with olfactory stimuli (S+/S−) as well as light (CS+) or tone (CS−) stimuli. Following extinction training, reinstatement of responding was induced by the ethanol-associated stimuli (S+/CS+). The mGlu2/3 receptor agonist LY379268 (0, 1, 3 and 5 mg/kg i.p.) and the mGlu8 receptor agonist (S)-3,4-DCPG (0, 5, 10 and 15 mg/kg i.p.) attenuated alcohol self-administration and reinstatement at doses that decreased also spontaneous locomotor activity. The results suggest that metabotropic glutamate receptors may have a role in the modulation of alcohol seeking and self-administration. However, further studies with ligands with fewer motor-suppressant side effects are needed. [Copyright &y& Elsevier]

Abstract: The purpose of the study was to investigate the effects of three different regimens of morphine treatment on subsequent voluntary ethanol drinking in alcohol-preferring AA (Alko Alcohol) rats. The rats were given morphine subcutaneously either intermittently on alternating days (15×10 mg/kg or 5×5–20 mg/kg in escalating doses) or subchronically on four consecutive days (3–20 mg/kg/d). Horizontal locomotor activity was monitored after challenges with additional morphine injections (3 mg/kg) ten days and six weeks after termination of the pretreatment to test if behavioral sensitization was induced by repeated morphine administration. Both intermittent pretreatments induced sensitized locomotor response after the first challenge, whereas subchronic injections did not. After the challenge the rats were given a free choice between tap water and 10% (v/v) ethanol solution for four weeks. The rats pretreated and challenged with morphine did not differ significantly in the acquisition of ethanol drinking from the saline-treated controls. In contrast, ethanol drinking was impaired during the first week of ethanol access in the saline-treated rats given a single morphine injection. The second morphine challenge given after the ethanol-drinking phase did not reveal sensitization in any of the groups. The results suggest that pattern of morphine administration rather than the dose or number of exposures to the drug is the most important factor in induction of behavioral sensitization, and that exposure to ethanol may interfere with this process. They also support earlier findings showing that acute morphine may suppress voluntary ethanol drinking, but failed to provide clear evidence for behavioral sensitization to morphine contributing to predilection towards ethanol in AA rats. [Copyright &y& Elsevier]

It has been suggested that opioid peptides play a role in the reinforcing effects of alcohol. The present study was designed to examine the function of the μ-opioid receptor system in rat lines selectively bred for alcohol preference (AA [Alko, Alcohol] rat line) and alcohol avoidance (ANA [Alko, Non-Alcohol] rat line). The functional coupling of μ-opioid receptors to G proteins was determined autoradiographically using Tyr-d-Ala-Gly-N(Me)Phe-Gly-ol-enkephalin-stimulated [35S]GTPγS binding in brain cryostat sections. The binding was significantly increased in the striatal patches and substantia nigra reticulata of the AA rats in comparison with that of the ANA rats. Within the AA rat line, there was a significant positive correlation between 3 mg/kg morphine-induced locomotor activity and activation of G-proteins in the substantia nigra compacta and nucleus accumbens core. These results of the selective breeding experiment suggest that brain region-specific differences in μ-opioid receptor function may correlate with innate differences in alcohol preference. [Copyright &y& Elsevier]

Ethanol is known to increase the release of dopamine in the nucleus accumbens. The question of whether this is a result of a direct or an indirect effect of ethanol on mesolimbic dopaminergic neurons was examined by investigating the extracellular levels of dopamine and its metabolites in the nucleus accumbens of alcohol-preferring AA (Alko Alcohol) and alcohol-avoiding ANA (Alko Non-Alcohol) rats after application of ethanol locally into either the nucleus accumbens or the ventral tegmental area with the use of reverse microdialysis. Application of ethanol (200, 400, or 800 mM in dialysate) into the nucleus accumbens, but not into the ventral tegmental area, temporarily increased the accumbal levels of dopamine in a dose-dependent manner. The ethanol-evoked increase in the level of extracellular dopamine was more prominent in AA rats than in ANA rats. Ethanol tended to suppress levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid. Because the concentrations of ethanol found to elevate the extracellular level of dopamine can hardly be considered pharmacologically relevant, the increase in accumbal dopamine levels after application of ethanol may be due to nonspecific membrane effects of ethanol. The findings support the suggestion that the increase in the extracellular level of dopamine in the nucleus accumbens after systemic administration of ethanol may involve other sites on dopamine neurons or even different neurotransmitter systems, rather than the action of ethanol at the mesolimbic dopaminergic terminals. [Copyright &y& Elsevier]

Abstract: The mechanisms involved in alcohol use disorder, a chronic relapsing brain disorder, are complex and involve various signalling systems in the brain. Recently, the orexigenic peptide ghrelin was shown to be required for alcohol-induced reward, an effect mediated via ghrelin receptors, GHS-R1A, at the level of the cholinergic-dopaminergic reward link. Moreover, ghrelin increases and GHR-R1A antagonists reduce moderate alcohol consumption in mice, and a single nucleotide polymorphism in the GHS-R1A gene has been associated with high alcohol consumption in humans. Therefore, GHS-R1A gene expression and alcohol intake were investigated in high, AA (Alko, Alcohol), versus low, ANA (Alko, Non-Alcohol), alcohol consuming rats as well as in Wistar rats. In the AA and ANA rats plasma ghrelin levels were also measured. GHS-R1A gene expression was increased in AA compared to ANA rats in nucleus accumbens, ventral tegmental area, amygdala, prefrontal cortex and hippocampus. A similar trend was observed in the ventral tegmental area of Wistar rats consuming high amounts of alcohol. Furthermore, the AA rats had significantly smaller reduction of plasma ghrelin levels over time, after several weeks of alcohol exposure, than had the ANA rats. The present study provides further evidence for that the ghrelin signalling system, in particular at the level of the mesocortocolimbic dopamine system, is involved in alcohol consumption, and thus possibly contributes to alcohol use disorder. Therefore the GHS-R1A may constitute a novel candidate for development of new treatment strategies for alcohol dependence. [Copyright &y& Elsevier]

Abstract: The aim of the present investigation was to compare the behavioural profiles in alcohol-preferring AA (Alko, alcohol) and alcohol-avoiding ANA (Alko, non-alcohol) rats. Twelve adult, alcohol-naïve male AA and ANA rats were tested in the recently established multivariate concentric square field (MCSF) test. The more traditional open field and elevated plus-maze tests were used as reference tests. Six weeks after the initial MCSF test, a repeated testing was used to explore differences in acquired recognition after a previous experience. The results revealed distinct differences between the two lines. The ANA rats were generally more active in the three tests. In the MCSF, parameters of risk taking and shelter seeking indicated differences between the two lines. The ANA rats had higher shelter seeking behaviour and less risk taking behaviour than the AA rats. Repeated exposure to the MCSF caused a general decrease in activity and reduction in the number of visits to the various zones, especially evident in the ANA rats. The ANA rats showed more shelter seeking than the AA rats and also more shelter seeking than in the first trial, supporting an “anxiety-like” profile in these rats. In conclusion, the parameters related to risk taking and shelter seeking revealed obvious differences between AA and ANA rats. The higher risk taking behaviour seen in the AA rats might relate to their innate propensity for high voluntary alcohol intake. The results are discussed in relation to the reported neurobiological differences and in relation to other alcohol-preferring and alcohol-avoiding rat lines. [Copyright &y& Elsevier]

Abstract: Glutamate and γ-amino-butyric acid (GABA) have been implicated in neuronal plasticity related to behavioral sensitization. In the present study, we examined morphine-induced changes in the extracellular concentrations of glutamate and GABA in the ventral tegmental area in alcohol-preferring Alko Alcohol (AA) and alcohol-avoiding Alko Non-Alcohol (ANA) rats that have previously been shown to differ in morphine-induced sensitization. The rats were given escalating doses (5–20 mg/kg) of morphine every other day for five days. This treatment produced behavioral sensitization to locomotor effects of morphine in AA, but not in ANA rats, when challenged with an additional injection of morphine (10 mg/kg) 10 days later. Morphine also increased the levels of glutamate in the ventral tegmental area only in AA rats, while no significant changes were found in the extracellular concentrations of GABA between the lines. Challenging the morphine-treated AA rats with ethanol (1.5 g/kg) did not modify the levels of glutamate or GABA. No changes in the concentrations of glutamate or GABA were seen in saline-treated AA and ANA rats after morphine challenge. These results render increased glutamate transmission in the ventral tegmental area a potential contributor to the higher susceptibility of AA rats to morphine-induced behavioral and neurochemical effects relative to ANA rats. [Copyright &y& Elsevier]

An atypical antipsychotic drug risperidone reduced ethanol drinking of ethanol-preferring Alko, Alcohol (AA) rats in a limited access paradigm. Its effect was transient at a dose known to preferentially antagonize the 5-HT2 receptors (0.1 mg/kg, s.c.), but long-lasting when the dose was increased to 1.0 mg/kg that also blocks dopamine D2 receptors. Risperidone also reduced dose-dependently locomotor activity and limited access saccharin intake of the AA rats, indicating that its effect on ethanol drinking was not selective. Risperidone at 0.1 mg/kg given before four successive daily ethanol-drinking sessions significantly reduced the ethanol intake. These data from an animal model of high ethanol intake suggest that risperidone should be tested in various populations of alcoholics for reducing ethanol consumption. [Copyright &y& Elsevier]

The purpose of the present study was to investigate the effects of repeated morphine and cocaine treatments on rotational behaviour in alcohol-preferring AA (Alko Alcohol) and alcohol-avoiding ANA (Alko Non-Alcohol) rats after unilateral 6-hydroxydopamine lesions of the ascending dopamine pathways. We administered saline, morphine (1 or 3 mg/kg) or cocaine (10 mg/kg) once daily for 4 days with an additional challenge 8 days after the repeated drug treatment. Ipsilateral rotations of the animals were monitored after each drug treatment. Both morphine (3 mg/kg but not 1 mg/kg) and cocaine induced more rotational behaviour in AA than in ANA rats over the 4-day drug treatment period. On Day 12, a challenge with 3 mg/kg morphine or cocaine induced significantly more rotations in the AA rats pretreated with morphine or cocaine when compared to saline-treated AA rats exposed to these drugs for the first time. This finding indicates that sensitization to the effects of morphine and cocaine occurs in the AA rats, while no clear sensitization was seen in the ANA rats. Collectively, these results suggest that morphine and cocaine activate the cerebral dopamine pathways to a greater extent in AA than in ANA rats. [Copyright &y& Elsevier]

We have earlier found that the histamine H3 receptor (H3R) antagonism diminishes motivational aspects of alcohol reinforcement in mice. Here we studied the role of H3Rs in cue-induced reinstatement of alcohol seeking in C57BL/6 J mice using two different H3R antagonists. Systemic administration of H3R antagonists attenuated cue-induced alcohol seeking suggesting that H3R antagonists may reduce alcohol craving. To understand how alcohol affects dopamine and histamine release, a microdialysis study was performed on C57BL/6 J mice and the levels of histamine, dopamine and dopamine metabolites were measured in the nucleus accumbens. Alcohol administration was combined with an H3R antagonist pretreatment to reveal whether modulation of H3R affects the effects of alcohol on neurotransmitter release. Alcohol significantly increased the release of dopamine in the nucleus accumbens but did not affect histamine release. Pretreatment with H3R antagonist ciproxifan did not modify the effect of alcohol on dopamine release. However, histamine release was markedly increased with ciproxifan. In conclusion, our findings demonstrate that H3R antagonism attenuates cue-induced reinstatement of alcohol seeking in mice. Alcohol alone does not affect histamine release in the nucleus accumbens but H3R antagonist instead increases histamine release significantly suggesting that the mechanism by which H3R antagonist inhibits alcohol seeking found in the present study and the decreased alcohol reinforcement, reward and consumption found earlier might include alterations in the histaminergic neurotransmission in the nucleus accumbens. These findings imply that selective antagonists of H3Rs could be a therapeutic strategy to prevent relapse and possibly diminish craving to alcohol use. This article is part of the Special Issue entitled ‘Histamine Receptors’. [ABSTRACT FROM AUTHOR]

The nonselective opioid receptor antagonist naltrexone is now used for the treatment of alcoholism, yet naltrexone׳s central mechanism of action remains poorly understood. One line of evidence suggests that opioid antagonists regulate alcohol drinking through interaction with the mesolimbic dopamine system. Hence, our goal here was to examine the role of the nucleus accumbens connectivity in alcohol reinforcement and naltrexone׳s actions using manganese-enhanced magnetic resonance imaging (MEMRI). Following long-term free-choice drinking of alcohol and water, AA (Alko Alcohol) rats received injections of MnCl 2 into the nucleus accumbens for activity-dependent tracing of accumbal connections. Immediately after the accumbal injections, rats were imaged using MEMRI, and then allowed to drink either alcohol or water for the next 24 h. Naltrexone was administered prior to the active dark period, and the second MEMRI was performed 24 h after the first scan. Comparison of signal intensity at 1 and 24 h after accumbal MnCl 2 injections revealed an ipsilateral continuum through the ventral pallidum, bed nucleus of the stria terminalis, globus pallidus, and lateral hypothalamus to the substantia nigra and ventral tegmental area. Activation was also seen in the rostral part of the insular cortex and regions of the prefrontal cortex. Alcohol drinking resulted in enhanced activation of these connections, whereas naltrexone suppressed alcohol-induced activity. These data support the involvement of the accumbal connections in alcohol reinforcement and mediation of naltrexone׳s suppressive effects on alcohol drinking through their deactivation. [ABSTRACT FROM AUTHOR]

Accumulating evidence continues to link certain aspects of the endogenous cannabinoid (EC) system with alcohol dependence, negative-reinforcement learning, and the modulation of stress responses. Specific alterations in brain regions that are related to stress and negative-reinforcement learning have been reported to exist in Cloninger type 1 and type 2 alcoholics. To study possible differences in profiles of EC systems between Cloninger type 1 (n = 9) and type 2 (n = 8) alcoholics and non-alcoholic control subjects (n = 10), we analyzed post-mortem amygdala and hippocampus brain samples for several ECs by quantitative liquid chromatography with triple quadrupole mass-spectrometric detection. A significant difference was found between these 3 groups in terms of EC profiles in the amygdala (p = 0.037). In particular, this difference was prominent for variations in docosahexaenoylethanolamide levels, which were significantly higher in type 1 alcoholics (p = 0.022) when compared to controls. There was also a large negative correlation between anandamide concentration and mGlu1/5 receptor density in the hippocampi of Cloninger type 1 alcoholics (R = -0.88, p = 0.002), which was not seen in Cloninger type 2 alcoholics or in controls. Although preliminary, and from relatively small diagnostic groups, these results suggest that the EC system profile may be altered in the hippocampus and amygdala of Cloninger type 1 alcoholics. [ABSTRACT FROM AUTHOR]

Abstract: Ethanol is known to directly interact with the glycine receptor (GlyR). GlyRs are membrane proteins and are constituted as either α-homomers or α-β heteromers with a subunit stoichiometry of 2α3β. Previous studies by our group have suggested a role for GlyRs and its endogenous ligands glycine and taurine in the mesolimbic dopamine activating and reinforcing effects of ethanol. Here we use quantitative PCR (qPCR) to compare the relative GlyR expression in Alko Alcohol/Non-Alcohol (AA/ANA) rats. These animals have been selectively bred to create distinct populations regarding alcohol consumption and preference, presumably mainly due to genetic differences. The aim of this study was to examine the relative gene expression of GlyR subunits (α1–3 and β) in different brain areas and relate it to alcohol consumption. The hypothesis was that AA/ANA rats are differently disposed to ethanol consumption due to their GlyR set-ups and/or compositions. Results from the present study indicate that α2 is the most widely expressed α-subunit in the forebrain regions and that the α2β-heteromer seems to be the most common subunit composition in this part of the CNS. Despite displaying different drinking behaviours the anticipated differences in mRNA expression were few. However, correlations found between alcohol consumption and/or preference and GlyR expression support a role for GlyRs in alcohol consumption. Tentative differences between AA and ANA animals related to GlyR transmission could therefore lie in, for example, the regulation of the levels of the endogenous ligand(s) for the receptor or in mechanisms downstream to GlyR activation. [Copyright &y& Elsevier]

Abstract: A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to compare endogenous cannabinoid levels in nematodes and in brains of rats and humans, with and without prior exposure to ethanol. After liquid–liquid extraction of the lipid fraction from homogenized samples, a reversed-phase sub 2μm column was used for separating analytes with an isocratic mobile phase. Deuterated internal standards were used in the analysis, and detection was made by triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). Ionization was performed with positive electrospray ionization (ESI). The nematode Caenorhabditis elegans fat-3 mutant, that lacks the necessary enzyme to produce arachidonic acid, the biologic precursor to 2-arachidonoyl glycerol and anandamide, was used as an analyte-free surrogate material for selectivity and calibration studies. The matrix effect was further investigated by in-source multiple reaction monitoring (IS-MRM) and standard addition studies. Selectivity studies demonstrated that the method was free from matrix effects. Good accuracy and precision were obtained for concentrations within the calibration range of 0.4–70nM and 40–11,000nM for monitored N-acylethanolamides (NAEs) and acyl glycerols, respectively. [Copyright &y& Elsevier]

Abstract: The endogenous cannabinoid (EC) system has been recently implicated in several neuropsychiatric disorders. This study analyzed post-mortem brain regions of Cloninger type 1 (n =9) and 2 (n =8) alcoholics and non-alcoholic controls (n =10) for ECs by quantitative liquid chromatography with triple quadrupole mass spectrometric detection. A significant difference was found in anandamide (AEA) levels in nucleus accumbens (NAcc) between the three groups (p =0.047). AEA levels were significantly lower when compared to controls in both perigenual anterior cingulate (p =0.017) and frontal cortices (p =0.018) of type 1 alcoholics. Similar trends were observed for dihomo-γ-linolenoyl ethanolamide and docosahexaenoyl ethanolamide, but not for 2-arachidonoylglycerol, palmitoyl ethanolamide, or oleoyl ethanolamide. Although preliminary, and from diagnostic groups with a relatively small number of subjects and substantially different mean ages for each group, these results suggest that the EC system may be hyperactive in type 2 alcoholics and hypoactive in type 1 alcoholics. [Copyright &y& Elsevier]

Excessive use of alcohol promotes the development of alcohol addiction, but the understanding of how alcohol-induced brain alterations lead to addiction remains limited. To further this understanding, we adopted an unbiased discovery strategy based on the principles of systems medicine. We used functional magnetic resonance imaging data from patients and animal models of alcohol addiction-like behaviors, and developed mathematical models of the 'relapse-prone' network states to identify brain sites and functional networks that can be selectively targeted by therapeutic interventions. Our systems level, non-local, and largely unbiased analyses converged on a few well-defined brain regions, with the insula emerging as one of the most consistent findings across studies. In proof-of-concept experiments we were able to demonstrate that it is possible to guide network dynamics towards increased resilience in animals but an initial translation into a clinical trial targeting the insula failed. Here, in a narrative review, we summarize the key experiments, methodological developments and knowledge gained from this complete round of a discovery cycle moving from identification of 'relapse-prone' network states in humans and animals to target validation and intervention trial. Future concerted efforts are necessary to gain a deeper understanding of insula function a in a state-dependent, circuit-specific and cell population perspective, and to develop the means for insula-directed interventions, before therapeutic targeting of this structure may become possible. Applying principles of systems biology we used functional MRI data from alcoholic patients and animal models to develop mathematical models of the 'relapse-prone' state of brain networks. In these network models the insula emerged as a key node that could be experimentally validated and refined in animals. Distinct network properties may provide biomarkers and therapeutic access points for alcohol addiction in humans. [Display omitted] • MRI provides objective measures of brain dysfunction in AUD. • These measures allow systematic cross-species translation between humans and animal models. • Unbiased analyses of MRI data implicate the insula as an integral part of a 'relapse-prone' network. • Insula neurons may coordinate meta-ensembles that bind multimodal external contingencies. • Activity of these meta-ensembles modulates alcohol-related behaviors. [ABSTRACT FROM AUTHOR]

Melanocortin (MC) peptides are suggested to play a role in opiate dependence, where they antagonise the addictive properties of opiates. To further study the involvement of the MCs in drug dependence, we analysed the effects of the MC4-receptor antagonist HS014 (1 nmol/rat), and the non-selective MC-receptor agonist MTII (1 nmol/rat), using i.c.v. administration, on ethanol intake in alcohol-preferring AA rats. The rats had access to ethanol during 40 days, resulting in a mean ethanol intake of 6.6 g/kg/day, before treatment. One group received only artificial cerebrospinal fluid solution. MTII caused a reduction in ethanol intake and ethanol preference, whereas HS014 was without effect. No effect on water intake was observed. A decrease in food intake was detected after MTII, whereas HS014 induced an increase in food intake. Analysis of dynorphin B and Met-enkephalin-Arg6Phe7 immunoreactive levels revealed that MTII and HS014 altered opioid peptide levels in several brain areas and the pituitary gland of the rats with an established ethanol intake. This is the first report showing that manipulation of the MC-receptor system changes ethanol intake in chronically ethanol-drinking AA rats. In addition, manipulation of the MC system modulates ethanol-induced changes in opioid peptide levels. [Copyright &y& Elsevier]