High Voluntary Alcohol Consumption Research Articles

Repeated Ethanol Exposure Alters DNA Methylation Status and Dynorphin/Kappa-Opioid Receptor Expression in Nucleus Accumbens of Alcohol-Preferring AA Rats.

Growing evidence suggests that epigenetic mechanisms, such as DNA methylation and demethylation, and histone modifications, are involved in the development of alcohol and drug addiction. However, studies of alcohol use disorder (AUD) that are focused on epigenetic DNA modifications and gene expression changes remain conflicting. Our aim was to study the effect of repeated ethanol consumption on epigenetic regulatory enzymes such as DNA methyltransferase and demethylase enzymes and whether those changes affected dynorphin/kappa-opioid receptor system in the Nucleus Accumbens (NAc). Two groups of male alcohol-preferring Alko Alcohol (AA) rats, rats which are selectively bred for high voluntary alcohol consumption and one group of male Wistar rats were used. The first group of AA rats had access to alcohol (10% ethanol solution) for 90 min on Mondays, Wednesdays and Fridays over a period of 3 weeks to establish a stable baseline of ethanol intake (AA-ethanol). The second group of AA rats (AA-water) and the Wistar rats (Wistar-water) were provided with water. Using qPCR, we found that voluntary alcohol drinking increased Dnmt1, −3a, and −3b mRNA levels and did not affect Tet family transcripts in the AA-ethanol group when compared with AA- and Wistar-water rats. DNMT and TET enzymatic activity measurements showed similar results to qPCR, where DNMT activity was increased in AA-ethanol group compared with AA-water and Wistar-water groups, with no statistically significant difference between groups in TET enzyme activity. In line with previous data, we found an increased percentage of global DNA methylation and hydroxymethylation in the AA-ethanol group compared with control rats. Finally, we investigated changes of selected candidate genes from dynorphin/kappa-opioid receptor system (Pdyn, Kor) and Dnmt3a genes that might be important in AUD-related behaviour. Our gene expression and promoter methylation analysis revealed a significant increase in the mRNA levels of Pdyn, Kor, and Dnmt3a in the AA-ethanol group, however, these changes can only be partially associate with the aberrant DNA methylation in promoter areas of the selected candidate genes. Thus, our findings suggest that the aberrant DNA methylation is rather one of the several mechanisms involved in gene expression regulation in AA rat model.

Further evidence for the involvement of the PPARγ system on alcohol intake and sensitivity in rodents.

Peroxisome Proliferator Activator receptors (PPARs) are intracellular receptors that function as transcription factors, which regulate specific metabolic and inflammatory processes. PPARs are broadly distributed in the body and are also expressed in the central nervous system, especially in areas involved in addiction-related behavioral responses. Recent studies support a role of PPARs in alcoholism and pioglitazone: a PPARγ agonist used for treatment of type 2 diabetes showed efficacy in reducing alcohol drinking, stress-induced relapse, and alcohol withdrawal syndrome in rats. In the current work, we tested the pharmacological effects of pioglitazone on binge-like alcohol consumption using an intermittent two-bottle choice paradigm in Wistar rats and on the "drinking in the dark" (DID) model in mice with selective deletion of PPARγ in neurons. Our data show that repeated administration of pioglitazone (10, 30mg/kg) reduces high voluntary alcohol consumption in Wistar rats. Pre-treatment with the selective PPARγ antagonist GW9662 (5mg/kg) completely prevented the effect of pioglitazone, demonstrating that its action is specifically mediated by activation of PPARγ. In line with this result, repeated administration of pioglitazone (30mg/kg) attenuated binge alcohol consumption in PPARγ(+/+) mice. Whereas in PPARγ(-/-) mice, which exhibit reduced alcohol consumption, pioglitazone had no effect. Of note, PPARγ(-/-) mice exhibited lower patterns of alcohol drinking without showing difference in sucrose (control) intake. Interestingly, PPARγ(-/-) mice displayed a higher sensitivity to the sedative and ataxic effect of alcohol compared with their wild-type counterpart. Collectively, these data suggest that PPARγ agonists, and specifically pioglitazone, could be potential therapeutics for the treatment of binge alcohol drinking.

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.

Chronic antidepressant treatment causes a selective reduction of mu-opioid receptor binding and functional coupling to G Proteins in the amygdala of fawn-hooded rats.

We have previously documented that chronic alcohol consumption or alcohol withdrawal affects mu-opioid receptor density and receptor-mediated G protein coupling in Fawn-Hooded (FH) rat brain, especially in mesolimbic regions. FH rats demonstrate comorbid depression and high voluntary alcohol consumption; treatment with standard antidepressants improves both facets of this phenotype. Accordingly, we sought to examine whether mu-opioid receptor binding and the receptor-mediated functional coupling to G protein is affected by this drug treatment. Using quantitative autoradiography, binding of mu-opioid receptors labeled by [125I]FK33,824 (D-Ala2,N-Me-Phe4,Met(O)5-ol enkephalin) and the coupling between receptors and G proteins determined by agonist-stimulated guanosine 5'-O -(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding was mapped throughout brain sections of FH rats after 10-day treatment with vehicle, desipramine, or sertraline. Both desipramine and sertraline produced significant decreases of [125I]FK33,824 binding in many brain regions; 13 of 20 measured regions for desipramine and 16 of 20 measured regions for sertraline. The coupling efficiency of mu-opioid receptors to G proteins was determined by an increase of [35S]GTPgammaS binding induced by stimulation with the mu-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (10 microM). In contrast to the receptor binding profile, functional coupling of receptors to G proteins was only significantly reduced in the amygdala, whereas it remained unchanged in other regions compared with control. The present findings suggest that antidepressants regulate opioid systems; however, this occurs differentially, and region-specific alteration of functional coupling of mu-opioid receptors to G proteins in the amygdala suggests that opioid function within the amygdala may be modulated by antidepressants.

Long-term alcohol self-administration and alcohol withdrawal differentially modulate microtubule-associated protein 2 (MAP2) gene expression in the rat brain

Chronic alcohol intoxication is known to produce neuronal degeneration in the central and peripheral nervous system of experimental animals and of humans. It is suggested that various components of the cytoskeleton undergo profound changes following chronic alcohol use and misuse. Here we studied the expression of the neuronal cytoskeletal microtubule-associated protein 2 (MAP2) following long-term alcohol consumption and subsequent alcohol withdrawal. Alcohol-preferring AA (Alko Alkohol) rats with a high voluntary alcohol consumption for a period of 16 months were compared with age-matched control rats without prior experience with alcohol. For comparison, in a second experiment, heterogeneous Wistar rats that also had voluntary access to alcohol for 8 months were examined following alcohol consumption and withdrawal. In situ hybridization and subsequent dot blot and Northern blot analysis for further quantification revealed that chronically alcoholized animals exhibit markedly decreased MAP2 mRNA levels in several parts of the extrapyramidal system (mainly in the caudate putamen, the substantia nigra pars compacta and the globus pallidus), the mesolimbic system, in several hypothalamic nuclei and in the nucleus inferior colliculus. Other areas such as the hippocampus, frontoparietal cortex and cerebellum were less affected by chronic alcohol intake, however, in these regions the MAP2 mRNA levels were increased during alcohol withdrawal. These results suggest that long-term alcohol self-administration affects central neurons involved in motor control via the influence on the integrity of the cytoskeleton and may thus induce motor dysfunction.

Lifetime prolongation in voluntary alcohol-consuming rats (SHR) treated with clofibrate

Clofibrate affects lipid and alcohol as well as drug and eicosanoid metabolism. Spontaneous hypertensive rats (SHR) further increase their high voluntary alcohol consumption during clofibrate feeding. The interaction of alcohol and clofibrate was studied in two long-term trials. Seventy-nine male SHR (aged 27 weeks) were offered increasing concentrations of ethanol, up to 30% (tap water ad lib), and 3 months later 0.5% clofibrate-food. Four groups were established: N, normal controls; NA, standard diet + alcohol; C, clofibrate feeding; and CA, clofibrate feeding + alcohol. Food intake, alcohol consumption, body weight, and laboratory values were recorded continuously. Life duration (weeks) after the start of the trial was 63.3 ± 3.3 in N, 73 ± 2.6 in NA, 77.7 ± 4.3 in C, and 90.3 ± 2.8 in CA. There were no alcohol-related liver findings in NA and CA. Most of the animals died of cardiac and renal failure. An increase of tumors in clofibrate-treated rats was not observed. Voluntary alcohol consumption or clofibrate feeding significantly lengthens lifetime, which is prolonged by 42% if ethanol is combined with clofibrate. This is obviously not mediated by the lipid lowering effect or an influence on body weight and blood pressure of either clofibrate or alcohol.

Dietary regulation of voluntary alcohol consumption in rats: Influence of a high protein diet and a methylene blue diet

The importance of glucose homeostatis for high voluntary alcohol consumption was studied in alcohol-preferring (AA) and alcohol-avoiding (ANA) rats fed either a control diet, a protein-rich diet or a control diet supplemented with methylene blue. AA rats on the control diet were found to receive 13.6% of their daily energy intake from alcohol. On the high-protein or methylène blue diet, the alcohol consumption of the AA rats was respectively 40% and 48% higher than on the control diet. The voluntary alcohol consumption of ANA rats corresponded to 0.8–2.3% of their daily energy intake irrespective of diet. The protein diet increased the blood glucose concentration of AA rats by 20% but no increase was observed after the methylene blue diet. The diets had no effect on the blood glucose levels of ANA rats. In AA rats, the protein diet reduced the hepatic concentration of the three major glucogenic amino acids (serine, glycine, alanine) on average by 24%, suggesting an increased utilization for gluconeogenesis. No such reduction was observed in AA rats on the methylene blue diet or in ANA rats on any diet. The utilization of amino acids for maintenance of glucose balance in AA rats is further supported by the observed negative correlation between plasma concentration of urea, the end product of amino acid catabolism, and the sum of the concentrations of the three glucogenic amino acids in the liver, and by the positive correlation between plasma urea and blood glucose concentration. Furthermore, in AA rats, but not in ANA rats, the concentration of alanine, the main amino acid used in gluconeogenesis, correlated negatively with the amount of alcohol consumed. These findings indicate that the maintenance of glucose homeostatis is important for high voluntary alcohol consumption.

A histochemical study of the distribution of aldehyde dehydrogenase activity in brain structures of rats with genetically different alcohol-related behaviour

Brain samples from rats genetically selected for high or low voluntary alcohol intake (AA and ANA strains) or for differences in alcohol-induced motor incoordination (AT and ANT strains) were analyzed by histochemistry for aldehyde dehydrogenase (EC 1.2.1.3; ALDH) activity in various CNS structures. All strains exhibited the highest ALDH activities in neurons of the mesencephalic tract of trigeminal nerve nucleus and in spinal cord motoneurons, while the lowest activities were observed in the somatosensory cortex. Although the general distribution pattern of ALDH activity was similar in the genetically selected strains, some potentially important differences were observed. AA rats with high voluntary alcohol consumption had lower ALDH activity (with acetaldehyde as substrate) in the neuropil of the olfactory tubercle but higher activity (with benzaldehyde as substrate) in the spinal cord motoneurons, Purkinje cells and capillary endothelium of the cerebellum as compared to the corresponding structures from the alcohol avoiding ANA rats. Alcohol-resistant AT rats had higher ALDH activity, with benzaldehyde, in most CNS structures than did the alcohol-sensitive ANT's, significantly so in the lamina II of the somatosensory cortex and the neurons of the lateral hypothalamic area. This relationship was also found with acetaldehyde as substrate in the neurons of the hypothalamic arcuate nuclei and in cerebellar capillaries, but the ANT's had the higher activity in the neurons of the cerebral cortex V lamina. We suggest that some of the differences observed may relate to the differences between the rat strains with respect to voluntary alcohol intake and alcohol-induced motor incoordination.