Does the transcription factor AP-2beta have an impact on the genetic and early environmental influence on ethanol consumption?

Genes involved in alcoholism have consensus sites for the transcription factor activator protein (TFAP) 2beta. In the present study, we investigated TFAP-2beta protein levels in the ethanol-preferring alko, alcohol (AA) and the ethanol-avoiding alko, non-alcohol (ANA) rat lines. Furthermore, basal and ethanol-induced TFAP-2beta levels were examined in Wistar rats exposed to different early postnatal environments that are known to affect later ethanol consumption. Taken together, we found differences in brainstem TFAP-2beta protein between the AA and ANA rats.

Modulation of voluntary ethanol consumption by beta-arrestin 2.

Beta-arrestin 2 is a multifunctional key component of the G protein-coupled receptor complex and is involved in mu-opiate and dopamine D2 receptor signaling, both of which are thought to mediate the rewarding effects of ethanol consumption. We identified elevated expression of the beta-arrestin 2 gene (Arrb2) in the striatum and the hippocampus of ethanol-preferring AA rats compared to their nonpreferring counterpart ANA line. Differential mRNA expression was accompanied by different levels of Arrb2 protein. The elevated expression was associated with a 7-marker haplotype in complete linkage disequilibrium, which segregated fully between the lines, and was unique to the preferring line. Furthermore, a single, distinct, and highly significant quantitative trait locus for Arrb2 expression in hippocampus and striatum was identified at the locus of this gene, providing evidence that genetic variation may affect a cis-regulatory mechanism for expression and regional control of Arrb2. These findings were functionally validated using mice lacking Arrb2, which displayed both reduced voluntary ethanol consumption and ethanol-induced psychomotor stimulation. Our results demonstrate that beta-arrestin 2 modulates acute responses to ethanol and is an important mediator of ethanol reward.

Glutathione-S-transferase expression in the brain: possible role in ethanol preference and longevity.

Identification of genes that are differentially expressed in rats bidirectionally selected for alcohol preference might reveal biological mechanisms underlying alcoholism or related phenotypes. Microarray analysis from medial prefrontal cortex (mPFC), a key brain region for drug reward, indicated increased expression of glutathione-S-transferases of the alpha (Gsta4) and mu (Gstm1-5) classes in ethanol-preferring AA rats compared with nonpreferring ANA rats. Real-time RT polymerase chain reaction (RT-PCR) analysis demonstrated approximately 2-fold higher Gsta4 transcript levels in several brain regions of ethanol-naive AA compared with ANA rats. Differences in mRNA levels were accompanied by differential levels of GSTA4 protein. We identified a novel haplotype variant in the rat Gsta4 gene, defined here as var3. Allele frequencies of var3 were markedly different between AA and ANA rats, 52% and 100%, respectively. Gsta4 expression was strongly correlated with the gene dose of var3, with approximately 60% of the variance in expression accounted for by genotype at this locus. The contribution of glutathione S-transferase expression to the ethanol-preferring phenotype is presently unclear. It could, however, underlie observed differences in life span between AA and ANA lines, prompting a utility of this animal model in aging research.

Gene expression and activity of specific opioid-degrading enzymes in different brain regions of the AA and ANA lines of rats.

There is increasing evidence that alcoholism runs in families suggesting that genetic factors may play a role. In support of this hypothesis, the alcohol-preferring (AA) and the alcohol-avoiding (ANA) rat lines have been developed through selective outbreeding. Numerous studies indicate that the endogenous opioid system may be involved in controlling ethanol consumption. Changes in opioid peptides and opioid receptors have been described after ethanol intake. But, the influence of ethanol on peptidolytic degradation of opioid peptides has been largely ignored, although the peptidase-mediated metabolism of neuropeptides is known as an important regulatory site of peptidergic transmission. Neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE) degrade neuropeptides, including enkephalin and are expressed in the brain. Furthermore, a good correspondence between the regional distribution of NEP and opioid receptors in rat brain has already been reported pointing to a possible role of NEP in regulating opioid peptides. For both enzymes studied, the gene expression pattern was found to be in good agreement with the corresponding enzyme activities in the brain regions investigated, showing the highest levels for both specific mRNAs and enzyme activities in the striatum. Differences in both measured parameters were detected in distinct brain regions of AA and ANA rats. Furthermore, in some brain regions discrepancies between ACE and NEP mRNA levels and the corresponding enzyme activities were observed. For example, in olfactory bulb and striatum such discrepancies were found for both enzymes studied. In tegmentum/colliculi a higher NEP gene expression in AA rats was associated with a higher NEP enzyme activity compared to the amounts found in ANA rats.

Urocortin 1 expression in five pairs of rat lines selectively bred for differences in alcohol drinking.

RATIONALE: There is accumulating evidence that the neuropeptide urocortin 1 (Ucn1) is involved in alcohol consumption. Thus far, however, most studies have been performed in mice. OBJECTIVES: The purpose of the present study was to characterize Ucn1 expression in rats selectively bred for either high or low alcohol intake. METHODS: Brains from naive male rats of five pairs of independently selected lines (iP/iNP, AA/ANA, HARF/LARF, HAD1/LAD1, and HAD2/LAD2) were analyzed by immunohistochemistry. RESULTS: Significant differences were found between iP/iNP, HARF/LARF, and HAD2/LAD2 in number of Ucn1-containing cells in the Edinger-Westphal (EW) nucleus (the main source of Ucn1 in the brain), whereas no significant differences were found between HAD1/LAD1 and AA/ANA. Similarly, significant differences in the optical density of Ucn1 immunoreactivity in EW were found between iP/iNP, HARF/LARF, and HAD2/LAD2, whereas no differences on this measure were found between HAD1/LAD1 and AA/ANA. In the lateral septum (LS, the main projection area of Ucn1-containing neurons in the rat), significant differences were found only between AA/ANA and HAD2/LAD2; however, a meta-analysis indicated that across all five lines, preferring animals had a significantly greater number of Ucn1-positive fibers than nonpreferring animals. CONCLUSIONS: These results provide evidence that, in rats, Ucn1 may be involved in regulation of alcohol intake, and that this regulation may occur through the Ucn1 projections to LS.

Intravenous heroin and ethanol self-administration by alcohol-preferring AA and alcohol-avoiding ANA rats.

The alcohol-preferring AA rats have previously been shown to drink more solution containing the opioid etonitazene than the alcohol-avoiding ANA rats. The present experiments were initiated to see whether the line difference in opioid and alcohol intake would persist if an intravenous (i.v.) route of self-administration is used. Following establishment of stable heroin responding (0.03 mg/kg per infusion), AA and ANA rats were first subjected to three within-session dose-response determinations during which they were allowed to respond for ascending heroin doses (0.0075, 0.015, 0.03, and 0.06 mg/kg per infusion) and then to one progressive-ratio schedule session. AA rats obtained more heroin infusions than ANAs during the first acquisition sessions but there were no significant differences between the lines either in their baseline heroin responding, across the ascending within-session doses, or on the progressive ratio probe. When, after additional heroin baseline sessions, ethanol (1.0 mg/kg per infusion) was substituted for heroin, AA rats initially increased their responding and showed stable rates for responding across ascending ethanol doses (2.0 and 4.0 mg/kg), whereas ANAs declined below their heroin baseline. These findings give evidence for only an initial line difference in i.v. opiate self-administration but for a sustained difference in i.v. ethanol self-administration, thus suggesting that the differential alcohol drinking of the AA and ANA rats is dependent at least partly on non-oral factors.

Enhanced morphine- and cocaine-induced behavioral sensitization in alcohol-preferring AA rats.

Locomotor stimulation and behavioral sensitization induced by acute and repeated treatment with alcohol, cocaine or morphine were studied in the alcohol-preferring AA (Alko, Alcohol), alcohol-avoiding ANA (Alko, Non-Alcohol) rats and non-selected Wistar rats. Daily treatment with alcohol (ethanol, 0.4 or 1.0 g/kg, IP) for 6 days had no effect on locomotor activity either in the AA or ANA rats. Acute cocaine (5, 10 or 20 mg/kg, IP) produced a locomotor stimulation in the animals of all lines studied, and there was no difference in this effect between the AA and ANA rats. During a 4-day repeated cocaine treatment, the AA rats became sensitized with the 10 mg/kg dose, while the ANA rats did not show any sensitization with this dose. With the 20 mg/kg cocaine dose, in addition to locomotor stimulation, the rats of all lines studied showed stereotyped behavior, which response was enhanced during repeated treatment. Morphine-induced locomotor stimulation was larger in the AA rats than in the ANA or Wistar rats both with 1.0 and 3.0 mg/kg doses and only the AA rats were sensitized during 4-day treatment with the 1 mg/kg dose. With the 3.0 mg/kg morphine dose, only the AA rats showed a weak sensitization evident only during the initial 30 min after morphine injection. As the drug-induced behavioral sensitization is an important factor in the development of drug addiction, it is possible that mechanisms underlying the enhanced susceptibility of the AA rats to morphine- and cocaine-induced sensitization contribute to the high intake of alcohol and other abused drugs by these animals.

GABAA receptor antagonism in the extended amygdala decreases ethanol self-administration in rats.

The present experiments examined the role of the extended amygdala GABAA receptors in the regulation of ethanol consumption in rats. The areas of the extended amygdala studied included the central nucleus of the amygdala, the bed nucleus of the stria terminalis, and the shell of the nucleus accumbens. The effects of bilateral microinjections of a competitive GABAA receptor antagonist, 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide (SR 95531), on ethanol consumption were assessed in Wistar rats that were trained to respond for oral ethanol (10% w/v) in a two-lever, free-choice operant task during 30-min sessions using a saccharin fading procedure. Injections of SR 95531 into the central amygdaloid nucleus decreased ethanol responding significantly at doses of 2 and 4 ng without affecting water responding. SR 95531 injections into the bed nucleus of the stria terminalis reduced ethanol responding significantly at the 8 ng and 16 ng dose, while only the 16 ng dose produced a significant effect in the shell of the nucleus accumbens. Cumulative response patterns showed that intra-amygdaloid injections did not disrupt the initiation of responding. Injections into the bed nucleus of the stria terminalis and the nucleus accumbens, however, suppressed both ethanol and water responding at the highest SR 95531 doses during the first minutes. These findings suggest that GABAA receptors in the extended amygdala may be involved in the mediation of some aspects of ethanol reward.

Site-specific NMDA receptor antagonists produce differential effects on cocaine self-administration in rats.

The effects of site-specific NMDA receptor antagonists on intravenous cocaine self-administration were examined in rats trained to self-administer cocaine (0.25 mg/infusion) on a fixed ratio (FR) 5 schedule with a 20-s time-out (TO) after each reinforcer. The non-competitive NMDA receptor antagonists, dizocilpine (MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate) (0.05-0.2 mg/kg i.p.) and memantine (1,3-dimethyl-5-amino-adamantane hydrochloride) (2.5-20 mg/kg i.p.), dose-dependently decreased cocaine self-administration, while the competitive NMDA receptor antagonist, CGP 39551 (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid carboxyethylester) (2.5-15 mg/kg i.p.), and the NMDA/glycine receptor antagonist, L-701,324 (7-chloro-4-hydroxy-3(3-phenoxy)-phenyl-2(H)quinolone) (1.25-10 mg/kg p.o.), were without effect. Under a progressive ratio (PR) schedule, dizocilpine (0.15 mg/kg i.p.) increased the number of cocaine infusions in a manner similar to increasing the unit dose of cocaine, suggestive of potentiation of cocaine reward. Conversely, memantine (10 mg/kg i.p.) produced rate-decreasing effects on the PR schedule. These results demonstrate that NMDA receptor antagonists acting at different modulatory sites of the NMDA receptor do not share dizocilpine's cocaine reward enhancing effects although they are all known to be effective blockers of NMDA receptor activity.

Alcohol drinking is reduced by a mu 1- but not by a delta-opioid receptor antagonist in alcohol-preferring rats.

To assess the roles of opioid receptor subtypes in voluntary alcohol drinking, alcohol-preferring AA (Alko, Alcohol) rats, non-deprived of food or water, were used in a paradigm where access to 10% alcohol solution was limited to 1-4-h sessions on every 2nd working day. The delta-opioid receptor antagonist naltrindole (1-5 mg/kg i.p. 15 min before the session) had no effect on alcohol drinking, while it attenuated the delta-opioid receptor agonist [D-Pen2, D-Pen5]enkephalin-induced locomotor stimulation. The mu1-opioid receptor antagonist naloxonazine (1-15 mg/kg i.p. 20 h before the session), at the largest dose, decreased alcohol drinking. It also decreased food intake. When naltrindole (1 mg/kg) and naloxonazine (15 mg/kg) were given prior to 3 consecutive sessions, the former had no effects at any session. Naloxonazine decreased alcohol consumption only in the 1st session, although the reduction of daily water intake became stronger during repeated administration. 4 days after the last drug administration, naloxonazine-treated animals consumed alcohol nearly twice as much as in the control session before any drug treatment. These data suggest that delta-opioid receptors are not involved in the regulation of alcohol drinking in AA rats. mu1-Opioid receptors may be involved in alcohol drinking, although the data suggest that even their prolonged blockade alone is insufficient to induce a sustained decrease in alcohol drinking.