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.

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.

A cluster of differentially expressed signal transduction genes identified by microarray analysis in a rat genetic model of alcoholism.

Analyzing gene expression patterns in genetic models of alcoholism may uncover previously unknown susceptibility genes, and point to novel targets for drug development. Here, we compared expression profiles in alcohol-preferring AA rats with the alcohol-avoiding counterpart ANA line, and unselected Wistar rats. Cingulate cortex, Nc. accumbens, amygdala and hippocampus of each line were analyzed using the Afymetrix RN U34 arrays and dChip 1.1 software. Analysis of line-specific expression revealed 48 differentially expressed genes between AA and ANA rats. Elevated hippocampal neuropeptide Y (NPY) was found in ANA rats in agreement with previous studies. A cluster of MAP-kinases indicating altered signal transduction was upregulated within the Nc. Accumbens of the AA line, and is of particular functional interest. Within the amygdala, a more loosely inter-related cluster of cytoskeleton-associated genes may point to structural abnormalities. The observed dysregulations may contribute to the alcohol-preferring phenotype.

Differential expression of NPY and its receptors in alcohol-preferring AA and alcohol-avoiding ANA rats.

BACKGROUND: Central neuropeptide Y (NPY) is known to control feeding and stress responses. Recently, it has been suggested that NPY also has a role in regulation of alcohol consumption. METHODS: NPY and NPY receptor expression in genetically selected alcohol-preferring (AA), alcohol-nonpreferring (ANA), and Wistar rats were investigated. Expression was assessed using in situ hybridization histochemistry with riboprobes specific for preproNPY, Y1, and Y2 receptors. Effects of central NPY administration on ethanol self-administration were also examined in AA, ANA, and Wistar rats by using oral operant self-administration. RESULTS: NPY mRNA expression was higher in ANA than in both AA and Wistar rats in the hippocampal CA region and dentate gyrus, whereas AA and Wistar did not differ from each other. No differences in NPY expression were found in the other regions analyzed: cingulate cortex, medial nucleus of the amygdala, arcuate, and paraventricular nuclei of the hypothalamus. Y1 receptor mRNA expression did not differ between the three lines. Y2 expression was higher in the dentate gyrus of both AA and ANA rats than in Wistar subjects. In the medial amygdala, Y2 mRNA was reduced in the AA line, compared to both ANA and Wistar rats. NPY injected intracerebroventricularly (1.5-3.0 nmol) did not affect operant ethanol self-administration in any of the three lines examined. CONCLUSION: The NPY system seems to differ in several respects between rat lines with different levels of alcohol preference. Differences observed within the hippocampus could be related to behavioral traits other than alcohol intake but it is also possible that elevated hippocampal expression of NPY in the ANA rats contributes to the low alcohol intake of this line. Aberrant NPY expression and/function within the amygdala complex could contribute to alcohol preference and constitute an anatomic substrate of the effects of NPY expression on alcohol intake observed previously in genetically modified animals.

Naltrexone suppresses ethanol intake in 6-hydroxydopamine-treated rats.

BACKGROUND: The suppressive effect of opioid antagonists, such as naltrexone, on ethanol intake has been suggested to be based on the interference with ethanol-induced stimulation of dopamine release in the nucleus accumbens. The aim of this study was to determine whether reduction of dopamine innervation to the nucleus accumbens with the neurotoxin 6-hydroxydopamine (6-OHDA) alters naltrexone-induced suppression of ethanol consumption. Because the mesolimbic dopaminergic neurons have also been implicated in ethanol reinforcement, the effects of 6-OHDA on the maintenance and acquisition of ethanol intake were also studied. METHODS: To damage accumbal terminals of the mesolimbic dopamine neurons, alcohol-preferring Alko Alcohol (AA) rats were given bilateral injections of 6-OHDA or vehicle into the nucleus accumbens after pretreatment with desipramine and pargyline. The effect of the lesion on the acquisition or maintenance of ethanol self-administration was studied in animals having continual access to ethanol solution (10% v/v) and water. Subsequently the effect of naltrexone on ethanol consumption was determined. RESULTS: Naltrexone (0.03-3.0 mg/kg subcutaneously) suppressed ethanol consumption in a dose-dependent manner both in 6-OHDA-treated and control animals given a daily 90-min access to ethanol solution. When the rats had continual access to ethanol, there was a clear day-to-day decline in ethanol intake during the first 5 days of the 7-day naltrexone treatment (10 mg/kg subcutaneously). 6-OHDA treatment had no effect on either the acquisition or maintenance of ethanol self-administration. Postmortem analysis of the brain dopamine content revealed approximately 92% depletion of dopamine in the nucleus accumbens of the 6-OHDA-treated rats. CONCLUSIONS: The suppressive effect of naltrexone does not depend on naltrexone's interaction with dopaminergic terminals in the nucleus accumbens. Furthermore, the role of the mesolimbic dopamine pathway is probably not central either in the acquisition or maintenance of ethanol self-administration in alcohol-preferring AA rats.

Effects of lifelong ethanol consumption on drinking behavior and motor impairment of alcohol-preferring AA and alcohol-avoiding ANA rats.

The effects of drinking ethanol throughout a lifetime on voluntary drinking behavior and ethanol-induced motor impairment were studied in alcohol-preferring AA (Alko, Alcohol) and alcohol-avoiding ANA (Alko, Non-Alcohol) rats of both sexes. At the age 3 months, the rats were tested for individual voluntary ethanol (10% vol./vol.) intake and ethanol-induced motor impairment (2 g/kg, i.p.). The rats were housed in group cages, half of them having 12% (vol./vol.) ethanol as the only source of fluid and the other half having free access to water. Food was always available for all animals. At the age of 23 months, their individual voluntary ethanol intake and ethanol-induced motor impairment were tested again. During forced drinking, the females of both strains consumed more ethanol than did the males. The ethanol consumption of the AA and ANA females and the ANA males increased significantly (P < .001) with age, but a slight decrease was seen in the ethanol consumption of the AA males. Time x strain interaction showed a significant (P < .05) difference in the ethanol consumption of male rats, with the AA males having a slight decrease in ethanol consumption with age, whereas the ANA males increased their ethanol consumption. After 19 months of forced ethanol exposure, AA males significantly decreased their individual voluntary ethanol consumption, and individual voluntary ethanol consumption by ethanol-exposed AA males was more pronounced (P < .001) than that of the AA rats that had free access to water (P < .05). For the female AA rats, those having free access to water significantly decreased their voluntary ethanol consumption (P < .05), but those having ethanol only did not. No significant changes in voluntary ethanol consumption with age or with different exposures were seen in the ANA rats. Body weights were higher in the groups having access to water than in the ethanol-only groups, but the differences were not significant within the AA and ANA strains. The ANA rats were significantly heavier in all groups. These results indicate that the voluntarily nondrinking ANA rats can drink almost as much ethanol as the voluntarily drinking AA rats when they are forced to drink ethanol and that lifelong forced ethanol drinking does not change their inherent drinking habits. When sensitivity to ethanol was measured with the tilting-plane test, the old AA female rats were more sensitive to ethanol than were the young ones. The young ANA females were more sensitive than the AA females when tested at 4 months. In males, aging did not produce any differences in ethanol sensitivity.

Neurobiological processes in alcohol addiction.

This article represents the proceedings of a symposium at the ISBRA Meeting in Yokohama, Japan. The chairs were A. D. Lê and K. Kiianmaa. The presentations were (1) Alcohol reward and aversion, by C. L. Cunningham; (2) The role of sensitization of neuronal mechanisms in ethanol self-administration, by J. A. Engel, M. Ericson, and B. Söderpalm; (3) Alcohol self-administration in dependent animals: Neurobiological mechanisms, by G. F. Koob, A. J. Roberts, and F. Weiss; (4) Stress and relapse to alcohol, by A. D. Lê; (5) Alcohol-preferring AA and alcohol-avoiding ANA rats differ in locomotor activation induced by repeated morphine injections, by P. Hyytiä, S. Janhunen, J. Mikkola, P. Bäckström, and K. Kiianmaa; and (6) Initial sensitivity and acute functional tolerance to the hypnotic effects of ethanol in mice genetically selected for mild and severe ethanol withdrawal convulsions, by I. Ponomarev and J. C. Crabbe.

Expression of c-Fos in Alko alcohol rats responding for ethanol in an operant paradigm.

BACKGROUND: Identification of the brain regions involved in ethanol administration is important for understanding the neurobiology of ethanol addiction. Animal studies with different brain mapping techniques found that voluntary ethanol self-administration leads to changes in activity of specific brain regions in patterns that only partially overlap with patterns of brain regions affected by involuntary (i.e., experimenter-administered) ethanol administration. As an extension of studies mapping changes in neural activity after voluntary ethanol drinking, this study analyzed expression of the inducible transcription factor c-Fos after ethanol consumption in an operant procedure. METHODS: AA (Alko alcohol) rats were trained to operantly respond for water, 0.2% saccharin, 0.2% saccharin/10% (w/v) ethanol, or 10% ethanol in a 30-min limited-access procedure. Animals were allowed to self-administer solutions for at least 40 ethanol response sessions and were killed 1.5 hr after beginning of the last session. Forty-seven brain regions were immunohistochemically analyzed for c-Fos expression. RESULTS: In this paradigm, ethanol dose-dependently increased c-Fos expression in the Edinger-Westphal nucleus (EW) and decreased expression in the dorsal tenia tecta compared with no-ethanol controls. No effects of saccharin on c-Fos expression were found. CONCLUSIONS: Our results extend previous findings of preferential sensitivity of EW to alcohol in voluntary self-administration procedures to operant responding for ethanol and warrant further investigation of ethanol's effects on the EW. The finding that ethanol attenuated c-Fos expression in the tenia tecta is novel. Taken together, these findings confirm that voluntary ethanol self-administration leads to changes in activity of a limited number of brain regions with previously unexamined roles in ethanol sensitivity and addiction.

Differential expression of diacylglycerol kinase iota and L18A mRNAs in the brains of alcohol-preferring AA and alcohol-avoiding ANA rats.

Ethanol preference and behavioral disinhibition in AA (alcohol accepting) animals is a behavioral constellation similar to that seen in human type II alcoholism, for which considerable genetic loading has been shown. In search of novel neural substrates for this phenotype, we compared gene expression in the cerebral cortex of the AA rat with two groups of control animals, the ANA (alcohol non-accepting) line and heterogeneous Wistar animals, by differential display RT-PCR. We identified two transcripts, ribosomal protein L18a mRNA and diacyglycerol kinase iota mRNA, which are differentially expressed between AA and ANA rats. Ribosomal protein L18A mRNA is evenly expressed throughout the brain, but strongly reduced in cortex of AA rats vs controls. Diacylglycerol kinase iota is exclusively found in the brain, and expressed in a distinct regional pattern. Its cortical expression is about 25% higher in AA than ANA rats. Differential display RT-PCR seems to provide a feasible strategy to identify previously unknown genes whose differential expression correlates with behavioral phenotypes related to dependence.

Suppression of ethanol responding by centrally administered CTOP and naltrindole in AA and Wistar rats.

BACKGROUND: Both mu- and delta-opioid receptors have been implicated in the reinforcing actions of ethanol. However, selective opioid receptor antagonists have not altered ethanol intake in all rodent strains consistently, which suggests that genotype may modulate their suppressive effects. Therefore, we tested the effects of the selective mu-antagonist D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and the selective delta-antagonist naltrindole in both high-drinking AA (Alko, Alcohol) and heterogeneous Wistar rats. METHODS: AA and Wistar rats were trained to respond for ethanol (10% w/v) in a two-lever operant condition by using a saccharin fading procedure. After stable baseline responding was established, rats were implanted stereotaxically either with a guide cannula above the lateral ventricle or with bilateral cannulas above the nucleus accumbens, basolateral amygdala, or ventral tegmental area. After postoperative recovery, AA and Wistar animals were tested after intracerebroventricular microinjections of either CTOP (0-3 microg) or naltrindole (0-30 microg) or subcutaneous injections of naloxone (0-1 g/kg), which was used as a reference antagonist. Effects of intracerebral microinjections of CTOP and naltrindole (both 0-500 ng) were tested only in Wistar rats. RESULTS: Subcutaneous naloxone and intracerebroventricular CTOP and naltrindole suppressed ethanol self-administration in a similar manner in AA and Wistar rats. Cumulative response patterns indicated that naloxone and naltrindole had no effect on the initiation of responding but suppressed it later during the session, whereas CTOP also affected initiation. In Wistar rats, naltrindole microinjections into both the nucleus accumbens and basolateral amygdala decreased ethanol responding, whereas CTOP was effective only in the amygdala. Injections of these antagonists into the ventral tegmental area had little effect on ethanol intake. CONCLUSIONS: The results confirm previous results which showed that both mu- and delta-opioid receptors are involved in the regulation of ethanol self-administration and indicate that genetic differences between AA and Wistar rats produced by selection do not modify the effects of opioid antagonists. The nucleus accumbens and the basolateral amygdala may be important central sites for the mediation of their suppressive effects.

The effects of nicotine on locomotor activity and dopamine overflow in the alcohol-preferring AA and alcohol-avoiding ANA rats.

The aim of the study was to investigate the importance of the interaction between central dopaminergic and cholinergic mechanisms for ethanol reinforcement. This was done by comparing the effects of nicotine on locomotor activity and release of dopamine in the nucleus accumbens of the alcohol-preferring Alko Alcohol (AA) and alcohol-avoiding alko non-alcohol (ANA) rats. Nicotine was administered acutely (0.25, 0.50 or 0.75 mg/kg, s.c.) or repeatedly once daily (0.5 mg/kg, s.c.) for 8 days. An acute dose of nicotine increased locomotor activity and the extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) measured with in vivo microdialysis suggesting stimulation of dopamine release by nicotine. No difference in the stimulation of locomotor activity or in the increase in the extracellular concentrations of dopamine or its metabolites by nicotine was found between the rat lines. The concentrations of nicotine in the plasma were also identical. The rats treated repeatedly with nicotine showed a progressive increase in locomotion. On the challenge day, 1 week after termination of nicotine or saline injections, rats previously treated with nicotine were activated more by nicotine than saline-treated rats. This behavioral sensitization was not accompanied by an increase in the amplitude of the neurochemical response to nicotine, but the duration of the increase in the levels of DOPAC was longer in the nicotine than saline-treated animals. The increases in locomotor activity and metabolite levels were, however, similar in both rat lines. These data suggest that differences in the interaction of central dopaminergic and cholinergic mechanisms probably do not contribute to the difference in ethanol self-administration between the AA and ANA rat lines.

Effects of continuous opioid receptor blockade on alcohol intake and up-regulation of opioid receptor subtype signalling in a genetic model of high alcohol drinking.

Effects of a continuous naloxone infusion via osmotic pumps on alcohol drinking and opioid receptor density and function in the high-drinking AA (Alko, Alcohol) rats were examined. AA rats were trained to drink 10% (v/v) ethanol in a 1-h limited access procedure and implanted with subcutaneous osmotic pumps delivering either saline, a low dose (0.3 mg/kg per hour), or a high dose (3.0 mg/kg per hour) of naloxone for 7 days. The pumps were then removed and alcohol, food and water intakes were measured for another 4 days. Compared with saline, both naloxone doses significantly suppressed 1-h alcohol intake during the 7-day infusion. The suppression was smaller than that by a bolus injection of the same daily dose 15 min before the session, although a complete blockade of morphine-induced antinociception was achieved even with the smaller naloxone infusion. Significant decreases were also seen in daily food and water intake during the first days, but they quickly returned to their previous baselines. After pump removal, rats of both naloxone-treated groups rapidly increased their alcohol drinking and reached the pretreatment baseline, while their food and water intakes significantly surpassed their baselines. Naloxone infusion at 3.0 mg/kg per hour for 7 days significantly decreased 24-h alcohol drinking without affecting alcohol preference. Twenty-four hours after pump removal, autoradiography with [3H]DAMGO, [3H]DPDPE and [3H]U-69,543 revealed an up-regulation of mu-, delta- and kappa-opioid receptor binding sites in many brain areas of these animals. This receptor up-regulation was functional, because receptor coupling to G-protein activation was enhanced by agonist ligands, as revealed by [35S]GTPgammaS autoradiography. A good correlation existed between ligand binding densities and G-protein activation for mu- and kappa-receptors in control and naloxone-treated brain sections. Furthermore, morphine-induced analgesia in a hot-plate test showed a leftward shift in the morphine dose-response curve after naloxone treatment. These results suggest that the usefulness of a chronic opioid antagonist dosing regime could be limited by nonspecific effects of the antagonist on ingestive behaviour, an up-regulation of opioid receptors with high antagonist doses, and the resulting supersensitivity to opioid agonists after the discontinuation of the treatment.

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.

Effects of morphine on metabolism of dopamine and serotonin in brains of alcohol-preferring AA and alcohol-avoiding ANA rats.

Morphine induces a larger locomotor stimulation in the alcohol-preferring AA rats than in the alcohol-avoiding ANA rats. We have now studied the acute effects of morphine (1 and 3 mg/kg) on metabolism of dopamine and serotonin (5-HT) in the dorsal and ventral striatum of the AA and ANA rats. The basal level of dopamine release, as reflected by the concentration of dopamine metabolite 3-methoxytyramine (3-MT), was lower in the caudate-putamen and nucleus accumbens of the AA rats than in the ANA rats. In the caudate-putamen, morphine increased dopamine metabolism and release more in the AA than in the ANA rats. In the nucleus accumbens and olfactory tubercle, the effects of morphine on dopamine metabolism and release did not differ between the rat lines. Morphine elevated the metabolism of 5-HT in the caudate-putamen and nucleus accumbens of the AA but not in those of the ANA rats. The results suggest that the larger morphine-induced psychomotor stimulation of the AA rats in comparison with the ANA rats is associated with the larger effect of morphine on dopamine metabolism in the caudate-putamen and 5-HT metabolism in the caudate-putamen and nucleus accumbens. Furthermore, low basal dopamine release may play a role in the high alcohol-preference of AA rats.

[3H]ethylketocyclazocine binding to brain opioid receptor subtypes in alcohol-preferring AA and alcohol-avoiding ANA rats.

We measured brain regional patterns of [3H]ethylketocyclazocine binding to brain opioid receptors in ethanol-naive alcohol-preferring Alko, Alcohol (AA) and alcohol-avoiding Alko, Non-Alcohol (ANA) rats, by using quantitative autoradiography. This agonist ligand labels all opioid receptor subtypes. The proportions of mu- and delta-opioid receptor binding were evaluated by displacing the mu- and delta-opioid receptor components by the peptides Tyr-D-Ala-Gly-N(Me)Phe-Gly-ol (DAMGO, 100 nM) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE, 100nM), respectively, the K-component being the naltrexone-sensitive binding left after removal of the above two components. The labeling patterns in the brains of the AA and ANA rats were consistent with the well-known distributions of the opioid receptor subtypes in nonselected rat strains and there was no major difference between the lines. The mu-opioid receptor binding was greater in the AA than ANA rats in several brain regions, most interestingly in the substantia nigra pars reticulata and striatal clusters with elevated shell/core ratios in the nucleus accumbens. The delta-opioid receptor binding did not differ between the lines, whereas the AA rats had more K-opioid receptors than the ANA rats in several brain regions, including limbic areas and basal ganglia. The observed results might indicate altered action of the opioidergic system on dopaminergic pathways in rats with differential alcohol preference.

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.

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.