Circadian drinking rhythms and blood alcohol levels in two rat lines developed for their alcohol consumption

The circadian drinking behavior of the AA (Alko Alcohol) and ANA (Alko Non-Alcohol) rat lines developed for high and low voluntary alcohol consumption was studied by using a microcomputer lick-counting system. The blood alcohol concentrations of AA rats were measured during the drinking peaks. The results showed that AA rats voluntarily drink intoxicating amounts of alcohol. The highest blood alcohol levels were about 25 mmol/l. Differences in circadian drinking rhythms between AA and ANA lines were also found. AA rats had a trimodal drinking pattern for alcohol intake during the dark period but ANA rats drank alcohol rather evenly throughout the period.     

Effects of repeated morphine treatment on metabolism of cerebral dopamine and serotonin in alcohol-preferring AA and alcohol-avoiding ANA rats

The alcohol-preferring AA (Alko Alcohol) rats are more rapidly sensitized to the locomotor activity-stimulating effects of small doses of morphine than the alcohol-avoiding ANA (Alko Non-Alcohol) rats. To study the involvement of dopaminergic and serotonergic transmission in this behaviour, the effects of acute morphine (1 mg/kg) challenge on the concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT, serotonin) and their metabolites were estimated in three dopaminergic areas in AA and ANA rats on the fourth day after a 3-day morphine or saline treatment. Acute administration of morphine enhanced DA metabolism in the caudate-putamen in the AA, but not in the ANA, rats; in the nucleus accumbens and in the olfactory tubercle the acute effect of morphine was similar in rats of both lines. Morphine pretreatment did not significantly enhance acute morphine's effects on DA metabolites in any of the brain areas studied in rats of either line. Acute administration of morphine enhanced brain 5-HT metabolism in the AA rats but not in the ANA rats, but after repeated treatment it induced no enhancement of 5-HT metabolism. With the methods used, no significant differences were found between the AA and ANA rats in the effects of repeated morphine on cerebral dopaminergic or serotonergic mechanisms which could account for the different behavioural sensitization found previously in rats of these lines. However, both monoamines studied might be involved in the acute locomotor stimulatory effects of morphine.     

Audiogenic immobility reaction and open-field behavior in AA and ANA rat lines

Individual predispositions in emotional reactivity have been suggested as factors involved in the development of alcoholism. To approach this problem, we assessed emotional reactivity in alcohol-naive animals from the alcohol-preferring (AA) and alcohol-avoiding (ANA) rat lines of Alko Ltd. AA rats are known to have higher brain levels of 5-hydroxytryptamine (5-HT) than ANA rats. Emotional reactivity was therefore assessed by an audiogenic immobility reaction (freezing), which is specifically sensitive to and shortened by depletion of 5-HT. The results showed that AA rats of both sexes displayed increased immobility reactions compared to the corresponding sex of the ANA rats. During the period of adaptation to the test cage ANA rats of both sexes showed increased locomotor activity compared to the corresponding sex of the AA rats. Levels of plasma corticosterone did not differ between the rat lines, either during resting or stressful conditions. The present results suggest that a passive, inhibited style of defensive behavior is associated with a high alcohol consumption.     

Synaptosomal sodium channels in rat lines selected for alcohol-related behaviors

Alcohol has been shown to inhibit veratridine- and batrachotoxin-stimulated sodium flux in rodent brain synaptosomes. In this study, binding of [3H]batrachotoxinin A 20-alpha-benzoate ([3H]BTX-B) and uptake of [14C]guanidine in cerebrocortical synaptosomes were measured in naive rats belonging to lines with high or low acute sensitivity to ethanol (ANT, Alcohol Non-Tolerant, vs. AT, Alcohol Tolerant, lines) and to lines with high or low voluntary alcohol consumption (AA, Alko Alcohol, vs. ANA, Alko Non-Alcohol, lines). Our rat line pairs did not differ in the equilibrium binding characteristics of [3H]BTX-B nor in the properties of [14C]guanidine uptake, suggesting that the genetic selection has not modulated the genes or the expression of the genes associated with the voltage-sensitive sodium channels.     

Binding of serotonergic ligands to brain membranes of alcohol-preferring AA and alcohol-avoiding ANA rats.

The alcohol-preferring AA rats have higher concentration of 5-hydroxytryptamine (5-HT) in the brain than the alcohol-avoiding ANA rats. In the present study, the 5-HT1, 5-HT2, and 5-HT3 receptors were studied with [3H]5-HT, [3H]ketanserin, and [3H]LY278584, respectively, in membrane homogenates from different brain regions of both rat lines using in vitro binding assays. No differences in the 5-HT1 and 5-HT2 receptor binding in the brainstem, hippocampus, frontal cortex, and hypothalamus or in the 5-HT3 receptor binding in the nucleus accumbens, amygdala, hippocampus, and frontal cortex were observed between the ethanol-naive animals of the rat lines. In rats given the opportunity to voluntarily consume alcohol, there was a tendency to increase 5-HT1 binding in the ANA rats, which tendency was, however, also found in their ethanol-naive controls subjected to the same handling and behavioral tests as the ethanol-experienced animals. The results do not, however, indicate that any genetic modifications of the 5-HT receptor-binding sites have occurred in the process of the selective breeding of AA and ANA rats for alcohol preference and avoidance, respectively.     

Taste reactivity in alcohol-preferring AA and alcohol-avoiding ANA rats.

Alcohol-preferring rats (Alko alcohol or AA) were tested for taste reactivity to water, sucrose, quinine, and a range of alcohol concentrations (5-40%) both before and after a period of continuous alcohol access. The alcohol-avoiding line of rats (Alko nonalcohol or ANA) was also tested for comparison. It was found that AA rats displayed greater ingestive reactivity to alcohol compared to ANA rats both before and after a three-week period of continuous access to 10% alcohol (during which time AA rats drank significantly more alcohol than ANA rats). AA rats also made significantly more ingestive responses to a 0.3 M sucrose solution and a 0.0005 M quinine solution. Differences between AA rats and ANA rats in aversive reactivity appeared only after the alcohol consumption tests; AA rats made significantly fewer aversive responses to the 30% and 40% concentrations after continuous alcohol access. AA rats also made significantly more aversive responses to the quinine solution. The results suggest that line differences between AA rats and ANA rats in the reactivity response to alcohol solutions have been selected in association with the original selection phenotype of alcohol consumption.     

AA and ANA rats exhibit the R100Q mutation in the GABAA receptor alpha 6 subunit.

The R100Q mutation in the gamma-aminobutyric acid type A (GABA(A)) receptor alpha(6) subunit was previously identified in Sardinian alcohol-preferring (sP) and Sardinian alcohol-nonpreferring (sNP) rats as a candidate gene influencing alcohol preference and sensitivity. The purpose of the current study was to determine to what extent this mutation and alcohol preference observed in the sP and sNP lines was present in other independently selected rat lines, including inbred alcohol-preferring (iP) and inbred alcohol-nonpreferring (iNP), high-alcohol-drinking 1 (HAD1) and low-alcohol-drinking 1 (LAD1), high-alcohol-drinking 2 (HAD2) and low-alcohol-drinking 2 (LAD2), and Alko Alcohol (AA) and Alko Non-Alcohol (ANA). Sequence analysis was first performed to screen for the R100Q mutation in several samples. Later, a genotyping assay was conducted to assess the frequency of the R100Q mutation in larger sample sizes. The R100Q mutation was identified only in the AA/ANA population, with a significantly (P<.0001) higher frequency in the alcohol-nonpreferring ANA line. The absence of the R100Q mutation in the other rat lines that were selectively bred for alcohol consumption and alcohol preference may be due to genetic diversity among the Wistar stocks used to develop the various lines.     

The effect of antagonists selective for mu- and delta-opioid receptor subtypes on alcohol consumption in C57BL/6 mice.

Several studies have demonstrated that non-selective opioid receptor antagonists effectively reduce alcohol consumption in both animal models and at the clinical level. However, research examining the contribution of specific opioid receptor subtypes to this effect has yielded conflicting results. Some of these studies have shown that the effect is contingent upon the action of mu receptors while others have suggested that delta receptors are primarily responsible. The data reported here re-examine this question using the alcohol-preferring C57BL/6 mice. The results of this experiment demonstrate that D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP), a mu-selective antagonist, and naltrindole, a delta-selective antagonist, are equally effective at reducing alcohol consumption in a limited access model compared to a saline control group. While there was no specific comparison of the effects of these drugs on alternative appetitive behavior, neither of these drugs had effects on measured off-session food or water consumption. The results of this experiment suggest that alcohol consumption is mediated by both mu- and delta-opioid receptor subtypes.     

Associations of corticosterone and testosterone with alcohol drinking in F2 populations derived from AA and ANA rat lines

In our previous studies on alcohol-preferring AA (Alko alcohol) and nonpreferring ANA (Alko nonalcohol) rats, we have observed that the AA rats exhibit lower endogenous levels of corticosterone, higher testosterone levels, and more frequent alcohol-induced testosterone elevations when compared with ANA rats. The objective of the present study was to get more conclusive evidence for the potential role of the hypothalamus-pituitary-adrenal and hypothalamus-pituitary-gonadal axes in alcohol drinking by using the F2 experimental design. Alcohol-preferring AA and alcohol-nonpreferring ANA rat lines were crossbred to form a F1 population from which the final F2 population was derived. Male animals were challenged with a priming alcohol dose after which a 3 weeks’ voluntary alcohol drinking period took place. After a washout period of 1 week, one-half of the 40 highest and 40 lowest alcohol drinkers were challenged with a second dose of alcohol and the other half with saline. Serum testosterone and corticosterone levels were measured before and during the test. Higher endogenous testosterone levels were detected in the rats of the high alcohol consumption group compared with the low consumption group. Also supporting the original AA/ANA line differences, a trend for lower endogenous corticosterone levels were measured in the high alcohol consumption group compared with the low consumption group. The alcohol challenge test after the drinking period resulted in a higher frequency (38%) of testosterone elevations in the high drinkers compared with the low drinkers (5%). The present data confirms the validity of the positive connections between testosterone elevation and increased alcohol drinking, as well as between testosterone reduction and decreased alcohol drinking, in AA and ANA rats.     

Regional central nervous system densities of delta-opioid receptors in alcohol-preferring P, alcohol-nonpreferring NP, and unselected Wistar rats.

The densities of delta-opioid receptors in the central nervous system of alcohol-naive, adult, male, alcohol-preferring P, alcohol-nonpreferring NP, and Wistar rats were examined with the use of quantitative autoradiography. Slides with coronal 20-microm sections through the regions of interest were incubated in 5 nM [3H]-[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) to label delta(1)-opioid receptor sites. Nonspecific binding was determined in the presence of 10 microM naloxone. Significant differences between the P and the NP rat lines were found in numerous cortical regions, the basolateral amygdala, and the posterior hippocampus, with 10%-20% lower [3H]-DPDPE binding found in the P line. In most regions examined, binding levels in the Wistar rats were intermediate between those of the P and the NP rats. Significantly lower [3H]-DPDPE binding levels in the P rat may indicate fewer delta(1)-opioid receptors or decreased binding affinity. The lower binding in certain limbic regions, such as the basolateral amygdala and posterior hippocampus, as well as cortical differences in the P rat may be associated with the divergent alcohol drinking behaviors found between the P and the NP lines.     

Differences in the sleep-wake patterns of the AA and ANA rat lines developed for high and low alcohol intake

The sleep-wake patterns of the AA and ANA rat lines, developed for high and low voluntary alcohol consumption by genetic selection, were studied at the age of 10 days with a movement sensitive mattress, and at the age of four months with a monitor for anesthesia and brain activity. The amount of REM sleep was significantly higher in the ANA rats than in the AA rats both as newborns and as adults. The total sleep times, however, were the same in both rat lines. These findings suggest that the differences in alcohol drinking observed in the AA and ANA rats may be related to REM sleep, possibly due to differences in cerebral monoaminergic activity.     

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.     

Reduction of alcohol drinking and upregulation of opioid receptors by oral naltrexone in AA rats.

Rats of the high-drinking AA line were given 1 mg/kg naltrexone (NTX) or vehicle orally with a stress-free procedure just before 1 h of access to 10% ethanol daily for 8 days and again, 8 h later on the first 7 days. Forebrain homogenate binding studies using 0.03-6.00 nM [3H] naloxone were conducted from 1 to 4 days following treatment. NTX significantly suppressed alcohol intake, with the effect becoming progressively greater over days and continuing during the post-treatment period. Saturation binding studies in brain homogenate revealed that NTX had increased the B(max) for opioid receptors by 93%, 74%, 49%, and 28%, respectively, from post-treatment days 1 to 4 without altering K(d). B(max) was negatively correlated (r=-0.510, p=0.008) with alcohol intake during the preceding hour, but in control rats, it was positively correlated with changes in alcohol intake over time (r=+0.790, p=0.020). These results are consistent with the hypothesis that opioid receptors mediate reinforcement from alcohol and that NTX reduces subsequent alcohol drinking by extinction. Opioid receptor upregulation can develop simultaneously with suppression of drinking and may partially counteract the clinical benefits from NTX in the treatment of alcoholism.     

Evidence of acetaldehyde-protein adduct formation in rat brain after lifelong consumption of ethanol

Acetaldehyde, the first metabolite of ethanol, has been shown to be capable of binding covalently to liver proteins in vivo, which may be responsible for a variety of toxic effects of ethanol. Acetaldehyde-protein adducts have previously been detected in the liver of patients and experimental animals with alcoholic liver disease. Although a role for acetaldehyde as a possible mediator of ethanol-induced neurotoxicity has also been previously suggested, the formation of protein-acetaldehyde adducts in brain has not been examined. This study was designed to examine the occurrence of acetaldehyde-protein adducts in rat brain after lifelong ethanol exposure. A total of 27 male rats from the alcohol-preferring (AA) and alcohol-avoiding (ANA) lines were used. Four ANA rats and five AA rats were fed 10-12% (v/v) ethanol for 21 months. Both young (n = 10) and old (n = 8) rats receiving water were used as controls. Samples from frontal cortex, cerebellum and liver were processed for immunohistochemical detection of acetaldehyde adducts. In four (two ANA, two AA rats) of the nine ethanol-exposed rats, weak or moderate positive reactions for acetaldehyde adducts could be detected both in the frontal cortex and cerebellum, whereas no such immunostaining was found in the remaining five ethanol-treated rats or in the control rats. The positive reaction was localized to the white matter and some large neurons in layers 4 and 5 of the frontal cortex, and to the molecular layer of the cerebellum. Interestingly, the strongest positive reactions were found among the ANA rats, which are known to display high acetaldehyde levels during ethanol oxidation. We suggest that acetaldehyde may be involved in ethanol-induced neurotoxicity in vivo through formation of adducts with brain proteins and macromolecules.     

Extracellular levels of dopamine in the nucleus accumbens in AA and ANA rats after reverse microdialysis of ethanol into the nucleus accumbens or ventral tegmental area.

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.     

Alcohol preference: association with reduced striatal nicotinic receptors

This study was designed to determine whether inherent alcohol preference is associated with differential expression of central nicotinic receptors. [(3)H]Cytisine and [(125)I]alpha-bungarotoxin binding-site, ligands selective for alpha4beta2 and alpha7 nicotinic receptor subtypes, respectively, were determined in various brain regions of alcohol-preferring (P) and non-preferring (NP) rats. Only the striatum of P rats had a reduction in the number of binding sites for both ligands, compared to NP rats. The data suggest a link between striatal nicotinic receptors and alcohol preference.     

Hypothalamic monoamines and food intake in alcohol-preferring AA and alcohol-avoiding ANA rats.

The concentrations and synthesis of monoamines in various hypothalamic nuclei and the influence of monoaminergic drugs on food intake were studied in two rat lines produced by selective outbreeding for voluntary high and low alcohol drinking. The hypothalamic nuclei of the alcohol-preferring AA rats contained slightly more serotonin than those of the alcohol-avoiding ANA rats, but the accumulation of 5-hydroxytryptophan after inhibition of aromatic amino acid decarboxylase was the same in both lines. There was no significant difference in the basal concentrations of catecholamines between the lines, but the accumulation of L-DOPA was significantly greater in the ANA than the AA rats, suggesting differences in catecholamine turnover. This difference was significant in the paraventricular nucleus, which is involved in the regulation of food intake. Clonidine (an alpha 2-agonist) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, a 5-HT1A agonist) induced hyperphagia and 1-(3-trifluoromethylphenyl)piperazine (TFMPP, a 5-HT1B agonist) induced hypophagia dose-dependently in both rat lines. Clonidine tended to be more potent in the ANA than the AA rats. Food intake following a 20-h fast was significantly lower in the ANA than AA rats. These results suggest that the alcohol-avoiding ANA and alcohol-preferring AA rats have different hypothalamic monoamine mechanisms controlling food intake, which could also partially account for their differential alcohol acceptance.     

Brain regional and adrenal monoamine concentrations and behavioral responses to stress in alcohol-preferring AA and alcohol-avoiding ANA rats

The concentrations of monoamines, precursors and metabolites in various brain regions and the levels of catecholamines in the adrenal glands were determined from naive rats of the AA and ANA lines, and from ones immediately after an escapable shock test. The brain determinations were made with a new step-gradient ion-pair elution method on a reversed phase column and coulometric detection. Several significant differences were observed in the amine concentrations, largely confirming and extending the findings made before the genetic revitalization of the lines: in particular, the AAs, unlike other alcohol-preferring rodents, had higher 5-hydroxytryptamine concentrations. The AA rats tended to have smaller changes than the ANAs in brain aminergic systems and had significantly less change in adrenal epinephrine and dopamine levels after the shock test. The AAs were consistently found to be less active than ANAs in this shock test and in a warm-water swim test, but whether this was a cause or an effect of their brain and adrenal changes could not be determined. Our behavioral results might suggest a reduced reaction of the alcohol-preferring rats to aversive stimulation.     

Ethanol as a neurochemical surrogate of conventional reinforcers: The dopamine-opioid link

Various lines of evidence support the view that ethanol is a neurochemical surrogate of conventional reinforcers, such as food and sex. In fact, ethanol activates central neuronal systems that utilize dopamine, opioids, and γ-aminobutyric acid (GABA) as neurotransmitters and also are activated by conventional reinforcers. These neurotransmitter systems are likely to mediate specific aspects of ethanol's reinforcing properties. Activation of the mesolimbic dopamine and endogenous opioid systems might be the substrate of the incentive and rewarding (ergotropic) properties of ethanol (arousal, euphoria, motor stimulation) and of the process of acquiring ethanol-related secondary reinforcers (incentive learning) and ethanol self-administration habits. Stimulation of the endogenous GABAergic system might mediate the sedative and drive-reducing (trophotropic) properties of ethanol. The dopamine and opioid systems are largely interconnected. Thus, pharmacological blockade of the endogenous opioid system by mu- or delta-opioid receptor antagonists prevents ethanol's activation of the dopamine system and reduces ethanol consumption. This interaction might contribute to naltrexone's effectiveness in reducing alcohol craving in humans.     

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.