Vasopressin and ethanol preference. II. Altered preference in two strains of diabetes insipidus rats and nephrogenic diabetes insipidus mice

In the first paper of this series, the influence of a single gene (di) for vasopressin deficiency on ethanol intake in rats was demonstrated. We studied preference for concentrations of ethanol between 2.2 and 10 percent versus tap water in Brattleboro rats homozygous for diabetes insipidus (di/di), heterozygous (di/+) or normal (+/+). The di/di rats, totally lacking in vasopressin, had greatly reduced preference scores for all concentrations of ethanol. Their intake of ethanol (g/day) was higher than heterozygotes or normals, but only when 2.2 percent ethanol was offered as a choice. Treatment with vasopressin or related peptides restored ethanol drinking to normal but also corrected water balance. In the experiments reported here, Roman High Avoidance (RHA) rats of three genotypes (+/+, di/+, and di/di) were also tested for ethanol intake and preference with similar but not identical results. Thus, the effects of the di gene are independent of the genetic background on which it is placed to at least some extent. Chlorothiazide, a drug unrelated to vasopressin, also normalized ethanol drinking and corrected water balance in di/di rats. In nephrogenic diabetes insipidus mice, there was a strong negative correlation between severity of polydipsia and preference for ethanol. Thus, no paradigm tested was effective in dissociating polydipsia from reduced ethanol preference and increased ethanol intake. While these results cannot exclude a possible regulatory role for endogenous vasopressin in ethanol preference drinking, they more strongly suggest that reduced preference for ethanol and increased ethanol intake are epiphenomena secondary to a polydipsic state.     

Vasopressin and ethanol preference. I. Effects of vasopressin and the fragment DGAVP on altered ethanol preference in Brattleboro diabetes insipidus rats

Preference for concentrations of ethanol between 2.2 and 10 percent versus tap water was studied in Brattleboro rats homozygous for diabetes insipidus (di/di), heterozygous (di/+) or normal (+/+). The di/di rats, totally lacking in vasopressin, had greatly reduced preference scores for all concentrations of ethanol. Their intake of ethanol (g/day) was higher than heterozygotes or normals, but only when 2.2 percent ethanol was offered as a choice. Administration of lysine vasopressin or the vasopressin fragment des-9-Glycinamide-[Arginine8] vasopressin (DGAVP) using osmotic minipumps enhanced ethanol preference scores, reduced ethanol (g/day) intake, and restored total daily fluid intake in di/di rats. When di/di and di/+ rats were first allowed to develop stable ethanol preference before treatment with DGAVP, the peptide had no effect on preference scores. Thus, no treatment was effective in dissociating polydipsia from reduced ethanol preference and increased ethanol intake. While these results cannot exclude a possible regulatory role for endogenous vasopressin in ethanol preference drinking, they more strongly suggest that reduced preference for ethanol and increased ethanol intake are epiphenomena secondary to a polydipsic state.     

Formation of alcohol motivation under blockade of protein synthesis by cycloheximide]

The influence of protein blocker cycloheximide on ethanol intake in rats under condition of development of alcohol motivation was studied. It was found that intracerebroventricular injection of cycloheximide caused inhibition of alcohol intake in rats which had free choice between water and 20% ethanol solution. The blockade action of cycloheximide on the development of alcohol motivation was dependent on initial preference of ethanol in rats and was more strong in rats with originally low preference for ethanol.     

Suppression of voluntary ethanol consumption in rats by gamma-butyrolactone

The effect of gamma-butyrolactone (GBL) on voluntary ethanol intake was studied in a group of Wistar rats in which a stable preference had been induced by exposure to increasing ethanol concentrations. These rats drank 60% of their daily fluid intake as 15% ethanol solution, corresponding to about 6 g ethanol/kg/day. GBL, injected intraperitoneally at the dose of 200 mg/kg, twice daily for 3 consecutive days, decreased ethanol intake by about 80% on the days of treatment, but did not reduce total fluid intake. Ethanol intake remained significantly reduced up to the 5th day following cessation of GBL administration. GBL, up to a concentration of 10(-3) M, inhibited neither alcohol-dehydrogenase nor aldehyde-dehydrogenase in rat liver homogenates, nor dopamine-beta-hydroxylase in homogenates of adrenal medulla or hypothalamus of rats. It is suggested that inhibition of firing in dopaminergic neurons mediates the suppressant effect of GBL on ethanol preference.     

Effect of Early Sucrose Diet on Ethanol Preference and Behavior in Male and Female Wistar Rats

The effect of adolescent sucrose diet on ethanol preference was studied in female and male Wistar rats. Our data show less pronounced ethanol preference in females. In males, pubertal exposure to sucrose was crucial for further ethanol preference, suggesting that cross-sensitization is more inherent to males than females, for whom the increased anxiety factor proved to be more significant. Finally, it was shown that in rats of both sexes habitual alcohol intake determins ethanol preference in the two-bottle test. Overall, our study demonstrates sex differences in ethanol preference as well as its anxiolytic properties.     

Neuropeptide-Y in the paraventricular nucleus increases ethanol self-administration

The paraventricular nucleus (PVN) of the hypothalamus is known to modulate feeding, obesity, and ethanol intake. Neuropeptide-Y (NPY), which is released endogenously by neurons projecting from the arcuate nucleus to the PVN, is one of the most potent stimulants of feeding behavior known. The role of NPY in the PVN on ethanol self-administration is unknown. To address this issue, rats were trained to self-administer ethanol via a sucrose fading procedure and injector guide cannulae aimed at the PVN were surgically implanted. Microinjections of NPY and NPY antagonists in the PVN were conducted prior to ethanol self-administration sessions. All doses of NPY significantly increased ethanol self-administration and preference, and decreased water intake. The NPY antagonist D-NPY partially reduced ethanol self-administration and completely blocked the effects of an intermediate dose of NPY (10 fmol) on ethanol intake, preference, and water intake. The competitive non-peptide Y1 receptor antagonist BIBP 3226 did not significantly alter ethanol self-administration or water intake when administered alone in the PVN but it completely blocked the effect of NPY (10 fmol) on ethanol intake. NPY infused in the PVN had no effect on ethanol self-administration when tested in rats that did not have a long history of ethanol self-administration. The doses of NPY tested produced no effect on food intake or body weight measured during the 24-h period after infusion in either ethanol-experienced or ethanol-inexperienced rats. These results indicate that elevation of NPY levels in the PVN potently increases ethanol self-administration and that this effect is mediated through NPY Y1 receptors.     

Unpredictable cold-immobilization stress effects on voluntary ethanol consumption in rats

The effects of exposure to a schedule of unpredictable cold-immobilization stress on voluntary ethanol consumption were examined. Following testing for ethanol preference, rats were divided into high, medium and low ethanol consuming groups on the basis of daily ethanol intake (g/kg/day) and exposed to immobilization stress over an 18 day period. Voluntary ethanol consumption was monitored during the stress period and for an additional 36 days post-stress. Results indicated a differential effect of stress on ethanol intake in that low ethanol consuming rats increased their ethanol intake during the stress period and maintained this increase throughout the entire post-stress period as compared to non-stressed controls. High ethanol consuming groups demonstrated a small (marginally significant) decrease in ethanol intake during the stress period as compared to baseline levels. No change in ethanol intake was observed for the medium ethanol consuming groups. The results suggest that unpredictable immobilization stress has a differential effect on ethanol intake depending upon pre-stress levels of ethanol consumption.     

Prenatal alcohol exposure alters enkephalin levels, without affecting ethanol preference

Pregnant dams were pair-fed a liquid diet containing 35% ethanol derived calories or isocaloric sucrose during the last two trimesters of pregnancy. No differences were observed in adult ethanol preference between fetal alcohol exposed (FAE) animals and pair-fed controls. However, Met- and Leu-enkephalin levels were significantly elevated in globus pallidus of adult FAE animals. Pituitary levels were unaffected.     

Effects of ethanol on locomotor and anxiety-like behaviors and the acquisition of ethanol intake in Lewis and spontaneously hypertensive rats

The purpose of the present study was to investigate whether Lewis (LEW) and spontaneously hypertensive rats (SHR), characterized in numerous behavioral tests as strains with high-anxiety and low-anxiety, respectively, could differ in their sensitivity to the effects of ethanol in the elevated plus maze (EPM) and the open field (OF), two classical models of anxiety/emotionality, as well as in the acquisition of ethanol drinking behavior. It was also of interest to examine the relationship between sweet and bitter fluids preference and ethanol intake. SHR and LEW rats were given saline or ethanol injections (0.6 or 1.2 g/kg, ip.) and tested in the EPM and OF. Subsequently the same animals were given continuous free choice between water and ethanol solution (2-8%). Additional groups of animals were exposed to a free-choice regimen between saccharin (0.002-0.09%) or quinine (0.0001-0.0015%) and water. The low dose of ethanol (0.6 g/kg) induced anxiolytic-like effects and intensive locomotor activation mainly in SHR rats tested in the OF arena. Overall, LEW counterparts were unaffected in OF test. In oral self-administration paradigm, SHR rats consumed significantly more ethanol than LEW rats. Concerning other solutions, SHR rats consumed large amounts of saccharin compared with LEW rats. These data indicate that the SHR preference for ethanol intake may be positively related to their differential sensitivity to the anxiolytic/stimulant effects of ethanol and to the sensitivity of this strain for saccharin reinforcement. In addition, these findings provide evidence that the SHR strain may represent a useful genetic and pharmacological tool to investigate ethanol drinking traits.     

Overexpression of dopamine D2 receptors reduces alcohol self-administration

The mechanism(s) underlying predisposition to alcohol abuse are poorly understood but may involve brain dopamine system(s). Here we used an adenoviral vector to deliver the dopamine D2 receptor (DRD2) gene into the nucleus accumbens of rats, previously trained to self-administer alcohol, and to assess if DRD2 levels regulated alcohol preference and intake. We show that increases in DRD2 (52%) were associated with marked reductions in alcohol preference (43%), and alcohol intake (64%) of ethanol preferring rats, which recovered as the DRD2, returned to baseline levels. In addition, this DRD2 overexpression similarly produced significant reductions in ethanol non-preferring rats, in both alcohol preference (16%) and alcohol intake (75%). This is the first evidence that overexpression of DRD2 reduces alcohol intake and suggests that high levels of DRD2 may be protective against alcohol abuse.     

Neuropeptide Y modulation of ethanol intake: effects of ethanol drinking history and genetic background

Intracerebroventricular administration of NPY suppresses ethanol intake in selectively bred alcohol-preferring rat lines, but not in rats selectively bred for low ethanol drinking or in unselected Wistar rats, when access to ethanol is limited to 2h/day. However, when rats undergo chronic (24h/day) ethanol drinking (or exposure to ethanol by vapor inhalation) and have periods of imposed ethanol abstinence, the reductions in ethanol drinking following NPY administration are enhanced in alcohol-preferring rats and are also observed in unselected Wistar rats. Thus, sensitivity to the effects of NPY on ethanol drinking appears to be altered by selective breeding for ethanol preference and by a prior history of chronic but intermittent exposure to ethanol.     

Evaluation of accelerated development of stable alcoholic motivation in rats for studying potential alcohol deterrents

The express technique reflecting an acquisition of a clear alcohol addiction during short-term voluntary alcoholization for further antialcoholic drugs testing was performed in male albino rats. By VARIMAX factor analysis of indexes related with preference of alcohol solutions with different tastes the conditions of short-term (2 months) voluntary alcoholization leading to persistent ethanol intake were studied. Isolation stress inducing a specific alcohol drive was excluded from rearing conditions. 0.1% saccharin solution in 15% ethanol was used for alcoholization. Statistical analysis revealed factor of "developed alcohol abuse" which may be detected in conditions of one-trail sweet ethanol intake after 3 days alcohol deprivation (similar to heavy drinking syndrome in humans). Using pharmacological drugs (pyrazidol, piracetam) validity of the method for specific drug design was confirmed.     

The effect of long-term, voluntary ethanol consumption on hepatic regeneration in rats

Previous studies have reported conflicting results regarding the effect of ethanol on hepatic regeneration. The purpose of the present study was to determine whether long-term, voluntary consumption of ethanol, within the range reported in humans, has an effect on hepatic regenerative activity in rats following partial hepatectomy. Ninety-four adult male Sprague-Dawley rats (n = 3-9/group) were studied. Based on the amount of 9% ethanol consumed over a 50-day period, low ethanol intake (0.1-1.9 g.kg-1.d-1) and high ethanol intake (2.0-4.0 g.kg-1.d-1) groups were identified. Control groups consisted of rats provided with propylene glycol in equivalent caloric amounts to the ethanol consumed by high ethanol intake rats (isocaloric group) and rats served water only (ad libitum group). An additional two groups from which ethanol was removed 5 days prior to surgery were also studied (low ethanol grace and high ethanol grace). Hepatic regeneration was determined by restitution of liver weight, [3H]thymidine incorporation into DNA, and [14C]leucine incorporation into protein 24, 48, and 72 h following partial (70%) hepatectomy. The results of the study revealed no significant differences in the rate of hepatic regeneration between low and high ethanol consuming rats or between either of these groups and isocaloric or ad-libitum fed control groups. Regeneration in low ethanol grace and high ethanol grace groups were also similar to each other and controls. Moreover, there was no correlation between mean ethanol consumption per rat and restitution of liver weight, [3H]thymidine incorporation into DNA, or [14C]leucine incorporation into protein by the regenerating liver (r = 0.0716, -0.1637, and 0.1395, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of kappa type opioids on ethanol drinking

The effects of the administration of the kappa agonist dynorphin1-17 and/or the kappa antagonist MR-2266-BS on ethanol preference was investigated using a paradigm by which rats develop alcohol preference. Administration of dynorphin shortly before or after the conditioning session (forced ethanol exposure) failed to affect later ethanol preference. However, dynorphin treatment prior to the first choice session reduced ethanol preference during the three consecutive testing days. This effect was reversed by the simultaneous administration of the kappa antagonist MR-2266-BS. The results of the present study provide further support for evidence of the involvement of dynorphinergic systems on drinking behavior and suggest that kappa-type opioid mechanisms may be involved in the consumption and development of preference to ethanol in rats.     

Leptin fails to reduce ethanol intake in Marchigian Sardinian alcohol-preferring rats

Leptin, a hormone secreted by the adipocytes and involved in feeding and energy balance control, has been proposed to modulate alcohol craving in mice and humans. This study evaluated whether leptin modulates alcohol intake in Marchigian Sardinian alcohol-preferring (msP) rats. Rats were offered 10% ethanol either 2h per day at the beginning of dark period of the 12:12h light/dark cycle, or 24h per day. Leptin was injected into the lateral ventricle (LV), the third ventricle (3V), or intraperitoneally (IP) once a day, 1h before the onset of the dark period. Neither acute nor chronic (9 days) leptin injections (1 or 8microg per rat) into the LV or 3V modified ethanol intake in male msP rats, offered ethanol 2h per day. Chronic LV injection of leptin (8 or 32 microg per rat in male rats and 8 or 16 microg per rat in female rats for 7 days), or chronic IP injections of leptin (1mg/kg in male rats for 5 days) failed to modify the intake of ethanol, offered 24h per day. Finally, chronic LV leptin injections (8 or 32 microg per rat for 12 days) did not modify ethanol intake in male msP rats, adapted to ad libitum access to ethanol and then tested after a 6-day period of ethanol deprivation. In contrast, in most of these conditions leptin significantly reduced food intake. These data do not support a role for leptin in alcohol intake, preference, or craving in msP rats.     

Alcohol motivation in rats under blockade of protein synthesis by cycloheximide

The influence of protein blocker cycloheximide on the ethanol intake in alcohol-dependent rats was studied. It was found that intracerebroventricular as well as perifornical area hypothalamus injection of cycloheximide caused inhibition of ethanol consumption in alcohol-dependent rats, persisting for 5-9 weeks. The blockade action of cycloheximide was more strong after its injection in phase of decreased ethanol intake. At the same time it was shown an increase in water and food intake. The blockade action of cycloheximide on ethanol intake was not found in alcohol-dependent rats. These data indicate that some protein substances take part in mechanisms of organization of alcohol motivation and dependence.     

Comparison of the anticonvulsant effects of opioid peptides and etorphine in rats after icv administration

The effect of acute icv administration of β-endorphin (5–160 μg), D-ala²-D-leu⁵-enkephalin (DADL; 5–160 μg), D-ala²-met-enkephalinamide (DAME; 10–160 μg), and etorphine (0.05–1.6 μg) on brain excitability was studied by measuring flurothyl seizure thresholds in rats. Each test compound produced a behavioral stupor characterized by muscle rigidity, exophthalmos, and the absence of spontaneous movement. Wet-dog shakes occured only after injection of the opioid peptides. All four compounds produced a dose-related increase in seizure threshold. Naloxone antagonized the behavioral and anticonvulsant effects; the increase in seizure threshold induced by β-endorphin was the most resistant to naloxone. These results indicate that the opioid peptides, in addition to their known EEG epileptogenic potential, are also anticonvulsant in the rat, thus raising the possibility of a dual action for the opioid peptides on central nervous system excitability.     

Effects of chronic administration of either ethanol or pentanol on rat duodenum morphology

The morphology of the rat duodenum after chronic treatment with 15% (v/v) ethanol and 4% (v/v) pentanol was studied. Male Wistar rats of experimental groups were given ethanol and pentanol for 15 weeks with food and fluid freely available. Ethanol-15% and 4% pentanol-fed rats showed a significantly reduced fluid and food intake as compared with control rats. The study of the mucosa indicated that the number of chronic inflammatory infiltrating (mononuclear cells) and goblet cells was higher in the groups of the ethanol- and pentanol-fed rats than in the control group. There was an increase in the thickness of the brush border in pentanol-fed rats. Intervillus adhesion was concurrently observed in the pentanol-fed rats but not in the control or ethanol-fed rats. After ethanol feeding many of the villi developed blebs at the apex of the villus or laterally on its upper half. These blebs generally remained intact. In contrast, after pentanol feeding no bleb formation was appreciated. The intake of ethanol and other short chain alcohols present in alcoholic beverages leads to mainfold disturbances on the rat duodenum. These findings suggest that the chronic ingestion of pentanol seems to promote cellular changes but less important than those observed after chronic ethanol ingestion.     

Effects of delta sleep-inducing peptide on electrophysiological parameters of sleep during alcohol withdrawal in rats

In rats with the persistent alcohol motivation the electrophysiological sleep pattern was studied during ethanol intake, after 24 and 48 hours of alcohol withdrawal. It was established that during the voluntary ethanol intake rats may be divided into two groups: with comparative deficit (1st group) and comparative abundance (2nd group) of REM sleep. Alcohol withdrawal caused differential alterations of sleep-wakefulness cycle: in the 1st group of rats REM sleep was more suppressed while in the 2nd group--more increased in comparison to those during ethanol intake. In all animals the SWS depression, increase of awakenings, the aggravation of falling asleep and decrease of sleep depth were observed. DSIP (0.1 mg/kg, i.p. 1 hour before sleep recording) was found to regulate sleep disorders caused by ethanol withdrawal. It makes the neuropeptide possible to be recommended for ethanol withdrawal syndrome treatment in clinical practice.