Temporal relationship of the induction of tolerance and physical dependence after continuous intoxication with maximum tolerable doses of ethanol in rats

Rats were treated by intragastric intubation of a 20% ethanol solution in doses of 9–15 g/kg in 3–5 fractions for 1–7 days. Both tolerance and physical dependence were demonstrated after this treatment with the maximum tolerable doses to only a few days. Tolerance was assessed by signs of severity of intoxication: coma, loss of righting reflex, ataxia-3, ataxia-2, ataxia-1, sedation, and neutrality. During withdrawal, as blood ethanol concentrations approached 100 mg/dl the ethanol dependence phase was characterized by the onset of signs and responses of progressive severity: hyperactivity, tremors, spastic rigidity, and spontaneous convulsive seizures. A significant degree of tolerance was demonstrated for all signs of intoxication after 4 days of treatment, but did not reach maximum level even after 7 days. The severity of the withdrawal reactions intensified progressively to a maximum intensity after 4 days of treatment when as many as 72% of animals exhibited severe withdrawal signs and reactions including convulsive seizures. These different time courses suggest that tolerance and physical dependence are mediated through different mechanisms.     

Effect of two iron-chelators,desferrioxamine and diethylenetriaminepentaacetic acid,on the development of tolerance to the physical dependence on ethanol in mice

Physical dependence on ethanol was induced in mice by exposing the animals to increasing concentrations of ethanol vapor for a period of 9 days. The degree of dependence was assessed by subjective scoring of a characteristic behavioral withdrawal syndrome upon cessation of ethanol administration. The daily administration of desferrioxamine (350 mg/kg, i.p.) or of diethylenetriaminepentaacetic acid (200 mg/kg, i.p.) during the ethanol treatment period reduced by 50% the behavioral withdrawal score. On the other hand, an iron-load of 250 mg/kg administered once prior to ethanol treatment and once during ethanol exposure caused a significant increase in the withdrawal score. While the degree of dependence was significantly lowered by the iron chelators, the state of tolerance after chronic ethanol treatment was not altered by these agents. However, the capacity of these ethanol-treated animals to develop rapid functional tolerance at termination of the chronic ethanol exposure was significantly enhanced by desferrioxamine and diethylenetriaminepentaacetic acid. The results are discussed in view of possible role of iron-induced peroxidation of neuronal membrane lipids in the development of tolerance to and dependence on, ethanol.     

Cyamemazine decreases ethanol intake in rats and convulsions during ethanol withdrawal syndrome in mice

The effect of cyamemazine a dopamine D₂ receptor antagonist on voluntary ethanol consumption in rats and on ethanol withdrawal in mice was examined. Male Sprague-Dawley rats were tested in a free choice (water and 10% ethanol) experiment and consumed 5 g/kg ethanol daily. Rats were treated daily IP with cyamemazine ( 0.5, 1, or 2 mg/kg) or acamprosate (100 mg/kg) during 2 weeks. Both acamprosate and 1 mg/kg cyamemazine significantly decreased ethanol intake by 45% without affecting either fluid or food intake. The lowest dose of cyamemazine had no effect on alcohol intake but increased food intake. The highest dose had no effect on any variables. During the post-treatment period, only 1 mg/kg cyamemazine decreased both ethanol and fluid intakes. Mice were made dependent on alcohol using a chocolate fluid diet containing increasing concentrations of alcohol and withdrawn after 9 days. Mice were treated with cyamemazine (1 or 0.5 mg/kg, respectively) or with the same doses of lorazepam acutely on the day of withdrawal or chronically (during alcohol treatment). Both chronic and acute cyamemazine and lorazepam treatments decreased convulsions during ethanol withdrawal. Both acute treatments decreased locomotor activity in control and alcohol dependent mice. Chronic treatment had no effect on locomotor activity. We suggest that cyamemazine could reduce alcohol consumption by antagonizing the activation of the dopaminergic pathways during the induction of alcohol dependence. The action of cyamemazine on 5-HT₃ receptors could also explain its effect on alcohol convulsions during withdrawal convulsions. Received: 19 October 1997/Final version: 6 April 1998     

Suppression by progabide of ethanol withdrawal syndrome in rats

Progabide, a specific and clinically used GABA receptor agonist, was tested for its ability to suppress ethanol withdrawal syndrome. Male rats were rendered physically dependent on ethanol by feeding for 12 days on a liquid diet in which ethanol isocalorically replaced dextrose. Progabide (100-400 mg/kg i.p.), administered 8 h after ethanol was withdrawn, produced a dose-related inhibition of both tremors and audiogenically induced seizures. A single dose of 400 mg/kg of progabide completely suppressed all ethanol withdrawal reactions. Seizures were more sensitive to the drug than tremors. The results support the view that a decrease in GABA transmission plays a role in ethanol withdrawal symptoms and suggest that progabide may be tested as a possible treatment of ethanol withdrawal syndrome in man.     

Gamma-Vinyl GABA Decreases Voluntary Alcohol Consumption in Alcohol-Preferring AA Rats

The effect of a GABA transaminase inhibitor, gamma-vinyl GABA, on the voluntary alcohol consumption of alcohol-preferring AA rats produced by selective breeding for high alcohol preference, was studied. The rats were first trained to voluntarily drink 10% (v/v) ethanol solution until their ethanol consumption stabilized. Gamma-vinyl GABA (100, 200 or 500 mg/kg) was then injected intraperitoneally in three groups of rats, with saline-injected animals serving as a control group. The rats continued to have a free choice between 10% ethanol and plain tap water for five days after the injection, and their ethanol, water and food consumptions were measured daily. Gamma-vinyl GABA decreased ethanol consumption by the rats in a dose-dependent way. The consumption remained significantly decreased for three days in the two groups receiving the highest doses, with only a small concomitant tendency to decreased food intake. The results suggest that an increase in brain GABA concentration decreases alcohol drinking, possibly through potentiation of the pharmacological action of ethanol.     

Effects of alcohol dependence on shock-induced fighting: action of muscimol and homotaurine.

We have applied the electroshock-induced fighting behavior to the study of experimental alcohol dependence. Adult Wistar rats were intoxicated chronically with ethanol (10 g/kg/24 h) for 13 days. Electroshock-induced fighting behavior was studied during chronic intoxication and withdrawal in comparison with normal rats receiving a water-carbohydrate solution isocaloric to ethanol. Rats were divided into groups receiving respectively muscimol (0.25 mg/kg), a GABAA agonist; homotaurine (140 mg/kg) a GABA mimetic; and physiological saline (10 ml/kg), intraperitoneally. During chronic intoxication, rats showed an increase in defensive-fighting behavior. Withdrawal accentuated the aggressive behavior and muscimol and homotaurine inhibited it. These results confirm the relevance of the electroshock-induced defensive fighting behavior test in chronic intoxication with alcohol, but to show the involvement of GABAergic transmission in the behavioral effects of alcohol withdrawal, additional experiments with other GABA mimetics and with GABA antagonists should be considered.     

Clonidine attenuates naloxone-induced opioid-withdrawal syndrome in cholestatic mice

Cholestasis is associated with elevated plasma level of endogenous opioid peptides. Naloxone-precipitated withdrawal syndrome has been described in a mouse model of acute cholestasis. Thus we aimed at determining whether central noradrenergic hyperactivity is involved in manifestation of naloxone-precipitated withdrawal syndrome in mice with obstructive cholestasis. Acute cholestasis was induced by bile duct resection in mice and physical dependence was observed by precipitating a withdrawal syndrome with naloxone (2 mg/kg, intraperitoneally) 5 days after induction of cholestasis. Administration of clonidine (0.1 mg/kg, intraperitoneally), an alpha2-adrenoceptor agonist, 15 min. before naloxone injection significantly alleviates withdrawal severity in cholestatic mice. However, pretreatment of animals with yohimbine (3 mg/kg, intraperitoneally), an alpha2-adrenoceptor antagonist, 15 min. before clonidine blocked this ameliorative effect of clonidine. The results of this study support the evidence for involvement of the alpha2-adrenoceptors in the withdrawal syndrome of cholestasis in a mouse model.     

Disposition of ethanol and its effect on rectal temperature in morphine-dependent, morphine-abstinent and morphine-naive rats

Rats were treated chronically with twice daily injections of morphine hydrochloride in gradually increasing doses from 20 to 200 mg/kg for 22 consecutive days. Rats in a control group were injected with 0.9% NaCl. At 1 hour after the last injection of morphine (dependent state) and at 3 and 16 days after abrupt withdrawal (abstinent state) the animals were injected intraperitoneally with 2.0 g/kg ethanol. Blood ethanol concentrations (tail blood) and rectal temperatures were determined at 30-60 min. intervals for up to 7 hours. The absorption of ethanol was slower in rats treated with morphine and the time taken to reach the end of the blood concentration curve was increased. This implies a slower turnover of ethanol in morphine-dependent and abstinent rats. At 16 days after withdrawal, the blood ethanol profiles were the same as in control rats not exposed to morphine. Injection of morphine (200 mg/kg) intolerant animals caused a pronounced hyperthermia which lasted for about 4 hours. Ethanol treatment rapidly counteracted the rise in body temperature. Morphine abstinent rats showed a hypothermic response to ethanol. The altered disposition of ethanol in acute withdrawal may result from physiological disturbances such as impaired fluid balance, dehydration, altered peripheral blood flow and poor nutritional status. There was no evidence for a faster rate of ethanol metabolism in the hypermetabolic state associated with morphine tolerance and dependence.     

Induction of physical dependence on and tolerance to ethanol in rats fed a new nutritionally complete and balanced liquid diet

Rats offered a nutritionally balanced and complete liquid diet containing 35% of energy as ethanol, 12% as fat, 21% as protein, and the balance as carbohydrate consumed greater than 9 g/kg ethanol after 10 days. Rats displayed signs of physical dependence and tolerance while showing a net gain in weight. Physical dependence was indicated by severe intensity of the following signs during withdrawal from ethanol: Muscle rigidity; tail tremors; caudal tremors; and general tremors. Severity of these signs reached a maximum intensity by 19 h after withdrawal of ethanol. Tolerance was assessed by performance on a moving belt after injection of an IP challenge dose of ethanol. Tolerance was exhibited by chronically treated rats as measured by significantly reduced time off the belt after 7 days. Concentrations of ethanol in blood were documented on selected mornings and were observed to increase. These data suggest that physical dependence and tolerance can be induced through voluntary consumption of ethanol by rats and without nutritional compromises or weight loss.     

Nicotine in alcohol deprivation increases alcohol operant self-administration during reinstatement

Tobacco and alcohol are highly co-abused by humans. Most experimental studies have evaluated ethanol consumption in animals exposed concomitantly to nicotine. However, little is known regarding the effects of nicotine administered during periods of alcohol deprivation. In the present study, adult male Wistar rats with an extended background of operant self-administration of ethanol were alcohol-deprived and treated with nicotine (0.1, 0.2, 0.4 and 0.8 mg/kg) or saline during five consecutive days in one chamber of a place conditioning apparatus. Nicotine-induced changes in locomotion were monitored daily, whereas the expression of place conditioning was studied the day after the last nicotine injection. Forty-eight hours after testing for conditioning, the animals resumed operant self-administration of ethanol and their alcohol intake was evaluated during the next 14 days. We observed that alcohol consumption was increased in animals treated with nicotine at doses of 0.2, 0.4 and 0.8 mg/kg but not in animals treated with the dose of 0.1 mg/kg or saline. Additionally, the dose of 0.8 mg/kg of nicotine not only induced persistent changes in alcohol self-administration but also produced conditioned place aversion and depressed locomotor activity. These results indicate that nicotine administration during the ethanol deprivation period can exacerbate the maintenance of alcohol consumption.     

Clonidine Attenuates Naloxone‐Induced Opioid‐Withdrawal Syndrome in Cholestatic Mice*

Abstract: Cholestasis is associated with elevated plasma level of endogenous opioid peptides. Naloxone‐precipitated withdrawal syndrome has been described in a mouse model of acute cholestasis. Thus we aimed at determining whether central noradrenergic hyperactivity is involved in manifestation of naloxone‐precipitated withdrawal syndrome in mice with obstructive cholestasis. Acute cholestasis was induced by bile duct resection in mice and physical dependence was observed by precipitating a withdrawal syndrome with naloxone (2 mg/kg, intraperitoneally) 5 days after induction of cholestasis. Administration of clonidine (0.1 mg/kg, intraperitoneally), an α₂‐adrenoceptor agonist, 15 min. before naloxone injection significantly alleviates withdrawal severity in cholestatic mice. However, pretreatment of animals with yohimbine (3 mg/kg, intraperitoneally), an α₂‐adrenoceptor antagonist, 15 min. before clonidine blocked this ameliorative effect of clonidine. The results of this study support the evidence for involvement of the α₂‐adrenoceptors in the withdrawal syndrome of cholestasis in a mouse model.     

Lithium inhibits the development of physical dependence to clonidine in mice

Based on our previous finding that chronic lithium treatment reduced naloxone-precipitated withdrawal syndrome in morphine-treated mice, the effect of chronic lithium treatment was evaluated on the development of dependence to clonidine. Dependence was induced by injection of either morphine (50, 50 and 75 mg/kg, intraperitoneally with 3 hr interval for 3 consecutive days), or clonidine (2 mg/kg/day, intraperitoneally for 10 days). Naloxone (4 mg/kg, intraperitoneally) precipitated withdrawal signs in both morphine- and clonidine-treated mice. Yohimbine (5 mg/kg, intraperitoneally) precipitated withdrawal signs in the clonidine-treated mice, similar to morphine withdrawal signs; but failed to precipitate any significant sign in the morphine-treated mice. Coadministration of lithium was carried out by adding lithium chloride to drinking water (600 mg/l for 20 days; 10 days before the beginning of clonidine administration and 17 days before the administration of morphine to allow the lithium concentration to reach steady-state). The results indicated that chronic lithium administration significantly attenuated the withdrawal signs, precipitated either by yohimbine or naloxone, in clonidine-treated mice. As a conclusion, clonidine withdrawal signs are very similar to opioid withdrawal signs, and lithium is able to prevent the development of physical dependence to clonidine.     

Nuclear factor-kappa-B inhibitor modulates the development of opioid dependence in a mouse model of naloxone-induced opioid withdrawal syndrome

The present study was designed to investigate the effect of diethyl dithiocarbamic acid sodium salt trihydrate (DDA), a selective inhibitor of nuclear factor-kappa-B, on the development of morphine dependence in a mouse model of naloxone-induced opioid withdrawal syndrome. Morphine (5 mg/kg, intraperitoneally) was administered twice daily for a period of 5 days, after which a single injection of naloxone (8 mg/kg, intraperitoneally) precipitated an opioid withdrawal syndrome in mice. Behavioral observations were made for a period of 30 min immediately after naloxone treatment. Withdrawal syndrome was quantitatively assessed in terms of withdrawal severity score and the frequency of jumping, rearing, forepaw licking, and circling. DDA markedly and dose-dependently (P<0.01) attenuated the morphine-naloxone-induced experimental opioid withdrawal syndrome. However, DDA administration did not alter locomotor activity, thus ruling out any sedative action of DDA per se. Further, DDA pretreatment did not alter the acute analgesic effect of morphine. The results suggest that nuclear factor-kappa-B is involved in the development of opioid dependence and the precipitation of its withdrawal syndrome, and thus, may serve as a viable pharmacological target to tackle the problem of opioid addiction.     

Potential involvement of tyrosine phosphatase and calpain-related pathways in opioid withdrawal syndrome in mice

The present study was designed to determine the effect of N'-[6,7-dichloro-4-(4-methoxy-phenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl] hydrazide (SJA 7019), a selective nonpeptide inhibitor of calpain, and sodium orthovanadate, a selective inhibitor of tyrosine phosphatase, on the development of physiological dependence, as assessed by precipitated morphine withdrawal behavior in mice. Subchronic morphine administration (5 mg/kg, intraperitoneally, twice daily for 5 days), followed by a single injection of naloxone was used to precipitate the opioid withdrawal syndrome in mice. Behavioral observations were made immediately after naloxone treatment. Withdrawal syndrome was assessed quantitatively in terms of the withdrawal severity score and the frequency of jumping, rearing, forepaw licking, and circling. Daily single administration of SJA 7019 (1.5, 3, and 6 mg/kg, intraperitoneally) or sodium orthovanadate (5, 10, and 20 mg/kg, intraperitoneally) was continued during the morphine treatment procedure. Administration of SJA 7019 as well as the sodium orthovanadate dose-dependently attenuated the naloxone-induced morphine withdrawal syndrome. Neither SJA 7019 nor sodium orthovanadate significantly affected locomotor activity or morphine-induced antinociception. Therefore, it may be concluded that treatment with SJA 7019 or sodium orthovanadate during the morphine exposure period attenuated the development of physiological dependence on morphine, possibly through mechanisms linked to activation of tyrosine phosphatase and calpain.     

Pineal gland and melatonin influence on chronic alcohol consumption by hamsters

Male golden hamsters preferentially consume alcohol solution when given a free-choice between water and the alcohol solution. The pineal gland has been implicated as influencing the predilection for the ethanol solution. Melatonin, a pineal hormone, was administered either daily for 11 weeks as a subcutaneous injection (25 μg/animal) or weekly as a subcutaneous beeswax implant (1 mg melatonin/24 mg beeswax) for 5 weeks to hamsters allowed a free-choice between water or a 10% ethanol solution. Food, water and alcohol consumptions were measured on a daily basis. Animals treated by daily injection with melatonin consumed slightly less ethanol than animals not given melatonin. In light-deprived animals given chronic implants of melatonin, alcohol consumption was reduced when compared to alcohol consumption by light-deprived hamsters not receiving melatonin. Melatonin treatment also resulted in reducing daily total fluid intake as well as ethanol consumption in light-deprived hamsters. The results indicate that the pineal gland may influence fluid consumption in the hamster, and indirectly alters the propensity of the hamster to consume alcohol.     

The new antiepileptic drug levetiracetam normalises chlordiazepoxide withdrawal-induced anxiety in mice

Some antiepileptic drugs have been used with success to counteract withdrawal symptoms following chronic use of sedatives, hypnotics or alcohol. We evaluated the potential of levetiracetam (Keppra), a new antiepileptic drug, to prevent benzodiazepine withdrawal in an animal model sensitive to the anxiogenic effect resulting from drug cessation. The effects of levetiracetam (17 and 54 mg/kg) given intraperitoneally (i.p.) were determined on anxiety induced in female NMRI mice by withdrawal from 21 days of chronic administration of chlordiazepoxide. Administration of chlordiazepoxide was i.p. twice daily, in increments of 2 mg/kg, from 10 up to 40 mg/kg. Anxiety was evaluated using an elevated plus-maze test 24-h after chlordiazepoxide withdrawal. Discontinuation of chronic chlordiazepoxide induced a significant anxiogenic profile in the plus-maze test mainly characterised by a decrease in open arm exploration. This effect was dose-dependently prevented by administration of levetiracetam during the withdrawal period. The highest dose tested (54 mg/kg) induced statistically significant effects on all variables recorded but had no effect upon plus-maze exploration in normal mice. This suggests that the observed effects are dependent upon the level of stress or anxiety of the animals. These results support potential efficacy of levetiracetam in the benzodiazepine withdrawal syndrome.     

Induction of physical dependence upon ethanol in rats using intravenous infusion

Intravenous infusions were used to produce physical dependence upon ethanol in rats. The procedure proved to be safe, rapid, and reliable. Ethanol (30% v/v) was administered over a 7-day period. The mean daily dose ranged from 10–14 g/kg/day. Control rats were exposed to a comparable procedure except that saline, rather than ethanol, was infused. All ethanol treated rats that survived the intoxication period (n=11) showed signs of physical dependence (moderate to severe3 n=8; mild, n=3) following ethanol withdrawal. Saline treated rats (n=8) did not show any of these symptoms. The most reliable ethanol withdrawal signs observed were: spontaneous seizure (n=7), audiogenic seizure (n=7), tremors (n=6), tail stiffening (n=10) and body rigidity (n=9). These symptoms were analyzed in terms of their hour of onset and hour of maximum intensity following ethanol withdrawal. Application of the intravenous method for the study of ethanol self-administration is discussed.     

Influence of clonazepam and carbamazepine on alcohol withdrawal syndrome, preference and development of tolerance to ethanol in rats.

The effects of clonazepam (0.3 and 1.0 mg/kg or 0.1 mg/kg, b.i.d., 5 days) and carbamazepine (50 and 100 mg/kg or 12.5 and 50 mg/kg b.i.d., 5 days) on alcohol withdrawal syndrome in rats were investigated. Moreover, the influence of clonazepam (0.3 mg/kg, single dose, or repeated doses for 8 days) and carbamazepine (50 mg/kg, single dose, or repeated doses for 8 days) on the development of tolerance to ethanol was also examined. To study the influence of clonazepam and carbamazepine on preference to ethanol, both drugs were administered for 5 days during the last week of the experiment, (clonazepam at 0.1 mg/kg, b.i.d., i.p. and carbamazepine at 12.5 mg/kg, b.i.d, i.p.). Clonazepam and carbamazepine administered at single doses as well as multiple doses diminished the symptoms of withdrawal syndrome. Clonazepam did not prevent the development of tolerance to sleep-inducing and hypothermal action of ethanol, while carbamazepine prevented the development of tolerance to hypnotic effect of ethanol. Carbamazepine clearly reduced preference to ethanol (significantly vs. the control group and vs. the baseline values). Clonazepam also diminished preference to alcohol, but only in comparison with baseline values.     

Inhibition, activation, destruction, and induction of drug-metabolizing enzymes by trichloroethylene.

A 10-day ethanol inhalation procedure without the use of pyrazole was used to produce physical dependence on ethanol in Sprague-Dawley rats. During this 10-day period, weight changes in the control animals reflected the weight changes in the ethanoltreated animals. At the end of the inhalation procedure the rate of ethanol clearance from the blood was 0.44 ± 0.2 mg/ml/hr in the dependent animals; also, signs of withdrawal were manifested in all ethanol vapor-treated animals, but not in control animals. Twenty-four hours after removal from the chamber, a 5-day drinking study was started. Control animals and physically dependent animals were each divided into two groups, one for diazepam (5 mg/ kg, orally) administration and one for vehicle administration. The results from the free-choice drinking study (water and ethanol solution, 7%) indicated that diazepam did not enhance ethanol consumption in animals which were physically dependent on ethanol but significantly suppressed ethanol drinking in nondependent animals. Also when examining the effect of physical dependence alone on postwithdrawal drinking, it appeared that no increase in ethanol preference was observed with animals under our experimental design.     

Effect of Ethanol Dependence and Withdrawal on the Catecholamines in Rat Brain and Heart

Abstract: To study whether ethanol dependence is accompanied by changes in the brain monoamines, male Sprague-Dawley rats were intubated intragastrically with 10% (w/v) ethanol solution 3 times daily (daily dose 8–11 g/kg) for 7–10 days. Control rats were intubated with similar volumes of tap water. The rats were killed 4–6 hrs (intoxicated group) or 16–18 hrs (withdrawal group) after the last administration of ethanol. At this time the withdrawn rats showed a stiff tail, spastic rigidity, piloerection, tremor, hyperexcitability, stereotyped head movements as well as convulsions. The dopamine and homovanillic acid concentrations as well as the disappearance rate of dopamine after synthesis blockade (α-methyl-p-tyrosine 250 mg/kg intraperitoneally 3 hrs) were similar in the brain of control, ethanol intoxicated and withdrawn rats. Chronic ethanol administration did not alter the concentration of noradrenaline in the brain or the heart of the rats. However, the disappearance rate of noradrenaline after synthesis blockade was accelerated by about 30–50% in the brain and heart of rats withdrawn from ethanol. Moreover, the concentration of noradrenaline was decreased by 40–60% in the brain and heart of rats which died in withdrawal convulsions. The experiments suggest that the noradrenergic mechanisms but not the dopaminergic mechanisms may be associated with the occurrence of withdrawal symptoms.