Fetal alcohol effects in long- and short-sleep mice: activity, passive avoidance, and in utero ethanol levels

Genetic differences in susceptibility to fetal alcohol effects (FAE) have been suggested by both human and animal studies. The Long-Sleep (LS) and Short-Sleep (SS) mouse lines, selectively bred for differences in ethanol-induced narcosis, provide a model for studying differential alcohol sensitivity in the etiology of FAE. LS and SS mice were intubated with either 2.9 g/kg (20% w/v) ethanol (E) or an isocaloric amount of sucrose (S) twice per day (6 hr apart) on Days 7 through 15 of pregnancy. An untreated control group (C) was maintained for each line. Offspring were fostered to lactating Rockland-Swiss mice at birth. LS offspring prenatally exposed to ethanol exhibited increased open-field activity relative to LS controls, but this effect was due to the overactivity of one litter. Activity for SS mice prenatally exposed to ethanol did not differ from control levels. Ethanol content in blood (280 mg/dl), amniotic fluid (258 mg/dl), and fetal tissue (230 mg/dl) did not differ in similarly treated LS and SS dams. In a second experiment, females were treated from Days 7 through 18 of gestation, and their offspring were tested for either open-field activity or passive avoidance learning. There were no group differences in open-field activity, but LS mice prenatally exposed to alcohol took more trials to reach a passive avoidance criterion than their controls, whereas similarly treated SS mice did not differ from controls. These results suggest that genetically-mediated sensitivity to ethanol influences susceptibility to FAE and that this may be task specific.     

Acute tolerance to the ataxic effects of ethanol in short-sleep (SS) and long-sleep (LS) mice

 The objective of this series of studies was to examine the relationship between alcohol sensitivity and the development of very rapid acute tolerance to alcohol in mice. In order to measure acute tolerance to alcohol, a behavioral test was developed using a rotorod. In the first study, mice selectively bred for resistance (short sleep, SS) or sensitivity (long sleep, LS) to the acute hypnotic effects of ethanol were used, as well as mice from the base population (heterogenous stock, HS). Mice were trained to run on the rotorod at a speed of 14 rpm to a criterion of 200 s, in four daily training sessions. On the test day, baseline measurements of rotorod performance were taken and mice were injected IP with alcohol in doses from 0 to 2.5 g/kg. Animals were tested at 1-min intervals for the first 5 min following injection, then at 5-min intervals for a total of 30 min. The results demonstrated that SS and HS mice developed tolerance within 10 min following the alcohol injections. LS mice did develop some acute tolerance, but at a much slower rate than the SS or HS mice. In the second study, the effects of intoxicated practice on the rates of acute tolerance development were examined in the SS, HS and LS mice at a dose of 2.0 g/kg alcohol. A total of ten groups of each strain were given a different number of practice trials (ranging from one to ten) on the rotorod prior to a final test session at 30 min post-injection. The results provide evidence that SS and HS mice are capable of developing acute tolerance independent of practice. That is, the group of animals injected at 0 time and tested ten times up to 30 min were no better at the 30-min time point than the group injected at 0 time and tested only once at 30 min. On the other hand, the LS mice showed a modest practice effect, developing additional tolerance to the ataxic effects of alcohol with increasing intoxicated practice. Overall, these studies demonstrated that mice can develop acute tolerance within minutes following alcohol exposure, and that this ability is correlated with the initial sensitivity to alcohol. Received: 12 July 1997 / Final version: 26 August 1997     

The rapid onset of tolerance to ataxic effects of ethanol in mice

We have developed a precise quantal method for assessing the sensitivity to ethanol in the mouse. Mice placed on a clamped stationary horizontal dowel are scored ataxi or not ataxic depending on whether they are able to remian on the dowel during a 30-s observation period. A threshold blood ethanol concentration is determined by assaying tail blood drawn immediately upon recovery from ethanol-induced ataxia. This threshold is quite reproducible within a population of Swiss-Webster mice (coefficient of variation 9%). The precision of this method allowed us to follow the onset of rapid tolerance during a series of sequential IP ethanol doses. Tolerance persisted overnight in the absence of ethanol, and was found not to increase further with additional ethanol exposure on 2 subsequent days. The observed tolerance was shown not to be due to circadian changes in ethanol sensitivity or repeated practice on the task, indicating a true tissue tolerance.     

Evidence that the selectively bred long- and short-sleep mouse lines display common narcotic reactions to many depressants

This report challenges the notion that the long-sleep and short-sleep selectively bred mouse lines display unique narcotic reactions to alcohols. First, we found that the specific ethanol sensitivity hypothesis is not supported by the relevant literature. Second, we found that much of the ambiguity with respect to this hypothesis concerns just pentobarbital. Consequently, the major intent of this paper was to further explore what effect pentobarbital had on these two mouse lines. Additionally, we examined the effects of barbital and ethanol. Our results for each of these compounds clearly indicate that when these mouse lines can be differentiated by particular doses the long-sleep animals always displayed greater narcotic reactions. In this inquiry only one sex was employed, and testing was always initiated at the same time of day. It is our contention that many of the equivocal findings that have been reported concerning pentobarbital are due to combining data from both sexes, circadian rhythmicity, and similar procedural variables.     

Antagonism of alcohol hypnosis by blockade of prostaglandin synthesis and activity: genotype and time course effects

Pretreatment of mice with prostaglandin synthetase inhibitors (PGSI's) antagonizes alcohol-induced behaviors. This study examined genetic and time course factors of this effect and studied the effects of a putative prostaglandin antagonist (SC-19220) on ethanol sleep time. Long Sleep (LS) and Short Sleep (SS) mice, lines bred for differential response to an hypnotic dose of ethanol, showed a four-fold difference in their dose-response curves for indomethacin antagonism of ethanol-induced hypnosis. Females of both lines required higher amounts of indomethacin relative to males. Indomethacin pretreated animals regained the righting response at a higher blood ethanol concentration than did saline pretreated animals. In addition, indomethacin pretreatment failed to alter the rate of ethanol disappearance from blood. In general, both lines showed effects with low doses of indomethacin at early time points and with high doses of indomethacin at later time points. Indomethacin did not antagonize ethanol-induced hypnosis if given after ethanol. In C3H mice, pretreatment with low doses of SC-19220, a dibenzoxazepine derivative, produced a significant decrease in ethanol sleep time, moderate doses produced no effect, and high levels increased sleep time. These results further substantiate and expand our previous reports. Possible mechanisms for the biphasic effects of indomethacin treatment are presented and discussed.     

Prostaglandin synthetase inhibitors antagonize the depressant effects of ethanol

Several studies indicate that ethanol may depress the central nervous system by altering neurotransmitter release. Evidence obtained from the peripheral nervous system suggests that prostaglandins act as negative feedback inhibitors of transmitter release. If a similar process occurs in the brain, then perhaps ethanol affects transmitter release via a mechanism involving prostaglandins. Prostaglandin synthetase inhibitors were administered to adult HS/Ibg male mice prior to intraperitoneal injection of a hypnotic dose of either ethanol, propanol, or t-butanol. A significant decrease in the length of alcohol sleep time was found: in the ethanol study, this was coupled with a significant increase in waking blood alcohol levels. These rresults indicate that inhibition of prostaglandin synthesis alters CNS sensitivity to the depressant effects of alcohol. When the same inhibitors were administered prior to other sedative hypnotics, i.e., pentobarbital and chloral hydrate, no effect was found. This suggests that prostaglandins may be specifically involved in the biochemical mechanism of alcohol depression.     

Differential effects of catecholamine antagonists on ethanol-induced excitation in mice

Catecholamine antagonists were assessed for their effects on ethanol-induced motor excitation. Motor excitation was measured in male Swiss-Webster mice using an open-field apparatus. Mice were treated with several doses of ethanol and at each dose, mice were pretreated with pimozide, a dopamine D2 antagonist, Schering 23390, a dopamine D1 antagonist, phenoxybenzamine, a noradrenergic alpha-1 antagonist, or yohimbine, a noradrenergic alpha-2 antagonist. Each mouse was subjected to only one dose regimen, and all injections were given IP. Ethanol produced an increase in locomotor activity. The degree to which pimozide attenuated ethanol excitation decreased with increasing ethanol dosage. At the highest dose of ethanol, pimozide increased ethanol excitation. Schering 23390 attenuated ethanol-induced excitation only at doses which affected motor activity per se. Phenoxybenzamine produced a dose-dependent reduction in ethanol excitation. Yohimbine had its greatest effects at the medium dose (4.0 mg/kg). These observations seem to indicate a role for both the dopamine D2 receptor and the noradrenergic alpha-1 receptor in ethanol-induced motor excitation.     

Differential sensitivity of long-sleep and short-sleep mice to high doses of cocaine

The cocaine sensitivity of male and female long-sleep (LS) and short-sleep (SS) mice, which have been selectively bred for differential ethanol-induced "sleep-time," was examined in a battery of behavioral and physiological tests. Differences between these two mouse lines were subtle and were seen primarily at high doses. At high doses, SS mice were more sensitive than LS mice, particularly to cocaine-induced hypothermia; however, significant hypothermia was not seen except at doses which were very near to the seizure threshold. During a 60-min test of locomotor activity, LS mice showed greater stimulation of Y-maze activity by 20 mg/kg cocaine than SS mice. Consistent with the finding of subtle differences in sensitivity to low doses of cocaine. LS and SS mice did not differ in sensitivity to cocaine inhibition of synaptosomal uptake of [3H]-dopamine, [3H]-norepinephrine or [3H]-5-hydroxytryptamine. However, consistent with the finding of differential sensitivity to high doses of cocaine, SS mice were more sensitive to the seizure-producing effects of the cocaine and lidocaine, a local anesthetic. It is hypothesized that the differential sensitivity of these mouse lines to high doses of cocaine is due to differential sensitivity to cocaine's actions on systems that regulate local anesthetic effects. Selective breeding for differential duration of alcohol-induced "sleep-time" may have resulted in differential ion channel structure or function in these mice.     

Neurobehavioral effects of alcohol in AMPA receptor subunit (GluR1) deficient mice

Of the ionotropic glutamatergic receptors, the NMDA receptor is clearly implicated in the acute and chronic effects of ethanol; however, the role of the AMPA receptor in mediating the effects of ethanol in vivo is as yet unclear. Using mice deficient in the AMPA receptor subunit GluR1 (GluR1-/- mice), we investigated whether the AMPA receptor had a significant role in mediating the effects of ethanol. GluR1-/- mice showed greater locomotor activity in a novel environment, but by the fifth day of repeated testing their activity was the same as that of wild-type mice. In contrast to their enhanced locomotor activity, on an accelerating rotarod GluR1-/- mice performed consistently worse than wild-types. With regard to the effects of ethanol on motor responses, GluR1-/- mice did not differ significantly from wild-type mice in ethanol's sedative or incoordinating effects. However, the GluR1-/- mice were insensitive to the hypothermic effects of a hypnotic dose of ethanol in contrast to wild-types; this effect was dissociable from the hypnotic effects of ethanol. Further, tolerance to ethanol developed equally for GluR1-/- mice versus wild-type mice. In terms of alcohol drinking behavior, compared to wild-types, GluR1-/- mice differed neither in the acquisition of voluntary ethanol consumption nor in stress-induced ethanol drinking, nor in the expression of an alcohol deprivation effect (ADE) which is used as a model of relapse-like drinking behavior. In summary, although the loss of a hypothermic effect of ethanol in GluR1-/- mice indicates a critical role for the AMPA receptors in this effect, the GluR1 subunit of the AMPA receptor does not seem to play a critical role in the etiology of alcohol dependence. However, changes observed in activity patterns may be related to the putative role of AMPA receptors in attention deficit hyperactivity disorder.     

Cocaine produces low dose locomotor depressant effects in mice

Cocaine produces several behavioral effects, most notably locomotor stimulation. Biochemically, cocaine is known to inhibit reuptake at the three monoamine transporter sites, and may have highest affinity at the serotonin transporter. Serotonin augmentation has been associated with decreases in behavioral activity, but cocaine has not been reported to produce behavioral depressant effects except at high doses which cause stereotypy and disruption of behavior. This study examined the effects of relatively low doses of cocaine, in the range of 0.1–10 mg/kg, on locomotor activity in C57BL/6J and DBA/2J mice. A biphasic dose-response curve was seen for both strains. At the lowest doses, activity was depressed. As the dose of cocaine increased, activity returned to baseline, and at the highest doses, increases in locomotor activity were found. DBA/2J mice were depressed at a lower dose of cocaine than were C57BL/6J mice; however, C57BL/6J mice showed locomotor depression over a broader range of doses. Activity was maximally depressed at 0.1 mg/kg for DBA/2J mice, and maximally depressed at 0.3 mg/kg for C57BL/6J mice. Thus, low doses of cocaine are shown to produce significant decreases in locomotor activity in two strains of mice. It is postulated that these low doses of cocaine which depress locomotor activity do so via inhibition of serotonin uptake, resulting in potentiation of serotonergic activity.     

Differential metabolism of 4-hydroxynonenal in liver,lung and brain of mice and rats

The lipid peroxidation end-product 4-hydroxynonenal (4-HNE) is generated in tissues during oxidative stress. As a reactive aldehyde, it forms Michael adducts with nucleophiles, a process that disrupts cellular functioning. Liver, lung and brain are highly sensitive to xenobiotic-induced oxidative stress and readily generate 4-HNE. In the present studies, we compared 4-HNE metabolism in these tissues, a process that protects against tissue injury. 4-HNE was degraded slowly in total homogenates and S9 fractions of mouse liver, lung and brain. In liver, but not lung or brain, NAD(P)+ and NAD(P)H markedly stimulated 4-HNE metabolism. Similar results were observed in rat S9 fractions from these tissues. In liver, lung and brain S9 fractions, 4-HNE formed protein adducts. When NADH was used to stimulate 4-HNE metabolism, the formation of protein adducts was suppressed in liver, but not lung or brain. In both mouse and rat tissues, 4-HNE was also metabolized by glutathione S-transferases. The greatest activity was noted in livers of mice and in lungs of rats; relatively low glutathione S-transferase activity was detected in brain. In mouse hepatocytes, 4-HNE was rapidly taken up and metabolized. Simultaneously, 4-HNE-protein adducts were formed, suggesting that 4-HNE metabolism in intact cells does not prevent protein modifications. These data demonstrate that, in contrast to liver, lung and brain have a limited capacity to metabolize 4-HNE. The persistence of 4-HNE in these tissues may increase the likelihood of tissue injury during oxidative stress.     

Influence of chlordiazepoxide on alcohol consumption in mice

In a free-choice situation, chlordiazepoxide (CDP; 12.5 or 25 mg/100 ml; groups B or C), when incorporated in ethanol solutions (2 to 20%, v/v), caused a significant decrease in ethanol preference index (P.I.). This was probably due to the combined CNS effects of both drugs rather than a taste effect, since the mice did not discriminate between aqueous CDP solutions and water. However, when the mice had prior exposure to ethanol and CDP was incorporated intermittently, no significant decreases in P.I. resulted. In a no-choice situation, ethanol intake was increased only on the first day of each intermittent incorporation of CDP (3 days for each 6-day cycle), being more persistent in group B (2 to 15% ethanol) than in group C (2 to 6.5%). Ethanol intake decreased in group C when alcohol concentrations exceeded 10%. The “first-day” CDP effect also occurred in the no-choice situation of an ethanolic liquid diet. Possible factors for this effect are discussed. Thus the effects of CDP on alcohol consumption in non-deprived mice vary with experimental designs.     

解酒保肝口服液对小鼠酒精中毒的影响

目的：观察解酒保肝口服液对小鼠醉酒实验,血清乙醇浓度和肝、胃组织乙醇脱氢酶活性的影响。方法：将生理盐水和将葛根,甘草等中药用水煎煮制成解酒保肝口服液灌服于小鼠后30min,灌服白酒,记录小鼠翻正反射消失（醉酒）至恢复（醒酒）所需时间（min),及24h内小鼠的死亡只数,另以相同操作连续6d后眼眶取血并处死动物,立即取出肝脏和胃,分别用生化比色法测定血肖乙醇浓度和肝、胃组织乙醇脱氢酶活性。结果：在醉酒实验中,。与对照组相比服用解酒保肝口服液组小鼠从饮酒到翻正反射消失（醉酒）的时间明显延长（P＜0．01）,醒酒时间明显缩短,且小鼠的死亡率明显降低（P＜0．05）,血清乙醇含量明显降低,肝脏ADH高于对照组, 结论：解酒保肝口服液具有解酒作用。     

Ethanol elimination rates in mice: Effects of gender,nutrition, and chronic ethanol treatment

Twenty male and 20 female mice of a heterogeneous stock were assigned to each of three groups. One group was administered ethanol in a liquid diet for 9 days, a second group was fed an isocalorically controlled diet containing no ethanol for the same length of time, and the third group was fed standard lab chow. Each animal was injected with a dose of ethanol equal to 3.5 g/kg body weight at the time corresponding to 6 hr post-withdrawal for the ethanol-treated group. Blood ethanol elimination rates were determined at 1, 2, and 3 hr post-injection. Neither gender, nutritional state, nor chronic ethanol treatment was found to affect ethanol elimination rates.     

Behavioral and physiological measures for studying ethanol dependence in mice

In the initial experiment, 20 male and 20 female heterogeneous stock (HS) mice were divided randomly into experimental and control groups. Ethanol was administered in liquid diet form to the experimental group in increasing dosages for a 9-day period followed by behavioral and physiological tests to determine differences between groups and genders 6 to 7 he post-withdrawal. Analysis of data showed significant differences between the ethanol-treated and control groups for at least six of the measures. In addition, significant gender differences and significant interactions between gender and treatment group were found for four of the measures; in all of these cases, females appeared to exhibit a less severe withdrawal syndrome than males. In a second experiment, no gender difference in rate of ethanol disappearance from the blood could be found in 36 HS mice tested at 1 to 3 hr post-withdrawal.     

The consequences of beer consumption in rats: acute anxiolytic and ataxic effects and withdrawal-induced anxiety

Abstract Rationale. Rats avidly consume standard off-the-shelf beer; however, the behavioural consequences of beer consumption in rodents have hardly been studied. Objectives. The present study examined the acute anxiolytic and ataxic effects of beer consumption in rats and the anxiogenic effects of withdrawal from free access to beer. Method. In experiment 1, male Wistar rats received 30 min access to "near-beer" each day (a malt beverage that looks and tastes like beer but which contains <0.5% ethanol). On the test day, for some rats, ethanol (either 2% or 4% v/v) was added to the near-beer to make it resemble standard (ethanol-containing) beer of "light" (2.5% beer) or full strength (4.5% beer). Immediately after this, rats were tested on their response to a predatory cue (a fabric collar that had been worn by a cat) and on an accelerating rotarod. In experiment 2, rats were trained in the same drinking paradigm as above and then tested on a further battery of anxiety tests. In experiment 3, rats were given continuous home cage access to either 4.5% beer or near-beer for 35 days. Half of the rats were then denied access to beer or near-beer for 24 h and then tested on the same anxiety test battery as in experiment 2. Results. Rats drinking 4.5% beer approached a predatory cue significantly more than those given near-beer, indicating an anxiolytic effect. In experiment 2, rats drinking 4.5% beer displayed less anxiety-like behaviour in the elevated plus maze and emergence tests but not in the social interaction test. Rats given 4.5% beer fell off the rotarod significantly faster than rats given near-beer, indicating an ataxic effect. Rats previously given 4.5% beer drank significantly less near-beer the following day, suggesting a moderate aversion the day after beer consumption. In experiment 3, rats denied access to 4.5% beer showed significantly less social interaction and took longer to emerge into an open field than controls. Conclusion. These results are the first to our knowledge to show that rats will consume beer at levels that produce clear effects on anxiety and on motor co-ordination, and that will eventually produce behavioural signs of withdrawal. Electronic Publication     

A rapid method to evaluate acute ethanol intoxication in mice

A simple technique for the evaluation of ethanol intoxication based on the ability of mice to remain on a bar suspended above an electrified grid is reported. The characteristics that make this model useful to measure ethanol induced intoxication include: (a) low variability; (b) high sensitivity; (c) rapidity; (d) requires no previous training of animals to be tested; (e) objective scoring which can be quantified; and (f) dose-dependent correlation between brain and blood ethanol levels and bar holding response.     

Behavioral effects of aminoadamantane class NMDA receptor antagonists on schedule-induced alcohol and self-administration of water in mice

Rationale Aminoadamantanes represent a class of NMDA glutamate receptor antagonists that reduce alcohol consumption and may prevent alcohol-induced neuronal adaptations and side effects.Objective Behavioral specificity of memantine and amantadine on alcohol drinking in a schedule-induced polydipsia (SIP) task was investigated in mice.Methods Male C57BL/6J mice were food-deprived and divided into four groups: 5% alcohol SIP, water SIP, 1 h limited access regulatory water drinking, and a control group to determine if either drug altered ethanol drinking. Behavioral specificity of memantine (5, 10, and 25 mg/kg, ip) and amantadine (20, 40, and 60 mg/kg, ip) was determined by comparing alterations in alcohol or water consumption in SIP and regulatory water drinking. Drug effects on SIP drinking-specific measures (grams per kilogram consumption) were also compared to nondrinking measures (locomotion, head-entries for food, and lick efficiency).Results Compared to saline, memantine reduced alcohol SIP drinking (10 and 25 mg/kg). Memantine increased locomotion during alcohol SIP (25 mg/kg) and during water SIP (5 and 25 mg/kg). In contrast, amantadine reduced both alcohol SIP (40 mg/kg) and water SIP (40 and 60 mg/kg). Both drugs reduced regulatory water consumption over the entire dose range tested. Blood alcohol concentrations indicated consumption of physiologically meaningful amounts of alcohol during SIP, and that changes in alcohol metabolism did not account for drug-induced reductions in alcohol drinking.Conclusions In addition to reducing alcohol drinking, both drugs had other behavioral effects that included reductions in regulatory drinking. These results suggest that the therapeutic utility of these drugs for ameliorating human alcohol addiction remains questionable.     

Differential regulation of cocaine-induced locomotor activity in inbred long-sleep and short-sleep mice by dopamine and serotonin systems

Acute injection of cocaine increases locomotor activity of inbred long-sleep (ILS) mice to a greater extent than inbred short-sleep (ISS) mice. Strain differences in dopamine and/or serotonin (5-HT) neurotransmission could underlie these behavioral differences. Here, we found that dopamine D1, 5-HT(2A) and 5-HT3 receptor antagonists reduced cocaine-stimulated activity selectively in ILS mice. In contrast, 5-HT transporter (SERT) or 5-HT(1A) receptor antagonists potentiated cocaine-stimulated activity in ISS, but not in ILS, mice; this potentiation in ISS mice was abolished by dopamine D1 receptor blockade. Thus, in ILS mice, cocaine-induced activation of D1, 5-HT(2A) or 5-HT3 receptors is sufficient to produce locomotor stimulation. In contrast, ISS mice require pharmacologically increased 5-HT levels, which appear to result in increased dopamine neurotransmission, for cocaine-induced activation. Our results demonstrate strain differences in dopamine/5-HT receptor subtypes and their interactions that contribute to the differential behavioral responsiveness of ILS and ISS mice to cocaine.     

Reinterpretation of the literature indicates differential sensitivities of long-sleep and short-sleep mice are not specific to alcohol

This paper reviews the findings and conclusions of the literature pertinent to the Long-Sleep and Short-Sleep selectively-bred lines of mice and challenges the widely-held notion that the selective breeding program was successful in separating alleles for specific sensitivities to just alcohol. Rather, it is argued that these lines of mice were selected for differing activity of a more general process. Recent evidence, as well as reevaluated previous evidence, indicates that Long-Sleep mice are more sensitive to the soporific effects of three major classes of CNS depressants (alcohols, barbiturates, and benzodiazepines), as well as many other anesthesia-inducing compounds (adenosine, chloral hydrate, trichloroethanol, paraldehyde, nitrous oxide, enflurane, and isoflurane). Further, much evidence also supports the conclusion that most of these hypnotic-depressants and anesthetics could exert their soporific influence by a potentiation of GABA activity. The other characteristic of interest in this regard is susceptibility to convulsions. Short-Sleep mice have significantly lower thresholds to both flurothyl-induced and bicuculline-induced convulsions, as well as being more likely to suffer from paroxysms during ethanol withdrawal.