Naltrexone Effects on Ethanol Drinking Acquisition and on Established Ethanol Consumption in C57BL/6J Mice

Naltrexone's success as a treatment agent for alcoholism seems to be due to its ability to reduce craving in abstinent, dependent individuals and to reduce the pleasure associated with subsequent intake. However, more study is needed to establish the optimal amount of time that naltrexone treatment should be continued. Little information seems to have been collected regarding the most effective dosing regimen for reducing alcohol craving and consumption, and the usefulness of opiate antagonists in the prevention of alcohol dependence in nonaddicts, rather than just as a treatment agent in addicted individuals, also deserves further study. The alcohol-preferring C57BL/6J (B6) mice were used to: (1) study naltrexone effects on consumption in established drinkers using an increasing dosing regimen, (2) study naltrexone effects on the acquisition of ethanol drinking, and (3) study the effects of chronic naltrexone from timed-release pellets on drinking in alcohol-naive mice. Naltrexone reduced ethanol preference in established drinkers, but its effects waned at increasing doses. Naltrexone slowed the acquisition of ethanol drinking, but was ineffective when readministered after a phase when ethanol was offered in the absence of naltrexone. Mice with chronic naltrexone pellets consumed greater amounts of ethanol and showed higher ethanol preference than did placebo-pelleted animals. The observed reduced efficacy of naltrexone with increasing dosage and chronic treatment may have been due to naltrexone-induced opiate receptor changes. Such changes are presumably more likely to occur when naltrexone doses remain high or perhaps accumulate. Thus, dose and frequency of administration may be important factors in determining naltrexone's effectiveness in treating alcohol dependence.     

Effects of Ethanol Consumption on Enriched Natural Killer Cells from C57BL/6 Mice

Ethanol (20% w/v) given to female, C57BL/6 mice in their drinking water was previously shown to suppress natural killer (NK) cell cytolytic activity in a mixed splenocyte population. The present study was designed to examine the hypothesis that ethanol consumption independently results in inhibition of NK cell cytolytic activity. Mice were given 20% w/v ethanol in the drinking water for 2 weeks, and splenic NK cells were enriched up to 88% based on surface expression of NK1.l. Cytolytic activity of these freshly enriched NK cells from ethanol-consuming mice against YAC-1 lymphoma cells was inhibited an average of 41% relative to water-drinking controls. Cytolytic activity of enriched NK cells from ethanol-consuming mice was stimulated to levels equal to control water-drinking mice after 16- to 18-hr incubation in 1000 units/ml recombinant interleukin 2. These data indicate that in vitro cytolytic activity of NK cells from ethanol-consuming mice is inhibited in the absence of other modulatory leukocytes.     

Ethanol Consumption and Taste Preferences in C57BL/6ByJ and 129/J Mice

Mice of the C57BL/6ByJ (B6) and 129/J (129) strains were offered different concentrations of taste solutions in 48-hr, two-bottle choice tests. In comparison with the 129 strain, the B6 strain had higher preferences for ethanol, sucrose, and citric acid. They had lower preferences for NaCl and similar preferences for capsaicin and quinine hydrochloride. These data are consistent with the hypothesis that the higher ethanol intake by B6 mice depends, in part, on higher hedonic attractiveness of its sweet taste component.     

Low-Level Hyperbaric Exposure Antagonizes Locomotor Effects of Ethanol and n-Propanol But Not Morphine in C57BL Mice

Low-level hyperbaric exposure antagonizes a broad range of behavioral effects of ethanol in a direct, reversible, and competitive manner. This study investigates the selectivity of the antagonism across other drugs. C57BL/6 mice were injected with saline, ethanol, n -propanol, or morphine sulfate, and then were exposed to 1 atmosphere absolute (ATA) air, 1 ATA helium-oxygen gas mixture (heliox), or 12 ATA heliox. Locomotor activity was measured from 10 to 40 min following injection, N -propanol produced a dose-dependent depression of locomotor activity from 1.0 g/kg. Morphine produced a dose-dependent stimulation of locomotor activity at doses of 3.75–12.0 mg/kg. Exposure to 12 ATA heliox significantly antagonized the locomotor depressant effects of 1.0 g/kg n -propanol and 2.5 g/kg ethanol, without significantly affecting blood concentrations of these drugs measured at 40 min postinjection. Exposure to 12 ATA heliox did not significantly antagonize the locomotor-stimulating effects of the two morphine doses tested (3.75 and 7.5 mg/kg). These findings suggest that exposure to 12 ATA heliox antagonizes the behavioral effects of intoxicant-anesthetic drugs like ethanol and n -propanol, which are believed to act via perturbation or allosteric modulation of functional proteins, but does not antagonize the effects of drugs like morphine, which act via more direct mechanisms. This demonstration of selective antagonism adds important support for the hypothesis that low-level hyperbaric exposure is a direct mechanistic ethanol antagonist, with characteristics similar to a competitive pharmacological antagonist.     

Diet and Ethanol Modulate Immune Responses in Young C57BL/6 Mice

Chronic ethanol (ETOH) ingestion adversely affects the immunocompetence of alcohol abusers. ETOH directly impairs host defense mechanisms and indirectly modulates immunocompetence by interfering with the nutritional status of the alcoholic. It is not clear from the current literature, however, to what extent ETOH, nutritional status, or the combination of the two factors modulates immune mechanisms in chronic alcoholics. To date, most animal studies investigating the immunotoxicity of ETOH have neglected the dietary factors, which may have masked additional immunotoxic effects of ETOH. To examine these dietary factors, we fed mice three liquid ETOH diets with different dietary sufficiencies for 7 weeks and investigated various immune responses. Spleen cell number and secretions of immunoreactive interleukin-2 and tumor necrosis factor were totally independent of the diet, being affected only by ETOH. Body, spleen, and thymus weights, interferon-γ secretion, and natural killer cell and phagocytic activities were modulated by ETOH as well as by diet. Natural killer cell and phagocytic activities were also directly affected by the nutritional quality of the diet. These results suggest that animal diets used in experimental studies of ETOH-induced immunomodulation must be planned and controlled carefully in order to single out the direct effects that ETOH has on the host defense system.     

Intravenous Ethanol Self-administration in C57BL/6J and DBA/2J Mice

Two strains of mice, C57BL/6J (B6) and DBA/2J (D2) were allowed to self-administer intravenous (iv) ethanol. These two strains were selected because they differ greatly in their preference for drinking ethanol solutions: 86 mice are preferrers, whereas D2 mice are avoiders of ethanol. Of interest was whether these strains would also differ in self-administration of iv ethanol when taste factors presumably do not influence consumption. Mice were trained with either 60, 75, or 90 mg/kg per infusion. Mice from both strains acquired nosepoking for all of these doses on an FR-3 schedule of reinforcement during 2-hr daily sessions. Additionally, mice in both strains acquired an equal preference for nosepoking on the side resulting in ethanol infusions, compared with the side that had no scheduled consequence, although B6 mice took somewhat more ethanol early in training than did D2 mice. Mice in both strains achieved equal levels of responding at the conclusion of training, when response rates had stabilized. A subset of animals were then tested at doses of ethanol ranging from 25 to 125 mg/kg per infusion. Although their responding tended to decrease over time regardless of changes in the unit dose of ethanol, these mice showed lower response rates for higher doses of ethanol, and less responding for saline than for ethanol. Together, these findings imply that iv ethanol has reinforcing properties in both these strains, despite the strain difference in preference for oral ethanol. Self-administration of iv ethanol in mice may prove a valuable addition to existing animal models for the study of ethanol reward.     

Circadian Timing of Ethanol Exposure Exerts Enduring Effects on Subsequent Ad Libitum Consumption in C57 Mice

Background:  There is a daily rhythm in the voluntary intake of ethanol in mice, with greatest consumption in the early night and lowest intake during the day. The role of daily timing of ethanol exposure on the development and control of long-term ethanol self-administration has been neglected. The present study examines these issues using C57BL/6J mice.
Methods:  Mice were repeatedly exposed to 10% ethanol for 2 hours early in the night or day for several weeks. Subsequently, ethanol was available at the opposite time (Expt 1) or 24 hours daily (Expts 1 and 2). Lick sensors recorded the patterns of drinking activity in Experiment 2.
Results:  Mice exposed to ethanol during the night drink more than mice exposed during the day. Prior history did not affect ethanol intake when the schedule was reversed. Under 24-hour exposure conditions, mice with a history of drinking during the night consumed significantly more than mice drinking during the day. The circadian patterns of drinking were not altered.
Conclusions:  These results demonstrate that the daily timing of ethanol exposure exerts enduring effects of self-administration of ethanol in mice. Understanding how circadian rhythms regulate ethanol consumption may be valuable for modifying subsequent intake.     

Ethanol Teratogenesis in Selectively Bred Long-Sleep and Short-Sleep Mice: A Comparison to Inbred C57BL/6J Mice

Sensitivity to alcohol may influence the severity of ethanol teratogenesis. To examine this hypothesis, the teratogenic effects of ethanol were compared in Long-Sleep (LS) and Short-Sleep (SS) mice, selectively bred for differences in ethanol-induced narcosis. Inbred C57BL/6J (B6) mice were included to confirm previously reported teratogenic effects using our own treatment regimen and standard assessment technique. Intragastric administration of ethanol (5.8 g/kg) on Days 9 and 10 of pregnancy resulted in growth retardation and an increase in prenatal mortality in LS litters but not in SS litters. Therefore, alcohol sensitivity plays a role in the severity of prenatal alcohol effects. B6 mice showed more ethanol teratogenicity than either LS or SS mice, even though maternal blood ethanol levels were similar across genotypes. This result suggests genetic variations other than alcohol sensitivity also influence ethanol teratogenesis.     

Characterization of the μ and δ Opioid Receptors in the Brain of the C57BL/6 and DBA/2 Mice, Selected for Their Differences in Voluntary Ethanol Consumption

Genetically determined differences in the activity of the hypothalamic β-endorphin system have been demonstrated between the C57BL/6 (alcohol-preferring) and DBA/2 (alcohol-aversive) inbred strains of mice. The present studies examined the distribution and density of the μ and δ receptors in specific brain regions that may mediate the rewarding and reinforcing effects of ethanol, using quantitative auto-radiography and the specific μ agonist FK 33–824 and 6 agonist DPDPE, in their iodinated form. ¹²⁵I-FK 33–824 recognizes a high affinity binding site in brain membrane preparations from both the C57BL/6 (K_d= 1.37 ± 0.22 nM; B_max= 80 ± 12.3 fmol/mg protein) and DBA/2 mice (K_d= 1.02 ± 0.16 nM; B_max= 39.5 ± 9.6 fmol/mg protein), whereas ¹²⁵I-DPDPE binds to a high affinity binding site in brain membranes from both the C57BL/6 (K_d= 1.08 ± 0.34 nM; B_max= 24.4 ± 4.5 fmol/mg protein) and DBA/2 mice (K_d= 0.68 ± 0.24 nM; B_max= 15.3 ± 3.7 fmol/mg protein). The auto-radiographic studies demonstrated differences in the density of the μ opioid receptors between the two strains of mice in brain nuclei that are not directly related to the brain reward system. However, strain-related differences in the density of δ opioid receptors were observed in regions of the limbic system known to mediate the positive reinforcing effects of many drugs of abuse. The density of δ receptors was significantly higher in the ventral tegmental area and nucleus accumbens of the C57BL/6 mice. The results of the present study support the hypothesis that genetically determined differences exist in the density of opioid receptors in distinct regions of the brain between the C57BL/6 and DBA/2 inbred strains of mice, which may play a role in controlling their voluntary ethanol consumption.     

The Effect of Chronic Poisoning with Carbon Tetrachloride on Voluntary Consumption of Ethanol by Mice

Mice were chronically poisoned with carbon tetrachloride injected twice weekly, intraperftoneally, in doses of 0.05-0.80 mg/g over 10 to 16 weeks. The poisoned animals, as compared to vehicle-injected controls, showed marginally significant increases in voluntary consumption of ethanol in a three-bottle choice (water, 10% and 20% ethanol continuously available), but not to a degree that could be considered alcoholic. The average daily intakes of ethanol remained within the range of variation for normal animals of this strain; there was no consistent shift in preference from 10% to 20% ethanol, and the distribution of drinking times within the 24-hour cycle remained normal. The relevance of these findings to the clinical problem of alcoholism is that the appetite for alcohol, a potential hepatotoxin, persists despite serious damage to the liver.     

Ethanol Consumption and Early Murine Retrovirus Infection Influence Liver, Heart, and Muscle Levels of Iron, Zinc, and Copper in C57BL/6 Mice

The relative and combined roles of ethanol and murine acquired immunodeficiency syndrome (MAIDS) on the mineral status (Fe, Zn, and Cu) of liver (storage site), heart, muscle (nutrient mobile sites) were investigated. C57BL/6 mice were randomly assigned to four groups: (a) uninfected mice fed isocaloric, adequate nutrient diet (NRC), (b) uninfected mice fed the NRC diet with 25% of energy derived from ethanol, (c) LP-BM5 retrovirus-infected mice fed the isocaloric NRC diet, and (d) retrovirus-infected mice fed the NRC diet with 25% of its energy derived from ethanol. The levels of Cu and Zn levels in the liver did not significantly change as a result of ethanol consumption. However hepatic Zn concentration was increased significantly in retrovirus-infected mice. This may be correlated to the increase in their liver weight. Ethanol administration significantly increased Fe concentration in the liver, yet significantly decreased concentration of Cu in the heart. Retrovirus infection alone, which had not proceeded to murine AIDS, resulted in a significant increase in heart Cu and Zn concentration as compared with uninfected mice. Retrovirus infection in C57BL/6 mice significantly increased Fe and Zn level/g of muscle. Early retrovirus infection alters tissue micronutrient levels, and may thus contribute to immunological changes.     

C57BL/6J Mice Are Not Suitable for Modeling Severe SARS-CoV-2 Beta and Gamma Variant Infection

SARS-CoV-2 variants, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) variants, have displayed increased transmissibility and, therefore, have been categorized as variants of concern (VOCs). The pervasiveness of VOCs suggests a high probability of future mutations that may lead to increased virulence. Prior reports have shown that VOC infection without expression of human angiotensin converting enzyme-2 receptor (hACE2) in mice is possible. We sought to understand if the increased transmissibility of VOCs can infect C57BL/6 mice without expression of hACE2 receptor required for entry of SARS-CoV-2 normally. We examined the ability of infection with Beta and Gamma variants to infect and cause both pathological and clinical changes consistent with severe COVID-19, including body weight changes, survival, subgenomic viral titer, lung histology on Hematoxylin and Eosin (H&E) staining, and viral protein expression as measured by immunohistochemistry staining of viral antigen (IHC). These methods were used to examine three groups of mice: C57BL6, Rag2-/-, and Ccr2-/- mice. We observed that these mice, infected with Beta and Gamma variants of SARS-CoV-2, did not show pathological changes as indicated by weight loss, altered survival, or significant lung pathology on H&E staining. Subgenomic qPCR and IHC staining for viral protein indicated that there was some evidence of infection but far below ACE2 transgenic mice, which showed clinical disease and pathologic changes consistent with ARDS. These data suggest that these variants replicate poorly even in the setting of profound immune deficiency.