Role of acetaldehyde in ethanol-induced elevation of the neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one in rats

Background: Systemic ethanol administration increases neuroactive steroid levels that increase ethanol sensitivity. Acetaldehyde is a biologically active compound that may contribute to behavioral and rewarding effects of ethanol. We investigated the role of acetaldehyde in ethanol‐induced elevations of 3α‐hydroxy‐5α‐pregnan‐20‐one (3α,5α‐THP) levels in cerebral cortex. Methods: Male Sprague–Dawley rats were administered ethanol, and plasma acetaldehyde concentrations were measured by gas chromatography to determine relevant concentrations. Rats were then administered acetaldehyde directly, acetaldehyde plus cyanamide to block its degradation, or ethanol in the presence of inhibitors of ethanol metabolism, to determine effects on 3α,5α‐THP levels in cerebral cortex. Results: Ethanol administration (2 g/kg) to rats results in a peak acetaldehyde concentration of 6‐7 μM at 10 minutes that remains stable for the duration of the time points tested. Direct administration of acetaldehyde eliciting this plasma concentration does not increase cerebral cortical 3α,5α‐THP levels, and inhibition of ethanol‐metabolizing enzymes to modify acetaldehyde formation does not alter ethanol‐induced 3α,5α‐THP levels. However, higher doses of acetaldehyde (75 and 100 mg/kg), in the presence of cyanamide to prevent its metabolism, are capable of increasing cortical 3α,5α‐THP levels. Conclusions: Physiological concentrations of acetaldehyde are not responsible for ethanol‐induced increases in 3α,5α‐THP, but a synergistic role for acetaldehyde with ethanol may contribute to increases in 3α,5α‐THP levels and ethanol sensitivity.     

Pregnenolone and ganaxolone reduce operant ethanol self-administration in alcohol-preferring p rats

Background: Neuroactive steroids modulate ethanol intake in several self‐administration models with variable effects. The purpose of this work was to examine the effects of the long‐acting synthetic GABAergic neurosteroid ganaxolone and the endogenous neurosteroid pregnenolone, a precursor of all GABAergic neuroactive steroids, on the maintenance of ethanol self‐administration in an animal model of elevated drinking—the alcohol‐preferring (P) rats. Methods: P rats were trained to self‐administer ethanol (15% v/v) versus water on a concurrent schedule of reinforcement, and the effects of ganaxolone (0 to 30 mg/kg, subcutaneous [SC]) and pregnenolone (0 to 75 mg/kg, intraperitoneal [IP]) were evaluated on the maintenance of ethanol self‐administration. After completion of self‐administration testing, doses of the neuroactive steroids that altered ethanol self‐administration were assessed on spontaneous locomotor activity. Finally, the effect of pregnenolone administration on cerebral cortical levels of the GABAergic neuroactive steroid (3α,5α)‐3‐hydroxypregnan‐20‐one (allopregnanolone, 3α,5α‐THP) was determined in both ethanol‐experienced and ethanol‐inexperienced P rats because pregnenolone is a precursor of these steroids. Results: Ganaxolone produced a dose‐dependent biphasic effect on ethanol reinforcement, as the lowest dose (1 mg/kg) increased and the highest dose (30 mg/kg) decreased ethanol‐reinforced responding. However, the highest ganaxolone dose also produced a nonspecific reduction in locomotor activity. Pregnenolone treatment significantly reduced ethanol self‐administration (50 and 75 mg/kg), without altering locomotor activity. Pregnenolone (50 mg/kg) produced a significant increase in cerebral cortical allopregnanolone levels. This increase was observed in the self‐administration trained animals, but not in ethanol‐naïve P rats. Conclusions: These results indicate that pregnenolone dose‐dependently reduces operant ethanol self‐administration in P rats without locomotor impairment, suggesting that it may have potential as a novel therapeutic for reducing chronic alcohol drinking in individuals that abuse alcohol.     

Ethanol-induced elevation of 3 alpha-hydroxy-5 alpha-pregnan-20-one does not modulate motor incoordination in rats

BACKGROUND: Ethanol administration elevates the levels of GABAergic neuroactive steroids in brain and contributes to some of its behavioral actions. In the present study, we investigated whether such elevation of GABAergic neuroactive steroids contributes to the motor incoordinating effects of ethanol. METHODS: Sprague-Dawley rats were administered ethanol (2 g/kg intraperitoneally) or saline, and the level of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) was measured across time in cerebral cortex and in various brain regions at the peak time by radioimmunoassay. To study whether increases in GABAergic neuroactive steroids are responsible for the motor incoordinating actions of ethanol, rats were subjected to chemical (5alpha-reductase inhibitor, finasteride) and surgical (adrenalectomy) manipulations before receiving ethanol (2 g/kg intraperitoneally) injections. The rats were then subjected to different paradigms to evaluate motor impairment including the Majchrowicz motor intoxication rating scale, Rotarod test, and aerial righting reflex task at different time points. RESULTS: The radioimmunoassay of 3alpha,5alpha-THP in different brain regions showed that ethanol increases 3alpha,5alpha-THP levels by 3- and 9-fold in cerebral cortex and hippocampus, respectively. There was no change in 3alpha,5alpha-THP levels in cerebellum and midbrain. The time course of 3alpha,5alpha-THP elevations in the cerebral cortex showed significant increases 20-min after ethanol injection with a peak at 60 min. In contrast, motor toxicity peaked between 5 and 10 min after ethanol injections and gradually decreased over time. Furthermore, adrenalectomy or pretreatment with finasteride (2 x 50 mg/kg, subcutaneously) did not reduce motor incoordinating effects of ethanol as assessed by the Majchrowicz intoxication rating score, Rotarod test, or aerial righting reflex task. CONCLUSIONS: Ethanol increases GABAergic neuroactive steroids in a time- and brain region-selective manner. The role of neuroactive steroids in alcohol action is specific for certain behaviors. Alcohol-induced deficits in motor coordination are not mediated by elevated neuroactive steroid biosynthesis.     

Zolpidem generalization and antagonism in male and female cynomolgus monkeys trained to discriminate 1.0 or 2.0 g/kg ethanol

Background: The subtypes of γ‐aminobutyric acid (GABA)_A receptors mediating the discriminative stimulus effects of ethanol in nonhuman primates are not completely identified. The GABA_A receptor positive modulator zolpidem has high, intermediate, and low activity at receptors containing α₁, α_2/3, and α₅ subunits, respectively, and partially generalizes from ethanol in several species. The partial inverse agonist Ro15‐4513 has the greatest affinity for α_4/6‐containing receptors, higher affinity for α₅‐ and lower, but equal, affinity for α₁‐ and α_2/3‐, containing GABA_A receptors, and antagonizes the discriminative stimulus effects of ethanol. Methods: This study assessed Ro15‐4513 antagonism of the generalization of zolpidem from ethanol in male (n = 9) and female (n = 8) cynomolgus monkeys (Macaca fascicularis) trained to discriminate 1.0 g/kg (n = 10) or 2.0 g/kg (n = 7) ethanol (i.g.) from water with a 30‐minute pretreatment interval. Results: Zolpidem (0.017 to 5.6 mg/kg, i.m.) completely generalized from ethanol (≥80% of total session responses on the ethanol‐appropriate lever) for 6/7 monkeys trained to discriminate 2.0 g/kg and 4/10 monkeys trained to discriminate 1.0 g/kg ethanol. Zolpidem partially generalized from 1.0 or 2.0 g/kg ethanol in 6/7 remaining monkeys. Ro15‐4513 (0.003 to 0.30 mg/kg, i.m., 5‐minute pretreatment) shifted the zolpidem dose–response curve to the right in all monkeys showing generalization. Analysis of apparent pK_B from antagonism tests suggested that the discriminative stimulus effects of ethanol common with zolpidem are mediated by low‐affinity Ro15‐4513 binding sites. Main effects of sex and training dose indicated greater potency of Ro15‐4513 in males and in monkeys trained to discriminate 1.0 g/kg ethanol. Conclusions: Ethanol and zolpidem share similar discriminative stimulus effects most likely through GABA_A receptors that contain α₁ subunits, however, antagonism by Ro15‐4513 of zolpidem generalization from the lower training dose of ethanol (1.0 g/kg) may involve additional zolpidem‐sensitive GABA_A receptor subtypes (e.g., α_2/3 and α₅).     

Stress and neurosteroids in adult and aged rats

—The progesterone derivative 3-hydroxy-5-pregnan-20 one (allopregnanolone/AP) and the deoxycorticosterone derivative 3-21-dihydroxy-5-pregnan-20 one (allotetrahydrodeoxycorticosterone/THDOC) are endogenous neuroactive steroids endowed with neuromodulatory actions in the central nervous system. Their best-characterized membrane–receptor-dependent action consists in the amplification of GABA-gated chloride currents mediated by specific interactions with the GABA_A receptor complex, which appears responsible for the pharmacological effects (anxiolytic, anticonvulsant, hypnotic/anaesthetic) of exogenously administered AP and THDOC. Several acute stress paradigms and different negative allosteric modulators (isoniazid and FG 7142) of GABA_A receptors time dependently increase brain and plasma concentrations of AP and THDOC only in intact or sham-operated but not in adrenalectomized–orchiectomized rats. These results suggest that acute stress and inhibitors of GABA_A receptors increase the brain and plasma neurosteroid concentrations via a reduction of the inhibitory action exerted by GABA on the hypothalamic–pituitary–adrenal axis. The comparison between the time course of the changes in GABA_A receptor function and of their behavioral correlates (proconflict behavior) and that of the changes of endogenous neuroactive steroids are consistent with the view that AP and THDOC may play a role in restoring the GABAergic tone to prestress conditions, by limiting the duration and the extent of its stress-induced reduction. The acute stress-elicited increase of AP and THDOC is observed in adult as well as in aged rats, which show a reduced basal GABAergic transmission and a greater response to the effect of stress in terms of their brain cortical neuroactive steroid concentrations than adult rats.     

Ethanol‐Induced Increase of Agouti‐Related Protein (AgRP) Immunoreactivity in the Arcuate Nucleus of the Hypothalamus of C57BL/6J,but not 129/SvJ,Inbred Mice

Background: The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor, pro‐opiomelanocortin (POMC). Previous research has shown that MC receptor (MCR) agonists reduce, and MCR antagonists increase, ethanol consumption in rats and mice. Consistently, genetic deletion of the endogenous MCR antagonist, agouti‐related protein (AgRP), causes reductions of ethanol‐reinforced lever pressing and binge‐like ethanol drinking in C57BL/6J mice. Ethanol also has direct effects on the central MC system, as chronic exposure to an ethanol‐containing diet causes significant reductions of α‐melanocyte stimulating hormone (α‐MSH) immunoreactivity in specific brain regions of Sprague‐Dawley rats. Together, these observations suggest that the central MC system modulates neurobiological responses to ethanol. To further characterize the role of the MC system in responses to ethanol, here we compared AgRP and α‐MSH immunoreactivity in response to an acute injection of saline or ethanol between high ethanol drinking C57BL/6J mice and moderate ethanol drinking 129/SvJ mice. Methods: Mice received an intraperitoneal (i.p.) injection of ethanol (1.5 g/kg or 3.5 g/kg; mixed in 0.9% saline) or an equivolume of 0.9% saline. Two hours after injection, animals were sacrificed and their brains were processed for AgRP and α‐MSH immunoreactivity. Results: Results indicated that acute ethanol administration triggered a dose‐dependent increase in AgRP immunoreactivity in the arcuate (ARC) of C57BL/6J mice, an effect that was not evident in the 129/SvJ strain. Although acute administration of ethanol did not influence α‐MSH immunoreactivity, C57BL/6J mice had significantly greater overall α‐MSH immunoreactivity in the ARC, dorsomedial, and lateral regions of the hypothalamus relative to the 129/SvJ strain. In contrast, C57BL/6J mice displayed significantly lower α‐MSH immunoreactivity in the medial amygdala. Conclusions: The results show that acute ethanol exposure has direct effects on endogenous AgRP activity in ethanol preferring C57BL/6J mice. It is suggested that ethanol‐induced increases in AgRP may be part of a positive feedback system that stimulates excessive binge‐like ethanol drinking in C57BL/6J mice. Inherent differences in α‐MSH immunoreactivity may contribute to differences in neurobiological responses to ethanol that are characteristically observed between the C57BL/6J and 129/SvJ inbred strains of mice.     

The μ opioid receptor is not involved in ethanol-stimulated dopamine release in the ventral striatum of C57BL/6J mice

Background: The mu opioid receptor (MOR) has previously been found to regulate ethanol‐stimulated dopamine release under some, but not all, conditions. A difference in ethanol‐evoked dopamine release between male and female mixed background C57BL/6J‐129SvEv mice led to questions about its ubiquitous role in these effects of ethanol. Using congenic C57BL/6J MOR knockout (KO) mice and C57BL/6J mice pretreated with an irreversible MOR antagonist, we investigated the function of this receptor in ethanol‐stimulated dopamine release. Methods: Microdialysis was used to monitor dopamine release and ethanol clearance in MOR ‐/‐, +/+, and +/? . male and female mice after intraperitoneal (i.p.) injections of 1.0, 2.0, and 3.0 g/kg ethanol (or saline). We also measured the increase in dopamine release after 5 mg/kg morphine (i.p.) in male and female MOR+/+ and ?/? mice. In a separate experiment, male C57BL/6J mice were pretreated with either the irreversible MOR antagonist beta funaltrexamine (BFNA) or vehicle, and dopamine levels were monitored after administration of 2 g/kg ethanol or 5 mg/kg morphine. Results: Although ethanol‐stimulated dopamine release at all the 3 doses of alcohol tested, there were no differences between MOR+/+, ?/?, and +/? mice in these effects. Female mice had a more prolonged effect compared to males at the 1 g/kg dose. Administration of 2 g/kg ethanol also caused a similar increase in dopamine levels in both saline‐pretreated and BFNA‐pretreated mice. Five mg/kg morphine caused a significant increase in dopamine levels in MOR+/+ mice but not in MOR?/? mice and in saline‐pretreated mice but not in BFNA‐pretreated mice. Intraperitoneal saline injections had a significant, albeit small and transient, effect on dopamine release when given in a volume equivalent to the ethanol doses, but not in a volume equivalent to the 5 mg/kg morphine dose. Ethanol pharmacokinetics were similar in all genotypes and both sexes at each dose and in both pretreatment groups. Conclusions: MOR is not involved in ethanol‐stimulated dopamine release in the ventral striatum of C57BL/6J mice.     

Ethanol self-administration and alterations in the livers of the cynomolgus monkey, Macaca fascicularis

Background: Most of the studies of alcoholic liver disease use models in which animals undergo involuntary administration of high amounts of ethanol and consume diets that are often high in polyunsaturated fatty acids. The objectives of this study were (1) to evaluate whether cynomolgus monkeys (Macaca fascicularis) drinking ethanol voluntarily and consuming a diet with moderate amounts of lipid would demonstrate any indices of alcoholic liver disease past the fatty liver stage and (2) to determine whether these alterations were accompanied by oxidative stress. Methods: Six adult male and 6 adult female cynomolgus monkeys were allowed to consume ethanol voluntarily for 18 to 19 months. Additional monkeys were maintained on the same consumption protocol, but were not provided with ethanol. During the course of the study, liver biopsy samples were monitored for lipid deposition and inflammation, serum for levels of liver enzymes, and urine for concentrations of the isoprostane (IsoP) metabolite, 2,3‐dinor‐5,6‐dihydro‐15‐F_2t‐IsoP, a biomarker for oxidative stress. Liver mitochondria were monitored for respiratory control and liver for concentrations of neutral lipids, adenine nucleotides, esterified F₂ isoprostanes, oxidized proteins, 4‐hydroxynonenal (HNE)‐protein adducts, and protein levels of cytochrome P‐450 2E1 and 3A4. Results: Ethanol consumption ranged from 0.9 to 4.05 g/kg/d over the period of the study. Serum levels of aspartate amino transferase were elevated in heavy‐consuming animals compared with those in ethanol‐naïve or moderate drinkers. Many of the ethanol consumers developed fatty liver and most showed loci of inflammation. Both hepatic energy charge and phosphorylation potential were decreased and NADH‐linked respiration was slightly, but significantly depressed in coupled mitochondria as a result of heavy ethanol consumption. The urinary concentrations of 2,3‐dinor‐5,6‐dihydro‐15‐F_2t‐IsoP increased as high as 33‐fold over that observed in ethanol‐abstinent animals. Liver cytochrome P‐450 2E1 concentrations increased in ethanol consumers, but there were no ethanol‐elicited increases in hepatic concentrations of the esterified F₂ isoprostanes, oxidized proteins, or HNE‐protein adducts. Conclusion: Our studies show that cynomolgus monkeys undergoing voluntary ethanol consumption for 1.5 years exhibit many of the features observed in the early stages of human alcoholic liver disease. Ethanol‐elicited fatty liver, inflammation, and elevated serum aspartate amino transferase were evident with a diet that contained modest amounts of polyunsaturated lipids. The dramatic increases in urinary IsoP demonstrated that the animals were being subjected to significant oxidative stress that correlated with their level of ethanol consumption.     

Soluble human p55 and p75 tumor necrosis factor receptors reverse spontaneous arthritis in transgenic mice expressing transmembrane tumor necrosis factor α

Objective

The roles of the transmembrane and secreted forms of tumor necrosis factor α (TNFα) in rheumatoid arthritis (RA) remain unclear. Agents used to inhibit TNFα have shown varying efficacy in RA patients, suggesting that anti‐TNFα agents possess dissimilar mechanisms of action, including the ability to neutralize transmembrane (tmTNFα) and secreted TNFα. In this study, TNFα‐knockout (TNFα‐KO) mice that were genetically altered to express elevated levels of tmTNFα were constructed to further understand the roles of the 17‐kd secreted, trimeric, and 26‐kd transmembrane forms of TNFα.

Methods

A speed‐congenic mating scheme was used to generate 3 unique strains of mice: 1) transgenic tmTgA86 mice overexpressing 26‐kd tmTNFα and also secreting 17‐kd trimeric TNFα (tmTNFα‐transgenic), 2) TNFα^−/− mice (TNFα‐KO), and 3) transgenic mice overexpressing tmTNFα backcrossed to TNFα‐KO mice (tmTNFα‐transgenic/TNFα‐KO). Mice were treated with phosphate buffered saline (as vehicle control), dexamethasone (as positive control), or modified recombinant human soluble TNF receptor (sTNFR) p55 or p75, and were assessed clinically and histopathologically for signs of inflammation and development of arthritis.

Results

The tmTNFα‐transgenic/TNFα‐KO mice were born with crinkled tails and spinal deformities similar to those in ankylosing spondylitis. By 2–4 weeks, these mice developed symmetric inflammatory arthritis, characterized by tissue swelling, pannus formation, and bone deformities. The tmTNFα‐transgenic mice also developed spontaneous‐onset arthritis, but at a slower rate (100% incidence by 10–12 weeks). Clinical and histologic progression of arthritis in the tmTNFα‐transgenic/TNFα‐KO mice was reduced by treatment with dexamethasone or with the p55 or p75 sTNFR (69% and 63% reduction in total histologic score, respectively).

Conclusion

These data show that arthritis is sufficiently initiated and maintained in tmTNFα‐transgenic/TNFα‐KO mice, and that it can be neutralized by recombinant human p55 or p75 sTNFR, resulting in amelioration of the biologic and subsequent histologic destructive effects of tmTNFα.

     

Variation in genes encoding the neuroactive steroid synthetic enzymes 5α-reductase type 1 and 3α-reductase type 2 is associated with alcohol dependence

Background: Studies of alcohol effects in rodents and in vitro implicate endogenous neuroactive steroids as key mediators of alcohol effects at GABA_A receptors. We used a case‐control sample to test the association with alcohol dependence (AD) of single nucleotide polymorphisms in the genes encoding two key enzymes required for the generation of endogenous neuroactive steroids: 5α–reductase, type I (5α‐R), and 3α‐hydroxysteroid dehydrogenase, type 2 (3α‐HSD), both of which are expressed in human brain. Methods: We focused on markers previously associated with a biological phenotype. For 5α‐R, we examined the synonymous SRD5A1 exon 1 SNP rs248793, which has been associated with the ratio of dihydrotestosterone to testosterone. For 3α‐HSD, we examined the nonsynonymous AKR1C3 SNP rs12529 (H5Q), which has been associated with bladder cancer. The SNPs were genotyped in a sample of 1,083 non‐Hispanic Caucasians including 552 controls and 531 subjects with AD. Results: The minor allele for both SNPs was more common among controls than subjects with AD: SRD5A1 rs248793 C‐allele (χ²(1) = 7.6, p = 0.006) and AKR1C3 rs12529 G‐allele (χ²(1) = 14.6, p = 0.0001). There was also an interaction of these alleles such that the “protective” effect of the minor allele at each marker for AD was conditional on the genotype of the second marker. Conclusions: We found evidence of an association with AD of polymorphisms in two genes encoding neuroactive steroid biosynthetic enzymes, providing indirect evidence that neuroactive steroids are important mediators of alcohol effects in humans.     

Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol

Background: Intermittent access (IA) to drugs of abuse, as opposed to continuous access, is hypothesized to induce a kindling‐type transition from moderate to escalated use, leading to dependence. Intermittent 24‐hour cycles of ethanol access and deprivation can generate high levels of voluntary ethanol drinking in rats. Methods: The current study uses C57BL/6J mice (B6) in an IA to 20% ethanol protocol to escalate ethanol drinking levels. Adult male and female B6 mice were given IA to 20% ethanol on alternating days of the week with water available ad libitum. Ethanol consumption during the initial 2 hours of access was compared with a short‐term, limited access “binge” drinking procedure, similar to drinking‐in‐the‐dark (DID). B6 mice were also assessed for ethanol dependence with handling‐induced convulsion, a reliable measure of withdrawal severity. Results: After 3 weeks, male mice given IA to ethanol achieved high stable levels of ethanol drinking in excess of 20 g/kg/24 h, reaching above 100 mg/dl blood ethanol concentrations, and showed a significantly higher ethanol preference than mice given continuous access to ethanol. Also, mice given IA drank about twice as much as DID mice in the initial 2‐hour access period. B6 mice that underwent the IA protocol for longer periods of time displayed more severe signs of alcohol withdrawal. Additionally, female B6 mice were given IA to ethanol and drank significantly more than males (ca. 30 g/kg/24 h). Discussion: The IA method in B6 mice is advantageous because it induces escalated, voluntary, and preferential per os ethanol intake, behavior that may mimic a cardinal feature of human alcohol dependence, though the exact nature and site of ethanol acting in the brain and blood as a result of IA has yet to be determined.     

Development of the Thyroid Hormone Receptor β‐Subtype Agonist KB‐141 : A Strategy for Body Weight Reduction and Lipid Lowering with Minimal Cardiac Side Effects

Few treatments for obesity exist and improvements for treatment of hyperlipidemia are still desirable. Thyroid hormone receptors (TRs) regulate body weight, adiposity, and cholesterol levels. However, thyroid hormones can have deleterious effects, particularly cardiac acceleration, that limits the use of hormones in the treatment of obesity. There is evidence that the TRβ subtype mediates lowering of blood cholesterol levels and possibly elevation of metabolic rate, whereas TRα appears to control heart rate. In studies, described in this review article, we examined the effects of selective TRβ activation on metabolic rate and heart rate in mice, rats and monkeys. T₃ had a greater effect on increasing heart rate in wild type (WT) than in TRα‐/‐ mice (ED₁₅ values of 34 and 469 nmol/kg/day, respectively). T₃ increased metabolic rate (MVO₂) in both WT and TRα‐/‐ mice, but the effect on TRα‐/‐ mice was less pronounced compared to WT mice. Stimulation of MVO₂ is mediated by both TRα and TRβ, but with different profiles. In cholesterol‐fed rats, KB‐141, a selective TRβ agonist, increased MVO₂ with a 10‐fold selectivity and lowered cholesterol with a 27‐fold selectivity vs. tachycardia. In primates, KB‐141 caused significant, cholesterol, Lp(a) and body weight reduction after 1 week of treatment with no effect on heart rate. These data suggest that selective TRβ agonists may represent a novel class of drugs for the treatment of obesity, hypercholesterolemia and elevated Lp(a), which may make them useful therapeutics for patients with metabolic syndrome.     

Effect of Ethanol on the Social Behavior of Group-Housed and Isolated Mice

The interaction between isolation and the response to ethanol was investigated with a social behavior test and a loss of righting reflex paradigm. The time a pair of mice spent in active social interaction, aggressive behavior, and avoidance-irritability showed an isolation-related increase, while locomotor activity was not affected by 0-10 days of isolation. Ethanol (0.8 g/kg) reduced the social interaction time in mice isolated for 10 days and 2.0 g/kg ethanol reduced it in all groups. Ethanol induced a locomotor stimulatory effect both in group-housed and isolated animals. An aggression-inducing effect of 0.8 g/kg ethanol was observed only in mice isolated for 5 days, while mice isolated for 10 days reduced their aggressive behavior after this dose of ethanol. The loss of righting reflex after 3.5 g/kg ethanol did not appear to vary with isolation. The results suggest that isolation alters some effects of ethanol qualitatively (e.g., its effects on aggressive behavior) but leaves other effects (e.g., its locomotor stimulant and hypnotic effects) unchanged.     

Period 2 Gene Deletion Abolishes β‐Endorphin Neuronal Response to Ethanol

Background: Ethanol exposure during early life has been shown to permanently alter the circadian expression of clock regulatory genes and the β‐endorphin precursor proopiomelanocortin (POMC) gene in the hypothalamus. Ethanol also alters the stress‐ and immune‐regulatory functions of β‐endorphin neurons in laboratory rodents. Our aim was to determine whether the circadian clock regulatory Per2 gene modulates the action of ethanol on β‐endorphin neurons in mice. Methods: Per2 mutant (mPer2^Brdml) and wild type (C57BL/6J) mice were used to determine the effect of Per2 mutation on ethanol‐regulated β‐endorphin neuronal activity during neonatal period using an in vitro mediobasal hypothalamic (MBH) cell culture model and an in vivo milk formula feeding animal model. The β‐endorphin neuronal activity following acute and chronic ethanol treatments was evaluated by measuring the peptide released from cultured cells or peptide levels in the MBH tissues, using enzyme‐linked immunosorbent assay (ELISA). Results: Per2 mutant mice showed a higher basal level of β‐endorphin release from cultured MBH cells and a moderate increase in the peptide content in the MBH in comparison with control mice. However, unlike wild type mice, Per2 mutant mice showed no stimulatory or inhibitory β‐endorphin‐secretory responses to acute and chronic ethanol challenges in vitro. Furthermore, Per2 mutant mice, but not wild type mice, failed to show the stimulatory and inhibitory responses of MBH β‐endorphin levels to acute and chronic ethanol challenges in vivo. Conclusions: These results suggest for the first time that the Per2 gene may be critically involved in regulating β‐endorphin neuronal function. Furthermore, the data revealed an involvement of the Per2 gene in regulating β‐endorphin neuronal responses to ethanol.     

Effects of Pregnanolone and Dehydroepiandrosterone on Ethanol Intake in Rats Administered Ethanol or Saline during Adolescence

Background: Adolescent alcohol use may contribute to long‐term changes in the receptors and neuroactive steroids that may mediate its effects and to subsequent alcohol abuse and dependence as an adult. Therefore, in this study, ethanol preference and intake as an adult were examined after adolescent ethanol or saline administration. In addition, ethanol intake in the same groups was examined after administration of 2 neuroactive steroids with modulatory effects at GABA_A receptors. Methods: Two groups of male Long‐Evans rats were administered 15 intraperitoneal (i.p.) injections of either ethanol (2 g/kg, 20% v/v) or saline between postnatal days 35 and 63. Starting on postnatal day 75, both groups were trained to consume 10% ethanol using a saccharin‐fading procedure, and ethanol intake and preference were measured after a series of manipulations involving food deprivation, changes in the duration of access to ethanol, and changes in the concentrations of ethanol presented. Following these manipulations, pregnanolone (1 to 10 mg/kg) and dehydroepiandrosterone (DHEA, 1 to 100 mg/kg) were administered prior to preference sessions with an 18% ethanol solution. Results: Adult ethanol preference and intake did not differ significantly in subjects treated with either saline or ethanol as adolescents during training, the substitution of other ethanol concentrations (3.2 to 32%), ad‐lib feeding, or moderate food deprivation. Pregnanolone administration altered the intake of both adolescent‐treated groups after the first injection of 3.2 mg/kg and after repeated injections with 10 mg/kg, a dose that produced sedation. In contrast, multiple doses of DHEA consistently decreased intake of an 18% ethanol concentration in both groups after repeated injections and 3 doses of DHEA (10, 32, and 56 mg/kg) administered with various ethanol concentrations dose‐dependently shifted the ethanol‐concentration curves for the volume and dosage of ethanol consumed downward. Conclusions: These results indicate that chronic intermittent ethanol (CIE) administration of 2 g/kg during adolescence did not alter preference or overall consumption of ethanol in outbred rats trained to drink ethanol as an adult under the conditions tested, and that DHEA may be more effective than pregnanolone at significantly decreasing ethanol consumption.     

Intermittent access to 20% ethanol induces high ethanol consumption in Long-Evans and Wistar rats

Background: There has been some difficulty getting standard laboratory rats to voluntarily consume large amounts of ethanol without the use of initiation procedures. It has previously been shown that standard laboratory rats will voluntarily consume high levels of ethanol if given intermittent‐access to 20% ethanol in a 2‐bottle‐choice setting [ Wise, Psychopharmacologia 29 (1973), 203 ]. In this study, we have further characterized this drinking model. Methods: Ethanol‐naïve Long–Evans rats were given intermittent‐access to 20% ethanol (three 24‐hour sessions per week). No sucrose fading was needed and water was always available ad libitum. Ethanol consumption, preference, and long‐term drinking behaviors were investigated. Furthermore, to pharmacologically validate the intermittent‐access 20% ethanol drinking paradigm, the efficacy of acamprosate and naltrexone in decreasing ethanol consumption were compared with those of groups given continuous‐access to 10 or 20% ethanol, respectively. Additionally, ethanol consumption was investigated in Wistar and out‐bred alcohol preferring (P) rats following intermittent‐access to 20% ethanol. Results: The intermittent‐access 20% ethanol 2‐bottle‐choice drinking paradigm led standard laboratory rats to escalate their ethanol intake over the first 5 to 6 drinking sessions, reaching stable baseline consumption of high amounts of ethanol (Long–Evans: 5.1 ± 0.6; Wistar: 5.8 ± 0.8 g/kg/24 h, respectively). Furthermore, the cycles of excessive drinking and abstinence led to an increase in ethanol preference and increased efficacy of both acamprosate and naltrexone in Long–Evans rats. P‐rats initiate drinking at a higher level than both Long–Evans and Wistar rats using the intermittent‐access 20% ethanol paradigm and showed a trend toward a further escalation in ethanol intake over time (mean ethanol intake: 6.3 ± 0.8 g/kg/24 h). Conclusion: Standard laboratory rats will voluntarily consume ethanol using the intermittent‐access 20% ethanol drinking paradigm without the use of any initiation procedures. This model promises to be a valuable tool in the alcohol research field.     

Reduced Sensitivity to Ethanol Reward, But Not Ethanol Aversion, in Mice Lacking 5=HT1B Receptors

Various serotonergic receptor systems are thought to influence the motivational effects of ethanol. This experiment characterized the acquisition of ethanol-induced conditioned taste aversion and ethanol-induced conditioned place preference in mutant knockout mice lacking 5-HT_1B receptors. In the taste conditioning procedure, adult homozygous knockout mice (-/-) and homozygous wild-type mice (+/+) received access to 0.2 M NaCl solution, followed immediately by intraperitoneal injection of 0 to 4 g/kg of ethanol. Ethanol produced dose-dependent conditioned taste aversion that was the same in both genotypes. In the place conditioning procedure, knockout and wild-type mice received six pairings of a tactile stimulus with ethanol (2 g/kg, ip). A different tactile stimulus was paired with saline. Ethanol produced increases in locomotor activity, with wild-type mice showing higher levels of ethanol-stimulated activity than knockout mice during conditioning trials 5 and 6. Wild-type mice demonstrated conditioned place preference for the ethanol-paired stimulus. In contrast, knockout mice showed no evidence of place conditioning. These results are generally consistent with an important role for serotonergic systems in ethanol reward and specifically indicate that 5-HT_1b receptors are important for ethanol's rewarding effects but not for ethanol's aversive effects.     

Neuroactive steroids and ethanol

This article presents the proceedings of a symposium presented at the International Society for Biomedical Research on Alcoholism 12th World Congress on Biomedical Alcohol Research, held in Heidelberg/Mannheim, Germany, from September 29 to October 2, 2004. The organizer and chairperson was Robert H. Purdy. The presentations were (1) Fetal ethanol-induced increase in brain levels of pregnenolone sulfate, by C. Fernando Valenzuela; (2) GABAergic neuroactive steroids after ethanol self-administration and relapse, by Patricia H. Janak; (3) Neuroactive steroid modulation of ethanol intake patterns in C57BL/6J mice, by Deborah A. Finn; (4) Role of neurosteroids in ethanol dependence and GABA_A receptor plasticity, by Giovanni Biggio; and (5) Alcohol and neuroactive steroid interactions in the menstrual cycle, by Torbjörn Bäckström.     

Drinking typography established by scheduled induction predicts chronic heavy drinking in a monkey model of ethanol self-administration

Background: We have developed an animal model of alcohol self‐administration that initially employs schedule‐induced polydipsia (SIP) to establish reliable ethanol consumption under open access (22 h/d) conditions with food and water concurrently available. SIP is an adjunctive behavior that is generated by constraining access to an important commodity (e.g., flavored food). The induction schedule and ethanol polydipsia generated under these conditions affords the opportunity to investigate the development of drinking typologies that lead to chronic, excessive alcohol consumption. Methods: Adult male cynomolgus monkeys (Macaca fascicularis) were induced to drink water and 4% (w/v in water) ethanol by a Fixed‐Time 300 seconds (FT‐300 seconds) schedule of banana‐flavored pellet delivery. The FT‐300 seconds schedule was in effect for 120 consecutive sessions, with daily induction doses increasing from 0.0 to 0.5 g/kg to 1.0 g/kg to 1.5 g/kg every 30 days. Following induction, the monkeys were allowed concurrent access to 4% (w/v) ethanol and water for 22 h/day for 12 months. Results: Drinking typographies during the induction of drinking 1.5 g/kg ethanol emerged that were highly predictive of the daily ethanol intake over the next 12 months. Specifically, the frequency in which monkeys ingested 1.5 g/kg ethanol without a 5‐minute lapse in drinking (defined as a bout of drinking) during induction strongly predicted (correlation 0.91) subsequent ethanol intake over the next 12 months of open access to ethanol. Blood ethanol during induction were highly correlated with intake and with drinking typography and ranged from 100 to 160 mg% when the monkeys drank their 1.5 g/kg dose in a single bout. Forty percent of the population became heavy drinkers (mean daily intakes >3.0 g/kg for 12 months) characterized by frequent “spree” drinking (intakes >4.0 g/kg/d). Conclusion: This model of ethanol self‐administration identifies early alcohol drinking typographies (gulping the equivalent of 6 drinks) that evolve into chronic heavy alcohol consumption in primates (drinking the equivalent of 16 to 20 drinks per day). The model may aid in identifying biological risks for establishing harmful alcohol drinking.     

Selected Line Difference in the Effects of Ethanol Dependence and Withdrawal on Allopregnanolone Levels and 5α-Reductase Enzyme Activity and Expression

Background: Allopregnanolone (ALLO) is a progesterone derivative that rapidly potentiates γ‐aminobutyric acid_A (GABA_A) receptor‐mediated inhibition and modulates symptoms of ethanol withdrawal. Because clinical and preclinical data indicate that ALLO levels are inversely related to symptoms of withdrawal, the present studies determined whether ethanol dependence and withdrawal differentially altered plasma and cortical ALLO levels in mice selectively bred for differences in ethanol withdrawal severity and determined whether the alterations in ALLO levels corresponded to a concomitant change in activity and expression of the biosynthetic enzyme 5α‐reductase. Methods: Male Withdrawal Seizure‐Prone (WSP) and ‐Resistant (WSR) mice were exposed to 72 hours ethanol vapor or air and euthanized at select times following removal from the inhalation chambers. Blood was collected for analysis of ALLO and corticosterone levels by radioimmunoassay. Dissected amygdala, hippocampus, midbrain, and cortex as well as adrenals were examined for 5α‐reductase enzyme activity and expression levels. Results: Plasma ALLO was decreased significantly only in WSP mice, and this corresponded to a decrease in adrenal 5α‐reductase expression. Cortical ALLO was decreased up to 54% in WSP mice and up to 46% in WSR mice, with a similar decrease in cortical 5α‐reductase activity during withdrawal in the lines. While cortical gene expression was significantly decreased during withdrawal in WSP mice, there was a 4‐fold increase in expression in the WSR line during withdrawal. Hippocampal 5α‐reductase activity and gene expression was decreased only in dependent WSP mice. Conclusions: These results suggest that there are line and brain regional differences in the regulation of the neurosteroid biosynthetic enzyme 5α‐reductase during ethanol dependence and withdrawal. In conjunction with the finding that WSP mice exhibit reduced sensitivity to ALLO during withdrawal, the present results are consistent with the hypothesis that genetic differences in ethanol withdrawal severity are due, in part, to modulatory effects of GABAergic neurosteroids such as ALLO.