Differential Effects of Ethanol on Serum GABAergic 3α,5α/3α,5β Neuroactive Steroids in Mice,Rats, Cynomolgus Monkeys,and Humans

Background: Acute ethanol administration increases plasma and brain levels of progesterone and deoxycorticosterone‐derived neuroactive steroids (3α,5α)‐3‐hydroxypregnan‐20‐one (3α,5α‐THP) and (3α,5α)‐3,21‐dihydroxypregnan‐20‐one (3α,5α‐THDOC) in rats. However, little is known about ethanol effects on GABAergic neuroactive steroids in mice, nonhuman primates, or humans. We investigated the effects of ethanol on plasma levels of 3α,5α‐ and 3α,5β‐reduced GABAergic neuroactive steroids derived from progesterone, deoxycorticosterone, dehydroepiandrosterone, and testosterone using gas chromatography‐mass spectrometry. Methods: Serum levels of GABAergic neuroactive steroids and pregnenolone were measured in male rats, C57BL/6J and DBA/2J mice, cynomolgus monkeys, and humans following ethanol administration. Rats and mice were injected with ethanol (0.8 to 2.0 g/kg), cynomolgus monkeys received ethanol (1.5 g/kg) intragastrically, and healthy men consumed a beverage containing 0.8 g/kg ethanol. Steroids were measured after 60 minutes in all species and also after 120 minutes in monkeys and humans. Results: Ethanol administration to rats increased levels of 3α,5α‐THP, 3α,5α‐THDOC, and pregnenolone at the doses of 1.5 g/kg (+228, +134, and +860%, respectively, p < 0.001) and 2.0 g/kg (+399, +174, and +1125%, respectively, p < 0.001), but not at the dose of 0.8 g/kg. Ethanol did not alter levels of the other neuroactive steroids. In contrast, C57BL/6J mice exhibited a 27% decrease in serum 3α,5α‐THP levels (p < 0.01), while DBA/2J mice showed no significant effect of ethanol, although both mouse strains exhibited substantial increases in precursor steroids. Ethanol did not alter any of the neuroactive steroids in cynomolgus monkeys at doses comparable to those studied in rats. Finally, no effect of ethanol (0.8 g/kg) was observed in men. Conclusions: These studies show clear species differences among rats, mice, and cynomolgus monkeys in the effects of ethanol administration on circulating neuroactive steroids. Rats are unique in their pronounced elevation of GABAergic neuroactive steroids, while this effect was not observed in mice or cynomolgus monkeys at comparable ethanol doses.     

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.     

The Reinforcing Effects of Ethanol Are Altered by the Endogenous Neurosteroid, Allopregnanolone

We examined the effect of systemic administration of the endog-enously occurring progesterone metabolite, allopregnanolone, on oral self-administration of ethanol by male rats. Rats were trained to perform an operant response for presentation of 0.1 ml of a solution of 10% ethanol in water using the sucrose fading technique. After acquisition of stable lever-press responding on a fixed-ratio 4 schedule, subjects received subcutaneous injections of 1,3, or 10 mg/kg of allopregnanolone, or vehicle, 20 min prior to the self-administration session. Pretreatment with 3 mg/kg, but not 1 or 10 mg/kg, increased the mean total number of lever press responses made to obtain ethanol, and therefore increased the mean total number of ethanol presentations. The number of responses and response rate were examined as a function of the number of “runs” within the 30-min session; a “run” was defined as a series of consecutive responses with an interresponse interval of <1 min. The increase in total responses after 3 mg/kg was due in part to an increased number of responses for the first run of the session, with no effect on response rates. However, the higher dose of 10 mg/kg decreased response rates within the first run. Thus, allopregnanolone alters ethanol-reinforced responding at concentrations lower than those that depress rates of responding. The effects of administration of the ben-zodiazepene, diazepam, were determined for comparison with those of the neurosteroid. The subcutaneous injection of 0.3, 1.0, or 3.0 mg/kg of diazepam did not produce any clear dose-dependent changes in measures of ethanol-reinforced operant responding, supporting the suggestion of differences in the contribution of the benzodiazepene and neurosteroid binding sites to GABA_A receptor function. The results indicate that exogenous administration of allopregnanolone dose-dependently alters ethanol-reinforced operant responding, and suggest that this endogenously occurring neurosteroid could mediate some of the reinforcing effects of ethanol.     

A Rationale for Allopregnanolone Treatment of Alcohol Use Disorders: Basic and Clinical Studies

For many years, research from around the world has suggested that the neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (allopregnanolone or 3α,5α-THP) may have therapeutic potential for treatment of various symptoms of alcohol use disorders (AUDs). In this critical review, we systematically address all the evidence that supports such a suggestion, delineate the etiologies of AUDs that are addressed by treatment with allopregnanolone or its precursor pregnenolone, and the rationale for treatment of various components of the disease based on basic science and clinical evidence. This review presents a theoretical framework for understanding how endogenous steroids that regulate the effects of stress, alcohol, and the innate immune system could play a key role in both the prevention and the treatment of AUDs. We further discuss cautions and limitations of allopregnanolone or pregnenolone therapy with suggestions regarding the management of risk and the potential for helping millions who suffer from AUDs.     

Behavioral Consequences of Repeated Nicotine During Adolescence in Alcohol-Preferring AA and Alcohol-Avoiding ANA Rats

Background: Epidemiological studies suggest that exposure to nicotine at adolescent age is associated with increased potential to use alcohol and that genetic predisposition may further increase the risk. The present study addressed adolescent vulnerability to repeated nicotine exposure and its influence on subsequent ethanol self‐administration by investigating interactions between nicotine‐induced behavioral sensitization and voluntary ethanol consumption in alcohol preferring AA (Alko Alcohol) and alcohol nonpreferring ANA (Alko Non‐Alcohol) rat lines selected for differential ethanol intake. Methods: Adolescent and adult rats received 10 injections of nicotine (0.5 mg/kg s.c.), given every second day from postnatal day (Pnd) 27 and 75, respectively. Nicotine‐induced (0.5 mg/kg) locomotor activity was measured acutely after the first injection, and after the repeated treatment with nicotine on Pnds 52 and 86 in the adolescent groups and on Pnd 99 in the adult groups. After this, acquisition of voluntary ethanol (10% v/v) consumption as well as nicotine‐induced (0.5 mg/kg) ethanol intake was measured in the AA rats. Results: Adolescent AA rats were more sensitive than adolescent ANA rats to the locomotor effects of nicotine. They were also stimulated more than adult AA rats, but such a difference was not found among ANA rats. Adolescent and adult rats did not differ in their susceptibility to nicotine‐induced behavioral sensitization. Genetic predisposition to ethanol self‐administration did not interact with development of behavioral sensitization in either adolescents or adults. Acquisition of ethanol intake was enhanced in the adolescent groups relative to the adult groups in a manner that was independent of the nicotine treatment. An increase in ethanol intake was found after challenging animals with nicotine, and this effect was enhanced in the nicotine‐treated adolescent group. Conclusions: These findings provide no or little support for the views that adolescent animals are more sensitive to the neurobehavioral effects of repeated exposure to nicotine and that exposure to nicotine in adolescence may contribute to enhanced vulnerability to ethanol abuse. Furthermore, genetic predisposition to high or low ethanol self‐administration does not seem to be a factor that influences individual vulnerability to the neurobehavioral effects of repeated administration of nicotine.     

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.     

Alteration of ethanol drinking in mice via modulation of the GABA(A) receptor with ganaxolone, finasteride, and gaboxadol

Background: Neurosteroids and other γ‐aminobutyric acid_A (GABA_A) receptor–modulating compounds have been shown to affect ethanol intake, although their mechanism remains unclear. This study examined how patterns of 24‐hour ethanol drinking in mice were altered with the synthetic GABAergic neurosteroid ganaxolone (GAN), with an inhibitor of neurosteroid synthesis (finasteride [FIN]), or a GABA_A receptor agonist with some selectivity at extrasynaptic receptors (gaboxadol HCL [THIP]). Methods: Male C57BL/6J mice had continuous access to a 10% v/v ethanol solution (10E) or water. Using lickometer chambers, drinking patterns were analyzed among mice treated in succession to GAN (0, 5, and 10 mg/kg), FIN (0 or 100 mg/kg), and THIP (0, 2, 4, 8, and 16 mg/kg). Results: GAN shifted drinking in a similar but extended manner to previous reports using low doses of the neurosteroid allopregnanolone (ALLO); drinking was increased in hour 1, decreased in hours 2 and 3, and increased in hours 4 and 5 postinjection. THIP (8 mg/kg) and FIN both decreased 10E drinking during the first 5 hours postinjection by 30 and 53%, respectively, while having no effect on or increasing water drinking, respectively. All 3 drugs altered the initiation of drinking sessions in a dose‐dependent fashion. FIN increased and GAN decreased time to first lick and first bout. THIP (8 mg/kg) decreased time to first lick but increased time to first bout and attenuated first bout size. Conclusions: The present findings support a role for the modulation of ethanol intake by neurosteroids and GABA_A receptor–acting compounds and provide hints as to how drinking patterns are shifted. The ability of THIP to alter 10E drinking suggests that extrasynaptic GABA_A receptors may be involved in the modulation of ethanol intake. Further, the consistent results with THIP to that seen previously with high doses of ALLO suggest that future studies should further examine the relationship between neurosteroids and extrasynaptic GABA_A receptors, which could provide a better understanding of the mechanism by which neurosteroids influence ethanol intake.     

Acute ethanol administration and acute allopregnanolone administration impair spatial memory in the Morris water task

BACKGROUND: Acute ethanol administration degrades performance on many learning and memory tasks, including tasks that are dependent on spatial information. One common test of spatial learning and memory is the Morris water task, a task that requires subjects to learn the spatial location of a submerged escape platform located in a pool of cloudy water. However, although some studies report that acute ethanol administration degrades spatial memory performance in the Morris task, other studies report no significant performance impairment. Acute ethanol administration also produces a dose- and time-dependent increase in the concentration of the endogenous neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) in rat brain. Given that ethanol and allopregnanolone are both gamma-aminobutyric acid type A receptor modulators, both drugs should produce similar degradations in spatial learning and memory. METHODS: Adult male rats were trained with either the spatial version or the nonspatial version of the Morris water task. After 4 days of training, the spatial or nonspatial memory performance of subjects was assessed after either an ethanol (1.0, 1.5, or 2.0 g/kg) or allopregnanolone (12.5, 17.0, or 20.0 mg/kg) challenge. RESULTS: Acute ethanol administration and acute allopregnanolone administration impaired spatial memory performance in a dose-dependent manner in the Morris water task. In addition, the impairment was selective in that neither acute ethanol nor acute allopregnanolone administration impaired nonspatial memory performance in the Morris water task. CONCLUSIONS: Acute ethanol administration and acute allopregnanolone administration impaired spatial memory performance but did not impair nonspatial memory performance in the Morris water task. These results demonstrate that both ethanol and allopregnanolone produce selective cognitive deficits that are not due to general sensory or motor deficits.     

Ethanol self-administration is regulated by CB1 receptors in the nucleus accumbens and ventral tegmental area in alcohol-preferring AA rats

Background: Endogenous cannabinoids and their receptors, CB1 receptors in particular, have been implicated in mediation of ethanol reinforcement. Previously, suppression of ethanol drinking by CB1 antagonists has been demonstrated in many experimental paradigms. However, the exact mechanism by which CB1 antagonists modulate ethanol drinking remains elusive. In the present study, we assessed the role of CB1 receptors within the key regions of the mesolimbic dopamine pathway, the nucleus accumbens (NAcc) and ventral tegmental area (VTA), in regulation of ethanol self‐administration. Methods: Adult male alcohol‐prefer AA rats were trained to self‐administer either 10% (w/v) ethanol or 0.1% (w/v) saccharin under an FR1 schedule during daily 30‐minute sessions. Following stable baseline responding, rats were tested after systemic administration of the CB1 antagonist SR141716A (0 to 10 mg/kg) and the agonist WIN55,212‐2 (0 to 2 mg/kg). Separate groups of rats were implanted with bilateral cannulas aimed at the NAcc or VTA, and tested after microinjections of SR141716A (0 to 3 μg) and WIN55,212‐2 (0 to 5 μg) into the NAcc or VTA. The highest intracerebral doses were tested also in rats responding for a 0.1% saccharin solution. Results: SR141617A dose‐dependently suppressed ethanol responding after systemic administration. Microinjections of SR141617A both into NAcc and VTA attenuated ethanol responding. In addition, intra‐NAcc injections of SR141617A suppressed saccharin intake. Although low doses of systemically given WIN55,212‐2 increased ethanol responding, no effects were seen after WIN55,212‐2 microinjections into NAcc or VTA. Conclusions: Bidirectional changes in ethanol self‐administration by the systematically administered CB1 agonist and antagonist show that ethanol reinforcement is controlled by CB1 receptors in alcohol‐preferring AA rats. Replication of the suppressive effects by CB1 antagonism in the NAcc and VTA suggests that endocannabinoids and their receptors mediate ethanol reinforcement through interaction with the mesolimbic dopamine pathway.     

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.     

Effects of Ethanol, MK-801, and Chlordiazepoxide on Locomotor Activity in Different Rat Lines: Dissociation of Locomotor Stimulation from Ethanol Preference

Several lines of research have suggested a link between the reward value of a drug and its ability to stimulate locomotion. One goal of the present study was to determine whether ethanol preferentially stimulates locomotor activity in lines of rat that show a preference for ethanol. A secondary goal was to determine the extent to which the benzodiazepine-like and NMDA antagonistic action of ethanol accounted for its effect on locomotor activity. To meet these goals, the effects of varying doses of ethanol (0.125-1.0 g/kg), MK-801 (0.1-0.3 mg/kg), and chlordiazepoxide (0.3-3 mg/kg) on locomotor activity were studied in several lines of rats that had been habituated to the testing procedure. The effect of low doses of ethanol on motor activity in the Alcohol-Preferring (P) and Fawn-Hooded rats, which show a strong ethanol preference, were similar to those of the alcohol-nonpreferring (NP), Flinders Sensitive Line, and Flinders Resistant Line rats. Only the Flinder Resistant Line rats showed a small, but significant increase in locomotor activity after the administration of ethanol. The highest dose of ethanol (1.0 g/kg) produced locomotor depression in all lines except the P and NP lines, which were not tested at this dose. These findings do not support a link between locomotor stimulation by ethanol and ethanol preference. In contrast, all lines exhibited locomotor stimulation after moderate (0.1-0.3 mg/kg) doses of MK-801, but did not exhibit increases in activity following any dose of chlordiazepoxide. These data indicate that the profiles of activity after MK-801 and chlordiazepoxide were distinct from that of ethanol in the various rat lines. Therefore, the effects of ethanol on locomotor activity cannot be accounted for by reference solely to its antagonist-like action at NMDA receptors and/or its agonist-like action at GABA/benzodiazepine receptors.     

Neuroactive steroid-serotonergic interaction: responses to an intravenous L-tryptophan challenge in women with premenstrual syndrome.

OBJECTIVE: To evaluate the circulating concentrations of the neuroactive steroids in response to an i.v. L-tryptophan (L-TP) challenge across the menstrual cycle in women with premenstrual syndrome (PMS) and in controls. METHOD: An i.v. L-TP challenge was administered eight times during 1 month to five women with prospectively documented PMS and five age- and body mass-matched controls. Progesterone, allopregnanolone pregnenolone and 3alpha-5alpha-tetrahydrocorticosterone were assessed 15 and 0 min before, and at 30, 60 and 90 min after the challenge, across the menstrual cycle. RESULTS: In response to L-TP challenge, only allopregnanolone concentrations were significantly increased across the cycle and this increase was of a greater magnitude in women with PMS. Pregnenolone and 3alpha-5alpha-tetrahydrocorticosterone concentrations were not affected in women with PMS or controls after L-TP challenge. CONCLUSIONS: The data provide evidence for possible interaction between the serotonergic system and the neuroactive steroid, allopregnanolone. Women with PMS demonstrated a more significant increase in allopregnanolone concentrations in response to L-TP challenge, which could be due to an initial low basal serotonergic tone in the luteal phase in the PMS group.     

Naltrexone Selectively Elevates GABAergic Neuroactive Steroid Levels in Heavy Drinkers With the ASP40 Allele of the OPRM1 Gene: A Pilot Investigation

Background: Preclinical studies have implicated GABAergic neurosteroids in behavioral responses to alcohol. Naltrexone is thought to blunt the reinforcing effects of alcohol, and a few studies have found that the effects of naltrexone are moderated by the Asn40Asp polymorphisms of the OPRM1 gene. The present study seeks to integrate these lines of research by testing (i) the moderating role of the functional Asn40Asp polymorphism of the OPRM1 gene on naltrexone‐induced alternations in GABAergic neurosteroid levels, namely (3α,5α)‐3‐hydroxypregnan‐20‐one (allopregnanolone, ALLO); and (ii) the combined effects of naltrexone or genotype with alcohol administration on neurosteroid levels in a sample of at‐risk drinkers. Methods: Participants were 32 (9 females) nontreatment‐seeking heavy drinkers who completed a placebo‐controlled laboratory study of naltrexone (50 mg/d for 3 days) and provided complete sets of serum samples for ALLO assays before and after alcohol administration under both naltrexone and placebo conditions. Results: Naltrexone treatment raised ALLO levels among carriers of the Asp40 allele, but not homozygotes for the Asn40 allele. The Asn40Asp polymorphism did not moderate effects of naltrexone on cortisol levels. Ethanol infusion modestly reduced ALLO levels in all subjects, independent of genotype or naltrexone exposure. Conclusions: Naltrexone increased ALLO levels among individuals with the Asn40Asp allele suggesting a potential neurosteroid contribution to the neuropharmacological effects of naltrexone among Asp40 carriers.     

Sex-dependent effect of a low neurosteroid environment and intrauterine growth restriction on foetal guinea pig brain development

Progesterone and its neuroactive metabolite, allopregnanolone, are present in high concentrations during pregnancy, but drop significantly following birth. Allopregnanolone influences foetal arousal and enhances cognitive and behavioural recovery following traumatic brain injury. Inhibition of allopregnanolone synthesis increases cell death in foetal animal brains with experimental hypoxia. We hypothesised that complications during pregnancy, such as early or preterm loss of placental steroids and intrauterine growth restriction (IUGR), would disrupt the foetal neurosteroid system, contributing to poor neurodevelopmental outcomes. This study aimed to investigate the effects of chronic inhibition of allopregnanolone synthesis before term and IUGR on developmental processes in the foetal brain. Guinea pig foetuses were experimentally growth restricted at mid-gestation and treated with finasteride, an inhibitor of allopregnanolone synthesis. Finasteride treatment reduced foetal brain allopregnanolone concentrations by up to 75% and was associated with a reduction in myelin basic protein (MBP) (P = 0.001) and an increase in glial fibrillary acidic protein expression in the subcortical white matter brain region (P < 0.001). IUGR resulted in decreased MBP expression (P < 0.01) and was associated with a reduction in the expression of steroidogenic enzyme 5α-reductase (5αR) type 2 in the foetal brain (P = 0.061). Brain levels of 5αR1 were higher in male foetuses (P = 0.008). Both IUGR and reduced foetal brain concentrations of allopregnanolone were associated with altered expression of myelination and glial cell markers within the developing foetal brain. The potential role of neurosteroids in protecting and regulating neurodevelopmental processes in the foetal brain may provide new directions for treatment of neurodevelopmental disorders in infants who are exposed to perinatal insults and pathologies.     

Low-dose stimulatory effects of ethanol during adolescence in rat lines selectively bred for high alcohol intake

Abstract : Background: The low‐dose stimulatory effect of ethanol (EtOH) in rats has been hypothesized to reflect its hedonic effects and to be associated with a genetic predisposition toward high alcohol preference. To test the hypothesis that phenotypes associated with high alcohol preference in adulthood are also present in adolescent rats at the time of onset of alcohol drinking, the current study examined the effects of EtOH on locomotor activity (LMA) during adolescence in lines of rats selectively bred for divergent alcohol intakes. Methods: Subjects were adolescent (31–40 days of age) rats from the alcohol‐preferring (P) and ‐nonpreferring (NP) lines and from the high–alcohol‐drinking (HAD) and low–alcohol‐drinking (LAD) replicate lines. On day 1, all subjects (n= 8–10/line/gender/dose) received intraperitoneal saline injections and were placed in the activity monitor for 30 min. On day 2, subjects received intraperitoneal saline or 0.25, 0.50, 0.75, 1.0, or 1.5 g EtOH/kg. Results: The LMA of male and female P rats was increased with low doses (0.25–0.75 g/kg) and decreased at the highest dose (1.5 g/kg) of EtOH. Similar effects were observed with low doses of EtOH on the LMA of HAD‐1 and HAD‐2 rats. None of the EtOH doses stimulated LMA in the NP, LAD‐1, or LAD‐2 rats, although all of the low–alcohol‐intake lines of rats showed decreased LMA at the highest dose of EtOH. Only the P rats among the high–alcohol‐consuming lines of rats showed decreased LMA at the highest dose of EtOH. Conclusion: Selective breeding for high alcohol consumption seems to be associated with increased sensitivity to the low‐dose stimulating effects of EtOH and reduced sensitivity to the high‐dose motor‐impairing effects of ethanol. The expression of these phenotypes emerges during adolescence by the age of onset of alcohol‐drinking behavior.     

Relationship between ethanol's acute locomotor effects and ethanol self-administration in male Long-Evans rats

Background: Human studies have suggested an important relationship between ethanol sensitivity and risk of alcoholism. These studies have led some to hypothesize that a low initial sensitivity to ethanol’s depressant effects and/or an elevated response to ethanol’s stimulant effects may represent important risk factors associated with the development of abusive drinking behavior. Unfortunately, elucidating neurobiologic mechanisms that may underlie these relationships between ethanol sensitivity and ethanol drinking have been hampered by difficulties in modeling some of these interactions in animals. In this study, we re‐examined some of these relationships in an outbred strain of rats using continuous access two‐bottle choice drinking and a limited‐access operant procedure that engenders pharmacologically relevant levels of ethanol intake and permits the discrete assessment of appetitive and consummatory measures of ethanol drinking behavior. Methods: Twenty‐three male Long‐Evans rats were habituated to a locomotor activity box and then tested for their response to a stimulant (0.5 g/kg) and depressant (1.5 g/kg) ethanol dose. Rats were then trained to complete a lever pressing requirement to gain access to 10% ethanol for 20‐minute sessions conducted 5 d/wk for 5 weeks. Appetitive behavior was assessed after 2.5 and 4.5 weeks using 20‐minute extinction trials in which ethanol was not presented and lever responses were recorded. Home‐cage ethanol preference was also assessed prior to and immediately following the 5‐week self‐administration regimen using a continuous access, two‐bottle choice procedure. Results: A significant increase in home‐cage ethanol preference was observed following the self‐administration procedure, however, neither measure of ethanol preference correlated with average daily ethanol intake during the operant self‐administration sessions or with initial sensitivity to ethanol’s stimulant or depressant effects. Notably, a significant negative correlation was observed between sensitivity to ethanol’s locomotor depressant effect and daily intake during the operant self‐administration sessions. No significant relationships were noted between sensitivity to ethanol’s locomotor effects and extinction responding. Conclusions: The results of these studies suggest that the well‐established relationship between a low level of response to ethanol and increased ethanol consumption reported in human studies can be observed in an outbred rodent strain using a limited‐access operant self‐administration procedure, but not with home‐cage ethanol drinking.     

The Alcohol Deprivation Effect in C57BL/6J Mice is Observed Using Operant Self-Administration Procedures and is Modulated by CRF-1 Receptor Signaling

Background: The alcohol deprivation effect (ADE) is characterized by transient excessive alcohol consumption upon reinstatement of ethanol following a period of ethanol deprivation. While this phenomenon has been observed in rats using both bottle drinking (consummatory behavior) and operant self‐administration (consummatory and appetitive “ethanol‐seeking” behavior) procedures, ADE studies in mice have primarily relied on bottle drinking measures. Furthermore, the neurochemical pathways that modulate the ADE are not well understood. Therefore, we determined whether the ADE can be observed in C57BL/6J mice using operant self‐administration procedures and if expression of the ADE is modulated by the corticotropin releasing factor‐1 (CRF‐1) receptor. Methods: C57BL/6J mice were trained in a 2‐hour operant self‐administration paradigm to lever press for 10% ethanol or water on separate response keys. Between operant sessions, mice had access to ethanol in their homecage. Once stable responding occurred, mice were deprived of ethanol for 4 days and were then retested with ethanol in the operant paradigm for 3 consecutive days. Next, to assess the role of the CRF‐1 receptor, mice were given intraperitoneal (i.p.) injection (0, 10, or 20 mg/kg) of the CRF‐1 receptor antagonist CP‐154,526 30 minutes before ADE testing. Additional experiments assessed (i) ADE responding in which the alternate response lever was inactive, (ii) the effects of CP‐154,526 on self‐administration of a 1% sucrose solution following 4 days of deprivation, and (iii) ADE responding in which mice did not received i.p. injections throughout the experiment. Results: Mice exhibited a significant increase in postdeprivation lever responding for ethanol with either a water reinforced or inactive alternate lever. Interestingly, i.p. injection of a 10 mg/kg dose of CP‐154,526 protected against the ADE while not affecting lever responding for a sucrose solution. Finally, baseline and deprivation‐induced increases of ethanol reinforced lever responding were greater in mice not given i.p. injections. Conclusions: The ADE in C57BL/6J mice can be modeled using the operant self‐administration paradigm and increased ethanol self‐administration associated with the ADE is modulated by CRF‐1 receptor signaling.     

Effects of allopregnanolone on the EEG of alcohol-preferring and alcohol-nonpreferring rats

BACKGROUND: Alcohol preferring (P) and alcohol-nonpreferring (NP) rats have been shown to have differing behavioral and electrophysiological responses to drugs that are positive modulators of the gamma-aminobutyric acid type A (GABA-A) receptor complex, such as ethanol and benzodiazepines. The neuroactive steroid allopregnanolone is also a positive modulator of GABA-A receptors; therefore, we hypothesized that P and NP rats would respond differently to intraperitoneally administered allopregnanolone. METHODS: Male P and NP rats were implanted with screw electrodes that overlay the frontal and parietal cortices and with a depth electrode aimed at the amygdala. Allopregnanolone (0.0-10.0 mg/kg ip) was administered 10 min before recording the EEG. RESULTS: Allopregnanolone increased high-frequency power (8-32 Hz) in the cortex and amygdala of both P rats and NP rats. In addition, allopregnanolone increased the predominant frequency of the cortical EEG in the 8 to 16 Hz bandwidth, decreased the predominant frequency in the 32 to 50 Hz bandwidth, and increased EEG variability (16-50 Hz). The effects of allopregnanolone were qualitatively similar in P and NP rats. However, P rats were more sensitive to low doses of allopregnanolone in cortex, whereas NP rats responded to lower doses of allopregnanolone in the amygdala. CONCLUSIONS: These data indicate that P and NP rats differ in their sensitivity to the EEG effects of allopregnanolone in the cortex and amygdala, which suggests that differences in GABAergic systems between P and NP rats may contribute to some of the differences observed in their behavioral repertoire.     

Regulation of operant oral ethanol self-administration: a dose-response curve study in rats

Background: Oral ethanol self‐administration procedures in rats are useful preclinical tools for the evaluation of potential new pharmacotherapies as well as for the investigation into the etiology of alcohol abuse disorders and addiction. Determination of the effects of a potential treatment on a full ethanol dose–response curve should be essential to predict its clinical efficacy. Unfortunately, this approach has not been fully explored because of the aversive taste reaction to moderate to high doses of ethanol, which may interfere with consumption. In this study, we set out to determine whether a meaningful dose–response curve for oral ethanol self‐administration can be obtained in rats. Methods: Long‐Evans rats were trained to self‐administer a 20% ethanol solution in an operant procedure following a history of excessive voluntary ethanol intake. After stabilization of ethanol self‐administration, the concentration of the solution was varied from 2.5 to 60% (v/v), and operant and drinking behaviors, as well as blood ethanol concentration (BEC), were evaluated following the self‐administration of a 20, 40, and 60% ethanol solution. Results: Varying the concentration of ethanol from 2.5 to 60% after the development of excessive ethanol consumption led to a typical inverted U‐shaped dose–response curve. Importantly, rats adapted their level and pattern of responding to changes in ethanol concentration to obtain a constant level of intake and BEC, suggesting that their operant behavior is mainly driven by the motivation to obtain a specific pharmacological effect of ethanol. Conclusion: This procedure can be a useful and straightforward tool for the evaluation of the effects of new potential pharmacotherapies for the treatment of alcohol abuse disorders.     

Suppression of Alcohol Intake after Administration of the Chinese Herbal Medicine, NPI-028, and Its Derivatives

The Chinese herbal medicine, NPI-028, has been used for centuries in China to counteract alcohol intoxication. The present study used a number of different experimental conditions to determine whether NPI-028 and its derivatives might selectively influence alcohol intake in rodents that naturally exhibit high alcohol intakes. It was determined that intraperitoneal (IP) injections of NPI-028 (0.5, 0.75, and 1.0 g/kg) suppressed alcohol intake by up to 30% in both alcohol-preferring P and Fawn-Hooded (FH) rats during a continuous access schedule. These injections did not significantly affect food or water intakes, nor did the highest dose of NPI-028 (1 g/kg) alter blood ethanol levels after an IP injection of 2.5 g/kg of ethanol. In P rats, it was found that NPI-028 was orally active with the dose of 1.5 g/kg having a greater effect on ethanol intake than the 1.0 g/kg dose; once again, food and water intakes were not significantly altered. In FH rats maintained on a limited access schedule (1 hr/day), alcohol intake was completely abolished by 1.5 g/kg of NPI-028. Chronic IP administration of NPI-028 (0.75 g/kg) for four consecutive days in FH rats maintained on a continuous access schedule did not lead to any diminution of its alcohol-suppressant effects. Thus, NPI-028 has significant effects on alcohol intake without much effect on water and food intake, and tolerance does not readily develop to these effects. The IP administration of a partially purified extract (NPI-031) of NPI-028, obtained by countercurrent chromatography, also dose-dependently suppressed ethanol intake in FH rats, but the highest dose (200 mg/kg) also significantly decreased food intake. Finally, the IP administration of puerarin (NPI-31G), an isoflavone isolated from NPI-031 by countercurrent chromatography, significantly reduced ethanol intake in FH rats without affecting food or water intake. Therefore, NPI-028 and one of its pure components, NPI-031G, selectively reduced ethanol intake in alcohol-preferring rats.