Effects of SR141716A on ethanol and sucrose self-administration

BACKGROUND: Previous studies have demonstrated that administration of central cannabinoid receptor (CB1) ligands can produce marked effects on ingestive behaviors. However, the possible relationship to ethanol self-administration has not been fully examined. The present series of experiments was designed to characterize further the role of CB1 receptors in appetitive and consummatory behaviors related to sucrose and ethanol. METHODS: To determine the relative contribution of CB1 receptors to ethanol seeking and consumption, a series of experiments was designed using the sipper-tube model. In this paradigm, the appetitive and consummatory phases of ethanol and sucrose self-administration are separated. In the appetitive phase, animals are required to complete a response requirement (16 lever presses) within 20 min. If the requirement is successfully completed, access to a sipper tube containing either sucrose or ethanol (consummatory phase) is made available for 20 min. RESULTS: In the ethanol condition, the CB1 receptor antagonist SR141716A (0.3-3.0 mg/kg, ip) produced dose-related decreases in the probability of response requirement completion without significantly affecting latency to first lever press or overall lever press rate. In the sucrose condition, SR141716A (0.3-3.0 mg/kg, ip) increased first lever press latency without affecting lever press rate. In the consummatory phase, SR141716A (0.3-3.0 mg/kg, ip) administration markedly decreased total intake and the total number of licks for both ethanol and sucrose. CONCLUSIONS: These data indicate that CB1 receptors are involved in mediating both appetitive and consummatory aspects of ingestive behaviors related to sucrose and ethanol.     

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.     

Endocannabinoid signaling via cannabinoid receptor 1 is involved in ethanol preference and its age-dependent decline in mice

Cannabinoids and ethanol can activate the same reward pathways, which could suggest endocannabinoid involvement in the rewarding effects of ethanol. The high ethanol preference of young (6-10 weeks) C57BL6J mice is reduced by the cannabinoid receptor 1 (CB1) antagonist SR141716A to levels observed in their CB1 knockout littermates or in old (26-48 weeks) wild-type mice, in both of which ethanol preference is unaffected by SR141716A. Similarly, SR141716A inhibits food intake in food-restricted young, but not old, wild-type mice. There are no age-dependent differences in the tissue levels of the endocannabinoids anandamide and 2-arachidonoylglycerol or the density of CB1 in the hypothalamus, limbic forebrain, amygdala, and cerebellum. CB1-stimulated guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]) binding is selectively reduced in the limbic forebrain of old compared with young wild-type mice. There is no age-dependent difference in G(i) or G(o) subunit protein expression in the limbic forebrain, and the selective reduction in GTP[gammaS] labeling in tissue from old mice is maintained in a receptorG protein reconstitution assay by using functional bovine brain G protein. These findings suggest that endocannabinoids acting at CB1 contribute to ethanol preference, and decreased coupling of CB1 to G proteins in the limbic forebrain by mechanisms other than altered receptor or G protein levels may be involved in the age-dependent decline in the appetite for both ethanol and food.     

Effects of ethanol and ethanol withdrawal on nociception in rats

The effect of acute and chronic administration of ethanol and ethanol withdrawal on a radiant heat tail-flick assay of nociception was examined in rats. Acute administration of ethanol (2.0 g/kg, i.p.) produced peak antinociception (68% of maximum) by 30 min, and effects were gone by 120 min. Cumulative doses of ethanol (0.5-2.0 g/kg, i.p.) produced dose-dependent increases in latencies to 49% of maximum. During chronic administration, a liquid diet containing ethanol (6.5%) was given for 10 days. Tail-flick latencies were measured on day 0 (baseline), day 2, 4, 6, 8, and 10 of chronic ethanol and at 3, 6, 12, and 36 hr after removal of ethanol. To test for behavioral tolerance, both between- and within-group designs were used. In both between- and within-group experiments, the antinociceptive effects of chronic ethanol peaked by day 4 of exposure to the liquid diet, and tolerance developed by day 10. When the liquid diet was removed, hyperalgesia was detected at 6 and 12 hr after withdrawal, and was gone by 36 hr after withdrawal. When cumulative doses of ethanol (0.5-2.0 g/kg) were administered starting 12 hr after withdrawal, ethanol (0.5 g/kg) fully reversed the hyperalgesia induced by ethanol withdrawal, even though this dose was without antinociceptive effect in the absence of withdrawal. Higher doses of ethanol during ethanol withdrawal did not increase tail-flick latencies over baseline. In summary: (1) ethanol produces antinociception when administered acutely or chronically; (2) tolerance to the antinociceptive effects develops during chronic administration; (3) ethanol withdrawal induced hyperalgesia, which was reversed by ethanol; and (4) repeated testing did not produce behavioral tolerance.     

Upregulation of cannabinoid type 1 receptors in dopamine D2 receptor knockout mice is reversed by chronic forced ethanol consumption

Background: The anatomical proximity of the cannabinoid type 1 (CNR1/CB1R) and the dopamine D2 receptors (DRD2), their ability to form CB1R–DRD2 heteromers, their opposing roles in locomotion, and their involvement in ethanol’s reinforcing and addictive properties prompted us to study the levels and distribution of CB1R after chronic ethanol intake, in the presence and absence of DRD2. Methods: We monitored the drinking patterns and locomotor activity of Drd2+/+ and Drd2?/? mice consuming either water or a 20% (v/v) ethanol solution (forced ethanol intake) for 6 months and used the selective CB1 receptor antagonist [³H]SR141716A to quantify CB1R levels in different brain regions with in vitro receptor autoradiography. Results: We found that the lack of DRD2 leads to a marked upregulation (approximately 2‐fold increase) of CB1R in the cerebral cortex, the caudate‐putamen, and the nucleus accumbens, which was reversed by chronic ethanol intake. Conclusions: The results suggest that DRD2‐mediated dopaminergic neurotransmission and chronic ethanol intake exert an inhibitory effect on cannabinoid receptor expression in cortical and striatal regions implicated in the reinforcing and addictive properties of ethanol.     

Nitrendipine blocks the nociceptive effects of chronically administered ethanol

BACKGROUND: Calcium channel blockers prevent the development of tolerance to ethanol and prevent seizures and other signs of ethanol withdrawal. Calcium channels may also mediate the development of tolerance to the antinociceptive effects of ethanol. METHODS: A radiant-heat tail-flick assay was used to assess the effects of nitrendipine, a dihydropyridine L-type calcium channel blocker, on ethanol-induced changes in nociception in rats. Chronic effects of ethanol were tested in four groups of rats, which received 10 days of exposure to a liquid diet. The groups received twice-daily injections of saline or nitrendipine (1, 5, or 10 mg/kg). Tail-flick latencies were measured on days 0 (baseline), 2, 4, 6, 8, and 10 and at 12 hr after removal of the liquid diet. RESULTS: Chronic exposure to ethanol produced maximal levels of antinociception by day 4 of exposure. The observed level of antinociception gradually decreased to baseline levels by day 8. Marked decreases in tail-flick latencies below baseline (hyperalgesia) were observed during ethanol withdrawal. Repeated administration of nitrendipine blocked the antinociceptive effects of chronic ethanol and prevented the hyperalgesia produced by ethanol withdrawal. The highest dose of nitrendipine (10 mg/kg, twice daily) reduced consumption of the ethanol diet. CONCLUSIONS: L-type calcium channel blockers block hyperalgesia during ethanol withdrawal and may mediate, at least in part, the antinociceptive effects of chronic ethanol.     

Voluntary ethanol drinking during the first three postnatal weeks in lines of rats selectively bred for divergent ethanol preference

BACKGROUND: Using a procedure first developed by Hall (1979), we examined ethanol self-administration in preweanling pups from Wistar rats and in lines of rats selectively bred for divergent ethanol preference (alcohol-preferring P, alcohol-nonpreferring NP, high-alcohol-drinking HAD-1 and -2, and low-alcohol-drinking LAD-2) to determine if factors contributing to high and low alcohol intakes are present early in development. METHODS: From postnatal days 5 to 20, nondeprived male and female rat pups received 30 min daily access to either water or a 15% (v/v) ethanol solution. In each daily session, pups were placed in a heated chamber containing Kimwipes soaked with a water or ethanol solution. Pups were weighed before and after each session, and intake levels were calculated as a percentage of body weight change. RESULTS: Similar to previous reports, Wistar pups exhibited over a 2-fold higher level of ethanol ingestion than water on postnatal days 9 through 14, with ethanol intakes approaching 3 g/kg body weight. When the drinking patterns of the selected lines were examined, only the HAD replicate lines showed a comparable preference for ethanol versus water during the preweanling period. The ethanol consumption of P, NP, and LAD lines was not consistently distinguishable from water intake levels. To reveal whether early ethanol exposure would influence later ethanol drinking behavior, a subset of HAD and LAD rat pups received free-choice ethanol access upon weaning. Although the divergent ethanol preference between high- and low-alcohol-drinking lines was evident within the first 4 days of access (>4 g/kg/day for HAD; <2 g/kg/day for LAD), preweanling ethanol exposure did not alter the acquisition or maintenance of ethanol drinking in either line. CONCLUSIONS: Overall, these results suggest that (a) the enhanced ethanol ingestion observed during the middle portion of the preweanling period is a robust phenomenon and generalizes across nonselected strains of rats, (b) selective breeding for divergent alcohol preference has arrested this age-specific effect in all but the HAD lines of rats, and (c) early ethanol exposure does not alter genetic dispositions for later high or low alcohol preference.     

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.     

Selective CB1 cannabinoid receptor antagonist, SR141716A, attenuates liver injury induced by Concanavalin A

Aim:  The aim of this study was to investigate the hepatoprotective activity of a selective cannabinoid receptor 1 (CB1) antagonist, SR141716A, in a Concanavalin A (Con A)-induced mouse liver injury model and to determine whether SR141716A has an effect on the production of inflammatory cytokines and chemokines induced by Con A.
Results:  Injection of Con A (20 mg/kg) to mice developed hepatitis determined by plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation and necrosis in the liver. Pretreatment with SR141716A (30 mg/kg) significantly reduced plasma AST and ALT level, protected against necrosis in the liver, and significantly reduced plasma cytokine and chemokine levels, including TNFα, IFN-γ, CXCL9, MIP1-α, and IL-10 and no change decreased in IL-4.
Conclusions:  The selective CB1 antagonist, SR141716A, exerts a hepatoprotective effect on Con A-induced liver injury in mice by attenuating the increase in cytokine and chemokine levels and inhibiting hepatocyte injury. These findings raise the possibility of using CB1 antagonists as anti-inflammatory drugs for treating hepatitis as well as other inflammatory diseases.     

Effects of benzodiazepines on acute and chronic ethanol-induced nociception in rats

BACKGROUND: Acute and chronic ethanol produces antinociception, and ethanol withdrawal induces hyperalgesia. METHODS: A radiant heat tail-flick assay was used to assess the effects of benzodiazepine ligands on ethanol-induced changes in nociception in rats. Acute activity of cumulative doses of ethanol (0.5-2.0 g/kg) and diazepam (0.1-10 mg/kg), a benzodiazepine agonist, was tested alone and after pretreatment with flumazenil (1.0-10 mg/kg), a benzodiazepine antagonist. Chronic effects of ethanol were tested in three groups of rats that received a liquid diet for 10 days. One group received ethanol alone; one group received ethanol and twice-daily injections of flumazenil (10 mg/kg); and one received a dextrin control diet. Acute withdrawal was tested at 12 hr after removal of the liquid diet. Effects of cumulative doses of diazepam (1.0-10 mg/kg) were tested during withdrawal (12 hr) in the ethanol-alone group. RESULTS: Acute doses of ethanol produced a small but significant degree of antinociception, which was fully suppressed by flumazenil. Acute doses of diazepam did not produce antinociception. Chronic exposure to ethanol produced antinociception on days 2 through 8. Tolerance developed by day 10, and hyperalgesia was seen 12 hr after removal of ethanol. Administration of diazepam or ethanol during withdrawal reversed the hyperalgesia induced by ethanol withdrawal. However, flumazenil (10-50 mg/kg) failed to reverse the antihyperalgesic effect of either diazepam or ethanol. No antinociception was seen in either the ethanol/flumazenil or dextrin control groups. CONCLUSIONS: These results suggest that the antinociceptive effects of both acute and chronic ethanol are at least partially mediated by GABA receptors, and that diazepam's antihyperalgesic effects may not be mediated by the GABA acid receptor.     

The endogenous cannabinoid, anandamide, activates the hypothalamo-pituitary-adrenal axis in CB1 cannabinoid receptor knockout mice

The purpose of this study was to investigate the effects of the endogenous cannabinoid arachidonoyl-ethanolamide, anandamide (AEA), on the activity of the hypothalamo-pituitary-adrenal (HPA) axis in cannabinoid receptor (CB(1) receptor) inactivated (KO) mice. A low dose (0.01 mg/kg i.p.) of AEA significantly increased plasma corticotropin (ACTH) and corticosterone concentrations in both wild-type (+/+) and in mutant (-/-) animals. In each case, hormone levels reached their peaks at 90 min after AEA administration. In a parallel experiment, AEA administration was preceded by the injection of SR 141716A (1.0 mg/kg), a selective and potent CB(1) receptor antagonist, or of capsazepine (5.0 mg/kg), a potent vanilloid receptor of type 1 (VR1) antagonist. The latter drugs did not prevent the effects of AEA on the HPA axis. Using Fos protein immunohistochemistry, we observed that the parvocellular part of the hypothalamic paraventricular nucleus (PVN) was activated as early as 45 min after AEA injection and reached peak levels after 60 min in both +/+ and -/- mice. Furthermore, the CB(1) and VR1 receptor antagonists did not block the effects of AEA on Fos immunoreactivity. The results strongly support the view that activation of the HPA axis produced by AEA possibly occurs via a currently unknown (CB(x)) cannabinoid receptor present in PVN.     

Free-choice alcohol consumption in mice after application of the appetite regulating peptide leptin

BACKGROUND: Leptin has been shown to regulate food intake and energy expenditure. Very recently, associations of elevated leptin plasma levels during alcohol withdrawal with alcohol craving have been observed in humans. Therefore, we tested the hypothesis that the application of exogenous leptin modulates voluntary alcohol consumption in mice. METHODS: Sixteen mice (129/Sv x C57BL/6J) were habituated to ethanol consumption over a time period of 3 months. After a basal 2-week free-choice drinking phase, mice were separated into two groups (n = 8) according to weight and alcohol consumption. They received recombinant leptin (1 mg/kg) versus saline intraperitoneally daily for 10 days. After 4 days of free-choice consumption of ethanol (16% v/v) versus water, ethanol was withdrawn at day 4 and replaced at day 6 to test the occurrence of an alcohol deprivation effects. Fluid intake was evaluated by controlling the weight of the drinking tubes daily. RESULTS: Free-choice ethanol consumption after withdrawal was significantly elevated in mice after intraperitoneal injection of 1 mg/kg leptin (alcohol deprivation effect), but not during basal drinking. CONCLUSION: We suggest that leptin may enhance motivation for alcohol consumption in habituated mice after alcohol withdrawal.     

Chronic intermittent ethanol exposure in early adolescent and adult male rats: effects on tolerance, social behavior, and ethanol intake

Background: Given the prevalence of alcohol use in adolescence, it is important to understand the consequences of chronic ethanol exposure during this critical period in development. The purpose of this study was to assess possible age‐related differences in susceptibility to tolerance development to ethanol‐induced sedation and withdrawal‐related anxiety, as well as voluntary ethanol intake after chronic exposure to relatively high doses of ethanol during adolescence or adulthood. Methods: Juvenile/adolescent and adult male Sprague‐Dawley rats were assigned to one of five 10‐day exposure conditions: chronic ethanol (4 g/kg every 48 hours), chronic saline (equivalent volume every 24 hours), chronic saline/acutely challenged with ethanol (4 g/kg on day 10), nonmanipulated/acutely challenged with ethanol (4 g/kg on day 10), or nonmanipulated. For assessment of tolerance development, duration of the loss of righting reflex (LORR) and blood ethanol concentrations (BECs) upon regaining of righting reflex (RORR) were tested on the first and last ethanol exposure days in the chronic ethanol group, with both saline and nonmanipulated animals likewise challenged on the last exposure day. Withdrawal‐induced anxiety was indexed in a social interaction test 24 hours after the last ethanol exposure, with ethanol‐naïve chronic saline and nonmanipulated animals serving as controls. Voluntary intake was assessed 48 hours after the chronic exposure period in chronic ethanol, chronic saline and nonmanipulated animals using an 8‐day 2 bottle choice, limited‐access ethanol intake procedure. Results: In general, adolescent animals showed shorter durations of LORR and higher BECs upon RORR than adults on the first and last ethanol exposure days, regardless of chronic exposure condition. Adults, but not adolescents, developed chronic tolerance to the sedative effects of ethanol, tolerance that appeared to be metabolic in nature. Social deficits were observed after chronic ethanol in both adolescents and adults. Adolescents drank significantly more ethanol than adults on a gram per kilogram basis, with intake uninfluenced by prior ethanol exposure at both ages. Conclusions: Adolescents and adults may differ in their ability and/or propensity to adapt to chronic ethanol exposure, with adults, but not adolescents, developing chronic metabolic tolerance. However, this chronic exposure regimen was sufficient to disrupt baseline levels of social behavior at both ages. Taken together, these results suggest that, despite the age‐related differences in tolerance development, adolescents are as susceptible as adults to consequences of chronic ethanol exposure, particularly in terms of disruptions in social behavior. Whether these effects would last into adulthood remains to be determined.     

Blockade of cannabinoid receptor 1 improves insulin resistance, lipid metabolism, and diabetic nephropathy in db/db mice

The endocannabinoid system is important in the pathogenesis of obesity-related metabolic disorders. However, the effect of inhibiting the endocannabinoid system in type 2 diabetic nephropathy is unclear. Therefore, we examined the effect of the cannabinoid (CB)1 receptor antagonist, SR141716, on insulin resistance and diabetic nephropathy in db/db mice. Six-week-old db/db mice were treated with the CB1-specific antagonist SR141716 (10 mg/kg · d) for 3 months. Treatment with SR141716 significantly improved insulin resistance and lipid abnormalities. Concomitantly, CB1 antagonism improved cardiac functional and morphological abnormality, hepatic steatosis, and phenotypic changes of adipocytes into small differentiated forms, associated with increased adiponectin expression and decreased lipid hydroperoxide levels. CB1 receptor was overexpressed in diabetic kidneys, especially in podocytes. Treatment with the SR141716 markedly decreased urinary albumin excretion and mesangial expansion and suppressed profibrotic and proinflammatory cytokine synthesis. Furthermore, SR141716 improved renal lipid metabolism and decreased urinary 8-isoprostane levels, renal lipid hydroperoxide content, and renal lipid content. In cultured podocytes, high-glucose stimulation increased CB1 receptor expression, and SR141716 treatment abolished high-glucose-induced up-regulation of collagen and plasminogen activator inhibitor-1 synthesis. Additionally, knockdown of CB1 receptor expression by stealth small interfering RNA abolished high-glucose-induced sterol-regulatory element-binding protein-1 expression in podocytes. These findings suggest that CB1 blockade improves insulin resistance and protect against renal injury through both metabolic and antifibrotic effects in type 2 diabetic nephropathy. Targeting CB1 blockade could therefore provide a new therapeutic target to prevent type 2 diabetic nephropathy.     

Effects of acamprosate on excitatory amino acids during multiple ethanol withdrawal periods

Background: Our previous studies on the effects of acamprosate on enhanced locomotion during repeated withdrawals are now extended to the effects of acamprosate on excitatory amino acids in the hippocampus during repeated ethanol withdrawals. Methods: In this study, Wistar rats were made ethanol dependent by 4 weeks of vapor inhalation. After this first cycle of chronic ethanol treatment, rats underwent repeated and alternate cycles of 24 hr withdrawals and 1 week of chronic ethanol treatment. The microdialysis technique was used together with high‐performance liquid chromatography and electrochemical detection to quantify different amino acids such as aspartate and glutamate. Results: An intraperitoneal administration of acamprosate (400 mg/kg) to naïve rats did not alter aspartate or glutamate levels compared with the saline groups. During the first cycle of ethanol withdrawal, the administration of acamprosate (400 mg/kg, intraperitoneally) 2 hr after the commencement of ethanol withdrawal decreased both aspartate and glutamate microdialysate levels when compared with their respective saline group. Acamprosate administration also significantly decreased glutamate levels during the third withdrawal compared with the saline group, whereas no changes were seen in aspartate levels. Conclusion: The results of this work demonstrate that acamprosate reduced the excitatory amino acid glutamate increase observed during repeated ethanol withdrawal. These effects of acamprosate may provide a protective mechanism against neurotoxicity by reducing excitatory amino acids, particularly glutamate.     

Restraint stress and ethanol consumption in two mouse strains

Background: This study examined the interaction between restraint stress and ethanol drinking in mice that consume low and high amounts of ethanol. Methods: Two strains of mice (129SVEV and C57BL/6J) underwent 1 hour of restraint stress twice per day for 4 days in the presence of a CRF‐1 receptor antagonist, a glucocorticoid receptor antagonist or vehicle. Ethanol preference and consumption were assessed using a two bottle choice design. In another study, mice were implanted with pellets containing corticosterone; ethanol preference and consumption were assessed using a two bottle choice design. Results: Restraint stress significantly increased ethanol preference and consumption in 129SVEV mice but not in C57BL/6J mice. Then 129SVEV mice underwent the identical stress procedure; however, mice received either the CRF‐1 receptor antagonist, R121919 (15 or 20 mg/kg, ip) or vehicle 30 minutes prior to stress. R121919 did not block the stress‐induced change in ethanol preference despite causing a significant blunting in the HPA axis. Negative results were also obtained using the CRF‐1 receptor antagonist, Antalarmin (20 mg/kg, ip). In another study, 129SVEV mice were administered either the glucocorticoid receptor antagonist Mifepristone (25, 50 or 100 μg/kg, ip) or vehicle under the same procedure. Mifepristone did not alter ethanol preference. Moreover, the three receptor antagonist did not alter nonstress ethanol consumption either. In the last study, both mouse strains underwent active or sham adrenalectomy, then pellets containing corticosterone or placebo were implanted and preference for ethanol versus water was tested. Corticosterone administration decreased ethanol consumption in a strain‐dependent manner. Conclusion: These data show the restraint model for stress can modestly increase ethanol consumption in 129SVEV mice but not in C57BL/6J mice. Pharmacologic manipulation of CRF and corticosterone did not blunt baseline or stress‐induced change in ethanol preference nor did administration of corticosterone mimic the effects of restraint stress on ethanol consumption. These findings suggest the mechanism responsible for increasing ethanol consumption in this model is independent of the HPA axis and extra‐hypothalamic CRF.     

Development of ethanol withdrawal-related sensitization and relapse drinking in mice selected for high- or low-ethanol preference

Background: Previous studies have shown that high alcohol consumption is associated with low withdrawal susceptibility, while at the same time, other studies have shown that exposure to ethanol vapor increases alcohol drinking in rats and mice. In the present studies, we sought to shed light on this seeming contradiction using mice selectively bred for High‐ (HAP) and Low‐ (LAP) Alcohol Preference, first, assessing these lines for differences in signs of ethanol withdrawal and second, for differences in the efficacy of intermittent alcohol vapor exposure on elevating subsequent ethanol intake. Methods: Experiment 1 examined whether these lines of mice differed in ethanol withdrawal‐induced CNS hyperexcitability and the development of sensitization to this effect following intermittent ethanol vapor exposure. Adult HAP and LAP lines (replicates 1 and 2), and the C3H/HeNcr inbred strain (included as a control genotype for comparison purposes) received intermittent exposure to ethanol vapor and were evaluated for ethanol withdrawal‐induced seizures assessed by scoring handling‐induced convulsions (HIC). Experiment 2 examined the influence of chronic intermittent ethanol exposure on voluntary ethanol drinking. Adult male and female HAP‐2 and LAP‐2 mice, along with male C57BL/6J (included as comparative controls) were trained to drink 10% ethanol using a limited access (2 h/d) 2‐bottle choice paradigm. After stable baseline daily intake was established, mice received chronic intermittent ethanol vapor exposure in inhalation chambers. Ethanol intake sessions resumed 72 hours after final ethanol (or air) exposure for 5 consecutive days. Results: Following chronic ethanol treatment, LAP mice exhibited overall greater withdrawal seizure activity compared with HAP mice. In Experiment 2, chronic ethanol exposure/withdrawal resulted in a significant increase in ethanol intake in male C57BL/6J, and modestly elevated intake in HAP‐2 male mice. Ethanol intake for male control mice did not change from baseline levels of intake. In contrast, HAP‐2 female and LAP‐2 mice of both sexes did not show changes in ethanol intake as a consequence of intermittent ethanol exposure. Conclusions: Overall, these results indicate that the magnitude of ethanol withdrawal‐related seizures is inversely related to inherited ethanol intake preference. Additionally, intermittent ethanol vapor exposure appears more likely to affect high‐drinking mice (C57BL/6J and HAP‐2) than low drinkers, although these animals are less affected by ethanol withdrawal.     

CB1 Receptor Blockade Decreases Ethanol Intake and Associated Neurochemical Changes in Fawn-Hooded Rats

Background:  This study was undertaken to identify the neurochemical changes underlying the attenuation of voluntary ethanol intake induced by the cannabinoid CB1 receptor antagonist AM251 in fawn-hooded rats.
Methods:  Rats were exposed to the 2-bottle-choice paradigm (ethanol 10% v/v or water) for 15 days. After this period, rats received AM251 (3 to 6 mg/kg, i.p.) or vehicle.
Results:  Voluntary ethanol intake decreased (30%) with the administration of incremental dosages of AM251 (3 mg/kg, 5 days and 6 mg/kg, 5 days) in rats with acquired high preferring ethanol consumption (>3.5 g of ethanol/kg/d). Ethanol intake significantly decreased proopiomelanocortin expression in the arcuate nucleus (38.31%) and μ-opioid-DAMGO-stimulated [³⁵S]-GTPγ binding in the caudate-putamen (40%), nucleus accumbens core (AccC) (32.87%), and shell (AccS) (34.21%). Moreover, ethanol intake increased tyrosine hydroxylase (TH) gene expression in the substantia nigra (24%) and ventral tegmental area (23%) and corticotrophin-releasing gene expression in the paraventricular hypothalamic nucleus (41.6%). The reduction of ethanol intake induced by AM251 was associated with blockade or significant reduction of the changes produced by ethanol in the expression of these genes in key regions related to drug dependence. Interestingly, treatment with AM251 reduced (20%) TH gene expression in rats drinking only water. In this respect, the action of AM251 in reducing TH gene expression may not be specific.
Conclusion:  Taken together, these results revealed that blockade of cannabinoid CB1 receptors (CB1r) decreased voluntary ethanol intake in ethanol-habituated rats by normalizing the neurochemical alterations induced by ethanol.     

The effects of short-term chronic ethanol intoxication and ethanol withdrawal on the molecular composition of the rat hippocampus by FT-IR spectroscopy

Background: The numerous adverse effects of ethanol abuse and ethanol withdrawal on biological systems are well documented. Conversely, the understanding of the molecular mechanisms underlying these pathological effects is still incomplete. This study was undertaken to investigate the effects of short‐term chronic ethanol administration and ethanol withdrawal on the molecular structure and function of hippocampal tissue, a brain region important for mnemonic processes and known to be highly susceptible to ethanol intoxication. Methods: Ethanol was administered to adult Wistar rats by intragastric intubation for 15 days with a stepwise increase in the daily dose from 6 to 12 g/kg body weight, with the highest dose delivered for the last 2 days only. The total daily dose of ethanol was divided into 3 equal portions administered 4 hours apart. Animals were sacrificed by decapitation at 4, 24, and 72 hours after the last ethanol administration to examine potential effects of ethanol intoxication and ethanol withdrawal. Ethanol‐related molecular changes were monitored by Fourier transform infrared (FT‐IR) spectroscopy. Results: Significant changes in the hippocampal content, structure, and function of lipids, proteins, and nucleic acids were recorded under ethanol intoxication. Seventy‐two hours after the cessation of ethanol administration, during the late phase of withdrawal, alterations in the macromolecules’ content and conformational changes in protein and nucleic acid structure ameliorated, while the changes in macromolecular ratios, lipid order, and dynamics aggravated. Conclusions: Our results suggest that 15 days of binge‐like drinking resulting in the high blood alcohol concentration (varying in the dose‐dependent manner between 253 and 606 mg/dl) produced a strong physical dependence manifested mainly by the changes in lipid profiles pointing toward withdrawal‐induced oxidative stress. These results show that ethanol withdrawal may cause equal to or even more severe brain damage than the ethanol itself, which should be considered when designing antialcohol therapies.     

MK-801 can exacerbate or attenuate behavioral alterations associated with neonatal alcohol exposure in the rat, depending on the timing of administration

BACKGROUND: We have reported that administration of MK-801, an NMDA receptor antagonist, during ethanol withdrawal in the developing rat attenuates ethanol's adverse effects on behavioral development. In the present study, we altered the timing of MK-801 delivery in relation to the last alcohol dose to determine if its protective effects were specific to the ethanol withdrawal phase. METHODS: Five groups of rats were artificially reared and exposed to alcohol in a binge-like manner on postnatal day (PD) 6, producing peak blood alcohol levels of 335 mg/dl that cleared to 0 mg/dl by 33 hours. Four groups received MK-801 at various times after alcohol treatment (0, 9, 21, or 33 hr post-ethanol). The fifth alcohol-treated group received saline. Two artificially reared control groups were included: one was injected with saline and the other injected with 0.5 mg/kg MK-801. Finally, a normally reared suckle control group was also included. Activity level and performance on a spatial discrimination reversal-learning task were evaluated at PD 18 and PD 40, respectively. RESULTS: Administration of MK-801 at the same time as ethanol treatment (0 hr) produced a high rate of mortality. Ethanol exposure on PD6 increased activity level relative to controls. Administration of MK-801 at 0 hr exacerbated this ethanol-induced overactivity, whereas administration of MK-801 at 21 and 33 hr reduced the severity of ethanol-related overactivity. Similarly, ethanol exposure on PD 6 significantly increased the number of errors committed on a spatial discrimination reversal-learning task. MK-801 injections 9 hrs after ethanol exacerbated this effect, whereas MK-801 treatment 33 hrs after ethanol attenuated this effect. Thus, MK-801 administration at the time of ethanol treatment was highly toxic, whereas during the withdrawal period it was protective. CONCLUSION: These data are consistent with the hypothesis that ethanol exposure in the neonatal rat inhibits the NMDA receptor, producing a subsequent rebound in NMDA receptor activation and possible excitotoxicity during withdrawal. Both the acute inhibitory effects of ethanol and the excitatory effects of withdrawal may contribute to fetal alcohol effects.