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.     

Effects of withdrawal from chronic intermittent ethanol vapor on the level and circadian periodicity of running-wheel activity in C57BL/6J and C3H/HeJ mice

Background: Alcohol withdrawal is associated with behavioral and chronobiological disturbances that may persist during protracted abstinence. We previously reported that C57BL/6J (B6) mice show marked but temporary reductions in running‐wheel activity, and normal free‐running circadian rhythms, following a 4‐day chronic intermittent ethanol (CIE) vapor exposure (16 hours of ethanol vapor exposure alternating with 8 hours of withdrawal). In the present experiments, we extend these observations in 2 ways: (i) by examining post‐CIE locomotor activity in C3H/HeJ (C3H) mice, an inbred strain characterized by high sensitivity to ethanol withdrawal, and (ii) by directly comparing the responses of B6 and C3H mice to a longer‐duration CIE protocol. Methods: In Experiment 1, C3H mice were exposed to the same 4‐day CIE protocol used in our previous study with B6 mice (referred to here as the 1‐cycle CIE protocol). In Experiment 2, C3H and B6 mice were exposed to 3 successive 4‐day CIE cycles, each separated by 2 days of withdrawal (the 3‐cycle CIE protocol). Running‐wheel activity was monitored prior to and following CIE, and post‐CIE activity was recorded in constant darkness to allow assessment of free‐running circadian period and phase. Results: C3H mice displayed pronounced reductions in running‐wheel activity that persisted for the duration of the recording period (up to 30 days) following both 1‐cycle (Experiment 1) and 3‐cycle (Experiment 2) CIE protocols. In contrast, B6 mice showed reductions in locomotor activity that persisted for about 1 week following the 3‐cycle CIE protocol, similar to the results of our previous study using a 1‐cycle protocol in this strain. Additionally, C3H mice showed significant shortening of free‐running period following the 3‐cycle, but not the 1‐cycle, CIE protocol, while B6 mice showed normal free‐running rhythms. Conclusions: These results reveal genetic differences in the persistence of ethanol withdrawal‐induced hypo‐locomotion. In addition, chronobiological alterations during extended abstinence may depend on both genetic susceptibility and an extended prior withdrawal history. The present data establish a novel experimental model for long‐term behavioral and circadian disruptions associated with ethanol withdrawal.     

Indexing Withdrawal in Mice: Matching Genotypes for Exposure in Studies Using Ethanol Vapor Inhalation

Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) mice have been bidirectionally selected for severity of handling-induced convulsions (HIC) following withdrawal from 72 hr of chronic ethanol vapor inhalation. During selection, daily injections of the alcohol dehydrogenase inhibitor, pyrazole, were used to enhance and stabilize blood ethanol concentrations (BEC). After 26 generations of selection, WSR mice show lower withdrawal BEC than WSP mice exposed to the same ethanol vapor concentrations. Because it is desirable to compare mice maintained at the same BEC to assess correlated responses to selection, this has necessitated exposing WSR mice to higher ethanol vapor concentrations than WSP mice to achieve matched chronic BEC. The experiments reported herein demonstrate two methods for producing matched withdrawal BEC: (1) by exposing mice to the same ethanol vapor concentration and varying the pyrazole dose; and (2) by administering only ethanol at different vapor concentrations and selecting some mice with approximately the same BEC. When exposed to the same ethanol vapor concentration, WSR mice given 1.0 mmol/kg pyrazole had withdrawal BEC equivalent to WSP mice given 0.75 mmol/kg pyrazole. However, WSP mice had much more severe withdrawal HIC than WSR mice. WSP and WSR mice metabolized ethanol at the same rate following withdrawal. The basis for the differential effectiveness of pyrazole is unknown. We also exposed mice to higher ethanol vapor concentrations in the absence of pyrazole. By exposing WSR mice to higher concentrations than WSP, roughly equivalent BEC on withdrawal was achieved. Because BEC are more variable in the absence of pyrazole, it was necessary to select animals of each genotype to achieve relatively matched BEC. Again, WSP mice had much more severe HIC on withdrawal than WSR.     

The Kindling Model of Alcohol Dependence: Similar Persistent Reduction in Seizure Threshold to Pentylenetetrazol in Animals Receiving Chronic Ethanol or Chronic Pentylenetetrazol

Rats on a chronic intermittent ethanol (CIE) regimen showed a persistent reduction in seizure threshold to the convulsant drug pentylenetetrazol (PTZ). CIE rats were given ethanol by intubation on an alternate day schedule and tested at selected intervals for seizure threshold with PTZ. A significant reduction in seizure threshold, a sign of withdrawal, was observed 20 hr after the first dose. The severity of withdrawal intensified on repetition of the ethanol administration and depression-hyperexcitability cycle, with the seizure threshold reaching a maximum decrease after 12 doses and remaining reduced up to 60 doses. The reduction in seizure threshold persisted for at least 40 days of no alcohol following the 60th dose. The long-lasting decrease in seizure threshold following CIE treatment resembled the “kindling” phenomenon produced by chronic administration of PTZ (25 mg/kg, 3 times/week). The CIE rats developed, in addition, a tolerance to the anticonvulsant action of ethanol, which occurred well after the decrease in PTZ seizure threshold, and a tolerance to the hypothermic effect of ethanol, which developed rapidly. PTZ kindled rats that had never been exposed to ethanol also exhibited tolerance to the hypothermic effect of ethanol. We propose that kindling contributes to the mechanism of the development of dependence on central nervous system depressants like benzodiazepines, barbiturates, and alcohol, drugs that act on the γ-aminobutyric acid-A receptor chloride ion channel complex. Repeated episodes of depression and withdrawal hyperexcitability are postulated to produce kindling during the repeated withdrawal episodes. In chronic ethanol abuse, the withdrawal stress/hyperexcitability would be repeated many times and lead to the development of dependence as measured by the persistent increase in withdrawal severity, manifested as heightened sensitivity to seizures (kindling). The long-lasting decreases in seizure threshold following both chronic pentylenetetrazol and chronic intermittent ethanol suggest the possibility that some common mechanisms, such as the involvement of γ-aminobutyric acid-A receptors, may underlie the two kindling phenomena.     

Chronic ethanol reduces nicotine-induced dopamine release in PC12 cells

Background: There is a high correlation between alcohol and nicotine use; that is, alcohol use is associated with high levels of smoking. One important aspect of nicotine addiction appears to be the activation of nicotinic acetylcholine receptors on dopaminergic neurons projecting from the ventral tegmental area to the nucleus accumbens. The release of dopamine from these neurons is thought to mediate, at least in part, the reward of nicotine consumption. If chronic alcohol consumption affects the amount of dopamine released in response to nicotine, it could contribute to the high level of smoking seen in alcoholics. Methods: We have used an in vitro model system to study the effects of chronic ethanol exposure on acute nicotine‐induced dopamine release and the withdrawal from ethanol. A pheochromocytoma cell line (PC12 cells) was exposed to ethanol for periods of 3 to 96 hr, followed by a 5 min exposure to nicotine. Dopamine released in response to nicotinic stimulation was measured by high‐pressure liquid chromatography. Results: Exposure of PC12 cells to chronic ethanol resulted in a time‐ and dose‐dependent inhibition of nicotine‐induced dopamine release. A moderate dose of ethanol (50 mM) resulted in a significant reduction in as little as 3 hr. The cells demonstrated a form of cross‐tolerance in that they showed diminished response to nicotine even though they had never been exposed to nicotine. After ethanol was withdrawn from the cells after a chronic exposure (96 hr), dopamine release slowly returned to normal levels but demonstrated a significant period of “overshoot” or hyperresponsiveness between 24 and 48 hr after withdrawal. Conclusions: These results show that chronic ethanol exposure decreases nicotine‐induced dopamine release and demonstrate a period of hyperresponsiveness during withdrawal from ethanol. These studies suggest potential interactions between chronic ethanol and nicotine that may provide insight into such phenomena as cross‐tolerance and increased use of nicotine by alcoholics.     

Differential dietary ethanol intake and blood ethanol levels in adolescent and adult rats: effects on anxiety-like behavior and seizure thresholds

Background: Adult rats exhibit increased anxiety‐like behavior after exposure to repeated cycles of chronic ethanol and withdrawal. While adolescent rats have differential responses to both acute and chronic ethanol treatments, the potential differences in the effects of repeated withdrawals in this population have yet to be determined. Methods: Male adult and adolescent rats received three 5‐day cycles of either a 4.5% or 7% ethanol diet (ED) separated by two 2‐day withdrawal periods. Five hours into the final withdrawal, rats were tested for social interaction (SI) deficits (an index of anxiety‐like behavior) and then assessed for seizure thresholds (audiogenic and bicuculline‐induced). Ethanol intake was monitored throughout, and blood ethanol concentrations (BEC) were obtained from a separate group of rats. Results: Adolescent rats have reduced SI during the final withdrawal from either ED and exhibit a greater reduction in SI compared to adult rats when exposed to a 7%ED. Audiogenic seizures were not increased during withdrawal from either ED in adult rats, but adolescent rats that received 7%ED displayed increased seizures. The bicuculline seizure thresholds were decreased in both ages exposed to a 7%ED, but only adolescent rats showed this decreased threshold after 4.5%ED. Ethanol intakes and BECs were higher in adolescent rats compared to similarly treated adults. However, ethanol intakes and BECs were comparable between 4.5%ED‐treated adolescent and 7%ED‐treated adult rats. Conclusions: Behavioral results from the 7%ED‐treated groups suggested that adolescent rats may be more vulnerable to repeated withdrawals from ethanol than adults; however, differences in ethanol intake and BECs may be at least in part responsible. When ethanol intakes and BECs were similar between 4.5%ED‐treated adolescent and 7%ED‐treated adult rats, behavioral effects were not different. Importantly, these data illustrated that adolescent rats can exhibit anxiety and reduced seizure thresholds following this repeated withdrawal paradigm.     

Genetic Correlations Between Initial Sensitivity to Ethanol and Brain cAMP Signaling in Inbred and Selectively Bred Mice

BACKGROUND: Several lines of evidence have suggested a role for cAMP (adenosine 3',5'-cyclic monophosphate) signaling in the acute and chronic effects of ethanol. This study investigated whether there is a genetic correlation between cAMP synthesis in the brain and the acute effects of ethanol [alcohol sensitivity or acute functional tolerance (AFT)]. METHODS: By using nine inbred strains of mice, we measured initial sensitivity and AFT to ethanol with a test of balance on a dowel. Initial sensitivity was defined by the blood ethanol concentration (BEC0) at the loss of balance on a dowel after an ethanol injection [1.75 g/kg intraperitoneally (ip)]. When mice were able to regain balance on the dowel, BEC1 was determined, and a second ethanol injection was given (2 g/kg ip). Upon final regaining of balance, BEC2 was determined. AFT was defined by the difference between BEC1 and BEC2 (AFT = DeltaBEC = BEC2 - BEC1). Cyclic AMP synthesis was measured in whole-cell preparations in the cerebellum and other brain areas of mice of the nine inbred strains. RESULTS: Significant differences in BEC0 and AFT were seen among the mice of the nine inbred strains. Cerebellar basal and forskolin- and isoproterenol-stimulated cAMP production differed significantly between the strains, and BEC0 was found to correlate significantly with forskolin- and isoproterenol-stimulated cAMP accumulation in the cerebellum (r = 0.70 and 0.94, respectively). When we measured cAMP production in mesencephalic and telencephalic tissue in three strains of mice that differed significantly in isoproterenol-stimulated cAMP accumulation in the cerebellum, significant differences between strains were found only in telencephalic tissue. The relative relationship between the rank order of the three strains for cAMP accumulation in the telencephalon and initial sensitivity to ethanol was identical to that seen with the cerebellum. However, AFT did not correlate with cAMP accumulation in the cerebellum or any other brain area tested. CONCLUSIONS: These results suggest that cAMP-generating systems of the cerebellum and possibly the brain areas contained in telencephalic tissues (e.g., basal ganglia) may have an important relationship to an animal's initial sensitivity to the incoordinating effects of ethanol.     

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.     

Differential effects of single versus repeated alcohol withdrawal on the expression of endocannabinoid system-related genes in the rat amygdala

Background: Endogenous cannabinoids such as anandamide and 2‐arachidonoylglycerol (2‐AG) exert important regulatory influences on neuronal signaling, participate in short‐ and long‐term forms of neuroplasticity, and modulate stress responses and affective behavior in part through the modulation of neurotransmission in the amygdala. Alcohol consumption alters brain endocannabinoid levels, and alcohol dependence is associated with dysregulated amygdalar function, stress responsivity, and affective control. Methods: The consequence of long‐term alcohol consumption on the expression of genes related to endocannabinoid signaling was investigated using quantitative RT‐PCR analyses of amygdala tissue. Two groups of ethanol (EtOH)‐exposed rats were generated by maintenance on an EtOH liquid diet (10%): the first group received continuous access to EtOH for 15 days, whereas the second group was given intermittent access to the EtOH diet (5 d/wk for 3 weeks). Control subjects were maintained on an isocaloric EtOH‐free liquid diet. To provide an initial profile of acute withdrawal, amygdala tissue was harvested following either 6 or 24 hours of EtOH withdrawal. Results: Acute EtOH withdrawal was associated with significant changes in mRNA expression for various components of the endogenous cannabinoid system in the amygdala. Specifically, reductions in mRNA expression for the primary clearance routes for anandamide and 2‐AG (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL], respectively) were evident, as were reductions in mRNA expression for CB₁, CB₂, and GPR55 receptors. Although similar alterations in FAAH mRNA were evident following either continuous or intermittent EtOH exposure, alterations in MAGL and cannabinoid receptor‐related mRNA (e.g., CB₁, CB₂, GPR55) were more pronounced following intermittent exposure. In general, greater withdrawal‐associated deficits in mRNA expression were evident following 24 versus 6 hours of withdrawal. No significant changes in mRNA expression for enzymes involved in 2‐AG biosynthesis (e.g., diacylglicerol lipase‐α/β) were found in any condition. Conclusions: These findings suggest that EtOH dependence and withdrawal are associated with dysregulated endocannabinoid signaling in the amygdala. These alterations may contribute to withdrawal‐related dysregulation of amygdalar neurotransmission.     

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.     

Characterization of an Automated Apparatus for Precise Control of Inhalation Chamber Ethanol Vapor and Blood Ethanol Concentrations

Inhalation chambers with a monitoring and control apparatus for ethanol vapor exposure of small animals were constructed. A thermal conductivity detector was employed for continuous measurement of inhalation chamber ethanol vapor concentration. The concentration was maintained within a very narrow range (+/- 1 mg/liter) by incorporating into the design a feedback loop which controls the ethanol pump. As expected, the blood ethanol concentrations (BEC) of male Sprague-Dawley rats were positively and linearly correlated to the chamber ethanol concentration. When rats were exposed for 24 hr to a chamber ethanol concentration of 17, 25, or 32 mg/liter, correspondingly low, moderate, or high mean blood ethanol levels were obtained. When a large population of this strain of rats (n = 121) was exposed to a constant ethanol vapor concentration for 14 days (25 mg/liter) considerable interindividual variation in blood levels occurred. There was also individual variation over time in the BEC of animals monitored. The mean +/- SD BEC was 189 +/- 90 mg/100 ml for this population and a gaussian-like distribution was obtained with regard to BEC. Behavior characteristic of alcohol withdrawal was observed in rats with BEC greater than 120 mg/100 ml after 3.5, 7, or 14 days of exposure. This apparatus and inhalation paradigm make possible the precise control of chamber ethanol concentration which markedly enhances control over both intra- and intersubject fluctuation in blood ethanol levels during alcohol exposure and the comprehensive examination of relationships between a wide range of blood ethanol concentrations and their physiological and biochemical effects.     

Intensity and Duration of Chronic Ethanol Exposure Is Critical for Subsequent Escalation of Voluntary Ethanol Drinking in Mice

Background: Excessive alcohol drinking continues to be an important health problem. Recent studies from our laboratory and others have demonstrated that animal models of ethanol dependence and relapse can contribute to understanding factors that contribute to excessive drinking. In this study, we tested the hypothesis that the amount and duration of ethanol exposure is critical for promoting the escalation in drinking by mice given access to ethanol in a limited access paradigm. Methods: We used several methods of chronic intermittent ethanol exposure in male C57BL/6J mice that would vary in the amount and duration of exposure to ethanol as indicated by blood ethanol concentrations (BEC). After establishing baseline drinking in the mice using a 2 hours, 2 bottle choice drinking paradigm, each study involved alternating between periods of ethanol exposure and periods of limited access to ethanol (1 cycle) for a total of 3 cycles. In Study 1, mice were allowed extended access (16 hours) to ethanol for oral consumption or remained in the home cage. In Study 2, the ethanol exposure consisted of intragastric gavage of increasing doses of ethanol or isocaloric sucrose as the control. Study 3 compared intragastric gavage combined with pyrazole, an alcohol dehydrogenase inhibitor, with vapor inhalation of ethanol using procedures known to lead to increased drinking in mice. Finally, Study 4 was a retrospective review of several studies conducted in our laboratory using inhalation procedures. The retrospective review encompassed a range of postvapor chamber BEC values and ethanol intakes that would allow a relationship between increased drinking and BEC to be examined. Results: Allowing mice to drink for longer periods of time did not cause increased drinking in subsequent limited access sessions. Likewise, gastric intubation of ethanol which produced high BEC (>300 mg/dl) with or without pyrazole did not increase drinking. Only the vapor inhalation procedure, which was associated with sustained BEC above 175 mg/dl for the entire exposure period resulted in increased drinking. The retrospective study provided further evidence that sustained BEC levels above 175 mg/dl was critical to the escalation in drinking. Conclusions: We found that the intensity (amount) and duration of ethanol exposure, indexed by BEC, is critical to produce increased drinking in mice. Specifically, BEC must regularly exceed 175 mg/dl for the escalation in drinking to occur. Future studies will examine neurobiological adaptations that may underlie the increased drinking behavior caused by chronic intermittent ethanol exposure.     

Effects of CB1 cannabinoid receptor blockade on ethanol preference after chronic ethanol administration

BACKGROUND: Chronic ethanol administration results in neurobiological alterations similar to those observed after chronic cannabinoid exposure. The purpose of this study was to investigate alcohol drinking and the withdrawal responses after pulmonary chronic alcoholization with intraperitoneal or oral administration of a cannabinoid CB1 receptor antagonist. METHODS: The cannabinoid receptor antagonist SR141716A, 1, 3 or 10 mg/kg/day intraperitoneally or orally, was administered to Wistar rats either during a 30-day chronic ethanol exposure or at the cessation of this procedure. Motility was recorded during 18 hr after the cessation of chronic alcoholization just before the beginning of the free-choice paradigm (water versus alcohol 10% v/v). RESULTS: A significant increase in ethanol preference was observed during the free-choice paradigm after chronic alcoholization with concurrent SR141716A administration (3 or 10 mg/kg/day). A significant decrease in withdrawal motility after administration of SR141716A was observed with only the highest dose (10 mg/kg/day). The administration of SR141716A, 3 or 10 mg/kg/day, after chronic pulmonary alcoholization significantly decreased the preference for alcohol. Finally, a significant decrease in ethanol preference was seen during the free-choice paradigm of nonalcoholized rats treated with SR141716A, 3 or 10 mg/kg/day, during 30 days before the free-choice paradigm. CONCLUSIONS: The concurrent administration of the CB1 antagonist together with the chronic alcoholization increases the preference for ethanol. Also, the administration of the CB1 antagonist after the chronic alcoholization or at the time of withdrawal drastically diminishes the ethanol preference.     

Lesions of the extended amygdala in C57BL/6J mice do not block the intermittent ethanol vapor-induced increase in ethanol consumption

Background: The central extended amygdala (cEA) which includes the central nucleus of the amygdala (CeA) and the lateral posterior bed nucleus of the stria terminalis (BNSTLP), has been proposed to play a key role in excessive ethanol consumption in humans (Koob and Le Moal, 2005 Nat Neurosci 8:1442). To examine this relationship, we used a murine model of ethanol dependence (Becker and Lopez, 2004 Alcohol Clin Exp Res 28:1829; Lopez and Becker, 2005 Psychopharmacology (Berl) 181:688) and compared animals with sham lesions and electrolytic lesions of the CeA and BNSTLP. Methods: Male C57BL/6J (B6) mice were first acclimated to a limited‐access 2‐bottle‐choice preference procedure. The access period began 3 hours into the dark phase of the light‐dark cycle and continued for 2 hours. Once acclimated (1 week), mice underwent chronic exposure to and intermittent withdrawal from ethanol vapor. The animals were then retested in the limited‐access 2‐bottle‐choice preference procedure. In some experiments, electrolytic and sham lesions of the CeA or BNSTLP were performed prior to initiating the 2‐bottle choice procedure. Results: In a series of 5 preliminary experiments, mice were randomly assigned either to the standard intermittent ethanol vapor procedure or to the standard procedure but with air in the vapor chamber (control). The air‐control procedure produced no change in ethanol intake when compared to baseline consumption. In contrast, intermittent ethanol vapor exposure increased ethanol consumption by almost 50%. The increase in consumption was associated with an increase in total fluid volume consumed and no change in ethanol preference. Lesions of both the BNSTLP and CeA significantly decreased baseline ethanol consumption, the former by decreasing fluid consumption and the latter by decreasing ethanol preference. Intermittent ethanol vapor exposure significantly increased consumption in both the BNSTLP‐ and CeA‐lesioned animals, largely by increasing the total volume of fluid consumed. Conclusions: The results obtained clearly demonstrate that the cEA has a role in the regulation of ethanol consumption in the limited‐access procedure. However, neither lesions of the CeA nor BNSTLP prevented the intermittent ethanol vapor‐induced increase in consumption. These data do not preclude some role of the cEA in the increased ethanol consumption following intermittent ethanol vapor exposure, but would suggest that other brain regions also must have a significant influence.     

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.     

Effects of lorazepam treatment for multiple ethanol withdrawals in mice

Background: Although many alcohol‐dependent patients present with a history of prior detoxifications, the efficacy and safety of pharmacotherapy in the context of multiple ethanol withdrawal experiences have not been extensively studied. The purpose of this study was to evaluate the ability of lorazepam treatment for multiple withdrawals to prevent or blunt the development/expression of sensitized central nervous system hyperexcitability during a subsequent untreated withdrawal episode. A mouse model of withdrawal sensitization involving repeated ethanol withdrawals was used. Methods: Adult male C3H/He mice were exposed to different patterns of chronic ethanol vapor in inhalation chambers. One group received four cycles of 16 hr of ethanol exposure separated by 8‐hr withdrawal periods, another group was tested after a single 16‐hr exposure period, and a final group served as ethanol‐naïve controls. These groups were further divided into lorazepam dosage (0.25–1.0 mg/kg) conditions. Lorazepam was administered 1 hr into each of the first three withdrawal cycles (or equivalent times); no drug injections were given during the final (fourth) withdrawal cycle. The ability of lorazepam treatment to alter development and expression of sensitized handling‐induced convulsions (HIC), as well as changes in pentylenetetrazol seizure threshold dosage during an untreated withdrawal episode, was examined. Separate animals were used to assess the effects of lorazepam treatment on blood ethanol clearance and plasma levels of the benzodiazepine during the test withdrawal cycle. Results: Lorazepam dose‐dependently reduced HIC activity during successive withdrawal cycles, and this resulted in attenuated expression of the sensitized HIC response during the acute phase of a subsequent untreated withdrawal episode. However, HIC activity was exacerbated at later time points during this final test withdrawal in mice that had received lorazepam treatment for earlier withdrawals. A similar pattern of results was obtained for changes in pentylenetetrazol seizure threshold dosage. These results do not seem to be due to pharmacokinetic factors, because peak blood ethanol levels, rate of ethanol elimination, and plasma levels of lorazepam did not significantly differ among groups during the final test withdrawal cycle. Conclusions: Blocking central nervous system hyperexcitability during repeated ethanol withdrawals with lorazepam effectively blunts the development and expression of sensitized seizure activity during the acute phase of a subsequent unmedicated withdrawal episode. At later time points, withdrawal‐related seizure activity was exacerbated, and this is possibly reflective of an interaction between protracted ethanol withdrawal and withdrawal from the benzodiazepine. The clinical implications of these findings suggest that repeated use of benzodiazepines for treatment of multiple ethanol withdrawals may have some initial beneficial effects, but such treatment may also place patients at increased risk of seizures at later time points.     

Increased ethanol drinking after repeated chronic ethanol exposure and withdrawal experience in C57BL/6 mice

BACKGROUND: The development of dependence may have significant motivational consequences regarding continued use and abuse of ethanol. We have developed a mouse model of ethanol dependence and repeated withdrawals that demonstrates sensitization of seizures and other symptoms of withdrawal. It is unclear whether such experience influences ethanol drinking behavior. The present series of experiments were designed to examine whether repeated cycles of chronic ethanol exposure and withdrawal has an impact on subsequent motivation to voluntarily self-administer ethanol. METHODS: With the use of a modified sucrose-fading procedure, adult male C57BL/6J mice were trained to drink 15% (v/v) ethanol in a limited access procedure (2 hr/day). The animals were not food or water deprived at any time during the experiments. Once stable baseline intake was established, mice were exposed to four cycles of 16 hr of ethanol vapor (or air) in inhalation chambers separated by 8-hr periods of withdrawal. At 32 hr after the last cycle of ethanol exposure, all mice were tested for ethanol intake under limited access conditions for 5 consecutive days. The animals then received a second series of chronic ethanol exposure and withdrawal followed by another 5-day test period for ethanol drinking. RESULTS: Stable daily baseline intake was established in mice that drank 15% ethanol combined with 5% sucrose (experiment 1), 15% ethanol alone (experiment 2), 5% sucrose alone (experiment 3), or 15% ethanol when presented as a choice with water (experiment 4). After repeated cycles of chronic ethanol exposure and withdrawal experience, consumption of ethanol solutions increased over baseline levels and in comparison with control (air-exposed) groups. However, sucrose consumption did not change in mice that were trained to drink 5% sucrose. The increase in ethanol consumption after chronic ethanol exposure and withdrawal experience resulted in a significant increase in resultant blood ethanol levels. CONCLUSIONS: Once the positive reinforcing properties of ethanol were established, chronic ethanol exposure and withdrawal experience resulted in a significant increase in voluntary ethanol drinking that yielded a >2-fold increase in resultant blood ethanol levels. This increase in ethanol intake occurred whether ethanol was presented in combination with sucrose, alone (unadulterated), or as a choice with tap water. Furthermore, this effect seems to be selective for ethanol in that animals that were trained to drink a sucrose solution did not exhibit a change in their intake after similar chronic ethanol exposure. As such, this model may be useful in studying the mechanisms and conditions in which chronic ethanol treatment influences motivation to resume drinking after a period of abstinence (relapse).     

Effects of Chronic Alcohol on Immunoreactive β-Endorphin Secretion from Hypothalamic Neurons in Primary Cultures: Evidence for Alcohol Tolerance, Withdrawal, and Sensitization Responses

Endogenous opioid peptides are known to be involved in the alcohol tolerance and dependence following alcohol abuse. However, the cellular mechanisms involved in the ethanol tolerance and dependence are not well established. We have previously shown that low concentrations of ethanol stimulate immunoreactive β-endorphin (IR-β-EP) release from the cultured hypothalamic neurons and that chronic ethanol exposure desensitizes these neurons to ethanol challenges. In this study, we determined the IR-β-EP response to increasing doses of ethanol during the desensitizing phase of moderate ethanol doses to test whether the cultured IR-β-EP-secreting neurons develop tolerance to ethanol following constant exposure. We also determined IR-β-EP responses following withdrawal from chronic ethanol challenge and compared the IR-β-EP secretory response to various doses of ethanol in ethanol-naive and ethanol-preexposed cultures. The IR-β-EP responses to increasing doses of ethanol (50-150 mM) were markedly reduced in the cultures preexposed to a 50 mM dose of ethanol when compared with those that were naive to ethanol. The ethanol-exposed cultures showed hypersecretion of IR-β-EP after removal from 48 hr of constant ethanol, as compared with ethanol-naive cultures. When ethanol-preexposed cultures were challenged with various doses of ethanol 4 days after ethanol withdrawal, the cultures showed higher IR-β-EP secretory responses than did the ethanol-naive cultures. These data suggest that IR-β-EP secretory neurons in primary cultures develop tolerance to chronic ethanol, show withdrawal response after removal of chronic ethanol exposure, and develop sensitization following repeated ethanol challenges.     

Chronic Tolerance to Ethanol in the N:NIH Rat

As the basis for selectively breeding rats from the N:NIH stock on the basis of differential development of tolerance to ethanol, chronic tolerance to the motor-disrupting effects of ethanol was examined. Male (n = 50) and female (n = 46) rats were administered 2.5 g/kg ethanol (i.p.) and the blood-ethanol concentration (BEC) at the time of regain of aerial righting reflex (RARR) was measured. The rats were then placed in inhalation chambers for 8 consecutive days and chronically exposed to ethanol. Thirty-two hours following the cessation of chronic ethanol exposure, the rats were again given a 2.5 g/kg dose of ethanol and the BEC at RARR was again measured. The amount of chronic ethanol tolerance developed, as measured by the difference in BEC at RARR prior to and after chronic ethanol administration, was widely and normally distributed for both male and female rats. Control rats, which did not receive chronic ethanol exposure between the two tests of RARR, did not show tolerance on this measure. A negative correlation (r = -0.46) was found between BEC at RARR prior to chronic ethanol treatment and the amount of chronic tolerance developed using the difference in BEC measure. This correlation suggested that the rats who were initially more sensitive to the effects of ethanol were more likely to display the greatest amount of chronic tolerance. However, using the residuals of the regression analysis of post-chronic ethanol BEC at RARR on prechronic BEC at RARR as an index of tolerance, this negative correlation was not found.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.