Sensitization, Duration, and Pharmacological Blockade of Anxiety-Like Behavior Following Repeated Ethanol Withdrawal in Adolescent and Adult Rats

Background: Repeated ethanol withdrawal sensitizes anxiety‐like behavior in adult rats and causes anxiety‐like behavior and decreased seizure thresholds in adolescent rats. Current experiments determined if adolescent rats exhibit sensitized anxiety‐like behavior, the duration of this effect, if drug pretreatments blocked these effects, and if these effects differed from those seen in adults. Methods: Male adolescent rats received three 5‐day cycles of 2.5% ethanol diet (ED) separated by two 2‐day withdrawal periods, continuous 15 days of 2.5%ED, or a single 5‐day cycle of 2.5%ED. Male adult rats received three 5‐day cycles of either 2.5% or 3.5%ED. These groups were tested 5 hours into the final withdrawal for social interaction (SI) deficits (an index of anxiety‐like behavior). Ethanol intake was monitored throughout and blood concentrations were obtained from separate groups of rats. Additionally, adolescent rats were tested for SI 1, 2, 7, 14, and 18 days and adults 1 and 2 days after the final withdrawal. Some adolescent rats were also pretreated with the CRF₁ antagonist CP‐154,526, the 5‐HT_1A agonist buspirone, or the benzodiazepine receptor antagonist flumazenil during the first 2 withdrawals. Results: SI was reduced in adolescent rats following repeated withdrawals of 2.5%ED while neither a continuous or single cycle ED exposure caused this effect. Adult rats also had reduced SI following repeated withdrawals from both 2.5% and 3.5%ED. This effect was present up to 1 week following the final withdrawal in adolescents but returned to baseline by 1 day in adults. CP‐154,526, buspirone, or flumazenil prevented this reduction in SI in adolescent rats. Conclusions: Adolescent rats exhibit sensitized anxiety‐like behavior following repeated withdrawals at ED concentrations similar to those used in adults. However, this effect is longer lasting in adolescent rats. Drugs modulating CRF, 5‐HT, or GABA systems during initial withdrawals prevent the development of anxiety‐like behavior otherwise manifest during a final withdrawal in adolescent rats.     

Modulation of ethanol withdrawal-induced anxiety-like behavior during later withdrawals by treatment of early withdrawals with benzodiazepine/gamma-aminobutyric acid ligands

BACKGROUND: Anxiety states, including those arising during acute or protracted withdrawal periods, may be precipitating factors in alcoholic relapse. Given the cyclical nature of ethanol withdrawal associated with repeated cycles of ethanol intake and abstinence in a pattern that often spans years, meaningful attempts to model ethanol withdrawal-associated anxiety should incorporate cycled ethanol treatments. The studies reported herein examined the effects of gamma-aminobutyric acid-modulating drugs on social interaction behavior-an established model of anxiety-in rats exposed to repeated cycles of ethanol treatment and withdrawal. METHODS: Rats were exposed to 8 to 12 g/kg/day ethanol during three 7-day dietary cycles (5 days on ethanol diet followed by 2 days on control diet). Ethanol was administered either at hour 4 of withdrawal after cessation of each of the first 2 ethanol cycles or during the final withdrawal only. In other groups, the early withdrawals were treated with alphaxalone, diazepam, PK11159, or flumazenil to block anxiety-like behavior during an untreated later (third) withdrawal. The benzodiazepine inverse agonist DMCM (methyl-6, 7-dymerhoxy-4-ethyl-beta-carboline-3-carboxylate) was also given repeatedly to determine whether it would sensitize anxiety-like behavior during a future withdrawal. Finally, the effects of all drugs on deficits in locomotor behavior were assessed. RESULTS: Pretreatment of earlier withdrawals with alphaxalone, diazepam, ethanol, or flumazenil reduced social interaction deficits during a later withdrawal, but pretreatment with PK11195 did not. In contrast, DMCM administered in lieu of early withdrawals increased social interaction deficits during an untreated later withdrawal. Locomotor deficits were significantly reversed only by the acute ethanol and diazepam treatment during the final withdrawal. CONCLUSIONS: Single-dose administration of drugs that enhance or diminish activity at benzodiazepine-gamma-aminobutyric acid- receptors during earlier withdrawals reduced or potentiated, respectively, anxiety-like behavior during later, drug-free withdrawals. These results support the potential of the novel strategy of using prophylactic therapy administered during early withdrawals to ameliorate symptoms of later withdrawals.     

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.     

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.     

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.     

The amygdala regulates the antianxiety sensitization effect of flumazenil during repeated chronic ethanol or repeated stress

BACKGROUND: The benzodiazepine receptor antagonist flumazenil reduces anxiety-like behavior and sensitization of anxiety-like behavior in various models of ethanol withdrawal in rodents. The mechanism and brain region(s) that account for this action of flumazenil remain unknown. This investigation explored the potential role of several brain regions (amygdala, raphe, inferior colliculus, nucleus accumbens, and paraventricular hypothalamus) for these actions of flumazenil. METHODS: Rats were surgically implanted with guide cannulae directed over the brain region of interest and then treated with an ethanol diet for three 7-day dietary cycles (5 days on ethanol diet followed by 2 days on control diet). At approximately 4 hours, flumazenil was administered intracranially into each of the first 2 withdrawals. Examinations of anxiety-like behavior followed 1 week later during a third withdrawal. In other animals, restraint stress sessions or intra-amygdala DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) injections, preceded by intraperitoneal flumazenil injections, were substituted for the first 2 ethanol treatment cycles to assess the potential anxiety-sensitizing action of stress or a benzodiazepine receptor inverse agonist, respectively. RESULTS: Flumazenil treatment of the amygdala during the first 2 withdrawals blocked the development of sensitized anxiety seen during a third withdrawal. Similar actions of flumazenil were found when stress sessions substituted for the first 2 cycles of ethanol exposure and withdrawal. Amygdala treatment with DMCM magnified the anxiety response to the single subthreshold chronic ethanol treatment, and prophylactic flumazenil blocked this effect. CONCLUSIONS: Intra-amygdala flumazenil inhibits the development of anxiety sensitized by repeated ethanol withdrawal, stress/ethanol withdrawal, or DMCM/ethanol withdrawal. These actions suggest that site-specific and persistent effects of flumazenil on gamma-aminobutyric acid-modulatory processes in this brain region are relevant to sensitized behavioral effects seen in alcoholism.     

Repeated Ethanol Withdrawal Delays Development of Focal Seizures in Hippocampal Kindling

Clinical studies report an increase in the prevalence of alcohol withdrawal-related seizures in patients with a history of multiple detoxifications. In order to investigate the alcohol withdrawal-related alterations in neural activity that lead to this increase in seizure propensity, basic researchers have examined both spontaneous and elicited seizures in animals undergoing withdrawal from chronic ethanol. This study was designed to further examine alcohol withdrawal-related seizure activity in a rodent model by assessing the development of electrical kindling after chronic ethanol exposure administered in multiple or single treatment episodes. Laboratory rats were exposed to either five periods of 3 days of ethanol, one 15-day period of continuous ethanol, or a period of control handling with no ethanol exposure. Ten days after a final withdrawal episode, all animals were surgically prepared with recording and stimulating electrodes. Twenty days after final withdrawal from ethanol or an equivalent period of similar handling, daily electrical stimulation of hippocampal area CA3 was initiated. Animals exposed to ethanol required more daily stimulations to become fully kindled than did ethanol-naive controls, with those animals experiencing five withdrawals requiring the most stimulations overall and more stimulations to progress from focal to generalized seizure behaviors. These results indicate that chronic ethanol exposure and withdrawal alter neuronal mechanisms important for hippocampal kindling in a manner that persists long after cessation of ethanol exposure, and they indicate that this effect is increased by exposure to repeated withdrawal episodes.     

Multiple Withdrawals from Chronic Ethanol "Kindles" Inferior Collicular Seizure Activity: Evidence for Kindling of Seizures Associated with Alcoholism

The present investigation tested the hypothesis that multiple withdrawals from chronic ethanol treatment "kindles" seizure activity. Two animal models of kindled seizure activity--electrical stimulation of the inferior collicular cortex or the amygdala--were used to evaluate this hypothesis. Four withdrawals from a 12-day ethanol-liquid diet regimen facilitated the seizure kindling rate in the inferior collicular cortex, when the stimulation was initiated 7 days after the last withdrawal. In contrast, four withdrawals from this chronic ethanol regimen significantly attenuated the rate of amygdaloid kindling. When the withdrawals were increased to six or 10 using a 5-day chronic ethanol treatment schedule, the kindling rate in the inferior collicular cortex proved directly proportional to the withdrawal number. Continuous ethanol exposure over the same period as the 10 withdrawal group also facilitated the inferior collicular kindling rate, but not to the extent found in the 10 withdrawal group. A before, 10 withdrawals from the 5-day chronic ethanol liquid diet treatment attenuated the rate of amygdaloid kindling. Thus, this kindling action of repeated ethanol withdrawals appears specific to seizures originating from the inferior collicular cortex, not the limbic system. These findings support a previous hypothesis for a kindling etiology of alcoholism related seizures.     

Accentuated decrease in social interaction in rats subjected to repeated ethanol withdrawals

BACKGROUND: Previous work has shown that repeated withdrawals from chronic ethanol exposure can kindle seizures in rodents. In this article, the effects of a three-cycle model of ethanol exposure and withdrawal on the social interaction test of anxiety are summarized. METHODS: Rats were exposed to ethanol (7% or 4.5%) diets over three periods of 5 days, with 2 days of withdrawal between cycles. Between 5 and 6 hr after the ethanol was removed, pairs of rats were placed in open field chambers for the assessment of social interaction behavior and locomotor activity. RESULTS: After the third cycle of ethanol (7%) presentation, both male and female rats exhibited lower social interaction behavior (more anxiety) and activity than after a single cycle. Rats exposed to a similar amount of ethanol but tested while ethanol was still available did not exhibit a reduction in social interaction. The decrease in social interaction was still present for up to 24 hr but had disappeared by 48 hr after ethanol was withdrawn. When rats were allowed 8 or 16 days to recover from the effects of the three-cycle protocol, a further exposure to 5 days of 7% ethanol diet resulted in a reduction in social interaction on withdrawal similar to that seen from the three-cycle protocol. In contrast, rats exposed continuously to 7% ethanol diet for 15 consecutive days exhibited higher levels of social interaction when maintained on control diet for 8 or 16 days and then reexposed to ethanol. Rats that were exposed to the three-cycle protocol and allowed 32 days to recover before being reexposed to ethanol still had a partial deficit in social interaction. Finally, animals subjected to repeated withdrawals from 4.5% ethanol exhibited a reduction in social interaction without a change in activity after the final withdrawal from ethanol, whereas rats exposed continuously to a 4.5% ethanol diet did not exhibit a reduction in social interaction or activity. Neither blood ethanol concentrations nor changes in body weight could account for these behavioral differences. CONCLUSION: Repeated withdrawal from ethanol can lead to accentuated or more persistent anxiety-like behavior in rats, as indicated by a decrease in social interaction. The withdrawal-induced decrease in locomotor activity is not accentuated by repeated withdrawals. This model of repeated withdrawals from ethanol may prove useful in defining the neurochemical basis of this accentuation.     

Effect of Repeated Ethanol Withdrawal on Glutamate Microdialysate in the Hippocampus

BACKGROUND: Our previous studies, which identified that ethanol withdrawal is associated with increases in glutamate microdialysate in the nucleus accumbens and reaches a maximum at 12 hr, have now been extended in order to assess whether repeated cycles of chronic ethanol intoxication followed by 12 hr withdrawal periods on three occasions alters glutamate release in the hippocampus of male rats. METHODS: In this study, the microdialysis technique has been used with the HPLC and electrochemical detection. RESULTS: During the first cycle of ethanol withdrawal, glutamate content increased significantly 8 hr after withdrawal (198.4% +/- 89.14%) by comparison with control rats. During the second period of ethanol withdrawal, 1 week after the initial withdrawal episode, glutamate microdialysate content increased significantly 10 hr after withdrawal, but to a much lower degree than in the first episode (179.08 +/- 25.68%), by comparison with control rats. During the third cycle of ethanol withdrawal, the concentration of glutamate in the hippocampus microdialysate did not significantly change at either of these time points except at 12 hr when glutamate was significantly decreased by comparison with control rats (52.09 +/- 14.38%). Apart from arginine, which was significantly decreased both at the cessation of alcoholization and during the 12 hr of the three withdrawal episodes, none of the other neurotransmitters assayed, aspartate, taurine, alanine, or GABA, showed any significant alteration. CONCLUSION: These results clearly indicate that elevated glutamate release during the first withdrawal episode is not paralleled in subsequent withdrawal episodes.     

Ethanol Tolerance and Withdrawal Responses in GABAA Receptor Alpha 6 Subunit Null Allele Mice and in Inbred C57BL/6J and Strain 129/SvJ Mice

We have been using a genetic strategy to define the contribution of specific candidate genes, such as those encoding subunits of the γ-aminobutyric acid type A receptor, to various ethanol sensitive responses. We have used the gene knockout approach in mouse embryonic stem cells to create mice in which the gene encoding the α6 subunit of the γ-aminobutyric acid type A receptor is rendered nonfunctional. In the present report, we provide a detailed characterization of several behavioral responses to ethanol in these null allele mice. In a separate series of experiments, behavioral response to ethanol was compared between two inbred strains of mice that are commonly used as background stock in knockout experiments, namely C57BL/6J and Strain 129/SvJ. Wild type (α6^+/+) and homozygous null allele (α6^?/?) mice did not differ to the ataxic effects of ethanol on acute functional tolerance (95.8 ± 8.7 vs. 98.8 ± 5.7 mg/dl ± SEM, respectively). Withdrawal hyperexcitability was assessed following chronic exposure to ethanol vapor (EtOH) or air (CONT) in inhalation chambers in a multiple withdrawal treatment paradigm. At the end of the last treatment cycle, mice were scored for handling induced convulsions (HIC). After adjusting for differences in blood ethanol concentration between genotypes at the end of the final treatment cycle, we observed a greater area under the 24-hr HIC curves in mice treated with ethanol (p < 0.0001) but did not detect an effect of genotype (α6^+/+/CONT 3.1 ± 2.0; α6^?/?/CONT 5.5 ± 2.5; α6^+/+/EtOH 30.1 ± 6.2; α6^?/?/EtOH 33.0 ± 5.8 mean units ± SEM). We also examined these mice for differences in protracted tolerance; at ～26 hr into the final withdrawal cycle, each mouse was injected with ethanol (3.5 mg/g body weight) and sleep time was measured. We detected a significant effect of treatment (p <0.001) with ethanol-treated mice demonstrating signs of tolerance as reflected by a reduction in duration of sleep time. However, effect of genotype was not significant (α6^+/+/CONT 57.4 ± 7.6; α6-/-/CONT 59.0 ± 7.6; 6^+/+/EtOH 34.8 ± 7.4; α6^?/?/EtOH 30.8 ± 5.6 min ± SEM). From these data we conclude that the α6 subunit of the GABA_A-R exerts little if any influence on acute functional tolerance, withdrawal hyperexcitability, or protracted tolerance. Strain 129/SvJ and C57BL/6J mice were also compared for acute functional tolerance and were found not to differ (96.3 ± 4.4 vs. 94.8 ±11.3 mg/dl ± SEM, respectively). Withdrawal hyperexcitability was assessed by comparing the area under the 24 hr HIC curves. Strain 129/SvJ mice displayed a much greater basal HIC response compared to C57BL/6J mice (19.8 ± 4.3 vs. 0.2 ± 0.2 mean units ± SEM, respectively); after adjusting for differences in blood ethanol concentration between strains at the end of the final ethanol treatment cycle, the HIC response was markedly enhanced by ethanol treatment in Strain 129/SvJ mice but not in C57BL/6J mice (50.4 ± 3.1 vs. 9.5 ± 5.4 mean units ± SEM, respectively). The effects of treatment (p <0.0001), strain (p <0.0001), and the interaction of strain with treatment (p < 0.01) were significant. Since many gene knockout mice are maintained on a mixed genetic background of Strain 129/SvJ and C57BL/6J, we conclude that significant differences in tests of withdrawal hyperexcitability may be confounded by the influence of genes that cosegregate with the gene targeted allele.     

Interactions of Stress and CRF in Ethanol‐Withdrawal Induced Anxiety in Adolescent and Adult Rats

Background: Repeated stress or administration of corticotropin‐releasing factor (CRF) prior to ethanol exposure sensitizes anxiety‐like behavior in adult rats. Current experiments determined whether adolescent rats were more sensitive to these challenges in sensitizing ethanol withdrawal‐induced anxiety and altering CRF levels in brain during withdrawal. Methods: Male adult and adolescent Sprague–Dawley rats were restraint stressed (1 hour) twice 1 week apart prior to a single 5‐day cycle of ethanol diet (ED; stress/withdrawal paradigm). Other rats received control diet (CD) and three 1‐hour restraint stress sessions. Rats were then tested 5, 24, or 48 hours after the final withdrawal for anxiety‐like behavior in the social interaction (SI) test. In other experiments, adolescent rats were given two microinjections of CRF icv 1 week apart followed by 5 days of either CD or ED and tested in social interaction 5 hours into withdrawal. Finally, CRF immunoreactivity was measured in the central nucleus of the amygdala (CeA) and paraventricular nucleus (PVN) after rats experienced control diet, repeated ethanol withdrawals, or stress/withdrawal. Results: Rats of both ages had reduced SI following the stress/withdrawal paradigm, and this effect recovered within 24 hours. Higher CRF doses were required to reduce SI in adolescents than previously reported in adults. CRF immunohistochemical levels were higher in the PVN and CeA of CD‐exposed adolescents. In adolescent rats, repeated ethanol withdrawals decreased CRF in the CeA but was not associated with decreased CRF cell number. There was no change in CRF from adult treatments. Conclusions: In the production of anxiety‐like behavior, adolescent rats have equal sensitivity with stress and lower sensitivity with CRF compared to adults. Further, adolescents had higher basal levels of CRF within the PVN and CeA and reduced CRF levels following repeated ethanol withdrawals. This reduced CRF within the CeA could indicate increased release of CRF, and future work will determine how this change relates to behavior.     

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.     

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).     

Alcohol withdrawal increases neuropeptide Y immunoreactivity in rat brain

BACKGROUND: Neuropeptide Y (NPY) is widely expressed in the brain and is known to affect consummatory behaviors including drinking alcohol as well as to play a role in seizures. We investigated the effects of a 4 day binge ethanol treatment model that is known to induce physical dependence and withdrawal seizures to determine the effects of ethanol dependence and withdrawal on NPY expression. METHODS: Male Sprague Dawley rats were treated with ethanol or control nutritionally complete diets by intragastric treatment three times per day for 2 or 4 days with an average daily dose of approximately 8 g/kg ethanol per day. Ethanol-fed rats treated for 4 days and then withdrawn for 24, 72, and 168 hr also were studied. Brains were perfused and sectioned for immunohistochemistry for NPY, phospho-cyclic adenosine monophosphate responsive element binding (pCREB), and other proteins. RESULTS: NPY immunoreactivity (NPY-IR) was found in several brain regions, with the hippocampus and cerebral cortex showing the most pronounced changes. NPY-IR was reduced by ethanol treatment in hippocampus and cortex, although at 72 hr of withdrawal there was a dramatic increase in NPY-IR in the hilus of the dentate gyrus and in CA3 and CA2 fields of hippocampus. Ethanol withdrawal seizures occurred around 12 to 24 hr of withdrawal, preceding the changes in NPY-IR at 72 hr. pCREB immunoreactivity (pCREB-IR) tended to decrease during ethanol treatment but showed a dramatic increase in dentate gyrus at 72 hr of withdrawal. Parvalbumin immunoreactivity indicated that some of the pCREB-IR and NPY-IR were within inhibitory interneuron basket cells of the hippocampal hilus. NPY-IR returned to control levels by 168 hr of withdrawal. CONCLUSIONS: These studies suggest that hippocampal NPY is reduced during the development of ethanol dependence. Ethanol withdrawal seizures precede a dramatic increase in hippocampal NPY-IR. Previous studies have suggested that NPY in the hippocampus reduces seizure activity and that NPY is induced by seizure activity. Thus, the increase in NPY-IR at 72 hr of withdrawal after binge ethanol treatment may be protective against prolonged withdrawal seizure activity.     

Nifedipine alleviates alterations in hippocampal kindling after repeated ethanol withdrawal

BACKGROUND: Current clinical treatment of alcohol detoxification commonly includes pharmacotherapy to lessen the potential for seizures, especially in those patients undergoing repeated treatment. Basic research continues to study the alcohol withdrawal-related "kindling" of seizures both to understand the mechanisms involved and to identify alternative treatments. Ethanol withdrawal has been shown to result in the delay of electrical kindling at several brain sites, which suggests a long-lasting disruption of neuronal function. METHODS: This study focused on the participation of the L-type voltage-gated calcium channels in this process by the treatment of animals during withdrawal with nifedipine, an agent that blocks these channels. Animals were randomly assigned to ethanol (ethanol-exposed/ethanol-naive) and drug treatment (nifedipine/vehicle) groups. Subjects receiving ethanol were exposed to five cycles of 3 days' ethanol exposure, with each exposure cycle separated by a 1-day withdrawal period. Drug treatment was administered twice during each withdrawal period. Twenty days after completion of ethanol exposure, animals received daily kindling stimulations to hippocampal area CA3 until the kindling criterion was attained. RESULTS: Ethanol-exposed animals that received vehicle treatment during ethanol withdrawal required more daily stimulations to become fully kindled than did ethanol-naive controls. This delay in seizure development was most pronounced in the progression from focal seizure behaviors to more generalized seizures. Animals that received the same ethanol exposure but that were treated with nifedipine required significantly fewer stimulations than did ethanol-exposed animals that received vehicle. Ethanol-exposed/ nifedipine-treated animals did not differ from ethanol-naive controls that received vehicle or nifedipine. CONCLUSIONS: Alcohol withdrawal-related alterations in seizure-sensitive neural circuitry such as the hippocampus persist long after cessation of ethanol exposure. Furthermore, the L-type voltage-gated calcium channels are involved in this effect in that blockade of these channels during acute withdrawal alleviates alterations in seizure mechanisms on a long-term basis.     

Alcohol Withdrawal Kindling: Mechanisms and Implications for Treatment

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was Larry P. Gonzalez. The presentations were (1) EEG indices of sensitization in a murine model of repeated ethanol withdrawals, by Lynn M. Veatch; (2) Long-term changes in central nervous system function after repeated alcohol withdrawals: Recommendations for the treatment of acute withdrawal, by Larry P. Gonzalez; (3) Differential regulation of GABA_A and NMDA receptors by repeated ethanol treatment in cultured mammalian neurons, by Maharaj K. Ticku; and (4) Involvement of GABA_A and NMDA receptors in alcohol withdrawal kindling: Implications for treatment, by Howard C. Becker.     

Only male mice show sensitization of handling-induced convulsions across repeated ethanol withdrawal cycles

BACKGROUND: Alcohol abuse, especially when experienced in multiple cycles of chronic abuse and withdrawal, leads to a sensitization of central nervous system hyperexcitability that may culminate in overt expression of seizures. In spite of the growing prevalence of alcohol abuse and dependence in females shown in recent epidemiologic studies, evidence of sexual dimorphism in the expression of alcohol withdrawal-induced seizures and the development of seizure sensitization following multiple cycles of ethanol (EtOH) exposure and withdrawal has not been examined in either animal models or in clinical reports. METHODS: Subjects in these experiments were male and female C3H/Hecr mice. The female mice were intact or ovariectomized, with ovariectomized mice receiving 17-beta-estradiol or placebo pellets. All mice were exposed to 4 cycles of exposure to 16-hour EtOH vapor, separated by 8-hour withdrawal periods. During each 8-hour withdrawal, hourly assessment of seizure propensity was assessed as handling-induced convulsions. Additional assessments were taken up to 72 hours after the final EtOH withdrawal cycle. RESULTS: Male and female mice showed similar seizure propensity during an initial withdrawal from chronic EtOH. Across subsequent withdrawal cycles, however, male mice exhibited a robust increase in seizure severity beginning with the third withdrawal cycle. In marked contrast, female mice failed to demonstrate sensitization of seizure severity. The lack of seizure sensitization following up to 4 cycles of alcohol exposure and withdrawal could not be explained by hormonal status (presence or absence of estrogen) or by sex differences in blood alcohol levels. CONCLUSIONS: Male and female mice exposed to the same number of cycles of EtOH withdrawal demonstrate differences in expression of seizures. Males show the typical sensitization of seizures, or kindling response, which has been reported clinically as well as in animal models, but females do not. The reason for the lack of seizure sensitization in female mice remains to be elucidated, but may be related to sex differences in alcohol effects on excitatory/inhibitory neurotransmission, rather than to hormonal or blood alcohol level differences.     

Lorazepam for seizure prophylaxis during high-dose busulfan administration

Seizure is a recognized complication of high-dose busulfan (BU) therapy and phenytoin (DPH) is widely used as prophylaxis. A number of adverse effects have been associated with DPH and it may also interfere with BU metabolism. We used lorazepam (median dose 0.022 mg/kg) i.v. or p.o. before each dose and for 24 h after the last dose of BU as seizure prophylaxis to 29 children undergoing hematopoietic stem cell transplantation. The regimen was well tolerated and drowsiness was the only significant side-effect. Twelve patients were able to receive the entire prophylaxis by mouth. No seizure developed during and within 48 h of BU. Concomitant pharmacokinetic studies showed no alternation of the absorption and clearance of BU during lorazepam administration. Lorazepam can be used as an alternative for seizure prophylaxis during high-dose BU treatment.     

Sensitivity to Strychnine Seizures Is Unaltered during Ethanol Withdrawal

Previous research from this laboratory has indicated that animals chronically exposed to ethanol and then withdrawn exhibit a variety of symptoms of central nervous system hyperexcitability that occur in unique clusters. These clusters of symptoms were observed to differ in their duration and in the time of onset relative to the time of ethanol withdrawal. In addition, the observed clusters of spontaneous seizure events in these animals were seen to correlate with differences in sensitivity to seizure-inducing treatments. These results suggest that seizure sensitivity during ethanol withdrawal may indicate the involvement of multiple, independent, neuronal mechanisms. To investigate this possibility further, the following study examined the sensitivity of ethanol-withdrawn animals to seizures induced by the glycine antagonist strychnine. Seizure sensitivity to strychnine was evaluated at the same times following ethanol withdrawal when animals were previously seen to show the differential occurrence of spontaneous seizure events and also differences in sensitivity to picrotoxin-induced seizures. Ethanol-withdrawn animals did not differ in their responses to strychnine compared with ethanol-naive controls at any of the times tested. This lack of change in seizures induced by antagonism of glycine inhibition occurred in spite of the increased sensitivity of similarly treated animals to picrotoxin-induced seizures at the same test times. These data suggest that chronic ethanol exposure and withdrawal may not significantly alter the function of glycinergic inhibitory neurotransmission and that the ethanol withdrawal syndrome is indicative of alterations in specific neuronal mechanisms rather than of a generalized state of central nervous system hyperexcitability.