Maury Cole

Excessive Ethanol Drinking Following a History of Dependence: Animal Model of Allostasis

Alcohol withdrawal symptoms, particularly negative emotional states, can persist for months following the removal of alcohol. These protracted withdrawal symptoms have been implicated as an important trigger of relapse to excessive drinking in alcoholics and may represent a long lasting shift in affective tone as a result of chronic alcohol exposure. It was shown previously that ethanol-dependent rats increased their operant responding for ethanol when tested during the first 12 hr after withdrawal. The purpose of the present experiments was to determine the persistence of this finding by examining operant oral ethanol self-administration in rats with a history of physical dependence upon ethanol, detoxified and then allowed a two week period of protracted abstinence. The results of these experiments indicate that operant responding for ethanol was enhanced during protracted abstinence by 30–100% and remained elevated for 4–8 weeks post acute withdrawal. These results have important implications for understanding the characteristics and mechanisms underlying vulnerability to relapse.

Dependence-induced increases in ethanol self-administration in mice are blocked by the CRF1 receptor antagonist antalarmin and by CRF1 receptor knockout

Models of dependence-induced increases in ethanol self-administration will be critical in increasing our understanding of the processes of addiction and relapse, underlying mechanisms, and potential therapeutics. One system that has received considerable attention recently is the CRF(1) system that may mediate the link between anxiety states and relapse drinking. C57BL/6J mice were trained to lever press for ethanol, were made dependent and then were allowed to self-administer ethanol following a period of abstinence. The effect of the CRF(1) antagonist, antalarmin, was examined on this abstinence-induced self-administration in a separate group of mice. Finally, dependence-induced changes in ethanol self-administration were examined in CRF(1) knockout and wild type mice. The results indicated that ethanol self-administration was increased following the induction of dependence, but only after a period of abstinence. This increase in ethanol self-administration was blocked by antalarmin. Furthermore, CRF(1) knockout mice did not display this increased ethanol self-administration following dependence and abstinence. These studies, using both a pharmacological and genetic approach, support a critical role for the CRF(1) system in ethanol self-administration following dependence. In addition, a model is presented that may be useful for studies examining underlying mechanisms of the ethanol addiction process as well as for testing potential therapeutics.

Alcohol Metabolism in Asian-American Men with Genetic Polymorphisms of Aldehyde Dehydrogenase

Epidemiologic studies have found that rates of alcohol use and alcoholism in persons of Asian descent are lower than rates in other ethnic groups. One possible reason is that about half of certain Asians, including Chinese, Japanese, and Korean persons, have a deficiency of the low-Km mitochondrial aldehyde dehydrogenase (ALDH2) isoenzyme, which is responsible for metabolizing acetaldehyde. A deficiency of ALDH2 results from inheritance of the mutant ALDH2*2 allele, a dominant mutation that exerts its effect both by reducing enzyme activity and increasing the turnover of this activity [1, 2]. After ingestion of alcohol, the faces of Asians with one or both alleles of ALDH2*2 become visibly flushed. Asians who are homozygous for ALDH2*1 generally lack visible alcohol-induced flushing or experience only a mild flush response. The dominance of the ALDH2 mutation, however, does not seem to be complete; phenotypic differences are associated with the three ALDH2 genotypes. Asians who are homozygous for ALDH2*2 drink very little alcohol [3], and no studies have found alcoholic persons with this genotype [4-8]. Asians who are heterozygous for ALDH2*2 drink less alcohol and are also less likely to be alcoholic compared with Asians with ALDH2*1 alleles, but they are not fully protected from alcoholism. Approximately 12% of alcoholic Asians have the ALDH2*1/2*2 genotype [5]. In the context of alcoholism or lower alcohol intake, Asian persons who are heterozygous for ALDH2*2 may be more vulnerable to alcohol-associated conditions, including liver disease [6-9], asthma [9], and esophageal cancer [10]. The three ALDH2 genotypes are also associated with variability in response to alcohol [11]. Asians who are homozygous for ALDH2*2 are very sensitive to alcohol and have tachycardia, hypotension, and vomiting after ingesting a moderate amount of alcohol. Asians who are heterozygous for ALDH2*2 are more sensitive to alcohol than Asians with ALDH2*1 alleles, although the response of the former is not necessarily aversive. Among Asians with an ALDH2 deficiency, differences in sensitivity to alcohol may be mediated by differences in alcohol metabolism, slower elimination of alcohol, or accumulation of acetaldehyde in the blood [12]. Some studies [13-17] have measured blood levels of alcohol or acetaldehyde after ingestion of alcohol in Asians who were known to have ALDH2 genotypes, but these studies had an inadequate sample size, did not include a placebo control, or did not control for use of alcohol and cigarettes (which can alter alcohol metabolism). We measured blood levels of alcohol and acetaldehyde after ingestion of alcoholic or placebo beverages in Asian-American men who underwent genotyping at the ALDH2 locus. Particular attention was given to matching the groups for age, height, weight, history of alcohol use, and history of smoking. Methods Asian-American men 21 to 25 years of age were recruited from advertisements in university newspapers for our randomized, double-blind, crossover study. They completed a questionnaire that solicited information on demographic characteristics; patterns of and problems with alcohol and drug use; and family history of alcohol, drug, and psychiatric problems. We excluded persons who completely abstained from alcohol, persons who had consumed more than 60 standard alcoholic drinks per month during the previous 6 months, and persons who reported that either biological parent was not of Chinese, Japanese, or Korean descent. Thirty-five men who did not have a personal or family history of alcohol dependence and who had no evidence of other substance dependence, major psychiatric disorders, or medical disorders gave informed consent to participate in two test sessions. The study was approved by the institutional review board at the Scripps Clinic and Research Foundation. Participants were asked to refrain from using alcohol, cigarettes, and other drugs (including aspirin, nonsteroidal anti-inflammatory agents, and anti-histamines) that might alter alcohol metabolism for 3 days before testing. On test days, each participant arrived at the clinic at 7:30 a.m. after an overnight fast. He then ate a low-fat breakfast (two slices of dry toast and juice), and an indwelling heparin lock was inserted for drawing blood. At the first session, blood was drawn and genotyping at the ALDH2 locus was done by using polymerase chain reaction of DNA and allele-specific oligonucleotide probes [1]. At 9:00 a.m., each participant was given a placebo beverage (3 mL of 95% alcohol in a reservoir on top of noncaffeinated, sugar-free soda) or 0.75 mL of 95% alcohol (0.56 g/kg of body weight) as a 20%-by-volume solution in the same mixer. The alcohol and placebo were ingested over 7 minutes through a placebo alcohol apparatus [18]. Blood was drawn to determine levels of alcohol and acetaldehyde before beverage ingestion and 15, 30, 45, 60, 90, 120, and 150 minutes after beverage ingestion. Blood alcohol concentrations were determined by using a modified alcohol dehydrogenase assay [19]. The rate of alcohol elimination (mg/kg per hour) was calculated from the slope of the pseudolinear decline of the blood alcohol concentration-time curve (usually from the 90-, 120-, and 150- minute samples) by using linear least-squares regression. Blood acetaldehyde levels were determined by using a modified fluorigenic high-performance liquid chromatographic assay [20] that had a detection sensitivity in the picomole range and intra-assay and interassay precisions of 2.4% and 3.7%, respectively. Statistical analyses, done by using SYSTAT software (SYSTAT, Inc., Evanston, Illinois), focused on differences between participants with ALDH2*1/2*1 and those with ALDH2*1/2*2. Demographic information, data on recent alcohol and cigarette use, peak blood alcohol concentration, time to peak blood alcohol concentration, volume of distribution, and rate of alcohol elimination were analyzed by using one-way analysis of variance; ALDH2 genotype was a between-participant variable. Data on blood alcohol concentration and acetaldehyde level were analyzed by using separate 2 8 analysis of variance for alcohol and placebo sessions; ALDH2 genotype was a between-participant variable, and time was a repeated measurement. Significant interactions were then analyzed by using post hoc comparisons with contrast matrices. The Bonferroni correction was used to limit the familywise error rate to 0.05 for comparisons among the placebo session time points and among the alcohol session time points. Results Genotyping for ALDH2 revealed 20 participants who had ALDH2*1/2*1 genotype, 13 who had ALDH2*1/2*2 genotype, and 2 who had ALDH2*2/2*2 genotype. Three participants (1 with ALDH2*1/2*2 genotype and the 2 with ALDH2*2/2*2 genotype) became ill after ingesting alcohol and were excluded from analyses because of missing data. One participant with ALDH2*1/2*2 genotype whose acetaldehyde levels exceeded 4 SDs from the mean (most likely as a result of instrumentation error) was also excluded from data analyses. The Table 1 shows demographic information and patterns of recent alcohol and cigarette use for the remaining 31 men. The ALDH2 genotype groups did not differ significantly for any of these variables; this reflects participant selection. Table 1. Demographic Information and Recent Patterns of Alcohol and Cigarette Use in 20 Asian-American Men with ALDH2*1/2*1 Genotype and 11 Asian-American Men with ALDH2*1/2*2 Genotype* Mean peak blood alcohol concentration SD was 81.3 12.48 mg/dL; the peak occurred 43.1 15.85 minutes after ingestion of alcohol. Mean volume of distribution was 0.718 0.1124 L/kg of body weight, and the mean rate of alcohol elimination was 97.8 33.97 mg/kg per hour. The ALDH2 genotype groups did not differ significantly for any of these variables. Mean blood alcohol concentrations for the alcohol session and mean acetaldehyde levels for the placebo and alcohol sessions, measured over time according to ALDH2 genotype, are shown in the (Figure 1). Figure 1. Mean (SD) blood levels of alcohol and acetaldehyde before and after ingestion of a placebo beverage containing 3 mL of 95% alcohol and an alcoholic beverage (0. n n P Analysis of variance revealed that the main effects for ALDH2 genotype and the interaction between ALDH2 genotype and time were not significant for the data on blood alcohol concentration from the alcohol session. Analysis of variance also revealed that the main effects for ALDH2 genotype and the interaction between ALDH2 genotype and time were significant for the data on acetaldehyde levels from the placebo sessions (ALDH2 genotype, P < 0.005; interaction between ALDH2 genotype and time, P < 0.013) and the alcohol sessions (ALDH2 genotype, P < 0.002; interaction between ALDH2 genotype and time, P < 0.001). Post hoc analyses with Bonferroni corrections revealed significant group differences 30, 45, and 60 minutes after placebo ingestion and 60, 90, 120, and 150 minutes after alcohol ingestion. Discussion We found that Asian-American men who were heterozygous for the ALDH2*2 allele did not differ from carefully matched men who were homozygous for the ALDH2*1 allele in measures of blood alcohol concentration overall or at any time after alcohol ingestion. These findings are consistent with the results of one study [14] but differ from those of other studies [15-17] in which persons with ALDH2*2 alleles had significantly slower rates of alcohol elimination than did persons with ALDH2*1 alleles. These discrepancies may result from group differences on important variables, especially history of alcohol use, that we controlled for. We also found that, despite equivalent blood alcohol concentrations, participants with ALDH2*1/2*2 genotype had significantly higher blood acetaldehyde levels after ingesting the alcohol beverage than did participants with ALDH2*1/2*1 genotype. These findings suggest that blood acetaldehyde levels rather than bloo

Operant behavior and alcohol levels in blood and brain of alcohol-dependent rats.

BACKGROUND The purpose of the present investigation was to more clearly define blood-alcohol parameters associated with alcohol dependence produced by alcohol vapor inhalation and alcohol-containing liquid diet. METHODS Alcohol levels in blood and brain were compared during and after 4 hours of acute alcohol vapor exposure; also, brain-alcohol levels were assessed in alcohol-exposed (14-day alcohol vapor) and alcohol-naïve rats during and after 4 hours of acute alcohol vapor exposure. A separate group of rats were implanted with i.v. catheters, made dependent on alcohol via vapor inhalation, and tested for operant alcohol responding; blood-alcohol levels (BALs) were measured throughout operant alcohol drinking sessions during alcohol withdrawal. A final group of rats consumed an alcohol-liquid diet until they were dependent, and those rats were then tested for operant behavior at various withdrawal time points; BALs were measured at different withdrawal time points and after operant sessions. RESULTS Blood- and brain-alcohol levels responded similarly to vapor, but brain-alcohol levels peaked at a higher point and more slowly returned to zero in alcohol-naïve rats relative to alcohol-exposed rats. Alcohol vapor exposure also produced an upward shift in subsequent operant alcohol responding and resultant BALs. Rats consumed large quantities of alcohol-liquid diet, most of it during the dark cycle, sufficient to produce high blood-alcohol levels and elevated operant alcohol responding when tested during withdrawal from liquid diet. CONCLUSIONS These results emphasize that the key determinants of excessive alcohol drinking behavior are the BAL range and pattern of chronic high-dose alcohol exposure.

Vapor Inhalation of Alcohol in Rats

Alcohol dependence constitutes a neuroadaptive state critical for understanding alcoholism, and various methods have been utilized to induce alcohol dependence in animals, one of which is alcohol vapor exposure. Alcohol vapor inhalation provides certain advantages over other chronic alcohol exposure procedures that share the ultimate goal of producing alcohol dependence in rats. Chronic alcohol vapor inhalation allows the experimenter to control the dose, duration, and pattern of alcohol exposure. Also, this procedure facilitates testing of somatic and motivational aspects of alcohol dependence. Chronic exposure to alcohol vapor produces increases in alcohol‐drinking behavior, increases in anxiety‐like behavior, and reward deficits in rats. Alcohol vapor inhalation as a laboratory protocol is flexible, and the parameters of this procedure can be adjusted to accommodate the specific aims of different experiments. This unit describes the options available to investigators using this procedure for dependence induction, when different options are more or less appropriate, and the implications of each. Curr. Protoc. Neurosci. 44:9.29.1‐9.29.19.

Periadolescent alcohol exposure has lasting effects on adult neurophysiological function in rats

Most individuals have their first experience with ethanol (EtOH) consumption as adolescents. Episodes of high EtOH drinking, lasting from hours to days (i.e. binges), are not uncommon. Thus, adolescent EtOH drinking has become a significant health concern due to the possible protracted effects of high doses of EtOH on behavior and the developing brain. This study assessed the effects of brief high levels of EtOH during periadolescence on subsequent behavior and electrophysiology in adult rats. Male Sprague-Dawley rats were exposed to EtOH vapor for 5 days (i.e. postnatal days 35-40) or 10 days (i.e. postnatal days 30-40) for 12 h/day. Locomotor activity, EEG activity, and event-related potentials (ERPs) were then assessed at 1 and 6-7 weeks post EtOH exposure. Significant differences in locomotor activity were not observed at 1 week or 6-7 weeks post-ethanol exposure. However, EtOH exposure did have long-term electrophysiological effects. EtOH exposure increased the frequency of the EEG in the 1-2 Hz range in the parietal cortex and the 16-32 Hz range in the hippocampus. EtOH exposure also increased hippocampal N2 amplitude, decreased hippocampal P3 amplitude, and decreased cortical and hippocampal P2 amplitudes. While these findings are generally similar to those reported following long-term ethanol exposure during adulthood, alcohol exposure during adolescence appears to produce more robust hippocampal effects following shorter periods of exposure. In addition, these data indicate that, in the absence of overt behavioral differences, there are long-lasting changes in the functional brain activity of adult rats briefly exposed to high levels of EtOH during the periadolescent period.

Recruitment of a Neuronal Ensemble in the Central Nucleus of the Amygdala Is Required for Alcohol Dependence

Abstinence from alcohol is associated with the recruitment of neurons in the central nucleus of the amygdala (CeA) in nondependent rats that binge drink alcohol and in alcohol-dependent rats. However, whether the recruitment of this neuronal ensemble in the CeA is causally related to excessive alcohol drinking or if it represents a consequence of excessive drinking remains unknown. We tested the hypothesis that the recruitment of a neuronal ensemble in the CeA during abstinence is required for excessive alcohol drinking in nondependent rats that binge drink alcohol and in alcohol-dependent rats. We found that inactivation of the CeA neuronal ensemble during abstinence significantly decreased alcohol drinking in both groups. In nondependent rats, the decrease in alcohol intake was transient and returned to normal the day after the injection. In dependent rats, inactivation of the neuronal ensemble with Daun02 produced a long-term decrease in alcohol drinking. Moreover, we observed a significant reduction of somatic withdrawal signs in dependent animals that were injected with Daun02 in the CeA. These results indicate that the recruitment of a neuronal ensemble in the CeA during abstinence from alcohol is causally related to excessive alcohol drinking in alcohol-dependent rats, whereas a similar neuronal ensemble only partially contributed to alcohol-binge-like drinking in nondependent rats. These results identify a critical neurobiological mechanism that may be required for the transition to alcohol dependence, suggesting that focusing on the neuronal ensemble in the CeA may lead to a better understanding of the etiology of alcohol use disorders and improve medication development. SIGNIFICANCE STATEMENT Alcohol dependence recruits neurons in the central nucleus of the amygdala (CeA). Here, we found that inactivation of a specific dependence-induced neuronal ensemble in the CeA reversed excessive alcohol drinking and somatic signs of alcohol dependence in rats. These results identify a critical neurobiological mechanism that is required for alcohol dependence, suggesting that targeting dependence neuronal ensembles may lead to a better understanding of the etiology of alcohol use disorders, with implications for diagnosis, prevention, and treatment.

Controlled and behaviorally relevant levels of oral ethanol intake in rhesus macaques using a flavorant-fade procedure.

BACKGROUND Flavorant-fading procedures can initiate and maintain oral ethanol intake in rodents. The present study developed a similar procedure to achieve controlled and behaviorally relevant levels of ethanol intake in monkeys. METHODS Male rhesus macaques (N = 13) were initially given the opportunity to consume 0.5 g/kg of a 1% (w/v) ethanol plus 4% (w/v) Tang solution in 1-hr limited-access sessions without the requirement of an operant response. Once consumption was stable at a particular concentration (%) and/or amount (g/kg), animals were given access to higher concentrations and/or amounts of ethanol. Animals were tested on a bimanual motor skill (BMS) task 20 and 90 min after consumption to assess behavioral impairment. Blood alcohol levels (BALs) were assessed after a session in which animals had the opportunity to consume up to 3.0 g/kg of 6% (w/v) ethanol. RESULTS The gradual fading up of higher concentrations and amounts of ethanol resulted in controlled and robust levels (>2.0 g/kg) of ethanol intake in half of the subjects. Increasing the concentration of the sweetener from 4 to 6% (w/v) was effective in initiating consumption in three animals. Two monkeys required the additional step of presenting the increased-sweetener solutions after a meal (postprandial consumption) to initiate significant ethanol intake. Animals were significantly impaired on the BMS task after consumption of 2.0, 2.5, and 3.0 g/kg of ethanol. Individual consumption ranging from 0.8 to 3.0 g/kg of ethanol produced BALs of 18 to 269 mg/dl. CONCLUSIONS The flavorant-fading procedure was effective in producing behaviorally relevant levels of ethanol consumption in rhesus macaques. This model facilitated a randomized-dose procedure to determine the behavioral effects of 0.5 to 3.0 g/kg of ethanol. This procedure therefore is of significant utility in determining behavioral or physiologic effects of specific doses of consumed ethanol in monkeys.

Exposure to chronic intermittent nicotine vapor induces nicotine dependence

Animal models of drug exposure are important tools for the study of the neurobiological mechanisms of nicotine dependence and as preclinical models for medication development. There are few non-invasive animal models of nicotine exposure and currently there is no known animal model of second-hand exposure to nicotine. We hypothesized that chronic administration of nicotine vapors would produce blood levels of nicotine in rodents that are clinically relevant to those observed in human smoking and that rodents exposed to nicotine vapors would develop dependence to nicotine. We developed a system that vaporizes nicotine in the air in a stable, reliable and consistent manner. Intermittent exposure to nicotine vapor (0.2mg/m(3)) for 8 or 14h per day for 7days produced a concentration of nicotine in the blood of 22ng/mL. Sixteen hours after removal from nicotine vapors, rats showed significant somatic withdrawal signs precipitated by mecamylamine (1.5mg/kg). These results provide a new rodent model of nicotine dependence using vapor administration that produces consistent levels of nicotine in the blood that are relevant for both heavy smoking and second-hand smoking, using a non-invasive technique that mimics the intermittent aspect and route of administration in humans.

Development of Individual Alcohol Inhalation Chambers for Mice: Validation in a Model of Prenatal Alcohol

BACKGROUND The purpose of this work was first to develop a system of individual chambers through which controlled delivery of alcohol vapors allows us to target specific blood alcohol levels (BALs) in mice without requiring the administration of an alcohol dehydrogenase inhibitor. As a proof of concept, we demonstrated that this new system could be used to expose pregnant BALB/c or C57BL/6 mice to alcohol and that the hypothalamic-pituitary-adrenal (HPA) axis of their mature offspring exhibited the well-known hyperactivity that has been previously documented in rats. METHODS A first series of experiments was designed to establish the parameters that resulted in specific BALs in nonpregnant adult male and female BALB/c as well as C57BL/6 mice that were exposed to various alcohol flow rates. Using information gathered from these experiments, we then chose a regimen of 6 hr of daily vapor exposure in pregnant mice to determine whether this regimen would alter the HPA axis activity of their mature offspring. Control dams were maintained in similar chambers but without alcohol. We first used control mice to assess plasma ACTH levels as a function of shock intensity as well as total duration of the shock session. The most suitable protocol was then used to measure shock-induced ACTH release in 2-month-old male and female offspring that were exposed to alcohol prenatally or not. RESULTS BALs increased as a function of the alcohol flow rates and remained within an acceptable range of homogeneity, consistency, and reproducibility over the desired periods of time. There were no sex differences in BALs while vapors were delivered. However, there was a strain difference in that BALB/c mice displayed slightly higher BALs than C57BL/6. Female mice also exhibited a slightly more pronounced decrease in BALs, compared with male mice, once removed from the drug. Measurement of plasma ACTH levels as a function of the intensity and duration of the shock sessions indicated that 0.3 mA intensity, 1-sec duration shocks at the rate of 2 shocks/min for 20 min provided the most reliable protocol. We then used the alcohol model in pregnant mice. Alcohol exposure did not interfere with maternal weights during gestation. When offspring were tested at 8 to 9 weeks of age, male and female BALB/c as well as female C57BL/6 mice that were exposed to alcohol vapors prenatally exhibited significantly higher shock-induced plasma ACTH levels, compared with controls of the same strain. CONCLUSIONS Collectively, our results indicate that the individual alcohol chamber system that we have developed offers a reliable means of exposing mice to alcohol so that they reach predetermined BALs in the absence of the pharmacological manipulations often used to influence alcohol metabolism in this species. This system, which is compatible with normal weight gains, was used to provide evidence that as previously demonstrated in rats, adult murine offspring of alcohol-treated dams exhibit a hyperactive HPA axis. The development of protocols for use in mice offers the possibility of investigating the influence of alcohol in mutant animals with manipulations of specific genes of interest.