Christopher L. Kliethermes

Anxiety-like behaviors following chronic ethanol exposure

Rodent models of ethanol withdrawal-induced anxiety have been used to explore the neurobiology underlying withdrawal and to evaluate the utility of therapeutic agents aimed at reducing withdrawal severity. Of the many tests of anxiety-like behavior, the elevated plus maze, light/dark box, and open field are the most commonly used. In general, ethanol withdrawal decreases most or all of the individual behaviors recorded in these tasks, indicating the occurrence of an anxiogenic-like effect of withdrawal in rodents, although these effects of withdrawal have not always been found. Potential problems with interpreting the effects of withdrawal as being indicative of an anxiety-like state include the effects of withdrawal on motivation to explore an apparatus, non-specific effects of withdrawal on locomotion, and the use of test parameters that have not been pharmacologically validated. For example, most of the published studies interpreted as having shown increased anxiety-like behavior during ethanol withdrawal have also observed concurrent decreases in locomotion. At a minimum, a given test of anxiety-like behavior during withdrawal should be responsive to the dose and duration of ethanol exposure that was used to produce physical dependence, and should not non-specifically decrease locomotion. In addition, standard anxiolytic drugs should ameliorate the anxiogenic-like effects of withdrawal, preferably in multiple tests of anxiety-like behavior.

Selection for contextual fear conditioning affects anxiety-like behaviors and gene expression.

Conditioned fear and anxiety‐like behaviors have many similarities at the neuroanatomical and pharmacological levels, but their genetic relationship is less well defined. We used short‐term selection for contextual fear conditioning (FC) to produce outbred mouse lines with robust genetic differences in FC. The high and low selected lines showed differences in fear learning that were stable across various training parameters and were not secondary to differences in sensitivity to the unconditioned stimulus (foot shock). They also showed a divergence in fear potentiated startle, indicating that differences induced by selection generalized to another measure of fear learning. However, there were no differences in performance in a Pavlovian approach conditioning task or the Morris water maze, indicating no change in general learning ability. The high fear learning line showed greater anxiety‐like behavior in the open field and zero maze, confirming a genetic relationship between FC and anxiety‐like behavior. Gene expression analysis of the amygdala and hippocampus identified genes that were differentially expressed between the two lines. Quantitative trait locus (QTL) analysis identified several chromosomal regions that may underlie the behavioral response to selection; cis‐acting expression QTL were identified in some of these regions, possibly identifying genes that underlie these behavioral QTL. These studies support the validity of a broad genetic construct that includes both learned fear and anxiety and provides a basis for further studies aimed at gene identification.

An Evolutionary Conserved Role for Anaplastic Lymphoma Kinase in Behavioral Responses to Ethanol

Anaplastic lymphoma kinase (Alk) is a gene expressed in the nervous system that encodes a receptor tyrosine kinase commonly known for its oncogenic function in various human cancers. We have determined that Alk is associated with altered behavioral responses to ethanol in the fruit fly Drosophila melanogaster, in mice, and in humans. Mutant flies containing transposon insertions in dAlk demonstrate increased resistance to the sedating effect of ethanol. Database analyses revealed that Alk expression levels in the brains of recombinant inbred mice are negatively correlated with ethanol-induced ataxia and ethanol consumption. We therefore tested Alk gene knockout mice and found that they sedate longer in response to high doses of ethanol and consume more ethanol than wild-type mice. Finally, sequencing of human ALK led to the discovery of four polymorphisms associated with a low level of response to ethanol, an intermediate phenotype that is predictive of future alcohol use disorders (AUDs). These results suggest that Alk plays an evolutionary conserved role in ethanol-related behaviors. Moreover, ALK may be a novel candidate gene conferring risk for AUDs as well as a potential target for pharmacological intervention.

Anxiety‐Like Behavior in Mice in Two Apparatuses During Withdrawal From Chronic Ethanol Vapor Inhalation

BACKGROUND Anxiety during ethanol withdrawal may be a factor in relapse to alcohol abuse and dependence. Animal models of ethanol withdrawal have typically used forced consumption of an ethanol-containing liquid diet to induce dependence. Ethanol vapor inhalation offers an advantage over liquid diet consumption in that the onset of withdrawal can be temporally controlled more precisely, allowing studies of the development of withdrawal symptoms. METHODS The purpose of the current study was to induce ethanol dependence in mice using an inhalation procedure and to assess withdrawal anxiety symptoms behaviorally in the elevated zero maze and in the light/dark box. Male and female mice were exposed to 3 days of ethanol vapors. Anxiety-like behavior was measured on the elevated zero maze and light/dark box at multiple time points during withdrawal. RESULTS Mice experiencing ethanol withdrawal demonstrated increased anxiety-like behaviors relative to control animals in both apparatuses. However, this finding was specific to the procedure used with the elevated zero maze and was strongly influenced by sex in the light/dark box. CONCLUSIONS Ethanol vapor inhalation appears to be a valid tool for the study of withdrawal-induced anxiety.

Validation of a modified mirrored chamber sensitive to anxiolytics and anxiogenics in mice

RationaleAnxiety is a common disorder in humans that exists in many forms, and animal models of human anxiety are typically employed for the discovery of anxiolytic drugs with human therapeutic potential.ObjectivesIdeally, animal models of anxiety are validated for the detection of both anxiogenic and anxiolytic effects, but most animal models can effectively only measure anxiolytic-like effects. As control animals typically spend small amounts of time in the aversive portion of an apparatus, decreases in time spent in this portion are difficult to detect.MethodsWe have modified an existing test of murine anxiety, the mirrored chamber, and have validated this test using several anxiolytic and anxiogenic drugs. In addition, nine mouse strains were compared on the elevated plus maze and modified mirrored chamber.ResultsIncreasing doses of ethanol, diazepam, and pentobarbital produced an anxiolytic-like profile while pentylenetetrazol (PTZ), d-amphetamine, and methyl-6, 7-dimethoxyl-4-ethyl-beta-carboline-3-carboxylate (DMCM) appeared anxiogenic. This modified test also dissociated drug effects on anxiety from those on activity for d-amphetamine and diazepam. The inbred mouse strains tested produced a similar range of scores for time spent on the open arms of the elevated plus maze and voluntary reentry time in the mirrored chamber, with an overall genetic correlation of 0.68.ConclusionsSince control animals reliably reentered the more aversive portion of the apparatus for 25% of the total time available, the modified mirrored chamber may be able to detect anxiogenic states produced by various stressors and drug withdrawal. Further, the strain differences detected suggest that the modified mirrored chamber will be a valuable tool in the discovery of the genetic bases of anxiety states and disorders.

Pharmacological and genetic influences on hole-board behaviors in mice.

Head dipping on a hole-board is frequently used as an indicator of exploratory tendencies in rodent studies. Drugs with diverse pharmacological properties alter head dipping suggesting that many neurotransmitter systems are involved in the expression of exploratory behavior. The aim of the current experiments was to determine the effects of several drugs from different classes on head dipping, and to compare the effects of some of these agents in lines of mice that have been selectively bred for divergent expression of head dipping on a hole-board. In the current experiments, the effects on head dipping of three doses each of fluoxetine, desipramine, GBR-12909, methamphetamine, pentylenetetrazol, and diazepam were evaluated in genetically heterogeneous mice. Most drugs altered the number of head dips in a predictable manner, but the effects on locomotion were generally as large as those seen for head dipping. Locomotion could completely account for the effects of fluoxetine and pentylenetetrazol, and to a lesser extent, diazepam. We have also developed replicate lines of mice selectively bred for high (High Exploratory Behavior: HEB) or low (Low Exploratory Behavior: LEB) head dipping on a hole-board and evaluated the effects of diazepam and methamphetamine on hole-board behaviors in these mice. Diazepam increased head dipping and locomotion equivalently in both lines of mice, but methamphetamine stimulated locomotion in HEB mice more than in LEB mice. These results broadly suggest that the effects of most drugs we tested are not specific for head dipping, since almost all drugs tested affected head dipping and locomotion equivalently. However, the results with the genetically heterogeneous mice and HEB and LEB mice suggest that some aspects of the dopaminergic system are involved in head dipping.

Drug reward and intake in lines of mice selectively bred for divergent exploration of a hole board apparatus

Individuals characterized as high‐novelty seekers are more likely to abuse drugs than are low‐novelty seekers, and it is possible that the biological substrates underlying novelty seeking and drug abuse are similar. We selectively bred replicate lines of mice from a B6D2 F3 hybrid stock for high exploratory behavior (HEB) or low exploratory behavior (LEB) as measured by the number of head dips on a hole board. To determine whether common genes might influence exploratory behavior and behaviors relevant to drug abuse, we tested HEB and LEB mice for conditioned place preference produced by ethanol and d‐amphetamine and also examined oral methamphetamine intake. After four generations of selection, HEB and LEB mice did not differ in the magnitude of place preference for ethanol, but LEB mice showed a greater place preference for an amphetamine‐paired location than did HEB mice. However, this difference did not replicate in mice tested from the fifth generation of selection. The selected lines also did not differ in sensitization to the locomotor stimulant effects of d‐amphetamine that developed across the conditioning trials. Finally, HEB and LEB mice consumed equivalently low amounts of methamphetamine. These results suggest that common genes do not influence head dipping and several behaviors potentially relevant to drug abuse.

Selection for pentobarbital withdrawal severity: correlated differences in withdrawal from other sedative drugs.

In mice, withdrawal from agents that depress central nervous system function, such as barbiturates and benzodiazepines, results in the production of a withdrawal syndrome, one feature of which is increased severity of handling induced convulsions (HICs). High and Low Pentobarbital Withdrawal mice (HPW and LPW) were selectively bred to display severe and mild pentobarbital withdrawal HICs, respectively. These mice provide a valuable means to assess genetic correlations between withdrawal from pentobarbital and other sedative agents. We tested HPW and LPW mice for severity of HICs elicited during withdrawal from ethanol, diazepam, and zolpidem, and measured consumption of and preference for pentobarbital solutions in HPW and LPW mice. HPW mice displayed greater HICs than LPW mice during ethanol and zolpidem withdrawal, but differed less robustly during diazepam withdrawal. LPW mice consumed more pentobarbital in a solution of a moderate concentration than did HPW mice, but did not consume more pentobarbital at a higher or lower concentration. These results indicate that some of the same genes that affect the severity of withdrawal from pentobarbital also influence ethanol and zolpidem withdrawal, but that diazepam withdrawal may be less influenced by these genes.

Home cage activity and ingestive behaviors in mice following chronic ethanol vapor inhalation

Although drug withdrawal may induce an anxiety-like state, decreased locomotion in tests of anxiety-like behavior is an almost universal finding in rodent studies of ethanol withdrawal. Decreased locomotion in many behavioral apparatus, either as a result of a withdrawal-induced lethargy, malaise, or reduced motivation to explore confounds interpreting the effects of withdrawal as specifically increasing an anxiety-like state. To address this issue, we measured home cage activity levels as well as food and water intake for 3 days prior to and up to 5 days after chronic ethanol vapor exposure in genetically heterogeneous mice. In the first experiment, ethanol-withdrawing WSC-2 mice drank less water than controls, but did not differ from controls on any other behavioral measure during the withdrawal assessments. When the dose of ethanol was elevated in a subsequent experiment in WSC-2 mice, a similar temporary decrease in food and water intake, but not in locomotion, was observed during withdrawal. These results differed from those of mice placed into activity monitors during peak withdrawal, which exhibited profoundly reduced activity levels compared to controls. Finally, home cage activity levels during withdrawal were only transiently decreased in a mouse line that has been selectively bred to display high ethanol withdrawal handling-induced convulsion severity (WSP mice). The reduction in food and water consumption seen in most experiments suggests that withdrawal may induce a temporary malaise-like state, but this state is not reflected in altered home cage activity levels. Further, even in a relatively severe mouse model of alcohol withdrawal, any decreases in general home cage activity are short-lived.

Food deprivation increases the low-dose locomotor stimulant response to ethanol in Drosophila melanogaster

Acute and chronic states of food deprivation result in increased sensitivity to a variety of natural reinforcers as well as to drugs of abuse. Food deprived animals show increased locomotor activity during periods of food deprivation, as well as increased locomotor stimulant responses to drugs of abuse, including cocaine, amphetamine, morphine, and ethanol, implying that drugs of abuse act in part on neural systems that underlie responses towards food. To determine whether this effect extends to an invertebrate, highly genetically tractable animal, the locomotor stimulant effects of low dose ethanol were assessed under a variety of feeding conditions in the fruit fly, Drosophila melanogaster. Food deprivation resulted in strain specific increases in ethanol-stimulated locomotor activity in most strains tested, although elevated baseline activity confounded interpretation in some strains. Experiments conducted with Canton S flies found that the effects of food deprivation on the locomotor stimulant response to ethanol increased with the duration of deprivation, and could be blocked by refeeding the flies with standard food or sucrose, but not yeast, immediately prior to the ethanol exposure. Life-span extending dietary depletion procedures or previous periods of food deprivation did not affect the response to ethanol, indicating that only animals in an acutely food deprived state are more sensitive to the stimulant effects of ethanol. These results suggest that increased sensitivity to the stimulant effects of some drugs of abuse might reflect an evolutionarily conserved neural mechanism that underlies behavioral responses to natural reinforcers and drugs of abuse. The identification of this mechanism, and the genes that underlie its development and function, will constitute a novel approach towards the study of alcohol abuse and dependence.