GABAergic regulation of locomotion before and during an ethanol exposure in Drosophila melanogaster.

Ethanol at low doses induces a locomotor stimulant response across a range of phylogenetically diverse species. In rodents, this response is commonly used as an index of ethanol's disinhibitory, anxiolytic, or reinforcing effects, and its expression is regulated by signaling through a number of conserved neurotransmitter systems. In the current experiments, we asked whether ethanol-induced locomotor stimulation in the fruit fly Drosophila melanogaster might be mediated by ionotropic GABA receptors. We measured basal and ethanol-stimulated locomotion in flies expressing RNAi directed against three known subunits of ionotropic GABA receptors, and also examined the effects of picrotoxin feeding on these behaviors. We found that RNAi-mediated knockdown of a subunit of fly ionotropic GABA receptors, RDL, in all neurons resulted in an increased ethanol-induced locomotor stimulant response, while knockdown of two other subunits, LCCH3 and GRD, did not affect the responses. The effect of pan neuronal RDL knockdown was recapitulated with selective RDL knockdown in cholinergic neurons, and increased ethanol-induced locomotor stimulation was also seen by feeding the GABAA antagonist picrotoxin to flies prior to behavioral testing. However, the increase in ethanol-stimulated locomotion in each of these experiments was largely accounted for by decreased baseline activity. Our results indicate that ionotropic GABA receptors might be a conserved mediator of the locomotor stimulant effects of ethanol, but that alternative experimental approaches will be necessary to disentangle effects of GABAergic manipulations on baseline and ethanol-stimulated locomotion in flies.

Pharmacological characterization of the forced swim test in Drosophila melanogaster.

The forced swim test is commonly used as a preclinical screen of antidepressant medication efficacy in rats and mice. Neckameyer and Nieto-Romero (Stress 18:254-66, 2015) adopted the forced swim test for use with the fruit fly Drosophila melanogaster and showed that behavior in this test is sensitive to several physiologically relevant stressors. However, whether this test might be sensitive to the effects of antidepressant medications or other compounds is unknown. In the current studies, we fed drugs to male and female flies that we expected to either decrease or increase the duration of immobility in the forced swim test, including fluoxetine, desipramine, picrotoxin, reserpine, 3-iodo-tyrosine, and ethanol. Fluoxetine was the only drug tested that affected behavior in this test, and surprisingly, the direction of the effect depended on the duration of feeding. Short-term (30 min) feeding of the drug prior to test resulted in the expected increase in latency to immobility, while a longer feeding duration (20-24 h) decreased this measure. These results suggest that the pharmacological profile of the fly FST is more restricted than that of the rat or mouse FST, and that the duration of drug exposure is an important consideration in pharmacological research using flies.

Somatosensory Perception of Running Shoe Mass may be influenced by Extended Wearing Time or Inclusion of a Personal Reference Shoe, Depending on Testing Method.

Consumers may purchase running shoes on the basis of their masses, yet little is known about shoe mass perceptual abilities. In this multi-part experiment, four groups of twenty-five young adult males (total n = 100) were challenged to gauge the relative masses of five unfamiliar running shoes. The four groups differed by the length of time they were given to wear the shoes (up to 1 minute versus 5 minutes) and whether or not they were able to use their own personal running shoes as a reference. After wearing each individual pair of shoes, participants provided perceived comfort and heaviness rankings using visual analogue scales (VAS). After wearing all five pairs of unfamiliar shoes, participants gave a verbal ranking of relative shoe mass. Participants also hefted the shoes with their hands and positioned them in order of relative mass. Extended wearing time improved overall verbal ranking accuracy, but did not improve mass perception accuracy as determined by comparing VAS heaviness rankings to actual shoe masses. Conversely, use of a personal reference shoe improved mass perception accuracy as determined by comparing VAS heaviness rankings to actual shoe masses, but did not improve overall verbal ranking accuracy. Hand perceptual scores were similar across the four groups, likely due to a ceiling effect. VAS comfort scores were unrelated to shoe masses. The results suggest that wearing time and reference shoes may influence mass perception by the lower limb in a context-specific manner.

An assessment of the utilization of the preclinical rodent model literature in clinical trials of putative therapeutics for the treatment of alcohol use disorders.

OBJECTIVE: Rodent models of Alcohol Use Disorders (AUD) are used extensively by preclinical researchers to develop new therapeutics for the treatment of AUD. Although these models play an important role in the development of novel, targeted therapeutics, their role in bringing therapeutics to clinical trials is unclear, as off-label use of existing medications not approved for the treatment of AUD is commonly seen in the clinic and clinical trials. METHOD: In the current study, we used the Clinicaltrials.gov database to obtain a list of drugs that have been tested for efficacy in a clinical trial between 1997 and 2017. We then conducted a set of literature searches to determine which of the 98 unique drugs we identified had shown efficacy in a rodent model of an AUD prior to being tested in a clinical trial. RESULTS: We found that slightly less than half of the drugs tested in clinical trials (48%) had shown prior efficacy in any rodent model of an AUD, while the remaining 52% of drugs were used off-label, or in some cases, following non-published studies. CONCLUSION: This study raises the question of how clinical researchers incorporate results from preclinical studies in the decision to bring a drug to a clinical trial. Our results underscore the need for ongoing communication among preclinical and clinical researchers.

Common genes regulate food and ethanol intake in Drosophila.

The abuse liability of alcohol (ethanol) is believed to result in part from its actions on neurobiological substrates that underlie the motivation toward food and other natural reinforcers, and a growing body of evidence indicates that these substrates are broadly conserved among animal phyla. Understanding the extent to which the substrates regulating ethanol and food intake overlap is an important step toward developing therapeutics that selectively reduce ethanol intake. In the current experiments, we measured food and ethanol intake in Recombinant Inbred (RI) lines of Drosophila melanogaster using several assays, and then calculated genetic correlations to estimate the degree to which common genes might underlie behavior in these assays. We found that food intake and ethanol intake as measured in the capillary assay are genetically correlated traits in D. melanogaster, as well as in a panel of 11 Drosophila species that we tested subsequently. RI line differences in food intake in a dyed food assay were genetically unrelated to ethanol intake in the capillary assay or to ethanol preference measured using an olfactory trap apparatus. Using publicly available gene expression data, we found that expression profiles across the RI lines of a number of genes (including the D2-like dopamine receptor, DOPA decarboxylase, and fruitless) correlated with the RI line differences in food and ethanol intake we measured, while the expression profiles of other genes, including NPF, and the NPF and 5-HT2 receptors, correlated only with ethanol intake or preference. Our results suggest that food and ethanol intake are regulated by some common genes in Drosophila, but that other genes regulate ethanol intake independently of food intake. These results have implications toward the development of therapeutics that preferentially reduce ethanol intake.

Conservation of the ethanol-induced locomotor stimulant response among arthropods.

Ethanol-induced locomotor stimulation has been variously described as reflective of the disinhibitory, euphoric, or reinforcing effects of ethanol and is commonly used as an index of acute ethanol sensitivity in rodents. The fruit fly Drosophila melanogaster also shows a locomotor stimulant response to ethanol that is believed to occur via conserved, ethanol-sensitive neurobiological mechanisms, but it is currently unknown whether this response is conserved among arthropod species or is idiosyncratic to D. melanogaster. The current experiments surveyed locomotor responses to ethanol in a phylogenetically diverse panel of insects and other arthropod species. A clear ethanol-induced locomotor stimulant response was seen in 9 of 13 Drosophilidae species tested, in 8 of 10 other species of insects, and in an arachnid (wolf spider) and a myriapod (millipede) species. Given the diverse phylogenies of the species that showed the response, these experiments support the hypothesis that locomotor stimulation is a conserved behavioral response to ethanol among arthropod species. Further comparative studies are needed to determine whether the specific neurobiological mechanisms known to underlie the stimulant response in D. melanogaster are conserved among arthropod and vertebrate species.

Somatosensory perception of running shoe mass.

Running shoes are often marketed based on mass. A total of 50 young adult males participated across two separate experiments to determine how well they could perceive the relative masses of five different running shoes using hands versus feet. For the foot portion, subjects were blindly fitted with the shoes and asked to rank their masses individually using visual analogue scales (VAS) and verbal rankings. For the hand portion, two different methods were used, one presenting all shoes simultaneously and the other presenting the shoes individually. Verbal accuracy and VAS scores correlated across subjects for the hand and foot, but accuracy in mass perception by the feet was 30% compared to 92% or 63% by the hand (depending on the method). These results indicate the foot perceives mass poorly compared to the hand, and that consumers' perception of shoe mass may come more from handling shoes versus wearing them.

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.

Insulin attenuates the acquisition and expression of ethanol-induced locomotor sensitization in DBA/2J mice.

AIM: Ethanol-induced locomotor sensitization is a behavioral manifestation of physiological responses to repeated ethanol exposures. While ethanol exerts direct effects on multiple neurotransmitter systems in the brain, ethanol-induced changes in metabolic state, including acute hyperglycemia and inhibition of insulin signaling, also have plausible roles in the expression of ethanol-related behaviors through direct and indirect effects on brain function. The current experiments examined whether insulin administration or the resultant hypoglycemia might attenuate the development of sensitization to the locomotor stimulant effect of ethanol. MAIN METHODS: Male and female DBA/2J mice received daily injections of 5 or 10 IU/kg insulin before or after a stimulating dose of ethanol and subsequent testing in an automated activity monitor. Blood glucose levels were determined upon the completion of the experiments. KEY FINDINGS: Insulin injected prior to ethanol blunted the acute stimulant response as well as the acquisition and expression of locomotor sensitization, while insulin given after ethanol did not affect the development of the sensitized response. In a separate experiment, mice given glucose concurrently with insulin developed ethanol-induced locomotor sensitization normally. SIGNIFICANCE: These experiments suggest that insulin attenuates the development of ethanol-induced locomotor sensitization, and that blood glucose levels can largely account for this effect. Further studies of the role of ethanol-induced metabolic states should provide novel information on the expression of ethanol-related behaviors.

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.

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.

Genetic independence of mouse measures of some aspects of novelty seeking.

High novelty seeking is a complex personality attribute correlated with risk for substance abuse. There are many putative mouse models of some aspects of novelty seeking, but little is known of genetic similarities among these models. To assess the genetic coherence of "novelty seeking," we compared the performance of 14 inbred strains of mice in five tests: activity in a novel environment, novel environment preference, head dipping on a hole-board, object preference, and a two-trial version of the spontaneous alternation task. Differences among strains were observed for all tasks, but performance in any given task was generally not predictive of performance in any other. To evaluate similarities among these tasks further, we selectively bred lines of mice for high or low head dipping on the hole-board. Similar to results from the inbred strain experiments, head dipping was not correlated with performance in the other measures but was genetically correlated with differences in locomotor activity. Using two approaches to estimating common genetic influences across tasks, we have found little evidence that these partial models of novelty seeking reflect the influence of common genes or measure a single, unified construct called novelty seeking. Based on the substantial influence of genetic factors, ease of implementation, and relative independence from general locomotion, head dipping on a hole-board is a good task to use in the domain of novelty seeking, but multiple tasks, including others not tested here, would be needed to capture the full genetic range of the behavioral domain.

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.

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 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.

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

RATIONALE: Anxiety 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. OBJECTIVES: Ideally, 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. METHODS: We 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. RESULTS: Increasing 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. CONCLUSIONS: Since 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.