The genetic relationships between ethanol preference, acute ethanol sensitivity, and ethanol tolerance in Drosophila melanogaster

The relationship between alcohol consumption, sensitivity, and tolerance is an important question that has been addressed in humans and rodent models. Studies have shown that alcohol consumption and risk of abuse may correlate with (1) increased sensitivity to the stimulant effects of alcohol, (2) decreased sensitivity to the depressant effects of alcohol, and (3) increased alcohol tolerance. However, many conflicting results have been observed. To complement these studies, we utilized a different organism and approach to analyze the relationship between ethanol consumption and other ethanol responses. Using a set of 20 Drosophila melanogaster mutants that were isolated for altered ethanol sensitivity, we measured ethanol-induced hyperactivity, ethanol sedation, sedation tolerance, and ethanol consumption preference. Ethanol preference showed a strong positive correlation with ethanol tolerance, consistent with some rodent and human studies, but not with ethanol hyperactivity or sedation. No pairwise correlations were observed between ethanol hyperactivity, sedation, and tolerance. The evolutionary conservation of the relationship between tolerance and ethanol consumption in flies, rodents, and humans indicates that there are fundamental biological mechanisms linking specific ethanol responses.     

The genetic relationships between ethanol preference,acute ethanol sensitivity,and ethanol tolerance in Drosophila melanogaster

The relationship between alcohol consumption, sensitivity, and tolerance is an important question that has been addressed in humans and rodent models. Studies have shown that alcohol consumption and risk of abuse may correlate with (1) increased sensitivity to the stimulant effects of alcohol, (2) decreased sensitivity to the depressant effects of alcohol, and (3) increased alcohol tolerance. However, many conflicting results have been observed. To complement these studies, we utilized a different organism and approach to analyze the relationship between ethanol consumption and other ethanol responses. Using a set of 20 Drosophila melanogaster mutants that were isolated for altered ethanol sensitivity, we measured ethanol-induced hyperactivity, ethanol sedation, sedation tolerance, and ethanol consumption preference. Ethanol preference showed a strong positive correlation with ethanol tolerance, consistent with some rodent and human studies, but not with ethanol hyperactivity or sedation. No pairwise correlations were observed between ethanol hyperactivity, sedation, and tolerance. The evolutionary conservation of the relationship between tolerance and ethanol consumption in flies, rodents, and humans indicates that there are fundamental biological mechanisms linking specific ethanol responses.     

Experimental reduction of codon bias in the Drosophila alcohol dehydrogenase gene results in decreased ethanol tolerance of adult flies

The ethanol tolerance of adult transgenic flies of Drosophila containing between zero and ten unpreferred synonymous mutations that reduced codon bias in the alcohol dehydrogenase (Adh) gene was assayed. As the amino acid sequences of the ADH protein were identical in the four genotypes assayed, differences in ethanol tolerance were due to differences in the abundance of ADH protein, presumably driven by the effects of codon bias on translational efficiency. The ethanol tolerance of genotypes decreased with the number of unpreferred synonymous mutations, and a positive correlation between ADH protein abundance and ethanol tolerance was observed. This work confirms that the fitness effects of unpreferred synonymous mutations that reduce codon bias in a highly expressed gene are experimentally measurable in Drosophila melanogaster.     

ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance

In Drosophila melanogaster, alcohol dehydrogenase (ADH) activity is essential for ethanol tolerance, but its role may not be restricted to alcohol metabolism alone. Here we describe ADH activity and Adh expression level upon selection for increased alcohol tolerance in different life-stages of D. melanogaster lines with two distinct Adh genotypes: Adh(FF) and Adh(SS). We demonstrate a positive within genotype response for increased alcohol tolerance. Life-stage dependent selection was observed in larvae only. A slight constitutive increase in adult ADH activity for all selection regimes and genotypes was observed, that was not paralleled by Adh expression. Larval Adh expression showed a constitutive increase, that was not reflected in ADH activity. Upon exposure to environmental ethanol, sex, selection regime life stage and genotype appear to have differential effects. Increased ADH activity accompanies increased ethanol tolerance in D. melanogaster but this increase is not paralleled by expression of the Adh gene.     

Acetic acid tolerance in Drosophila is a prerequisite for ethanol tolerance

Acetic acid tolerance compared with ethanol tolerance of Drosophila simulans and six Drosophila melanogaster strains shows a curvilinear relation with apparent asymptotic hyperbolic profile. The upper limit of acetic acid tolerance is lower than that for ethanol. We compared strains which had pairwise identical alcohol dehydrogenase (ADH) coding regions but different genetic backgrounds. A positive regression existed for ethanol tolerance on ADH activity. Adh-null mutants with very low ethanol tolerances had appreciable acetic acid tolerances and as a consequence did not fit the curve. ADH-F and ADH-S strains selected for high ethanol tolerances had the ability to tolerate high ethanol concentrations even after selection had been relaxed for several years. These selected lines tolerated higher acetic acid concentrations than the non-selected original strains. We propose that intake of high concentrations of ethanol and oxidation into acetic acid induces esterification of ethanol and acetic acid into ethylacetate. This cannot take place after the intake of acetic acid only, which also gives a lower energy yield.     

Micro-evolution in a wine cellar population: An historical perspective

The population ofDrosophila melanogaster inside the wine cellar of Chateau Tahbilk of Victoria, Australia was found by McKenzie and Parsons (1974) to have undergone microevolution for greater alcohol tolerance when compared to the neighboring population outside the cellar. This triggered additional studies at Tahbilk, and at other wine cellars throughout the world. The contributions and interactions of researchers and the development of ideas on the ecology and genetics of this unique experimental system are traced. Although the ADH-F/ADH-S polymorphism was found to be maintained by selection in the Tahbilk populations, the selection is not significantly associated with alcohol tolerance. The environment inside the Tahbilk wine cellar is not as rich in ethanol as was originally anticipated, and selection that affects the alcohol dehydrogenase polymorphism may be more concerned with the relative efficiency with which ethanol is used as a nutrient byD. melanogaster. The synthesis and modification of lipids, particularly in membranes, appears to be important to alcohol tolerance. The studies of the Tahbilk population are at a crossroad. New experimental approaches promise to provide the keys to the selection that maintains the alcohol dehydrogenase polymorphism, and to factors that are important to alcohol tolerance and stress adaptation. From these research foundations at Tahbilk very significant contributions to our future understanding of the genetic processes of evolution can be made.     

Circadian modulation of acute alcohol sensitivity but not acute tolerance in Drosophila

An increased understanding of the factors affecting behavioral and neurological responses to alcohol and alcohol physiology is necessary given the tremendous toll alcohol abuse and alcoholism exert on individuals and society. At the behavioral and molecular levels, the response to alcohol appears remarkably conserved from Drosophila to humans, suggesting that investigations across model species can provide insight into the identification of common modulatory factors. We investigated the interaction between the circadian clock and alcohol sensitivity, alcohol tolerance, and alcohol absorbance in Drosophila melanogaster. Using a loss-of-righting reflex (LoRR) assay, we found that flies exhibit a circadian rhythm in the LoRR, with the greatest sensitivity to alcohol occurring from mid to late night, corresponding to the flies' inactive phase. As predicted, a circadian rhythm in the LoRR was absent in circadian mutant flies and under conditions in which the circadian clock was nonfunctional. Circadian modulation of the response to alcohol was not due to circadian regulation of alcohol absorbance. Similar to other animals, Drosophila develop acute and chronic tolerance to alcohol upon repeat exposures. We found that the circadian clock did not modulate the development of acute alcohol tolerance measured as the difference in sensitivity to alcohol between na?ve and pre-exposed flies. Thus, the circadian clock modulates some, but not all, of the behavioral responses to alcohol exposure, suggesting that specific mechanisms underlie the observed circadian modulation of LoRR rather than global cellular circadian regulation. This study provides valuable new insights in our understanding of the circadian modulation of alcohol-induced behaviors that ultimately could facilitate preventative measures in combating alcohol abuse and alcoholism.     

Selection for high alcohol preference drinking in mice results in heightened sensitivity and rapid development of acute functional tolerance to alcohol's ataxic effects

Propensity to develop acute functional (or within session) tolerance to alcohol (ethanol) may influence the amount of alcohol consumed, with higher drinking associated with greater acute functional tolerance (AFT). The goal of this study was to assess this potential correlated response between alcohol preference and AFT in second and third replicate lines of mice selectively bred for high (HAP2 and HAP3) and low (LAP2 and LAP3) alcohol preference drinking. Male and female mice were tested for development of AFT on a static dowel task, which requires that animals maintain balance on a wooden dowel in order to prevent falling. On test day, each mouse received one (1.75 g/kg; Experiment 1) or two (1.75 and 2.0 g/kg; Experiment 2) injections of ethanol; an initial administration before being placed on the dowel and in Experiment 2, an additional administration after the first regain of balance on the dowel. Blood samples were taken immediately after loss of balance [when blood ethanol concentrations (BECs) were rising] and at recovery (during falling BECs) in Experiment 1, and after first and second recovery in Experiment 2. It was found that HAP mice fell from the dowel significantly earlier and at lower BECs than LAP mice following the initial injection of ethanol and were therefore more sensitive to its early effects. Furthermore, Experiment 1 detected significantly greater AFT development (BECfalling ? BECrising) in HAP mice when compared with LAP mice, which occurred within ～30 min, supporting our hypothesis. However, AFT was not different between lines in Experiment 2, indicating that ～30–60 min following alcohol administration, AFT development was similar in both lines. These data show that high alcohol drinking genetically associates with both high initial sensitivity and very early tolerance to the ataxic effects of ethanol.     

Direct and correlated responses to selection for larval ethanol tolerance in Drosophila melanogaster

Ethanol is an important larval resource and toxin for natural Drosophila melanogaster populations, and ethanol tolerance is genetically variable within and among populations. If ethanol‐tolerant genotypes have relatively low fitness in the absence of ethanol, as suggested by the results of an earlier study, genetic variation for ethanol tolerance could be maintained by variation in ethanol levels among breeding sites. I selected for ethanol tolerance in large laboratory populations by maintaining flies on ethanol‐supplemented media. After 90 generations, the populations were compared with control populations in egg‐to‐adult survival and development rate on ethanol‐supplemented and unsupplemented food. When compared on ethanol‐supplemented food, the ethanol‐selected populations had higher survival and faster development than the control populations, but on unsupplemented food, the populations did not differ in either trait. These results give no evidence for a ‘trade‐off’ between the ability to survive and develop rapidly in the presence of ethanol and the ability to do so in its absence. The effect of physiological induction of ethanol tolerance by exposing eggs to ethanol was also investigated; exposing eggs to ethanol strongly increased subsequent larval survival on ethanol‐supplemented food, but did not affect survival on regular food, and slowed development on both ethanol‐supplemented and regular food, partly by delaying egg hatch.     

Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids

The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits.     

Influence of the biogenic amine tyramine on ethanol‐induced behaviors in Drosophila

The biogenic amine tyramine has been implicated in drug‐induced behavior. The Drosophila inactive mutant is characterized by reduced tyramine and octopamine levels and is defective in cocaine sensitization. To test whether there is an overlap in the use of the amine neurotransmitter system in ethanol‐ and cocaine‐induced behaviors, mutant analyses were extended to the phenotypic characterization of inactive and other mutants effecting the tyramine and octopamine neurotransmitter system. The inactive mutant displays increased ethanol sensitivity and is impaired in the initial startle response upon ethanol application. Furthermore, this mutant fails to regulate its alcohol‐induced hyperactivity properly. In contrast to the defects seen after cocaine application, inactive mutants develop normal ethanol tolerance and sensitize to the locomotor activating effect of ethanol. The tyramine‐β‐hydroxylase mutant (TβH) with increased tyramine and depleted octopamine levels displays normal ethanol sensitivity, a startle repression, and hyperactivates more in response to ethanol. In addition, TβH mutants fail to develop a tolerance to the hyperactivating effect of ethanol. Ethanol‐induced sensitization does not seem to be impaired in either mutant, suggesting that tyramine is not required for this process. The comparative analysis of the phenotypes associated with inactive and TβH mutants suggests that the fine tuning of ethanol‐induced hyperactivity can be correlated with different tyramine levels. Defects in other aspects of ethanol‐induced behaviors might be due to different molecules or mechanisms. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

HERITABLE VARIATION IN ETHANOL TOLERANCE AND ITS ASSOCIATION WITH BIOCHEMICAL TRAITS IN DROSOPHILA MELANOGASTER

To help elucidate mechanisms of larval ethanol tolerance seven isochromosomal lines of Drosophila melanogaster with different second chromosomes were fed a growth-limiting concentration of ethanol (4.5% v/v) and examined for associations between growth traits and biochemical characteristics that had previously been implicated in the determination of tolerance variation. Repeated measures of survival and development time over four generations verified the inherited nature of these traits. Significant variation among the lines were evident for flux from ethanol into lipid, for activity levels of alcohol dehydrogenase and glycerol-3-phosphate oxidase (GPO), and for levels of long chain and unsaturated fatty acids. A high degree of positive association occurred among the variables. A partial correlation analysis controlling for performance of the lines on ethanol-free medium revealed a strong association between the degree of long chain fatty acid content and line survival when ethanol was fed. The correlation between GPO activity and survival in an ethanol environment appeared to depend on the association of GPO activity with long chain fatty acid content. The positive correlations of flux from ethanol into lipid with many of the other variables suggested that the ADH pathway influenced the level of ethanol tolerance. These associations are all consistent with the hypothesis that the lipid content of body tissues, especially the levels of long chain and unsaturated fatty acids in cell membranes, may have an important influence on both spatial and interspecific variation in the ethanol tolerance of larvae.     

Analysis of Drosophila nervous system development following an early,brief exposure to ethanol

The effects of ethanol on neural function and development have been studied extensively, motivated in part by the addictive properties of alcohol and the neurodevelopmental deficits that arise in children with fetal alcohol spectrum disorder (FASD). Absent from this research area is a genetically tractable system to study the effects of early ethanol exposure on later neurodevelopmental and behavioral phenotypes. Here, we used embryos of the fruit fly, Drosophila melanogaster, as a model system to investigate the neuronal defects that arise after an early exposure to ethanol. We found several disruptions of neural development and morphology following a brief ethanol exposure during embryogenesis and subsequent changes in larval behavior. Altogether, this study establishes a new system to examine the effects of alcohol exposure in embryos and the potential to conduct large‐scale genetics screens to uncover novel factors that sensitize or protect neurons to the effects of alcohol.     

Arginine vasopressin deficient Brattleboro rats fail to develop tolerance to the hypothermic effects of ethanol

We have tested the hypothesis that animals with reduced levels of arginine vasopressin (AVP) would show reduced tolerance to ethanol. Brattleboro rats either heterozygous or homozygous for the diabetes insipidus (DI) trait and normal Sprague-Dawley rats were exposed to ethanol vapor for 21 days. Two days later, tolerance was evaluated by monitoring body temperature reductions after intraperitoneal injection of 2 g/kg (20% w/v) ethanol. Under the same conditions of chronic ethanol exposure, Sprague-Dawley rats, but not Brattleboro rats, displayed tolerance to the hypothermic effects of intraperitoneal ethanol. This phenomenon did not appear to be related to differences in ethanol metabolism or blood alcohol levels in Brattleboro rats. These data support a possible role for AVP in the development or maintenance of tolerance.     

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.     

X射线和ENU诱变的Adh基因对黑腹果蝇醇耐受性的杂合效应

为研究部分显性的机制,用１２个黑腹果蝇醇脱氢酶（ＤＡＤＨ）基因内无效突变（Ａｄｈ＾ｎ）体作为材料。这些已知序列伯突变体「包括单碱基置换或基因内小段缺失（９￣１６个碱基）」,用来分析肽的合成,二聚体的形成和杂二聚体酶活性。杂合体中酶的部分表达和很广范围显性表达（从几乎完全陷性到高度显性）具多重机制。在１０％乙醇的高度胁迫下,所有１２个Ａｄｈ＾ｎ型果蝇中都观察到醇耐受性的部分显性表达。无效「突变表达的     

Direct Evidence That Genetic Variation in Glycerol-3-Phosphate and Malate Dehydrogenase Genes (Gpdh and Mdh1) Affects Adult Ethanol Tolerance in Drosophila melanogaster

Many studies of alcohol adaptation in Drosophila melanogaster have focused on the Adh polymorphism, yet the metabolic elimination of alcohol should involve many enzymes and pathways. Here we evaluate the effects of glycerol-3-phosphate dehydrogenase (Gpdh) and cytosolic malate dehydrogenase (Mdh1) genotype activity on adult tolerance to ethanol. We have created a set of P-element-excision-derived Gpdh, Mdh1, and Adh alleles that generate a range of activity phenotypes from full to zero activity. Comparisons of paired Gpdh genotypes possessing 10 and 60% normal activity and 66 and 100% normal activity show significant effects where higher activity increases tolerance. Mdh1 null allele homozygotes show reductions in tolerance. We use piggyBac FLP–FRT site-specific recombination to create deletions and duplications of Gpdh. Duplications show an increase of 50% in activity and an increase of adult tolerance to ethanol exposure. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection related to adaptation to alcohols. Finally, we examine the interactions between activity genotypes for Gpdh, Mdh1, and Adh. We find no significant interlocus interactions. Observations on Mdh1 in both Gpdh and Adh backgrounds demonstrate significant increases in ethanol tolerance with partial reductions (50%) in cytosolic MDH activity. This observation strongly suggests the operation of pyruvate–malate and, in particular, pyruvate–citrate cycling in adaptation to alcohol exposure. We propose that an understanding of the evolution of tolerance to alcohols will require a system-level approach, rather than a focus on single enzymes.     

Tolerance to 1-pentene-3-ol and to 1-pentene-3-one in relation to alcohol dehydrogenase (ADH) and aldo keto reductase (AKR) activities inDrosophila melanogaster

The detoxification of 1-pentene-3-ol (pentenol) and 1-pentene-3-one (pentenone) by Drosophila melanogaster adult flies has been studied in two homozygous lines for the AdhF and AdhS alleles (LRC lines), in their respective lines selected for tolerance to ethanol (LRSe lines) and in a homozygous strain for the Adhn4 null allele. For each line, the genotype and sex LDs50 of both compounds were estimated. Then, in order to explain the differences in LD50, both alcohol dehydrogenase (ADH) and aldo keto reductase (AKR) activities were assayed. In addition, the effects of pentenone on AKR activity were also studied. Our results show that ADH-positive flies exhibit a much higher sensitivity to pentenol than ADH-null flies. However, both ADH-positive and ADH-null flies show a similar tolerance to pentenone. Our results show that flies selected for improving tolerance to ethanol also have increased tolerance to pentenol (FF and SS flies) and pentenone (SS flies). However, this improved ability to tolerate pentenol and/or pentenone cannot be explained by changes in ADH or AKR activities. On the other hand, we have observed a beneficial effect of pentenol, but not of pentenone, in n4 flies. We also show that AKR activity is not modified by the administration of pentenone. These results suggest that, in the absence of ADH activity, pentenol may be transformed into a compound that is less toxic than pentenone and that pentenone itself might also be transformed into a less toxic compound.