• Energy Research

Alcohol (ethanol) abuse remains to be a leading cause of medical, including mental, problems throughout the world. Whether alcohol consumption leads to chronic use, and subsequent alcohol dependency and abuse is known to be influenced by the acute effects of this drug. Numerous factors may influence how alcohol administered acutely affects the individual. For example, the mechanisms engaged by drugs of abuse, e.g. cocaine as well as alcohol, have been shown to overlap with those underlying circadian rhythm, and conversely, the effects of these drugs may be dependent upon the time of day of their consumption. To investigate the interaction between circadian rhythm and alcohol, here we employ a simple vertebrate model organism that was previously successfully utilized in other aspects of alcohol research, the zebrafish. We expose zebrafish to alcohol for 20 min in the morning or in the evening, and analyze the effects of this treatment by comparing 1% (vol/vol) alcohol-treated and control (alcohol naive) zebrafish. We record numerous swim path parameters, and report, for the first time, that the time of day of alcohol administration differentially affects certain behavioral parameters, enhancing some while blunting others. Our results suggest a complex interaction between circadian dependent and alcohol engaged mechanisms, findings that represent both practical complications as well as opportunities for understanding how alcohol affects brain function and behavior of vertebrates.

Ethanol affects numerous neurobiological processes depending upon the developmental stage at which it reaches the vertebrate embryo. Exposure time dependency may explain the variable severity and manifestation of life-long symptoms observed in fetal alcohol spectrum disorder (FASD) patients. Characterization of behavioural deficits will help us understand developmental stage-dependency and its underlying biological mechanisms. Here we highlight pioneering studies that model FASD using zebrafish, including those that demonstrated developmental stage-dependency of alcohol effects on some behaviours. We also succinctly review the more expansive mammalian literature, briefly discuss potential developmental stage dependent biological mechanisms alcohol alters, and review some of the disadvantages of mammalian systems versus the zebrafish. We stress that the temporal control of alcohol administration in the externally developing zebrafish gives unprecedented precision and is a major advantage of this species over other model organisms employed so far. We also emphasize that the zebrafish is well suited for high throughput screening and will allow systematic exploration of embryonic-stage dependent alcohol effects via mutagenesis and drug screens.

Several studies have demonstrated translational potential of the zebrafish in modeling fetal alcohol spectrum disorders (FASDs), including the less severe forms of this disease. Short exposure to even low doses of alcohol during embryonic development has been shown to disrupt behavior, alter neurochemistry, and expression of neuronal markers and glial cell phenotypes in zebrafish. However, no study to date has systematically analyzed the potential morphological effects of the short- and low-dose embryonic alcohol exposure regimen used before with zebrafish to model milder forms of human FASD. In this study, we use this previously used embryonic alcohol exposure regimen. We immerse intact zebrafish eggs of AB strain and of a genetically variable wild-type population for 2 h into 1% or 0% (vol/vol) ethanol bath at one of five developmental stages (8, 16, 24, 32, or 40 h postfertilization). At 8 days postfertilization, we quantify body length and width and eye diameter of the larvae. We report nonsignificant effects of embryonic alcohol exposure used at all developmental stages in both populations of zebrafish. Our results confirm that visual perception or motor function is unlikely to have contributed to previously reported behavioral abnormalities resulting from embryonic alcohol exposure in zebrafish.

Fetal Alcohol Spectrum Disorders (FASD) represent a worldwide problem. The severity and types of symptoms of FASD vary, which may be due to the genotype of the fetus and the developmental stage at which the fetus is exposed to alcohol. The most prevalent forms of FASD present less severe symptoms, including behavioral and cognitive abnormalities, and arise from exposure to low amounts of alcohol consumed infrequently. Treating or diagnosing FASD patients has been difficult because we do not understand the mechanisms underlying FASD. Animal models, including the zebrafish, have been suggested to answer this question. Here, we present a proof of concept analysis studying the behavioral effects of embryonic alcohol exposure in one-week old juvenile zebrafish. We exposed zebrafish embryos at one of five developmental stages (8, 16, 24, 32, or 40 hour post-fertilization) to 0% (control) or 1% (vol/vol) ethanol for 2 h, and tested the behavior of these fish at their age of 7-9 days post-fertilization. We employed two genetically distinct zebrafish populations, a quasi-inbred AB derivative strain, and a genetically variable WT population. We report significant developmental time and genotype dependent effects of alcohol on certain measures of motor function and/or anxiety-like responses. For example, we found embryonic alcohol exposed AB fish to swim faster, vary their speed more, stop moving more often and turn less compared to control fish, alcohol induced changes that were absent or less robust in WT fish. We conclude that our results open new avenues to the identification of genetic mechanisms that mediate or influence alcohol induced developmental alteration of brain function and behavior, which, on the long run, may allow us to identify diagnostic biomarkers and treatment options for human FASD.

The zebrafish is becoming increasingly utilized in behavioral neuroscience as it appears to strike a good compromise between practical simplicity and system complexity. Particularly in alcohol (ethanol) research, the zebrafish has been employed as a translationally relevant model organism. However, the majority of studies investigating the effects of alcohol on brain function and behavior has used adult zebrafish. In the current study, we utilize 6-8 post-fertilization day old larval zebrafish (fry) to investigate the effects of a 40 min-long, acute, immersion into the alcohol bath. We measure the behavioral responses of the fry during the immersion session in relatively large arenas, the petri dish, instead of the often employed 96 well plate, and report on significant modification of behavior induced by alcohol. For example, we found the intermediate dose of alcohol (0.5%, vol/vol) to exert a stimulant effect manifesting as slight elevation of swim speed, robust increase of turning, temporal variability of swim speed and turning, and diminished frequency of staying immobile. We also found the high dose of 1% alcohol to elicit an opposite response, a sedative effect. This biphasic dose response of alcohol mimics what has been found in mammals, including humans, and thus we conclude that a few day-old zebrafish fry may be a cost effective and efficient tool with which one can screen for small molecules or mutations with alcohol-effect modifying properties.

Zebrafish have become a popular animal model for studying the development of alcohol addiction. Several behavioral paradigms for studying alcohol addiction have been developed for zebrafish, including conditioned place preference, alcohol-induced tolerance, and withdrawal. However, alcohol choice preference tasks have not been established in zebrafish as of yet. The ability of zebrafish to detect alcohol in their environment is required in alcohol choice or preference tasks. To our knowledge, it is currently unknown whether zebrafish are able to detect alcohol in their environment immediately following bath immersion. In the current study, we analyzed the time course of alcohol-induced behavioral changes of zebrafish while being immersed in alcohol solution in a 1.5 L tank. We recorded each trial in high-definition and quantified behavioral responses using automated video tracking-based and manual observation-based methods to quantify temporal changes in alcohol-induced behaviors. As alcohol is known to require several minutes of bath immersion to reach the brain in zebrafish, we argued that behavioral responses before this time point would prove zebrafish's ability to detect this substance in the water. Our results show that a 60-min exposure to 1% alcohol alters behavioral responses in a time-dependent manner. Notably, alcohol exposure significantly increased absolute turn angle, decreased distance to bottom, and variance of distance to bottom within the first 3 min immediately following exposure, a response that occurred before alcohol could reach the brain of the subjects in measurable amounts. These results imply that zebrafish are able to detect alcohol in their environment immediately following immersion into the drug solution.

Zebrafish have become a popular animal model for behavioural pharmacology due to their small size, rapid development, and amenability to high throughput behavioural drug screens. Furthermore, water-soluble compounds can be administered via immersion of the fish in the drug solution, which provides a non-invasive drug delivery method. Numerous studies have demonstrated stimulant effects of alcohol. Diazepam and caffeine, on the other hand have been found to have inhibitory effect on locomotor activity in zebrafish. However, the time-dependent changes induced by these psychoactive drugs are rarely reported, and potential drug interactions have not been examined in zebrafish, despite the translational relevance of this question. In the current study, we examine time- and dose-dependent changes in zebrafish following exposure to caffeine, diazepam, and ethanol quantifying four different behavioural parameters over a 30min recording session. We subsequently analyze potential drug-drug interactions by co-administering the three drugs in different combinations. Our time-course and dose-response analyses for each of the three drugs represent so far the most detailed studies available serving as a foundation for future psychopharmacology experiments with zebrafish. Furthermore, we report significant interactions between the three drugs corroborating findings obtained with rodent models as well as in humans, providing translational relevance for the zebrafish model.