Lasting alterations induced in glial cell phenotypes by short exposure to alcohol during embryonic development in zebrafish.

Despite the known teratogenic effects of alcohol (ethanol) on the developing human fetus, the prevalence of fetal alcohol spectrum disorder (FASD) is not decreasing. Appropriate treatment for this life-long disease has not been developed, and even diagnostic biomarkers are unavailable. FASD remains a large unmet medical need. Numerous animal models have been developed to mimic FASD and study potential underlying biological mechanisms. However, most of these models focused on neuronal phenotypes. Given that glial cells represent the majority of cells in the vertebrate brain, and given the increasingly appreciated roles they play in a myriad of neuronal functions as well as CNS disorders, we decided to investigate potential embryonic alcohol exposure induced changes in them. Building upon a previously introduced zebrafish model of milder and most prevalent forms of FASD, we investigated the effect of a 2-hour-long exposure to alcohol (1% vol/vol bath concentration) employed at the 24th hour postfertilization stage of development of zebrafish on a number of glial cell-related phenotypes. We studied oligodendrocyte, astrocyte as well as microglia-related phenotypes using immunohistochemistry, lipid, and enzyme activity analyses. We report significant changes in wide-spread glial cell phenotypes induced by embryonic alcohol exposure in the zebrafish brain and conclude that the zebrafish will advance our understanding of the mechanisms of this devastating disorder.

Lasting changes induced by mild alcohol exposure during embryonic development in BDNF, NCAM and synaptophysin-positive neurons quantified in adult zebrafish.

Fetal alcohol spectrum disorder is one of the leading causes of mental health issues worldwide. Analysis of zebrafish exposed to alcohol during embryonic development confirmed that even low concentrations of alcohol for a short period of time may have lasting behavioral consequences at the adult or old age. The mechanism of this alteration has not been studied. Here, we immersed zebrafish embryos into 1% alcohol solution (vol/vol%) at 24 hr post-fertilization (hpf) for 2 hr and analyzed potential changes using immunohistochemistry. We measured the number of BDNF (brain-derived neurotrophic factor) and NCAM (neuronal cell adhesion molecule)-positive neurons and the intensity of synaptophysin staining in eight brain regions: lateral zone of the dorsal telencephalic area, medial zone of the dorsal telencephalic area, dorsal nucleus of the ventral telencephalic area, ventral nucleus of the ventral telencephalic area, parvocellular preoptic nucleus, ventral habenular nucleus, corpus cerebella and inferior reticular formation. We found embryonic alcohol exposure to significantly reduce the number of BDNF- and NCAM-positive cells in all brain areas studied as compared to control. We also found alcohol to significantly reduce the intensity of synaptophysin staining in all brain areas except the cerebellum and preoptic area. These neuroanatomical changes correlated with previously demonstrated reduction of social behavior in embryonic alcohol-exposed zebrafish, raising the possibility of a causal link. Given the evolutionary conservation across fish and mammals, we emphasize the implication of our current study for human health: even small amount of alcohol consumption may be unsafe during pregnancy.

Lasting effects of mild embryonic ethanol exposure on voltage-gated ion channels in adult zebrafish brain.

The zebrafish is increasingly well utilized in alcohol research, particularly in modeling human fetal alcohol spectrum disorders (FASD). FASD results from alcohol reaching the developing fetus intra utero, a completely preventable yet prevalent and devastating life-long disorder. The hope with animal models, including the zebrafish, is to discover the mechanisms underlying this disease, which may aid treatment and diagnosis. In the past, we developed an embryonic alcohol exposure regimen that is aimed at mimicking the milder, and most prevalent, forms of FASD in zebrafish. We have found numerous lasting alterations in behavior, neurochemistry, neuronal markers and glial cell phenotypes in this zebrafish FASD model. Using the same model (2 h long bath immersion of 24 h post-fertilization old zebrafish eggs into 1% vol/vol ethanol), here we conduct a proof of concept analysis of voltage-gated cation channels, investigating potential embryonic alcohol induced changes in L-, T- and N- type Ca++ and the SCN1A Na+ channels using Western blot followed by immunohistochemical analysis of the same channels in the pallium and cerebellum of the zebrafish brain. We report significant reduction of expression in all four channel proteins using both methods. We conclude that reduced voltage-gated cation channel expression induced by short and low dose exposure to alcohol during embryonic development of zebrafish may contribute to the previously demonstrated lasting behavioral and neurobiological changes.

Short exposure to low concentrations of alcohol during embryonic development has only subtle and strain- dependent effect on the levels of five amino acid neurotransmitters in zebrafish.

The zebrafish has been successfully employed to model and study the effects of embryonic alcohol exposure. Short exposure to low alcohol concentrations during embryonic development has been shown to significantly disrupt social behavior as well as the dopaminergic and serotoninergic systems in zebrafish. However, analysis of potential effects of embryonic alcohol exposure on other amino acid neurotransmitter systems has not been performed. Here we analyzed neurochemicals obtained from adult AB and TU strain zebrafish that were immersed in 0.00% (control), 0.25%, 0.50%, 0.75% or 1.00% alcohol solution (vol/vol%) at 24 h post-fertilization for 2 h. From whole brain extracts, we quantified glutamate, aspartate, glycine, taurine and GABA levels using high performance liquid chromatography (HPLC). We found embryonic alcohol exposure not to have any significant effect on the levels of glutamate, aspartate, glycine and GABA in both AB and TU zebrafish. AB zebrafish showed a significant elevation of taurine levels, but only in the highest alcohol dose group compared to control. These results, albeit mainly negative, together with prior findings suggest that behavioral abnormalities resulting from embryonic alcohol exposure described before for AB zebrafish may primarily be due to altered dopaminergic and serotoninergic mechanisms. Furthermore, a Principal Component Analysis conducted with all neurochemicals tested in this and in our prior study, found a strain-dependent correlation structure response to embryonic alcohol treatment, confirming that embryonic alcohol effects may be genotype dependent.

The Importance of Holding Water: Salinity and Chemosensory Cues Affect Zebrafish Behavior.

The zebrafish is becoming a popular model organism for studying numerous biological phenomena. Among these are brain function and behavior, including social behavior. Although usually neglected, few studies have already demonstrated that even trivial factors, such as features of the holding water may alter zebrafish behavior. In this study, we employed a 2 × 2 between-subject experimental design, exposing zebrafish to water of either high or low salinity and with chemosensory/olfactory cues of conspecifics either present or absent (while maintaining pH, temperature, nitrate, nitrite, and ammonia levels constant). We presented moving images of conspecifics to experimental zebrafish and analyzed their behavioral responses using video tracking. We found significant interaction between salinity and olfactory cues. For example, zebrafish exposed to their home tank water (high salinity with chemosensory/olfactory cues present) stayed significantly closer to the bottom of their tank compared with fish exposed to the other water conditions, and fish exposed to water with chemosensory/olfactory cues significantly reduced their turns compared with fish exposed to water without chemosensory/olfactory cues. These differences signify the impact environmental factors, for example, fluctuations in salinity level and presence or absence of chemosensory/olfactory cues, may have on zebrafish behavior. We conclude that maintaining stable environmental conditions and specifying and reporting them precisely are important for reducing error variation and for making results across independent studies more comparable.

Strain dependent neurochemical changes induced by embryonic alcohol exposure in zebrafish.

Fetal Alcohol Spectrum Disorder (FASD) is a preventable disease of the child resulting from alcohol (ethanol) consumption by pregnant women. Despite being preventable, FASD represents a prevalent problem throughout the world. Embryonic alcohol induced abnormalities in behavioral responses to social stimuli have been shown in humans and zebrafish. The neurobiological mechanisms underlying the abnormalities remain obscured. Here we start a mechanistic analysis by investigating the effect of embryonic alcohol exposure on the neurochemistry of zebrafish. The differing severity of symptoms seen in FASD may be partially due to genetic factors. To explore such genetic effects, here we analyzed two distinct zebrafish strains: AB and TU. Zebrafish were exposed to one of the following concentrations of alcohol, 0.00%, 0.25%, 0.50%, 0.75%, or 1.00% (vol/vol %) at 24 hours post-fertilization (hpf) for 2h. From whole brain extracts we analyzed the amount of neurotransmitters dopamine and serotonin and their metabolites across 4 different developmental time points: 15, 40, 70 and 102 days post-fertilization (dpf) using high performance liquid chromatography (HPLC). AB zebrafish exhibited a significant dose dependent embryonic alcohol exposure effect which increased in robustness with age. However, TU showed no such concentration effect: the levels of neurochemicals remained mainly unaltered by embryonic alcohol exposure in all age groups. We also analyzed the amount of alcohol reaching the embryo in the two strains and ruled out the possibility that TU has a more protective chorion. We conclude that the uncovered strain differences are due to genetic differences that protect TU from the deleterious effects of embryonic alcohol exposure.

Maturation of shoaling in two zebrafish strains: a behavioral and neurochemical analysis.

Abnormal social behavior is a hallmark of several human neuropsychiatric and neurodevelopmental disorders for which appropriate treatment is lacking. The zebrafish has been proposed as a tool with which these disorders may be modeled and their mechanisms analyzed. A potential starting point of such analyses is the identification of genetic differences between distinct zebrafish strains. Here we compare AB and TU, two well established zebrafish strains, and characterize the developmental trajectories of their shoaling (social) behavior and of the levels of dopamine, serotonin as well as a metabolite of each of these neurotransmitters, DOPAC and 5HIAA from whole brain extracts. Using a novel video-tracking software application, we demonstrate significant strain dependent changes in the maturation of shoaling between day 7 and day 87 post-fertilization. Using high-precision liquid chromatography specifically adapted to zebrafish, we uncover a significant age×strain interaction in dopamine and DOPAC that apparently correlates well with the behavioral differences found between the strains. We also report on strain differences in serotonin and 5HIAA. We discuss possible mechanistic analyses that will address causality and conclude that zebrafish will be a useful tool with which the neurobiological and genetic bases of social behavior may be analyzed in vertebrates.