Embryonic Alcohol Research Articles

Embryonic alcohol exposure promotes long-term effects on cerebral glutamate transport of adult zebrafish

Ethanol is a widely consumed substance throughout the world. During development it can substantially damage the human fetus, whereas the developing brain is particularly vulnerable. The brain damage induced by prenatal alcohol exposure may lead to a variety of long-lasting behavioral and neurochemical problems. However, there are no data concerning the effects of developmental ethanol exposure on the glutamatergic system, where extracellular glutamate acts as signaling molecule. Here we investigated the effect of ethanol exposure for 2h (concentrations of 0.0%, 0.1%, 0.25%, 0.50%, and 1.00%) in embryos at 24h post-fertilization (hpf) by measuring the functionality of glutamate transporters in the brain of adult (4 months) zebrafish. However, ethanol 0.1%, 0.25% and 0.50% decreased transport of glutamate to 81.96%, 60.65% and 45.91% respectively, when compared with the control group. Interestingly, 1.00% was able to inhibit the transport activity to 68.85%. In response to the embryonic alcohol exposure, we found impairment in the function of cerebral glutamate transport in adult fish, contributing to long-term alteration in the homeostasis glutamatergic signaling.

Behavioral Responses to Novelty or to a Predator Stimulus Are Not Altered in Adult Zebrafish by Early Embryonic Alcohol Exposure.

Fetal alcohol spectrum disorders (FASD) may vary in symptoms and severity. In the milder and more prevalent forms of the disease, behavioral abnormalities may include impaired social behavior, for example, difficulty interpreting social cues. Patients with FASD remain often undiagnosed due to lack of biomarkers, and treatment is unavailable because the mechanisms of the disease are not yet understood. Animal models have been proposed to facilitate addressing these problems. More recently, short exposure of the zebrafish embryo to low concentrations of alcohol was shown to lead to significant and lasting impairment of behavior in response to social stimuli. The impairment may be the result of abnormal social behavior or altered fear/anxiety. The goal of the current study was to investigate the latter. Here, we employed the alcohol exposure regimen used previously (exposure of 24th hour postfertilization embryos to 0.00, 0.25, 0.50, 0.75, or 1.00% vol/vol alcohol for 2hours), allowed the fish to reach adulthood, and measured the behavioral responses of these adults to a novel tank (anxiety-related behaviors) as well as to an animated image of a sympatric predator of zebrafish (fear-related behaviors). We found behavioral responses of embryonic alcohol-exposed adult fish to remain statistically indistinguishable from those of controls, suggesting unaltered anxiety and fear in the embryonic alcohol-treated fish. Given that motor and perceptual function was previously shown to be also unaltered in the adults after embryonic alcohol exposure, our current results suggest that the impaired response of these fish to social stimuli may be the result of abnormal social behavior.

Impairment of social behaviour persists two years after embryonic alcohol exposure in zebrafish: A model of fetal alcohol spectrum disorders

Zebrafish naturally form social groups called shoals. Previously, we have shown that submerging zebrafish eggs into low concentrations of alcohol (0.00, 0.25, 0.50, 0.75 and 1.00 vol/vol% external bath concentration) during development (24h post-fertilization) for two hours resulted in impaired shoaling response in seven month old young adult zebrafish. Here we investigate whether this embryonic alcohol exposure induced behavioural deficit persists to older age. Zebrafish embryos were exposed either to fresh system water (control) or to 1% alcohol for two hours, 24h after fertilization, and were raised in a high-density tank system. Social behaviour was tested by presenting the experimental fish with a computer animated group of zebrafish images, while automated tracking software measured their behaviour. Control fish were found to respond strongly to animated conspecific images by reducing their distanceand remaining close to the images during image presentation, embryonic alcohol treated fish did not. Our results suggest that the impaired shoaling response of the alcohol exposed fish was not due to altered motor function or visual perception, but likely to a central nervous system alteration affecting social behaviour itself. We found the effects of embryonic alcohol exposure on social behaviour not to diminish with age, a result that demonstrates the deleterious and potentially life-long consequences of exposure to even small amount of alcohol during embryonic development in vertebrates.

Embryonic alcohol exposure: Towards the development of a zebrafish model of fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorder (FASD) is a devastating disease of the brain caused by exposure to alcohol during prenatal development. Its prevalence exceeds 1%. The majority of FASD cases represent the milder forms of the disease which often remain undiagnosed, and even when diagnosed treatment options for the patient are limited due to lack of information about the mechanisms that underlie the disease. The zebrafish has been proposed as a model organism for exploring the mechanisms of FASD. Our laboratory has been studying the effects of low doses of alcohol during embryonic development in the zebrafish. This review discusses the methods of alcohol exposure, its effects on behavioral performance including social behavior and learning, and the potential underlying biological mechanisms in zebrafish. It is based upon a recent keynote address delivered by the author, and it focuses on findings obtained mainly in his own laboratory. It paints a promising future of this small vertebrate in FASD research.

Embryonic alcohol exposure impairs the dopaminergic system and social behavioral responses in adult zebrafish.

Background:The zebrafish is a powerful neurobehavioral genetics tool with which complex human brain disorders including alcohol abuse and fetal alcohol spectrum disorders may be modeled and investigated. Zebrafish innately form social groups called shoals. Previously, it has been demonstrated that a single bath exposure (24 hours postfertilization) to low doses of alcohol (0, 0.25, 0.50, 0.75, and 1% vol/vol) for a short duration (2 hours) leads to impaired group forming, or shoaling, in adult zebrafish.Methods:In the current study, we immersed zebrafish eggs in a low concentration of alcohol (0.5% or 1% vol/vol) for 2 hours at 24 hours postfertilization and let the fish grow and reach adulthood. In addition to quantifying the behavioral response of the adult fish to an animated shoal, we also measured the amount of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid from whole brain extracts of these fish using high-pressure liquid chromatograph.Results:Here we confirm that embryonic alcohol exposure makes adult zebrafish increase their distance from the shoal stimulus in a dose-dependent manner. We also show that the shoal stimulus increases the amount of dopamine and 3,4-dihydroxyphenylacetic acid in the brain of control zebrafish but not in fish previously exposed to alcohol during their embryonic development.Conclusions:We speculate that one of the mechanisms that may explain the embryonic alcohol-induced impaired shoaling response in zebrafish is dysfunction of reward mechanisms subserved by the dopaminergic system.

Embryonic alcohol exposure impairs associative learning performance in adult zebrafish

The zebrafish has been proposed for modeling fetal alcohol spectrum disorders (FASD). Previous FASD research with zebrafish employed high concentrations of alcohol and/or long exposure periods. Here, we exposed zebrafish eggs to low doses of alcohol (0, 0.25, 0.50, 0.75 and 1.0% (vol/vol); external bath application of which 1/20th may reach the inside of the egg) at 16-h post-fertilization (hpf) and only for a short duration (2h) in the hope to avoid gross morphological aberrations and to mimic the more frequent FASD exposure levels. Upon reaching adulthood the exposed and control zebrafish were tested for their associative learning performance in a plus-maze. Embryonic alcohol exposure led to no gross anatomical abnormalities and did not increase mortality. Unexposed (control) zebrafish showed excellent acquisition of association between a conditioned visual stimulus (CS) and food reward, demonstrated by their preference for the target zone of the maze that contained the CS during a probe trial in the absence of reward. However, alcohol-exposed fish showed no such preference and performed indistinguishable from random chance. Locomotor activity during training and the probe trial or the amount of food consumed during training did not differ between the embryonic alcohol exposed and unexposed (control) fish, suggesting that the impaired learning performance found was unlikely to be caused by altered motivation or motor function. Our results suggest that even very small amounts of alcohol reaching the embryo for only a short duration of time may have long lasting deleterious effects on cognitive function in vertebrates.

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 24hours 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 102days 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.

Embryonic alcohol exposure impairs shoaling and the dopaminergic and serotoninergic systems in zebrafish

Embryonic alcohol exposure impairs shoaling and the dopaminergic and serotoninergic systems in zebrafish

Alcohol-Induced Morphological Deficits in the Development of Octavolateral Organs of the Zebrafish (Danio rerio)

Prenatal alcohol exposure is known to have many profound detrimental effects on human fetal development (fetal alcohol spectrum disorders), which may manifest as lifelong disabilities. However, how alcohol affects the auditory/vestibular system is still largely unknown. This is the first study to investigate morphological effects of alcohol on the developing octavolateral system (the inner ear and lateral line) using the zebrafish, Danio rerio. Zebrafish embryos of 2 hours post fertilization (hpf) were treated in 2% alcohol for 48 hours and screened at 72 hpf for morphological defects of the inner ear and lateral line. Octavolateral organs from both alcohol-treated and control zebrafish were examined using light, confocal, and scanning electron microscopy. We observed several otolith phenotypes for alcohol-treated zebrafish including zero, one, two abnormal, two normal, and multiple otoliths. Results of this study show that alcohol treatment during early development impairs the inner ear (smaller ear, abnormal otoliths, and fewer sensory hair cells) and the lateral line (smaller neuromasts, fewer neuromasts and hair cells per neuromast, and shorter kinocilia of hair cells). Early embryonic alcohol exposure may also result in defects in hearing, balance, and hydrodynamic function of zebrafish.

Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers

Children exposed to alcohol in utero have significantly delayed gross and fine motor skills, as well as deficiencies in reflex development. The reasons that underlie the motor deficits caused by ethanol (EtOH) exposure remain to be fully elucidated. The present study was undertaken to investigate the effects of embryonic alcohol exposure (1.5%, 2% and 2.5% EtOH) on motor neuron and muscle fiber morphology in 3days post fertilization (dpf) larval zebrafish. EtOH treated fish exhibited morphological deformities and fewer bouts of swimming in response to touch, compared with untreated fish. Immunolabelling with anti-acetylated tubulin indicated that fish exposed to 2.5% EtOH had significantly higher rates of motor neuron axon defects. Immunolabelling of primary and secondary motor neurons, using znp-1 and zn-8, revealed that fish exposed to 2% and 2.5% EtOH exhibited significantly higher rates of primary and secondary motor neuron axon defects compared to controls. Examination of red and white muscle fibers revealed that fish exposed to EtOH had significantly smaller fibers compared with controls. These findings indicate that motor neuron and muscle fiber morphology is affected by early alcohol exposure in zebrafish embryos, and that this may be related to deficits in locomotion.

Ethanol exposure alters zebrafish development: A novel model of fetal alcohol syndrome

Prenatal exposure to alcohol has been shown to produce the overt physical and behavioral symptoms known as fetal alcohol syndrome (FAS) in humans. Also, it is believed that low concentrations and/or short durations of alcohol exposure can produce more subtle effects. The purpose of this study was to investigate the effects of embryonic ethanol exposure on the zebrafish ( Danio rerio) in order to determine whether this species is a viable animal model for studying FAS. Fertilized embryos were reared in varying concentrations of ethanol (1.5% and 2.9%) and exposure times (e.g., 0–8, 6–24, 12–24, and 48–72 h postfertilization; hpf); anatomical measures including eye diameter and heart rate were compared across groups. Results found that at the highest concentration of ethanol (2.9%), there were more abnormal physical distortions and significantly higher mortality rates than any other group. Embryos exposed to ethanol for a shorter duration period (0–8 hpf) at a concentration of 1.5% exhibited more subtle effects such as significantly smaller eye diameter and lower heart rate than controls. These results indicate that embryonic alcohol exposure affects external and internal physical development and that the severity of these effects is a function of both the amount of ethanol and the timing of ethanol exposure. Thus, the zebrafish represents a useful model for examining basic questions about the effects of embryonic exposure to ethanol on development.

Effects of ethanol and hydrogen peroxide on mouse limb bud mesenchyme differentiation and cell death.

Many of the morphological defects associated with embryonic alcohol exposure are a result of cell death. During limb development, ethanol administration produces cell death in the limb and digital defects, including postaxial ectrodactyly. Because an accumulation of reactive oxygen species (ROS) is produced in adult and embryonic tissues by ethanol exposure, this investigation examines the possibility that ethanol-induced cell death in the limb is a result of ROS. Using an in vitro primary culture of limb mesenchyme, the effects of hydrogen peroxide (H2O2) and ethanol on cell death and differentiation were examined. In addition, a dichlorofluorescein diacetate assay was performed to determine the relative intracellular ROS levels after exposure to several concentrations of ethanol and H2O2. Exposure of 1 to 100 microM H2O2 resulted in a 1.08-1.21 times control increase in cartilage matrix accumulation. Cell death was increased 1.69-2.76 times the untreated control value. Production of ROS ranged from 1.25-1.51 times untreated controls. Ethanol exposure of 0.25 to 1.00% (v/v) did not affect cartilage matrix accumulation but resulted in an increase of cell death (1.45-2.31 times untreated control). Intracellular ROS levels after ethanol exposure increased 1.08-1.15 times control but were lower than that produced by 1 microM H2O2. On the basis of the correlation between ROS level produced by H2O2, it was concluded that ethanol-induced cell death in limb mesenchyme is a result of a non-ROS-mediated mechanism. Therefore, in addition to ethanol-induced cell death mediated by ROS reported in the literature, ethanol-induced cell death can be induced in limb mesenchyme by mechanisms that are not dependent upon ROS.

Holoprosencephaly as a possible embryonic alcohol effect

Three mothers of infants with holoprosencephaly consumed alcohol heavily in pregnancy. We postulate that early alcohol exposure is a possible cause of their malformation. The 3 mothers consumed alcohol only in the first trimester but the first mother continued to take chlordiazepoxide and imipramine throughout the pregnancy. Her infant had an alobar holoprosencephaly associated with a median cleft lip, ocular hypotelorism, and a flat nose. The other infants had semilobar holoprosencephaly and hydrocephalus. These latter 2 infants did not show the characteristic facies of the fetal alcohol syndrome. G-band chromosome studies were normal in all 3 infants. The association of holoprosencephaly with alcohol exposure during pregnancy in humans has been mentioned only briefly, although this malformation has been induced by alcohol in animals. These 3 infants may support the hypothesis that acute or subacute heavy alcohol exposure early in pregnancy could lead to holoprosencephaly without the necessity of a chronic alcohol exposure and without necessarily causing the typical facial findings of the fetal alcohol syndrome.

Holoprosencephaly as a possible embryonic alcohol effect: Another observation

Holoprosencephaly as a possible embryonic alcohol effect: Another observation

Alcohol teratogenicity in the human: A detailed assessment of specificity, critical period, and threshold

Though the occurrence of alcohol-related birth defects is well documented in the human and in animal models, definition of specificity, critical period, and dose-response threshold with a precision adequate for clinical risk assessment and management has been lacking. Data from a cohort of 359 neonates, from a large prospective observational study in which chronic alcohol problems and maternal drinking were assessed during pregnancy and standardized neonatal examinations were blinded for prenatal information, were analyzed with the use of multivariate techniques, with uniform control for confounding by eight factors. Craniofacial abnormalities were found to be definitively related to prenatal alcohol exposure in a dose-response manner (p < 0.001); a significant, but less striking, relationship was observed for other anomalies (p < 0.01). The critical period for alcohol teratogenicity was confirmed to be around the time of conception. Risk for anatomic abnormalities in the offspring was clearly defined among the 5.6% of infants whose mothers drank more than three ounces of absolute alcohol, that is, more than six drinks, per day. Because of a trend toward an increase in craniofacial abnormalities with increasing embryonic alcohol exposure at lower levels, a clear threshold could not be defined. These are not experimental data and the results should not be overinterpreted. Nevertheless, pending further studies with larger samples, the findings suggest that to completely avoid alcohol-related anatomic abnormalities, advice to discontinue drinking or at least to reduce it to a minimal level before conception is clinically appropriate.