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.     

Early embryonic ethanol exposure impairs shoaling and the dopaminergic and serotoninergic systems in adult zebrafish

Fetal alcohol syndrome (FAS) is a devastating disorder accompanied by numerous morphological and behavioral abnormalities. Human FAS has been modeled in laboratory animals including the zebrafish. Recently, embryonic exposure to low doses of ethanol has been shown to impair behavior without any gross morphological alterations in zebrafish. The exposed zebrafish showed reduced responses to animated conspecific images. The effect of embryonic ethanol exposure, however, has not been investigated in a real shoal and the potential mechanisms underlying the behavioral impairment are also unknown. Here we show that a 2 h long immersion in 0.25% and 0.50% (vol/vol) alcohol at 24 h post fertilization significantly increases the distance among members of freely swimming groups of zebrafish when measured at 70 days post fertilization. We also show that this impaired behavior is accompanied by reduced levels of dopamine, DOPAC, serotonin and 5HIAA as quantified by HPLC from whole brain extracts. Our results demonstrate that even very low concentrations of alcohol applied for a short period of time during the development of zebrafish can impair behavior and brain function. We argue that the observed behavioral impairment is not likely to be due to altered performance capabilities, e.g. motor function or perception, but possibly to social behavior itself. We also argue that our neurochemical data represent the first step towards understanding the mechanisms of this abnormality in zebrafish, which may lead to better modeling of, and ultimately perhaps better therapies for human FAS.     

Eye-specific gene expression following embryonic ethanol exposure in zebrafish: Roles for heat shock factor 1

The mechanisms through which ethanol exposure results in developmental defects remain unclear. We used the zebrafish model to elucidate eye-specific mechanisms that underlie ethanol-mediated microphthalmia (reduced eye size), through time-series microarray analysis of gene expression within eyes of embryos exposed to 1.5% ethanol. 62 genes were differentially expressed (DE) in ethanol-treated as compared to control eyes sampled during retinal neurogenesis (24–48 h post-fertilization). The EDGE (extraction of differential gene expression) algorithm identified >3000 genes DE over developmental time in ethanol-exposed eyes as compared to controls. The DE lists included several genes indicating a mis-regulated cellular stress response due to ethanol exposure. Combined treatment with sub-threshold levels of ethanol and a morpholino targeting heat shock factor 1 mRNA resulted in microphthalmia, suggesting convergent molecular pathways. Thermal preconditioning partially prevented ethanol-mediated microphthalmia while maintaining Hsf-1 expression. These data suggest roles for reduced Hsf-1 in mediating microphthalmic effects of embryonic ethanol exposure.     

Effects of embryonic exposure to ethanol on zebrafish visual function

Across a variety of species, including humans, it has been shown that embryos exposed to ethanol display eye abnormalities as well as deficiencies in visual physiology and behavior. The purpose of this study was to examine the effects of embryonic exposure to ethanol on visual function in zebrafish. Visual function was assessed physiologically, via electroretinogram (ERG) recordings, and behaviorally, by measuring visual acuity with the optomotor response. Zebrafish larvae were exposed to 1.5% ethanol at various times during development, including the period of maximal eye development. The results show that ethanol effects on visual function were most pronounced when exposure occurred during eye development. ERG recordings from ethanol-exposed larvae differed from normal subjects both in shape of the response waveform and in visual thresholds under both light and dark adaptation; the differences were more pronounced under lower levels of adaptation. Also, ethanol-exposed larvae displayed lower visual acuity as determined from the optomotor response. These results indicate embryonic ethanol exposure affects visual function particularly when exposure occurs during eye development. In addition, these findings illustrate the usefulness of the zebrafish as a viable animal model for studying Fetal Alcohol Syndrome (FAS).     

Opioid but not nonopioid stress-induced analgesia is enhanced following prenatal exposure to ethanol

Two neurochemically distinct forms of stress-induced analgesia were examined in adult rats following prenatal ethanol exposure. Rats were exposed to ethanol during the last 2 weeks of gestation through a liquid diet presented to the dams. Analgesia testing was conducted when the offspring were 150–210 days of age. Two forms of footshock stress were administered; one that resulted in a naloxone-sensitive (opioid-mediated) analgesia and one that resulted in a naloxone-insensitive (nonopioid) form of analgesia. Rats prenatally exposed to ethanol demonstrated significantly enhanced opioid-mediated analgesia, but unaltered nonpoioid analgesia compared to controls. These results confirm previous findings that prenatal exposure to ethanol leads to long-term alterations in responding to some, but not all forms of stress. The possibility that prenatal exposure to ethanol leads to perturbations in the endogenous opioid systems is discussed.     

Long-term effects of fetal ethanol exposure on pituitary-adrenal response to stress

Pregnant female rats were fed either a 5.0–5.5% w/v ethanol-containing liquid diet ad lib or pair-fed the isocaloric control diet during gestation weeks 2 and 3. At 75–105 days of age, female offspring of the ethanol-treated dams showed significantly greater corticosterone responses than pair-fed- or normally-derived offspring to the stress of cardiac puncture or of noise and shaking, while pituitary-adrenal responses to exposure to a novel environment, cold or 2–3 days of fasting were normal. Adrenal sensitivity to ACTH in dexamethasone-suppressed adult offspring was unaffected by the prenatal treatment. The results demonstrate that fetal ethanol exposure enhances adult pituitary-adrenal responses to certain stressors, including alcohol as demonstrated previously, and suggest that the long-term effects may be mediated by developmental actions of alcohol on central neural mechanisms involved in the regulation of this neuroendocrine system.     

Neonatal ethanol exposure produces a hyperalgesia that extends into adolescence,and is associated with increased analgesic and rewarding properties of nicotine in rats

Rationale Drug exposure during CNS development may alter subsequent dependence liability. We postulated that early alcohol exposure might produce persistent alterations in responses to noxious stimuli. Because relief of physical discomfort may be negatively reinforcing, changes in responses to noxious stimuli produced by early alcohol exposure may increase the rewarding properties of nicotine, a potent analgesic. Such factors may contribute to the high level of alcohol and nicotine co-abuse in humans.Objectives The purpose of this study was to determine whether neonatal ethanol exposure in rats altered responses to noxious stimuli, and whether nicotine would then be more rewarding to the alcohol-exposed offspring, perhaps via its analgesic actions.Methods Neonatal rats received ethanol by gavage (5.0 or 6.5 g/kg) on postnatal days (PND) 9–13. An iso-caloric control group was also included. Rats were then tested to assess responsiveness to a mild noxious heat stimulus, as measured in the tail-flick assay (PND 14 and PND 28), for their response to acute analgesic injections of either nicotine or ethanol (PND 28), and for nicotine induced conditioned place preference (CPP) (PND 36).Results Neonatal ethanol exposure produced hyperalgesia during the first 24 h after alcohol withdrawal (PND 14) that continued through PND 28. The analgesic effects of 12.5 g/kg nicotine were enhanced approximately 2-fold in adolescent rats with previous ethanol histories, relative to controls. These ethanol-exposed rats also showed a significant CPP to nicotine, whereas controls showed no CPP.Conclusions Persistent decreases in tail-flick response latencies suggestive of hyperalgesia were observed following neonatal ethanol exposure in the rat. These changes were accompanied by increases in the analgesic and place-conditioning effects of nicotine in adolescence. If similar effects occur in humans, prenatal alcohol exposure may play a role in an increased risk for the rewarding effects and dependence liability of nicotine later in life.     

Viability and sensorimotor development of mice exposed to prenatal short-term ethanol

The identification of the fetal alcohol syndrome in man has led to an abundant, but sometimes contradictory body of research examining the effects of prenatal ethanol exposure in animals. The present study examines the early sensorimotor development of heterogenous stock mice after ethanol exposure during one of two time periods in gestation, days 8–12, or days 14–18. Animals received either a liquid diet containing 20% ethanol derived calories, an isocalorically balanced yoked control diet or a lab chow control diet. Results indicated no apparent effects due to administration of alcohol during the middle of gestation, but a marked decrease in offspring viability, birthweight, and growth due to administration during the last part of gestation. The late gestation treatment group also showed a slight delay in the rate of development of the grasping and horizontal screen tasks. Overall, animals that survived showed relatively normal sensorimotor development.     

Strain-dependent effects of developmental ethanol exposure in zebrafish

Developmental ethanol exposure from maternal consumption of alcoholic beverages and many other consumer products has been linked to developmental abnormalities in humans and animal models. The sensitivity of an individual to ethanol-induced perturbation of developmental processes is strongly influenced by genetic factors. In this study, we show that there are strain- and dose-dependent differences in sensitivity to developmental ethanol exposure in zebrafish (Danio rerio), suggesting that genetic variation within regulatory factors, influencing critical developmental pathways, is responsible for these differences. Embryos/larvae from genetically distinct strains of zebrafish [Ekkwill (EK), AB, and Tuebingen (TU)] were treated with different concentrations of ethanol. Embryo/larval survival, neurocranial and craniofacial skeletal development, and CNS cell death were analyzed. EK was the most resistant strain to the embryolethal effects of ethanol exposure but had the greatest increase in ethanol-induced cell death. AB survival was affected moderately, as were the neurocranial and craniofacial skeletal structures and ethanol-induced cell death. TU had the lowest survival rate but was the most resistant to alterations in neurocranial and craniofacial skeletal elements. No single strain is the most sensitive or the most resistant to any of the phenotypes examined, suggesting that alcohol influences each of these pathways independently. Further analysis of the molecular and biochemical pathways underlying the strain-dependent differences reported herein could lead to a significant advancement in our mechanistic understanding of the teratogenic effects of ethanol in humans.     

Teratogenic effects of ethanol in the Quackenbush Special mouse

The intragastric exposure of QS mice to alcohol both under short-term (6-day period) (3.0 g/kg, but not 1.5 g/kg, body weight/day through gestation day (GD) 7 to GD 12) and long-term (chronic) (15% ethanol in drinking water beginning several weeks before mating and continuing into pregnancy) conditions reduced the weight, size, and protein content of GD 12 embryos, and the weight of GD 18 embryos. The incidence of brachydactyly with delayed ossification was also significantly greater in embryos chronically exposed to alcohol than in controls (45% vs. 6.7%). The short-term and long-term exposure regimens produced incidences of only 1% and 5.8%, respectively, of forelimb ectrodactyly in GD 18 embryos. It was concluded that alcohol exerts embryo growth retarding effects in pregnant QS mice without inducing a high incidence of skeletal defects. Thus, the QS mouse could serve as an excellent model to resolve the mechanisms whereby alcohol induces pre- and post-natal growth restrictions during pregnancy.     

Protective effect of pyruvate against ethanol-induced apoptotic neurodegeneration in the developing rat brain

Exposure to alcohol during the early stages of brain development can lead to neurological disorders in the CNS. Apoptotic neurodegeneration due to ethanol exposure is a main feature of alcoholism. Exposure of developing animals to alcohol (during the growth spurt period in particular) elicits apoptotic neuronal death and causes fetal alcohol effects (FAE) or fetal alcohol syndrome (FAS). A single episode of ethanol intoxication (at 5 g/kg) in a seven-day-old developing rat can activate the apoptotic cascade, leading to widespread neuronal death in the brain. In the present study, we investigated the potential protective effect of pyruvate against ethanol-induced neuroapoptosis. After 4 h, a single dose of ethanol induced upregulation of Bax, release of mitochondrial cytochrome-c into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1), all of which promote apoptosis. These effects were all reversed by co-treatment with pyruvate at a well-tolerated dosage (1000 mg/kg). Histopathology performed at 24 and 48 h with Fluoro-Jade-B and cresyl violet stains showed that pyruvate significantly reduced the number of dead cells in the cerebral cortex, hippocampus and thalamus. Immunohistochemical analysis at 24 h confirmed that ethanol-induced cell death is both apoptotic and inhibited by pyruvate. These findings suggest that pyruvate treatment attenuates ethanol-induced neuronal cell loss in the developing rat brain and holds promise as a safe therapeutic and neuroprotective agent in the treatment of neurodegenerative disorders in newborns and infants.     

Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish

Ethanol is a well-established developmental toxicant; however, the mechanism(s) of this toxicity remains unclear. Zebrafish are becoming an important model system for the evaluation of chemical and drug toxicity. In this study, zebrafish embryos were utilized to compare the developmental toxicity resulting from either ethanol or acetaldehyde exposure. Embryos were exposed to waterborne ethanol concentrations for various lengths of time but encompassed the earliest stages of embryogenesis. The waterborne ethanol concentration that causes 50% mortality (LC₅₀) following a 45-h ethanol exposure was approximately 340 mM (1.98% v/v). A number of reproducible endpoints resulted from ethanol exposure and included pericardial edema, yolk sac edema, axial malformations, otolith defects, delayed development, and axial blistering. When the exposure period was reduced, similar signs of toxicity were produced at nearly identical ethanol concentrations. To estimate the embryonic dose following a given waterborne ethanol concentration, a kinetic alcohol dehydrogenase (ADH) assay was adapted. The average embryonic ethanol dose was calculated to be a fraction of the waterborne concentration. Embryos exposed to waterborne acetaldehyde resulted in similar, but not identical, endpoints as those induced by ethanol. Embryos were however, almost three orders of magnitude more sensitive to acetaldehyde than to ethanol. Ethanol and acetaldehyde both negatively impact embryonic development; however, ethanol is more teratogenic based on teratogenic indices (TIs). These results demonstrate that the zebrafish model will provide an opportunity to further evaluate the mechanism of action of ethanol on vertebrate development.     

Trophic and proliferative perturbations of in vivo/in vitro cephalic neural crest cells after ethanol exposure are prevented by neurotrophin 3

Neural crest cells (NCCs), a transient population that migrates from the developing neural tube, distributes through the embryo and differentiates into many derivatives, are clearly involved in the damage induced by prenatal exposure to ethanol. The aim of this work was to evaluate alterations of trophic parameters of in vivo (in ovo) and in vitro NCCs exposed to teratogenic ethanol doses, and their possible prevention by trophic factor treatment.Chick embryos of 24-30 h of incubation were treated during 10 h with 100 mM ethanol, or 40 ng/ml Neurotrophin 3 (NT3), or 10 ng/ml Ciliary Neurotrophic Factor (CNTF), or ethanol plus NT3 or CNTF, or defined medium; then the topographic distribution of NCC apoptosis was assessed using a whole-mount acridine orange supravital method. Cultures of cephalic NCCs were exposed to the same ethanol or NT3, or CNTF treatments, or ethanol plus one of both trophic factors, or N2 medium. A viability assay was performed using the calcein-ethidium test, apoptosis was evaluated with the TUNEL test, and proliferative capacity after BrdU labeling.After direct exposure of embryos to 100 mM ethanol for 10 h, a high level of NCC apoptosis was coincident with the abnormal closure of the neural tube. These anomalies were prevented in embryos exposed to ethanol plus NT3 but not with CNTF. In NCC cultures, high cell mortality and a diminution of proliferative activity were observed after 3 h of ethanol treatment. Incubation with ethanol plus NT3 (but not with CNTF) prevented NCC mortality as well as a fall in NCC proliferation.The consequences of direct exposure to ethanol expand data from our and other laboratories, supporting current opinion on the potential risk of alcohol ingestion (even at low doses and/or during a short time), in any period of pregnancy or lactation. Our in vivo/in vitro model encourages us to examine the pathogenic mechanism(s) of the ethanol-exposed embryo as well as the use of trophic factors for the treatment and/or prevention of anomalies induced by prenatal alcohol.     

Behavioral effects of prenatal ethanol exposure and differential early experience in rats

Offspring of rats that were intubated with ethanol during Days 10–14 of gestation and offspring in two control groups were compared on measures of growth, viability, and performance in behavioral tasks. Influences of postnatal environment were assessed by using fostering-cross fostering procedures and by providing different postweaning housing conditions. Results were that offspring from dams treated with ethanol displayed prenatal and postnatal growth deficiency as well as increased postnatal mortality. In the open field, offspring in the Ethanol group were more active than those in the other two groups. Ethanol offspring were also more active in the Y maze and made more avoidance responses and correct discriminations. Early experience as manipulated by the fostering-cross fostering procedures and post-weaning rearing conditions had no impact on the effects of prenatal ethanol on offspring growth, viability, or behavior.     

The effects of ethanol on CNS development in the chick embryo

Human and animal studies show that the central nervous system (CNS) is particularly vulnerable to developmental exposure to alcohol across all stages of development. New critical periods of ethanol sensitivity continue to be defined. The aim of this study was to further examine the stage-specific effects of ethanol on CNS development using a relatively simple programme of neuronal migration and differentiation, the chick embryo spinal cord, and treating at the immediate post-neurulation stage. Embryos (HH-stage 10–12) were explanted into shell-less culture and treated with ethanol (20 μl/40%) or saline (20 μl). At 6,12, 24 and 48 h post-treatment specimens were processed for resin histology. In addition, levels of cell death were analysed using Lysotracker Red, neural crest cell migration patterns were examined using HNK-1 staining and effects on DNA synthesis were evaluated on autoradiographs prepared 1 h after exposure to ³H-TdR. This treatment protocol produced significant growth retardation in ethanol specimens examined at 48 h post-treatment. This effect was shown to involve increased levels of cell death, perturbation of DNA synthesis and an abnormal translocation and subsequent loss of cells into the neural tube lumen. No gross malformations were observed. We conclude that these results further highlight the stage-specific effects of ethanol on neurodevelopment.     

Prenatal ethanol exposure decreases hippocampal H-vinylidene kainic acid binding in 45-day-old rats

The effect of prenatal ethanol exposure on the kainate-sensitive subtype of glutamate receptor binding sites was studied using in vitro ³H-vinylidene kainic acid (VKA) autoradiography. Pregnant Sprague-Dawley rats were fed a liquid diet containing either 3.35% or 6.7% ethanol throughout gestation. Pair-fed dams received isocalorically matched liquid diets and a lab chow ad lib group served as control for paired feeding. At 45 days of age, the offspring were sacrificed and their brains analyzed for specific ³H-VKA binding. Compared to pair-fed controls, specific ³H-VKA binding was reduced by 13% to 32% in dorsal and ventral hippocampal CA₃ stratum lucidum, entorhinal cortex and cerebellum of 45-day-old rats whose mothers consumed either 3.35% or 6.7% ethanol diets. The binding site reductions were statistically significant only in the ventral hippocampal formation and entorhinal cortex of the 3.35% ethanol diet group rats. Saturation of binding studies in the ventral hippocampal formation of 3.35% ethanol rats indicated that the decrease in specific ³H-VKA binding was due to a decrease in the total number of binding sites. Given the excitatory effect of kainic acid on the spontaneous firing rate of hippocampal CA₃ pyramidal neurons, the reduction of kainate-sensitive glutamate binding in this region is consistent with the electrophysiological observation of decreased spontaneous activity of CA₃ pyramidal neurons in fetal alcohol rats.     

Interaction of ethanol with retinol and retinoic acid in RAR β and GAP-43 expression

Fetal ethanol exposure has many detrimental effects on neural development, which possibly occurs through ethanol-induced disruption of the function of vitamin A. In LAN-5 neuroblastoma cells, retinol (10⁻⁶ M) and retinoic acid (RA; 10⁻⁵–10⁻⁶ M) increased RAR β mRNA expression. Ethanol downregulated RAR β levels, even in the presence of retinol. RAR β mRNA expression was decreased by ethanol in the presence of 10⁻⁶ M RA, but not 10⁻⁵ M RA. With cycloheximide (CX), RA still stimulated RAR β mRNA, but the effect of ethanol was abolished. The mRNA expression of GAP-43, an important factor in neural development, increased with 10⁻⁶ M retinol and 10⁻⁵–10⁻⁹ M RA. Ethanol decreased GAP-43 mRNA expression in the presence or absence of retinol. Ethanol was without effect on GAP-43 mRNA at 10⁻⁵ M RA, but did lower the levels at 10⁻⁶ and 10⁻⁷ M RA. CX prevented the effects of both RA and ethanol on GAP-43 mRNA. These studies provide support for the hypothesis that retinoid function is altered by ethanol.