Fetal Alcohol Syndrome (FAS) is the most widely recognized consequence of prenatal alcohol exposure and is a leading cause of mental retardation 
. FAS is characterized by three developmental anomalies: 1) prenatal and/or postnatal growth retardation; 2) a distinct facial appearance; and 3) central nervous system (CNS) dysfunction 
. However, FAS is not the only result of prenatal alcohol exposure. Prenatal ethanol exposure results in a continuum of developmental anomalies leading to a spectrum of physical, mental and behavioral deficits, often with lifelong implications. The term Fetal Alcohol Spectrum Disorder (FASD) is used to describe these disorders. The total prevalence of FASD in the United States has been estimated to be as high as 2–5% 
. In addition, low levels of fetal alcohol exposure may result in subtle neurodevelopmental consequences that are not necessarily identified as FASD, yet result in susceptibility to a range of neuropsychiatric and behavioral disorders including attention deficit hyperactivity disorder, depression, psychosis, drug addiction and alcoholism 
An individual’s increased susceptibility to these neuropsychological and behavioral disorders is perhaps due in part to fetal ethanol exposure and to the individual’s genetic background. Genetics plays a significant role in an individual’s sensitivity to ethanol, and fetal exposure may exacerbate neurodevelopmental deficits resulting from certain genetic disorders 
. Genetic disorders associated with mutations in the MED12
gene provide an interesting example. MED12, also known as TRAP230, is a component of Mediator, an evolutionarily conserved multi-protein complex that bridges gene regulatory regions with RNA polymerase, affecting the regulation of a large number of genes 
. One potential example of fetal ethanol exposure influencing the prognosis of a genetic disorder is Opitz-Kaveggia syndrome, an X-linked mental retardation disorder resulting from mutations in MED12 
. Fetal alcohol exposure could aggravate the neuropsychiatric disorders associated with this syndrome, although the mechanisms involved are not currently understood.
Several hypotheses have been advanced for mechanisms by which fetal ethanol exposure leads to the reduced proliferation, neuronal dysfunction and cell death suggested to be causes of the clinically observed behavioral phenotype. These include: 1) depriving neurons of activity-dependent trophic factors by antagonizing NMDA receptors and potentiating GABAA receptors, thus, triggering apoptosis; 2) disruption of midline serotonergic neurons; 3) interference with L1 CAM function; 4) oxidative stress and free radical damage; 5) disruption of growth factor signaling (e.g., IGF); and 6) interference with neuronal metabolic enzymes 
. The deleterious effect of ethanol on fetal brain development may proceed by any one or more of these mechanisms, or others yet to be discovered. In all scenarios, fetal alcohol exposure leads to reduced proliferation, neuronal dysfunction and/or cell death, with some groups of neurons being more sensitive to ethanol than others. The spectrum of mental and behavioral abnormalities associated with FASD is thought to arise as a result of this differential sensitivity, which could produce a pathogenic or suggestive phenotype for diagnosis 
Recently, improvements in imaging technologies have enabled investigators and clinicians to visualize structural changes in the brain, such as agenesis of the corpus callosum, thought to arise from high levels of fetal alcohol exposure 
. While these new technologies are exciting, higher resolution is required to answer critical questions regarding mechanisms and consequences of differential cellular responses to ethanol.
We are particularly interested in the potential effects of fetal alcohol exposure on the development of oxytocin expressing cells of the neuroendocrine hypothalamus, and the impact of oxytocin system dysfunction on the susceptibility to neurobehavioral disorders. This interest arises from the fact that the oxytocin system is known to mediate a wide range of social interactions, many of which are perturbed in individuals with FASD 
. However, a direct link between the oxytocin system and FASD has not been established. Zebrafish are being utilized for these experiments because unlike other vertebrate models their anatomy and development allows for direct visualization of alterations in neuronal development in an organism that can be subsequently tested for resulting changes in behavior. The zebrafish oxytocin-like
) gene produces the neurohormone Isotocin, which is closely related to mammalian oxytocin 
. Thus, we hypothesized that embryonic ethanol exposure would disrupt development of the oxtl
system in zebrafish and lead to subsequent alterations in behavior.
In order to directly visualize the effect of ethanol exposure in intact developing brains at the cellular level, we utilized transgenic zebrafish that express green fluorescent protein (GFP) in specific subsets of neurons. Beyond their external fertilization and optical clarity, zebrafish present several additional experimental advantages for investigating perturbations of brain development. First, a large collection of transgenic lines that express fluorescent proteins in highly restricted neuronal populations has been generated by several laboratories 
. Second, zebrafish are amenable to genetic analyses, and a number of mutant strains are readily available (http://zebrafish.org/zirc
). Third, the larva and adults display a wide range of basal behaviors that are relevant for alcohol studies including memory, addiction and social behavior 
. Finally, due to the small size of the zebrafish embryo, the entire nervous system can be observed as it develops.
In this study, four well-characterized transgenic lines were utilized to evaluate the hypothesis that specific subsets of neurons are sensitive to ethanol exposure. Our results demonstrate that zebrafish are well suited for visual detection of sensitive developmental periods of specific groups of ethanol-sensitive neurons. Accordingly, zebrafish provide an excellent model in which to comprehensively map ethanol dose, duration and stage of exposure to alterations in brain development at a cellular level. In addition, the effects of ethanol were evaluated in med12y82
embryos, which carry a mutation in the transcriptional Mediator component, Med12 
. The results of these experiments suggest a highly productive new avenue for investigating the interaction between genetic factors and ethanol exposure. Finally, in order to test the idea that ethanol perturbs the development of the oxytocinergic system, we generated a transgenic line, which expresses GFP from the zebrafish oxtl
promoter. Contrary to our expectations, ethanol treatment did not disrupt oxytocinergic system development but instead induced oxtl
expression in the posterior hindbrain.