Background: Prenatal ethanol exposure (PNEE) causes irreversible intellectual and behavioral disabilities, clinically known as fetal alcohol spectrum disorder. Few neuropathologic studies of human brain exist. Hypotheses: First, markers of oxidative stress persist following PNEE. Second, PNEE is associated with inhibitory and excitatory neuron changes. Methods: Human brain autopsies (153) with known PNEE were reviewed; 18 cases (fetus to adult) and controls were selected. Oxidative stress and neuronal differentiation markers were used for immunohistochemistry. Results: There were no obvious differences between control and PNEE brains using oxidative stress markers. In human PNEE brains, glutamatergic neurons were reduced 15.96 % and 18.03% in dentate gyrus and temporal cortex, respectively. GABAergic neurons reactive for parvalbumin were reduced in all hippocampal regions (CA1= 57.86%, CA3= 65.15%, and DG= 53.39%) and temporal cortex (44.13%) in all age groups. Conclusion: GABAergic neuron reduction in human following PNEE could explain motor and behavior distractibility in FASD individuals.

Alcohol consumption during pregnancy can result in fetal alcohol spectrum disorders (FASD), which encompass a range of physical, behavioral, learning, emotional and social disturbances. Many mechanisms for this array of alcohol-derived fetal injuries have been proposed, but none fully accounts for the deficiencies observed. Alcohol is a ubiquitous drug that may affect the brain at any or all stages of development and at multiple sites; regional differences in vulnerability of different brain structures during different periods of exposure have been demonstrated. This study investigates possible mechanisms for the alcohol induced neurodevelopment damage seen as a result of prenatal alcohol exposure, and also includes evaluation of a potential intervention strategy (glutamine). These experiments all utilized the sheep model, which has distinct advantages over the rodent model for third trimester-equivalent studies (a time of increased vulnerability to the effects of alcohol). The fetal hippocampal formation (pyramidal cells in the CA1 and CA2/3 fields and granule cells of the dentate gyrus) and olfactory bulb (mitral cells) have been altered in response to alcohol exposure in rodent model studies. This study examined the effects on the fetal hippocampal formation and olfactory bulb in response to all three trimester-equivalent alcohol exposure in the sheep model, a species in which the third trimester-equivalent occurs in utero (as opposed to post-natal as occurs in the rodent). It is known that both maternal and fetal cortisol levels increase in response to alcohol. The role of cortisol in mediating fetal cerebellar Purkinje cell loss (known to occur with alcohol exposure) was analyzed. Lastly, the availability of circulating amino acids, both maternal and fetal, in response to alcohol are reported. The results of administration of a single acute dose of glutamine to the ewe, concurrent with alcohol, was evaluated for its ability to prevent the amino acid and pH perturbations known to occur in response to alcohol.

Prenatal alcohol exposure (PAE) is associated with a range of physical, growth, and neurobehavioral deficits characteristic of individuals with fetal alcohol spectrum disorders (FASD). Although declarative memory impairment is a key feature of the neurocognitive profile of FASD, the mechanisms underlying this deficit require further clarification. The aim of this cross-sectional research was to examine, both directly and indirectly (via bottom-up and top-down processes), a critical cognitive mechanism that supports successful declarative memory functioning (viz., memory encoding), in children with FASD. Data were collected from a sample (N = 88) of South African children with and without PAE. In Study I, I used a blocked design functional magnetic resonance imaging (fMRI) paradigm to investigate neural activation during visual perception, a lower-order cognitive process essential to memory encoding. The task elicited bilateral category-specific activation during the visual perception of objects and scenes in all participants. The absence of between-group differences suggests that functional recruitment of brain regions during basic visual perception is less susceptible to the effects of PAE than during higher-order processes supporting memory encoding. In Study II, I used an event-related fMRI paradigm to investigate neural activation during memory encoding itself. All participants demonstrated similar memory performance accuracy and recruited extensive bilateral networks during memory encoding. However, participants with a diagnosis of fetal alcohol syndrome (FAS) or partial FAS (PFAS) activated additional regions associated with attentional function. Within the FAS/PFAS group, higher exposure levels were associated with smaller activation increases in the parahippocampal gyri and greater activation increases in the right hippocampal formation during encoding. Data from this study therefore suggest that children with FAS/PFAS recruited more extensive neural resources to support successful memory encoding during this task. In Study III, I used a behavioral source memory paradigm to investigate higher-order executive processes essential for memory encoding. Despite similar recognition accuracy across all diagnostic groups, participants in the FAS/PFAS group showed impaired memory for source details. This pattern of impairment was only partially mediated by working memory performance. These three studies provide novel clarification of the neural and cognitive mechanisms underlying declarative memory impairments in children with FASD.

This dissertation is based on an ethnographic study of Fetal Alcohol Spectrum Disorders (FASD) and the racial, cultural and political considerations that shape the meaning of diagnosis for Alaska Native individuals and families in Anchorage, Alaska. During the period from August 6, 2010 to through August 5, 2011, I worked with foster families and extended natural families living with and supporting individuals diagnosed with FASD. Documenting the experiences of families in their interactions with clinical, state, tribal and non-profit institutions, I sought to understand how a diagnosis of FASD structures opportunities, outcomes and everyday life experiences across several critical life domains, including health, education, employment, kinship and identity. Family narratives and experiences are highlighted to illustrate the ways in which difference is reproduced in everyday public understanding and clinical practice.

Maternal alcohol consumption during pregnancy is a public health concern due to the possible lifelong physical and cognitive effects in offspring. Prevention of alcohol-exposed pregnancies (AEP) should begin preconceptionally, either by preventing unintended pregnancies or by discouraging alcohol consumption in women who are at-risk for pregnancy. The purpose of this dissertation is to utilize the Theory of Planned Behavior's construct of perceived behavioral control (PBC)--including perceived power and control beliefs--to guide the measurement and understanding of two behaviors related to AEP among non-pregnant women: birth control use and binge drinking. For the first specific aim--to estimate the prevalence of alcohol-exposed pregnancies--a secondary data analysis was conducted using surveillance data from North Dakota and South Dakota women who have had a child with FAS. The FAS prevalence estimates (per 1,000 live births) in both states (ND=0.8/1,000; SD=0.9/1,000) were found to be higher than that calculated from national averages (0.7/1,000) using a comparable surveillance methodology. The goal of Specific Aim 2 was to determine risk for AEP among a random group of women, while Specific Aim 3 determined the control beliefs and perceived power to using birth control and decreasing binge drinking levels, and Specific Aim 4 focused on relating PBC of these two behaviors to behavioral intentions. Data for aims 2-4 were derived from a mailed, cross-sectional survey of 190 non-pregnant women randomly chosen from an electronic health records system in the upper Midwest. Of the 190 women included in the analyses, eight (6.6%) were binge drinking while being at-risk for pregnancy (i.e., being sexually active but not always using an effective form of birth control) (Specific Aim 2). This is lower than national estimates. For Specific Aim 3, there were high direct PBC scores for both birth control and binge drinking, and there was a positive correlation between birth control direct and indirect scores (although a negative correlation between binge drinking direct and indirect scores). Finally, Specific Aim 4 uncovered high intentions to both use birth control and to not binge drink. Also, the direct birth control PBC measure was significantly associated with birth control intention when controlling for other variables, although neither PBC nor intention appeared to be associated with actual birth control behavior. For binge drinking, the intention score and the direct measure of PBC were significantly associated with one another; as well, the direct measure of PBC and intention were both significantly associated with actual binge drinking behavior. Therefore, the relationship between PBC and intention was validated for both behaviors, and the association between PBC, intention, and actual behavior was indicated for binge drinking. Overall, the study both supported and disagreed with previous research, indicating that additional research with this theory and topic matter is necessary.

Background: Fetal alcohol syndrome (FAS) is a devastating disorder whose mechanisms may be best investigated using animal models. Here we present a novel zebrafish FAS model to investigate the effects of low to moderate alcohol exposure during early development on learning. Methods: At 24-hours postfertilization zebrafish embryos were exposed to low doses of ethanol (external concentrations = 0.00, 0.25, 0.50, 0.75 and 1.00% vol/vol) for a very short duration (2 hours). Upon adulthood associative learning in the zebrafish was tested in a plus maze. Results: This exposure led to no gross anatomical abnormalities or increased morbidity or mortality. Overall activity was not significantly affected by embryonic ethanol exposure. A trend towards a dose-dependent decrease in learning and memory performance was observed. Conclusions: We suggest that zebrafish will be an appropriate model with which one can analyze the behavioural effects of embryonic alcohol exposure and the mechanisms of the ensuing abnormalities.

Background: Fetal alcohol syndrome (FAS) is a devastating disorder whose mechanisms may be best investigated using animal models. Here we present a novel zebrafish FAS model to investigate the effects of low to moderate alcohol exposure during early development on learning. Methods: At 24-hours postfertilization zebrafish embryos were exposed to low doses of ethanol (external concentrations = 0.00, 0.25, 0.50, 0.75 and 1.00% vol/vol) for a very short duration (2 hours). Upon adulthood associative learning in the zebrafish was tested in a plus maze. Results: This exposure led to no gross anatomical abnormalities or increased morbidity or mortality. Overall activity was not significantly affected by embryonic ethanol exposure. A trend towards a dose-dependent decrease in learning and memory performance was observed. Conclusions: We suggest that zebrafish will be an appropriate model with which one can analyze the behavioural effects of embryonic alcohol exposure and the mechanisms of the ensuing abnormalities.

Fetal alcohol exposure is the most common known cause of preventable mental retardation, yet we know little about how microglia respond to, or are affected by, alcohol in vivo. Using an acute (single day) model of moderate (3 g/kg) to severe (5 g/kg) alcohol exposure in postnatal day (P) 7 or P8 mice we have found that alcohol-induced cortical neuroapoptosis is closely correlated in space and time with the appearance of activated microglia near dead cells. Microglia found in close proximity to dying neurons selectively engulfed those that were in later stages of apoptosis. Remarkably, most dead cells were cleared and microglia began to deactivate within 1-2 days of the initial insult. Coincident with microglial activation and deactivation, in the 5 g/kg alcohol model, there was a transient but substantial increase in pro-inflammatory factor (PIFs) expression. Work in BAX-null mice demonstrated that microglial activation and PIF expression were linked to BAX-dependent neuroapoptosis. As such, the level of microglial activation scaled with alcohol-induced cell death. Therefore, acute alcohol exposure in the developing cortex causes transient microglial activation and mobilization, promoting clearance of dead cells and tissue recovery. Moreover, cortical microglia show a remarkable capacity to rapidly deactivate following even severe neurodegenerative insults in the developing brain. Given that alcohol exposure on either P7 or P8 induced comparable levels of neuroapoptosis and microglial activation, we hypothesized that alcohol exposure on two sequential days (P7 and P8) would exacerbate neuroapoptosis and extend microglial activation. Instead, we found that the period of neuroapoptosis and microglial activation was similar after one day of alcohol exposure on P7 or after two days of exposure on P7 and P8. This was true for both the moderate and severe alcohol paradigms. Potentially, the low levels of cell death produced by the second day of injection may be due to neuroprotective mechanisms elicited by the first day of alcohol injection. In support of this idea, a preliminary microarray analysis of cortical gene expression 12 and 24 h after 5 g/kg alcohol exposure shows a decrease in expression of several pro-apoptotic factors and an increase in the expression of pro-survival factors, including neurotrophins. Of particular interest, BDNF, which has previously been shown to inhibit alcohol-induced neuroapoptosis, showed an eight-fold increase in expression at 24 h following 5 g/kg alcohol exposure and in situ hybridization showed strong BDNF expression near cortical regions with high levels of cell death. Future studies will be needed to extend the analysis of microglial activation states in this two-day injection model and to further explore the possibility that BDNF expression by microglia enacts neuroprotective mechanisms against a second insult. Finally, work in cell culture has suggested that chronic alcohol exposure may potentiate or inhibit microglial phagocytosis of dead cells. These studies raise the possibility that alcohol may directly affect microglial mobility which is important for their surveillance and synaptic remodeling functions. Therefore, we measured the effect of increasing doses of alcohol (0, 0.25, 0.5 and 1%) on microglial migration, branch motility, and morphology in dissociated BV-2 cell cultures and in acutely isolated neonatal (P5-6) brain slices. The results indicate that alcohol dose-dependently inhibits microglial migration and ruffling in cell culture, but in brain slices even high alcohol concentrations (0.5%) only reduce microglial branch motility by ~2%. When combined with our evidence for efficient microglial phagocytic clearance of dead cells in the neonatal cortex, these data suggest that while there is a measurable effect of acute alcohol exposure on microglial mobility, it does not impede microglia from performing their surveillance and phagocytic functions in vivo.

Since fetal alcohol syndrome was first reported, studies have demonstrated a range of perinatal/developmental abnormalities that fall under the umbrella term fetal alcohol spectrum disorders. Of these, low birth weight in exposed children is among the most commonly observed and widely accepted. However, in the past, assertion of an association between prenatal alcohol exposure and preterm birth was controversial. Methodological difficulties may have contributed to failure to consistently detect such a relationship. However, new evidence suggests that pregnancy drinking may be a major contributor to extreme, but not mild prematurity. Extreme prematurity is a major cause of severe perinatal morbidity and mortality. If recent findings are confirmed, it suggests that extreme prematurity might be reduced by eliminating prenatal alcohol exposure.

Maternal binge alcohol consumption has been linked to congenital birth defects in the fetus. Said defects include abnormalities in heart development, a category of disease referred to as Congenital Heart Disease. Given the prevalence of Congenital Heart Disease, with a study showing around 49.9% of women having at least participated in binge alcohol consumption at least once during the early stages of their pregnancy and Congenital Heart Disease being linked to various complications in adulthood, this is a topic relevant to the clinical setting. Alcohol consumption has been linked to decreases in DNA methylation, which generally increases transcriptional expression of nearby genes. This thesis will focus on how alcohol affects the genomic-wide epigenetics of the embryonic heart with the aim of identifying specific genes and sites within those genes that are affected by alcohol exposure in utero. We hypothesize that embryonic mouse hearts exposed to ethanol will show a differential methylation pattern characteristic of hypomethylation versus control hearts not exposed to ethanol. To test this hypothesis, we used oral gavage to administer ethanol to pregnant mice at embryonic age E9.5 (a time associated with heart chamber formation). Maternal mice were sacrificed at E11.5, embryonic hearts were removed, and DNA was extracted for further experimentation with whole genome bisulfite sequencing. Analysis of whole genome bisulfite sequencing data showed a slight trend towards hypomethylation but suggested no significant changes in the overall methylation pattern in embryonic mouse hearts at the genomic level, but we have independently identified several genes whose expression is depressed in the embryonic mouse following a single maternal binge ethanol dose at E9.5, and thus we are investigating potential alcohol-induced DNA methylation alterations in specific target genes of interest. Future investigations into gene and site-specific DNA methylation profiles as well as other epigenetic modifications should prove useful in our quest to learn how maternal alcohol consumption causes cardiac malformations leading to congenital heart disease.