Fetal Alcohol Spectrum Disorders (FASD) represent a wide range of

developmental conditions caused by in utero ethanol exposure. Today, FASD affects nearly 1% of the total population, a number likely to be underestimated. Cognitive, perceptual and behavioral deficits following prenatal ethanol exposure arise from underlying neurobiological damage in developing brain structures, including the neocortex, which is involved in higher level cognitive and behavioral function. Current research indicates that alcohol-mediated alterations to epigenetic function could underlie some aspects of FASD, and that these changes may be heritable (Govorko et al., 2012; Bekdash et al., 2013). To determine the impact of prenatal ethanol exposure (PrEE) on functional organization of the neocortex, we first generated an FASD mouse model in CD-1 mice. Preliminary work with this model demonstrated a correlation between gross anatomical changes, and altered gene expression patterns of RZRβ, Cad8 and Id2 at birth along with extensive disruption in neocortical targeting within the neocortex of PrEE offspring (El Shawa et al., 2013; Abbott et al., in review). We extend these findings transgenerationally, identifying numerous stable modifications transmitted via the male germline to unexposed offspring including significant upregulation of RZRβ and Id2, as well as downregulated ephrin A5 expression in cortex. Associated with this change was a significant decrease in global DNA methylation levels in cortex. Absolute methylation levels of CpG islands in promoter regions of RZRβ and Id2 revealed significant hypomethylation in PrEE mice and their offspring. These assays established a strong inverse correlation between gene expression and DNA hypomethylation. Expression of DNMTs 1, 3A and 3B was suppressed in PrEE cortex, providing further insight into possible points at which ethanol perturbs DNA methylation, ultimately resulting in altered gene transcription. The anatomical and behavioral phenotype described in the first generation PrEE mice were also observed in the subsequent generations. These alterations in brain development could be responsible for cognitive, sensorimotor and behavioral deficits seen in our model and children with FASD and may generate phenotypes in the offspring of exposed humans. Thus, understanding the epigenetic means by which this phenotype is generated may reveal novel targets for therapeutic intervention of FASD.

Alcohol exposure during fetal development is the leading known preventable cause of mental retardation in the western world (Abel et al., 1995; Abel et al., 1987; Stratton et al., 1996). Animal models and human studies reveal deficits in sensory-processing, behavior, motor learning, spatial functioning, and increased anxiety as a result of prenatal ethanol exposure (PrEE; Glavas et al., 2001; Kalberg et al., 2006; Hellemans et al., 2010; Carr et al., 2010; Hamilton et al., 2010). Work in the Huffman Laboratory has demonstrated how PrEE can induce abnormal neocortical gene expression, mistargeting of intraneocortical connections, changes to brain anatomy, and altered behavior in the first filial (F1, directly exposed) generation (El Shawa et al., 2013), as well as ethanol’s ability to induce epigenetic modifications (Abbott et al., 2017). If PrEE-induced epigenetic modifications contribute to the observed abnormal gene expression in PrEE animals, then it is plausible that PrEE related conditions could be heritable across generations without further exposure. By extending the metrics used to assess the first generation, we discovered stable modifications to brain anatomy, connectivity and behavior that is detectable across three generations. Many of the anatomical and connectivity related phenotypes are detectable at birth, and behavior is altered across all three generations when measured in prepubescent mice. Cataloging the effects that persist across multiple generations provides strong evidence for ethanol-induced epigenetic changes during development and provides insights into some of the mechanisms influencing heritability. The final study of this dissertation assesses the general perception of safe dietary behavior during pregnancy including alcohol. Responses demonstrated a lack of understanding about the danger of items containing teratogens like BPA, mercury and prescription pain medications and revealed a misconception about the safety of periodic wine drinking throughout pregnancy. The safety of alcohol as scored on our survey approximated CDC statistics of actual consumption during pregnancy. It is clear that increased education about the dangers of teratogen consumption, such as alcohol, during pregnancy is critical for the health and well being of future generations

Prenatal ethanol exposure (PrEE) is a leading cause of preventable birth defects and neurobehavioral disorders; PrEE can lead to Fetal Alcohol Spectrum Disorders (FASD). Clinical profiles of patients with FASD indicate damage to the central nervous system (CNS), including the neocortex, may underlie the varied, cognitive deficits associated with FASD. Recent preclinical evidence has suggested that PrEE may induce epigenetic dysregulation in offspring, producing adverse heritable, transgenerational behavioral effects, however the neurobiological underpinnings remain unclear. Preventive or therapeutic treatments for FASD have not yet been discovered and need to reflect the neurobiological damage inflicted by PrEE as well as the abstinence-resistant population. Additionally, although much research has focused on maternal PrEE, much less is known about the impact of paternal ethanol exposure (PatEE) and how development of the neocortex may be disrupted by preconception alcohol use by fathers. The research presented within this dissertation attempts to fill these gaps in knowledge using three, distinct research designs and multi-level neurobiological and behavioral approaches. First, in Chapter 1, we assessed the full extent of behavioral disruptions that are passed several generations following PrEE and attempted to find novel mechanistic links within the CNS using a transgenerational mouse model of FASD. We found multi-modality behavioral abnormalities that extend several generations and novel CNS anomalies in directly exposed offspring. Second, in Chapter 2, we investigated how co-administration of choline could prevent PrEE-induced neurobehavioral deficits. We found choline has a robust protective effect on several parameters of neocortical development and behaviors that are normally disrupted by PrEE in a mouse model. Lastly, in Chapter 3, we described the impact of preconception ethanol exposure on early postnatal development of the neocortex in an novel PatEE mouse model. We found that PatEE induced several alterations within the developing CNS including altered cortical gene expression as well as patterns of early sensory area connectivity. Overall, the results gleaned from these studies provide key descriptive, mechanistic, and therapeutic information on the still unclear etiology of FASD, as well as novel information on how paternal alcohol use may impact future offspring. Conclusions drawn from this dissertation have clear implications for the substantial impact of alcohol on human health.

This dissertation provides a discussion of the effects of maternal consumption of environmental toxins, and will hopefully contribute to the prevention and understanding of developmental disorders and physiological deficits. Developing systems are particularly susceptible to toxic insults, and small changes in utero can result in long-term deficits. Chapter one of this dissertation reviews the potential teratogenicity of nicotine, alcohol, caffeine, MeHg, PCBs, BPA, and tap water contaminants, so as to characterize the current body of literature detailing the effects and implications of prenatal exposure to toxins. In chapter two, research on maternal consumption habits is presented, with an emphasis on commonly-consumed, potentially-teratogenic substances. Occurrences and frequencies of maternal intake of healthy and unhealthy foods, beverages, and medications in a population of predominantly Hispanic women in Southern California were assessed using the Food, Beverage, and Medication Intake Questionnaire (FBMIQ). The described study reveals that a proportion of pregnant women consumed BPA, MeHg, caffeine, and alcohol at varied levels during pregnancy. The following chapters provide an in-depth analysis of the postnatal effects of a particular neuroteratogen, nicotine, which has been shown to impart various detrimental postnatal effects on exposed offspring. A CD-1 mouse model of prenatal nicotine exposure (PNE) was used to analyze aspects of the brain and neocortex that may underly some of the cognitive and behavioral phenotypes seen with PNE. Analyses included postnatal measurements of brain weight, brain widths and lengths, development of neocortical circuitry, and cortical thickness measures. Exposed mice were found to exhibit reduced brain and body weights at birth, a phenotype that recovered by postnatal day 10. No changes in neocortical circuity or thickness in sensory and motor areas were found. PNE also resulted in persistent behavioral effects, including increased anxiety and deficits in sensorimotor integration abilities, in six month old females. Such analyses describe immediate and long-lasting postnatal effects of prenatal nicotine exposure, underscoring the importance of abstaining from nicotine during pregnancy. Hopefully, the works detailed in this dissertation will provide a foundation upon which future researchers can build a better understanding of how prenatal exposures contribute to developmental deficits.

Exposure to alcohol during early development can induce a multitude of long-lasting developmental abnormalities in exposed individuals due to the teratogenicity associated with alcohol exposure. These abnormalities are characterized as Fetal Alcohol Spectrum Disorders (FASD); which range from physical, behavioral, and cognitive impairs due to alcohol exposure before birth. Maintaining up-to-date and extensive characterization profiles of prenatal ethanol exposure (PrEE) and lactational ethanol exposure (LEE) is crucial for understanding the wide ranging impacts of pre-, peri- and postnatal ethanol exposure. Our laboratory has identified many abnormalities associated with PrEE and LEE. In summary, our results indicate that PrEE and LEE measurably impact brain and behavioral development of affected offspring . In Chapter 1, we explore the heritability of FASD in our mouse model. We previously published on heritable phenotypes in our PrEE mice (Abbott et al., 2018), and this chapter is an extension of the earlier work. Specifically, we document heritable, transgenerational changes in the gross development and behavior in PrEE offspring, in the absence of subsequent alcohol exposure. Our study investigates PrEE-induced effects in directly exposed first- (F1), indirectly exposed second- (F2), and non-exposed third- filial (F3) generations. We report reduced brain and body weights in F1-F3 due to PrEE. In addition we report thinning of the cortex in prelimbic, auditory, and visual cortices. Lastly, we report altered social behavior for F1 and F2 PrEE mice with a return to control levels at F3. In Chapter 2, we established and validated a novel mouse model of LEE where we exposed mice via ethanol contaminated breast milk during the brain growth spurt. This novel model of LEE demonstrated sustained deficits in gross neural development and behavior. Specifically, we note a thinning of the frontal cortex compared to age matched controls, potential reductions in dendritic density within the medial frontal cortex, and altered behavior due to the exposure during lactation. Furthermore, Chapter 3 explores the genomic, epigenomic, and behavioral associated with LEE. This chapter is an extension of our previous work in LEE. We explored mRNA expression of cortical patterning genes, global DNA methylation via 5-methylcytosine (5mC) levels, and further characterized behavior in LEE. Through our approaches we report reductions in gross development, increased gene expression of Id2, and altered behavior due to LEE. In summary, this dissertation includes three projects, presented as chapters. Chapter 1 has been published previously in a more complete form as the data from the manuscript was divided between two dissertations; chapters 2 and 3 will each be published as separate papers. In the body of work represented in these three chapters, we report sustained, observable neural and behavioral deficits in the affected offspring associated with prenatal or lactational ethanol exposure during early development. Prenatal ethanol exposure not only induces significant deficits in the directly exposed offspring, as shown in many reports from the Huffman Laboratory. Our data demonstrate heritable, transgenerational change to at least the third filial generation in PrEE mice. While early postnatal exposure to ethanol via breast milk during lactation and nursing does not produce the severity of phenotypes associated with PrEE, LEE does cause both neuroanatomical and behavioral effects in the affected offspring. Although this research is not directly applied science, our data strongly suggests that both pregnant and nursing women should avoid alcohol consumption.