Hippocampal transcriptome analysis following environmental enrichment in a mouse model of fetal alcohol spectrum disorder
ABSTRACT: Maternal alcohol consumption during pregnancy results in a spectrum of lifelong behavioral and cognitive deficits collectively known as Fetal Alcohol Spectrum Disorders (FASD). FASD is a major health burden in most societies, there is no cure, and the molecular mechanism involved in its development is poorly understood. Human neurodevelopment is a continuum that extends over two decades after birth, with the potential to influence outcomes both prenatally and postnatally. Here, we experimentally investigate if positive postnatal environment enrichment ameliorates behavioral deficits caused by ethanol exposure. Furthermore, we assessed if this modulation is associated with alterations in hippocampal gene expression. To accomplish this, we used a binge model of ethanol exposure followed by environmental enrichment in C57BL/6 mice to generate four groups of animals: (1) control mice raised in standard conditions, (2) mice raised in enriched environments, (3) ethanol-exposed mice raised in standard conditions, and (4) ethanol-exposed mice raised in enriched environments. The environmental enrichment includes larger home cages with more individuals for social interaction, regular exposure to novel items, and access to running wheels. Ethanol exposure results in anxiety-like behavior (light-dark box) as well as learning and memory deficits (Barnes maze) that are at least partially ameliorated by enrichment. Environmental enrichment also improves performance for individuals not exposed to ethanol. Ethanol exposure induces changes in adult hippocampal gene expression (RNA-Seq). Some of the changes in adult hippocampal gene expression following ethanol exposure are reversed by environmental enrichment. The results offer a potential mechanism of behavioral deficits caused by ethanol exposure, including the potential for amelioration after an FASD diagnosis. Overall design: Hippocampal RNA profiles of adult mice that had been prenatally exposed to alcohol and/or postnatal environmental enrichment as well as non-exposed controls were generated by sequencing, using Illumina HiSeq.
Project description:The neurodevelopmental fetal alcohol spectrum disorder (FASD) is characterized by cognitive and behavioral deficits in the offspring. Conferring the deficits to the next generation would increase overall FASD disease burden and prevention of this transmission could be highly significant. Prior studies showed the reversal of these behavioral deficits by low dose thyroxine (T4) supplementation to the ethanol-consuming mothers. Here we aim to identify whether prenatal ethanol (PE) exposure impairs hippocampus-dependent learning and memory in the second-generation (F2) progeny, and whether T4 administration to the ethanol-consuming dam can prevent it. Sprague-Dawley (S) dams received control diets (ad libitum and nutritional control) or ethanol containing liquid diet with and without simultaneous T4 (0.3mg/L diet) administration. Their offspring (SS F1) were mated with naive Brown Norway (B) males and females generating the SB F2 and BS F2 progeny. Hippocampus-dependent contextual fear memory and hippocampal expression of the thyroid hormone-regulated type 3 deiodinase, (Dio3) and neurogranin (Nrgn) were assessed. SS F1 PE-exposed females and their SB F2 progeny exhibited fear memory deficits. T4 administration to the mothers of F1 females reversed these deficits. Although SS F1 PE-exposed males also experienced fear memory deficit, this was neither transmitted to their BS F2 offspring nor reversed by prenatal T4 treatment. Hippocampal Dio3 and Nrgn expression showed similar pattern of changes. Grandmaternal ethanol consumption during pregnancy affects fear memory of the matrilineal second-generation progeny. Low dose T4 supplementation prevents this process likely via altering allele-specific and total expression of Dio3 in the hippocampus.
Project description:Fetal alcohol spectrum disorder (FASD) is characterized by developmental and behavioral deficits caused by maternal drinking during pregnancy. Children born with FASD often face additional stresses, including maternal separation, that add yet additional deficits. The mechanism associated with this interaction is not known. We have used a mouse model for prenatal ethanol exposure and maternal separation to demonstrate that the combination of the two treatments results in more than additive deficits. Furthermore, the behavioral deficits are associated with changes in hippocampal gene expression that persist into adulthood. What initiates and maintains these changes remains to be established and forms the focus of this report. Specifically, MeDIP-Seq was used to assess if changes in promoter DNA methylation are affected by exposure to prenatal ethanol and maternal separation including its relationship to gene expression. The novel results show that different sets of genes implicated by promoter DNA methylation are affected by both treatments independently, and a relatively unique set of genes are affected by the combination of the two treatments. Prenatal ethanol exposure leads to altered promoter DNA methylation at genes important for transcriptional regulation. Maternal separation leads to changes at genes important for histone methylation and immune response, and the combination of two treatments results in DNA methylation changes at genes important for neuronal migration and immune response. Our dual results from the same hippocampal samples suggest there is minimal complementarity between changes in promoter DNA methylation and gene expression, although genes involved tend to be critical for brain development and function. While remaining to be validated, such results argue that mechanisms beyond promoter DNA methylation must be involved in lasting gene expression alterations leading to behavioral deficits implicated in FASD. They may facilitate early and reliable diagnosis, as well as novel strategies for the amelioration of FASD-related deficits.
Project description:Prenatal exposure to ethanol induces aberrant tangential migration of corticopetal GABAergic interneurons, and long-term alterations in the form and function of the prefrontal cortex. We have hypothesized that interneuronopathy contributes significantly to the pathoetiology of fetal alcohol spectrum disorders (FASD). Activity-dependent tangential migration of GABAergic cortical neurons is driven by depolarizing responses to ambient GABA present in the cortical enclave. We found that ethanol exposure potentiates the depolarizing action of GABA in GABAergic cortical interneurons of the embryonic mouse brain. Pharmacological antagonism of the cotransporter NKCC1 mitigated ethanol-induced potentiation of GABA depolarization and prevented aberrant patterns of tangential migration induced by ethanol in vitro. In a model of FASD, maternal bumetanide treatment prevented interneuronopathy in the prefrontal cortex of ethanol exposed offspring, including deficits in behavioral flexibility. These findings position interneuronopathy as a mechanism of FASD symptomatology, and posit NKCC1 as a pharmacological target for the management of FASD.
Project description:Prenatal ethanol exposure can produce structural and functional deficits in the brain and result in Fetal Alcohol Spectrum Disorder (FASD). In rodent models acute exposure to a high concentration of alcohol causes increased apoptosis in the developing brain. A single causal molecular switch that signals for this increase in apoptosis has yet to be identified. The protein p53 has been suggested to play a pivotal role in enabling cells to engage in pro-apoptotic processes, and thus figures prominently as a hub molecule in the intracellular cascade of responses elicited by alcohol exposure. In the present study we examined the effect of ethanol-induced cellular and molecular responses in primary somatosensory cortex (SI) and hippocampus of 7-day-old wild-type (WT) and p53-knockout (KO) mice. We quantified apoptosis by active caspase-3 immunohistochemistry and ApopTag™ labeling, then determined total RNA expression levels in laminae of SI and hippocampal subregions. Immunohistochemical results confirmed increased incidence of apoptotic cells in both regions in WT and KO mice following ethanol exposure. The lack of p53 was not protective in these brain regions. Molecular analyses revealed a heterogeneous response to ethanol exposure that varied depending on the subregion, and which may go undetected using a global approach. Gene network analyses suggest that the presence or absence of p53 alters neuronal function and synaptic modifications following ethanol exposure, in addition to playing a classic role in cell cycle signaling. Thus, p53 may function in a way that underlies the intellectual and behavioral deficits observed in FASD.
Project description:Fetal alcohol spectrum disorders (FASD) are common, seen in 1-5% of the population in the United States and Canada. Regrettably, children diagnosed with FASD are not likely to remain with their biological parents, facing early maternal separation and foster placements throughout childhood. We have modeled FASD in mice via prenatal alcohol exposure and further induce early life stress through maternal separation. We report an association between adult hippocampal gene expression and prenatal and postnatal treatment that is related to behavioral changes. Clustering of expression profiles through weighted gene co-expression network analysis (WGCNA) identifies a set of transcripts associated with anxiety-like behavior as well as treatment group. Genes in this module are overrepresented by genes involved in transcriptional regulation and other pathways related to neurodevelopment. Interestingly, one member of this module, Polr2a, polymerase (RNA) II (DNA directed) polypeptide A, is downregulated by the combination of prenatal ethanol and postnatal stress in an RNA-Seq experiment and qPCR validation. Together, transcriptional control is implicated as a potential underlying mechanism leading to anxiety-like behavior via environmental insults. Greater understanding of the role of prenatal alcohol exposure and postnatal stress in altering the hippocampal transcriptome in the hippocampus is warranted. Further research is required to elucidate the mechanism involved and use this insight towards early diagnosis and amelioration strategies involving children born with FASD. Overall design: Hippocampal RNA profiles of adult mice that had been prenatally exposed to alcohol and/or postnatal maternal separation stress as well as non-exposed controls were generated by sequencing, using Illumina HiSeq.
Project description:Deficits in sensory processing in Fetal Alcohol Spectrum Disorders (FASD) implicate dysfunction in the somatosensory cortex. However, the effects of prenatal ethanol exposure on the development of this region await elucidation. Here, we used an established mouse model of FASD with binge-type ethanol exposure from embryonic day 13.5-16.5 to investigate the effects of prenatal ethanol exposure on pyramidal neurons in the somatosensory cortex. Specifically, we focused on the radial migration of primordial pyramidal neurons during embryonic corticogenesis and their morphology and function during active synaptogenesis in early postnatal development. We found that prenatal ethanol exposure resulted in aberrant radial migration, particularly affecting the populations of postmitotic pyramidal neurons. In addition, there was an enduring effect of prenatal ethanol exposure on glutamate-mediated synaptic transmission in layer V/VI pyramidal neurons. This persisted beyond a transient decrease in pyramidal neuron dendritic complexity that was evident only during early postnatal development. Adolescent mice exposed prenatally to ethanol also displayed decreased tactile sensitivity, as revealed by a modified adhesive tape removal assay. Our findings demonstrate the persistent effects of binge-type in utero ethanol exposure on pyramidal neuron form and function and ultimately sensory processing, the latter being reminiscent of that seen in individuals with FASD.
Project description:Human birth defects are highly variable and this phenotypic variability can be influenced by both the environment and genetics. However, the synergistic interactions between these two variables are not well understood. Fetal alcohol spectrum disorders (FASD) is the umbrella term used to describe the wide range of deleterious outcomes following prenatal alcohol exposure. Although FASD are caused by prenatal ethanol exposure, FASD are thought to be genetically modulated, although the genes regulating sensitivity to ethanol teratogenesis are largely unknown. To identify potential ethanol-sensitive genes, we tested five known craniofacial mutants for ethanol sensitivity: cyp26b1, gata3, pdgfra, smad5 and smoothened. We found that only platelet-derived growth factor receptor alpha (pdgfra) interacted with ethanol during zebrafish craniofacial development. Analysis of the PDGF family in a human FASD genome-wide dataset links PDGFRA to craniofacial phenotypes in FASD, prompting a mechanistic understanding of this interaction. In zebrafish, untreated pdgfra mutants have cleft palate due to defective neural crest cell migration, whereas pdgfra heterozygotes develop normally. Ethanol-exposed pdgfra mutants have profound craniofacial defects that include the loss of the palatal skeleton and hypoplasia of the pharyngeal skeleton. Furthermore, ethanol treatment revealed latent haploinsufficiency, causing palatal defects in ?62% of pdgfra heterozygotes. Neural crest apoptosis partially underlies these ethanol-induced defects in pdgfra mutants, demonstrating a protective role for Pdgfra. This protective role is mediated by the PI3K/mTOR pathway. Collectively, our results suggest a model where combined genetic and environmental inhibition of PI3K/mTOR signaling leads to variability within FASD.
Project description:Fetal alcohol spectrum disorder (FASD) is a common developmental behavioral disorder caused by maternal drinking during pregnancy. Children born with FASD often face additional stress, particularly maternal separation that adds yet additional deficits. The mechanism associated with this phenomenon is not known. Using a mouse model, prenatal ethanol exposure and maternal separation stress have resulted in behavioral deficits and the combination of treatments results in more than additive effects. In addition, behavioral alterations are associated with changes in hippocampal gene expression that persist into adulthood. What initiates and maintains these changes remains to be established and forms the focus of this research. Specifically, MeDIP-Seq was used to assess how changes in promoter DNA methylation are affected by the combination of prenatal ethanol exposure and maternal separation stress with the potential to affect gene expression. The novel results show different sets of genes implicated by promoter DNA methylation affected by both treatments independently, and a relatively unique set of genes affected by the combination of treatments. Prenatal ethanol exposure leads to altered promoter DNA methylation at genes important for brain function and transcriptional regulation. Maternal separation stress leads to changes at genes important for histone methylation and immune response, and the combination of two treatments results in DNA methylation changes at genes important for neuronal migration and immune response. Our dual results on gene expression and DNA methylation from the same samples have allowed comparison of the two observations. There is minimal reciprocal overlap between changes in promoter DNA methylation and gene expression, although overlapping genes tend to be critical for brain development and function. These results suggest that epigenetic mechanisms beyond promoter DNA methylation must be involved in lasting gene expression alterations leading to behavioral deficits implicated in FASD. Overall design: Hippocampal DNA methylation profiles of adult mice that had been prenatally exposed to ethanol and/or postnatal maternal separation stress as well as non-exposed controls were generated by MeDIP-Seq
Project description:In utero alcohol, or ethanol (EtOH), exposure produces developmental abnormalities in the brain of the fetus, which can result in lifelong behavioral abnormalities. Fetal alcohol spectrum disorders (FASD) is a term used to describe a range of adverse developmental conditions caused by EtOH exposure during gestation. Children diagnosed with FASD potentially exhibit a host of phenotypes including growth retardation, facial dysmorphology, central nervous system anomalies, abnormal behavior, and cognitive deficits. Previous research suggests that abnormal gene expression and circuitry in the neocortex may underlie reported disabilities of learning, memory, and behavior resulting from early exposure to alcohol (J Neurosci, 33, 2013, 18893).Here, we utilize a mouse model of FASD to examine effects of prenatal EtOH exposure (PrEE), on brain anatomy in newborn (postnatal day [P]0), weanling (P20), and early adult (P50) mice. We correlate abnormal cortical and subcortical anatomy with atypical behavior in adult P50 PrEE mice. In this model, experimental dams self-administered a 25% EtOH solution throughout gestation (gestational days 0 to 19, day of birth), generating the exposure to the offspring.Results from these experiments reveal long-term alterations to cortical anatomy, including atypical developmental cortical thinning, and abnormal subcortical development as a result of in utero EtOH exposure. Furthermore, offspring exposed to EtOH during the prenatal period performed poorly on behavioral tasks measuring sensorimotor integration and anxiety.Insight from this study will help provide new information on developmental trajectories of PrEE and the biological etiologies of abnormal behavior in people diagnosed with FASD.