Morphological alteration in rat hippocampal neuronal dendrites following chronic binge prenatal alcohol exposure.

Prenatal alcohol exposure (PAE) may result in Fetal Alcohol Spectrum Disorders (FASD). The hippocampus has been recognized as a vulnerable target to alcohol-induced developmental damage. However, the effect of prenatal exposure to alcohol on dendritic morphological adaptations throughout the hippocampal fields in the developing brain still remains largely unknown in the context of FASD. We hypothesized that chronic binge alcohol exposure during pregnancy alters dendrite arborization throughout the developing rat hippocampus. Pregnant Sprague-Dawley rats were assigned to either a pair-fed control (PF-Cont) or a binge alcohol (Alcohol) treatment group. Alcohol dams were acclimatized via a once-daily orogastric gavage of 4.5 g/kg alcohol from gestational day (GD) 5-10 and progressed to 6 g/kg alcohol from GD 11-21. Pair-fed dams similarly received isocaloric maltose dextrin. After parturition, all dams received an ad libitum diet and nursed their offspring until postnatal day (PND) 10 when the pup brains were collected for morphological analysis. PAE increased dendritic arborization and complexities of CA1, CA2/3, and DG neurons in the PND 10 rat hippocampus. The number of primary dendrites, total dendritic length, and number of dendritic branches were significantly increased following PAE, and Sholl analysis revealed significantly more intersections of the dendritic processes in PND 10 offspring following PAE compared with those in the PF-Cont group. We conclude that chronic binge PAE significantly alters hippocampal dendritic morphology in the developing hippocampus. We conjecture that this morphological alteration in postnatal rat hippocampal dendrites following chronic binge prenatal alcohol exposure may play a critical role in FASD neurobiological phenotypes.

Chronic Binge Alcohol Exposure During Pregnancy Alters mTOR System in Rat Fetal Hippocampus.

BACKGROUND: Gestational alcohol exposure can contribute to fetal alcohol spectrum disorders (FASD), an array of cognitive, behavioral, and physical developmental impairments. Mammalian target of rapamycin (mTOR) plays a key role in regulating protein synthesis in response to neuronal activity, thereby modulating synaptic plasticity and long-term memory formation in the brain. Based on our previous quantitative mass spectrometry proteomic studies, we hypothesized that gestational chronic binge alcohol exposure alters mTOR signaling and downstream pathways in the fetal hippocampus. METHODS: Pregnant Sprague-Dawley rats were assigned to either a pair-fed control (PF-Cont) or a binge alcohol (Alcohol) treatment group. Alcohol dams were acclimatized via a once-daily orogastric gavage of 4.5 g/kg alcohol (peak BAC, 216 mg/dl) from GD 5-10 and progressed to 6 g/kg alcohol (peak BAC, 289 mg/dl) from GD 11-21. Pair-fed dams similarly received isocaloric maltose dextrin. RESULTS: In the Alcohol group, following this exposure paradigm, fetal body weight and crown-rump length were decreased. The phosphorylation level of mTOR (P-mTOR) in the fetal hippocampus was decreased in the Alcohol group compared with controls. Alcohol exposure resulted in dysregulation of fetal hippocampal mTORC1 signaling, as evidenced by an increase in total 4E-BP1 expression. Phosphorylation levels of 4E-BP1 and p70 S6K were also increased following alcohol exposure. P-mTOR and P-4E-BP1 were exclusively detected in the dentate gyrus and oriens layer of the fetal hippocampus, respectively. DEPTOR and RICTOR expression levels in the fetal hippocampus were increased; however, RAPTOR was not altered by chronic binge alcohol exposure. CONCLUSION: We conclude that chronic binge alcohol exposure during pregnancy alters mTORC1 signaling pathway in the fetal hippocampus. We conjecture that this dysregulation of mTOR protein expression, its activity, and downstream proteins may play a critical role in FASD neurobiological phenotypes.

Distribution of Phosphatidylethanol in Maternal and Fetal Compartments After Chronic Gestational Binge Alcohol Exposure.

BACKGROUND: Phosphatidylethanol (PEth) is a promising biomarker for gestational alcohol exposure. Studies show PEth accumulation in maternal and fetal blood following alcohol exposure; however, distribution of specific PEth homologues (16:0/18:1, 16:0/18:2, 16:0/20:4) in maternal and fetal blood is unknown. Additionally, PEth levels in highly vulnerable FASD targets in maternal and fetal compartments remain unexplored. We hypothesized that all 3 major PEth homologues will be detectable in the maternal and fetal blood, the maternal uterine artery (a reproductive tissue that delivers oxygen and nutrients to fetoplacental unit), and fetal brain regions following gestational binge alcohol exposure and that homologue distribution profiles will be tissue-specific. METHODS: Pregnant rats received once-daily orogastric gavage of alcohol (Alcohol; BAC 216 mg/dl@4.5g/kg/d; BAC 289 mg/dl@6g/kg/d) or iso-caloric maltose dextrin (Pair-fed control) from gestation days (GD) 5 to 20 or 21. Following chronic exposure, maternal and fetal tissues were analyzed for PEth homologue concentrations utilizing LC-MS/MS technology. RESULTS: All 3 PEth homologues were detected in alcohol-exposed maternal blood, fetal blood, maternal uterine artery, and fetal brain regions (hippocampus, cerebral cortex, and cerebellum). In both maternal and fetal blood, respectively, PEth 16:0/18:2 was more abundant compared to 16:0/18:1 (p < 0.0001,~66%,↑; p = 0.0159, 20.4%↑) and 16:0/20:4 (p = 0.0072,~25%↑; p = 0.0187, 19.4%↑). Maternal PEth 16:0/20:4 was ~ 42% higher than 16:0/18:1 (p = 0.0015). Maternal PEth 16:0/18:2 and 16:0/20:4 were ~ 25%↑ and ~ 20%↑ higher than in fetal blood (p < 0.05). No homologue differences were detected in the maternal uterine artery. In all fetal brain regions, PEth 16:0/18:1 was significantly higher (p < 0.0001) than 16:0/18:2 (~48 to 78%↑) and 16:0/20:4 (~31 to 62%↑) concentrations. PEth 16:0/20:4 was ~ 18% higher than 16:0/18:1 (p < 0.05) in the fetal hippocampus and cortex. CONCLUSION: All major PEth homologues were detected in maternal and fetal blood following chronic gestational binge alcohol exposure; homologue distribution profiles were tissue-specific. This study also provides insights into PEth accumulation in critical FASD targets, specifically the maternal uterine artery and fetal brain.

Hippocampal transcriptome reveals novel targets of FASD pathogenesis.

INTRODUCTION: Prenatal alcohol exposure can contribute to fetal alcohol spectrum disorders (FASD), characterized by a myriad of developmental impairments affecting behavior and cognition. Studies show that many of these functional impairments are associated with the hippocampus, a structure exhibiting exquisite vulnerability to developmental alcohol exposure and critically implicated in learning and memory; however, mechanisms underlying alcohol-induced hippocampal deficits remain poorly understood. By utilizing a high-throughput RNA-sequencing (RNA-seq) approach to address the neurobiological and molecular basis of prenatal alcohol-induced hippocampal functional deficits, we hypothesized that chronic binge prenatal alcohol exposure alters gene expression and global molecular pathways in the fetal hippocampus. METHODS: Timed-pregnant Sprague-Dawley rats were randomly assigned to a pair-fed control (PF) or binge alcohol (ALC) treatment group on gestational day (GD) 4. ALC dams acclimatized from GDs 5-10 with a daily treatment of 4.5 g/kg alcohol and subsequently received 6 g/kg on GDs 11-20. PF dams received a once daily maltose dextrin gavage on GDs 5-20, isocalorically matching ALC counterparts. On GD 21, bilateral hippocampi were dissected, flash frozen, and stored at -80° C. Total RNA was then isolated from homogenized tissues. Samples were normalized to ~4nM and pooled equally. Sequencing was performed by Illumina NextSeq 500 on a 75 cycle, single-end sequencing run. RESULTS: RNA-seq identified 13,388 genes, of these, 76 genes showed a significant difference (p < 0.05, log2 fold change ≥2) in expression between the PF and ALC groups. Forty-nine genes showed sex-dependent dysregulation; IPA analysis showed among female offspring, dysregulated pathways included proline and citrulline biosynthesis, whereas in males, xenobiotic metabolism signaling and alaninine biosynthesis etc. were altered. CONCLUSION: We conclude that chronic binge alcohol exposure during pregnancy dysregulates fetal hippocampal gene expression in a sex-specific manner. Identification of subtle, transcriptome-level dysregulation in hippocampal molecular pathways offers potential mechanistic insights underlying FASD pathogenesis.

Mechanisms Underlying Chronic Binge Alcohol Exposure-Induced Uterine Artery Dysfunction in Pregnant Rat.

BACKGROUND: A cardinal feature of fetal alcohol syndrome is growth restriction. Maternal uterine artery adaptations to pregnancy correlate with birthweight and survival. We hypothesized that gestational binge alcohol exposure impairs maternal uterine vascular function, affecting endothelial nitric oxide (NO)-mediated vasodilation. METHODS: Pregnant rats grouped as pair-fed control or binge alcohol exposed received a once-daily, orogastric gavage of isocaloric maltose-dextrin or alcohol, respectively. On gestational day 20, primary uterine arteries were isolated, cannulated, and connected to a pressure transducer, and functional studies were conducted by dual-chamber arteriography. Uterine arteries maintained at constant intramural pressure (90 mm Hg) were maximally constricted with thromboxane, and a dose-response for acetylcholine (Ach) was recorded. RESULTS: The alcohol group exhibited significantly impaired endothelium-dependent, Ach-induced uterine artery relaxation (↓∼30%). Subsequently, a dose-response was recorded following inhibition of endothelium-derived hyperpolarizing factor (apamin and TRAM-34) and prostacyclin (indomethacin). Ach-induced relaxation in the pair-fed control decreased by ~46%, and interestingly, relaxation in alcohol group further decreased by an additional ~48%, demonstrating that gestational binge alcohol impairs the NO system in the primary uterine artery. An endothelium-independent sodium nitroprusside effect was not observed. Immunoblotting indicated that alcohol decreased the level of endothelial excitatory P-Ser1177 endothelial NO synthase (eNOS) (p < 0.05) and total eNOS expression (p < 0.05) compared to both the normal and pair-fed controls. P-Ser1177 eNOS level was also confirmed by immunofluorescence imaging. CONCLUSIONS: This is the first study to demonstrate maternal binge alcohol consumption during pregnancy disrupts uterine artery vascular function via impairment of the eNOS vasodilatory system.

Regional dysregulation of taurine and related amino acids in the fetal rat brain following gestational alcohol exposure.

The fetal brain exhibits exquisite alcohol-induced regional neuronal vulnerability. A candidate mechanism for alcohol-mediated brain deficits is disruption of amino acid (AA) bioavailability. AAs are vitally important for proper neurodevelopment, as they comprise the most abundant neurotransmitters in the brain and act as neurotransmitter precursors, nitric oxide donors, antioxidants, and neurotrophic factors, which induce synaptogenesis, neuronal proliferation, and migration. We hypothesized that gestational alcohol alters brain AA concentrations, disrupts AAs associated with neuropathogenesis, and that alterations are region-specific. We assigned pregnant Sprague-Dawley rats to either a pair-fed control or a binge alcohol treatment group on gestational day (GD) 4. Alcohol animals were acclimatized via a once-daily orogastric gavage of a 4.5 g/kg alcohol dose from GD 5-10, and progressed to a 6 g/kg alcohol dose from GD 11-20. Pair-fed animals received isocaloric maltose dextrin (once daily; GD 5-20). Fetal cerebral cortex, cerebellum, and hippocampus were collected on GD 21. Following collection, Fluorometric High Performance Liquid Chromatography (HPLC) involving pre-column derivatization with o-phthaldialdehyde quantified regional content of 22 AAs. Chronic binge alcohol administration to pregnant dams regionally altered AA concentrations in all three structures, with the cerebral cortex exhibiting the least vulnerability and the hippocampus exhibiting maximal vulnerability. We conjecture that the AA imbalances observed in this study are critically implicated in pathological and compensatory processes occurring in the brain in response to gestational alcohol exposure.