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• Epigenetic modifications change global gene expression in response to environmental factors.

Background Prenatal alcohol exposure (PAE) can result in physical and neurocognitive deficits that are collectively termed "fetal alcohol spectrum disorders" (FASD). Although FASD is associated with lifelong intellectual disability, the mechanisms mediating the emergence of secondary mental health and physical disabilities are poorly understood. Based on our previous data showing that maternal ethanol (EtOH) exposure in mice resulted in an immediate reduction in cranially directed fetal blood flow, we hypothesized that such exposure would also result in persistent alterations in cranially directed blood flow in the prenatally alcohol-exposed (PAE) adult. We also hypothesized that PAE adults exposed to an acute cerebrovascular insult would exhibit more brain damage and neurobehavioral impairment compared to non-PAE adult controls. Methods Pregnant C57BL/6 mice were exposed to EtOH, 3 g/kg, or water by intragastric gavage. Blood flow in carotid, renal, and femoral arteries was assessed by ultrasound imaging in PAE and control adults at 3, 6, and 12 months of age. To mimic ischemic stroke in young adult populations, 3-month-old PAE and control animals were subject to transient middle cerebral artery occlusion (MCAo) and subsequently assessed for behavioral recovery, stroke infarct volume, and brain cytokine profiles. Results PAE resulted in a significant age-related decrease in blood acceleration in adult mice, specifically in the carotid artery. A unilateral transient MCAo resulted in equivalent cortico-striatal damage in both PAE and control adults. However, PAE adult mice exhibited significantly decreased poststroke behavioral recovery compared to controls. Conclusions Our data collectively show that PAE adult mice exhibit a persistent, long-term loss of cranially directed blood flow, and decreased capacity to compensate for brain trauma due to acute-onset adult diseases like ischemic stroke.

The 2012 meeting of the Fetal Alcohol Spectrum Disorders Study Group (FASDSG) focused on the development and ethics of biomarkers for fetal alcohol exposure. This one-day international conference brought students and trainees together with clinicians and researchers to discuss the latest research on FASD. One keynote speaker discussed the value of profiling epigenetic modifications in readily available fetal tissues to diagnose fetal exposure to environmental agents, while the second speaker discussed the ethics of biomarker development within the context of core principles of justice, autonomy, beneficence and non-maleficence. Three sessions of short data talks informed the audience of research advances with particular emphasis on the diagnosis of FASD. Other activities included updates on FASD-related activities by representatives of government agencies, a report on the implementation FASD-related diagnostic criteria in the fifth edition of the Diagnostic and Statistical Manual (DSM-5) of the American Psychiatric Association and a networking lunch, and the presentation of the “Merit Award” to Dr. Nathan Muraski for his work on behavioural outcomes of fetal alcohol exposure. The capstone of the meeting was the presentation of the “Henri Rosett” award to Dr. Denis Viljoen, in recognition of his role in raising awareness about the incidence of FASD in South Africa and in promoting FASD prevention and treatment programs as chairperson and chief executive officer of the Foundation for Alcohol Related Research (FARR).

Identification of the transcriptional networks disrupted by prenatal ethanol exposure remains a core requirement to better understanding the molecular mechanisms of alcohol-induced teratogenesis. In this regard, quantitative reverse-transcriptase polymerase chain reaction (qPCR) has emerged as an essential technique in our efforts to characterize alterations in gene expression brought on by exposure to alcohol. However, many publications continue to report the utilization of inappropriate methods of qPCR normalization, and for many in vitro models, no consistent set of empirically tested normalization controls have been identified. In the present study, we sought to identify a group of candidate reference genes for use within studies of alcohol exposed embryonic, placental, and neurosphere stem cells under both conditions maintaining stemness as well as throughout in vitro differentiation. To this end, we surveyed the recent literature and compiled a short list of fourteen candidate genes commonly used as normalization controls in qPCR studies of gene expression. This list included: Actb, B2m, Gapdh, Gusb, H2afz, Hk2, Hmbs, Hprt, Mrpl1, Pgk1, Ppia, Sdha, Tbp, and Ywhaz. From these studies, we find no single candidate gene was consistently refractory to the influence of alcohol nor completely stable throughout in vitro differentiation. Accordingly, we propose normalizing qPCR measurements to the geometric mean C(T) values obtained for three independent reference mRNAs as a reliable method to accurately interpret qPCR data and assess alterations in gene expression within alcohol treated cultures. Highlighting the importance of careful and empirical reference gene selection, the commonly used reference gene Actb was often amongst the least stable candidate genes tested. In fact, it would not serve as a valid normalization control in many cases. Data presented here will aid in the design of future experiments using stem cells to study the transcriptional processes driving differentiation, and model the developmental impact of teratogens.

Fetal Alcohol Spectrum Disorders (FASD) might be 10-15 times more prevalent among foster/adopted children compared to the general population; however, many of these children remain undiagnosed or misdiagnosed. The lack of confirmed prenatal alcohol exposure (PAE) may be a key barrier to diagnosis. Our sample included 681 patients evaluated for FASD, according to the University of Washington 4-Digit Diagnostic Code, at a pediatric specialty clinic. Guardianship status and other patient characteristics were evaluated by multinomial logistic regression as potential predictors of being classified into one of the following FASD groups: 1) full or partial Fetal Alcohol Syndrome (FAS/pFAS; n = 97); 2) Static Encephalopathy/Alcohol-Exposed (SE/AE) or Neurobehavioral Disorder/Alcohol-Exposed (ND/AE) (n = 135); and 3) some features of FASD (equivalent to pFAS, SE/AE or ND/AE phenotypes) but unknown PAE (n = 449). Median age at assessment was 7.0 years, non-Hispanic White constituted the predominant racial/ethnic group (49.5%), and the majority (81.8%) lacked involvement from a biological parent/relative. Many patients (66.0%) had some features of FASD but lacked reliable PAE information. Children classified into the 'some features/unknown PAE' group had higher median age of assessment (8 years) compared to other groups (6 years; p 0.001). No association was observed between race/ethnicity or child's sex and FASD outcomes (p 0.05). Adopted/foster children were 2.8 times as likely (95% CI: 1.6; 4.8) to be classified into the 'some features/unknown PAE' group compared to children living with a parent/relative after adjusting for covariates. This study's findings indicate that adopted/foster children are more likely to have unknown PAE and not receive a FASD diagnosis, potentially denying them access to specialized services, treatment, and rehabilitation.

Prenatal alcohol exposure can result in a range of physical, neuropathological, and behavioral alterations, collectively termed fetal alcohol spectrum disorders (FASD). We have shown that supplementation with the nutrient choline reduces the severity of developmental alcohol-associated deficits in hippocampal-dependent behaviors and normalizes some aspects of hippocampal cholinergic development and DNA methylation patterns. Alcohol's developmental effects may also be mediated, in part, by altering microRNAs (miRNAs) that serve as negative regulators of gene translation. To determine whether choline supplementation alters ethanol's long-lasting effects on miRNAs, Sprague-Dawley rats were exposed to 5.25 g/kg/day ethanol from postnatal days (PD) 4-9 via intubation; controls received sham intubations. Subjects were treated with choline chloride (100 mg/kg/day) or saline vehicle subcutaneously (s.c.) from PD 4-21. On PD 22, subjects were sacrificed, and RNA was isolated from the hippocampus. MiRNA expression was assessed with TaqMan Human MicroRNA Panel Low-Density Arrays. Ethanol significantly increased miRNA expression variance, an effect that was attenuated with choline supplementation. Cluster analysis of stably expressed miRNAs that exceeded an ANOVA p 0.05 criterion indicated that for both male and female offspring, control and ethanol-exposed groups were most dissimilar from each other, with choline-supplemented groups in between. MiRNAs that expressed an average 2-fold change due to ethanol exposure were further analyzed to identify which ethanol-sensitive miRNAs were protected by choline supplementation. We found that at a false discovery rate (FDR)-adjusted criterion of p 0.05, miR-200c was induced by ethanol exposure and that choline prevented this effect. Collectively, our data show that choline supplementation can normalize disturbances in miRNA expression following developmental alcohol exposure and can protect specific miRNAs from induction by ethanol. These findings have important implications for the mechanisms by which choline may serve as a potential treatment for FASD.

Fetal alcohol spectrum disorders are a leading cause of intellectual disability worldwide. Previous studies have shown that developmental ethanol exposure results in loss of microRNAs (miRNAs), including miR-9, and loss of these miRNAs, in turn, mediates some of ethanol's teratogenic effects in the developing brain. We previously found that ethanol increased methylation at the miR-9-2 encoding gene locus in mouse fetal neural stem cells (NSC), advancing a mechanism for epigenetic silencing of this locus and consequently, miR-9 loss in NSCs. Therefore, we assessed the role of the BAF (BRG1/BRM-Associated Factor) complex, which disassembles nucleosomes to facilitate access to chromatin, as an epigenetic mediator of ethanol's effects on miR-9. Chromatin immunoprecipitation and DNAse I-hypersensitivity analyses showed that the BAF complex was associated with both transcriptionally accessible and heterochromatic regions of the miR-9-2 locus, and that disintegration of the BAF complex by combined knockdown of BAF170 and BAF155 resulted in a significant decrease in miR-9. We hypothesized that ethanol exposure would result in loss of BAF-complex function at the miR-9-2 locus. However, ethanol exposure significantly increased mRNA transcripts for maturation-associated BAF-complex members BAF170, SS18, ARID2, BAF60a, BRM/BAF190b, and BAF53b. Ethanol also significantly increased BAF-complex binding within an intron containing a CpG island and in the terminal exon encoding precursor (pre)-miR-9-2. These data suggest that the BAF complex may adaptively respond to ethanol exposure to protect against a complete loss of miR-9-2 in fetal NSCs. Chromatin remodeling factors may adapt to the presence of a teratogen, to maintain transcription of critical miRNA regulatory pathways.

Poverty, conflict, chemical contaminants, and substance abuse are examples of environmental conditions that are associated with malnutrition, stress, vulnerability to obesity, cancer, psychiatric and other diseases, and diminished life expectancy. We have come to expect that conditions such as these result in deleterious consequences for target populations. However, what if the effects of these conditions transcend generations? What if grandparents’ adverse life experiences influence health and disease susceptibility of their grandchildren or even great-grandchildren? What are the implications for the practice of medicine and social justice? Evidence from human and animal studies in the rapidly growing field of transgenerational epigenetics suggests that the inherited consequences of adverse ancestral life experiences are real. Biology can be programmed in one generation to produce long-lasting, multigenerational maladaptation. Periods of embryonic development and epigenetic programing, which modify the output of genes, are specifically vulnerable to environmental perturbations. This chapter discusses the somewhat checkered history of enquiry into the heritability of acquired traits, along with more recent experimental and epidemiological data that validates the phenomenon of transgenerational epigenetics. Last, we focus on specific examples of environmental perturbations, diet, exposure to toxic chemicals, and addiction that can be modeled in the laboratory, to show how transgenerational epigenetic mechanisms may underlie human susceptibility to disease.

Ethanol (EtOH) and nicotine are often co-abused. However, their combined effects on fetal neural development, particularly on fetal neural stem cells (NSCs), which generate most neurons of the adult brain during the second trimester of pregnancy, are poorly understood. We previously showed that EtOH influenced NSC maturation in part, by suppressing the expression of specific microRNAs (miRNAs). Here, we tested in fetal NSCs the extent to which EtOH and nicotine coregulated known EtOH-sensitive (miR-9, miR-21, miR-153, and miR-335), a nicotine-sensitive miRNA (miR-140-3p), and mRNAs for nicotinic acetylcholine receptor (nAChR) subunits. Additionally, we tested the extent to which these effects were nAChR dependent.Gestational day 12.5 mouse fetal murine cerebral cortical-derived neurosphere cultures were exposed to EtOH, nicotine, and mecamylamine, a noncompetitive nAChR antagonist, individually or in combination, for short (24 hour) and long (5 day) periods, to mimic exposure during the in vivo period of neurogenesis. Levels of miRNAs, miRNA-regulated transcripts, and nAChR subunit mRNAs were assessed by quantitative reverse transcription polymerase chain reaction.EtOH suppressed the expression of known EtOH-sensitive miRNAs and miR-140-3p, while nicotine at concentrations attained by cigarette smokers induced a dose-related increase in these miRNAs. Nicotine's effect was blocked by EtOH and by mecamylamine. Finally, EtOH decreased the expression of nAChR subunit mRNAs and, like mecamylamine, prevented the nicotine-associated increase in α4 and β2 nAChR transcripts.EtOH and nicotine exert mutually antagonistic, nAChR-mediated effects on teratogen-sensitive miRNAs in fetal NSCs. These data suggest that concurrent exposure to EtOH and nicotine disrupts miRNA regulatory networks that are important for NSC maturation.

Ethanol consumption during pregnancy can lead to brain, craniofacial, cardiovascular and limb defects that are collectively termed Fetal Alcohol Spectrum Disorders (FASD) (Abel, 1984; Jones et al., 1973). FASD is a leading non-genetic cause of mental retardation (Abel, 1995). Significant numbers of pregnant women continue to consume ethanol into the second trimester (SAMHSA, 2009), a critical period for fetal neurogenesis and brain angiogenesis. FASD continues to be a significant public health problem, with an estimated incidence of 2–5% of the US population (May et al., 2009). Significant research has focused on the fetal neural basis for the emergence of behavioral deficits associated with maternal ethanol exposure. These studies showed that ethanol directly interferes with several aspects of neural development, including the biology of neural stem cells (Camarillo and Miranda, 2008; Santillano et al., 2005; Vangipuram et al., 2008), neuronal migration (reviewed in (Bearer, 2001)), and the survival and maturation of differentiating neurons (reviewed in (Goodlett and Horn, 2001)). By promoting death (West et al., 2001) and disrupting synaptic connectivity (Carta et al., 2006) of cerebellar neurons for example, ethanol may promote the appearance behavioral deficits like gait disturbances. However, ethanol’s neural effects could additionally be mediated by disturbances within non-neural tissue including developing brain vasculature (Parnell et al., 2007). During the second trimester period, a network of blood vessels within the sub-arachnoid space give rise to microvessels that invade the fetal brain (Norman and O’Kusky, 1986) during the same time period that neural stem cells generate most of the neurons of the brain. This emergent vasculature supports nutrition needs and endocrine control of fetal growth (Fowden and Forhead, 2009) and promotes neural development (Tam and Watts, 2010). Previous research showed that ethanol exposure during the murine first trimester-equivalent period produced a persistent alteration in cardiac physiology (Serrano et al., 2010), which persisted into the critical second trimester window for brain development. In ovine models, chronic fetal ethanol exposure can increase cerebral blood flow under acidaemic and hypercapnic conditions (Parnell et al., 2007) and second trimester exposure alters subsequent cerebral vascular responses to hypoxia and to vasodilatory hormones like VIP (Mayock et al., 2007; Ngai et al., 2008). However, until recently it has been technically difficult to visualize fetal blood flow, particularly in smaller arteries. Modern high-resolution ultrasonography makes possible repeated, non-invasive and real-time analysis of blood flow in sub-millimeter sized vessels like the murine fetal middle cerebral arteries, making it feasible to ask questions about the direct impact of adverse environmental factors like maternal ethanol consumption on fetal cerebral blood flow. In this study, we used this technology to test the hypothesis that single as well as repeated binge-like episodes of maternal ethanol exposure, which are particularly damaging to the fetus (Maier and West, 2001), both immediately and persistently alter cranially-directed fetal blood flow during a period which encompasses blood vessel and neuron formation in the developing brain.

Spinal cord injury (SCI) is medically and socioeconomically debilitating. Currently, there is a paucity of effective therapies that promote regeneration at the injury site, and limited understanding of mechanisms that can be utilized to therapeutically manipulate spinal cord plasticity. MicroRNAs (miRNAs) constitute novel targets for therapeutic intervention to promote repair and regeneration. Microarray comparisons of the injury sites of contused and sham rat spinal cords, harvested 4 and 14 days following SCI, showed that 32 miRNAs, including miR124, miR129, and miR1, were significantly down-regulated, whereas SNORD2, a translation-initiation factor, was induced. Additionally, three miRNAs including miR21 were significantly induced, indicating adaptive induction of an anti-apoptotic response in the injured cord. Validation of miRNA expression by qRT-PCR and in situ hybridization assays revealed that the influence of SCI on miRNA expression persists up to 14 days and expands both anteriorly and caudally beyond the lesion site. Specifically, changes in miR129-2 and miR146a expression significantly explained the variability in initial injury severity, suggesting that these specific miRNAs may serve as biomarkers and therapeutic targets for SCI. Moreover, the pattern of miRNA changes coincided spatially and temporally with the appearance of SOX2, nestin, and REST immunoreactivity, suggesting that aberrant expression of these miRNAs may not only reflect the emergence of stem cell niches, but also the reemergence in surviving neurons of a pre-neuronal phenotype. Finally, bioinformatics analysis of validated miRNA-targeted genes indicates that miRNA dysregulation may explain apoptosis susceptibility and aberrant cell cycle associated with a loss of neuronal identity, which underlies the pathogenesis of secondary SCI.

The current study examined micro RNA (miRNAs) clusters from the maternal plasma to determine their association with preterm birth (PTB) and infant birth outcomes. A subsample of 42 participants who spontaneously delivered either preterm (≤37 weeks) or term was selected from a parent sample of 515 pregnant Mexican American women. Plasma samples and prenatal data were collected at a single mid-gestation time point (22–24 weeks’ gestation) and birth outcomes were obtained from medical records after delivery. Circulating miRNAs were analyzed by qPCR. When miRNAs were grouped according to chromosomal cluster rather than expression level, individual miRNAs correlated strongly with other individual miRNAs within their respective genomic locus. miRNAs from the c19mc cluster negatively correlated with c14mc miRNAs, and this relationship was more pronounced in PTB. Clusters c14mc was negatively associated with length of gestation; while the c19mc was positively associated with length of gestation and infant head circumference. Together, these findings suggest that groups of miRNAs from common chromosomal clusters, rather than individual miRNAs, operate as co-regulated groups of signaling molecules to coordinate length of gestation and infant outcomes. From this evidence, differences in cluster-wide expression of miRNAs are involved in spontaneous PTB.

Background: In utero exposure to ethanol can result in severe fetal brain defects. Previous studies showed that ethanol induces apoptosis in differentiated cortical neurons. However, we know little about ethanol's effects on proliferating embryonic cortical progenitors. This study investigated the impact of ethanol exposure on the Fas/Apo-1/CD95 suicide receptor pathway, and on the survival of proliferating cortical neuroepithelial progenitors. Methods: Murine embryonic-derived primary cortical neuroepithelial cells were maintained as neurosphere cultures and exposed to a dose range of ethanol for periods ranging from 1 to 5 days. Programmed cell death was measured by 4 independent means (Annexin-V staining, caspase activation, DNA fragmentation, and autophagic vacuole formation). Surface Fas/Apo-1 suicide receptor expression was measured by flow cytometry. Expression of Fas/Apo-1-associated DISC-complex genes was measured by quantitative polymerase chain reaction. Results: Ethanol exposure did not substantially increase apoptosis, necrosis, or surface Fas/Apo-1 expression. Moreover, ethanol significantly decreased caspase activation and autophagic activity. Finally, ethanol exposure induced mRNA expression of genes that constitute the death receptor complex. Conclusions: This study provides surprising evidence that ethanol does not induce either programmed cell death or necrosis of immature progenitors during neurogenesis, although ethanol may render neural progenitors susceptible to future apoptotic insults. Furthermore, our novel observation that ethanol suppresses autophagy is consistent with a hypothesis that ethanol promotes premature neural progenitor maturation. Taken together with our previous data regarding the role of the Fas/Apo-1 receptor in neural development, we conclude that ethanol disrupts basic proliferation and differentiation machinery rather than initiating cell death per se.

Heavy alcohol consumption during pregnancy can cause significant mental retardation and brain damage. We recently showed that ethanol depletes reserve cerebral cortical stem cell capacity. Moreover, proliferating neuroepithelial cells exposed to ethanol were resistant to subsequent retinoic acid-induced differentiation. Emerging evidence suggests that cytokines play a crucial growth-promoting role in the developing neural tube.We cultured murine cortical neurosphere cultures in control or ethanol-supplemented mitogenic medium, to mimic alcohol exposure during the period of neuroepithelial proliferation. Cultures were then treated with a step-wise mitogen-withdrawal, integrin-activation model to mimic subsequent phases of neuronal migration and early differentiation. We examined the impact of alcohol exposure during neurogenesis on the secretion of inflammatory and growth-promoting cytokines.Cortical neurosphere cultures exhibit increasingly complex differentiation phenotypes in response to step-wise mitogen-withdrawal and laminin exposure. Some inflammation-modulating cytokines were secreted independent of differentiation state. However, chemotactic cytokines were specifically secreted at high levels, as a function of differentiation stage. monocyte chemotactic protein-1, vascular endothelial growth factor-A, and interleukin (IL)-10 were coordinately decreased during differentiation compared with neuroepithelial proliferation, while granulocyte macrophage-colony stimulating factor (GM-CSF) was induced during differentiation, compared with the neuroepithelial proliferation period. Ethanol exposure during the period of neuroepithelial proliferation prevented the early differentiation-induced increase in GM-CSF while inducing differentiation-associated increase in IL-12 secretion.Embryonic cerebral cortical neuroepithelial-derived precursors secrete high levels of several angiogenic and neural-growth-promoting cytokines as they differentiate into neurons. Our data collectively suggest that ethanol exposure during the period of neuroepithelial proliferation significantly disrupts cytokine signals that are required for the support of emerging neurovascular networks, and the maintenance of neural stem cell beds.

Background In recent years, we have come to recognize that a multitude of in utero exposures have the capacity to induce the development of congenital and metabolic defects. As most of these encounters manifest their effects beyond the window of exposure, deciphering the mechanisms of teratogenesis is incredibly difficult. For many agents, altered epigenetic programming has become suspect in transmitting the lasting signature of exposure leading to dysgenesis. However, while several chemicals can perturb chromatin structure acutely, for many agents (particularly alcohol) it remains unclear if these modifications represent transient responses to exposure or heritable lesions leading to pathology. Results Here, we report that mice encountering an acute exposure to alcohol on gestational Day-7 exhibit significant alterations in chromatin structure (histone 3 lysine 9 dimethylation, lysine 9 acetylation, and lysine 27 trimethylation) at Day-17, and that these changes strongly correlate with the development of craniofacial and central nervous system defects. Using a neural cortical stem cell model, we find that the epigenetic changes arising as a consequence of alcohol exposure are heavily dependent on the gene under investigation, the dose of alcohol encountered, and that the signatures arising acutely differ significantly from those observed after a 4-day recovery period. Importantly, the changes observed post-recovery are consistent with those modeled in vivo, and associate with alterations in transcripts encoding multiple homeobox genes directing neurogenesis. Unexpectedly, we do not observe a correlation between alcohol-induced changes in chromatin structure and alterations in transcription. Interestingly, the majority of epigenetic changes observed occur in marks associated with repressive chromatin structure, and we identify correlative disruptions in transcripts encoding Dnmt1, Eed, Ehmt2 (G9a), EzH2, Kdm1a, Kdm4c, Setdb1, Sod3, Tet1 and Uhrf1. Conclusions These observations suggest that the immediate and long-term impacts of alcohol exposure on chromatin structure are distinct, and hint at the existence of a possible coordinated epigenetic response to ethanol during development. Collectively, our results indicate that alcohol-induced modifications to chromatin structure persist beyond the window of exposure, and likely contribute to the development of fetal alcohol syndrome-associated congenital abnormalities. Electronic supplementary material The online version of this article (doi:10.1186/s13072-015-0031-7) contains supplementary material, which is available to authorized users.

In animal models, middle-aged females sustain greater ischemia-induced infarction as compared to adult females. This age difference in infarct severity is associated with reduced functional capacity of astrocytes, a critical neural support cell. The impaired response of astrocytes following stroke in middle-aged females may be related to epigenetic alterations, including histone acetylation or methylation. The present study measured the activity of enzymes that regulate histone acetylation and methylation in cerebral cortical astrocytes of adult (6 month) and middle-aged (11+ month) female rats 48 h following middle cerebral artery occlusion. H3K4 histone methyltransferase activity was decreased in astrocytes from middle-aged females. The next experiment therefore examined H3K4me3 (transcriptional enhancer) and H3K9me3 (transcriptional repressor) in astrocytes from adult and middle-aged females using ChIP-seq analysis. Adult females had more enriched H3K4me3 peaks (304 vs. 26) at transcriptional start sites and fewer H3K9me3 enriched peaks than middle-aged females (4 vs. 22), indicating a pattern of less active chromatin in astrocytes in the older group following ischemia. DAVID clustering analysis of H3K4me3 enriched genes found several functional categories, including cell motility, regulation of apoptosis and the vascular endothelial growth factor (VEGF) pathway. H3K4me3 was enriched at the miR-17-20 cluster and VEGFa, and analysis of a separate set of astrocytes confirmed that VEGF protein expression and miR-20 mRNA expression were significantly greater following ischemia in adult females compared to middle-aged females. These data indicate that astrocytes display less active chromatin with aging and provide new insight into possible mechanisms for differences in stroke severity observed during aging.

Using spinally transected rats, research has shown that neurons within the L4-S2 spinal cord are sensitive to response-outcome (instrumental) relations. This learning depends on a form of N-methyl-D-aspartate (NMDA)-mediated plasticity. Instrumental training enables subsequent learning, and this effect has been linked to the expression of brain-derived neurotrophic factor. Rats given uncontrollable stimulation later exhibit impaired instrumental learning, and this deficit lasts up to 48 hr. The induction of the deficit can be blocked by prior training with controllable shock, the concurrent presentation of a tonic stimulus that induces antinociception, or pretreatment with an NMDA or gamma-aminobutyric acid-A antagonist. The expression of the deficit depends on a kappa opioid. Uncontrollable stimulation enhances mechanical reactivity (allodynia), and treatments that induce allodynia (e.g., inflammation) inhibit learning. In intact animals, descending serotonergic neurons exert a protective effect that blocks the adverse consequences of uncontrollable stimulation. Uncontrollable, but not controllable, stimulation impairs the recovery of function after a contusion injury.

Following spinal transection of the upper thoracic spinal cord, male Sprague-Dawley rats given legshock whenever a hindlimb is extended learn to maintain the leg in a flexed position. The region of the cord that mediates this instrumental learning was isolated using neuroanatomical tracing, localized infusion of lidocaine, and surgical transections. DiI and Fluoro-Gold microinjection at the site of shock application labeled motor neuron bodies of lamina IX in the lower lumbar region. Local application of the Na-super++ channel blocker lidocaine disrupted learning when it was applied over a region extending from the lower lumbar (L3) to upper sacral (S2) cord. The drug had no effect rostral or caudal to this region. Surgical transections as low as L4 had no effect on learning. Learning also survived a dual transection at L4 and S3, but not L4 and S2. The results suggest that the essential neural circuit lies between L4 and S3.

Six-day ‘binge’ ethanol intoxication postnatal days (PD) 4–9 delays up-regulation of γ-aminobutyric acid type A receptors (GABAARs) in developing rat septal neurons [Dev. Brain Res. 130 (2001) 25]. This distortion occurs during synaptogenesis and could contribute to cognitive dysfunction in fetal alcohol syndrome (FAS). Here, we asked two questions concerning requirements for vulnerability to GABAAR blunting by ethanol. First, we asked whether receptor blunting required PD 4–9 ethanol exposure in rat pups and found that just a brief 2-day exposure (PD 8–9) was as effective as all 6 days. However, 2-day exposure on PD 4–5 was ineffective, showing that ‘binge’ timing was important. We also asked whether ‘binge’ exposure directly inhibited intrinsic processes of septal neurons and could blunt GABAARs on cells maturing outside the brain. Embryonic septal neurons grown in serum-free dispersed culture developed extensive dendritic arborizations, spontaneous synaptic activity and robust whole-cell GABAAR function, but surprisingly, did not show developmental up-regulation of GABAARs like septal neurons maturing in vivo [Brain Res. 810 (1998) 100]. Furthermore, age-matched 6-day ‘binge’ ethanol exposure did not blunt GABAAR function in septal neurons in vitro. These results suggest developmental mechanisms driving up-regulation of GABAAR function in septal neurons in vivo briefly becomes vulnerable to ethanol insult in early postnatal life. While septal neurons express comparable functional GABAARs whether maturing in vivo or in vitro, vulnerability to ethanol-induced receptor blunting requires elements of an intact brain environment not replicated in culture.

This commentary discusses the impact of the manuscript by Zhou et al., titled "Alcohol Alters DNA Methylation Patterns and Inhibits Neural Stem Cell Differentiation," published in the April 2011 issue of Alcoholism: Clinical and Experimental Research (volume 35, issue 4, pages 1-12). In this manuscript, the authors present intriguing evidence from a genome scale analysis of promoter DNA methylation patterns in a class of neural crest stem cells associated with dorsal root ganglia, showing that ethanol essentially prevents epigenetic programming associated with neural stem cell differentiation. This manuscript presents several interesting and novel pieces of data and raises important questions for future research. The implications of these data for our understanding of the etiology of fetal alcohol spectrum disorders are discussed.

Uncontrollable nociceptive stimulation adversely affects recovery in spinally contused rats. Spinal cord injury (SCI) results in altered microRNA (miRNA) expression both at, and distal to the lesion site. We hypothesized that uncontrollable nociception further influences SCI-sensitive miRNAs and associated gene targets, potentially explaining the progression of maladaptive plasticity. Our data validated previously described sensitivity of miRNAs to SCI alone. Moreover, following SCI, intermittent noxious stimulation decreased expression of miR124 in dorsal spinal cord 24 hrs after stimulation and increased expression of miR129-2 in dorsal, and miR1 in ventral spinal cord at 7 days. We also found that brain-derived neurotrophic factor (BDNF) mRNA expression was significantly down-regulated 1 day after SCI alone, and significantly more so, after SCI followed by tailshock. Insulin-like growth factor-1 (IGF-1) mRNA expression was significantly increased at both 1 and 7 days post-SCI, and significantly more so, 7 days post-SCI with shock. MiR1 expression was positively and significantly correlated with IGF-1, but not BDNF mRNA expression. Further, stepwise linear regression analysis indicated that a significant proportion of the changes in BDNF and IGF-1 mRNA expression were explained by variance in two groups of miRNAs, implying co-regulation. Collectively, these data show that uncontrollable nociception which activates sensorimotor circuits distal to the injury site, influences SCI-miRNAs and target mRNAs within the lesion site. SCI-sensitive miRNAs may well mediate adverse consequences of uncontrolled sensorimotor activation on functional recovery. However, their sensitivity to distal sensory input also implicates these miRNAs as candidate targets for the management of SCI and neuropathic pain.

MicroRNAs (miRNAs) are a class of small nonprotein-coding RNAs (ncRNAs) that have been shown to promote the degradation of target messenger RNAs and inhibit the translation of networks of protein-coding genes to control the development of cells and tissues, and facilitate their adaptation to environmental forces. In this chapter, we will discuss recent data that show that miRNAs are an important component of the epigenetic landscape that regulates the transcription as well as the translation of protein-coding gene networks. We will discuss the evidence that implicates miRNAs in both developmental and adult effects of alcohol consumption. Understanding the interactions of this novel class of ncRNAs with the epigenome will be important for understanding the etiology of alcohol teratology and addiction as well as potential new treatment strategies.

We previously showed that peripheral noxious input after spinal cord injury (SCI) inhibits beneficial spinal plasticity and impairs recovery of locomotor and bladder functions. These observations suggest that noxious input may similarly affect the development and maintenance of chronic neuropathic pain, an important consequence of SCI. In adult rats with a moderate contusion SCI, we investigated the effect of noxious tail stimulation, administered one day after SCI, on mechanical withdrawal responses to von Frey stimuli from 1 to 28 days, post-treatment. In addition, because the pro-inflammatory cytokine tumor necrosis factor α (TNFα) is implicated in numerous injury-induced processes including pain hypersensitivity, we assessed the temporal and spatial expression of TNFα, TNF receptors, and several downstream signaling targets after stimulation. Our results showed that unlike sham surgery or SCI only, nociceptive stimulation following SCI induced mechanical sensitivity by 24 hours. These behavioral changes were accompanied by increased expression of TNFα. Cellular assessments of downstream targets of TNFα revealed that nociceptive stimulation increased the expression of caspase 8 and the active subunit (12 kDa) of caspase 3 at a time point consistent with the onset of mechanical allodynia, indicative of active apoptosis. In addition, immunohistochemical analysis revealed distinct morphological signs of apoptosis in neurons and microglia at 24 hours post-stimulation. Interestingly, expression of the inflammatory mediator NFκB was unaltered by nociceptive stimulation. These results suggest that noxious input caudal to the level of SCI can increase the onset and expression of behavioral responses indicative of pain, potentially involving TNFα signaling.

Consumption of alcohol during pregnancy can be severely detrimental to the development of the brain in fetuses. This study explores the usage of optical coherence tomography (OCT) to the study the effects of maternal consumption of ethanol on brain development in mouse fetu ses. On gestational day 14.5 , fetuses were collected and fixed in 4% paraformaldehyde. A swept-source OCT (SSOCT) system was used to acquire 3D images of the brain of ethanol-exposed and control fetuses. The volume of right and left brain ventricles were measured and used to compare between ethanol-exposed and cont rol fetuses. A total of 5 fetuses were used for each of the two groups. The average volumes of the right and left ventricles were measured to be 0.35 and 0.15 mm 3 for ethanol-exposed and control fetuses, respectively. The results demonstrated that there is an alcohol-induced developmental delay in mouse fetal brains. Keywords: alcohol exposure, mouse embryo brain, ve ntricle volume, OCT, ultrasound imaging

Background Plasma or circulating miRNAs (cirmiRNAs) have potential diagnostic value as biomarkers for a range of diseases. Based on observations that ethanol (EtOH) altered intracellular miRNAs during development, we tested the hypothesis that plasma miRNAs were biomarkers for maternal alcohol exposure, and for past in utero exposure, in the neonate. Methods Pregnant sheep were exposed to a binge model of EtOH consumption resulting in an average peak blood alcohol content of 243 mg/dl, for a third-trimester-equivalent period from gestational day 4 (GD4) to GD132. MiRNA profiles were assessed by quantitative PCR analysis in plasma, erythrocyte, and leukocytes obtained from nonpregnant ewes, and plasma from pregnant ewes 24 hours following the last binge EtOH episode, and from newborn lambs, at birth on ~GD147. Results Pregnant ewe and newborn lamb cirmiRNA profiles were similar to each other and different from nonpregnant female plasma, erythrocyte, or leukocyte miRNAs. Significant changes in cirmiRNA profiles were observed in the EtOH-exposed ewe and, at birth, in the in utero, EtOH-exposed lamb. CirmiRNAs including miR-9, -15b, -19b, and -20a were sensitive and specific measures of EtOH exposure in both pregnant ewe and newborn lamb. Additionally, EtOH exposure altered guide-to-passenger strand cirmiRNA ratios in the pregnant ewe, but not in the lamb. Conclusions Shared profiles between pregnant dam and neonate suggest possible maternal-fetal miRNA transfer. CirmiRNAs are biomarkers for alcohol exposure during pregnancy, in both mother and neonate, and may constitute an important shared endocrine biomarker that is vulnerable to the maternal environment.

Background: Age and sex have significant effects on stroke outcomes, however, the precise mechanisms by which these factors affect stroke pathophysiology remains unclear. Small non-coding RNA (miRNA) found in circulation have been used successfully as biomarkers and mechanistic targets for chronic and acute diseases. The present study investigated the impact of age and sex on microRNA expression following ischemic stroke. Methods: Adult (6 mo) and middle aged (11-12 mo) female and male rats were subject to middle cerebral artery occlusion (MCAo) using Endothelin-1. Blood samples were drawn at 2d and 5d post stroke. All animals were terminated at 5d and the brains processed for infarct analysis by standard histological procedures. Total RNA isolated from serum was subject to QPCR amplification for serum microRNAs (168) and 7 reference microRNAs (Exiqon focus panels). Normalized miRNA values were analyzed by Genesifter program using a two-way ANOVA with Benjamini and Hochberg corrections for false discovery rates.. Results: Normalized infarct volume was significantly smaller in adult females (0.12+) as compared to middle-aged females (0.4; p Conclusion: Overall, the pattern of circulating miRNA expression suggests an early influence of age in stroke pathology, with a later emergence of sex as a factor for stroke severity. Furthermore, a small cohort of miRNAs were found to discriminate severe and mild stroke infarction.

Alcohol, though widely consumed, can be addictive, toxic, tumorigenic, and teratogenic. The biology of alcoholism and its consequences is often viewed as insurmountably complex. However, recent data showing the involvement of microRNAs (miRNAs) in alcohol addiction and pathology provides evidence for a new layer of cellular regulation that may explain and integrate the complexity of alcohol’s effects. MiRNAs are small, non-protein-coding RNA molecules that control networks of protein-coding genes by translation repression and by other more recently discovered mechanisms, including epigenetics and endocrine signaling. In this chapter, we will focus mainly on the brain effects of ethanol and discuss the small but growing literature that shows that common miRNAs mediate aspects of alcoholism and the emergence of alcohol-associated pathology and teratology. We will also examine mechanisms that regulate miRNA biogenesis and function, which are potentially vulnerable to drugs of abuse such as alcohol.

MicroRNAs (miRNAs) are members of a large class of non–protein-coding RNA (ncRNA) molecules that represent a significant, but until recently unappreciated, layer of cellular regulation. Assessment of the generation and function of miRNAs suggests that these ncRNAs are vulnerable to interference from genetic, epigenetic, and environmental factors. A small but rapidly expanding body of studies using a variety of animal- and cell culture–based experimental models also has shown that miRNAs are important targets of alcohol during fetal development and that their dysregulation likely plays a significant role in the etiology of fetal alcohol spectrum disorders (FASD). Accordingly, an analysis of the regulation and function of these miRNAs may yield important clues to the management of FASD.

Infection with the Daniel strain of Theiler’s murine encephalomyelitis (TMEV-DA) virus induces persistent demyelinating lesions in mice and serves as a model for multiple sclerosis. During the acute phase of the disease, however, viral infection leads to cell death in vivo. Viral-induced death may result directly from viral infection of neural cells, or indirectly, by activation of the immune system. To examine the direct effects of TMEV infection on neural cells, myelinated explant cultures of the murine cerebellum were infected with 105 pfu of TMEV-DA for periods ranging from 1 to 72 h. Our results indicate that TMEV-DA replicates in cultured neural tissue. Initially, viral antigen is localized to a few isolated neural cells. However, within 72 h antigen was observed in multiple foci that included damaged cells and extracellular debris. Viral infection led to a rapid and cyclical induction of necrosis with a time period that was consistent with the lytic phase of the viral life-cycle. Simultaneously, we observed an increase in apoptosis 48 h post-infection. Electron micrographic analysis indicated that viral-infected cultures contained cells with fragmented nuclei and condensed cytoplasm, characteristic of apoptosis. The localization of apoptosis to the cerebellar granule cell layer, identified these cells as presumptive granule neurons. Viral infection, however, did not lead to myelin damage, though damaged axons were visible in TMEV-infected cultures. These results suggest that during the acute phase of infection, TMEV targets neural cells for apoptosis without directly disrupting myelin. Myelin damage may therefore result from the activation of the immune system.

Introduction: Animal studies modeling fetal alcohol syndrome have demonstrated that developmental exposure to alcohol is associated with decreased brain weight and significant neuronal loss in multiple regions of the developing brain. Our previous data suggest that the Fas/Apo [apoptosis]-1 receptor is transiently expressed in the developing cerebral cortex during the peak period of naturally occurring apoptotic cell death and maximum sensitivity to alcohol. Therefore, we hypothesized that ethanol increases the expression of suicide receptors such as Fas/Apo-1 in the developing fetal cerebral cortex and leads to an upregulation or extension of the normal period of apoptosis and consequent disorganization of the neural circuitry. Methods: Ethanol was administered in one of four doses (120, 320, 630, and 950 mg/dl) to organotypic explant cultures of the developing cerebral cortex established from postnatal day 2 rats and maintained for 6 days in vitro. The number of cells expressing Fas/Apo-1 receptor mRNA was counted. Apoptosis was measured by the use of two independent assays; a cell death enzyme-linked immunosorbent assay for DNA fragmentation and flow cytometric analysis of Annexin-V binding to phosphatidylserine externalized to the outer leaflet of the plasma membrane. Necrosis was also estimated by two independent measures, the amount of lactate dehydrogenase released into culture medium and flow cytometric analysis of cells that were positive for both Annexin-V and propidium iodide. Results: A significantly larger number of developing cortical cells expressed Fas/Apo-1 mRNA at the lower doses (120 and 320 mg/dl) than at the higher doses (630 and 950 mg/dl). Furthermore, ethanol induced apoptosis in a dose-related manner, with peak apoptosis observed at a dose of 630 mg/dl in the case of DNA fragmentation and at 630 and 950 mg/dl in the case of phosphatidylserine translocation to the outer leaflet of the plasma membrane. Ethanol did not induce necrosis at any of the administered doses of ethanol. Conclusions: Our data suggest that ethanol induces a susceptibility to apoptotic signals at low doses by upregulating the expression of mRNAs for cytotoxic receptors such as Fas/Apo-1 in the developing cerebral cortex. However, ethanol itself specifically induces apoptosis in the developing cerebral cortex only at higher doses.

Neural tube defects (NTDs), although prevalent and easily diagnosed, are etiologically heterogeneous, rendering mechanistic interpretation problematic. To date, there is evidence that mammalian neural tube closure (NTC) initiates and fuses intermittently at four discrete locations. Disruption of this process at any of these four sites may lead to a region-specific NTDs, possibly arising through closure site-specific genetic mechanisms. Although recent efforts have focused on elucidating the genetic components of NTDs, a void persists regarding gene identification in closure site-specific neural tissue. To this end, experiments were conducted to identify neural tube closure site-specific genes that might confer regional sensitivity to teratogen-induced NTDs. Using an inbred mouse strain (SWV/Fnn) with a high susceptibility to VPA- induced NTDs that specifically targets and disrupts NTC between the prosencephalon and mesencephalon region (future fore/midbrain; neural tube closure site II), we identified a VPA-sensitive closure site II-specific clone. Sequencing of this clone from an SWV neural tube cDNA library confirmed that it encodes the r1 subunit of the cell cycle enzyme ribonucleotide reductase (RNR). The abundance of rnr-r1 mRNA was significantly increased in response to VPA drug treatment. This upregulated expression was accompanied by a significant decrease in cellular proliferation in the closure site II neural tube region of the embryos, as determined by ELISA cellular proliferation assays performed on BrdU-pulsed neuroepithelial cells in vivo. We hypothesize that rnr-r1 plays a critical role in the development of VPA-induced exencephaly. Teratology 61:305–313, 2000. © 2000 Wiley-Liss, Inc.

The developing cerebral cortex undergoes overlapping periods of neurogenesis, suicide, and differentiation to generate the mature cortical plate. The following experiments examined the role of the gonadal hormone estrogen in comparison to the neurotrophins, in the regulation of p53-dependent cortical cell fate. To synchronize choices between neurogenesis, apoptosis, and neural differentiation, embryonic rat cerebral cortical neuroblasts were conditionally immortalized with the SV40 large T antigen containing the tsA58/U19 temperature-sensitive mutations. At the nonpermissive temperature, cessation of large T antigen expression was accompanied by induction of p53, as well as the p53-dependent proteins, wild-type p53-activated fragment-1/Cdk (cyclin-dependent kinase)-interacting protein-1 (p21/Waf1), Bcl (B-cell lymphoma)-associated protein (Bax), and murine double minute 2 (MDM2), that lead to cell cycle-arrest, suicide, and p53 inhibition, respectively. Simultaneously, neuroblasts exit cell cycle and die apoptotically or differentiate primarily into astrocytes and immature postmitotic neuroblasts. At the nonpermissive temperature, estrogen specifically induced an antagonist-independent increase in phosphorylated p53 expression, while increasing p21/Waf1 and decreasing Bax. Coincidentally, estrogen rapidly increased and then decreased MDM2 relative to controls, suggesting temporal modulation of p53 function. Both estrogen and neurotrophins prevented DNA fragmentation, a marker for apoptosis. However, estrogen also induced a transient increase in released lactate dehydrogenase, suggesting that estrogen simultaneously induced rapid cell death in a subpopulation of cells. In contrast to the neurotrophins, estrogen also increased cell proliferation. Both estrogen and the neurotrophins supported neuronal differentiation. However, in contrast to the neurotrophins, estrogen only supported the expression of a subset of oligodendrocytic markers. These results suggest that estrogen and the neurotrophins support overlapping and distinct aspects of differentiation in the developing cerebral cortex.

Photic entrainment of mammalian circadian rhythms occurs because the pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus is endowed with a rhythmic sensitivity to photic signals conveyed by the retinohypothalamic tract. Since brain-derived neurotrophic factor (BDNF) has been implicated in the functional modulation of other retinal targets, the rat SCN was examined for expression and cellular distribution of this neurotrophin and TrkB, the tyrosine kinase receptor that preferentially binds BDNF. The rat SCN was found to express the mature BDNF peptide and mRNA by Western blotting, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR) analyses. BDNF-immunoreactivity and hybridization signal for its mRNA were coextensively localized within a number of SCN cells throughout the rostrocaudal axis of each nucleus. In addition, some cells intercalated within the optic chiasm were distinguished by expression of BDNF immunoreactivity or mRNA. Immunostaining for the TrkB receptor was also evident in the SCN within terminals or fibers predominantly located along the SCN/optic chiasm interface and within scattered perikarya near the medial border of each nucleus. Combined in situ hybridization and immunocytochemical analysis revealed that BDNF mRNA-expressing cells within the ventrolateral SCN were often closely apposed to TrkB-positive fibers extending from the optic chiasm. These findings raise the possibility that target-derived interactions between BDNF and TrkB receptors could play a role in the circadian modulation of SCN pacemaker sensitivity to photic input transmitted by the retinohypothalamic tract.

Ethanol exposure during development leads to alterations in neuronal differentiation and profound neuronal loss in multiple regions of the developing brain. Although differentiating Purkinje cells of the cerebellum are particularly vulnerable to ethanol exposure, the mechanisms that ameliorate ethanol-induced Purkinje cell loss have not been well defined. Previous research indicates that glial-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β family, promotes the survival of several neuronal populations, including cerebellar Purkinje cells. Therefore, we examined whether GDNF could attenuate ethanol-induced Purkinje cell loss in an in vitro model system using calbindin-D28k-immunoreactivity as a specific marker for Purkinje cells. We found that ethanol led to a significant dose-related decline in calbindin-D28k-immunoreactive cells in explant cultures of the developing cerebellum. However, concurrent administration of GDNF led to a significant rescue of calbindin-D28k-immunoreactive cells. Therefore, our results suggest that GDNF prevents ethanol-associated Purkinje cell loss. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 835–847, 1997

While the incidence of cognitive disorders such as Alzheimer’s disease (AD) is increasing among the ageing populations of Europe and North America, few therapeutic agents have been identified for effective disease management. Substantial recent evidence suggests that the neurological circuits targeted by AD are also sensitive to gonadal hormones such as oestrogen. This review examines the evidence for hormone replacement therapy as a possible strategy for the management of ageing-related cognitive disorders, emphasising possible cellular/molecular actions of oestrogen. We examine the recent patent literature for promising trends in hormone-related therapeutics, especially advances related to the development of tissue-selective oestrogenic compounds. We also review the strategy of combining oestrogen with other compounds to increase their therapeutic efficacy.

Background Fetal alcohol exposure produces multiorgan defects, making it difficult to identify underlying etiological mechanisms. However, recent evidence for ethanol (EtOH) sensitivity of the miRNA miR-9 suggests one mechanism, whereby EtOH broadly influences development. We hypothesized that loss of miR-9 function recapitulates aspects of EtOH teratology. Methods Zebrafish embryos were exposed to EtOH during gastrulation, or injected with anti-miR-9 or nonsense control morpholinos during the 2-cell stage of development and collected between 24 and 72 hours postfertilization (hpf). We also assessed the expression of developmentally important, and known miR-9 targets, FGFR-1, FOXP2, and the nontargeted transcript, MECP2. Methylation at CpG islands of mammalian miR-9 genes was assessed in fetal murine neural stem cells (mNSCs) by methylation-specific PCR, and miRNA processing assessed by qRT-PCR for pre-miR-9 transcripts. Results EtOH treatment and miR-9 knockdown resulted in similar cranial defects including microcephaly. Additionally, EtOH transiently suppressed miR-9, as well as FGFR-1 and FOXP2, and alterations in miR-9 expression were correlated with severity of EtOH-induced teratology. In mNSCs, EtOH increased CpG dinucleotide methylation at the miR-9-2 locus and accumulation of pre-miR-9-3. Conclusions EtOH exerts regulatory control at multiple levels of miR-9 biogenesis. Moreover, early embryonic loss of miR-9 function recapitulated the severe range of teratology associated with developmental EtOH exposure. EtOH also disrupts the relationship between miR-9 and target gene expression, suggesting a nuanced relationship between EtOH and miRNA regulatory networks in the developing embryo. The implications of these data for the expression and function of mature miR-9 warrant further investigation.

Alcohol consumption during pregnancy can be severely damage to the brain development in fetuses. This study investigates the effects of maternal ethanol consumption on brain development in mice embryos. Pregnant mice at gestational day 12.5 were intragastrically gavaged with ethanol (3g/Kg bwt) twice daily for three consecutive days. On gestational day 14.5, fetuses were collected and fixed in 4% paraformaldehyde and imaged using a swept-source optical coherence tomography (SSOCT) system. 3D images of the mice embryo brain were obtained and the volumes of the left and right ventricles of the brain were measured. The average volumes of the left and the right volumes of 5 embryos each alcohol-exposed and control embryos were measured to be 0.35 and 0.15 mm 3 , respectively. The results suggest that the left and right ventricle volumes of brain are much larger in the alcohol-exposed embryos as compared to control embryos indicating alcohol-induced developmental delay.

The developing fetal brain is vulnerable to a variety of environmental agents including maternal ethanol consumption. Preclinical studies on the development and amelioration of fetal teratology would be significantly facilitated by the application of high resolution imaging technologies like optical coherence tomography (OCT) and high-frequency ultrasound (US). This study investigates the ability of these imaging technologies to measure the effects of maternal ethanol exposure on brain development, ex vivo, in fetal mice. Pregnant mice at gestational day 12.5 were administered ethanol (3 g/Kg b.wt.) or water by intragastric gavage, twice daily for three consecutive days. On gestational day 14.5, fetuses were collected and imaged. Three-dimensional images of the mice fetus brains were obtained by OCT and high-resolution US, and the volumes of the left and right ventricles of the brain were measured. Ethanol-exposed fetuses exhibited a statistically significant, 2-fold increase in average left and right ventricular volumes compared with the ventricular volume of control fetuses, with OCT-derived measures of 0.38 and 0.18 mm 3 , respectively, whereas the boundaries of the fetal mouse lateral ventricles were not clearly definable with US imaging. Our results indicate that OCT is a useful technology for assessing ventriculomegaly accompanying alcohol-induced developmental delay. This study clearly demonstrated advantages of using OCT for quantitative assessment of embryonic development compared with US imaging.

Estrogen profoundly affects the organization of the nervous system. Its receptor, a nuclear transcription factor, is widely expressed in the developing forebrain. Earlier work established that forebrain estrogen target neurons coexpress nerve growth factor (NGF) receptors and receptor mRNA. The present study examined the regulation of forebrain estrogen receptors by NGF, using organotypic cultures of the developing cerebral cortex and basal forebrain. NGF significantly increased nuclear estrogen binding in cortical but not basal forebrain explants. Both cortical and basal forebrain explant cultures express the NGF receptor, TrkA. However, our earlier observation that developing cortical neurons, unlike basal forebrain neurons, widely coexpress mRNAs for NGF and its cognate receptors, suggests that in the present study cortical neuronal responses to exogenous NGF may have been primed by autocrine mechanisms. Alterations in nuclear estrogen binding but not estrogen receptor mRNA levels suggests that NGF may regulate cortical estrogen receptors posttranscriptionally.

Fetal alcohol spectrum disorders (FASD) are difficult to diagnose since many heavily exposed infants, at risk for intellectual disability, do not exhibit craniofacial dysmorphology or growth deficits. Consequently, there is a need for biomarkers that predict disability. In both animal models and human studies, alcohol exposure during pregnancy resulted in significant alterations in circulating microRNAs (miRNAs) in maternal blood. In the current study, we asked if changes in plasma miRNAs in alcohol-exposed pregnant mothers, either alone or in conjunction with other clinical variables, could predict infant outcomes. Sixty-eight pregnant women at two perinatal care clinics in western Ukraine were recruited into the study. Detailed health and alcohol consumption histories, and 2nd and 3rd trimester blood samples were obtained. Birth cohort infants were assessed by a geneticist and classified as unexposed (UE), heavily prenatally exposed and affected (HEa) or heavily exposed but apparently unaffected (HEua). MiRNAs were assessed in plasma samples using qRT-PCR arrays. ANOVA models identified 11 miRNAs that were all significantly elevated in maternal plasma from the HEa group relative to HEua and UE groups. In a random forest analysis classification model, a combination of high variance miRNAs, smoking history and socioeconomic status classified membership in HEa and UE groups, with a misclassification rate of 13%. The RFA model also classified 17% of the HEua group as UE-like, whereas 83% were HEa-like, at least at one stage of pregnancy. Collectively our data indicate that maternal plasma miRNAs predict infant outcomes, and may be useful to classify difficult-to-diagnose FASD subpopulations.

Background From studies using a diverse range of model organisms, we now acknowledge that epigenetic changes to chromatin structure provide a plausible link between environmental teratogens and alterations in gene expression leading to disease. Observations from a number of independent laboratories indicate that ethanol (EtOH) has the capacity to act as a powerful epigenetic disruptor and potentially derail the coordinated processes of cellular differentiation. In this study, we sought to examine whether primary neurospheres cultured under conditions maintaining stemness were susceptible to alcohol-induced alterations in the histone code. We focused our studies on trimethylated histone 3 lysine 4 and trimethylated histone 3 lysine 27, as these are 2 of the most prominent posttranslational histone modifications regulating stem cell maintenance and neural differentiation. Methods Primary neurosphere cultures were maintained under conditions promoting the stem cell state and treated with EtOH for 5 days. Control and EtOH-treated cellular extracts were examined using a combination of quantitative RT-PCR and chromatin immunoprecipitation techniques. Results We find that the regulatory regions of genes controlling both neural precursor cell identity and processes of differentiation exhibited significant declines in the enrichment of the chromatin marks examined. Despite these widespread changes in chromatin structure, only a small subset of genes including Dlx2, Fabp7, Nestin, Olig2, and Pax6 displayed EtOH-induced alterations in transcription. Unexpectedly, the majority of chromatin-modifying enzymes examined including members of the Polycomb Repressive Complex displayed minimal changes in expression and localization. Only transcripts encoding Dnmt1, Uhrf1, Ehmt1, Ash2 l, Wdr5, and Kdm1b exhibited significant differences. Conclusions Our results indicate that primary neurospheres maintained as stem cells in vitro are susceptible to alcohol-induced perturbation of the histone code and errors in the epigenetic program. These observations indicate that alterations to chromatin structure may represent a crucial component of alcohol teratogenesis and progress toward a better understanding of the developmental origins of fetal alcohol spectrum disorders.

Stem cells play a vital role in the development, maintenance, and repair of tissues and organs, and hold significant promise for tissue repair following injury and disease, and for the cure of degenerative diseases. However, fulfilling the therapeutic potential of stem cell therapy requires that we understand and effectively manage cell-, tissue-, and even organism-specific programs that guide stem cell maturation. In this chapter, I briefly highlight evidence for a core network of genes that control stem cell renewal and maturation. I then focus on emerging evidence for a novel and evolutionary diverse layer of cell regulation, termed “epigenetics”, that controls the fate of stem cells. I also explore emerging evidence for a vast regulatory network of non-protein-coding RNA molecules (ncRNAs) that participate in stem cell renewal and differentiation, by coordinating DNA replication, controlling gene transcription and nuclear chromatin structure and by controlling the translation of cellular mRNAs. The integrative role of epigenetics and ncRNA networks makes these particularly valuable, albeit complex, tools for stem cell programming.

Recent work has shown that estrogen receptor mRNA and protein co-localize with neurotrophin receptor systems in the developing basal forebrain. In the present study we examined the potential for reciprocal regulation of estrogen and neurotrophin receptor systems by their ligands in a prototypical neurotrophin target, the PC12 cell. using in situ hybridization histochemistry, RT-PCR and a modified nuclear exchange assay, we found both estrogen receptor mRNA and estrogen binding in PC12 cells. Moreover, while estrogen binding was relatively low in naive PC12 cells, long-term exposure to NGF enhanced estrogen binding in these cells by sixfold. Furthermore, concurrent exposure to estrogen and NGF receptor mRNAs deifferentially regulated the expression of the two NGF receptor mRNAs. The expression of trkA mRNA was up-regulated, while p75NGFR mRNA was down-regulated transiently. The present data indicate that NGF may increase neuronal sensitivity to estrogen, and that estrogen, by differentially regulating p75NGFR and trkA mRNA, may alter the ratio fo the two NGF receptors, and, conseuqnetly, neurotrophin responsivity. In view of the widespread co-localization of estrogen and neurotrophin receptor systems in the developing CNS, the reciprocal regulation of these receptor systems by NGF and estrogen may have important implications for processes governing neural maturation and the maintenance of neural funciton. 1994 John Wiley & Sons, Inc.

The annual meeting of the Fetal Alcohol Spectrum Disorders Study Group (FASDSG) was held on June 26, 2010 in San Antonio, TX, as a satellite of the Research Society on Alcoholism meeting. The FASDSG membership includes clinical, basic, and social scientists who meet to discuss recent advances and issues in Fetal Alcohol Spectrum Disorder (FASD) research. The central theme of the meeting was “Glia and Neurons: Teamwork in Pathology and Therapy.” Alcohol disruption of neuron development and alcohol-induced neurodegeneration is central to the pathology and clinical expression of FASD. The active role of glia as perpetrator, victim, or bystander in neurotoxicology and neurodegenerative processes has emerged at the forefront of adult central nervous system (CNS) disorders and therapy. Glia– and neuron–glial interactions hold the potential to elucidate causes and offer treatment of FASD as well. Growing evidence indicates that neurons and glia are direct targets of alcohol, but may also be vulnerable to molecules produced in peripheral systems as a result of alcohol exposure. Diagnostics and therapies can take advantage of these processes and biomarkers, and these may be applicable to CNS pathology in FASD. Two keynote speakers, Howard E. Gendelman, M.D., and Ernest M. Graham, M.D, addressed the role of glia and neuroinflammation in brain development and neurodegeneration. The invited speakers and FASDSG members discussed new paradigms in CNS development and discuss new strategies for understanding and treating neurodegenerative disease. Members of the FASDSG provided updates on new findings through presentation of breaking research in the FASt data sessions. Representatives of national agencies provided updates on programs, activities, and funding priorities. The Henry Rosett Award was presented to R. Louise Floyd, R.N., D.S.N., for her career contributions to the field of fetal alcohol research. The Student and Postdoctoral Fellow Research Merit Award was presented to Shonagh O’Leary-Moore, Ph.D., for her contributions to the field as a young investigator.

We previously showed that middle-aged female rats sustain a larger infarct following experimental stroke as compared to younger female rats, and paradoxically, estrogen treatment to the older group is neurotoxic. Plasma and brain insulin-like growth factor-1 (IGF-1) levels decrease with age. However, IGF-1 infusion following stroke, prevents estrogen neurotoxicity in middle-aged female rats. IGF1 is neuroprotective and well tolerated, but also has potentially undesirable side effects. We hypothesized that microRNAs (miRNAs) that target the IGF-1 signaling family for translation repression could be alternatively suppressed to promote IGF-1-like neuroprotection. Here, we report that two conserved IGF pathway regulatory microRNAs, Let7f and miR1, can be inhibited to mimic and even extend the neuroprotection afforded by IGF-1. Anti-mir1 treatment, as late as 4 hours following ischemia, significantly reduced cortical infarct volume in adult female rats, while anti-Let7 robustly reduced both cortical and striatal infarcts, and preserved sensorimotor function and interhemispheric neural integration. No neuroprotection was observed in animals treated with a brain specific miRNA unrelated to IGF-1 (anti-miR124). Remarkably, anti-Let7f was only effective in intact females but not males or ovariectomized females indicating that the gonadal steroid environment critically modifies miRNA action. Let7f is preferentially expressed in microglia in the ischemic hemisphere and confirmed in ex vivo cultures of microglia obtained from the cortex. While IGF-1 was undetectable in microglia harvested from the non-ischemic hemisphere, IGF-1 was expressed by microglia obtained from the ischemic cortex and was further elevated by anti-Let7f treatment. Collectively these data support a novel miRNA-based therapeutic strategy for neuroprotection following stroke.

Development and survival of neurons in the central nervous system are dependent on the activity of a variety of endogenous neurotrophic agents. Using combined isotopic and nonisotopic in situ hybridization histochemistry, we have found that subsets of neurons within the developing forebrain coexpress the mRNAs for both neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3) and their receptors (p75NGFR, TrkA, and TrkB). The colocalization of mRNA for neurotrophin receptors and their ligands in presumptive neurotrophin target neurons suggests the potential for autocrine and paracrine mechanisms of action during development. Such mechanisms may ensure the onset of differentiation and survival of specific subsets of neurons prior to and following target innervation.

Ethanol exerts complex effects on human physiology and health. Ethanol is not only addictive, but it is also a fetal teratogen, an adult neurotoxin, and an etiologic agent in hepatic and cardiovascular disease, inflammation, bone loss, and fracture susceptibility. A large number of genes and signaling mechanisms have been implicated in ethanol's deleterious effects leading to the suggestion that ethanol is a "dirty drug." An important question is, are there cellular "master-switches" that can explain these pleiotropic effects of ethanol? MicroRNAs (miRNAs) have been recently identified as master regulators of the cellular transcriptome and proteome. miRNAs play an increasingly appreciated and crucial role in shaping the differentiation and function of tissues and organs in both health and disease. This critical review discusses new evidence showing that ethanol-sensitive miRNAs are indeed regulatory master-switches. More specifically, miRNAs control the development of tolerance, a crucial component of ethanol addiction. Other drugs of abuse also target some ethanol-sensitive miRNAs suggesting that common biochemical mechanisms underlie addiction. This review also discusses evidence that miRNAs mediate several ethanol pathologies, including disruption of neural stem cell proliferation and differentiation in the exposed fetus, gut leakiness that contributes to endotoxemia and alcoholic liver disease, and possibly also hepatocellular carcinomas and other gastrointestinal cancers. Finally, this review provides a perspective on emerging investigations into potential roles of miRNAs as mediators of ethanol's effects on inflammation and fracture healing, as well as the potential for miRNAs as diagnostic biomarkers and as targets for therapeutic interventions for alcohol-related disorders.

The distribution of estrogen receptor mRNA expression was studied in the developing rat cerebral cortex by in situ hybridization histochemistry. We used a specific, nonisotopically (digoxigenin) labeled, synthetic oligodeoxyribonucleotide complementary to a 48 base sequence in the region of the estrogen-binding domain of rat uterine estrogen receptor cDNA. During development, estrogen receptor mRNA was observed in all forebrain regions previously reported to bind estrogen, as determined by steroid autoradiography or nuclear binding assay. In the developing cerebral cortex, estrogen receptor mRNA was extensively expressed in the ventricular zone, primitive plexiform layer, and immature cortical plate at least as early as embryonic day 16. During the first 3 postnatal weeks, cortical mRNA expression was increasingly restricted to the upper third of the cerebral cortex and to the neurons of the cortical subplate (layer VIb/VII) and decreased to low levels by postnatal day 28. In the cerebral cortex, the spatial distribution of estrogen receptor mRNA expression overlapped that reported for the encoded protein. The extensive distribution of estrogen receptor mRNA throughout the late prenatal and early postnatal cerebral cortex points to an important role for estrogen in the differentiation and maturation of the cerebral cortex.