Glia and fetal alcohol syndrome.

Glial cells and their interactions with neurons play vital roles during the ontogeny of the nervous system and in the adult brain. Alcohol intake during pregnancy can cause mental retardation and neurobehavioral disorders as well as fetal alcohol syndrome (FAS). Clinical and experimental evidence indicate that in utero alcohol exposure induces structural and functional abnormalities in gliogenesis and in glial-neuronal interactions, suggesting a potential role of glial cells on ethanol-induced developmental brain abnormalities. In vivo studies have shown ethanol-associated alterations in the migration of neurons and radial glial as well as in astrogliogenesis and myelin development. In astrocytes in primary culture, ethanol has been found to (1) impair cell growth and differentiation, (2) decrease the levels of glialfibrillary acidic protein or GFAP (an astrocyte marker) and its gene expression and (3) interfere with the stimulatory effect of trophic factors affecting their release and receptor expression. Evidence also suggests that ethanol affects intracellular protein trafficking, which may mediate some effects of ethanol on astroglial cells. These findings suggest that glial cells are target of ethanol toxicity during brain development and may underlie the neurodevelopmental abnormalities observed after in utero alcohol exposure and in FAS.     

Mechanisms involved in central nervous system dysfunctions induced by prenatal ethanol exposure

Clinical and experimental evidence has demonstrated that ethanol is a teratogen, and that its consumption during pregnancy induces harmful effects on the developing foetus that leads to foetal alcohol syndrome (FAS). Central nervous system dysfunctions are the most severe and permanent consequence of maternal alcohol intake and can occur in absence of gross morphological defects associated with FAS. Mental retardation and long-term cognitive and behavioural deficits are some of the problems commonly found in children of women who were moderate or heavy drinkers during pregnancy. Experimental evidence demonstrates that alcohol interferes with many molecular, neurochemical and cellular events occurring during the normal development of the brain. Some brain areas are more affected than others and, even within a given region, some cell populations are more vulnerable than others. The neocortex, hippocampus and cerebellum are especially susceptible to alcohol and have been associated with the behavioural deficits. For example, alcohol exposure during the development of neocortex increases natural apoptosis and induces cell necrosis. These effects may be associated with ethanol-induced alterations in both neurotrophic support, and the expression of cell adhesion molecules, which may affect cell-cell interactions and cell survival. Experimental evidence also shows that alcohol disrupts radial glial and astroglial development which may lead to alterations in cell migration and neuronal survival and differentiation. Impairment of several neurotransmitter systems and/or their receptors, as well as changes in the endocrine environment during brain development, are also important factors involved in the neurodevelopmental liabilities observed after in utero alcohol exposure.     

How does alcohol impair neuronal migration?

Maternal alcohol consumption during pregnancy can cause serious birth defects, of which fetal alcohol syndrome (FAS) is the most devastating. Recognized by characteristic craniofacial abnormalities and growth deficiency, this condition produces severe alcohol-induced damage in the developing brain. FAS children experience ataxia; deficits in intellectual functioning; and difficulties in learning, memory, problem solving, and attention. Multiple aspects of central nervous system development can be affected by alcohol exposure, but the most striking abnormalities are neuronal and glial migration. Little is known about cellular mechanisms by which alcohol affects the migration of immature neurons. Recently, it has been found that Ca(2+) signaling and cyclic nucleotide signaling are the central targets of the action of alcohol in neuronal cell migration. Most importantly, the aberrant migration of immature neurons caused by alcohol exposure is significantly ameliorated by controlling the activity of these second-messenger pathways. In this Mini-Review, we first describe how alcohol exposure impairs the migration of cerebellar granule cells and then discuss the signaling mechanisms involved.     

Fetal alcohol spectrum disorders: a practical clinical approach to diagnosis

In utero exposure to alcohol can have numerous adverse effects on a developing fetus. These effects represent a spectrum of structural anomalies and neurocognitive and behavioral disabilities that have recently been termed fetal alcohol spectrum disorders (FASD). Children at the most severe end of this spectrum and displaying the complete phenotype of characteristic facial anomalies, growth retardation and developmental abnormalities of the central nervous system are defined as having fetal alcohol syndrome (FAS). While FAS is the most readily clinically recognized form of FASD, other categories within the continuum of adverse effects due to prenatal alcohol exposure are becoming better defined. These include partial fetal alcohol syndrome (PFAS), alcohol-related birth defects (ARBD) and alcohol-related neurodevelopmental disorder (ARND). As more is learned regarding the exact manifestations of alcohol on brain development, these classifications may be expanded and/or refined. Because FASD represents a major public health concern, early recognition of at-risk children is important for initiating interventional strategies. Thus, the purpose of this report is to educate practicing physicians about the recognizable phenotypes of FASD in order to accurately identify these children and implement the most appropriate management plans.     

The protective effect of neuronal nitric oxide synthase (nNOS) against alcohol toxicity depends upon the NO-cGMP-PKG pathway and NF-kappaB

Fetal alcohol syndrome (FAS) stems from maternal alcohol abuse during pregnancy and is an important cause of mental retardation and hyperactivity in children. In the developing brain, alcohol can kill neurons, leading to microencephaly. However, due to their genetic makeup, some individuals are less vulnerable than others to alcohol's neurotoxic effects. Animal studies have demonstrated that one particular gene, neuronal nitric oxide synthase (nNOS), protects developing neurons in vivo against alcohol-induced death. We utilized pharmacologic techniques to demonstrate that nNOS protects neurons against alcohol toxicity by activating the NO-cGMP-PKG signaling pathway. Cerebellar granule cell cultures derived from mice carrying a null mutation for nNOS (nNOS-/- mice) were substantially more vulnerable than cultures from wild-type mice to alcohol-induced cell death. However, activation of the pathway at sites downstream of nNOS protected the cultures against alcohol toxicity. Conversely, blockade of the pathway rendered wild-type cultures vulnerable to alcohol-induced death. We further identified NF-kappaB as the downstream effector through which nNOS and the NO-cGMP-PKG pathway signal their neuroprotective effects. Tumor necrosis factor-alpha (TNF-alpha), which activates NF-kappaB, ameliorated alcohol-induced cell death in nNOS-/- and wild-type cultures, while an NF-kappaB inhibitor (NFi) blocked the protective effects of TNF-alpha and worsened alcohol-induced cell death. Furthermore, NFi blocked the protective effects of NO-cGMP-PKG pathway activators, demonstrating that NF-kappaB is downstream of the NO-cGMP-PKG pathway. As wild-type neurons matured in culture, they became resistant to alcohol toxicity. However, this maturation-dependent alcohol resistance did not occur in nNOS-/- mice and could be reversed in wild-type mice with NFi, demonstrating that nitric oxide and NF-kappaB are crucial for the development of alcohol resistance with age. Thus, nNOS protects developing neurons against alcohol toxicity by activating the NO-cGMP-PKG-NF-kappaB pathway and is crucial for the acquisition of maturation-dependent alcohol resistance.     

Developmental neurotoxicity of toluene: in vivo and in vitro effects on astroglial cells

Toluene, an inexpensive and available industrial solvent, has become increasingly popular as a drug of abuse. Inhaling toluene leads to a feeling of euphoria and several reports have shown that children born to women who had abused toluene during pregnancy present a syndrome (toluene embryopathy or fetal solvent syndrome) that is characterized by CNS effects (e.g. microencephaly), growth retardation and facial dysmorphologies. The characteristics of the fetal solvent syndrome are very similar to those observed in the fetal alcohol syndrome. As exposure of rats to ethanol during the brain growth spurt has been shown to cause microencephaly and to affect glial cell proliferation and maturation, the present study examines the effects of toluene administration (250, 500 and 750 mg/kg) in neonatal rats from postnatal day 4 to 10. This treatment resulted in a significant decrease in both brain and body weights, and in a significant reduction of levels of glial fibrillary acidic protein, but not of neuron-specific enolase in rat brain. In vitro experiments demonstrate that pharmacologically relevant concentrations of toluene (250-1,000 microM) significantly inhibit proliferation of rat cortical astrocytes without causing overt cytotoxicity. These results indicate that toluene does not cause selective microencephaly; however, it affects brain weight, and appears to target developing astrocytes, possibly by inhibiting their proliferation.     

The study of brain function impairment in fetal alcohol syndrome: Some fruitful directions for research

Fetal alcohol syndrome (FAS) is the most prevalent known preventable health hazard to the human fetus by a noxious agent. It is associated with impairments of the central nervous system that are expressed in the forms of mental retardation of varying severity, learning disabilities, attentional deficits and an increased vulnerability to stress. Results of psychophysiological studies of the effects of ethanol on the central nervous system are reviewed, with the aim of exploring how conclusions derived from them can serve as testable hypotheses in FAS research. The experimental methods used in such studies are examined for their applicability to FAS research. It is concluded that FAS research effort will benefit from the inclusion of psychophysiological studies.     

Consequences of prenatal toxin exposure for mental health in children and adolescents

Drug use during pregnancy is common and the developing foetus may be exposed to a range of environmental toxins that have long-term consequences for neurodevelopment. We conducted a systematic review of the literature to explore the results of longitudinal cohort studies that have examined this question. Out of 2,977 abstracts identified, 7 previous systematic reviews and 95 original articles met further selection criteria. These mostly addressed the neurodevelopmental effects of exposure to lead, polychlorinated biphenyls, mercury, cocaine, alcohol, marijuana, cigarettes and antidepressants. Radiation, opiates, steroids, amphetamines and caffeine have received much less attention. Findings are difficult to interpret because risk factors tend to cluster together and interact. However, some findings are consistent. Lead and PCB’s have a general effect on brain development, whilst marijuana and alcohol appear to have long-term effects specifically on attentional skills. The effects of alcohol increase with maternal age and binge drinking is more important than average intake. The effects of cocaine diminish with age and are largely mediated through psychosocial factors, whilst the relation between smoking and later delinquency is largely mediated by genetically inherited factors. Exposure to toxins during pregnancy may constitute an important but relatively unacknowledged cause of child psychiatric morbidity. Electronic Supplementary Material Supplementary Table 1 for this article is available at (doi:) and is accessible for authorised users only     

Effects of prenatal alcohol exposure on the hippocampus: spatial behavior, electrophysiology, and neuroanatomy

Prenatal exposure to alcohol can result in fetal alcohol syndrome (FAS), characterized by growth retardation, facial dysmorphologies, and a host of neurobehavioral impairments. Neurobehavioral effects in FAS, and in alcohol-related neurodevelopmental disorder, include poor learning and memory, attentional deficits, and motor dysfunction. Many of these behavioral deficits can be modeled in rodents. This paper reviews the literature suggesting that many fetal alcohol effects result, at least in part, from teratogenic effects of alcohol on the hippocampus. Neurobehavioral studies show that animals exposed prenatally to alcohol are impaired in many of the same spatial learning and memory tasks sensitive to hippocampal damage, including T-mazes, the Morris water maze, and the radial arm maze. Direct evidence for hippocampal involvement is provided by neuroanatomical studies of the hippocampus documenting reduced numbers of neurons, lower dendritic spine density on pyramidal neurons, and decreased morphological plasticity after environmental enrichment in rats exposed prenatally to alcohol. Electrophysiological studies also demonstrate changes in synaptic activity in in vitro hippocampal brain slices isolated from prenatal alcohol-exposed animals. Considered together, these observations demonstrate that prenatal exposure to alcohol can result in abnormal hippocampal development and function. Such studies provide a better understanding of neurological deficits associated with FAS in humans, and may also contribute to the development of strategies to ameliorate the effects of prenatal alcohol exposure on behavior.     

Altered organization of cortical interneurons in rats exposed to ethanol during neonatal life

The fetal alcohol syndrome (FAS) is a known cause of mental retardation in humans. Studies based on experimental models of FAS have demonstrated deep alterations of the cerebral cortex. Here, the anatomical organization of cortical interneurons immunoreactive for different calcium binding proteins has been studied in adult rats exposed to alcohol inhalation during the first week of postnatal life. The main finding is represented by an increase of calretinin neurons in ethanol-treated animals compared to controls and by a corresponding decrease of calbindin neurons. The radial distribution of these neurons was also modified in ethanol-treated cases. These changes were evident both in the primary motor and somatosensory area. No significant differences were found in the number and distribution of parvalbumin interneurons. The functional implications of these data and their significance for FAS are discussed.     

Nicotinamide Inhibits Ethanol-Induced Caspase-3 and PARP-1 Over-activation and Subsequent Neurodegeneration in the Developing Mouse Cerebellum

Fetal alcohol spectrum disorder (FASD) is the principal preventable cause of mental retardation in the western countries resulting from alcohol exposure during pregnancy. Ethanol-induced massive neuronal cell death occurs mainly in immature neurons during the brain growth spurt period. The cerebellum is one of the brain areas that are most sensitive to ethanol neurotoxicity. Currently, there is no effective treatment that targets the causes of these disorders and efficient treatments to counteract or reverse FASD are desirable. In this study, we investigated the effects of nicotinamide on ethanol-induced neuronal cell death in the developing cerebellum. Subcutaneous administration of ethanol in postnatal 4-day-old mice induced an over-activation of caspase-3 and PARP-1 followed by a massive neurodegeneration in the developing cerebellum. Interestingly, treatment with nicotinamide, immediately or 2 h after ethanol exposure, diminished caspase-3 and PARP-1 over-activation and reduced ethanol-induced neurodegeneration. Conversely, treatment with 3-aminobenzadine, a specific PARP-1 inhibitor, was able to completely block PARP-1 activation, but not caspase-3 activation or ethanol-induced neurodegeneration in the developing cerebellum. Our results showed that nicotinamide reduces ethanol-induced neuronal cell death and inhibits both caspase-3 and PARP-1 alcohol-induced activation in the developing cerebellum, suggesting that nicotinamide might be a promising and safe neuroprotective agent for treating FASD and other neurodegenerative disorders in the developing brain that shares similar cell death pathways.     

Mechanisms of Ethanol-Induced Death of Cerebellar Granule Cells

Maternal ethanol exposure during pregnancy may cause fetal alcohol spectrum disorders (FASD). FASD is the leading cause of mental retardation. The most deleterious effect of fetal alcohol exposure is inducing neuroapoptosis in the developing brain. Ethanol-induced loss of neurons in the central nervous system underlies many of the behavioral deficits observed in FASD. The cerebellum is one of the brain areas that are most susceptible to ethanol during development. Ethanol exposure causes a loss of both cerebellar Purkinje cells and granule cells. This review focuses on the toxic effect of ethanol on cerebellar granule cells (CGC) and the underlying mechanisms. Both in vitro and in vivo studies indicate that ethanol induces apoptotic death of CGC. The vulnerability of CGC to ethanol-induced death diminishes over time as neurons mature. Several mechanisms for ethanol-induced apoptosis of CGC have been suggested. These include inhibition of N-methyl-d-aspartate receptors, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, disturbance of potassium channel currents, thiamine deficiency, and disruption of translational regulation. Cultures of CGC provide an excellent system to investigate cellular/molecular mechanisms of ethanol-induced neurodegeneration and to evaluate interventional strategies. This review will also discuss the approaches leading to neuroprotection against ethanol-induced neuroapoptosis.     

Difference in susceptibility to teratogenic effects of alcohol in discordant twins exposed to alcohol during the second half of gestation

A pair of discordant twins exposed to heavy maternal alcohol consumption only during the second half of pregnancy is reported. Apparently, differences in susceptibility to the dysmorphogenic influence of ethanol caused 1 twin to be more severely affected than the other. One twin had prenatal growth retardation, neonatal withdrawal symptoms, delay in both motor and cognitive function during the first year of life, slowing of background activity on electroencephalography, and cortical and central brain atrophy on computed tomography. Catch-up growth occurred during the postnatal period for the affected twin. The other twin was normal at the end of the follow-up at age 17 months. It seems that exposure to alcohol during the second half of pregnancy greatly increases the risk for brain damage but not lasting postnatal growth retardation. Minor abnormalities can also be caused during the second half of pregnancy. Genetic factors may have been important in determining the differences in fetal susceptibility to alcohol exposure.     

Voxel-based morphometric analyses of the brain in children and adolescents prenatally exposed to alcohol

Children of mothers who abuse alcohol during pregnancy can suffer varying degrees of neurological abnormality, cognitive impairment, and behavioral problems, and in the worst case, are diagnosed with fetal alcohol syndrome (FAS). The purpose of the present study was to localize brain abnormalities in a group of children and adolescents prenatally exposed to alcohol using high resolution, 3D structural MRI data and whole-brain voxel-based morphometry (VBM). Data were collected for 21 children and adolescents with histories of prenatal alcohol exposure (ALC) and 21 normally developing individuals. Statistical parametric maps revealed abnormalities most prominent in the left hemisphere perisylvian cortices of the temporal and parietal lobes where the ALC patients tended to have too much gray matter and not enough white matter. These results provide further support for dysmorphology in temporo-parietal cortices above and beyond the overall microcephaly that results from severe prenatal alcohol exposure.     

The importance of alcohol misuse, malnutrition and genetic susceptibility on brain growth and plasticity

The "dyad: alcoholic mother and foetus" is a very complex entity in which several elements such as genes, metabolism, diet, drugs and social habits play a role at different stages in the development of the fetal brain damage. The literature on the effects of alcohol consumption on the developing brain is extensive but very few evidences have been reported regarding the combined neurotoxic effects of poor nutrition and alcohol consumption. The consequences of ethanol intake alone or combined with poor maternal nutrition appear to be severe and life-long. Alcohol exerts its neurotoxic effects on the developing brain directly by acting on fetal brain tissues, and indirectly either by interfering with placental physiology or by impairing the mother's physiology. Alcohol misuse in pregnancy is also frequently associated with other conditions that can potentially increase the brain damage such as poor nutrition and smoking. This article reviews the effects of poor nutrition and alcohol misuse during pregnancy on the development of the fetal brain and discusses the cumulative effects of these two environmental factors and their interaction with maternal and fetal genetic make-ups.     

Glutamate and GABA receptor dysfunction in the fetal alcohol syndrome

The brain damaging effects of ethanol, as the most disabling component of the fetal alcohol syndrome (FAS), have been recognized for three decades, but the mechanism underlying these effects has remained elusive. Recently, we discovered that ethanol triggers widespread apoptotic neurodegeneration throughout the developing brain when administered to infant rodents during the period of synaptogenesis, also known as the brain growth spurt period. These findings provide a more likely explanation than has heretofore been available for the reduced brain mass and lifelong neurobehavioral disturbances associated with the human FAS. We propose that a dual mechanism—blockade of NMDA glutamate receptors and hyperactivation of GABA_A receptors—mediates ethanol's apoptogenic action, based on established evidence that ethanol has both NMDA antagonist and GABAmimetic properties, and our recent finding that other drugs with either NMDA antagonist or GABAmimetic properties robustly trigger apoptotic neurodegeneration in the developing brain. The brain growth spurt occurs in different species at different times relative to birth. In rats and mice it is a postnatal event, but in humans it extends from the sixth month of gestation to several years after birth. Thus, there is a period in pre and postnatal human development, lasting for several years, during which immature CNS neurons are prone to commit suicide if exposed to intoxicating concentrations of drugs with NMDA antagonist or GABAmimetic properties. These findings are important, not only because of their relevance to the FAS, but because there are many agents in the human environment, other than ethanol, that have NMDA antagonist or GABAmimetic properties. Such agents include drugs that may be abused by pregnant mothers [ethanol, phencyclidine (angel dust), ketamine (Special K), nitrous oxide (laughing gas), barbiturates, benzodiazepines], and many medicinals used in obstetric and pediatric neurology (anticonvulsants), and anesthesiology (all general anesthetics are either NMDA antagonists or GABAmimetics).     

Inhibition of muscarinic receptor-induced proliferation of astroglial cells by ethanol: mechanisms and implications for the fetal alcohol syndrome

In utero exposure to ethanol is deleterious to fetal brain development. Children born with the fetal alcohol syndrome (FAS) display a number of abnormalities, the most significant of which are central nervous system (CNS) dysfunctions, such as microencephaly and mental retardation. An interaction of ethanol with glial cells, particularly astrocytes, has been suggested to contribute to the developmental neurotoxicity of this alcohol. At low concentrations (10-100 mM) ethanol inhibits the proliferation of astroglial cells in vitro, particularly when stimulated by acetycholine through muscarinic M3 receptors. Of the several signal transduction pathways activated by these receptors in astrocytes or astrocytoma cells, which are involved in mitogenic signaling, only some (e.g. protein kinase C (PKC) zeta, p70S6 kinase) appear to be targeted by ethanol at the same low concentrations which effectively inhibit proliferation. Inhibition of astroglial proliferation by ethanol may contribute to the microencephaly seen in FAS.     

Metabolic and genetic factors contributing to alcohol induced effects and fetal alcohol syndrome

Alcohol-related damages on newborns and infants include a wide variety of complications from facial anomalies to neurodevelopmental delay, known as fetal alcohol syndrome (FAS). However, only less than 10% of women drinking alcohol during pregnancy have children with FAS. Understanding the risk factors increasing the probability for newborn exposed in utero to alcohol to develop FAS is therefore a key issue. The involvement of genetics as a one risk factor in FAS has been suggested by animal models and by molecular epidemiological studies on different populations, bearing allelic variants for those enzymes, such as ADH e CYP2E1, involved in ethanol metabolism. Indeed, one of the major factors determining the peak blood alcohol exposure to the fetus is the metabolic activity of the mother, in addition to placental and fetal metabolism, explaining, at least partially, the risk of FAS. The different rates of ethanol metabolism may be the result of genetic polymorphisms, the most relevant of which have been described in the paper.     

Single alcohol exposure in early life damages hippocampal stem/progenitor cells and reduces adult neurogenesis

Alcohol exposure during pregnancy may cause fetal alcohol syndrome (FAS), characterized by impaired cognitive functions. Neurogenesis occurs in the adult hippocampus and is functionally associated with learning, memory, and mood disorders. However, whether early postnatal exposure to alcohol impairs neurogenesis and through which mechanisms it occurs is poorly understood. Here, we report that a single episode of alcohol exposure in postnatal day 7 (P7) decreases neurogenesis in the adult hippocampus. Furthermore, we demonstrate a co-localization of glial fibrillar acidic protein, nestin, and vimentin with activated caspase-3 12 h after ethanol treatment. Finally, we show that the number of primary neurospheres derived from the hippocampi of alcohol-exposed mice is reduced compared to controls. These findings suggest that alcohol exposure in postnatal mice reduces the pool of neural stem/progenitor cells in the DG, and subsequently results in a decrease of adult neurogenesis. This may explain certain aspects of impaired hippocampal functions in FAS.