Using choleratoxin/antitoxin immunohistochemistry, this study examined the effects of in vitro alcohol exposure on the morphology of cell plasma membranes in mixed fetal rat cortical cultures, and assessed the neuroprotective effects of exogenous monosialoganglioside (GM1). Gangliosides are involved in critical biological functions, including maintenance of membrane integrity. Plasma membranes are directly affected by alcohol exposure through multiple mechanisms. Results indicate that exposure to alcohol altered plasma membrane morphology as assessed by staining for the surface distribution of membrane GM1. Pretreatment with endogenous GM1 ameliorated the alcohol-induced alterations.

Ethanol, a developmental neurotoxin, alters plasma membranes' physicochemical properties affecting embryogenesis, cell migration, differentiation, and synaptogenesis. In a previous study using a model for fetal alcohol effects, GM1 ganglioside treatment was shown to reduce ethanol-induced accumulation of endogenous GM1 and fatty acid ethyl esters in rat fetuses. The present study was initiated to define further the in utero effects of ethanol and the capacity of GM1 treatment to ameliorate such effects. Wistar dams were exposed to ethanol (intragastrically) on gestation day (GD) 7 and GD8 and GD13 and GD14. GM1 ganglioside (10 mg/kg, im) was given 24 hr before ethanol administration. Cortical cultures were derived from GD15 and GD20 fetuses. GM1, which is highly localized on the cellular plasma membrane outer surface of CNS cells, was used as a marker molecule to assess cell integrity. Cholera toxin/antitoxin/fluorescence immunohistochemistry was used to localize GM1. Results indicate that the brief in utero exposure to ethanol affected cell growth and morphology. A marked retardation of cell development and arborization was observed as early as 24 hr after plating. Ethanol-exposed cells evidenced considerably altered GM1 localization. Such alterations likely reflect losses of membrane integrity. These in utero ethanol-induced pathologies are remarkably diminished in cultures derived from ethanol-exposed fetuses of dams treated with GM1.

We present an hypothesis integrating epidemiological, clinical case, and basic biomedical research to explain why only relatively few women who drink alcohol during pregnancy give birth to children with alcohol-related birth defects (ARBDs), in particular, Fetal Alcohol Syndrome (FAS). We argue that specific sociobehavioral risk factors, e.g., low socioeconomic status, are permissive for FAS in that they provide the context for increased vulnerability. We illustrate how these permissive factors are related to biological factors, e.g., decreased antioxidant status, which in conjunction with alcohol, provoke FAS/ARBDs in vulnerable fetuses. We propose an integrative heuristic model hypothesizing that these permissive and provocative factors increase the likelihood of FAS/ARBDs because they potentiate two related mechanisms of alcohol-induced teratogenesis, specifically, maternal/fetal hypoxia and free radical formation.

Maternal alcohol abuse is known to produce retardation in brain maturation and brain functions. Using cultured glial cells as a model system to study these effects of alcohol we found an alcohol antagonizing property for manganese (Mn). Mn was added to the alcohol diet (MnCl2 25 mg/l of 20% v/v ethanol) of pregnant rats. Glial cells were cultured during 4 weeks from cortical brain cells of pups born to these mothers. Several biochemical parameters were examined: protein levels, enzymatic markers of glial cell maturation (enolase and glutamine synthetase), superoxide dismutase a scavenger of free radicals produced during alcohol degradation. The results were compared to appropriate controls. A beneficent effect of Mn was observed for the pups weight which was no more significantly different from the control values. Protein levels, enolase and glutamine synthetase activities were increased mainly during the proliferative period when Mn was added to the alcohol diet compared to the only alcohol treated animals. This Mn effect was not found for superoxide dismutase in cultured glial cells but exists in the total brain of the 2 week-old offspring. In the total 2 and 4 week-old brain the alcohol induced decrease of enolase and glutamine synthetase was also antagonized by the Mn supplementation. Our data suggest that Mn may act as a factor overcoming at least partially some aspects of alcohol induced retardation of nerve cell development.

Fetal alcohol exposure has multiple deleterious effects on brain development, and represents a leading known cause of mental retardation. This review of the effects of alcohol exposure on the developing brain evaluates results from human, animal and in vitro studies, but focuses on key research issues, including possible mechanisms of damage. Factors that affect the risk and severity of fetal alcohol damage also are considered.

The biochemical mechanism of alcohol teratogenicity is not known. We have demonstrated that alcohol administration to pregnant rats during gestation days (GD) 6 and 7 and/or 13 and 14 leads to significant accumulation of ethyl esters of long chain fatty acids (FAEEs) in both maternal and fetal organs. This observation extends the report of Bearer et al. (Pediat Res 31: 492-495, 1992) who detected the presence of metabolites in maternal and fetal organs of pregnant C57B1/6J mice exposed to alcohol on GD 7 and/or GD 14. The ethyl esters of arachidonic, linoleic, oleic, stearic and palmitic acids were major metabolites detected in both maternal and fetal organs. It was also demonstrated that detectable levels of FAEEs remain 14 days (GD 20) after initial exposure to alcohol on GD 7. Ganglioside GM1 treatment 1 and 24 hr prior to alcohol exposure on both GD 7 and/or GD 14 reduced the accumulation of FAEEs. A similar regimen was shown to prevent development of tolerance to alcohol in the adult pups exposed to alcohol in utero in our previous studies. Thus, the ganglioside GM1 may have therapeutic value in reducing neurobehavioral effects of alcohol exposure in utero.

We have investigated the effect of in utero ethanol exposure and ganglioside GM1 pretreatment on the endogenous ganglioside profile of the rat fetal brain. Prenatal ethanol exposure on gestation day (GD) 7 and GD8 and/or GD13 and GD14 leads to a very significant increase in the ganglioside GM1 content in at least 50% of the pup brains when assayed on GD20. This treatment protocol also results in significant decrease in the content of polysialogangliosides GD1a, GT1b, and GQ1b. GM1 treatment of pregnant dams before ethanol administration prevented this alteration in pup brain ganglioside profile. Ganglioside GM1 pretreatment appears to block the cellular membrane changes associated with fetal alcohol effects and thereby minimizes alterations in brain maturation and associated behavioral dysfunction.

The adaptation (tolerance) to chronic EtOH exposure was explained by the development of resistance to the disordering of the membrane phospholipids (PL). This phenomenon may be associated with changes in enzymes such as phospholipase A2 (PLA2) that govern PL metabolism. The data presented here, using the mouse inhalation model, supports and confirms previously reported findings that chronic exposure to EtOH substantially increased PLA2 activity in synaptosomal preparations from rat brain. We have previously reported that pretreatment with ganglioside GM1 reduced the intoxicating effect of EtOH in mice. The present study indicates that GM1 pretreatment both in vivo and in vitro reduced the EtOH-induced activation of PLA2 in synaptosomal preparations. Thus GM1 may exert its neuroprotective effects by influencing deacylation/reacylation of membrane phospholipids.

Alcohol exerts its pharmacological effects in adult brain by altering the physicochemical properties of cellular plasma membranes. Although alcohol does induce changes in membrane lipid composition, studies to relate these alterations to the development of behavioral tolerance to alcohol and the withdrawal effects have been unsuccessful. Actions of alcohol on developing brain are even more complex. Some of the reported effects include inhibition of embryogenesis, cell migration, and differentiation, including synaptogenesis. Gangliosides have neuroprotective action against a variety of neural insults (e.g., mechanical injury, drug toxicity, or hypoxic insult). This review addresses the role and significance of gangliosides in the CNS pathophysiology of alcohol exposure, as well as the effect of changes in endogenous gangliosides on membrane structure and function. We also describe the role of exogenous gangliosides in prevention of alcohol (acute and/or chronic)-induced CNS (prenatal and postnatal) neurotoxicity through their action on cellular plasma membranes. We propose that ganglioside's neuroprotective effects against alcohol neurotoxicity involve protection and restoration of plasma membrane structure (proteins and lipids) and thereby its function (ionic homeostasis, neurotransmitter receptor-mediated signal transduction). Thus gangliosides may have potential therapeutic use in treatment of alcohol-related problems.

The demonstration that ganglioside GM1 pretreatment reduced the ethanol induced neurobehavioral effects in adult pups exposed to ethanol in utero, prompted study to examine whether GM1 crosses the placenta and penetrates fetal tissues. The present results indicate that 3H-galactose labeled GM1 not only passes the placenta but also served as a substrate for the synthesis of polysialogangliosides, and remained in various tissues up to 48 h after maternal (3H)-GM1 administration.