Abstract
BackgroundDeveloping brain is a major target for alcohol’s actions and neurological/functional abnormalities include microencephaly, reduced frontal cortex, mental retardation and attention-deficits. Previous studies have shown that ethanol altered the lateral ventricular neuroepithelial cell proliferation. However, the effect of ethanol on subventricular basal progenitors which generate majority of the cortical layers is not known.MethodsWe utilized spontaneously immortalized rat brain neuroblasts obtained from cultures of 18-day-old fetal rat cerebral cortices using in vitro ethanol exposures and an in utero binge model. In the in vitro acute model, cells were exposed to 86 mM ethanol for 8, 12 and 24 h. The second in vitro model comprised of chronic intermittent ethanol (CIE) exposure which consisted of 14 h of ethanol treatment followed by 10 h of withdrawal with three repetitions.ResultsE18 neuroblasts expressing Tbr2 representing immature basal progenitors displayed significant reduction of proliferation in response to ethanol in both the models. The decreased proliferation was accompanied by absence of apoptosis or autophagy as illustrated by FACS analysis and expression of apoptotic and autophagic markers. The BrdU incorporation assay indicated that ethanol enhanced the accumulation of cells at G1 with reduced cell number in S phase. In addition, the ethanol-inhibited basal neuroblasts proliferation was connected to decrease in cyclin D1 and Rb phosphorylation indicating cell cycle arrest. Further, in utero ethanol exposure in pregnant rats during E15-E18 significantly decreased Tbr2 and cyclin D1 positive cell number in cerebral cortex of embryos as assessed by cell sorting analysis by flow cytometry.ConclusionsAltogether, the current findings demonstrate that ethanol impacts the expansion of basal progenitors by inducing cytostasis that might explain the anomalies of cortico-cerebral development associated with fetal alcohol syndrome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-016-0225-8) contains supplementary material, which is available to authorized users.
Highlights
Developing brain is a major target for alcohol’s actions and neurological/functional abnormalities include microencephaly, reduced frontal cortex, mental retardation and attention-deficits
embryonic day-18 (E18) neuroblasts represent immature basal progenitor which on differentiation express neuronal markers To ensure that the cells used were of neuroblastic phenotype and exhibit respective characteristics, early passage of cells used throughout the study along with performing an initial characterization for various immature and mature neuronal markers
We performed immunoblotting for T-box brain protein 2 (Tbr2)/EOMES, which is a marker for subventricular basal progenitors of neocortex
Summary
Developing brain is a major target for alcohol’s actions and neurological/functional abnormalities include microencephaly, reduced frontal cortex, mental retardation and attention-deficits. Intrauterine ethanol exposure damages developing brain cells (such as neural crest, neural progenitors, radial glia and newly born neurons) causing structural brain malformations leading to several functional, behavioral and cognitive impairments including but not limited to poor memory, hyperactivity, attention deficits, poor judgment and reasoning, learning problems and impulsivity [5,6,7]. Alcohol exerts its teratogenic effects on various parts of the developing brain including cerebral cortex, cerebellum, basal ganglia, corpus callosum and hippocampus. Studies have shown that prenatal ethanol exposure during the sensitive period of cortex development decreases the genesis of cortical neurons as well as the demise of newly
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