Ethanol exposure during neurogenesis induces persistent effects on neural maturation: evidence from an ex vivo model of fetal cerebral cortical neuroepithelial progenitor maturation

Abstract

Ethanol is a significant neuroteratogen. We previously used fetal cortical-derived neurosphere cultures as an ex vivo model of the second trimester ventricular neuroepithelium, and showed that ethanol directly induced fetal stem and progenitor cell proliferation and maturation without inducing death. However, ethanol is defined as a teratogen because of its capacity to persistently disrupt neural maturation beyond a specific exposure period. We therefore utilized a simplified neuronal maturation paradigm to examine the immediate and persistent changes in neuronal migration following ethanol exposure during the phase of neuroepithelial proliferation. Our data indicate that mRNA transcripts for migration-associated genes RhoA, Paxillin (Pxn), and CDC42 were immediately induced following ethanol exposure, whereas dynein light chain, LC8-type 1 (DYNLL1), and growth-associated protein (Gap)-43 were suppressed. With the exception of Gap43, ethanol did not induce persistent changes in the other mRNAs, suggesting that ethanol had an activational, rather than organizational, impact on migration-associated mRNAs. However, despite this lack of persistent effects on these mRNAs, ethanol exposure during the proliferation period significantly increased subsequent neuronal migration. Moreover, differentiating neurons, pretreated with ethanol during the proliferation phase, exhibited reduced neurite branching and an increased length of primary neurites, indicating a persistent destabilization of neuronal maturation. Collectively, our data indicate that ethanol-exposed proliferating neuroepithelial precursors exhibit subsequent differentiation-associated increases in migratory behavior, independent of mRNA transcript levels. These data help explain the increased incidence of cerebral cortical neuronal heterotopias associated with the fetal alcohol syndrome.

Figure 1

Neurosphere model of the fetal ventricular neuroepithelium and mitogen-withdrawal model of neuronal differentiation. (a–c) In the presence of mitogenic factors, FGF, EGF, and LIF, neuroepithelial cells proliferate to form neurospheres [the proliferation/VZ (ventricular zone) condition]. (a) Phase contrast image of neurospheres derived from gestational day 12.5 mouse cortex. (b–c) Sample neurosphere stained with DAPI (b) to visualize nuclei and immunolabeled for nestin (c) showing that neurospheres cells are immature. (d–f) Partial withdrawal of mitogenic factors, EGF and LIF, and addition of laminin cause neurosphere-derived neuroepithelial cells to become adherent and migrate away from the parental neurosphere (d and e, phase contrast image), assume a bipolar morphology (e) and express immunoreactivity for neurofilament protein (f), the early differentiation or SVZ (subventricular zone) condition. (g–h) Complete withdrawal of mitogenic factors, EGF, FGF, and LIF, results in the appearance of multipolar cells (g) that express immunoreactivity for neurofilament protein (h), the late differentiation or CP (cortical plate) condition. (i) Schematic of the experimental paradigm to examine the acute and persistent effects of ethanol exposure during the neurogenesis period. Scale bars: 50 μm.

Figure 2

Real-time RT-PCR analyses of the expression of DYNLL1/Pin, RhoA, Pxn, and CDC42 mRNA transcripts during the proliferation phase or early differentiation phase in control cultures, or following ethanol exposure for 5 days, at 320 mg/dl during the proliferation period. Data are expressed as mean of the log₁₀ Pfaffl ratio ± SEM based on three independent replicate experiments.

Figure 3

Real-time RT-PCR analyses of the expression of Gap43 and Stmn1 mRNA transcripts during the proliferation phase or early differentiation phase in control neurosphere cultures, or following ethanol exposure for 5 days, at 320 mg/dl during the proliferation period. Data are expressed as mean of the log₁₀ Pfaffl ratio ± SEM based on three independent replicate experiments.

Figure 4

Assessment of neuronal migration following ethanol exposure, at 320 mg/dl for 5 days, during the proliferation period. (a, c) Quantitative graphical representation of shows that ethanol preexposure during the proliferation period significantly induces neuronal migration during both early (a) and late (c) differentiation phases. (b, d) Representative photomicrographs showing increased migration in ethanol-pretreated cultures, compared to control cultures. Control and ethanol-treated neurospheres were cultured in tissue culture inserts containing 8-μm pores (arrow in d). Scale bar: 50 μm.

Figure 5

Assessment of neurite growth following ethanol exposure at 320 mg/dl for 5 days, during the proliferation period. (a) The number of primary neurites was regulated by differentiation state, but not by ethanol preexposure. (b–c) Ethanol preexposure during the proliferation phase did not significantly increase primary neurite length in the early differentiation phase (b), but induced a significant increase in primary neurite length in the late differentiation condition (c). (d) Ethanol preexposure during the proliferation phase slightly, but not statistically significantly, decreased the length of second-order neurites in the late differentiation condition. However, (e) ethanol preexposure decreased the proportion of cells that expressed second-order neurite branches in the late differentiation condition.