Ethanol consumption during early pregnancy alters the disposition of tangentially migrating GABAergic interneurons in the fetal cortex

Abstract

Consumption of alcohol (ethanol) during pregnancy can lead to developmental defects in the offspring, the most devastating being the constellation of symptoms collectively referred to as fetal alcohol syndrome (FAS). In the brain, a hallmark of FAS is abnormal cerebral cortical morphology consistent with insult during corticogenesis. Here, we report that exposure to a relatively low level of ethanol in utero (average maternal and fetal blood alcohol level of 25 mg/dl) promotes premature tangential migration into the cortical anlage of primordial GABAergic interneurons, including those originating in the medial ganglionic eminence (MGE). This ethanol-induced effect was evident in vivo at embryonic day 14.5 (E14.5) in GAD67 knock-in and BAC-Lhx6 embryos, as well as in vitro in isotypic telencephalic slice cocultures obtained from E14.5 embryos exposed to ethanol in utero. Analysis of heterotypic cocultures indicated that both cell-intrinsic and -extrinsic factors contribute to the aberrant migratory profile of MGE-derived cells. In this light, we provide evidence for an interaction between ethanol exposure in utero and the embryonic GABAergic system. Exposure to ethanol in utero elevated the ambient level of GABA and increased the sensitivity to GABA of MGE-derived cells. Our results uncovered for the first time an effect of ethanol consumption during pregnancy on the embryonic development of GABAergic cortical interneurons. We propose that ethanol exerts its effect on the tangential migration of GABAergic interneurons extrinsically by modulating extracellular levels of GABA and intrinsically by altering GABA(A) receptor function.

Figure 1.

The mouse model of low-level chronic ethanol consumption used to examine the effect of in utero ethanol exposure on corticogenesis increases the size of embryos. A, B, Images of control (A1) and 2% ethanol-exposed (A2) embryos and brains (B) at E14.5. C, The body and brain weight were significantly increased with ethanol exposure (Student's t test; *p < 0.05) without altering the brain to body weight ratio (Student's t test; p = 0.96) as measured in the BAC-Lhx6 embryos (control, n = 8; ethanol, n = 9). Data are expressed as mean ± SEM. Asterisks denote a statistical difference compared with control. D, BAL was not significantly different between dams and embryos at time of killing within treatment groups (Student's t test; GAD67, n = 3, p = 0.86; BAC-Lhx6, n = 10, p = 0.44; GFP β-act, n = 7, p = 0.70) or between treatment groups (GAD67, 28.94 ± 1.97 mg/dl; BAC-Lhx6, 25.03 ± 1.59 mg/dl; GFP β-act, 24.95 ± 1.36 mg/dl; ANOVA, p = 0.42). Data are expressed as mean ± SEM. GAD67, GAD67-GFP transgenic mice; GFP β-act, GFP under the control of a β-actin/CMV promoter/enhancer sequence.

Figure 2.

Ethanol consumption during pregnancy increases the number of GABA-immunoreactive cells in the neocortex. A, Representative GABA-immunoreactive E14.5 telencephalic sections obtained from control (n = 10) and ethanol exposed in utero (n = 7) wild-type littermates of BAC-Lhx6 litters. Note that immunostaining for GABA is clearly evident in the cells in the marginal, intermediate, and subventricular zones of the neocortex. Scale bar, 100 μm. B, C, Ethanol exposure increased the density of GABA-immunopositive cells throughout the extent of the neocortex as seen in cortical bins (B) and in layer density (C). Asterisks denote a significant difference from control (Student's t test; p < 0.01).

Figure 3.

A, B, Representative sections from E14.5 GAD67-GFP knock-in control (A; n = 22) and ethanol-treated (B; n = 18) embryos. A2 and B2 are enlargements of the neocortex in A1 and B1, respectively. C, Exposure to ethanol in utero increased the number of GAD67-GFP cells throughout the cortical layers (Student's t test; MZ–CP, p < 0.001; IZ, p = 0.003; SVZ–VZ, p < 0.001). D, However, the ratio between the cortical layers is unaltered (Student's t test; MZ–CP, p = 0.26; IZ, p = 0.12; SVZ–VZ, p = 0.92). Data are expressed as mean density ± SEM. Asterisks denote a significant difference from control (Student's t test, p < 0.005).

Figure 4.

Exposure to ethanol in utero increases cell differentiation. A, B, Representative 20-μm-thick cryosections of E14.5 brains stained with an antibody against BrdU obtained from control (A) and ethanol-exposed (B) wild-type embryos. Scale bar, 100 μm. C, Individual cells were monitored in still images taken every 5 min for 12 h in isotypic slice cocultures in which an MGE explant from a GFP β-actin embryo was transplanted onto the MGE of a wild-type littermate. Arrowheads show an example of migrating MGE-derived cells. D, Cells monitored in slices from in utero ethanol exposed embryos (n = 142; before CSJ, 37.52 ± 1.56 μm/h; after CSJ, 37.65 ± 1.08 μm/hr) migrated faster than those from control slices (n = 288; before CSJ, 16.52 ± 0.57 μm/h; after CSJ, 16.58 ± 0.77 μm/hr) both before and after the CSJ (ethanol, n = 142). Data are expressed as mean ± SEM. Asterisks indicate a significant difference from control (Student's t test; p < 0.005).

Figure 5.

Exposure to ethanol in utero increases the number of MGE-derived cells in the neocortex without altering the ratio in Bac-Lhx6 mice. A–D, Representative cryosections from E14.5 Bac-Lhx6 embryos obtained from control (A1; n = 17) or ethanol-treated (A2; n = 23) embryos. GFP+/MGE cells were counted within the cortical layers (B, C) and within 100-μm-wide cortical bins to reflect the distance from the CSJ (D) of cryosections obtained from control (black bars) and ethanol-treated (gray bars) embryos. Ethanol exposure significantly increased the number of GFP-immunopositive cells throughout the extent of the neocortex (B, D; Student's t test; p < 0.01). However, the ratio of GFP-positive cells was not altered (C; Student's t test; VZ–SVZ, p = 0.89; IZ, p = 0.35; CP–MZ, p = 0.41). Data are expressed as mean ± SEM. Asterisks denote a significant difference from control.

Figure 6.

Exposure to ethanol in utero increases the differentiation of MGE-derived cells in BAC-Lhx6 mice. A, B, Cryosections of control (n = 8) and ethanol-treated embryos (n = 9) immunostained for NeuN (A1, B1) and GFP (A2, B2). A3 and B3 are overlays of control and ethanol-treated sections, respectively. Scale bar, 100 μm. Arrows are examples of double-labeled cells. C, The ratio of GFP-immunopositive cells that were colabeled with NeuN was increased with ethanol treatment (control, 0.28 ± 0.04; ethanol, 0.43 ± 0.03; Student's t test, p < 0.05), whereas the ratio of cell GFP-immunopositive-only cells were decreased with in utero ethanol exposure (control, 0.72 ± 0.04; ethanol, 0.57 ± 0.03; Student's t test, p < 0.05). Data are expressed as mean ± SEM. Asterisks indicate a significant difference from control.

Figure 7.

Both cell-intrinsic and -extrinsic factors contribute to the effect of increased tangential migration of MGE-derived cells seen after exposure to ethanol in utero. A, Table depicting the heterotypic slice cocultures obtained from GFP β-actin embryos used in this experiment. B, Crossing indices of slice cocultures obtained from control, ethanol, and mixed cocultures. Data are expressed as mean ± SEM. Asterisks indicate a significant difference (ANOVA, p < 0.05).

Figure 8.

The GABAergic system contributes extrinsic mechanisms that are altered with ethanol exposure in utero. Crossing index of isotypic slice cocultures obtained from GFP β-actin embryos demonstrates that blocking the GABA_A receptor with bicuculline (Bicu) or sequestering ambient GABA with a GABA-specific antibody blocks the effect of ethanol. Addition of 2 μm GABA, similar to the ambient level of GABA observed in ethanol-exposed embryos increases the crossing index to levels that are indistinguishable from that of ethanol-treated slice cocultures. Data are expressed as mean ± SEM. Asterisks denote a significant difference (***different from control, ethanol + Bicu, ethanol + GABA; **different from control and ethanol + Bicu only; ANOVA and Holm–Sidak Method, p < 0.05). EtOH, Ethanol.

Figure 9.

Prenatal ethanol exposure increases ambient GABA. A–D, Application of 20 μm bicuculline (Bicu) to GFP+/MGE cells recorded from the MGE (A, C) and the IZ (B, D) of control and in utero ethanol-exposed acute BAC-Lhx6 slices, respectively. E, Mean bicuculline-induced outward current from cells recorded from the neocortex (cortex; 14.87 ± 2.02 pA; n = 16) of control and the MGE (n = 22) and neocortex (n = 21) of in utero ethanol-exposed slices (MGE, 31.55 ± 4.5 pA; neocortex, 35.70 ± 2.35 pA; ANOVA, *p < 0.05). F, Example of a GABA concentration–response curve (black circles and regression line) obtained from a GFP+/MGE cell in the neocortex of an in utero ethanol-exposed slice and the bicuculline-induced current (gray circle) used to calculate the concentration of ambient GABA. G, Calculations based on bicuculline-induced outward current recorded from cells in the cortex of control (0.43 ± 0.11 μm) and the MGE (2.47 ± 0.24 μm) and neocortex (2.73 ± 0.57 μm) of in utero ethanol-exposed slices reveal a significant difference in ambient GABA concentration (ANOVA, *p < 0.01).

Figure 10.

GFP+/MGE cells recorded in slices obtained from BAC-Lhx6 in utero ethanol-exposed embryos are more sensitive to GABA than those recorded within control slices. A–D, Responses to application of 100 μm GABA to GFP+/MGE cells recorded from the MGE (A, B) and neocortex (C, D) of control and in utero ethanol exposed acute BAC-Lhx6 slices, respectively. E, Average response of GFP+/MGE cells recorded within the MGE and the IZ of the neocortex in slices from control and in utero ethanol-exposed E14.5 embryos (control MGE, 25.25 ± 4.16 pA; control cortex, 148.85 ± 19.66 pA; ethanol MGE, 156.20 ± 10.72 pA; ethanol cortex, 138.06 ± 12.64 pA; ANOVA, p < 0.001). Data are expressed as mean amplitude ± SEM. Asterisks denote a significant difference from control MGE (p < 0.001). F, GABA concentration–response curves for GFP+/MGE cells recorded in the MGE and IZ of the neocortex of acute slices obtained from control and in utero ethanol-treated E14.5 BAC-Lhx6 embryos normalized to the max amplitude of cells recorded in the MGE of control slices.