Binge-like Prenatal Ethanol Exposure Causes Impaired Cellular Differentiation in the Embryonic Forebrain and Synaptic and Behavioral Defects in Adult Mice

Abstract

An embryo's in-utero exposure to ethanol due to a mother's alcohol drinking results in a range of deficits in the child that are collectively termed fetal alcohol spectrum disorders (FASDs). Prenatal ethanol exposure is one of the leading causes of preventable intellectual disability. Its neurobehavioral underpinnings warrant systematic research. We investigated the immediate effects on embryos of acute prenatal ethanol exposure during gestational days (GDs) and the influence of such exposure on persistent neurobehavioral deficits in adult offspring. We administered pregnant C57BL/6J mice with ethanol (1.75 g/kg) (GDE) or saline (GDS) intraperitoneally (i.p.) at 0 h and again at 2 h intervals on GD 8 and GD 12. Subsequently, we assessed apoptosis, differentiation, and signaling events in embryo forebrains (E13.5; GD13.5). Long-lasting effects of GDE were evaluated via a behavioral test battery. We also determined the long-term potentiation and synaptic plasticity-related protein expression in adult hippocampal tissue. GDE caused apoptosis, inhibited differentiation, and reduced pERK and pCREB signaling and the expression of transcription factors Pax6 and Lhx2. GDE caused persistent spatial and social investigation memory deficits compared with saline controls, regardless of sex. Interestingly, GDE adult mice exhibited enhanced repetitive and anxiety-like behavior, irrespective of sex. GDE reduced synaptic plasticity-related protein expression and caused hippocampal synaptic plasticity (LTP and LTD) deficits in adult offspring. These findings demonstrate that binge-like ethanol exposure at the GD8 and GD12 developmental stages causes defects in pERK-pCREB signaling and reduces the expression of Pax6 and Lhx2, leading to impaired cellular differentiation during the embryonic stage. In the adult stage, binge-like ethanol exposure caused persistent synaptic and behavioral abnormalities in adult mice. Furthermore, the findings suggest that combining ethanol exposure at two sensitive stages (GD8 and GD12) causes deficits in synaptic plasticity-associated proteins (Arc, Egr1, Fgf1, GluR1, and GluN1), leading to persistent FASD-like neurobehavioral deficits in mice.

Keywords: FASD; alcohol; brain; cognition; development; disabilities; electrophysiology; gestation; obsessive-compulsive disorder; psychiatric disorders; synaptic plasticity.

Figure 1

GDE enhances apoptosis and reduces proliferation in GD13.5 embryo forebrain tissues. Experimental design indicates developmental age, timing of ethanol exposure, and various analyses at early (GD13.5) and adult stages (a). Pregnant mice were exposed to 1.75 g/kg ethanol or saline i.p. at 0 h and again at 2 h on GD8 and GD12. Cleaved caspase-3 (CC3), a nucleoprotein expressed in proliferative cells (Ki67); (b) phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated cAMP-response element binding protein (pCREB), Paired Box 6 (Pax6), and LIM homeobox 2 (Lhx2); (c) levels were evaluated in forebrain tissues obtained from GD13.5 embryos by Western blot analysis. The protein samples were equally loaded, confirmed with Ponceau S staining, and normalized to β-actin or total proteins. Error bars, SEM (* p < 0.05 vs. GDS group; n = 8 embryos/group) (one-way ANOVA).

Figure 2

GDE-caused spatial memory and spatial recognition memory abnormalities in adult mice. SM was evaluated with a Y-maze in adult male and female mice exposed to GDS or GDE. The discrimination ratios (preference for the novel arm over the familiar other arm [novel/(novel + other)] for arm entries (a) and dwell time (time spent in each arm) (b) were determined for GDS and GDE mice 24 h after the first encounter with the partially opened maze. The percentages of mice choosing the novel arm as the first choice (c) are shown for GDS and GDE mice 24 h after the first encounter with the partially opened maze. The percentages of time spent in social investigation are shown (d) for the GDS and GDE groups 24 h after the first encounter by the same juvenile mice. Error bars, SEM (* p < 0.05 vs. GDS male group, # p < 0.05 vs. GDS female group; n = 8 mice/group/behavior).

Figure 3

GDE-caused repetitive behavioral abnormalities in adult mice. The percentage of nestlets shredded was calculated by determining the weight difference divided by the starting weight in adult male and female mice exposed to GDS or GDE (a). The number of marbles buried (>50% marble enclosed by bedding) was recorded in adult male and female mice exposed to GDS or GDE (b). Error bars, SEM (* p < 0.05 vs. GDS male group, # p < 0.05 vs. GDS female group; n = 8 mice/group/behavior).

Figure 4

GDE-enhanced anxiety-like behaviors in adult male and female mice. Anxiety-like behavior in the elevated plus maze was calculated by determining the percentage of open arm entries (a), the percentage of open arm time (b), and closed arm entries (c) for adult males and females. Error bars, SEM (* p < 0.05 vs. GDS male group, # p < 0.05 vs. GDS female group; n = 8 mice/group).

Figure 5

GDE-caused LTP and LTD impairments in adult mice. Input/output relationship plots of HP slices from GDS and GDE mice (a). The average field excitatory postsynaptic potentials (fEPSP) slope at various time points obtained from GDS and GDE adult male mice (b). For each slice, the fEPSP slopes were normalized against the average slope over the 10 min recording period before LTP induction. The arrows show the time of theta burst stimulation (TBS) (4 pulses at 100 Hz; the bursts repeated at 5 Hz, and each tetanus included three different 10-burst trains separated by 15 s). The bar graph indicates the average fEPSP slopes at multiple time points after TBS for the GDS and GDE groups (c). The average fEPSP slope at various time points obtained from GDS and GDE adult male mice after LTD induced by low-frequency stimulation (LFS) (d). The fEPSP slopes were normalized to the average value 10 min before LFS stimulation. A bar graph indicates a combined plot of the averages of fEPSP slopes at 75 min and shows the absence of LTD induced by LFS in GDE mice compared with GDS mice (e). Error bars, SEM (* p < 0.05 vs. GDS group; n = 5 mice/group; 10 slices/group).

Figure 6

Decreased pCREB, Egr1, Fgf1, Arc (a), GluR1, and GluN1 (b) protein levels in adult mouse HP tissues in response to GDE. HP nuclear or membrane fractions were subjected to Western blot analysis. The protein samples were equally loaded, confirmed with Ponceau S staining, and normalized to β-actin and total proteins. Error bars, SEM (* p < 0.05 vs. GDS group; n = 8 mice/group).