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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: Alcohol. 2020 Feb 28;85:153–164. doi: 10.1016/j.alcohol.2020.01.005

Prenatal alcohol exposure in the second trimester-equivalent increases the seizure susceptibility in developing rats

Sue J Cho 1, Jamila Newton 1, Tengfei Li 2, Padmini Khandai 1, George Luta 2, David M Lovinger 3, Prosper N’Gouemo 1,*
PMCID: PMC7340347  NIHMSID: NIHMS1586227  PMID: 32114257

Abstract

We have previously reported that prenatal alcohol exposure (PAE) in the 2nd trimester-equivalent of gestation is associated with increased N-methyl-D-aspartate (NMDA)-induced generalized tonic-clonic seizures (GTCSs) prevalence in postpartum developing rats. Whether the 1st trimester-equivalent of gestation is also a vulnerable period for developing GTCSs following PAE is unknown. Here, we investigated the effects of a single episode of PAE at embryonic day 8 (E8, in the 1st trimester-equivalent) or E18 (in the 2nd trimester-equivalent) on NMDA-induced seizures in developing rats at postnatal day 7 (P7, the equivalent of preterm newborns) and P15 (the equivalent of term infants). Pregnant Sprague-Dawley rats were given a single oral dose of ethanol (5 g/kg body weight) at E8 or E18 and the postpartum rats were tested for the susceptibility to NMDA-induced seizures at either P7 or P15. NMDA-induced seizures consisted of wild running-like behavior (WRLB), flexion seizures (FSs), clonic seizures (CSs), GTCSs and tonic seizures (TSs); these seizures were observed in both control-treated and PAE-treated, male and female, P7 and P15 rats. Quantification reveals that the overall prevalence of CSs, GTCSs and TSs occurrence were significantly increased in the E18-PAE group compared to E8-PAE group, adjusting for sex and postnatal day. Furthermore, the overall prevalence of FSs and TSs occurrence were significantly increased in PAE-treated males compared to females, adjusting for embryonic stage and postnatal day. The overall prevalence of WRLB and FSs occurrence were also increased in PAE-P7 rats compared to PAE-P15 rats, adjusting for sex and embryonic stage. We conclude that the susceptibility to develop GTCSs was higher when PAE occured in the 2nd rather than the 1st trimester-equivalent of gestation.

Keywords: prenatal alcohol exposure, neonatal seizures, generalized tonic-clonic seizures, N-Methyl-D-aspartate, preterm newborns, term infants

Introduction

Epilepsy is a common feature of the fetal alcohol spectrum disorder (FASD); infants who had been exposed in utero to alcohol (i.e., prenatal alcohol exposure or PAE) were up to 20 times more likely to develop seizures and epilepsy after birth (Bell et al., 2010; Cassano, Koepsell, & Farwell, 1990; Jarmasz, Basalah, Chudley, & Del Bigio, 2017; Nicita et al., 2014; O’Malley & Barr, 1998; Pierog, Chandavasu, & Wexler, 1977; Robe, Gromisch, & Iosub, 1981; Spohr & Steinhausen, 1987). The prevalence for the occurrence of seizures and epilepsy was higher when drinking alcohol occurred in the 1st trimester, 2nd trimester or throughout all trimesters, but not in the 3rd trimester (Bell et al., 2010; Sun et al., 2009). Time at risk for these PAE-induced neonatal seizures starts soon after birth in human term infants (Bell et al., 2010; Nicita et al., 2014; Sun et al., 2009). Generalized tonic-clonic seizures (GTCSs) are the most prevalent type of neonatal seizures among FASD patients (Bell et al., 2010; Nicita et al., 2014; Sun et al., 2009; Vestergaard et al., 2005). Although alcohol exposure in utero plays an important role in the etiology of PAE-related seizures, current knowledge of the underlying pathogenic mechaninsms is limited (Bird et al., 2015; Brady et al., 2013; Iqbal, Dringenberg, Brien, & Reynolds, 2004; Samudio-Riuz et al., 2010). Debate still exists as to whether alcohol exposure in the 1st or 2nd trimester poses the greater susceptibility for developing neonatal seizures in FASD patients (Bell et al., 2010; Jarmasz et al., 2017; Sun et al., 2009). Furthermore, it is important to determine the timing of PAE in relation to the enhanced seizure susceptibility to identify the potential period of vulnerability since most pregnancies are unintended and not recognized until after the 4th week of gestation (Alvik, Heyerdahl, Haldorsen, & Lindemann, 2006).

Seizures and epilepsies in children shared three main mechanisms including: i) over-activation of NMDA receptors, ii) defect of phasic GABA transmission characterized by a loss of GABA-mediated inhibition, and iii) excess of tonic GABA activation, in which GABA produces tonic activation of GABAA receptors (Gataullina, Bienvenu, Nabbout, Hurerfeld, & Dulac, 2019). Thus, activation of NMDA receptors was chosen to trigger seizures in postpartum rats because GABAergic signaling is mainly excitatory in embryonic and early post-natal life (Khazipov et al., 2004). In the PAE model we developed, a single dose of N-methyl-D-aspartate (NMDA) increased the prevalence for the occurrence of GTCSs in postnatal day 7–35 (P7–P35) rats which had been prenatally exposed to a single binge-like episode at embryonic day 18 (E18) during the 2nd trimester-equivalent of gestation; the highest prevalence of GTCSs occurred at P7 (Cho, Lovinger, & N’Gouemo, 2017). To elucidate whether PAE during the 1st trimester-equivalent also predisposes postpartum rats to seizures to the same extent as the 2nd trimester-equivalent, we investigated the effects of a single PAE episode at two different gestation time points, including E8 (during the 1st trimester-equivalent) and E18, on NMDA-induced seizures in both postpartum male and female developing rats, at two distinct ages, P7 and P15, corresponding to preterm newborns and term infants, respectively (Avishai-Eliner, Brunson, Sandman, & Baram, 2002; Pellock, Arzimanoglou, D’Cruz, & Holmes, 2017; Romijin, Hoffman, & Gramsbergen, 1991; Sengupta, 2013).

Material and methods

Animals.

Timed-pregnant Sprague-Dawley rats (dams) were purchased from Taconic Farms (Germantown, New York, United States) and housed individually during the remainder of their pregnancy terms in standard polycarbonate cages with standard chow and water available ad libitum. The rats were housed in a temperature- and humidity-controlled room with a 12/12-hour light/dark cycle. From the time of birth (defined as P0), the pups remained with their mothers until P7 or P15. All experimental procedures were approved by Georgetown University Animal Care and Use Committee and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (National Research Council, 2001).

Ethanol administration.

On E8 or E18, dams were randomly assigned to receive either a single dose of ethanol (5 g/kg body weight) or control vehicle by gavage. Ethyl alcohol (95%, U.S.P., The Warner-Gram Company, Cockeysville, Maryland, United States) was freshly prepared as 30% (vol/vol) ethanol mixed with Isomil-milk-based infant formula (Abbott Laboratories, Abbott Park, Illinois, United States). Dams in the control-treated group received isovolumetric and isocaloric solution of Isomil without ethanol. Following administration of ethanol, dams were closely monitored for 4 h for signs of intoxication. The behavioral signs of ethanol intoxication were determined based on a well-described intoxication scale (Majchrowicz, 1975; Faingold, 2008): 0 Neutrality, 1 Sedation, 2 Ataxia 1 or absence of signs of intoxication or withdrawal; 1 Sedation; 2 Ataxia 1 characterized by the lowest degree of gait impairment; 3 Ataxia 2 corresponding to an intermediate degree of gait impairment; 4 Ataxia 3 characterized by a marked level of intoxication or recovery of the righting reflex; 5 Loss of righting reflex; and 6 Coma or absence of movements, eye closure, and absence of the eye blink reflex. A single episode of PAE either at E8 or E18 did not alter the duration of gestation, the average size of the litter, or the ratio of males to females in the litter.

Blood alcohol concentrations.

In a separate set of experiments, blood alcohol concentrations (BACs) were measured at E8 or E18, from dams (n=4) and their fetuses (n=20). Two hours after administering the ethanol dose, blood samples (ranging from 0.1–1 mL in volume) were extracted by intracardiac puncture. The blood samples were then centrifuged for 10 minutes at 1000 x g, and the plasma was isolated and stored at −80°C. Alcohol levels were measured using an Analox model AM1 analyzer (Analox Instruments, London, UK).

Seizure testing.

To precipitate seizures in rats, NMDA (Sigma Chemicals, St. Louis, Missouri, United States) was systemically administered. On P7 or P15, NMDA was dissolved in 0.9% NaCl and injected intraperitoneally at a dose of 5 or 20 mg/kg body weight, respectively, based on exploratory evaluations and previous studies (Cho, Lovinger, & N’Gouemo, 2017; Mares & Velisek, 1992). Two to five P7 or P15 rats per litter were used. The rats were then placed in clear Plexiglas boxes and monitored for 60 min for the occurrence of seizures. To compensate for the weak thermoregulation of P7 rats, their body temperatures were maintained at 33±2°C using a heating pad and a heat therapy pump (Model TP700; Stryker Medical, Portage, Missouri, United States). In P7 rats, NMDA induced sequential behaviors consisting of stereotypy and seizures. Stereotypy included sigmoidal tail movement (or tail twisting) and caudal biting. Seizures consisted of wild running-like behavior (WRLB), flexion seizures (FSs, characterized by tonic hyperflexion of the head, neck, spine and tail), GTCSs (bouncing tonic-clonic seizures experienced while lying on the belly or side) and tonic seizures (TSs, characterized by hindlimb extension). The seizure severity was classified as follows: stage 0 no response; stage 1 WRLB; stage 2 FSs; stage 3 GTCSs; stage 4 TSs. In postpartum P15 rats, NMDA-induced seizures were characterized by the emergence of clonic seizures (CSs, characterized by clonic movements of the forelimbs) in addition to seizure phenotypes seen in P7 rats; the seizure severity was classified as follows: stage 0 no response; stage 1 WRLB; stage 2 FSs; stage 3 CSs; stage 4 GTCSs; stage 5 TSs (Cho, Lovinger, & N’Gouemo, 2017; Mares & Velisek, 1992). In some cases, NMDA administration was lethal. We used the acute NMDA-induced seizure model because seizures induced by this method develop gradually over a period of approximately 3–20 minutes (in contrast, seizures induced by electrical stimulation have an abrupt onset, precluding our ability to measure latency). Following NMDA injections, animals that did not develop WRLB, FSs, CSs, GTCSs, and TSs within 60 minutes were considered “seizure resistant”, due to an elevated seizure threshold; therefore, the seizure latency was recorded as 60 minutes to allow the use of all observations in the statistical analysis. For each animal, the prevalence of WRLB, FSs, CSs, GTCSs, and TSs were recorded. The time interval between NMDA injection and the onset of the first WRLB, FSs, CSs, GTCSs or TSs episode was recorded and defined as WRLB, FSs, CSs, GTCSs or TSs latency, respectively. For each animal, the seizure severity score was also recorded and was representative of the most severe seizure endpoint.

Data analysis.

Comparisons were made between P7 and P15 rats because the phenotypes of NMDA-induced seizures (except for clonic seizures) were similar in these animals. We used statistical models with generalized estimating equations (GEE) with an exchangeable working correlation structure, to account for the potential correlation of the outcomes of rats from the same litter. The seizure prevalence, seizure latency, seizure severity or mortality are the outcome variables; embryonic stage (E8 vs. E18) and postnatal day (P7 vs. P15) are the factors of interest; and sex (male vs. female) is the covariate (Diggle et al., 2002; Liang & Zegler, 1986). We also explored the presence of any 2-way or 3-way interactions between embryonic day, postnatal status, and sex. To analyze binary outcomes (prevalence, mortality), we used logistic regression models and calculated odds ratio (OR) with 95% confidence intervals (CI). For a given seizure phenotype, the OR was calculated by dividing the odds in the PAE-treated group by the odds in the control-treated group. For continuous outcomes (seizure latency), we used analysis of variance models with GEE and the estimated mean difference (with 95% CI) between two groups was obtained by subtraction. To analyze ordinal outcomes (seizure severity), a cumulative logistic regression model with GEE was used and we calculated OR (with 95% CI) as follows: 1 odds of seizure severity 0 divided by odds of seizure severity 1–4, 2 odds of seizure severity 0–1 divided by odds of seizure severity 2–4, 3 odds of seizure severity 0–2 divided by odds of seizure severity 3–4, and 4 odds of seizure severity 0–3 divided odds seizure severity 4. In exploratory evaluations, we analyzed seizure prevalence, seizure latency, seizure severity or mortality within subgroups defined by a combination of postnatal day and sex. We estimated the effects of E8 and E18 for the control-treated group and the PAE-treated group separately, and compared the PAE-treated group to the control-treated group for E8 and E18 separately. All effect estimates are adjusted estimates. Four litters were used for each group and the number of postpartum rats in each group was reported in parentheses within the bar graphs. Data are presented as the prevalence for the occurrence of behavioral phenotypes (WRLB, FSs, CSs, GTCSs, TSs, mortality), mean (±S.E.M.) for BACs and seizure latency, and median (±median absolute deviation) for seizure severity and alcohol intoxication. All analyses were conducted using SAS software (SAS Institute, Inc., Cary, North Carolina, United States). The cumulative logistic regression models with GEE were run using SAS macro GEEORD (Gao, Schwartz, Preisser, & Perin, 2017). Differences were considered statistically significant when p < 0.05.

Results

Pregnant female rats were given a single, oral dose of ethanol (5g/kg body weight) or control vehicle on E8, the first trimester-equivalent, or E18, the 2nd trimester-equivalent; the behavioral levels of intoxication were similar in the E8 group and E18 group (Table 1). Ethanol administration at either time point during gestation led to a significant increase in BACs in both the dams and the embryos, indicating that ethanol readily crosses the placental barrier (Table 1). PAE at E8 or E18 had no effect on neither the number of rats per litter, nor the sex ratio of male/female rats in a litter (Table 1). At the dose of 5 mg/kg, NMDA did not induce seizures in P15 rats, while the 20 mg/kg dose was lethal in P7 rats.

Table 1.

Ethanol intoxication and blood alcohol levels were measured as described in Methods. Prenatal alcohol exposure at either E8 or E18 led to high blood alcohol levels in dams and their embryos.

E8
 E18
CON PAE CON PAE
Alcohol intoxication levels
 Dams n/a 2.0 ± 0.55 (n = 16) n/a 2.0 ± 0.59 (n = 16)
Blood alcohol levels (g/dL)
 Dams n/a 0.28 ± 0.02 (n = 4) n/a 0.26 ± 0.02 (n = 4)
 Embryos n/a 0.28 ± 0.02 (n = 20,4litters) n/a 0.26 ± 0.02 (n = 20,4litters)
 Litter 12.25 ± 0.58 12.19 ± 0.40 12.25 ± 0.49 12.0 ± 0.35
 Size (n = 16) (n = 16) (n = 16) (n = 16)
 Ratio male/female pups 1.15 1.09 1.18 1.09
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n/a: not applicable.

Quantification revealed that the E18-PAE group had an increased prevalence for the occurrence of CSs, GTCSs and TSs compared to the E8-PAE group, adjusting for sex and postpartum day (Table 2). The latency to develop CSs was significantly decreased in the E18-PAE group compared to the E8-PAE group, adjusting for sex and postpartum day (Table 3). Furthermore, the prevalence for the occurrence of FSs and TSs were significantly increased in postpartum males compared to females, adjusting for embryonic stage and postpartum day (Table 2). The increased prevalence for the occurrence of FSs in postpartum males was associated with decreased seizure latency, adjusting for embryonic stage and postpartum day (Table 3). We also found that the prevalence for the occurrence of WRLB and FSs were significantly increased in PAE-P7 rats compared to PAE-P15 rats, adjusting for sex and embryonic stage (Table 2). The increased prevalence for the occurrence of WRLB and FSs were associated with decreased seizure latency, adjusting for sex and embryonic stage (Table 3)

Table 2.

Overall effect of PAE on the prevalence of the occurrence of NMDA-induced seizures and mortalityDetails about NMDA-induced seizures and data analyses are provided in Methods. Postpartum P7 rats had 9.37 and 9.06 times higher odds to exhibit WRLB and FSs, respectively, than P15 rats. Analysis also showed that postpartum male rats had 2.23 and 2.41 times higher odds to develop FSs and TSs, respectively, than females. Furthermore, postpartum rats subjected to PAE at E8 had 0.37, 0.52, and 0.6 times lower odds to develop CSs, GTCSs, and TSs, respectively than at E18.

OR 95% CI Z score p-value
Wild running-like behavior (WRLB)
 Sex (male vs. female) 1.66 0.89 3.09 1.60 0.1100
 Embryonic stage (E8 vs. E18) 0.61 0.32 1.16 −1.51 0.1308
 Postnatal day (P7> P15) 9.37 3.89 22.55 4.99 <0.0001
Flexion seizures (FSs)
 Sex (male > female) 2.23 1.13 439 233 0.0200
 Embryonic stage (E8 vs. E18) 0.65 0.33 1.27 −1.26 0.2060
 Postnatal day (P7> P15) 9.06 444 18.48 6.06 <0.0001
Clonic seizures (CSs)
 Sex (male vs. female) 1.59 0.80 3.17 131 0.1900
 Embryonic stage (E8 < E18 0.37 0.22 0.63 −3.66 0.0003
Generalized tonic-clonic seizures (GTCSs)
 Sex (male vs, female) 1.45 0.78 2.71 1.17 0.2425
 Embryonic stage (E8 < E18) 0.52 0.31 0.87 −2.50 0.0125
 Postnatal day (P7vs.P15) 1.24 0.74 207 0.82 0.4129
Tonic seizures (TSs)
 Sex (male > female) 2.41 1.30 447 2.79 0.0052
 Embryonic stage (E8 < E18) 0.60 0.40 091 −2.42 0.0154
 Postnatal day (P7vs. P15) 0.82 0.5ct2 1.31 −0.82 0.4138
Mortality
 Sex (male vs. female) 1.06 0.50 225 0.15 0.8792
 Embryonic stage (E8 vs. E18) 0.60 0.30 1.20 −1.44 0.1509
 Postnatal day (P7vs. P15) 0.71 0.35 1.43 −0.96 0.3358
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Table 3.

Overall effect of PAE on the latency of NMDA-induced seizures Details about NMDA-induced seizures and data analyses are provided in Methods. Postpartum P15 rats have longer WRLB and FSs latencies than P7 rats. In addition, postpartum female rats have longer FSs latency than males. Furthermore, postpartum rats subjected to PAE at E8 have longer CSs and GTCSs latencies than at E18. Sex slightly altered the prevalence for the occurrence of TSs, while embryonic stage and postnatal day had no effect.

Difference SE estimate SE 95% CI Z score p-value
Wild running-like behavior (WRLB)
 Sex (male vs. female) −281 2.292 −7.30 1.68 −1.23 0.2202
 Embryonic stage (E8 vs. E18) 4.21 2.662 −1.00 9.43 1.58 0.1135
 Postnatal day (P7< P15) −18.53 2.534 −23.50 −13.57 −7.31 0.0001
Flexion seizures (FSs)
 Sex (male < female) −5.68 2.472 −10.53 −0.84 −2.30 0.0216
 Embryonic stage (E8 vs. E18) 2,50 2,545 −2.48 7.49 0.98 0.3250
 Postnatal day (P7< P15) −19.03 2.357 −23.65 −14.41 −8.07 <0.0001
Clonic seizures (CSs)
 Sex (male vs. female) −4.77 3.016 −10.68 1.14 −1.58 0.1139
 Embryonic stage (E8 > E18 7.54 2.600 2.45 12.64 2.90 0.0037
Generalized tonic-clonic seizures (GTCSs)
 Sex (male vs. female) −2.84 2.898 −8.52 2.84 −0.98 0.3266
 Embryonic stage (E8 vs. E18) 4.46 2.447 −0.33 9.26 1.82 0.0682
 Postnatal day (P7> P15) −3.46 2.533 −8.42 1.52 −1.37 0.1720
Tonic seizures (TSs)
 Sex (male vs. female) −0.403 2.206 −8.35 0.30 −1.83 0.0680
 Embryonic stage (E8 vs. E18) 3.21 1.929 −0.57 6.99 1,66 0.0963
 Postnatal day (P7vs. P15) 0.97 2.025 −3.00 4.94 0.48 0.6333
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Analysis also revealed that male P7 rats had 2.6 times (OR=2.6, 95% CI: [1.26, 4.41], p=0.007) or 5.57 times (OR=5.57, 95% CI: [1.35, 22.95], p=0.017) higher odds to develop GTCSs when PAE occurred at E8 or E18, respectively, when compared to the control-treated group (Fig. 1E). The increased prevalence for the occurrence of GTCSs was associated with a decrease in the latency of the first episode of GTCSs in both the E8 group (difference=−5.68, 95% CI: [−8.28, −3.07], p<0.0001; Fig. 1F) and the E18 group (difference=−17.80, 95% CI: [−28.94, −6.66], p=0.002; Fig. 1F), when compared to the control-treated groups. The PAE-treated E8 groups also had longer seizure latency than the PAE-treated E18 groups for both GTCSs (difference=13.36, 95% CI: [3.20, 23.53], p=0.01, Fig. 1F) and TSs (difference=12.49, 95% CI: [2.11, 22.86], p=0.018). However, PAE at either E8 or E18 did not alter the prevalence for the occurrence or latencies of WRLB (Fig. 1A,B) and FSs (Fig. 1C,D), when compared to the control-treated group. Quantification also showed that male P7 rats had 2.94 times (OR=2.94, 95% CI: [1.18, 7.30], p=0.009) higher odds to have increased seizure severity if PAE occurred at E18 (but not at E8), compared to the control-treated group (Fig. 3A). No change in the rate of mortality was observed in the E8 group (CON: 11.8%, PAE: 12.5%) and E18 group (CON: 25%, PAE: 25%).

Figure 1.

Figure 1.

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Effects of prenatal alcohol exposure (PAE) on NMDA-induced seizures in postnatal male P7 rats. A single dose of PAE at embryonic day 8 (E8) or 18 (E18) did not affect wild running-like behavior (WRLB; A,B) and flexion seizures (FSs; C,D). PAE at either E8 or E18 significantly increased the prevalence to develop generalized tonic-clonic seizures (GTCSs) and decreased GTCSs latency (E,F). PAE did not alter the expression of NMDA-induced tonic seizures (TSs; G,H). Data are shown as the prevalence for the occurrence of WRLB, FSs, GTCSs, and TSs, and mean±S.E.M. for the seizure latency. Four litters in each group were used and the total number of postpartum rats is indicated within the bar graph. Details about seizure induction and data analyses are provided in Methods. * P < 0.05, **P < 0.01, ****P <0.0001.

Figure 3.

Figure 3.

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Effects of prenatal alcohol exposure (PAE) on the severity of NMDA-induced seizures in postnatal P7 rats. A single dose of PAE at embryonic day 18 (E18) but not E8 enhanced the severity of NMDA-induced seizures in male (A) but not in female (B) postpartum P7 rats. Data are shown as the score median±median absolute deviation (that was reported when appropriate). Note that in males, the median absolute deviation was 0 although no similar seizure severity score was recorded. Four litters in each group were used and the total number of postpartum rats is indicated within the bar graph. Details about seizure induction and data analyses are provided in Methods. **P <0.01.

Following PAE at E18, female P7 rats were 3.56 times (OR=3.56, 95% CI: [1.37, 9.22], p=0.009) more likely to develop GTCSs compared to the control-treated group (Fig. 2E) and the latency to GTCSs was significantly decreased (difference=−10.74, 95% CI: [−18.58, −2.90], p=0.007, Fig. 2F). In the E8 group, PAE significantly decreased the latency to GTCSs (difference=−11.90, 95% CI: [−22.56, −1.24], p=0.029, Fig.2F), but nonsignificantly (p=0.159) altered the odds to develop GTCSs prevalence (Fig.2E). PAE also did not alter the prevalence for the occurrence and latencies of WRLB, FSs, GTCSs, and TSs, when compared to the control-treated groups (Fig. 2A–D,G,H). Quantification also shows that PAE at E18 or E8 nonsignificantly increased the median score of seizure severity; no significant change was noted when comparing the seizure severity between the PAE-E18 group and PAE-E8 group (Fig. 3B). A nonsignificant change in the rate of mortality was observed in the E8 group (CON: 11.8%, PAE: 2.2%) and E18 group (CON: 10%, PAE: 21.1%).

Figure 2.

Figure 2.

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Effects of prenatal alcohol exposure (PAE) on NMDA-induced seizures in postnatal female P7 rats. A single dose of PAE at embryonic day 8 (E8) or 18 (E18) did not affect wild running-like behavior (WRLB; A,B) and flexion seizures (FSs; C,D). PAE at E8 nonsignificantly increased the prevalence for the occurrence of generalized tonic-clonic seizures (GTCSs) but significantly decreased GTCSs latency (E,F). PAE at E18 significantly increased GTCSs prevalence and decreased GTCSs latency (E, F). PAE did not alter the expression of NMDA-induced tonic seizures (TSs, G,H). Data are shown as the prevalence for the occurrence of WRLB, FSs, GTCSs, and TSs, and mean±S.E.M. for the seizure latency. Four litters in each group were used and the total number of postpartum rats is indicated within the bar graph. Details about seizure induction and data analyses are provided in Methods. *P < 0.05, **P < 0.01.

Overall, the data from postpartum P7 rats suggest a strong and a modest increase in the prevalence of the occurrence of NMDA-induced seizures in males and females, respectively, when exposed to ethanol during the 1st or 2nd trimester-equivalent of gestation. Thus, seizures in FASD patients may be better modeled in postpartum P15 rats. Interestingly, NMDA-induced seizures in these P15 rats were characterized by the occurrence of CSs, in addition to other seizure phenotypes seen in P7 rats.

In the male P15 rats, there was a significant increase in the prevalence for the occurrence of all seizure phenotypes if the rats had been exposed to ethanol at E18 rather than E8. When PAE occurred at E18 (but not E8), male P15 rats had 2.53 times (OR=2.53, 95% CI: [1.05, 6.10], p=0.039) higher odds to develop WRLB, and had decreased seizure latency (difference=−10.54, (95% CI: [−19.88, −1.20], p=0.027), when compared to the control-treated groups (Fig. 4A and B). Additionally, the latency of WRLB was significantly decreased in the PAE-treated E18 group compared to the PAE-treated E8 group (difference=10.68, 95% CI: [1.39, 19.96], p=0.024; Fig. 4B). Quantification also showed that when PAE occurred at E18 (but not at E8), male P15 rats had 2.53 times (OR=2.53, 95% CI: [1.05, 6.10], p=0.039) higher odds to develop FSs, when compared to the control-treated group; no change was found in the latency of FSs compared to the control-treated groups (Fig. 4C,D). We also examined the prevalence for the occurrence of CSs in postpartum male rats. When PAE occurred at E18 (but not E8), male P15 rats had 4.51 times (OR=4.51, 95% CI: [1.39, 14.67], p=0.012) higher odds to develop of CSs, when compared to the control-treated group (Fig. 4E). The increased prevalence for the occurrence of CSs was associated with a decrease in the latency of the first episode of CSs in the PAE-treated group (difference=−13.02, 95% CI: [−22.36, −3.68], p=0.006, Fig. 4F). Further analysis revealed that the prevalence for the occurrence of CSs in the PAE-E18 group was significantly increased compared to the PAE-E8 group (difference=9.85, 95% CI: [2.30, 17.41], p=0.011, Fig. 4E); this effect was associated with reduced seizure latency (difference=9.85, 95% CI: [2.30, 17.41], p=0.011). We also examined the expression of GTCSs in postpartum male rats. When PAE occurred at E18, male P15 rats had 3.69 times (OR=3.69[2.12, 6.41], p < 0.0001) higher odds to develop GTCSs, when compared to the control-treated group; however, the odds of having GTCSs if PAE occurred at E8 were nonsignificantly (p=0.1179) increased (Fig. 4G). Change in the prevalence for the occurrence of GTCSs was associated with a decrease in the latency of GTCSs in both the PAE-treated E8 group (difference=−11.70, 95% CI: [−21.85, −1.55], p=0.024) and PAE-treated E18 group (difference= −7.76, 95% CI: [−12.18, −3.35], p=0.001), compared to the control-treated groups (Fig. 4H). The timing of alcohol exposure during gestation also did not alter the prevalence and latency of GTCSs (Fig. 4G,H). We also examined the expression of TSs in postpartum male rats. When PAE occurred at E18, male P15 rats had 2.89 (OR=2.89, 95% CI: [1.80, 4.63], p<0.0001) higher odds to develop TSs compared to the control-treated group; however, the odds of developing TSs were nonsignificantly (p=0.0501) increased if PAE occurred at E8, when compared to control-treated group (Fig. 6A). The increased seizure susceptibility was associated with a reduced latency of TSs in both the E8 PAE-treated group (difference=−10.40, 95% CI: [−15.08, −5.72], p < 0.0001) and E18 PAE-treated group (difference= −6.01[−8.37, −3.65], p<0.0001), when compared to the control-treated groups (Fig. 6B). Quantification also showed that PAE at E18 (but not E8) significantly (OR=0.26, 95% CI: [0.17, 0.41], p < 0.0001) increased the seizure severity (Fig. 6C). No change in the rate of mortality was observed in the E8 group (CON: 20%, PAE: 20%) and E18 group (CON: 33.33%, PAE: 38.9%).

Figure 4.

Figure 4.

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Effects of prenatal alcohol exposure (PAE) on NMDA-induced seizures in postpartum male P15 rats. A single dose of PAE at embryonic day 18 (E18) but not E8 significantly increased the prevalence for the occurrence of WRLB (A), FSs (C), CSs (E), and GTCSs (G); this effect was associated with a decreased seizure latency of WRLB (B), CSs (F), and GTCSs (H). The prevalence for the occurrence of CSs also was significantly increased at E18 compared to E8 (E); this effect was associated with reduced seizure latency was significantly reduced at E18 compared to E8 (F). Data are shown as the percentage for the occurrence of a given seizure phenotype, and mean±S.E.M. for the seizure latency. Four litters in each group were used and the total number of postpartum rats is indicated within the bar graph. Details about seizure induction and data analyses are provided in Methods. *P < 0.05, **P < 0.01, ***P ≤ 0.001, ****P < 0.0001.

Figure 6.

Figure 6.

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Effects of prenatal alcohol exposure (PAE) on NMDA-induced tonic seizures (TSs) and seizure severity in both male and female P15 rats. In males, a single dose of PAE at embryonic day 18 (E18) significantly increased the prevalence for the occurrence of TSs (A), and reduced the seizure latency (B); at E8, however, PAE significantly decreased TSs latency (B) but nonsignificantly increased the prevalence for the occurrence seizure (A). PAE at E18 (but not E8) also significantly increased the severity of NMDA-induced seizures in males (C). In females, PAE at either E18 or E8 did not alter the prevalence of TSs (D), latency to TSs (E), and the seizure severity (F). Note that in control-treated E8 females, the median absolute deviation was 0 although no similar seizure severity score was recorded. Data are shown as the percentage of prevalence for the occurrence of TSs, mean±S.E.M. for TSs latency, and score median±median absolute deviation for seizure severity. Four litters in each group were used and the total number of postpartum rats is indicated within the bar graph. Details about seizure induction and data analyses are provided in Methods. ****P < 0.0001.

In the female P15 rats, PAE did not alter the prevalence for the occurrence and latency of WRLB (Fig. 5A,B), FSs (Fig. 5C,D), GTCSs (Fig. 5G,H), and TSs (Fig. 6D,E), when compared to control-treated groups. PAE at E8 significantly decreased CSs latency (difference=−10.92, 95% CI: [−15.05, −6.78], p < 0.0001), but did not alter the prevalence of occurrence of this seizure phenotype; non-significant changes in CSs prevalence and latency were observed if PAE occurred at E18 (Fig. 5E,F). Furthermore, quantification also showed that PAE did not alter the seizure severity (Fig. 6F). In control-treated female P15 rats, the odds of developing of WRLB was significantly decreased (OR=0.22, 95% CI: [0.06, 0.78], p=0.019) at E8 compared to E18 (Fig. 5A); this effect was associated with increased WRLB latency (difference=19.10, 95% CI:[5.36, 32.84], p=0.006), Fig. 5B). No change in the rate of mortality was observed if PAE occurred at E8 (CON: 15.8%, PAE: 21.1%) or E18 (CON: 26.7%, PAE: 29.4%).

Figure 5.

Figure 5.

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Effects of prenatal alcohol exposure (PAE) on NMDA-induced seizures in postnatal female P15 rats. A single dose of PAE at either embryonic day 18 (E18) or E8 did not alter the prevalence of the occurrence of WRLB (A), FSs (C), CSs (E), and GTCS (G). PAE at E8 but not E18 significantly reduced the latency to CSs (F). PAE significantly increased the prevalence for the occurrence of CSs at E18 compared to E8 (E). The prevalence of the occurrence of WRLB was significantly increased in control-treated P15 at E18, when compared to E8 (A); this effect was associated with decreased WRLB latency (B). Data are shown as the prevalence of the occurrence of WRLB, FSs and GTCSs, and mean±S.E.M. for the seizure latency. Four litters in each group were used and the total number of postpartum rats is indicated within the bar graph. Details about seizure induction and data analyses are provided in Methods. *P < 0.05, **P < 0.01, ****P < 0.0001.

Discussion

Here, we report that a single episode of PAE in the 2nd trimester-equivalent of pregnancy enhanced the susceptibility to develop NMDA-induced CSs, GTCSs, and TSs in all postpartum rats compared to the 1st trimester-equivalent. PAE also significantly increased the prevalence for the occurrence of WRLB and FSs in all postpartum P7 rats compared to P15 rats, and the increased seizure susceptibility was associated with decreased seizure latency. The prevalence for the occurrence of FSs and TSs were also significantly increased in all postpartum males compared to females. Furthermore, PAE in the 2nd (but not the 1st) trimester-equivalent significantly increased the prevalence for the occurrence of CSs, GTCSs, and TSs in postpartum P15 rats. The prevalence for the occurrence of CSs also was increased in P15 rats subjected to PAE in the 2nd trimester-equivalent compared to the 1st trimester. These findings suggest a timing-, age-, and sex-difference in the impact of PAE on the prevalence of NMDA-induced seizure occurrence in developing rats, in which timing of PAE would account for the difference in the prevalence for the occurrence of CSs, GTCSs, and TSs, while postpartum day would account for the difference in the prevalence for the occurrence of WRLB and FSs. Furthermore, these findings suggest that PAE facilitates the propagation of seizure activity in neural networks that underlie NMDA-induced seizures. We also found that postpartum P7 rats have a reduced threshold-dose of NMDA-induced seizures compared to P15 rats. These findings suggest that P7 rats are more susceptible to seizures than P15 rats, which is consistent with clinical reports that the prevalence for the occurrence of neonatal seizures in preterm newborns is higher than in term infants (Cowan, 2002; Mizrahi & Watanabe, 2005; Pisani et al., 2012; Pisani & Spagnoli, 2019).

In our PAE model, the rat fetus’ BAC matches BAC (0.25 g/dL) measured in newborns prenatally exposed to alcohol 60 min after birth (Kvigne, Randall, Simanton, Brenneman, & Welty, 2012). In addition, BAC in dams matches the estimated BAC (0.27 g/dL) in women taken about 30 min before giving birth to newborns with FASD; at birth the newborn experienced alcohol withdrawal symptoms (Kvigne et al., 2012; Nichols, 1967). These findings indicate that BACs in our rat PAE model mimic those reported in the human condition. Our PAE model is designed to model the increased GTCSs susceptibility in newborns and infants induced by a single binge-drinking episode by a pregnant woman during the 1st or 2nd trimester of gestation. Interestingly, a single episode of binge drinking is also sufficient to produce FASD in humans (Floyd & Sidhu, 2004). PAE during the 1st trimester or all of the three trimesters is associated with an 18% prevalence of occurrence of seizures in FASD patients (Bell et al., 2010). However, a large scale epidemiological study revealed that binge alcohol drinking during the 2nd trimester led to a 3-fold increase in neonatal seizures (Sun et al., 2009); suggesting that 2nd trimester exposure may be the most vulnerable period for alcohol-induced epileptogenesis in FASD patients. Remarkably, when comparing PAE during the 1st and 2nd trimester equivalent, we found that a single episode of PAE during the 2nd but not the 1st trimester-equivalent increased the prevalence of occurrence of NMDA-induced CSs, GTCSs and TSs in postpartum rats. We also found a sex-related difference, with postpartum males having higher susceptibility to develop TSs than females. Interestingly, sex-difference in the prevalence of GTCSs have also been reported in the FASD population as these seizures were seen in 44% and 25% of males and females, respectively (Nicita et al., 2014). However, in this clinical study, no attempt was made to evaluate changes associated with the timing of alcohol exposure during pregnancy. Although the extent to which a single PAE episode in the 3rd trimester-equivalent alter NMDA-induced seizures in developing rats was not evaluated is this study, evidence indicates PAE during the 3rd trimester is not associated with an increase in the prevalence of seizures and epilepsy in FASD patients (Bell et al., 2010). In contrast, a single PAE episode on P4 or a repeated PAE episode on P4-P9 in the 3rd trimester-equivalent reduced the threshold for pentylenetetrazole-induced seizures in juveniles but not adult rats, suggesting increased seizure susceptibility (Bonthius, Woodhouse, Bonthius, Taggard, & Lothman, 2001).

A report indicates that PAE is also associated with increased prevalence for the occurrence of partial complex seizures in the FASD patients (Bell et al., 2010). These seizures can be modeled by NMDA-induced CSs, which are reminiscent of seizures initiated in the limbic system. Accordingly, we found that the susceptibility to develop CSs is increased when PAE occurred in the 2nd trimester-equivalent; this effect is associated with a reduced latency to develop CSs in postpartum male rats. These findings suggest that the susceptibility to develop CSs is heightened when PAE occurs during the 2nd trimester-equivalent and that postpartum males are more vulnerable than females. Systemic administration of NMDA to developing rats (ca. P7–P21) induces a unique age-dependent seizure phenotype that is characterized by the occurrence of FSs; no FSs are triggered in >P21 rats (Cho, Lovinger, & N’Gouemo, 2017; Kabova, Liptakova, Slamberova, Pometlova, & Velisek, 1991; Mares & Velisek, 1992). NMDA-induced FSs in developing rats are believed to mimic infantile spams in humans (Mares and Velisek, 1992; Kabova et al., 1999). Whether infantile spams occur in FASD patients remains unknown. Here, we found that for PAE the odds to develop FSs were 9 times higher in P7 rats compared to P15 rats, suggesting that P7 rats may be more prone to develop spasms than P15 rats. In this study, we also found that the overall effect of PAE on the prevalence for the occurrence of WRLB was 9 times higher in P7 rats compared to P15 rats. The clinical correlates of WRLB are unknown; nevertheless, WRLB may represent a pre-convulsive state.

The mechanisms underlying the enhanced seizure susceptibility following PAE are not fully understood. Nevertheless, reports show changes in the expression of both glutamate and GABA receptors in FASD models (Bird et al., 2015; Brady et al, 2013; Iqbal et al., 2004; Samudio-Riuz et al 2010). Altered calcium signaling in the inferior colliculus may also play a role in the pathophysiology of seizures following PAE (N’Gouemo, unpublished observations). Furthermore, alcohol exposure at P4 during the 3rd trimester equivalent at the time of brain growth spurt has been reported to promote the development of limbic seizures, suggesting that changes in mechanisms underlying early brain development may contribute to enhance the seizure susceptibility (Bonthius, Woodhouse, Bonthius, Taggard, & Lothman, 2001). Accordingly, alcohol exposure downregulates several genes implicated in the development of the central nervous system (Halder et al., 2015). The timing of PAE and related neuronal abnormalities may also have an impact on brain excitability. Accordingly, craniofacial and neuronal tube abnormalities are thought to possibly be related to alcohol consumption in the 1st trimester of gestation (Petrelli, Weinberg, & Hicks, 2018). Moreover, abnormal glial and neuronal generation, proliferation, and migration are linked to alcohol use in the 2nd trimester, while abnormal synaptogenesis, increased apoptosis, and necrosis occur following alcohol exposure during the 3rd trimester of gestation (Petrelli et al., 2018). In our model, it is likely that PAE during the 2nd trimester-equivalent of gestation may have a tremendous impact on GTCSs susceptibility by altering glial and neuronal processing.

In the present study, NMDA-induced seizures were characterized by the occurrence of CSs in postpartum P15 rats; the expression of these seizures was transient such that no CSs were observed in postpartum >P15 rats (Cho, Lovinger, & N’Gouemo, 2017; Mares & Velisek, 1992). We also noted that PAE did not alter the prevalence for the occurrence of GTCSs in female P15 rats, but increased the prevalence for the occurrence of GTCSs in female P21 rats (Cho et al., 2017; present study). We should mention that data analyses were performed using different methods in both studies, which may explain some differences, such as decreased GTCSs latency found in both male and female P7 rats in the present study but not in the previous report (Cho, Lovinger, & N’Gouemo, 2017). Overall, we report a consistent increase in the prevalence for the occurrence of GTCSs in postpartum male rats exposed to PAE during the 2nd trimester-equivalent. Thus, our model may be suitable for investigating the cellular and molecular mechanisms underlying PAE-related GTCSs that begin during infancy in humans.

As a limitation, our interpretation should consider taking into account the relatively small to moderate sample size used in this study. Consequently, our study may not have enough statistical power to detect relatively small differences between groups of interest. We also did not adjust for multiple testing to further reduce the statistical power for comparisons between groups. Thus, the results of our study should be considered when designing future larger studies that will be appropriately powered to detect small differences that are of preclinical importance.

In conclusion, the present study reports that PAE have sex-, timing- and age-differential effects on NMDA-induced seizure susceptibility in postpartum rats, with males being more susceptible to develop seizures than females, P7 rats being more prone to develop seizures than P15 rats. Furthermore, the susceptibility to develop seizures is higher when PAE occurs in the 2nd than in the 1st trimester-equivalent.

Table 4.

Effects of PAE on the severity of NMDA-induced seizures Details about NMDA-induced seizures and data analyses are provided in Methods. Sex and embryonic stage have no effect on the seizure severity in P7 rats. Furthermore, embryonic stage slightly altered the seizure severity in P15 rats, while sex had no effect.

OR 95% CI p-value
P7 rats
 Sex (male vs. female) 0.67 0.37 1.22 0.1915
 Embryonic stage (E8 vs. E18) 1.36 0.75 2.48 0.3115
P15 rats
 Sex (male vs. female) 0.79 0.44 1.42 0.4302
 Embryonic stage (E8 vs. E18) 1.86 0.99 3.50 0.0543
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Research Highlights.

  • Prenatal alcohol exposure in the first and second trimester exacerbates seizures in developing rats

  • Prenatal alcohol exposure in second trimester increases the incidence of clonic/tonic seizures

  • Prenatal alcohol exposure in second trimester increases flexion/tonic seizures in postpartum males

Acknowledgements

This publication was funded in part by the National Institutes of Health Public Health Service grants (AA027171 and AA027660 to P.N.) and the NIAAA Division of Intramural Clinical Biological Research (Z1A AA000407 to D.M.L.). NIAAA has no further role in the study design and the decision to publish the findings. This publication’s contents are the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

Footnotes

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Statement of Interest

The authors have no conflicts of interest.

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