Maternal and Offspring Data
A total of 11 AL, 11 PF1,2, and 10 PNEE1,2 dams were used for generation of experimental offspring. All pregnant dams continued to gain body weight throughout gestation, regardless of diet, F(2, 23) = 1.22, p = 0.342 (Table 1). Furthermore, there was no significant difference in litter size, F(2, 28) = 0.15, p = 0.863, or gestation length, F(2, 22) = 0.73, p = 0.492. The average BEC was 145.32 ± 5.31 mg/dl for the EtOH treated dams. These BEC levels are similar to those found in previously published studies (Christie et al., 2005).
Table 1. Maternal and Offspring Data
|% Weight gain||Length of gestation (days)||Litter size (pup #)||BEC (mg/dl)||PD 2/3 (g)a||PD 50 to 70 (g)b,c|
|AL||29.95 ± 4.07||22.20 ± 0.20||15.18 ± 1.19||–||8.68 ± 0.28||8.26 ± 0.18||395.12 ± 9.10||260.58 ± 9.10|
|PF1,2||23.71 ± 4.06||22.00 ± 0.21||14.40 ± 1.12||–||7.03 ± 0.20||7.12 ± 0.18||345.38 ± 10.41||250.93 ± 9.70|
|PNEE1,2||31.84 ± 4.85||22.30 ± 0.15||15.20 ± 1.21||145.31 ± 5.31||7.40 ± 0.18||6.92 ± 0.39||388.54 ± 10.03||228.27 ± 11.32|
Offspring were weighed across the postnatal period until their experimental use in adulthood (Table 1). Following birth, weights taken on the litter cull date (PD 3/4) were significantly affected by prenatal treatment, F(2, 81) = 22.93, p < 0.001, but not gender, F(1, 81) = 1.83, p > 0.251. PNEE1,2 (p < 0.001; n = 25) and PF1,2 (p < 0.001; n = 28) offspring had significantly reduced body weights compared to AL (n = 34). At the time of electrophysiological experimentation, there was a main effect of prenatal treatment, F(2, 81) = 5.02, p = 0.008, and gender, F(1, 81) = 253.95, p < 0.001, on body weight. Additionally, there was a significant interaction between prenatal treatment and gender, F(2, 81) = 5.184, p = 0.008, on body weight. Upon assessment, AL males (n = 17) weighed more than PF1,2 males (p = 0.007; n = 13) at adulthood (i.e., PD 50 to 70). Additionally, males (n = 44) weighed significantly more than females (n = 43, p < 0.001), a common gender difference at this age (River, 2012).
To determine whether prenatal EtOH exposure alters the response of the adult dentate gyrus to acute EtOH application, hippocampal slices from all 3 prenatal treatment groups were exposed to various concentrations of EtOH (0, 20, or 50 mM) prior to LTP induction. Table 2 provides a summary of all LTP data.
Table 2. Offspring Numbers and Long-Term Potentiation Data
|Gender||Prenatal treatment group||Acute ethanol (EtOH) concentration (mM)||Slice (n), animal (a), litter (l) number||Long-term potentiation|
|Male||AL||0||n = 9, a = 7, l = 6||60.02 ± 9.18|
|20||n = 8, a = 5, l = 3||16.72 ± 4.73|
|50||n = 9, a = 7, l = 4||−1.30 ± 4.11|
|PF1,2||0||n = 8, a = 6, l = 3||23.78 ± 3.71|
|20||n = 8, a = 7, l = 4||9.75 ± 2.80|
|50||n = 7, a = 6, l = 3||3.13 ± 4.07|
|PNEE1,2||0||n = 8, a = 7, l = 4||22.68 ± 5.10|
|20||n = 7, a = 6, l = 4||21.72 ± 8.86|
|50||n = 7, a = 7, l = 4||12.73 ± 6.77|
|Female||AL||0||n = 9, a = 8, l = 5||46.12 ± 5.13|
|20||n = 7, a = 6, l = 1||18.61 ± 3.45|
|50||n = 9, a = 6, l = 4||0.06 ± 4.05|
|PF1,2||0||n = 9, a = 6, l = 4||40.84 ± 5.60|
|20||n = 6, a = 5, l = 2||21.33 ± 5.04|
|50||n = 8, a = 7, l = 3||24.67 ± 6.25|
|PNEE1,2||0||n = 10, a = 7, l = 5||37.38 ± 6.28|
|20||n = 9, a = 4, l = 2||25.34 ± 5.90|
|50||n = 8, a = 5, l = 2||16.91 ± 4.53|
Statistical analyses revealed a main effect of acute EtOH application, F(2, 128) = 42.79, p < 0.001, as EtOH significantly reduced LTP in a concentration-dependent manner (0 mM vs. 20 mM, p < 0.001; 0 mM vs. 50 mM, p < 0.001; 20 mM vs. 50 mM, p = 0.009) and a main effect of gender, F(1, 128) = 6.58, p = 0.011, indicating higher levels of LTP in females compared to males (p = 0.009). There was no main effect of prenatal treatment found; however, there was a significant interaction between prenatal treatment and gender, F(2, 128) = 4.77, p = 0.010, and between prenatal treatment and acute EtOH application, F(4, 128) = 8.26, p < 0.001. Each interaction is presented below.
The interaction between prenatal treatment and acute EtOH exposure indicates that prenatal treatment groups were affected differentially by acute EtOH in adulthood. In the absence of acute EtOH (0 mM), a robust and persistent enhancement of the fEPSP was observed in AL control offspring (53% change in the fEPSP 60 minutes following TBS). However, LTP levels were significantly smaller in PNEE1,2 (42% reduction, p = 0.001) and PF1,2 (38% reduction, p = 0.006) offspring than in AL offspring (Fig. 2). LTP levels did not differ between PF1,2 and PNEE1,2 offspring (p = 0.999).
Figure 2. Prenatal ethanol exposure (PNEE) results in long-term alterations in long-term potentiation (LTP). (A) Time course of LTP, prior to and after induction (at time 0 minute). (B) LTP was reduced in both PNEE1,2 and pair-fed (PF)1,2 offspring compared to ad libitum (AL) offspring. Insert illustrates samples of traces obtained from corresponding groups; field excitatory postsynaptic potentials (fEPSPs) recorded before (black) or 1 hour (gray) after conditioning stimulation are superimposed. *corresponds to significance level p < 0.05 from AL. Data presented as mean fEPSP slope (% change relative to preconditioning responses) ± SEM.
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When EtOH was administered acutely to the hippocampal slice prior to and during LTP induction, 20 mM EtOH significantly reduced LTP in AL offspring (67% reduction from 0 mM, p < 0.001), whereas 50 mM EtOH completely blocked LTP, t(34) = 0.222, p = 0.825; 101% reduction from 0 mM, p < 0.001. Furthermore, LTP was significantly affected by the acute application of EtOH in a concentration-dependent manner (20 mM vs. 50 mM, p = 0.032; Fig. 3A).
Figure 3. Effects of acute ethanol (EtOH) application on long-term potentiation (LTP). EtOH (black horizontal bar) was administered acutely to hippocampal slices prior to and during LTP induction. (A) Time course of LTP in slices from ad libitum offspring (white). LTP was reduced by 20 mM EtOH (triangle) and blocked by 50 mM EtOH (square). (B) Time course of LTP in slices from pair-fed (PF)1,2 offspring (gray). LTP was attenuated following application of 20 and 50 mM EtOH (triangle and square, respectively). (C) Time course of LTP in slices from prenatal EtOH exposure (PNEE)1,2 offspring (black). LTP in PNEE1,2 offspring was not affected by acute EtOH application. (D) Summary of LTP experiments examining the interactions between prenatal treatment and acute EtOH application. (E) Samples of traces obtained from corresponding groups; field excitatory postsynaptic potentials (fEPSPs) recorded before (black) or 1 hour (gray) after conditioning stimulation are superimposed. Symbols represent acute EtOH concentration, whereas colors represent prenatal treatment group. Black bar signifies the presence of EtOH for 15 minutes. *corresponds to significance level p < 0.05 from 0 mM EtOH slices within the respected prenatal treatment group. Data presented as mean fEPSP slope (% change relative to preconditioning responses) ± SEM.
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In contrast to the observations in AL offspring, 50 mM EtOH failed to block LTP in PF1,2, t(28) = −2.817, p < 0.001, Fig. 3B, or PNEE1,2, t(28) = −4.059, p < 0.001, Fig. 3C, offspring. However, LTP levels did not significantly differ across the prenatal treatment groups (PF1,2 vs. AL, p = 0.144; PNEE1,2 vs. AL, p = 0.124; PNEE1,2 vs. PF1,2, p = 1.00). Similarly, LTP levels did not significantly differ across prenatal treatment groups following acute exposure to 20 mM EtOH (PF1,2 vs. AL, p = 0.999; PNEE1,2 vs. AL, p = 0.999; PNEE1,2 vs. PF1,2, p = 1.00; Fig. 3D).
Within PF1,2 offspring, 20 mM (55% reduction from PF1,2 0 mM, p = 0.048) and 50 mM (55% reduction from PF1,2 0 mM, p = 0.038) EtOH had similar effects on LTP, significantly attenuating LTP as compared to levels observed in the absence of EtOH (0 mM; Fig. 3B). LTP levels did not differ following 20 and 50 mM application (p = 1.00). Within PNEE1,2 offspring, LTP levels were not significantly affected by acute EtOH exposure (20 mM: 22% reduction from PNEE1,2 0 mM, p = 0.937; 50 mM: 51% reduction from PNEE1,2 0 mM, p = 0.109; Fig. 3C).
The interaction between prenatal treatment and gender indicates that male and female offspring were differently affected by prenatal treatment. Post hoc analysis revealed that LTP levels were significantly larger in PF1,2 females as compared to PF1,2 males (p = 0.003; data not shown). It is important to note that the interpretation of this finding is limited as it does not take into consideration the effects of acute EtOH application. It is important to note that although no other significant gender differences were found, it does appear as though prenatal treatment and acute EtOH application had less of a negative effect on LTP levels in females than in males (see Table 2 for a breakdown of LTP levels).
To test whether acute EtOH exposure affected slice health, I/O function was recorded at the end of each LTP recording. I/O curves were generated from the application of increasing stimulus pulse width. In all slices, the slope of the fEPSP significantly increased with increasing stimulation, repeated measures ANOVA, F(8, 920) = 1,871.59, p < 0.001. There was no main effect of prenatal treatment, acute EtOH exposure, or gender. Furthermore, there were no significant interactions between any of the variables, demonstrating that the attenuation of LTP by acute application of EtOH is not due to a rundown in the health of the slice (Fig. 4).
Figure 4. Input/output curves. (A) Output population response as a function of pulse width for all prenatal treatment groups and acute ethanol (EtOH) concentrations. Neither prenatal treatment nor acute EtOH application had any effect on the population response. Data presented as mean field excitatory postsynaptic potential (fEPSP) slope (% change relative to the response induced from the fifth pulse). (B) Samples of traces obtained from corresponding groups; fEPSPs recorded at each test pulse are superimposed. Symbols represent acute EtOH concentration, whereas colors represent prenatal treatment group.
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