Article Figures & Data
Figures
- Figure 1.
Schematic illustration of the study design. Timed-pregnant female mice were administered either ethanol or an isocaloric/isovolumetric amount of sucrose by gavage from G10 to G18. Offspring were then administered the same treatment (ethanol or sucrose) by gavage from P4 to P14. Male offspring were food-restricted and tested for attention behavior using the 5-CSRTT from P60 to P193 (the age of the oldest mouse to complete testing). The same cohort of male offspring was then tested for electrophysiological function of medial prefrontal layer VI pyramidal neurons between P225 and P273. The coronal slice diagram was modified from Paxinos and Franklin, 2001. Timelines are not drawn to scale.
- Figure 2.
Developmental ethanol exposure impairs performance on an attention task in adulthood. Adult male offspring were trained on the 5-CSRTT for visual attention. Training began with the light stimulus duration set to 8 s, and each mouse was required to achieve the criteria of (i) 60 trials completed in 60 min, (ii) >80% accuracy, and (iii) <20% omissions for three of four consecutive days to advance to the next-lowest stimulus duration. The number of days required to meet criteria at each stimulus duration is shown in A, where the dotted line represents the minimum of 3 days. Mice that were administered ethanol during development required more days to reach criteria than mice that were administered sucrose during development, both during initial training on the task and also at the lowest stimulus duration that required the highest attentional demand (two-way repeated-measures ANOVA, effect of developmental treatment, p = 0.01; effect of stimulus duration, p < 0.0001; interaction, p = 0.001; Bonferroni’s post hoc test at 8 s, *p = 0.04, and at 1 s, §p < 0.0001). All remaining data are shown as the mean performance for all days up to and including the day on which each mouse met training criteria for each stimulus duration. B, Mice that were administered ethanol during development required more time to complete 60 trials at the initial 8-s stimulus duration (effect of developmental treatment, p = 0.1; effect of stimulus duration, p < 0.0001; interaction, p < 0.0001; Bonferroni’s post hoc test at 8 s, §p < 0.0001). Mice that were administered ethanol showed lower accuracy at the initial 8-s stimulus duration (C, effect of developmental treatment, p = 0.6; effect of stimulus duration, p < 0.0001; interaction, p = 0.009; Bonferroni’s post hoc test at 8 s, ‡p = 0.005), and also showed greater omissions, which was most pronounced at lower stimulus durations (D, effect of developmental treatment, p = 0.01; effect of stimulus duration, p < 0.0001; interaction, p = 0.003; Bonferroni’s post hoc test at 1.2 s, *p = 0.04, and at 1 s, §p < 0.0001). E, The number of premature responses per session was affected by stimulus duration (p < 0.0001) but not by developmental treatment (p = 0.4). F, The latency to make correct responses also was affected by stimulus duration (p < 0.0001) but not by developmental treatment (p = 0.9). All data are shown as mean + 1 SEM.
- Figure 3.
Developmental ethanol exposure impairs performance on the 5-CSRTT even when mice are considered to be trained. Data are shown as means for the 3 days on which each mouse met the training criteria for each stimulus duration. A, Mice that were administered ethanol during development required more time to complete 60 trials at the 8-s stimulus duration than mice that were administered sucrose during development (two-way repeated-measures ANOVA; effect of developmental treatment, p = 0.3; effect of stimulus duration, p < 0.0001; interaction, p = 0.01; Bonferroni’s post hoc test at 8 s, ‡p = 0.001). B, Mice that were administered ethanol during development committed more errors of omission when trained on the task, and this effect was most prominent at lower stimulus durations that required higher attentional demand (effect of developmental treatment, p = 0.03; effect of stimulus duration, p < 0.0001; interaction, p = 0.04; Bonferroni’s post hoc test at 1.6 s, *p = 0.02, and at 1 s, *p = 0.04). All data are shown as mean + 1 SEM.
- Figure 4.
Developmental ethanol exposure decreases the intrinsic excitability of adult medial prefrontal layer VI pyramidal neurons. A, Neurons from mice that were administered ethanol during development required more current to reach action potential threshold from rest (rheobase) than neurons from mice that were administered sucrose during development (two-tailed unpaired t test, *p = 0.009). B, The input–output curve is shifted to the right in neurons from mice that were administered ethanol during development (two-way repeated-measures ANOVA; interaction between effects of current and developmental treatment, p < 0.0001; effect of developmental treatment within each indicated segment, *p < 0.04). Representative action potential trains elicited by 100-pA current steps are shown in C for one neuron from each developmental treatment group. D, AHP amplitude at the end of the action potential trains elicited by 100- and 250-pA current steps is greater in neurons from mice that were administered ethanol during development (two-way repeated-measures ANOVA on log-transformed data, p = 0.02; Mann–Whitney U test on raw data for each current step, p < 0.04). Representative AHP traces are shown on the right for one neuron from each developmental treatment group. All data are shown as mean ± 1 SEM.
- Figure 5.
Developmental ethanol exposure increases nicotinic receptor function in adult medial prefrontal layer VI pyramidal neurons. A, The peak inward current response to 1 mm acetylcholine (15 s in the presence of 200 nm atropine) was significantly greater in neurons from mice that were administered ethanol during development than in neurons from mice that were administered sucrose during development (two-tailed unpaired t test, *p = 0.01). Exemplary voltage-clamp traces are shown on the right for one neuron from each developmental treatment group. B, For neurons that had been induced to fire action potentials by current injection, further nicotinic stimulation with 1 mm acetylcholine (15 s in the presence of 200 nm atropine) increased firing frequency to a greater degree in neurons from mice that were administered ethanol during development (Mann–Whitney U test, ‡p = 0.008). Exemplary current-clamp traces are shown on the right for one neuron from each developmental treatment group. The instantaneous firing frequency for this experiment is plotted against time in C1, where a significant effect of developmental treatment was observed during the acetylcholine response period (two-way ANOVA, §p < 0.0001). Firing frequency peaked at a greater magnitude (C2, Mann–Whitney U test, *p = 0.01) and occurred at an earlier time (C3, two-tailed unpaired t test, *p = 0.01) in neurons from mice that were administered ethanol during development. Acetylcholine applications are indicated on all traces by a gray bar. All data are shown as mean ± 1 SEM.
- Figure 6.
Exemplary traces of recorded glutamatergic sEPSCs. A, Exemplary traces are shown for one neuron from the sucrose (A1) and ethanol (A2) developmental treatment groups held at –75 mV in voltage-clamp mode. For each neuron, traces of approximately 10 s in length are shown at the top, and four individual exemplary sEPSCs are shown at the bottom. B, The average of 200 representative EPSC traces is shown for neurons from the sucrose (blue) and ethanol (red) developmental treatment groups. Data for the frequency, amplitude, and kinetics of sEPSCs in this study are shown in Table 4.
- Figure 7.
Developmental ethanol exposure increases AMPA receptor function in adult medial prefrontal layer VI pyramidal neurons. A, The peak inward current response to 2 μm (S)-AMPA (15 s) was not significantly different between neurons from mice that were administered ethanol during development and neurons from mice that were administered sucrose during development (Mann–Whitney U test, p = 0.1). Exemplary voltage-clamp traces are shown on the right for one neuron from each developmental treatment group. B, For neurons that had been induced to fire action potentials by current injection, further glutamatergic stimulation with 2 μm (S)-AMPA (15 s) increased firing frequency to a greater degree in neurons from mice that were administered ethanol during development (two-tailed unpaired t test, *p = 0.04). Exemplary current-clamp traces are shown on the right for one neuron from each developmental treatment group. The instantaneous firing frequency for this experiment is plotted against time in C1, where a significant effect of developmental treatment was observed during the (S)-AMPA response period (two-way repeated-measures ANOVA, *p = 0.02). The peak firing frequency was not significantly different between developmental treatment groups (C2, two-tailed unpaired t test, p = 0.6) although it did occur at an earlier time in neurons from mice that were administered ethanol during development (C3, two-tailed unpaired t test, *p = 0.047). AMPA applications are indicated on all traces by a gray bar. All data are shown as mean ± 1 SEM.
Tables
Line Location Type of test p-value a Table 2 Two-tailed Mann-Whitney U test (gestation length) 0.3 b Table 2 Two-tailed unpaired t test (litter size) 0.9 c Table 2 Bonferroni’s post hoc test (body weight at P4) 1.0 d Table 2 Bonferroni’s post hoc test (body weight at P14) 1.0 e Table 2 Bonferroni’s post hoc test (body weight at P21) 1.0 f Table 2 Bonferroni’s post hoc test (body weight at P28) 1.0 g Table 2 Bonferroni’s post hoc test (body weight at P60) 1.0 h Fig. 2A Two-way repeated-measures ANOVA (stimulus duration) F(7,203) = 13.2; p < 0.0001 i Fig. 2A Two-way repeated-measures ANOVA (treatment) F(1,29) = 6.9; p = 0.01 j Fig. 2A Bonferroni’s post hoc test at 8 s p = 0.04 k Fig. 2A Bonferroni’s post hoc test at 1 s p = 0.0001 l Fig. 2B Two-way repeated-measures ANOVA (stimulus duration) F(7,203) = 178.2; p < 0.0001 m Fig. 2B Two-way repeated-measures ANOVA (treatment) F(1,29) = 2.3; p = 0.1 n Fig. 2B Two-way repeated-measures ANOVA (stimulus duration X treatment) F(7,203) = 5.3; p < 0.0001 o Fig. 2B Bonferroni’s post hoc test at 8 s p <0.0001 p Fig. 2C Two-way repeated-measures ANOVA (stimulus duration) F(7,203) = 26.7, p < 0.0001 q Fig. 2C Two-way repeated-measures ANOVA (treatment) F(1,29) = 0.2; p = 0.6 r Fig. 2C Two-way repeated-measures ANOVA (stimulus duration × treatment) F(7,203) = 2.8; p = 0.009 s Fig. 2C Bonferroni’s post hoc test at 8 s p = 0.005 t Fig. 2D Two-way repeated-measures ANOVA (stimulus duration) F(7,203) = 38.4; p < 0.0001 u Fig. 2D Two-way repeated-measures ANOVA (treatment) F(1,29) = 7.1; p = 0.01 v Fig. 2D Two-way repeated-measures ANOVA (stimulus duration × treatment) F(7,203) = 3.2; p = 0.003 w Fig. 2D Bonferroni’s post hoc test at 1.2 s p = 0.04 x Fig. 2D Bonferroni’s post hoc test at 1.0 s p < 0.0001 y Fig. 2E Two-way repeated-measures ANOVA (stimulus duration) F(7,203) = 58.6; p < 0.0001 z Fig. 2E Two-way repeated-measures ANOVA (treatment) F(1,29) = 0.8; p = 0.4 aa Fig. 2F Two-way repeated-measures ANOVA (stimulus duration) F(7,203) = 430.1; p < 0.0001 ab Fig. 2F Two-way repeated-measures ANOVA (treatment) F(1,29) = 0.01; p = 0.9 ac Fig. 3A Two-way repeated-measures ANOVA (stimulus duration) F(7,203) = 73.1; p < 0.0001 ad Fig. 3A Two-way repeated-measures ANOVA (treatment) F(1,29) = 1.3; p = 0.3 ae Fig. 3A Two-way repeated-measures ANOVA (stimulus duration × treatment) F(7,203) = 2.5; p = 0.02) af Fig. 3A Bonferroni’s post hoc test at 8 s p = 0.001 ag Fig. 3B Two-way repeated-measures ANOVA (stimulus duration) F(7,203) = 42.8; p < 0.0001 ah Fig. 3B Two-way repeated-measures ANOVA (treatment) F(1,29) = 5.6; p = 0.03 ai Fig. 3B Bonferroni’s post hoc test at 1.6 s p = 0.02 aj Fig. 3B Bonferroni’s post hoc test at 1.0 s p = 0.04 ak Table 3 Two-tailed Mann-Whitney U test (capacitance) p = 0.002 al Table 3 Two-tailed Mann-Whitney U test (input resistance) p = 0.09 am Table 3 Two-tailed Mann-Whitney U test (resting membrane potential) p = 0.5 an Table 3 Two-tailed Mann-Whitney U test (spike amplitude) p = 0.8 ao Fig. 4A Two-tailed unpaired t test p = 0.009 ap Fig. 4B Two-way repeated-measures ANOVA (current injected × treatment) F(10,1930) = 4.7; p < 0.0001 aq Fig. 4B (rising phase) Two-way repeated-measures ANOVA (current injected) F(3,579) = 922.1; p < 0.0001 ar Fig. 4B (rising phase) Two-way repeated-measures ANOVA (treatment) F(1,193) = 4.9; p = 0.03 as Fig. 4B (rising phase) Two-way repeated-measures ANOVA (current injected × treatment) F(3,579) = 2.3; p = 0.07 at Fig. 4B (descending phase) Two-way repeated-measures ANOVA (current injected) F(3,579) = 144.3; p < 0.0001 au Fig. 4B (descending phase) Two-way repeated-measures ANOVA (treatment) F(1,193) = 4.2, p = 0.04 av Fig. 4B (descending phase) Two-way repeated-measures ANOVA (current injected × treatment) F(3,579) = 0.3; p = 0.8 aw Fig. 4D Two-way repeated-measures ANOVA on log-transformed data (current injected) F(1,174) = 56.4; p < 0.0001 ax Fig. 4D Two-way repeated-measures ANOVA on log-transformed data (treatment) F(1,174) = 5.2; p = 0.02 ay Fig. 4D Two-way repeated-measures ANOVA on log-transformed data (current injected × treatment) F(1,174) = 0.04; p = 0.8 az Fig. 4D Two-tailed Mann-Whitney U test (at 100 pA) p = 0.03 ba Fig. 4D Two-tailed Mann-Whitney U test (at 250 pA) p = 0.04 bb Fig. 5A Two-tailed unpaired t test p = 0.01 bc Fig. 5B Two-tailed Mann-Whitney U test p = 0.008 bd Fig. 5C1 Two-way repeated-measures ANOVA (time) F(11,1419) = 30.5; p < 0.0001 be Fig. 5C1 Two-way repeated-measures ANOVA (treatment) F(1,1419) = 35.8; p < 0.0001 bf Fig. 5C2 Two-tailed unpaired t test p = 0.01 bg Fig. 5C3 Two-tailed Mann-Whitney U test p = 0.01 bh Table 4 Two-tailed Mann-Whitney U test (frequency) p = 0.6 bi Table 4 Two-tailed Mann-Whitney U test (amplitude) p = 0.08 bj Table 4 Two-tailed Mann-Whitney U test (10–90 rise) p = 0.0008 bk Table 4 Two-tailed Mann-Whitney U test (10–90 slope) p = 0.02 bl Table 4 Two-tailed Mann-Whitney U test (decay) p = 0.9 bm Fig. 7A Two-tailed Mann-Whitney U test p = 0.1 bn Fig. 7B Two-tailed unpaired t test p = 0.04 bo Fig. 7C1 Two-way repeated-measures ANOVA (time) F(11,311) = 4.0; p < 0.0001 bp Fig. 7C1 Two-way repeated-measures ANOVA (treatment) F(1,311) = 5.4; p = 0.02 bq Fig. 7C2 Two-tailed unpaired t test p = 0.6 br Fig. 7C3 Two-tailed unpaired t test p = 0.047 bs Table 5 Two-tailed Pearson correlation coefficient As indicated bt Table 6 Two-tailed Pearson correlation coefficient As indicated Characteristic Sucrose Ethanol p-value Number of litters 8 9 Gestation length (d) 19.9 ± 0.1 20.3 ± 0.2 0.3a Litter size (number of pups at P4) 8.4 ± 0.9 8.2 ± 0.6 0.9b Offspring body weight (g) P4 (female and male) 2.8 ± 0.1 2.9 ± 0.1 1.0c P14 (female and male) 7.1 ± 0.2 6.9 ± 0.1 1.0c P21 (male only) 9.8 ± 0.6 9.6 ± 0.2 1.0c P28 (male only) 16.5 ± 0.9 16.7 ± 0.3 1.0c P60 (male only) 24.6 ± 0.8 24.7 ± 0.2 1.0c Data are presented as litter mean ± 1 SEM. Data sets were analyzed by aMann–Whitney U test, btwo-tailed unpaired t test, or cBonferroni’s post hoc test.
Characteristic Sucrose Ethanol p-value Number of mice 14 16 Number of neurons 104 114 Capacitance (pF) 56.9 ± 0.9 53.4 ± 0.9 0.002* Input resistance (MΩ) 228.7 ± 7.5 240.6 ± 7.8 0.09 Resting membrane potential (mV) –78.7 ± 0.5 –78.3 ± 0.4 0.5 Spike amplitude (mV) 95.0 ± 0.5 94.7 ± 0.5 0.8 Data are presented as mean ± 1 SEM for neurons within each data set. Data sets were analyzed by Mann–Whitney U test. *Statistically significant (p < 0.05).
Characteristic Sucrose Ethanol p-value Number of mice 14 16 Number of neurons 98 104 Frequency (Hz) 0.65 ± 0.06 0.68 ± 0.05 0.6 Amplitude (pA) 11.4 ± 0.3 12.4 ± 0.4 0.08 10–90 Rise (ms) 2.7 ± 0.1 2.3 ± 0.1 0.0008* 10–90 Slope (pA/ms) –5.5 ± 0.3 –7.2 ± 0.5 0.02* Decay (ms) 4.9 ± 0.2 5.0 ± 0.2 0.9 Data are presented as mean ± 1 SEM for neurons within each data set. Data sets were analyzed by Mann–Whitney U test. *Statistically significant (p < 0.05).
- Table 5.
Correlation analysis comparing electrophysiological properties of prefrontal layer VI pyramidal neurons and accuracy percentage at the 8-s stimulus duration in the 5-CSRTT.
Correlation versus accuracy Sucrose Ethanol Pearson r p-value Pearson r p-value Resting membrane potential –0.37 0.19 0.08 0.78 Capacitance 0.28 0.34 –0.09 0.73 Input resistance –0.41 0.14 –0.01 0.99 Spike amplitude 0.25 0.38 0.29 0.27 Rheobase 0.51 0.06 0.22 0.52 Receptor-mediated inward currents Nicotinic 0.61 0.02 0.21 0.43 Muscarinic 0.03 0.92 0.46 0.07 AMPA glutamatergic –0.06 0.87 –0.16 0.77 Receptor-mediated stimulation of firing neurons Nicotinic 0.51 0.06 0.08 0.78 Muscarinic 0.41 0.17 0.27 0.31 AMPA glutamatergic 0.15 0.72 –0.25 0.64 - Table 6.
Correlation analysis comparing electrophysiological properties of prefrontal layer VI pyramidal neurons and percentage of omissions at the 1-s stimulus duration in the 5-CSRTT.
Correlation versus omissions Sucrose Ethanol Pearson r p-value Pearson r p-value Resting membrane potential 0.46 0.09 0.25 0.35 Capacitance 0.31 0.27 0.23 0.40 Input resistance 0.02 0.95 0.25 0.35 Spike amplitude 0.33 0.24 0.33 0.21 Rheobase –0.17 0.55 –0.01 0.98 Receptor-mediated inward currents Nicotinic –0.52 0.05 0.03 0.93 Muscarinic 0.09 0.77 –0.08 0.78 AMPA glutamatergic 0.51 0.16 0.31 0.55 Receptor-mediated stimulation of firing neurons Nicotinic –0.58 0.03 0.02 0.93 Muscarinic –0.28 0.36 0.14 0.62 AMPA glutamatergic 0.10 0.81 0.27 0.61
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