Examination of Diurnal Variation and Sex Differences in Hippocampal Neurophysiology and Spatial Memory

LTP magnitude is greater at night compared with the day, regardless of sex. A, Average slopes of fEPSPs in response to increasing stimulation of the Schaffer collaterals (time-of-day × sex interaction: p < 0.001). Note that significant sex differences in input-output responses were observed only at 180 μA (p = 0.041), 190 μA (p = 0.042), and 200 μA (p = 0.035), regardless of time-of-day, as indicated by horizontal line above stimulation intensities. Data were plotted for two time points and both sexes: male day, n = 13 slices from 3 mice; male night, n =10 slices from 3 mice; female day, n = 14 slices from 3 mice; female night, n = 12 slices from 3 mice. B, Example fEPSPs from two male mice used to generate input-output curves in A. C, Average slopes of fEPSPs before and after a HFS (100 Hz, 0.5 s, 2×; t = 20 min) to Schaffer collaterals (time-of-day: *p = 0.003; means ± SEMs at 60 min, night: 1.451 ± 0.176; day: 1.286 ± 0.141). Data were plotted for two time points and both sexes: male day, n = 7 slices from 3 mice; male night, n = 8 slices from 3 mice; female day, n = 10 slices from 3 mice; female night, n = 9 slices from 3 mice. D, Example fEPSPs from two female mice used to produce LTP in C. In all plots, blue codes recordings at night and orange codes recordings during the day. ♀ = female. ♂ = male. LTP, Long-term potentiation; fEPSPs, field EPSPs; SEM, standard error of the mean; HFS, high-frequency tetanus. All statistical tests were performed with a three-way linear mixed model (input-output curves) or three-way, RM-ANOVA (LTP). Data are shown as means ± SEMs. For a detailed statistical summary, see Extended Data Table 1-1.

Action potential-mediated inhibition onto CA1 pyramidal neurons is greater during the day compared with night, regardless of sex. A, Example traces of IPSCs onto CA1 pyramidal neurons. Scale bars represent 100 pA and 1000 ms. B, Cumulative probability distribution plots for the IEI of sIPSCs. C, Estimated marginal means and confidence intervals of the IEI of sIPSCs; time-of-day: *p = 0.003; pooled EMM [95% confidence intervals] for night, 87.491 [82.215, 90.180] ms and day, 78.050 [72.164, 87.145] ms. D, Cumulative probability distribution plots for the amplitude of sIPSCs. E, Estimated marginal means and confidence intervals of the amplitude of sIPSCs; time-of-day: *p = 0.008; pooled EMM [95% confidence intervals] for day, 41.697 [37.523, 46.334] pA and night, 36.066 [35.424, 36.720] pA. sIPSCs were measured from five male mice during the day (n = 16 cells), five male mice at night (n = 28 cells), five female mice during the day (n = 18 cells), and five female mice at night (n = 17 cells). In all plots, blue codes recordings at night and orange codes recordings during the day. ♀ = female. ♂ = male. EMM, estimated marginal means; IEI, interevent interval; sIPSCs, spontaneous IPSCs. All statistical tests were performed with a two-way GEE model. Data were shown as EMM ± confidence intervals. For a detailed statistical summary, see Extended Data Table 1-1.

Action potential-independent inhibition onto CA1 pyramidal neurons is influenced by sex and time-of-day. A, Example traces of spontaneous, mIPSCs onto CA1 pyramidal neurons. Scale bars represent 100 pA and 500 ms. B, Cumulative probability distribution plots for the IEI of mIPSCs. C, Estimated marginal means and confidence intervals of the IEI of mIPSCs (interaction, p = 0.068). D, Cumulative probability distribution plots for the amplitude of mIPSCs. E, Estimated marginal means and confidence intervals of the amplitude of mIPSCs (interaction: *p = 0.038). Note that significant sex differences were observed during the day (p = 0.006) but not at night (p = 0.594). mIPSCs were measured from male five mice during the day (n = 12 cells), five male mice at night (n = 13 cells), five female mice during the day (n = 12 cells), and five female mice at night (n = 14 cells). In all plots, blue codes recordings at night and orange codes recordings during the day. ♀ = female. ♂ = male. EMM, estimated marginal means; IEI, interevent interval; mIPSCs, miniature IPSCs. All statistical tests were performed with a two-way GEE model followed by Wald χ² pairwise comparisons. Data were shown as EMM ± confidence intervals. For a detailed statistical summary, see Extended Data Table 1-1.

Action potential-mediated excitation onto CA1 pyramidal neurons depends on sex. A, Example traces of spontaneous sEPSCs onto CA1 pyramidal neurons. Scale bars represent 40 pA and 1000 ms. B, Cumulative probability distribution plots for the IEI of sEPSCs. C, Estimated marginal means ± confidence intervals of the IEI of sEPSCs; sex: *p = 0.022; pooled EMM [95% confidence intervals] for males, 469.002 [426.705, 515.530] ms and females, 311.536 [222.006, 436.968] ms. Note that differences in sEPSC IEIs recorded during the day (455.037 [410.344, 506.913] ms) and night (321.107 [230.600, 449.839] ms) failed to reach statistical significance (time-of-day: p = 0.052). D, Cumulative probability distribution plots for the amplitude of sEPSCs. E, EMM and confidence intervals of the amplitude of sEPSCs; sex: *p = 0.020; pooled EMM [95% confidence intervals] for females, 24.952 [23.898, 26.046] pA and males, 23.578 [23.111, 24.058] pA. sEPSCs were recorded from five male mice during the day (n = 17 cells), five male mice at night (n = 16 cells), five female mice during the day (n = 16 cells), and five female mice at night (n = 18 cells). In all plots, blue codes recordings at night and orange codes recordings during the day. ♀ = female. ♂ = male. EMM, estimated marginal means; IEI, interevent interval; sEPSCs, spontaneous EPSCs. All statistical tests were performed with a two-way GEE model. Data were shown as EMM ± confidence intervals. For a detailed statistical summary, see Extended Data Table 1-1.

Action potential-independent excitation onto CA1 pyramidal neurons is dependent on sex and time-of-day. A, Example traces of spontaneous mEPSCs onto CA1 pyramidal neurons. Scale bars represent 20 pA and 500 ms. B, Cumulative probability distribution plots for the IEI of mEPSCs. C, Estimated marginal means and confidence intervals of the IEI of mEPSCs (interaction: *p = 0.021). Note that significant time-of-day differences were observed in males (*p = 0.002) but not in females (p = 0.765). D, Probability distribution plots for the amplitude of mEPSCs. E, EMM and confidence intervals of the amplitude of mEPSCs (no significant effects). Spontaneous mEPSCs were recorded from four male mice during the day (n = 11 cells), three male mice at night (n = 13 cells), three female mice during the day (n = 14 cells), and four female mice at night (n = 13 cells). In all plots, blue codes recordings at night and orange codes recordings during the day. ♀ = female. ♂ = male. EMM, estimated marginal means; IEI, interevent interval; mEPSCs, miniature EPSCs. All statistical tests were performed with a two-way GEE model followed by Wald χ² pairwise comparisons. Data were shown as EMM ± confidence intervals. For a detailed statistical summary, see Extended Data Table 1-1.

Excitability of CA1 pyramidal neurons depends on time-of-day but not sex. A, Schematic locations of CA1 pyramidal neurons across the anterior-posterior axis in reference to the Allen Brain Atlas. Cells were considered “anterior” if recorded in hippocampal slices that corresponded to plate 48 or lower in the Allen brain atlas, and “posterior” if they corresponded to plate 49 or higher. B, Average number of action potentials fired in response to increasing depolarizing current injections in neurons recorded from anterior slices from 14 mice at night (n = 33 cells) and 11 mice during the day (n = 33 cells; time-of-day: *p = 0.046). C, Scatterplot of individual values with mean ± SEM of baseline membrane potentials of neurons recorded from anterior slices (time-of-day: ns p = 0.535). D, Example traces of a 240-pA current step response in anterior slices from two male mice recorded during the day and night. Scale bars represent 20 mV and 100 ms. E, Average number of action potentials fired in response to increasing depolarizing current injections in neurons recorded from posterior slices from 11 mice at night (n = 27 cells) and 11 mice during the day (n = 27 cells; time-of-day: ns p = 0.484). F, Scatterplot of individual values with mean ± SEM of baseline membrane potentials of neurons recorded from posterior slices (time-of-day: *p = 0.044). G, Example traces of a 240-pA current step response in posterior slices from two male mice recorded during the day and night. Scale bars represent 20 mV and 100 ms. In all plots, blue codes recordings at night and orange codes recordings during the day. SEM, standard error of the mean. All statistical tests were performed with a two-way RM-ANOVA. Data were shown as means ± SEMs. For a detailed statistical summary, see Extended Data Table 1-1.