Extended Data Fig. 1: Cortical pyramidal neurons exhibit diurnal variations in EGABAA.
From: Intracellular chloride regulation mediates local sleep pressure in the cortex
Activating Cl−-permeable receptors in current clamp, and estimating the equilibrium potential of GABAARs (EGABAA) or glycine receptors (EGlycine) under different recording conditions, supports the conclusion that cortical pyramidal neurons exhibit diurnal variations in intracellular chloride regulation. a. EGABAA shows diurnal variation in both juvenile and adult somatosensory cortex. EGABAA was more depolarized at ZT15 than at ZT3 in L5 pyramidal neurons from mice aged 4–5 weeks (left; *p = 0.015, unpaired t-test; t = 2.66; df = 21; d = 1.16; blue: 8 neurons, 8 slices, 2 animals; orange: 15 neurons, 15 slices, 3 animals) and in mice aged 8–12 weeks (right; *p = 0.016, unpaired t-test; t = 2.74; df = 14; d = 1.38; blue: 7 neurons, 7 slices, 5 animals; orange: 9 neurons, 9 slices, 5 animals; Multi group comparison: p = 0.0008 for ZT time effect, p = 0.27 for age effect, p = 0.75 for interaction between ZT time and age effect; 2-way Anova). b. The proportion of L2/3 pyramidal neurons in brain slices from auditory cortex that exhibited a depolarizing GABAAR response at ZT3 and ZT15 (left; p = 0.3, Fisher’s exact test; blue: 10 neurons, 10 slices, 7 animals; orange: 7 neurons, 7 slices, 4 animals). EGABAA in L2/3 pyramidal neurons of auditory cortex was more depolarized at ZT15 than at ZT3 (right; **p = 0.006, unpaired t-test; t = 2.97; df = 28; d = 1.09; blue: 13 neurons, 13 slices, 7 animals; orange: 17 neurons, 17 slices, 7 animals). EGABAA data from Fig. 1. c. The same pattern was observed when action potential activity was blocked by applying TTX throughout the recordings. A higher proportion of L5 pyramidal neurons exhibited a depolarizing GABAAR response at ZT15, compared to ZT3 (left; *p = 0.026, Fisher’s exact test; blue: 8 neurons, 8 slices, 4 animals; orange: 14 neurons, 14 slices, 5 animals). EGABAA was more depolarized at ZT15 than at ZT3 when action potential activity was blocked throughout the recordings (*p = 0.0109, unpaired t-test; t = 2.708; df = 31; d = 0.95; blue: 14 neurons, 14 slices, 7 animals; orange: 19 neurons, 19 slices, 7 animals). EGABAA data from Fig. 1. d. Activating Cl−-permeable glycine receptors, in the presence of TTX and the GABAB receptor blocker CGP55845 (CGP), revealed the same pattern. A higher proportion of L5 pyramidal neurons exhibited a depolarizing glycine response at ZT15 than at ZT3 (left; **p = 0.0097, Fisher’s exact test; blue: 6 neurons, 6 slices, 3 animals; orange: 8 neurons, 8 slices, 4 animals). EGlycine was more depolarized at ZT15 than at ZT3 (right; **p = 0.0033, unpaired t-test; t = 3.663; df = 12; d = 1.98; blue: 6 neurons, 6 slices, 3 animals; orange: 8 neurons, 8 slices, 4 animals). e. The same pattern was observed when GABAA receptors, GABAB receptors and action potential activity were all blocked. A higher proportion of L5 pyramidal neurons exhibited a depolarizing glycine response at ZT15 than at ZT3 (left; **p = 0.0097, Fisher’s exact test; blue: 6 neurons, 6 slices, 3 animals; orange: 8 neurons, 8 slices, 4 animals). EGlycine was more depolarized at ZT15 than at ZT3 (right; **p = 0.0026, unpaired t-test with Welch correction; t = 4.313; df = 8; d = 2.04; blue: 6 neurons, 3 animals; orange: 8 neurons, 4 animals). Data represent mean ± sem. f. Across all recordings conditions (a total of n = 57 neurons from 57 slices, 28 animals at ZT3 and 75 neurons from 75 slices, 30 animals at ZT15), the estimated difference in EGABAA/EGlycine between ZT3 and ZT15 was 11.1 mV (left; ***p < 0.0001, unpaired t-test with Welch correction; t = 7.98; df = 115; d = 1.29). Using the Nernst equation and treating EGABAA/EGlycine as an estimate of the equilibrium potential for Cl−, this would indicate a shift in [Cl−]i of approximately 4.4 mM, (right; ***p < 0.0001, Mann-Whitney Test; d = 1.08). Center line: median, box limits: 25th and 75th percentile, whiskers: non-outlier min and max, outlier: 1.5 x interquartile range away from the bottom or top of the box. Data represent mean ± sem. All tests are two sided.
