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Intracellular chloride regulation mediates local sleep pressure in the cortex

Nature Neuroscience volume 26, pages 64–78 (2023)Cite this article

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Abstract

Extended wakefulness is associated with reduced performance and the build-up of sleep pressure. In the cortex, this manifests as changes in network activity. These changes show local variation depending on the waking experience, and their underlying mechanisms represent targets for overcoming the effects of tiredness. Here, we reveal a central role for intracellular chloride regulation, which sets the strength of postsynaptic inhibition via GABAA receptors in cortical pyramidal neurons. Wakefulness results in depolarizing shifts in the equilibrium potential for GABAA receptors, reflecting local activity-dependent processes during waking and involving changes in chloride cotransporter activity. These changes underlie electrophysiological and behavioral markers of local sleep pressure within the cortex, including the levels of slow-wave activity during non-rapid eye movement sleep and low-frequency oscillatory activity and reduced performance levels in the sleep-deprived awake state. These findings identify chloride regulation as a crucial link between sleep–wake history, cortical activity and behavior.

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Fig. 1: Cortical pyramidal neurons exhibit diurnal variations in EGABAA.
Fig. 2: Preceding sleep–wake history determines EGABAA in cortical pyramidal neurons.
Fig. 3: Cortical [Cl−]i regulation determines the level of local SWA during NREM sleep.
Fig. 4: Sleep–wake-dependent EGABAA determines how easily a cortical neuron is recruited to spike.
Fig. 5: Depolarized EGABAA boosts NREM SWA by enhancing cortical neuronal recruitment in vivo.
Fig. 6: [Cl−]i regulation underlies local low-frequency cortical oscillations in the sleep-deprived awake state.
Fig. 7: [Cl−]i regulation affects the levels of local NREM SWA observed with SD.
Fig. 8: Cortical [Cl−]i regulation underlies performance levels in the sleep-deprived awake state.

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Data availability

Example raw signals are included within the manuscript. Unprocessed electrophysiological recording data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

The code for automated vigilance scoring is available in open-source format at https://github.com/paulbrodersen/somnotate66. The code for the neuronal network simulation is available in open-source format at https://gist.github.com/paulbrodersen/b6ce791a927b7e7e714e6a322d90e2b7.

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Acknowledgements

We thank the laboratory of C.J.A. for advice and comments, the laboratory of V.V.V. for support with in vivo electrophysiology experiments and S. Peirson and A. Jagannath for commenting on the manuscript. We thank E. Tyler, L. Kravitz, L. Petrucco and Scidraw.io for providing the mouse drawings. The research leading to these results received funding from a Sir Henry Wellcome Postdoctoral Fellowship 206500/Z/17/Z (H.A.), a St. John’s College Junior Research Fellowship (H.A.), the European Research Council under grant agreement 617670 (C.J.A.), MRC project MR/S01134X/1 and Wellcome Trust project 106174/Z/14/Z. For the purpose of Open Access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission.

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Authors and Affiliations

  1. Department of Pharmacology, University of Oxford, Oxford, UK

    Hannah Alfonsa, Richard J. Burman, Paul J. N. Brodersen, Sarah E. Newey, Kashif Mahfooz & Colin J. Akerman

  2. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK

    Tomoko Yamagata & Vladyslav V. Vyazovskiy

  3. Department of Experimental Psychology, University of Oxford, Oxford, UK

    Marios C. Panayi & David M. Bannerman

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  1. Hannah Alfonsa
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Contributions

H.A. and C.J.A. conceptualized the project. H.A. and C.J.A. designed the in vitro experiments. H.A., C.J.A. and V.V.V. designed the in vivo electrophysiology experiments. H.A., C.J.A., V.V.V., M.C.P. and D.M.B. designed the behavioral experiments. H.A. performed and analyzed the in vitro electrophysiology experiments. R.J.B. performed in vivo gramicidin perforated patch recordings. H.A. and S.E.N. performed the western blotting experiments. H.A. performed and analyzed the in vivo electrophysiology experiments. K.M. performed the in utero electroporations. T.Y. assisted with the optogenetics experiments. H.A. performed and analyzed the behavioral experiments. P.J.N.B. performed the neuronal network modeling and wrote the automated sleep scoring algorithm. H.A. and C.J.A. wrote the manuscript with input from all authors.

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Correspondence to Hannah Alfonsa or Colin J. Akerman.

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Extended data

Extended Data Fig. 1 Cortical pyramidal neurons exhibit diurnal variations in EGABAA.

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.

Source data

Extended Data Fig. 2 Diurnal and sleep-wake dependent variations in EGABAA alter the GABAAR driving force.

a. The GABAAR driving force was calculated as the difference between EGABAA and the neuron’s resting membrane potential. The GABAAR driving force was significantly more depolarizing at ZT15 than at ZT3 in L5 pyramidal neurons (left; **p = 0.003, Mann-Whitney test; d = 1.08; blue: 17 neurons, 17 slices, 8 animals; orange: 21 neurons, 21 slices, 8 animals). b. The GABAAR driving force was more depolarizing at ZT3-SD when sleep pressure was high, than at ZT3 when sleep pressure was low (***p < 0.0001, unpaired t-test; t = 5.57; df = 25; d = 2.22; red: 10 neurons, 10 slices, 4 animals; blue: same data as ‘a’). c. Mice had their whiskers trimmed unilaterally at ZT3, when EGABAA is normally hyperpolarized, and were then subjected to 3 hours of SD (as in Fig. 2i). The GABAAR driving force was more depolarizing in the hemisphere contralateral to the intact whisker input, compared to the hemisphere contralateral to the trimmed whisker input (**p = 0.0078, Wilcoxon matched-pairs signed-ranks test; d = 1.38; 8 neuronal pairs, 8 slices, 3 animals). Data represent mean ± sem. All tests are two sided.

Source data

Extended Data Fig. 3 Levels of NREM SWA reflect recent sleep-wake history and indicate sleep pressure.

a. At ZT15, when mice have mainly been awake during the preceding 3 hours, spectral power shows higher levels of NREM SWA. At ZT3, when mice have mainly been asleep during the preceding 3 hours, lower levels of NREM SWA are observed (*p = 0.0137, Wilcoxon matched-pairs signed-ranks test; d = 1.49; blue: 10 days, 6 animals; orange: 10 days, 6 animals). SWA was monitored by frontal EEG and spectral power recorded over a two-hour period was normalized to the mean 12 hours light period. b. In a similar manner, mice at ZT0 that have mainly been awake during the preceding 3 hours show higher levels of NREM SWA compared to mice at ZT3, which have mainly been asleep during the preceding 3 hours (*p = 0.0241, unpaired t-test with Welch’s correction; t = 2.996; df = 6; d = 1.73; black: 6 days, 6 animals; grey: 6 days, 6 animals). SWA was monitored by LFP via a tungsten wire targeted to L5 of primary somatosensory cortex (S1) and spectral power recorded over a two-hour period was normalized to the mean 12 hours light period. Data represent mean ± sem. All tests are two sided.

Source data

Extended Data Fig. 4 Sleep-wake history affects phospho-NKCC1 levels in layer 5 pyramidal neurons.

a. Confocal microscope images of immunofluorescence detected using a rabbit polyclonal antibody raised against a linear diphosphopeptide corresponding to Thr212 and Thr217 of NKCC1 (‘pNKCC1’; right) in L5 neurons that underwent in vivo shRNA knockdown of NKCC1 (‘shRNA-NKCC1’; bottom) or received control shRNA (‘shRNA-control’; top). Co-expression of TdTomato (‘TdTom’; left) was used to identify neurons transfected with shRNA. Scale bar: 10 μm b. Compared to surrounding non-transfected cells, the pNKCC1 signal was significantly reduced in shRNA-NKCC1 L5 neurons (light blue: p < 0.0001, Wilcoxon signed rank test; d = 1.27; 60 neurons, 4 slices, 4 animals), but not in shRNA-control neurons (grey: p = 0.21, one sample t-test; t = 1.27; df = 59; d = 0.16; 60 neurons, 3 slices, 3 animals), and the two groups were significantly different (p < 0.0001, Mann-Whitney test; d = 1.27). This supports the conclusion that the pNKCC1 antibody signal reflects NKCC1 levels. Central line: mean; error bar: sem c. Immunofluorescence signals for a neuron-specific marker (‘NeuN; left) and pNKCC1 (right) in layer 5 somatosensory cortex from mice 3 hours after light onset under control conditions (top; ‘ZT3’) or after a 3-hour sleep deprivation protocol (bottom; ‘ZT3-SD’). Scale bar: 10 μm. d. Cumulative frequency plots of fluorescence (in arbitrary units) show higher pNKCC1 signal in the ZT3-SD condition compared to ZT3 (right; ***p < 0.0001, Kolmogorov-Smirnov test; blue: 480 neurons, 6 slices, 3 animals; red: 480 neurons, 6 slices, 3 animals). This was not the case for the NeuN signal in the same population of neurons (left; p = 0.11, Kolmogorov-Smirnov test). All tests are two sided.

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Extended Data Fig. 5 Spectral power during different vigilance states following bumetanide or VU infusion.

a-b. [Cl−]i was manipulated at different time points by locally infusing blockers of NKCC1 (bumetanide) or KCC2 (VU) into S1. Spectral power from REM (‘a’) and wake (‘b’) LFP following infusion of vehicle or bumetanide during the early light period (left), vehicle or bumetanide during the late light period (middle), and vehicle or VU during the late light period (right). No differences were observed in theta frequency (5–10 Hz) during REM sleep following early bumetanide infusion (left; p = 0.615, paired t-test; t = 0.54; df = 5; d = 0.22; black and blue: 6 trials, 5 animals), late bumetanide infusion (middle; p = 0.234, paired t-test; t = 1.35; df = 5; d = 0.55; black and blue: 6 trials, 5 animals), or late VU infusion (right; p = 0.356, paired t-test; t = 1.02; df = 5; d = 0.41; black and red: 6 trials, 4 animals). Data represent mean ± sem. All tests are two sided.

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Extended Data Fig. 6 Local NREM SWA and vigilance state distribution following bumetanide or VU infusion.

[Cl−]i was manipulated at different time points by locally infusing blockers of NKCC1 (bumetanide) or KCC2 (VU) into S1. a. Population data showing NREM SWA derived from the S1 LFP (that is local) following infusion in mice that received vehicle or bumetanide during the early light period (left; black and blue: 6 trials, 5 animals), vehicle or bumetanide during the late light period (middle; black and blue: 6 trials, 5 animals), and vehicle or VU during the late light period (right; black and red: 6 trials, 4 animals). b-d. Population data on vigilance states (that is global state) showing the proportion of time spent in NREM sleep (left), REM sleep (middle), and wake (right) following local infusion of vehicle or bumetanide during the early light period (b, black and blue: 5 trials, 5 animals), vehicle or bumetanide during the late light period (c, black and blue: 5 trials, 5 animals), and vehicle or VU during the late light period (d, black and red: 5 trials, 4 animals). Data are plotted in 1-hour intervals. Data represent mean ± sem.

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Extended Data Fig. 7 The effects of optical Cl− loading on NREM SWA and neuronal recruitment during ON and OFF periods.

a. Multi-unit activity from an animal expressing halorhodopsin in pyramidal neurons of S1, during which a 10 s period of light was delivered during NREM sleep (left). Opsin activation was confirmed by decreased spike rate during light activation (right; ***p < 0.0001, one sample t-test; t = 7.88; df = 39; d = 1.26; 39 trials, 12 days, 5 animals). b. Multi-unit activity from an animal expressing archaerhodopsin (‘Arch’; ***p < 0.0001, Wilcoxon signed rank test; d = 1.42; 32 trials, 8 days, 5 animals). c. Normalized SWA before (low [Cl−]i) and after (high [Cl−]i) halorhodopsin activation (5 s time bins) shows recovery kinetics following Cl− loading (39 trials, 14 days, 5 animals). d. Normalized spike rate before and after halorhodopsin activation (5 s time bins) shows recovery kinetics following Cl− loading (26 trials, 11 days, 5 animals). e. Normalized SWA before (low [Cl−]i) and after (‘Arch control’) archaerhodopsin activation (34 trials, 10 days, 5 animals). f. Normalized spike rate before and after archaerhodopsin activation (26 trials, 8 days, 5 animals). g. Representative LFP trace (0.5–12 Hz band pass filtered) during NREM sleep, recorded before (low [Cl−]i; top) and after (high [Cl−]i; bottom) halorhodopsin activation. Spikes are marked with dots. h. Spikes plotted on the Hilbert-transform of the filtered LFP signal. The OFF period was defined as the upward phase (30–150о angle) of high-amplitude SWA waveforms (>30% of baseline). i. Spike rate during the OFF phase of SWA was reduced after halorhodopsin activation (*p = 0.0178, one sample t-test; t = 2.538; df = 25; d = 0.5; 26 trials, 13 days, 5 animals). j. No change in spike rate after the Arch control (p = 0.6238, one sample t-test; t = 0.496; df = 29; d = 0.09; 30 trials, 8 days, 5 animals). k. Normalized spike rate (ON/OFF) during SWA was higher after halorhodopsin activation (***p < 0.0001, Wilcoxon matched-pairs signed rank test; d = 0.6; 25 trials, 11 days, 5 animals). l. No change in normalized spike rate (ON/OFF) during SWA recorded after the Arch control (p = 0.948, Wilcoxon matched-pairs signed-ranks test; d = 0.01; 29 trials, 8 days, 5 animals). Data represent mean ± sem. All tests are two sided.

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Extended Data Fig. 8 [Cl−]i regulation underlies local low-frequency cortical oscillations in the sleep-deprived awake state.

Data from Fig. 6 is replotted by normalizing the LFP and EEG spectral power in the 3rd hour of the sleep-deprived awake state, to the preceding 12 h baseline. Continuous awake LFP and EEG recordings were used to monitor local and global spectral power, respectively. Mice experienced a 3-hour SD protocol at the beginning of the light period (ZT0 to ZT3), during which [Cl−]i was manipulated by locally infusing blockers of NKCC1 or KCC2 into S1. a. The awake LFP normalized to 12 h baseline (left) revealed an increase in low-frequency cortical oscillations (2–6 Hz; black: 15 animals). The level of low-frequency oscillations was reduced by local infusion of bumetanide (blue versus black, *p = 0.0116, paired t-test; t = 3.582; df = 6; d = 1.35; blue: 7 animals, black: 7 animals) and increased by VU (red versus black, **p = 0.0078, Wilcoxon matched-pairs signed-ranks test; d = 0.89; red: 8 animals, black: 8 animals,). These manipulations did not affect the frontal EEG (right; blue versus black, p = 0.5122, paired t-test; t = 0.6965; df = 6; d = 0.26; blue: 7 animals, black: 7 animals; red versus black, p = 0.4467, paired t-test; t = 0.8255; df = 5; d = 0.34; red: 6 animals, black: 6 animals). b. To test the relationship of the low-frequency oscillations to activity-dependent processes during SD, whiskers were trimmed unilaterally just before the animal experienced the 3-hour SD protocol. Either vehicle control (Veh) or VU was infused unilaterally during SD. The awake LFP normalized to 12 h baseline (left) revealed that whisker trimming prevented the increase in local low-frequency cortical oscillations (blue versus black, ***p = 0.0007, paired t-test; t = 6.452; df = 6; d = 2.44; blue: 7 animals, black: 7 animals). This effect could be rescued by VU infusion into S1 (red versus blue, *p = 0.0122, unpaired t-test with Welch correction; t = 3.541; df = 6; d = 1.4; red: 7 animals). Neither whisker trimming nor S1 infusion affected the increase in low-frequency oscillations detected in the frontal EEG (right; blue versus black, p = 0.0873, paired t-test; t = 2.041; df = 6; d = 0.77; red versus blue p = 0.6495, unpaired t-test; t = 0.47; df = 12; d = 0.9). Data represent mean ± sem. All tests are two sided.

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Extended Data Fig. 9 Spectral power during different vigilance states after SD, time course of local NREM SWA after SD, and time course of vigilance state distribution after SD.

Mice experienced a 3-hour SD protocol at the beginning of the light period (ZT0 to ZT3), during which [Cl−]i was manipulated by locally infusing blockers of NKCC1 or KCC2 into S1. a-b. LFP spectral power during REM sleep (‘a’) and wake (‘b’) after SD. No differences were observed in theta frequency (5–10 Hz) during REM sleep with either bumetanide (left; p = 0.985, paired t-test; t = 0.02; df = 5; d = 0.01; black and blue: 6 trials, 5 animals) or VU (right; p = 0.5625, Wilcoxon matched-pairs signed-ranks test; d = 0.4; black and red: 6 trials, 4 animals). In addition, when we compared ZT3 and ZT3-SD vehicle control animals, there was also no difference in theta frequency during REM (p = 0.233, unpaired t-test; t = 1.23; df = 18; d = 0.55). c. Time course of NREM SWA after SD, as derived from the S1 LFP (black: 12 trials, 9 animals; blue: 6 trials, 5 animals; red: 6 trials, 4 animals). d. Time course of vigilance states (that is global state) showing the proportion of time spent in NREM sleep (top), REM sleep (middle), and wake (bottom) after SD (black: 10 trials, 9 animals; blue: 5 trials, 5 animals, red: 5 trials, 4 animals). Data represent mean ± sem. All tests are two sided.

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Extended Data Fig. 10 Activity-dependent regulation of cortical [Cl−]i determines the high levels of local NREM SWA associated with sleep deprivation.

a. Whiskers were trimmed unilaterally at ZT3, when [Cl−]i is normally low, and mice were then subjected to 3 hours of SD (left). NREM SWA was then analysed during the first 2 hours of sleep following SD. Example LFP signals (middle) show SWA recorded from S1 contralateral to intact whiskers (‘intact’; black) and S1 contralateral to the trimmed whiskers (‘trimmed’; blue). Data was collected from the same animal, with an interval of at least 2 days. The S1 contralateral to trimmed whiskers showed reduced SWA during NREM sleep (right; ***p = 0.0004, one sample t-test; t = 8.506; df = 5; d = 3.47; 6 animals). b. Using the same paradigm, [Cl−]i was raised in S1 contralateral to the trimmed whiskers by local infusion of the KCC2 blocker, VU (horizontal bar indicates period of infusion). Other conventions as in ‘a’. Pharmacologically raising [Cl−]i reversed the reduction in SWA associated with whisker trimming (right; **p = 0.0025, one sample t-test; t = 5.583; df = 5; d = 2.28; 6 animals). Data represent mean ± sem. All tests are two sided.

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Alfonsa, H., Burman, R.J., Brodersen, P.J.N. et al. Intracellular chloride regulation mediates local sleep pressure in the cortex. Nat Neurosci 26, 64–78 (2023). https://doi.org/10.1038/s41593-022-01214-2

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