Research Article

Changes in the composition of brain interstitial ions control the sleep-wake cycle

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Science  29 Apr 2016:
Vol. 352, Issue 6285, pp. 550-555
DOI: 10.1126/science.aad4821
  • Fig. 1 Neuromodulators increase [K+]e concentration in the absence of neuronal activity.

    (A) Representative traces of [K+]e shifts before, during, and after administering the neuromodulator cocktail. Scale bar, x = 5 min, y = 0.2 mM [K+]e. (B) Thirty-second binned averages of shifts before and after neuromodulator cocktail. Arrow indicates time of cocktail administration. (C and D) Summary of [K+]e increase after neuromodulator cocktail application in slices ± TTX [paired t test. (C) –TTX, t(11) = 5.871, P = 0.0001; (D) +TTX, t(21) = 11.69, P < 0.0001]. (Inset) Representative field excitatory postsynaptic potential (fEPSP) recordings before and after application of TTX. Scale bar, 10 ms. (E) Summarized shifts at 10 min after changing perfusion solution. n = 6 cocktail-free ACSF, 12 cocktail, 10 ACSF+TTX, and 22 (cocktail+TTX) slices. One-way analysis of variance (ANOVA), F(3,46) = 25.94, P < 0.0001. Post-hoc Tukey test, **P < 0.01, ***P < 0.001. (F) Representative trace of large [K+]e spike after metabolic stress by using iodoacetate (IA). Trace includes pre-cocktail baseline, ACSF + cocktail, ACSF + IA, and return to baseline ACSF. (Right) Magnified trace showing [K+]e increases in slices treated with the neuromodulator cocktail (±TTX) followed by IA. Scale bars, x = 5 min, y = 0.2 mM [K+]e. Mean (black circle) ± SEM.

  • Fig. 2 Extracellular K+ is higher during wakefulness.

    (A and B) Representative ECoG, [K+]e, and EMG recordings with a data summary of state transitions in (A) sleep-to-awake or (B) awake-to-sleep. Initial [K+]e concentrations are shown to the left, and the 1- to 4-Hz power is displayed above so as to illustrate state-dependent shifts, binned at 10 s for clarity. n = 34 transitions; one-way, repeated meaures ANOVA, F(3,99) = 9.536, P < 0.0001. Tukey post-hoc multiple comparisons test, ***P < 0.001. Scale bar, x = 20 s, y = 0.1 mM [K+]e; ECoG = 0.75 mV; EMG = 0.3 mV (A) and 1 mV (B). (C) Representative recording of awake-to-isoflurane transitions recorded during the natural awake period (ZT16 to ZT20); n = 11 animals. One-way repeated measures ANOVA, F(2,20) = 35.61, P < 0.0001. Post-hoc Tukey test, ***P < 0.001. Scale bar, x = 1.5 min, y = 0.15 mM [K+]e, 0.75 mV EMG and ECoG. (D) Microdialysis samples collected from freely moving mice during their awake (ZT14 to ZT20) or sleeping (ZT2 to ZT8) period, and under isoflurane anesthesia. (Left) Schematic illustrating setup and inflow [K+] gradient, with representative ISM trace and sample no-net flux method plot used for [K+]e estimate. (Right) Summary of [K+]e by state. n = 12 awake, 12 sleep, and 13 isoflurane animals. One-way ANOVA, F(2,34) = 8.055, P = 0.0014. Post-hoc Tukey test, *P < 0.05, **P < 0.01. (E) Comparison of all state-dependent transitions. One-way ANOVA comparing mean shifts by group, F(5,161) = 16.61, P < 0.0001. Post-hoc Tukey test, *P < 0.05, **P < 0.01. Mean (black circle) ± SEM.

  • Fig. 3 Extracellular Ca2+ decreases during wakefulness.

    (A and B) Representative ECoG, EMG, and [Ca2+]e recordings in (A) sleep-to-awake and (B) awake-to-sleep. Initial [Ca2+]e is listed to the left with 1- to 4-Hz power presented above. n = 28 transitions. One-way, repeated measures ANOVA, F(3,81) = 39.91, P < 0.0001. Post-hoc Tukey test, ***P < 0.001. Scale bars, x = 20 s (A) and 40 s (B); y, [Ca2+]e = 0.05 mM, 0.75 mV EMG/ECoG. (C) Representative recording of isoflurane induction and recovery (ZT16 to ZT20) and data summary. n = 11 animals. One-way repeated measures ANOVA, F(2,20) = 18.52, P < 0.0001. Post-hoc Tukey test, ***P < 0.001. Scale bar, x = 6 min, y = [Ca2+]e = 0.2 mM, 0.5 mV EMG/ECoG. (D) Schematic of microdialysis collection. Individual sleep (light blue) and awake (gray) data are pooled and compared with isoflurane. n = 8 awake, 8 sleep, and 8 isoflurane animals. Two-tailed t test of isoflurane versus non-isoflurane, t(22) = 3.420, P = 0.003, **P < 0.01. (E) Comparisons of [Ca2+]e shifts from the awake-to-sleep, isoflurane, CNQX, and CNQX + isoflurane. n = 8 animals; CNQX and CNQX + isoflurane one-way ANOVA comparison of state-dependent shifts, F(4,117) = 5.824, P = 0.0003; Post-hoc Tukey test, *P < 0.05, **P < 0.01. Mean (black circle) ± SEM.

  • Fig. 4 Extracellular Mg2+ decreases during wakefulness.

    (A and B) Representative state transitions between sleep and awake ECoG, EMG, and Mg2+-sensitive microelectrodes, with data summary. Initial [Mg2+]e is given to the left of the ISM trace. n = 73 sleep-to-awake transitions (A) and 58 awake-to-sleep transitions (B). One-way ANOVA, F(3, 258) = 28.13, P < 0.0001. Post-hoc Tukey test, ***P < 0.001. Scale bars, x = 20 s, y = 0.05 mM [Mg2+]e, 0.33 mV EMG/ECoG. (C) Representative awake-to-isoflurane recording (ZT16 to ZT20) and summary of changes. n = 11 animals. One-way repeated measures ANOVA, F(2,20) = 28.13, P < 0.0001. Post-hoc Tukey test, *P < 0.05, ***P < 0.001. Scale bar, x = 5 min, y = 0.25 mM [Mg2+]e, 0.6 mV EMG/ECoG. (D) Schematic of microdialysis collection with data summary. Representative Mg2+-ISM and no-net flux calculation are shown. Sleep (light blue) and awake (gray) are pooled and compared with isoflurane. n = 8 awake, 8 sleep, and 8 isoflurane animals. Two-tailed t test of isoflurane versus pre-isoflurane, t(16) = 2.427, P = 0.0274 (*P < 0.05). (E) Comparison of state-dependent [Mg2+]e shifts from awake-to-sleep, isoflurane, CNQX, and awake + CNQX to isoflurane + CNQX. n = 6 awake-to-CNQX animals and 6 awake + CNQX-to-isoflurane + CNQX animals. One-way ANOVA of relative state-dependent shifts, F(4,167) = 27.31, P < 0.0001. Post-hoc Tukey test, **P < 0.01, ***P < 0.001. Mean (black circle) ± SEM.

  • Fig. 5 Imposing changes in extracellular ion concentrations alter local activity, extracellular space, and behavioral state.

    (A) Schematic of double-cranial window recording setup. Symmetrically positioned, separate cranial windows over opposing somatosensory cortices were prepared with ECoGs simultaneously recorded in both hemispheres. (B and C) Representative ECoG recordings in (B) sleeping (ZT4 to ZT8), and (C) awake (ZT16 to ZT20) mice. (Top) Representative recordings in ACSF mimicking natural state-dependent interstitial ion composition. (Bottom) Recordings after change of left-hemisphere ACSF to (B) awake-inducing or (C) sleep-inducing ACSF. (Right) Summary of 1- to 4-Hz ECoG power shift in the left hemisphere, normalized to the right, after change to (B) awake-inducing or (C) sleep-inducing ACSF. Paired t test of 1- to 4-Hz power shifts, (A) t(8) = 3.530, P = 0.008; (B) t(6) = 3.091, P = 0.0214. *P < 0.05, **P < 0.01. Scale bar, x = 4 min, y = 10%. (D) TMA+ traces of shifts in extracellular space volume (α) after switch from sleep to awake-inducing ACSF (top trace) or awake to sleep-inducing ACSF (bottom trace). Higher amplitude equals decreased dilution of TMA+ and smaller extracellular space. Data are summarized to the right. Paired t test, awake-inducing, n = 16 animals; t(15) = 11.04, P < 0.0001; sleep-inducing, n = 11 animals; t(10) = 8.95, P < 0.0001, **P < 0.01. Scale bars, x = 2 min, y = 2 mV. (E) Schematic of cisterna-magna infusion and wire EEG/EMG recording setup. (F and G) Representative traces showing EEG and EMG activity before, during, and after a 0.3- to 0.5-μl min−1 infusion of modified (F) awake-inducing or (G) sleep-inducing ACSF into the cisterna magna. The 1- to 4-Hz relative power (percentage of 1 to 32 Hz) is presented in averaged 10-min bins below. Infusion was run between (F) ZT5.5 and ZT7 (gray bar) and (G) ZT15 and ZT16.5 (purple bar) during sleep and awake periods, respectively. Scale bar, x = 30 min, y = 0.5 mV (F) and 1mV (G).

Supplementary Materials

  • Changes in the composition of brain interstitial ions control the sleep-wake cycle

    Fengfei Ding, John O’Donnell, Qiwu Xu, Ning Kang, Nanna Goldman, Maiken Nedergaard

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
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