Sleep drives metabolite clearance from the adult brain

Abstract

The conservation of sleep across all animal species suggests that sleep serves a vital function. We here report that sleep has a critical function in ensuring metabolic homeostasis. Using real-time assessments of tetramethylammonium diffusion and two-photon imaging in live mice, we show that natural sleep or anesthesia are associated with a 60% increase in the interstitial space, resulting in a striking increase in convective exchange of cerebrospinal fluid with interstitial fluid. In turn, convective fluxes of interstitial fluid increased the rate of β-amyloid clearance during sleep. Thus, the restorative function of sleep may be a consequence of the enhanced removal of potentially neurotoxic waste products that accumulate in the awake central nervous system.

Fig. 1. Wakefulness suppresses influx of CSF tracers

(A) Diagram of experimental setup used for two-photon imaging of CSF tracer movement in real time. To avoid disturbing the state of brain activity, a cannula with dual ports was implanted in the cisterna magna for injection of CSF tracers. ECoG and EMG were recorded to monitor the state of brain activity. (B) Three-dimensional (3D) vectorized reconstruction of the distribution of CSF tracers injected in a sleeping mouse and then again after the mouse was awakened. The vasculature was visualized by means of cascade blue-dextran administered via the femoral vein. FITC-dextran (green) was first injected in the cisterna magna in a sleeping mouse and visualized by collecting repeated stacks of z-steps. Thirty min later, the mouse was awakened by gently moving its tail, and Texas red-dextran (red) was administered 15 min later. The experiments were performed mostly asleep (12 to 2 p.m.). The arrow points to penetrating arteries. (C) Comparison of time-dependent CSF influx in sleep versus awake. Tracer influx was quantified 100 μm below the cortical surface; n = 6 mice; *P < 0.05, two-way ANOVA with Bonferroni test. (Right) The tracer intensity within the two arousal states at the 30-min time point was compared. **P < 0.01, t test. (D) ECoG and EMG recordings acquired during sleep and after the mouse was awakened. Power spectrum analysis of all the animals analyzed in the two arousal states (n = 6 mice; *P < 0.05, t test). (E) 3D reconstruction of CSF tracer influx into the mouse cortex. FITC-dextran was first injected in the awake stage, and cortical influx was visualized by means of two-photon excitation for 30 min. The mouse was then anesthetized with ketamine/xylazine (intraperitoneally), and Texas red-dextran was injected intra-cisternally 15 min later. The vasculature was visualized by means of cascade blue-dextran. Arrows point to penetrating arteries. (F) Comparison of time-dependent CSF influx in awake versus ketamine/xylazine anesthesia; n = 6 mice; *P < 0.05, two-way ANOVA with Bonferroni test. (Right) The tracer intensity during the two arousal states at the 30-min time point was compared. **P < 0.01, t test. (G) ECoG and EMG recordings in the awake mouse and after administration of ketamine/xylazine. Power spectrum analysis of all the animals analyzed in the two arousal states; n = 6 mice; *P < 0.05, t test.

Fig. 2. Real-time TMA⁺ iontophoretic quantification of the volume of the extracellular space in cortex

(A) TMA⁺ was delivered with an ion-tophoresis microelectrode during continuous recordings by a TMA⁺-sensitive microelectrode located a distance of ~150 μm away. The electrodes were filled with Alexa488 and Alexa568, respectively, so that their distance could be determined with two-photon excitation (insert over objective). A smaller extracellular space results in reduced TMA⁺ dilution, reflected by higher levels of detected TMA⁺. (B) The extracellular space is consistently smaller (α) in awake than in sleeping mice, whereas the tortuosity remained unchanged (λ); n = 4 to 6 mice; **P < 0.01, t test. (C) Power spectrum analysis of ECoG recordings; n = 6 mice; *P < 0.05, t test. (D) The extracellular space was consistently smaller in the awake state than after administration of ketamine/xylazine in paired recordings within the same mouse, whereas tortuosity did not change after anesthesia; n = 10 mice; **P < 0.01, t test. (Bottom) TMA measurements obtained during the two arousal states compared for each animal. (E) Power spectrum analysis of ECoG; n = 6 mice; *P < 0.05, t test.

Fig. 3. Sleep improves clearance of Aβ

(A). Time-disappearance curves of ¹²⁵I-Aβ_1-40 after its injection into the frontal cortex in awake (orange triangles), sleeping (green diamonds), and anesthetized (red squares, ketamine/xylazine) mice. (B) Rate constants derived from the clearance curves. (C) Time-disappearance curves of ¹⁴C-inulin after its injection into the frontal cortex of awake (orange triangles), sleeping (green diamonds), and anesthetized (red squares, ketamine/xylazine) mice. (D) Rate constants derived from the clearance curves. A total of 77 mice were included in the analysis: 25 awake, 29 asleep, and 23 anesthetized, with 3 to 6 mice per time point. *P < 0.05 compared with awake, ANOVA with Bonferroni test.

Fig. 4. Adrenergic inhibition increases CSF influx in awake mice

(A) CSF tracer influx before and after intracisternal administration of a cocktail of adrenergic receptor antagonists. FITC-dextran (yellow, 3 kD) was first injected in the cisterna magna in the awake mouse, and cortical tracer influx was visualized by means of two-photon excitation for 30 min. The adrenergic receptor antagonists (prazosin, atipamezole, and propranolol, each 2 mM) were then slowly infused via the cisterna magna cannula for 15 min followed by injection of Texas red-dextran (purple, 3 kD). The 3D reconstruction depicts CSF influx 15 min after the tracers were injected in cisterna magna. The vasculature was visualized by means of cascade blue-dextran. Arrows point to penetrating arteries. (B) Comparison of tracer influx as a function of time before and after administration of adrenergic receptor antagonists. Tracer influx was quantified in the optical section located 100 μm below the cortical surface; n = 6 mice; *P < 0.05, two-way ANOVA with Bonferroni test. (Right) The tracer intensity during the two arousal states at the 30-min time point was compared. **P < 0.01, t test. (C) Comparison of the interstitial concentration of NE in cortex during head-restraining versus unrestrained (before and after), as well as after ketamine/xylazine anesthesia. Microdialysis samples were collected for 1 hour each and analyzed by using high-performance liquid chromatography. **P < 0.01, one-way ANOVA with Bonferroni test. (D) TMA⁺ iontophoretic quantification of the volume of the extracellular space before and after adrenergic inhibition; n = 4 to 8 mice; **P < 0.01, t test. (E) Power spectrum analysis, n = 7 mice; **P < 0.01, one-way ANOVA with Bonferroni test.