(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.

(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.

(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.

(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.