Fig. 6: ENT2-mediated adenosine release mediates the enhancement of glymphatic flow induced by 40 Hz light flickering.

a, b Levels of extracellular adenosine quantified by UPLC in the CSF collected from the cisterna magna of anesthetized WT mice (a) or ENT2-KO mice (b) 30 min after exposure either to control light or 40 Hz light flickering for 30 min (n = 5–7 mice/group, mean ± SEM in the bar graphs, *P < 0.05, ns, not significant, unpaired Student’s t-test). c, d Representative western blot (c, out of 3 similar experiments) and quantification (d) of ENT2 and β-tubulin densities 30 min after exposure to normal light or 40 Hz light flickering during 30 min (n = 6 mice/group, mean ± SEM in the bar graphs, **P < 0.01, unpaired Student’s t-test). e Representative photographs of fluorescence in coronal brain sections collected 30 min after injection of the Y39-1 fluorescent tracer in the cisterna magna of WT mice (top two rows) or ENT2-KO mice (bottom two rows), previously exposed either to normal light or 40 Hz light flickering during 30 min; numbers indicate the anteroposterior distance from bregma in mm (scale bars, 1 mm). f, g Quantification of intracisternally injected Y39-1 MPI (in arbitrary units, a.u.) and of the fluorescent area (expressed as % of section area), showed that exposure to 40 Hz light flickering increased the glymphatic influx in WT mice, but not in ENT2-KO mice; analysis was performed in six sections per animal (n = 5–7 mice/group, mean ± SEM in the bar graphs, *P < 0.05, ***P < 0.001, ns, not significant, one-way ANOVA with Tukey’s multiple comparison test). h Representative in vivo two-photon images (100 μm below the cortical surface) repeatedly scanned at 10-min intervals showing that after 30 min of exposure to 40 Hz light flickering, the parenchymal distribution of Y39-1 (following its intracisternal injection) was not significantly different compared to ENT2-KO mice exposed to normal light (scale bars, 50 μm). i, j Quantification of intracisternally injected Y39-1 fluorescence intensity (arbitrary units, a.u.) in images 80–100 μm below the cortical surface showing that ENT2-KO mice did not display an increased parenchymal distribution triggered by 40 Hz light flickering (n = 3–4 mice/group, **P < 0.01, ns, not significant, two-way repeated measures ANOVA). k Average intensity of AQP4 staining centered on the vasculature in the cerebral cortex after exposure to either normal light or 40 Hz light flickering in ENT2-KO mice, with mean ± SEM indicated by the thick line (mean) with shading (SEM). l The average AQP4 polarization index increased after 40 Hz light flickering; the quantification of AQP4 polarization was carried out upon setting the baseline as the average intensity within a 10 μm range, from –20 μm to –10 μm relative to the point of peak fluorescence (n = 5–6 mice/group, mean ± SEM in the bar graph, **P < 0.01, ns, not significant, one-way ANOVA with Tukey’s multiple comparison test). m Representative images of vasomotion after 40 Hz light flickering in ENT2-KO mice. n Vasomotion was measured from ascending arteries and the increased vasomotion after 40 Hz light flickering observed in WT mice was abolished in ENT2-KO mice (n = 5–7 mice/group, mean ± SEM in the bar graphs, ns, not significant, one-way ANOVA with Tukey’s multiple comparison test).