Fig. 1: 40 Hz light flickering enhances glymphatic influx and efflux measured by CSF fluorescence tracing.

a Schematic protocol of the measurement of cerebrospinal fluid influx after exposure for 30 min to control light (500 lux) or light flickering (white light, illuminance of 3000 lux, irradiance of 1.10 mW/cm² at 20 cm distance, 50% duty cycle) at frequencies of either 40 Hz or 80 Hz, followed by injecting the fluorescent CSF tracer (Y39-1) into the cisterna magna; after 30 min mice were sacrificed and Y39-1 fluorescence was measured in coronal brain sections. b Representative images showing that 40 Hz light flickering increased the parenchymal distribution of Y39-1 at 30 min after intracisternal injection; numbers indicate the anteroposterior distance from bregma in mm (scale bars, 1 mm). c, d Quantification of intracisternally injected Y39-1 mean pixel intensity (MPI, in arbitrary units, a.u.) and fluorescent area (expressed as % of section area) in whole sections 30 min after exposure to 40 Hz or 80 Hz light flickering during 30 min; analysis was performed on six sections per animal (n = 8 mice normal light, n = 6 mice 40 Hz, n = 6 mice 80 Hz, mean ± SEM in the bar graphs, ****P < 0.0001, ns, not significant, one-way ANOVA with Tukey’s multiple comparison test). e Representative images showing that 40 Hz light flickering at 3000 lux increased glymphatic influx, with similar effects at 4000 lux, compared to DC light stimulation at 3000 lux; 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 fluorescent area (expressed as % of section area) in whole sections; analysis was performed in six sections per animal (n = 6 mice/group, mean ± SEM in the bar graphs, *P < 0.05, ns, not significant, one-way ANOVA with Tukey’s multiple comparison test). h Schematic protocol of the measurement of CSF efflux after exposure for 30 min to control light or light flickering at frequencies of either 20, 40, or 80 Hz, followed by injecting the fluorescent CSF tracer (Y39-1) intrastriatally; after 60 min mice were sacrificed and Y39-1 fluorescence was measured in coronal brain sections. i Representative images showing that 40 Hz light flickering selectively decreased the Y39-1 fluorescence in the brain parenchyma; numbers indicate the anteroposterior distance from bregma in mm (scale bars, 1 mm). Quantification of the area (expressed as % of section area) covered by Y39-1 in coronal brain sections (j) and mean fluorescence (k) after exposure to 20, 40 or 80 Hz light flickering; analysis was performed in five sections per animal (n = 6–8 mice/group, mean ± SEM in the bar graphs, **P < 0.01, ns, not significant, one-way ANOVA with Tukey’s multiple comparison test). l Representative images showing that 40 Hz light flickering selectively increased the Y39-1 fluorescence in the deep cervical lymph nodes (scale bar, 100 μm). m Quantification of the area (expressed as % of section area) covered by Y39-1 in the deep cervical lymph nodes after exposure to 20, 40 or 80 Hz light flickering (mean ± SEM in the bar graphs, **P < 0.01, ns, not significant, one-way ANOVA with Tukey’s multiple comparison test).