In the original work, Nedergaard's group showed that glia, the brain's non-neuronal cells, control the flow of CSF through channels in their cell membranes. "If we delete the channels in glial cells, the flow almost stops," Nedergaard says. Because the transport of fluid across cell membranes requires a lot of energy, Nedergaard and her team had a hunch that the brain would not be able to both clean and process sensory information at the same time and decided to test whether the activity of the glymphatic system changed during sleep. Lulu Xie, the new study's first author, spent the next 2 years
Once Xie was sure the mice were asleep, based on their EEG brain activity, she injected a green dye into their CSF through a catheterlike device in their necks. After half an hour, she awakened them by touching their tails and injected a red dye that the two-photon microscope could easily distinguish from the green. By
A comparison of the volume of space between nerve cells while the mice were awake and asleep revealed that the glial channels carrying CSF expanded by 60 percent when the mice were asleep. The team also injected labeled β amyloid proteins into the brains of sleeping mice and awake mice and found that during sleep, CSF cleared away this "dirt" outside of the cells twice as quickly — "like a dishwasher," Nedergaard says. Such proteins can aggregate as pathogenic plaques inside cells and are associated with Alzheimer's disease, she says.