Single-nanotube tracking reveals the nanoscale organization of the extracellular space in the live brain

The brain is a dynamic structure with the extracellular space (ECS) taking up almost a quarter of its volume(1,2). Signalling molecules, neurotransmitters and nutrients transit via the ECS, which constitutes a key microenvironment for cellular communication(3) and the clearance of toxic metabolites. The spatial organization of the ECS varies during sleep4, development5 and aging(6) and is probably altered in neuropsychiatric and degenerative diseases(7), as inferred from electron microscopy(8,9) and macroscopic biophysical investigations(2,10). Here we show an approach to directly observe the local ECS structures and rheology in brain tissue using super-resolution imaging. We inject single-walled carbon nanotubes into rat cerebroventricles and follow the near-infrared emission of individual nanotubes as they diffuse inside the ECS for tens of minutes in acute slices. Because of the interplay between the nanotube geometry and the ECS local environment, we can extract information about the dimensions and local viscosity of the ECS. We find a striking diversity of ECS dimensions down to 40 nm, and as well as of local viscosity values. Moreover, by chemically altering the extracellular matrix of the brains of live animals before nanotube injection, we reveal that the rheological properties of the ECS are affected, but these alterations are local and inhomogeneous at the nanoscale.