Microfiberoptic fluorescence photobleaching reveals size-dependent macromolecule diffusion in extracellular space deep in brain

Diffusion in brain extracellular space (ECS) is important for nonsynaptic intercellular communication, extracellular ionic buffering, and delivery of drugs and metabolites. We measured macromolecular diffusion in normally light-inaccessible regions of mouse brain by microfiberoptic epifluorescence photobleaching, in which a fiberoptic with a micron-size tip is introduced deep in brain tissue. In brain cortex, the diffusion of a noninteracting molecule [fluorescein isothiocyanate (FITC)-dextran, 70 kDa] was slowed 4.5 ± 0.5-fold compared with its diffusion in water (D_o/D), and was depth-independent down to 800 μm from the brain surface. Diffusion was significantly accelerated (D_o/D of 2.9±0.3) in mice lacking the glial water channel aquaporin-4. FITC-dextran diffusion varied greatly in different regions of brain, with D_o/D of 3.5 ± 0.3 in hippocampus and 7.4 ± 0.3 in thalamus. Remarkably, D_o/D in deep brain was strongly dependent on solute size, whereas diffusion in cortex changed little with solute size. Mathematical modeling of ECS diffusion required nonuniform ECS dimensions in deep brain, which we call "heterometricity, " to account for the size-dependent diffusion. Our results provide the first data on molecular diffusion in ECS deep in brain in vivo and demonstrate previously unrecognized hindrance and heterometricity for diffusion of large macromolecules in deep brain.