Nanoscale diffusion in the synaptic cleft and beyond measured with time-resolved fluorescence anisotropy imaging

Neural activity relies on molecular diffusion within nanoscopic spaces outside and inside nerve cells, such as synaptic clefts or dendritic spines. Measuring diffusion on this small scale in situ has not hitherto been possible, yet this knowledge is critical for understanding the dynamics of molecular events and electric currents that shape physiological signals throughout the brain. Here we advance time-resolved fluorescence anisotropy imaging combined with two-photon excitation microscopy to map nanoscale diffusivity in ex vivo brain slices. We find that in the brain interstitial gaps small molecules move on average similar to 30% slower than in a free medium whereas inside neuronal dendrites this retardation is similar to 70%. In the synaptic cleft free nanodiffusion is decelerated by similar to 46%. These quantities provide previously unattainable basic constrains for the receptor actions of released neurotransmitters, the electrical conductance of the brain interstitial space and the limiting rate of molecular interactions or conformational changes in the synaptic microenvironment.