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

Abstract

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 ~30% slower than in a free medium whereas inside neuronal dendrites this retardation is ~70%. In the synaptic cleft free nanodiffusion is decelerated by ~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.

Time-Resolved Integrative Optical Imaging of Diffusion during Spreading Depression

“…Volume fraction is the proportion of brain tissue volume occupied by the ECS and primarily governs concentration of molecules released into the ECS. Diffusion permeability Θ = D D , a ratio of the effective diffusion coefficient D to its value in an obstacle-free medium D, describes how much a diffusion-mediated process is slowed down in the ECS by obstacles represented by the cells and their various appendages (8), combined with other effects such as viscosity (9) or restricted diffusion (1). One additional parameter, κ, accounts for nonspecific clearance proportional to the concentration and describes removal of marker molecules over time.…”

Stochastic, structural and functional factors influencing AMPA and NMDA synaptic response variability: a review

“…Most of these factors essentially produce variable influences on the free diffusion of glutamate [62] by changing the diffusion coefficient (see definition of γ in eqn 2).…”

Electrodiffusion phenomena in neuroscience: a neglected companion

“…an effective increase in the diffusion path compared to a free medium, ranging between λ=1.4-1.6 (reviewed in 31 ). Thirdly, even on the nanoscale (no cellular obstacles) the extracellular ion movement is decelerated by 30-50% compared to a free medium 34 , likely due to microscopic steric hindrance (fixed or mobile molecular obstacles to diffusion) and viscous interactions 35 . An 6 additional influence may come from the extracellular matrix, be it a straightforward hindrance to diffusion 31 or the electrostatic interactions with ions 36 .…”