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.
Highlights
Introduction3D-diagram with Chem3D-Ultra) excited with polarised light (left: red arrows, polarisation plane; green arrow, excitation plane) moving and rotating over time Δt before emitting in the original excitation plane (yellow arrow); emission recorded through analysers IP(t) and I⊥(t). (b) Example: AF350 fluorescence anisotropy time course r (t) solution, as
3D-diagram with Chem3D-Ultra) excited with polarised light moving and rotating over time Δt before emitting in the original excitation plane; emission recorded through analysers IP(t) and I⊥(t). (b) Example: AF350 fluorescence anisotropy time course r (t) solution, as
We asked how the TR-FAIM-measured rotational diffusivity of AF350 DR is related to its translational diffusivity DT, over a range of medium viscosities
Summary
3D-diagram with Chem3D-Ultra) excited with polarised light (left: red arrows, polarisation plane; green arrow, excitation plane) moving and rotating over time Δt before emitting in the original excitation plane (yellow arrow); emission recorded through analysers IP(t) and I⊥(t). (b) Example: AF350 fluorescence anisotropy time course r (t) solution, as. (d) DT plotted against θ−1 (rotational diffusion coefficient; Methods) in aqueous solutions of varied viscosity: w/w percentage concentrations of glycerol (MW 92, left) and dextran (MW ~30 K, right) are shown; dotted lines, best-fit linear regression (Methods). High-resolution fluorescence imaging in organised tissue usually requires a femtosecond-pulse infra-red laser, to ensure highly confocal two-photon excitation (2PE) by ballistic photons, with no concomitant light scattering[21]. Bearing this in mind, we attempted to develop an approach that would enable the mapping of nanoscale molecular diffusivity in the mammalian brain tissue, inside and outside nerve cell compartments, in the established preparation of acute hippocampal slices under full electrophysiological control
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