Time-Resolved Integrative Optical Imaging of Diffusion during Spreading Depression

Abstract

ABSTRACTAn improved version of Integrative Optical Imaging method has been developed which substantially increases the time resolution of diffusion measurements. We present a theory for Time-Resolved Integrative Optical Imaging (TR-IOI) that incorporates time-dependent effective diffusion coefficient in homogeneous anisotropic media and time-dependent nonspecific linear clearance. The method was applied to measure the very fast changes in extracellular diffusion that occur during spreading depression in rat hippocampal slices. We were able to achieve time resolution of approximately one second, an improvement of at least ten times compared to the standard methods for extracellular diffusion measurement. We have found that diffusion of a small fluorescent extracellular marker (MW 3000) completely stopped during the maximum DC shift associated with the spreading depression wave, then gradually resumed over several minutes afterward. The effect of spreading depression on extracellular space is much larger than previously estimated by other methods with lower time resolution.