Two-Photon Sodium Imaging in Dendritic Spines

Abstract

Neuronal activity causes substantial postsynaptic Na+ transients. However, the physiological consequences of such Na+ transients remain largely unknown. High-resolution Na+ imaging is pivotal to study these questions, and, up to now, two-photon imaging with the fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) has been the method of choice. When introduced into individual neurons in acute tissue slices, SBFI enables measurements of [Na+]i transients in dendrites and spines. This technique is also suitable for the determination of [Na+]i transients in other cell types, such as glial cells, with high spatial resolution, and is likely to be useful for measurement of Na+ signals in vivo. This protocol provides guidelines and tips for measuring dynamic Na+ concentrations within neurons, as well as an in situ calibration procedure. Because the basic properties of SBFI inside a cell differ significantly from the same properties in vitro, in situ calibrations are required to obtain meaningful experimental results.