Two-photon sodium imaging in dendritic spines

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.