Dual video microscopic imaging of membrane potential and cytosolic calciumof immunoidentified embryonic rat cortical cells

Membrane potential (MP) and cytosolic Ca2+ (Ca-c(2+)) constitute important components involved in the physiological regulation of a myriad of cell fun ctions in eukaryotic organisms. In particular, during development of the ce ntral nervous system, both properties are thought to be important in the re gulation of cell cycle, cell migration, cell differentiation, cell-cell com munication, and naturally occurring cell death. However, obtaining insight into the precise relationship between these two parameters of cell function is relatively limited either by technical difficulties inherent in using e lectrical recordings of membrane properties in conjunction with optical ima ging of single cells or by employing optical imaging of either one or anoth er property alone. Here, we describe in detail a novel strategy to record c hanges in both MP and Ca-c(2+) from many intact single cells in a noninvasi ve manner using digital video microscopy. This method involves double-loadi ng the cells with voltage- and calcium-sensitive fluorescent indicator dyes , green oxonol, and fura-2, which can be sequentially excited with a mercur y are lamp filtered at appropriate wavelengths and their resulting emission s can be captured with an intensified charged-coupled device camera at Is i ntervals. As an example of the utility of dual-recording strategy, we prese nt data on a distinct functional expression of excitable membrane and cytop lasmic calcium properties in and differentiating embryonic rat cerebral cor tical cells.