Abstract
This protocol describes a method for efficient chemical synthesis of an analog of inositol-1,4,5-trisphosphate (IP(3)) hexakis acetoxymethyl ester having an ortho-nitroveratryl photochemical caging group on the 6-hydroxyl position. The six esters render the probe membrane permeant, such that it can be loaded into intact living cells in vitro or in vivo. Inside cells, the caged IP(3) is inert until activated by two-photon excitation at 720 nm. The photoliberated signaling molecule can mobilize release of Ca(2+) from intracellular stores on the endoplasmic reticulum. When co-loaded with the fluorescent Ca(2+) indicator rhod-2, one laser can be used for stimulating and monitoring intracellular Ca(2+) signaling with single-cell resolution. This protocol has chemistry and biology sections; the former describes the organic synthesis of the caged IP(3), which requires 12 d, and the latter an application to a day-long study of astrocyte-regulated neuronal function in living brain slices acutely isolated from rats. As Ca(2+) is the single most important intracellular second messenger and the IP(3)-Ca(2+) signaling cascade is used by many cells to produce increases in Ca(2+) concentration, this method should be widely applicable for the study of a variety of physiological processes in intact biological systems.