Confocal imaging and local photolysis of caged compounds: Dual probes of synaptic function

Chemical signals generated at synapses are highly limited in both spatial range and time course, so that experiments studying such signals must measure and manipulate them in both these dimensions. We describe an optical system that combines confocal laser scanning microscopy, to measure such signals, with focal photolysis of caged compounds. This system can elevate neurotransmitter and second messenger levels in femtoliter volumes of single dendrites within a millisecond. The method is readily combined with whole-cell patch-clamp measurements of electrical signals in brain slices. In cerebellar Purkinje cells, photolysis of caged IP₃ causes spatially restricted intracellular release of Ca²⁺, and photolysis of a caged Ca²⁺ compound locally opens Ca²⁺-dependent K⁺ channels. Furthermore, localized photolysis of the caged neurotransmitter GABA transiently activates GABA receptors. The use of focal uncaging can yield new information about the spatial range of signaling actions at synapses.