Detection of ecto-ATPase activity in synaptic plasma membranes for studying extracellular ATP-induced signal transduction

Abstract

This paper describes protocol for the correlated light and electron microscopical histochemical visualization of the reaction product of ecto-ATPase activity in brain. The protocol employs a biochemically optimized incubation medium to adjust the appropriate kinetical parameters for detection of the histochemical reaction product by means of confocal laser scanning and electron microscopy. The reaction product is formed when the liberated inorganic phosphate is captured in histochemical reaction by the cerium ions. Confocal microscopy is performed in reflectance mode due to sufficient reflectance properties of the cerium-containing reaction product. Using this procedure, the prominent reaction of ecto-ATPase activity is readily detectable in hippocampus and cerebellum at sites where ATP is supposed to act as a synaptic neurotransmitter: on synapses of neurons in the pyramidal cell layer of hippocampus, in the granule cell layer of the dentate gyrus, and around synapse-containing areas in the granule cell layer of cerebellum. Reaction product is seen in close association with both pre- and postsynaptic membranes and exclusively extracellularly. Specificity of the visualization is justified in control experiments with diethyl pyrocarbonate, specific inhibitor of ecto-ATPase. The procedure is easy to perform, sensitive, and reproducible. It is recommended as a valuable tool in the arsenal of biochemical, immunochemical, and physiological techniques in studying signal transduction induced by extracellular ATP. Themes: Cellular and molecular biology Topics: Staining, tracing, and imaging techniques