This project aims to determine the distribution of diffusible components at chemical synapses. This work is significant because of the relationship between the localization and movement of mobile constituents and their role in synaptic transmission. To attain the necessary spatial and temporal resolution, this study combines three major technological advances: 1) rapid freezing of unfixed tissues in order to achieve a time resolution of 1-2 msec and to limit ice damage; 2) cryosectioning to prepare thin, unstained sections in which diffusion of even very mobile components is controlled; and 3) quantitative, element-specific x-ray imaging in a computerized analytical electron microscope to obtain simultaneous quantitation and localization of tissue components. This approach is being applied to two synaptic preparations from the central nervous system. Experiments on the predominantly cholinergic synaptosomes from the optic lobe of the squid are designed to characterize the biochemically-active synaptosomes which synthesize acetylcholine (ACh) and release this transmitter in response to Ca-dependent depolarization. Morphological and compositional data have identified at least two populations of structures which are candidates for viable synaptosomes. This preparation is also being used, in conjunction with an antimony-labeled ACh analog, to determine where ACh is taken up and stored in cholinergic synaptosomes. Elemental imaging and analysis of the molecular layer of mouse cerebellum indicate that, in certain physiological states, potassium and calcium may be characteristically distributed among different areas that correspond to axons, dendrites, and glia. The detailed relationship between diffusible ions and other synaptic components, as well as how these relationships change during synaptic activity, is now under investigation.