This project will utilize sophisticated optical imaging methods to explore activity-dependent regulation of both presynaptic and postsynaptic structures in rat hippocampus. The fluorescent marker FM 1-43 will be used to study the recycling vesicles of presynaptic boutons in cell culture preparations. Lipophillic carbocyanine stains will be used to study dendrites and spines in cell cultures and tissue slices. Fluorescence will be visualized in liver preparations using laser-scanning fluorescence microscopy (transmission, confocal and two-photon) while neural activity is manipulated by electric field stimulation and by drugs. Pharmacological analysis will be used to test for the involvement of specific receptor- medicated signaling pathways in structural dynamics. Retrospective immunohistochemistry and electron microscopy will be used for cell-type identification and to improve interpretation of fine structural events detected by fluorescence in live specimens. Specific questions about interpretation of fine structural events detected by fluorescence in live specimens. Specific questions about basic presynaptic and postsynaptic mechanisms and their use-dependent plasticities will be addressed as indicated below. 1. Presynaptic vesicle pool dynamics. A. Basic release and recycling mechanisms: (i) How long do synaptic vesicles remain open after transmitter release? Does open time depend on stimulation intensity? (ii) Do all synaptic vesicles take up FM 1-43 during transmitter release? (iii) How are vesicles compartmentalized during recycling? (iv) How do vesicle pool dynamics vary from synapse to synapse of the same and different cell types. B. Regulation and plasticity of synaptic vesicle pools. (i) How stable are synaptic vesicle pools over periods of hours to days? (ii) How does electrical activity influence the synaptic vesicle pool? (iii) Do presynaptic autoreceptors or adrenergic receptors modulate pool size or dynamic parameters? (iv) How do specific vesicle proteins influence the size, dynamics and regulation of vesicle pools? 2. Postsynaptic dendrite spine dynamics. (i) How stable are dendritic spine and branchlet structures over hours an days in mature, steady-state cultures? (ii) How is dendrite structure influenced by electrical activity? What signaling mechanisms link dendritic structure to activity? These studies of basic mechanisms of synaptic function and synapse structural plasticity should contribute to understanding and treating mental retardation, learning disorders and epilepsies. They may also help in devising means to promote recovery of function after nervous system trauma and stroke.