An understanding of the complex relationship between neural structure and function is central to a wide range of problems in neurobiology research. Progress in this area has until recently been hampered by technical difficulties associated with light microscopy in living brain tissue. The recent introduction of 2-photon laser scanning microscopy (LSM) together with the development of new vital fluorescent indicator dyes has the potential to revolutionize our understanding of brain function in health and disease. The 2-photon LSM utilizes long- wavelength excitation light that penetrates deep into brain tissue and, through a quantum interaction of photons, only produces fluorophore excitation at the focal point. Because excitation does not occur in out- of-focus tissue and emitted photons are efficiently detected, problems of scattering, bleaching, and phototoxicity are greatly reduced. The true power of 2-photon microscopy to neurobiology will undoubtedly be realized by a combination of this technique with electrophysiology in intact brain preparations, allowing the visualization of second messenger system activity by reported dyes in concert with electrophysiological recording and manipulations. We propose to assemble a 2-photon LSM system on a upright microscopy platform with water immersion objectives and integrated electrophysiology equipment to exploit the potential of 2-photon LSM for vital neurobiology. Our Major Users are NIH-funded neurobiologists with extensive imaging and electrophysiology experience and have all made significant contributions to their specific areas of expertise. We plan to use the requested equipment to examine early stages of brain development and the emergence of neuronal circuits, axon pathfinding and synaptogenesis, dendritic differentiation, synaptic release mechanisms, the actions of dopamine and nicotine in the brain, and the mechanisms of olfactory signal transduction. The Technical Consultant on this proposal, r. Rafael Yuste, is a pioneer in the application of 2-photon microscopy and calcium imaging to neurobiology, and will greatly assist in the construction and operation of the proposed facility, 2-photon LSM, because of its unique non-invasive properties and high spatiotemporal resolution in the intact living preparations studied by the Major Users, rep[resents the only way that many questions of critical importance to the Users' research can be addressed. The requested equipment would be located in a central facility in the Department of Neurology at the Health Sciences Campus of Columbia University, and thus facility would be strongly supported by the Neurology Department and the extensive community of NIH-funded investigators in the Center for Neurobiology and Behavior.
