Elio Raviola - US grants

DESCRIPTION (Adapted from applicant's abstract): The overall goal of this project is to combine transgenic technology with microscopy and electrophysiology to study circuitry of the mouse retina. The use of transgenic mice to generate reporter gene products that identify neurons allows a correlation of physiology and morphology in a known subset of mammalian cells. This is a powerful technique that has been applied currently to DAergic amacrine cells. Two other transgenic lines, rod bipolars and alpha ganglion cells, have also been labeled. In the previous funding period, the applicant discovered a spontaneous bursting pattern in DAergic amacrine cells. One specific aim is to fully understand the mechanisms that modulate DAergic amacrine cells. Using RT-PCR, the applicant plans to identify the receptor subunits contained in DAergic amacrine cells and then use electrophysiological experiments to describe transmitter currents. Ultrastructural immunocytochemistry will be used to localize receptor subunits on the surface of DAergic amacrine cells. The second specific aim is to expand these approaches to other neurons in the retina. cDNA libraries will be generated from specific cell types using RT-PCR and reporter genes for cell identification. The applicant plans to characterize these neurons either in the intact retina or the retinal slice. In the continuing development of this specific aim, the applicant plans to generate new lines of transgenic animals in which different populations of neurons are labeled.

Five experienced electron microscopists belonging to the same department submit a proposal to replace two outdated electron microscopes with a new instrument. All investigators heavily rely upon electron microscopy in their research activities. These include studies on: 1. Processing of visual information by the mammalian retina: retinal neurons are penetrated by microelectrodes, their light responses are recorded, and, after intracellular delivery of an ultrastructural tracer, their synaptic connections are studied with the electron microscope. The electric signals generated by each individual cell are thus correlated to its inputs and outputs. 2. Molecular structure of gap junctions and tight junctions: the electron microscope is used in a morphological assay for the purity of the junctional preparations, to localize junctional epitopes in situ using immunocytochemistry and, finally, in combination with X-ray diffraction, to determine the molecular structure of gap junctions to atomic resolution. 3. Role of extracellular matrix (ECM) in morphogenesis: the electron microscope is used to analyze the interaction of the cytoskeleton with ECM and the induction by ECM of epithelial- mesenchymal transformation. 4. Process of damage and repair of the gastric mucosa: the electron microscope is used to study the mechanism of repair after injury and the effects of experimental maneuvers that favor the restoration process. The investigators are presently using two fifteen year old microscopes that need continuous repairs and have long breakdown periods; the moment is near that they will come to a complete halt. They would like to substitute for them a new, all-purpose, transmission electron microscope, that combines a routine high resolution performance with great speed in obtaining micrographs at low magnification.

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