Certain amphipathic dyes, when bound to the membranes of neurons, cardiac and skeletal muscle, glands, and other cells, behave as molecular indicators of membrane potential. The optical properties of these molecules vary linearly with potential and may be used to monitor action potentials, synaptic potentials, or other changes in membrane voltage from a large number of site at once, without the use of electrodes. The project will continue to develop more sensitive probes, add to our understanding of the mechanisms by which they transduce voltage, and extend the technology associated with their use. Our present intention is to use these molecular voltmeters for optical recording of membrane potential from functional arrays of ganglion cells in retinae of teleost fishes, from otherwise inaccessible regions of single neurons such as nerve terminals and dendritic arborizations, and from many sites simultaneously in small ensembles of neurons in tissue culture. First, we will use our newly completed high resolution system for Multiple Site Optical Recording of Transmembrane Voltage MSORTV), capable of monitoring changes in membrane potential from as many as 464 loci at one, to identify and visualize functional units within the retinal ganglion cell mosaic of bony fishes when their double cone system of photoreceptors is estimated with linearly polarized light having its electric vector alternating rapidly between two orthogonal directions. These experiments should permit the detection of polarization-opponent retinal ganglion cells. Second, we will exploit the laboratory's ability to monitor electrical activity in the intact nerve terminals of vertebrates in order to study the mechanism of excitation-secretion coupling in a fast secretory system, and also to understand how change in the invasion of the highly ramified terminal arborization of monocellular neurons can be used to modulate the release of neuropeptides in the neurohypophyses of mammals. Third, the new MSORTV system will be used to record membrane potential changes from fine processes of single neurons in order to determine the electrical properties , both active and passive, of neuronal structures which are not penetrable by electrodes and are frequently too far away, electrically, for their activity to be reflected in the somata. Finally, we will use high resolution multiple site optical recording to continue our study of the spatial and temporal patterning of activity in truly simple nervous systems, small ensembles of synaptically connected Aplysia central neurons maintained in culture, by monitoring electrical activity in all of their cells simultaneously.
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