Potentiometric probes are dyes which, 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 sites at once, without the use of electrodes. For more than twenty years our laboratory has pioneered the technology for using potentiometric probes, and developed new optical methods for use in cellular neurophysiology, including a 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 once. We will now apply these techniques to the study of the nervous system at three levels of integration: the stimulus-coupled release of peptides from nerve terminals, the action potential s invasion of a highly ramified nerve terminal arborization, and the complete analysis of the ensemble behavior of an intact mammalian neural network. First, we will use light scattering methods, together with fluorescent calcium indicators and voltage sensitive dyes to examine the hypothesis that the triggered release of calcium from intraterminal stores is required for the release of neuropeptides in mammals (and that the stores may be the vesicles themselves.) Second, we will use potentiometric dyes to record how the action potential s invasion of a nerve terminal arbor may be modulated by intrinsic control mechanisms, including the temporal patterning of activity, and the local release of an inhibitory neurotransmitter (gamma-aminobutyric acid). Finally, we will use these molecular voltmeters to monitor the electrical activity of all of the neurons in a mammalian simple nervous system that is uniquely amenable to analysis by optical means, the submucous plexus of the Guinea-pig ileum, and adapt a novel analytical tool (the gravitational transformation) to the task of elucidating completely the ensemble behavior of an intact neural network.
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