Brian M. Salzberg - US grants

Certain substances, when bound to the membranes of neurons, cardiac and skeletal muscle, salivary acini, and other cells, behave as molecular indicators of membrane potential. The optical properties of these molecules, most notably fluorescence and absorbance, vary in a linear fashion with potential and may, therefore, be used to monitor action potentials, synaptic potentials, or other changes in membrane voltage from a large number of sites at once, without the necessity of using electrodes. We propose to develop more sensitive probes, to extend the technology associated with their use, and to employ these molecular voltmeters for optical recording of membrane potential from hitherto inaccessible regions of single neurons such as axon an neuroendocrine terminals and axonal and dendritic processes and from many sites simultaneously in small assemblages of neurons and in electrical syncitia, in order to study the spatial and temporal patterning of activity. First, we intend to use a computer based system for Multiple Site Optical Recording of Transmembrane Voltage (MSORTV), already constructed and capable of monitoring changes in membrane potential from as many as 124 loci at once, to record patterns of electrical activity throughout syncitia (such as glandular tissue), and to study the properties of truly simple nervous systems -- small artificially constructed ensembles of synaptically connected invertebrate central neurons maintained in culture -- by recording electrical activiy optically from all of their components simultaneously. Second, we propose to use this appratus, with an Argon ion laser light source, to record membrane potential changes from fine processes of single neurons in situ, within an invertebrate neuropil, and isolated in tissue culture. These structures are not penetrable by microelectrodes and are frequently too far away, electrically, for their activity to be reflected in the somata. Finally, we expect to exploit the optical properties of potentiometric probes, and our multiple site optical recording capability, to detect potential changes in vertebrate nerve terminals, and to correlate alterations in the shape of the nerve terminal action potential with the release of neuropeptides.

DESCRIPTION (provided by applicant): Secretion is one of the most ubiquitous of cellular processes, and the elucidation of its mechanism(s) remains a challenge to cell physiologists. It is becoming increasingly clear, however, that quantal, or vesicular release of neurotransmitters, neuropeptides, neuromodulators, and hormones proceeds differently in different cell types, and that the mammalian neurohypophysis is an excellent model for rapid-release neurosecretory systems including presynaptic terminals. We are now able to monitor, using optical methods having sub-millisecond time resolution, both the electrical events and cytoplasmic calcium changes in nerve terminals of vertebrates, as well as a sequence of intrinsic optical changes (IOC's) that are related directly to the secretory event. Thus, we possess an exceptional array of tools with which to study excitation-secretion (E-S) coupling, and to provide a more complete understanding of synaptic transmission in higher animals, and of the secretory event in general. First, we will use moderate angular resolution light scattering methods, together with fluorescent calcium indicators, voltage sensitive dyes, immuno-gold labeling, electron microscopy, and electron energy loss spectroscopy (EELS) 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 secretory granules themselves.) We will also examine several alternative explanations for the origin of the IOC's, including the hypothesis that a pH-dependent solubilization of secretory granule contents, mediated by a rise in intraterminal calcium, precedes exocytosis and generates a large and rapid change in light scattering. Second, we will use the neurohypophyses of transgenic mice expressing the pH-sensitive Green Fluorescent Protein, ecliptic-pHluorin, for high time-resolution studies of neuropeptide secretion. Finally, we will characterize extensively the changes in intraterminal calcium during E-S coupling in mammalian nerve terminals, and we will identify the sources and sinks of this critical second messenger, and their specific roles before, during and after exocytosis. These experiments should have a major impact, and should add to our understanding of synaptic transmission, and its failure or malfunction in human neurological disease.