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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS016824-18
Application #
2637884
Study Section
Physiology Study Section (PHY)
Program Officer
Baughman, Robert W
Project Start
1980-12-01
Project End
2001-04-30
Budget Start
1998-07-01
Budget End
1999-04-30
Support Year
18
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Physiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Fisher, Jonathan A N; Salzberg, Brian M (2015) Two-Photon Excitation of Fluorescent Voltage-Sensitive Dyes: Monitoring Membrane Potential in the Infrared. Adv Exp Med Biol 859:427-53
Salzberg, Brian M; Muschol, Martin; Kosterin, Paul et al. (2012) Measuring intrinsic optical signals from Mammalian nerve terminals. Cold Spring Harb Protoc 2012:
Kosterin, P; Obaid, A L; Salzberg, B M (2010) Long-lasting intrinsic optical changes observed in the neurointermediate lobe of the mouse pituitary reflect volume changes in cells of the pars intermedia. Neuroendocrinology 92:158-67
Fisher, Jonathan A N; Barchi, Jonathan R; Welle, Cristin G et al. (2008) Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ. J Neurophysiol 99:1545-53
Fisher, Jonathan A N; Salzberg, Brian M; Yodh, Arjun G (2005) Near infrared two-photon excitation cross-sections of voltage-sensitive dyes. J Neurosci Methods 148:94-102
Obaid, A L; Nelson, M E; Lindstrom, J et al. (2005) Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system. J Exp Biol 208:2981-3001
Salama, G; Choi, B-R; Azour, G et al. (2005) Properties of new, long-wavelength, voltage-sensitive dyes in the heart. J Membr Biol 208:125-40
Salzberg, B M; Kosterin, P V; Muschol, M et al. (2005) An ultra-stable non-coherent light source for optical measurements in neuroscience and cell physiology. J Neurosci Methods 141:165-9
Obaid, A L; Loew, L M; Wuskell, J P et al. (2004) Novel naphthylstyryl-pyridium potentiometric dyes offer advantages for neural network analysis. J Neurosci Methods 134:179-90
Muschol, Martin; Kosterin, Paul; Ichikawa, Michinori et al. (2003) Activity-dependent depression of excitability and calcium transients in the neurohypophysis suggests a model of ""stuttering conduction"". J Neurosci 23:11352-62

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