1985 |
Fishman, Harvey M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Fluctuation Phenomena and Ion Movements in Membranes @ University of Texas Medical Br Galveston
The ultimate objective of this project is to obtain a physical description of the molecular processes underlying ion movements during excitation of a nerve membrane. The short-term objectives are to obtain information on the fundamental properties of K and Na conduction in the model squid nerve preparation. The questions to be addressed are the following: 1) Are the elementary processes at equilibrium? 2) Are their kinetics linear or nonlinear? 3) Are their kinetics dependent on ion species or on concentration? 4) How many conducting states are involved? The measurement techniques used in these studies are centered around an internal axial-electrode voltage-clamp system, which has been improved significantly with respect to background noise. Rapid transfer function determinations are made by using synchronized, Fourier-synthesized pseudorandom signals as perturbations superposed on step clamps of membrane potential in combination with fast Fourier transform computations. The complex admittance is obtained in the frequency range 12.5-5000 Hz, where the ion conduction properties dominate the admittance. Linear analysis is carried out by curve fitting the complex admittance data using linear models. Power spectra of spontaneous current fluctuations are also obtained under the same conditions as the admittance determinations and in the same axons. Model fits of power spectra yield kinetic parameters which are compared and used with the admittance data to provide the information necessary to resolve the above questions. In the second part of the project, the origin, statistical properties and interactions of noise generating processes in isolated components of the squid axon will be studied. These measurements will provide information that will enable more effective use of noise data in discriminating between microscopic models of the elementary ion-current processes. Glass micropipets will be used to isolate and record the conduction properties of axoplasm, portions of the subaxolemmal filamentous network, and axolemma channels with and without the Schwann cell layer.
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0.984 |
1985 — 1986 |
Fishman, Harvey M |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Stochastic and Kinetic Processes in Membrane Function @ University of Texas Medical Br Galveston
Ion transport and conduction processes in single cell membrane preparations from epithelial, nerve, muscle and secretory tissues will be studied by advanced quantitative measurements of the kinetic and stochastic phenomena that are necessary to understand physiological functions in these tissues. The control of Na and K permeability by intracellular Ca++ and H+ ions will be examined in isolated patches of apical and basolateral membrane from a kidney cell line in tissue culture. The way in which H+ affects ouabain-sensitive fluxes and its effect on the kinetics of the Na pump will be determined by flux measurements in dialyzed squid axon. The exocytosis process in secretory cells will be characterized by membrane capacitance changes related to vesicle fusion and by transient potentials and altered membrane properties connected with release. The origin of anomalous K current rectification in molluscan neurons will be studied by relaxation, fluctuation and single channel current measurements to determine whether rectification arises from voltage-dependent channel lifetimes or a nonlinear single channel conductance. Ion channel properties that account for delayed, outward K current rectification and are responsible for control of repolarization of the cardiac action potential will be measured and described in single cardiac cells from bullfrog atria. The sequence of activation of Ca channels will be examined in snail neurons and PC-12 cells. The relative effects of potential and Ca current upon inactivation will be studied. Conduction in open channels and regulation of the numbers of functional channels will also be investigated. Patch and whole cell voltage clamp methods will be used. The transmembrane ionic current generated by the Na/K ATPase in isolated frog cardiac cells will be characterized in both atrial and pacemaker cells. Voltage-dependent ion conductances will be identified in hippocampal pyramidal neurons of guinea pig and the action of acetylcholine, noradrenaline, dopamine on the conductances determined. Synaptic effects in a cultured sympathetic cell line (NG108) will be evaluated using complex impedance measurements from the soma and dendritic regions.
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0.984 |
1993 — 2004 |
Fishman, Harvey M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cellular Mechanisms of Axonal Repair and Degeneration @ University of Texas Medical Br Galveston
Survival of lesioned axons is critically dependent upon an ability to repair the damage (seal off small hole or cut ends) within minutes to hours after lesioning. However, very little is currently known about the cellular mechanisms responsible for short-term repair of damaged axons. To obtain such data, we have used several unmyelinated and myelinated, both invertebrate and vertebrate, axons to demonstrate that the cut ends of severed axons are sealed by a tightly-packed plug of vesicles, some of which arise from endocytosis of the axolemma. We now propose to describe specific cellular/ biochemical/ molecular/ biophysical mechanisms responsible for the sealing of a small axonal hole or a complete axonal transection using data obtained from various techniques, including photomicroscopy (DIC, confocal fluorescence), electron microscopy, vibrating probe measures of injury current, ID and 2D SDS gels or Western transfers, and patch voltage clamp analysis of ion channels and transporters in membranes obtained from injury-induced vesicles. More specifically, we now propose to determine (1) the Ca2+ distribution in axons ms to hrs after a lesion (small hole or complete transection), (2) the origins and mechanisms of formation of vesicles that seal a lesioned (small hole or completely transected) axon, (3) the mechanisms that move vesicles to an axonal lesion site (small hole or complete transection) (4) the mechanisms by which vesicles seal small holes or completely transected cut axonal ends, and (5) whether the same cellular mechanisms seal small holes vs complete transections in a given axon or seal similar lesions in axons with different characteristics. These studies should be significant for several reasons (1) data on cellular repair mechanisms and the consequences of their failure should be of general interest to cell biologists and neurobiologists, (2) data on cellular mechanisms for sealing of lesioned (small hole or completely transected) axons might lead to procedures which increase axonal survival after injury, and (3) lack of repair in the axon studied may be helpful as a simple model of complicated neurodegenerative processes in mammalian fibers.
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0.984 |