Andrea J. Yool - US grants

DESCRIPTION (Verbatim from the Applicant's Abstract): The proposed research uses a disarmed Herpes viral vector carrying the marker gene for Green Fluorescent Protein (GFP) to mediate antisense knockdown of the K-Ca channel, rslo, of rat cerebellar Purkinje neurons. Preliminary data show that antisense knockdown of rslo causes a measurable decrease in the abundance of K-Ca channels in Purkinje neurons, and that viral-driven antisense against another K+ channel beta subunit (Kvbeta1.1) dramatically affects whole cell firing patterns, thus demonstrating the feasibility of the viral vector antisense approach. Expression of the alpha subunit of the K-Ca channel rslo is regulated in cerebellum by depolarization and Ca2+ entry in a temporal pattern that is comparable in vivo and in vitro. Electrophysiology, molecular biology, immunocytochemistry and fluorescence imaging are used to analyze the consequences of viral infection, molecular antisense manipulations, and to evaluate the role of the K-Ca channel in the generation of the classic pacemaker firing pattern of Purkinje neurons. Aim 1 uses viral-driven antisense against the alpha sequence to analyze the function of K-Ca conductance in generating pacemaker firing. Aim 2 analyzes the expression of the rslo beta subunit in cerebellum, and uses viral-mediated overexpression and underexpression to dissect the contribution of the beta subunit to the maturation of the pacemaker firing pattern. The disarmed viral vector is a powerful new approach for manipulating gene expression in neurons in vivo and in vitro. A number of human neurological developmental disorders affect the cerebellum; its extended postnatal developmental period constitutes a window of vulnerability to clinical agents with neurological side effects. The rat provides a useful model for studying postnatal cerebellar development and neuropathological damage, and may provide insights into methods to manipulate developmental abnormalities in the cerebellum.

0080843
Gordon

The functioning of the nervous system depends on an intricate
network of connections between cells. Much of the communication
occurs at tiny physical contacts between cells called synapses. How
molecular components of synapses come to be organized together is an
important problem within neuroscience. The current project will
examine how influences from motor neurons direct the localization of
synaptic molecules on muscle cells. It is hypothesized that calcium
channels on the muscle cells mediate the signaling from the neurons
and that local entry of calcium through these channels directs the
local accumulation of synaptic molecules. In order to test this
hypothesis, muscle cells in tissue culture will be stimulated with
signaling molecules derived from neurons, and the calcium channels
will be manipulated with pharmacological agents. Both local calcium
entry and the local accumulation of synaptic molecules will be
assayed.
The results of the proposed work will further our understanding of
the molecular signaling mechanisms that control the formation,
maintenance, and modulation of synapses. Synapses provide the
nervous system with something akin to what transistors provide
computers, pathways of communication that can be modulated to obtain
different results. As such, an understanding of how synapses can be
modulated has profound implications for learning and memory.

water channel; membrane channels; protein structure function; biological signal transduction; protein binding; water flow; protein kinase A; nucleotides; cations; gene mutation; tetraethylammonium compound; membrane permeability; electrical conductance; electrophysiology; mutant; intermolecular interaction; phosphorylation; cyclic GMP; cyclic AMP; Xenopus oocyte; site directed mutagenesis; voltage /patch clamp; genetic manipulation; tissue /cell culture;

DESCRIPTION (provided by applicant): The proposed program will emphasize interdisciplinary predoctoral training in neuroscience within the framework of the existing Committee on Neuroscience at the University of Arizona. The program will support predoctoral trainees during the initial two years of study towards the Ph.D. degree and will focus on the fundamental concepts of neuroscience that are essential regardless of the eventual area of specialization. The training faculty will consist of 35 members of the interdepartmental Committee on Neuroscience, who were selected on the basis of their distinguished records in research and training. Training faculty members hold appointments in 18 different departments on the main campus and College of Medicine, which are contiguous. Research strengths in several disciplines are represented, including developmental neuroscience, insect neurobiology, motor control, cognition, learning and memory, speech production, and neuropharmacology. Based upon the current size of the training faculty and their level of research activity, we are requesting 6 predoctoral training positions, with two years of support being offered to the most promising new students seeking training in neuroscience. All trainees will complete a common set of core requirements, and may choose to seek the Ph.D. degree either through the interdepartmental doctoral Program in Neuroscience (in which all of the training faculty participate) or through one of the departmental doctoral programs in which the training faculty participate, including Cell Biology & Anatomy, Molecular & Cellular Biology, Psychology, Physiological Sciences and Pharmacology. Facilities for research training include the extensive equipment and expertise in individual faculty laboratories, as well as comprehensive shared resources at the University of Arizona.