Dean O Smith - US grants

health science research support; medical education;

The goal of this proposal is to determine the mechanisms underlying age-related changes in synaptic physiology at the neuromuscular junction. Acetylcholine (ACh) levels decrease with age. This manifests a change in the balance between synthesis and degradation. Thus, using sensitive gas chromatography/mass spectrometry, the rates of ACh synthesis and degradation will be measured to determine the nature of this age-related change. Intracellular Ca2+ levels may also be altered during aging. This will be tested using electrophysiological techniques. The kinetics of Ca2+ entry and clearance will be estimated from frequency histograms of synaptic delays. The relative amounts of Ca2+ entry will be measured using focal extracellular electrodes following pharmacological blocking of nerve-terminal K+ currents. The rate of clearance of intracellular Ca2+ will be estimated by measuring the time course for the decay of synaptic facilitation. Moreover, the sensitivity of the release mechanism to Ca2+ will be estimated from data relating quantal release to extracellular Ca2+ concentrations. Proteins weighing 125, 145, and 210 kDa are present in nerve terminals of 10-month but not 28-month rats. The 145 and 210 kDA molecules are quite probably neurofilament proteins. Using gel electrophoresis and immunoblot techniques, this identity will be tested. Similar techniques will be used to ascertain whether the failure to detect these proteins in aged rats is due to impaired axonal transport from the cell body or enhanced Ca2+-dependent degradation. In the postsynaptic membrane, there is an age-related increase in low-affinity, slow channel open time ACh receptors. Patch-clamp methods will be used to determine whether there are corresponding changes in mean channel conductance. Since these changes in receptor properties are similar to those following functional denervation, focal recording techniques will be used to test whether all nerve terminals are functional in the end plates of aged animals. These studies should elucidate the underlying causes of reduced ACh levels, altered synaptic efficacy, and changing end-plate structure during aging.

The goal of the proposed research is to determine the mechanisms underlying failure of action potentials to invade every axon branch in the presynaptic terminal arborization. Intracellular Ca++ may regulate axon excitability. Using ion-selective electrodes and intracellular recording techniques, the extent of intracellular Ca++ accumulation, the effects of injecting Ca++ or EGTA, the relative contribution of Ca++ currents during repetitive action potentials, and the amount of intracellular Na+ accumulation under conditions known to cause conduction block will be measured. Axonal adenylate concentrations increase during repetitive stimulation and may also affect conduction. Using sensitive biochemical techniques, the amounts of adenylate and adenosine released by nerve and muscle and the amounts taken up by the nerve will be assayed. The effects of these compounds on axon excitability will be tested using patch-clamp recording techniques. Recent experiments have shown clearly that action potentials do not propagate to the terminal region of motor axons at the mouse neuromuscular junction; the terminal portions are depolarized due to passive current invasion beyond a more proximal site of conduction block. Using Nomarski optics to visualize terminal branches, terminal depolarization and synaptic responses will be recorded with patch-clamp electrodes to determine if the site of blockage varies and whether transmitter release by individual terminal branches is graded or all-or-none. To interpret the significance of any alterations in terminal excitability or passive depolarization, the extent of the synaptic contact zone in the terminals will be determined from serial sections obtained with the electron microscope. These studies should elucidate the underlying mechanisms and the extent of variable nerve terminal invasion by the action potential; this is a fundamental issue, for variable terminal activation could modulate the divergence of information throughout the nervous system and the effectiveness of single synapses.

The long-term goal is to determine mechanisms leading to failure of an action potential to activate all of the axonal targets. In pursuit of this goal, regulation of calcium current flowing in motor-nerve terminals in response to depolarization by an action potential will be analyzed. Exogenous ATP and adenosine cause nerve-terminal calcium currents to decrease. Nonhydrolyzable analogues of ATP will be tested to determine whether ATP must first be converted to adenosine to reduce calcium current. Pertussis toxin, cAMP analogues, and phorbol esters will be used to ascertain whether second messengers are involved. The effects of adenosine will be tested in the presence of extracellular Ba2+ to determine whether a decreased calcium current is secondary to adenosine activation of a K+ current. ACh also causes nerve-terminal calcium currents to decrease. Whether this involves nicotinic or muscarinic receptors will be tested. The roles of second messengers and activation of K+ currents will also be analyzed. The calcium current duration becomes shorter as stimulation frequency increases. Whether this is due to inhibition resulting from accumulated extracellular adenosine will be tested. Calcium-dependent calcium channel inactivation will be tested by measuring the relationship between calcium current duration and extracellular calcium, the effects of equimolar substitution of Ba2+ or Sr2+ for calcium, and the time course of recovery-rich membrane patches or immobilized antibodies as probes, ACh, ATP, and calcitonin gene-related peptide (CGRP) release will be measured as the calcium-current duration is varied. A postsynaptic counterpart to presynaptic inhibition of synaptic activation, namely rapid desensitization of glutamate receptors on motoneuron cell bodies, will be characterized by measuring voltage and agonist dose dependence, sensitivity to exogenous glutamate levels, and recovery times using patch-clamp techniques. these and previous studies have utilized embryonic cells. Similar experiments will use motoneurons from adult tissue to determine whether rapid desensitization changes during development.

[unreadable] DESCRIPTION (provided by applicant): Building upon the network foundation established by BRIN, INBRE proposes to expand and to develop Hawaii's competitive biomedical research capacity. The expansion will center on three thematic projects exploring the cellular basis of immunological and neurological diseases from the perspective of immunology, cell biology, and developmental biology. Each project will be led by a well-established senior investigator who will mentor junior investigators at both the lead and the affiliated baccalaureate institutions. This will extend into the state's community colleges where participating faculty will collaborate with established researchers at the lead institution. Each investigator - senior, junior, and Outreach - will recruit and mentor undergraduate and graduate students as well. The development will concentrate on not only individual research careers but also the network's overall approach to competitive research. This involves the establishment of rigorous standards and performance expectations coupled with attentive mentoring to assist network investigators and students in meeting these challenging criteria. Organizationally, INBRE will [unreadable] consist of four major cores: Administrative, Research, Bioinformatics, and Outreach. The Administrative [unreadable] Core will provide overall administrative support, including maintenance of the network's web site, evaluation efforts, and training and mentoring workshops and seminars. The Research Core will be comprised of the three thematic research projects with a Core leader to ensure project coordination. The Outreach Core extends the research thrust into the community colleges by promoting research and student participation through collaborations with the lead and the baccalaureate institutions. The Bioinformatics Core will also provide unique opportunities by emphasizing academic work force development. To facilitate this, the Core will be housed within the University of Hawaii at Manoa's Department of Information and Computer Sciences. [unreadable] [unreadable]