Ellen F. Barrett, Ph.D. - US grants

Our laboratory is studying axonal and synaptic electrophysiology, using new techniques for intracellular recording from vertebrate peripheral myelinated axons and motor nerve terminals. We plan to see if the prolonged depolarizing afterpotential we discovered in frog and lizard axons also occurs in mammalian axons. We plan to study the ionic currents in motor nerve terminals, and to correlate these currents with the rate of transmitter release from these terminals. We will also study how various cholinergic drugs (agonists, antagonists, acetylcholinesterase inhibitors) affect the electrical properties of motor nerve terminals.

A group of investigators representing several departments and disciplines at the University of Miami School of Medicine seeks funds to establish a shared confocal laser-scanning microscope facility. This University has many PHS-supported biomedical investigators in need of a high resolution confocal microscope. No confocal microscope facility exists within a 300 mile radius of Miami. In this proposal five groups of major users describe how the optical sectioning and high resolution provided by the requested confocal microscope would benefit their NIH-supported research programs. These programs include studies of permeation pathways through the myelin sheath, specificity of synaptic connections, regulation of acetylcholinesterase expression at the neuromuscular junction, interaction between immune killer cells and their targets, and the effects of spinal cord injury on bone growth and innervation. The descriptions of these projects include images of relevant tissues acquired during confocal microscope demonstrations here, showing the feasibility of the proposed projects and the scientific advantages conferred by confocal microscopy. We document why the requested Bio-Rad system is optimal for these projects. All the major users' projects require the ability of the Bio-Rad system to image simultaneously at least two dyes within the same specimen. We outline an organizational plan to ensure the efficient and skillful operation, long-term maintenance and financial stability of the facility. This plan also ensures that other PHS-supported investigators (nine of whose needs for the confocal microscope are summarized in the Appendix) will have adequate access to the facility. This revised application also addresses the concerns expressed by reviewers of our initial application submitted in 1992.

This application is to study mechanisms of Ca2+ metabolism and transmitter release in vertebrate (lizard, mouse) nerve terminals innervating skeletal muscle. Stimulation-induced changes in cytosolic and mitochondrial [Ca2+] will be measured by monitoring fluorescence changes of indicator dyes at high spatial and temporal resolution with a confocal laser-scanning microscope. Phasic and asynchronous transmitter release will be measured electrophysiologically by recording end-plate potentials or voltage-clamped end-plate currents in the underlying muscle fiber. One group of experiments will study the relationship between intraterminal [Ca2+] ([Ca2+]i) and transmitter release, elevating [Ca2+]i by uncaging photolabile Ca2+ chelators. These experiments will test the hypothesis that quantal release from motor terminals result from two parallel mechanisms with differing Ca2+ affinities. Experiments using similar techniques will study the temperature sensitivity of the synaptic delay, to determine whether the interval between Ca2+ entry and the onset of transmitter release is Ca2+- dependent, and whether it is controlled by processes with low (e.g. diffusion) or high energy barriers. Another group of experiments will use simultaneous imaging of cytosolic and mitochondrial [Ca2+] and application of various inhibitors to study Ca2+ sequestering and extruding mechanisms in motor nerve terminals. These experiments will test two hypotheses. The first is that mitochondria sequester a major portion of the Ca2+ that enters the terminal during repetitive stimulation and effectively "clamp" cytosolic [Ca2+] at a slightly elevated plateau level. The second hypothesis is that slow release of Ca2+ from mitochondria following stimulation has a major role in producing post-tetanic potentiation for transmitter release. We will also investigate mechanisms underlying the heterogeneity with which different boutons of the same motor terminal handle Ca2+ loads from trains of action potentials.

DESCRIPTION (provided by applicant): This is a revised application for continued support to study mitochondrial contributions to Ca2+ regulation in wild type and mutant SOD1G93A mouse motor nerve terminals. Changes in cytosolic and intramitochondrial [Ca2+] evoked by repetitive stimulation will be measured by imaging the fluorescence of indicator dyes. Phasic (end-plate potentials) and asynchronous quantal transmitter release will be recorded electrophysiologically, and vesicular recycling measured using styryl dyes. We have shown that mitochondrial uptake of Ca2+ is critically important for sustaining neuromuscular transmission during high-frequency stimulation. Proposed experiments will investigate the mechanisms by which motor terminal mitochondria take up, store and extrude this Ca2+ load. Using a novel permeabilized motor terminal preparation we will test how certain cytosolic components affect the affinity of mitochondrial Ca2+ uptake. We will test the role of inorganic phosphate in intramitochondrial Ca+ buffering, and investigate the linkage between mitochondrial Ca2+ extrusion and cytosolic [Na+]. The impact of Ca2+ uptake on mitochondrial energy metabolism will be investigated using measurements of stimulation-induced changes in mitochondrial membrane potential and changes in NADH and FAD autofluorescence. Another series of experiments will investigate the linkage between abnormal mitochondrial [Ca2+] handling and abnormal transmitter release in a mouse model of familial amyotrophic lateral sclerosis (ALS, SOD1G93A). Embryonic SOD1G93A motoneurons are especially vulnerable to nitric oxide (NO)-induced death. NO is produced in active muscle, so we will investigate whether the function of SOD1G93A motor terminals is more susceptible to NO-induced disruption than that of wild-type terminals, to test the hypothesis that NO contributes to functional deficits and degeneration of motor terminals in these mice. The proposed studies will thus probe fundamental mechanisms concerning how motor terminal mitochondria handle physiological Ca2+ loads, and investigate how Ca2+ dysregulation and NO contribute to motor terminal dysfunction in SOD1G93A motor terminals.

DESCRIPTION (provided by applicant): Motor nerve terminals are especially vulnerable to ischemic stress. The proposed experiments will test the hypothesis that even at early ages motor terminals in mice that overexpress a mutant human superoxide dismutase I (SODIG93A, a model of familial amyotrophic lateral sclerosis) are more vulnerable to stress than terminals in mice that overexpress wild-type human superoxide dismutase (hSOD1). We will test three stresses that might sometimes be encountered by motor terminals in vivo: (1) hindlimb ischemia/reperfusion stress in vivo, (2) hypoxia/ reoxygenation stress in vitro, and (3) in vivo intense stimulation of a single motor nerve. Structural integrity of the stressed motor terminals will be assessed by a fluorescence endplate occupancy assay, testing the extent to which labeled skeletal muscle endplates in fast and slow muscles are occupied by an innervating motor nerve terminal. Preliminary results indicate that both the ischemic and stimulation stresses increase endplate denervation. The function of motor terminals will be assessed during and/or after the stress by measuring resting and stimulation-induced changes in cytosolic and mitochondrial [Ca2+] and mitochondrial membrane potential using fluorescent indicators, and by measuring quantal transmitter release using electrophysiological recording. Preliminary results show disruptions in all these functional parameters during the hypoxia/ reoxygenation stress. Other experiments will test whether stresses that damage motor terminals also produce immunohistochemical signs of damage in the parent motoneurons. We will also test whether agents shown to be neuroprotective for motoneurons (e.g. vascular endothelial growth factor, VEGF; insulin-like growth factor, IGF-1) can protect motor nerve terminals during these stresses.