This project aims to develop, improve, and exploit new molecules, mostly genetically targetable, for measuring and/or manipulating neuronal messengers and signals such as calcium, glutamate, GABA, action potentials, surface exposure of selected proteins, singlet oxygen, protein-protein proximity over tens of nm, turnover of key synaptic proteins at steady-state vs. conditions of new synapse formation, and activity of extracellular proteases during stroke. Calcium indicators genetically targeted to channel mouths or synaptic proteins will be used to investigate privileged nanodomains of calcium, long postulated but not directly measured. Fluorescent protein (FP)-based indicators of glutamate should enable quantitative dissection of pre- vs. postsynaptic components of excitatory synapse modulation, localization of excitatory signaling, and mechanisms of excitotoxicity. Analogous indicators of gamma-aminobutyrate (GABA) would extend such analyses to inhibitory synapses. Channels that sensitize neurons to fire action potentials in response to red/near-infrared light or magnetic field transients would allow noninvasive transcranial stimulation of neurons in behaving animals. FPs excitable by red/near-infrared would greatly improve detection of gene expression and protein fate deeper inside intact tissues and organisms, due to greater penetration and lesser autofluorescence at long wavelengths. Labeling of extracellular hexahistidine motifs by fluorescent chelators of zinc offers fast detection of the surface exposure of designated proteins, useful for understanding cycling of proteins at synapses and during capacitative calcium entry. FP-based generators and sensors of singlet oxygen should enable assays of protein-protein proximity over distances of many tens of nm, the size of large supramolecular complexes. Turnover of synaptic proteins will be investigated with photoconvertible FPs and two-color pulse-chase labeling of tetracysteine motifs, seeking proteins that are incorporated mainly into nascent synapses and can serve as molecular markers of new synapse formation. Activatable cell-penetrating peptides that accumulate where matrix metalloproteinases are active will be used to probe the role of these enzymes in stroke models. Relevance: New molecular tools will be developed for analyzing key molecules and signals. Such tools will help elucidate mechanisms of neuronal information processing, communication, lifecycles, control of behavior, and pathological injury.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37NS027177-25
Application #
8403604
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Talley, Edmund M
Project Start
1989-09-01
Project End
2014-12-31
Budget Start
2013-01-01
Budget End
2014-12-31
Support Year
25
Fiscal Year
2013
Total Cost
$622,748
Indirect Cost
$220,975
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Nabavi, Sadegh; Fox, Rocky; Proulx, Christophe D et al. (2014) Engineering a memory with LTD and LTP. Nature 511:348-52
Davalos, Dimitrios; Baeten, Kim M; Whitney, Michael A et al. (2014) Early detection of thrombin activity in neuroinflammatory disease. Ann Neurol 75:303-8
Inagaki, Hidehiko K; Jung, Yonil; Hoopfer, Eric D et al. (2014) Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nat Methods 11:325-32
Lin, John Y; Sann, Sharon B; Zhou, Keming et al. (2013) Optogenetic inhibition of synaptic release with chromophore-assisted light inactivation (CALI). Neuron 79:241-53
Whitney, Michael; Savariar, Elamprakash N; Friedman, Beth et al. (2013) Ratiometric activatable cell-penetrating peptides provide rapid in vivo readout of thrombin activation. Angew Chem Int Ed Engl 52:325-30
Lin, John Y (2013) Production and validation of recombinant adeno-associated virus for channelrhodopsin expression in neurons. Methods Mol Biol 998:401-15
Tsien, Roger Y (2013) Very long-term memories may be stored in the pattern of holes in the perineuronal net. Proc Natl Acad Sci U S A 110:12456-61
Lin, John Y; Knutsen, Per Magne; Muller, Arnaud et al. (2013) ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat Neurosci 16:1499-508
Butko, Margaret T; Savas, Jeffrey N; Friedman, Beth et al. (2013) In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation. Proc Natl Acad Sci U S A 110:E726-35
Miller, Evan W; Lin, John Y; Frady, E Paxon et al. (2012) Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires. Proc Natl Acad Sci U S A 109:2114-9

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