Zinc is a metal ion with many functions in the body, including roles in DNA recognition, recognition, enzymatic catalysis, and neural function. Among these, the role(s) of zinc in the brain are the least well understood, yet evidence is mounting that zinc plays a role in nerve cell death following stroke, Alzheimer's disease, epilepsy, and other forms of brain damage. The major goal of this proposal is to develop a group of optical probes for studying zinc in neural tissue by fluorescence microscopy employing our fluorescence-based biosensor technology. The fluorescence-based sensing technology utilizes the high selectivity and affinity of human apo-carbonic anhydrase for zinc; upon binding Zn(II), a covalent or noncovalent label on the protein exhibits changes in fluorescence intensity, wavelength, lifetime, and/or anisotropy. Fluorescence anisotropy microscopy offers significant advantages over wavelength-ratiometric and lifetime-based microscopies. The demonstrated advantages of this approach compared to other fluorescence probes for zinc are thousand-fold better sensitivity (down to picomolar zinc); insensitivity to Mg(II) at 50 mM, Ca at 10 mM, and most other metals; excitation at visible wavelengths, and high efficiency.
Specific aims i nclude quantitative imaging of zinc release from mossy fiber boutons in the hippocampus, imaging intracellular free zinc levels and fluxes in cultured cells, observing zinc release in vivo with a microdialysis technique, and developing probes for multiphoton-excitation microscopy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS038585-01A1
Application #
6051089
Study Section
Special Emphasis Panel (ZRG1-MDCN-3 (01))
Program Officer
Michel, Mary E
Project Start
1999-12-17
Project End
2003-11-30
Budget Start
1999-12-17
Budget End
2000-11-30
Support Year
1
Fiscal Year
2000
Total Cost
$291,814
Indirect Cost
Name
University of Maryland Baltimore
Department
Biochemistry
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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