QUANTITATIVE IMAGING OF ZINC METABOLISM IN THE BRAIN

锌是一种在体内具有多种功能的金属离子，包括DNA识别、识别、酶催化和神经功能。其中，锌在大脑中的作用(S)被了解得最少，然而越来越多的证据表明，锌在中风、阿尔茨海默病、癫痫和其他形式的脑损伤后的神经细胞死亡中发挥作用。这项建议的主要目标是利用我们的荧光生物传感器技术，开发一组用于研究神经组织中锌的荧光显微镜下的光学探针。这种基于荧光的传感技术利用了人脱氢碳酸氢酶对锌的高选择性和亲和力；当结合锌(II)时，蛋白质上的共价或非共价标记显示出荧光强度、波长、寿命和/或各向异性的变化。荧光各向异性显微镜比波长比显微镜和基于寿命的显微镜具有显著的优势。与其他锌荧光探针相比，这种方法的优点是灵敏度提高了数千倍(低至皮摩尔锌)；对50 mm的镁(II)、10 mm的钙以及大多数其他金属不敏感；在可见光波长下激发，并且效率高。具体目标包括定量成像从海马区苔藓纤维环释放的锌，成像培养细胞内游离锌的水平和通量，用微透析技术观察体内锌的释放，以及开发多光子激发显微镜的探针。

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 include 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.