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J. Biol. Chem., Vol. 281, Issue 25, 17420-17431, June 23, 2006

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Two-photon Imaging of Glutathione Levels in Intact Brain Indicates Enhanced Redox Buffering in Developing Neurons and Cells at the Cerebrospinal Fluid and Blood-Brain Interface^*

Xiaojian Sun, Andy Y. Shih¹, Helge C. Johannssen², Heidi Erb, Ping Li, and Timothy H. Murphy, Supported by the Canadian Stroke Network and is an MSFHR Senior Scholar³

From the Departments of Psychiatry and Physiology, Kinsmen Laboratory of Neurological Research and Brain Research Centre, University of British Columbia, Vancouver V6T 1Z3, British Columbia, Canada

Glutathione is the major cellular thiol present in mammalian cells and is critical for maintenance of redox homeostasis. However, current assay systems for glutathione lack application to intact animal tissues. To map the levels of glutathione in intact brain with cellular resolution (acute tissue slices and live animals), we have used two-photon imaging of monochlorobimane fluorescence, a selective enzyme-mediated marker for reduced glutathione. Previously, in vitro experiments using purified components and cultured glial cells attributed cellular monochlorobimane fluorescence to a glutathione S-transferase-dependent reaction with GSH. Our results indicate that cells at the cerebrospinal fluid or blood-brain interface, such as lateral ventricle ependymal cells (2.73 ± 0.56 mM; glutathione), meningeal cells (1.45 ± 0.09 mM), and astroglia (0.91 ± 0.08 mM), contain high levels of glutathione. In comparison, layer II cortical neurons contained 20% (0.21 ± 0.02 mM) the glutathione content of nearby astrocytes. Neuronal glutathione labeling increased 250% by the addition of the cell-permeable glutathione precursor N-acetylcysteine indicating that the monochlorobimane level or glutathione S-transferase activity within neurons was not limiting. Regional mapping showed that glutathione was highest in cells lining the lateral ventricles, specifically ependymal cells and the subventricular zone, suggesting a possible function for glutathione in oxidant homeostasis of developing neuronal progenitors. Consistently, developing neurons in the subgranular zone of dentate gyrus contained 3-fold more glutathione than older neurons found in the neighboring granular layer. In conclusion, mapping of glutathione levels in intact brain demonstrates a unique role for enhanced redox potential in developing neurons and cells at the cerebrospinal fluid and blood-brain interface.

Received for publication, February 17, 2006 , and in revised form, April 17, 2006.

^* This work was supported in part by a Canadian Institutes of Health Research training grant (to X. S.), a Canadian Institutes of Health Research Canadian Graduate Scholarship (to A. Y. S.), and an operating grant from the Heart and Stroke Foundation of British Columbia and Yukon (to T. H. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a Michael Smith Foundation for Health Research (MSFHR) studentship.

² Present address: Dept. of Neurophysiology, Brain Research Institute, University of Zurich, Zurich, Switzerland.

³ To whom correspondence should be addressed: 4N1-2255 Wesbrook Mall, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-0705; Fax: 604-822-7981; E-mail: thmurphy{at}interchange.ubc.ca.

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