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Clomeleon Biosensor-Mauslinien zur optophysiologischen Bestimmung der intrazellulären Chloridkonzentration in Nervenzellen

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Schleich,  Wolfram
Department of Cell Physiology, Max Planck Institute for Medical Research, Max Planck Society;

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Schleich, W. (2006). Clomeleon Biosensor-Mauslinien zur optophysiologischen Bestimmung der intrazellulären Chloridkonzentration in Nervenzellen. PhD Thesis, Naturwissenschaftlich-Mathematische Gesamtfakultät der Ruprecht-Karls-Universität Heidelberg, Heidelberg.


Cite as: http://hdl.handle.net/11858/00-001M-0000-002A-EE78-0
Abstract
Chloride is of eminent relevance for neural signal processing. Its intracellular concentration determines whether GABA or glycine excite or inhibit neurons. Despite its physiological relevance only a few techniques are available to measure intracellular chloride concentrations. Clomeleon is a ratiometric genetically encoded indicator comprising a fusion of cyan and yellow fluorescent protein that allows noninvasive chloride measurements in living tissue. The work presented here deals with strategies to make this indicator available in biosensor mouse lines and to test their applicability by investigating the developmental dependence of chloride concentration in neurons. Seven mouse lines that express the indicator under control of Thy1 promotor have been generated. Each line exhibits a distinct expression pattern. Generally widespread and high expression of the indicator is observed. These lines are well suited for population imaging. Single-cell imaging is challenging due to the dense expression but became feasible by applying a quantitative approach that allowed for the determination of autofluorescence background by comparison with wildtype tissue. By considering these constraints the postulated decrease in intracellular chloride of neurons could be shown using acute slices. Starting from a concentration of 21 mM at p5 the concentration drops to 6 mM at p20 in CA1 neurons. That means an inversion of a GABAergic response around p14. Furthermore a universal chloride indicator mouse line was engineered making use of the ubiquitously active ROSA26 promotor and a loxP-flanked stop-cassette. This mouse line allows for expression of Clomeleon in a genetically defined cell population by a simple breeding scheme. This concept harbors enormous possibilities to improve imaging conditions. On the one side all collected light originates from the same genetically identified population augmenting the significance of population experiments. On the other side this concept increases the chance to obtain a sparse labeling facilitating background acquisition so vital for single-cell measurements. Due to the low expression levels in neural tissue, intensity based measurements are hardly feasible. The employment of 2P FLIM holds promise to conduct experiments despite low fluorescence. We pursued a similar labeling concept by combining virus-mediated gene transfer and techniques of conditional gene expression. We succeeded in achieving high levels of Clomelon expression in genetically defined neurons of an activator mouse line by infecting with the non-toxic AAV1/2 virus. This situation provides best conditions for single cell imaging. We showed that chronic expression of Clomelon in biosensor mouse lines enables successful non-invasive optophysiological recordings of intracellular chloride concentrations. This establishes a new tool for answering many questions that were experimentally inaccessible up to now.