Abstract
We constructed a novel optical indicator for chloride ions by fusing the chloride-sensitive yellow fluorescent protein with the chloride-insensitive cyan fluorescent protein. The ratio of FRET-dependent emission of these fluorophores varied in proportion to the concentration of Cl and was used to measure intracellular chloride concentration ([Cl-]i) in cultured hippocampal neurons. [Cl-]i decreased during neuronal development, consistent with the shift from excitation to inhibition during maturation of GABAergic synapses. Focal activation of GABAA receptors caused large changes in [Cl-]i that could underlie use-dependent depression of GABA-dependent synaptic transmission. GABA-induced changes in somatic [Cl-]i spread into dendrites, suggesting that [Cl-]i can signal the location of synaptic activity. This genetically encoded indicator will permit new approaches ranging from high-throughput drug screening to direct recordings of synaptic Cl- signals in vivo.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Cells, Cultured
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Chlorides / analysis
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Chlorides / metabolism*
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Dendrites / metabolism
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Energy Transfer
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Fluorescent Dyes
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Hippocampus / cytology
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Hippocampus / metabolism*
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Hydrogen-Ion Concentration
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Intracellular Fluid / chemistry
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Intracellular Fluid / metabolism
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Neurons / cytology
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Neurons / metabolism*
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Rats
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Rats, Sprague-Dawley
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Receptors, GABA-A / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism*
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Signal Transduction / physiology
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Spectrometry, Fluorescence
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Synapses / metabolism
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Synaptic Transmission / physiology
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Transfection
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gamma-Aminobutyric Acid / metabolism
Substances
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Bacterial Proteins
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Chlorides
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Fluorescent Dyes
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Luminescent Proteins
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Receptors, GABA-A
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Recombinant Fusion Proteins
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clomeleon
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yellow fluorescent protein, Bacteria
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gamma-Aminobutyric Acid