A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons.
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.
Kuner
T
T
Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Augustine
G J
GJ
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002712:Chlorides / Q000032:analysis / Q000378:metabolism*; D003712:Dendrites / Q000378:metabolism; D004735:Energy Transfer; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013569:Synapses / Q000378:metabolism; D009435:Synaptic Transmission / Q000502:physiology; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D001426:Bacterial Proteins; D002712:Chlorides; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; C415371:clomeleon; C054562:yellow fluorescent protein, Bacteria; D005680:gamma-Aminobutyric Acid