Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters.
Genetically encoded sensors of glutamate concentration are based on FRET between cyan and yellow fluorescent proteins bracketing a bacterial glutamate-binding protein. Such sensors have yet to find quantitative applications in neurons, because of poor response amplitude in physiological buffers or when expressed on the neuronal cell surface. We have improved our glutamate-sensing fluorescent reporter (GluSnFR) by systematic optimization of linker sequences and glutamate affinities. Using SuperGluSnFR, which exhibits a 6.2-fold increase in response magnitude over the original GluSnFR, we demonstrate quantitative optical measurements of the time course of synaptic glutamate release, spillover, and reuptake in cultured hippocampal neurons with centisecond temporal and spine-sized spatial resolution. During burst firing, functionally significant spillover persists for hundreds of milliseconds. These glutamate levels appear sufficient to prime NMDA receptors, potentially affecting dendritic spike initiation and computation. Stimulation frequency-dependent modulation of spillover suggests a mechanism for nonsynaptic neuronal communication.
Hires
Samuel Andrew
SA
Graduate Program in Neurosciences and Department of Pharmacology and Howard Hughes Medical Institute, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093.
Zhu
Yongling
Y
Tsien
Roger Y
RY
D000595:Amino Acid Sequence; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D009435:Synaptic Transmission
D018698:Glutamic Acid