Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters

Samuel Andrew Hires, Yongling Zhu, Roger Y. Tsien

Research output: Contribution to journalArticlepeer-review

232 Scopus citations

Abstract

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 glutamatesensing 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.

Original languageEnglish (US)
Pages (from-to)4411-4416
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number11
DOIs
StatePublished - Mar 18 2008

Keywords

  • Fluorescence resonance energy transfer
  • Hippocampal neurons
  • Synaptic release

ASJC Scopus subject areas

  • General

Fingerprint

Dive into the research topics of 'Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters'. Together they form a unique fingerprint.

Cite this