Imaging of glutamate in brain slices using FRET sensors.
The neurotransmitter glutamate is the mediator of excitatory neurotransmission in the brain. Release of this signaling molecule is carefully controlled by multiple mechanisms, yet the methods available to measure released glutamate have been limited in spatial and/or temporal domains. We have developed a novel technique to visualize glutamate release in brain slices using three purified fluorescence (Forster) energy resonance transfer (FRET)-based glutamate sensor proteins. Using a simple loading protocol, the FRET sensor proteins diffuse deeply into the extracellular space and remain functional for many tens of minutes. This allows imaging of glutamate release in brain slices with simultaneous electrophysiological recordings and provides temporal and spatial resolution not previously possible. Using this glutamate FRET sensor loading and imaging protocol, we show that changes in network excitability and glutamate re-uptake alter evoked glutamate transients and produce correlated changes in evoked-cortical field potentials. Given the sophisticated advantages of brain slices for electrophysiological and imaging protocols, the ability to perform real-time imaging of glutamate in slices should lead to key insights in brain function relevant to plasticity, development and pathology. This technique also provides a unique assay of network activity that compliments alternative techniques such as voltage-sensitive dyes and multi-electrode arrays.
Dulla
Chris
C
Department of Neurology and Neurological Sciences, Stanford University School of Medicine, 300 Pasteur Drive, Room M016, Stanford, CA 94305-5122, USA. dulla@stanford.edu
Tani
Hiroaki
H
Okumoto
Sakiko
S
Frommer
Wolf B
WB
Reimer
Rich J
RJ
Huguenard
John R
JR
D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D001921:Brain / Q000033:anatomy & histology*; D001923:Brain Chemistry; D002138:Calibration; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D018755:GABA Agonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D007202:Indicators and Reagents; D008297:Male; D009415:Nerve Net / Q000033:anatomy & histology; D011724:Pyridazines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins
D005456:Fluorescent Dyes; D018755:GABA Agonists; D007202:Indicators and Reagents; D011724:Pyridazines; D011994:Recombinant Proteins; D018698:Glutamic Acid; C049853:gabazine