Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters. Proc Natl Acad Sci U S A

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.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2008; 105(11):4411-6. DOI: 10.1073/pnas.0712008105
Source: PubMed


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.

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Available from: Yongling Zhu, Jan 17, 2014
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    • "In the best DEBP-based glutamate sensors, however, DEBP has been modified in the hinge region between the domains (Hires et al. 2008; Marvin et al. 2013), raising the possibility that the capture and release of glutamate by wild-type DEBP involves only a local rather than global structural change. "
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    • "We observed that activation of extrasynaptic NMDARs enhanced striatal correlated network activity and increased dispersion of correlated pairs of neurons in the network (Fig. 3). These results are consistent with increases in extrasynaptic interactions and promotion of network synchrony observed following spillover of glutamate (Mitchell et al., 2007; Harris and Pettit, 2008; Hires et al., 2008; Chalifoux and Carter, 2011). By blocking extrasynaptic NMDAR we could evaluate effects of synaptic NMDAR activation on neuronal activity and network patterns (Figs. 3 and 4). "
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    • "Linkers can have dramatic effects on sensor responses (Deuschle et al., 2005b; Hires et al., 2008; Takanaga et al., 2008). Therefore, five different linkers were inserted between the two PAS domains in dPAS110. "
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