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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    • "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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    ABSTRACT: N-methyl-D-aspartate receptors (NMDAR) are crucial for the function of excitatory neurotransmission and are present at the synapse and on the extrasynaptic membrane. The major nucleus of the basal ganglia, striatum, receives a large glutamatergic excitatory input carrying information about movements and associated sensory stimulation for its proper function. Such bombardment of glutamate synaptic release results in a large extracellular concentration of glutamate that can overcome the neuronal and glial uptake homeostatic systems therefore allowing the stimulation of extrasynaptic glutamate receptors. Here we have studied the participation of their extrasynaptic type in cortically evoked responses or in the presence of NMDARs stimulation. We report that extrasynaptic NMDAR blocker memantine, reduced in a dose-dependent manner cortically induced NMDA excitatory currents in striatal neurons (recorded in zero-Mg(++) plus DNQX 10μM). Moreover, memantine (2-4μM) significantly reduced the NMDAR-dependent membrane potential oscillations called up and down states. Recordings of neuronal striatal networks with a fluorescent calcium indicator or with multielectrode arrays (MEA) also showed that memantine reduced in a dose-dependent manner, NMDA-induced excitatory currents and network behavior. We used multielectrode arrays (MEA) to grow segregated cortical and striatal neurons. Once synaptic contacts were developed (>21DIV) recordings of extracellular activity confirmed the cortical drive of spontaneous synchronous discharges in both compartments. After severing connections between compartments, active striatal neurons in the presence of memantine (1μM) and CNQX (10μM) were predominantly fast spiking interneurons (FSI). The significance of extrasynaptic receptors in the regulation of striatal function and neuronal network activity is evident.
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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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    • "However, distance dependency of FRET may not always be easily deconvolved from orientation dependency. For example, while developing a unimolecular sensor for glutamate based on the conformational change of a glutamate-binding protein, one group observed a 4-fold change in dynamic range between versions that differed in length by only one amino acid (Hires et al., 2008). They ascribed this change in sensitivity of energy transfer to minute differences in orientation and distance, which were enhanced by the rigidity of the peptide in between the fluorescent proteins. "
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