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

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.fluorescence resonance energy transfer ͉ hippocampal neurons ͉ synaptic release G lutamate is the primary excitatory neurotransmitter in the brain, and precise measurement of its spatiotemporal pattern of synaptic release and propagation would provide insight into diverse brain processes, including synaptic crosstalk, cerebral ischemia, and mechanisms of learning and memory. In hippocampal slices, synaptic glutamate spillover to the dendrite and neighboring synapses induces homeostatic regulation of glutamate release through extrasynaptic mGluR activation (1), limits synaptic independence (2), lengthens EPSC durations (3), and permits heterosynaptic LTP/LTD (4). Spillover is a primary means of chemical neurotransmission between mitral cells in the rat olfactory bulb (5) and between climbing fibers and molecular layer interneurons in cerebral cortex (6). Estimates of glutamate concentration and dynamics in synaptic, extrasynaptic, and extracellular compartments have been made by NMDAR antagonist displacement (7), glutamate uptake inhibitor application (2), whole ''sniffer'' cells (8), outside-out ''sniffer'' patch electrodes (9, 10), patch recording of astrocyte synaptically evoked transporter currents (STCs) (11), enzymatically coupled electrochemical probes (12), enzymatically coupled metabolite imaging (13), and other methods. Although each method provided a new perspective on glutamate action, all were hampered by a lack of resolution in the spatial or temporal domains because of single-site measurement, reliance on partially coupled or confounded currents, desensitizing receptors, or indirect and slow secondary cascades.Recently, the glutamate reporters glutamate-sensing fluorescent reporter (GluSnFR) ʈ (14) and fluorescent indicator protein for glutamate (FLIPE) (15) were constructed by linear genetic fusions of the glutamate periplasmic binding protein GltI (also known as ybeJ) ...

Aβ induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss

“…1073/pnas.1306832110/-/DCSupplemental. glutamate sensor system (SuperGluSnFR) (17) to detect the local concentration of glutamate contiguous to astrocytes after exposure to Aβ peptides. To ensure the close apposition of the sensor probe (consisting of GluSnFR-transfected HEK 293 cells) to the astrocytes, the sensor cells were genetically engineered to coexpress neuroligin (18) (Fig.…”

Reporting neural activity with genetically encoded calcium indicators

“…Discrepancies are likely due to differences in the biochemical milieu of the assay . Ionic strength can dramatically affect the maximum ratio change of FRET reporters (Hires et al, 2008) or the apparent affinity of the ligand-binding domain. Calmodulin's calcium affinity is reduced~3-fold when assayed in 150 mM vs. 100 mM [KCl] (Linse et al, 1991), and both affinity and apparent cooperativity are reduced with increasing [Mg 2+ ] (Ogawa and Tanokura, 1984).…”

“…Alternative FRET pairs using GFP and tdTomato or other coral-derived FPs may have greater brightness, tissue penetration, and photostability (Shaner et al, 2005). In both cases, substitution of these proteins may necessitate sensor re-optimization, as emission dipoles and linker effects can be starkly different even with subtly different FRET pairs (Hires et al, 2008). For single-FP GECIs, the extinction coefficient and quantum yield of the saturated state of G-CaMP2 (Tallini et al, 2006) could potentially be increased to the level of EGFP.…”

Fluorescence resonance energy transfer imaging of PKC signalling in living cells using genetically encoded fluorescent probes

“…This can be achieved by using circular permutated fluorescent proteins ( Nagai et al 2004). Another approach is to systematically alter the length of the linker between the probes and the sensor region (Hires et al 2008). Finally, the FRET efficiency generally increases when red-shifted probes are used (Goedhart et al 2007).…”

“…The conformation of a protein can be studied by sandwiching the protein of interest between a donor and an acceptor. It may be worthwhile to optimize the efficiency and dynamic range by optimizing the linker length (Hires et al 2008) or the conformation of the fluorescent protein (Nagai et al 2004). …”