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158served as the glutamate binding protein, and the small-molecule fluorophore was attached near the binding pocket (Figure 1A). Changes in the fluo-rescence signal occur upon glutamate binding because of the environmental sensitivity of the fluo-rophore component. By screening of candidates, several indicators with high affinity and large signal amplitude were obtained (Figure 1A).EOS labels extrasynaptic sites in acute brain slices and the brain in vivo. Changes in EOS fluo-rescence induced by glutamate spillover were imaged by two-photon microscopy. In response to synaptic Figure 1　Imaging glutamate spillover with EOS.A: (Top) Structure of EOS. Glutamate binding domain (S1S2) of GluA2 subunit was isolated and the fluorophore was labeled. (Bottom) Dose-response relationship of fluorescence intensity of EOS against glutamate. B: Increases in fluorescence intensity of EOS in response to afferent stimulation in the acute cerebellar slice. C: Spatio-temporal cluster of glutamate release induces a local increase in extrasynaptic glutamate concentration. Modified from Okubo Proc Natl Acad Sci U S A 2010 and Okubo Folia Pharmacol Jpn 2014.Fluorescence imaging of metabotropic glutamate receptor (mGluR) signalingIn this review, I will discuss fluorescence imaging of brain functions with examples from my studies. My work has focused on the molecular dynamics of mGluR signaling at synapses10). Glutamatergic transmission is mediated not only by ionotropic glutamate receptors, but also by G protein-coupled mGluRs. There are eight types of mGluRs divided into group I (mGluR1 and mGluR5), group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7 and mGluR8). Group II and III mGluRs are mainly distributed at presynaptic terminals and involved in the inhibition of neurotransmitter release via Gi/o protein signaling. On the other hand, group I mGluRs are postsynaptic receptors that mediate the production of inositol 1,4,5-trisphosphate (IP3) via Gq protein to induce Ca2+ release from the endo-plasmic reticulum (ER) via the IP3 receptor. This postsynaptic mGluR-IP3-Ca2+ signaling is involved in many important synaptic functions including synaptic plasticity. Furthermore, group I mGluR- targeted drugs exert various psychiatric effects. Although the functional significance of group I mGluRs is obvious, mGluR-targeted therapeutics has not been available so far. This is mainly due to a lack of knowledge about the detailed mechanism of synaptic mGluR signaling. To address this issue, the investigation of the dynamics of molecular signaling in situ is essential. Therefore, I set out to develop techniques for the direct imaging of gluta-mate9, 11), IP312-14) and Ca2+ within the ER15 at synapses in brain tissue.Clustered input-dependent glutamate spillovermGluRs are localized to the perisynaptic regions that border the synaptic clefts. Therefore, gluta-mate spillover from the synaptic clefts is required for the activation of mGluRs. However, the charac-teristics of the glutamate spillover were unclear. Therefore, I decided to directly analyze glutamate spillover by glutamate imaging.To image glutamate dynamics, a fluorescent glutamate indicator named Glutamate (E) Optical Sensor (EOS) was developed9, 16) (Figure 1). EOS is a hybrid type indicator consisting of a glutamate binding protein and a small-molecule fluorophore. The extracellular domain of the glutamate receptor