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　An intricate interplay of complex spatio-temporal events underlies brain functions. Therefore, clarifying these dynamic processes is indispensable for revealing the mechanisms of brain functions. Fluorescence imaging is a powerful technique for visualizing cellular and molecular dynamics in the brain. Recent developments in fluorescent indicators and specialized optics have advanced research in the field of neuroscience. In this review, I will exemplify the power and beauty of fluorescence imaging by discussing my work focusing on the molecular dynamics of metabotropic glutamate receptor (mGluR) signaling at the synapse. By developing novel fluorescent indicators for glutamate, inositol 1,4,5-trisphosphate and Ca2+ within the endoplasmic reticulum, I succeeded in imaging the spatio-temporal dynamics of synaptic mGluR signaling, which led to the identification of novel mechanisms of mGluR-mediated glutamatergic neurotransmission. These discoveries highlight the importance of the development and application of novel fluorescence imaging techniques for the investigation of brain functions.Juntendo Medical Journal2022. 68(2), 157-162Key words: calcium, fluorescence microscope, glutamate, inositol 1,4,5-trisphosphate, metabotropic glutamate receptorMini ReviewsInvestigation of Brain Functions with Fluorescence Imaging TechniquesIntroductionComplex spatio-temporal processes are the foun-dation of the information-processing power of the brain. For example, the dynamic interplay of different brain regions is the basis of the regulation of physical and mental states. Spatio-temporal firing patterns of neuronal networks in the brain encode sensory inputs and behavioral outputs. Elaborate molecular dynamics at synapses is essen-tial for synaptic transmission, the elemental basis of brain functions. Therefore, the visualization of spatio-temporal dynamics is key to understanding the mechanisms of brain functions. Fluorescence microscopy is a powerful technique for imaging cellular and molecular dynamics in living samples. Various fluorescent molecules including small-mol-ecule fluorophores and fluorescent proteins have been used as biocompatible dyes for specific labeling of molecules of interest. Furthermore, fluo-Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of MedicineCorresponding author: Yohei Okubo (ORCiD: 0000-0001-7611-3237)Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of MedicineHongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, JapanTEL: +81-3-5802-1035　E-mail: y.okubo.xq@juntendo.ac.jp〔Received　Dec. 9, 2021〕〔Accepted　Jan. 5, 2022〕J-STAGE Advance published date: Apr. 15, 2022Copyright © 2022 The Juntendo Medical Society. This is an open access article distributed under the terms of Creative Commons Attribution License (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original source is properly credited. doi: 10.14789/jmj.JMJ21-0051-OTrescent molecules have been functionalized as fluo-rescent indicators for imaging the dynamics of various events such as local Ca2+ signaling at synapses1). Imaging within the intact brain tissue is crucial to the study of brain functions. However, light scattering by brain tissue is a major obstacle for conventional fluorescence microscopy. Develop-ment of two-photon microscopy2) solved this problem and allowed high-resolution imaging deep within the brain tissue in vivo3). Recent progress in fluorescent indicators and optics have revolution-ized the neurosciences by enabling long-awaited analyses such as the imaging of synaptic plasticity at single-synapse resolution4, 5), the spatio-temporal analysis of firing patterns of hundreds of neurons in vivo6), the identification of circuit rewiring upon learning7), and the direct imaging of neurotrans-mitter dynamics8, 9). The continuous development of fluorescence imaging techniques has been advancing research into brain functions.157Yohei OKUBO