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160Surprisingly, an AMPAR antagonist also signifi-cantly blocked IP3 production (Figure 3B). These results indicate a cooperative contribution of mGluR and AMPAR to IP3 production. I then inves-tigated the cellular components mediating this cooperativity and found that intracellular loading with a G protein inhibitor and a Ca2+ chelator blocked IP3 production. This strongly suggests that AMPAR-dependent Ca2+ influx via voltage-gated Ca2+ channels enhances mGluR-G protein-depen-dent IP3 production (Figure 2). This crosstalk between mGluR and AMPAR can function as a physiological booster of IP3 signaling at synapses.Diffusion-dependent refilling of Ca2+ in the ERActivation of mGluRs induces Ca2+ release from the ER via IP3 receptors. Because the ER has a relatively small volume and limited Ca2+ buffering capacity compared with the cytosol at synapses, Ca2+ release inevitably results in the depletion of Ca2+ within the ER. Therefore, ER Ca2+ refilling mechanisms are a critical determinant of the interval of synaptic mGluR signaling. However, ER Ca2+ refilling mechanisms at synapses remained elusive due to a lack of indicators for imaging intra-luminal Ca2+ dynamics within the ER. To resolve this problem, novel genetically encoded Ca2+ indica-tors (GECIs) termed calcium-measuring organ-elle-entrapped protein indicators (CEPIAs) were developed15, 18) (Figure 4A). The Ca2+ concentra-tion within the ER can reach millimolar amounts. Therefore, GECIs for cytosolic Ca2+ cannot be applied, and indicators with appropriately low affinity for Ca2+ are required. Through the muta-genesis of previously developed cytosolic GECIs, indicators that cover the ER Ca2+ concentration range were obtained (Figure 4A). The brightness and large signal amplitude of the new CEPIA enabled unprecedented high-resolution imaging.G-CEPIA1er was expressed in neurons in acute brain slices and imaged with two-photon micros-copy. G-CEPIA1er was observed throughout the dendrites and spines, consistent with the distribu-tion of the ER network in neurons. Transient decreases in G-CEPIA1er fluorescence were observed in an input-specific manner (Figure 4B). This suggested local ER Ca2+ depletion mediated by mGluR signaling followed by ER Ca2+ refilling. The conventional view of refilling mechanisms posits a Figure 4　Imaging Ca2+ dynamics within the ER with CEPIA.A: Ca2+ affinity of CEPIA was optimized for Ca2+ concentrations within the ER. B: Plot of G-CEPIA1er fluorescence intensity and corresponding grayscale image upon afferent stimulation indicate local ER Ca2+ depletion followed by refilling upon synaptic mGluR signaling in dendrites of a neuron. C: ER Ca2+ refilling mediated by lateral diffusion of Ca2+ within the ER network. A SERCA inhibitor (cyclopiazonic acid, CPA) had a negligible effect on recovery dynamics of ER Ca2+ concentration. Modified from Okubo J Neurosci 2015.major contribution to Ca2+ reuptake by sarco/endo-plasmic reticulum Ca2+-ATPase (SERCA). However, lateral diffusion of Ca2+ within the contiguous ER network may also have an important role, espe-cially given the local depletion within the small dendritic compartment. Indeed, diffusional dynamics