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inputs, local increases in extrasynaptic glutamate concentration were detected (Figure 1B). To induce detectable glutamate spillover, repetitive inputs of high frequency (50-100 Hz) were necessary. Further-more, while stimulation of a single axon was insuf-ficient to induce glutamate spillover, stimulation of an axon bundle locally induced glutamate spillover that was sufficient to activate mGluRs. These results indicate that the spatial and temporal summation of glutamate release is essential for the glutamate spillover to activate mGluRs (Figure 1C). Therefore, not a single synaptic event, but a spatio-temporal cluster of glutamate release events is the unit of synaptic mGluR signaling (Figure 2).Cooperative production of IP3During mGluR activation, ionotropic glutamate receptors, such as the α-amino-3-hydroxy-5-methyl -4-isoxazolepropionic acid receptor (AMPAR), expressed within the synaptic cleft, are also acti-vated. However, it was unclear how this simulta-neous activation of different types of glutamate receptors affected mGluR-IP3-Ca2+ signaling at synapses. I conjectured that IP3 might play a key role in the potential crosstalk between mGluRs and AMPARs.IP3 was imaged with GFP-PHD, a genetically Figure 2　Synaptic mGluR signaling mechanisms revealed by fluorescence imaging.Glutamate spillover induced by spatio-temporal cluster of inputs forms the unit of synaptic mGluR signaling. Crosstalk between mGluR and AMPAR in IP3 production is a physiological booster of synaptic mGluR signaling. The ER functions as a Ca2+ store for the rapid and efficient redistribution of Ca2+ upon synaptic mGluR signaling. Modified from Okubo Folia Pharmacol Jpn 2014.encoded IP3 indicator12, 13, 17) (Figure 3A). GFP-PHD is a fusion protein of green fluorescent protein (GFP) and the pleckstrin homology (PH) domain. The PH domain binds to phosphatidylinositol 4,5- bisphosphate (PIP2) in the plasma membrane. The PH domain also binds to IP3 with 20-fold higher affinity. Therefore, GFP-PHD translocates from the plasma membrane to the cytosol in response to increases in IP3 concentration (Figure 3A). By imaging this translocation of GFP-PHD, we can analyze IP3 dynamics.GFP-PHD was expressed in a neuron in acute brain slices using a viral vector and imaged with two-photon microscopy. Translocation of GFP-PHD was monitored by the increase in fluorescence intensity in the cytosol of fine dendrites. IP3 produc-tion was observed upon the repetitive stimulation of axon bundles, consistent with a requirement for glutamate spillover. This IP3 production was blocked by an mGluR antagonist, as expected (Figure 3B). Figure 3　Imaging IP3 production with GFP-PHD.A: Schematic of IP3 imaging by GFP-PHD. GFP-PHD translocates from the plasma membrane to the cytosol upon IP3 production. B: IP3 production cooperatively mediated by mGluR and AMPAR. Both mGluR and AMPAR antagonists blocked synaptic IP3 production. Modified from Okubo J Neurosci 2004 and Okubo Antioxid Redox Signal 2011.159