Astrocytes play key roles in the central nervous system and regulate local blood flow and synaptic transmission via intracellular calcium (Ca2+) signaling. Astrocytic Ca2+ signals are generated by multiple pathways: Ca2+ release from the endoplasmic reticulum (ER) via the inositol 1, 4, 5-trisphosphate receptor (IP3R) and Ca2+ influx through various Ca2+ channels on the plasma membrane. However, the Ca2+ channels involved in astrocytic Ca2+ homeostasis or signaling have not been fully characterized. Here, we demonstrate that spontaneous astrocytic Ca2+ transients in cultured hippocampal astrocytes were induced by cooperation between the Ca2+ release from the ER and the Ca2+ influx through store-operated calcium channels (SOCCs) on the plasma membrane. Ca2+ imaging with plasma membrane targeted GCaMP6f revealed that spontaneous astroglial Ca2+ transients were impaired by pharmacological blockade of not only Ca2+ release through IP(3)Rs, but also Ca2+ influx through SOCCs. Loss of SOCC activity resulted in the depletion of ER Ca2+, suggesting that SOCCs are activated without store depletion in hippocampal astrocytes. Our findings indicate that sustained SOCC activity, together with that of the sarco-endoplasmic reticulum Ca2+-ATPase, contribute to the maintenance of astrocytic Ca2+ store levels, ultimately enabling astrocytic Ca2+ signaling. (C) 2017 Elsevier Inc. All rights reserved.

Astrocytes regulate hippocampal synaptic plasticity by the Ca2+ dependent release of the N-methyl D-aspartate receptor (NMDAR) co-agonist D-serine. Previous evidence indicated that D-serine release would be regulated by the intracellular Ca2+ release channel IP3 receptor (IP3R), however, genetic deletion of IP(3)R2, the putative astrocytic IP3R subtype, had no impact on synaptic plasticity or transmission. Although IP(3)R2 is widely believed to be the only functional IP3R in astrocytes, three IP3R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP3R and the contribution of the three IP3R subtypes to Ca2+ signalling. As a proxy for gliotransmission, we found that long-term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP3R blocker heparin, and rescued by exogenous D-serine, indicating that astrocytic IP(3)Rs regulate D-serine release. To explore which IP3R subtypes are functional in astrocytes, we used pharmacology and two-photon Ca2+ imaging of hippocampal slices from transgenic mice (IP(3)R2(-/-) and IP(3)R2(-/-); 3(-/-)). This approach revealed that underneath IP(3)R2-mediated global Ca2+ events are an overlooked class of IP3R-mediated local events, occurring in astroglial processes. Notably, multiple IP(3)Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca2+ release by astrocytic IP(3)Rs.

Calcium (Ca2+) is a versatile intracellular second messenger that operates in various signaling pathways leading to multiple biological outputs. The diversity of spatiotemporal patterns of Ca2+ signals, generated by the coordination of Ca2+ influx from the extracellular space and Ca2+ release from the intracellular Ca2+ store the endoplasmic reticulum (ER), is considered to underlie the diversity of biological outputs caused by a single signaling molecule. However, such Ca2+ signaling diversity has not been well described because of technical limitations. Here, we describe a new method to report Ca2+ signals at subcellular resolution. We report that OER-GCaMP6f, a genetically encoded Ca2+ indicator (GECI) targeted to the outer ER membrane, can monitor Ca2+ release from the ER at higher spatiotemporal resolution than conventional GCaMP6f. OER-GCaMP6f was used for in vivo Ca2+ imaging of C elegans. We also found that the spontaneous Ca2+ elevation in cultured astrocytes reported by OER-GCaMP6f showed a distinct spatiotemporal pattern from that monitored by plasma membrane-targeted GCaMP6f (Lck-GCaMP6f); less frequent Ca2+ signal was detected by OER-GCaMP6f, in spite of the fact that Ca2+ release from the ER plays important roles in astrocytes. These findings suggest that targeting of GECI5 to the ER outer membrane enables sensitive detection of Ca2+ release from the ER at subcellular resolution, avoiding the diffusion of GECI and Ca2+. Our results indicate that Ca2+ imaging with OER-GCaMP6f in combination with Lck-GCaMP6f can contribute to describing the diversity of Ca2+ signals, by enabling dissection of Ca2+ signals at subcellular resolution. (C) 2016 Elsevier Inc. All rights reserved.

細胞膜の構成要素である脂質・タンパク質は流体としての性質を持ち，側方拡散運動により自由に動く．自由拡散運動により脂質・タンパク質は均一に分布すると予想されるが，実際の細胞膜では膜分子の分布は一様ではない．シナプス後膜には神経伝達物質受容体が高密度で局在し，効率の良い神経伝達を可能にしている．いかにして神経細胞は，自由拡散運動に逆らって受容体の密度勾配を形成・維持し，シナプス伝達の制御を行っているのか？ 我々は，ほ乳類の中枢神経系のGABA作動性シナプスに注目し，「量子ドット1分子イメージング」という技術で細胞膜上の受容体1分子のふるまいを「見る」ことにより取り組んで来た．神経細胞膜上のGABAA受容体の動きを1分子レベルで追跡したところ，小胞体からのIP3誘導カルシウム放出（IICR）が，GABAA受容体を動きにくくし，抑制性シナプスの中でのGABAA受容体の安定性を高めていることが分かった．さらに，GABAA受容体の安定性を高めるためには，IP3受容体に加えて代謝型グルタミン酸受容体（mGluR）とリン酸化酵素プロテインキナーゼCの活性化が必要であることも明らかになった．これまでの研究で，グルタミン酸はNMDA型グルタミン酸受容体（NMDAR）を活性化し，細胞外から細胞内へ大量のカルシウムを流入させることにより，GABAA受容体を動きやすくすることが知られていた．一方，今回解明したメカニズムでは，同じグルタミン酸とカルシウムというシグナル物質が，mGluRとIP3受容体という全く異なる受容体を介して，逆にGABAA受容体を動きにくくし，安定性を高める働きをしていることが明らかになった．これはグルタミン酸とカルシウムというGABA作動性シナプス（GABAA受容体が機能するシナプス）の制御に関与するシグナル物質が，従来知られていた役割とは正反対の役割も担っていることを示す．

GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABA(A) receptor (GABA(A)R) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3 receptor-dependent calcium release and protein kinase C to promote GABA(A)R clustering and GABAergic transmission. This GABA(A)R stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses.

Astrocytes produce a complex repertoire of Ca2+ events that coordinate their major functions, The principle of Ca2+ events integration in astrocytes, however, is unknown. Here we analyze whole Ca2+ events, which were defined as spatiotemporally interconnected transient Ca2+ increases. Using such analysis in single hippocampal astrocytes in culture and in slices we found that spreads and durations of Ca2+ events follow power law distributions, a fingerprint of scale-free systems. A mathematical model demonstrated that such Ca2+ dynamics can arise from intracellular inositol-3-phosphate diffusion. The power law exponent (a) was decreased by activation of metabotropic glutamate receptors (mGluRs) either by specific receptor agonist or by low frequency stimulation of glutamatergic fibers in hippocampal slices. Decrease in a indicated an increase in proportion of large Ca2+ events. Notably, mGluRs activation did not increase the frequency of whole Ca2+ events. This result suggests that neuronal activity does not trigger new Ca2+ events in astrocytes (detectable by our methods), but modulates the properties of existing ones. Thus, our results provide a new perspective on how astrocyte responds to neuronal activity by changing its Ca2+ dynamics, which might further affect local network by triggering release of gliotransmitters and by modulating local blood flow. (C) 2014 Elsevier Ltd. All rights reserved.

This unit describes the method that we have developed to clarify endogenous mGluR5 (metabotropic glutamate receptors 5) dynamics in astrocytes by single-particle tracking using quantum dots (QD-SPT). QD-SPT has been a powerful tool to examine the contribution of neurotransmitter receptor dynamics to synaptic plasticity. Neurotransmitter receptors are also expressed in astrocytes, the most abundant form of glial cell in the brain. mGluR5s, which evoke intracellular Ca2+ signals upon receiving glutamate, contribute to the modulation of synaptic transmission efficacy and local blood flow by astrocytes. QD-SPT has previously revealed that the regulation of the lateral diffusion of mGluR5 on the plasma membrane is important for local Ca2+ signaling in astrocytes. Determining how mGluR5 dynamics are regulated in response to neuronal input would enable a better understanding of neuron-astrocyte communication in future studies. © 2014 by John Wiley and Sons, Inc.

Monitoring the pattern of intracellular Ca2+ signals that control many diverse cellular processes is essential for understanding regulatory mechanisms of cellular functions. Various genetically encoded Ca2+ indicators (GECIs) are used for monitoring intracellular Ca2+ changes under several types of microscope systems. However, it has not yet been explored which microscopic system is ideal for long-term imaging of the spatiotemporal patterns of Ca2+ signals using GECIs. We here compared the Ca2+ signals reported by a fluorescence resonance energy transfer (FRET)-based ratiometric GECI, yellow cameleon 3.60 (YC3.60), stably expressed in DT40 B lymphocytes, using three different imaging systems. These systems included a wide-field fluorescent microscope, a multipoint scanning confocal system, and a single-point scanning confocal system. The degree of photobleaching and the signal-to-noise ratio of YC3.60 in DT40 cells were highly dependent on the fluorescence excitation method, although the total illumination energy was maintained at a constant level within each of the imaging systems. More strikingly, the Ca2+ responses evoked by B-cell antigen receptor stimulation in YC3.60-expressing DT40 cells were different among the imaging systems, and markedly affected by the illumination power used. Our results suggest that optimization of the imaging system, including illumination and acquisition conditions, is crucial for accurate visualization of intracellular Ca2+ signals. (C) 2013 Elsevier Inc. All rights reserved.

Ca2+ release via inositol 1,4,5-trisphosphate (IP3) receptors (IP(3)Rs) plays a crucial role in astrocyte functions such as modulation of neuronal activity and regulation of local blood flow in the cerebral cortex and hippocampus. Bergmann glia are unipolar cerebellar astrocytes that release Ca2+ through IP(3)Rs in response to the activation of G(q)-coupled receptors. The composition of the three subtypes of IP3R is a factor that determines the spatiotemporal pattern of Ca2+ release. However, the functional expression of IP3R subtypes and their contribution to Ca2+ release in Bergmann glia remain controversial. In this study, we first characterized the Ca2+ response in Bergmann glia to noradrenaline and histamine stimulation in organotypic cultures of the mouse cerebellum using a Ca2+ indicator, Inverse-Pericam, and found that Bergmann glial processes exhibit a higher agonist-induced Ca2+ indicator response than the soma. Furthermore, we performed Ca2+ imaging using mutant mice lacking each IP3R subtype. This revealed that Bergmann glia lacking type 2 IP3R exhibited reduced responses to noradrenaline or histamine compared with wild-type Bergmann glia and Bergmann glia with other genotypes, suggesting that type 2 IP3R is the major functional IP3R subtype involved in agonist-induced Ca2+ release in Bergmann glia, although types 1 and 3 IP3R could also contribute to rapid agonist-induced [Ca2+](i) elevation in the processes. (C) 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

The flux of neurotransmitter receptors in and out of synapses depends on receptor interaction with scaffolding molecules. However, the crowd of transmembrane proteins and the rich cytoskeletal environment may constitute obstacles to the diffusion of receptors within the synapse. To address this question, we studied the membrane diffusion of the gamma-aminobutyric acid type A receptor (GABA(A)R) subunits clustered (gamma 2) or not (alpha 5) at inhibitory synapses in rat hippocampal dissociated neurons. Relative to the extrasynaptic region, gamma 2 and alpha 5 showed reduced diffusion and increased confinement at both inhibitory and excitatory synapses but they dwelled for a short time at excitatory synapses. In contrast, gamma 2 was similar to 3-fold more confined and dwelled similar to 3-fold longer in inhibitory synapses than alpha 5, indicating faster synaptic escape of alpha 5. Furthermore, using a gephyrin dominant-negative approach, we showed that the increased residency time of gamma 2 at inhibitory synapses was due to receptor-scaffold interactions. As shown for GABA(A)R, the excitatory glutamate receptor 2 subunit ( GluA2) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) had lower mobility in both excitatory and inhibitory synapses but a higher residency time at excitatory synapses. Therefore barriers impose significant diffusion constraints onto receptors at synapses where they accumulate or not. Our data further reveal that the confinement and the dwell time but not the diffusion coefficient report on the synapse specific sorting, trapping and accumulation of receptors.

Ca2+ microdomains or locally restricted Ca2+ increases in the cell have recently been reported to regulate many essential physiological events. Ca2+ increases through the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/Ca2+ release channels contribute to the formation of a class of such Ca2+ microdomains, which were often observed and referred to as Ca2+ puffs in their isolated states. In this report, we visualized IP3-evoked Ca2+ microdomains in histamine-stimulated intact HeLa cells using a total internal reflection fluorescence microscope, and quantitatively characterized the spatial profile by fitting recorded images to a two-dimensional Gaussian distribution. Ca2+ concentration profiles were marginally spatially anisotropic, with the size increasing linearly even after the amplitude began to decline. We found the event centroid drifted with an apparent diffusion coefficient of 4.20 +/- 0.50 mu m(2)/s, which is significantly larger than those estimated for IP(3)Rs. The sites of maximal Ca2+ increase, rather than initiation or termination sites, were detected repeatedly at the same location. These results indicate that Ca2+ microdomains in intact HeLa cell are generated from spatially distributed multiple IP3R clusters or Ca2+ puff sites, rather than a single IP3R cluster reported in cells loaded with Ca2+ buffers.

Metabotropic glutamate receptor (mGluR)-dependent calcium ion (Ca2+) signaling in astrocytic processes regulates synaptic transmission and local blood flow essential for brain function. However, because of difficulties in imaging astrocytic processes, the subcellular spatial organization of mGluR-dependent Ca2+ signaling is not well characterized and its regulatory mechanism remains unclear. Using genetically encoded Ca2+ indicators, we showed that despite global stimulation by an mGluR agonist, astrocyte processes intrinsically exhibited a marked enrichment of Ca2+ responses. Immunocytochemistry indicated that these polarized Ca2+ responses could be attributed to increased density of surface mGluR5 on processes relative to the soma. Single-particle tracking of surface mGluR5 dynamics revealed a membrane barrier that blocked the movement of mGluR5 between the processes and the soma. Overexpression of mGluR or expression of its carboxyl terminus enabled diffusion of mGluR5 between the soma and the processes, disrupting the polarization of mGluR5 and of mGluR-dependent Ca2+ signaling. Together, our results demonstrate an mGluR5-selective diffusion barrier between processes and soma that compartmentalized mGluR Ca2+ signaling in astrocytes and may allow control of synaptic and vascular activity in specific subcellular domains.

The activity-dependent modulation of GABA-A receptor (GABA(A)R) clustering at synapses controls inhibitory synaptic transmission. Several lines of evidence suggest that gephyrin, an inhibitory synaptic scaffold protein, is a critical factor in the regulation of GABA(A)R clustering during inhibitory synaptic plasticity induced by neuronal excitation. In this study, we tested this hypothesis by studying relative gephyrin dynamics and GABA(A)R declustering during excitatory activity. Surprisingly, we found that gephyrin dispersal is not essential for GABA(A)R declustering during excitatory activity. In cultured hippocampal neurons, quantitative immunocytochemistry showed that the dispersal of synaptic GABA(A)Rs accompanied with neuronal excitation evoked by 4-aminopyridine (4AP) or N-methyl-D-aspartic acid (NMDA) precedes that of gephyrin. Single-particle tracking of quantum dot labeled-GABA(A)Rs revealed that excitation-induced enhancement of GABA(A)R lateral mobility also occurred before the shrinkage of gephyrin clusters. Physical inhibition of GABA(A)R lateral diffusion on the cell surface and inhibition of a Ca2+ dependent phosphatase, calcineurin, completely eliminated the 4AP-induced decrease in gephyrin cluster size, but not the NMDA-induced decrease in cluster size, suggesting the existence of two different mechanisms of gephyrin declustering during activity-dependent plasticity, a GABA(A)R-dependent regulatory mechanism and a GABA(A)R-independent one. Our results also indicate that GABA(A)R mobility and clustering after sustained excitatory activity is independent of gephyrin.

Inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) is an intracellular Ca2+ release channel that plays crucial roles in the functions of Purkinje cells. The dynamics of IP(3)R1 on the endoplasmic reticulum membrane and the distribution of IP(3)R1 in neurons are thought to be important for the spatial regulation of Ca2+ release. In this study, we analyzed the lateral diffusion of IP(3)R1 in Purkinje cells in cerebellar slice cultures using fluorescence recovery after photobleaching. In the dendrites of Purkinje cells, IP(3)R1 showed lateral diffusion with an effective diffusion constant of approximately 0.30 mu m2/s, and the diffusion of IP(3)R1 was negatively regulated by actin filaments. We found that actin filaments were also involved in the regulation of IP(3)R1 diffusion in the spine of Purkinje cells. Glutamate or quisqualic acid stimulation, which activates glutamate receptors and leads to a Ca2+ transient in Purkinje cells, decreased the diffusion of IP(3)R1 and increased the density of actin in spines. These findings indicate that the neuronal activity-dependent augmentation of actin contributes to the stabilization of IP(3)R1 in spines.

An activity-dependent change in synaptic efficacy is a central tenet in learning, memory, and pathological states of neuronal excitability. The lateral diffusion dynamics of neurotransmitter receptors are one of the important parameters regulating synaptic efficacy. We report here that neuronal activity modifies diffusion properties of type-A GABA receptors (GABA(A)R) in cultured hippocampal neurons: enhanced excitatory synaptic activity decreases the cluster size of GABA(A)Rs and reduces GABAergic mIPSC. Single-particle tracking of the GABA(A)R gamma 2 subunit labeled with quantum dots reveals that the diffusion coefficient and the synaptic confinement domain size of GABA(A)R increases in parallel with neuronal activity, depending on Ca2+ influx and calcineurin activity. These results indicate that GABA(A)R diffusion dynamics are directly linked to rapid and plastic modifications of inhibitory synaptic transmission in response to changes in intracellular Ca2+, concentration. This transient activity-dependent reduction of inhibition would favor the onset of LTP during conditioning.

Zhang et al. and Maximov et al. [S. Zhang, A. Mizutani, C. Hisatsune, T. Higo, H. Bannai, T. Nakayama, M. Hattori, and K. Mikoshiba, Protein 4.1 N is required for translocation of inositol 1,4,5-trisphosphate receptor type I to the basolateral membrane domain in polarized Madin-Darby canine kidney cells, J. Biol. Chem. 278 (2003) 4048-4056; A. Maximov, T. S. Tang. and I. Bezprozvanny, Association of the type I inositol (1,4,5)-trisphosphate receptor with 4.1 N protein in neurons, Mol. Cell. Neurosci. 22 (2003) 271-283.] reported that 4. 1 N is a binding partner of inositol 1,4,5-trisphosphate receptor type I (IP(3)R1), however the binding site of IP(3)R1 differed: the former determined the C-terminal 14 amino acids of the cytoplasmic tail (CTT14aa) as the binding site.. while the latter assigned another segment, cytoplasmic tail middle 1 (CTM1). To solve this discrepancy, we performed immunoprecipitation and found that both the segments had binding activity to 4.1N. Both segments also interfered the 4.1N-regulated IP3R1 diffusion in neuronal dendrites. However, IP(3)R1 lacking the CTT14aa (IP(3)R1-ACTT14aa) does not bind to 4. 1 N [S. Zhang, A. Mizutani, C. Hisatsune, T. Higo, H. Bannai, T. Nakayama, M. Hattori, and K. Mikoshiba, Protein 4. IN is required for translocation of inositol 1,4,5-trisphosphate receptor type I to the basolateral. membrane domain in polarized Madin-Darby canine kidney cells, J. Biol. Chem. 2 78 (2003) 4048-4056.] and its diffusion constant is larger than that of IP(3)R1 full-length in neuronal dendrites [K. Fukatsu, H. Bannai.. S. Zhang, H. Nakamura, T. Inoue, and K. Mikoshiba, Lateral diffusion of inositol 1,4,5-trisphosphate receptor type I is regulated by actin filaments and 4. 1 N in neuronal dendrites, J. Biol. Chem. 279 (2004) 48976-48982.]. We conclude that both the CTT14aa and CTM1 sequences can bind to 4.1N in peptide fragment forms. However, we propose that the responsible binding site for 4.1 N binding in full-length tetramer form of IP(3)R1 is CTT14aa. (c) 2006 Elsevier Inc. All rights reserved.

This protocol describes a sensitive approach to tracking the motion of membrane molecules such as lipids and proteins with molecular resolution in live cells. This technique makes use of fluorescent semiconductor nanocrystals, quantum dots (QDs), as a probe to detect membrane molecules of interest. The photostability and brightness of QDs allow them to be tracked at a single particle level for longer periods than previous fluorophores, such as fluorescent proteins and organic dyes. QDs are bound to the extracellular part of the object to be followed, and their movements can be recorded with a fluorescence microscope equipped with a spectral lamp and a sensitive cooled charge-coupled device camera. The experimental procedure described for neurons below takes about 45 min. This technique is applicable to various cultured cells.

The inositol 1,4,5-trisphosphate (IP3) receptor (IPsR) Ca2+ channel plays pivotal roles in many aspects of physiological and pathological events. It was previously reported that IP3R forms clusters on the endoplasmic reticulum when cytosolic Ca2+ concentration ([Ca2+](C)) is elevated. However, the molecular mechanism of IP,R clustering remains largely unknown, and thus its physiological significance is far from clear. In this study we found that the time course of clustering of green fluorescent protein-tagged IP3R type 1 (GFP-IP(3)R1), evoked by IP3-generating agonists, did not correlate with [Ca2+](C) but seemed compatible with cytoplasmic IP3 concentration. IP3 production alone induced GFP-Ip(3)R1 clustering in the absence of a significant increase in [Ca2+](C) but elevated [Ca2+](C) without IP3 production did not. Moreover IP(3)R1 mutants that do not undergo an IP3-induced conformational change failed to form clusters. Thus, IP3R clustering is induced by its IP3-induced conformational change to the open state. We also found that GFP-Ip(3)R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity. This is the first example of ligand-induced clustering of a ligand-gated channel protein.

mRNA transport and local translation in the neuronal dendrite is implicated in the induction of synaptic plasticity. Recently, we cloned an RNA-interacting protein, SYNCRIP ( heterogeneous nuclear ribonuclear protein Q1/NSAP1), that is suggested to be important for the stabilization of mRNA. We report here that SYNCRIP is a component of mRNA granules in rat hippocampal neurons. SYNCRIP was mainly found at cell bodies, but punctate expression patterns in the proximal dendrite were also seen. Time-lapse analysis in living neurons revealed that the granules labeled with fluorescent protein-tagged SYNCRIP were transported bi-directionally within the dendrite at similar to0.05 mum/s. Treatment of neurons with nocodazole significantly inhibited the movement of green fluorescent protein-SYNCRIP-positive granules, indicating that the transport of SYNCRIP-containing granules is dependent on microtubules. The distribution of SYN-CRIP-containing granules overlapped with that of dendritic RNAs and elongation factor 1alpha. SYNCRIP was also found to be co-transported with green fluorescent protein-tagged human staufen1 and the 3'-untranslated region of inositol 1,4,5-trisphosphate receptor type 1 mRNA. These results suggest that SYNCRIP is transported within the dendrite as a component of mRNA granules and raise the possibility that mRNA turnover in mRNA granules and the regulation of local protein synthesis in neuronal dendrites may involve SYNCRIP.

Inositol 1,4,5-trisphosphate receptor type1 (IP(3)R1) plays an important role in neuronal functions; however, the lateral diffusion of IP(3)R1 on the endoplasmic reticulum membrane and its regulation in the living neurons remain unknown. We expressed green fluorescent protein-tagged IP(3)R1 in cultured rat hippocampal neurons and observed the lateral diffusion by the fluorescence recovery after photobleaching technique. IP(3)R1 showed lateral diffusion with an effective diffusion constant of similar to 0.3 mum(2)/s. Depletion of actin filaments increased the diffusion constant of IP(3)R1, suggesting that the diffusion of IP(3)R1 is regulated negatively through actin filaments. We also found that protein 4.1N, which binds to IP(3)R1 and contains an actin-spectrin-binding region, was responsible for this actin regulation of the IP(3)R1 diffusion constant. Overexpression of dominant-negative 4.1N and blockade of 4.1N binding to IP3R1 increased the IP(3)R1 diffusion constant. The diffusion of IP3R type 3 (IP(3)R3), one of the isoforms of IP(3)Rs lacking the binding ability to 4.1N, was not dependent on actin filaments but became dependent on actin filaments after the addition of a 4.1N-binding sequence. These data suggest that 4.1N serves as a linker protein between IP(3)R1 and actin filaments. This actin filament-dependent regulation of IP(3)R1 diffusion may be important for the spatiotemporal regulation of intracellular Ca2+ signaling.

The type 1 mositol 1,4,5-trisphosphate receptor (IP(3)R1) is an intracellular Ca2+ channel protein that plays crucial roles in generating complex Ca2+ signalling patterns. Ip(3)R1 consists of three domains: a ligand-binding domain, a regulatory domain and a channel domain. In order to investigate the function of these domains in its gating machinery and the physiological significance of specific cleavage by caspase 3 that is observed in cells undergoing apoptosis, we utilized various IP(3)R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP(3)R1 or IP(3)R1 lacking the ligand-binding domain in HeLa. and COS-7 cells had little effect on cells' responsiveness to an IP3-generating agonist ATP and Ca2+ leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP-IP(3)R1-casp) or the channel domain alone (GFP-IP(3)R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca2+ concentration. Interestingly, store-operated (-like) Ca2+ entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca2+ stores of cells expressing GFP-IP(3)R1-casp or GFP-IP(3)R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca2+. We therefore propose that the channel domain of IP(3)R1 tends to remain open and that the large regulatory domain of IP(3)R1 is necessary to keep the channel domain closed. Thus cleavage of IP(3)R1 by caspase 3 may contribute to the increased cytosolic Ca2+ concentration often observed in cells undergoing apoptosis. Finally, GFP-IP(3)R1-casp or GFP-IP(3)R1-ES can be used as a novel tool to deplete intracellular Ca2+ stores.

Although spatially restricted Ca2+ release from the endoplasmic reticulum (ER) through intracellular Ca2+ channels plays important roles in various neuronal activities, the accurate distribution and dynamics of ER in the dendrite of living neurons still remain unknown. To elucidate these, we expressed fluorescent protein-tagged ER proteins in cultured mouse hippocampal neurons, and monitored their movements using time-lapse microscopy. We report here that a sub-compartment of ER forms in relatively large vesicles that are capable, similarly to the reticular ER, of taking up and releasing Ca2+. The vesicular sub-compartment of ER moved rapidly along the dendrites in both anterograde and retrograde directions at a velocity of 0.2-0.3 mum/second. Depletion of microtubules, overexpression of dominant-negative kinesin and kinesin depletion by antisense DNA reduced the number and velocity of the moving vesicles, suggesting that kinesin may drive the transport of the vesicular sub-compartment of ER along microtubules in the dendrite. Rapid transport of the Ca2+-releasable sub-compartment of ER might contribute to rapid supply of fresh ER proteins to the distal part of the dendrite, or to the spatial regulation of intracellular Ca2+ signaling.

Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) using a yeast two-hybrid system. 4.1N and IP(3)R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP(3)R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP(3)R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 3057330585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP(3)R1 is necessary and sufficient for the localization of IP(3)R1 at the basolateral membrane domain. A fragment of the IP(3)R1-binding region of 4.1N blocks the localization of co-expressed IP3R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP3R1 translocation to the basolateral membrane domain in polarized MDCK cells.

The changes in the bending pattern of flagella induced by an increased intracellular Ca2+ concentration are caused by changes in the pattern and velocity of microtubule sliding. However, the mechanism by which Ca2+ regulates microtubule sliding in flagella has been unclear To elucidate it, we studied the effects of Ca2+ on microtubule sliding in reactivated sea urchin sperm flagella that were beating under imposed head vibration, We found that the maximum microtubule sliding velocity obtainable by imposed vibration, which was about 170-180 rad/second in the presence of 250 mu M MgATP and <10(-9) M Ca2+, was decreased by 10(-6)-10(-5) M Ca2+ by about 15-20%. Similar decrease of the sliding velocity was observed at 54 and 27 mu M MgATP. The Ca2+-induced decrease of the sliding velocity was due mainly to a decrease in the reverse bend angle. When the plane of beat was artificially rotated by rotating the plane of vibration of the pipette that held the sperm head, the asymmetric bending pattern also rotated at 10(-5) M Ca2+ as well as at <10(-9) M Ca2+. The rotation of the bending pattern was observed at MgATP higher than 54 mu M (similar to 100 mu M ATP). These results indicate that the Ca2+-induced decrease of the sliding velocity is mediated by a rotatable component or components (probably the central pair) at high MgATP, but is not due to specific dynein arms on particular doublets, We further investigated the effects of a mild trypsin treatment and of trifluoperazine on the Ca2+-induced decrease in sliding velocity. Axonemes treated for 3 minutes with a low concentration (0.1 mu g/ml) of trypsin beat with a more symmetrical waveform than before the treatment. Also, their microtubule sliding velocity and reverse bend angle were not affected by high Ca2+ concentrations. Trifluoperazine (25-50 mu M) had no effect on the decrease of the sliding velocity in beating flagella at 10(-5) M Ca2+. However, the flagella that had been 'quiescent' at 10(-4) M Ca2+ resumed asymmetrical beating following an application of 10-50 mu M trifluoperazine. In such beating flagella, both the sliding velocity and the reverse bend angle mere close to their respective values at 10(-5) M Ca2+. Trypsin treatment induced a similar recovery of beating in quiescent flagella at 10(-4) M Ca2+, albeit with a more symmetrical waveform. These results provide first evidence that, at least at ATP concentrations higher than similar to 100 mu M, 10(-6)-10(-5) M Ca2+ decreases the maximum sliding velocity of microtubules in beating flagella through a trypsin-sensitive regulatory mechanism which possibly involves the central pair apparatus. They also suggest that calmodulin may be associated with the mechanism underlying flagellar quiescence induced by 10(-4) M Ca2+.

In the spinal cord, most inhibitory synapses have a mixed glycine-GABA phenotype. Using a pharmacological approach, we report an NMDAR activity-dependent regulation of the mobility of GlyRs but not GABA(A)Rs at inhibitory synapses in cultured rat spinal cord neurons. The NMDAR-induced decrease in GlyR lateral diffusion was correlated with an increase in receptor cluster number and glycinergic mlPSC amplitude. Changes in GlyR diffusion properties occurred rapidly and before the changes in the number of synaptic receptors. Regulation of synaptic GlyR content occurred without change in the amount of gephyrin. Moreover, NMDAR-dependent regulation of GlyR lateral diffusion required calcium influx and calcium release from stores. Therefore, excitation may increase GlyR levels at synapses by a calcium-mediated increase in postsynaptic GlyR trapping involving regulation of receptor-scaffold interactions. This provides a mechanism for a rapid homeostatic regulation of the inhibitory glycinergic component at mixed glycine-GABA synapses in response to increased NMDA excitatory transmission.

細胞内カルシウムイオン(Ca2+)の増減「Ca2+シグナル」は、細胞の外界からの情報を適切な生理応答へと変換する役割を担う、普遍的な細胞内シグナルである。しかし、同じCa2+という伝達物質を用いて複数のメッセージを生命暗号としてコードし、多様な生理機能の誘導を可能にする機構については未解明の点が多い。先行研究において我々は、「Local Ca2+センサー」を作成し、従来法では判別できなかったカルシウムシグナルの由来（細胞内からの放出か、細胞外からの流入か）を特定し、Ca2+シグナルの多様性を記述するための新解析法を確立している。今年度は、同一細胞で２カ所同時でのCa2+シグナル測定を可能にするために、波長の異なるセンサーを利用して細胞膜とER膜の同時カルシウムイメージングの技術を確立した。細胞膜に標的したRCaMP2 (Lck-RCaMP2)とER膜に標的したGCaMP6f (OER-GCaMP6f)を同一細胞で、同時に観察できる測定系を確立した。広い視野をもつCMOSカメラとイメージスプリッティング光学系の組み合わせにより、時空間的に高い解像度で、Ca2+シグナルの発生パターンを記録することができた。この測定系を用いてHeLa細胞のHistamine刺激に対する応答を観察したところ、細胞膜に標的したセンサーは長期間持続したCa2+シグナルを示すのに対し、ER膜標的センサーは速やかなCa2+シグナルの低下を示した。この結果は、同じ刺激をうけても、Ca2+シグナルは細胞の局所ドメインごとに異なるを示すということを意味している。

分子の動きを１分子解像度で解析できる「量子ドット１分子イメージング法」は、細胞膜に現れる細胞・分子レベルの異常を検出するための、画期的なブレイクスルーである。本研究では、この膜分子動態に着目し、老化脳タンパク質が引き起こす「異常な膜分子動態」を検出し、それらが神経細胞毒性を及ぼすシグナルカスケードを分子レベルで明らかにすることを目指している。今年度は、正常な神経細胞に発現する「生理的タウ」が神経機能に果たす役割について調べた。タウ欠損マウス由来の海馬初代培養神経細胞の膜分子動態に現れる異常を、量子ドット１分子イメージング法を用いて解析した。量子ドットで神経機能に関わる機能膜タンパク質（神経伝達物質受容体、Gタンパク質共役型受容体、細胞接着因子、トランスポーター、リガンド受容体）をラベルし、１分子解像度で細胞膜上での分子動態を追跡したところ、タウ欠損神経細胞の樹状突起・細胞体において、特定の膜分子の動態が増加することがわかった。タウ欠損により興奮性シナプスの長期抑圧（LTD）が失われることが先行研究により報告されている。その分子機構を明らかにするために、LTD誘導時のAMPA受容体分子の１分子の動きと、AMPA受容体のエンドサイトーシス量を測定した。１分子イメージングにより解析したところ、タウ欠損マウスと野生型のAMPA受容体の拡散ダイナミクスが異なることがわかった。また、内在性AMPA受容体のエンドサイトーシス量を定量化するために、pH感受性蛍光色素Acidifluorを利用した新規エンドサイトーシス定量化法を開発した。

ドイツのグリア研究コンソーシアムと連携し、日独若手研究者の共同研究を目的とした国際グリア若手の会（YoungGlia）を2015年度から立ち上げた。2015年度は日本、2016年度はドイツでYoungGliaを行い、研究提案、審査を経て計11件採択した。助成グループは1-2年間の共同研究を行った。相互交流に必要な旅費、滞在費を助成した。2017年度には報告会を日本で行った。この会にはカナダ、アメリカからの若手研究者も招聘し、YoungGliaを日独の二国間から多国間への取り組みへと発展させた。若手研究者同士が自由な発想に基づき、共同研究提案を行うことは画期的である。

グリア細胞の存在は多くの生物種で知られているが，ほ乳類以外の動物において神経系で，脳機能の制御に積極的に関わるか否かは明らかにされていない．本研究では，が透明で遺伝子組換え動物の作成が容易であり，逃避行動に関わる神経回路に関する知見が豊富なゼブラフィッシュの利点を生かし，グリア細胞におけるカルシウムシグナルが個体の行動制御を司る可能性を明らかにした。細胞膜に標的したGCaMP6f(LCK-GCaMP6f)をGFAPプロモータ制御下で発現するゼブラフィッシュを用いて受精後3-7日稚魚脳のin vivoイメージングを行い，視蓋，後脳，脊髄のグリア細胞で自発的なカルシウムシグナルが存在することを確認した．また、逃避行動を誘起する聴覚刺激(500 Hz)を与えたところ，後脳のグリア細胞では自発的カルシウムシグナルとは異なる，より長く広範囲に広がるカルシウムシグナルが観察された．この結果は、魚類でも神経活動に伴ってグリア細胞の活動が誘起されることを示唆している．また、Young gliaの枠組みで、デュッセルフ大学のGriemsmaan博士と共同で、げっ歯類アストロサイトAMPA受容体の１分子イメージング実験を行った。さらに、ER標的型のGCaMP6fを開発し、げっ歯類海馬アストロサイトにおいて自発的カルシウムシグナルイメージングを行ったところ、実はERからのカルシウム放出よりもカルシウム流入の方が多いことがわかった(Niwa et al. 2016). このカルシウム流入は正体はStore operated Ca2+ channel (SOCC)を介しておこっており、海馬アストロサイトではERが枯渇しない状態でもSOCCが恒常的に活性化され、ERのカルシウム量を一定に保つことに貢献していることが示された(Sakuragi et al. 2017)。

ほ乳類の脳においてグリア細胞の一種アストロサイトは，神経細胞の生存や発達機能発現のためのサポートに加えて，カルシウムシグナル依存的にシナプス伝達効率や局所脳血流の制御を司る．グリア細胞の存在は多くの生物種で知られているが，ほ乳類以外の動物において，グリア細胞が成熟した神経系において脳機能の制御に積極的に関わるか否かはほとんど明らかにされていない．本研究は，ゼブラフィッシュをモデル生物として，ほ乳類のアストロサイト同様魚類のグリア細胞が，成熟した神経系においてカルシウムシグナル依存的に神経伝達や動物の行動を制御できるかを検討し，グリア細胞の役割の進化的意義を探ることを目的とする．本年度は計画通り，ゼブラフィッシュグリア細胞におけるカルシウムシグナル検出法の確立を行った．名古屋大学の小田洋一教授，谷本昌志助教の指導のもと，逆行性ラベルやサカナのライブイメージング等，基本となる手技を獲得した．遺伝子コード型カルシウム指示薬(GPCI)をグリア細胞特異的に発現するゼブラフィッシュ系統を作成するために，東島眞一准教授（生理学研究所）および川上浩一教授（遺伝学研究所）との共同研究を開始した．サカナの遺伝子組み換えに必要なベクターを入手し，本研究に特化したGPCIの改変を行った．また，本研究のもう一つの柱は，光学顕微鏡の分解能を越えた「超解像STED顕微鏡」を利用したシナプスーグリア細胞相互作用形態の観察である．そのために，STED顕微鏡の専門家であるボルドー大学のValentin Nagerl教授を招聘し，ゼブラフィッシュの脳イメージングに適用するためのアドバイスを得た．

シナプス伝達の強さを決めるファクターとして,近年,細胞膜上における受容体の側方拡散が注目されている.本研究では,細胞外からのカルシウム流入とカルシニュリンの活性化により誘導されるGABA_A受容体の側方拡散の促進が,抑制性シナプス可塑性の開始点であることを示した.また,細胞内カルシウム貯蔵庫からのカルシウム放出も,タンパク質リン酸化によりGABA_A受容体の側方拡散を抑制している可能性を提示した.

IP_3受容体は細胞内小胞体からのカルシウム放出を担うチャネルの一つであり,その機能は時間的空間的に厳密に制御されている.IP_3受容体の局在がカルシウム放出の空間的制御に寄与していると予想されているが,カルシウムシグナルとIP_3受容体の局在を関連づけた研究はこれまで行われていない.MDCK細胞のIP_3受容体タイプ1は,sub-confluent状態では細胞全体のER膜上に存在しconfluent状態ではER膜から離れて細胞膜近傍に局在することが知られているので,IP_3受容体の位置変化にともなって"量子的IP_3依存的カルシウム放出"(Ca^<2+> puff)の位置もsub-confluentとconfluentで変化するか否かを,全反射顕微鏡を利用したカルシウムイメージング法で調べた.実験初期の段階で,MDCK細胞はconfluencyに関わらず自発的なカルシウム流入を常に行っていることを見いだした.このカルシウム流入は細胞外カルシウム濃度依存的であり,Ca^<2+> puffと同様量子的に観察された.Ca^<2+> puffをこの量子的カルシウム流入と区別するために,本研究では細胞外カルシウム非存在下でCa^<2+> puffの測定を行った.カルシウム指示薬にFluo-4,細胞膜のラベルにoctadecyl rhodamine B chlorideを用いることにより,Ca^<2+> puffの細胞の位置情報が得られる実験系を確立した.現在実験で得られたデータをもとに細胞膜とCa^<2+> puffがおこる場所との関係を解析し,sub-confluentとconfluentで差があるかどうかを統計的に検討している.また,MDCK細胞における自発的な量子的カルシウム流入は新しい知見であり,腎臓におけるカルシウムの再吸収に関わる可能性が考えられる.このためカルシウム流入を担うチャネルを同定するために,薬理学的実験を行っている.