Schizophrenia is a chronic and disabling psychiatric disorder characterized by disturbances of thought, cognition, and behavior. Despite massive research efforts to date, the etiology and pathophysiology of schizophrenia remain largely unknown. The difficulty of brain research is largely a result of complex interactions between contributory factors at different scales: susceptible gene variants (molecular scale), synaptopathies (synaptic, dendritic, and cell scales), and alterations in neuronal circuits (circuit scale), which together result in behavioral manifestations (individual scale). It is likely that each scale affects the others, from the microscale to the mesoscale to the macroscale, and vice versa. Thus, to consider the intricate complexity of schizophrenia across multiple layers, we introduce a multi-scale, hierarchical view of the nature of this disorder, focusing especially onN-methyl-D-aspartate-type glutamate receptors (NMDARs). The reason for placing emphasis on NMDAR is its clinical relevance to schizophrenia, as well as its diverse functions in neurons, including the robust supralinear synaptic integration provided byN-methyl-D-aspartate-type glutamate (NMDA) spikes and the Ca(2+)permeability of the NMDAR, which facilitates synaptic plasticity via various calcium-dependent proteins. Here, we review recent evidence implicating NMDARs in the pathophysiology of schizophrenia from the multi-scale perspective. We also discuss recent advances from optical techniques, which provide a powerful tool for uncovering the mechanisms of NMDAR synaptic pathology and their relationships, with subsequent behavioral manifestations.

Recording the electrical activity of multiple neurons simultaneously would greatly facilitate studies on the function of neuronal circuits. The combination of the fast scanning by random-access multiphoton microscopy (RAMP) and the latest two-photon-compatiblehigh-performance fluorescent genetically encoded voltage indicators (GEVIs) has enabled action potential detection in deep layers in in vivo brain. However, neuron connectivity analysis on optically recorded action potentials from multiple neurons in brain tissue has yet to be achieved. With high expression of a two-photon-compatible GEVI, ASAP3, via in utero electroporation and RAMP, we achieved voltage recording of spontaneous activities from multiple neurons in brain slice. We provide evidence for the developmental changes in intralaminar horizontal connections in somatosensory cortex layer 2/3 with a greater sensitivity than calcium imaging. This method thus enables investigation of neuronal network connectivity at the cellular resolution in brain tissue.

HER2 is overexpressed in 25-30% of breast cancers, and approximately 30% of HER2-positive breast cancers metastasize to the brain. Although the incidence of brain metastasis in HER2-positive breast cancer is high, previous studies have been mainly based on cell lines of the triple-negative subtype, and the molecular mechanisms of brain metastasis in HER2-positive breast cancer are unclear. In the present study, we performed intracranial injection using nine HER2-positive breast cancer cell lines to evaluate their proliferative activity in brain tissue. Our results show that UACC-893 and MDA-MB-453 cells rapidly proliferated in the brain parenchyma, while the other seven cell lines moderately or slowly proliferated. Among these nine cell lines, the proliferative activity in brain tissue was not correlated with either the HER2 level or the HER2 phosphorylation status. To extract signature genes associated with brain colonization, we conducted microarray analysis and found that these two cell lines shared 138 gene expression patterns. Moreover, some of these genes were correlated with poor prognosis in HER2-positive breast cancer patients. Our findings might be helpful for further studying brain metastasis in HER2-positive breast cancer.

RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein (Arc) and inositol 1,4,5-trisphosphate receptor type 1 (Ip3r1) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF-FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells.

© 2020, Springer Science+Business Media, LLC, part of Springer Nature. Quantum dot-single-particle tracking (QD-SPT) is a super-resolution imaging technique that uses semiconductor nanocrystal quantum dots as fluorescent probes and is a powerful tool for analyzing protein and lipid behavior in the plasma membrane. Recent QD-SPT experiments have provided critical insight into the mechanism and physiological relevance of membrane self-organization in neurons and astrocytes in the brain. The mobility of some membrane molecules may become abnormal in cellular models of epilepsy and Alzheimer’s disease. Based on these findings, we propose that the behavior of membrane molecules reflects the condition of neurons in pathological disease states. In this chapter, we describe the latest, simple QD-SPT technique, which is feasible with epifluorescence microscopy and dissociated cell cultures.

Sonic hedgehog (SHH) is important for organogenesis during development. Recent studies have indicated that SHH is also involved in the proliferation and transformation of astrocytes to the reactive phenotype. However, the mechanisms underlying these are unknown. Involvement of SHH signaling in calcium (Ca) signaling has not been extensively studied. Here, we report that SHH and Smoothened agonist (SAG), an activator of the signaling receptor Smoothened (SMO) in the SHH pathway, activate Ca oscillations in cultured murine hippocampal astrocytes. The response was rapid, on a minute time scale, indicating a noncanonical pathway activity. Pertussis toxin blocked the SAG effect, indicating an involvement of a G(i) coupled to SMO. Depletion of extracellular ATP by apyrase, an ATP-degrading enzyme, inhibited the SAG-mediated activation of Ca oscillations. These results indicate that SAG increases extracellular ATP levels by activating ATP release from astrocytes, resulting in Ca oscillation activation. We hypothesize that SHH activates SMO-coupled Gi in astrocytes, causing ATP release and activation of G(q/11)-coupled P2 receptors on the same cell or surrounding astrocytes. Transcription factor activities are often modulated by Ca patterns; therefore, SHH signaling may trigger changes in astrocytes by activating Ca oscillations. This enhancement of Ca oscillations by SHH signaling may occur in astrocytes in the brain in vivo because we also observed it in hippocampal brain slices. In summary, SHH and SAG enhance Ca oscillations in hippocampal astrocytes, G(i) mediates SAG-induced Ca oscillations downstream of SMO, and ATP-permeable channels may promote the ATP release that activates Ca oscillations in astrocytes.

The astrocyte, one of the glial cells, plays many functional roles. These include provision of nutrients from blood vessels to neurons, supply of neurotransmitters and support of blood-brain barrier (BBB) integrity. Astrocytes are known to support the integrity of BBB through maintenance of the tight junction between endothelial cells of blood vessels. However, evidence of its direct contribution to BBB is lacking owing to technical limitations. In this study, astrocytic endfeet covering blood vessels were removed by the laser ablation method with two photon laser scanning microscopy in in vivo mouse brain, and the re-covering of blood vessels with the astrocytic endfeet was observed in about half of the cases. Blood vessels kept their integrity without astrocytic endfoot covers: leakage of plasma marker dyes, Evans Blue or dextran-conjugated fluorescein, was not observed from stripped blood vessels, while ablation of vascular walls induced extravasation of Evans Blue. These results suggest that the astrocytic endfeet covering blood vessels do not contribute to the immediate BBB barrier.

Calmodulin-dependent protein kinase II (CaMKII) and calmodulin (CaM) play essential roles insynaptic plasticity, which is an elementary process of learning and memory. In this study, fluorescencecorrelation spectroscopy (FCS) revealed diffusion properties of CaM, CaMKII alpha and CaMKII beta proteins inhuman embryonic kidney 293 (HEK293) cells and hippocampal neurons. A simultaneous multiple-point FCS recording system was developed on a random-access two-photon microscope, which facilitatedefficient analysis of molecular dynamics in neuronal compartments. The diffusion of CaM in neuronswas slower than that in HEK293 cells at rest, while the diffusion in stimulated neurons was acceleratedand indistinguishable from that in HEK293 cells. This implied that activity-dependent binding partnersof CaM exist in neurons, which slow down the diffusion at rest. Diffusion properties of CaMKII alpha and beta proteins implied that major populations of these proteins exist as holoenzymatic forms. Upon stimulationof neurons, the diffusion of CaMKII alpha and beta proteins became faster with reduced particle brightness, indicating drastic structural changes of the proteins such as dismissal from holoenzyme structure andfurther fragmentation. (c) 2018 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.

TI Workbench is a software package that serves as a control and analysis center for cellular imaging and electrophysiological experiments. It is unique among general-purpose software packages where it integrates the control of cellular imaging and electrophysiological devices, as well as sophisticated data analyses, which provides superior usability in imaging experiments combined with electrophysiology. During the development over the last 20 years, the range of supported image acquisition devices has expanded from cooled charge-coupled device (CCD) cameras to multi-photon microscope systems. In this review, I outline the concept of TI Workbench together with its unique functions and features derived from ideas emerging during daily experiments in my own lab and in those of my collaborators over the last 20 years. TI Workbench includes standard functions required for time-lapse multicolor fluorescence imaging and electrophysiological experiments, in addition to specialized functions such as random-scan or conventional raster-scan two-photon microscopy packages and fluorescence life time imaging (FLIM) utilities. Data analysis modules, e.g. digital data filters for temporal waveforms of time-lapse image data and electrophysiology and for 2-D image data, and fluorescence correlation spectroscopy (FCS) analysis functions, are well integrated with data acquisition functions. A notebook function holds formatted text, graphs, image and movie data altogether, which are linked to the actual data files. TI Workbench uses Igor Pro software as a back-end output for publishing. In addition, TI Workbench imports several different formats of image and electrophysiology data, serving as a general-purpose data analysis software package.

Cyclin-dependent kinase 5 (Cdk5) is recognized as a unique member among other Cdks due to its versatile roles in many biochemical processes in the nervous system. The proper development of neuronal dendrites is required for the formation of complex neural networks providing the physiological basis of various neuronal functions. We previously reported that sparse dendrites were observed on cultured Cdk5-null Purkinje cells and Purkinje cells in Wnt1(cre)-mediated Cdk5 conditional knockout (KO) mice. In the present study, we generated L7(cre)-mediated p35; p39 double KO (L7(cre)-p35(f/f); p39(-/-)) mice whose Cdk5 activity was eliminated specifically in Purkinje cells of the developing cerebellum. Consequently, these mice exhibited defective Purkinje cell migration, motor coordination deficiency and a Purkinje dendritic abnormality similar to what we have observed before, suggesting that dendritic growth of Purkinje cells was cell-autonomous in vivo. We found that mixed and overlay cultures of WT cerebellar cells rescued the dendritic deficits in Cdk5-null Purkinje cells, however, indicating that Purkinje cell dendritic development was also supported by non-cell-autonomous factors. We then again rescued these abnormalities in vitro by applying exogenous brain-derived neurotrophic factor (BDNF). Based on the results from culture experiments, we attempted to rescue the developmental defects of Purkinje cells in L7(cre)-p35(f/f); p39(-/-) mice by using a TrkB agonist. We observed partial rescue of morphological defects of dendritic structures of Purkinje cells. These results suggest that Cdk5 activity is required for Purkinje cell dendritic growth in cell-autonomous and non-cell-autonomous manners. (c) 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1175-1187, 2017

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Keratinocytes undergo a unique type of programmed cell death known as cornification, which leads to the formation of the stratum corneum (SC), the main physical barrier of the epidermis. A defective epidermal barrier is a hallmark of the two most common inflammatory skin disorders, psoriasis, and atopic dermatitis. However, the detailed molecular mechanisms of skin barrier formation are not yet fully understood. Here, we showed that downregulation of phospholipase C (PLC) d 1, a Ca 2+-mobilizing and phosphoinositide-metabolizing enzyme abundantly expressed in the epidermis, impairs the barrier functions of the SC. PLCd 1 downregulation also impairs localization of tight junction proteins. Loss of PLCd 1 leads to a decrease in intracellular Ca 2+ concentrations and nuclear factor of activated T cells activity, along with hyperactivation of p38 mitogen-activated protein kinase (MAPK) and inactivation of RhoA. Treatment with a p38 MAPK inhibitor reverses the barrier defects caused by PLCd 1 downregulation. Interestingly, this treatment also attenuates psoriasis-like skin inflammation in imiquimod-treated mice. These findings demonstrate that PLCd 1 is essential for epidermal barrier integrity. This study also suggests a possible link between PLCd 1 downregulation, p38 MAPK hyperactivation, and barrier defects in psoriasis-like skin inflammation.

Growing evidence suggests that excess glucocorticoids (GCs) exposure during the pregnancy results in behavioral abnormality in offspring. Although research using animal models has demonstrated that systemic GCs treatment impairs development of fetal brain, direct impact of GCs on the phenotype of embryonic neural stem/progenitor cells (eNSPCs) and its mechanism has not been fully understood. Here, we investigated the effect of chronic GCs exposure on cell proliferation, differentiation, and survival of eNSPCs in vitro. Corticosterone (CORT, a murine GC) treatment did not affect the proliferation of eNSPCs. On the other hand, decreased expression of neuronal, synaptic, and astroglial marker proteins were observed when the differentiation of eNSPCs was induced in the presence of CORT. CORT also reduced the survival rate of eNSPCs after the differentiation. Moreover, CORT inhibited extracellular signal-regulated kinase (ERK) and phosphatidylinosito13-kinase/Akt (PI3K/Akt) signaling pathways, which were activated during cell differentiation of eNSPCs. Inhibiting these signaling pathways reduced neural differentiation and eNSPCs viability, indicating their essential roles in the eNSPCs differentiation. Furthermore, IGF-I, a potent PI3K/Akt and ERK signaling stimulator, partially restored the adverse effect of CORT on eNSPCs, suggesting a possible involvement of the repression of these intracellular signaling in the GCs-caused eNSPCs impairment. (C) 2016 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Orexins (hypocretins) are neuropeptides that regulate multiple homeostatic processes, including reward and arousal, in part by exciting serotonergic dorsal raphe neurons, the major source of forebrain serotonin. Here, using mouse brain slices, we found that, instead of simply depolarizing these neurons, orexin-A altered the spike encoding process by increasing the postspike afterhyperpolarization (AHP) via two distinct mechanisms. This orexin-enhanced AHP (oeAHP) was mediated by both OX1 and OX2 receptors, required Ca2+ influx, reversed near E-K, and decayed with two components, the faster of which resulted from enhanced SK channel activation, whereas the slower component decayed like a slow AHP (sAHP), but was not blocked by UCL2077, an antagonist of sAHPs in some neurons. Intracellular phospholipase C inhibition (U73122) blocked the entire oeAHP, but neither component was sensitive to PKC inhibition or altered PKA signaling, unlike classical sAHPs. The enhanced SK current did not depend on IP3-mediated Ca2+ release but resulted from A-current inhibition and the resultant spike broadening, which increased Ca2+ influx and Ca2+ -induced-Ca2+ release, whereas the slower component was insensitive to these factors. Functionally, the oeAHP slowed and stabilized orexin-induced firing compared with firing produced by a virtual orexin conductance lacking the oeAHP. The oeAHP also reduced steady-state firing rate and firing fidelity in response to stimulation, without affecting the initial rate or fidelity. Collectively, these findings reveal a new orexin action in serotonergic raphe neurons and suggest that, when orexin is released during arousal and reward, it enhances the spike encoding of phasic over tonic inputs, such as those related to sensory, motor, and reward events.

© 2016 Elsevier Inc. All rights reserved. Alzheimer's disease (AD) is one of the best known neurodegenerative diseases; it causes dementia and its pathological features include accumulation of amyloid β (Aβ) and neurofibrillary tangles (NFTs) in the brain. Elevated Cdk5 activity and CRMP2 phosphorylation have been reported in the brains of AD model mice at the early stage of the disease, but the significance thereof in human AD remains unelucidated. We have recently reported that Aβ accumulation in the cerebellum of AD model APPswe/PS1dE9 (APP/PS1) mice, and cerebellar dysfunctions, such as impairment of motor coordination ability and long-term depression (LTD) induction, at the pre-Aβ accumulation stage. In the present study, we found increased phosphorylation levels of CRMP2 as well as increased p35 protein levels in the cerebellum of APP/PS1 mice. Interestingly, we show that pioglitazone, an agonist of peroxisome proliferator-activated receptor γ, normalized the p35 protein and CRMP2 phosphorylation levels in the cerebellum. Impaired motor coordination ability and LTD in APP/PS1 mice were ameliorated by pioglitazone treatment at the pre-Aβ accumulation stage. These results suggest a correlation between CRMP2 phosphorylation and AD pathophysiology, and indicate the effectiveness of pioglitazone treatment at the pre-Aβ accumulation stage in AD model mice.

Background: Knowledge about the distribution, strength, and direction of synaptic connections within neuronal networks are crucial for understanding brain function. Electrophysiology using multiple electrodes provides a very high temporal resolution, but does not yield sufficient spatial information for resolving neuronal connection topology. Optical recording techniques using single-cell resolution have provided promise for providing spatial information. Although calcium imaging from hundreds of neurons has provided a novel view of the neural connections within the network, the kinetics of calcium responses are not fast enough to resolve each action potential event with high fidelity. Therefore, it is not possible to detect the direction of neuronal connections.New method: We took advantage of the fast kinetics and large dynamic range of the DiO/DPA combination of voltage sensitive dye and the fast scan speed of a custom-made random-access two-photon microscope to resolve each action potential event from multiple neurons in culture.Results: Long-duration recording up to 100 min from cultured hippocampal neurons yielded sufficient numbers of spike events for analyzing synaptic connections. Cross-correlation analysis of neuron pairs clearly distinguished synaptically connected neuron pairs with the connection direction.Comparison with existing method: The long duration recording of action potentials with voltage-sensitive dye utilized in the present study is much longer than in previous studies. Simultaneous optical voltage and calcium measurements revealed that voltage-sensitive dye is able to detect firing events more reliably than calcium indicators.Conclusions: This novel method reveals a new view of the functional structure of neuronal networks. (C) 2016 Elsevier B.V. All rights reserved.

Intracellular trafficking of receptor proteins is essential for neurons to detect various extracellular factors during the formation and refinement of neural circuits. However, the precise mechanisms underlying the trafficking of neurotrophin receptors to synapses remain elusive. Here, we demonstrate that a brain-enriched sorting nexin, ARHGAP33, is a new type of regulator for the intracellular trafficking of TrkB, a high-affinity receptor for brain-derived neurotrophic factor. ARHGAP33 knockout (KO) mice exhibit reduced expression of synaptic TrkB, impaired spine development and neuropsychiatric disorder-related behavioural abnormalities. These deficits are rescued by specific pharmacological enhancement of TrkB signalling in ARHGAP33 KO mice. Mechanistically, ARHGAP33 interacts with SORT1 to cooperatively regulate TrkB trafficking. Human ARHGAP33 is associated with brain phenotypes and reduced SORT1 expression is found in patients with schizophrenia. We propose that ARHGAP33/SORT1-mediated TrkB trafficking is essential for synapse development and that the dysfunction of this mechanism may be a new molecular pathology of neuropsychiatric disorders.

Proper density and morphology of dendritic spines are important for higher brain functions such as learning and memory. However, our knowledge about molecular mechanisms that regulate the development and maintenance of dendritic spines is limited. We recently reported that cyclin-dependent kinase 5 (Cdk5) is required for the development and maintenance of dendritic spines of cortical neurons in the mouse brain. Previous in vitro studies have suggested the involvement of Cdk5 substrates in the formation of dendritic spines; however, their role in spine development has not been tested in vivo. Here, we demonstrate that Cdk5 phosphorylates collapsin response mediator protein 2 (CRMP2) in the dendritic spines of cultured hippocampal neurons and in vivo in the mouse brain. When we eliminated CRMP2 phosphorylation in CRMP2(KI/KI) mice, the densities of dendritic spines significantly decreased in hippocampal CA1 pyramidal neurons in the mouse brain. These results indicate that phosphorylation of CRMP2 by Cdk5 is important for dendritic spine development in cortical neurons in the mouse hippocampus.

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report(1), the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

The diffusion of materials from systemic circulation to the central nervous system (CNS) is restricted by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). Choroid plexus epithelial cells (CPECs) of the brain ventricles constitute the BCSFB and regulate the infiltration of plasma proteins as well as immune cells into the interstitium of the CNS. The barrier function is altered in pathologic conditions. However, the regulatory mechanism of BCSFB is not fully understood. Here, we investigated the function of transient receptor potential vanilloid 4 (TRPV4), a polymodally gated divalent cation channel that is highly expressed in CPECs. TRPV4 was localized broadly on the apical membrane in swine CPECs, in contrast with an intense ciliary localization found on other cell types. Treatment with the TRPV4-specific agonist, GSK1016790A (GSK; EC50 34 nM), induced a robust calcium influx and an immediate serine/threonine protein phosphorylation. The agonist treatment induced a marked decrease in the amount of filamentous actin and disintegrated the cell junctions in 10-20 minutes. In contrast, inhibition of the basal TRPV4 activity with the TRPV4-specific antagonist, HC067047 (HC; IC50 74 nM), reduced the basolateral-to-apical transport of alpha-2-macroglobulin (A2M). Overall, this study demonstrated a novel physiologic function of TRPV4 in the regulation of BCSFB permeability.

Mitochondrial thermodynamics is the key to understand cellular activities related to homeostasis and energy balance. Here, we report the first ratiometric fluorescent molecular probe (Mito-RTP) that is selectively localized in the mitochondria and visualize the temperature. We confirmed that Mito-RTP could work as a ratiometric thermometer in a cuvette and living cells.

Previous studies have implicated the role of Purkinje cells in motor learning and the underlying mechanisms have also been identified in great detail during the last decades. Here we report that cyclin-dependent kinase 5 (Cdk5)/p35 in Purkinje cell also contributes to synaptic plasticity. We previously showed that p35(-/-) (p35 KO) mice exhibited a subtle abnormality in brain structure and impaired spatial learning and memory. Further behavioral analysis showed that p35 KO mice had a motor coordination defect, suggesting that p35, one of the activators of Cdk5, together with Cdk5 may play an important role in cerebellar motor learning. Therefore, we created Purkinje cell-specific conditional Cdk5/p35 knockout (L7-p35 cKO) mice, analyzed the cerebellar histology and Purkinje cell morphology of these mice, evaluated their performance with balance beam and rota-rod test, and performed electrophysiological recordings to assess long-term synaptic plasticity. Our analyses showed that Purkinje cell-specific deletion of Cdk5/p35 resulted in no changes in Purkinje cell morphology but severely impaired motor coordination. Furthermore, disrupted cerebellar long-term synaptic plasticity was observed at the parallel fiber-Purkinje cell synapse in L7-p35 cKO mice. These results indicate that Cdk5/p35 is required for motor learning and involved in long-term synaptic plasticity.

Alzheimer's disease (AD) is a neurodegenerative disorder that represents the most common type of dementia among elderly people. Amyloid beta (Aβ) peptides in extracellular Aβ plaques, produced from the amyloid precursor protein (APP) via sequential processing by β- and γ-secretases, impair hippocampal synaptic plasticity, and cause cognitive dysfunction in AD patients. Here, we report that Aβ peptides also impair another form of synaptic plasticity; cerebellar long-term depression (LTD). In the cerebellum of commonly used AD mouse model, APPswe/PS1dE9 mice, Aβ plaques were detected from 8 months and profound accumulation of Aβ plaques was observed at 18 months of age. Biochemical analysis revealed relatively high levels of APP protein and Aβ in the cerebellum of APPswe/PS1dE9 mice. At pre-Aβ accumulation stage, LTD induction, and motor coordination are disturbed. These results indicate that soluble Aβ oligomers disturb LTD induction and cerebellar function in AD mouse model. © 2014 International Society for Neurochemistry.

Downregulation of brain-derived neurotrophic factor (BDNF), a member of neurotrophin family, has been implicated in psychiatric diseases including schizophrenia. However, detailed mechanisms of its reduction in patients with schizophrenia remain unclear. Here, using cultured cortical neurons, we monitored BDNF mRNA levels following acute application of phencyclidine [PCP; an N-methyl-d-aspartate (NMDA) receptor blocker], which is known to produce schizophrenia-like symptoms. We found that PCP rapidly caused a reduction in total amount of BDNF transcripts without effect on cell viability, while mRNA levels of nerve growth factor was intact. Actinomycin-D (ActD), an RNA synthesis inhibitor, decreased total BDNF mRNA levels similar to PCP, and coapplication of ActD with PCP did not show further reduction in BDNF mRNA compared with solo application of each drug. Among BDNF exons I, IV, and VI, the exon IV, which is positively regulated by neuronal activity, was highly sensitive to PCP. Furthermore, PCP inactivated cAMP response element-binding protein (CREB; a regulator of transcriptional activity of exon IV). The inactivation of CREB was also achieved by an inhibitor for Ca2+/calmodulin kinase II (CaMKII), although coapplication with PCP induced no further inhibition on the CREB activity. It is possible that PCP decreases BDNF transcription via blocking the NMDA receptor/CaMKII/CREB signaling. Synapse 68:257-265, 2014. (c) 2014 Wiley Periodicals, Inc.

Several lines of evidence demonstrate that oxidative stress is involved in the pathogenesis of neurodegenerative diseases, including Parkinson's disease. Potent antioxidants may therefore be effective in the treatment of such diseases. Cabergoline, a dopamine D2 receptor agonist and antiparkinson drug, has been studied using several cell types including mesencephalic neurons, and is recognized as a potent radical scavenger. Here, we examined whether cabergoline exerts neuroprotective effects against oxidative stress through a receptor-mediated mechanism in cultured cortical neurons. We found that neuronal death induced by H2O2 exposure was inhibited by pretreatment with cabergoline, while this protective effect was eliminated in the presence of a dopamine D2 receptor inhibitor, spiperone. Activation of ERK1/2 by H2O2 was suppressed by cabergoline, and an ERK signaling pathway inhibitor, U0126, similarly protected cortical neurons from cell death. This suggested the ERK signaling pathway has a critical role in cabergoline-mediated neuroprotection. Furthermore, increased extracellular levels of glutamate induced by H2O2, which might contribute to ERK activation, were reduced by cabergoline, while inhibitors for NMDA receptor or L-type Ca2+ channel demonstrated a survival effect against H2O2. Interestingly, we found that cabergoline increased expression levels of glutamate transporters such as EAAC1. Taken together, these results suggest that cabergoline has a protective effect on cortical neurons via a receptor-mediated mechanism including repression of ERK1/2 activation and extracellular glutamate accumulation induced by H2O2.

Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Forster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics.

Cilia have crucial roles in various developmental and physiological events. Previously, we reported that choroid plexus epithelial cells (CPECs) have multiple, nonmotile 9+0 cilia, but the cilia exhibit transient motility with variable axonemal arrangements in the neonatal period. These features make these cilia unique, as they do not fit in to the traditional categories of primary or motile cilia, and their physiological roles remain elusive. To address this issue, we studied ciliary motility on CPECs through development, with particular interest in the embryonic period. In the fetal choroid plexus of the lateral ventricles, the proportion of cells with motile cilia and their beat frequency increased over time. The ciliary motility profiles peaked near the day of birth, and gradually declined in the two weeks thereafter. The dynamic changes in ciliary motility correlated with changes in Dnahc11 expression. We demonstrated previously that the ciliary motility at P2 was insufficient to produce detectable fluid flow
thus it appears that CPEC cilia do not produce fluid flow at any point during development. Together, our results suggest that a temporally regulated, unique function of CPEC cilia may exist during the perinatal period. © 2013 Wiley Periodicals, Inc.

The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C-3-Glu2C(14), and a lysine-based lipid, Lys-C-3-Glu2C(14), with two different types of counterion, chloride ion (Cl-) and trifluoroacetic acid (TFA(-)), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C-3-Glu(2)C(14) with TEA(-) induced a rate of transfection in primary cultured neurons higher than that of Lys-C-3-Glu2C(14) using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C-3-Glu2C(14) with TFA(-) showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C-3-Glu2C(14) with TFA(-) is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity.

Primary cilia function as specialized compartments for signal transduction. The stereotyped structure and signaling function of cilia inextricably depend on the selective segregation of molecules in cilia. However, the fundamental principles governing the access of soluble proteins to primary cilia remain unresolved. We developed a methodology termed 'chemically inducible diffusion trap at cilia' to visualize the diffusion process of a series of fluorescent proteins ranging in size from 3.2 nm to 7.9 nm into primary cilia. We found that the interior of the cilium was accessible to proteins as large as 7.9 nm. The kinetics of ciliary accumulation of this panel of proteins was exponentially limited by their Stokes radii. Quantitative modeling suggests that the diffusion barrier operates as a molecular sieve at the base of cilia. Our study presents a set of powerful, generally applicable tools for the quantitative monitoring of ciliary protein diffusion under both physiological and pathological conditions.

初期化を阻害する転写因子が分化を促進する. 京都大学プレスリリース. 2013-04-02.

Background: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group. Methods: Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000. Results: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity. Conclusion: The gene delivery efficiency of amino acid-based cationic assemblies is influenced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions. © 2013 Sarker et al, publisher and licensee Dove Medical Press Ltd.

Transcription factors (TFs) are able to regulate differentiation-related processes, including dedifferentiation and direct conversion, through the regulation of cell type-specific transcriptional profiles. However, the functional interactions between the TFs regulating different transcriptional profiles are not well understood. Here, we show that the TFs capable of inducing cell type-specific transcriptional profiles prevent the dedifferentiation induced by TFs for pluripotency. Of the large number of TFs expressed in a neural-lineage cell line, we identified a subset of TFs that, when overexpressed, strongly interfered with the dedifferentiation triggered by the procedure to generate induced pluripotent stem cells. This interference occurred through a maintenance mechanism of the cell type-specific transcriptional profile. Strikingly, the maintenance activity of the interfering TF set was strong enough to induce the cell line-specific transcriptional profile when overexpressed in a heterologous cell type. In addition, the TFs that interfered with dedifferentiation in hepatic-lineage cells involved TFs with known induction activity for hepatic-lineage cells. Our results suggest that dedifferentiation suppresses a cell type-specific transcriptional profile, which is primarily maintained by a small subset of TFs capable of inducing direct conversion. We anticipate that this functional correlation might be applicable in various cell types and might facilitate the identification of TFs with induction activity in efforts to understand differentiation.

The powerful strategy of "intracellular click reaction" was used to retain a chemical Ca2+ indicator in the cytosol. Specifically, a novel clickable Ca2+ indicator "N-3-fura-2 AM" was coupled with dibenzylcyclooctyl-modified biomacromolecules via copper-free click reaction in living cells and Ca2+ oscillation was observed for an extended period of time.

The standardized extract of the St. John's wort plant (Hypericum perforatum) is commonly used to treat mild to moderate depression. Its active constituent is hyperforin, a phloroglucinol derivative that reduces the reuptake of serotonin and norepinephrine by increasing intracellular Na+ concentration through the activation of nonselective cationic TRPC6 channels. TRPC6 channels are also Ca2+-permeable, resulting in intracellular Ca2+ elevations. Indeed, hyperforin activates TRPC6-mediated currents and Ca2+ transients in rat PC12 cells, which induce their differentiation, mimicking the neurotrophic effect of nerve growth factor. Here, we show that hyperforin modulates dendritic spine morphology in CA1 and CA3 pyramidal neurons of hippocampal slice cultures through the activation of TRPC6 channels. Hyperforin also evoked intracellular Ca2+ transients and depolarizing inward currents sensitive to the TRPC channel blocker La3+, thus resembling the actions of the neurotrophin brain-derived neurotrophic factor (BDNF) in hippocampal pyramidal neurons. These results suggest that the antidepressant actions of St. John's wort are mediated by a mechanism similar to that engaged by BDNF. (c) 2012 Wiley Periodicals, Inc.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its kinase activity is dependent upon its association with either of the activating subunits p35 or p39, which are mainly expressed in neurons. We previously reported that Cdk5 knockout (KO) mice exhibit perinatal lethality, defective neuronal migration, and abnormal positioning of neurons in the facial motor nucleus and inferior olive in the hindbrain and Purkinje cells (PCs) in the cerebellum. In this study, we focused on the analysis of the role of Cdk5 in cerebellar development. For this purpose we generated midbrain-hindbrain-specific Cdk5 conditional knockout (MHB-Cdk5 KO) mice because the cerebellum develops postnatally, whereas Cdk5 KO mice die perinatally. Histological analysis of the MHB-Cdk5 KO mice revealed a significant size reduction of the cerebellum. In addition, profound disturbance of inward migration of granule cells (GC) was observed in the developing cerebellum. A normal dendritic development of the Purkinje cells (PCs) was disturbed in MHB-Cdk5 KO mice. Cultured Cdk5-null PCs showed similar dendritic abnormalities. These results indicate that Cdk5/p35 plays an important role in neuronal migration of PCs and GCs and dendrite formation of PCs in cerebellar development. (c) 2012 Elsevier Inc. All rights reserved.

Background: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group.
Methods: Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine (TM) 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine (TM) 2000.
Results: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity.
Conclusion: The gene delivery efficiency of amino acid-based cationic assemblies is influenced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions.

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.

Cilia are structurally and functionally diverse organelles, whose malfunction leads to ciliopathies. While recent studies have uncovered common ciliary transport mechanisms, limited information is available on the proteome of cilia, particularly that of sensory subtypes, which could provide insight into their functional and developmental diversities. In the present study, we performed proteomic analysis of unique, multiple 9+0 cilia in choroid plexus epithelial cells (CPECs). The analysis of juvenile swine CPEC cilia identified 868 proteins. Among them, 396 were shared with the proteome of 9+0 photoreceptor cilia (outer segment), whereas only 152 were shared with the proteome of 9+2 cilia and flagella. Various signaling molecules were enriched in a CPEC-specific ciliome subset, implicating multiplicity of sensory functions. The ciliome also included molecules for ciliary motility such as Rsph9. In CPECs from juvenile swine or adult mouse, Rsph9 was localized to a subpopulation of cilia, whereas they were non-motile. Live imaging of mouse choroid plexus revealed that neonatal CPEC cilia could beat vigorously, and the motility waned and was lost within 1-2 weeks. The beating characteristics of neonatal CPEC cilia were variable and different from those of typical 9+2 cilia of ependyma, yet an Efhc1-mediated mechanism to regulate the beating frequency was shared in both types of cilia. Notably, ultrastructural analysis revealed the presence of not only 9+0 but also 9+2 and atypical ciliary subtypes in neonatal CPEC. Overall, these results identified both conserved and variable components of sensory cilia, and demonstrated a novel mode of ciliary development in mammals. (C) 2012. Published by The Company of Biologists Ltd.

Molecular mechanisms involved in the strengthening and formation of synapses include the activation and repression of specific genes or subsets of genes by epigenetic modifications that do not alter the genetic code itself. Chromatin modifications mediated by histone acetylation have been shown to be critical for synaptic plasticity at hippocampal excitatory synapses and hippocampal-dependent memory formation. Considering that brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity and behavioral adaptations, it is not surprising that regulation of this gene is subject to histone acetylation changes during synaptic plasticity and hippocampal-dependent memory formation. Whether the effects of BDNF on dendritic spines and quantal transmitter release require histone modifications remains less known. By using two different inhibitors of histone deacetylases (HDACs), we describe here that their activity is required for BDNF to increase dendritic spine density and excitatory quantal transmitter release onto CA1 pyramidal neurons in hippocampal slice cultures. These results suggest that histone acetylation/deacetylation is a critical step in the modulation of hippocampal synapses by BDNF. Thus, mechanisms ofepigenetic modulation of synapse formation and function are novel targets to consider for the amelioration of symptoms of intellectual disabilities and neurodegenerative disorders associated with cognitive and memory deficits. (C) 2011 Wiley Periodicals, Inc.

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.

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.

Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here. we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-(L)) and an alternatively initiated or spliced (Bcap-(S)) mRNA, and little is known about the differential functions of the BCAP-L and BCAP-(S) proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-(L) enhanced IL-6 and IL-10 production but not TNF-alpha production in TLR ligand-stimulated macrophages. We propose that BCAP-(L) (but not BCAP-(S)) is a negative regulator of the TLR-mediated host defense system in macrophages. (C) 2010 Elsevier Inc. All rights reserved.

Considerable epidemiological evidence indicates that dietary consumption of moderate levels of polyphenols decreases both the incidence of cardiovascular disease and the mortality associated with myocardial infarction. Molecular mechanisms of this cardiovascular protection remain uncertain but can involve changes in rates of nitric oxide (NO) generation by endothelial nitric oxide synthase (eNOS). We examined the vascular responses to quercetin using a combination of biochemical and vessel function criteria. Quercetin treatment for 30 min enhanced relaxation of rat aortic ring segments. Moreover, the addition of L-NAME (100 mu M) or charybdotoxin (ChTx) blocked quercetin-mediated vasorelaxation thus demonstrating the effect was partially dependent on NOS and endothelium-derived hyperpolarizing factor (EDHF). Additionally, bovine aortic endothelial cells (BAEC) treated with quercetin showed a rapid increase of intracellular Ca2+ concentrations as well as a dose- and time-dependent stimulation of eNOS phosphorylation with a concomitant increase in NO production. These results demonstrate that quercetin-mediated stimulation of eNOS phosphorylation increases NO bioavailability in endothelial cells and can thus play a role in the vascular protective effects associated with improved endothelial cell function. (C) 2010 Elsevier Inc. All rights reserved.

Li Y, Calfa G, Inoue T, Amaral MD, Pozzo-Miller L. Activity-dependent release of endogenous BDNF from mossy fibers evokes a TRPC3 current and Ca2+ elevations in CA3 pyramidal neurons. J Neurophysiol 103: 2846-2856, 2010. First published March 10, 2010; doi: 10.1152/jn.01140.2009. Multiple studies have demonstrated that brain-derived neurotrophic factor (BDNF) is a potent modulator of neuronal structure and function in the hippocampus. However, the majority of studies to date have relied on the application of recombinant BDNF. We herein report that endogenous BDNF, released via theta burst stimulation of mossy fibers (MF), elicits a slowly developing cationic current and intracellular Ca2+ elevations in CA3 pyramidal neurons with the same pharmacological profile of the transient receptor potential canonical 3 (TRPC3)-mediated I-BDNF activated in CA1 neurons by brief localized applications of recombinant BDNF. Indeed, sensitivity to both the extracellular BDNF scavenger tropomyosin-related kinase B (TrkB)-IgG and small hairpin interference RNA-mediated TRPC3 channel knockdown confirms the identity of this conductance as such, henceforth-denoted MF-I-BDNF. Consistent with such activity-dependent release of BDNF, these MF-I-BDNF responses were insensitive to manipulations of extracellular Zn2+ concentration. Brief theta burst stimulation of MFs induced a long-lasting depression in the amplitude of excitatory postsynaptic currents (EPSCs) mediated by both AMPA and N-methyl-D-aspartate (NMDA) receptors without changes in the NMDA receptor/AMPA receptor ratio, suggesting a reduction in neurotransmitter release. This depression of NMDAR-mediated EPSCs required activity-dependent release of endogenous BDNF from MFs and activation of Trk receptors, as it was sensitive to the extracellular BDNF scavenger TrkB-IgG and the tyrosine kinase inhibitor k-252b. These results uncovered the most immediate response to endogenously released-native -BDNF in hippocampal neurons and lend further credence to the relevance of BDNF signaling for synaptic function in the hippocampus.

The accumulation of autophagosomes within axons is often observed in axonopathies associated with various neurological disorders, including those following excitotoxic insults. Nevertheless, the life cycle of autophagosomes in axons is not well understood. In the present study, we used microexplant cultures of cerebellar granule cells from GFP-LC3 transgenic mice to perform time-lapse imaging of LC3-positive dots in identified axons. Since these GFP-LC3 dots were never observed in granule cells on an Atg5-null background, they were considered to represent autophagosomes. Under physiological conditions, the autophagosomes showed bidirectional and saltatory movement with a bias towards one direction. Such vectorial movement was largely blocked by the dynein motor inhibitor EHNA (erythro-9-[3-(2-hydroxynonyl)] adenine), suggesting that the autophagosomes moved towards the soma, where most lysosomes are located. Interestingly, the application of the glutamate analog N-methyl-D-aspartic acid (NMDA) as an excitotoxin increased the number of autophagosomes in axons, while it did not significantly change its movement characteristics. These results suggest that autophagosomes play important roles in axons and are dynamically regulated under physiological and pathological conditions.

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression-related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression-related behaviour. The NR2A subunit of the NMDA receptor is tyrosine-phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr-1325-Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock-in mice show antidepressant-like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock-in mice, DARPP-32 phosphorylation at Thr 34, which is important for the regulation of depression-related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src-induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor-mediated downstream signalling to modulate depression-related behaviour. The EMBO Journal (2009) 28, 3717-3729. doi: 10.1038/emboj.2009.300; Published online 15 October 2009

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.

Ca2+ is known to have important roles in sperm chemotaxis, although the relationship between intracellular Ca2+ concentration ([Ca2+](i)) and modulation of the swimming and chemotactic behavior of spermatozoa has not been elucidated. Using a highspeed Ca2+ imaging system, we examined the chemotactic behavior and [Ca2+](i) in individual ascidian sperm cells exhibiting chemotactic responses toward sperm activating and attracting factor (SAAF), a chemoattractant released by eggs. In this study, we found that transient [Ca2+](i) increased in the flagellum (Ca2+ bursts) concomitantly with a change in the swimming direction in an SAAF gradient field. During the initial phase of the Ca2+ bursts, the flagellum of the spermatozoon exhibited highly asymmetric waveforms enabling the quick turning of the swimming path. However, the flagellum subsequently changed to symmetric beating, causing the spermatozoon to swim straight. Interestingly, during such responses, [Ca2+](i) remained higher than the basal level, indicating that the series of responses was not simply determined by Ca2+ concentrations. Also, we found that Ca2+ bursts were consistently evoked at points at which the spermatozoon attained around a temporally minimal value for a given SAAF concentration. We concluded that Ca2+ bursts induced around a local minimal SAAF concentration trigger a sequence of flagellar responses comprising quick turning followed by straight swimming to direct spermatozoa efficiently toward eggs.

Here, we show that cultured Purkinje cells from inositol 1,4,5-trisphosphate receptor type 1 knock-out (IP(3)R1KO) mice exhibited abnormal dendritic morphology. Interestingly, despite the huge amount of IP(3)R1 expression in Purkinje cells, IP(3)R1 in granule cells, not in the Purkinje cells, was responsible for the shape of Purkinje cell dendrites. We also found that BDNF application rescued the dendritic abnormality of IP(3)R1KO Purkinje cells, and that the increase in BDNF expression in response to activation of AMPA receptor (AMPAR) and metabotropic glutamate receptor (mGluR) was impaired in IP(3)R1KO cerebellar granule cells. In addition, we observed abnormalities in the dendritic morphology of Purkinje cells and in the ultrastructure of parallel fiber-Purkinje cell (PF-PC) synapses in IP(3)R1KO mice in vivo. We concluded that activation of AMPAR and mGluR increases BDNF expression through IP(3)R1-mediated signaling in cerebellar granule cells, which contributes to the dendritic outgrowth of Purkinje cells intercellularly, possibly by modifying PF-PC synaptic efficacy.

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate(IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+-induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.

Changes in synaptic efficacy at the parallel fiber (PF)-Purkinje cell (PC) synapse are postulated to be a cellular basis for motor learning. Although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (STP) lasting for tens of minutes, have not been discussed to date. Here we report that this synapse shows an apparent STP reliably by a periodic burst pattern of homosynaptic stimulation. This STP is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic Ca2+ reduction by BAPTA injection or in P/Q-type Ca channel knockout cerebella. This novel type of synaptic plasticity at the PF-PC synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Changes in synaptic efficacy at the parallel fiber (PF)-Purkinje cell (PC) synapse are postulated to be a cellular basis for motor learning. Although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (STP) lasting for tens of minutes, have not been discussed to date. Here we report that this synapse shows an apparent STP reliably by a periodic burst pattern of homosynaptic stimulation. This STP is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic Ca2+ reduction by BAPTA injection or in P/Q-type Ca channel knockout cerebella. This novel type of synaptic plasticity at the PF-PC synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

We previously reported a primitive cell fraction derived from human circulating CD14(+) monocytes, named monocyte-derived multipotential cells (MOMC), that can differentiate along mesenchymal lineages, including bone, cartilage, fat, skeletal muscle and cardiac muscle. In this study, we investigated whether MOMC can differentiate into the neuronal lineage. MOMC were fluorescently labelled and cocultivated with a primary culture of rat neurons for up to 4 weeks. The protein and gene expressions of neuron-specific markers in the human MOMC were evaluated over time using immunohistochemistry, in situ hybridization and reverse transcription followed by PCR. Shortly after cocultivation with rat neurons, nearly all the MOMC expressed early neuroectodermal markers, Mash1, Neurogenin2 and NeuroD, together with nestin, an intermediate filament expressed in neurogenesis. After 14 days of coculture, a subpopulation of MOMC displayed a multipolar morphology with elongated neurites and expressed mature neuron-specific markers, including neurofilament, microtubule-associated protein type 2, beta 3-tubulin, NeuN and Hu. Transdifferentiation of monocytes into the neuroectodermal lineage was shown by the simultaneous expression of proneural markers and CD45/CD14 early in the differentiation process. The cocultivated MOMC retained their proliferative capacity for at least 16 days. Finally, the neuronal differentiation of MOMC was observed when they were cultured with neurons without cell-to-cell contact. The capacity of MOMC to differentiate into both mesodermal and neuroectodermal lineages suggests that circulating CD14(+) monocytes are more multipotential than previously thought.

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.

Previously, we reported a unique CD14(+)CD45(+)CD34(+) type I collagen(+) cell fraction derived from human circulating CD14(+) monocytes, named monocyte-derived mesenchymal progenitors (MOMPs). These primitive cells differentiate along mesenchymal lineages, including bone, cartilage, fat, and skeletal muscle. Here, we demonstrate that CD14(+) monocytes generate MOMPs that differentiate into cardiomyocytes. MOMPs labeled with a fluorescent marker and co-cultivated with rat cardiomyocytes for 4 weeks expressed the cardiomyocyte-specific transcription factors Nkx2.5, GATA-4, eHAND, and MEF2 and the hematopoietic/monocytic markers CD45 and CD14 within 10 days and retained their proliferative capacity for up to 16 days. A subpopulation of MOMPs subsequently expressed the cardiomyocyte-specific markers alpha-sarcomeric actinin, troponin I, and atrial natriuretic peptide on day 21. Furthermore, fluorescence-labeled, spontaneously beating cells that formed gap junctions with adjacent rat cardiomyocytes appeared in these cultures and these cells exhibited electrophysiological properties typical of ventricular myocytes. The co-cultivation of human MOMPs with rat GFP-tagged cardiomyocytes resulted in the generation of human cardiomyocytes lacking green fluorescent protein (GFP) staining, suggesting that our observations could not solely be explained by cell fusion. Our results demonstrate for the first time that human circulating CD14(+) monocytes include progenitors capable of proliferating and differentiating along the cardiomyogenic lineage via their differentiation into MOMPs.

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP3) receptors (IP(3)R2 and IP(3)R3, respectively) from mouse lung and found a novel alternative splicing segment, SIm2, at 176-208 of IP3R2. The long form (IP(3)R2 SIm2+) was dominant, but the short form (IP(3)R2 SIm2-) was detected in all tissues examined. IP(3)R2 SIm2- has neither IP3 binding activity nor Ca2+ releasing activity. In addition to its reticular distribution, IP(3)R2 SIm2+ is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca2+ ionophore stimulation, most of the IP(3)R2 SIm2+ is in clusters. IP(3)R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca2+ ionophore stimulation. IP(3)R2 SIm2- does not form clusters in either resting or stimulated cells. IP3 binding-deficient site-directed mutants of IP(3)R2 SIm2+ and IP(3)R3 fail to form clusters, indicating that IP3 binding is involved in the cluster formation by these isoforms. Coexpression of IP(3)R2 SIm2+ prevents stimulus-induced IP3R clustering, suggesting that IP(3)R2 SIm2- functions as a negative coordinator of stimulus-induced IP3R clustering. Expression of IP(3)R2 SIm2- in CHO-K1 cells significantly reduced ATP-induced Ca2+ entry, but not Ca2+ release, suggesting that the novel splice variant of IP(3)R2 specifically influences the dynamics of the sustained phase of Ca2+ signals.

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na+/K+ ATPase (Na+ pump) or Na+/Ca2+ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na+/K+ ATPase and Na+/Ca2+ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na+](i) and Na+ pump current (I-p), it was shown that an ouabain-high sensitive Na+/K+ ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for alpha1-subunit and alpha3-subunit of the Na+/K+ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast alpha2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na+/Ca2+ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca2+ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca2+](i) oscillations by blocking of Na+/K+ ATPase, suggesting the functional coupling with Na+/Ca2+ exchanger. From these results and RT-PCR analysis, we conclude that alpha2-subunit Na+/K+ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes. (C) 2004 Elsevier Ltd. All rights reserved.

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.

Efficiency of synaptic transmission within the neocortex is regulated throughout life by experience and activity. Periods of correlated or uncorrelated presynaptic and postsynaptic activity lead to enduring changes in synaptic efficiency [long-term potentiation (LTP) and long-term depression (LTD), respectively]. The initial plasticity triggering event is thought to be a precipitous rise in postsynaptic intracellular calcium, with higher levels inducing LTP and more moderate levels inducing LTD. We used a pairing protocol in visual cortical brain slices from young guinea pigs with whole-cell recording and calcium imaging to compare the kinetic profiles of calcium signals generated in response to individual pairings along with the cumulative calcium wave and plasticity outcome. The identical pairing protocol applied to layer 2/3 pyramidal neurons results in different plasticity outcomes between cells. These differences are not attributable to variations in the conditioning protocol, cellular properties, inter-animal variability, animal age, differences in spike timing between the synaptic response and spikes, washout of plasticity factors, recruitment of inhibition, or activation of different afferents. The different plasticity outcomes are reliably predicted by individual intracellular calcium transients in the dendrites after the first few pairings. In addition to the differences in the individual calcium transients, the cumulative calcium wave that spreads to the soma also has a different profile for cells that undergo LTP versus LTD. We conclude that there are biological differences between like-type cells in the dendritic calcium signals generated by coincident synaptic input and spiking that determine the sign of the plasticity response after brief associations.

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.

Dysfunction of the hypocretin/orexin (Hcrt/Orx) peptide system is closely linked to the sleep disorder narcolepsy, suggesting that it is also central to the normal regulation of sleep and wakefulness. Indeed, Hcrt/Orx peptides produce long-lasting excitation of arousal-related neurons, including those in the laterodorsal tegmentum (LDT) and the dorsal raphe (DR), although the mechanisms underlying these actions are not understood. Since Hcrt/Orx mobilizes intracellular calcium ([Ca2+](i)) in cells transfected with orexin receptors and since receptor-mediated Ca2+ transients are ubiquitous signaling mechanisms, we investigated whether Hcrt/Orx regulates [Ca2+](i) in the LDT and DR. Changes in [Ca2+](i) were monitored by fluorescence changes of fura-2 AM loaded cells in young mouse brain slices. We found Hcrt/Orx (Orexin-A, 30-1,000 nM) evoked long-lasting increases in [Ca2+](i) with differing temporal profiles ranging from spiking to smooth plateaus. A fragment of Hcrt/Orx (16-33) failed to evoke changes in [Ca2+](i) and changes were not blocked by TTX or ionotropic glutamate receptor antagonists, suggesting they resulted from specific activation of postsynaptic orexin receptors. Unlike orexin receptor - transfected cells, Hcrt/Orx-responses were not attenuated by depletion of Ca2+ stores with cyclopiazonic acid (CPA; 3- 30 muM), thapsigargin (3 muM), or ryanodine (20 muM), although store-depletion by either CPA or ryanodine blocked Ca2+ mobilization by the metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD; 30 muM). In contrast, Hcrt/Orx responses were strongly attenuated by lowering extracellular Ca2+ (similar to20 muM) but were not inhibited by concentrations of KB-R7943 ( 10 muM) selective for blockade of sodium/calcium exchange. Nifedipine ( 10 muM), inhibited Hcrt/Orx responses but was more effective at abolishing spiking than plateau responses. Bay K 8644 (5-10 muM), an L-type calcium channel agonist, potentiated responses. Finally, responses were attenuated by inhibitors of protein kinase C (PKC) but not by inhibitors of adenylyl cyclase. Collectively, our findings indicate that Hcrt/Orx signaling in the reticular activating system involves elevation of [Ca2+](i) by a PKC-involved influx of Ca2+ across the plasma membrane, in part, via L-type calcium channels. Thus the physiological release of Hcrt/Orx may help regulate Ca2+-dependent processes such as gene expression and NO production in the LDT and DR in relation with behavioral state. Accordingly, the loss of Hcrt/Orx signaling in narcolepsy would be expected to disrupt calcium-dependent processes in these and other target structures.

There are several lines of evidence showing that synaptic activity regulates the level of expression of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) in neurons. In this study, we examined the effect of chronic activity blockade on the localization and level of IP(3)R1 expression in cultured hippocampal neurons. We found that chronic blockade of NMDA receptors (NMDARs), one of the major Ca2+-permeable ion channels, increased the number of neurons that express a high level of IP(3)R1 without any apparent changes in its intracellular localization. Interestingly, this up-regulation was time-dependent; there was no clear change in IP(3)R1 expression level up to day 5 of the NMDAR blockade, but expression increased at day 6, and the increased expression level persisted for at least a week. The up-regulation of IP(3)R1 depended on transcription and protein synthesis and required cAMP-dependent protein kinase activity. Moreover, although most of the control neurons did not respond to the metabotropic glutamate receptor (mGluR) stimulation, the 2-amino-5-phosphonopentanoic acid-treated neurons with high IP(3)R1 expression became sensitive to mGluR stimulation. Furthermore, we also found that hippocampal neurons transiently overexpressing green fluorescent protein-tagged IP(3)R1 released Ca2+ in response to mGluR and muscarinic acetylcholine receptor stimulation. These findings suggested that chronic NMDAR blockade increased the IP(3)R1 expression and enhanced sensitivity to mGluR stimulation. The change in IP(3)R1 expression level in response to alteration of synaptic activity may be an important determinant of the sensitivity of Ca2+ stores to G-protein-coupled receptor stimulation and would help to maintain intracellular Ca2+ homeostasis in hippocampal neurons.

Various hormonal stimuli and growth factors activate the mammalian canonical transient receptor potential ( TRPC) channel through phospholipase C ( PLC) activation. However, the precise mechanism of the regulation of TRPC channel activity remains unknown. Here, we provide the first evidence that direct tyrosine phosphorylation by Src family protein-tyrosine kinases (PTKs) is a novel mechanism for modulating TRPC6 channel activity. We found that TRPC6 is tyrosine-phosphorylated in COS-7 cells when coexpressed with Fyn, a member of the Src family PTKs. We also found that Fyn interacts with TRPC6 and that the interaction is mediated by the SH2 domain of Fyn and the N-terminal region of TRPC6 in a phosphorylation-independent manner. In addition, we demonstrated the physical association of TRPC6 with Fyn in the mammalian brain. Moreover, we showed that stimulation of the epidermal growth factor receptor induced rapid tyrosine phosphorylation of TRPC6 in COS-7 cells. This epidermal growth factor-induced tyrosine phosphorylation of TRPC6 was significantly blocked by PP2, a specific inhibitor of Src family PTKs, and by a dominant negative form of Fyn, suggesting that the direct phosphorylation of TRPC6 by Src family PTKs could be caused by physiological stimulation. Furthermore, using single channel recording, we showed that Fyn modulates TRPC6 channel activity via tyrosine phosphorylation. Thus, our findings demonstrated that tyrosine phosphorylation by Src family PTKs is a novel regulatory mechanism of TRPC6 channel activity.

Three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R1, IP(3)R2, and IP(3)R3) Ca2+ release channel share basic properties but differ in terms of regulation. To what extent they contribute to complex Ca2+ signaling, such as Ca2+ oscillations, remains largely unknown. Here we show that HeLa cells express comparable amounts of IP(3)R1 and IP(3)R3, but knockdown by RNA interference of each subtype results in dramatically distinct Ca2+ signaling patterns. Knockdown of IP(3)R1 significantly decreases total Ca2+ signals and terminates Ca2+ oscillations. Conversely, knockdown of IP(3)R3 leads to more robust and long lasting Ca2+ oscillations than in controls. Effects of IP(3)R3 knockdown are surprisingly similar in COS-7 cells that predominantly (> 90% of total IP3R) express IP(3)R3, suggesting that IP(3)R3 functions as an anti-Ca2+-oscillatory unit without contributing to peak amplitude of Ca2+ signals, irrespective of its relative expression level. Therefore, differential expression of the IP3R subtype is critical for various forms of Ca2+ signaling, and, particularly, IP(3)R1 and IP(3)R3 have opposite roles in generating Ca2+ oscillations.

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.

Ca2+ and Na+ play important roles in neurons, such as in synaptic plasticity. Their concentrations in neurons change dynamically in response to synaptic inputs, but their kinetics have not been compared directly. Here, we show the mechanisms and dynamics of Ca2+ and Na+ transients by simultaneous monitoring in Purkinje cell dendrites in mouse cerebellar slices. High frequency parallel fibre stimulation (50 Hz, 3-50-times) depolarized Purkinje cells, and Ca2+ transients were observed at the anatomically expected sites. The magnitude of the Ca2+ transients increased linearly with increasing numbers of parallel fibre inputs. With 50 stimuli, Ca2+ transients lasted for seconds, and the peak [Ca2+] reached similar to100 muM, which was much higher than that reported previously, although it was still confined to a part of the dendrite. In contrast, Na+ transients were sustained for tens of seconds and diffused away from the stimulated site. Pharmacological interventions revealed that Na+ influx through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and Ca2+ influx through P-type Ca channels were essential players, that AMPA receptors did not operate as a Ca2+ influx pathway and that Ca2+ release from intracellular stores through inositol trisphosphate receptors or ryanodine receptors did not contribute greatly to the large Ca2+ transients.

Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora B, RhoA, and the GAP MgcRac-GAP have been implicated in cytokinesis, but their underlying mechanisms of action have remained unclear. Here, we show that MgcRacGAP colocalizes with Aurora B and RhoA, but not Rac1/Cdc42, at the midbody. We also report that Aurora B phosphorylates MgcRacGAP on serine residues and that this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase-defective mutant of Aurora B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylation-deficient MgcRacGAP-S387A mutant, but not phosphorylation-mimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces polyploidy. Together, these findings indicate that during cytokinesis, MgcRacGAP, previously known as a GAP for Rac/ Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora B.

To investigate the role in synaptic plasticity of Ca2+ released from intracellular Ca2+ stores, mice lacking the inositol 1,4,5-trisphosphate type 1 receptor were developed and the physiological properties, long-term potentiation, and long-term depression of their hippocampal CA1 neurons were examined. There were no significant differences in basic synaptic functions, such as membrane properties and the input/output relationship, between homozygote mutant and wild-type mice. Enhanced paired-pulse facilitation at inter-pulse intervals of less than 60 ms and enhanced post-tetanic potentiation were observed in the mutant mice, suggesting that the presynaptic mechanism was altered by the absence of the inositol 1,4,5-trisphosphate type 1 receptor. Long-term potentiation in the field-excitatory postsynaptic potentials induced by tetanus (100 Hz, 1 s) and the excitatory postsynaptic currents induced by paired stimulation in hippocampal CA1 pyramidal neurons under whole-cell clamp conditions were significantly greater in mutant mice than in wild-type mice. Homosynaptic long-term depression of CA1 synaptic responses induced by low-frequency stimulation (1 Hz, 500 pulses) was not significantly different, but heterosynaptic depression of the non-associated pathway induced by tetanus was blocked in the mutant mice. Both long-term potentiation and long-term depression in mutant mice were completely dependent on N-methyl-D-aspartate receptor activity. To rule out the possibility of an effect compensating for the lack of the inositol 1,4,5-trisphosphate type 1 receptor occurring during development, an anti-inositol 1,4,5-trisphosphate type 1 receptor monoclonal antibody that blocks receptor function was diffused into the wild-type cell through a patch pipette, and the effect of acute block of inositol 1,4,5-trisphosphate type 1 receptor on long-term potentiation was examined. Significant enhancement of long-term potentiation was observed compared with after control immunoglobulin G injection, suggesting that developmental redundancy was not responsible for the increase in long-term potentiation amplitude observed in the mutant mouse. The properties of channels that could be involved in long-term potentiation induction were examined using whole-cell recording. N-methyi-D-aspartate currents were significantly larger in mutant mice than in wild-type mice only between holding potentials of -60 and -80 mV. We conclude that inositol 1,4,5-trisphosphate type 1 receptor activity is not essential for the induction of synaptic plasticity in hippocampal CA1 neurons, but appears to negatively regulate long-term potentiation induction by mild modulation of channel activities. (C) 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Long-term depression (LTD) of synaptic transmission is induced by low-frequency stimulation (LFS) of afferents lasting for a long time, typically for 10-15 min, in neocortical and hippocampal slices. It is suggested that calcineurin, Ca2+/calmodulin-dependent protein phosphatase, plays a role in the induction of LTD, based on the results that pharmacological or genetic manipulation of calcineurin activity interfered in its induction. However, questions as to why it takes so long to induce LTD and in which compartment of neurons calcineurin is activated remain unanswered. With a fluorescent indicator for calcineurin activity, we visualized the spatiotemporal pattern of its activation in living neurons in layer II/III of visual cortical slices of rats during the LFS of layer IV that induced LTD of synaptic responses. During LFS, the fluorescence intensity gradually increased with a latency of a few minutes in dendrites and soma of neurons, and remained increased during the whole observation period (10-25 min) after LFS. The onset latency of the increase in the soma was slower than that in the distal dendritic region. The LFS-induced rise in fluorescence was not observed in neurons which were loaded with inhibitors of calcineurin, indicating that the intensity of fluorescence reflects calcineurin activity. Control stimulation at 0.05 Hz and theta-burst stimulation did not significantly change the intensity of fluorescence. Only LFS-type inputs effectively activate calcineurin in postsynaptic neurons in an augmenting manner, and such a time-consuming activation of calcineurin may be a reason why long-lasting LFS is necessary for the induction of LTD.

Electrophysiological studies have shown that decreases in extracellular Ca2+ concentration and increases in extracellular K+ concentration cause non-synaptic seizure-like neuronal activity in the hippocampus. Using the calcium imaging technique, we investigated the role of intracellular Ca2+ concentration in the cause of non-synaptic seizure-like neuronal activity. Rat hippocampal slice culture was loaded with calcium indicator dye. Non-synaptic seizure-like neuronal activity was induced by low Ca2+/high K+ solution. The calcium imaging, with a confocal laser scanning microscope and extracellular field recording were made simultaneously. Pharmacological inhibitors were used to examine the mechanism of the intracellular Ca2+ changes. As a result, intracellular Ca2+ oscillation occurred in neurons in a nonuniform fashion after replacing the solution, and then, gradual massive synchronization occurred in the large population. This massive synchronized Ca2+ oscillation propagated transversely through the CA1 area at a velocity of 4.2-6.8 mm/min. A negative shift, which reflects seizure-like neuronal activity, was observed in extracellular field recording, corresponding to the synchronized intracellular Ca2+ oscillation. Voltage-dependent calcium channel blocker and gap junction blocker inhibited this synchronization and the propagation.
In conclusion, using the calcium-imaging technique, we demonstrated dynamic changes in synchronization and propagation of intracellular Ca2+ oscillation, which corresponded to nonsynaptic epileptiform activity. The role of gap junction as a contributing mechanism was speculated. (C) 2002 Elsevier Science B.V. All rights reserved.

The endoplasmic reticulum (ER) is the major membranous component present throughout the axon. Although other membranous structures such as synaptic vesicles are known to move via fast axonal transport, the dynamics of ER in the axon still remains unknown. To study the dynamics of ER in the axon, we have directly visualized the movement of two ER-specific membrane proteins, the sarcoplasmic/endoplasmic reticulum calcium-ATPase and the inositol 1,4,5-trisphosphate receptor, both of which were tagged with green fluorescence protein (GFP) and expressed in cultured chick dorsal root ganglion neurons. In contrast to GFP-tagged synaptophysin that moved as vesicles at 1 mum/sec predominantly in the anterograde direction in the typical style of fast axonal transport, the two ER proteins did not move in a discrete vesicular form. Their movement determined by the fluorescence recovery after photobleaching technique was bi-directional, 10-fold slower (similar to0.1 mum/sec), and temperature-sensitive. The rate of movement of ER was also sensitive to low doses of vinblastine and nocodazole that did not affect the rate of synaptophysin-GFP, further suggesting that it is also distinct from the well-documented movement of membranous vesicles in its relation with microtubules. J. Neurosci. Res. 65:236-246, 2001. (C) 2001 Wiley-Liss, Inc.

Nitric oxide synthase (NOS)-containing mesopontine cholinergic (MPCh) neurons of the laterodorsal tegmental nucleus (LDT) are hypothesized to drive the behavioral states of waking and REM sleep through a tonic increase in firing rate which begins before and is maintained throughout these states. In principle, increased firing could elevate intracellular calcium levels and regulate numerous cellular processes including excitability, gene expression, and the activity of neuronal NOS in a state-dependent manner. We investigated whether repetitive firing, evoked by current injection and N-methyl-D-aspartate (NMDA) receptor activation, produces somatic and proximal dendritic [Ca2+](i) transients and whether these transients are modulated by serotonin, a transmitter thought to play a critical role in regulating the state-dependent firing of MPCh neurons. [Ca2+](i) was monitored optically from neurons filled with Ca2+ indicators in guinea pig brain slices while measuring membrane potential with sharp microelectrodes or patch pipettes. Neither hyperpolarizing current steps nor subthreshold depolarizing steps altered [Ca2+](i). In contrast, suprathreshold currents caused large and rapid increases in [Ca2+](i) that were related to firing rate. TTX (1 mu M) strongly attenuated this relation. Addition of tetraethylammonium (TEA, 20 mM), which resulted in Ca2+ spiking on depolarization, restored the change in [Ca2+](i) to pre-TTX levels. Suprathreshold doses of NMDA also produced increases in [Ca2+](i) that were reduced by up to 60% by TTX. Application of 5-HT, which hyperpolarized LDT neurons without detectable changes in [Ca2+](i), suppressed both current- and NMDA-evoked increases in [Ca2+](i) by reducing the number of evoked spikes and by inhibiting spike-evoked Ca2+ transients by similar to 40% in the soma and proximal dendrites. This inhibition was accompanied by a subtle increase in the spike repolarization rate and a decrease in spike width, as expected for inhibition of high-threshold Ca2+ currents in these neurons. NADPH-diaphorase histochemistry confirmed that recorded cells were NOS-containing. These findings indicate the prime role of action potentials in elevating [Ca2+](i) in NOS-containing MPCh neurons. Moreover, they demonstrate that serotonin can inhibit somatic and proximal dendritic [Ca2+](i) increases both indirectly by reducing firing rate and directly by decreasing the spike-evoked transients. Functionally, these data suggest that spike-evoked Ca2+ signals in MPCh neurons should be largest during REM sleep when serotonin inputs are expected to be lowest even if equivalent firing rates are reached during waking. Such Ca2+ signals may function to trigger Ca2+-dependent processes including cfos expression and nitric oxide production in a REM-specific manner.

A variety of receptors coupled to the heterotrimeric guanine nucleotide-binding protein G(q/11) stimulate intracellular Ca2+ release through inositol (1,4,5)-trisphosphate (IP3) formation. We previously reported that tyrosine phosphorylation of the alpha subunit of the G(q/11) protein by protein tyrosine kinases (PTKs) regulates the activation of G(q/11) protein. Here we show that protein tyrosine phosphatases (PTPs) are also essential for G(q/11) protein activation. We find that G(q/11) protein-coupled receptor-mediated formation of IP3 is blocked by PTP inhibitors as well as PTK inhibitors. These inhibitors act prior to G(q/11) protein activation. Tyrosine phosphorylation of the alpha subunit of G(q/11) appears to inhibit its interaction with receptors. Thus, PTP is required for controlling the level of tyrosine phosphorylation of the alpha subunit of G(q/11) to promote its interaction with receptors. Therefore, we conclude that PTKs and PTPs co-operate to proceed activation cycle of the G(q/11) protein through tyrosine phosphorylation and de-phosphorylation of the alpha subunit of G(q/11).

Recently, mice with a disrupted inositol trisphosphate (IP3) receptor type 1 allele were produced by gene targeting. To examine the role of IP3 receptor type 1 in the regulation of intracellular calcium concentration ([Ca2+](i)) of glomerular cells, [Ca2+](i) was measured with furs 2-acetoxymethyl-ester in the superfused glomeruli from homozygous and wild-type mice. [Ca2+](i) was determined in calcium-free medium before and after the addition of 10(-7) M endothelin-1 (ET-1) and 10(-6) M angiotensin II (AngII). The expression of mRNA of IP3 receptor isoforms and hormone receptors in the glomeruli from these animals also was measured by quantitative reverse transcription-PCR with specific primers for IP3 receptor isoforms (types 1, 2, and 3), AngII receptor type 1, and ET receptors (types A and B. Tn homozygous mutants, the shorter mRNA of IP3 receptor type II which lacks the first exon, is transcribed. Basal [Ca2+](i) and the responses to ET-1 and AngII in homozygous mutants (ET-1, 55 +/- 7 nM to 73 +/- 7 nM; AngII, 66 +/- 6 to 91 +/- 8 nM) were significantly Lower than those in the wild-type mice (ET-1, 93 +/- 13 nM to 162 +/- 13 nM; AngII, 87 +/- 7 to 147 +/- 9 nM; P < 0.05 for both hormones) without significant changes in mRNA expression of hormone receptors. The results with quantitative reverse transcription-PCR also revealed that mRNA expression of the IP3 receptor gene family was not significantly different between the two groups. The present study clearly shows that IP3 receptor type 1 plays a major role in the regulation of [Ca2+](i) in the glomeruli and that lack of an isoform of IP3 receptor in the glomeruli does not induce expression of the other isoforms of the IP3 receptor.

To investigate the physiological consequences of the increase in spine density induced by estradiol in pyramidal neurons of the hippocampus, He performed simultaneous whole cell recordings and Ca2+ imaging in CAI neuron spines and dendrites in hippocampal slices. Four- to eight-days in vitro slice cultures were exposed to 17 beta-estradiol (EST) for an additional 4- to 8-day period, and spine density was assessed by confocal microscopy of DiI-labeled CAI pyramidal neurons. Spine density was doubled in both apical and basal dendrites of the CA1 region in EST-treated slices; consistently, a reduction in cell input resistance was observed in EST-treated CAI neurons. Double immunofluorescence staining of presynaptic (synaptophysin) and postsynaptic (alpha-subunit of CaMKII) proteins showed an increase in synaptic density after EST treatment. The slops of the input/output curves of both alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate: (NMDA) postsynaptic currents were steeper in EST-treated CAI neurons, consistent with the observed increase in synapse density. To characterize NMDA-dependent synaptic currents and dendritic Ca2+ transients during Schaffer collaterals stimulation, neurons were maintained at +40 mV in the presence of nimodipine, picrotoxin, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). No differences in resting spine or dendritic Ca2+ levels were observed between control and EST-treated CAI neurons. Intracellular Ca transients during afferent stimulation exhibited a faster slope and reached higher levels in spines than in adjacent dendrites. Peak Ca2+ levels were larger in both spines and dendrites of EST-treated CAI neurons. Ca2+ gradients between spine heads and dendrites during afferent stimulation were also larger in EST-treated neurons. Both spine and dendritic Ca2+ transients during afferent stimulation were reversibly blocked by D,L-2-amino-5-phosphonovaleric acid (D,L-APV. The increase in spine density and the enhanced NMDA-dependent Ca2+ signals in spines and dendrites induced by EST may underlie a threshold reduction for induction of NMDA-dependent synaptic plasticity in the hippocampus.

Changes in [Ca2+](i) are an essential factor regulating egg activation. Matured ascidian eggs are arrested at metaphase I, and two series of [Ca2+](i) transients have been observed after fertilization: Ca2+ waves just after fertilization (Series I) and [Ca2+](i) oscillation between the first and second polar body extrusion (Series II). We investigated mechanisms involved in the elevation of [Ca2+](i) and the role of the [Ca2+](i) transients during egg activation in Ciona savignyi. The monoclonal antibody 18A10 against IP3 receptor type 1, which inhibits IP3-induced Ca2+ release in hamster and mouse eggs, did not show substantial inhibitory effects on series I or egg deformation, whereas Series II and the first cell division were inhibited by the antibody. Ruthenium red, an inhibitor of ryanodine receptor-mediated Ca2+ release, had no apparent effect of [Ca2+](i) transients and other events related to the egg activation. Microinjection of IP3 into unfertilized eggs induced [Ca2+](i) transients similar to those seen in Series I, whereas injection of cyclic ADP ribose, an agonist of ryanodine receptors, rarely induced [Ca2+](i) transient. Adenophostin B, a potent nonmetabolizable agonist of IP3 receptors, induced [Ca2+](i) oscillations which continued after first polar body extrusion, without separation to two series, and led to extrusion of first and second polar bodies. These results suggest that Series II is driven by the mouse type 1-like IP3 receptor while Series I seems to be mediated by another type of IP3 receptor. Injection of IP3 only induced the first polar body extrusion and the egg was arrested at metaphase II even when a higher amount of IP3 was injected. On the other hand, reinjection of IP3 after the first polar body extrusion led to emission of the second polar body. Thus, Series I and II of [Ca2+](i) transients are likely to be required for metaphase-anaphase transition in meiosis. (C) 1998 Academic Press.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) acts as a Ca2+ release channel on internal Ca2+ stores. Type 1 IP3R(IP(3)R1) is enriched in growth cones of neurons in chick dorsal root ganglia. Depletion of internal Ca2+ stores and inhibition of IP3 signaling with drugs inhibited neurite extension. Microinjection of. heparin, a competitive IP3R blocker, induced neurite retraction. Acute Localized Loss of function of IP(3)R1 in the growth cone induced by chromophore-assisted Laser inactivation resulted in growth arrest and neurite retraction. IP3-induced Ca2+ release in growth cones appears to have a crucial role in control of nerve growth.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ channel that releases Ca2+ from internal Ca2+ stores in response to InsP3. Although InsP3R is highly expressed in various regions of the mammalian brain, the functional role of this receptor has not been clarified. We show here that cerebellar slices prepared from mice with a disrupted InsP3R type 1 gene, which is predominantly expressed in Purkinje cells, completely lack long-term depression (LTD), a model of synaptic plasticity in the cerebellum. Moreover, a specific antibody against InsP3R1, introduced into wild-type Purkinje cells through patch pipettes, blocked the induction of LTD. These data indicate that, in addition to Ca2+ influx through Ca2+ channels on the plasma membrane, Ca2+ release from InsP3R plays an essential role in the induction of LTD, suggesting a physiological importance for InsP3R in Purkinje cells.

The inositol 1,4,5-trisphosphate (IP3) receptor is a calcium ion channel involved in the release of free Ca2+ from intracellular stores. For analysis of the role of IP3-induced Ca2+ release (IICR) on patterning of the embryonic body, monoclonal antibodies that inhibit IICR were produced. Injection of these blocking antibodies into the ventral part of early Xenopus embryos induced modest dorsal differentiation. A close correlation between IICR blocking potencies and ectopic dorsal axis induction frequency suggests that an active IP3-Ca2+ signal may participate in the modulation of ventral differentiation.

The N-methyl-D-aspartate (NMDA) receptor plays important roles in synaptic plasticity and brain development. The NMDA receptor subunits have large intracellular domains in the COOH-terminal region that may interact with signal-transducing proteins. By using the yeast two-hybrid system, we found that calmodulin interacts with the COOH terminus of the NR1 subunit and inactivates the channels in a Ca2+-dependent manner, Here we show that protein kinase C (PKC)-mediated phosphorylation on serine residues of NR1 decreases its affinity for calmodulin. This suggests that PKC-mediated phosphorylation of NR1 prevents calmodulin from binding to the NR1 subunit and thereby inhibits the inactivation of NMDA receptors by calmodulin, In addition, we show that stimulation of metabotropic glutamate receptor 1 alpha, which potentiates NMDA channels through PKC, decreases the ability of NR1 to bind to calmodulin, Thus, our data provide clues to understanding the basis of cross-talk between two types of receptors, metabotropic glutamate receptors and the NR1 subunit, in NMDA channel potentiation.

To study the development of the calcium release mechanism, we examined the temporal and spatial expression of inositol 1,4,5-trisphosphate receptor (IP3R) during the oogenesis and meiotic maturation of Xenopus laevis. Observation of a series of fixed samples by immunofluoresence microscopy revealed a relocalization of Xenopus IP3R (XIP3R)-positive structures during meiotic maturation. We visualized the endoplasmic reticulum (ER) using ER-sensitive dye, DiI, by observation under confocal laser scanning microscopy. Time-lapse visualization of the living oocytes revealed that while the ER of immature fully grown oocytes underwent relatively little movement, the ER of maturing oocytes and mature eggs moved rapidly. A possible role for the increase of ER mobility in the dynamic redistribution of XIP3R during oocyte maturation is also discussed. (C) 1997 Academic Press.

Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium transients that localized to cleavage furrows and propagated along the furrows as calcium waves. The calcium transients at the cleavage furrows were observed at each cleavage furrow at least until blastula stage. The velocity of the calcium waves at the first cleavage furrow was similar to 3 mu m/s, which was much slower than that associated with fertilization/egg activation. These calcium waves traveled only along the cleavage furrows and not in the direction orthogonal to the furrows. These observations imply that there exists an intracellular calcium-releasing activity specifically associated with cleavage furrows. The calcium waves occurred in the absence of extracellular calcium and were inhibited in embryos injected with heparin, an inositol 1,4,5-trisphosphate (InsP(3)) receptor antagonist. These results suggest that InsP(3) receptor-mediated calcium mobilization plays an essential role in calcium wave formation at the cleavage furrows.

THE inositol 1,4,5-trisphosphate (InsP(3)) receptor acts as an InsP(3)-gated Ca2+ release channel in a variety of cell types(1,2). Type 1 InsP(3) receptor (IP(3)R1) is the major neuronal member of the IP(3)R family in the central nervous system(3,4), predominantly enriched in cerebellar Purkinje cells but also concentrated in neurons in the hippocampal CA1 region, caudate-putamen, and cerebral cortex(5,6). Here we report that most IP(3)R1-deficient mice generated by gene targeting die in utero, and born animals have severe ataxia and tonic or tonic-clonic seizures and die by the weaning period. An electroencephalogram showed that they suffer from epilepsy, indicating that IP(3)R1 is essential for proper brain function. However, observation by light microscope of the haematoxylin-eosin staining of the brain and peripheral tissues of IP(3)R1-deficient mice showed no abnormality, and the unique electrophysiological properties of the cerebellar Purkinje cells of IP(3)R1-deficient mice were not severely impaired.

1. Primary-cultured cerebellar Purkinje cells (PCs) from mouse embryos were whole cell voltage clamped, and L-glutamate (Glu) was applied iontophoretically to the dendrite. Long-term depression (LTD) of Glu-evoked currents was induced through the conjunction of repeated depolarizations and Glu applications.
2. Thapsigargin, a specific inhibitor of Ca2+-ATPase on the endoplasmic reticulum, and ryanodine and ruthenium red, inhibitors of the ryanodine receptor, blocked the induction of LTD.
3. Thapsigargin and ryanodine alone did not affect influx of Ca2+ through voltage-gated Ca2+ channels and inward currents evoked by Glu applications.
4. Our results suggest that Ca2+ release from internal stores, particularly from ryanodine-sensitive stores, is necessary for the induction of LTD in cultured PCs.

In the vertebrate nervous system, glutamate (Glu) receptors are generally known to cause depolarizing responses. We report here a novel type of Glu response in Purkinje neurons of mouse cerebellar slices, namely glutamate-induced hyperpolarization (GH). This response is not due to activation of inhibitory interneurons, because application of tetrodotoxin (TTX), bicuculline, or strychnine did not abolish GH. In addition, GH persisted in a Ca2+-free or a low-Cl- solution, which rules out the involvement of g(K(Ca)) or GABA(A) mechanisms. Quisqualate (Quis) and trans-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), which are potent and selective agonists, respectively, for the metabotropic Glu receptor (mGluR), failed to induce GH. L-2-Amino-4-phosphonobutyric acid (L-AP4) was also ineffective. Simultaneous recording of electrical activity and intracellular Ca2+ concentration ([Ca2+]i) showed that GH was not accompanied by [Ca2+]i changes. Voltage clamp experiments showed that GH is due to reduction of a tonically active conductance with a reversal potential around 0 mV. Two possible mechanisms are suggested for GH: (1) changes in the desensitized steady state of ionotropic Glu receptors, or (2) a novel Glu-mediated mechanism.

Mice homozygous for targeted disruption of the α1A subunit of the voltage gated Ca channel (PQKO) show fatal ataxia and dystonia. We investigated properties of the cerebellar Purkinje cell (PC) in PQKO (P14-20) using acute cerebellar slices. PCs were patch-clamped at whole-cell mode and electrophysiologic/Ca imaging studies were performed in response to synaptic stimuli and somatic depolarization as well.In comparison with wild type littermates (WT), the magnitude of Ca transient (CaT) in PQKO PC induced by a train of parallel fiber stimuli (PF) was much smaller, whereas CaT evoked by climbing fiber (CF) was as large as that in WTs.Pharmacological studies revealed that compared to WT PF- and CF-EPSC in PQKO were more sensitive to w-conotoxin, an N-type Ca channel blocker, whereas deporalization-induced dendritic CaT was more sensitive to low-dose Ni²⁺, a T-type channel blocker. Overall, contribution of L-type channel was very little in PQKO and WT. These data suggest that the defect in P/Q-type Ca channel is not well conpensated at least in part, resulting in compromised Ca signaling in PC dendrites, and that the compensation mechanism is different between pre- and post synaptic sites in PCs. [Jpn J Physiol 55 Suppl:S148 (2005)]

Long-term depression (LTD) of the parallel fiber (PF)–Purkinje cell (PC) synapse is an underlying mechanism by which motor coordination and learning are regulated. Elevation of cytoplasmic calcium in the PC dendrites is a critical event for LTD induction. To investigate the relationship between the calcium signal pattern and the LTD induction, we performed calcium imaging from PCs in acute cerebellar slices. LTD was induced by ten trains of 50Hz x 5 bursts of parallel fiber stimuli at 1Hz in PCs from 2 weeks old mice, but not from 4 weeks old mice. This PF short burst stimuli (50Hz x 5) produced a biphasic calcium transient in dendrites of PCs, first peak of which reflects calcium influx and second one does calcium release from intracellular calcium stores. By comparing amplitudes of the first and second calcium transients, we found a correlation between calcium release activity and LTD amplitude in PCs from 2 weeks old mice. This finding unveils an importance of calcium release especially in synaptic maturation in the developing cerebellar cortex. [Jpn J Physiol 55 Suppl:S149 (2005)]

IP3 is an important second messenger to release Ca2+ from internal store. IP3 receptor (IP3R) works as an IP3-induced Ca2+ release channel. We studied the role of type 1 IP3R (IP(3)R1) in neural plasticity. We found that IP(3)R1 is necessary for the induction of long-term depression (LTD) by using cerebellar slices of IP(3)R1-deficient mice. We also studied the neural plasticity of the hippocampus. While no differences were seen between mutant and wild-type mice in terms of the mean magnitude of the long-term potentiation (LTP) or LTD induced by a standard tetanus or low-frequency afferent stimulation (LFS), a short tetanus protocol evoked much greater LTP amplitude in the IP(3)R1-deficient mice than the control. In addition, DP or LTP suppression was attenuated in the mutant mice, the mean magnitude of the responses after delivery of LFS or tetanus being significantly greater than in wild-type mice. These results suggested that IP(3)R1 is involved in LTP, DP and UP suppression, but is not essential for LTD. In addition, IP(3)R1 was involved in the determination of polarity and input specificity of activity-induced synaptic modification in hippocampal CA1 pyramidal cells. We studied the electrophysiological activity and learning behavior of ryanodine receptor (type 3; RyR3) deficient mice. When LTP was evoked using high-frequency stimulation in the CA1 hippocampal neuron, the increase in the initial slope of field excitatory postsynaptic potentials (EPSPs) was not significantly different between homozygous and wild mice, although the increase in the homozygous mice tended to be greater than in the wild mice. With near-threshold stimulation, however, LTP was not induced in wild-type mice but was induced in homozygous mice. In the probe trial test, in which the platform was removed after the training schedule, ryanodine receptor type 3-deficient mice spent significantly more time than the wild-type mice in the quadrant where the platform had been. (C) 2003 Published by Elsevier B.V.

生後3〜4月のSCA1マウス及びコントロールの野生型マウスより小脳切片を定法により作成し,正立顕微鏡下で直視化にてプルキンエ細胞の細胞体よりパッチクランプ法により電気記録をとり,同時にカルシウム色素bis-fura-2を細胞内に導入し,冷却CCDカメラを用いて細胞内カルシウム濃度を測定した.顕著に萎縮したSCA1マウスのプルキンエ細胞においても電気生理学的特徴の多くは保存されていたことから,これらは単にプルキンエ細胞の樹状突起の形態的特徴に基づくものではなく,他の要素によることがわかった.SCA1マウスプルキンエ細胞では平行線維刺激によるカルシウム放出が起こり易くなっているのみならず,野生型では見られない登上線維刺激によるカルシウム放出も観察された.IP3R1の発現量が減少していることとあわせると,プルキンエ細胞樹状突起におけるカルシウム放出能はIP3R1の発現量と相反していることになる,プルキンエ細胞樹状突起におけるカルシウム放出はIP3R1が担うが,SCA1マウスにおいて放出能とIP3R1発現量には逆の相関が示唆された

神経細胞の興奮に伴うカルシウム変動はスパイン一つ一つを見分けられる解像度でとらえられるようになってきた.一方,カルシウム流入・排出機構の分子的実体も明らかになりつつある.ER(endoplasmic reticulum)やミトコンドリアが担うカルシウム貯蔵・放出機構の実態の解明も進みつつある.神経細胞におけるこれらの研究の進展について概説した

Inositol 1,4,5-trisphosphate (IP3) is a second messenger which induces Ca2+ release from the intracellular store sites (1-3, 6, 7, 25, 37-39, 41-43). This IP3-induced Ca2+ release (IICR) is mediated by an intracellular Ca2+ release channel, IP3 receptor (IP3R) (Fig. 1). IP3R was first characterized in 1979 as a protein called P-400 which was abundant in normal mice but almost absent in Purkinje cell-deficient mutant mice, long before the importance of IP3, as a second messenger to release Ca2+ from the intracellular compartment, was recognized (25-29, 44-47). Our group and others cloned the cDNAs of three different types of IP3R from various species (types 1, 2, and 3) (4, 9, 19, 30, 62, 68, 71, 75). The basic structure of all these types is composed of three functional domains: the ligand-binding domain (N-terminal portion) (74), the modulatory domain (middle portion), and the channel domain (C-terminal portion) (74). The modulatory domain contains various sites for modulator-binding (ATP, calmodulin, Ca2+, etc.) and phosphorylation (PKA, PRC, CaMKII), which are important for crosstalk with other signal transduction systems (37-39: 47-43). The channel domain contains six membrane-spanning segments and one putative "pore"-forming segment (36). IP3R belongs to ion channel superfamily including voltage-sensitive and nucleotide-gated channels (37, 39, 42). Through new techniques in bioscience evidence has accumulated that IP3R plays an important role in various physiological cell functions. Here, we describe recent findings on the structure of IP3R and also summarize its role in cell function.

マウスMBP遺伝子の組織特異的転写には,-120付近に存在するNF1 (nuclear factor 1)配合配列が重要な役割を担っている.マウス脳より多数のNF1様因子のcDNAクローン(NF1-B)を単離し,その構造と機能を解析した.NF1-Bはいずれも約530個のアミノ酸をコードすることができ,構造上CTN/NF1と部分的に類似性を有するが,それとは異なる遺伝子産物である.またポリペプチド鎖のN端と中央部に変異しやすい部分を持つ,cDNAより作られた蛋白質は,MBPのNF1部位に結合し転写活性化能を示した.NF1-B RNAは脳,特に小脳に多く含まれており,MBP遺伝子の組織特異的転写制御に重要な役割を果たしていると考えられる

マウスMBP遺伝子の組織特異的転写調節機構を解析する目的でin vitroでの組織特異的転写を再現する無細胞転写系を確立した.マウスの脳由来の核抽出液は,肝やHeLa細胞の抽出液に比べ,優先的にMBPプロモーターを転写した.MBPプロモーターの主要な部分は-253から+60の中に見い出され,-53を境に上流(DR)と下流(PR)の転写領域に分割される.DRとPRは互いに協調し,より強い組織特異的転写活性を発揮すると考えられる

本研究は申請者の開発したランダムアクセス型2光子励起顕微鏡システムを用いて、これまで困難だった遊離タンパク質の動態を直接計測することを目指す。多点で同時に蛍光相関分光法(FCS)を適用することにより樹状突起の細胞質中を高速に移動するタンパク質分子の動態を捉え、シナプスと核を連絡するシグナル系の実態を明らかにすることを目的とする。これまで細胞質内を拡散する遊離型タンパク質は拡散速度の桁違いの大きさのため直接的な動態の計測は困難だった。蛍光相関分光法(FCS)は溶液中での一分子の振る舞いを計測する手法で、これを細胞内に適応することにより細胞質内の遊離タンパク質の挙動を追跡できる。しかし従来のFCS装置は細胞内の一点からしか記録できず、細胞内の様々な部位における機能タンパク質の動態解析を行うには制約があった。特に神経細胞は細胞内の機能局在性が高く、同時多点からのFCS解析は非常に多くの情報をもたらすことが期待される。本研究では新規に開発した2光子励起顕微鏡システムを用いカルモジュリン、CamKII、Ca結合タンパク質(calbindin等)などシナプス機能に重大な機能分子のスパイン内外での動態を検出することを試みた。本年度は初代培養神経細胞を用いて樹状突起およびスパイン内のタンパク質のFCS計測を試みた。GFP発現プラスミドあるいはGFP融合CamKIIタンパク質発現プラスミドをマウス由来初代培養神経細胞に発現させ細胞質の複数点から蛍光強度変化を10kHz以上の時解像度で取得することに成功した。自己相関関数よりタンパク質の細胞局所での拡散係数が異なることを見いだした。

新規に開発した2光子励起顕微鏡システムを用い多点で同時に蛍光相関分光法(FCS)を適用することにより神経細胞樹状突起の細胞質中を高速に移動するタンパク質分子の動態を捉えることを目的とした。GFP発現プラスミドあるいはGFP融合CamKIIタンパク質発現プラスミドをマウス由来初代培養神経細胞に発現させ細胞質の複数点から蛍光強度変化の記録を10kHz以上の時間解像度で取得しFCS解析することに成功した。

これまでに培養海馬神経細胞の樹状突起で1型IP3受容体に特異的なアクチン骨格依存的な側方拡散制御を見いだした。IP3受容体がシナプス可塑性に重要な働きをしている小脳プルキンエ細胞のスパインでもこの様な機構が働いていれば、アクチン骨格を介して活動依存的にIP3受容体の側方拡散の度合いを変化させ、スパインにIP3受容体を止めおくことによリスパイン内部のCa2+放出活性を調節し、ひいてはシナプス可塑性を制御している可能性がある。これを検討するためにプルキンエ細胞樹状突起に蛍光タンパクを融合した小胞体膜タンパクを遺伝子銃を用い発現させ、二光子励起顕微鏡を用い蛍光退色回復(FRAP)法により、小胞体膜タンパクの移動速度を解析した。
プルキンエ細胞樹状突起でのIP3受容体各タイプとSERCAタンパクの拡散速度を測定した。内在性の1型IP3受容体の影響を避けるため、3型IP3受容体の拡散速度の計測のためには1型IP3受容体ノックアウトマウスを用いた。IP3受容体の拡散速度はSRCAより遅く、やはリアクチン骨格依存的であったが、海馬とは異なり1型と3型のIP3受容体の間の差はなかった。この結果は、IP3受容体は樹状突起幹部と同様に、アクチン骨格依存的な側方拡散制御機構があるが海馬神経細胞とは異なる機構によることを示唆する(投稿準備中)。
次にスパインと樹状突起間でのIP3受容体とSERCAタンパクの移動速度を検討した。そのために異なる蛍光波長の蛍光タンパクをそれぞれのER膜タンパクに融合した遺伝子を同時に小脳切片中のプルキンエ細胞に発現させ、両者のスパインと樹状突起間での拡散速度の差を検討した。その結果、IP3受容体はスパインでもSERCAより拡散速度が遅いことが明らかとなった(投稿準備中)。更に、この拡散速度の差に活動依存性があることが明らかになりつつある。

スパイン内部でのアクチン骨格再構成に伴いIP3受容体のスパインへの動員に変化が起これば、スパイン内部でのCa^<2+>放出活性の増減に直接影響し、ひいてはシナプス可塑性を制御する可能性がある。これを検討するためにマウス小脳切片中のプルキンエ細胞樹状突起に蛍光タンパクを融合した小胞体膜タンパクを遺伝子銃を用い発現させ、二光子励起顕微鏡を用い蛍光退色回復(FRAP)法により、スパインと樹状突起間の小胞体膜タンパクの移動速度を解析した。樹状突起スパインでも樹状突起幹部と同様にIP3受容体とSERCAタンパクの移動速度に差があることが明らかになった。
IP3受容体欠損マウスのプルキンエ細胞樹状突起の形態の観察から、IP3受容体の機能がプルキンエ細胞樹状突起の形態形成に大きく関与していることを明らかにした(Hisatsune, et al.)。種々の実験結果から、プルキンエ細胞樹状突起の形態形成に関与しているのは、顆粒細胞側に発現している1型IP3受容体であることが判明した。顆粒細胞のIP3受容体の活性により顆粒細胞からのBDNF放出が制御されており、BDNFがプルキンエ細胞の形態を決定づけていた。1型IP3受容体はシナプス後部のプルキンエ細胞のみならず、シナプス前部の顆粒細胞でも重要な機能を担っていることが明らかになった。
また、IP3受容体のIP3結合部分と蛍光タンパクのFRETを基にしたIP3指示薬を新規に開発した(Matsu-ura, et. al.)。神経細胞樹状突起内でのIP3動態の測定には非常に興味が持たれており、強力なツールとして用いることが出来る。

細胞内Ca^<2+>放出チャンネルである1型イノシトール3リン酸受容体(IP3R1)は小脳プルキンエ細胞に非常に豊富に発現しており、ERからのCa^<2+>放出の主要な機能を担っている。プルキンエ細胞樹状突起でのCa^<2+>動態はきわめて複雑な時間,空間的特性をもっており、細胞内メカニズムを厳密かつ精妙に制御している。平行線維入力時のプルキンエ細胞樹状突起のCa^<2+>動態において、細胞外からの流入・細胞内放出機構それぞれの関与の度合いを詳細に検討した。50Hz、3〜8発の刺激で見られるCa^<2+>の流入と細胞内からの放出による二相性のCa^<2+>上昇は、刺激回数を増加すると一相性となり、持続時間は刺激回数に比例し、50発刺激ではCa^<2+>上昇は6〜8秒程度持続し、Ca^<2+>濃度は200μMにまで上昇していると推定された。この大きなCa^<2+>上昇はほとんどがCa^<2+>流入によるものであり、更にNa^+濃度とCa^<2+>濃度変化を比較した結果、P型Caチャンネルを通ったCa^<2+>がその本態であることを明らかにした(Kuruma et al.,2003)。
更に小脳皮質の生後発達に伴うCa^<2+>放出活性の変化を検討するため、異なる日齢のマウスを用いて二相性Ca^<2+>上昇を観察した。2〜3週齢のマウスのプルキンエ細胞ではCa^<2+>放出が相対的に大きく、4週齢以降では逆にCa^<2+>流入の方が大きかった。すなわち平行線維刺激で誘起されるプルキンエ細胞樹状突起のCa^<2+>動態は2週前後の幼弱なマウスにおいてはCa^<2+>放出がCa^<2+>流入よりも支配的であり、成長に伴ってそれが逆転することが示された。2週と4週齢のマウスを用いて平行線維・プルキンエ細胞シナプスのLTDの起こりやすさの差異を検討した。2週齢では平行線維刺激のみでLTDを起こす場合が見られ、同時に測定したCa^<2+>放出活性の大きさとLTDの度合いに相関が見られた。同じ刺激方法では4週齢ではLTDは見られなかった(投稿準備中)。以上の結果から、小脳皮質が発達・成熟をする2週齢前後では、シナプス結合の可塑的変化や成熟化にシナプス後部のCa^<2+>放出能が重要な役割を果たしているという結論に至った。

本研究課題は,研究代表者が見出した単球由来多能性細胞(monocyte-derived multipotent cell ; MOMC)を組織修復・再生医療へと応用するための基礎研究である。そのため,(1)MOMCの網羅的遺伝子発現解析による多分化能を規定する遺伝子の同定,(2)MOMCの機能的な心筋,神経,血管内皮への分化能,(3)造血幹細胞増幅能,の3つの点について検討した。
単球系細胞の中で唯一多分化能を有するMOMCに選択的に発現する遺伝子の中に分化能にかかわる遺伝子が含まれる可能性を想定し,Genechipを用いた網羅的遺伝子発現解析手法を用いてMOMCに選択的に発現する6つの遺伝子(DLG3、Myo10,SEPT3,EFNA3,ATF-1,SAP30)を同定した。MOMCは特定の分化誘導培養条件により骨,軟骨,骨格筋,脂肪などの間葉系細胞へと分化することが知られていたが,ラット胎児由来の心筋および脳の一次培養細胞との共培養により,少なくともin vitroで心筋,神経の系統への分化傾向を示すことが明らかとなった。また,各種血管新生因子存在下でMOMCが血管内皮様の形態を示す細胞へと分化し,それらは遺伝子・蛋白発現,機能(ヒスタミン刺激でのvon Willebrand因子放出,低酸素暴露によるVEGFR1遺伝子発現の上昇,Matrigel中での管腔構造形成)のいずについても成熟血管内皮細胞と区別できなかった。さらに,免疫不全マウス腫瘍血管新生モデル,ラット脳虚血モデルへのMOMC移植により,in vivoでの血管内皮への分化を確認した。一方,MOMCはin vitroでヒト造血幹細胞の増幅を支持したが,未分化性を維持する活性は低く,むしろ分化段階のすすんだ血球前駆細胞を増やした。
以上より,MOMCは幹細胞移植による組織修復・再生療法に適した多能性細胞であることが確認された。今後,難治性疾患に対する自己MOMC移植療法の早期実現が望まれる。

本研究では小脳プルキンエ細胞樹状突起のCa^<2+>動態が非常に多彩でかつ局所性に富むものであることをNa^+動態と比較しつつ明らかにした。またCa^<2+>放出活性をCa^<2+>流入と分離して記録することで、Ca^<2+>放出能が小脳発達とともに変化することを明らかにした。小脳とは異なり、海馬錐体細胞における神経可塑性ではIP3受容体は補助的な役割を演じていることがわかったが、神経活動依存的なIP3受容体の発現調節を示すことで複雑なCa^<2+>制御機構の一端を明らかにした。IP3受容体がのっているERそのものの一部が樹状突起中をダイナミックに移動することを示し、またIP3受容体のER膜上での側方移動も細胞骨格依存的に制御されていることを明らかにした。これらの知見は、神経細胞樹状突起のCa^<2+>シグナル制御機構の一部としてのCa^<2+>放出調節機構ひとつをとっても非常に複雑な機構が存在することを示唆している。Ca^<2+>放出を担うIP3受容体の構造と機能の相関を精密に知ることも、Ca^<2+>放出調節機構を解明する上で必須であり、今回IP3受容体の構造・機能相関をさらに詳細に明らかにしたことは重要な前進であると考える。刺激入力時のCa^<2+>上昇のシグナル下流に位置する各種機能タンパク質の修飾変化を知ることは、Ca^<2+>シグナルの機能を推し量る上で非常に重要である。神経細胞内でカルシニューリン活性の可視化に成功したことはその他の機能タンパク質の活性を観察する第一歩であり、今後の発展が期待される。
以上、本研究により神経細胞内でのCa^<2+>動態、特にCa^<2+>放出機構について多様な制御機構が明らかとなった。これら多角的な研究成果は神経細胞樹状突起内でのCa^<2+>動態の果たす多種多様な生理機能における役割を解明する上で重要な手がかりを与えた。これらの知見をもとにして本研究の目的として揚げたスパインレベルでの可視化に今後取り組みたい。

細胞内Ca^<2+>放出チャンネルである1型イノシトール3リン酸受容体(IP3R1)は小脳プルキンエ細胞に豊富に発現している。細胞内Ca^<2+>濃度の増加はプルキンエ細胞でのシナプス可塑性である長期抑圧現象(LTD)に必須であることは知られていたが、IP3R1がLTDに必須であることを、IP3R1の阻害抗体とIP3R1ノックアウトマウスを用いて示した。生後変性、脱落していく、1型小脳脊髄変性症(SCA1)モデルマウスのプルキンエ細胞においてIP3R1の発現量は減少しているが、シナプス刺激によるCa^<2+>放出はむしろ活性化していた。これらの知見は、プルキンエ細胞樹状突起でのCa^<2+>動態は複雑な時間・空間的特性をもって細胞内メカニズムを制御していることを示唆している。プルキンエ細胞樹状突起のCa^<2+>動態をさらに詳細に検討した。平行線維を50Hz、3〜8発刺激するとCa^<2+>の流入と細胞内からの放出が二相性に見られた。刺激回数を増加するとCa^<2+>上昇は一相性となり、持続時間は刺激回数に比例し、50発刺激では持続時間は6〜8秒、濃度は200μMに達した。IP3R1欠損マウスおよびIP3受容体阻害剤を用いて、この大きなCa^<2+>上昇はほとんどがCa^<2+>流入によることを示し、AMPA受容体阻害剤やCa^<2+>チャンネル阻害剤を用いNa^+とCa^<2+>の濃度変化を測定した結果、P型Ca^<2+>チャンネルがその主役であることが明らかとなった。プルキンエ細胞のAMPA受容体はCa^<2+>を通さないことが分子生物学的研究から示唆されているが、ここでは機能的に示すことができた。

ERは細胞内に網状にはりめぐらされた構造物であり、培養細胞においては細胞骨格系をレールとしてダイナミックに動いていることが近年示されている。神経細胞においても、神経軸索、樹状突起内に高度に発達したERのネットワークが存在している。神経軸索内では神経終末までの長い構造にわたって細胞機能を維持するために様々なレベルの軸策内輸送があることが知られているが、ERの構造はいかなる仕組みで維持されているかは不明であった.我々はER上にあるタンパク質に蛍光ラベルする事により、世界に先駆けて神経軸索内でのERのダイナミックな動きを可視化し、速い軸策輸送と遅い軸索輸送の中間の速度で両方向に連続的に動いていることを明らかにした.ERは細胞内カルシウム貯蔵部位であるから、神経軸索内をカルシウム貯蔵部位がダイナミックに移動していることを意味し、神経軸索の機能を考える上で重要な知見であるといえる。神経細胞の樹状突起もまた非常に極性のある細胞構造であり、神経細胞に投射するシナプス情報を集約し、制御している。樹状突起(シナプス後部)でのERからのカルシウム放出はシナプス可塑性といった高度な神経機能を制御する要素である.我々は,初代培養マウス海馬由来神経細胞の樹状突起において,小胞体の一部はvesicle状で微小管依存的に高速で輸送されていることを見い出し、その動態を詳細に解析した。また、vesicle状のERもやはりIP3によるカルシウム放出能があることを示し、さらにこの輸送にはキネシンモータータンパク質が関わっていることを証明した。以上の成果を論文にまとめて現在投稿中である。

細胞内Ca^<2+>放出チャンネルである1型イノシトール3リン酸受容体(IP3R1)は小脳プルキンエ細胞に豊富に発現している。細胞内Ca^<2+>濃度の増加はプルキンエ細胞でのシナプス可塑性である長期抑圧現象(LTD)に必須であることは知られていたが、IP3R1がLTDに必須であることを、IP3R1の阻害抗体とIP3R1ノックアウトマウスを用いて示した。生後変性、脱落していく、1型小脳脊髄変性症(SCA1)モデルマウスのプルキンエ細胞においてIP3R1の発現量は減少しているが、シナプス刺激によるCa^<2+>放出はむしろ活性化していた。これらの知見は、プルキンエ細胞樹状突起でのCa^<2+>動態は複雑な時間・空間的特性をもって細胞内メカニズムを制御していることを示唆している。プルキンエ細胞樹状突起のCa^<2+>動態をさらに詳細に検討した。平行線維を50Hz、3〜8発刺激するとCa^<2+>の流入と細胞内からの放出が二相性に見られた。刺激回数を増加するとCa^<2+>上昇は一相性となり、持続時間は刺激回数に比例し、50発刺激では持続時間は6〜8秒、濃度は200μMに達した。IP3R1欠損マウスおよびIP3受容体阻害剤を用いて、この大きなCa^<2+>上昇はほとんどがCa^<2+>流入によることを示し、AMPA受容体阻害剤やCa^<2+>チャンネル阻害剤を用いNa^+とCa^<2+>の濃度変化を測定した結果、P型Ca^<2+>チャンネルがその主役であることが明らかとなった。プルキンエ細胞のAMPA受容体はCa^<2+>を通さないことが分子生物学的研究から示唆されているが、ここでは機能的に示すことができた。

細胞内Ca^<2+>放出チャンネルである1型イノシトール3リン酸受容体(IP3R1)は小脳プルキンエ細胞に豊富に発現している。細胞内Ca^<2+>濃度の増加はプルキンエ細胞でのシナプス可塑性である長期抑圧現象(LTD)に必須であることは知られていたが、IP3R1がLTDに必須であることを、IP3R1の阻害抗体とIP3R1ノックアウトマウスを用いて示した。生後変性、脱落していく、1型小脳脊髄変性症(SCA1)モデルマウスのプルキンエ細胞においてIP3R1の発現量は減少しているが、シナプス刺激によるCa^<2+>放出はむしろ活性化していた。これらの知見は、プルキンエ細胞樹状突起でのCa^<2+>動態は複雑な時間・空間的特性をもって細胞内メカニズムを制御していることを示唆している。プルキンエ細胞樹状突起のCa^<2+>動態をさらに詳細に検討した。平行線維を50Hz、3〜8発刺激するとCa^<2+>の流入と細胞内からの放出が二相性に見られた。刺激回数を増加するとCa^<2+>上昇は一相性となり、持続時問は刺激回数に比例し、50発刺激では持続時間は6〜8秒、濃度は200μMに達した。IP3R1欠損マウスおよびIP3受容体阻害剤を用いて、この大きなCa^<2+>上昇はほとんどがCa^<2+>流入によることを示し、AMPA受容体阻害剤やCa^<2+>チャンネル阻害剤を用いNa^+とCa^<2+>の濃度変化を測定した結果、P型Ca^<2+>チャンネルがその主役であることが明らかとなった。プルキンエ細胞のAMPA受容体はCa^<2+>を通さないことが分子生物学的研究から示唆されているが、ここでは機能的に示すことができた。

(1) 神経突起伸長と成長円錐の形態変化を、各部位でIP3受容体、各種骨格蛋白質、カルモジュリン関連蛋白質をレーザー光により欠落させた際の変化の解析を行った。
(2) IP3受容体の単一イオンチャネルを解析。形質発現させた受容体蛋白質を、特異抗体をカップルさせたアフィニティ・カラムを用いて精製した。
(3) 蛍光測光・画像処理システムを用いた細胞内Ca2+動態を解析。タイプ別に発現する細胞種を用いて、ホルモン・神経伝達物質・成長因子などの刺激を与えたときのCa2+蛍光指示薬を用いたCa2+動態解析をした。
(4) IP3受容体をノックアウトしたマウスの各種細胞生物学的解析を行う。
(5) IP3受容体、PI代謝回転の阻害の胚軸形成に対する効果の検討; 以下に述べるIP3受容体、PI-代謝回転、Protein Kinase C(PKC)のブロッカーを用いて、初期胚背側あるいは腹側の細胞に注入し、発生に対する影響を検討中である。すでにIP3レセプター抗体はアフリカツメガエルの受精、卵の活性化やその他のIP3依存性の現象を抑制することを明らかにしている。各々のブロッカーの作用を検討することにより、胚軸形成における、PI-シグナル系のIP3カスケードとPKCカスケードの各々の役割を、より明確にできる。

運動記憶の細胞レベルでの表現型であると考えられている、小脳プルキンエ細胞の平行線維入力に対する長期抑圧(LTD)における細胞内Ca^<2+>放出機構の果たす役割を解明しつつ、プルキンエ細胞内での細胞内Ca^<2+>動員機構の生理的意義を検討した。プリキンエ細胞内に非常に高濃度に存在する細胞内Ca^<2+>放出機構であるIP3受容体および、Ca^<2+>誘導性Ca^<2+>放出チャンネルであるリアノジン受容体がLTD形成に必須であることを、初代培養を用いたLTD実験系を確立し、明らかにした(J.Neurophysiol.1995)。また、タイプ1IP3受容体ノックアウトマウスを作成することに成功し、同受容体が欠失していてもプルキンエ細胞の基本的な膜興奮特性に影響がないことを明らかにした(Nature,1996)。報告書は同変異体マウスにおいて顕著な小脳性失調が認められるにも関わらず、プルキンエ細胞そのものの顕著な形態学的及び膜興奮特性の明らかな変化が認められないことから、IP3受容体と長期抑圧現象そのものとの関連の解析を行い、IP3受容体は長期抑圧現象に必須であるとの結果を得た(投稿中)。

神経細胞にはこのIP3以外にも種々のイノシトールポリリン酸代謝産物が存在することからその重要性が考えられてきたが、特異的結合蛋白質が未同定であったため生理的機能の詳細は明らかではなかった。我々はイノシトールポリリン酸(IP_4,IP_5,IP_6)結合タンパク質として小脳よりシナプトタグミン精製することに成功した。シナプトタグミンの構造的特徴は膜貫通領域を一ヵ所持ちC末端の細胞質側にはプロテインキナーゼCのC2調節領域とホモロジーを有する配列が2ヵ所存在しそれぞれC2A,C2Bドメインと呼ばれている。C2Bドメインはカルシウム非依存的にリン脂質およびイノシトールポリリン酸(イノシトール-1、3、4、5-四リン酸(IP_4)、イノシトール-1、3、4、5、6-五リン酸(IP_5)、イノシトール六リン酸(IP_6))を結合した。これらの結果から我々は二つのC2ドメインが神経伝達物質の放出の過程で異なる役割を持つものと考え、各々のドメインに対する特異的抗体を作製した。次に我々はイカの巨大軸索のプレシナプスにこれらの抗体およびイノシトールポリリン酸(IP_4,IP_5,IP_6)をマイクロインジェクションすることにより以下の結論を得た。(i)シナプトタグミンのC2Aドメインは神経伝達物質放出の際のカルシウムセンサーとして機能しシナプス小胞とプレシナプス膜の融合の過程に関与すること、また(ii)C2Bドメインにはイノシトールポリリン酸が結合することによりC2Aドメインを介する膜融合の過程を阻害する(イノシトールポリリン酸が神経伝達物質放出の負の制御因子をして作用する)。さらに(iii)C2BドメインはClathrin assembly protein,AP-2を結合することによりシナプス小胞のリサイクリングの過程にも関与することが示唆された。

神経細胞にはこのIP3以外にも種々のイノシト-ルポリリン酸代謝産物が存在することからその重要性が考えられてきたが、特異的結合蛋白質が未同定であったため生理的機能の詳細は明らかではなかった。我々はイノシト-ルポリリン酸(IP_4,IP_5,IP_6)結合タンパク質として小脳よりシナプトタグミン精製することに成功した。シナプトタグミンの構造的特徴は膜貫通領域を一ヵ所持ちC末端の細胞質側にはプロテインキナ-ゼCのC2調節領域とホモロジ-を有する配列が2ヵ所存在しそれぞれC2A,C2Bドメインと呼ばれている。C2Bドメインはカルシウム非依存的にリン脂質およびイノシト-ルポリリン酸(イノシト-ル-1、3、4、5-四リン酸(IP_4)、イノシト-ル-1、3、4、5、6-五リン酸(IP_5)、イノシト-ル六リン酸(IP_6))を結合した。これらの結果から我々は二つのC2ドメインが神経伝達物質の放出の過程で異なる役割を持つものと考え、各々のドメインに対する特異的抗体を作製した。次に我々はイカの巨大軸索のプレシナプスにこれらの抗体およびイノシト-ルポリリン酸(IP_4,IP_5,IP_6)をマイクロインジェクションすることにより以下の結論を得た。(i)シナプトタグミンのC2Aドメインは神経伝達物質放出の際のカルシウムセンサ-として機能しシナプス小胞とプレシナプス膜の融合の過程に関与すること、ま...

運動記憶の細胞レベルでの表現型であると考えられている、小脳プルキンエ細胞の平行線維入力に対する長期抑圧(LTD)における細胞内Ca2+放出機構の果たす役割を解明しつつ、プルキンエ細胞内での細胞内Ca2+動員機構の生理的意義を検討した。プルキンエ細胞内に非常に高濃度に存在する細胞内Ca2+放出機構であるIP3受容体および、Ca2+誘導性Ca2+放出チャンネルであるリアノジン受容体がLTD形成に必須であることを、初代培養を用いたLTD実験系を確立し、明らかにした(J.Neurophysiol.1995)。また、タイプ1IP3受容体ノックアウトマウスを作成することに成功し、同受容体が欠失していてもプルキンエ細胞の基本的な膜興奮特性には影響がないことを明らかにした(Nature,1996)。報告者は同変異体マウスにおいて顕著な小脳性失調が認められるにも関わらず、プルキンエ細胞そのものの顕著な形態学的及び膜興奮特性の明らかな変化が認められないことから、IP3受容体と長期抑圧現象そのものとの関連の解析を行い。IP3受容体は長期抑圧現象に必須であるとの結果を得た(投稿準備中)。

運動記憶の細胞レベルでの表現型であると考えられている、小脳プルキンエ細胞の平行線維入力に対する長期抑圧現象(LTD)における細胞内Ca^<2+>放出機構の果たす役割を解明しつつ、プルキンエ細胞内での細胞内Ca^<2+>動員機構の生理的意義を検討した。プルキンエ細胞内に非常に高濃度に存在する細胞内Ca^<2+>放出機構であるIP3受容体および、Ca^<2+>誘導性Ca^<2+>放出チャンネルであるリアノジン受容体がLTD形成に必須であることを、初代培養を用いたLTD実験系を確立し、明らかにした(J. Neurophysiol. 1995)。また、タイプ1IP3受容体ノックアウトマウスを作成することに成功し、同受容体が欠失していてもプルキンエ細胞の基本的な膜興奮特性には影響がないことを明らかにした(Nature, 1996)。報告者は同変異体マウスにおいて顕著な小脳性失調が認められるにも関わらず、プルキンエ細胞そのものの顕著な形態学的及び膜興奮特性の明らかな変化が認められないことから、IP3受容体欠失と長期抑圧現象そのものとの関連の解析に着手している。

蛍光相関分光法（FCS）を細胞内に適応することにより細胞質内の遊離タンパク質の挙動を追跡できる。多点に蛍光相関分光法（FCS）を適用することにより神経細胞局所の細胞質中を高速に移動するタンパク質分子の動態を捉え、シナプスと核を連絡するシグナル系の実態を明らかにすることを目的とする。本研究ではランダムスキャン型2光子励起顕微鏡システムを用いカルモジュリン、CamKIIなどシナプス機能に重大な機能分子のスパイン内外での動態を検出することを試みた。その結果初代培養神経細胞内でのタンパク質拡散の様態を示すデータが得られた。また、解析法を開発し、データモデルに基づく拡散を精密に解析した。

脳の熱産生の主体である神経細胞やグリア細胞個々の熱産生の実体はよくわかっていない。本研究はこれまで手がついていなかった、脳内部の熱産生の理解を、細胞あるいは細胞内局所レベルでの温度計測によって深め、脳内の熱動態およびその病態理解への基礎的知見を得ることを目的とした。蛍光寿命変化を温度の指標として用いることを検討した。Rhodamine Bはミトコンドリアの状態により脂質膜の内外に局在を変化させることが知られていた。蛍光寿命変化は局在の変化に影響を受けないと予想したが、脂質中と水相では温度による蛍光寿命特性が大きく異なることが判明した。また、ポリマー被包温度プローブは粒子径がホールセル・パッチクランプ法で細胞に導入するには大きすぎることが判明し、現在粒子径の小さいポリマー被覆温度プローブの作製を検討している。

蛍光相関分光法（FCS）を細胞内に適応することにより細胞質内の遊離タンパク質の挙動を追跡できる。多点に蛍光相関分光法（FCS）を適用することにより神経細胞局所の細胞質中を高速に移動するタンパク質分子の動態を捉え、シナプスと核を連絡するシグナル系の実態を明らかにすることを目的とする。本研究では新規に開発した2光子励起顕微鏡システムを用いカルモジュリン、CamKIIなどシナプス機能に重大な機能分子のスパイン内外での動態を検出することを試みた。その結果初代培養神経細胞内でのタンパク質拡散の様態を示すデータが得られつつある。また、解析法を開発しつつ、データモデルに基づく拡散を精密に解析した。

蛍光相関分光法（FCS）を細胞内に適応することにより細胞質内の遊離タンパク質の挙動を追跡できる。多点に蛍光相関分光法（FCS）を適用することにより神経細胞局所の細胞質中を高速に移動するタンパク質分子の動態を捉え、シナプスと核を連絡するシグナル系の実態を明らかにすることを目的とする。本研究では新規に開発した2光子励起顕微鏡システムを用いカルモジュリン、CamKIIなどシナプス機能に重大な機能分子のスパイン内外での動態を検出することを試みた。その結果初代培養神経細胞内でのタンパク質拡散の様態を示すデータが得られつつある。また、解析法を開発しつつ、データモデルに基づく拡散を精密に解析した。

本研究は申請者の開発したランダムアクセス型２光子励起顕微鏡システムを用いて、これまで困難だった遊離タンパク質の動態を直接計測することを目指す。多点で同時に蛍光相関分光法（FCS）を適用することにより樹状突起の細胞質中を高速に移動するタンパク質分子の動態を捉え、シナプスと核を連絡するシグナル系の実態を明らかにすることを目的とする。これまで細胞質内を拡散する遊離型タンパク質は拡散速度の桁違いの大きさのため直接的な動態の計測は困難だった。蛍光相関分光法（FCS)は溶液中での一分子の振る舞いを計測する手法で、これを細胞内に適応することにより細胞質内の遊離タンパク質の挙動を追跡できる。しかし従来のFCS装置は細胞内の一点からしか記録できず、細胞内の様々な部位における機能タンパク質の動態解析を行うには制約があった。特に神経細胞は細胞内の機能局在性が高く、同時多点からのFCS解析は非常に多くの情報をもたらすことが期待される。本研究では新規に開発した2光子励起顕微鏡システムを用いカルモジュリン、CamKII、Ca結合タンパク質(calbindin等)などシナプス機能に重大な機能分子のスパイン内外での動態を検出することを試みた。その結果初代培養神経細胞内でのタンパク質拡散の様態を示すデータが得られつつある。また、解析法を開発しつつ、データモデルに基づく拡散を精密に解析した。同顕微鏡の高速ランダムスキャンは膜電位色素の蛍光変化を多点で高速に記録することが可能で、これにより複数神経細胞の活動電位を光学的に計測することが可能になる。初代培養神経細胞に電位依存性色素を導入する方法を開発し、さらに膜電位および細胞内カルシウム濃度の同時記録系を立ち上げ、実際に計測を行った。その結果、カルシウム濃度計測では時間分解能が不足して測定できなかった神経細胞同士のシナプス結合の向きが膜電位計測により測定できることが明らかとなった。

本研究は申請者の開発したランダムアクセス型２光子励起顕微鏡システムを用いて、これまで困難だった遊離タンパク質の動態を直接計測することを目指す。多点で同時に蛍光相関分光法（FCS）を適用することにより樹状突起の細胞質中を高速に移動するタンパク質分子の動態を捉え、シナプスと核を連絡するシグナル系の実態を明らかにすることを目的とする。これまで細胞質内を拡散する遊離型タンパク質は拡散速度の桁違いの大きさのため直接的な動態の計測は困難だった。蛍光相関分光法（FCS)は溶液中での一分子の振る舞いを計測する手法で、これを細胞内に適応することにより細胞質内の遊離タンパク質の挙動を追跡できる。しかし従来のFCS装置は細胞内の一点からしか記録できず、細胞内の様々な部位における機能タンパク質の動態解析を行うには制約があった。特に神経細胞は細胞内の機能局在性が高く、同時多点からのFCS解析は非常に多くの情報をもたらすことが期待される。本研究では新規に開発した2光子励起顕微鏡システムを用いカルモジュリン、CamKII、Ca結合タンパク質(calbindin等)などシナプス機能に重大な機能分子のスパイン内外での動態を検出することを試みた。その結果初代培養神経細胞内でのタンパク質拡散の様態を示すデータが得られつつある。また、解析法を開発しつつ、データモデルに基づく拡散を精密に解析した。同顕微鏡の高速ランダムスキャンは膜電位色素の蛍光変化を多点で高速に記録することが可能で、これにより複数神経細胞の活動電位を光学的に計測することが可能になる。初代培養神経細胞に電位依存性色素を導入する方法を開発し、さらに膜電位および細胞内カルシウム濃度の同時記録系を立ち上げ、実際に計測を行った。その結果、カルシウム濃度計測では時間分解能が不足して測定できなかった神経細胞同士のシナプス結合の向きが膜電位計測により測定できることが明らかとなった。

通常の二光子励起顕微鏡で使われるガルバノミラーによるXY両軸のスキャンではなく、直交する２つのAODデバイスによるXYスキャンを用いることにより、従来型では不可能であったランダムスキャンを可能とし、非常に低侵襲で高速な（数10 kHz以上）観測を可能とする新型二光子励起顕微鏡を開発した。AODによるスキャン方式では２次元空間上の複数の点から測定できるので、神経細胞の多数のスパインおよび樹状突起中の情報を収集できる。またランダムスキャン方式により、必要最小限のスポットにのみ励起光を照射するので、従来型のXYスキャンあるいはラインスキャンの場合に大きな問題であった細胞のダメージが大きく軽減される。また、記録部は光電子増倍管を光子計数モードで動作させるので熱ノイズをキャンセルすることができ、低ノイズでの記録が期待される。本年度は蛍光相関分光法（FCS）をリアルタイムで適用するソフトウェアを実装し、細胞質でのタンパク分子蛍光分子の挙動を測定することによりシグナル分子の挙動を精密に測定することを試みた。その結果、蛍光ビーズを用いて自己相関を計測することができた。小分子蛍光色素では自己相関がまだうまくとれないので光学系の調整を進めている。また、高速ランダムスキャンを生かして多点膜電位計測を試みている。本年度は電位依存性色素の効率的取り込み法の確立を目指した。最終的に遺伝子銃を用いた電位依存性色素注入法が有効であることが明らかになった。

通常の二光子励起顕微鏡で使われるガルバノミラーによるXY両軸のスキャンではなく、直交する２つのAODデバイスによるXYスキャンを用いることにより、従来型では不可能であったランダムスキャンを可能とし、非常に低侵襲で高速な（数10 kHz以上）観測を可能とする新型二光子励起顕微鏡を開発した。AODによるスキャン方式では２次元空間上の複数の点から測定できるので、神経細胞の多数のスパインおよび樹状突起中の情報を収集できる。またランダムスキャン方式により、必要最小限のスポットにのみ励起光を照射するので、従来型のXYスキャンあるいはラインスキャンの場合に大きな問題であった細胞のダメージが大きく軽減される。また、記録部は光電子増倍管を光子計数モードで動作させるので熱ノイズをキャンセルすることができ、低ノイズでの記録が期待される。本年度は蛍光相関分光法（FCS）をリアルタイムで適用するソフトウェアを実装し、細胞質でのタンパク分子蛍光分子の挙動を測定することによりシグナル分子の挙動を精密に測定することを試みた。その結果、蛍光ビーズを用いて自己相関を計測することができた。小分子蛍光色素では自己相関がまだうまくとれないので光学系の調整を進めている。また、高速ランダムスキャンを生かして多点膜電位計測を試みている。本年度は電位依存性色素の効率的取り込み法の確立を目指した。最終的に遺伝子銃を用いた電位依存性色素注入法が有効であることが明らかになった。

通常の二光子励起顕微鏡で使われるガルバノミラーによるXY両軸のスキャンではなく、直交する２つのAODデバイスによるXYスキャンを用いることにより、従来型では不可能であったランダムスキャンを可能とし、非常に低侵襲で高速な（数10 kHz以上）観測を可能とする新型二光子励起顕微鏡を開発した。本年度はフェムト秒レーザーの波形調節機構（プレチャープ光学系、ＡＯＭデバイスの制御系）、レーザー導入系、AODデバイスの制御系、及び光子計測モードで動作させる光電子増倍管の制御系を完成した。また、高速に焦点面を変化させるためのピエゾ素子を用いた焦点可変装置を導入した。AODによるスキャン方式では２次元空間上の複数の点から測定できるので、神経細胞の多数のスパインおよび樹状突起中の情報を収集できる。またランダムスキャン方式により、必要最小限のスポットにのみ励起光を照射するので、従来型のXYスキャンあるいはラインスキャンの場合に大きな問題であった細胞のダメージが大きく軽減される。また、記録部は光電子増倍管を光子計数モードで動作させるので熱ノイズをキャンセルすることができ、低ノイズでの記録が期待される。今後は本装置に蛍光相関分光法（FCS）をリアルタイムで適用するソフトウェアを実装し、細胞質でのタンパク分子蛍光分子の挙動を測定することによりシグナル分子の挙動を精密に測定することを試みる。

シナプス可塑性の機構を解明することは脳の高次機能を理解するうえで不可欠である。遺伝子発現変化を介して数日あるいはそれ以上持続するシナプスの長期可塑性には核での遺伝子発現変化を促すためにシナプス後部から核への情報伝達機構が存在すると考えられている。この情報伝達の媒体としては、PKA, MAPキナーゼ, CaMキナーゼ群の活性化による核内転写因子の調節経路が注目されているが、本研究ではRIPにより特異的切断を受ける一群の膜タンパクによる、細胞内での増幅ステップを含まない新しい情報伝達系をとりあげた。RIPを受けることが知られているタンパク質のうち、シナプス後部に存在するものとしてN-cadherin、E-cadherinを選び、これらが実際にシナプス後部で刺激依存的に切断されC末断片が細胞質へと放出される様態を観察してきた。これまでに海馬初代培養神経細胞において刺激依存的なRIPがシナプス後部で働いており、この機構は標的タンパク質選択的であることを明らかにしてきた。本年度は更に詳細にN-cadherinの神経細胞活動依存的な切断と核移行を解析するために、N-cadherin遺伝子を改変してRIPによる切断を受けない変異遺伝子を作製した。この変異遺伝子を培養細胞にて発現しRIPによる切断が実際に起こらないことを確認した。今後はこの変異型N-cadherinを用いて、詳細に活動依存的、非依存的RIPを定量的に検討したい。また切断されたC末断片がどのような経過をたどり核に到達するかを検討してゆく。

シナプス可塑性の機構を解明することは脳の高次機能を理解するうえで不可欠である。遺伝子発現変化を介して数日あるいはそれ以上持続するシナプスの長期可塑性には核での遺伝子発現変化を促すためにシナプス後部から核への情報伝達機構が存在すると考えられている。この情報伝達の媒体としては、PKA, MAPキナーゼ, CaMキナーゼ群の活性化による核内転写因子の調節経路が注目されているが、本研究ではRIPにより特異的切断を受ける一群の膜タンパクによる、細胞内での増幅ステップを含まない新しい情報伝達系をとりあげた。RIPを受けることが知られているタンパク質のうち、シナプス後部に存在するものとしてアミロイド前駆体タンパク(APP)、Alcadeinα、N-cadherin、E-cadherinを選び、これらが実際にシナプス後部で刺激依存的に切断されC末断片が細胞質へと放出される様態を観察した。マウス海馬から調製した初代培養神経細胞を用い、免疫組織化学によりこれらタンパク質が実際にシナプスに存在することを確認した後、これらタンパク質と蛍光タンパク質の融合タンパク質を発現するプラスミドベクターを初代培養神経細胞にて発現し、これらタンパク質がシナプス後部に局在することを更に確認した。シナプス後部に蛍光蛋白質との融合タンパク質が発現している状態で、神経細胞を刺激し、蛍光強度の変化を観察した。刺激にはKチャンネル阻害薬の4-APを用いた。その結果、N-cadherinのシグナルは刺激後10分程でシナプス後部から減少し、核の蛍光シグナルは増強した。E-cadherinは逆に刺激後シナプス後部で増加が見られた。APPとAlcadeinαでは刺激による変化は認められなかった。以上の結果から、刺激依存的なRIPがシナプス後部で働いており、この機構は標的タンパク質選択的であることがわかった。RIPによる膜タンパクのダイナミックなターンオーバーがこの実験系で検討できることが明らかとなった。今後はこの知見をもとに、詳細に活動依存的、非依存的RIPを定量的に検討し、また切断されたC末断片がどのような経過をたどり核に到達するかを検討したい。

シナプス可塑性の機構を解明することは脳の高次機能を理解するうえで不可欠である。遺伝子発現変化を介して数日あるいはそれ以上持続するシナプスの長期可塑性には核での遺伝子発現変化を促すためにシナプス後部から核への情報伝達機構が存在すると考えられている。この情報伝達の媒体としては、PKA, MAPキナーゼ, CaMキナーゼ群の活性化による核内転写因子の調節経路が注目されているが、本研究ではRIPにより特異的切断を受ける一群の膜タンパクによる、細胞内での増幅ステップを含まない新しい情報伝達系をとりあげた。RIPを受けることが知られているタンパク質のうち、シナプス後部に存在するものとしてアミロイド前駆体タンパク(APP)、Alcadeinα、N-cadherin、E-cadherinを選び、これらが実際にシナプス後部で刺激依存的に切断されC末断片が細胞質へと放出される様態を観察した。マウス海馬から調製した初代培養神経細胞を用い、免疫組織化学によりこれらタンパク質が実際にシナプスに存在することを確認した後、これらタンパク質と蛍光タンパク質の融合タンパク質を発現するプラスミドベクターを初代培養神経細胞にて発現し、これらタンパク質がシナプス後部に局在することを更に確認した。シナプス後部に蛍光蛋白質との融合タンパク質が発現している状態で、神経細胞を刺激し、蛍光強度の変化を観察した。刺激にはKチャンネル阻害薬の4-APを用いた。その結果、N-cadherinのシグナルは刺激後10分程でシナプス後部から減少し、核の蛍光シグナルは増強した。E-cadherinは逆に刺激後シナプス後部で増加が見られた。APPとAlcadeinαでは刺激による変化は認められなかった。以上の結果から、刺激依存的なRIPがシナプス後部で働いており、この機構は標的タンパク質選択的であることがわかった。RIPによる膜タンパクのダイナミックなターンオーバーがこの実験系で検討できることが明らかとなった。今後はこの知見をもとに、詳細に活動依存的、非依存的RIPを定量的に検討し、また切断されたC末断片がどのような経過をたどり核に到達するかを検討したい。