情報伝達とその調節 Signal Transduction and Modulation
P3-1-23 培養神経回路網における確率共鳴 Stochastic resonance in cultured neural networks ○庭野真理子1, 棚元亮1, 堀田耕司1, 岡浩太郎1 ○Mariko Niwano1, Ryo Tanamoto1, Kohji Hotta1, Kotaro Oka1 慶應義塾大学大学院　理工学研究科　基礎理工学専攻　生命システム情報専修　生物物理・神経情報学1 Graduate School of Science and Technology, Keio University, Yokohama,1 Stochastic resonance (SR) is a noise-induced phenomenon whereby signal detection can be improved by additive noise in nonlinear systems. Previous studies have also shown that SR could be found in hippocampal CA1 neurons in brain slices (Stacey and Durand, 2000, 2001). Furthermore, hypothesis that effects of coupling between each pair of neurons on the signal to noise ratio (SNR) have also been tested in a number of neural network models. Despite the successful prediction of the theory about significant effect on SR, there has been no experimental test of how SR were demonstrated in neural networks and whether SNR of a single cell and in the entire network in fact can be enhanced by additive noise, when subthreshold signals are applied. To investigate what kind of feature of network activity can enhance SR of single cells and in neural networks, we performed subthreshold periodic current input stimulations and white noise in cultures of rat hippocampal neurons with combined a specific culture device, using Indium tin oxide (ITO) glass, for stimulating and monitoring larger number of neurons by calcium imaging. As a result, we found some neurons in the network exhibited SR at an optimal noise intensity, and blocking excitatory synapses decreased the number of neurons occurring SR and the SNR of them. By estimating the effect of functional connectivity on SR in single cells and in the network, we found that funtional connections between neurons significantly influence SR in single cells and also in the entire network. Our results suggest that SNR of a single cell and also in the entire network can indeed be enhanced by applied noise and controlled by functional synaptic connections.	P3-1-24 神経前駆細胞及び脳腫瘍においてNemo-like kinaseは転写因子Gli1をリン酸化してヘッジホッグシグナルを抑制する Nemo-like kinase blocks Hedgehog signaling by phosphorylating Gli1 transcription factor in neural progenitor cells and brain tumor ○石谷太1, 清水誠之1, 石谷閑1 ○Tohru Ishitani1, Nobuyuki Shimizu1, Shizuka Ishitani1 九州大・生医研・細胞統御1 MIB, Kyushu Univ, Fukuoka1 The Hedgehog (Hh) signaling pathway mediated by the transcription factor Gli1 controls the proliferation of neural progenitor cells during the central nervous system development. Aberrant activation of the Hh-Gli1 pathway also induces the neural progenitor cell hyper-proliferation and initiates the formation of brain tumor, such as medulloblastoma. However, regulation of the transcriptional activity of Gli1 is poorly understood. Here we show that MAPK-related Nemo-like kinase (NLK) negatively regulates Gli1-dependent transcriptional activation. RNAi-mediated NLK knockdown enhances endogenous Hh signaling activity in a medulloblastoma cell line ONS76. In contrast, overexpression of NLK reduces Hh signaling activity and induces the expression of the neuronal markers in ONS76 cells. NLK binds to and phosphorylates Gli1. NLK-phosphorylated Gli1 is impaired in its ability to form a complex with histone deacetylase HDAC1, which promotes Gli1 transcriptional activity by deacetylating it. We also demonstrated that inhibition of NLK function in zebrafish results in hyperactivation of the Hh-Gli1 pathway in hindbrain and cerebellum, resulting in an enhancement of the hindbrain neural progenitor cell generation and the cerebellar granule cell progenitor proliferation. These defects are related to the negative regulation of the Hh-Gli1 pathway by NLK. Our results both define a new function for NLK and reveal a novel mode of regulation in the Hh signaling pathway in neural progenitor cells and brain tumors.
P3-1-25 ヒトiPS細胞から分化させた神経細胞の蛍光イメージングを用いた特徴づけ Characterization of neurons derived from human iPS cells with calcium dynamics ○塩谷晃弘1, 山中龍1, 新藤豊1, 木藤古孝行2, 堀田耕司1, 岡浩太郎1 ○Akihiro Enya1, Ryu Yamanaka1, Yutaka Shindo1, Takayuki Kitogo2, Kohji Hotta1, Kotaro Oka1 慶應義塾大学　理工学研究科　基礎理工学専攻1, 株式会社リプロセル2 Department of Biosciences and Informatics, Faculty of Science and Technology, KEIO UNIVERSITY, YOKOHAMA, JAPAN1, Reprocell Inc, YOKOHAMA, JAPAN2 Recently, development of methods for reprogramming by introducing Yamanaka factors into adult human somatic cells, established induced pluripotent stem cells (iPS cells). In neuroscience field, iPS cells enable us to study human neural development and its function. Approach using iPS cells has been useful to generate in vitro models for several genetic conditions, inherited and sporadic forms of human degenerative neurologic diseases, such as Alzheimer's disease, and Parkinson disease. However, iPS cells have various properties in each cell line as tendency of differentiation, even established from same somatic cells. Furthermore, even established iPS cells, it could not induce safe differentiation. Therefore, it is necessary to evaluate safety and neural property on applications of iPS cell to the neuroscience research. In most studies, criterions of neural differentiation of iPS cells are determined by protein marker. Functional criterions have not been clearly provided yet. In this study, we combine the results of immunocytochemistry imaging and calcium dynamics induced by drug administration in neurons derived from iPS cells, tried to characterize individual cells. The cells obtained from Reprocell Inc. (Japan), were differentiated according to the protocol. At the stage of 14-21days from sowing, we stained neurons with calcium indicator fluo-8, and measured intracellular calcium concentration induced by administration of dopamine, glutamate and potassium chloride. As the result, we found two phase calcium dynamics induced by glutamate. Immediately after glutamate administration, calcium transient was observed. Then after 3 minutes from administration, sustained calcium increase occurred. Almost all neurons (~98%) showed this type of calcium responses by glutamate stimulus, 2% of these cells presented calcium transient rise by dopamine administration.	P3-1-26 新規FRET型蛍光プローブによって明らかにされた神経細胞でのカルシウムによるCdc42の活性調節機構の解明 A calcium-dependent mechanism for the transformation of input patterns into Cdc42 activity in neurons revealed by novel fluorescent indicators ○太田裕作1, 岸俊輔1, 並木繁行1, 廣瀬謙造1 ○Yusaku Ohta1, Syunsuke Kishi1, Shigeyuki Namiki1, Kenzo Hirose1 東京大院・医・神経生物1 Dept. Neurobiol., Grad. Sch. Med., Univ. Tokyo1 Cdc42, a member of the Rho GTPase family, has an important role in regulating the morphology of neurons by regulating the organization of the actin cytoskeleton in response to various input patterns. However, it is still unclear whether or not different patterns of neural input cause different activity levels of Cdc42. And if so, it is still unknown how the level of Cdc42 activity evoked by neural input is determined. To solve these problems, we have constructed a new fluorescent indicator for Cdc42 activity based on FRET between GFP and tetramethylrhodamine (TMR). By using this indicator, we measured the levels of Cdc42 activity in response to various input patterns in cultured rat cortical neurons. We found that Cdc42 activity was dependent on the number and frequency of stimuli in the ranges of 1-40 trains and 1-10 Hz, respectively. Furthermore, by measuring the changes in intracellular calcium response using the calcium indicator Fluo-4FF, we found that the activity level of Cdc42 was dependent on both the amplitude and duration of the changes in calcium response. These results indicate that neurons determine the level of Cdc42 activity according to the extent of changes in the intracellular calcium response evoked by synaptic inputs, and thereby make it possible to respond appropriately to various input patterns.
P3-1-27 ノルアドレナリンはβ受容体を介して扁桃体中心核内側亜核への抑制性入力を増強させる Noradrenaline Dramatically Increases GABAergic Inputs Through Beta Adrenoceptor Activation in Medial Division of the Central Nucleus of Amygdala ○山本純偉1, 高橋由香里2, 渡部文子2, 田中誠1, 加藤総夫2 ○Sumii Yamamoto1, Yukari Takahashi2, Ayako Watabe2, Makoto Tanaka1, Fusao Kato2 筑波大学医学医療系1, 慈恵会医科大学神経生理2 Dep Anesthesiol, Faculty of Medicine, Univ Tsukuba, Tsukuba1, Lab Neurophysiol, Dept Neurosci, Jikei Univ Sch Med, Tokyo2 The central nucleus of amygdala (CeA) plays essencial role in emotional memory formation. Of the subnuclei composition the CeA, neurons in the medial division of the central nucleus of amygdala (CeM) receive intranuclear inputs from the lateral and lateral-capsular division of CeA (CeL and CeC, respectively) as well as inputs from the basolateral amygdala (BLA) and project to the hypothalamus and brain stem nuclei involeved in fear-related behavioral, autonomic and endocrinal responses. Recently, accumulated lines of evidence indicate noradrenaline (NA), intra-amygdala release of which is increased in response to nociception, plays facilitatory role in emotional memory formation but its mechanism of effect remains largely unidentified. In order to identify the role of NA in modulation of the CeA network activity, we analyzed the effects of exogenously applied NA on spontaneous inhibitory postsynaptic current (sIPSC) in the presence CNQX and APV in the CeM from 3- to 8- week old mouse brain slice. Bath application of NA (50 μM) dramatically and reversibly increased sIPSC frequency (143%±70%, n=12, P<0.05) and amplitude (118%±63%, n=12, P<0.05) in a manner sensitive to tetrodotoxin (TTX, 1 μM) and propranolol (40 μM). Highly selective α2 agonist dexmedetomidine did not affect frequency and amplitude of sIPSC. A subset of CeA neurons were depolarized by NA and increased intracellular Ca 2+ concentration as visualized with Fluo-4 and confocal Ca 2+ imaging. Altogether, it is likely that NA directly excites CeL/CeM GABAergic neurons, which results in increased spontaneous GABAergic inputs to CeM neurons to strongly modulate the output from the amygdala network to other brain nuclei.	P3-1-28 ラット海馬神経細胞におけるGABAによる二元的なマグネシウム動員機構 Dual Mechanism Mediates Magnesium Mobilization Triggered by GABA in Rat Hippocampal Neurons ○山中龍1, 新藤豊1, 堀田耕司1, 鈴木孝治2, 岡浩太郎1 ○Ryu Yamanaka1, Yutaka Shindo1, Kohji Hotta1, Koji Suzuki2, Kotaro Oka1 慶應義塾大学大学院　理工学研究科　基礎理工学専攻1, 慶應義塾大学大学院　理工学研究科　総合デザイン工学専攻2 School of Fundamental Science and Technology, Graduate School of Science and Technology, Keio University, Yokohama, Kanagawa, Japan.1, Center for Science and Technology for Designing Functions, School of Integrated Design Engineering, Graduate School of Science and Technology, Keio University, Yokohama, Kanagawa, Japan.2 Athough the magnesium ion (Mg2+) is involved in a wide variety of biochemical reactions and physiological functions, the function of intracellular Mg2+ in neurons is still largely unknown. In this study, we investigated effects of the inhibitory neurotransmitter GABA on intracellular Mg2+ concentration by using KMG-104, a highly selective fluorescent Mg2+ probe. Administration of GABA to hippocampal neurons induced an intracellular Mg2+ increase. Mainly, GABA receptors are classified ionotropic GABAA receptors and metabotropic GABAB receptors. Interestingly, both GABAA and GABAB receptors agonists triggered the intracellular Mg2+ increase. GABAA receptor agonist muscimol induced an intracellular Mg2+ increase in normal and Mg2+-free medium, but did not in Cl-free medium. Furthermore, pre-administration of FCCP, which induced Mg2+ release from mitochondria, suppressed the muscimol-induced intracellular Mg2+ increase. The muscimol-induced intracellular Mg2+ increase is not accompanied by depolarization of mitochondria. GABAB receptor agonist baclofen induced intracellular Mg2+ increase, which was suppresseded by Na+/Mg2+ exchanger inhibitors. The selective PKC inhibitor reduced the GABA-induced intracellular Mg2+ increase but the selective PKA inhibitor did not. In addition, the application of the PKC activator induced an intracellular Mg2+ increase. Therefore, our findings indicate that GABA induced the intracellular Mg2+ increase results from the activation of the Na+/Mg2+ exchanger via PKC. Taken together, GABA induced the activation of GABAA receptor, which induced the release of Mg2+ from mitochondria via Cl- without the depolarization of mitochondria, and the activation of GABAB receptor, which induced the influx of Mg2+ via Na+/Mg2+ exchanger modulated by PKC. This work is supported by Grants-in-Aid for Scientific Research (A) (24240045).
P3-1-29 レーザーアンケージングによるカントールコーディングにおける空間入力様式の依存性 The dependency for spatial input pattern in Cantor coding using laser uncaging system ○上條中庸1, 山口裕2, 福島康弘3, 津田一郎2,4, 塚田稔1, 相原威1 ○Tadanobu Kamijo1, Yutaka Yamaguti2, Yasuhiro Fukushima3, Ichiro Tsuda2,4, Minoru Tsukada1, Takeshi Aihara1 玉川大学大学院脳情報研究科1, 北海道大学大学院理学院数学専攻2, 川崎医療福祉大学医療福祉学部3, 北海道大学電子科学研究所4 Brain Science Inst, Tamagawa Univ, Tokyo1, Dept of Math, Hokkaido Univ.2, Dept of Medical Welfare, Kawasaki Univ of Medical Welfare3, Research Institute for Electronic Science, Hokkaido Univ4 In humans, the hippocampus is a necessary organ for the formation of episodic memory, and recent studies in rodents also showed the involvement of hippocampal cells in the processing of context-dependent and/or sequentially represented episodic like memories. It is an important theme for the investigation of memory systems to clarify how information is coded and represented in hippocampus. The CA3 area is characterized by a distinct biological neural network, which has a recurrent (feedback) connection. We hypothesized that the hippocampal CA3 network forms a context of time sequence, while CA1 maps the spatio-temporal context to its synaptic weight space. However, the coding property of spatial clustering and its self-similarity has not been experimentally reported in the input-output relation of hippocampal CA1 neurons. To clarify how the information of spatiotemporal sequence of the hippocampal CA3 affects the postsynaptic membrane potentials of single pyramidal cells in the hippocampal CA1, two type stimulations were applied and the post-synaptic membrane potentials were recorded using the patch-clamp recording method. First, spatio-temporal stimuli were delivered to Schaffer collaterals through a pair of glass electrodes. Second, spatio-temporal stimuli were applied onto the four dendritic sites by using high-speed laser uncaging system. The input-output relations were sequentially analyzed by applying the measure; 'spatial clustering index. The membrane potentials were hierarchically clustered in a self-similar manner to the input sequences. The property was significantly observed at two and three time-history steps. Furthermore, to investigate the detail properties of the spatio-temporal processing in CA1 pyramidal cell, the stimuli of the spatio-temporal pattern were applied to dendritic sites using high-speed uncaging system. The experimental results are discussed in relation to theoretical results of Cantor coding.	P3-1-30 シナプス外NMDA受容体の分布様式と機能発現 Distribution pattern and function of extrasynaptic N-methyl-D-aspartate receptor ○福永優子1, 前田博毅1, 山本悠貴1, 有賀理瑛1, 桃田菜央1, 宮澤淳夫1 ○Yuko Fukunaga1, Hiroki Maeda1, Yuki Yamamoto1, Rie Ariga1, Nao Momota1, Atsuo Miyazawa1 兵庫県立大学大学院　生命理学研究科　細胞構造学1 Laboratory of Cell Biochemistry, Graduate School of Life Science, University of Hyogo1 The NMDA receptors located at the synapses facilitate the cell survival whereas the NMDA receptors distributed at the extrasynaptic sites, by contrast, involve in cell death as results of high dose-glutamate treatment or ischemia episodes. The relationship between the distribution pattern and the function of the extrasynaptic NMDA receptors remains largely unknown. In order to analyze the distribution pattern of the extrasynaptic NMDA receptors, we have previously established to label only extrasynaptic NMDA receptors using the primary cultured hippocampal neurons. The extrasynaptic NMDA receptors of hippocampal neurons were labeled with 10 nm-colloidal gold particles under the detergent-free condition and observed by scanning electron microscopy. The observations of the sample stained with this method demonstrated that the sizes of the extrasynaptic NMDA receptor clusters of 2 weeks in vitro (2 WIV) hippocampal neurons were larger than that of 1 WIV hippocampal neurons. On the other hand, the total number of the NMDA receptors distributed on the non-synaptic area of cytoplasmic membrane of the neuronal processes was not different between 1 WIV neurons and 2 WIV neurons. The analysis of intracellular calcium concentration in hippocampal neurons at 1 WIV and 2 WIV suggested that the size of clusters consisting of the NMDA receptors might be important for the global cytosolic calcium transient through the extrasynaptic NMDA receptors. In addition, the sensitivity against NMDA toxicity at 2 WIV was higher than that at 1 WIV. Altogether, the cluster size of extrasynaptic NMDA receptors enough to increase the global cytosolic calcium concentration may be required for induction of the cell death.
P3-1-31 初代培養神経細胞におけるオートファジー誘導とToll様受容体の関連 Toll-like receptor stimulation induces autophagy in primary neurons ○砂堀毅彦1, 内山安男1 ○Takehiko Sunabori1, Yasuo Uchiyama1 順天堂大学医学部神経生物学・形態学講座1 Dept Cell Biol and Neurosci, Juntendo Univ, Tokyo1 Recent genetic studies have shown that mice lacking an autophagy-related gene (Atg5 or Atg7) cannot survive longer than 12 hours after birth because of nutrient shortage. Moreover, tissue-specific impairment of autophagy in central nervous system (CNS) causes massive loss of neurons, resulting in neurodegeneration. Although autophagy generally prevents cell death as described, our study using conditional Atg7-deficient mice in CNS tissue demonstrated the presence of autophagic neuron death in the hippocampus after neonatal hypoxic-ischemic (H-I) brain injury. On the other hand, we have recently discovered that Toll-like receptor (TLR) 2-deficient mice also show a protective effect against hippocampal neurons after H-I injury. However, the relationship between autophagosome formation and TLR-signaling pathway remains obscure. Therefore, we stimulated TLR2 with a specific agonist, Pam3CSK4, and observed autophagosome formation in primary neurons. The ratios of the amounts of LC3-II to LC3-I were significantly increased by Pam3CSK4-stimulation, like Rapamycin (an autophagy inducer)-stimulation, against primary neurons, compared to the non-treated control. In addition, GFP-LC3 dots appeared abundantly in primary neurons when treated with Pam3CSK4 and Rapamycin, respectively. These results suggest that TLR-stimulation can induce autophagy in primary neurons.	P3-1-32 Dendritic NMDA-spikes in thalamocortical neurons ○Sigita Augustinaite1,2, Bernd Kuhn1, Paul Johannes Helm3, Paul Heggelund2 Okinawa Institute of Science and Technology Graduate University1, Institute of Basic Medical Sciences, University of Oslo2 Various dendritic integration mechanisms regulate the output from single neurons. Dendritic plateau potentials termed NMDA-spikes, first discovered in cortical pyramidal neurons, provide supralinear integration of temporally clustered synaptic inputs on thin and distal dendrites and thereby increase the impact of these inputs on the soma. The more specific functional role of NMDA-spikes has been difficult to clarify partly due to the complex circuitry of cortical neurons. We investigated dendritic potentials in thalamocortical (TC) neurons in the dorsal lateral geniculate nucleus (dLGN), which participate in a considerably simpler circuit. TC neurons receive their primary afferent input from retina and send their output to visual cortex. Cortex, in turn, sends massive feedback to dLGN that regulates the output of TC neurons. The glutamatergic retinal and cortical inputs are segregated in the dendrites of TC neurons: retinal synapses are located proximally, while cortical synapses are mainly located distally. Because of T-type Ca2+ conductance, TC neurons can operate in two membrane potential dependent modes: "burst mode" during hyperpolarization, when Ca2+ bursts largely disrupt thalamocortical transmission, and "tonic mode" during depolarization, which inactivates Ca2+ bursts and provide reliable transmission of retinal signals to cortex. We studied dendritic processing in TC neurons with combined 2-photon calcium imaging and somatic whole-cell recording of response to local dendritic glutamate stimulation. We found that NMDA-spikes can be elicited locally at distal dendrites of TC neurons during both, hyperpolarized and depolarized membrane potentials. This suggests that NMDA-spikes in TC neurons may be an important mechanism through which cortical feedback can regulate the neuronal output by switching the functional mode from burst to tonic, and facilitate retinal signal transfer during the tonic mode.

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