見て調べる神経回路機能のダイナミクス：再編成と変調 Optical approaches to neuronal circuit dynamics in health and disease
S3-4-2-1 化学物質と神経回路再編成(行動からエピジェネティクスまで) Chemical induced reorganization of neural circuit during development - from behavior to epigenetics ○五十嵐勝秀1, 冨永貴志2, 古川佑介1, 大塚まき1, 森山紀子1, 菅野純1, 種村健太郎3 ○Katsuhide Igarashi1, Takashi Tominaga2, Yusuke Furukawa1, Maki Otsuka1, Noriko Moriyama1, Jun Kanno1, Kentaro Tanemura3 国立医薬品食品衛生研究所・安全性生物試験研究センター・毒性部1, 徳島文理大・香川薬・神経科学研2, 東北大院・農・動物生殖科学3 Div Cellular & Molecular Toxicol, NIHS, Tokyo, Japan1, Inst Neurosci., Kagawa Sch Pharm Sci, Tokushima Bunri Univ., JAPAN2, Lab Animal Reproduction, Grad Sch Agr Sci, Tohoku Univ., Sendai, Japan3 There are numerous chemicals in our living environment. They include pharmaceutical components, foods or various industrial products and so on. Therefore, it is difficult to imagine life without contacting them. Many chemicals can influence the signaling pathways in central nervous system. Several lines of evidences imply that brief transient exposure to these chemicals during the brain developmental stage may eventually lead to the late-onset brain functional defects. In order to search such chemicals that can induce signaling system disruptions on the course of the brain development, we constructed a behavioral test battery for mice. In this symposium, we would like to show the effect of early exposure of domoic acid and triazolam as the actual examples that can cause behavioral abnormality at adulthood with neonatal exposure to the chemicals. Domoic acid, a shellfish poison, activates glutamic acid pathway, caused the poor learning and memory performance with hyperactive tendency, and triazolam, a hypnotic drug which activates GABAergic pathway, caused the low-performance spatial memory. We would like to emphasize that our behavior screenings show that only brief exposure to the chemicals that cause transient disruption of signaling systems in the developmental stage actually induced late-onset defects of brain functions. For the next step, it is important to elucidate the background mechanisms how those kinds of late-onset defects are established. To that end, the means to visualize the functional neuronal circuits are extremely useful. Therefore, we also would like to show how to utilize the voltage sensitive dye (VSD) imaging technique for the analysis of the chemical induced late-onset functional defects of brain. In addition, we will show that our effort to reveal the epigenetic change in hippocampus, especially about DNA methylation.	S3-4-2-2 神経回路動作の網羅的定量解析-バルプロ酸による神経回路動作変容の解明 Optical assay of abnormal neuronal circuit dynamics: Effect of prenatal exposure to valproic acid ○冨永貴志1, 冨永洋子1, 五十嵐勝秀2, 種村健太郎3, 菅野純2, 中島欽一4 ○Takashi Tominaga1, Yoko Tominaga1, Katsuhide Igarashi2, Kentaro Tanemura3, Jun Kanno2, Kinichi Nakshima4 徳島文理大学香川薬学部1, 国立医薬品食品衛生研・安全性生物試験研究センター・毒性部2, 東北大院・農・動物生殖科学3, 奈良先端大・バイオサイエンス・分子神経分化制御4 Tokushima Bunri University, Kagawa Sch Pharm Sci1, Div Cellular & Molecular Toxicol, NIHS, Tokyo, Japan2, Lab Animal Reproduction, Grad Sch Agr Sci, Tohoku Univ., Sendai, Japan3, Lab Mol Neurosci, NAIST, Nara, Japan4 Voltage-sensitive dye (VSD) was developed in the 1960s to measure fast membrane potential changes in excitable membranes by using optical devices. Development of a large-scale imager allowed for the visualization of membrane potential events of a neural circuit. VSD imaging could permit observation of membrane potential activities at many levels of neuronal structures, but the practical application of VSD has been limited by a poor signal-to-noise ratio (SNR) due to the small fractional changes of VSD. However, because of the advances in optics and imagers, single-photon wide-field VSD imaging has been improved to visualize various types of neural activity and has been used as a quantitative tool to visualize the excitatory and inhibitory activity of neural circuits, especially in in vitro specimens. Because the VSD imaging method allows us to see modifications in the dynamics of membrane potential activity in neuronal circuits, it should be the best strategy to assay abnormal neuronal circuit dynamics, such as the disruption of excitatory/inhibitory (E/I) balance and wide-range correlations among the brain areas. Disruption of those factors has been thought to be a major cause of brain dysfunctions in large-scale networks relating to neuropsychiatric disorder, such as autism spectrum disorders (ASDs). Here, we present an example of using VSD imaging to assess the effect of maternal exposure to an antiepileptic, valproic acid (VPA). VPA has attracted attention recently because children born from mothers treated with VPA are known to have an increased risk of ASD-like cognitive deficiencies. Hippocampal slice preparations of VPA-treated mice showed reduced sensitivity to a GABAA receptor inhibitor. We hypothesized that this resulted from reduced inhibitory activation in VPA-treated mice, accompanied by reduced excitatory activation. We will further discuss the use of VSD imaging as a robust and quantitative assay tool to detect possible disruption of brain functions.
S3-4-2-3 Ex vivo単離脳イメージングによる辺縁系ネットワークの大規模解析 Whole-scale voltage imaging of limbic network using isolated brain preparation ○梶原利一1, 高島一郎2, 冨永貴志3 ○Riichi Kajiwara1, Ichiro Takashima2, Takashi Tominaga3 産業技術総合研究所・バイオメディカル研究部門1, 産業技術総合研究所・ヒューマンライフテクノロジー研究部門2, 徳島文理大・香川薬・神経科学研3 Biomed RI, AIST, Tsukuba1, Human Tech RI, AIST, Tsukuba2, Inst Neurosci., Kagawa Sch Pharm Sci, Tokushima Bunri Univ., Sanuki3 The 'limbic system' is considered as a crucial structure for the neuralplasticity caused by learning and memory behavior. Therefore the neural circuitry of limbic system is the primary target of functional modifications relating to the higher cognitive dysfunctions caused by various factors. The in vitro brain slice preparations have been used for the purpose so often, due to the difficulty in recording neural activities from deep brain structures under in vivo conditions. Particularly, hippocampal slice preparations are more commonly used to investigate the plasticity on a cellular / synaptic level. However, the functional connectivity should also be examined in much larger scales. The isolated whole brain preparation, in which multi-synaptic circuits and the intracellular activity they generate are well preserved, can be useful to examine such large scale neural circuitry. In the presentation, we describe about the whole scale optical imaging of limbic network using this unique preparation. This experimental approach combines the advantages of the in vivo experimental condition with those of in vitro slice preparations, i.e. an intact synaptic network, excellent mechanical stability, and control over the ionic and biochemical extracellular environment. In particular, it provides easy access to brain areas of the limbic system and preserved the neuronal network of the entorhinal-hippocampal loop. Here we present example data obtained from this preparation in combination with optical imaging of voltage-sensitive dyes. We visualized the propagation pattern of neural activity following olfactory nerve stimulation, and demonstrated that piriform and entorhinal activities converge in the amygdaloid cortex. This convergence may allow the amygdaloid cortex to integrate olfactory sensation with information retained or processed in the entorhinal cortex.	S3-4-2-4 Concepts and applications of genetically encoded voltage indicators ○Thomas Knöpfel1 RIKEN Brain Science Institute1 Protein-based fluorescent probes of neuronal activity are at the core of emerging approaches to study the dynamics of neuronal circuits that are composed of heterologous cell types. The rationale behind our large effort to develop genetically encoded voltage indicators lies in the fact that these probes allow us and others to move beyond the electrophysiological analysis of individual or small numbers of cells without neglecting cellular diversity or compromising temporal resolution. Work in our and other laboratories during the last 15 years resulted in a new generation of voltage-sensitive fluorescent proteins (VSFPs) based on the voltage-sensing domain (VSD) of Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP). To this end, we explored different design principle families for these engineered proteins and characterized numerous mutational variants for each facility. We demonstrated that recent versions of VSFPs can report membrane voltage signals in isolated neurons, brain slices and living mice. The most advanced probes enable the optical recording of action potentials from individual neurons in single sweeps and voltage imaging of population activity, including synchronized activities in the gamma frequency band, from defined cell populations in acute brain slices. In living mice, VSFPs afford sufficient SNR for probing sensory-evoked responses and enables univocal detection of spontaneous electrical population activity in somato-sensory cortex during light anesthesia or quiet alertness. Along with the ability to target specific genetically-defined cell populations, VSFPs open a new experimental window for the study of the interaction dynamics of neuronal assemblies, facilitate the investigation of information processing mechanisms of the brain, such as the circuit operations involved in sensing our environment and generation of body movements, but will also be applicable to directly visualize cognitive functions.
S3-4-2-5 光計測による海馬周辺皮質機能的結合の生後発達過程解析 Optical analysis of development of functional projections to medial entorhinal cortex ○小金澤紀子1,2, , 飯島敏夫3, 白尾智明1 ○Noriko Koganezawa1,2, Cathrin B Canto2, Toshio Iijima3, Tomoaki Shirao1, Menno P Witter2 群馬大学大学院 医学系研究科 神経薬理学1, ノルウェー科学技術大学2, 東北大院・生命科学・脳情報処理3 Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine1, Kavli Institute for Systems Neuroscience and Centre for the Biology of Memory, NTNU, Trondheim, Norway2, Systems Neuroscience, Department of Developmental Biology and Neurosciences, Graduate School of life Sciences Tohoku University, Sendai, Japan3 The entorhinal cortex and hippocampus, as well as the presubiculum (PrS) and parasubiculum (PaS) contain spatially modulated neurons. In medial entorhinal cortex (MEC) there are position-coding grid cells, in hippocampus place cells are found, and in pre- and parasubiculum the majority of spatially tuned cells are head direction cells. PaS and PrS have been postulated to provide directional information to MEC and directional input has been suggested to be important for stable grid cell properties in MEC. Grid cells in MEC develop from P16 onwards, whereas headdirection cells in PrS are already present and stable at that age (Langston et al., 2010). It is not known however whether functional connectivity between these structures exists at those early postnatal stages. One way to study the possible relevance of the interactions between these areas and the neuron-types within, is to look when neurons become functionally connected, locally as well as between areas. We investigated when functional inputs to MEC from PrS or PaS develop postnatally by using optical imaging with a voltage sensitive dye (RH-795) in slices taken from postnatal day (P) 5-P61 old rats. Monosynaptic connectivity from PaS and PrS to MEC was seen from P9 onwards. From P14/15 on, reactivity of MEC neurons receiving para- and presubicular inputs became more or less adultlike. The maturation of the efficacy of both inputs is paralleled by changes in electrophysiological and morphological properties of the respective target neurons. In conclusion monosynaptic projections from PaS and PrS to MEC become functional and adultlike before the emergence of grid cells in MEC around P16.

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