電気生理学的手法 Electrophysiology
P3-2-247 全自動パッチクランプシステム、Qpatch HTX、におけるマルチホールテクノロジーを用いたリガンド作動性イオンチャネルに関する生物物理学および薬理学的基礎検討 QPatch HTX: Biophysical and pharmacological characterization of ligand-gated ion channels in multi-hole mode ○竹内啓太1 ○Keita Takeuchi1, M. Knirke Jensen2, Rikke Schrøder2, Hervør L Olsen2, Rasmus B Jacobsen2, Jeffrey Webber3, Søren Friis2, Dorthe Nielsen2, Mette T Christensen2, Morten Sunesen2 ソフィオンバイオサイエンス株式会社1 Sophion Bioscience K.K., Honjo-shi, Japan1, Sophion Bioscience A/S, Ballerup, Denmark2, Sophion Bioscience, INC, NJ, United States3 The multi-hole patch technology for the QPatch enables gigaseal recordings of up to ten cells patch-clamped on a single measurement site. In this set of experiments, we have convincingly demonstrated that multi-hole QPatch experiments of fast desensitizing ligand-gated ion channels perform as well as single-hole QPatch experiments with respect to both biophysical and pharmacological characteristics.In the QPlate X, the ten patch holes have a relatively wide spatial distribution to avoid intercellular contact and downstream space clamp issues. The wide spatial distribution could, on the other hand, potentially slow down the liquid exchange times (Tau of activation). The rise times of the two fast desensitizing ligand-gated ion channels glutamate receptor 5 (GluR5) and nicotinic acetylcholine receptor α1 (nAChR α1), were therefore investigated on the QPatch HTX in multi-hole mode. Compared to single-hole recordings, the results showed that the liquid exchange times were not affected for any of the tested ligand-gated ion channels.Furthermore, we examined the GluR5, the nAChR α1, the acid-sensing ion channel 1a (ASIC1a), and the anionic γ-aminobutyric acid receptor A (GABA-A) with regards to agonist kinetics, reversal potential and pharmacological properties on the QPatch HTX in multi-hole mode and compared to results obtained in the classical single-hole mode. All data clearly demonstrate that while the amplitude of the elicited ion channel current is multiplied by a factor of 7-10, other significant features of these fast desensitizing ion channels remain unaltered. Our QPatch HTX data demonstrates the expected biophysical as well as pharmacological properties for these ligand-gated ion channels, when patched in multi-hole mode but with an unprecedented combination of high throughput and high quality recordings.	P3-2-248 高密度CMOSアレイ上の神経回路網の解析へ向けた分散培養神経細胞の細胞体位置の推定及び手法 Estimation of Neuronal Somata Location and Methods toward Neuronal Network Analysis of Dissociated Cultured Neurons on High-density CMOS Microelectrode Arrays ○三田毅1, 神崎亮平1, 高橋宏知1 ○Takeshi Mita1, Douglas Bakkum2, Urs Frey3, Andreas Hierlemann2, Ryohei Kanzaki1, Hirokazu Takahashi1 東京大学大学院 情報理工学系研究科1, チューリッヒ工科大学2, 理化学研究所3 Graduate school of information science and technology, University of Tokyo1, ETH Zurich, Basel, Switzerland2, RIKEN, Kobe, Japan3 We study networks of dissociated cultured neurons by taking advantage of a high-density CMOS microelectrode array. High-density CMOS microelectrode arrays are a tool for extracellular electrophysiology, similar to Multi-electrode Arrays, but featuring a higher spatial resolution by placing ten thousand electrodes in about 4 mm2 area. With this high spatial resolution, we are able to localize neurons without any optical imaging technique. Additionally, we are able to observe propagation of action potentials within the network. The high-density CMOS microelectrode array allows for detailed investigation of dynamics of neuronal networks in vitro. We show results towards the investigation of dynamics of neuronal networks: (1) estimation of locations of neuronal somata; (2) assignment of inhibitory neurons; (3) investigation of neurons in dissociated cultures at single-neuron level and network level; and additionally (4) stimulation of arbitrarily selected single somata.
P3-2-249 EToS4: 全面的に改良された高精度スパイクソーティングシステムの新バージョン EToS4: new version of accurate spike sorting system based on totally improved algorithms ○竹川高志1, 礒村宜和2, 深井朋樹1 ○Takashi Takekawa1, Yoshikazu Isomura2, Tomoki Fukai1 理研・脳総研・脳回路機能理論1, 玉川大学・脳総研2 Lab for Neural Circuit Theory, RIKEN BSI, Wako1, Brain Science Institute, Tamagawa University, Machida2 Simultaneous observations of multiple neurons' activities using extracellular recording with multi-channel electrodes is widely used for clarifying the brain information processes. The recorded signals contain the spike events of a number of adjacent or distant neurons, and must be sorted correctly into spike trains of individual neurons. We have developed high performance spike sorting system and published continuous improvement version (EToS1 and EToS3). EToS3 has been appreciated as one of the best spike sorting system. However, EToS3 has some difficulty in parameter setting and non-stationary data. Our new spike sorting system (EToS4) outputs clearer isolated clusters than EToS3 without parameter setting even if spike waveforms are non-stational and/or small amplitude. In fact, final number of isolated neurons and total spikes by EToS4 could be about twice of these by EToS3. Moreover, EToS4 includes visualization tool for post-processing manual clustering.	P3-2-250 High-resolution recording and analysis of complex spike responses in cerebellar Purkinje cells with monoplanar and multiplanar dendrites ○Marie Engelene J. Obien1, Andreas Hierlemann2, Urs Frey1 Frey Initiative Research Unit, RIKEN Quantitative Biology Center, Kobe, Japan1, Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland2 In the mammalian cerebellum, Purkinje cell (PC) dendrites undergo a remarkable change in arborization during the third postnatal week. At this stage, PC dendrites transform from being multiplanar to monoplanar (Kaneko, et. al, PLoS ONE, 2011). This is the same period, when multiple climbing fibers (CFs) innervating a PC compete until only one CF remains. We examine the functional changes in the PC during this phase by looking at spontaneous and evoked activities of PCs in acutely prepared cerebellar slices with a focus on complex spikes, since they can be triggered by CF activation. Our approach includes the use of a CMOS-based high-density microelectrode array (HDMEA) featuring 11,011 planar electrodes with 126 reconfigurable channels. Since CF activation is needed to evoke dendritic activity, the HDMEA setup is augmented with an external metal electrode for extracellular stimulation of the cerebellar white matter or granular cell layer. We combine this with the conventional patch clamp to be able to achieve simultaneous intra- and extracellular recording. With this system, it is possible to scan for interesting activity, to identify single cells, and to record with subcellular spatial resolution, which allows for analysis of the distribution and dynamics of action potentials in the somatic and dendritic regions of a PC. In our experiments, we have confirmed that complex spiking in the majority of the mature PCs (above P20) that we recorded from precedes a brief period of silence, which is rarely the case for immature PCs (below P18). Moreover, complex spikes that occur with dendritic spikes often terminate somatic bursts in mature PCs, while immature PCs exhibit tonic firing including dendritic and complex spikes. Backpropagation of action potentials to the proximal dendrites is also visible. These observations suggest that the HDMEA setup provides a solution to analyze the origin of and relationship between dendritic and complex spikes in mature and immature PCs.

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