先端技術 2
Technique 2
O3-6-2-1
光制御型イオンチャネルの開発
Engineering genetically encoded highperformance photoswichable ion channel

○角田‐熱田京子1, 角田聡1
○Kyoko Tsunoda-Atsuta1, Sonja Minniberger1, Arend Vogt1, Peter Hegemann1, Satoshi Tsunoda1
フンボルト大学.生物物理部門1
Humboldt University, Institute of biology, Experimental biophysics1

Optogenetics has been recognized in cell biology. It is the combination of genetic and optical methods to control specific events in targeted cells or in living animals with the high temporal and special precision (millisecond-timescale). Acid-sensing ion channels (ASICs) are proton-activated sodium channels. These are found in neuron and implicated in astounding range of physiological functions such as pain and taste transduction, learning and memory. ASICs are activated by extracellar acidification and permeate Na+ mainly. The currents are quite larger compare with ChR. But they do respond to light application. Here we attempt to engineer a genetically encoded photoswichable ion channel by combining ASICs with H+ pump rhodopsin. Resently, a new rhodopsin was found from Chlorella vurgaris (CvRh). It's a light dependent proton pomp showing similarities to bacteriorhodopsin. This pump is activated by green light (the max wavelength of 560nm). We co-expressed 3 types of ASICs (ASIC1a, ASIC2a and ASIC3) together with CvRh in Xenopus laevis Oocyte and characterized this system by electropysiology. Upon illumination, extracellar pH was lowered due to transported proton by CvRh and all ASICs which have different kinetics were successfully activated. The amplitude of each channel activity were very large, depending on membrane voltage and light intensity. ASIC2a showed the slowest light response, and ASIC3 was the fastest. ASIC1a drops into an inactivation state after the first activation. The recovery of peak amplitude depends on waiting time. ASIC1a needs over 2 min, while ASIC3 needed 20 s for full recovery.Currently, we are transferring the system into neurons to control the neuronal behavior with light. Instead of ASICs, other proton sensitive ion channel could be employed to control the neuronal system with light. This dual component strategy is the new and could be a strong tool for optogenetics field.
O3-6-2-2
マウス嗅球における自発神経活動のイメージング
In vivo two-photon Ca2+ imaging of spontaneous neuronal activity in the mouse olfactory bulb

○岩田遼1, 今井猛1,2
○Ryo Iwata1, Takeshi Imai1,2
理研CDB・感覚神経回路形成研究チーム1, さきがけ・科学技術振興機構2
Laboratory for Sensory Circuit Formation, RIKEN CDB, Kobe1, PRESTO, JST2

Even in the absence of odors, some olfactory sensory neurons (OSNs) in isolated olfactory epithelium (OE) are excited by mechanical stimuli. It has been assumed that these OSNs may detect airflow passing through the nasal cavity. However, it remains unknown whether such responses are limited to a specialized population of OSNs or are general properties of OSNs. To examine this mechanical response in vivo, we generated transgenic mice that express the calcium indicator GCaMP3 in OSNs. OSNs project axons to the olfactory bulb (OB), where each of ~1000 glomeruli receives axon terminals of OSNs expressing a single type of odorant receptors. With two-photon microscopy, ~100 glomeruli on the dorsal side of OB were imaged in anesthetized mice. To artificially produce airflow through the nasal cavity, their trachea was suctioned by a computer-controlled air pump. Although the inhaled air did not contain odors, the artificial inhalation alone induced widespread and diverse responses in glomeruli. (1) 20-50% of glomeruli showed activation, as expected from previous electrophysiological studies in OE. (2) 10-30% of glomeruli showed inactivation, which had not been described in OE. (3) The remaining glomeruli showed no response. In addition, the degree of activation and inactivation was different between glomeruli and unique to each glomerulus. The temporal kinetics of the mechanical response was largely similar between activated glomeruli and between inactivated glomeruli. Our observations suggest that the airflow can produce widespread and differential effects on glomerular neuronal activity. We will discuss possible roles of the responses in activity-dependent olfactory circuit formation.
O3-6-2-3
線虫C. elegansの匂い応答行動のための新規神経アルゴリズム ―統合型顕微鏡システムによる解析
A neuronal algorithm for sensory behavior in C. elegans revealed by a highly integrated microscope system

○谷本悠生1, 山添萌子1, 藤田幸輔1, 川添有哉1, 宮西洋輔1, 費仙鳳2, 橋本浩一2, 木村幸太郎1
○Yuki Tanimoto1, Akiko Yamazoe1, Kosuke Fujita1, Yuya Kawazoe1, Yosuke Miyanishi1, Xianfeng Fei2, Koichi Hashimoto2, Kotaro Kimura1
大阪大院・理・生物科学1, 東北大院・情報科学・システム情報科学2
Dept. of Biol. Sci., Grad. Sch. of Sci., Osaka Univ., Osaka, Japan1, Grad. Sch. of Sys. Info. Sci., Tohoku Univ., Sendai, Japan2

An animal's nervous system processes sensory information into appropriate behavioral response. To elucidate the neural mechanisms involved in this information processing, the behavioral and neural responses to controlled sensory inputs should be quantified. For this, we have developed a novel integrated microscope system to simultaneously monitor neural activity and behavioral output in a model animal, the nematode C. elegans, under odor stimulation at varying concentrations. In this system, an animal is maintained at the center of the view field of a Ca2+ imaging microscope by using an auto-tracking motorized stage [1]. Using this system, we found that the opposing activities of 2 types of sensory neurons play significant roles in efficient odor avoidance behavior along the concentration gradient. Integrated behavioral and optophysiological analyses revealed that a temporal increment of concentration of the repulsive odor 2-nonanone activated a pair of ASH sensory neurons to change the animal's migratory direction randomly. In contrast, a temporal decrement of concentration of the odor activated another pair of AWB sensory neurons to continuously migrate in the present direction. Thus, by using the integrated microscope system, we have revealed a neuronal algorithm that process temporal information into directional behavioral response. To further elucidate the neural mechanisms, such as the integration of opposing sensory inputs, we are currently trying to analyze the downstream neural circuits by using this system. Such integrated analysis will allow us to comprehensively understand the function of a small neural circuit as a whole. [1] Maru et al., IEEE/SICE Int. Symp. Sys. Integr. Proc., 2011
O3-6-2-4
オプトジェネティクスへの応用を目指した光スイッチ型カリウムチャネルの作製
Engineering novel light-gated K+ channels for optogenetics application

○角田聡1, 角田京子1
○Satoshi Tsunoda1, Kyoko Tsunoda1, Iris Ceyisakar1, Peter Hegemann1
Institute fuer Biologie, Humboldt Universitaet zu Berlin1
Dept Experimental biophysics, Humboldt Uni. Berlin, Germany1

Light-regulated ion channels have enormous potential for neuronal research and for other biological studies such as muscle cells or calcium signaling where specific ions play important roles. Channelrhodopsins (ChRs) from green algae are light-gated cation channels which is responsible for phototaxis in the organisms and are recognized as a strong research tools because it can depolarize the membrane potentials simply by light resulting in trigger of action potentials in cultured cells, tissues and even moving animals after expressing them. Although owing great advantages, application of ChRs are limited by following reasons. (1) cation conductivity of ChRs are very small, 40-200 times smaller than common cation channels. Conductivity directly determines efficiency of light-induced depolarization. Thus high expression of ChRs are needed for sufficient membrane depolarization. (2) Very low ion selectivity. ChRs are non-selective cation channel toward H+, Na+, K+ and C2+.We present here an attempt to engineer light-gated K+ channels. There are several K+ channels which are regulated by pH. On the other hand light-gated pumping rhodopsin such as bacteriorhodopsin transports protons and thus alters local pH on membrane surface. These two ion transporters were coupled together and expressed in Xenopus laevis oocyte to test the functions electrophysiologicaly. Upon illumination of 550 nm light, we have observed proton pumping activity of the rhodopsin which is followed by activation of pH sensitive-K+ channel. This indicates that successful opening of K+ channels by light via the rhodopsin. After switching off light, K+ channel was immediately closed because pH shift by rhodopsin was diminished. This principle could be applied in neurons and be a promising technique to repolarize and silence neurons simply by light.
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