This work was supported by the World Class Institute (WCI) program of the National Research Foundation of Korea funded by Ministry of Education, Science, and Technology of Korea Grant WCI 2009-003 and Korea Institute of Science and Technology Institutional Program Project 2E24210. Sungmoo Lee was supported by Global Ph.D. Fellowship program (NRF-2013H1A2A1033344) of the National Research Foundation (NRF) under the Ministry of Education (MOE, Korea).

<ol>
	<li>Salzberg, B. M., Davila, H. V., Cohen, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+of+impulses+in+individual+neurones+of+an+invertebrate+central+nervous+system.">Optical recording of impulses in individual neurones of an invertebrate central nervous system.</a> Nature. 246, 508-509 (1973).</li><li>Cohen, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+axon+fluorescence+during+activity:+molecular+probes+of+membrane+potential.">Changes in axon fluorescence during activity: molecular probes of membrane potential.</a> J. Membrane Biol. 19, 1-36 (1974).</li><li>Tasaki, I., Warashina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye-membrane+interaction+and+its+changes+during+nerve+excitation.">Dye-membrane interaction and its changes during nerve excitation.</a> Photochem Photobiol. 24, 191-207 (1976).</li><li>Grinvald, A., Hildesheim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSDI:+a+new+era+in+functional+imaging+of+cortical+dynamics.">VSDI: a new era in functional imaging of cortical dynamics.</a> Nat Rev Neurosci. 5, 874-885 (2004).</li><li>Jin, L., Han, Z., Platisa, J., Wooltorton, J. R., Cohen, L. B., Pieribone, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+action+potentials+and+subthreshold+electrical+events+imaged+in+neurons+with+a+fluorescent+protein+voltage+probe.">Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.</a> Neuron. 75, 779-785 (2012).</li><li>Cao, G., Platisa, J., Pieribone, V. A., Raccuglia, D., Kunst, M., Nitabach, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+targeted+optical+electrophysiology+in+intact+neural+circuits.">Genetically targeted optical electrophysiology in intact neural circuits.</a> Cell. 154, 904-913 (2013).</li><li>St-Pierre, F., Marshall, J. D., Yang, Y., Gong, Y., Schnitzer, M. J., Lin, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-fidelity+optical+reporting+of+neuronal+electrical+activity+with+an+ultrafast+fluorescent+voltage+sensor.">High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor.</a> Nat Neurosci. 17, 884-889 (2014).</li><li>Piao, H. H., Rajakumar, D., Kang, B. E., Kim, E. H., Baker, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+mutagenesis+of+the+voltage-sensing+domain+enables+the+optical+resolution+of+action+potentials+firing+at+60+Hz+by+a+genetically+encoded+fluorescent+sensor+of+membrane+potential.">Combinatorial mutagenesis of the voltage-sensing domain enables the optical resolution of action potentials firing at 60 Hz by a genetically encoded fluorescent sensor of membrane potential.</a> J Neurosci. 35, 372-385 (2015).</li><li>Jung, A., Garcia, J. E., Kim, E., Yoon, B. J., Baker, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linker+length+and+fusion+site+composition+improve+the+optical+signal+of+genetically+encoded+fluorescent+voltage+sensors.">Linker length and fusion site composition improve the optical signal of genetically encoded fluorescent voltage sensors.</a> Neurophoton. 2, 021012 (2015).</li><li>Dimitrov, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+characterization+of+an+enhanced+fluorescent+protein+voltage+sensor.">Engineering and characterization of an enhanced fluorescent protein voltage sensor.</a> PLoS One. 2, e440 (2007).</li><li>Lam, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+FRET+dynamic+range+with+bright+green+and+red+fluorescent+proteins.">Improving FRET dynamic range with bright green and red fluorescent proteins.</a> Nat Methods. 9, 1005-1012 (2012).</li><li>Sung, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+fast+fluorescent+protein+voltage+sensors+by+optimizing+FRET+interactions.">Developing fast fluorescent protein voltage sensors by optimizing FRET interactions.</a> PLoS One. 10, e0141585 (2015).</li><li>Kralj, J. M., Douglass, A. D., Hochbaum, D. R., Maclaurin, D., Cohen, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+of+action+potentials+in+mammalian+neurons+using+a+microbial+rhodopsin.">Optical recording of action potentials in mammalian neurons using a microbial rhodopsin.</a> Nat Methods. 9, 90-95 (2012).</li><li>Flytzanis, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archaerhodopsin+variants+with+enhanced+voltage-sensitive+fluorescence+in+mammalian+and+Caenorhabditis+elegans+neurons.">Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons.</a> Nat Commun. 5, 4894 (2014).</li><li>Hochbaum, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-optical+electrophysiology+in+mammalian+neurons+using+engineered+microbial+rhodopsins.">All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.</a> Nat Methods. 11, 825-833 (2014).</li><li>Gong, Y., Wagner, M. J., Zhong Li, J., Schnitzer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+spiking+in+brain+tissue+using+FRET-opsin+protein+voltage+sensors.">Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors.</a> Nat Commun. 5, 3674 (2014).</li><li>Zou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+and+fast+multicoloured+voltage+reporters+via+electrochromic+FRET.">Bright and fast multicoloured voltage reporters via electrochromic FRET.</a> Nat Commun. 5, 4625 (2014).</li><li>Chanda, B., Blunck, R., Faria, L. C., Schweizer, F. E., Mody, I., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+approach+to+measuring+electrical+activity+in+genetically+specified+neurons.">A hybrid approach to measuring electrical activity in genetically specified neurons.</a> Nat Neurosci. 8, 1619-1626 (2005).</li><li>Wang, D., McMahon, S., Zhang, Z., Jackson, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+voltage+sensor+imaging+of+electrical+activity+from+neurons+in+hippocampal+slices+from+transgenic+mice.">Hybrid voltage sensor imaging of electrical activity from neurons in hippocampal slices from transgenic mice.</a> J Neurophysiol. 108, 3147-3160 (2012).</li><li>Weigel, S., Flisikowska, T., Schnieke, A., Luksch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+voltage+sensor+imaging+of+eGFP-F+expressing+neurons+in+chicken+midbrain+slices.">Hybrid voltage sensor imaging of eGFP-F expressing neurons in chicken midbrain slices.</a> J Neurosci Methods. 233, 28-33 (2014).</li><li>Waters, J. C., Sluder, G., Wolf, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-Cell+Fluorescence+Imaging.">Live-Cell Fluorescence Imaging.</a> Methods in Cell Biology Volume 81. , 115-140 (2007).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1, 2406-2415 (2006).</li><li>Beaudoin, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+pyramidal+neurons+from+the+early+postnatal+mouse+hippocampus+and+cortex.">Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex.</a> Nat Protoc. 7, 1741-1754 (2012).</li><li>Jiang, M., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Ca2+-phosphate+transfection+efficiency+in+low-density+neuronal+cultures.">High Ca2+-phosphate transfection efficiency in low-density neuronal cultures.</a> Nat Protoc. 1, 695-700 (2006).</li><li>Molleman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Patch clamping: an introductory guide to patch clamp electrophysiology. , 101-102 (2003).</li><li>Osorio, N., Delmas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+recording+from+enteric+neurons+in+situ.">Patch clamp recording from enteric neurons in situ.</a> Nat Protoc. 6, 15-27 (2010).</li><li>Schroder, M., Kaufman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+unfolded+protein+response.">The mammalian unfolded protein response.</a> Annu Rev Biochem. 74, 739-789 (2005).</li><li>Wilt, B. A., Fitzgerald, J. E., Schnitzer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photon+shot+noise+limits+on+optical+detection+of+neuronal+spikes+and+estimation+of+spike+timing.">Photon shot noise limits on optical detection of neuronal spikes and estimation of spike timing.</a> Biophys J. 104, 51-62 (2013).</li><li>Lundby, A., Mutoh, H., Dimitrov, D., Akemann, W., Knopfel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+genetically+encodable+fluorescent+voltage+sensor+exploiting+fast+Ci-VSP+voltage-sensing+movements.">Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements.</a> PLoS One. 3, e2514 (2008).</li><li>Peterka, D. S., Takahashi, H., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+voltage+in+neurons.">Imaging voltage in neurons.</a> Neuron. 69, 9-21 (2011).</li></ol>

著者らは、開示することは何もありません。

神経系は、いくつかの異なる方法で電圧を使用して、阻害は、シナプス入力がわずかな脱分極および比較的大きな電圧変化における活動電位の結果を引き起こし、若干の過分極を引き起こします。 GEVIsによって膜電位の変化を測定する能力は、同時に神経回路のいくつかの成分を分析の有望な可能性を提供しています。この報告書では、GEVIsを使用して、膜電位のイメージングの変更のため?...

この論文の主要な焦点は、遺伝的にコード化された蛍光タンパク質を用いたin vitroでの膜電位の変化の光学イメージングを実証することです。膜電位のイメージングの変化は、神経回路の活動を研究のエキサイティングな可能性を提供しています。蛍光強度の変化、膜電位の結果の変化は、カメラの各画素は、神経活動の非侵入測定を可能にする代理電極になったとき。 40年以上にわたり、有機電位感受性色素は、1-4膜電位の変化を観察するために有用でした。しかし、これらの染料は、細胞特異性を欠きます。また、いくつかの細胞型は染色が困難です。遺伝的にコードされた電圧インジケータ（GEVIs）蛍光電位感受性プローブ​​を発現特に検討される細胞を有することにより、これらの制限を克服します。 GEVIsの3つのクラスがあります。 GEVIの最初のクラスは、VOを使用しています単一の蛍光タンパク質（FP）5-9または蛍光共鳴エネルギー移動（FRET）ペア10-12のいずれかの電圧センシングホスファターゼからltage感知ドメイン。センサの第二のクラスは、蛍光指示薬直接13-15として、またはエレクトロクロミックFRET 16,17を介して微生物のロドプシンを使用しています。第三のクラスは、2つのコンポーネント、膜アンカーFPおよび膜結合消光染料18-20である第2の成分である遺伝的要素を利用しています。第二および第三のクラスは 、in vitroおよびスライス実験19,20 内のために有用であるが、センサーの唯一の最初のクラスは現在、in vivoでの 6を分析するのに有用です。 この報告では 、in vitroでGEVIsの最初のクラス（ 図1）を使用して、膜電位のイメージングを実証します。電圧センサのこの最初のクラスは、in vivoイメージングに移行するのが最も簡単です。 GEVIs U以来FPに融合された電圧センシングドメインは約センサのロドプシンクラスより明るい50倍されるtilizing、それらはむしろ非常に強力なレーザを必要とするよりも、アーク灯照明を用いて撮像することができます。明るさの格差の別の結果は、GEVIsの最初のクラスを簡単に脳の自家蛍光を超えることができるということです。ロドプシンベースのプローブはできません。センサの第三のクラスは、ファーストクラスと同じくらい明るいですが、in vivoで投与することが困難である化学消光剤の添加を必要とします。 我々は、したがって、単一のFP（Bongwoori）8とFRETペア（ナビ2）12からなるプローブとプローブの取得のデモンストレーションを行います。ナビ2.242とナビ2.244という名前の緑色蛍光ドナー、クローバーから成る11、および赤色蛍光アクセプター、mRuby2、 - VSFP-CR（クローバー-mRuby2電位感受性蛍光タンパク質）の蝶のバージョンでは、FRETがこのレポートで構築されています<s&gt; 12アップ。録音のこれらのタイプの導入は、研究者に提供できる情報GEVIsの種類のより良い理解を与える必要があります。 <img alt="図1" src="/files/ftp_upload/53566/53566fig1.jpg" /> 図1.この報告書 （A） で撮像された遺伝的にコードされた電圧インジケータ（GEVIs）膜貫通電圧センシングドメインおよび蛍光タンパク質を持つGEVIベースのモノFPの二種類 。 （B）の膜貫通電圧センシングドメインで構成されるFRETベースGEVI、FRETドナーとアクセプター。 <a href="https://www.jove.com/files/ftp_upload/53566/53566fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

<table border="0" cellpadding="0" cellspacing="0" width="923">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Inverted Microscope</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">IX71</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">60X objective lens (numerical aperture = 1.35)</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">UPLSAPO 60XO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Excitation filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">&#160;FF02-472/30</td>
			<td rowspan="3" style="width:187px;">For voltage imaging of super ecliptic pHluorin in Bongwoori</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF495-Di03-25x36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF01-497/LP</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">75W Xenon arc lamp</td>
			<td style="width:165px;">CAIRN</td>
			<td style="width:203px;">OptoSource Illuminator</td>
			<td style="width:187px;">LEDs and lasers are also effective light sources</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Slow speed CCD camera</td>
			<td style="width:165px;">Hitachi</td>
			<td style="width:203px;">KP-D20BU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dual port camera adaptor</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:203px;">U-DPCAD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">High speed CCD camera</td>
			<td style="width:165px;">RedShirtImaging, LLC</td>
			<td style="width:203px;">NeuroCCD-SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Image splitter</td>
			<td style="width:165px;">CAIRN</td>
			<td style="width:203px;">Optosplit 2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Excitation filter</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF01-475/23-25</td>
			<td rowspan="5" style="width:187px;">For voltage imaging of FRET pair based GEVI consisting of Clover and mRuby2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF495-Di03-25x36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Chroma</td>
			<td style="width:203px;">ET520/40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dichroic mirror</td>
			<td style="width:165px;">Semrock</td>
			<td style="width:203px;">FF560-FDi01-25X36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Emission filter</td>
			<td style="width:165px;">Chroma</td>
			<td style="width:203px;">ET645/75</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Vibration isolation system</td>
			<td style="width:165px;">Kinetic systems</td>
			<td style="width:203px;">250BM-IC, 5702E-3036-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Patching chamber</td>
			<td style="width:165px;">Warner instruments</td>
			<td style="width:203px;">RC-26G, 64-0235</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">#0 Micro Coverglass (22x40mm)</td>
			<td style="width:165px;">Electron Microscopy Sciences</td>
			<td style="width:203px;">72198-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Temperature controller</td>
			<td style="width:165px;">Warner instruments</td>
			<td style="width:203px;">TC-344B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">#0 (0.08~0.13mm) - 10mm diameter glass coverslip</td>
			<td style="width:165px;">Ted Pella</td>
			<td style="width:203px;">260366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Lipofection agent</td>
			<td style="width:165px;">Life Technologies</td>
			<td style="width:203px;">11668-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Calcium phosphate reagent</td>
			<td style="width:165px;">Clontech - Takara</td>
			<td style="width:203px;">631312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Patch clamp amplifier</td>
			<td style="width:165px;">HEKA</td>
			<td style="width:203px;">EPC 10 USB amplifier</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Multi-channel data acquisition software</td>
			<td style="width:165px;">HEKA</td>
			<td style="width:203px;">Patchmaster</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Image acquisition and analysis software</td>
			<td style="width:165px;">RedShirtImaging</td>
			<td style="width:203px;">Neuroplex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Spreadsheet application software</td>
			<td style="width:165px;">Microsoft</td>
			<td style="width:203px;">Microsoft Excel 2010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Data analysis software</td>
			<td style="width:165px;">OriginLab</td>
			<td style="width:203px;">OriginPro 8.6.0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Demagnifier</td>
			<td style="width:165px;">Qioptiq LINOS</td>
			<td style="width:203px;">Optem standard camera coupler 0.38x SC38 J clamp</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Confocal microscope</td>
			<td style="width:165px;">Nikon</td>
			<td style="width:203px;">Nikon A1R confocal microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Anti-fade reagent</td>
			<td style="width:165px;">Life Technologies</td>
			<td style="width:203px;">P36930</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging membrane potential with two types of genetically encoded fluorescent voltage sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

一過性にトランスフェクトされた細胞は、有意な蛍光強度の変化と原形質膜発現の程度を示すことができます。でも同じカバースリップ上でいくつかのセルは、内部蛍光のレベルを変化させる必要があります。これは、細胞によって吸収されるトランスフェクション剤の使用量が最も可能性が高いです。時折、あまりにも多くの式は（明るい、高い内部蛍光で、細胞?...

Watch this Scientific Journal Video about Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors at JoVE.com

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

遺伝的にコードされた蛍光電圧センサーの2種類のイメージング膜電位

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

imaging-membrane-potential-with-two-types-genetically-encoded

Research

JoVE Journal

Neuroscience

10.6K ビュー.  Seoul National University. A method for imaging changes in membrane potential using genetically encoded voltage indicators is described. 

ビデオ: Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

電位感受性色素を持つカニ口と胃の神経節における神経細胞の光学的イメージング

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

神経科学

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

ライブラット皮質ニューロンのミトコンドリア膜電位と活性酸素種の定量

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM染料を用いて培養小脳顆粒神経細胞のシナプス小胞プールの補充の定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

遺伝的にコードされたカルシウムセンサーを発現する鋤鼻器のスライス標本のイメージング神経細胞応答

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

同時に電圧クランプとアンペロとドーパミン放出の単一細胞計測

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

グルコース感知を勉強する成体マウス由来の膜視床下部腹内側部のニューロンの電位色素イメージング

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

イメージング細胞内Ca<sup&gt; 2+</sup遺伝的にエンコードされたカルシウム指標を用いて成体マウスからの線条体アスト​​ロサイト&gt;シグナル

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

マウス脊髄における細胞ダイナミクスの二光子イメージング

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

ニューロンに遺伝的にコードされた非天然アミノ酸を用いた神経タンパク質の光制御

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.