Name: isolation of sensory neurons of aplysia californica for patch clamp recordings of glutamatergic currents
Uploaded: 2013-07-10T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents at JoVE.com

NIH P40 OD010952、Korein財団、SLCとATKのメイタグフェローシップへマイアミフェローシップ大学によって資金を供給。作者は感謝アメフラシのための国家資源のスタッフを認めるだけでなく、図のための顕微鏡写真を提供したローレンSimonitisとハンナペック、。

1。細胞調製<ol><li> 1日目に、動物の重さと麻酔。 <ol><li> 30グラム、1キロの動物を計量。等張のMgCl 1:1海水の5-10動物ボリュームで麻酔。このような添付airstone付き電動アクアリウムエアーポンプとしてエアレーションと1時間6H 2 0。 </li></ol></li><li>解剖の供給を準備します。 <ol><li>このような生地のピンとしてステンレス鋼ストレートピン、クリーンな解剖トレイを組み立てます。 +ペ?...

<ol>
	<li>Buttner, N., Siegelbaum, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+modulation+of+a+hyperpolarization-activated+Cl(-)+current+in+Aplysia+sensory+neurons+by+SCP(B)+and+FMRFamide.">Antagonistic modulation of a hyperpolarization-activated Cl(-) current in Aplysia sensory neurons by SCP(B) and FMRFamide.</a> J. Neurophysiol. 90 (2), 586-598 (2003).</li><li>Byrne, J. H., Castellucci, V. F., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+individual+mechanoreceptor+sensory+neurons+to+defensive+gill-withdrawal+reflex+in+Aplysia.">Contribution of individual mechanoreceptor sensory neurons to defensive gill-withdrawal reflex in Aplysia.</a> J. Neurophysiol. 41 (2), 418-431 (1978).</li><li>Carlson, S. L., Fieber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+evidence+that+D-Aspartate+activates+a+current+distinct+from+ionotropic+glutamate+receptor+currents+in+Aplysia+californica.">Physiological evidence that D-Aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica.</a> J. Neurophysiol. 106 (4), 1629-1636 (2011).</li><li>Carlson, S. L., Kempsell, A. T., Fieber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+evidence+that+D-Aspartate+activates+a+current+distinct+from+ionotropic+glutamate+receptor+currents+in+Aplysia+californica.">Pharmacological evidence that D-Aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica.</a> Br. Behav. 2 (4), 391-401 (2012).</li><li>Fieber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Na++and+Ca2++currents+in+bag+cells+of+sexually+immature+Aplysia+californica.">Characterization of Na+ and Ca2+ currents in bag cells of sexually immature Aplysia californica.</a> J. Exp. Biol. 201 (5), 745-754 (1998).</li><li>Fieber, L. .. A., Carlson, S. L., Capo, T. R., Schmale, M. C. <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+D-Aspartate+ion+currents+in+the+Aplysia+nervous+system+with+aging.">Changes in D-Aspartate ion currents in the Aplysia nervous system with aging.</a> Br. Res. 1343, 28-36 (2010).</li><li>Glansman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+in+Aplysia:+The+Cheshire+Cat+of+neurobiology.">Habituation in Aplysia: The Cheshire Cat of neurobiology.</a> Neurobiol. Learn. Mem. 92, 147-154 (2009).</li><li>Hawkins, R. D., Abrams, T. W., Carew, T. J., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cellular+Mechanism+of+Classical+Conditioning+in+Aplysia:+Activity-Dependent+Amplification+of+Presynaptic+Facilitation.">A Cellular Mechanism of Classical Conditioning in Aplysia: Activity-Dependent Amplification of Presynaptic Facilitation.</a> Science, New. 219 (4583), 400-405 (1983).</li><li>Ichinose, M., Sawada, M., Maeno, T., McAdoo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+acetylcholine+on+ventrocaudal+sensory+neurons+in+the+pleural+ganglia+of+Aplysia.">Effect of acetylcholine on ventrocaudal sensory neurons in the pleural ganglia of Aplysia.</a> Cell Mol. Neurobiol. 9, 233-245 (1989).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cellular basis of behavior. , (1976).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+memory+storage:+a+dialog+between+nerves+and+synapses.">The molecular biology of memory storage: a dialog between nerves and synapses.</a> Science. 294 (5544), 1030-1038 (2001).</li><li>Kupfermann, I., Castellucci, V., Pinsker, H., Kandel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+correlates+of+habituation+and+habituation+of+the+gill-withdrawal+reflex+in+Aplysia.">Neuronal correlates of habituation and habituation of the gill-withdrawal reflex in Aplysia.</a> Science. 167 (3926), 1743-1745 (1970).</li><li>Magoski, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+an+Aplysia+bag-cell+neuron+cation+channel+by+closely+associated+protein+kinase+A+and+a+protein+phosphatase.">Regulation of an Aplysia bag-cell neuron cation channel by closely associated protein kinase A and a protein phosphatase.</a> J. Neurosci. 24 (30), 6833-6841 (2004).</li><li>R, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+transcriptome+of+Aplysia:+neuronal+compartments+and+circuitry.">Neuronal transcriptome of Aplysia: neuronal compartments and circuitry.</a> Cell. 127, 1453-1467 (2006).</li><li>Nick, T. A., Kaczmarek, L. K., Carew, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+currents+underlying+developmental+regulation+of+repetitive+firing+in+Aplysia+bag+cell+neurons.">Ionic currents underlying developmental regulation of repetitive firing in Aplysia bag cell neurons.</a> J. Neurosci. 16 (23), 7583-7598 (1996).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-channel recording. , (1995).</li><li>Tam, A. K., Geiger, J. E., Hung, A. Y., Groten, C. J., Magoski, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+Ca2++current+contributes+to+a+prolonged+depolarization+in+Aplysia+bag+cell+neurons.">Persistent Ca2+ current contributes to a prolonged depolarization in Aplysia bag cell neurons.</a> J. Neurophysiol. 102 (6), 3753-3765 (2009).</li><li>Schacher, S., Proshansky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+regeneration+by+Aplysia+neurons+in+dissociated+cell+culture:+modulation+by+Aplysia+hemolymph+and+the+presence+of+the+initial+axonal+segment.">Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment.</a> J. Neurosci. 3 (12), 2403-2413 (1983).</li><li>Walters, E. T., Byrne, J. H., Carew, T. J., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanoafferent+neurons+innervating+tail+of+Aplysia.+I.+Response+properties+and+synaptic+connections.">Mechanoafferent neurons innervating tail of Aplysia. I. Response properties and synaptic connections.</a> J. Neurophysiol. 50 (6), 1522-1542 (1983).</li><li>Wilson, G. F., Richardson, F. C., Fisher, T. E., Olivera, B. M., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+Ca(2+)-sensitive+nonspecific+cation+channel+underlying+prolonged+repetitive+firing+in+Aplysia+neurons.">Identification and characterization of a Ca(2+)-sensitive nonspecific cation channel underlying prolonged repetitive firing in Aplysia neurons.</a> J. Neurosci. 16 (11), 3661-3671 (1996).</li><li>White, B. H., Nick, T. A., Carew, T. J., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+regulates+a+vesicular+class+of+calcium+channels+in+the+bag+cell+neurons+of+Aplysia.">Protein kinase C regulates a vesicular class of calcium channels in the bag cell neurons of Aplysia.</a> J. Neurophysiol. 80 (5), 2514-2520 (1998).</li><li>Wilson, G. F., Magoski, N. S., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+a+calcium-sensitive+nonspecific+cation+channel+by+closely+associated+protein+kinase+and+phosphatase+activities.">Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities.</a> Proc. Natl. Acad. Sci. U.S.A. 95 (18), 10938-10943 (1998).</li><li>Zhao, Y., Wang, D. O., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aplysia+sensory-motor+neuronal+cell+cultures.">Preparation of Aplysia sensory-motor neuronal cell cultures.</a> J. Vis. Exp. (28), e1355 (2009).</li></ol>

解離技術はグリアや他の未確認の細胞の小さな数字が点在50〜100孤立したニューロンを含む感覚ニューロンの文化を生み出す、ここで説明。プロトコルの中で最も重要なステップは、神経、酵素溶液中に残存する時間であり、そして、個々の細胞にクラスターを分解する消化細胞塊の解離をフリック。酵素消化（ステップ1.8）が使用可能温度で最適化する必要があります。 23°遅い振とうしな...

海洋opistobranch軟体動物、 アメフラシは 、何十年もの間、有用な神経生物学的モデルとなっています。これは最高の馴化と古典的条件7,8のモデルとして知られています。このモデルでは学習と記憶に関する研究は、彼がArvidカールソンとポールグリーンガード10と共有賞で、エリックR.カンデル2000年に生理学·医学ノーベル賞を受賞した。この無脊椎動物の神経系の要素が付加左神経と筋肉との動物から解剖されている削減の準備からの電気録音を、関与する研究は、 アメフラシの個々の神経細胞の役割を解明に役立っています。しかしアメフラシでの学習を構成する精密な分子メカニズムの識別は、多くの場合、個々のドナー動物から1つずつ取得し別の技術、感覚ニューロンと運動ニューロンの長期共培養を採用し、培養皿21でシナプスを形成させ。 我々は、他の1,3,6、14、15、16は、名目上均質なニューロンのクラスタの解離短期間培養物を作るためにニューロンは、このモデルで対象とすることができる同定ならびに長期の実験では、その耐久性の容易さを利用しているている私たちは、パッチクランプ構成で電圧クランプの下にイオン電流を勉強。多くのアメフラシニューロンは長続き実験操作のための時間を可能にするためにパッチクランプを繰り返しラウンドに立ち上がる。技術は、腹部の神経節の神経分泌袋細胞、解離我々はここで説明する胸膜と頬神経節の感覚ニューロンとしてニューロンのためではなく、非常に大規模なニューロン&gt;60μmの径などのL7またはR2などに便利です腹部神経節。我々は他の場所に記載感覚ニューロン共培養とは異なり、私たちの文化でアメフラシ血清を使用しない。この手順を使用して得られたほとんどのニューロンはtに対してプロセスがなくてもなります彼は全体のセル電圧記録を促進する文化の中で最初の48時間、しかし、その後、新芽や栄養素、および/または成長因子の不足から、死ぬ前におよそ14日間軸索と他のプロセスを詳しく説明します。 この手法は、頬や胸膜神経の生理的に文書化された地域から皿当たり50〜100のニューロンの初代培養を生成します。このプロトコルは、多数の実験は、動物ごとに複製が必要な実験で単一細胞生理学の側面を研究する研究者にとって有用です。それは一致した治療群と対照培養物から研究その利益を可能にして、左右hemigangliaに標的細胞の解剖学的分離に起因する文化のペアを生成します。 プロトコルは、頬神経の頬Sクラスター（BSC）ニューロン、および胸膜神経節の胸膜ventrocaudal（PVC）のニューロンを対象としています。これらの細胞は、全体セル電圧の記録および表示ローバスに適したサイズであるTグルタミン酸応答。議論の方法論は、 アメフラシ神経系の中で最も神経に適しています。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Artificial seawater ASW</td>
 <td>Sigma-Aldrich</td>
 <td>assorted</td>
 <td>(mM): 417 NaCl, 10 KCl, 10 CaCl2 (2 H2O), 5MgCl2 (6H2O), 15 HEPES-NaOH, pH 7.6</td>
 </tr>
 <tr>
 <td>Intracellular solution</td>
 <td>Sigma</td>
 <td>assorted</td>
 <td>(mM): 450 KCl, 2.9 CaCl2 (2 H2O), 2.5 MgCl2 (6 H2O), 5 Na2ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4</td>
 </tr>
 <tr>
 <td>Poly-D-lysine</td>
 <td>Sigma</td>
 <td>P6407</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>penicillin/streptomycin added to ASW at 1:100</td>
 <td>Lonzo Walkersville, Inc.</td>
 <td>17-603E</td>
 <td>5,000 Units/ml penicillin plus 5,000 mg/ml streptomycin</td>
 </tr>
 <tr>
 <td>Neutral dispase II</td>
 <td>Roche Diagnostics</td>
 <td>10165859001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>hyaluronidase</td>
 <td>Sigma-Aldrich</td>
 <td>H4272</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>collagenase type XI</td>
 <td>Sigma-Aldrich</td>
 <td>C9407</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glutamate (L-Glu)</td>
 <td>Sigma-Aldrich</td>
 <td>49601-100G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-Aspartate (D-Asp)</td>
 <td>Sigma-Aldrich</td>
 <td>11200-10G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>N-methyl-D-aspartate (NMDA)</td>
 <td>Biomol</td>
 <td>100002-268</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Asp</td>
 <td>Sigma</td>
 <td>A6683-25G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA)</td>
 <td>Sigma</td>
 <td>A6816-5MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glu R antagonists</td>
 <td>various</td>
 <td>various</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>agar</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>kynurenate</td>
 <td>Sigma-Aldrich</td>
 <td>61250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>APV</td>
 <td>Sigma-Aldrich</td>
 <td>A5282</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DL-2-Amino-5-phosphonopentanoic acid (NMDAR antagonist)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2-propanol</td>
 <td>VWRSP</td>
 <td>BDH1133</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chloriding solution</td>
 <td>Sigma</td>
 <td>assorted</td>
 <td>25 g FeCl3 + 25 ml concentrated HCl + 50 ml H2O</td>
 </tr>
 <tr>
 <td>Sylgard silicone 2-part polymer</td>
 <td>World Precision Instruments (WPI)</td>
 <td>SYL184</td>
 <td>Provides pin-out surface for small dissection dishes</td>
 </tr>
 <tr>
 <td>0-40x zoom magnification microscope for dissections</td>
 <td>Wild</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Techniquip 150 Watts Fiber Optic Illuminator</td>
 <td>Microoptics of Florida</td>
 <td>TQ FOI-150</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RotoMix 50800 orbital mixer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nikon Diaphot inverted phase-contrast microscope with 4x, 20x (optional) &amp; 40x objectives</td>
 <td>SR Research Ltd.</td>
 <td>Eyelink II</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tektronix digital oscilloscope</td>
 <td>SR Research Ltd.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pClamp 10 data acquisition and analysis software</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PC with Windows XP or higher operating system</td>
 <td>PC Solutions</td>
 <td>&nbsp;</td>
 <td>Thinkserver with solid state hard drives (80GB) and low noise monitors</td>
 </tr>
 <tr>
 <td>Flaming/Brown P87 micropipette puller</td>
 <td>Sutter Instruments, Novato, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon Instruments Axopatch 200B clamp amplifier with a capacitance compensation range of 1-1000 pF; preamplifier</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon instruments electrode holder assembly for Axopatch 200B preamplifier</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>CV203BU</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digidata 1200 A/D converter</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Picospritzer, powered by N2 adjustable for pressure and duration</td>
 <td>Parker Hannifin, Cleveland, OH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TMC Micro-G Vibration isolation table</td>
 <td>Ametek</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Faraday cage</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>custom manufacture</td>
 </tr>
 <tr>
 <td>Burleigh Piezoelectric Clamshell Micromanipulators</td>
 <td>Burleigh Instruments; Thorlabs</td>
 <td>&nbsp;</td>
 <td>presently PCS-5000; -6000 series + mounts</td>
 </tr>
 <tr>
 <td>Narishige M-152 manual manipulators (for perfusion system and picospritzer)</td>
 <td>Narishige USA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Filament pipette glass,1.5 mm OD, 0.84 mm ID -</td>
 <td>WPI</td>
 <td>1B150-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3 inch length</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ag/AgCl half cell</td>
 <td>WPI</td>
 <td>EP4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes, 35-2097 BD Falcon* Centrifuge Tubes</td>
 <td>VWRSP</td>
 <td>21008-918</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Angled Scissors</td>
 <td>Fine Science Tools</td>
 <td>15006-09</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dumostar Fine forceps</td>
 <td>Fine Science Tools</td>
 <td>11295-00</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>35 mm falcon tissue culture dishes</td>
 <td>VWRSP</td>
 <td>25382-064</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>falcon 150 x 25 mm tissue culture dishes; 1013</td>
 <td>VWRSP</td>
 <td>1013</td>
 <td>also can be made into small dissection dishes with sylgard</td>
 </tr>
 <tr>
 <td>sylgard</td>
 <td>WPI</td>
 <td>SYL184</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>animal dissection tray</td>
 <td>various</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes, 35-2097 BD Falcon</td>
 <td>VWRSP</td>
 <td>21008-918</td>
 <td>For 6-bore gravity-fed perfusion system</td>
 </tr>
 <tr>
 <td>Aluminum clips with screw hole ends</td>
 <td>hardware store</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>23 gauge needles (manually file off points)</td>
 <td>VWRSP</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>Polyethylene tubing 0.022"ID x 0.042"OD; 427411</td>
 <td>Becton-Dickinson</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>H-7 pipette stand/holder for microcap perfusion array</td>
 <td>Narishige USA</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>one-way valves</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>Drummond Microcaps 1 &mu;l</td>
 <td>VWRSP</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>18 gauge needles for suction (filed off points)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polyethylene tubing</td>
 <td>Cole Parmer</td>
 <td>4.27436E+11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>fine dissection pins</td>
 <td>Fine Science Tools</td>
 <td>26002-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>capillary tubes</td>
 <td>Kimble 71900-100</td>
 <td>&nbsp;</td>
 <td>fire-polished and U-shaped in a Bunsen burner flame and filled with 3% agar in ECS</td>
 </tr>
 <tr>
 <td>modeling clay</td>
 <td>craft store</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>dish holder for microscope stage with isolated ground bath</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Custom manufacture</td>
 </tr>
 <tr>
 <td>pasteur pipettes</td>
 <td>VWRSP</td>
 <td>14672-412</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pipette bulbs</td>
 <td>VWRSP</td>
 <td>53283-911</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>acrodisk syringe filters</td>
 <td>VWRSP</td>
 <td>28144-040</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>thick-walled 1.5 mm diameter borosilicate filament glass</td>
 <td>WPI</td>
 <td>1B150F-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>High purity nitrogen cylinder and bifurcating regulator</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Tables 1-3. Lists of Reagents, Materials, and Equipment.

isolation of sensory neurons of aplysia californica for patch clamp recordings of glutamatergic currents

海洋腹足類軟体動物アメフラシカリフォルニは、学習と記憶の研究において特に重要で、神経系機能のモデルとして由緒ある歴史を持っています。このような研究のための一般的な準備は、感覚と運動ニューロンを最小限解剖動物、または個々の感覚と運動ニューロンの技術的に精巧な神経細胞の共培養にそのまま残されているものです。あまり一般的では公称均質ニューロンの小さなクラスターは短期培養における単一細胞に解離されている孤立したニューロンの準備です。そのような単離された細胞は、パッチクランプ技術、およびこれらのコンダクタンスの目標変調を用いたイオン電流の生物物理学的特徴付けに有用である。このような培養物を調製するためのプロトコルが記載されている。プロトコルは胸膜と頬神経の容易に識別グルタミン酸感覚ニューロンを活用して、それらの解離と最小限のメンテナンスを説明I血清を含まない数日間のn文化。

このプロトコルにおいて対象となる神経節内の感覚ニューロンの位置は、BSC及びPVCニューロンは、 図1に示されている。 BSCのニューロンは頬神経節の腹側に2対称の楕円形のクラスター、そのまま神経節（ 図1A）で頬塊から離れて対向する面に配置されています。 PVCニューロンは中心軸（ 図1B）に向かって胸膜神経節の背面の周りをラップする二国間の?...

Watch this Scientific Journal Video about Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents at JoVE.com 

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents (Video) | JoVE 

我々は、海洋アメフラシの神経系の解剖を説明<em&gt;アメフラシ</em&gt;麻酔の後、短期·組織培養のための神経細胞、およびパッチクランプ法を介した単一細胞イオン電流の録音の分離。

の感覚ニューロンの分離<em&gt;アメフラシカリフォルニ</em&gt;グルタミン酸作動電流のパッチクランプ記録のために

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Research

JoVE Journal

Neuroscience

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals (Video) | JoVE 

の電極の作製と注入アメフラシカリフォルニ無傷、自由に振る舞う動物における多チャンネル神経と筋肉の録音のための

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation (Video) | JoVE 

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons (Video) | JoVE 

Sf9細胞でとアメフラシ感覚ニューロンにおけるPKC転座のライブイメージング

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish (Video) | JoVE 

内耳有毛細胞におけるパッチクランプ記録は、単離されたゼブラフィッシュから

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes (Video) | JoVE 

<sub&gt; 1</sub&gt; F<sub&gt; O</sub亜ミトコンドリアの小胞膜のパッチクランプ記録を実行するための&gt; ATPアーゼ小胞の準備とテクニック

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica (Video) | JoVE 

体外における筋モータープールのために運動ニューロンの識別<em&gt;アメフラシカリフォルニ</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells (Video) | JoVE 

胚ゼブラフィッシュマウスナー細胞からパッチクランプ記録

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor (Video) | JoVE 

無傷の神経上皮におけるマウス嗅細胞の穿孔パッチクランプ記録：同定された嗅覚受容体を発現するニューロンの機能解析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell Patch-clamp Recordings in Brain Slices (Video) | JoVE 

脳スライスにおける全細胞パッチクランプ記録

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons (Video) | JoVE 

黒質ドーパミンニューロンの細胞体樹状突起のドメインからの亜細胞パッチクランプ記録

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...