This work is supported by a National Science Foundation award (1145581) to LWG.

注：イリノイ大学シカゴ校の動物実験委員会によって承認された全体の手順は、国立衛生研究所のガイドラインに従って実施した。 1。ソリューションおよび培養培地の準備<ol><li> 6ヶ月までのために-20℃で、すべてのソリューションにしてください。 3ヶ月まで4℃で培養培地にしてく​​ださい。 </li><li> DMEM中で100mlにITSX（インスリン - トランスフェリン - セレンサプリメント）1ペン連鎖球菌溶液および1ミリリットルを混合することにより、培養培地100mlのを準備します。 </li><li> 100のCaCl 2、および50mM EDTAを2.5ミリリットルの2.5ミリリットル、250ミリリットルのDMEMを混合して酵素溶液を調製します。 </li><li> DMEMを225ミリリットル、FBSを25ミリリットル、アルブミン625 mg及び625 mgのトリプシン阻害剤を混合して不活性化溶液を調製する。 </li><li> 154のNaCl、5.6のKCl、5.0mMのHEPES、3.6 mMの炭酸水素ナトリウム、5.6 mMグルコースのロックのソリューションを準備します。 NaOHで7.3にpHを調整する。 </li><li>50 mg / mlののポリ-D-リジン（PDL）溶液を調製する。被覆カバースリップに1mg / mlの最終濃度を使用する。 </li><li> 140mMのNaCl、5mMのKCl、2mMのCaCl 2を 、1mMのMgCl 2、10mMのHEPES-NaOHを、および10 mMグルコースの細胞外溶液を調製する。 〜310ナノモル/ kgで浸透圧をNaOHで7.3にpHを調整する。 </li><li> 50のNaCl、100mMのTEACL、5mMのKCl、2mMの塩化カルシウム、1mMのMgCl 2、および10mMのHEPESのピペット溶液を調製します。 〜290ナノモル/ kgで浸透圧をNaOHで7.3にpHを調整する。 </li></ol> 2副腎の分離<ol><li>大小の解剖ハサミとピンセットの2組のペアをオートクレーブ。全体のプロセスの間に手袋を着用し、氷のバケツに解剖トレイを置きます。 </li><li>日0-3で取られたマウスを使用してください。断頭続いて加圧CO 2タンクで動物を安楽死させる。 </li><li>チェルビから背骨以下の子犬の背中を削減するハサミの小さいセットを活用<html>尾側領域にcalは。皮膚の下に表面的に切断され、体腔内に刺していないようにしてください。 </li><li>離れて背骨から次に、剥離皮膚筋系は脊柱の明確な見解を持って露出するように。ここから、頸髄でtransectionalカットを行い、その後、背骨の両側に沿って2つの平行カットを行う。 </li><li>動物の本体を保持する鉗子の一組では、脊柱をつかみ、腎臓が露出するまで、背骨をバック剥離する鉗子の他のペアを使用しています。 </li><li>このとき、腎臓の上に副腎を可視化。その後、慎重にロックの溶液を充填したコニカルチューブに各動物から2腺を配置します。 注：たまに腺が鉗子に固執します。この場合は、静かに鉗子からロックの溶液に腺の除去を助けるために鉗子の別のペアを使用する。腺は、最大2時間この状態に保つことができる。 </li></ol>E &quot;&gt; 3。組織消化<ol><li>一方、気泡使用前に15分間、5％CO 2 + 95％のO 2との酵素溶液。平衡化したソリューションは、ピンクがかった/オレンジ色に鮮やかなピンク色の電源が入るか確認します。 </li><li> 20〜25単位/ mlの最終濃度で新鮮に泡立て酵素液にパパインを追加します。パパイン酵素溶液中、37℃で40〜60分間、各動物から腺の各ペアをインキュベートする。 </li><li>各チューブに不活性化溶液の75％を加え、さらに10分間37℃でインキュベートする。このインキュベーションは、パパインの酵素活性を不活性化する。 </li><li>不活性化溶液で10分間インキュベーションした後、穏やかに新たに標識されたチューブに腺を転送して腺を培養培地で3回洗浄する。 </li></ol> 4。細胞解離とめっき<ol><li>洗浄後、培養培地を200μlに2腺を配置し、 静かに腺を滴定し、ダウン200μlのピペットを用いてピペットチップを通して組織の大部分が溶液中にもはや存在するまで。 NOTE：滴定すると、200μlのピペットの先端から下向きの力でチューブの底腺を維持し、穏やかにゆっくりと組織に係合する。高速または突然のピペッティング決して遅く、連続ストロークを使用するように注意してください。 </li><li> 450μlの最大の培地の全量を、追加の250μlの培養培地を追加します。 </li><li>プレートを60×15mmの培養皿に入れ、7つ12ミリメートルPDLプレコーティングしたカバースリップを、それぞれ上にこの細胞を含む溶液の60μlの。 </li><li>一度メッキ、細胞生存率のための環境を確保するために30分間、5％CO 2の定常流/供給しながら37℃に設定され、維持さインキュベーターに皿を置く。インキュベーションの後、静かに培養皿に予め温め培地の5ミリリットルを追加し、24時間インキュベーターに戻し、皿を返す細胞アタッシュケースの前にD録音。 注：典型的には、細胞は、最大4日間、電気生理学的記録のために使用することができる。 </li></ol> 5。細胞の同定およびギガオームシール形成<ol><li>細胞外液中に12mmのカバーガラスを配置します。全体副腎が細胞調製のために使用されるので、ベージュ/茶色がかったカラーリングと皮質細胞（細胞をクロム親和よりも小さく、調光器）と、培養中の近くの円形のフォーム（ 図1）と非常に明るいクロム親和細胞（通常はを混ぜる。 </li><li>プログラム可能なP-97プラーを使用して、4つの段階でパッチピペットを引き出します。 「ブローアウェイ」をするためにこのワックスをピペットチップの内部から先端をガラスの容量を低減するのに役立ち、その後火ポリッシュれる、溶けたロウにピペットチップを浸し。 NOTE：パッチピペット溶液で満たされたときに、パッチピペットは、典型的には約2MΩの抵抗を有する。 </li><li>数m内の細胞に近いパッチピペットアプローチicrons。セルに接続された構成を実現するには、セルに近いパッチピペットのアプローチの間にわずかな細胞変形を探します。 GΩシールが達成されるまで、穏やかな吸引を適用します。 </li></ol> 6セル接続型静電容量録音<ol><li> GΩシールが明らかにされると、EPC-7プラスパッチクランプアンプと二相のアナログロックインアンプ（機器の添付の表を参照）を使用することにより、セルに接続された録音を行う。 50 mVの（実効値）の振幅とロックインアンプから20kHzの周波数で正弦波を適用します。 </li><li> 1ミリ秒の時定数と24デシベルで、ロックインアンプの出力フィルタを設定してください。ロックインアンプなどの穏やかな吸引パルスによって生成される過渡的な静電容量の変化のみパッチコンダクタンス（再）トレースになし投影とパッチ容量（イム）トレースに表示されている（ 図2）の位相を設定します。 注：再、システムのキャリブレーションの詳細をご覧下さいference 19。 </li><li> 「下」の手順（ 図3）によって、一般的な静電容量の変化を識別します。 注：固体記録は、単一の小胞を内在化を示している多くの下向きの手順で「階段」タイプの似ている、と見ることになります。動作電流は典型的には、ほとんどの細胞から記録することができるので、パッチ適用プロセスは、機械的刺激として働く。 </li><li>そのファイル内の/月/年、自身の個別の番号（XX）として、各セルに接続された記録：個別の日を含むフォルダなど、ファイルに名前を付けます。 </li></ol>

<ol>
	<li>Rettig, J., 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=Emerging+roles+of+presynaptic+proteins+in+Ca++-triggered+exocytosis.">Emerging roles of presynaptic proteins in Ca++-triggered exocytosis.</a> Science. 298, 781-785 (2002).</li><li>Gong, L. W., 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=Phosphatidylinositol+phosphate+kinase+type+I+gamma+regulates+dynamics+of+large+dense-core+vesicle+fusion.">Phosphatidylinositol phosphate kinase type I gamma regulates dynamics of large dense-core vesicle fusion.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 5204-5209 (2005).</li><li>Lindau, M., Alvarez de Toledo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fusion+pore.">The fusion pore.</a> Biochimica et Biophysica Acta. 1641, 167-173 (2003).</li><li>Albillos, A., 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=The+exocytotic+event+in+chromaffin+cells+revealed+by+patch+amperometry.">The exocytotic event in chromaffin cells revealed by patch amperometry.</a> Nature. 389, 509-512 (1997).</li><li>Lollike, K., Borregaard, N., Lindau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacitance+flickers+and+pseudoflickers+of+small+granules,+measured+in+the+cell-attached+configuration.">Capacitance flickers and pseudoflickers of small granules, measured in the cell-attached configuration.</a> Biophys J. 75, 53-59 (1998).</li><li>Lollike, K., Borregaard, N., Lindau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+exocytotic+fusion+pore+of+small+granules+has+a+conductance+similar+to+an+ion+channel.">The exocytotic fusion pore of small granules has a conductance similar to an ion channel.</a> The Journal of cell biology. 129, 99-104 (1995).</li><li>Dernick, G., Gong, L. W., Tabares, L., Alvarez de Toledo, G., Lindau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+amperometry:+high-resolution+measurements+of+single-vesicle+fusion+and+release.">Patch amperometry: high-resolution measurements of single-vesicle fusion and release.</a> Nat Methods. 2, 699-708 (2005).</li><li>Neher, E., Marty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrete+changes+of+cell+membrane+capacitance+observed+under+conditions+of+enhanced+secretion+in+bovine+adrenal+chromaffin+cells.">Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 79, 6712-6716 (1982).</li><li>Gong, L. W., de Toledo, G. A., Lindau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exocytotic+catecholamine+release+is+not+associated+with+cation+flux+through+channels+in+the+vesicle+membrane+but+Na++influx+through+the+fusion+pore.">Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore.</a> Nature Cell Biology. 9, 915-922 (2007).</li><li>Gong, L. W., Hafez, I., Alvarez de Toledo, G., Lindau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+vesicles+membrane+area+is+regulated+in+tandem+with+quantal+size+in+chromaffin+cells.">Secretory vesicles membrane area is regulated in tandem with quantal size in chromaffin cells.</a> The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 23, 7917-7921 (2003).</li><li>Yao, L. H., 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=Actin+polymerization+does+not+provide+direct+mechanical+forces+for+vesicle+fission+during+clathrin-mediated+endocytosis.">Actin polymerization does not provide direct mechanical forces for vesicle fission during clathrin-mediated endocytosis.</a> The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 33, 15793-15798 (2013).</li><li>Yao, L. H., 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=Synaptotagmin+1+is+necessary+for+the+Ca2++dependence+of+clathrin-mediated+endocytosis.">Synaptotagmin 1 is necessary for the Ca2+ dependence of clathrin-mediated endocytosis.</a> The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 32, 3778-3785 (2012).</li><li>McMahon, H. T., Boucrot, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+and+physiological+functions+of+clathrin-mediated+endocytosis.">Molecular mechanism and physiological functions of clathrin-mediated endocytosis.</a> Nature reviews. Molecular Cell Biology. 12, 517-533 (2011).</li><li>Murthy, V. N., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+the+presynaptic+terminal.">Cell biology of the presynaptic terminal.</a> Annu Rev Neurosci. 26, 701-728 (2003).</li><li>Granseth, B., Odermatt, B., Royle, S. J., Lagnado, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-mediated+endocytosis+is+the+dominant+mechanism+of+vesicle+retrieval+at+hippocampal+synapses.">Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.</a> Neuron. 51, 773-786 (2006).</li><li>Rosenboom, H., Lindau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exo-endocytosis+and+closing+of+the+fission+pore+during+endocytosis+in+single+pituitary+nerve+terminals+internally+perfused+with+high+calcium+concentrations.">Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 5267-5271 (1994).</li><li>MacDonald, P. E., Eliasson, L., Rorsman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+increases+endocytotic+vesicle+size+and+accelerates+membrane+fission+in+insulin-secreting+INS-1+cells.">Calcium increases endocytotic vesicle size and accelerates membrane fission in insulin-secreting INS-1 cells.</a> Journal of Cell Science. 118, 5911-5920 (2005).</li><li>Zhao, Y., Fang, Q., Straub, S. G., Lindau, M., Sharp, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+inhibition+of+endocytosis:+novel+roles+for+noradrenaline+and+G+protein+G(z).">Hormonal inhibition of endocytosis: novel roles for noradrenaline and G protein G(z).</a> J Physiol. 588, 3499-3509 (2010).</li><li>Debus, K., Lindau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+patch+capacitance+recordings+and+of+fusion+pore+conductances+in+small+vesicles.">Resolution of patch capacitance recordings and of fusion pore conductances in small vesicles.</a> Biophys J. 78, 2983-2997 (2000).</li></ol>

著者は、彼らが競合する金融利害がないことを宣言します。

セルに接続された静電容量測定に成功し、高品質での録音を得るためには、いくつかの重要なステップが必要です：副腎から調製1）生存し、健康な細胞;カバースリップの2）、PDLコーティング; 3）ギガオームシール形成; 4）システムのノイズレベル;および5）位相補正。 動物を手術のために、一つは、潜在的な修正は、最も適した器用に外科的アプローチを調整し、解剖...

シナプス伝達は、神経伝達物質を含むシナプス小胞のエキソサイトーシスによって媒介され、これらの小胞は、長期的にはニューロンの通信を維持する神経末端内のローカルエンドサイトーシスのリサイクルを受けなければならない。脳内のシナプス伝達の重要な役割を考えると、シナプス小胞サイクルを構成している分子機構を理解することは、全体としてセルラ通信でより良い理解に向けて不可欠な基盤である。細胞モデル系の中では、副腎髄質細胞は、シナプス小胞のリサイクルの根底にある分子機構に最も決定的な洞察のいくつかを提供してきました。エキソサイトーシス、神経伝達物質放出の最終段階は、非常に学び、副腎クロマフィン細胞1,2を使用することによって検討されている。実際には、分泌顆粒の形成、ターゲティング、ドッキングおよび融合を調整する分子の選手のほとんどが原因のdivの適用に同定されているクロマフィン細胞1におけるERSEテクニック。さらに、エキソサイトーシスに関与するタンパク質合成機構の単一小胞の解決を可能にする機会を提供することにより、クロム親和性細胞は、小胞融合3の問題に対処するための強力なモデルのままです。 細胞結合型の静電容量の測定は、第3エキソサイトーシスの間に単一小胞の融合を解決に利用した。直径60nmのセル接続構成4-7のパッチクランプ技術を用いて細胞膜アドミタンス測定によって検出されることが実証されている〜と小さい小胞のエキソサイトーシス。アドミタンスは、回路またはデバイスは、電流が流れることを可能にする方法を簡単の尺度として定義される。それは、インピーダンスの逆数である。このように、アドミタンス測定値は、膜キャパシタンスの理解を提供する。これは、原形質膜への小胞の膜を組み込むことによって達成される。この取り込みは、表面の変化を明らかにエリア8。各融合する小胞は、膜キャパシタンス9,10段階的増加を引き起こす。また、このアドミタンス測定は、膜コンダクタンスとエキソサイトーシス事象3時の融合孔コンダクタンスを提供しています。この技術は、エキソサイトーシスの間に単一小胞の動態を同定することをユニークなツールを提供してきたように、私たちの研究室は最近、単一の小胞11,12のエンドサイトーシスを検出するために、この概念を適用しております。 私たちの具体的な関心は、多くの細胞の基本的なハウスキーピング部品13として、および神経端子14,15におけるシナプス小胞エンドサイトーシスのための主要な経路と考えられてきたクラスリン依存性エンドサイトーシス（CME）、である。 CMEは、生物学的に重要であることが知られているが、その動態は、単数エンドサイトーシスイベントを監視する技術的な制限のためによく理解されていないままである。マフィン細胞と神経細胞1との間のエキソサイトーシスの機構の類似性を考えると、PLはクロマフィン細胞での分裂メカニズムはおそらくニューロンにシナプス小胞エンドサイトーシスに適用することができることがausible。細胞結合型の静電容量の測定は、個別のエンドサイトーシスイベントを監視し、ほとんどの方法は、解決できない核分裂反応速度を分析するために利用されてきた。私達のセルに接続された記録では、20kHzの正弦波は、保持電位上に重畳され、出力電流は、二相ロックインアンプから他のチャネルでのチャネル及び膜キャパシタンスに膜コンダクタンスに分離される16 18。膜コンダクタンスと静電容量の変化から、人はおそらく小胞ピンチオフする前に細胞膜に内在化小胞をつなぐ管状膜ネックに対応核分裂細孔の動態を計算することができます。総称して、この技術は私たちにCMEの中に小胞分裂の調節メカニズムを検討する機会を与えてくれます。

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			<td height="61" style="height:61px;width:88px;">Name of Material/ Equipment</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:88px;">Poly-D-Lysine</td>
			<td rowspan="2" style="width:88px;">Sigma</td>
			<td rowspan="2" style="width:88px;">P0899</td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
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		<tr height="21">
			<td height="21" style="height:21px;width:88px;">DMEM&#160;&#160;</td>
			<td style="width:88px;"></td>
			<td rowspan="2" style="width:88px;">15066024</td>
			<td rowspan="2" style="width:88px;">Keep out of UV</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:88px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:88px;">Life Technologies</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Cover Glass</td>
			<td style="width:88px;">Carolina Biological</td>
			<td style="width:88px;">633029</td>
			<td style="width:88px;">12mm</td>
		</tr>
		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">Penicillin Streptomycin</td>
			<td rowspan="2" style="width:88px;">Life Technologies</td>
			<td rowspan="2" style="width:88px;">15140122</td>
			<td rowspan="2" style="width:88px;">100mL</td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Insulin-Trans-Sel-X</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">51500056</td>
			<td style="width:88px;">Only thaw on ICE!</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Papain</td>
			<td style="width:88px;">Worthington</td>
			<td style="width:88px;">39S11614</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">EPC-7 plus patch amplifier</td>
			<td rowspan="2" style="width:88px;">HEKA</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BNC-2090 data acquisition board</td>
			<td rowspan="2" style="width:88px;">National Instruments</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Igor data acquisition software</td>
			<td style="width:88px;">Wavemetrics</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">P-97 pipette puller</td>
			<td style="width:88px;">Sutter Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Microforge</td>
			<td style="width:88px;">Scientific Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="60">
			<td height="161" rowspan="3" style="height:161px;width:88px;">Borosilicate glass capillaries</td>
			<td rowspan="3" style="width:88px;">Sutter Instruments</td>
			<td rowspan="3" style="width:88px;">B150-110-10</td>
			<td style="width:88px;">Outer diameter-1.5 mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Inner diameter 1.10 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Length- 10 cm</td>
		</tr>
	</tbody>
</table>

methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

細胞生存率およびギガオームのシールの質は、細胞結合型静電容量記録の品質を決定する上で重要である。したがって、前の電気生理学的記録に効果的かつ効率的な細胞培養を調達することが重要であり、典型的な生細胞は、 図1に示されている。練習時間が高品質でギガオームのシールを達成するのに役立つであろう。プロトコルステップ5.3で説明したようにパッチピペットが...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

マウス副腎クロマフィン細胞における細胞 - 添付容量測定のための方法

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

Research

JoVE Journal

Neuroscience

8.7K ビュー.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

ビデオ: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

超音波ガイド下のマウスの前脳へのマイクロインジェクション子宮内で E9.5で

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

神経科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

複数のマウスの神経解剖学的磁気共鳴イメージング

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

開発マウス網膜の in vivoエレクトロポレーションで

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

パッチクランプキャパシタンス測定とCa 2 +</sup網膜スライスにおける単一神経終末で>イメージング

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

マウスモデルにおける静脈内自己投与方法

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

NF-κB依存性神経新生の調節および分析のための方法

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

マウス坐骨神経に生体内電気生理学的測定値の

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

発生中のマウス胚性皮質における有糸分裂のライブイメージング

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

てんかんのマウスモデルにおける熱誘発発作の行動スクリーニング

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

タンパク質分析のためのマウス網膜における光受容体細胞区画の単離のための2つの剥離方法

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...