この作品は、ウェインS. Sossinに健康研究（CIHR）グラントのカナダの協会によってサポートされていました。キャロルA.ファラーはデラフォンルシェルシュエンサンテケベック（FRSQ）とコンラッドハリントンの交わりからポスドクの受信者です。著者は、グラフィックの概要とヘルプのためのビデオ撮影とマデリーンリッチモンドプールのヘルプはジョアンナブジー、マーガレットヘイスティングスとマーガレットLabbanに感謝します。

1。 Sf9細胞の単層培養の準備とメンテナンス<ol><li> Sf9細胞の培養と維持管理は、組織培養フードで実行されます。 </li><li> Sf9細胞はスポドプテラフルギペルダの卵巣細胞（Sf21細胞）から誘導されるとインビトロジェンからグレースのメディアで凍結した細胞として購入することができます。 </li><li>補ったグレイス昆虫培地、（1X）、〜30％ウシ胎児血清（FBS）斜め首が75 cm 2の細胞培養フラスコ内で追加された。の代わりに8 mLの</li><li> （約1〜2分）前後にチューブを移動して37℃のウォーターバス中で急速にSf9細胞の凍結チューブを融解する。 </li><li>均等に細胞を分散さは75 cm 2の細胞培養フラスコと優しく手で岩の板にSf9細胞を移す。 </li><li>細胞が接続されるまで、27で1〜2時間℃でインキュベートする。 </li><li>古い培地を除去し、30％FBSをfreshGraceの昆虫培地10mLに置き換えます。 </li><li>細胞がコンフルエントになるまで27℃でインキュベートする。 </li><li>単層培養を維持するためには、Sf9細胞のコンフルエントなフラスコから古い培地を除去し、10％FBSをグレース昆虫培地5 mLを加える。 </li><li>軽くフラスコの側にSf9細胞を分離するために数回タップします。 </li><li>ピペッティングしながらフラスコの壁を噴霧、一度ゆっくりと上下に10 mLのピペットとピペッター、ピペットメディアを使用する。滅菌済み15 mLのPolystereneチューブに細胞を移す。 </li><li>新しい75センチメートル2細胞培養フラスコと優しく手で岩の板に10％FBS（対数増殖期を維持するために1:5希釈）とグレース昆虫培地を8mLにSf9細胞懸濁液2 mlを加え。 </li><li>細胞がコンフルエントになるまで27℃でインキュベートする。 </li></ol> 2。 Sf9細胞で蛍光標識PKCの発現<ol><li>ライブイメージングの実験、文化35ミリメートルでSf9細胞マテックガラス底培養disheswith 14mmのガラス表面と0.16〜0.19ミリメートルのカバーガラスの厚さ。 </li><li>蛍光タンパク質でタグ付けされたPKCSは、以下に詳述する変更とメーカーのCellfectin IIのトランスフェクション試薬、以下の推奨事項を使用したSf9細胞で発現されています。 </li><li> 1日目：前に細胞を播種するために、少なくとも30分間、10％FBSとグレース昆虫培地で各マテックの料理を扱う。 </li><li>血球計を使用して、ステップ11（上記）からSf9細胞を数える。 </li><li> 150μLの全体積の各マテックのガラスカバースリップ上にプレートを0.05 × 10 6個の細胞。細胞は室温で30分間添付することができます。 </li><li>グレースの2mLを徐々に細胞を脱着しないように、各皿を1 mLに10％FBSを添加している追加。 </li><li> 27℃で一晩。 </li><li>この時点から、プラスミドDNAをSf9細胞のトランスフェクションのための製造業者の勧告を使用してください。 </li><li> 2日目：Cellfectin IIのトランスフェクション試薬を用いて蛍光標識およびPKCSトランスフェSf9細胞。私たちはしばしば、このシステムで共発現している（プラスミド構築のための詳細は以前に2,5を記載されている）強化された緑色蛍光タンパク質（EGFP）と単量体の赤色蛍光タンパク質（MRFP）でタグ付けされたPKCS。一度に蛍光タンパク質の発現は約70〜80％の細胞に発現している72時間のトランスフェクション後に得られます。つの蛍光タンパク質の同時発現は、約30％共発現細胞にトランスフェクション効率を低下させる。 PKC EGFP -タグ付きおよびPKC MRFP -タグを共発現したとき、最適なタンパク質発現のためのMRFP -タグ融合タンパク質のためのより多くのプラスミドDNAを2倍使用してください。 </li><li>実行ライブイメージング48〜72時間トランスフェクション後（最適なタンパク質発現のために、72時間を待つ）。 </li></ol> 3。 アメフラシ感覚ニューロンに蛍光標識PKCの発現<ol><li> アメフラシ神経細胞培養の調製は、趙と同僚の4でビデオの記事に記載されている。 </li><li>蛍光タンパク質でタグ付けされたPKCSは、以下に詳述するマイクロインジェクションの手順を使用してアメフラシ感覚ニューロンに発現されています。 </li><li>蒸留水の1％ファストグリーン（FCF）のストック溶液を調製、注射器と28ミリメートルシリンジフィルター（0.20μm）を使用してフィルタ。 -20℃で分注しストア</li><li>感覚ニューロンのマイクロインジェクションは、神経細胞を分離した後、1日または2日目に行ったが、我々は2日待っていると、カバースリップに優れた細胞接着を可能にすることに気付くことができます。 </li><li>感覚ニューロンの単離後2日目に、ファストグリーンのアリコートを解凍し、注射器と28ミリメートルシリンジフィルター（0.20μm）を使用して再度フィルタ。 </li><li> 0.5パーセントファストグリーンを含む蒸留水でプラスミドDNAの溶液を調製します。 μg/μLの0.4という高いプラスミドDNAの濃度を使用することができますが、これらの値より上にすることは推奨されません。 </li><li> 1 mLの注射器と4mmシリンジフィルター（0.20μm）を用いてプラスミドDNA /ファストグリーンソリューションをフィルタリングします。 </li><li>マイクを使って15分間16110 × gでプラスミドDNA /ファストグリーンソリューションを遠心rocentrifuge。 </li><li>微小電極プラーを使用して神経細胞のマイクロインジェクション（サッターフレイミング/ブラウンマイクロピペットプラー、モデルP - 97）のために鋭い電極を準備します。ボックスのフィラメントを使用して、1ミリメートル、外径、内径0.75ミリメートルと内部のフィラメント（世界精密機器がTW100F - 4）で薄壁ガラスキャピラリーを用いて長さ100 mmのガラス微小電極を引き出します。マイクロインジェクションの電極を引き出す方法の詳細についてはサター電極の料理本を参照してください。 </li><li> microloaderピペットチップ（エッペンドルフCA32950 - 050）を用いてプラスミドDNA /ファストグリーン溶液2μLを各電極を埋める。 </li><li>マイクロインジェクションの駅にアメフラシ神経細胞の培養を含むマテックガラス底の培養皿に移す。 </li><li>マイクロインジェクションステーションの構成は次のとおりです。自家製の防振台（キネティックシステムの防振ワークステーション）、外部のハロゲン照明とステレオスコープ、マイクロマニピュレーター（サターハクスリーウォールタイプのマニピュレーターに培養皿を保持するために、大きなガラスのステージをその空気ポンプ（世界の精密機器PV820空気圧ピコ-ポンプ）に接続されている微小電極ホルダー、）三軸の粗と超微細位置決めの両方を可能にする。空気ポンプの圧力入力は、圧縮された窒素のタンクに接続されています。 </li><li>微小電極ホルダーに微小電極を挿入します。 </li><li>ステレオ顕微鏡下での表示、細胞核内にマイクロピペットの先端を挿入します。 </li><li>核がuniformely緑色になるまで、窒素（/ 2の20ポンド期間に10〜100ミリ秒）の短い圧力パルスを実現。 </li><li>室温で4時間細胞をインキュベートし、℃で使用するまで4℃で保管。 24時間後に注射 - 最適なPKC転座の場合は、ライブイメージングの実験20を実行してください。 </li></ol> 4。 Sf9細胞をリビングにし、 アメフラシ感覚ニューロンにおけるPKC転座の可視化<ol><li>イメージングのために、我々は、Axiovert 200でツァイスLSM510倒立型共焦点顕微鏡を使用してください。 </li><li>イメージングプロトコルは、次の点以外はSf9細胞用とアメフラシ感覚神経でも同じです： アメフラシ感覚ニューロンは、X40の油浸対物レンズで撮像されているのに対し、Sf9細胞は、X63油浸対物レンズで撮像されています。 Sf9細胞は、 アメフラシ感覚ニューロンが18から20 o Cで台を維持室温でイメージングされたのに対し、26〜27 ° Cの時に維持温度制御チャンバー内に撮像されています</li><li>我々は、EGFPタンパク質とMRFPタンパク質でタグ付けされた画像のPKCにHeNeレーザー（543nmで励起）でタグ付けされた画像のPKCに50％のレーザー出力で30mWのアルゴンレーザー（488nmの励起）を使用します。アルゴンやヘリウムネオンレーザーの線は4％に減衰しており、事前の画像処理やピンホールにはそれぞれ50％の伝送出力が1に調整されます。 </li><li>イメージング段階で安定培養皿を持っているし、動きや振動を避けることが重要です。 </li><li>静かに皿に1 mLの全容量を残して10 mLのピペットを用いてディッシュから培地を1mLを削除します。 </li><li>核を見ることができる細胞の中央部で撮影してください。 </li><li>画像は30秒ごとに取る10月20日共焦点画像の時系列を設定します。 </li><li>時系列を開始します。 </li><li>写真2枚が（30秒の時点の後に）撮影された後、軽く薬を1 mL（2X濃度）P1000ピペットを用いて細胞の上にドロップしてドロップを追加。薬剤は、約30秒間連続して追加されます。 </li><li>一部の薬は、他の人が唯一の最適な5〜10分後の治療（動画1と動画2）である遅い転座を誘発しながら（60秒の時点で）すぐに次の薬物治療に表示できる高速な転座を誘発する。 </li><li>薬剤が洗い流さする必要がある場合は、皿の片側に1つのピペットで静かに洗浄バッファーをピペッティングし、反対側でメディアをペッティング、洗浄を行うために2 × 10 mLのピペットを使用してください。 </li><li>洗浄が効果的と薬剤の効果は可逆的であるかにされている場合、PKCは30〜60秒以内に細胞質ゾルに戻ります。 </li><li> LSMファイルとしてムービーを保存します。 </li><li> LSMのファイルを開くと画像が前と後の薬物治療定量化するためにNIHの画像Jソフトウェアを使用してください。定量化は別の場所で1記載されている。 </li></ol>

<ol>
	<li>Farah, C. A., Nagakura, I., Weatherill, D., Fan, X. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+role+for+phosphatidic+acid+in+the+translocation+of+the+novel+protein+kinase+C+Apl+II+in+Aplysia+neurons.">Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons.</a> Mol Cell Biol. 28, 4719-4733 (2008).</li><li>Zhao, Y., Leal, K., Abi-Farah, C., Martin, K. C., Sossin, W. S. &. a. m. p. ;. a. m. p., Klein, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.Isoform+specificity+of+PKC+translocation+in+living+Aplysia+sensory+neurons+and+a+role+for+Ca2+-dependent+PKC+APL+I+in+the+induction+of+intermediate-term+facilitation.">M.Isoform specificity of PKC translocation in living Aplysia sensory neurons and a role for Ca2+-dependent PKC APL I in the induction of intermediate-term facilitation.</a> J Neurosci. 26, 8847-8856 (2006).</li><li>Farah, C. A., Weatherill, D., Dunn, T. W. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC+differentially+translocates+during+spaced+and+massed+training+in+Aplysia.">PKC differentially translocates during spaced and massed training in Aplysia.</a> J Neurosci. 29, 10281-10286 (2009).</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. , (2009).</li><li>Manseau, F., Fan, X., Hueftlein, T., Sossin, W. S., Castellucci, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-independent+PKC+Apl+II+mediates+the+serotonin+induced+facilitation+at+depressed+synapses+in+Aplysia.">Ca2+-independent PKC Apl II mediates the serotonin induced facilitation at depressed synapses in Aplysia.</a> J. Neuroscience. 21, 1247-1256 (2001).</li></ol>

利害の衝突は宣言されません。

我々は、Sf9細胞やアメフラシ感覚ニューロンのリアルタイムで蛍光標識PKCの画像の転座に手法を記載している。 Sf9細胞は、培養するため、画像PKC転座にシンプルなシステムを提供し、それらをトランスフェクトするのはとても簡単です。対照的に、 アメフラシ感覚ニューロンのマイクロインジェクションは、習得には時間がかかります。まで数カ月に。この技法に関連する問?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sf9 frozen cells</td>
<td>Spodoptera frugiperda ovarian cells</td>
<td>Invitrogen</td>
<td>B825-01</td>
<td>&nbsp;</td>
<tr>
<td>Grace&#x2019;s Insect Medium, Supplemented (1X), liquid</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11605-094</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Corning 75cm2 canted neck cell culture flask</td>
<td>Tool</td>
<td>Corning</td>
<td>430720</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>CanSera</td>
<td>CS-C08-500</td>
<td>Supplement Grace's media with FBS prior to use</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309604</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309602</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431229</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431212</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Glass Bottom Dish</td>
<td>Tool</td>
<td>Mattek</td>
<td>P35G-1.5-14-C</td>
<td>Use to culture Sf9 cells prior to transfection and imaging</td>
</tr>
<tr>
<td>Cellfectin II Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10362-100</td>
<td>Used to express fluorescently tagged PKCs in Sf9 cells</td>
</tr>
<tr>
<td>Fast Green FCF</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F-7252</td>
<td>Use to visualize microinjection of plasmid DNA solution into Aplysia sensory neurons</td>
</tr>
<tr>
<td>Thin wall glass capillaries</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>Use to make microelectrodes for microinjection</td>
</tr>
<tr>
<td>Microloader pipette tip</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>CA32950-050</td>
<td>Autoclave before using to fill up the microelectrodes for microinjection</td>
</tr>
<tr>
<td>PV820 Pneumatic PicoPump </td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
<td>Part of microinjection station</td>
</tr>
<tr>
<td>Microelectrode holder</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>MPH6S</td>
<td>Use to hold microelectrode </td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tool</td>
<td>Sutter</td>
<td>MP-85</td>
<td>Use to position microelectrode in the three-axis</td>
</tr>		</tbody>
		</table>
		</div>

live-imaging of pkc translocation in sf9 cells and in aplysia sensory neurons

プロテインキナーゼCS（PKCには）そのような細胞の成長と学習と記憶のような細胞プロセスの様々な調節において中心的役割を果たすスレオニンキナーゼセリンです。古典的PKCに（α、βI、βII、γ）、新規のI型は、PKCS（ε、η）、小説II型PKCの（δ、θ）、そして非定型PKCの（ζとι：脊椎動物におけるPKCアイソフォームの4つの既知の家族が暮らしている）。古典的なPKCSは、 カルシウムによって活性化されている小説のPKCがDAGにより活性化されるが、Ca 2 +の -独立している間に+とdiacylclycerol（DAG）、。非定型PKCには、Ca 2 +もDAGのどちらによって活性化されています。 アメフラシカリフォルニ 、記憶形成を研究する我々のモデルシステムでは、という三神経系特異的PKCアイソフォームの主要な各クラスから1つ、従来のPKC APL私、小説のI型PKC APL IIと非定型PKC APL IIIがあります。 PKCには、脂質活性化キナーゼであるため、細胞外シグナルに応答して、古典と小説PKCの活性化は、しばしば細胞質から形質膜へのPKC転座と相関している。そのため、生きた細胞でリアルタイムでPKCトランスロケーションを可視化するPKCの活性化につながるシグナル伝達経路を解明するための貴重なツールとなっています。私たちはシナプス可塑性の異なる型を媒介するため、異なる条件下で転位するPKCの、異なるアイソフォームを確立し、PKC APL IIの、セロトニン（5HT）アクティベーションのためのDAGとホスファチジン酸の両方の生産（PA）を必要とするために例えば、このテクニックは、許可されている転座1-2。重要なのは、同じニューロンを可視化する機能は、繰り返し私たちは、例えば、絶妙の詳細3におけるPKC応答の脱感作を測定することができました。このビデオでは、我々はSf9細胞培養の準備の各ステップを示す、 アメフラシ感覚ニューロンの培養物に反応してSf9細胞やアメフラシ感覚神経とPKC転座のライブイメージングの蛍光標識のPKCS発現する別のビデオの第4条に記載されているレーザー走査顕微鏡を用いて異なる活性剤。

Watch this Scientific Journal Video about Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons at JoVE.com

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

このビデオでは、我々は、蛍光標識のPKCS使用して生きた細胞内PKC転座の可視化を実証する。

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 ...

live-imaging-pkc-translocation-sf9-cells-aplysia-sensory

Research

JoVE Journal

Neuroscience

11.9K ビュー.  McGill University. このビデオでは、我々は、蛍光標識のPKCS使用して生きた細胞内PKC転座の可視化を実証する。

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

開発ゼブラフィッシュ前脳におけるクローン関連の神経前駆細胞のタイムラプスライブイメージング

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

ニューロモデュレーションとミトコンドリア輸送：長時間にわたって海馬ニューロンのライブイメージング

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

無傷の感覚器官の前駆細胞の生細胞イメージングショウジョウバエ蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅神経細胞のレンチウイルスを介した遺伝子操作と可視化 in vivoで</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

嗅神経細胞の繊毛のケージド化合物のフラッシュフォトリシス

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

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

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

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

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

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 ...

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

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

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

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

における嗅覚受容ニューロンのホールマウント免疫標識<em&gt;ショウジョウバエ</em&gt;アンテナ

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

方法は、標的とマウスにおけるアイソ気道-神経支配感覚ニューロンへ

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...