この作品は、NIHの助成金NS065183とラトガースロバートウッドジョンソンメディカルスクールからの起動の資金によってサポートされています。

1。馴化培地およびアストロサイトニューロン共培養ラットアストロサイト培養物を調製。 <ol><li> （BSSレシピについては表1を参照）解剖バッファを用意し、4℃で保存し、使用直前まで。 </li><li> 500ミリリットルのビーカー内のイソフルランで新生ラットの仔（P0-P2）を麻酔。 </li><li>子犬が動かないときは、70％エタノールでスプレーし、首を切る。 </li><li>脳を削除します。 <ol><li>デュモン＃5ピンセットでしっかりと頭を保持し、皮膚や頭蓋骨を通して正中切開を作るために細かいハサミを使用しています。 </li><li>側面に頭蓋骨を反射することによって、脳を公開、冷BSSを含む皿に脳を削除します。 </li></ol></li><li>解剖スコープの下で間脳および脳幹から大脳半球を分離する。慎重にピンセットの1組で組織を安定化させ、静かに鉗子の別のペアで髄膜を離れて引っ張ることで、すべての髄膜を除去します。 </li><li>すべての半球を収集IN 1 60mmディッシュ。できる限り細かく、ミンチ組織を小さなハサミを使用して。その後、50ミリリットルコニカルチューブにみじん切りの組織を移す。 </li><li>脳に1％のDNase Iと2.5％トリプシンとBSSの12ミリリットル（15ミリリットルの全容量）の1.5ミリリットルの1.5ミリリットルを追加します。 37℃で15分間、振盪水浴中でインキュベート良好な混合を確実にするために、チューブを手で5分毎に旋回される。 </li><li>新しい50ミリリットルコニカルチューブの上に70μmの細胞濾過器上清を移す。この上清に3ミリリットルFBSを追加します。 </li><li>残りの部分に、13.5 BSSミリリットルと1.5ミリリットル、2.5％トリプシンを追加し、さらに15分間揺れ水浴中でインキュベートする。 </li><li>セルストレーナーを通して残りの上清を移し、ステップ1.8から上清を兼ね備えています。 </li><li>細胞をペレットに5分間1,000 rpmで遠心分離します。 </li><li>グリアメディアの5ミリリットルに再懸濁ペレット。上清を残りの細胞をペレット化し、再び遠心分離することができる。 </li><li>血球計数器を用いて細胞を数える。 </li><li>プレートCEL1×10 7センチあたり150 2フラスコの密度でLS。 </li><li>めっき後の新鮮なグリアメディアデーにメディアを変更します。その後、グリアメディアで週二回、細胞を養う。 </li><li>細胞が&gt; 80％の集密度（めっき後約10日）に達したときに、90％ウマ血清および10％DMSO中で細胞をダウン凍結し、液体窒素中で株式を保つ。細胞は、バイアル当たり2×10 6で凍結されています。約5バイアルを各フラスコから凍結することができる。 </li><li>約10〜14日間の海馬の文化を設定する前に、グリア細胞は、凍結ストックから播種する。私たちは通常、海馬ニューロンの90カバーグラス（ウェル当たり3 15ミリメートルラウンドカバーガラス）の成長をサポートするのに十分である5 6ウェルプレートを、プレート。我々はまた、トランスフェクションのためのコンディショニングN2.1のメディアに使用されるグリア細胞の追加の八から十60ミリメートル料理をプレート。神経培養前日に、細胞がNB27の培地を供給する必要があります。アストロサイトは、理想的に目で&gt; 90％コンフルエントであるべきポイントです。大幅に凍結するとアストログリア培養におけるミクログリアの数が減少していることを、私たちを見つける。ミクログリアは、アストログリア培養物中に存在する場合に増加した神経毒性が観察されているので、私たちは常に神経細胞との共培養のために凍結ストックから調製したアストログリアの文化を使用しています。 </li></ol> 2。神経培養<ol><li>ミリQ水2Xの最初すすぐことによって清浄なガラス製カバースリップ。次いで、これらを24時間、濃硝酸に浸漬し、2時間の合計よりも少なくとも5回milliQ水ですすぐ。カバーガラスを6時間225℃のオーブンで焼成して乾燥し、滅菌する。 </li><li>転送は、滅菌後60ミリメートル皿にカバースリップ。共培養の際にグリア細胞とは別の神経細胞を維持するために、各カバースリップの外側のエッジ付近の滅菌パラフィンの4ドットを配置します。 </li><li> 1ミリグラム/ 37℃インキュベーター内の0.1 Mホウ酸緩衝溶液中で一晩、ポリ-L-リシンでコートしたカバー。 </li><li>次の日、無菌メートルで二度カバース​​リップを洗う少なくとも15分間ずつilliQ水。次いで、これらを37℃のインキュベーター中でプレート培地中で一晩インキュベートする。 </li><li>気胸が続くイソフルラン麻酔による妊娠ラット（E18）を安楽死させると、断頭により胚性ラットを安楽死させる。脳を取り出して、上記のように大脳半球を解剖する。 </li><li>大脳半球から海馬を解剖する。 15ミリリットルコニカルチューブにすべての海馬を置き、15分間37℃で0.25％トリプシン（0.5ミリリットル、2.5％トリプシン、4.5ミリリットルBSS）で消化する。 </li><li>消化された海馬は、5分間ずつ、BSS 3Xですすぐ。次いで、それらをガラスパスツールピペットで磨砕し、細胞密度を血球計を用いて決定される。ニューロンを60 mmディッシュ当たり2×10 5細胞の密度で播種する。 </li><li>めっき後約2〜4時間後、転送がニューロンはグリアに向けて下向きにして、アストログリア細胞を含むディッシュに装着ニューロンとカバースリップ。 DIV3で、シトシンアラビノシドを追加5μmの最終濃度で細胞はグリア増殖を停止します。 </li></ol> 3。カルシウムトランスフェクション<ol><li>グリアトランスフェクションの前日に、条件N2.1メディア。 </li><li>トランスフェクションの日に、グリアのオフエアコンN2.1メディアを取り、新たな皿に移す。転送はエアコンN2.1メディアへのニューロンとカバースリップ。 10月30日分間インキュベーター内で平衡化してみましょう。 </li><li>滅菌チューブの1に設定するには、DNAの1-4μgを全量100μlのための2MのCaCl 2の12.5μL、及び滅菌H 2 Oを兼ね備えています。チューブの第二のセットに、2X HBSを100μlを加える。 </li><li>数秒間毎回ボルテックス、 塩化カルシウム/ DNA混合物を含むチューブに一度2X HBSの⅛量（12.5μL）を追加します。チューブは15分間座ってすることができます。 </li><li> 15分のインキュベーション後、細胞にトランスフェクション混合物を滴下する。 1〜1.5時間インキュベートする。砂のような沈殿物の層が見えるはずです10×対物レンズを用いて顕微鏡下で。 </li><li>インキュベーション後、温かいHBS洗浄緩衝液で2回カバースリップをすすぐ。 </li><li>グリアとオリジナル料理にカバースリップを返し、最終濃度0.5mMにキヌレン酸を追加します。 </li><li>トランスフェクションしたニューロンは、ライブ画像化や、早くも次の日などの免疫細胞化学のために処理することができる。ニューロンは、トランスフェクション後3週間まで生き残ることができる。 </li></ol>

<ol>
	<li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> J. Neurosci. 30, 6171-6177 (2010).</li><li>Zeitelhofer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transfection+of+mammalian+neurons+via+nucleofection.">High-efficiency transfection of mammalian neurons via nucleofection.</a> Nat. Protoc. 2, 1692-1704 (2007).</li><li>Janas, J., Skowronski, J., Van Aelst, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+delivery+of+RNAi+in+hippocampal+neurons.">Lentiviral delivery of RNAi in hippocampal neurons.</a> Method Enzymol. 406, 593-605 (2006).</li><li>Graham, F. L., vander Eb, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+assay+of+infectivity+of+human+adenovirus+5+DNA.">A new technique for the assay of infectivity of human adenovirus 5 DNA.</a> Virology. 52, 456-467 (1973).</li><li>Craig, A. M., Banker, G. a. G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfecting+cultured+neurons.">Transfecting cultured neurons.</a> Culturing Nerve Cells. , (1998).</li><li>Alavian, K. N., 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=Bcl-xL+regulates+metabolic+efficiency+of+neurons+through+interaction+with+the+mitochondrial+F1FO+ATP+synthase.">Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase.</a> Nat. Cell Biol. 13, 1224-1233 (2011).</li><li>Zhang, Y., 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=Modulation+of+synaptic+function+by+VAC14,+a+protein+that+regulates+the+phosphoinositides+PI(3,5)P(2)+and+PI(5)P.">Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P(2) and PI(5)P.</a> EMBO J. 31, 3442-3456 (2012).</li><li>Dudek, H., Ghosh, A., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+phosphate+transfection+of+DNA+into+neurons+in+primary+culture.">Calcium phosphate transfection of DNA into neurons in primary culture.</a> Curr. Protoc. Neurosci. Chapter 3, Unit 3 11 (2001).</li><li>Goslin, K. A. H., Banker, G., Banker, G. a. G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Hippocampal+Neurons+in+Low-Density+Culture.">Rat Hippocampal Neurons in Low-Density Culture.</a> Culturing Nerve Cells. , (1998).</li><li>Kohrmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+convenient,+and+effective+method+to+transiently+transfect+primary+hippocampal+neurons.">Fast, convenient, and effective method to transiently transfect primary hippocampal neurons.</a> J. Neurosci. Res. 58, 831-835 (1999).</li><li>Jiang, M., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Ca2+-phosphate+transfection+efficiency+in+low-density+neuronal+cultures.">High Ca2+-phosphate transfection efficiency in low-density neuronal cultures.</a> Nat. Protoc. 1, 695-700 (2006).</li><li>Goetze, B., Grunewald, B., Baldassa, S., Kiebler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+controlled+formation+of+a+DNA/calcium+phosphate+coprecipitate:+application+for+transfection+of+mature+hippocampal+neurons.">Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons.</a> J. Neurobiol. 60, 517-525 (2004).</li></ol>

興味の競合が宣言されていない。

慎重に、一貫して成功したトランスフェクション10,11のために制御する必要があるいくつかの重要なパラメータがあります。リン酸カルシウムトランスフェクションのための最も重要なパラメータは、我々の手で、通常7.10から7.15の間で変化する2×HBS、pH値である。私たちは、pH計との違いを説明するために0.05刻みでpH値を有する株式の3つのバッチを作るお勧めします。別の方法とし?...

一次ニューロンは、それらが有糸分裂後で、微小環境の変化に非常に敏感であるようにトランスフェクトする最も困難な細胞タイプの一つである。これらの細胞1における外来遺伝子および短期ヘアピンRNA（shRNAの）の発現のための方法の4一般的に使用されるタイプがあります。それぞれ独自の長所と短所があります。細胞はトランスフェクションのためのキュベットに移しなければならないので、例えば、エレクトロポレーションは、通常、新たに単離されたニューロン2上で実行される。ウイルス感染は通常、非常に高い効率3を達成するが、事業者のためのより多くの労働集約的で危険であることができます。多くの脂質媒介トランスフェクション試薬は、ニューロンおよび細胞毒性の異なるレベルにおける成功の度合いは様々で、市販されている。 リン酸カルシウムトランスフェクションは、神経細胞に外来遺伝子を導入するための、便利で経済的な方法を表す。方法は、第一の哺乳類セルへのアデノウイルスDNAを導入するために使用されたグラハムとファンデEB（1973）によるLS 4。トランスフェクションは、リン酸緩衝液中の組換えDNAと塩化カルシウムを混合することにより行った。これは、徐々に細胞の単層上に滴下したとき、細胞表面に付着し、DNA /リン酸カルシウム沈殿物の形成は、エンドサイトーシスによって取り込まれることができ、最終的に核に5を入力してください。このプロセスは、標的細胞における導入外来遺伝子の発現につながる。 0.5〜5％、6〜8の間のリン酸カルシウムトランスフェクション範囲の典型的な効率。しかしながら、慎重な最適化お​​よび実験プロトコルの一貫性のある実行と、それは約50％のトランスフェクション効率を達成することができる。ここでは、サンドイッチフォーマット9にアストログリア細胞と共培養された初代海馬ニューロンのリン酸カルシウムトランスフェクション、私たちの詳細なプロトコルを記述します。

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	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:284px;">Dumont #5 forceps</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">11251-20</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Fine scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">14090-09</td>
			<td>straight / sharp 8.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Vannas-Tubinger spring scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">15008-08</td>
			<td>angled / sharp / 8.5 cm / 5 mm cutting edge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Standard pattern forceps</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">11000-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Student surgical scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">91401-14</td>
			<td>blunt / 14.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Spring scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">15006-09</td>
			<td>angled to side / 9 cm / 10 mm cutting edge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Isoflurane</td>
			<td style="width:207px;">Webster Veterinary</td>
			<td style="width:119px;">14043-225-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Poly-L-lysine</td>
			<td>Sigma</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM</td>
			<td>Sigma</td>
			<td>M2279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">100 mM Sodium pyruvate</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">S8636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Glucose</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x HBSS</td>
			<td>Sigma</td>
			<td>H4385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">1 M HEPES, pH 7.3</td>
			<td>Gibco/Invitrogen</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX</td>
			<td>Gibco/Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal Media</td>
			<td>Gibco/Invitrogen</td>
			<td>21130-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement (50x)</td>
			<td>Gibco/Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 Supplement</td>
			<td>Gibco/Invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ovalbumin</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco/Invitrogen</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">2.5% Trypsin</td>
			<td>Gibco/Invitrogen</td>
			<td style="width:119px;">15090-046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">DNase I</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Cytosine arabinoside</td>
			<td style="width:207px;">Calbiochem/EMD Millipore</td>
			<td align="right" style="width:119px;">251010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Kynurenic acid</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">K3375</td>
			<td></td>
		</tr>
	</tbody>
</table>

calcium phosphate transfection of primary hippocampal neurons

リン酸カルシウム沈殿法は、培養細胞のトランスフェクションのため便利で経済的な方法である。最適化により、一次ニューロンのような困難なトランスフェ細胞上で、この方法を使用することが可能である。ここでは、アストログリア細胞と共培養した海馬神経細胞のリン酸カルシウムトランスフェクションのための私達の詳細なプロトコルを記述します。

トランスフェクションの異なるパラメータを最適化し、注意深く実験毎に制御される場合には、50％までのトランスフェクション効率を得ることができる。 図1は DIV4でGFPでトランスフェクトされたニューロンのフィールドを示している。フィールドが16をトランスフェクトした、そのうち28ニューロンの合計が含まれています。これは、50％以上の効率を表しています。同じカバ?...

Watch this Scientific Journal Video about Calcium Phosphate Transfection of Primary Hippocampal Neurons at JoVE.com

Calcium Phosphate Transfection of Primary Hippocampal Neurons

初代海馬ニューロンのリン酸カルシウムトランスフェクション

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

calcium-phosphate-transfection-of-primary-hippocampal-neurons

Research

JoVE Journal

Neuroscience

25.1K ビュー.  Rutgers University. リン酸カルシウム沈殿法は、培養細胞のトランスフェクションのため便利で経済的な方法である。最適化により、一次ニューロンのような困難なトランスフェ細胞上で、この方法を使用することが可能である。ここでは、アストログリア細胞と共培養した海馬神経細胞のリン酸カルシウムトランスフェクションのための私達の詳細なプロトコルを記述し?...

ビデオ: Calcium Phosphate Transfection of Primary Hippocampal Neurons

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofectionとマウス胎児海馬および皮質ニューロンの初代培養

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

神経科学

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

胎児マウスから海馬ニューロンの単離および培養

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

疾患の病因を研究するツールとしてのニューロンへの抗体のトランスフェクション

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

同時電気生理学的記録および視交叉上核ニューロンのカルシウムイメージング

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

構造化照明顕微鏡を用いてラット初代海馬ニューロンの樹状突起棘を画像化

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

マウスドーパミン作動性ニューロンの初代培養

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

細胞内のCaの蛍光と生物発光イメージング<sup&gt; 2+</sup&gt;高齢者における海馬ニューロン

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

低密度初代海馬ニューロンの文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

培養海馬神経細胞のシナプス小胞エンドサイトーシスを測定

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

線虫の行動で個々 のニューロンのレシオ メトリック カルシウム イメージング

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

初代海馬ニューロンのリン酸カルシウムトランスフェクション

要約

さらに動画を探す

この動画の章

Nucleofectionとマウス胎児海馬および皮質ニューロンの初代培養

胎児マウスから海馬ニューロンの単離および培養

疾患の病因を研究するツールとしてのニューロンへの抗体のトランスフェクション

同時電気生理学的記録および視交叉上核ニューロンのカルシウムイメージング

構造化照明顕微鏡を用いてラット初代海馬ニューロンの樹状突起棘を画像化

マウスドーパミン作動性ニューロンの初代培養

細胞内のCaの蛍光と生物発光イメージング

低密度初代海馬ニューロンの文化

培養海馬神経細胞のシナプス小胞エンドサイトーシスを測定

線虫の行動で個々のニューロンのレシオメトリックカルシウムイメージング

初代海馬ニューロンのリン酸カルシウムトランスフェクション

要約

さらに動画を探す

この動画の章

Nucleofectionとマウス胎児海馬および皮質ニューロンの初代培養

胎児マウスから海馬ニューロンの単離および培養

疾患の病因を研究するツールとしてのニューロンへの抗体のトランスフェクション

同時電気生理学的記録および視交叉上核ニューロンのカルシウムイメージング

構造化照明顕微鏡を用いてラット初代海馬ニューロンの樹状突起棘を画像化

マウスドーパミン作動性ニューロンの初代培養

細胞内のCaの蛍光と生物発光イメージング

低密度初代海馬ニューロンの文化

培養海馬神経細胞のシナプス小胞エンドサイトーシスを測定

線虫の行動で個々 のニューロンのレシオ メトリック カルシウム イメージング

線虫の行動で個々のニューロンのレシオメトリックカルシウムイメージング