私たちは、技術支援のためのLihong陰に感謝しています。 NVDはNARSAD若手研究賞を受賞であり、また、NIH（5 K99 MH095825-02）からの助成金によってサポートされています。 Fishellラボでの研究は、国立衛生研究所、国立精神衛生研究所（5 R01 MH095147-02、5 R01 MH071679-09）、神経疾患·脳卒中研究所（5 R01 NS081297-02、1 P01 NS074972によってサポートされている-01A1）とシモンズ財団。

全ての動物は、医学のNYUの学校の施設内動物管理使用委員会の規則やガイドラインに沿って処理した。 マウス系統 TACONICによって提供スイスウェブスター雌マウスをこれらの実験に使用した。具体的に表層のニューロンを標的化するために、E15.5胚を使用した。 注：この作品で使用されたプラスミド（DLX5 / 6.eGFPプラスミド3μgを/μl）を、標準的なクローニング技術を使用して生成された。 eGFPの cDNAはDLX5 / 6-Pminは、ポリAプラスミドにクローニングした。このプラスミドは、要求（demarn02@nyumc.org）に応じて入手可能です。 マイクロインジェクションピペットの作製<ol><li> 860に引っ張りヒート＃2を設定してください。 </li><li>フィラメントに近いガラスキャピラリーを配置し、固定します。ピペットの外径（OD）が約30ミクロンであることを確認してください。 </li><li> 「プル」ボタンを押します。 </li><li>慎重にキャピラリーを削除します。底部が針である。上部を捨てる。 </li></ol> 2麻酔手順<ol><li>イソフルラン収納室を準備します。注：イソフルランは、その高速動作や副作用の発生率が低い、好ましい麻酔薬である。ペントバルビタールは、別のオプションです。しかしながら、この薬物の耐性は可変であり、イソフルランよりも高い死亡率につながる可能性がある。 </li><li> 0.5-1リットル/分のO 2中4から5パーセントにイソフルランの流れを設定します。 </li><li>ヘッドアダプタ用のバルブが閉じているのに対し、室内用のバルブが開いていることを確認してください。 </li><li>チャンバー内に妊娠したマウスを置きます。 </li><li>マウスは麻酔の影響下で行くことを許可します。これは、約2〜3分かかります。呼吸数をチェックしてください。マウスが過呼吸始めた場合、イソフルランの投与量を下げる。 </li><li>マウスを麻酔した後は、速やかにチャンバーから取り外します。加熱パッド上で腹側を上に置き、私手術全体にイソフルランを供給していきますマスクで頭をnsert。ヘッドアダプタピースを接続するチューブにチャンバーからイソフルランの流れを切り替えるようにしてください。各眼内の眼潤滑剤のドロップを入れて、目を閉じて。 </li><li>低体温症を最小限にするために36℃度に加熱パッドを設定してください。 </li><li>後足と尾をつまんで足と尾反射の不在を確認してください。 </li></ol> 3。手術<ol><li> 70％エタノールで腹部を覆っている皮膚を清潔に。 </li><li>上向きに皮膚を引っ張って鉗子を使用してください。各手術後、洗剤と水ですべての楽器を洗浄し、72℃のCO / Nで滅菌した。 </li><li>乳腺の3番目と4番目のペアの間の中間に始まる正中線に沿って小さな垂直切開（約2cm）を作成します。細かい手術はさみを使用してください。腹壁を損傷しないように注意してください。 </li><li>静かに腹膜から皮膚を分離する。 </li><li>動脈を可視化腹壁上の静脈。腹腔を露出させ（血管を避ける）腹膜を介して他の2センチメートル垂直の切開を加えます。 </li><li>動脈をクランプ避け各側の皮膚や腹壁をクランプ。動脈を誤ってパンクしている場合は、頸椎脱臼またはイソフルラン過剰投与を行うことで、すぐに動物を犠牲に：注意してください。 </li><li>前37℃に滅菌PBSを温める。キムワイプ湿ったPBSでクランプをカバー。 </li><li> PBSで露光され、腹腔に潤いを与える。手術を行いながら、この手順を数回繰り返します。 </li><li>ラウンド鉗子を使用して、静かにキャビティからの胚チェーン（E15.5）を引く。ウェットティッシュ上の鎖の残りをしましょう​​。最初の子宮の半分を露出することから始めます。その中の胚をエレクトロポレーションされた後、後半では動作した後、内部に戻ってそれを持って来る。 </li></ol> 4。エレクトロポレーション<ol><li>胚を操作するために丸いピンセットを使用し続ける。 </li><li> 6側脳室をsualize。正中線（ 図1b）に沿って走る側脳室。 </li><li>注射の間、それを可視化する1：20混合物のためのDNA溶液に：ファストグリーン（10％w / vの株式）を追加します。蒸留水で標準マキシプレップ精製プロトコルから得られたペレットを溶解させてDNA溶液を調製します。 </li><li>ファストグリーンでDNA溶液1ミリリットル（DLX5 / 6-EGFP、3μgの/μl）を注入するためにプランジャーで前に引っ張らマイクロピペットを使用してください。ピペットをロードする前に、針の先端の最後に肩の先頭から6ミリメートルの長さを残すように細かい鉗子＃5（デュモン）でマイクロピペットの先端をカットします。丸いピンセットで胚の頭部を持ってください。正中線の近くに位置し、側脳室を目指して45°の角度でピペットを用いて、脳を入力してください。針が心室を貫通する場合には、DNAを注入するプランジャーを使用したように、ゆっくりとピペットを撤回。心室はグリーンに点灯したとき解決策は、それを埋めるために開始されます。この時点で、ピペットを移動を停止し、DNAの1ミリリットルを注入。静かに、ピペットを撤回。 </li><li> 950ミリ秒だけ離れた50ミリ秒の5 45VパルスにCUY21のエレクトロを設定します。 </li><li> E15.5胚のために5mmのパドル電極を使用してください。効率的な電流伝達を確実にするために、PBS中の電極を浸し。 </li><li> DNA溶液を含む心室にプラスのパドルを胚の頭の周りの電極パドルを配置します。少し神経節隆起を通して腹側に電流を作成するために、負の1のに対して正のパドルを下げる。この操作は錐体細胞における異所性発現を最小限に抑えることができます。 </li><li>電極を配置した後、一度フットペダルを押して、パルスを供給。 </li><li>電極を取り外し、きれいなそれらを拭いてください。繰り返します各胚を4.6から4.9を繰り返します。 </li><li>すべての胚をエレクトロ後、腹腔内に3mlのPBSを注ぎ、腹腔に戻す。 </li><li>第1の縫合湾曲した針と5-0絹縫合した後、皮膚と腹壁。傷口にリドカイン（4％）を適用します。鎮痛のためのアスピリンのイントラ腹膜120mgの/ kgのを管理します。 注：全体の手順は、20分以内に行われるべきである。それは、初心者は、短い動作時間を維持するために、いくつかの胚をエレクトロポことをお勧めします。長時間の手術は胚の生存率を減少させる。 </li><li>麻酔管から動物を取り出し、拭い紙に加熱パッド上で回復を可能にします。 </li><li> 、ケージに動物を返し、37℃で加熱パッド上にそれを維持し、健康状態を監視します。動物は一般的によく手術に耐え、それらが麻酔管から除去された後すぐにゆっくりと移動し始める。過度の出血や無気力が認められた場合、動物実験委員会は、頸椎脱臼または麻酔の過剰摂取などの安楽死の手続きを承認し実行することで、すぐに動物を生け贄に捧げる。 </li><li>ビバにケージを返すバクテ。女性は自然に出産します。 </li></ol>

<ol>
	<li>Fishell, G., Rudy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+inhibition+within+the+telencephalon:+&amp;#34;where+the+wild+things+are&amp;#34;.">Mechanisms of inhibition within the telencephalon: &amp;#34;where the wild things are&amp;#34;.</a> Annual review of neuroscience. 34, 535-567 (2011).</li><li>Stenman, J., Toresson, H., Campbell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+distinct+progenitor+populations+in+the+lateral+ganglionic+eminence:+implications+for+striatal+and+olfactory+bulb+neurogenesis.">Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis.</a> J Neurosci. 23, 167-174 (2003).</li><li>Eisenstat, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DLX-1,+DLX-2,+and+DLX-5+expression+define+distinct+stages+of+basal+forebrain+differentiation.">DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation.</a> J Comp Neurol. 414, 217-237 (1999).</li><li>Batista-Brito, R., Fishell, 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+developmental+integration+of+cortical+interneurons+into+a+functional+network.">The developmental integration of cortical interneurons into a functional network.</a> Curr Top Dev Biol. 87, 81-118 (2009).</li><li>Miyoshi, G., Butt, S. J., Takebayashi, H., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologically+distinct+temporal+cohorts+of+cortical+interneurons+arise+from+telencephalic+Olig2-expressing+precursors.">Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors.</a> J Neurosci. 27, 7786-7798 (2007).</li><li>Miyoshi, G., 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=Genetic+fate+mapping+reveals+that+the+caudal+ganglionic+eminence+produces+a+large+and+diverse+population+of+superficial+cortical+interneurons.">Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.</a> J Neurosci. 30, 1582-1594 (2010).</li><li>Bulfone, 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+mouse+Dlx-2+(Tes-1)+gene+is+expressed+in+spatially+restricted+domains+of+the+forebrain,+face+and+limbs+in+midgestation+mouse+embryos.">The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos.</a> Mechanisms of development. 40, 129-140 (1993).</li><li>Bulfone, 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=Spatially+restricted+expression+of+Dlx-1,+Dlx-2+(Tes-1),+Gbx-2,+and+Wnt-3+in+the+embryonic+day+12.5+mouse+forebrain+defines+potential+transverse+and+longitudinal+segmental+boundaries.">Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries.</a> J Neurosci. 13, 3155-3172 (1993).</li><li>Robinson, G. W., Wray, S., Mahon, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+expression+of+a+member+of+a+new+family+of+murine+Distal-less+homeobox+genes+in+the+developing+forebrain.">Spatially restricted expression of a member of a new family of murine Distal-less homeobox genes in the developing forebrain.</a> The New biologist. 3, 1183-1194 (1991).</li><li>Close, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satb1+is+an+activity-modulated+transcription+factor+required+for+the+terminal+differentiation+and+connectivity+of+medial+ganglionic+eminence-derived+cortical+interneurons.">Satb1 is an activity-modulated transcription factor required for the terminal differentiation and connectivity of medial ganglionic eminence-derived cortical interneurons.</a> J Neurosci. 32, 17690-17705 (2012).</li><li>De Marco Garcia, N. V., Karayannis, T., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+is+required+for+the+development+of+specific+cortical+interneuron+subtypes.">Neuronal activity is required for the development of specific cortical interneuron subtypes.</a> Nature. 472, 351-355 (2011).</li><li>Petros, T. J., Rebsam, A., Mason, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+and+ex+vivo+electroporation+for+gene+expression+in+mouse+retinal+ganglion+cells.">In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.</a> Journal of visualized experiments : JoVE. (31), (2009).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> Journal of visualized experiments : JoVE. (6), e239 (2007).</li></ol>

著者らは、開示することは何もない。

技術の限界この技術は、細胞プロセスの細胞自律的な分析を可能にするが、それは集団分析には適していない。エレクトロポレーションは、脳あたりにエレクトロ未満千細胞と非常に疎である。結果として、この技術は、CGE由来のニューロンの遺伝子操作から生じる行動の結果を評価するために使用することができない。 エレクトロポレーションは?...

皮質GABA作動性介在ニューロンの大部分は、内側と尾側神経節隆起（MGEそれぞれとCGE）をという名前の2つの一過性の胚の構造から発生4。パルブアルブミンおよびソマトスタチンを発現するニューロンは、ニューロンは、CGEから発信表現カルレチニン（CR）、Vasointestinalペプチド（VIP）およびリーリン（再）に対し、MGEに由来する。これらのニューロンのサブタイプは、その誕生日によって区別す​​ることができる。 MGE派生サブタイプは胎生9.5（E9.5）とE16.5 5,6の間に生まれている。これとは対照的に、CGE派生介在ニューロンは、それらの生産がE15.5 6でピークに達してE12.5からE18.5を通して生まれている。この後半生まれの人口の遺伝的標的化は、しかし、とらえどころのないままになります。 ネズミ遠位レス（デラックス）の遺伝子はもっぱら発展途上腹側前脳3で表現される。 GABA作動性介在ニューロンと線条体投射ニューロンではなく、皮質錐体細胞がDlx1を表現、初期の発達段階3での2,5、及び6つの遺伝子。実際、 デラックス遺伝子は、すべてのGABA作動性前駆細胞におけるMGEとCGE脳室下帯（SVZ）で表現される。これらの遺伝子の発現は、有糸分裂後の段階7-9でのサブタイプを選択するために制限となる。以前の実験的証拠は、DLX5 / 6エンハンサーエレメントは、トランスジェニックマウスモデル2におけるGABA作動性系統の選択的ターゲティングを可能にすることを示した。私たちは、発生中のマウス脳におけるエピソーム発現の文脈におけるこれらのエンハンサーエレメントの1つの使用を試験した。私たちは、サブブルースクリプト（BS）骨格プラスミド（ 図1）における最小プロモーターおよび強化緑色蛍光タンパク質（EGFP）と共にDLX5 / 6エンハンサーエレメントをクローニングした。私たちは、選択的にCrイオン、VIPおよび再サブタイプ3,8,10を標的とするためにE15.5での子宮内電気穿孔中によりプラスミドを導入しました。私たちの技術は容易にスパースエレクトロポレーションを可能にする焦がす細胞の形態学的特徴の再構築。また、皮質GABA作動性ニューロンにおける遺伝子発現の非常に高いレベルの機能研究を可能にします。私たちは、いくつかの野生型およびドミナントネガティブ遺伝子11を用いた機能研究の損失と利得を実施しました。

<table border="0" cellpadding="0" cellspacing="0" width="1106">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Electroporator&#160; with pedal</td>
			<td style="width:227px;">Protech International</td>
			<td style="width:164px;">CUY21</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5mm paddle electrodes&#160;</td>
			<td style="width:227px;">Protech International</td>
			<td style="width:164px;">CUY650P5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad&#160;</td>
			<td style="width:227px;">Kent Scientific</td>
			<td>DCT-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Sutter Instruments P30 Puller&#160;</td>
			<td>Sutter Instruments</td>
			<td>3282322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluovac Anesthesia Systems</td>
			<td>&#160;Harvard Apparatus</td>
			<td>726425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Delicate Operating Scissors 4.75&#34; Straight Sharp/Sharp</td>
			<td>Roboz</td>
			<td>&#160;RS-6702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Silk Black Braid 18&#34; C-1 Box 36&#160;</td>
			<td>Roboz</td>
			<td>SUT-1073-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Clip Applying Forceps 5.5&#34;</td>
			<td>Roboz</td>
			<td>&#160;RS-5410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 Clamp scissors</td>
			<td>Roboz</td>
			<td>&#160;RC-4894</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Holding forceps&#160;</td>
			<td style="width:227px;">Fine Science Tools</td>
			<td>11031-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass capillary tubing&#160;</td>
			<td>&#160;FHC</td>
			<td>27-30-0</td>
			<td>Borosil 1.0mm OD x 0.75mm ID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green</td>
			<td>Sigma-Aldrich</td>
			<td>F7258&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile PBS</td>
			<td>Life Technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
	</tbody>
</table>

subtype-selective electroporation of cortical interneurons

中枢神経系（CNS）成熟の研究は、ニューロン集団の遺伝的標的化に依存しています。しかし、特定のニューロンのサブタイプに関心のある遺伝子の発現を制限するタスクが原因特定のプロモーターエレメントの相対的希少性に著しく困難なことが証明されている。 GABA作動性ニューロンは、大規模な遺伝的および形態的多様性ニューロン集団を構成している。確かに、GABA作動性介在ニューロンの11を超える異なるサブタイプは、マウスの大脳皮質1に特徴付けられている。ここでは、GABA作動性集団の選択的標的化のために適合したプロトコルを提示する。私たちは、ホメオボックス転写のエンハンサーエレメントを使用することによりGABA作動性ニューロンのサブタイプ選択的ターゲティングを達成し子宮内電気穿孔法で特定の遺伝子の発現を介して駆動するためにDLX5とDlx6、ショウジョウバエ遠位レス（DLL）遺伝子2,3のホモログを、要因。

私たちは、成熟ニューロンの細胞型特異的ターゲティングを達成するために、 子宮内エレクトロポレーション技術を適合される。 CGE由来のニューロンにおけるEGFPの発現を駆動するためには、CGE由来のニューロンの大部分が生成される段階、DLX5 / 6エンハンサーエレメントを使用し、E15.5に私達の注射を制限した。私たちは、P8とP15 11（ 図1および図2）<...

Watch this Scientific Journal Video about Subtype-selective Electroporation of Cortical Interneurons at JoVE.com

Subtype-selective Electroporation of Cortical Interneurons

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

皮質介在ニューロンのサブタイプ選択的エレクトロポレーション

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

subtype-selective-electroporation-of-cortical-interneurons

Research

JoVE Journal

Neuroscience

8.7K ビュー.  New York University School of Medicine. This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

ビデオ: Subtype-selective Electroporation of Cortical Interneurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

子宮内エレクトロポレーションは、皮質ニューロンのサブセットで、遺伝子機能の研究のために初代神経培養した

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

神経科学

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

皮質ネットワークの開発で機能的カルシウムイメージング

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

マウス大脳皮質アストロサイトの単離および培養

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

新生児の脳室下帯のエレクトロ

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

exの子宮内エレクトロポレーションと全半球植：早期皮質の発達についての研究のための簡単な実験方法

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

新生児軟膜表面エレクトロ

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

へのガイド<em&gt;インビボ</emOptogenetically同定された皮質抑制性介在から&gt;シングル単位記録

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

の記録細胞内の電圧応答のための電気生理学的手法<em&gt;ショウジョウバエ</em&gt;光刺激に感光体と介在ニューロン<em&gt;インビボ</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

平坦化された哺乳類の皮質皮質モジュールの可視化

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...