我々は彼らの助けをカリーヌKindbeiter、ミュリエルBozon、およびFlorieレイノーに感謝します。仕事はCNRS、ANR依田プログラムとLabex DevWeCanでサポートされています。

1日目 1。 E12.5マウス胚からの脊髄床板（FP）の解剖 注意：全体の手順は、無菌状態を使用する必要があります。これは、汚染を避けるために切開フード下で切開を行うことが好ましい。フードの表面をエタノールで洗浄する必要があります。すべての解剖器具は、滅菌シャーレに滅菌され、維持されなければならない。液体（媒体、切開媒体）を閉じたまま、氷浴中に入れなければならない。各胚は、個々の新鮮なドロップで採取します。これは組織保存のために重要である。解剖はデュモン＃5鉗子を用いて行われる。後脳に損傷せずにお料理、グリップ臍帯やヘッドとの間に胚を転送します。それが正常解剖を完了するために、脊髄を損傷しないことが重要です。調製冷リン酸緩衝生理食塩水（PBS）、冷ハンクス平衡塩溶液（HBSS） -6.5％グルコース及び室温神経基礎培地。 1.1脊髄解剖動物は、欧州指令次のセンター国立デラルシェルシュ科学研究の動物ケアのガイドラインに従って処理する。安楽死の方法は、首に圧力を適用し、脳から脊柱を脱臼からなる、頸椎脱臼である。この方法は、マウス動物モデルのために推奨され、迅速かつ効率的な方法で行うことができる。 <ol><li>妊娠（つや消し次E0.5 =初日）のE12.5の妊娠マウスを安楽死させる。マウス腹側を上にして置きます。 </li><li> 70％エタノールで腹部を浸す。アドソンのピンセットで腹部の皮膚をつまんで、外科はさみで皮膚と腹膜層を通ってカット。切開を行い、腹腔を露出させ、マウスの両側にカットするために使用します。 </li><li>胚の文字列は、マウスの各側に存在している。 UTEの1ホーンを把握2胚および外装部品から始め、それを解剖する間の組織内RUS。氷上で冷P​​BSで100ミリメートルペトリ皿にそのまま子宮を転送します。 </li><li>胚体外組織から胚を抽出します。鉗子の2ペアが（濃い赤）胎盤組織をつかみ、組織を引き裂き、子宮嚢からの胚を取り除くためにそっと引き出します。 </li></ol> 注意：より良い保全のために、彼らの子宮嚢1つずつから胚を収集します。 <ol start="5"><li>次のステップは、双眼虫眼鏡や解剖フードの下で行われている。冷HBSS-6.5％グルコースの低下に1胚を置きます。 </li><li>鉗子を使用して、胚を首を切る。 </li></ol> 注：下顎と後脳の間で切断、後脳の後部には、次のステップのために重要である。 <ol start="7"><li>実験者が直面する前部と胚の腹面を下にして置きます。 </li><li>肌をつまむ鉗子の1組とし、他のペアを持つ胚の皮膚を引き離す。 </li></ol> 注：このステップは、皮膚が完全に胚の前部から削除されるまで繰り返されなければならない。 <ol start="9"><li>左に胚前側に回します。 1ピンセットで前部に胚を「突き止める」。他の鉗子で皮膚をつかみ、胚の吻側側に引きます。脊髄はそれからアクセス可能です。 </li><li>まだ脊髄を包む髄膜をカットする鉗子の片側を使用しています。脊髄は、今開いている。 </li><li>右に胚の前方側に回します。 </li><li>胚の子宮頸側から脊髄から組織を切り離す。脊髄（鉗子は、脊髄損傷を避けるために閉じ保持する必要があります）の下に鉗子をスライドさせます。鉗子は、脊髄の下に見えるようにする必要があります。脊髄の下の小さなロータリー運動は切り離すのに十分である組織および後根神経節（DRG）を囲む。 </li></ol> 注意：このように、次のステップの間にそれを破壊する確率を高め、脊髄に重みを追加添付周囲の組織を残す。 <ol start="13"><li>冷HBSS-6.5％グルコースの新鮮なドロップで孤立した脊髄を置きます。 </li><li>上部と前部側の髄膜離れて実験者から、平らな位置に脊髄を置きます。 </li><li> 1ピンセットで残りの後脳を使用して脊髄を「突き止める」、第二の鉗子は髄膜をつかむと。脊髄の吻側側から髄膜をはがす。 </li></ol> 注：運動は脊髄のいずれかの破壊を避けるために低速で一定でなければならない。振動運動は、髄膜の剥離を容易にすることができる。 1.2 FP郭清<ol><li> 1ピンセットで後脳ピンチ。 </li><li> FPは、中央TRAです組織のnsparent一部。メスを使用すると、底板を切り出す。 </li></ol> 注：同じ品質で両サイドをカットし、吻側側から開始して、交互に右側と底板の左側を切った。 <ol start="3"><li>室温でNeurobasal培地中で解剖し、FPを節約。 </li></ol> 2。 FP文化 注：すべてのステップは、組織培養フード内で無菌条件下で実施すべきである。新鮮な培地と新たに解凍サプリメントおよび試薬を使用してください。滅菌ガラスカバースリップ、暖かい神経基礎+ B27、血漿およびHBSS-トロンビンを準備します。 2.1 FP文化<ol><li>各カバースリップの中央に100ミリメートルペトリ皿ピペット20μlの血漿中でいくつかの滅菌カバースリップを配置。 </li><li>二から四のFP各血漿ドロップでの転送と次のプラズマ降下に20μlのHBSS-トロンビンを加え、丁寧に混ぜ合わせます。カバースリップを保つ血漿の凝固を可能にするために、室温で10分以上である。 </li></ol> 注意：一部の解剖例における完全な解剖以上の場合の譲渡2のFPを。ピペットチップでゆっくりと混ぜて、FP外植片との接触を避ける。 血漿クロットは、凝固の印として数μmを後退させる。凝固は、血漿血餅の分離を回避するために、途中で停止してはいけません。 <ol start="3"><li> 24ウェルプレートの各カバースリップを転送したNeurobasal + B27の500μlを添加する。 37℃で48時間インキュベートする</li></ol> 3日目 3。 FP調整培地（FP センチ ）収穫 注：収穫は、組織培養フード内で無菌条件下で実施すべきである。上清を培養ペリ中に汚染されているか、床板（意味浮遊している場合、彼らは死亡している可能性がある場合OD）は、この井戸からのFP CMを収穫しないでください。 3.1のFP CM収穫 <ol><li>丁寧に収穫各ウェルからの培地。 </li></ol> 注：プラズマ重合トロンビンミックスまたはFPフラグメントをピペットないでください。 <ol start="2"><li> -80℃で100μlアリコートを保存</li></ol> 4日目 4。 E12.5マウス胚から脊髄交連ニューロンの解剖 注：この製剤は、無菌条件下で実施する必要はない。胚を収集するために、上記の手順に従ってください。冷PBS、冷HBSS-6.5％グルコースと冷たい神経基礎を準備します。 4.1脊髄解剖<ol><li>鉗子を使用して、胚を首を切る。 </li></ol> 注：下顎と後脳の間で切断、後脳の後部には、のために重要である次のステップ。 <ol start="2"><li>実験者が直面する前部と新鮮なドロップに胚腹面を下にして置きます。 </li><li> 1鉗子で胚の皮膚をつまんで、他の1で皮膚を引き離す。 </li></ol> 注：このステップは、皮膚が完全に胚の前部から削除されるまで繰り返されなければならない。 <ol start="4"><li>左に胚前側に回します。 1ピンセットで前部に胚を「突き止める」。他の鉗子で、肌を把握し、脊髄を露出させ、胚の吻側側に引きます。 </li><li>まだ脊髄を包む髄膜をカットする鉗子の片側を使用しています。脊髄は、現在公開さとオープンされている。 </li><li>右に胚前側に配置します。 </li><li>胚の子宮頸側から脊髄から組織を切り離す。脊髄（閉じた鉗子を保つ）の下に鉗子をスライドさせます。のためにCEPSは、脊髄の下に表示する必要があります。脊髄の下の小さなロータリーの動きは、隣接する組織と後根神経節（DRG）を取り外すのに十分である。 </li><li>冷HBSS-6.5％グルコースの新しいドロップで孤立した脊髄を転送します。 </li><li>上部と前部側の髄膜離れて実験者から、平らな位置に脊髄を置きます。 </li><li>二鉗子は髄膜をつかむと、1ピンセットで残りの後脳を使用して脊髄を「突き止める」。脊髄の吻側側から髄膜をはがす。 </li></ol> 4.2交連ニューロン解剖<ol><li> 1ピンセットで後脳ピンチ。 </li><li>交連ニューロンは、脊髄（背側）の最外側部​​に位置している。細かいメスで、この横方向の部分を切り出す。 </li></ol> 注記：脊髄の背側部分は、セルdの違いによって、腹側部分から区別することができるensity、腹側の部分は、より不透明であること。 注：同じ品質で両サイドをカットし、吻側側から開始して、交互に右側のと左側の小さな長さをカット。 <ol start="3"><li>すぐに使用氷上Neurobasal培地中で解剖した組織を節約する。 </li></ol> 5。 FPのCMと交連組織の処置 注記：この手順では、無菌状態を必要としません。組織は、個々の1.5mlチューブに収集することができる必要があります。凍結されたFPのCMは、一度だけ使用されるべきである。複数の再凍結は避けてください。暖かいFP センチを調製し（37°C）。 交連ニューロンの5.1治療<ol><li>小さなハサミで交連組織を引き裂く。 </li></ol> 注：可能な限り小さく、組織を切断し、細胞のアクセス可能性を増強する。 <ol start="2"><li>かなり異なる実験条件の制御処理およびFP CM処理の間の組織を配布します。 </li><li> 4℃で1分間、800×gで組織片を遠心分離</li><li>チューブ間の再分配を確認してください。 </li></ol> 注：断片まで再必要な再配布遠心分離機が均等に分散している場合。 <ol start="5"><li>上清を取り除きます。 </li><li>組織に暖かいFP CMとコントロール治療を追加します。 </li></ol> 注意：ボリュームがペレットの量に調整する必要があり、少なくとも二回ペレットの量でなければなりません。 <ol start="7"><li> 30分間37℃でインキュベートする。 </li></ol>注意：ゆっくり10〜15分毎に振る<ol start="8"><li> 1分間800×gで遠心分離し、治療を取り外します。 </li><li>溶解バッファーを追加します。 </li></ol> 注：ボリュームがAに調整する必要がありますペレットのマウントとは、少なくとも二回ペレットの量でなければなりません。 <ol start="10"><li>アップピペット上下ペレットを再懸濁する。 </li></ol> 注：渦を使用することもできる。 <ol start="11"><li>氷上で15分間インキュベートする。 </li><li> 4℃で15分間14,000 xgで遠心分離</li><li>上清を保持し、ブラッドフォードアッセイ9とタンパク質の量を測定する。 </li><li> 6Xレムリ·バッファを追加します。 </li><li>タンパク質含量を変性させ、5分間95℃でインキュベートする。 </li></ol> 注：サンプルは、直ちに使用することができ、または-80℃で保存することができる。 <ol start="16"><li>ウエスタンブロット10による治療の効果を評価する。 </li></ol> 注：総タンパク質の少なくとも50μgの各レーンのためにロードする必要があります。

<ol>
	<li>Nawabi, H., Castellani, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+commissures+in+the+central+nervous+system:+how+to+cross+the+midline.">Axonal commissures in the central nervous system: how to cross the midline.</a> Cell. Mol. Life Sci. 68, 2539-2553 (2011).</li><li>Placzek, M., Briscoe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+floor+plate:+multiple+cells,+multiple+signals.">The floor plate: multiple cells, multiple signals.</a> Nat. Rev. Neurosci. 6, 230-240 (2005).</li><li>Chedotal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+tales+of+the+midline.">Further tales of the midline.</a> Curr. Opin. Neurobiol. 21, 68-75 (1016).</li><li>Nawabi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+midline+switch+of+receptor+processing+regulates+commissural+axon+guidance+in+vertebrates.">A midline switch of receptor processing regulates commissural axon guidance in vertebrates.</a> Genes Dev. 24, 396-410 (2010).</li><li>Ruiz de Almodovar, C., 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=VEGF+mediates+commissural+axon+chemoattraction+through+its+receptor+Flk1.">VEGF mediates commissural axon chemoattraction through its receptor Flk1.</a> Neuron. 70, 966-978 (2011).</li><li>Charoy, C., 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=gdnf+activates+midline+repulsion+by+Semaphorin3B+via+NCAM+during+commissural+axon+guidance.">gdnf activates midline repulsion by Semaphorin3B via NCAM during commissural axon guidance.</a> Neuron. 75, 1051-1066 (2012).</li><li>Kiecker, C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+organizers+in+patterning+the+nervous+system.">The role of organizers in patterning the nervous system.</a> Annu. Rev. Neurosci. 35, 347-367 (2012).</li><li>Le Dreau, G., Marti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+patterning+of+the+neural+tube:+a+tale+of+three+signals.">Dorsal-ventral patterning of the neural tube: a tale of three signals.</a> Dev. Neurobiol. 72, 1471-1481 (2012).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J. Vis. Exp. , (2010).</li><li>Salinas, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+collaborates+with+netrin-1+to+guide+axons+in+the+spinal+cord.">The morphogen sonic hedgehog collaborates with netrin-1 to guide axons in the spinal cord.</a> Trends Neurosci. 26, 641-643 (2003).</li><li>Bechara, 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=FAK-MAPK-dependent+adhesion+disassembly+downstream+of+L1+contributes+to+semaphorin3A-induced+collapse.">FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse.</a> EMBO J. 27, 1549-1562 (2008).</li></ol>

著者らは、開示することは何もない

フロアプレート馴化培地の生産は床板にリリース信号の生物学的特性を評価するための効率的かつ簡単な方法を提供します。 プラズマトロンビンマトリックスは、組織の生存のための優れた条件を提供しています。それにもかかわらず、この豊かな環境は、実験の種類によっては制限がある可能性があります。このように、底板組織はまた、アガロースマトリックスで?...

床プレートは、前駆細胞と分裂後の細胞系譜と制御軸索ナビゲーション7,8の仕様で重要な役割を果たして、開発脊髄のよく知られたパターンニングセンターです。 FP CMを作るためにここに記載の実験方法は、細胞のパターニングと生存の細胞と軸索の移行のために、研究者は発生過程の様々な文脈でのフロアプレート由来のシグナルの機能的特性を評価することができます。 このようなFP cmの使用を例示するために、交連ニューロンを含む背側脊髄組織は、解剖dilaceratedおよびFP センチメートルで刺激する。組織は、次いで、ウェスタンブロット分析のために処理することができる。これは、床版リリースされた信号により、軸索誘導機構の規制を調査できます。 dilacerated新鮮な組織の治療方法は、目の中の細胞の微小環境を保存する大きな利点を保持しているE組織。従ってFP センチによる治療または治療のいずれかのタイプの結果は、細胞及び組織培養条件におけるよりもより生理学的な方法で評価する。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS REQUIRED</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, Ca2+/Mg2+- free</td>
			<td>Invitrogen</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G-7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Invitrogen</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td>Sigma</td>
			<td>P-3266</td>
			<td>1mg/ml (Reconstitute in H2O and filtrate)</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T-4648</td>
			<td>10mg/ml(Reconstitute in H2O and filtrate)</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50X</td>
		</tr>
		<tr>
			<td>Hepes (260g/mol)</td>
			<td>Sigma</td>
			<td>H-7006</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E-5513</td>
			<td>0,5M pH8</td>
		</tr>
		<tr>
			<td>MgCL2</td>
			<td>Euromedex</td>
			<td>2189</td>
			<td>1M</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL</td>
			<td>Sigma</td>
			<td>I-3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Protease inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>04 693 116 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate (Na3VO4)</td>
			<td>Sigma</td>
			<td>S-6508</td>
			<td></td>
		</tr>
		<tr>
			<td>distilled H2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Reagents required</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>TOOLS AND MATERIAL</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Fine Tips forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>micro knives</td>
			<td>Fine Science Tools</td>
			<td>10136-14</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Tools and material</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			<table border="1">
				<tbody>
					<tr>
						<td colspan="2">Dissection and FP culture</td>
					</tr>
					<tr>
						<td>Ethanol 70%</td>
						<td></td>
					</tr>
					<tr>
						<td>dissection hood</td>
						<td></td>
					</tr>
					<tr>
						<td>dissection microscope</td>
						<td></td>
					</tr>
					<tr>
						<td>Petri dishes</td>
						<td></td>
					</tr>
					<tr>
						<td>glass coverslips</td>
						<td>18mm x 18mm</td>
					</tr>
					<tr>
						<td>Bunsen gas burner</td>
						<td></td>
					</tr>
					<tr>
						<td>24 well plate</td>
						<td></td>
					</tr>
					<tr>
						<td>tissue culture incubator (37&#176;C, 5% CO2, humidity controlled)</td>
						<td></td>
					</tr>
					<tr>
						<td>Centrifuge</td>
						<td></td>
					</tr>
					<tr>
						<td colspan="2">RECIPES</td>
					</tr>
					<tr>
						<td colspan="2">FP culture media</td>
					</tr>
					<tr>
						<td>Neurobasal</td>
						<td></td>
					</tr>
					<tr>
						<td>B27</td>
						<td>1/50e dilution from stock</td>
					</tr>
					<tr>
						<td colspan="2">HBSS - Thrombin</td>
					</tr>
					<tr>
						<td>HBSS</td>
						<td>1 ml</td>
					</tr>
					<tr>
						<td>Thrombin 10mg/ml</td>
						<td>30 &#956;l</td>
					</tr>
					<tr>
						<td colspan="2">Lysis Buffer pH7,4</td>
					</tr>
					<tr>
						<td>Hepes (260 g/mol)</td>
						<td>25mM</td>
					</tr>
					<tr>
						<td>EDTA (pH8)</td>
						<td>5mM</td>
					</tr>
					<tr>
						<td>MgCL2</td>
						<td>1mM</td>
					</tr>
					<tr>
						<td>Glycerol</td>
						<td>10%</td>
					</tr>
					<tr>
						<td>IGEPAL (NP40)</td>
						<td>0.10%</td>
					</tr>
					<tr>
						<td>Protease Inhibitor Cocktail</td>
						<td>1X</td>
					</tr>
					<tr>
						<td>Sodium Orthovanadate</td>
						<td>0,2M</td>
					</tr>
					<tr>
						<td>H2O</td>
						<td></td>
					</tr>
					<tr>
						<td colspan="2">Laemmli Buffer</td>
					</tr>
					<tr>
						<td>Tris 0,5M, pH 6,8</td>
						<td>375 mM</td>
					</tr>
					<tr>
						<td>SDS</td>
						<td>6%</td>
					</tr>
					<tr>
						<td>Glycerol</td>
						<td>60%</td>
					</tr>
					<tr>
						<td>DTT</td>
						<td>0,6M</td>
					</tr>
					<tr>
						<td>Bromophenol blue</td>
						<td></td>
					</tr>
					<tr>
						<td>H2O</td>
						<td></td>
					</tr>
				</tbody>
			</table>
			</td>
		</tr>
	</tbody>
</table>

culture of isolated floor plate tissue and production of conditioned medium to assess functional properties of floor plate-released signals

開発中に、前駆細胞と有糸分裂後のニューロンは、彼らの運命を制御し、隣接する地域からの信号を受信する。床プレートは、腹の位置に上衣運河の内側を覆うグリア細胞のグループです。床プレートは、脊髄内の細胞系譜のパターニングに貢献キーモルフォゲンを表現しています。後期発生段階で、床版は、交連軸索1により同側軸索と制御正中線交差の交差を防止するためのバリアとして機能し、脊髄内の軸索のナビゲーションを調整する。これらの機能は、様々な指導の手がかりの分泌によって達成される。他の人が地元の指導の手がかりに軸索2,3の感度を調節するために、ガイダンス受容体と下流のシグナル伝達を制御しながら、これらの手がかりの中には、成長している軸索のための誘引と忌避剤として機能します。ここでは、DEVE様々なフロアプレート由来シグナルの性質を調査可能にする方法を説明フロアプレート調整培地（FP センチ）4-6の生産に基づいてlopmentalコンテキスト。次に、軸索ガイダンスのコンテキストでこのFP cm の使用を例示。まず、脊髄をE12.5でのマウス胚から単離され、フロアプレートを解剖し、血漿トロンビンマトリックス（ 図1）で培養される。第2日後に、交連組織は、E12.5胚から摘出粉砕し、FPのCMにさらされている。第三に、組織は、交連のマーカーのウェスタンブロット分析のために処理される。

交連組織はFP センチにより処理し、試料を、ウェスタンブロットにおける受容体レベルの分析のために処理した。 FP cm のアプリケーションは、ガイダンス受容体のレベルを増加させる（ 図2）を横切った後正中線忌避Semaphorin3Bに交連軸索の感受性を媒介するPlexinA1を示した。これは、FPが発表した信号がPlexinA1レベル4,6を制御することを明らかにした。 ...

Watch this Scientific Journal Video about Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals at JoVE.com

Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

フロア·プレートは、脊髄を現像前駆細胞への拡散信号を提供し、ニューロン分化の重要な構造である。我々は、床プレート馴化培地を生成するために、新鮮な脊髄組織に適用し、そして生化学ことにより、目的のタンパク質上の影響を評価するための方法を記載している。

床板リリースされた信号の機能的特性を評価するために馴化培地離さ床板組織と生産の文化

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of ...

culture-isolated-floor-plate-tissue-production-conditioned-medium-to

Research

JoVE Journal

Neuroscience

7.9K ビュー.  University of Lyon 1. フロア·プレートは、脊髄を現像前駆細胞への拡散信号を提供し、ニューロン分化の重要な構造である。我々は、床プレート馴化培地を生成するために、新鮮な脊髄組織に適用し、そして生化学ことにより、目的のタンパク質上の影響を評価するための方法を記載している。 

ビデオ: Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

培養皿内の機能モーターユニット：脊髄外植片と筋細胞の共培養

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

神経科学

Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), magnetoencephalography or functional magnetic-resonance imaging. These techniques have either inherently low temporal or low spatial resolution, and suffer from low signal-to-noise ratio and/or poor high-frequency sensitivity. Thus, they are suboptimal for exploring the short-lived spatio-temporal dynamics of many of the underlying brain processes. In contrast, the invasive technique of electrocorticography (ECoG) provides brain signals that have an exceptionally high signal-to-noise ratio, less susceptibility to artifacts than EEG, and a high spatial and temporal resolution (i.e., &lt;1 cm/&lt;1 millisecond, respectively). ECoG involves measurement of electrical brain signals using electrodes that are implanted subdurally on the surface of the brain. Recent studies have shown that ECoG amplitudes in certain frequency bands carry substantial information about task-related activity, such as motor execution and planning1, auditory processing2 and visual-spatial attention3. Most of this information is captured in the high gamma range (around 70-110 Hz). Thus, gamma activity has been proposed as a robust and general indicator of local cortical function1-5. ECoG can also reveal functional connectivity and resolve finer task-related spatial-temporal dynamics, thereby advancing our understanding of large-scale cortical processes. It has especially proven useful for advancing brain-computer interfacing (BCI) technology for decoding a user's intentions to enhance or improve communication6 and control7. Nevertheless, human ECoG data are often hard to obtain because of the risks and limitations of the invasive procedures involved, and the need to record within the constraints of clinical settings. Still, clinical monitoring to localize epileptic foci offers a unique and valuable opportunity to collect human ECoG data. We describe our methods for collecting recording ECoG, and demonstrate how to use these signals for important real-time applications such as clinical mapping and brain-computer interfacing. Our example uses the BCI2000 software platform8,9 and the SIGFRIED10 method, an application for real-time mapping of brain functions. This procedure yields information that clinicians can subsequently use to guide the complex and laborious process of functional mapping by electrical stimulation.
Prerequisites and Planning:
Patients with drug-resistant partial epilepsy may be candidates for resective surgery of an epileptic focus to minimize the frequency of seizures. Prior to resection, the patients undergo monitoring using subdural electrodes for two purposes: first, to localize the epileptic focus, and second, to identify nearby critical brain areas (i.e., eloquent cortex) where resection could result in long-term functional deficits. To implant electrodes, a craniotomy is performed to open the skull. Then, electrode grids and/or strips are placed on the cortex, usually beneath the dura. A typical grid has a set of 8 x 8 platinum-iridium electrodes of 4 mm diameter (2.3 mm exposed surface) embedded in silicon with an inter-electrode distance of 1cm. A strip typically contains 4 or 6 such electrodes in a single line. The locations for these grids/strips are planned by a team of neurologists and neurosurgeons, and are based on previous EEG monitoring, on a structural MRI of the patient's brain, and on relevant factors of the patient's history. Continuous recording over a period of 5-12 days serves to localize epileptic foci, and electrical stimulation via the implanted electrodes allows clinicians to map eloquent cortex. At the end of the monitoring period, explantation of the electrodes and therapeutic resection are performed together in one procedure.
In addition to its primary clinical purpose, invasive monitoring also provides a unique opportunity to acquire human ECoG data for neuroscientific research. The decision to include a prospective patient in the research is based on the planned location of their electrodes, on the patient's performance scores on neuropsychological assessments, and on their informed consent, which is predicated on their understanding that participation in research is optional and is not related to their treatment. As with all research involving human subjects, the research protocol must be approved by the hospital's institutional review board. The decision to perform individual experimental tasks is made day-by-day, and is contingent on the patient's endurance and willingness to participate. Some or all of the experiments may be prevented by problems with the clinical state of the patient, such as post-operative facial swelling, temporary aphasia, frequent seizures, post-ictal fatigue and confusion, and more general pain or discomfort.
At the Epilepsy Monitoring Unit at Albany Medical Center in Albany, New York, clinical monitoring is implemented around the clock using a 192-channel Nihon-Kohden Neurofax monitoring system. Research recordings are made in collaboration with the Wadsworth Center of the New York State Department of Health in Albany. Signals from the ECoG electrodes are fed simultaneously to the research and the clinical systems via splitter connectors. To ensure that the clinical and research systems do not interfere with each other, the two systems typically use separate grounds. In fact, an epidural strip of electrodes is sometimes implanted to provide a ground for the clinical system. Whether research or clinical recording system, the grounding electrode is chosen to be distant from the predicted epileptic focus and from cortical areas of interest for the research. Our research system consists of eight synchronized 16-channel g.USBamp amplifier/digitizer units (g.tec, Graz, Austria). These were chosen because they are safety-rated and FDA-approved for invasive recordings, they have a very low noise-floor in the high-frequency range in which the signals of interest are found, and they come with an SDK that allows them to be integrated with custom-written research software. In order to capture the high-gamma signal accurately, we acquire signals at 1200Hz sampling rate-considerably higher than that of the typical EEG experiment or that of many clinical monitoring systems. A built-in low-pass filter automatically prevents aliasing of signals higher than the digitizer can capture. The patient's eye gaze is tracked using a monitor with a built-in Tobii T-60 eye-tracking system (Tobii Tech., Stockholm, Sweden). Additional accessories such as joystick, bluetooth Wiimote (Nintendo Co.), data-glove (5th Dimension Technologies), keyboard, microphone, headphones, or video camera are connected depending on the requirements of the particular experiment.
Data collection, stimulus presentation, synchronization with the different input/output accessories, and real-time analysis and visualization are accomplished using our BCI2000 software8,9. BCI2000 is a freely available general-purpose software system for real-time biosignal data acquisition, processing and feedback. It includes an array of pre-built modules that can be flexibly configured for many different purposes, and that can be extended by researchers' own code in C++, MATLAB or Python. BCI2000 consists of four modules that communicate with each other via a network-capable protocol: a Source module that handles the acquisition of brain signals from one of 19 different hardware systems from different manufacturers; a Signal Processing module that extracts relevant ECoG features and translates them into output signals; an Application module that delivers stimuli and feedback to the subject; and the Operator module that provides a graphical interface to the investigator.
A number of different experiments may be conducted with any given patient. The priority of experiments will be determined by the location of the particular patient's electrodes. However, we usually begin our experimentation using the SIGFRIED (SIGnal modeling For Realtime Identification and Event Detection) mapping method, which detects and displays significant task-related activity in real time. The resulting functional map allows us to further tailor subsequent experimental protocols and may also prove as a useful starting point for traditional mapping by electrocortical stimulation (ECS).
Although ECS mapping remains the gold standard for predicting the clinical outcome of resection, the process of ECS mapping is time consuming and also has other problems, such as after-discharges or seizures. Thus, a passive functional mapping technique may prove valuable in providing an initial estimate of the locus of eloquent cortex, which may then be confirmed and refined by ECS. The results from our passive SIGFRIED mapping technique have been shown to exhibit substantial concurrence with the results derived using ECS mapping10.
The protocol described in this paper establishes a general methodology for gathering human ECoG data, before proceeding to illustrate how experiments can be initiated using the BCI2000 software platform. Finally, as a specific example, we describe how to perform passive functional mapping using the BCI2000-based SIGFRIED system.

Recording Human Electrocorticographic ECoG Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.

神経科学の研究とリアルタイム機能皮質マッピングのためのヒトElectrocorticographic（ECOG）信号を記録

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

の解離、文化、および分析<em&gt;アフリカツメガエル</em&gt;胚性網膜組織

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

細胞培養およびひよこ胚における軸索誘導受容体のダイナミクス評価におけるpHluorinの使用

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

閉ループ神経のロボット実験は、神経回路網の計算特性を試験すること

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

脳組織における測定細胞外イオンの信号のための、二連と同心微小電極

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

原子間力顕微鏡、インパクトインデント、およびレオを使用した脳組織のマルチスケール力学的性質を特徴づけます

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

動的なを促進するために齧歯動物のミクログリアの分離と培養分岐無血清培地の形態

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Presynapse Formation Assay Using Presynapse Organizer Beads and &ldquo;Neuron Ball&rdquo; Culture

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.

Presynapse Formation Assay Using Presynapse Organizer Beads and &amp;ldquo;Neuron Ball&amp;rdquo; Culture

Presynapse formation is a dynamic process including accumulation of synaptic proteins in proper order. In this method, presynapse formation is triggered by beads conjugated with a presynapse organizer protein on axonal sheets of &#8220;neuron ball&#8221; culture, so that accumulation of synaptic proteins is easy to be analyzed during presynapse formation.

シナプス前オーガナイザービーズと「ニューロンボール」培養を用いたシナプス形成アッセイ

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

胚性マウス小脳から単離されたニューロンの一次培養

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...