The authors sincerely thank the Canadian Institutes of Health Research MOP 130258 and the Star Foundation for Children&#8217;s Health Research, along with the Molly Towell Foundation, for the provision of the research facility and financial support. The authors also sincerely thank Dr. Kinkead Richard for manuscript proofreading and advice.

この方法は、ラバル大学の動物倫理委員会（プロトコル＃2012から170）で許可されている動物対象、の使用を必要としました。 1.セットアップと準備<ol><li>ソリューション<ol><li>以下のレシピ7,17に従ってaCSFのストック溶液を準備します。濃度の変動と他のレシピは文献で入手可能です。 1ヶ月まで4℃で保存原液。 <ol><li>塩溶液：塩化ナトリウム（最終129 mM）のの75.39グラムを追加します。 KCl（最終3.35ミリモル）の2.5グラム。 NaH 2 PO 4 0.81gの（最終0.58ミリモル）。 MgCl 2を2.33gの（最終1.15ミリモル）。塩化カルシウムの1.85グラム（1.26ミリモル）。蒸留水で1リットルに記入後、蒸留水800mlに溶解します。 </li><li>重炭酸塩溶液：のNaHCO 3（21 mMの最終）の17.65グラムを追加します。その後、蒸留水で1リットルに記入蒸留水900mlに溶解します。重炭酸塩濃度の変動は、pHの変化をもたらします。 </li><li>グルコース溶液：54.06を追加グルコースのグラム（30mMの最終）。蒸留水で500ミリリットルに記入後、蒸留水400mlに溶解します。 </li></ol></li><li>塩溶液100ml、重炭酸塩溶液100ml、蒸留水1L中のグルコース溶液50mlを希釈することによりaCSFのを準備します。使用するまで4℃で保存します。 </li><li>温暖化とそれが破断するまでガラス管を延伸してガラス電極を準備します。使用前に、先端ダウンサンド。電極は、それがきれいに留まるように多くの時間を再利用することができます。 </li></ol></li><li>実験のセットアップ 注：セットアップの詳細は、図2に示されています。 <ol><li>平均化、データ収集システム、温度調節器とポンプを動かし、アンプの電源をオンにします。生の信号（2.5 kHzの）のデジタル変換、アナログのサンプリング・レート、信号増幅（ゲイン= 10,000）、濾過（高しきい値、5 kHzの低閾値、10 Hz）を確認してください。 </li><li>カルボゲンでaCSFのバブルとそれをボトルを記入した（95％のO 2 </suB&gt;、5％CO 2）。 /分4ミリリットルで記録チャンバー（容量5ml）中のbubbled-と温め-aCSFの流れを誘導します。 ℃〜26（±1）で記録チャンバー内の温度を保持します。 pHは、これらの標準条件で7.4にする必要があります。 </li><li>解剖室の近くに50ミリリットル注射器でRTとカーボゲン-バブリングaCSFの50mlのを保管してください。 </li><li>コンピュータの電源をオンにし、記録ソフトウェアを起動します。 </li></ol></li></ol> 2.解剖<ol><li>計量し、視覚的に（女性は短い肛門性器の距離を持っている一方で男性は、長い肛門性器の距離を持っている、女性の性器はピンクですしながら、性器は、多くの場合、暗い男性）動物の性別を決定します。 </li><li> - 、注射（/ kgを4 mlでequitensine）19または揮発性麻酔薬（イソフルラン20またはエーテル21）の吸入cryoanesthesia（5分18完全4氷で動物を浸す）：以下のいずれかのオプションにより、新生児げっ歯類を麻酔。 ConfiRM足引っ込め反射の欠如によって麻酔の適切な面。 </li><li>ベンチに動物を置き、腹面を下に。セクション（皮膚や頭蓋骨を通して見える）ブレグマのレベルでメスで冠状に頭の吻側部分。動物が麻酔された直後に、この手順を実行します。 </li><li>第身体冠状前方メンバー下メスで。 </li><li>外科手術と薄いはさみやペンチで皮膚、筋肉、脂肪組織や臓器を削除します。骨はこの年齢でソフトであるため、神経系に損傷を与えないように注意が必要です。ベンチでこの手順を実行します。 </li><li>解剖室で準備を置きます。準備に酸素シリンジに保存されているaCSFのを使用してください。この時点から、記録の最後に、顕微鏡を使用しています。 </li><li>準備の背面には、外科的な薄いハサミやペンチを有する媒体軸に沿って尾の部分に吻側から頭蓋骨と脊椎骨を切りました。カット頭蓋骨を開きますおよび神経組織を露出させるために椎骨。準備に酸素するために注射器でstrored aCSFのを使用してください。 </li><li>ペンチで、くも膜、神経組織の表面を覆う薄い組織を除去します。神経組織に対する軟膜血管を保持します。準備に酸素シリンジに保存されているaCSFのを使用してください。 </li><li>背面を下にして準備を置き、慎重に所定の位置に準備を保持しながらはさみで神経と結合組織を切断することにより、脳幹および脊髄をダウンさせます。背部アプローチも可能です。できるだけ長く根や神経をしてください。脳幹と脊髄を分離するために骨を削除します。準備に酸素シリンジに保存されているaCSFのを使用してください。 </li><li>小脳を取り外し、メスでそれらを区画することにより吻側構造を残り。準備に酸素するために、針に保存されているaCSFのを使用してください。 </li><li>必要に応じてexperimenに応じてタルデザイン、下小脳動脈22の冠状断面の前部によってメスで橋を削除します。全体の橋を維持することは、ラットでリズムを遅くし、完全にマウスでそれを阻害することに注意してください。橋の唯一の尾の部分を含んで準備は、C4のルートに安定した周波数での呼吸のような活性を示します。 </li></ol> 3.録音<ol><li>録音室、腹顔アップで準備を置きます。脊髄と脳幹の吻側 - 大部分の最下部にあるピンと準備を修正。 </li><li>部分的にaCSFのに電極を埋めるために、注射器のピストンを引き出して - ：その針によって電極に結合された注射器を用いて、電極（225〜150μmのガラス先端直径）でうつ病を誘発します。 </li><li>マイクロマニピュレーターを使用して、慎重に第腹側根の1に近い電極を配置します。他の腹側根も呼吸様活性（ 電子を提示することができます。グラム。、XII脳神経、C1前根）。 </li><li>優しく神経細根を吸引するためにピストンを引き抜くことにより、シリンジを介して電極槽内のうつ病を誘発します。その後、慎重に脊髄に対してそれを適用するために電極を移動します。 注：理想的には細根の大きさは、電極開口部の大きさに一致するため、電極の内部と外部の区画の間にシールを作成します。差動増幅器は、一般に、記録のために使用されるので、電極の内部と記録チャンバーとの間の開口部は、信号の品質を低下させ、それが困難なバックグラウンドノイズを除去することができます。 </li><li>記録を開始します。 </li><li>正常酸素条件下で調製することによって製造さリズムを記録する（ すなわち、aCSFのは、カルボゲンを通気：95％のO 2および5％CO 2）で少なくとも20分の準備のベースラインパラメータを決定します。 </li><li>にカーボゲンバブリングaCSFのから灌流を切り替えます刺激aCSFの15分間（ すなわち、低酸素刺激のためのCO 2、95％N 2及び5％をバブリング）。実験プロトコルに依存した露光時間を変更します。記録と動物のデータシート上の露光時間を記録します。 aCSFのボトルやチューブの外側にガス拡散を避けるために、可能な限りチャンバとの間にステンレス鋼管を使用してください。 </li><li>回収記録のために少なくとも15分間、標準カーボゲンバブリングaCSFのに戻って灌流を切り替えます。記録と動物のデータシートでこれを記録します。 </li><li>録音を終了します。 </li></ol> 4.統計分析<ol><li>統合された信号から（バースト/分で表される）、毎分バースト数と周波数、及びベースライン（MVで表される）バーストのピークとの間の差として、振幅、持続時間などのバースト期間を計算します（複数で表される）、バーストの最後に始まり、CUR下の領域としてバースト領域統合された信号（S・mVので表される）（ 図3）にバーストのVEの。 </li><li>各条件について録音の最後の5分間の平均値として、周波数、振幅、正常酸素下でのバースト期間およびバースト領域（ベースライン）、低酸素（刺激）および低酸素後（ポスト刺激）回復条件を計算します。記録チャンバーに潅流およびaCSFの均質化を切り替えるとの間に遅延があるため、各条件の最初の部分を分析しないでください。 </li><li>その後、対応する記録のためのベースライン値のパーセンテージとして、刺激（ すなわち、低酸素症）およびポスト刺激（ すなわち、低酸素後）回復値を表現。平均±SDとして結果を表現</li></ol>

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S., Denavit-Saubie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+NMDA+receptors+in+the+respiratory+phase+transition+is+different+in+the+adult+guinea+pig+in+vivo+and+in+the+isolated+brain+stem+preparation.">Involvement of NMDA receptors in the respiratory phase transition is different in the adult guinea pig in vivo and in the isolated brain stem preparation.</a> J Neurophysiol. 74, 770-778 (1995).</li><li>Otsuka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+volatile+anesthetics+on+respiratory+activity+and+chemosensitivity+in+the+isolated+brainstem-spinal+cord+of+the+newborn+rat.">Effects of volatile anesthetics on respiratory activity and chemosensitivity in the isolated brainstem-spinal cord of the newborn rat.</a> Hokkaido Igaku Zasshi. 73, 117-136 (1998).</li><li>Gestreau, 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=Task2+potassium+channels+set+central+respiratory+CO2+and+O2+sensitivity.">Task2 potassium channels set central respiratory CO2 and O2 sensitivity.</a> PNAS. 107, 2325-2330 (2010).</li><li>Caravagna, C., Soliz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+and+MEK+molecular+pathways+are+involved+in+the+erythropoietin-mediated+regulation+of+the+central+respiratory+command.">PI3K and MEK molecular pathways are involved in the erythropoietin-mediated regulation of the central respiratory command.</a> Respir Physiol Neurobiol. 206C, 36-40 (2014).</li><li>Tree, K., Caravagna, C., Hilaire, G., Peyronnet, J., Cayetanot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anandamide+centrally+depresses+the+respiratory+rhythm+generator+of+neonatal+mice.">Anandamide centrally depresses the respiratory rhythm generator of neonatal mice.</a> Neuroscience. 170, 1098-1109 (2010).</li><li>Arata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+activity+of+the+neonatal+dorsolateral+pons+in+vitro.">Respiratory activity of the neonatal dorsolateral pons in vitro.</a> Respir Physiol Neurobiol. 168, 144-152 (2009).</li><li>Onimaru, H., Homma, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+functional+neuron+group+for+respiratory+rhythm+generation+in+the+ventral+medulla.">A novel functional neuron group for respiratory rhythm generation in the ventral medulla.</a> J Neurosci. 23, 1478-1486 (2003).</li><li>St-John, W. M., Paton, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizations+of+eupnea,+apneusis+and+gasping+in+a+perfused+rat+preparation.">Characterizations of eupnea, apneusis and gasping in a perfused rat preparation.</a> Respir Physiol. 123, 201-213 (2000).</li></ol>

The authors have no competing financial interests or conflicts of interests to disclose.

呼吸活性の正確な定量化が困難な場合があります。実際、呼吸は自動および自主両方にすることができます関数であり、それは環境、身体のニーズ、感情的な状態や行動に応じて変調されます。この技術の利点は、呼吸のコマンドを生成する原因神経要素の単離であります。したがって、脳幹・脊髄調製物とプレチスモグラフィの電気生理学的記録は、それぞれ、in vitroおよびin vivoで?...

呼吸は、二原子酸素（O 2）取り込みおよび二酸化炭素（CO 2）の除去を可能にする、脳によって制御される、複雑で重要な活動です。中央呼吸ドライブは、両方の哺乳類1、両生類2、爬虫類3、鳥4と魚5に脳幹に位置する複雑なネットワークによって生成されます。呼吸の研究は、生体内で処理することができたとしても、正確なメカニズムの調査は、呼吸制御ネットワークへの直接アクセスを必要とします。この目的を達成するために、エイドリアンとBuytendijkは脳幹表面記録に鰓換気5に関連して生成されたリズムを配置された電極れる減少金魚の準備を開発しました。このアプローチは、その後、新生児げっ歯類で使用するために1984年6で鈴江によって適応されました。この準備の出現は、呼吸器、神経生物学における重要な進歩をもたらしました。それは比較的単純であるので、この技術は時間提示しましたEREは、リズミカルな運動行動や新生児げっ歯類におけるその起源の基本的な調査の幅広い影響を受けやすいです。 この方法の全体的な目標は、吸気活動の神経相関、呼吸器ネットワークによって生成される仮想の呼吸と呼ばれる呼吸のようなリズムを記録することです。この方法は、野生型7と8トランスジェニック動物の両方における呼吸変動または薬理学的に吸気応答を標的、研究目的の広い範囲で使用することができます。記録された信号の生理学的な関連性についての実験は、感覚求心性なしに、低温で行われ、およびaCSFの中でグルコースとO 2の濃度が高い条件下では、問題が提起されていることを考えます。 in vivoおよびin vitro条件の間に明確な違いがありますが（ 例えば 、吸気バーストの周波数）は実際には存在することを残っています呼吸ネットワーク6のコア要素は、それが可能な重要な恒常性維持機能9,10に関連した強固なリズムを研究することを可能にします。 開発し、この技術の使用の理論的根拠は特に新生児で、 生体内でほとんどアクセス可能な呼吸器ネットワークの脳幹要素への直接アクセスを容易にすることです。脳幹は、厳密に制御された条件の下に置かれている：記録リズムが肺や頸動脈体からの末梢の求心性入力によって変調されていない、研究では、中央呼吸ドライブ自体11に集中することができます。したがって、このアクセ​​スは、刺激を適用し、出力信号を記録するために利用されます。録音をプレチスモグラフィとは対照的に、呼吸リズムは、それが困難な正確な刺激を適用すること、身体（ 例えば 、肺の膨満、周辺化学センサー）を通じてそのすべてのコンポーネントによって変調されます。 におけるewbornラット、プロトコルは、人工脳脊髄液（aCSFの）中で維持孤立脳幹および短縮型脊髄の4番目の前根信号を、記録で構成されています。脳幹・脊髄調製物によって生成されたリズムが吸気信号 9に連結されている個々の遅いバーストで構成されています。 7 -絶縁脳幹・脊髄調製物は、簡単に4（P4 P0）に出 ​​生後0日目からラットで記録すること。このアプローチは、一般に呼吸器ネットワークの低酸素応答、また高炭酸ガス血症、アシドーシスまたは薬物に対する応答を評価するために使用されます。急性低酸素症のプロトコルがここに提示されます。この刺激は、aCSFの中のO 2の撤退によって得られます。このアプローチは、一般的に低酸素傷害に対する耐性と反応性を評価するために使用されます。プロトコルは、低酸素曝露（ 図1）12 の端まで最初の分からリズムうつ病を誘発します。この凹部が逆になります低酸素後の回復12の間。実験計画については、それは脳幹の吻側部に位置橋は、リズム発生器8に対する阻害作用を有することに注意することが重要です。このように、完全な脳幹と吻側脊髄の調製物は、下のリズムを表示します。記録のために単離されたサンプル中の橋を含めると、実験13の目標に応じて決定されます。延髄ネットワーク上の橋影響の研究結果14を比較するために橋ととせずに録音を必要とするであろう。また、この手法の利点の一つは、脳および/ ​​または間脳領域15,16を含むように製剤の吻側部分を拡張することが可能先斗、延髄呼吸ネットワーク上のこれらの領域の効果を評価することの可能性があります。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sylgard</td>
			<td>Sigma Aldrich</td>
			<td>761036-5EA</td>
			<td>Use under hood</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>SOD002</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bioshop</td>
			<td>POC888</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Bioshop</td>
			<td>CCL444</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Bioshop</td>
			<td>MAG510</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Bioshop</td>
			<td>SPM306</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Bioshop</td>
			<td>GLU501</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Linde</td>
			<td>343-02-0006&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction electrode</td>
			<td>A-M Systems, Everett, WA, USA</td>
			<td>model 573000</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential AC amplifier</td>
			<td>A-M Systems, Everett, WA, USA</td>
			<td>model 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Moving averager</td>
			<td>CWE, Ardmore, PA, USA</td>
			<td>model MA-821</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition system</td>
			<td>Dataq Instruments, Akron, OH, USA</td>
			<td>model DI-720</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart software</td>
			<td>ADInstruments, Colorado Springs, CO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism sofware</td>
			<td>Graphpad, La Jolla, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection chamber</td>
			<td>Plastic box (e.g. petri box) will do</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recording chamber</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Kanetec, Bensenville, IL, USA</td>
			<td>MB</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instrument Inc, Sarasota, FL, USA</td>
			<td>KITE-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Kanetec, Bensenville, IL, USA</td>
			<td>MB</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson, Middleton, WI, USA</td>
			<td>MINIPULS 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophysiology on isolated brainstem-spinal cord preparations from newborn rodents allows neural respiratory network output recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

冒頭で述べたように、この技術の最も重要な利点の一つは、脳幹への直接のアクセスは、様々な刺激を適用することです。一例として、低酸素状態は、ここで適用した。 図1。ABは両方正常酸素および低酸素条件下で、完全なプロトコルの記録を表示する。 図1.CE、すなわち酸素正常状態（に記録されたリズムを表示し、aCSFの、95％のO 2および5％でバブ...

Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

新生児げっ歯類から単離された脳幹・脊髄調製物の電気生理学は、ニューラルネットワーク呼吸器の出力の記録を可能にします

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

electrophysiology-on-isolated-brainstem-spinal-cord-preparations-from

Research

JoVE Journal

Neuroscience

8.5K ビュー.  Laval University. The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

ビデオ: Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

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

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

胚の脊髄から交連ニューロンの解剖と文化

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

神経科学

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髄電気生理学II：細胞外吸引電極の作製

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress has been made in our understanding of differences in response properties of neurons in different cortical layers5-8, yet there is still a great deal left to learn about whether and how neuronal populations encode information in a laminar-specific manner.
Existing multi-electrode array techniques, although informative for measuring responses across many millimeters of cortical space along the cortical surface, are unsuitable to approach the issue of laminar cortical circuits. Here, we present our method for setting up and recording individual neurons and local field potentials (LFPs) across cortical layers of primary visual cortex (V1) utilizing multi-contact laminar electrodes (Figure 1; Plextrode U-Probe, Plexon Inc).
The methods included are recording device construction, identification of cortical layers, and identification of receptive fields of individual neurons. To identify cortical layers, we measure the evoked response potentials (ERPs) of the LFP time-series using full-field flashed stimuli. We then perform current-source density (CSD) analysis to identify the polarity inversion accompanied by the sink-source configuration at the base of layer 4 (the sink is inside layer 4, subsequently referred to as granular layer9-12). Current-source density is useful because it provides an index of the location, direction, and density of transmembrane current flow, allowing us to accurately position electrodes to record from all layers in a single penetration6, 11, 12.

A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers.

マルチ連絡ラミナーの電極の録音を使用してローカルネットワークの処理を検討

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

動作げっ歯類の可動シリコンプローブによるニューロンの大規模なレコーディング

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行性蛍光標識は、特定された神経細胞から標的細胞外シングルユニット記録を可能にします<em生体内で&gt;</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

無傷のゴキブリ神経系の神経回路の録音

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

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 ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

マイクロ電極アレイにおけるニューロン細胞培養の刺激に対するネットワーク応答の時間依存的増加

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Thy1を用いた一次体性感覚野における神経活動のイメージング-GCaMP6s トランスジェニック マウス

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

新生児げっ歯類から単離された脳幹・脊髄調製物の電気生理学は、ニューラルネットワーク呼吸器の出力の記録を可能にします

要約

さらに動画を探す

この動画の章

胚の脊髄から交連ニューロンの解剖と文化

脊髄電気生理学II：細胞外吸引電極の作製

マルチ連絡ラミナーの電極の録音を使用してローカルネットワークの処理を検討

動作げっ歯類の可動シリコンプローブによるニューロンの大規模なレコーディング

逆行性蛍光標識は、特定された神経細胞から標的細胞外シングルユニット記録を可能にします

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

無傷のゴキブリ神経系の神経回路の録音

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

マイクロ電極アレイにおけるニューロン細胞培養の刺激に対するネットワーク応答の時間依存的増加

Thy1を用いた一次体性感覚野における神経活動のイメージング-GCaMP6s トランスジェニックマウス

新生児げっ歯類から単離された脳幹・脊髄調製物の電気生理学は、ニューラルネットワーク呼吸器の出力の記録を可能にします

要約

さらに動画を探す

この動画の章

胚の脊髄から交連ニューロンの解剖と文化

脊髄電気生理学II：細胞外吸引電極の作製

マルチ連絡ラミナーの電極の録音を使用してローカルネットワークの処理を検討

動作げっ歯類の可動シリコンプローブによるニューロンの大規模なレコーディング

逆行性蛍光標識は、特定された神経細胞から標的細胞外シングルユニット記録を可能にします

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

無傷のゴキブリ神経系の神経回路の録音

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

マイクロ電極アレイにおけるニューロン細胞培養の刺激に対するネットワーク応答の時間依存的増加

Thy1を用いた一次体性感覚野における神経活動のイメージング-GCaMP6s トランスジェニック マウス

Thy1を用いた一次体性感覚野における神経活動のイメージング-GCaMP6s トランスジェニックマウス