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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<li>Caravagna, C., Soliz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PI3K+and+MEK+molecular+pathways+are+involved+in+the+erythropoietin-mediated+regulation+of+the+central+respiratory+command%5BTitle%5D)+AND+%22Respir+Physiol+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((Anandamide+centrally+depresses+the+respiratory+rhythm+generator+of+neonatal+mice%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Respiratory+activity+of+the+neonatal+dorsolateral+pons+in+vitro%5BTitle%5D)+AND+%22Respir+Physiol+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((A+novel+functional+neuron+group+for+respiratory+rhythm+generation+in+the+ventral+medulla%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Characterizations+of+eupnea%2C+apneusis+and+gasping+in+a+perfused+rat+preparation%5BTitle%5D)+AND+%22Respir+Physiol%22%5BJournal%5D)">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><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>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioshop</td><td>CCL444</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioshop</td><td>MAG510</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Bioshop</td><td>SOB999</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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&amp;nbsp;</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

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 ...

神経科学

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髄電気生理学

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

生物学

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

等電点電気泳動脳の状態の誘導は、神経細胞の興奮に内因性シナプス活性の影響を調査するために、<em&gt;インビボ</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

新しい方法を用いた自由に動く新生児ラットの脳波記録

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;インビボ</em&gt;麻酔ラットにおけるHyperdirect経路からの局所場ポテンシャルとマルチユニット活性の電気生理学的記録

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

皮質局所電界電位、心電図、筋電図、呼吸リズム自由行動ラットからの同時録音

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

リズミカルにアクティブな準備体外新生児齧歯動物脳幹-脊髄損傷で薄くスライス

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

マウスにおける脳幹誘発応答オーディオメトリーにおけるデータ獲得と分析

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

C-FOSタンパク質免疫組織学的検出：特定の生理反応に関与する中央経路のマーカーとして有用ツール<em&gt;インビボ</em&gt;と<em&gt; ex vivoで</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

<meta charset="utf8"></head><body>マウス脳深部構造のための角度付きシリコン多電極記録

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many brain regions. However, recording neuronal activity from deep caudal structures like the brainstem using multi-channel probes remains challenging. A significant concern is finding a trajectory for probe insertion that avoids large blood vessels, such as the superior sagittal venous sinus and the transverse venous sinus. Injuring these large veins can cause extensive bleeding, damage to the underlying brain tissue, and potentially death. This approach describes targeting brainstem structures by coupling anterior coordinates with an angled approach, allowing the recording probe to penetrate the brain below high-risk vascular structures. Compared to a strictly vertical approach, the angled approach maximizes the number of brain regions that can be targeted. Using this strategy, the ventrolateral periaqueductal gray (vlPAG), a brainstem region associated with REM sleep, can be reproducibly and reliably accessed to obtain single-unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia. The ability to record neuronal activity in the vlPAG and surrounding nuclei with high temporal resolution is a step forward in advancing the understanding of the relationship between REM sleep and anesthesia.

<meta charset="utf8"></head><body>著者スポットライト:マウスにおける麻酔誘発睡眠経路とニューロン興奮性の調査

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

頭部固定マウスの脳幹からの急性単一ユニット多電極記録

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many ...