The authors thank Marin Manuel and Olivia Goldman-Szwajkajzer for their help in taking the photographs. The authors also thank Arjun Masukar and Tobias Bock for proofreading the manuscript. Financial supports were provided by the Agence Nationale pour la Recherche (HYPER-MND, ANR-2010-BLAN-1429-01), the NIH-NINDS (R01NS077863), the Thierry Latran Foundation (OHEX Project), the French association for myopathy (grant number 16026) and Target ALS are gratefully acknowledged. Felix Leroy was the recipient of a &#34;Contrat Doctoral&#34; from the Ecole Normale Sup&#233;rieure, Cachan.

実験は、欧州指令（609分の86 / CEEと2010から63-UE）とフランスの法律に従って行われ、パリデカルト大学の倫理委員会によって承認されました。 1.脊髄スライスの準備<ol><li>毎日、以下の溶液を調製するか、事前に1日。場合は95％O 2および5％CO 2で一晩、バブルを維持し、密閉瓶で冷蔵保管してください。 <ol><li> 低のNa +人工脳脊髄液（ACSF）を準備：3のKCl、1 mMののNaH 2 PO 4、230mMのスクロース、26 mMの炭酸水素ナトリウム、0.8mMのCaCl 2を、8のMgCl 2、25 mMグルコース、0.4 mMのアスコルビン酸、 1 mMののNa-kynurenate、2 mMのピルビン酸ナトリウム。 95％O 2および5％CO 2（pH7.4）でバブル。 Na-kynurenateは、多くの場合、溶解することが困難であるので、材料の表に記載されているものを購入することを確認します。 </li><li> K-グルコン酸ソリューを準備ン：KOHでpH7.4に調整130mMのK-グルコン酸塩、15mMのKClを、0.05 mMのEGTA、20mMのHEPES、25 mMグルコース、1mMのNaをkynurenate、2 mMのピルビン酸ナトリウム、。 </li><li> 準備ACSF：130のNaCl、2.5mMのKClを、2mMのCaCl 2を、1mMのMgCl 2、1mMののNaH 2 PO 4、26 mMの炭酸水素ナトリウム、25 mMグルコース、0.4mMのアスコルビン酸、2mMのナトリウムピルビン酸。 95％O 2および5％CO 2（pH7.4）でバブル。 </li><li>解剖の開始前に、K-グルコン酸溶液を80ミリリットルで2％寒天を溶解し、60℃で保温。 </li></ol></li><li> 心臓内灌流 <ol><li> P2からP11までの年齢に至るまで、雌雄のマウスにこの準備を行います。 </li><li> 25mMのペントバルビタールナトリウム（50mg / kg）の0.1mlを腹腔内注射してマウスを麻酔。 </li><li>針やテープを使用して、シリコンを充填した大型シャーレに背の上でマウスを固定化します。解剖顕微鏡を使用して、解剖の残りの電子。 </li><li>胸骨の先端を持ち、胸を持ち上げ、細かいハサミを使用して、振動板をカット。その後、心臓を露出するためにリブを切断することにより、両側の胸を開きます。 </li><li> 27G針で左心室を穿刺する前に、右心房をカットします。 </li><li>氷のように冷たい低いのNa + ACSFで灌流します。 30秒後に、低いのNa + ACSFは、心房から流出見られるべきです。ナトリウムの低い量はスパイクから細胞を防止し、解剖時に細胞死を減少させます。 </li><li>血液が排出される際に肝臓が黄色になるまで解剖顕微鏡下で心臓に針を保持し続けます。 </li></ol></li><li> 脊髄の解剖 <ol><li>動物の首をはねるとそのお腹の上に置きました。 </li><li>素早くバック（ 図1A1）の皮膚を除去。肩を介して2つのカットを行い、胸のケージ（ 図1A2）を下って行きます。その後リットルとしてコードをカット動物の下部からリブの始まりと脊柱を分離するために、尾部でできるだけOW。再び動物を裏返し、まだリブに取り付けられた内臓を取り除きます。 </li><li>別の脊柱を移し、より小さなシリコンで満たされたペトリ皿と背側を上（ 図1A）、それを保持するために4虫ピンを使用します。 </li><li>背側の椎弓切除を行い、吻側端（ 図1B）から脊髄を露出させながら、連続的に（約4℃）カーボゲンバブリングACSFで動物を灌流。これを行うには、骨と脊髄の間の微妙なハサミの先端を挿入し、白質から離れて滞在することを確認して、吻側端から少しずつ骨を切りました。それぞれの側に代替すでに（ 図1B1）カット骨のバンドを離れて保つためにピンセットを使用している間。 </li><li>利用可能な最小春のはさみとピンセットを使用して、硬膜とカットを持ち上げますはさみで脊髄を損傷しないようにするために、両側に硬膜の緩い部分を保持したまま。吻側 - 尾側軸に沿って切断。 注：硬膜は、半透明の連続膜です。この年齢で軟膜はあまりにも脆く、解剖やスライシング（ 図1B2）の間に離れて来ます。 </li><li>硬膜は、吻側側から、使用に優しくハーフカット脊柱によって形成された溝の左側にコードをプッシュし、右側の腹側と背側根をカットする鈍いガラスやプラスチックの先端を除去されると、彼らは（少なくとも数mm、 図1B2）コードを入力する場所から遠いです。 </li><li>常に頭側から尾側に行く、左側の操作を繰り返します。左利きの場合は、左側から開始し、右側に移動します。 </li></ol></li><li> 寒天に埋め込みます <ol><li>脊柱の外にコードをスリップ。突き止めるために小さい虫ピンを使用しますコード背面を上にしてと繊細にはまだ（ 図1C1）それに接続されている膜の任意の部分を削除します。 </li><li>洗浄後は、両端（ 図1C2）をトリミングします。コードを操作し、その向き（ 図1C2の矢印）を注意することは、コードの前部に曲がった虫ピンを挿入します。その後、氷冷K-グルコン酸溶液に脊髄を転送します。 注：このソリューションは、CSFの細胞内の組成を模倣し、運動ニューロンが26をカットされます一度浸透圧ショックで死ぬから細胞を防ぐことができます。 </li><li>脊髄がイントラセルラーソリューション内になると、乾燥槽の外に寒天を入れたビーカーを取り、氷と水の混合物でそれをクールダウン。 </li><li>温度を測定しながら攪拌してください。温度が到達すると38°Cは、虫ピンでそれを保持し、脊髄を浸すとダウンそれを吻側側に配置します。脊髄は、STのようであることを確認してくださいraight可能な限り、離れて尾部、壁からやや上向き（ 図1D1）。 </li><li>寒天は、可能な限り迅速に固化することを可能にするために氷と水の混合物中にビーカーを残します。それは場所にとどまり、コードが直（ 図1D1）可能な限りであることを確認してください。 </li></ol></li><li> スライス <ol><li>固化した後、ブロックのベースはコード（ 図1D2の矢印）の腰部に35°の角度になるように脊髄を含む寒天ブロックをカット。背面は、ベース（ 図1D2）から反対されるべきです。 注：これは、彼らが終了し、そこから前根に運動ニューロンプールの連続性を維持するための手順における重要なステップです。 </li><li>シアノアクリレート接着剤を使用して、ビブラのチャンバ内にブロックを接着。維持するために、K-グルコン酸溶液中に浸し、酔っぱらった冷凍K-グルコン酸溶液を加え（2℃以下）チルド風呂。 </li><li> （その曲率と大きい直径によって識別）腰部の400μmの厚さにスライス - 350をカットします。その正しい向き（1.4.1を参照。）で、腹面に背側から切り取り、スライス連続より吻側を作るためにブレードを使用しています。一般的に、2mm以上を拡張する前根と4-5の適切なスライスがあります。 10°の角度、70 Hzの振動周波数とスライスの10mm /分の速度：次のパラメータを使用します。 </li></ol></li><li> インキュベーション <ol><li> 34℃でACSFにスライスを転送します。約30分後、RTにスライスを冷却し、記録セッションを開始します。 </li></ol></li></ol><img alt="図1" src="/files/ftp_upload/54525/54525fig1.jpg" /> 図1.解剖 A1：脊柱を露出させるために背中の皮膚の除去A2：脊柱を解放するために肩や肋骨の切断B1：脊椎のコラムはシリコンで満たされたペトリ皿（背側を上、左尾側）に固定B2：脊髄露出し、解剖と同じC1：脊髄孤立。（左吻側側）C2：埋め込みする準備ができて脊髄（腹側まで、吻側側左）。吻側側の小さい虫ピンに注意してくださいD1：寒天ビーカー（吻側側ウ、底に面した腹側）で脊髄D2：埋め込まれた脊髄と寒天ブロックをカット。 35°の角度にブロックのベースと腰膨大（矢印）と脊髄のフォームを注意してください。スケールバー1センチメートル。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54525/54525fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 2.チャンバー内にスライスを配置します注：Veはntral根は可変サイズです。 <ol><li>事前に40から170ミクロンまでの範囲の先端径を有する様々なピペットの箱を用意します。吸引ピペットを準備するには、長いテーパーで、多くのピペットを準備します。ダイヤモンドナイフを使用して、異なる位置で切断してください。その後、解剖顕微鏡下で、ピンセットで先端を押すことでそれを破ります。 </li><li>記録顕微鏡から室を削除し、解剖顕微鏡下に置きます。 </li><li>吸引刺激電極上に搭載されるのに十分な長さ（2mm以上）の腹側のルートを含むスライスを選択します。前根とスライス上方（ 図2A）の適切な方向を選択し、スライス（ 図2B）の周りに寒天の残りの部分を残しながら微妙に前根の周りに寒天をカット。 注：寒天は、スライスよりもより強固であるため、これはスライスのアンカーのスレッドが寒天上ではなくスライス上に静止することができますので、Anchorは、組織を損傷することはありません。スライスのアンカーのスレッドが（ 図2Cに赤で示されている）運動ニューロンプールの上にあることを確認します。 </li><li>顕微鏡上にバックチャンバーをマウントし、継続的に1の割合でACSFで記録チャンバーを灌流 - 室温で、2ml /分。ガラスピペットACSFを充填したシリンジに接続を使用して、前根（ 図2Cの矢印）のいずれかを吸います。前根の良い刺激を達成するために、ピペットチップは、前根の周りにタイトにする必要があります。一方の極には、刺激電極と他の浴中で1つ（またはパッチクランプ電極基準に接続されている）であるべきです。 </li><li>所望の細胞型のパッチクランプ記録を達成し、previsouly 10記載されているように前根刺激の効果を記録。 <ol><li>ここでは、データ収集のためのアンプを使用しています。 3 kHzでホールセル記録をフィルタリングします。 10kHzでデジタル化します。 curreにブリッジ抵抗を補償NT-クランプモード。 </li></ol></li></ol><img alt="図2" src="/files/ftp_upload/54525/54525fig2.jpg" /> 図2.前根の準備 ：上を向いて前根と寒天中に埋め込まれた腰椎脊髄スライスB：寒天から解放された前根と腰椎脊髄スライスC：しっかりと前根（矢印）の周りに配置され刺激電極と腰椎脊髄スライス。マウスレポーターR26 トム・17。スケールバー1ミリメートルと交配chrna2-Creをマウス28で赤色蛍光を発現しているレンショウ細胞の位置に注意してください。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54525/54525fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

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D., Zytnicki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+intrinsic+hyperexcitability+does+not+contribute+to+motoneuron+degeneration+in+amyotrophic+lateral+sclerosis.">Early intrinsic hyperexcitability does not contribute to motoneuron degeneration in amyotrophic lateral sclerosis.</a> Elife. 3, (2014).</li><li>Leroy, F., Lamotte d'Incamps, B., Zytnicki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+currents+dynamically+set+the+recruitment+and+firing+properties+of+F-type+motoneurons+in+neonatal+mice.">Potassium currents dynamically set the recruitment and firing properties of F-type motoneurons in neonatal mice.</a> J Neurophysiol. 114 (3), 1963-1973 (2015).</li><li>Lamotte d'Incamps, B., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+composition+and+kinetics+of+the+Renshaw+cell+heteromeric+nicotinic+receptors.">Subunit composition and kinetics of the Renshaw cell heteromeric nicotinic receptors.</a> Biochem Pharmacol. 86 (8), 1114-1121 (2013).</li><li>Lamotte d'Incamps, B., Krejci, E., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+shaping+the+slow+nicotinic+synaptic+current+at+the+motoneuron-renshaw+cell+synapse.">Mechanisms shaping the slow nicotinic synaptic current at the motoneuron-renshaw cell synapse.</a> J Neurosci. 32 (24), 8413-8423 (2012).</li><li>Dugue, G. P., Dumoulin, A., Triller, A., Dieudonne, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-dependent+use+of+co-released+inhibitory+transmitters+at+central+synapses.">Target-dependent use of co-released inhibitory transmitters at central synapses.</a> J Neurosci. 25 (28), 6490-6498 (2005).</li><li>Mentis, G. Z., Siembab, V. C., Zerda, R., O'Donovan, M. J., Alvarez, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+afferent+synapses+on+developing+and+adult+Renshaw+cells.">Primary afferent synapses on developing and adult Renshaw cells.</a> J Neurosci. 26 (51), 13297-13310 (2006).</li><li>Perry, S., 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=Firing+properties+of+Renshaw+cells+defined+by+Chrna2+are+modulated+by+hyperpolarizing+and+small+conductance+ion+currents+Ih+and+ISK.">Firing properties of Renshaw cells defined by Chrna2 are modulated by hyperpolarizing and small conductance ion currents Ih and ISK.</a> Eur J Neurosci. 41 (7), 889-900 (2015).</li><li>Thurbon, D., Luscher, H. R., Hofstetter, T., Redman, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+electrical+properties+of+ventral+horn+neurons+in+rat+spinal+cord+slices.">Passive electrical properties of ventral horn neurons in rat spinal cord slices.</a> J Neurophysiol. 79 (5), 2485-2502 (1998).</li><li>Zengel, J. E., Reid, S. A., Sypert, G. W., Munson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electrical+properties+and+prediction+of+motor-unit+type+of+medial+gastrocnemius+motoneurons+in+the+cat.">Membrane electrical properties and prediction of motor-unit type of medial gastrocnemius motoneurons in the cat.</a> J Neurophysiol. 53 (5), 1323-1344 (1985).</li><li>Cooper, S., Sherington, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gower's+tract+and+spinal+border+cells.">Gower's tract and spinal border cells.</a> Brain. 63, 123-124 (1940).</li><li>Morin, F., Schwartz, H. G., O'Leary, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+of+the+spinothalamic+and+related+tracts.">Experimental study of the spinothalamic and related tracts.</a> Acta Psychiatr Neurol Scand. 26 (3-4), 371-396 (1951).</li><li>Sengul, G., Fu, Y., Yu, Y., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+projections+to+the+cerebellum+in+the+mouse.">Spinal cord projections to the cerebellum in the mouse.</a> Brain Struct Funct. 220 (5), 2997-3009 (2015).</li><li>Russier, M., Carlier, E., Ankri, N., Fronzaroli, L., Debanne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A-,+T-,+and+H-type+currents+shape+intrinsic+firing+of+developing+rat+abducens+motoneurons.">A-, T-, and H-type currents shape intrinsic firing of developing rat abducens motoneurons.</a> J Physiol. 549 (Pt 1), 21-36 (2003).</li><li>Dourado, M., Sargent, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+nicotinic+receptors+underlying+Renshaw+cell+excitation+by+alpha-motor+neurons+in+neonatal+rat+spinal+cord).">Properties of nicotinic receptors underlying Renshaw cell excitation by alpha-motor neurons in neonatal rat spinal cord).</a> J Neurophysiol. 87 (6), 3117-3125 (2002).</li><li>Mitra, P., Brownstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+spinal+cord+slice+preparation+for+recording+from+lumbar+motoneurons+of+the+adult+mouse.">An in vitro spinal cord slice preparation for recording from lumbar motoneurons of the adult mouse.</a> J Neurophysiol. 107 (2), 728-741 (2012).</li><li>Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurotoxicity+of+excitatory+amino+acids+is+produced+by+passive+chloride+influx.">The neurotoxicity of excitatory amino acids is produced by passive chloride influx.</a> J Neurosci. 5 (6), 1483-1489 (1985).</li><li>Olney, J. W., Price, M. T., Samson, L., Labruyere, 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+role+of+specific+ions+in+glutamate+neurotoxicity.">The role of specific ions in glutamate neurotoxicity.</a> Neurosci Lett. 65 (1), 65-71 (1986).</li></ol>

The authors have nothing to disclose.

Oblique slicing of the spinal cord is important since it allows for unilateral stimulation of motoneuron pools and Renshaw cells at a single vertebral segment in a reliable, comprehensive&#160;and specific way. Furthermore, it allows for a quick, elegant&#160;and non-ambiguous identification of recorded motoneurons. Next, we will highlight&#160;the advantages of this&#160;technique compared to other slice preparation methods, and then we will stress out&#160;the most common pitfalls to avoid while performing this procedure.
Most studies using transverse slices base their identification on intrinsic electrical properties 3,14,15,29. However, these vary greatly between motoneuron pools as well as between motoneuron subtypes within a given pool (alpha-, beta- and gamma-motoneurons30). Therefore, studies using size parameters are likely to exclude smaller gamma-motoneurons. On the other hand, Cooper and Sherrington described in 1940 a group of large nerve cells in the ventro-lateral grey matter of the lumbar spinal cord of monkeys and cats which had crossed ascending axons31,32. In addition to being close to the motoneuron&#160;somas, they noted that they appeared histologically indistinguishable from motoneurons. A recent article confirmed the existence of such cells in the mouse33. Due to the large size of these cells and their location, they could have been mistakenly included in former studies. Our identification criterion excludes putative spinal border cells, since their axon does not project in the ventral root, while allowing reliable identification of all motoneurons regardless of their&#160;size. Furthermore, analyzing the intrinsic electrical properties, (e.g., input resistance), is more time consuming than the simple observation of an antidromic action potential.
In addition to using intrinsic electrical properties, some studies using&#160;transverse slices, confirm their identification by performing post&#160;hoc analysis of the molecular marker for motoneurons (such as Islet-1/216) or by visualizing the morphology using biocytin labelings14,17. Such identifications are however tedious and do not provide a direct identification. They are therefore rarely systematically performed.
Some studies tried to make use of mouse lines expressing a genetically-encoded motoneuron&#160;fluorescent marker&#160;(Hb9-GFP mouse line18, ChAT-EGFP transgenic mice,&#160;which express eGFP in cholinergic cells including motoneurons19, or G85R SOD1-YFP transgenic mice, which strongly express the YFP fusion protein in motoneurons17). However, because the genetic marker expression is usually conditioned in time and since the slices are usually taken from embryos or juveniles, the expression of the marker may not be adequate at the age the study is performed. Other studies have used retrograde agent injection to visualize the motoneuron&#160;pool34. We also performed Cholera toxin beta injections into the soleus or the EDL (personal observation). Such labeling techniques are of great value to label a particular motoneuron&#160;pool but require tedious surgery a few days prior to the experiment. Furthermore, the young age at which the slice experiments are being done, makes it significantly harder to specifically target the muscles. Finally, there is a real concern of perturbing the motoneuron by injecting an exogenous agent.
Under optimal conditions we were able to obtain antidromic action potentials in virtually all of the recorded motoneurons. The 35&#176; angle used is critical to obtain ventral roots of sufficient length. In conclusion, the oblique slice preparation offers a quick and reliable way to identify every motoneuron, which is superior to other identification techniques.
As stated in the introduction, only two studies reported successful stimulation of the ventral roots in transverse slices. The first study recorded cervical neurons in the rat and stimulated the white matter where the ventral root stub emerges4. They successfully induced antidromic action potential in 85% of the recorded neurons. We believe their high success rate relied on the fact that at the cervical level, the axons and somas of motoneurons are on the same plane. This is not the case in more caudal slices. Even at the cervical level, their transverse slices only retained stubs of the ventral roots, which are less reliable to stimulate. The second study stimulated the chick embryo dorso-lumbar segment5. However,&#160;they used voltage sensitive dye recording, which lacked the spatial resolution to indicate the success of inducing an antidromic action potentials at the single cell level.
Our slice preparation also offers a superior way to stimulate Renshaw cells as stated in a recent paper28. Using the oblique slices, they were able to record monosynaptic Renshaw cells responses in 46% of the cases, which was a major improvement from the 10% success rate encountered using transverse slices35. Although we could not find a similar comparison for the stimulation of the motoneuron&#160;pools, this observation in the Renshaw cell leads us to believe that motoneurons also retain more connectivity.
Oblique slicing of the spinal cord is a highly demanding technique that requires much practice before reliably obtaining a preparation from which one can record. Using very fine and sharp tweezers and micro-scissors is essential. The time spent dissecting can be quite long (up to 1 hr) as long as the entire procedure is conducted in chilled carbogen-bubbled dissection medium. While some authors did not acknowledge any benefit of intracardiac perfusion in the preparation of slices36, we decided to maintain this step of the procedure. It is hard to objectively measure the benefit of intracardiac perfusion for the quality of the recordings, especially because it varies with age.
The most critical factor is the mouse age. We successfully recorded from mice between P2 and P11 using the above protocol. Past this age, the spinal cord myelin becomes too dense and the biggest cells (the motoneurons) die before slicing, most likely due to oxygen deprivation. After that window, we found it increasingly difficult to obtain healthy slices. Recently, new techniques have been reported to obtain healthy slices in older animals 17,36. They used extra additives to their dissecting and recording medium such as supplementary sources of energy (ethyl-pyruvate), lower Na+ and Cl- concentrations to prevent oncosis of the cells37,38 and polyethylene glycol to limit the effects of the extensive membrane transections that occur during slicing. With such enhancements, they were able to record from up to 6 month old animals.
In the future it would be interesting to attempt oblique slice preparations in adult animals to combine the advantages of the two techniques. Combining the two protocols should prove relatively easy and will provide a reliable tool to confirm motoneuron identity and study the population targeted by axon collaterals of motoneurons in adult mice. Additionally, one could try obtaining oblique slices in the embryo mouse. Noteworthy, our preparation also retains the dorsal roots, which can be stimulated to study the activation monosynaptic Ia innervation to the motoneurons, as well as the activation of dorsal spinal cord neuronal populations. In conclusion, ventral root stimulations offer a reliable tool to confirm motoneuron identity and to study the population targeted by motoneuron axon collaterals.

歴史的に、シャープな電極を用いた運動ニューロンの記録は、例えば、ネコやネズミ1などの大型動物やマウス2で分離された全脊髄のin vivoで行われました。 SOMAの必要なシールのような運動ニューロンへの直接アクセスを呼びかけ1980年代にパッチクランプ記録技術の出現は、視覚的な指導の下で達成されます。したがって、脊髄スライス標本は容易に1990年代初頭3は達成されています。しかし、初期のスライス標本は、多くの場合、前根を刺激することができませんでした。我々の知る限り、唯一の二つの研究は、横断スライスにおける腹側根の成功刺激を報告している、とどれもマウス4,5から得られませんでした。 運動ニューロンプールはその前根を逸脱軸索を保持する - この記事では、新生児マウス（P11 P2）の実行可能な脊髄スライスを達成するための手法を提示します。ベントRALルート刺激は同じ前根から出た運動ニューロンプールのSOMAのに戻し逆行性活動電位をトリガします。また、運動ニューロン担保対象、他の運動ニューロン6-10とレンショウ細胞11-13を励起します 。唯一の運動ニューロンは、前根ダウンその軸索を送っているので、我々は運動ニューロン10を識別physiologicalyするための簡単で確実な方法として、逆行性活動電位の記録を使用します。 運動ニューロンのアイデンティティを確認するために、潜在的に非包括的または誤解を招く電気生理学的および形態学的な規準を使用することに加えて、脊髄運動ニューロン上の最近の研究はまた、面倒で時間のかかる事後染色16に依存していました。このような識別は、通常は記録された細胞のサンプルに対して実行されます。他の特定の戦略は、運動ニューロンの内因性蛍光を発現するマウス系統に依存しています<sup&gt; 17-19。研究はすでにトランスジェニックマウスラインを使用して、必要な場合は、マーカーの発現はまだ変数であるか、しかし、遺伝的にコードされたマーカーを使用すると、若い年齢で難しいかもしれません。また、逆行性活動電位の記録は、セル記録の開始からすべてのマウスに日常的に行うことができます。ネコ、ラットおよびマウスで無傷の脊髄の準備に取り組んで実験者は、確実に1950年の1,2,20,21ため、このような識別技術を使用しています。最適な条件では、記録された運動ニューロンの事実上すべてから逆行性活動電位を誘発することができました。 また、前根刺激は確実に他の運動ニューロン22,23、またはそれらの標的を励起するために使用することができます。レンショウ細胞10,24,25。ここでは、運動ニューロンのSOMAのからの逆行性活動電位記録の形で前根刺激の用途だけでなく、レンショウ細胞の興奮を提示します。

<table><tbody><tr><td>Na-kynurenate</td><td>ABCAM</td><td>ab120256</td><td>dissolves better then other brands</td></tr><tr><td>KCl</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>sucrose&amp;nbsp;</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>G Biosciences</td><td>R040</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Quality Biological</td><td>351-033-721</td><td></td></tr><tr><td>glucose&amp;nbsp;</td><td>Sigma</td><td>G5767</td><td></td></tr><tr><td>ascorbic acid&amp;nbsp;</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>Na-pyruvate&amp;nbsp;</td><td>Sigma</td><td>P2250</td><td></td></tr><tr><td>K-gluconate&amp;nbsp;</td><td>Sigma</td><td>P1847</td><td></td></tr><tr><td>EGTA&amp;nbsp;</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Agar</td><td>Sigma</td><td>A9799</td><td></td></tr><tr><td>QX-314</td><td>Alomone</td><td>Q150</td><td></td></tr><tr><td>Mg-ATP</td><td>Sigma</td><td>A9187</td><td></td></tr><tr><td>CsOH</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>Na-GTP</td><td>Sigma</td><td>51120</td><td></td></tr><tr><td>gluconic acid</td><td>Sigma</td><td>G1951</td><td></td></tr><tr><td>Cesium hydroxide solution</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>KOH</td><td>Sigma</td><td>P5958</td><td></td></tr><tr><td>Vannas Spring Scissors - 2.5mm&amp;nbsp;</td><td>FST</td><td>15000-08</td><td>only use for cutting the dura, might get damaged if cutting bones</td></tr><tr><td>Stimulator</td><td>A-M Systems</td><td>Isolated Pulse Stimulator Model 2100</td><td></td></tr><tr><td>Vibratome</td><td>Campden</td><td>Vibrating Microtome 7000 - Model 7000smz-2</td><td></td></tr></tbody></table>

the preparation of oblique spinal cord slices for ventral root stimulation

脊髄切片からの電気生理学的記録は、携帯電話からネットワーク特性に問題の広い範囲を調査する価値のある技術であることが証明されています。私たちは、若いマウス（ - P11 P2）の脊髄の実行可能な斜めのスライスを準備する方法を示しています。この準備では、運動ニューロンは脊髄の前根から出てくる彼らの軸索を保持します。これらの軸索の刺激は脊髄内の運動ニューロンのSOMAの、エキサイティングな運動ニューロン担保に侵入バック伝搬する活動電位を誘発します。逆行性活動電位の記録は、他の識別方法を上回る運動ニューロンのアイデンティティを、特徴付けるため、即時の決定的かつエレガントな方法です。また、運動ニューロンの担保を刺激することは、他の運動ニューロンまたはRとして、脊髄内の運動ニューロンの担保対象を励起するためのシンプルで信頼性の高い方法であります細胞をenshaw。このプロトコルでは、我々は前根刺激に起因する、逆行運動ニューロンのSOMAのからの記録だけでなく、レンショウ細胞の興奮を提示します。

Confirmation of Motoneuron Identity Using Antidromic Action Potentials
Cell targeting
Motoneurons are found in the ventral horn (visible in red in Figure 2C). Start from the bundle of axons forming the ventral root and go up until the bundle disperses fully and one starts seeing large cells (long soma axis, above&#160;20 &#181;m). Achieve whole-cell recording of a round healthy looking cell using an electrode of initial resistance of 3 to 4 M&#937;. The internal solution used contained: 140 mM K-gluconate, 6 mM KCl, 10 mM HEPES, 1 mM EGTA, 0.1 mM CaCl2, 4 mM Mg-ATP, 0.3 mM Na2GTP. Adjust the pH to 7.3 with KOH, and the osmolarity to 285 - 295 mOsm by adding distilled water.
Confirmation of the motoneuron identity
Once whole-cell recording is achieved, apply a single biphasic stimulation of the ventral root (1 -&#160;50 V, 0.1 -&#160;0.3 msec) to elicit an antidromic action potential in the recorded cells of the ventral cord. Figures 3A1 and 3A2 show antidromic action potentials in voltage-clamp or current-clamp, respectively. When increasing the stimulation intensity from 1 to 5 V (Figure 3A1) and from 10 to 15 V (Figure 3A2) the antidromic action potential appears in an all-or-nothing fashion with a latency of 0.9 msec and 1.1 msec, respectively. Given that only motoneurons send their axons through the ventral roots, the antidromic action potential is a proof of the cell identity.
In a minority of motoneurons (about 10%), ventral root stimulation failed to elicit antidromic action potential but rather elicited orthodromic action potentials. Figures 3B1 and 3B2 show orthodromic action potentials in voltage-clamp or current-clamp, respectively. When increasing the stimulation from 20 to 30 V (Figure 3B1) and from 25 to 40 V (Figure 3B2), an action potential appears, following a previous excitatory post-synaptic current (EPSC) or excitatory post-synaptic potential (EPSP). The latencies of the action potentials are longer (5.1 msec and 5.3 msec, respectively). In these two cells, stimulating the ventral root elicited a feed-forward excitation (motoneurons send axon collaterals to themselves and to other motoneurons) that was strong enough to elicit an orthodromic action potential. In such cells, the failure to elicit an antidromic action potential (characterized by its all-or-nothing fashion and a latency shorter than 5 msec) does not allow us to conclude that these cells are motoneurons. Note that the required stimulation intensity to elicit&#160;feed-forward excitation is higher than the one needed to elicit an antidromic action potential. All stimulations shown here are 0.1 msec but sometimes, longer stimulation (up to 0.3 msec) may be required to elicit an antidromic action potential.
<img alt="Figure 3" src="/files/ftp_upload/54525/54525fig3.jpg" /> 
Figure 3. Motoneuron&#160;Responses to Ventral Root Stimulation 
A1: 1 V (red trace) and 5 V (black trace) stimulations. A2: 10 V (red trace) and 15 V (black trace) stimulations. B1: 20 V (red trace) and 30 V (black trace) stimulations. B2: 25 V (red trace) and 40 V (black trace) stimulations. Black dots indicate stimulus timing. Double arrowheads emphasize the difference in latencies between ortho- and antidromic spikes. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54525/54525fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Renshaw Cell Stimulation
Cell targeting
Renshaw cells are found in the ventral horn27,28. Visualize the axon bundles with differential interference contrast (DIC) imaging using a 20X and&#160;a 63X objectives. Start from the bundle of axons forming the ventral root and go up until the axons start to disperse but are still discernible (Figure 4A, arrows). Aim for the cells of intermediate size (about 10 - 15 &#181;m in diameter, Figure 4A, arrowheads). Achieve whole-cell recording of a round healthy-looking cell using an electrode of initial resistance of 5 to 7 M&#937;. Depending on the experiment, we used either Cs+-based or K+-based internal solutions10. The Cs-based solution prevented large potassium currents that were visible around -45 mV. In some experiments, we also added 5 mM QX-314 to block the sodium channels responsible for spiking in the recorded cell. The Cs+-based solution contains: 125 mM Cs-gluconate, 5 mM QX-314 Cl, 10 mM HEPES, 10 mM EGTA, 1 mM CaCl2, 4 mM Mg-ATP, and 0.4 mM Na-GTP, with the pH adjusted to 7.3 with CsOH. The K+-based solution should be used to analyze,&#160;both in voltage- and current-clamp, the Renshaw cell responses to ventral root stimulation in conditions approximating as much as possible the physiological ones. This solution contains:&#160;125 mM K-gluconate, 10 mM HEPES, 1 mM EGTA, 0.1 mM CaCl2, 4 mM Mg-ATP, 0.4 mM Na-GTP, with the pH adjusted to 7.3 with KOH. The stimulus intensity varied between 10 and 100 V and its duration varied between 50 and 300 &#181;sec.&#160;Bipolar pulses were used in all cases.
Response of the Renshaw cells to ventral root stimulation
Stimulation of the ventral root triggers the corelease of glutamate and acetylcholine from motoneuron&#160;collaterals onto the Renshaw cells10. Feed-forward inhibition mediated by GABA and glycine is also recruited10. Figure 4B displays responses to the single stimulation of the ventral root. The cell was recorded in a voltage-clamp mode with the cesium-based solution, and maintained at a&#160;voltage of -45 mV. QX-314 in the intracellular solution prevented the cell from firing and GABA and glycine responses were blocked by 3 &#181;M gabazine and 1 &#181;M strychnine, respectively. The inward synaptic current in Figure 4B is the sum of the glutamatergic and nicotinic currents.
<img alt="Figure 4" src="/files/ftp_upload/54525/54525fig4.jpg" /> 
Figure 4. Renshaw Cell Stimulation from the Ventral Root 
A: Images of the Renshaw cell pool acquired using an oblique condenser in the same slice taken at two different magnifications (20X and 40X objectives). Note the motoneuron axons merging into the ventral root (arrow). Putative Renshaw cells are indicated with arrowheads. Scale bars: 100 &#181;m in A1, 50 &#181;m in A2. B: Voltage-clamp recordings of a Renshaw cell following stimulation of the ventral root (black dot). The red trace is the average of the grey ones. Holding voltage was set at -45 mV. QX-314 in the intra-cellular solution prevented the cell from firing and GABA and glycine responses were blocked by 3 &#181;M gabazine and 1 &#181;M strychnine, respectively. Black dot indicates stimulus timing. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54525/54525fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation at JoVE.com

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

前根刺激のための斜め脊髄スライスの調製

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

the-preparation-oblique-spinal-cord-slices-for-ventral-root

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

9.4K ビュー.  Centre de Neurophysique, Physiologie et Pathologie, Université Paris Descartes. We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

ビデオ: The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

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

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

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

ヤツメウナギ網様体軸索の急性解離は、個別の機能シナプス前終末のリリース顔膜から録音を有効にする方法

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

モーターエンドプレートに沿って筋肉内注射：直接運動ニューロンへのシャトルトレーサーへの低侵襲アプローチ

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

ゼブラフィッシュ<em&gt;その場での</em&gt;脊髄脊髄感覚運動ニューロンからの電気生理学的記録のための準備

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

膠様質ニューロンの細胞パッチク ランプ記録のため急性脊髄スライスの準備

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

生体内ニューロン回路接続を模倣するためのインビトロウェッジスライスの準備

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

微小電極アレイを用いた脊髄侵害受容回路におけるネットワーク活動の記録

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an ...

脊髄刺激に対する運動ニューロン応答の理解

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

著者スポットライト:脊髄刺激の進歩 - パッチクランプ電気生理学による運動ニューロンの細胞応答の探索 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

脊髄刺激時の運動ニューロンにおける全細胞パッチクランプ記録のための脊髄スライスの ex vivo 調製

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...

前根刺激のための斜め脊髄スライスの調製

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

ヤツメウナギ網様体軸索の急性解離は、個別の機能シナプス前終末のリリース顔膜から録音を有効にする方法

モーターエンドプレートに沿って筋肉内注射：直接運動ニューロンへのシャトルトレーサーへの低侵襲アプローチ

ゼブラフィッシュ

膠様質ニューロンの細胞パッチクランプ記録のため急性脊髄スライスの準備

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

生体内ニューロン回路接続を模倣するためのインビトロウェッジスライスの準備

微小電極アレイを用いた脊髄侵害受容回路におけるネットワーク活動の記録

脊髄刺激時の運動ニューロンにおける全細胞パッチクランプ記録のための脊髄スライスの ex vivo 調製

前根刺激のための斜め脊髄スライスの調製

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

ヤツメウナギ網様体軸索の急性解離は、個別の機能シナプス前終末のリリース顔膜から録音を有効にする方法

モーターエンドプレートに沿って筋肉内注射：直接運動ニューロンへのシャトルトレーサーへの低侵襲アプローチ

ゼブラフィッシュ

膠様質ニューロンの細胞パッチク ランプ記録のため急性脊髄スライスの準備

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

生体内ニューロン回路接続を模倣するためのインビトロウェッジスライスの準備

微小電極アレイを用いた脊髄侵害受容回路におけるネットワーク活動の記録

脊髄刺激時の運動ニューロンにおける全細胞パッチクランプ記録のための脊髄スライスの ex vivo 調製

膠様質ニューロンの細胞パッチクランプ記録のため急性脊髄スライスの準備