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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 >NaH2PO4</td>
			<td >Sigma</td>
			<td >P5655</td>
			<td></td>
		</tr>
		<tr >
			<td >sucrose&#160;</td>
			<td >Sigma</td>
			<td >S9378</td>
			<td></td>
		</tr>
		<tr >
			<td >NaHCO3&#160;</td>
			<td >Sigma</td>
			<td >S6014</td>
			<td></td>
		</tr>
		<tr >
			<td >CaCl2&#160;</td>
			<td >G Biosciences</td>
			<td >R040</td>
			<td></td>
		</tr>
		<tr >
			<td >MgCl2&#160;</td>
			<td >Quality Biological</td>
			<td >351-033-721</td>
			<td></td>
		</tr>
		<tr >
			<td >glucose&#160;</td>
			<td >Sigma</td>
			<td >G5767</td>
			<td></td>
		</tr>
		<tr >
			<td >ascorbic acid&#160;</td>
			<td >Sigma</td>
			<td >A5960</td>
			<td></td>
		</tr>
		<tr >
			<td >Na-pyruvate&#160;</td>
			<td >Sigma</td>
			<td >P2250</td>
			<td></td>
		</tr>
		<tr >
			<td >K-gluconate&#160;</td>
			<td >Sigma</td>
			<td >P1847</td>
			<td></td>
		</tr>
		<tr >
			<td >EGTA&#160;</td>
			<td >Sigma</td>
			<td >E3889</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES&#160;</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&#160;</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 colspan="2">Isolated Pulse Stimulator Model 2100</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

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

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人の全体のマウントの準備ショウジョウバエ</emジャイアント繊維染料注入のための>腹神経索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

げっ歯類の内側嗅内皮質の背腹軸機構の調査のための矢状スライスの作成

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

幼虫のゼブラフィッシュにおける脊髄切断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

縦断のために脊髄商工を移植するための手順<em&gt;インビボ</emマウス脊髄の&gt;イメージング

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

成体マウスの脊髄組織からの初代混合グリア培養の調製

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

げっ歯類での後根神経節の脊髄および単離の油圧押出

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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 ...

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 ...

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 ...