我々は、この方法の確立の際に有用な議論のためのマンフレッドHECKMANNに感謝します。さらに、我々はビデオを生成するサポートのための技術支援やフランク·シューベルトのためクラウディアソマーに感謝します。 01EO1002とイエナ大学病院の臨床研究のための学際センター（IZKF）：仕事は、連邦教育研究省（BMBF）、ドイツ、FKZによってサポートされていました。

以下のプロトコルに記載されている全ての実験手順は、チューリンゲン州当局（テゥーリンガーLandesamtエリーゼVerbraucherschutz、Reg.-Nr. 02-044/12）によって承認された。 1。実験に向けた準備<ol><li>吸引電極の作製<ol><li> 例えば 、標準のパッチ電極マイクロピペットプラー、標準ホウケイ酸ガラスキャピラリーを使用してマイクロピペットを引き出します。 </li><li>ブレーキダイヤモンドファイルを使用して（背側根の直径よりもわずかに大きい）0.5〜1ミリメートルの直径を傾けるための電極。 </li><li>熱ポリッシュは、それが標準的な実験トーチインチ吸引する際には、後根に害を与えないように先端が適切であろう。 </li><li>負圧を適用することができる介してシリンジに接続された電極ホルダにガラスフィラメントを取り付ける。 </li></ol></li><li>溶液の調製<ol><li>マウス用の注射麻酔：0.75ミリリットルケタミン10パーセント、0.24ミリリットルキシラジン2％Aを混ぜるND 5ミリリットル0.9％生理食塩水。溶液10μl/ gの体重（BW）は、腹腔内（ip）注射する。 </li><li>人工脳脊髄液（ACSF）：（単位：mm）再蒸留水に希釈し、次の塩：NaClの134;塩化カリウム3、KH 2 PO 4 1.25、硫酸マグネシウム4•H 2 O 2、 炭酸水素ナトリウム25、 塩化カルシウム2、D-グルコース10。 0.003％の最終濃度までH 2 O 2を追加する。常に新鮮に調製した溶液を使用してください。 </li></ol></li><li>録画設定します（ 図2）の調製<ol><li>データをデジタル化するためのPCインターフェースにアンプを接続します。 </li><li> PCのハードドライブ上の方形パルス刺激とデータ収集をトリガするために、PCベースのプログラムまたはアナログタイマを使用。 </li><li>刺激と記録用として標準的なガラス電極ホルダに接続塩素化silverwiresを使用してください。 </li><li>ステールのまわり、それぞれの刺激、記録と参照電極のための3つのマニピュレータを組み立てるマウスについてのotacticフレーム、全ての電極は、後で準備脊髄へのアクセス権を持つように。 </li><li>負圧を適用するリンゴであると電極ホルダにチューブや注射器を接続します。 </li></ol></li></ol> 2。記録手順のための動物実験や動物の準備のための一般的なコメント<ol><li>それぞれの制度的動物管理使用委員会のガイドラインに従って、マウスを使用して、すべての実験を行う。動物の苦痛を最小化していることを確実にする、深い麻酔下で、すべての手術との録音を行う。 </li><li> （;上記のように調製した溶液を10μl/ G BWのそれぞれ125mgのケタミン及びキシラジン、のG BWあたり8mgの）ケタミン/キシラジンの腹腔内注射することによって動物を麻酔。長期記録時に必要な場合、追加の注射は腹腔内または筋肉にすることができる 注：0.05〜0.1の反復筋肉内注射を上部大腿部におけるケタミン/キシラジン溶液のmlを適切であることが証明されている最大3時間、深麻酔で動物を維持する。 </li><li>麻酔下ながら乾燥を防ぐために、目に獣医軟膏を使用してください。 </li><li>定位フレームに動物の頭部を固定し、実験中、動物の体温を制御するために、直腸プローブと反射的ループで加熱パッドを使用しています。定位フレーム中の脊髄の固定は必要ありません。 </li><li>準備を始める前に、マウスの足の指の間に瞬目反射および単収縮による麻酔の深さを確認してください。反射は廃止すべき。 </li><li>メスを用いて明確な運用のフィールドを取得するために腰椎の領域を下げる胸部上部レベルから脊髄上記の正中線に沿って皮膚を開きます。皮膚切開を行うためにハサミを使用しないでください。慎重に下にある組織から皮膚を失う。その後の工程の間に0.9％生理食塩水で湿らせた傷を保つ。 </li><li>小刀やはさみを使って胸椎レベルに腰椎から脊椎の両側に腱および結合組織を切断する。 </李&gt; <li>小さなニッパーを使って脊椎の周りの棘突起および結合組織の残りの部分を削除します。 </li><li>慎重に解剖顕微鏡下で腰椎レベル（L4/L5）から始まるニッパーで脊椎割れ。脊椎と脊髄の間の空間にニッパーの先端を突き出すと離れて骨片を持ち上げます。硬膜を害すると脊髄に圧力を避けるためにしないでください。どちらも、成功のために重要である。全体の手順の間、湿らせた脊髄に保管してください。半ば胸部のレベルに進みます。 </li></ol> 3。後根およびDRPの記録を分離します（図2） <ol><li>慎重に曲がった先端を細い針（30 G）を使用して硬膜を開きます。これから加湿のためのaCSFを使用してください。 </li><li>ゲージを使用して、可能な限り後根を分離し、可能な限り遠位として2隣接根を切る。活発な分離の間に根を引っ張ると、実験の成功に影響を与えます。 </li><li> DOに吸引電極を下に下げるRSALルーツ。液体は後根を吸引するのに役立ちますので、可能な限り、脊髄に限りのaCSFを追加します。 </li><li>シリンジを介して負圧を印加することによって一方のガラスピペット内に1後根の切断端を吸う。必要に応じて、細い針を使用し、電極開口部の前面に後根を移動します。 </li><li>後根は、吸引電極内の乾燥にある場合、慎重に吸引しながら、ピペットを十分のaCSFで満たされるまで、その先端にいくつかのaCSFを追加します。 </li><li>後根が吸い込まれた後、脊髄から電極先端を高めています。ピペットチップや脊髄短絡記録/刺激電極の間には &quot;水の橋」と注意してください。 </li><li>繰り返します同側隣接するルートのための3.3から3.6を繰り返します。 </li><li>後根にできるだけ近い参照電極を配置し、aCSFのを適用することで湿らせ、それを維持する。 </li><li>録画設定を確立することによって、1ルートがすでに刺激のために選択され、録音用です。二後根から記録が電流クランプモードで行われている間、電圧を段階的に増やします。一つは、DRPを表す遅く、長期的な上向きのたわみが続いている短い下方への撓みが発生することがあります。 </li><li> （ - 0.1ヘルツ、0.3ヘルツ、3 kHzの間のフィルタで30スイープ/後根少なくとも20）のレベルを最大上、いくつかのスイープを記録するための刺激電圧を調整します。 </li><li> DRPの時間依存合計に関する情報を取得するために3つの刺激（100ヘルツ）の短い列車を記録します。 </li><li>追加の対側記録に記録し、刺激部位を切り替える。 </li><li>動物を手順に耐えることを意図するものではない。まだ深麻酔下にある間断頭により最後の記録の後に動物を犠牲に（ケタミン/キシラジン、上記参照、ステップ2.2）。 </li></ol> 4。データ解析<ol><li>解析プログラムにデータを転送（ 例えばシグマプロット、イゴールプロ、またはMATLAB）。 </li><li>平均トレースFRを計算するOM、20〜30スイープ。 </li><li>さらに分析するため（ 図3）、（DRPのピーク振幅ベースラインからの）電圧振れの最大振幅をとる。 </li><li>その後のポテンシャルの時間依存合計の測定値を得るために3パルスと単一パルスの後に、DRPのピーク振幅の比を計算します。 </li></ol>

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著者らは、競合する経済的利益を宣言していません。

in vivoでの神経活動やシナプス電位の細胞外および細胞内電気生理学的記録は、CNS神経機能と病態生理を調査中の最新技術である。脊髄統合は、運動機能などの四肢の動きやマルチモーダル感覚知覚するために重要である。シナプス前抑制は、感覚入力への適切な対応を確保すること、この計算プロセスにおける1重要なメカニズムである。のIa求心性線維上のGABA作動性シナプス?...

シナプス前抑制は、脊髄の中で最も強力な阻害メカニズムの1つです。これは、シナプス後膜電位および運動ニューロン1-3の興奮性を変更することなく、monosynaptically興奮運動ニューロンにおける興奮性シナプス後電位（EPSPS）を阻害する。感覚シナプス前繊維にGABA作動性AXO-軸索シナプスによって誘導された一次求心性脱分極（PAD）は、基本的なメカニズム4-7（もFigure1aを参照）である。これらのシナプスは、GABA Aと GABA B受容体（GABA A R及びGABA B R） ​​を含んでいる。 GABA A Rの活性は、ローカルイオン分布にPADを誘発クロライドコンダクタンスの増加をもたらす。この脱分極をブロック軸索終末に活動電位の伝播と減少したのCa 2 +流入と伝達物質放出の減少をもたらす彼らの強さを軽減します。 GABA B受容体の活性化はないなしトンPADに寄与せず、+流入は、それによってシナプス前抑制を増強するCa 2 +の減 ​​少をもたらす。 GABA A Rの活性化が短期阻害に関与しているように見える一方で、GABA B Rは、長期8-10変調に関与している。 PADとシナプス前抑制の大部分を占めるGABAに加えて、他の送信機システムはまた調節する可能性があり、この機構11,12に寄与する。 シナプス前抑制における病理学的変化は、欠陥のあるGABA作動性伝達17によって媒介運動過剰興奮性と末梢性炎症および神経障害性疼痛13,14、ならびに異常な中枢性疼痛処理15、脊髄損傷16、およびCNS疾患、例えば 、いくつかの疾患状態において重要であると思わ18。従って、シナプス前抑制を推定するin vivoでの脊髄レベルに関する実験病理学的状態を調査する価値がある</em&gt;。 PADは、脊髄におけるシナプス前抑制の直接的な尺度を提供するボリューム行わ電位を生じさせる。これらの電位は、後根電位（DRP）と呼ばれ、隣接する背側根7の刺激後に脊髄後根から測定することができる。 DRPの最初の測定値は、猫やカエル19で報告されており、集中的に、1970年代初頭3,4,20,21にエクルズ、シュミット、そして他の人が猫で研究した。ネコおよびラット22 23 DRP のインビボ記録は、広く使用されてきたが、マウスでの測定は、ほぼ独占的にex vivoで単離された脊髄調製物15,24に行われている。ここでは、完全な生物体におけるシナプス前抑制の直接測定を可能に生体内で麻酔したマウスでは、DRPを記録する方法を説明している。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass tubing (inner diameter 1.16 mm)</td>
			<td>Science Products (Hofheim, Germany)</td>
			<td>GB200F-10</td>
			<td>Other glass tubing might also be suitable</td>
		</tr>
		<tr>
			<td>Superfusion solution (sterile, 0,9% NaCl)</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>3570350</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Germany)</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine)</td>
			<td>Bayer Animal Health GmbH (Leverkusen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamin 10%</td>
			<td>Medistar GmbH (Ascheberg, Germany)</td>
			<td>KETAMIN 10%</td>
			<td></td>
		</tr>
		<tr>
			<td>30G micro needle/ Sterican</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Geramny)</td>
		</tr>
		<tr>
			<td>Salts for aCSF</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Diverse</td>
			<td></td>
		</tr>
		<tr>
			<td>S88 Dual Output Square Pulse</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>S88X</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>SIU5 RF Transformer Isolation Unit</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>SIU-V</td>
			<td></td>
		</tr>
		<tr>
			<td>InstruTECH LIH 8+8</td>
			<td>HEKA (Lambrecht, Deutschland)</td>
			<td>LIH 8+8 + Patchmaster software</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition&#160;</td>
		</tr>
		<tr>
			<td>Universal amplifier</td>
			<td>npi (Tamm, Deutschland)</td>
			<td>ELC-03X</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus (Tokyo, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>MPC-200/MPC-325</td>
			<td>Mechanical micromanipulators also possible</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Stoelting (Wood Dale, USA)</td>
			<td>50300V</td>
			<td></td>
		</tr>
		<tr>
			<td>Intra-/extracellular recording electrode holder</td>
			<td>Harvard Apparatus (Holliston, USA)</td>
			<td>641227</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo

シナプス前抑制は、脊髄の中で最も強力な阻害メカニズムの1つです。基本的な生理学的なメカニズムは、GABA作動性AXO-軸索シナプス（一次求心性脱分極）が介在する一次求心性線維の脱分極である。一次求心性脱分極の強度は、後根（後根電位、DRP）で体積を行っ電位を記録することによって測定することができる。シナプス前抑制の病理学的変化は、特定の疼痛症状の異常な中央演算処理において、モータ過剰興奮のいくつかの疾患において重要である。ここでは、マウスにおいてインビボで DRPを記録する方法を記載している。麻酔した動物および吸引電極を用いて記録手順における脊髄後根の調製について説明する。この方法は、GABA作動性、DRPを測定し、それによって生きているマウスでは脊髄シナプス前抑制を推定することができます。トランスジェニックマウスモデルとの組み合わせで、DRP記録月SE疾患関連脊椎の病態生理学を研究するための強力なツールとしてRVEは、 インビボ記録は、 例えば、同時記録又は脊柱上のネットワークおよび末梢神経の刺激によるDRPの誘導の操作の可能性をex vivoで単離された脊髄調製物と比較していくつかの利点を有する。

典型的なDRPトレースを図3に示す。著名な刺激アーティファクトは通常は短い下方への撓みが続いている。その後、DRPを表す遅い、長期持続上向き偏向は、明確に区別される。録音の一部では、脊髄後根反射は、DRPの上に小さなスパイクとして表示されます。刺激電圧が過剰になると正常な野生型マウスでは、後根反射は最も頻繁に現れる。後根反射神経がこの準備に再現性よく?...

Watch this Scientific Journal Video about Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo at JoVE.com

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABA作動性シナプス前抑制は、モータと脊髄ネットワークでの感覚信号の統合のための重要な脊髄の強力な阻害のメカニズムです。基礎となる一次求心性の脱分極は、後根電位（DRP）の記録により測定することができる。ここでは、マウスにおけるDRP のインビボ記録する方法を実証する。

後根潜在的記録によりマウスにおける脊髄シナプス前抑制を測定する<em&gt;インビボ</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

measuring-spinal-presynaptic-inhibition-mice-dorsal-root-potential

Research

JoVE Journal

Neuroscience

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

13.9K ビュー.  Jena University Hospital, Jena, Germany.  GABA作動性シナプス前抑制は、モータと脊髄ネットワークでの感覚信号の統合のための重要な脊髄の強力な阻害のメカニズムです。基礎となる一次求心性の脱分極は、後根電位（DRP）の記録により測定することができる。ここでは、マウスにおけるDRP のインビボ記録する方法を実証する。 

ビデオ: Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

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

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

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

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

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

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

神経科学

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同時<em&gt; ex vivoで</emによる二つの網膜の&gt;機能テスト<em&gt; in vivoで</em&gt;電図システム

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

マウスで睡眠を測定するためのポリグラフ記録手順

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

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 ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

後根神経節インジェクションおよび後根感覚軸索再生のためのモデルとして傷害をつぶします

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

腰部後根神経節のアクセスと豚注入の椎

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

成体マウス後根神経節の調査の哺乳類の軸索の再生: In Vivoエレクトロポレーション

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

インビボシングルファイバー記録と無傷の後部根神経を用いて伝導障害のメカニズムを調べる

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

ドーサルルート神経節マクロファージの迅速な分離

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.