<p class="jove_content"&gt;この作品は、国立衛生研究所の神経疾患や脳卒中の国立研究所の学内プログラムによってサポートされていました。また、メソッドには、図3のデータへの彼の以前の貢献のために博士ジョージMentisに感謝します。</p

蛍光デキストランは、解剖学的ツールとして、イメージング神経活動1-4使用されています。フィールドら 、（2009）4は、モデル系として脊髄を中心とした軸索路にイオンと電位感受性色素を適用するためのプロトコルを公開しました。ここでは、 インビトロ optophysiologicalおよび形態学的研究のためにカット腹根、後根または脊髄のいずれかの神経管に蛍光及び/又はイオン感受性色素を適用するための詳細な手順について説明します。 1。タイプ-IとType-IIピペットテーパーチップとピペットを生成するためにアルコールランプ7の炎の上にポリエチレンチューブの二つの短いセクション（PE90、クレイ·アダムスブランド）を引いて起動します。 Oneピペット（タイプI）がしっかりとターゲットルートまたは管を（;内径0.2〜0.4ミリメートル：0.1〜0.3ミリメートル外径）を保持できる小型チップで（3-7 mm）を短くする必要があります。次に、第二のピペット（タイプを引く-II）より長い（8-12 cm）の薄型化、シャフトと非常に微細なチップ（外径：0.2〜0.3ミリメートル、内径に挿入することができます0.1〜0.2ミリメートル）タイプ-Iは限りピペットその先端。第二シンナーピペットは、Type-Iピペットからそれぞれ人工脳脊髄液（ACSF）と色素溶液を吸引し、導入するために使用されます。 2。タイプ-Iピペットの位置決め解剖と脊髄の8,9を分離し、ロード·プロセスを通して冷やしたACSF（〜16°C）で灌流浴に入れてください。タイプ-Iは、そのバックの開口部が容易にフレキシブルチューブを介してシリンジ（1ミリリットルU-100インスリン注射器、ベクトン·ディッキンソンまたは同等の）に接続することができるように電極ホルダー（H1/12電極ホルダー、ナリシゲ）にピペット（PharMedを置くBPT、コール·パーマー、＃AY242002、10センチ）（図1A-B）。 3。染料のアプリケーション<ol><li>シリンジを使用して、最初の描画ACSFは、Type-Iにピペット（ 図1B、2A）は満たされるように軸索路またはルート（ 図2B）が続いている。フレキシブルチューブは、その後、開口部から削除する必要があります。タイプ-IIピペットは、別の注射器に接続されているとタイプ-Iは軸索路またはルートホールディングピペットに挿入されます。残留ACSFだけで軸索路（; 2C-D図1C）を覆うようにII型ピペット、吸引ACSFに接続された注射器を使用します。その後I型ピペットからタイプ-IIピペットを取り消すことができます。軸索管や神経（ &quot;良い印鑑&quot;のトラブルシューティングのセクション3を参照）で良好なシールを確認するために数分間ACSFレベルを監視します。 </li><li> ACSFの染料または再蒸留水で0.2％トリトンX-100を溶解させ、気泡を含まないように注意を払いながら、タイプ-IIピペットにそれを描画します。 I型ピペットに、残留ACSF溶液（ 図2E）に色素を含有するII型ピペットを挿入します。徐々に放出する穏やかな陽圧（ 図2F）を使用して、ACSFに染料。気泡を導入するか、先端の内側から標的組織の変位を引き起こすことがないように注意してください。染料のに十分な量（染料溶液の3-5μL）がリリースされた後、タイプ-IIピペットを取り消すことができます。 </li></ol> 4。インキュベーション実験の種類に応じて6〜20時間の間に暗闇の中で組織をインキュベートします。一度十分な時間が穏やかにイメージングまたは組織の準備ができて組織を残して組織から電極を引き離し、充填に許可されています。 トラブルシューティング<ol><li>標的組織は、Type-Iピペットの先端に簡単に入力するか、一度内部に緩んでいない場合は、先端径サイズが間違っています。この問題が発生した場合、引き出して、別のピペットを使用しています。 </li><li> I型ピペットにACSFに染料を追加する前に、十分なACSFは、それがで到達できるように残っていることを確認してくださいタイプ-IIピペットチップ（ 図2D）。染料が追加されると、それ以外の場合は、エアギャップが存在します。この問題が発生した場合は、染料を追加し、ピペットから軸索路を削除して、Type-IIにピペットで到達するのに十分なACSFでそれを戻って描画されません。 </li><li>タイプ-Iピペット増加ACSFのレベルは、それがタイプIIピペットで吸引した後なら、それは組織のシールが良好でないことを意味します。異なる電極を使用して、それ以外の色素が標識手順の中で希釈されます。 </li><li>染料を注入しながら気泡がType-Iピペットに導入されている場合は、バブルにType-IIピペットチップの近くを進めるゆっくりと付属のシリンジを用いて弱い負圧を印加することによって気泡を排出します。 </li></ol> 5。代表的な結果法の適用を説明するために、我々は同時にロード運動ニューロン、感覚求心性、3つの異なるDEXTと脊髄介在蛍光色素（ 図3A）結合を実行しました。 図2に示すように。ニューロンの3B、3つのクラスは、ラベルされています。感覚求心性（赤）、腹索（緑）と運動ニューロン（青）に突出介在。 <img alt="図1" src="/files/ftp_upload/4008/4008fig1.jpg" /> 図1。ピペットタイプ-IとType-IIアセンブリを示す模式図 。 （A）タイプ-Iピペット（ライトブルー）は（C）は背面の開口部が容易に柔軟なエラストマーチューブ（B）に接続することができていると先のType-IIにピペットを挿入することができるように電極ホルダーに配置されます。 <img alt="図2" src="/files/ftp_upload/4008/4008fig2.jpg" /> 図2。蛍光色素とニューロンのロード·プロセスを示す模式図 。 （A）タイプ-Iピペットを充填する前根の近くに配置されています。 （B）吸引は、ルートに続いてピペットにACSFを描画するためのピペットに適用されます。 （C-D）タイプ-Iピペットのバックエンドからの柔軟なエラストマーチューブを外し、タイプ-IIピペットに吸引を適用することにより、ACSFの量を減らすことができます。 （E）タイプ-Iピペット内の残りのACSFに溶解した染料を格納しているType-IIピペットの先端を挿入します。 （EF）を徐々に染料でI型ピペットを記入して、Type-IIにピペットを削除します。 <img alt="図3" src="/files/ftp_upload/4008/4008fig3.jpg" /> 図3。孤立したマウスの脊髄への充填手順の適用 。私はタイプの異なる脊髄神経のクラスを塗りつぶすために使用されるピペット（A）の回路図は、3を示す。運動ニューロン（青、カスケードブルーデキストラン）と仙骨感覚求心性（赤、テキサスレッドデキストラン）をそれぞれ、腹側と背側根を通して埋めた。フルオレセインデキストランは、その軸索は腹索（VF）を介して登る脊髄介在（緑）をロードするために使用された。各デキストラン染料の1ミリグラム（10,000 MW、Molecular Probes社）トリトンX-100（B）0.2％を含む6μLの蒸留水に溶解した。標識された感覚の求心性はいくつかの反対側の突起を有する主に同側であることをAに説明したようにニューロンが戻って標識された脊髄の仙髄からセクションの共焦点画像は、注意してください。標識された介在は、塗りの側（矢印頭部）に反対であるのに対し、ラベルの付いた運動ニューロンは、充填前根と同側である。キャリブレーションバーは200μm。

<ol>
	<li>Nance, D. M., Burns, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dextrans+as+sensitive+anterograde+neuroanatomical+tracers:+applications+and+pitfalls.">Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: applications and pitfalls.</a> Brain Res. Bull. 25, 139-145 (1990).</li><li>Glover, J. C., Petursdottir, G., Jansen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dextran-amines+used+as+axonal+tracers+in+the+nervous+system+of+the+chicken+embryo.">Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo.</a> J. Neurosci. Methods. 18, 243-254 (1986).</li><li>McPherson, D. R., McClellan, A. D., O'Donovan, M. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+functional+synaptic+connections+between+medullary+reticulospinal+neurons+and+lumbar+descending+commissural+interneurons+in+the+neonatal+mouse.">Organization of functional synaptic connections between medullary reticulospinal neurons and lumbar descending commissural interneurons in the neonatal mouse.</a> J. Neurosci. 31, 4731-4742 (2011).</li></ol>

<p class="jove_content"&gt;私たちは、開示することは何もありません。</p

<p class="jove_content"&gt;ここでは神経細胞、神経や脊髄路に色素を導入するための簡単​​かつ費用対効果の高いプロトコルを記述します。このメソッドは、ロードプロセスの間、高濃度の色素溶液に識別解剖学的経路を公開することが含まれます。 microinjections、バス·アプリケーションおよびエレクトロポレーション法に比べて少し背景を持つターゲット·サイトの逆行性標識法で、この結果。しかし、この方?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td colspan="2"<strong&gt;材料名</strong</td&gt;<td<strong&gt;会社概要</strong</td&gt;<td<strong&gt;カタログ番号</strong</td&gt;<td<strong&gt;コメント</strong</td&gt;</tr&gt;<tr&gt;<td rowspan="2"&gt;チューブ</td&gt;<td&gt; PE90（IDxOD：0.034 &quot;x0.050&quot;;壁Thikness：0.008 &quot;）</td&gt;<td&gt;クレイ·アダムスブランドIntermedic</td&gt;<td&gt; 427421</td&gt;<td&gt;タイプIとタイプIIピペット用</td&gt;</tr&gt;<tr&gt;<td&gt; NSF-51（IDxOD：1/16x1/8、壁Thikness：1/32）</td&gt;<td&gt; PharMed BPT、コール·パーマー</td&gt;<td&gt; AY242002</td&gt;<td&gt;フレキシブルチューブ用</td&gt;</tr&gt;<tr&gt;<td&gt;シリンジ</td&gt;<td&gt; 1ミリリットルのインスリンシリンジU-100</td&gt;<td&gt;ベクトン·ディッキンソン</td&gt;<td&gt; 329650</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;ニードル</td&gt;<td&gt; 19G X1-1/2 &quot;メタルハブニードル</td&gt;<td&gt; MONOJECT</td&gt;<td&gt; 200136</td&gt;<td&gt;シリンジにタイプIIのピペットを接続する</td&gt;</tr&gt;<tr&gt;<td&gt;</td&gt;<td&gt;アルコールランプ</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;コック</td&gt;<td男性ルアースリップアダプタを搭載した&gt;三方活栓</td&gt;<td&gt;バクスターヘルスケア（株）</td&gt;<td&gt; 2C6241</td&gt;<td&gt;注射器を使用します。</td&gt;</tr&gt;<tr&gt;<td rowspan="3"&gt;ホルダー</td&gt;<td&gt; H-1電極ホルダー</td&gt;<td&gt;ナリシゲ</td&gt;<td&gt; H-1/12</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;マグネットスタンド</td&gt;<td&gt;ナリシゲ</td&gt;<td&gt; GJ-8</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;マニピュレーター</td&gt;<td&gt;ナリシゲ</td&gt;<td&gt; M-3333</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;ピンセット</td&gt;<td&gt; S＆Tデュモンスイス00632から11</td&gt;<td&gt;デュモン</td&gt;<td&gt; JF-5 TC</td&gt;<td&gt;</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"&gt; *これらの提案した材料である。互換性のある任意の製品に置き換えることができます</p

retrograde loading of nerves, tracts, and spinal roots with fluorescent dyes

ニューロンの逆行性標識はまた、ニューロンの3月6日に、カルシウムや電位感受性色素をロードするために使用されている標準的な解剖学的方法1,2です。一般的に、色素が固体結晶として、あるいはガラスピペットを使用してローカルの圧力注入により適用されます。しかし、これはいくつかの時間が染料拡散するために必要な場合は特に、染料と還元の標識強度の希釈になる可能性があります。ここでは、色素溶液を充填したポリエチレン吸引ピペットを用いて神経細胞に蛍光灯やイオン感受性色素を導入するためのシンプルで低コストの手法を示しています。このメソッドは、ロード処理全体の軸索と接触している染料の高濃度を維持するための信頼できる方法を提供しています。

Watch this Scientific Journal Video about Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes at JoVE.com

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

我々は、ニューロンまたはポリエチレン吸引ピペットを使用して、任意の神経管に蛍光カルシウム感受性色素を高濃度に導入するための簡単​​かつ低コストの手法を説明します。

神経、地帯、および蛍光色素で脊髄根の逆行ローディング

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

retrograde-loading-nerves-tracts-spinal-roots-with-fluorescent

Research

JoVE Journal

Neuroscience

12.2K ビュー.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. 我々は、ニューロンまたはポリエチレン吸引ピペットを使用して、任意の神経管に蛍光カルシウム感受性色素を高濃度に導入するための簡単​​かつ低コストの手法を説明します。

ビデオ: Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

スチリル染料の光変換を使用して、シナプス小胞プールの勉強

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

神経科学

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

電位感受性色素を持つカニ口と胃の神経節における神経細胞の光学的イメージング

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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 ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

重度の脊髄損傷後のフィブリンおよび成長因子カクテル神経幹細胞の生存と分化の促進

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

蛍光色素で大人のゼブラフィッシュの網膜神経節細胞の逆行性標識

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

マウス坐骨神経に生体内電気生理学的測定値の

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

遺伝的にコードされた蛍光電圧センサーの2種類のイメージング膜電位

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

マウスにおける横隔膜の運動ニューロンの逆行性神経解剖学的トレーシング

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

脊髄とトレッドミル上のアクティビティ ベースのトレーニング負傷 Wistar ラット

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.