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どんな研究が幼児の倫理的承認を行うことができると書かれた通知の前に親の同意が求められている必要があります。 本研究では倫理的な承認は、大学病院の倫理委員会から入手し、親の同意を書面通知、各手順の前に得られた。れましたこの研究は、ヘルシンキ宣言および医薬品の臨床試験の実施に関するガイドラインの定める基準に適合さ。 1。データの収集 - セットアップ<ol><li>肌が10〜20電極配置のシステム（図1A）にしたがって準備中後の乳児の頭の上に16個使い捨てEEG銀/塩化銀カップ電極の最小値を置きます。より包括的なカバレッジは、埋め込み電極による脳波キャップを使用して達成することができます。 EEGキャップを使用すると、プロセスはより速く、より混乱の少ない、電極を頭の上にキャップを配置する前に、誘導性ゲルを用いて調製され、特にとなる。使い捨ての脳波電極のアプリケーションが必要です。より多くの時間とスキルが、より良い記録で、一般的な結果。乳児へのアクセスが制限されている場合は、電極の数を減らすことを検討し、常に（Czに、CPZとFCZ）正中線の電極を使用してください。記録用参照電極としてFCZを使用してください。 </li><li>電極/皮膚電気的結合を最適化するために脳波の導電性ペーストを使用してください。 </li><li>場所でそれらを保持するために、電極上に弾性ネットを置きます。 </li><li>電極は、電気的干渉を最小限に抑えるために一緒につながる接続します。 </li><li>胸や頭の上に接地電極を配置。 </li><li>肌が（ 図1B）準備中後の両脚の大腿二頭筋のEMG電極を配置。 </li><li>肌が（胸の左側に一方の電極、右上の1と脳波と同じ接地電極を使用して）準備中後のボディにECGの活動の場のリード1 ECG電極を記録するには。 </li><li>呼吸を測定するために腹部の動きのトランスデューサを配置。 </li><li>徒歩でのパルスオキシメータのプローブを配置酸素飽和度と心拍数を測定する。プローブが所定の位置に固定されていることと、連続的な信号がドロップアウトすることなく記録されていることを確認してください。オキシメータのプローブを使用すると、刺激しようと足の反対側である足の上に配置する必要があります。 </li><li>顔の表情でその変更が記録できるように乳児の顔をフレームに三脚に取り付けられたビデオカメラを設定します。 </li><li>カメラのフレーム内の発光ダイオード（LED）を配置します。刺激が脳波、筋電図とビデオ録画を同期させるために提示されるときに点滅するように、LEDは、タイミング回路にリンクされています。 </li></ol> 2。データの収集 - 記録<ol><li>ビデオ録画を開始します。 </li><li>パルスオキシメトリーの記録を開始します。 </li><li> EEG / EMG記録を開始します。 </li><li>乳児が確定するまで待ちます。 </li><li>ヒールランスと手動でイベントマーク脳波と筋電図記録を行うかのように足をホールド。この時代は、脳波コントロールの背景のセクションを識別するために使用されます。とEMG。 </li><li>軽くかかとの表面に対する腱ハンマーのアームに取り付けられているゴム栓をタップしてタッチの刺激を適用します。パルスオキシメータに接続されていない足を刺激する。乳児が刺激されるとEEG / EMGおよびビデオ録画は、刺激が行われたときに、録音の時間を特定するために、イベントマークする必要があります。タッチの刺激は、電子的に記録装置への刺激をつなぐ腱ハンマーにインピーダンスヘッドを取り付けることにより、イベントをマークすることができます。ビデオ録画は、LEDの点滅により、イベントマークです。繰り返しタッチを適用することができると刺激が体のさまざまな領域に適用することができる、肩、すなわち</li><li>バネ付きのブレードが解放されたときにそれが皮膚に接触しないように90度でランセットを回転し、足に対してそれを置くことで、非有害なコントロールの刺激を適用します。このイベントは、tの上面に取り付けられた加速度計を使用して、時間をロックすることができます彼ランス。加速度計は、ブレードが解放されたときに発生する振動を検出します。 </li><li>新生児室における臨床実践に基づいて臨床的に本質的なヒールランスを実行します。脳波活動がヒールランスを行う前に決済されるまで待ちます。ヒールランスのタイムロックが制御刺激の場合と同じ方法で行うことができます。ヒールランスに続いて、記録された応答が槍にもっぱら起因していることを確認するために少なくとも30秒間足を絞るしないでください。 </li><li>血液の必要量が収集されていることを確認し、臨床分析用サンプルを準備する。 </li><li>データを保存し、すべての録音を停止します。 </li><li>乳児の人口統計情報と実験の詳細と入力それらの安全な保管と将来の参考のためにデータベースへの記録。 </li><li>乳幼児の必要なサンプルでは、​​この手順を繰り返します。この例では乳児の数= 23。 </li></ol> 3。脳波データの解析<ol><li>それぞれのタッチ、コントロールとランス刺激と背景脳波に対応する1.7秒の脳波エポックを作成します。エポックは、各イベントの前に0.6秒を開始する必要があります。各モダリティに対応したエポックの数は同じでなければなりません。 </li><li>ベースラインは、平均ベースラインの信号と高域が0.1 Hzでそれらをフィルタリング差し引くことによって紀元を修正。 </li><li>さらなる分析のためにCPZまたはCzにで記録されたエポックを考慮すると動きのアーチファクトによって汚染されていた新紀元を除外する。動きのアーチファクトは、50ms以内で50μVよりも大きい振幅の変化として定義されています。 </li><li>触覚の潜在的な（すなわち脳波活動が触覚刺激に関連）を識別するために50〜300ミリ秒のポスト刺激と行動の主成分分析（PCA）との間のこの時間間隔にジッタレイテンシを補正するためのトレースを合わせます。変数にエポックを考慮すると時間は、観測を指しています。 PCAは、基本的な波形にEEGエポックと呼ば​​れる、主要なコンポーネントを（分解パソコン）と時間の点で信号の振幅の体系的変化を表しています。 </li><li>触覚の可能性を表しているPCかを判断する最初の2台のPCのそれぞれの重みに一元配置分散分析（ANOVA）を実行します。これは、その重み脳波背景に比べて以下の触覚刺激有意に大きかったPCになります。 </li><li> 300と700ミリ秒の後の刺激とこの時間間隔でPCAを行うとの間のレイテンシジッタを補正するためのトレースを合わせます。 </li><li>侵害受容固有のポテンシャルを表しているPCかを判断する最初の2台のPCのそれぞれの重みに一元配置ANOVAを実行します。これは、その重みれた非常に大きく、以下の侵害刺激触覚刺激と背景脳波に比べてPCになります。 </li></ol> 4。 EMGデータの解析<ol><li>二乗平均平方根（RMS）コントロールのための最初の1000ミリ秒後の刺激とランスの刺激で、EMG信号のを計算する。 </li><li>でt -検定を実行RMSは、侵害受容性特有の脊髄反射の撤退を決定する値。 </li></ol> 5。代表的な結果<img alt="図1" src="/files/ftp_upload/3118/3118fig1.jpg" /> <st栄&gt;図1（A）（i）は図と脳波記録（国際10月20日電極配置のシステムを変更）するための電極配置の（ⅱ）写真;。大腿二頭筋でEMGの記録のための電極配置の（B）図。 <img alt="図2" src="/files/ftp_upload/3118/3118fig2.jpg" /> 図2（A）はCzでの感覚の可能性の例としては、3児の接触によって誘発、（B）CZにおける侵害受容性固有性の例としては、3児の有害ランスによって誘発。 <img alt="図3" src="/files/ftp_upload/3118/3118fig3.jpg" /> 図3。Czでの刺激のモダリティのPCの重さ（平均± SEM）の依存性。刺激開始後50〜300ミリ秒の間に得られたPCは、触覚の可能性を表し、刺激開始後300〜700ミリ秒の間に得られたPCは、侵害受容性固有のポテンシャルを表しています。パソコン（太線）は壮大な平均値でオーバーレイされます個々のトレースが記載時間間隔で整列された後、すべての刺激の種類（背景脳波、タッチ、有害ランス）を介して得られた。 <img alt="図4" src="/files/ftp_upload/3118/3118fig4.jpg" /> 図4（i）の有害ヒールランスとヒールの（ⅱ）タッチ後の幼児1における筋電図活動の（）の例。 （B）平均（± SE）EMG有害なヒールと非有害なタッチの刺激後の乳児の二乗平均平方根EMG活動。

<ol>
	<li>Fitzgerald, M., Walker, 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=Infant+pain+management:+a+developmental+neurobiological+approach.">Infant pain management: a developmental neurobiological approach.</a> Nat. Clin. Pract. Neurol. 5 (1), 35-35 (2009).</li><li>Slater, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+sucrose+as+an+analgesic+drug+for+procedural+pain+in+newborn+infants:+a+randomised+controlled+trial.">Oral sucrose as an analgesic drug for procedural pain in newborn infants: a randomised controlled trial.</a> Lancet. 376 (9748), 1225-1225 (2010).</li><li>Slater, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evoked+potentials+generated+by+noxious+stimulation+in+the+human+infant+brain.">Evoked potentials generated by noxious stimulation in the human infant brain.</a> Eur. J. Pain. 14 (3), 321-321 (2010).</li><li>Slater, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+infants+display+increased+noxious-evoked+neuronal+activity+in+the+brain+compared+to+healthy+age-matched+term-born+infants.">Premature infants display increased noxious-evoked neuronal activity in the brain compared to healthy age-matched term-born infants.</a> Neuroimage. 52 (2), 583-583 (2010).</li></ol>

著者らは、開示することは何もないことを宣言。

このビデオでは、触覚や侵害刺激により誘発される電気生理学的反応が、、脳波と筋電図記録を用いて、ヒト乳児を特徴とする方法を示しています。このタイプの研究は、人間の痛みの処理の開発と可塑性を理解する助けになる、と乳児の痛み1、2の改良された臨床的評価と治療につながる。 これらの実験の成功は、学際的なチームの密接な協力が必要です。専門は新生児学、臨床神経生理学、生命工学、発達神経科学の分野で要求される。 それは、これらの実験が実施されているときに赤ちゃんの世話が最も高い優先順位を取ることが不可欠です。ヒールランスを行う研究看護師や医師が乳児の健康に責任を持っており、実験が臨床の現場に準拠して行われていることを確認する必要があります。病院倫理委員会とwritteから承認nは親の同意は、これらの研究を行うことが必要です。 ヒールランスは、臨床的に必要なイベントであり、研究の目的のために繰り返すことはできません。それは記録が臨床実習3、4と干渉しない信頼性の高い堅牢な方法で収集されることが不可欠です。特に、それは時間がロックするのに使用される方法は、刺激が信頼性が高く、臨床血液のコレクションを妨げないことが重要です。 それが記録された活動が、特に有害事象と乳児のケア2の他のではない側面によって惹起されることを確かめることが重要であるような乳児の痛みのすべての措置は、必ずしも間接です。これは、データの慎重な技術的設計と後処理を介して達成することができます。

electrophysiological measurements and analysis of nociception in human infants

痛みは不快な感覚および感情的な経験です。幼児は口頭での経験を報告できないので、痛みの評価の現在の方法は、そのような表情で泣いて、身体の動きや変化などの行動や生理的な身体の反応に基づいています。これらの措置は、乳児は有害刺激に続く応答をマウントすることを実証しながら、彼らは限られて：彼らは確実に脳の中央感覚処理にリンクされていない可能性があります皮質体性および自律運動経路の活性化に基づいています。中枢神経系の有害事象にどのように応答するかの知識は、侵害受容情報と痛みが新生児にどのように処理されるかに洞察を提供することができます。 入院の乳児の血液を抽出するために使用される踵穿刺の手順では、乳児期に痛みを研究するユニークな機会を提供しています。このビデオでは、脳波（EEG）と筋電図検査（EMG）は、この手順をCAに時間ロック方法を説明します。nは、脳と脊髄の侵害受容活動を調査するために使用される。 乳児の疼痛の測定には、この統合的なアプローチは効果的で敏感な臨床測定ツールへの道を開く可能性を秘めています。

Watch this Scientific Journal Video about Electrophysiological Measurements and Analysis of Nociception in Human Infants at JoVE.com

Electrophysiological Measurements and Analysis of Nociception in Human Infants

幼児は口頭での経験を報告できないので、乳児の疼痛の評価と治療が困難です。このビデオでは、乳児神経系からの有害事象に対する反応を測定するために使用できる定量的な電気生理学的手法や分析手法を説明します。

電気生理学的測定とヒト幼児における痛覚の分析

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

electrophysiological-measurements-analysis-nociception-human

Research

JoVE Journal

Neuroscience

17.2K ビュー.  University College London. 幼児は口頭での経験を報告できないので、乳児の疼痛の評価と治療が困難です。このビデオでは、乳児神経系からの有害事象に対する反応を測定するために使用できる定量的な電気生理学的手法や分析手法を説明します。

ビデオ: Electrophysiological Measurements and Analysis of Nociception in Human Infants

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

蛾嗅覚系の電気生理学的測定

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

神経科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

開発ゼブラフィッシュ前脳におけるクローン関連の神経前駆細胞のタイムラプスライブイメージング

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography (dEEG)

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any other current brain imaging technology1,2, is being used increasingly in the study of social cognitive functioning in infants and adults. While dEEG is enabling researchers to examine brain activity patterns with unprecedented levels of sensitivity, conventional EEG recording systems continue to face certain limitations, including 1) poor spatial resolution and source localization3,4,2) the physical discomfort for test subjects of enduring the individual application of numerous electrodes to the surface of the scalp, and 3) the complexity for researchers of learning to use multiple software packages to collect and process data. Here we present an overview of an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults. Although several analytical software techniques can be used to establish indirect indices of source localization to improve the spatial resolution of dEEG, the HydroCel Geodesic Sensor Net (HCGSN) by Electrical Geodesics, Inc. (EGI), a dense sensory array that maintains equal distances among adjacent recording electrodes on all surfaces of the scalp, further enhances spatial resolution4,5,6 compared to standard dEEG systems. The sponge-based HCGSN can be applied rapidly and without scalp abrasion, making it ideal for use with adults7,8, children9,10,11, and infants12, in both research and clinical4,5,6,13,14,15 settings. This feature allows for considerable cost and time savings by decreasing the average net application time compared to other dEEG systems. Moreover, the HCGSN includes unified, seamless software applications for all phases of data, greatly simplifying the collection, processing, and analysis of dEEG data.
The HCGSN features a low-profile electrode pedestal, which, when filled with electrolyte solution, creates a sealed microenvironment and an electrode-scalp interface. In all Geodesic dEEG systems, EEG sensors detect changes in voltage originating from the participant's scalp, along with a small amount of electrical noise originating from the room environment. Electrical signals from all sensors of the Geodesic sensor net are received simultaneously by the amplifier, where they are automatically processed, packaged, and sent to the data-acquisition computer (DAC). Once received by the DAC, scalp electrical activity can be isolated from artifacts for analysis using the filtering and artifact detection tools included in the EGI software. Typically, the HCGSN can be used continuously for only up to two hours because the electrolyte solution dries out over time, gradually decreasing the quality of the scalp-electrode interface.
In the Parent-Infant Research Lab at the University of Toronto, we are using dEEG to study social cognitive processes including memory, emotion, goals, intentionality, anticipation, and executive functioning in both adult and infant participants.

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography dEEG

Dense array electroencephalography is being used increasingly to study social cognitive functions in infants and adults. Here we present an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults.

（密な配列の脳波を用いた乳児と大人で社会的認知を調査 D EEG）

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

に温痛覚を評価するためのローカルおよびグローバルメソッド<em&gt;ショウジョウバエ</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

マウス脊髄における条件遺伝子操作のためのウイルスベクターの定位注射

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

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 ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

冷痛覚のための新規アッセイ<em&gt;ショウジョウバエ</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step.
Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers.
Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution.
Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

ひと脳脊髄液中の Endopeptidomic の分析のための試料作製

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神経クラッシュ損傷のラットモデルにおける機能的評価のための3D運動学的解析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...