この作品は、SFB 604、DFGミズーリ1421/2-1とKrebshilfe 107089（HM）によりサポートされていました。 ASは、子供の腫瘍財団（米国ニューヨーク州）からの若手研究者賞を受賞です。

本研究は、ドイツ連邦共和国の動物保護法（Tierschutzgesetzデル·ドイツ連邦共和国）に従って行ったし、食品安全·消費者保護（テゥーリンガーLandesamtエリーゼLebensmittelsicherheitウントVerbraucherschutz）がチューリンゲン州政府によって承認された。 1。測定の設定<ol><li>イソフルラン/ O 2吸入によりマウスを麻酔-麻酔3％の開始のために、100％酸素中のメンテナンスを2％イソフルラン（ 図1）。このような動きの反射神経と低悪性度の痛みに対する感度のテストのような単純な反射神経をテストすることによって、十分な麻酔を確認してください。手順は、通常、合計でわずか30分/動物がかかるため、麻酔中の乾燥を防ぐために、目に軟膏を使用することが推奨されますが、必須ではない。生存実験の場合には、術後疼痛を管理するためのタイムリーな長い作用する鎮痛薬を投与する。 </li><li>電気かみそりで後肢をカバー毛皮を剃ると動物が鎮痛下に既にある間、市販の脱毛クリームで脱毛を行います。作業中は病原体を含まない状態を維持するために、無菌手袋を着用し、常に慎重に70％エタノールで洗浄楽器を使用しています。 </li><li>フィードバック制御された加熱プレートおよび直腸サーモプローブ（ 図2）により制御体温の安定性。必要に応じて、無菌実験環境を維持するために、動物の間の熱板をカバーするために、滅菌ドレープを使用してください。さらに、組織の損傷を回避するために40°Cを≤ために加熱温度を制限するために統合されたセンサを介して電子的に制御される加熱プレートを使用することが推奨される。 <img alt="図1" fo:content-width="4in" fo:src="/files/ftp_upload/51181/51181fig1highres.jpg" src="/files/ftp_upload/51181/51181fig1.jpg" /> 図1。経験剃毛後肢で麻酔マウスを示すerimentalセットアップ。 </li><li>重要なパラメータとして心拍数を監視する心電図（ECG）記録を取る。各前肢の皮膚の下に一方の電極と首領域内の皮膚の下に一つの電極を次のようにECG記録のための3つの電極を取り付けます。 </li><li>最適な導電率/抵抗転写用コンタクトゲルを使用して行わリング電極。 （黒色で表示）感知電極は、腓腹筋がその最大直径を有する位置に配置されている。 （赤色で表示）参照電極は単に感知電極の下に配置されている。 注意：機器の詳細については、「素材の表」を参照してください。 </li><li>近位および遠位の坐骨神経刺激とリード電極との間に所定のが、一貫性のある距離の測定の正確な位置をマークします。実験的な提案は：検知電極から4mmの距離では、遠位の刺激が行われる。感知電極へ16mmの距離では、近位刺激（ 図2）を実施する。 </li></ol><img alt="図2" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig2highres.jpg" src="/files/ftp_upload/51181/51181fig2.jpg" /> 図2。直前の測定値の先頭に実験状況を示す代表的な画像。白い矢印は、センシング（黒）と基準（赤）左後肢の腓腹筋の電極の位置を示している。針電極による刺激は、黒検知電極に関連して定義された位置で行われる。遠位刺激のポイント（左後肢に「DS」でブラックマーク）は、検知電極から4ミリメートルの距離が、近位の刺激（「PS」でブラックマーク）の場所が離れて16ミリメートルである。右後肢の赤い線は、約を示しています<html>坐骨神経の解剖学コースをimate。さらに、関連する後肢の筋肉の大まかな位置はランドマークとして表示されます。アスタリスクは、直腸サーマルプローブを示している。 2。測定<ol><li>原則：単極使い捨て28 G針電極により0.1ミリ秒の持続時間の繰り返し生成された単一の方形波パルスで神経刺激のシリーズを実行します（繰り返しレート200ミリ、 図3（a）参照 ）。オフラインデータ分析のために、同時にによる神経刺激に対する神経筋機能応答曲線（ 図3B）と一緒に刺激信号を取得することが推奨される。平均その後のデータ解析のための最大応答のシリーズ（「代表的な神経筋機能応答」）。信頼性の高いデータ、レコードおよびそれ以降平均刺激部位および動物あたり少なくとも3つの独立した、最適な応答曲線を生成するため。 p_upload/51181/51181fig3highres.jpg &quot;SRC =&quot; / files/ftp_upload/51181/51181fig3.jpg &quot;/&gt; データ収集と分析のための図3。手順（概略図）。繰り返し発生するパルスは、針電極を介して、坐骨神経（ 図3A上段）に適用されます。同時に、いくつかの対応する神経応答曲線（ 図3Aの下段）に記録されている。平均化され、拡大された場合には、刺激パルスに起因する神経筋応答曲線（ 図3（b）の上段）は、次の特性（ 図3（b）の下段）を示しています。信号応答のレイテンシだけでなく、信号の持続時間と振幅を示したことができているその後の計算および統計を得ることが。不規則な信号伝導および/または準最適な録音は通常は1の正と負たわみや変形信号よりも様々な信号の変形につながる減少した振幅（ 図3C）と（二峰形状）。 </li><li>決定された位置での針状電極（ 図2の「PS」）と近位の刺激を実行します。 <ol><li>最大振幅が最適な記録条件を達成するために、刺激の処理に同時実際の応答曲線を可視化。そうすることにより、実験者は直ちに応答曲線の形状並びに振幅の大きさを評価することができる。 </li><li>必要に応じて、若干、慎重に位置および/または坐骨神経に関して刺激針の角度を操作します。刺激条件のこの穏やかな最適化が可能な最大値と、典型的な二相性形状（ 図4）の応答曲線を一定の振幅に達することができます。 </li></ol></li><li>決定された位置（ 図2の「DS」）に針電極を遠位刺激を実行します。 </li><li> completio後に測定のN、それは胸骨横臥位を維持するのに十分な意識を取り戻したまでは別々のケージにテストされ、マウスを転送します。それは完全に麻酔から回復するまで無人と他の動物の会社で動物を放置しないでください。術後疼痛を管理するためのタイムリーな長い演技鎮痛薬を管理します。非ステロイド性抗炎症薬（NSAID）およびオピオイドの全身投与は、1〜3日に推奨される。 </li><li>代わりに、首の脱臼などによって動物のためのこれ以上の痛み、せずに、迅速かつ人道的なやり方で、まだ麻酔したマウスを生け贄に捧げる。 </li></ol><img alt="図4" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig4highres.jpg" src="/files/ftp_upload/51181/51181fig4.jpg" /> 図4。最大振幅とCMAPの録音を決定するためのイラスト。完全な登録シリーズが発表されている。 （A </st最小限のCMAP応答を持つ栄&gt;）挿入ポイント。 （b）は、わずかな刺激針の動きは、最大振幅のCMAP記録をもたらす。 （C）針配置の追加の変更がほぼ最大振幅を含む様々な振幅のCMAPの録音を生産する。ほぼ最大振幅のシリアルCMAPの録音と（d）の刺激針の交換。注意：CMAP振幅の典型的な減少量は、最適な刺激部位12、13で反復刺激中に発生する可能性があります。アスタリスクは、平均化に描かれた最大振幅でCMAP記録を示している。 3。分析<ol><li>適切なソフトウェアパッケージ（ 例えば、AtisaPro）を用いて、代表的な神経筋機能応答データセットに基づいて神経伝導パラメータを抽出する。 注：特別な注意を払って時間に関連するデータ判定を処理してくださいので、化合モーターアクションポットの変曲点決定差動（CMAP）開始と終了が困難な場合があります。検証、再現性の手順は、発症後および終了前の信号たわみに接線が使用されている。図3（b）に示す。 </li><li> &#39;信号の待ち時間&#39;と &#39;CMAP&#39;値を計算します。 <ol><li>レイテンシーCMAP持続時間 &#39;&#39;初期陰性波のキャンプ発症の間の時間間隔がベースラインに戻った当初のに対し、刺激とCMAP発症の間の時間遅延を表すが呼び出されます &#39;。遠位および近位の刺激部位間の距離と共に伝導速度を計算するために遠位端と近位待ち時間との差を使用する。 刺激部位間の伝導速度=レイテンシー/距離 </li><li> CMAP &#39;（化合モータ活動電位）振幅（mV単位で与えられる）CMAP信号の最大の正および負の折り返し点との間の大きさを表す。 CMAP = value &#39;の正の方向転換点」 -値&#39;負のターンアラウンドPOINT &#39; </li></ol></li></ol>

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著者らは、開示することは何もありません。

記載されたプロトコールは、関心のある神経を露出することなく、麻酔したマウスに坐骨神経伝導特性を決定するための簡単​​で信頼できる方法を提供する。それにもかかわらず、この実験手順は、針穿刺による組織損傷の原因となる。したがって、録音を終えた後、動物を犠牲にする合理的な選択肢である。しかし、従来の記録に神経の露出を必要とする他のより侵襲的な処置と比較し?...

電気生理学的測定は、臨床および実験室環境の両方で、末梢神経の機能的完全性を調査するための不可欠なツールである。ヒトでは、神経筋障害および神経障害の多くは、診断電気生理学的測定に依存しています。伝導速度、または信号の電位振幅のような神経の特性を測定することにより、末梢神経疾患の大まかな起源を特徴付けることが可能である。 神経伝導速度は髄鞘では有効になって急速な信号伝播に大きく依存している。そのため、脱髄プロセスは、一般伝導速度を4減のショー。化合モータ活動電位（CMAP） -機能軸索の数と相関が-かなり5に減少軸索損傷の指標である。 したがって、電気生理学的方法により末梢神経損傷の病因そのような遺伝性神経障害6,7、糖尿病性神経障害8,9、慢性炎症性脱髄性多発ニューロパシー（CIDP）10、または代謝性ニューロパシー11として、識別することができる。 通常、ヒトへの適用に腓腹または尺骨神経上の非侵襲的記録が好ましい。そして運動の感覚系の小口径の軸索 - マウスにおいて、坐骨神経の神経性、大型の両方を含む末梢神経系（PNS）の最大の神経を分析することは簡単である。 ここに示されているように手順がそのままマウスの末梢神経に電気生理学に関連するすべての標準的な値を測定するためには、迅速、簡単かつ信頼できる方法である。保存生物から録音を取ることによって、神経環境の生理学的条件が保証されています。

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Concentric Needle Electrodes (Stimulation)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td style="width:119px;">9013S0901</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital Ring Electrodes (Recording)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td>9013S0302</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ToM - Tower of Measurement (A/D converter)</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AtisaPro, Data acquisition &#38; analysis software</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HSE-Stimulator T</td>
			<td colspan="2">Hugo Sachs Elektronik, Hugstetten, Germany</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo electrophysiological measurements on mouse sciatic nerves

電気生理学的研究は、基礎となる病態生理1の理解を、一緒に神経病理学的技術と、様々な神経筋疾患の合理的な分類を可能にし、助けている。ここでは、 生体内でのマウスの坐骨神経に電気生理学的研究を行うための方法が記載されている。 動物は約30分/動物を取り、測定中に試験したマウスと邪魔されずに作業環境のための鎮痛を確保するために、イソフルランで麻酔する。 37℃の一定の体温は、加熱プレートによって保持され、連続的に、直腸サーモプローブ2によって測定される。さらに、心電図（ECG）が日常的に連続的に調査した動物の生理学的状態を監視するために、測定中に記録される。 電気生理学的記録は、坐骨神経、目の最大の神経で実行されます運動の感覚の両方繊維路でマウス後肢を供給E、末梢神経系（PNS）。我々のプロトコルでは、坐骨神経は、その場に残り、したがって、抽出または露出されている必要はありませんが、実際の録音と一緒に不利な神経の炎症のない測定が可能。適切な針電極3を使用して、我々は腓腹筋で検出電極を用いて送信ポテンシャルを登録し、近位および遠位の神経刺激の両方を行う。データ処理の後、信頼性が高く、神経伝導速度（NCV）および化合モータ活動電位（CMAP）のために高度に一貫した値、総末梢神経機能の定量化のための重要なパラメータは、達成することができる。

<html>各性別の6匹の動物：我々は、この研究のため、合計で12匹のマウスの坐骨神経にin vivoでの電気生理学的測定のシリーズを行った。測定は、提示されたプロトコルを用いて実施し、以下の結果が配信された。 両方の雄および雌のマウスは、約20m /秒の平均坐骨神経伝導速度（ 図5）を表示する。これは、文献中の他の測定値と一致する。さらに、雄?...

Watch this Scientific Journal Video about In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves at JoVE.com

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

インビボでの神経伝導特性の測定は、神経筋疾患の種々の動物モデルを特徴づける強力なツールを例示する。ここでは、麻酔したマウスの坐骨神経に対する電気生理学的分析を実施することのできる、簡単で信頼性の高いプロトコルを提示する。

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 ...

in-vivo-electrophysiological-measurements-on-mouse-sciatic-nerves

Research

JoVE Journal

Neuroscience

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

33.1K ビュー.  Fritz Lipmann Institute.  インビボでの神経伝導特性の測定は、神経筋疾患の種々の動物モデルを特徴づける強力なツールを例示する。ここでは、麻酔したマウスの坐骨神経に対する電気生理学的分析を実施することのできる、簡単で信頼性の高いプロトコルを提示する。 

ビデオ: In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

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 ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

同時電気生理学的記録および視交叉上核ニューロンのカルシウムイメージング

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

エレクトロポレーションによる齧歯類坐骨神経内のシュワン細胞へのインビボ遺伝子導入で

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

単一の坐骨神経とフローサイトメトリーを使用して単一のマウスの後根神経節における免疫系細胞の解析

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

生体内で運動ニューロンの変性症のマウスモデルの前肢からの複合筋活動電位の電気生理学的測定

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

ゼブラフィッシュ稚魚における側線有毛細胞の生体内カルシウム イメージング

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...