我々は生命のファンド、オクラホマ大学学部研究機会プログラム、およびこの作業に資金を提供するための動物の部に感謝。我々はまた、編集のヘルプは博士マリエルのHoefnagelsに感謝。

1。プリレコーディング、ライブアニマル準備<ol><li>そのホームの瓶からサソリを削除し、あらかじめ冷却したガラス容器に入れて、次に、2分間の最低凍結環境（-5℃）にサソリを含むjarファイルを置きます。この時間は、年齢、サイズ、種類、研究の下で特定のサソリの等に応じて異なる場合があります。動物は動かずになるとしかし、一般的に、凍結環境フェーズは完了です。 </li><li> 2分後、冷たいガラス容器から固定化サソリを削除し、顕微鏡スライド上に腹側を上に置きます。スティンガー、尾、足、および成形可能な粘土（油ベース、バンアーケンPlastalina）とpedipalpsを固定します。 </li><li>サソリのpectinesを置くためのプラットフォームを作る。櫛の大きさに応じて、プラットフォームの長さと幅を調整します。一般的に、私たちのプラットフォームは、顕微鏡のカバーガラスで作られたと約10（L）X 18（W）mmであるされています。 </li><li>に合わせてテープを（必要に応じて）トリミング、ホタテガイプラットフォームに両面粘着テープを付着する。 </li><li> pectinalチャンバーを作るために、3〜4 mmの深さに予備溶融ワックス（我々は100％蜜蝋のチップを使用）に、プラットフォームの四辺のうち3つを置く。私たちは、ホットプレート上にあるガラスのペトリ皿内でチップを配置することにより、ワックスを溶かす。 pectinal室の端のワックスが室温に冷却されると、これは高さ1 mm程度のワックスの壁を作成する必要があります。 </li><li>動物では、pectinal挿入のポイントを見つけ、そしてそれにちょうど後方のプラットフォームのアンワックスエッジ（テープ側を上に）配置します。その後、ゆっくりと動物の脚を抑制するために使用される粘土に対してその左右の周縁を押して、プラットフォームを（それは後で移動することがないように）安定する。 </li><li>チャンバー内の櫛またはpectinesを慎重に配置する先の細いピンセットを使用してください。具体的には、鉗子を使用して、pectinal背骨に沿って櫛状突起を押したまま、ゆっくりとカバーガラスの下から櫛状突起を引き出すと接着プラットフォームの床に下ろします。慎重にpectinal背骨に圧力を適用することにより、ホタテガイからテープへの結合を確立する。 </li><li>現在のプラットフォームのアンワックスエッジに溶けたワックスを（我々は予備加熱された金属製のへらを使って）適用する。目的は、櫛を確保し、完全に将来の鉱物油を導入するためのチャンバーを囲むために、2つあります。それが過熱してワックスで櫛状突起を破損することが可能であるとして、動物の櫛状突起上のワックスを適用するときは注意してください。 </li><li>櫛室の建設は完了です。次に、2つのテールセグメントの間に銀線を挿入することにより、動物の血リンパと不関電極の接続を確立する。 </li></ol> 2。同時細胞外記録法と化学的刺激<ol><li>我々は細胞外、先端録音を行う前に動物の24時間を準備し、これは、他の理由（固定位置まで、すなわち動物の調整）の間で、特定の（感覚子に依存する）先端径のカスタムメイクの刺激のピペットに時間が可能になります。我々はそのような仕様を達成するためにマイクロピペットプラーを使用してください。理想的には、ピペットの直径はsensillar細孔径より≈が2μmより大きいです。私たちは、それぞれの動物のためにそれに応じて私たちのマイクロピペットの仕様を調整する。 </li><li>記録前の1時間は、我々は、石油で櫛状突起を水没、pectinal室にミネラルオイルを5μlを加える。その後、我々は、長距離の目標と落射照明を行っていると高倍率の顕微鏡、下に動物を配置します。 </li><li>化学的にペグ感覚器を刺激するために、我々は、石油の媒体を介して水味物質（すなわちクエン酸と塩）を紹介します。最初に、我々は興奮剤のピペットに興奮剤を注入するマイクロピペットフィラーを使用してください。注：すべての刺激のソリューションは、電気伝導度のための電解質が含まれている必要があります。 </li><li>次に、マイクロマニピュレータ（記録のリグで）で覚せい剤のピペットを配置した後、刺激のピペットの鈍い、開放端に記録電極を挿入する。金属電極と電解刺激のソリューションとの間の接触を確保することが重要です。 </li><li>準備が電気的に（不関電極を介して）に接地されると、単純に油媒体を介してとsensillar孔上にピペットを下げます。 pectinal感覚子あたりの化学的刺激の持続時間は同じくらい30回分から、わずか1秒に変えることができます。一般的に、我々は、ピペットごとに複数のペグ（&gt; 30）をサンプリングすることができます。覚せい剤のピペットは分破片で目詰まりした場合に、単に新しい、未使用のピペット用詰まったピペットを交換する。 </li></ol> 3。実験の数日に動物を準備<ol><li>動物が長期間にわたって使用する場合、我々はpectinalチャンバーを再利用し、各レコーディングセッションのために鉱物油の滴を置き換えることをお勧めします。ある特定の日のレコーディングセッションが完了したら、セットアップの記録から動物を削除し、鉱物油の洗浄を行います。 </li><li>仕様fically、チャンバーにミネラルオイルの別のドロップ（5μl）を追加し、すべての油を取り除く。これは、記録セッション中にピペットから漏れた可能性がある残留刺激の存在を、最小限に抑えることができます。 </li><li>次のセッションの前に、前述のように石油の一滴を追加。 </li></ol> 4。代表的な結果： 少なくとも3:1のN比：（特定のアンプを、例えば、ハードウェア、等をデジタル化する）使用する記録装置に応じて、代表的な結果は、Sと細胞外記録です。 それは極性（水ベース）化学物質の味物質8を提供するための培地を提供するので、鉱物油室の成功の建設は、個々の感覚器に化学的刺激を単離するために必要です。すべてにおいてあまりにも少量の油またはnoneがある場合は、刺激のソリューションは、化学的刺激8の長期、制御されていない試合を引き起こす可能性があるか、その近隣の感覚器に記録された感覚子から広がっていく。

<ol>
	<li>Hodgson, E. S., Lettvin, J. Y., Roeder, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+a+Primary+Chemoreceptor+unit.">Physiology of a Primary Chemoreceptor unit.</a> Science. 12, 417-418 (1955).</li><li>Newland, P. L., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+processing+in+the+insect+nervous+system.">Taste processing in the insect nervous system.</a> Methods in Insect Sensory Neuroscience. , 289-318 (2005).</li><li>Foelix, R. F., M&#252;ller-Vorholt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+scorpion+sensory+organs+II.+Pecten+sensilla.">The fine structure of scorpion sensory organs II. Pecten sensilla.</a> Bull. Br. Arachnol. Soc. 6, 68-74 (1983).</li><li>Carthy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+structure+and+function+of+the+sensory+pegs+on+the+scorpion+pecten.">Fine structure and function of the sensory pegs on the scorpion pecten.</a> Experientia. 22, 89-91 (1966).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+chemosensory+peg+sensilla+on+the+pectines+of+scorpions.">Response properties of chemosensory peg sensilla on the pectines of scorpions.</a> J. Comp. Physiol. A. 181, 291-300 (1997).</li><li>Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pectine+organs+of+the+scorpion,+Vaejovis+spinigerus:+Structure+and+(glomerular)+central+projections.">The pectine organs of the scorpion, Vaejovis spinigerus: Structure and (glomerular) central projections.</a> Arthropod Struct. Dev. 37, 67-80 (2008).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+evidence+of+synaptic+interactions+within+chemosensory+sensilla+of+scorpion+pectines.">Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines.</a> J. Comp. Physiol. A. 181, 301-307 (1997).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+examining+scorpion+peg+sensilla:+the+mineral+oil+flood+technique.">A new approach to examining scorpion peg sensilla: the mineral oil flood technique.</a> J. Arachnol. 37, 379-382 (2009).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+tip-recording+method+to+test+scorpion+pecten+chemoresponses+to+water-soluble+stimulants.">A new tip-recording method to test scorpion pecten chemoresponses to water-soluble stimulants.</a> J. Neurosci Methods. , .</li><li>Haupt, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+sucrose+perception+in+the+honey+bee+(Apis+mellifera+L.):+behaviour+and+electrophysiology.">Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology.</a> J. Comp. Physiol. A. 190, 735-745 (2004).</li><li>Bland, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensilla+on+the+antennae,+mouthparts,+and+body+of+the+larva+of+the+alfalfa+weevil,+hypera+postica+(gyllenhal)+(Coleoptera+:+Curculionidae).">Sensilla on the antennae, mouthparts, and body of the larva of the alfalfa weevil, hypera postica (gyllenhal) (Coleoptera : Curculionidae).</a> Int. J. Insect Morphol. Embryol. 12, 261-272 (1983).</li><li>Cate, H. S., Derby, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooded+Sensilla+Homologues:+Structural+Variations+of+a+Widely+Distributed+Bimodal+Chemomechanosensillum.">Hooded Sensilla Homologues: Structural Variations of a Widely Distributed Bimodal Chemomechanosensillum.</a> J. Comp. Neurol. 444, 345-357 (2002).</li></ol>

我々は、これまでの2誌、ノウルトンとGaffin（2009）とノウルトンとGaffin（印刷中）でこれらのメソッドを公開しています。

上記のプロトコルは、電気生理学的研究のために砂漠草原サソリ（Paruroctonus utahensisを ）準備する方法について説明します。具体的には、油の下にpectinal化学感覚ニューロンの制御された細胞外の先端 - 記録用チャンバーを構築する方法を示します。油と水が混合しないため、単一の感覚器に化学的刺激を（水ベースの刺激剤との）分離することが可能です。それは、P.ことを強調しておきたいutahensis、比較的小さな動物（≈2.5〜5センチメートル）であり、より大きな動物にこのプロトコルを適用すると、このような動物のプラットフォーム、チャンバー、および適用される油の量の大きさなど、多くのサイズの調整を必要とする可能性があります。我々は、水ベースの化学覚せい剤の制御された送達のために必要な油の最少量をテストするパイロット研究を実施することをお勧めします。 さらに、我々はpectinal感覚器8内のベースラインの神経活動のオイルの効果は認められなかった。この前chemoresponsivenessを評価するだけでなく、他のモデルシステムのために確認されるべきである。 このメソッドは、感覚分野における感覚器の間でchemoresponse相互作用の効果をテストする別の方法と組み合わせて使用​​することができます。例えば、それは近隣の感覚子9から我々のベースレコード（タングステン電極を介して）として1つの感覚子からtip -記録（油下）に可能です。このような記録の構成は、化学的にone感覚子を刺激しているかどうかを評価するために使用することがベースに記録された感覚子の神経活動に影響を与えます。これまでに、誰もサソリpectinal感覚器のためにこれをテストしていない、そしてそれは同様に他の化学的感覚のシステムの未解決の問題のまま。 要約すると、我々は、石油の進歩節足動物味覚の神経基盤に電気生理学的研究の範囲で先端の記録方法を考える。この原稿では、我々はこの方法のために動物を準備するためのプロトコルを提供し、我々はそれが末梢感覚神経系のさらなる研究のための確固たる基盤を提供する願っています。

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<td>Lift-N-Press tab</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>16082</td>
<td>Doubled-sided adhesive</td>
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<td>MicroFil</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>MF34G</td>
<td>Micropipette filler</td>
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<td>Standard Glass Capillaries</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>1B100F-6</td>
<td>Glass pipettes</td>
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<td>Microelectrode Holder</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc. </td>
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electrophysiology of scorpion peg sensilla

我々は、電気生理学的に短く、ペグのような感覚感覚器3,4を調査するため、既存の先端記録技術の1,2の変更を説明します。すべてのサソリの半ば腹側表面にメカノと化学的感覚ペグ感覚器5,6の密なフィールドがあるpectinesという二つの付属です。これらの感覚器のchemoresponsivenessを評価するための一つの方法は、揮発性の匂いが感覚フィールド5,7に導入される細胞外に感覚子内の神経活動を記録するためのタングステン電極を使用しています。この方法の制限は、低速なデータ収集と制御されていない刺激の導入、そしてペグのフィールドからの削除などがあります。これらの制限を克服するために、我々は、個々のpeg感覚器8,9に水ベースのtastantsを提供を通す媒体として非極性鉱物油を使用する新しい先端記録技術を開発した。我々は成功し、クエン酸、エタノール、および塩にsensillar chemoresponsesを取得するためにこのメソッドを適用している。ここではそのような研究9の実験的なプロトコルについて説明します。我々はこの新しい方法は昆虫10、11、甲殻類12のものも含めて他の節足動物化学的感覚のシステムの応答特性を研究するために有用かもしれないと思う。

Watch this Scientific Journal Video about Electrophysiology of Scorpion Peg Sensilla at JoVE.com

Electrophysiology of Scorpion Peg Sensilla

この記事では、ミネラルオイル下外、先端録音を経由して個々の感覚器に化学的刺激を単離するための電気生理学的方法を説明します。

スコーピオンペグ感覚器の電気生理学

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

electrophysiology-of-scorpion-peg-sensilla

Research

JoVE Journal

Neuroscience

8.9K ビュー.  University of Oklahoma. この記事では、ミネラルオイル下外、先端録音を経由して個々の感覚器に化学的刺激を単離するための電気生理学的方法を説明します。

ビデオ: Electrophysiology of Scorpion Peg Sensilla

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

齧歯類の脳における同時fMRIと電気生理学

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

神経科学

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髄電気生理学II：細胞外吸引電極の作製

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

NRCCパッチクランプチップ上で細胞の培養と電気生理学

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

用いて、化合物の生理活性をスクリーニングするためのペアNanoinjectionと電気生理学的アッセイキイロショウジョウバエジャイアントファイバシステム

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

電気生理学および高分解能二光子顕微鏡用マウス前庭感覚上皮の切り抜き半無傷準備

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

から電気生理学的記録<em&gt;ショウジョウバエ</em&gt; Labellar味感覚器

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

リアルタイム電気生理学：プローブ神経ダイナミクスとその先にクローズド·ループ·プロトコルの使用

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

新生児げっ歯類から単離された脳幹・脊髄調製物の電気生理学は、ニューラルネットワーク呼吸器の出力の記録を可能にします

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

マルチ電極アレイ上のらせん神経節ニューロン外植片培養と電気生理学

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

電気生理学およびカルシウムイメージングのための急性神経組織の長期のインキュベーション

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...