はじめ生物は、脅威刺激や環境の変化に対応する行動の広い範囲を示さなければならない。自律神経応答はよく交感神経系が生理的に内部環境（;ニコルズら 、2001。カーペンター、1976）現金れた戦いまたは飛行応答として脊椎動物で研究されている。それは、生物がストレス応答を調節し、差し迫った攻撃や環境の変化に反応するように動作を変更することができる生理学的な変化を介してです。証拠は、心臓血管系がしっかりとこのように応答を応答し、実装するために内部環境を可能にする呼吸器系に接続されていることを示します。それはよく呼吸器と心臓血管系の自律神経調節が差し迫って行動反応に必要な特定の標的組織への酸素の可用性と栄養素を調節することができることが知られている。 脊椎動物では、自律神経と生理反応は非常によく発展であり、複雑な無脊椎動物では、同様に開発された応答システムを持っているというのはよくあることに思われる（。Schapker ら 、2002; Zavarzin、1941）。脊椎動物と無脊椎動物が体系的に非常に異なっていても、高度に開発された無脊椎動物では、戦いまたは飛行の方法で対応する迅速な心血管や呼吸器系の応答を必要とする。多くの無脊​​椎動物の場合は、交感神経のような生理反応を定量化することができる。 1927年にまで遡る、非常に初期の研究の多くは、特に（;オルロフ、1927; Zavarzin、1941年Alexandrowicz、1932）一般的に節足動物を使用して、無脊椎動物で実施されている。 ザリガニは、迅速な行動の広い範囲と同様に評価し、環境刺激に応答する能力を示すことが知られている。非常に初期の研究は、防衛の姿勢の即時かつ迅速な応答（;ハクスリー、1880;シュラーノワら 、2006;。Wiersma、1961ベーテ、1897）の交感神経のような応答を指摘している。ビートとリズムが中枢神経系（;山岸と広瀬、1997;。山岸ら 、1997;ウィルケンズ、1999 Alexandrowicz、1932）によって制御されるので、大人のザリガニの心臓は、神経性です。日常的に、動物の心拍数（HR）の測定は、興奮性と内部環境の準備状況の直接的な指標を提供します。知覚にかかわる刺激（Listerman ら 、2000）で提示されたときザリガニでは、HRの増加は、防御姿勢中に記載されています。神経系の応答のこのタイプは、イセエビ（矢沢と勝山、2001）に示された。 換気システムはまたneuronally用の特別な付属物と呼ばれるscaphognathitesのポンプ作用（メンデルソン、1971）により鰓での酸素摂取量の責任換気中枢パターン発生器（VPG）で制御されます。シングルscaphognathiteは前方両端にそれぞれの鰓室に常駐し、リズミカルな動き（パーストル、1968）により鰓を介して水を描画します。 VPGは、心臓と同じように、多くの要因によって調整することができる。カニの前の仕事、 がんのマジスターは、心臓と呼吸のリズムを示し、コマンドのニューロン（ウィルキンスら 、1974）を介して中央制御することで変更できます。 Cuadras、1979、1980; Li ら 、2000;。Listerman らに加えて、VPGの活性は、内部応答と社会的相互作用や甲殻類の環境変化（Burmistrovとシュラーノワ、1996での変更で変更することが知られている。 、2000;マクマホンとウィルケンズ、1983; Schapker ら 、2002;。シュラーノワら 、2002;。ウィルケンズ、1976）。としてザリガニに見られるように、呼吸活動（VR）は、予期しない外部からの刺激（。。シュラーノワら 、2002。シュラーノワら 、1993）中に記録することができる内部状態とVRの変化によって異なる場合があります。 HRとVR対策の信頼性と実現可能性のために、頼まれるため、将来の問題は無限大です。前述のように、仕事は社会的相互作用と環境障害時の自律神経応答を調べるために行われています。興味深いことに、多くの分野はそのような自然なホルモンの変化時の自律神経応答の検査および/または生体で起こる他の生理学的プロセスとして探求されて残されています。可能な将来の方向性はまた、交配や嗅覚化学受信中に交感神経のような応答を検査することができる。最も重要なことは、生理学的反応は、彼らが内部で自分自身の準備を進めているとき、動物は、常に行動的に応答しないので、行動観察に比べて環境への生物応答に大きな洞察を提供することがあります。従って、生理学的測定は、生物の全体的な変化を説明するかもしれない生物の内部状態を評価するための生物学的なインデックスを提供します。 の方法ザリガニの配線を始める前に、配線の準備が必要です。十分な長ザリガニに挿入し、十分にインピーダンス検出器（UFI、モデル2991）に到達するために、2つの絶縁ステンレス鋼線をカット（AMシステムズ、Carlsburg、WA直径0.005インチとコーティング0.008インチで）。甲羅に挿入される両方のワイヤの両端に火によって絶縁（〜0.5 mm）を取り外します。動物の外側の塗装されていないワイヤが短絡さインピーダンスのレコーディングを防ぐためにリコートする必要があるとしてあまりにも多くの断熱材を削除しないように注意してください。その後、ピンセットを使用すると甲羅に配置するための90の角度で線の焼き付けられた端を曲げる。これは内臓にダメージを与える可能性があるため、ワイヤの挿入部分が長すぎないことを確認してください。プラスチックチューブ（電線保護される）にワイヤを挿入するの実現可能性については、それは緩く一緒にワイヤーをねじるのが最善です。記録の心臓と換気率の場合、上記の手順を繰り返すことは二回目とワイヤーの2つのセットがプラスチック製のチューブに配置する前に一緒に緩く編みにすることができます。記録のためのどのインピーダンス検出器に接続するどの配線混同しないように心臓や換気率のためにワイヤを指定してください。配線にラベルを付けたり、初めに他のより短い一組をカットするのが最善です。 都合良くかつ安全にザリガニを配線するには、それは、人および/またはザリガニ（図1）に傷害を除去するために濡れた布や紙タオルで爪や足を（背中と露出片側を残して）囲むことをお勧めします。 <img src="/files/ftp_upload/1594/1594fig1.jpg" style="float:left;" alt="図1" />図1。ザリガニのchelipedsと足の巻き方。濡れたペーパータオルでザリガニをラップすると、人とザリガニに傷害を防ぐことができます。折り返しがchelipedsや足を動かすためにザリガニの能力を妨げることになります。 甲羅の厚さは、内臓への損傷を引き起こす可能性が、このように鰓室で開始することにより、過度の圧力がザリガニを以下のように殺すために可能性が少ない各ザリガニと過剰な力で変化するので、配線プロセスを開始するには、それは、鰓のチャンバー内に開始するのが最適です胸部チャンバーと比較して。最初の穴は鰓室（。。Schapker ら 、2002すなわち、換気率を監視するprebranchial室）の吻側領域でキューティクルの下に挿入する電線のための十分なメスとだけ大規模な細かい点を用いて行われる。大きな穴は、過剰な体液の損失を持ち、適切にシールされず、これは指定された穴に残っているのワイヤの確率が増加します。ワイヤーが配置されると、ワイヤの上に接着剤（シアノアクリレートエステル）とアクセラレータ（HobbyTown米国、レキシントン、ケンタッキー州）の少量をドロップします。速乾性接着剤の使用は、動物の取り扱いのストレスを軽減。これは、ザリガニに対して毒性が強いと死亡の原因となりますので、キューティクルにアクセルのわずかな量を配置するように注意してください。すぐにペーパータオルで余分なアクセルを拭き取ることが最善です。鰓室第二ワイヤー、これらの手順を繰り返します。これら2本のワイヤが各scaphognathite運動（図2）の間に強い正確なインピーダンス測定を保証するためにscaphognathite（呼吸器）をまたがるように配置されます。正確な記録を取得するには、第2ワイヤは鰓室の外部と胸部チャンバー内に配置されていないことを確認してください。 <img src="/files/ftp_upload/1594/1594fig2.jpg" style="float:left;" alt="図2" />図2。鰓室内のステンレス鋼線の配置。甲羅上の白い点は、キューティクルの下に置かれるステンレス鋼線のための点を表します。ワイヤーは、ダイナミックインピーダンスを通してscaphognathiteと対策の動きにまたがる。 胸部室内の配線の心拍数のステップは、線が吻側 - 尾側の配置（図3）で直接心臓とスパンで背甲の下に心が配置されている以外は上記と同じです。この配置は、それぞれの心臓の収縮（Listerman ら 、2001）の間に強い正確なインピーダンス測定を保証します。 <img src="/files/ftp_upload/1594/1594fig3.jpg" style="float:left;" alt="図3" />図3。胸部チャンバー内のステンレス鋼線の配置。甲羅上の白い点は、キューティクルの下に置かれるステンレス鋼線のための点を表します。ワイヤは、吻側 - 尾側配置の中心をまたがる。これは、ダイナミックインピーダンスを介して心臓の収縮の対策が可能になります。 一度にすべての4本のワイヤの配置が完了すると、（イーストマン、Eを乾燥5分、瞬間接着剤を混ぜる梅毒に感染した）とコートそれぞれの場所だけでなく、配線用の穴。ワイヤーとドリル穴が適切に網羅されると、ザリガニの後ろにプラスチックチューブを保持するために、残りのエポキシ樹脂を使用。これは、配線が将来ハンドリングの穴から引き出されることが保証されます。エポキシはエポキシはもはやタッチに付箋がなくなるまで水のない容器でザリガニを、乾燥させ保持または配置するために数分かかりますので。エポキシが乾燥したら、余分な化学物質を除去し、水生環境に戻す配置する水でザリガニを洗う。ザリガニの健康を確保するため、それは以前に削除されない化学薬品を除去するために約1時間で水を変更するのが最善です。前述の研究者から指摘、我々は、その長期化処理が3日に24時間の持続時間のために当然ストレスを生理学的測定値を変化させる発見（ウィルキンスら 、1985。）ので、動物はどんな実験前に2〜3日放置してください。 心電図を記録するには、ザリガニからインピーダンス検出器への配線を接続する。心臓（測定HR）をまたがる2本のワイヤがscaphognathiteにまたがる2本のワイヤーのための単一のインピーダンス検出器と同様に（測定VR）に接続する必要があります。インピーダンス検出器は2つのステンレス鋼線との間の動的な抵抗を測定します。これらの信号はPowerLab/4SPインタフェース（ADインスツルメンツ）を介してPowerLabにオンライン検出器を接続して記録されます。すべてのイベントが測定さとPowerLabのChartソフトウェアのバージョン5.5.6（ADインスツルメンツ、オーストラリア）で校正されます。開始するには、4 kHzで取得率を設定します。大きなたわみは、ハートビートや換気率を表します。各HR / VRを数えるのを容易にするために、平滑化関数は、データセットに適用できます。 100ポイントを平滑化されている場合（すなわち、平均）結果のトレースは、ハートビートを描く予定。心拍数は、10秒間隔で、各ビートの直接カウントにより決定し、分あたりのビート（BPM）に変換することができます。

<ol>
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A. 124, 531-538 (1999).</li><li>Yamagishi, H., Ando, H., Makioka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+heartbeat+in+the+primitive+crustacean+Triops+longicaudatus.">Myogenic heartbeat in the primitive crustacean Triops longicaudatus.</a> Biol. Bull. 193, 350-358 (1997).</li><li>Yazawa, T., Katsuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+repetitive+cardiac+slowdown+in+the+freely+moving+spiny+lobster,+Panulirus+japonicas.">Spontaneous and repetitive cardiac slowdown in the freely moving spiny lobster, Panulirus japonicas.</a> J. Comp. Physiol. 187, 817-824 (2001).</li><li>Zavarzin, A. A., Zavarzin, A. A., Izbrannye, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ocherki po evol utsionnoj gistologii nervnoj sistemy (Essays on the evolutionary histology of the nervous system). III, (1950).</li></ol>

<table border="1">
<tbody>
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
</tr>	
<tr>
<td>Scalpel (Stainless steel surgical blades No. 11 for No. 3 handle)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Stainless steel wire (diameter 0.005 inches with the coating 0.008 inches; A-M Systems, Carlsburg, WA)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Impedance detector (UFI, model 2991)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Plastic tubing (Tygon Tubing ID .0812, OD .1492, Cole-Parmer Instrument Company)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fast-drying glue (cyanoacrylate ester, HobbyTown USA, Lexington, KY)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Accelerator (HobbyTown USA, Lexington, KY)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Adhesive (Eastman, 5-min drying epoxy)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">PowerLab via a PowerLab/4SP interface (AD Instruments)</a>.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">PowerLab Chart software version 5.5.6 (AD Instruments, Australia)</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">Chart software version 3.5.6 (AD Instruments, Australia)</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tbody>
		</table>

measures of heart and ventilatory rates in freely moving crayfish

恐怖、飛行や戦い応答は、切迫した捕食者の攻撃を受けて、その環境の生物の意識を調べるための基本的な生理学的な基礎としての役割を果たす。脊椎動物のそれと同一の無脊椎動物の保有は、自律神経系かどうかそれが知られていないが、証拠は無脊椎動物の内部環境と与えられた刺激に行動的に行動する準備ができて生物を規制する交感神経のようなレスポンスを持って示しています。さらに、この生理的応答がうまくいくように測定したところ、動物の内部状態の生物学的指標として機能するbeできます。生理学的応答の測定は、心血管系および呼吸器系の協調制御する中枢神経系の変化を介して直接内部と外部のストレス要因に関連することができます。具体的には、監視心臓や換気率は、常に行動的に観察されないストレス反応の定量化可能な対策を提供します。ザリガニは、録音の可能性だけでなく、ザリガニの形態が知られている豊かな歴史、1888年にハクスリーにさかのぼる、とよく研究された典型的な行動が原因で心臓や呼吸速度の測定のための良いモデル生物である。

Watch this Scientific Journal Video about Measures of Heart and Ventilatory Rates in Freely Moving Crayfish at JoVE.com

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

無脊椎動物は、脊椎動物のために記載したものと同様の自律交感神経のような応答を示しています。心血管系および換気システムの調整は、生体内部の状態を評価するための生物学的指標の測定が可能になります。

自由にザリガニを移動でハートと換気率の措置

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an ...

measures-of-heart-and-ventilatory-rates-in-freely-moving-crayfish

Research

JoVE Journal

Biology

15.2K ビュー.  University of Kentucky. 無脊椎動物は、脊椎動物のために記載したものと同様の自律交感神経のような応答を示しています。心血管系および換気システムの調整は、生体内部の状態を評価するための生物学的指標の測定が可能になります。

ビデオ: Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

ラット心臓における培養組織の移植

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

生物学

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

自由に動物を振る舞うの神経回路のアクティベーションの記録

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

の心臓の鼓動の可視化ショウジョウバエ</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

ザリガニ開閉筋肉のNMJにおいて歴史的なビューと生理学のデモンストレーション

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

の電子顕微鏡のための胚の調製ショウジョウバエ胎児心臓の管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

電離放射線は、次のγH2AXの空間分布の評価

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

無傷骨格筋線維内のカルシウムスパークスの評価

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNAの崩壊料金の測定で<em&gt;サッカロマイセス·セレビシエ</em&gt;使い方<em&gt; rpb1-1</em&gt;株

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

以下のための遺伝学的および生化学的アプローチ<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;タンパク質のオリゴマーの評価：リアノジン受容体のケーススタディ

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...