保健研究、カナダ脳卒中ネットワーク、および医学研究のためのアルバータヘリテージ財団のカナダの協会からの補助金によって支え。

Rotorod歩行装置 rotorodは、35センチメートル地上に固定された直径4 cm、回転筒であり、透明なプレキシグラスで囲まれた。 rotorodのシリンダーは、トラクションを容易にテクスチャラバーコーティングで覆われていた。装置の床は、動物が落ちた時に怪我を防ぐために泡の層で覆われていた。小さな電気モーターは、ゴム製のベルトを介してrotorodをオンにするパワーを提供する。 rotorodは一度ごとに5秒を回転するように設定されたビデオの録画と運動学高速デジタルビデオカメラは1秒の1000のシャッタースピードを使用して、動物を撮影するために使用された。動物がrotorodの上を歩いている間に正面、側面、および背面ビューが撮影されました。二腕ニコン、MII冷光源と光ファイバーライトの1つのセットが記録中に十分な照明を提供するために使用されていました。テープはSony DSRを20 DV CAMデッキを使用してフレームごとに解析した。ビデオフレームは、フレームグラバやMacintoshコンピュータを使用して捕獲された。 手順と分析のテスト 6 - OHDAと対照ラットの歩行動作の違いを評価するために、手術後の日15からビデオレコード - 30（病変からの回復がほぼ完了であることが予想されるかもしれない後）は、2つの独立によって調べた実験条件にブラインドオブザーバー。黒質線条体6 - OHDAの病変は左前部と後肢の持続的な障害の結果、右半球で誘導された。評価尺度は、速度にラットの姿勢と前肢のステップ運動（Whishawら、2003）を開発した。ステッピングの5つのコンポーネントが評価されました。 <ol><li> 姿勢は 、姿勢の評価は、ドラム上のラットの位置を調べることによって行われた。 </li><li> リフトとリリース 、足がドラムから解除されたステッピングモータサイクルの部分。 </li><li> 、ラットは前方に移動するように手足がドラムの上に開催されたステップのサイクルの一部を運ぶ ; </li><li> 事前に 、手足がドラムとの接触を回復するために拡張された範囲の部分、および</li><li>前足がドラムに戻って配置された配置 、。 </li></ol>以下の基準は通常の動きを定義するために使用された。 <ul><li> 姿勢：ラットの体が上に保持ヘッドとドラムに配向した。 </li><li> 持ち上げて離す：肩、肘、手首はドラムの表面から足を上げる屈曲されています。 </li><li> キャリー：足は、ドラムの表面上に保持され、数字は大まかに屈曲され、数字のヒントは、体の正中線に集中している。アドバンス：肢は桁が延長し、開位置にsemiflexed位置から移動すると前方に拡張されます。 </li><li> 配置：肢は肩、肘、手首で拡張され、数字はラットののほぼ中央に位置する数字2（正中線からの桁の秒）で、前方とドラムの表面上に足を置くために開かれボディ。 </li></ul>それぞれの動きは、3点尺度で評価した：1 = /運動は= /動きがわずかに異常な登場対照ラット、0.5のこと似ている、と0 = /動きが（Whishawら、2003。）明らかに異常だ。この評価スケールは、そして五つのステップ術後日に各ラットからの動き1、3、5、7、9、および15を評価するために使用されていました。 five対策からスコアはそれぞれの日に各ラットを平均し、結果が繰り返される尺度として日でANOVAを行った。 6 - OHDAと対照ラットで歩いrotorodの概要運動評価尺度は、定性的な運動解析のための術後のテストセッションに適用されました。テストセッション中に、6 - OHDAの病変群では対照群、F（1,8）= 33.2、P &lt;0.001よりも有意に低い点数を受けたが、試験日の有意な効果はなかった、またそこには重要なグループだったテストデーの相互作用による。ステッピングのスコアは、別々の変数（;コントロールラット、右足対左前足6 - OHDAのラットの病変の同側の前肢対病変に対側前肢）のような前肢と分析した。前肢、F（1,40）= 15.8、P &lt;0.01の有意な効果があった、と前肢、F（1,40）= 15.0、P &lt;0.01でグループの有意な相互作用があった。フォローアップt検定は6 - OHDA群の反対側から病変前肢は同側から病変前肢または差は認められなかった対照ラットの前肢よりも有意に低いスコアを受け取ったことを示している。運動のコンポーネントは6 - OHDAのグループによって得られた貧しい人々のスコア、姿勢の施策に寄与したかを判断するために、と6 - OHDA群の同側および対側前肢からステップサイクルの個々のコンポーネントは、対照ラットから得られた値と比較した。 6 - OHDAのグループは、姿勢と対側肢によるステッピングすべてのコンポーネントで異なっていたが、同側の肢のコンポーネントのどれもが（P &lt;0.05）。 コントロールと6 - OHDAラットにおける姿勢の質的および記述的な違い対照ラットでは、ヘッドが水平位置に保持され、サポート前肢と後肢がサポートするアーチを形成するドラムをまたいている。 6 - OHDAラットは、通常、対照ラットの同側の手足や手足のために発生したよりも高い位置にあるドラムの表面上での障害がより緊密に配置前肢（視聴者が直面している）と（と対照ラットのビデオを見て頭で歩いて姿勢のスコアは&quot;1&quot;、および&quot;0&quot;）のスコアを持つ6 - OHDAの病変の動物。一般的に、姿勢の異常がラットの頭部の相対的な位置から一目で見ることができます。 前肢ステッピング制御シーケンスでは、ステッピング前肢は肩、肘、手首を曲げると持ち上げている。手足が解除されると、数字は大まかに屈曲され、四肢はよくドラムの表面上に持ち上げられます。手足が進んだとき、それは肩、肘、手首で拡張され、それが配置されている数字が拡張され、オープンされています。対照ラットと比較して、病変四肢に反対DA欠乏ラットのすべてのコンポーネントの振幅は減衰する。肢は、後でドラムを解放未満を持ち上げられ、そして制御前肢よりも短い距離を進んでいる。さらに、手足が解除されたときに、桁がより屈曲している、と手足が配置されている場合、数字が少ない対照ラットのそれよりも延長して開設されています。それがドラムの上部に達すると、コントロールの前肢が解除され、手首は足を持ち上げて屈曲されており、前方に、そして手首は、四肢の進歩のように拡張されます。これとは対照的に、病変の前肢に反対6 - OHDAのラットのは、ドラムの動きによって、さらに尾側に運ばれ、そのため、まだ拡張手首で後で解放します。手首の屈曲は、受動的であると思われると数字がドラムの動きによって後方に運ばれ、前肢が前方に置かれたとき、足と腕がドラム表面から明確に解除されていないため発生します。対照ラットの前肢は前方にも拡張し、数字は四肢の進歩として開きます。手足が配置されているとして、それは数字4,3、および2で連続的に続いて、ドラム最初の表面に接触する5桁で、アルペジオの動きで配置されます。これとは対照的に、6 - OHDAのラットの肢が解除され、拡張少なく、数字が少ない拡張され、そして彼らは&quot;スラップ&quot;とほぼ並行してドラムの表面に接触される。ビデオは&quot;1&quot;としてスコア化した対照動物の前肢のステップの動き、そして主に&quot;0&quot;としてスコア化した6 - OHDAの病変の動物の前肢のステップの動きを示しています。 ステッピング後肢 6 - OHDAラットの反対から病変後肢の障害もあった。コントロール後肢は後方に移動すると、それは、回転ドラムに対してプッシュする表示され、それがそうするように、つま先が外側に回転する。手足が進むにつれ、足が外側に揺動する。 6 - OHDAのラットの後肢は、ドラム回転と直接後方に移動し、どちらかといえば、少し内側になります。手足が進むにつれ、足が直接繰り越されます。コントロールラットスイング外側に、それが高度であるため、足の指が足首よりも高く保持されているの麓、およびつま先は延長。コントロールのラットの位置、つま先5接点の間に最初のドラムと重量の面は、つま先と手のひらに分散されます。対照的に、キャリーと配置中に屈曲されている6 - OHDAのラットのつま先は、アルペジオの動きなしで行われます。対照ラットのつま先は足指5のような逆アルペジオパターンでリリースされた第1及びつま先1持ち上げられ、最後に解除されます。足首は、キャリーの移動中に、その曲げた姿勢を維持しながら、この動きは、6 - OHDAの病変のラットではありませんでした。再び6 - OHDAのラットの事前運動が減衰する、少ないexorotated肢で、以下の解除、および対照ラットのそれよりも小さいが進んだ。コントロールラットは彼らのつま先を置くように、彼らは、ラットの体内への5点横方向に相対的なようにつま先を延長して開設されています。つま先5接点第一ラットはつま先の先端にその重量を支持するように残りの足の指が続くドラム。足が繰り越されているように6 - OHDAのラットの場合は、つま先をやや屈曲され、つま先は足首までの水平方向の相対的に低くなって、足がドラム上に配置され足の指の小さい開口部があります。ストライドの長さは、また、対照ラットのそれよりやや短いです。 CONTの違いを定量化するために前肢がそうであったようにROLおよび6 - OHDA後肢の動き、リフトとリリースのコンポーネントを、運ぶ、進歩、および配置は、3点尺度で評価された。対策は、術後20日目に作られたビデオの記録から作られた、と三段階のサイクルは、各ラットについて評価した。重要なグループの違いは、F（1,8）= 47.6、P &lt;0.001があった。コンポーネントのフォローアップt検定は6 - OHDAラットの成分がよりも対照ラットのすべてのコンポーネントが有意に高いスコアを受け取ったことを示している。ビデオは&quot;1&quot;としてスコア化した対照動物の後肢のステッピング運動を示しています。ビデオに示すように6 - OHDAの後肢のステッピング運動は主に&quot;0&quot;のスコアで評価された。

<ol>
	<li>Chang, J. W., Wachtel, S. R., Young, D., Kang, U. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+anatomical+characterization+of+forepaw+adjusting+steps+in+rat+models+of+Parkinson's+disease:+studies+on+medial+forebrain+bundle+and+striatal+lesions.">Biochemical and anatomical characterization of forepaw adjusting steps in rat models of Parkinson's disease: studies on medial forebrain bundle and striatal lesions.</a> Neuroscience. 88, 617-628 (1999).</li><li>Metz, G. A., Tse, A., Ballermann, M., Smith, L. K., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unilateral+6-OHDA+rat+model+of+Parkinson's+disease+revisited:+an+electromyographic+and+behavioural+analysis.">The unilateral 6-OHDA rat model of Parkinson's disease revisited: an electromyographic and behavioural analysis.</a> Eur J Neurosci. 22, 735-744 (2005).</li><li>Miklyaeva, E. I., Castaneda, E., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+deficits+in+unilateral+dopamine-depleted+rats:+impairments+in+movement+and+posture+and+compensatory+adjustments.">Skilled reaching deficits in unilateral dopamine-depleted rats: impairments in movement and posture and compensatory adjustments.</a> J Neurosci. 14, 7148-7158 (1994).</li><li>Miklyaeva, E. I., Martens, D. J., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairments+and+compensatory+adjustments+in+spontaneous+movement+after+unilateral+dopamine+depletion+in+rats.">Impairments and compensatory adjustments in spontaneous movement after unilateral dopamine depletion in rats.</a> Brain Res. 68, 23-40 (1995).</li><li>Morrissey, T. K., Pellis, S. M., Pellis, V. C., Teitelbaum, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seemingly+paradoxical+jumping+in+cataleptic+haloperidol-treated+rats+is+triggered+by+postural+instability.">Seemingly paradoxical jumping in cataleptic haloperidol-treated rats is triggered by postural instability.</a> Behav Brain Res. 35, 195-207 (1989).</li><li>Muir, G. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+locomotor+recovery+following+corticospinal+tract+lesions:+measurement+of+ground+reaction+forces+during+overground+locomotion+in+rats.">Complete locomotor recovery following corticospinal tract lesions: measurement of ground reaction forces during overground locomotion in rats.</a> Behav Brain Res. 103, 45-53 (1999).</li><li>Muir, G. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ground+reaction+forces+in+locomoting+hemiparkinsonian+rats:+a+definitive+test+for+impairments+and+compensations.">Ground reaction forces in locomoting hemiparkinsonian rats: a definitive test for impairments and compensations.</a> Exp Brain Res. 126, 307-314 (1999).</li><li>M, O. l. s. s. o. n., Nikkhah, G., Bentlage, C., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forelimb+akinesia+in+the+rat+Parkinson+model:+differential+effects+of+dopamine+agonists+and+nigral+transplants+as+assessed+by+a+new+stepping+test.">Forelimb akinesia in the rat Parkinson model: differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test.</a> J Neurosci. 15, 3863-3875 (1995).</li><li>Schallert, T., DeRyck, M., Whishaw, I. Q., Ramirez, V. D., Teitelbaum, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+bracing+reactions+and+their+control+by+atropine+and+LDOPA+in+an+animal+analog+of+Parkinsonism.">Excessive bracing reactions and their control by atropine and LDOPA in an animal analog of Parkinsonism.</a> Exp Neurol. 64, 33-43 (1979).</li><li>Schallert, T., Norton, D., Jones, 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=A+clinically+relevant+unilateral+rat+model+of+Parkinsonian+akinesia.">A clinically relevant unilateral rat model of Parkinsonian akinesia.</a> J Neural Transpl Plasticity. 3, 332-333 (1992).</li><li>Whishaw, I. Q., Gorny, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arpeggio+and+fractionated+digit+movements+used+in+prehension+by+rats.">Arpeggio and fractionated digit movements used in prehension by rats.</a> Behav Brain Res. 60, 15-24 (1994).</li><li>Whishaw, I. Q., O'Connor, W. T., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contributions+of+motor+cortex,+nigrostriatal+dopamine+and+caudate-putamen+to+skilled+forelimb+use+in+the+rat.">The contributions of motor cortex, nigrostriatal dopamine and caudate-putamen to skilled forelimb use in the rat.</a> Brain. 109, 805-843 (1986).</li><li>Whishaw, I. Q., Pellis, S. M., Gorny, B., Kolb, B., Tetzlaff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximal+and+distal+impairments+in+rat+forelimb+use+in+reaching+follow+unilateral+pyramidal+tract+lesions.">Proximal and distal impairments in rat forelimb use in reaching follow unilateral pyramidal tract lesions.</a> Behav Brain Res. 56, 59-76 (1993).</li><li>Whishaw, I. Q., Woodward, N. C., Miklyaeva, E., Pellis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+limb+use+by+control+rats+and+unilateral+DA-depleted+rats+in+the+Montoya+staircase+test:+movements,+impairments+and+compensatory+strategies.">Analysis of limb use by control rats and unilateral DA-depleted rats in the Montoya staircase test: movements, impairments and compensatory strategies.</a> Behav Brain Res. 89, 167-177 (1997).</li></ol>

本研究の目的は、齧歯類の運動の技能のためのrotorod試験は定性的な姿勢の検査、前肢のステッピングと神経系負傷したラットに、ステッピング後肢に使用できるかどうかを調べることであった。分析のために、一方的なDAの枯渇と対照ラットとラットでは、正面側面と後部ビューから記録された映像だった。姿勢と前肢の動きの評価尺度は、ステップ運動が慢性的に以下の手術を障害することが示された。?...

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified, and another reference was included.
Additional reference:
15. Whishaw IQ, Li K, Whishaw PA, Gorny B, Metz GA. Distinct forelimb and hind limb stepping impairments in unilateral dopamine-depleted rats: use of the rotorod as a method for the qualitative analysis of skilled walking. J Neurosci Meth 126, 13-23 (2003).
Revised Abstract:
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Additional Discussion Paragraph:
A more detailed presentation of the present study has been made (Whishaw et al, 2003), but the present study presents the video support describing the stepping movement in the good and affected limbs of unilateral dopamine-depleted rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicated that whereas the DA-depleted rats could use the limbs contralateral to the lesion for support, they received minimal use for shifting weight. The results of this study indicate that the rotorod task, in addition to providing quantitative measures of motor impairments, can also provide insights into the qualitative impairments.
Original Abstract:
The rotorod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration that an animal stays up on the drum as a function of drum speed. Here we report that the task provides a rich source of information about qualitative aspects of walking movements. Because movements are performed in a fixed location, they can readily be examined using high-speed video recording methods. The present study was undertaken to examine the potential of the rotorod to reveal qualitative changes in the walking movements of hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). Beginning on the day following surgery and then periodically over the next two months, the rats were filmed from frontal, lateral, and posterior views as they walked on the rotorod. Behavior was analyzed by frame-by-frame replay of the video records. Rating scales of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in their posture and in the use of the limbs contralateral to the DA-depletion. The contralateral limbs not only displayed postural and movement abnormalities, they participated less in initiating and sustaining propulsion than did the ipsilateral limbs. These findings not only reveal new deficits secondary to unilateral DA-depletion, but also show that the rotorod can provide a robust tool for the qualitative analysis of movement.

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified and another reference was included.

Erratum: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

use of rotorod as a method for the qualitative analysis of walking in rat

動物が回転ドラムの上を歩くれるrotorod試験は、広く実験用げっ歯類で、モータの状態を評価するために使用されます。パフォーマンスは、動物がドラム速度の関数としてドラム上にとどまること期間によって測定されます。ここでは、タスクが歩行運動の質的側面に関する情報の豊富な源を提供することを報告。動きが固定された場所で実行されるため、それらは容易に、高速ビデオ録画の方法を使用して調べることができます。本研究は、半パーキンソンアナログラットの歩行運動の質的変化を明らかにするrotorodの可能性を調べるために行わ黒質線条体ドーパミン（DAを枯渇させるために右の黒質線条束に6 - ヒドロキシドーパミン（6 - OHDA）の注入によって製作されました）。定期的に今後2か月間に​​、手術の翌日に開始し、ラットは、彼らがrotorodに歩いて、正面側面、および後のビューから撮影された。動作は、ビデオ記録のフレーム毎の再生によって分析した。行動のステップの評価尺度は、ヘミパーキンソンのラットは慢性的に彼らの姿勢やDA -枯渇に対側肢の使用で損なわれたことを示した。対側の手足だけでなく姿勢や動きの異常を表示する、彼らは同側の手足に比べ推進を開始し、維持するには以下が参加。これらの知見だけでなく、DA -枯渇一方的に二次新しい欠損を明らかにするだけでなく、rotorodは運動の定性分析のための堅牢なツールを提供できることを示している。

Watch this Scientific Journal Video about Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat at JoVE.com

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

rotorod試験は半パーキンソンアナログラットの歩行運動の運動状態を評価するために使用されます。

ラットの歩行の質的分析のための方法としてRotorodの使用

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

use-rotorod-as-method-for-qualitative-analysis-walking

Research

JoVE Journal

Biology

12.9K ビュー.  University of Lethbridge. rotorod試験は半パーキンソンアナログラットの歩行運動の運動状態を評価するために使用されます。

ビデオ: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

生物学

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

ラットに到達熟練前肢の構造：運動評価尺度

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

タスクを歩くラダーラング：スコアリングシステムとその実用化。

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

出芽酵母のタンパク質濃度の関数としての形態学的表現型の開発の分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

モデル遺伝生物として使用するためのメダカ胚のマイクロインジェクション

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

イメージングのための簡単​​な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

微生物システム生物学のケモスタットの使用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

家禽生産のサンプルからの細菌群集の定性的および定量的評価のためのハイブリッドDNA抽出法

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

ラットの外側尾静脈から血液をサンプリング

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...