EY015748、EY017964（MEZ）、およびEY019517（ASV）：この作品は、国立衛生研究所からの補助金によって賄われていた。この作品はまた、眼科部門及びセントラルニューヨークのライオンズに失明無制限グラントを防止するための研究によってサポートされていました。我々はまた、動物の彼の優れたケアのためのこのビデオに出演するにはステップのために、私たちの動物技術者、マシュー·メリーニに感謝したいと思います。

動物ケア アフリカツメガエルは、本研究で用いたオタマジャクシがアップステート医科大学動物実験委員会および実験動物の管理と使用に関する指針によって承認された手順に従って上昇し、治療、成長させたツメガエル 。 1前の行動セットアップ：オタマジャクシ<ol><li>市販のソースから受精アフリカツメガエル胚を入手するか、以前に記載したように、6 in vitroで 、卵母細胞を受精。 </li><li> 20のKCl;; 20のCaCl 2、50mMのHEPES 0.1X MMR [10×Marcの修正リンゲル（MMR）は、溶液の在庫（10 mMのMgCl 2を含む60ミリメートルペトリ皿に18˚Cで神経板期胚への胞胚を置く。 1 MのNaCl、50μg/ mlのゲンタマイシン抗生物質]）、pH7.5に調整し、オートクレーブにかけ、室温で保存した。毎日0.1X MMRを変更し、デッド胚を削除します。 </li><li>胚はNieuwkoopとフェーバー段階に到達したら〜27から30 7、目を切り替える抗生物質なしの0.1X MMRへのE胚腸細菌の増殖を可能とする。 </li><li>彼らは供給ステージ（ステージ45）に到達するまで、オタマジャクシを育て、0.1X MMRで満たさ100ミリメートルペトリ皿に移動します。介入の日に0.1X MMRを変える、一週間、一日おきに、彼らにイラクサ粉末上清フィード。 </li><li> 100ミリメートルペトリ皿中の1週間後、カエルの水で満たされた半ガロンのタンクに動物を移動し、[0.5グラム/ Lの瞬間、海と2 mMリン酸ナトリウム二塩基性（のNa 2 HPO 4：141.96 MW）のpH 6.8]。ステップ2.4​​のように、彼らを養うが、水が濁っている場合には、水を週に2〜3回だけを変更してください。ライトは6時にオンにして、12時間のlight/12明暗サイクルで動物を維持する。 </li><li>試験前に、少なくとも12時間、O / N、実験台のアンダーパッドのような白い表面の試験対象と半ガロンタンクを配置します。 </li><li>ステージ45から50の試験オタマジャクシは以下の手順3で説明。 </li></ol> 2前行動セットアップ：技術設備T <ol><li>行動分析のための実験室の面積を取っておく。これは、標準的な蛍光灯の静かな、低トラフィックエリアである必要があります。 </li><li>動物のためのテストタンクを準備します。 ネストされた、半ガロン試験槽は2つの部分から構成されています。水やオタマジャクシを開催する内槽;および視覚刺激と外槽。 <ol><li>内槽の場合は、シャーピー、一番下から5cmと外側の角に小さなマークを作る。これは水ライン（ 図1A、緑の点線）である。 </li><li>また、内槽のために、シルガードエラストマーのような不活性化合物と（コーナーと中央）タンク内のdivetsを埋める。注：これらは記入されていない場合は、オタマジャクシは、これらの分野に残るでしょう</li><li>外槽の場合は、1黒い絶縁テープでタンクと白のティッシュペーパー図1Bとの残りの半分の外のちょうど半分をカバーしています。 </li></ol></li><li>上のタンクを配置ターンテーブルを接種する。 </li><li>セットアップウェブカメラ/三脚には、テスト用のタンクの上になるように。 図1Cに示すように、検査部位の視覚化を可能にするようにカメラを調整する。 </li><li> 5をインストールしたQuickTime PlayerソフトウェアでコンピュータにWebカメラを接続してください。カメラの電源を入れます。 QuickTime Playerのでは、ファイルの下に、「新しい動画撮影を。」を選択行動分析のセットアップがすぐにコンピュータ上で表示されます。 </li><li>オタマジャクシ動作に影響を与える可能性があり、外部の合図を低減、ならびに、水の表面での反射光を低減するためにセットアップ全体にわたって軽量綿布ドレープ。 </li><li> 35〜50のCD / m 2で布措置に基づく輝度いることを確認してください。 </li></ol> 3。行動アッセイ<ol><li>カエルの水でステップ1.2.1で行われた5cmのウォーターマーク、内側テスト給油して下さい。 </li><li>小さなネットを使用して、ゆっくりと内部テストタンクに動物を移動します。 </li><li>オープンQコンピュータと、ガイドとして外槽の使用についてuickTimeソフトウェアは、カメラのテストエリアを視覚化し、記録することができることを確認してください。 </li><li>カメラで紙や記録の一部に、動物の名前、日付と時刻を書き込みます。 </li><li>右図2に、タンクの黒い面と外槽に試験水槽を配置します。 </li><li>タンクを配置した直後に動画撮影を開始し、2分間のタイマーをセットします。 </li><li>タイマービープ音は、内部試験水槽を削除すると、外槽を180°回転させ、外槽の内側にインナー試験水槽を配置します。タイマーを開始する。注：黒側は、現在、他の側に配置する必要があります。 </li><li> 10の試験の合計ステップ3.7、8回繰り返します。各試行をオフにチェックし、ガイドとして、図2を使用してください。 </li><li> 2別の日に行動分析を繰り返します。 </li></ol> 4。網膜軸索切断<ol><li>彼らが応答しなくなるまで0.02％トリカイン動物を配置＃3ピンセットで尾のピンチに。 </li><li> 0.1X MMR中の1％アガロースを融解し、次いで60ミリメートルのペトリ皿に入れる。いったん冷却し、アガロース中に小さな長方形のディボットを作り、動物が部分的に液体に沈められるように、少し0.02％トリカインでディボットにオタマジャクシを追加します。 </li><li>ピアース25 G針で目の背部の後ろに45度の角度で肌、反対側の鉗子に対して動物をブレースながら。 </li><li>視神経の隣にある静脈をチョキチョキしないように注意しながら、慎重に、＃5ピンセットで穴に達する視神経を切り取ると邪魔にそれを反転。重度の出血が原因で誤って動脈を切り取るに存在する場合には、すぐに安楽死させるために、2％トリカイン動物を配置します。注意：トリカイン液2％がヒトにしびれを引き起こす可能性があります。このソリューションは、手袋をして取り扱ってください。 </li><li>動物を回復するために、手術後、0.7X MMRで100ミリメートルペトリ皿と20分間50μg/ mlのゲンタマイシン動物を配置します。 NEXT、カエルの水で回収タンクにオタマジャクシを移す。てみよう動物の残りのO / N </li><li>翌朝、（3.1から3.9ステップ）上記のような視覚機能をテストします。 </li></ol> 5。結果の分析<ol><li>試験が終了した後、オタマジャクシは、タンクの黒い面に留まる時間を測定する。 <ol><li>手動で時刻を表示し、頻繁に一時停止を可能にするソフトウェアを使用して動画を表示する。映像の表示時間を見てください。内槽が正常に外槽の内側に入れ子にし、カメラのビューで二乗されたときである2分間の裁判の開始を書き留めます。 </li><li>動物が上の泳いでいる水槽のどちら側を定義するためにオタマジャクシの目の位置を使用してください。両眼が黒/白のラインを越えた場合にのみ、他にタンクの片側からクロスオーバー定義します。 </li></ol></li><li>動物は、黒側に費やすことを（秒単位）の各区間の始まりと終わりを書き留めます。一時停止して再正確さを保証するためにビデオを巻き。各試行内の秒の合計。 </li><li>付属の行動のためのワークシートに入力これらの番号を。に費や​​した合計秒 - ワークシートには、動物が（120 [秒で合計時間で合計120秒のトライアル·アンド·ディバイドから、タンクの黒い面に費やす時間を差し引くことにより、白側に費やした時間の割合を計算します黒側）/ 120秒]。全10試行以上のワークシートの平均は、この比率。 </li><li>日の間試験は（P≤0.05）が有意であるかどうかを判断するためにスチューデントのt検定、対の、両側分布を使用してください。 </li></ol>

<ol>
	<li>Maurer, C. M., Huang, Y. Y., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+zebrafish+oculomotor+behavior+to+model+human+disorders.">Application of zebrafish oculomotor behavior to model human disorders.</a> Rev Neurosci. 22 (1), 5-16 (2011).</li><li>Solessio, E., Scheraga, D., Engbretson, G. A., Knox, B. E., Barlow, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+temporal+properties+of+the+rod+pathway+in+larval+Xenopus.">Circadian modulation of temporal properties of the rod pathway in larval Xenopus.</a> J Neurophysiol. 92 (5), 2672-2684 (2004).</li><li>Moriya, T., Kito, K., Miyashita, Y., Asami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+for+background+color+of+the+Xenopus+laevis+tadpole.">Preference for background color of the Xenopus laevis tadpole.</a> J Exp Zool. 276 (5), 335-344 (1996).</li><li>Viczian, A. S., Solessio, E. C., Lyou, Y., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+eyes+from+pluripotent+cells.">Generation of functional eyes from pluripotent cells.</a> PLoS Biol. 7 (8), (2009).</li><li>Choi, R. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+degeneration+following+rod+ablation+in+a+reversible+model+of+retinal+degeneration.">Cone degeneration following rod ablation in a reversible model of retinal degeneration.</a> Invest Ophthalmol Vis Sci. 52 (1), 364-373 (2011).</li><li>Viczian, A. S., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+determination+using+the+animal+cap+transplant+(ACT)+assay+in+Xenopus+laevis.">Tissue determination using the animal cap transplant (ACT) assay in Xenopus laevis.</a> J Vis Exp. 16 (39), (2010).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal table of Xenopus laevis (Daudin) : a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Blackiston, D., Shomrat, T., Nicolas, C. L., Granata, C., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second-generation+device+for+automated+training+and+quantitative+behavior+analyses+of+molecularly-tractable+model+organisms.">A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms.</a> PLoS One. 5 (12), (2010).</li><li>Rosemberg, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+spatio-temporal+behavior+of+zebrafish+in+the+open+tank+paradigm+after+a+short-period+confinement+into+dark+and+bright+environments.">Differences in spatio-temporal behavior of zebrafish in the open tank paradigm after a short-period confinement into dark and bright environments.</a> PLoS One. 6 (5), (2011).</li><li>Dong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Avoidance+in+Xenopus+Tadpoles+is+Correlated+With+the+Maturation+of+Visual+Responses+in+the+Optic+Tectum.">Visual Avoidance in Xenopus Tadpoles is Correlated With the Maturation of Visual Responses in the Optic Tectum.</a> J Neurophysiol. 101 (2), 803-815 (2009).</li><li>Lan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin+Elicits+Retinal+Fate+In+Xenopus+Animal+Cap+Embryonic+Stem+Cells.">Noggin Elicits Retinal Fate In Xenopus Animal Cap Embryonic Stem Cells.</a> Stem Cells. 27 (9), 2146-2152 (2009).</li><li>De Robertis, E. M., Kuroda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+patterning+and+neural+induction+in+Xenopus+embryos.">Dorsal-ventral patterning and neural induction in Xenopus embryos.</a> Annu Rev Cell Dev Biol. 20, 285-308 (2004).</li></ol>

The authors have nothing to disclose.

ここでは簡単に最小​​限の訓練を受けた研究室の担当者が週の下で実行することができる簡単な視覚誘導行動分析を報告する。他のアッセイは、動物の行動に特化した設備や専門知識を必要としますが、このアッセイは、視覚的な機能を決定するために簡単なテストを可能にする。別の行動分析、ビジュアル回避アッセイは、蓋がアフリカツメガエル 10内視知覚に貢献する?...

アフリカツメガエルは、眼の形成を研究するためのモデル生物として用いられてきた。目はビジュアル開発と機能に影響を与える遺伝子または経路をテストするための一週間も経たないうちに満期まで成長し、急速に開発しています。視覚機能をテストするには、視運動と微細運動応答は、それぞれ1,2、ゼブラフィッシュおよびアフリカツメガエルのオタマジャクシで使用されている。 アフリカツメガエルのオタマジャクシの目はゼブラフィッシュよりも比較的小さいので、このアッセイは、 アフリカツメガエルの微妙なテールフリップと眼球運動の挙動を検出するために、特殊な装置を使用し、訓練された人材を必要とします。 アフリカツメガエルにおけるより堅牢な動作がここに記述されている3、白背景、とのタンクで泳ぐための好みです。半分黒/ハーフ白タンクにオタマジャクシを配置するときに、事前に変成オタマジャクシはすぐにタンクの白側に泳ぐ。我々は以前、多能性細胞由来かどうかを判断するためにこのアッセイを使用目は4機能的であった。ここでは、変態前のアフリカツメガエルのオタマジャクシの視覚機能をテストするために使用され得るこのアッセイの詳細なバージョンを、報告する。 それだけで、ほとんどのアフリカツメガエルの研究室で発見搭載したデジタルビデオカメラ、デジタルカメラのソフトウェアや標準的な装置を必要とするので、このアッセイは、微細運動応答アッセイよりも簡単である。また、記録された応答は、集計結果に特別なトレーニングを必要としません。当社の代表的な結果は、二重の網膜軸索切断を受けたオタマジャクシの同じグループは、タンクの周りにランダムに泳ぐことを示している。また、片方の眼は、視覚的応答を試験することができる方法を示す、代表的なオタマジャクシから行動アッセイの結果が含まれている。アッセイの間に取得された数値が挿入され、分析することができるようにワークシートが含まれている。このワークシートは、試験したオタマジャクシは、視覚的応答を有するか否かを決定するために使用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1/2-gal. Flex-Tank with Cover</td>
			<td style="width:95px;">eNasco</td>
			<td style="width:119px;">SB19271M</td>
			<td style="width:225px;">size: 5-3/8&#34; x 7&#34; x 3-3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Black electrical tape</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">White tissue paper</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large Inoculating Turntable</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">50809-022</td>
			<td>size: Dia 114.3 x&#160; H 76.2 mm (4 1/2&#160; x&#160; 3&#34;)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Durasorb Underpad</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">82004-836</td>
			<td>size: 43.2 x 60.1 cm (17 x 24&#34;)</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Kim wipe</td>
			<td style="width:95px;">Krackeler Scientific, Inc.</td>
			<td style="width:119px;">1945-34155-CS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard Tripod</td>
			<td style="width:95px;"></td>
			<td style="width:119px;">various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iSight camera or webcam</td>
			<td style="width:95px;">Apple</td>
			<td style="width:119px;">M8817LL/A</td>
			<td>Good for larger tadpoles but small ones are difficult to see</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Portable computer</td>
			<td style="width:95px;">Apple/PC</td>
			<td style="width:119px;">various</td>
			<td>We used a 13&#34; MacBook, 2 GHz Intel Core 2 Duo running MacOSX Lion 10.7.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MiniDV Handycam Camcorder</td>
			<td style="width:95px;">SONY</td>
			<td style="width:119px;">DCR-HC42</td>
			<td>Connected by firewire to the computer with a 6-conductor and 4-conductor alpha FireWire 400&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Handycam Station</td>
			<td style="width:95px;">SONY</td>
			<td style="width:119px;">DCRA-C121</td>
			<td>This can be used for connecting firewire to camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QuickTime Player software</td>
			<td style="width:95px;">Quicktime</td>
			<td style="width:119px;">Version 10.1</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">26 Gauge Needle (5/8&#34; length)</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">BD305115</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Dumont #5 Forceps Biology</td>
			<td style="width:95px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Disposables</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Gentamicin Sulfate [50 mg/ml]</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">17-528Z</td>
			<td>Stored at room temperature</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sylgard 184 Silicone Elastomer</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">NC9644388</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Instant Ocean</td>
			<td style="width:95px;">Doctors Foster and Smith</td>
			<td style="width:119px;">CD-116528</td>
			<td>Stock solution = 100 g/L stored at room temperature</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:119px;">S0876</td>
			<td>Stock solution = 0.4 M stored at room temperature</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Tricaine (Ethyl-3-aminobenzoate methanesulfonate salt)</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:119px;">A5040</td>
			<td>Stock solution = 10% stored at -20&#730;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">In vitro fertilized embryos</td>
			<td style="width:95px;">eNasco</td>
			<td style="width:119px;">LM00490MX</td>
			<td>100 embryos/unit</td>
		</tr>
	</tbody>
</table>

a simple behavioral assay for testing visual function in xenopus laevis

カエルのオタマジャクシにおける視覚機能の測定は、 アフリカツメガエル 、生きた動物で失明のためのスクリーニングが可能となる。視運動性応答は試験した全ての脊椎動物で観察されている視覚ベース、反射的な動作です。従ってテールフリップ応答が微妙応答を記録するための訓練を受けた技術者を必要とする別の尺度として使用したオタマジャクシの目が小さい。私たちは、オタマジャクシは黒と白の両サイドにタンク内に置かれたとき、タンクの白い側に泳ぐことを好むという事実に基づいて、代替行動アッセイを開発した。ここで紹介するアッセイは、容易に測定された応答を作成し、安価な、簡単な代替手段です。セットアップは、ほとんどのアフリカツメガエルの研究室で容易に利用可能、三脚、ウェブカメラと、ネストされたテスト用のタンクで構成されています。この記事では、前と視神経を切断した後、オタマジャクシの挙動を示すのムービーが含まれています。片目の機能をテストするために、我々はまた、REPREを含むそれぞれの目には連続した日に、網膜軸索切断を受けているオタマジャクシのsentative結果。今後の研究では、一度に多くのオタマジャクシのビジョンをテストするためにこのアッセイの自動化されたバージョンを開発することもできます。

以前の報告は、我々はオタマジャクシのどちらかの目の視覚機能をテストするために、このアッセイを変更した、変態前のアフリカツメガエルのオタマジャクシは、白/黒のタンクの白側に泳ぐと、このアッセイと呼ばれることを好むことを背景色嗜好のアッセイ3を示している週間足らず。このようにして、彼らの目は、組織学的検査のために収集することができる。 <p clas...

Watch this Scientific Journal Video about A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis at JoVE.com

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

アフリカツメガエルのオタマジャクシは黒/白のタンクの白側水泳を好むツメガエル 。この現象は、それらの視覚によって案内される。この動作に基づいて、我々はオタマジャクシの視覚機能をテストするための簡単​​な分析を提示する。

テスト視機能のための簡単​​な行動アッセイ<em&gt;アフリカツメガエル</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

a-simple-behavioral-assay-for-testing-visual-function-xenopus

Research

JoVE Journal

Neuroscience

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

9.7K ビュー.  Center for Vision Research, SUNY Eye Institute, Upstate Medical University.  アフリカツメガエルのオタマジャクシは黒/白のタンクの白側水泳を好むツメガエル 。この現象は、それらの視覚によって案内される。この動作に基づいて、我々はオタマジャクシの視覚機能をテストするための簡単​​な分析を提示する。 

ビデオ: A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

春雨とカッペリーニの処理試験：ラットとマウスの器用な前足の機能の簡単な定量的指標

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

神経科学

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

ラットのショ糖の渇望のインキュベーションを評価するための一般的な方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

からの神経外植文化<em&gt;アフリカツメガエル</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

の解離、文化、および分析<em&gt;アフリカツメガエル</em&gt;胚性網膜組織

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

海馬のインスリンおよびマイクロインジェクション<emインビボ&gt;</em空間記憶テスト中&gt;マイクロダイアリシス

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

鳴禽類には聴覚フィードバックを操作するための軽量、ヘッドフォンベースのシステム

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同時<em&gt; ex vivoで</emによる二つの網膜の&gt;機能テスト<em&gt; in vivoで</em&gt;電図システム

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

でMARCMベースのクローンのMechanosensationための行動アッセイ<em&gt;キイロショウジョウバエ</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

の嗅球にカルシウム変化をモニタリングを通じて温度によって誘発される神経活動を記録<em&gt;アフリカツメガエル</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

準備やアフリカツメガエルの視蓋ニューロンの全体セル パッチ ・ クランプ記録のためのプロトコル

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

テスト視機能のための簡単​​な行動アッセイ

テスト視機能のための簡単な行動アッセイ