KPMはEU FP7（RETICIRC）によってサポートされていました。

1。魚類の繁殖胚は保持され、隆起した標準的な条件（ブランド2002年）の下や日後受精（DPF）の開発によると上演された。 5 DPFにおける大人と幼虫を測定に利用された。 2。実験手順<ol><li>楽器の準備</li></ol>幼虫：魚の幼虫を3％に埋め込まれて予め温めておいた（28℃）体の動きを防ぐために、メチルセルロース。胚は、突出した画面に直面して、VisioTrackerに背側を上にして置かれた。アダルト魚：魚は300mg / lで、MS-222、固定装置に嵌め込まれVisioTrackerに配置で簡単に麻酔した。測定が開始された前に、彼らは、1〜2分間放置した後に回復する。 <ol start="2"><li>刺激パターンの生成</li></ol>魚の周りを回転し、垂直白と黒の正弦波格子から成る刺激パターンが作成されましたプロプライエタリなソフトウェアパッケージを使用して。彼らは、波形、コントラスト、輝度、角速度と空間周波数に応じたソフトウェアパッケージを介して変調された可能性があります。パターンはVisioTracker内に含まれているデジタル光プロジェクターを使ってスクリーンに投影された。魚の眼とスクリーンの間のおおよその距離がスクリーンで4.5 cm、および投写サイズは水平360度、垂直方向に55度であった。仔魚は、刺激の方向は、サッカードの頻度を減らすために0.33 Hzの周波数で変化した。成魚は一方向に刺激されたと一般的に鼻に時空目速度が（MuellerとNeuhauss、2010を参照）有意に低いとほぼ一定であるため、唯一の一時的なツー鼻方向に刺激眼は、と考えられていた。 <ol start="3"><li>眼球運動の記録</li></ol>魚の頭の明視野像、赤外線ビデオカメラに供給した。魚の赤外線照明は、fを行った下のROM。カメラはそれぞれ、5フレーム/秒（幼虫）または12.5フレーム/秒（成人）の速度で画像を記録した。画像は自動的に処理され修正され、目の形のために平滑化される。横軸の関係で眼の向きが自動的に決定されたと目の速度はプロプライエタリなソフトウェア·パッケージによって算出した。魚の小さな動きは、ソフトウェアによって自動的に補正した。すべての記録および分析をリアルタイムで実現しました。 3。ポスト·実験データ処理<ol><li>眼球速度の生の測定値が遅い位相速度を抽出するために、サッカーのために濾過した。 </li><li>眼球運動フィルタリング眼のベロシティ·カーブは7フレームのスライディングウィンドウを使用して、実行中の平均によって平滑化された。 </li><li>瞳の速度は同一のスティミュラス条件を持つフレームにわたって平均した。 </li><li>仔魚は、眼球速度は両眼で平均化された。 </li></ol> 4。代表の結果： 幼虫のゼブラフィッシュでは、 バンパー変異が選ばれました。この変異体では、水晶体上皮細胞が減少し、レンズの大きさとレンズの異所性の場所につながる、hyperproliferate。これらの形態学的変化は、コントラスト感度の大幅な削減と視力（Schonthaler ら 、2010）によって反射される。図1は、バンパー変異体対野生型の兄弟のコントラスト感度の違いを示していますバンパー変異体はますます刺激コントラストが減少するにつれて、目の速度を調整するために失敗する。類推すると、刺激の空間周波数が高くなると、刺激のストライプ幅が小さくなる、すなわち、 バンパー変異体は、同様に視力低下（図2）を証明する成体ゼブラフィッシュ映像設備への依存性環境条件では、lのパフォーマンスは30分間、自分の水槽の水に含まれるアルコールの濃度を変化させることに魚を施し、その後、様々な刺激条件下での視運動応答を測定することによって調べた。成体ゼブラフィッシュは増加アルコール濃度（図3）で維持コントラスト感度の著しい減少を示しています。魚が増加してアルコール濃度（図4）で処理したときの空間周波数の広い範囲にわたって全体的な眼の速度の類似の用量依存的な減少が観察された。アルコール治療は、さらに用量依存的に増加した刺激速度（図5）に代表される、より厳しい作業で眼球運動パフォーマンスが低下します。 <img alt="図1" src="/files/ftp_upload/3556/3556fig1.jpg" /> 図1：ゼブラフィッシュ幼生アイ速度は刺激コントラストに依存しています。 10 バンパー変異体および10は、野生型の兄弟は、sを変化させながら5 DPFで分析した<html> timulusストライプコントラスト条件。グラフは、平均的な眼の速度±1 SEMを示しています。 <img alt="図2" src="/files/ftp_upload/3556/3556fig2.jpg" /> 図2：ゼブラフィッシュ幼生アイ速度は空間周波数に依存します。 10 バンパー変異体および10は、野生型兄弟は5 DPFで異なる刺激ストライプ幅に供し、前述のように分析した。グラフは、平均的な眼の速度±1 SEMを示しています。 <img alt="図3" src="/files/ftp_upload/3556/3556fig3.jpg" /> 図3：コントラスト感度でアダルトゼブラショーアルコール濃度依存性の減少。成体ゼブラフィッシュを30分間示されており、様々な刺激ストライプコントラスト条件下で分析したアルコール濃度を変化させて維持した。グラフは、平均的な時間的·ツー·鼻眼速度±グループごとに9魚（：N = 11、対照群を除く）の1 SEMを示しています。 E 4は &quot;src =&quot; / files/ftp_upload/3556/3556fig4.jpg &quot;/&gt; 図4刺激ストライプ幅の広い範囲にわたって全体的な眼球運動でアダルトゼブラショーアルコール濃度依存性の減少。成体ゼブラフィッシュを30分間示されており、様々な刺激ストライプ幅の条件下で分析したアルコール濃度を変化させて維持した。グラフは、平均的な時間的·ツー·鼻眼速度±グループごとに9魚（：N = 11、対照群を除く）の1 SEMを示しています。 <img alt="図5" src="/files/ftp_upload/3556/3556fig5.jpg" /> 図5刺激速度の広い範囲全体眼球運動におけるアダルトゼブラショーアルコール濃度依存的な減少。成体ゼブラフィッシュを30分間示されており、刺激速度条件を変えることで分析としてアルコール濃度を変化させて維持した。グラフは、平均的な時間的·ツー·鼻眼速度±対照群を除く各群9魚（1 SEMを示しています。n = 11）で。

<ol>
	<li>Bahadori, R., Rinner, O., Schonthaler, H. B., Biehlmaier, O., Makhankov, Y. V., Paris, R., Jagadeeswaran, P., Neuhauss, S. C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fade+out+mutant+is+a+novel+genetic+model+for+Hermansky-Pudlak+syndrome.">The fade out mutant is a novel genetic model for Hermansky-Pudlak syndrome.</a> Investigative Optical and Visual Sciences. 47, 4523-4531 (2006).</li><li>Beck, J. C., Gilland, E., Baker, R., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instrumentation+for+measuring+oculomotor+performance+and+plasticity+in+larval+organisms.">Instrumentation for measuring oculomotor performance and plasticity in larval organisms.</a> Methods in Cell Biol. 76, 383-411 (2004).</li><li>Brand, M. G. M., N&#252;sslein-Vollhard, C., N&#252;sslein-Vollhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+and+raising+Zebrafish.">Keeping and raising Zebrafish.</a> Zebrafish, Practical Approach Series. , 7-37 (2002).</li><li>Easter, S. S., Nicola, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+vision+in+the+zebrafish+(Danio+rerio).">The development of vision in the zebrafish (Danio rerio).</a> Dev. Biol. 180, 646-663 (1996).</li><li>Easter, S. S., Nicola, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+eye+movements+in+the+zebrafish+(Danio+rerio).">The development of eye movements in the zebrafish (Danio rerio).</a> Dev. Psychobiol. 31, 267-276 (1997).</li><li>Haug, M. F., Biehlmaier, O., Mueller, K. P., Neuhauss, S. C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+acuity+in+zebrafish:+behavior+and+histology.">Visual acuity in zebrafish: behavior and histology.</a> Frontiers in Zoology. 7, 8-8 (2010).</li><li>Huang, Y., Neuhauss, S. C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+optokinetic+response+in+zebrafish+and+its+applications.">The optokinetic response in zebrafish and its applications.</a> Frontiers in Bioscience. 13, 1899-1916 (2008).</li><li>Major, G., Baker, R., Aksaya, E., Mensh, B., Seung, H. S., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+and+tuning+by+visual+feedback+of+the+stability+of+a+neural+integrator.">Plasticity and tuning by visual feedback of the stability of a neural integrator.</a> Proc Natl Acad Sci U.S.A. 101, 7739-7744 (2004).</li><li>Mueller, K. P., Neuhauss, S. C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Measurements+of+the+optokinetic+response+in+adult+fish.">Quantitative Measurements of the optokinetic response in adult fish.</a> J. Neurosci. Meth. 186, 29-34 (2010).</li><li>Neuhauss, S. C. F., Biehlmaier, O., Seeliger, M. W., Das, T., Kohler, K., Harris, W. A., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+disorders+of+vision+revealed+by+a+behavioral+screen+of+400+essential+loci+in+zebrafish.">Genetic disorders of vision revealed by a behavioral screen of 400 essential loci in zebrafish.</a> J. Neurosci. 19, 8603-8615 (1999).</li><li>Qian, H., Zhu, Y., Ramsey, D. J., Chappell, R. L., Dowling, J. E., Ripps, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+asymmetries+in+the+optokinetic+response+of+larval+zebrafish.">Directional asymmetries in the optokinetic response of larval zebrafish.</a> Zebrafish. 2, 189-196 (2005).</li><li>Renninger, S. L., Gesemann, M., Neuhauss, S. C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+arrestin+confers+cone+vision+of+high+temporal+resolution+in+zebrafish+larvae.">Cone arrestin confers cone vision of high temporal resolution in zebrafish larvae.</a> Eur. J. Neurosci. 33, 658-667 (2011).</li><li>Rick, J. M., Horschke, I., Neuhauss, S. C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optokinetic+behavior+is+reversed+in+achiasmatic+mutant+zebrafish+larvae.">Optokinetic behavior is reversed in achiasmatic mutant zebrafish larvae.</a> Curr. Biol. 10, 595-598 (2000).</li><li>Rinner, O., Rick, J. M., Neuhauss, S. C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+sensitivity,+spatial+and+temporal+tuning+of+the+larval+zebrafish+optokinetic+response.">Contrast sensitivity, spatial and temporal tuning of the larval zebrafish optokinetic response.</a> Invest. Ophthalmol. Vis. Sci. 46, 137-142 .</li><li>Rinner, O., Makhankov, Y. V., Biehlmaier, O. S., Neuhauss, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+cone+specific+kinase+GRK7+in+larval+zebrafish+leads+to+impaired+cone-response+recovery+and+delayed+dark+adaptation.">Knockdown of cone specific kinase GRK7 in larval zebrafish leads to impaired cone-response recovery and delayed dark adaptation.</a> Neuron. 47, 231-242 (2005).</li><li>Roeser, T., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visuomotor+behaviors+in+larval+zebrafish+after+GFP-guided+laser+ablation+of+the+optic+tectum.">Visuomotor behaviors in larval zebrafish after GFP-guided laser ablation of the optic tectum.</a> J. Neurosci. 23, 3726-3734 (2003).</li><li>Schonthaler, H., Fleisch, V. C., Biehlmaier, O., Makhankov, Y. M., Rinner, O., Bahadori, R., Schwarz, H., Neuhauss, S. C. F., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+mutant+leberkn+del/vam6+resembles+human+multi-systemic+disorders+caused+by+aberrant+trafficking+of+endosomal+vesicles.">The zebrafish mutant leberkn del/vam6 resembles human multi-systemic disorders caused by aberrant trafficking of endosomal vesicles.</a> Development. 135, 387-399 (2008).</li><li>Schonthaler, H. B., Franz-Odendaal, T. A., Hodel, C., Gehring, I., Schwarz, H., Neuhauss, S. C. F., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+mutant+bumper+shows+a+hyperproliferation+of+lens+epithelial+cells+and+fibre+cell+degeneration+leading+to+functional+blindness.">The zebrafish mutant bumper shows a hyperproliferation of lens epithelial cells and fibre cell degeneration leading to functional blindness.</a> Mech. Dev. 127, 203-219 (2010).</li><li>Schonthaler, H. B., Lampert, J. M., Lintig, J. v. o. n., Schwarz, H., Geisler, R., Neuhauss, S. C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+the+pmel17+gene+leads+to+defects+in+melanosome+biogenesis+and+alterations+the+visual+system+in+the+zebrafish+mutant+fading+vision.">A mutation in the pmel17 gene leads to defects in melanosome biogenesis and alterations the visual system in the zebrafish mutant fading vision.</a> Developmental Biology. 284, 231-242 (2005).</li></ol>

オリバーのDR SchnaedelbachとHolger D. Russigは、この記事で使用したビジュアル性能トラッキングシステムを生成東証システムズ社の従業員である。この記事の産生は、東証システムズ社が主催した。ステファンCF Neuhuassは、販売システムごとに東証システムによって報酬を受け取るにはチューリッヒ大学の従業員です。

視覚機能の研究のためOKRの重要性は、長い時間（イースター＆ニコラ1996年、1997年）のために科学界で認識されており、本当にパラダイムを定量化する試みは、10年以上前によく始めている。イースターとニコラ（1996）は、眼球運動のビデオ録画を手動で分析した縞模様のドラムを回転させるモーターとシステムを開発しました。このシステムでは、再配置が頻繁マニュアルを必要と魚類胚の固定化の欠如に苦しんで、そして非常に困難で目の追跡動きを検出することができる。ステップが前方に複数の可変コンピュータ生成刺激提示（; Rinner ら 、2005A Roeser＆バイアー、2003）を考慮するために、ビデオ投影縞模様のドラムの使用でした。 ビデオテープに録画録音のほとんどはマニュアル、フレーム毎の分析は非常に面倒であることが証明されて、観察者バイアス（ベックらによって阻まある程度2004年）。リアルタイムで自動化された分析は、行動のフィードバック学習のメカニズム（主ら 、2004）の使用を許可することが示唆された。赤外線照明と周波数制御回転刺激の使用は、Beck らによって開拓されています（2004）。ただし、システムは唯一の幼虫のためにそこに使用されてきた説明、および分析はオフラインで行われた。さらに、VisioTrackerそれによって実験の過程で、より高い柔軟性と自発的な影響力をできるように、実験中に刺激を変更するなどの刺激を完全に制御することができます。また、VisioTrackerで使用されるデジタル刺激の作成 ​​はストライプ刺激ドラム（ベックら 、2004）の慣性質量の加速で前に述べた問題点を克服した。 メチルセルロースによる幼虫の拘束が大幅に眼球の動きを妨げないとゼブラフィッシュ幸福上の任意の長期的な効果を持っていません。魚の幼虫は正常であった皮膚を介して酸素供給が加齢に伴って需要（Qian ら、2005）には不十分になるまで、数日間メチルセルロースに埋め込 ​​ま維持した。 成魚の拘束方法は、動物にも同様に簡単です。急速に別の1のための試験動物を交換するためのオプションと組み合わせる実験期間が短いが、さらにシステムの正動物福祉の側面に追加されます。鰓が連続水によって洗い流されるので、それはスパイク眼球運動と視覚の性能への影響を研究するために選択した任意の化学物質と水に便利です。同様に、ウォッシュアウトの実験は、実験動物の間を処理しなくても追加することができます。 ビデオ画像内のピクセルノイズが目の位置と角速度の高精度の測定を可能にする、独自のVisioTrackerソフトウェアのアルゴリズムを平滑化することにより、最小限に抑えられました。さらに、統計的容易にする解析、ソフトウェアは一定の速度で発生し、実験的な文に寄与しないサッカード運動を除外。 7ビデオフレーム上の速度曲線の平均化は、後の分析を容易にした。 VisioTrackerは、多くの様々な研究分野に新たな次元を開きます。システムとその前任者はすでにそのような視力、コントラスト感度および明順応（Rinner ら 、2005A、Schonthaler ら 、2010）などのパラメータを用いて、ゼブラフィッシュ幼生における視覚性能を定量化するために成功裏に使用されている、の機能解析変異体ゼブラフィッシュ幼生におけるコーン視覚伝達カスケードのメンバーの操作後に感光体（例えばRinner ら 、2005B、Renninger ら、2011）、または視覚欠陥の分析（例えばSchonthaler ら、2005、2008;。Bahadoriら、2006）。視覚系の形態学的および機能的成熟の相互依存その視力が主ですが、完全には幼虫の段階で感光体の間隔（ハウグら 、2010）によって限定されるものではない表示するOKR測定によって研究されてきた。 VisioTrackerは大人のゼブラフィッシュと他の同様の大きさの魚種（ミューラーとNeuhauss（2010）、本報告書）における視覚機能を分析することも同様に適しています。 それは調査されるべき物質が成魚のエラを周囲の水の流れに加えられるかもしれないそれによって毒性や薬理学などの研究分野でシステムを利用することも考えられる。さらに、VisioTrackerの汎用性は、視覚機能、神経回路機能の開発、または感覚運動制御（黄＆Neuhauss、2008年のレビューを参照）のontogeneticsの例については、より徹底した分析を可能にします。

<table border="1"><tbody><tr><th>試薬の名称</th><th>会社</th><th>カタログ番号</th></tr><tr><td>メチルセルロース</td><td>シグマアルドリッチ</td><td> M0387 </td></tr><tr><td> 3 - アミノ安息香酸エチルメタンスルホネート（MS-222） </td><td>シグマアルドリッチ</td><td> E10521 </td></tr><tr><td> 35mmの細胞培養皿</td><td>コー​​ニング</td><td> 430165 </td></tr><tr><td>血清ピペット</td><td>グライナーバイオ1 </td><td> 612 361 </td></tr><tr><td> VisioTracker </td><td>東証システム</td><td> 302060 </td></tr></tbody></table>

visiotracker, an innovative automated approach to oculomotor analysis

視覚系の発達と機能への調査では、操作する、引き出すのは簡単、堅牢でシンプルなビジュアル性能の定量化ビヘイビア·モデルを必要としている。適切なモデルは、視運動反応（OKR）、その高選択値に起因するすべての脊椎動物に存在する反射的な行動で発見されている。 OKRは急速リセットサッカーと交互に目の遅い刺激、次の動きを伴います。この現象の測定が容易に起因し、その早期かつ安定した発症までゼブラフィッシュ幼生、およびゼブラフィッシュ遺伝学についての十分な知識の恩恵を受け、（完全に96時間後に受精（HPF）の後に開発された）待遇モデルの数十年1のために行われるこの分野での生物。一方、成魚で同様のメカニズムの分析は特に薬理学的および毒物学的なアプリケーションでは、重要性を増しています。 ここでは、完全に自動化された、高throughpuをVisioTrackerを記述ビジュアルパフォーマンスの定量分析のためのtシステム。システムは、教授ステファンNeuhaussのグループで行われた研究に基づいており、東証システムによって再設計されました。それは、高解像度のズームレンズを搭載し、高品質のビデオカメラで監視小魚用の固定装置で構成されています。魚コンテナはコンピュータによって生成された刺激パターンを投影することができる上に、ドラムスクリーンに囲まれています。眼球運動を記録し、自動的にリアルタイムでVisioTrackerソフトウェアパッケージによって分析される。 データ分析は、このような低速および高速位相持続時間、運動サイクルの頻度、遅い相ゲイン、視力、コントラスト感度などのパラメータの即時認識を可能にします。 典型的な結果は、例えば、野生型の形態に明らかな変化を示していない視覚系突然変異体の迅速な同定、または薬理学的又は毒性と変異原性の定量的効果の決定を可能にする視覚システムのパフォーマンス上のエージェント。

Watch this Scientific Journal Video about VisioTracker, an Innovative Automated Approach to Oculomotor Analysis at JoVE.com

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

VisioTrackerは、眼球運動の記録に基づいて、幼虫や小さな成魚の視覚性能を定量的に分析するための自​​動化されたシステムです。それはそのような視覚系の発達と機能、薬理学、神経回路の研究や感覚統合などの分野で高スループットの研究を可能にし、視覚刺激のプロパティおよびリアルタイム分析を完全に制御しています。

VisioTracker、眼球運動解析への革新的な自動化されたアプローチ

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

visiotracker-an-innovative-automated-approach-to-oculomotor-analysis

Research

JoVE Journal

Neuroscience

11.0K ビュー.  University of Zurich. VisioTrackerは、眼球運動の記録に基づいて、幼虫や小さな成魚の視覚性能を定量的に分析するための自​​動化されたシステムです。それはそのような視覚系の発達と機能、薬理学、神経回路の研究や感覚統合などの分野で高スループットの研究を可能にし、視覚刺激のプロパティおよびリアルタイム分析を完全に制御しています。

ビデオ: VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

複数のスケールでデジタル化された神経形態の自動化されたショールの分析

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

神経科学

VisualEyes: A Modular Software System for Oculomotor Experimentation

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and dysfunctional brain.1 However, development of a platform to stimulate and store eye movements can require substantial programming, time and costs. Many systems do not offer the flexibility to program numerous stimuli for a variety of experimental needs. However, the VisualEyes System has a flexible architecture, allowing the operator to choose any background and foreground stimulus, program one or two screens for tandem or opposing eye movements and stimulate the left and right eye independently. This system can significantly reduce the programming development time needed to conduct an oculomotor study. The VisualEyes System will be discussed in three parts: 1) the oculomotor recording device to acquire eye movement responses, 2) the VisualEyes software written in LabView, to generate an array of stimuli and store responses as text files and 3) offline data analysis. Eye movements can be recorded by several types of instrumentation such as: a limbus tracking system, a sclera search coil, or a video image system. Typical eye movement stimuli such as saccadic steps, vergent ramps and vergent steps with the corresponding responses will be shown. In this video report, we demonstrate the flexibility of a system to create numerous visual stimuli and record eye movements that can be utilized by basic scientists and clinicians to study healthy as well as clinical populations.

Neural control and cognitive processes can be studied through eye movements. The VisualEyes software allows an operator to program stimuli on two computer screens independently using a simple, custom scripting language. The system can stimulate tandem eye movements (saccades and smooth pursuit) or opposing eye movements (vergence) or any combination.

VisualEyes：眼球運動実験のためのモジュラ式のソフトウェアシステム

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

脳スライスビオチン：<em&gt;生体外</emアダルトニューロンにおいて地域固有の細胞膜のタンパク質輸送を測定するために&gt;の取り組み

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

微小電極アレイへの3Dエンジニア神経文化のインタフェース：革新的な<em&gt;インビトロ</em&gt;実験モデル

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

後根神経節ニューロンのアライメントや髄鞘形成を強化するためのアプローチ

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

の記録細胞内の電圧応答のための電気生理学的手法<em&gt;ショウジョウバエ</em&gt;光刺激に感光体と介在ニューロン<em&gt;インビボ</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

の自動解析<em&gt; C。エレガンス</emセレストソフトウェアを使用&gt;スイム挙動

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

絞扼性損傷マウス自動歩行分析

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

検出技術その場で革新的な RNA 側坐齧歯動物の核内ドーパミン D1-アルファ受容体の同定

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Ex Vivoラット脳切片を用いた神経変性疾患の主要なイベントを勉強する方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Methods Article

VisioTracker、眼球運動解析への革新的な自動化されたアプローチ