We thank the staff in the Ecology Branch, Toxicology Branch and Environmental Chemistry Branch of the U.S. Geological Survey, Columbia Environmental Research Center for technical and analytical assistance. Funding provided in part by Teck American Incorporated through an agreement with the US Environmental Protection Agency (USEPA) Region 10 with funds provided by USEPA to US Geological Survey (USGS) through the Department of Interior Central Hazmat Fund.

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior.">Behavior.</a> Fundamentals of Aquatic Toxicology: Methods and Applications. , 221-256 (1985).</li><li>Calfee, R. D., 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=Acute+sensitivity+of+white+sturgeon+(Acipenser+transmontanus)+and+rainbow+trout+(Onchorhynchus+mykiss)+to+copper,+cadmium,+or+zinc+in+water-only+laboratory+exposures.">Acute sensitivity of white sturgeon (Acipenser transmontanus) and rainbow trout (Onchorhynchus mykiss) to copper, cadmium, or zinc in water-only laboratory exposures.</a> Environ Toxicol Chem. 33 (10), 2259-2272 (2014).</li><li>Little, E. E., Calfee, R. 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汚染物質への暴露に行動の変化は、多くの場合、サブ致死毒性のエンドポイントとして使用されるが、測定することが困難な場合があります。一般的に、行動応答は、目視観察し、処理するために多くの時間を必要とする手動によるデータ分析により測定されています。技術を進めるとともにしかし、水泳の活動を定量化するための方法は、ビデオ撮影18および運動解析や処理および分析時間を短縮し、デジタルトラッキングソフトウェアを使用することに焦点を当てています。ビデオの分析の間ので、チョウザメの遊泳行動を分析するためのより効果的かつ効率的な方法を提供し、映像データの記録や魚のトラッキングソフトウェアの使用を消費手動で非常に時間があったであろう水泳変数を定量化、データを捕獲しました。手順は、魚の遊泳行動を強調したが、このような両生類や水生無脊椎動物のような他の生物のためにカスタマイズすることは簡単な変更を必要とするであろう。どのような行動endpoinに応じて、TSは、実験的なデザインとカメラシステムは、ちょうど約あらゆる市販の追跡ソフトウェアパッケージで使用するために開発することができ、アドレス指定されています。 この方法は、溶解した銅を用いて実証するが、他の水性汚染物質又は温度又は酸素含有量等の特性に適用可能です。本論文で開発し、提示されたプロトコルは、記録装置のような単純なデジタルビデオカメラを利用しました。デジタルファイルは、容易にコンピュータに転送し、運動解析ソフトウェアにアップロードされます。方法は絶えず変更され、定量化プロセスを合理化するために洗練されています。ビデオ品質を追跡するために、各個々の魚を識別するために解析ソフトウェアために高解像度であることが不可欠です。データファイルを処理しようとすると魚とのコントラストしない任意の背景には、問題が発生します。 2次元ビデオトラッキングと別の一般的な問題は、個人を識別しています水泳のパスが渡ります。これは、パスの横断中に各魚を識別し、ソフトウェア内経路セグメントを連結することによって、手動で補正することができます。あるいは、総活性は、グループの平均として各反復室から決定することができます。 1魚をそれぞれ含むものといくつかの個々のチャンバは、個々の魚の動きを計算するために、同じ視野で撮影することができます。 現在、我々は、高精細デジタルビデオ記録装置（HD-DVR）にリンクされている露光室以上のオーバーヘッドの監視カメラのシリーズを使用してアップグレードしました。しかし、高解像度のMPEG-4ビデオを録画することができます任意のカメラシステムの使用が動作します。 HD-DVRは、指定した時刻に記録するように設定し、7日間までプログラムすることができます。この手オフの自動化のアプローチは、魚の行動を損なう可能性外乱を最小限に抑えながら、一貫性を維持するために、同時に複数のビデオのキャプチャを可能にします。 HD-DVRのsysteMSは、そのようにファイルの転送は比較的簡単である内部ネットワークに接続されています。自動化されたカメラシステムは、遊泳行動を定量化するためのはるかに改善された技術ですが、まだ毒性試験中に行動障害を文書化するためなどの追加支援情報を提供するために視覚的な観察を行うことが有益です。 1960年代初頭19,20,21にさかのぼる金属への暴露に起因する文献の文書化変更された魚の行動の長い歴史があります。銅はブルーギル22でこのような活動低下などの活動レベルの変化（ ブルーギル属macrochirus rafinesque）とカワマス23（ イワナ fontinalis）の運動と摂食活動の変化を引き起こすことが示されています。少なくともいくつかの稚魚の捕食者を検出し、回避するために、匂いの感覚に頼る、および銅誘導性の化学感覚の剥奪は、アラーム化学物質24,25,26の検出に関連した行動に影響を与える可能性があります</sup&gt;。嗅上皮は、それによって、見当識障害につながる可能性があり感覚のメカニズム、行動の回避、低減し供給および嗅覚27に案内され、他の行動に影響を与えることにより、銅暴露に破損しています。これらの変更された行動は、エクスポージャーの間に観察されたものと一致しました。 白チョウザメの遊泳行動が大幅に銅水溶液濃度に致死量以下の露光時に影響を受けました。これらの結果は、動作が銅の亜致死濃度で影響を受け、毒性ストレスの指標として使用することができる方法を示しています。ビデオベースの分析では、遊泳行動を定量化するのに有効証明し、また銅にさらさチョウザメに深刻な影響の定性的な視覚的なドキュメントを務めていました。解析ソフトウェアは、他の様々な行動のエンドポイントを定量化することが可能です。リストについては、 表1を参照してください。露光システムは、各エンドポイントに対処するために変更することができますリアルタイムモードと関心のさまざまな汚染物質への曝露に関連した動作の違いを定量化するために使用することができます。 水生毒性学的研究における行動のエンドポイントの使用がますます採用されており、適応行動機能は、環境傷害9の決意に重要であるため、汚染物質の影響を評価する際に考慮すべきです。魚の行動への環境汚染物質の影響は、標準的な方法の不在下で亜致死エンドポイントを扱う場合は特に、多くの場合、主観的かつ挑戦的です.. 厳密に監視することができ、これらの方法を使用して定量化活性を水泳、生物に最小限のストレスで非破壊的であり、繰り返すことができます。水泳の動作は、標準的な毒性試験5の感度を拡大するテストプロトコルに組み込まれるべきである亜致死毒性の有効かつ一貫性の指標です。

汚染物質の曝露は行動にどのように影響するかを理解することは時間に非常に困難かつ主観的です。行動は、一般的に、中枢神経系を介して動作と、生き残る成長し、再現するために生物を有効に明白な、観察可能な、全身の一連の活動として定義されています。生存率1と比較した場合に起因する毒物への暴露に行動の変化は、環境ストレス、多くの場合、10から100の間の倍の感度の最も敏感な指標の一つです。これらの研究の大半は、スイミングアクティビティ、換気、および魚の2,3,4の採餌行動を調べました。水泳の活動は、毒性試験5中の汚染物質に反応して行動変化を決定する上で最も頻繁に使用される亜致死のエンドポイントです。水泳変数は、周波数や旅の移動、速度と距離の期間、頻度およびターンの角度、位置水柱中や水泳のパターンを含みます。水泳活性があります遊泳行動の有効な手段水生毒性の本の基礎にランド6により、第9章で提案された基準に基づいて、毒性物質に対する感受性を評価します。 本稿では、早期開発の様々な段階での早期のライフステージ白チョウザメへの銅の毒性（ シロチョウザメ ）水のみの遊泳行動を評価したチョウザメする相対的なエクスポージャー方法の例として、毒性学的研究を提示し、水泳を定量する方法を示しています動作。 以前の研究では、有害な行動反応は銅に急性および慢性暴露の最初の数日の間に早期発症と明らかにしたと暴露時間と濃度の7,8,9の上に次第により厳しいなりました。これらの行動反応の発症の大きさとタイミングが長期生存を制限する可能性が十分であり、したがってrecruiに影響与え懸念されますtment障害10。正確に金属露出方法および手順に、この感度の意義を解釈するために銅濃度を基準に行動の減損の時間経過と程度を定量化するために開発されました。 行動機能と開発のための試験プロトコルは、露光チャンバ内でチョウザメのオーバーヘッドビデオサンプルを分析することによって確立しました。ビデオサンプルは、暴露期間を通して銅処理間発達と機能の定性的な評価を提供します。行動と避難所を追究活動を行うようにと協調、平衡、および呼吸を泳いで、避難所のシーク、無気力に影響を与える減損の時系列を特徴づけるために時間を決定するために、水性銅濃度に対するエクスポージャーの間に評価しました。また、ビデオ録画を定量的に文書化する自発的な水泳ACTIの目的のために複製それぞれからサブサンプリングされたチョウザメで作られていましたVITY。これらのエンドポイントは、持続時間の測定が含まれるか、または時間が移動費やし、遊泳速度及び距離は、商業的に利用可能なデジタル画像分析ソフトウェアパッケージを用いて5,11を旅しました。このソフトウェアは、視野内の各画像の輪郭を定義し、このことから、各画像の重心を定義します。ソフトウェアは、運動の経路を決定するために、フレームの連続によってフレーム内の各重心の位置を追跡することができます。 動物福祉法の規定（9 CFR）の最終規則の該当するすべてのセクションで、文化と実験中の試験生物の人道的な扱いのためのすべての施設のガイドラインを遵守し、この研究。研究の終了時に、すべての魚は、米国地質調査所のための制度的動物実験委員会、コロンビア環境研究センターによって承認されたように、適切なガイドラインに従って安楽死させました。

<table border="0" cellpadding="0" cellspacing="0" width="885">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">copper II sulfate pentahydrate</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">contaminant of concern</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">syringe dispenser</td>
			<td style="width:285px;">Hamilton MicroLab 600 Series</td>
			<td style="width:119px;"></td>
			<td>apparatus to spike chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">2-L volumetric flask</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td>container for holding stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">24-1.5 L glass jars</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td>test chamber for 2 dph sturgeon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">video camera</td>
			<td style="width:285px;">Sony Handycam HDR-CX550V</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">digital tracking software</td>
			<td style="width:285px;">Noldus Ethovision</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">3-17&#34; flat screen monitors</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">24 surveillance cameras</td>
			<td style="width:285px;">Model CL101</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">3-16 channel digital recording devices</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">DO meter</td>
			<td style="width:285px;">YSI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">pH meter</td>
			<td style="width:285px;">Orion 940</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">ph probe</td>
			<td style="width:285px;">Orion&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">ammonia meter</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">ammonia probe</td>
			<td style="width:285px;">Orion</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">chiller unit</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">recirculating water pump</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying fish swimming behavior in response to acute exposure of aqueous copper using computer assisted video and digital image analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction. However, these responses may be subtle, and measurement can be challenging. Using juvenile white sturgeon (Acipenser transmontanus) with copper exposures, this paper illustrates techniques used for quantifying behavioral responses using computer assisted video and digital image analysis. In previous studies severe impairments in swimming behavior were observed among early life stage white sturgeon during acute and chronic exposures to copper. Sturgeon behavior was rapidly impaired and to the extent that survival in the field would be jeopardized, as fish would be swept downstream, or readily captured by predators. The objectives of this investigation were to illustrate protocols to quantify swimming activity during a series of acute copper exposures to determine time to effect during early lifestage development, and to understand the significance of these responses relative to survival of these vulnerable early lifestage fish. With mortality being on a time continuum, determining when copper first affects swimming ability helps us to understand the implications for population level effects. The techniques used are readily adaptable to experimental designs with other organisms and stressors.

手動で処理する視覚観測データは、2日後に孵化（DPH）チョウザメ（ 図2）で開始曝露のわずか72時間後に増加銅濃度とともに増加パーセントの異常を示しました。ビデオサンプルは、チョウザメの遊泳行動（動画2）上の銅暴露の極端な影響を文書化し、その結果としての行動障害を定義する際に支援します。別の例では、30 DPHでのチョウザメは、死亡率のみに基づいて40.3μg/ Lを96時間半数致死影響濃度（LC50）と銅の露出に敏感であることが判明しました。しかし、平衡および固定化の損失の亜致死行動エンドポイントは、死亡率と一緒に含まれている場合2.4〜5.0μgの/ Lの範囲の推定96時間中央値は影響濃度（EC50）での感度が高くなります。記録された行動異常のビデオドキュメントこれらの亜致死影響を捕獲し、さらに検証人間観察露光中。水泳活動を分析する場合、デジタルトラッキングソフトウェアの使用は、後処理時間を減少させました。魚の遊泳速度は、時間が動いて過ごした、との距離はすべて大幅に増加銅濃度で（ 図3）減少し 、トリップアドバイザー。水泳経路も増加銅濃度（ 図4）で還元しました。 <img alt="図1" src="/files/ftp_upload/53477/53477fig1.jpg" /> 図1：試験原液濃度目標濃度に基づいて、スプレッドシートのテンプレートを使用して決定した希釈毒性試験のための化学の株式の準備を決定するために使用されるスプレッドシートのテンプレートのコンピュータのスクリーンショット <a href="https://www.jove.com/files/ftp_upload/53477/53477fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 図2：希釈のセットアップと実験デザイン白チョウザメのさまざまなライフステージが銅に曝露しました。露光のために使用される希釈のサイズは、魚の大きさに基づいて選択しました。 （a）は初期ライフステージのチョウザメは、大きな希釈のセットアップを使用して露光した小希釈のセットアップおよび（b）は 、古いライフステージのチョウザメを用いて露光しました。 <img alt="図3" src="/files/ftp_upload/53477/53477fig3.jpg" /> 図3：初期生活段階の白チョウザメ（ シロチョウザメ ）72時間の銅暴露（[DPH]魚30日の孵化後から始まる ） から 、 行動結果をスイミング 。白チョウザメDPH 30の間での移動の水泳活動のエンドポイントは、（a）の期間; （b）は、水泳のスピード。そして（c </移動strong&gt;の）距離は96時間さらさ白チョウザメの中銅濃度の増加に伴って減少しました。アスタリスクは対照からの有意差を示し、エラーバーは標準偏差を表します。 <img alt="図4" src="/files/ftp_upload/53477/53477fig4.jpg" /> 図4：初期生活段階の白チョウザメ（シロチョウザメ）の結果96時間の銅暴露（2日目の孵化後[DPH]魚で始まる）の72時間後の平衡および固定化の損失を示す2 DPHで白チョウザメを存続。銅濃度の増加に伴って96時間暴露。アスタリスクは対照からの有意差を示し、エラーバーは標準偏差を表します。 <img alt="図5" src="/files/ftp_upload/53477/53477fig5.jpg" /> 図5：初期生活段階の白チョウザメ（チョウザメ属transmoから例水泳パス結果ntanus）96時間銅暴露（30日の孵化後[DPH]魚）と凝視。チョウザメの水泳経路が（N = 5魚）と（b）高い治療から（50（a）の制御の反復からのデジタルトラッキングソフトウェアを使用してデジタル化96時間曝露後μgの/ L）の反復（n = 3の魚）。不活性であったため、いくつかの魚の遊泳経路の数は、チャンバ内の魚の存在の数を表すものではありません。 <a href="https://www.jove.com/files/ftp_upload/53477/53477fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 定量化可能なパラメータ </td><td> 観測パラメータ </td></tr><tr><td>速度</td><td>無気力/多動</td></tr><tr><td>距離移動</td><td>均衡の喪失</td></tr><tr><td>ゾーンで過ごした時間の長さ</td><td>痙攣/振戦/逆さまダウン</td></tr><tr><td>選択したゾーン間の時間生物の移動のゾーン遷移数</td><td>水柱内の位置</td></tr><tr><td>見出し興味のある点に向かって、動物のパスの偏差をポイント計算</td><td>呼吸（速い/遅いです） </td></tr><tr><td>見出しは、選択されたボディポイントの見出しを算出し、 </td><td>着色</td></tr><tr><td> 2サンプル間の見出しに角度差を回し</td><td>隠蔽</td></tr><tr><td>角速度計算サンプル間隔によって回転角度を分割することにより</td><td></td></tr><tr><td>移動距離によって旋回角度を分割することにより蛇行計算しました。異なる速度で移動する動物で回し比較するために使用されます</td><td></td></tr><tr><td>時間が動いて過ごしました</td><td></td></tr><tr><td>モビリティでもセントた場合、動物のように検出された完全なエリアが変更されている期間を状態は、計算しますER点は同じまま</td><td></td></tr><tr><td>選択されたボディポイントが360°の累積回転角を有するとき、回転一回転が終了します。無視される閾値未満の反対方向に回転。 </td><td></td></tr><tr><td>移動度は、中心点が同じであっても、検出された動物の完全な領域の移動度の割合を連続計算します。 </td><td></td></tr><tr><td>間の距離全ての関係者と選択した受信機との間の距離を被験者は、計算します</td><td></td></tr><tr><td>俳優であるか、または受信機に近接していないする期間を近接は、計算します</td><td></td></tr><tr><td>相対運動</td><td></td></tr><tr><td>ネット加重移動-（ポジティブ）に俳優の動きとそれらの間の距離によって重み付け（負の）受信機、から</td><td></td></tr><tr><td>加重移動から、moveme<html>それらの間の距離によって重み付け受信機から俳優のntは、。 </td><td></td></tr><tr><td>それらの間の距離によって重み付け受信機に俳優の動き対加重移動、 </td><td></td></tr><tr><td> 2試用コントロール要素のイベント、または1つの要素内の間の試用制御state-期間</td><td></td></tr><tr><td>あなたが裁判コントロールに定義されている要素内のイベントが発生した裁判制御イベントモーメント。 </td><td></td></tr></tbody></table> 表1：デジタル追跡ソフトウェアを介して定量化行動のエンドポイントは、これらのエンドポイントは、個人またはグループで使用され、また、露光時の行動障害を視覚的に観察チェックのリストとして機能することができます。 <img alt="ビデオ1" src="/files/ftp_upload/53477/53477movie1.jpg" /> /www.jove.com/files/ftp_upload/53477/53477video1.avi &quot;&gt; ビデオ1：影響を受けた白チョウザメが示す異常行動のビジュアル定義（右クリックしてダウンロード）毎日観察チェックリストは、異常を文書化するために使用されたザ・。。平衡および固定化の損失は、曝露の間に観察された最も一般的な異常であった。Calfee らから変更された。7 <img alt="動画2" src="/files/ftp_upload/53477/53477movie2.jpg" /> <a href="https://www.jove.com/files/ftp_upload/53477/53477video2.avi">ビデオ2：白チョウザメの遊泳行動の例を強調ビジュアルドキュメント。 （右クリックしてダウンロード）</a>白チョウザメ水泳活性が大幅に増加銅濃度への暴露で還元しました。このビデオに描かチョウザメは、コントロール、中低、および96時間銅水溶液の終わりに高い治療からのものです暴露。 白チョウザメはまだ生きていたが、魚が大幅にコントロールと比較して治療が損なわれた明白です。 Calfee らから変更。7

Watch this Scientific Journal Video about Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis at JoVE.com

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

コンピュータ支援ビデオとデジタル画像解析を用いた銅水溶液の急性暴露に応答して、魚の遊泳行動を定量化

魚の行動に環境汚染物質の影響を測定することは、亜致死エンドポイントを扱う場合は特に、多くの場合、主観的かつ挑戦的です。我々は、中および銅の種々の濃度に対する96時間の急性曝露後初期生活段階白チョウザメ（ シロチョウザメ ）の遊泳行動を定量化するためのビデオ技術を含む方法が記載されています。

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction.  ...

quantifying-fish-swimming-behavior-response-to-acute-exposure-aqueous

Research

JoVE Journal

Neuroscience

11.1K ビュー.  US Geological Survey. 魚の行動に環境汚染物質の影響を測定することは、亜致死エンドポイントを扱う場合は特に、多くの場合、主観的かつ挑戦的です。我々は、中および銅の種々の濃度に対する96時間の急性曝露後初期生活段階白チョウザメ（ シロチョウザメ ）の遊泳行動を定量化するためのビデオ技術を含む方法が記載されています。 

ビデオ: Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

神経科学

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

コンピュータ支援多電極パッチクランプシステム

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

自由水泳弱電気魚の長期的な行動の追跡

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

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 ...

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

UV反射構造を検出してのSailFinモリーを使用して、メイト優先でその重要性を決定するためのアッセイ<em&gt; Poecilia latipinna</em

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

のシナプス変調を分析<em&gt;キイロショウジョウバエ</em&gt;長時間の光照射後の感光体

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

マイクロ電極アレイにおけるニューロン細胞培養の刺激に対するネットワーク応答の時間依存的増加

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

カメの中で脳神経のIn Vitro刺激を用いて眼球運動

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

嗅覚ニューロンの光刺激に対する応答のショウジョウバエ幼虫の行動を追跡

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

麻酔下ラットの中枢神経系の操作への応答での腎交感神経活動の急性変化の定量

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...