私は動物行動の統合に関する研究（CISAB）のためのセンターからクリストファーエヴァンスとダレンバークに感謝します。

ジャッキードラゴンズ ジャッキートカゲ（A. muricatusは ）気候制御キャプティブの施設で開催されました。 エンクロージャは、60 cm 3を測定する加工ガラスのテラリウムで構成された。エンクロージャは、シドニーの砂の基板、ネイティブ葉、および天然木製の止まり木でいっぱいだった。エンクロージャは、被験者がすべて表示するモニターの中央に配置するために許可されている可動台車の上に置かれた。 シミュレートされた夏の状態の間に10Dの光周期、および夏時間の前半の時間の間加熱ランプ（125W）と十分な紫外線（UV）照明（300W）の両方で提供された：トカゲは、14Lの下に置かれた。トカゲはまた水アドリブで提供された、葉コオロギ（Achetusドメス ） は 、非実験的なセッション中にビタミンを振りかけ、そして日常的にイベルメクチンの希釈用量を使用して寄生虫の虫の食った。 再生に使用する機器 3つのビデオ再生のリグは、刺激の提示（図1参照）に使用されます。中央のリグは、ドットの刺激を含んでいるのiMac（アップルコンピュータ社）で構成されています。つの追加リグは、再生の刺激を（すなわち、無脊椎動物）に含まれる刺激提示用のFinal Cut Pro HDを搭載したiMacで構成されています。 デジタル信号は、ビデオ再生用のデジタル-アナログ変換のための高度なデジタルビデオコンバーター（カノープス© GT ADVC110、メルボルン、ビクトリア、オーストラリア）に送られ、ソニーTrintron（ソニー株式会社、品川区、東京日本）モニター（シリーズに表示されます第PVM-14M2A/PVM-14L2/PVM-14N5A）。キヤノン（モデルNo。MV650i）22倍光学ズーム（キャノン株式会社、日本）とデジタルビデオカメラを直接テスト領域の上にマウントされていると、レコードを直接ソニー（ソニー株式会社、品川区、東京日本）ビデオカセットレコーダーの刺激に対する応答（シリアル番号SLV - EZ717AS）。 記録された画像は、カラービューファインダー（NEC、シリアル番号1892、神奈川県、日本）に送信されます。ドット表示の開始から7.5秒で、電子的に発生する音のマーカーは、カノープス® ADVC110とVHSへの記録に送信されます。ビデオの再生は、PAL - DV規格（;水平解像度575ライン、25フレームS - 1 5:1の圧縮）のために調整されます。 ランダムドットKinematograms（RDKs） RDKsはVPixx V1.79（VPixx V1.79、VPixx Technologies社2002）を使用して開発することができます。 30 × 20度のフィールドを持つデザインの刺激は、5 × 5画素を測定するそれぞれのドットで、200ドットで埋める。 128様々な刺激を生成する、一貫性の8つのレベルと二つの方向を組み合わせた速度の8つのバリエーションで、ドット刺激を構築する。 速度パラメータは毎秒度（° / s）で設計されていた。速度変動は0.5、2.5、5、10、20、40、80、および160℃/ sであった。コヒーレンスの割合は、0、2.5、5、10、20、40、80、および左右どちらかの方向の動きで100％であった。 モーションJPEG - Bのファイルとしてそれぞれのシーケンスをエクスポートする。ただし、機能の配列RDKsを設計することができます。 各シーケンスは、持続時間の合計15秒です。シーケンスは、試験速度の変数と一致する0％のコヒーレンスの2.5秒から始まります。この期間は、現在のトライアルを開始します。これは、速度とコヒーレンスの組み合わせの変化の5秒が続いている。シーケンスは適切な方向に100％の7.5 Sが完成させた。 アップルのFinal Cut Pro HD（アップルコンピュータ社）のすべてのモーションJPEG - Bのシーケンスと編集をインポートします。それぞれの間隔で音マーカーを追加します。このマーカーは、VHSへの唯一の音であり、刺激提示の変更が発生した場合の動作をスコアリングするときにキューとして使用されます。 Final Cut Proのムービーとして128の各シーケンスをエクスポート。 一次的強化 - ミルワーム 実験時のブロックの試験ごとに5件の試験（16試験）の無作為化強化スケジュールの上に置いて科目。主な強化は、ミルワーム（チャイゴミムシダマシ）です。 二次的強化 - アニメーション無脊椎動物 二次的強化は、一次強化子とペアになるです。コオロギ、2）ダニ、及び3）スパイダー1）：そのようなコンピュータアニメーション無脊椎動物の3つのタイプのような生物学的に意味のある強化子を、作成。 初期の7.5 Sはまだ背景のと、長さが15秒間光V8.3を（NewTek社（株）、サンアントニオ、テキサス州、米国）を使用してアニメーションを作成し、適切な画面に無脊椎動物の7.5 Sが続きます。 フォーEAHクリップにJPEGとしてインポートする自然植生の背景をtograph。個々のJPEG画像のシーケンス（375フレーム）に各シーケンスをエクスポート、およびビデオの再生のためのFinal Cut Pro HDを使用してムービーファイルに圧縮する。コンディショニングのパラダイムに慣れを防ぐために、50のユニークなクリップを設計する。 一般的な手順 表示モニターの間に被写体を置きます。 各科目のRDK刺激の提示をランダム化します。 八日で16試行のブロック内の刺激を示す。各試験は30〜60秒から様々なintertrial間隔で15秒です。速度をテストの反射0％コヒーレンスの2.5秒に分割し、各試行の間隔を、スピードと一貫性をテストする5秒、および速度と一致する適切な方向に100％の7.5 Sが続きます。 この5秒クリティカル期間中、被験者は、対応する必要があります。応答するために左または右のモニターに向かって向きや待ち時間などに依存する措置を記録する。 一次的強化子唯一の5つランダムな試行を使用してアニメーション無脊椎動物で各試験を強化する。各ブロックの試験が完了した時点で、テストの領域から対象のエンクロージャを削除し、別の筐体を導入する。

<ol>
	<li>Burghardt, G. M., Gans, C., Tinkle, 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=Learning+processes+in+reptiles.">Learning processes in reptiles.</a> Biology of the reptilia. 7, 555-681 (1977).</li><li>VPixx, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Instruction Manual. VPixx Techniologies Inc. , 96 (2002).</li></ol>

資金は、マッコーリー大学のCISABと心理学科大学院賞、およびオーストラリアの研究評議会によってサポートされていました。研究は、マッコーリー大学動物倫理委員会（2003/014）とニューサウスウェールズ州国立公園とオーストラリアの野生生物サービス（S11024）の倫理指針の中で行われました。

トカゲの種の感覚能力をテストすることは困難であった、そして主に退屈で骨の折れるプロセスの両方をされています。これらの困難は、正しい補強剤を見つける、あるいは機能的なプロトコルを確立することに由来する。ここで、私はトカゲの運動感覚をテストすることに成功プロトコルを確立した。また、この手順では、他の視覚特性だけでなく、他の感覚のメカニズムをテストでは適用できるかもし?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>iMac</td>
<td>Computer</td>
<td>Apple Computers, Inc.</td>
<td>&nbsp;</td>
<td>Three systems</td>
</tr>
<tr>
<td>Canopus&#174;</td>
<td>Digital-to-analog converter</td>
<td>Canopus&#174;</td>
<td>ADVC110</td>
<td>&nbsp;</td>
<tr>
<td>Sony Trintron</td>
<td>Colour viewfinder</td>
<td>Sony Corporation</td>
<td>PVM-14M2A, PVM-14L2, PVM-14N5A</td>
<td>Three monitors</td>
</tr>
<tr>
<td>Canon</td>
<td>Digital camcorder</td>
<td>Cannon Inc.</td>
<td>MV650i</td>
<td>Records behavior</td>
</tr>
<tr>
<td>NEC</td>
<td>Colour viewfinder</td>
<td>NEC</td>
<td>1892</td>
<td>View responses</td>
</tr>
<tr>
<td>Sony VCR</td>
<td>VHS recorder</td>
<td>Sony Corporation</td>
<td>SLV-EZ717AS</td>
<td>Record behavior to VHS</td>
</tr>
<tr>
<td>VHS tape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Generic VHS</td>
<td>Recording media</td>
</tr>
<tr>
<td>VPixx</td>
<td>Software program</td>
<td>VPixx Technologies, Inc.</td>
<td>v1.79</td>
<td>To create RDKs</td>
</tr>
<tr>
<td>Final Cut Pro</td>
<td>Software program</td>
<td>Apple Computer, Inc.</td>
<td>HD</td>
<td>To create playback sequences</td>
</tr>
<tr>
<td>Lightwave&#174; 3D</td>
<td>Software program</td>
<td>NewTek Inc.</td>
<td>v8.3</td>
<td>To create secondary reinforcers</td>
</tr>
<tr>
<td>Mealworm</td>
<td>Tenebrio molitor</td>
<td>Pisces Enterprises LTD</td>
<td>&nbsp;</td>
<td>Primary reinforcer</td>
</tr>
<tr>
<td>Cricket</td>
<td>Achetus domesticus</td>
<td>Pisces Enterprises LTD</td>
<td>&nbsp;</td>
<td>Food for husbandry</td>
</tr>
<tr>
<td>Terrariums</td>
<td>Glass</td>
<td>Crystal Palace Aquariums</td>
<td>&nbsp;</td>
<td>Enclosure material for lizards (60 x 60 x 60 cm)</td>
</tr>
<tr>
<td>Globes</td>
<td>Spotone</td>
<td>Phillips</td>
<td>&nbsp;</td>
<td>125W, Heat lamps for lizards</td>
</tr>
<tr>
<td>Ultraviolet globes</td>
<td>Ultra-Vitalux</td>
<td>Osram</td>
<td>&nbsp;</td>
<td> 300W, UV lamps for lizards</td>
</tr>
<tr>
<td>Herptivite</td>
<td>&nbsp;</td>
<td>Rep-Cal Research Labs</td>
<td>&nbsp;</td>
<td>Vitamins: beta carotene, calcium, and vitamin D3</td>
</tr>		</tbody>
		</table>
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testing visual sensitivity to the speed and direction of motion in lizards

任意の種の視覚的な感度をテストする行動、進化、および生態学についての基本的な情報を提供します。しかし、視覚システムの特定の機能をテストする機能的なアプリケーションのためのより多くの経験的な証拠を提供しています。感覚系の調査は、感覚能力、学習と記憶の機能に関する情報を提供し、逸脱を測定するために知られているベースラインの動作（Burghardt、1977）を確立します。しかし、哺乳類や鳥類のシステムとは異なり、爬虫類の種の学習と記憶のためのテストは困難です。さらに、感覚能力の心理物理学的指標としてオペラントパラダイムを使用すると同様として困難です。歴史的に、爬虫類の種が原因動機に関連する問題、生理学、代謝、および基本的な生物学的特性のコンディショニング試験に十分に対応してきた。ここで、私は、小説のモデルのトカゲの種を使用するオペラントパラダイムを示すジャッキー竜（Amphibolurus muricatus）と顕著なスピードと動きの特性に周辺機器の感度をテストする方法について説明します。このメソッドは、評価、学習とトカゲの感覚能力に革新的なアプローチを使用しています。私はランダムドットkinematogramsの使用（RDKs）速度に対する感度を測定し、一貫した方向に移動するドットの割合を変更することにより、信号強度のレベルを操作するために採用しています。 RDKsは、生物学的に意味のある刺激を表し、視覚システムに係合する、人間や他の動物の感度を測定するために使われる古典的な心理物理学的ツールであることはありません。ここで、RDKsは3つのビデオ再生のシステムを使用してトカゲに表示されます。トカゲは、彼らが動いているようにドットを感知方向（左または右）を選択することです。適切な方向の選択は、コンピュータアニメーション無脊椎動物でシミュレー生物学的に重要な獲物の刺激によって補強されている。

Watch this Scientific Journal Video about Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards at JoVE.com

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

ランダムドットkinematogramsとコンピュータで生成された無脊椎動物のビデオ再生を採用したオペラント条件づけパラダイムを使用して、トカゲの視覚感度のテスト。

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

testing-visual-sensitivity-to-speed-direction-motion

Research

JoVE Journal

Biology

11.4K ビュー.  Macquarie University. ランダムドットkinematogramsとコンピュータで生成された無脊椎動物のビデオ再生を採用したオペラント条件づけパラダイムを使用して、トカゲの視覚感度のテスト。

ビデオ: Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

マイクロインジェクションを使用してアブラムシのウイルス伝播の生理学的障壁のテスト

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

生物学

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

成体マウスでの嗅覚行動試験

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

マウスの錐体光受容体からの単一細胞サクション録音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

ゼブラフィッシュの視覚機能を研究するために視運動性応答を使用して

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

インスリン注射と成人のインスリン感受性を測定する血リンパの抽出キイロショウジョウバエ</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

ショウジョウバエにおける温度嗜好挙動とその概日リズムの設計と解析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

細胞特異的にLINC複合体を破壊するマウスモデルの検証

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

ライブの一次繊毛で高速超解像顕微鏡の応用

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

蚊を殺虫化合物小説の毒性試験のプロトコル

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...