著者らは、バックグラウンドミュージックを提供するために、ビデオ処理とエリック·トーマス、音楽科、コルビー大学と技術支援のために、Qiuli王、言語リソースセンター、コルビー·カレッジに感謝したいと思います。このプロジェクトは、研究資源、INBRE（P20RR016463-12）ナショナル·センターからの助成、一般的な医科学研究所（P20のGM103423-12）、健康科学課グラント、コルビー·カレッジ（STA）の国民研究所によってサポートされていました。 JLとLWM夏奨学生基金、コルビー大学からの補助金によってサポートされていました。

1 ショウジョウバエ驚愕誘発性歩行アッセイ<ol><li>薬物治療<ol><li> CO 2を使用して、1〜3日齢のオスのハエの希望数を（およそ8-12）固定化し、薬剤を補充食品を含むバイアルに輸送することが落ち着いた。注：別の麻酔薬、例えばエーテルまたは氷が計数および取り扱いを可能にするためにハエを鎮静するために使用することができます。 </li><li>ハエは（食品に付着したばかりハエを防ぐために）水平にバイアルを20分間（またはリカバリまで）、鎮静から回復した後、12時間の暗闇の中で直立バイアルを配置することができ、25℃で12時間の明インキュベーター実験の残りのために。 </li></ol></li><li>実験のセットアップ<ol><li>油性マジックでバイアルの周りに円をマークすることで、6.33センチメートルの3等分この二重バイアルセットアップを分割します。 </li><li>薬物曝露の3日後、転送が底バイアル中に麻酔なしで飛ぶとすぐにバイアルを置く開口部に。明確なテープと一緒に2つのバイアルをテープで固定します。 </li><li>ハエは15分間、新しい環境に順応できるようにします。 </li><li>場所バイアル白地に、ビュー内のタイマによる二重バイアル装置から適切な距離にデジタルカメラを設置。装置全体は、単一の画像フレームで表示され、すべてのハエが焦点になっていることを確認してください。試験間一貫したフレームを維持するために、カメラやバイアルの位置をマーク。 </li></ol></li><li>モビリティアッセイ<ol><li>明らかに、カメラビューに試行番号、薬物治療、タイマーを表示する。 </li><li>しっかりとカウンターに対する二重バイアル装置を3回タップすると、すべてのハエがバイアルの底に落ちることを確認してください。同時にタイマーを開始。 </li><li> 1分間ごとに5秒間、装置の写真を撮る。注：または、ビデオがキャプチャされ、測定のための適切な間隔で一時停止することができた。 </li><li>ハエが1分間邪魔されずに回復することを許可します。 </li><li&gt;各試行の間に1分間の回復時間をさらに2回繰り返します。注：各装置は、データ収集を完了するために5分を取るべきである。裁判の間、タッピングの同じような力を維持します。複数（少なくとも3）装置は、容易に同時に取り扱うことができる。 </li></ol></li><li>データ分析<ol><li>写真を確認し、時間をかけて各セクションのハエの数を記録する。時間をかけて各セクションのハエの割合を計算します。注：あまりにも多くのハエは、実験を通して死ぬ場合は、補償するために、元の試行番号をスケールアップすることが可能であり、例えば、関心のある2または3の時点で同一のハエで、この手順全体を繰り返して3日目、5、および7死亡率。データを比較するために、適切な統計分析を使用してください。 </li></ol></li></ol> 2 ショウジョウバエ自発歩行アッセイ<ol><li>食品の調製<ol><li> 15脱イオン水mlの希望ロテノンインスタントショウジョウバエ培地の3グラムを再構成（または関心のある別の薬剤）の投与量。 </li><li>食品混合物はしっかりした（約5分）となっていたら、慎重にメーカーへの高約1cmを透明のチューブ（5ミリメートル×65 mm）を供給すべき食品をロードします。慎重に食品中に垂直にチューブを配置し、それらがチューブ内の食品を用いて除去することができるまで、それらをねじることによって管に食物を注入された薬剤を追加します。注：これは、真空を作成するために、管の開口部に指を置くと便利です。食べ物は気泡が含まれているか、ハエがままになることがように表面に凹凸があってはなりません。 </li></ol></li><li>実験の設定<ol><li>食品最寄りのチューブの端部にプラスチックキャップを置きます。強制的にプッシュされた場合、それがバイアル内に気泡を作成することができますよう、可能な限り少ない管上にプラスチックキャップを押してください。 </li><li>落ち着いた1日齢の雄は、CO 2を使用して飛行し、慎重に1オスが絵筆を各チューブに飛んで挿入します。所望の試行回数に応じて繰り返す。 </li><li>プラグ大きな店買ったコットンボールから手をロールバックすることができ、小さなコットンボールを持つ食物からチューブ最も遠い、の終わり。 </li><li>ハエは15分間水平にチューブを回復し、すべてのハエが生きて、アクティブであることを確認できるようにします。 DAMにチューブを挿入し、すべての管がDAMに対する相対同じ位置にあることを確認してください。注：これは、管の端部が監視されているように、バイアル瓶の中央モニタリングの領域とに配置する、または側面にすべてのバイアルをプッシュすることが可能である。注：この方法のバリエーションのための説明を参照してください。 </li></ol></li><li>データ収集<ol><li> 12時間の暗闇の中でDAMを置き、12時間の明インキュベーターを25℃に設定してください。データ収集システムにDAMを接続します。 DAMソフトウェアを開いて、好みの下で10分のビンの長さを選択します。データ収集を開始し、番組が7日間のデータを収集できる。注：必要に応じてビンの長さを調整することができる。 </li><li>データ分析 注：プロセスデータは、長期自発運動の尺度として分あたりのカウントを得る。 <ol><li>選択入力データをクリックして開くDAMファイルスキャンプログラムとアクセスモニタデータ。 </li><li>適切なモニターの範囲を選択し、10分間隔にビンの長さを選択します。 </li><li>出力ファイル·タイプは、チャネルファイルを選択します。デフォルトとして他のすべてのオプションのままにしておきます。 </li><li>スキャンデータをクリックして、指定したフォルダに保存します。 </li><li>分あたりのカウントを取得するために、概日データ解析ソフトウェアでデータをインポート。注：データ解析Clocklabソフトウェアが一般的に使用されるために。その他のオプションも用意しています。 </li></ol></li></ol></li></ol>

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著者らは、開示することは何もない。

本研究では、パーキンソン病のロテノン誘発性ショウジョウバエモデルにおける長期的な自発運動および短期驚愕誘発性運動の両方を測定するための2つの手順を説明します。 Oneはまた、パーキンソン病などをモデル化することが知られている他の薬理学的薬剤、パラコート14、パーキンソン病などの遺伝的モデルが、α-シヌクレイン変異体15、</s...

ロコモーションの不備は、パーキンソン病の主な症状であり、主に黒質1のドーパミン作動性ニューロンの劣化によって引き起こされる。ロテノンは、 ショウジョウバエ 2-6のパーキンソン運動障害をモデル化するために広く研究されているケトン殺虫剤である。ロテノンは、最終的に細胞死7を引き起こす酸化的リン酸化経路を遮断することによって酸化的損傷を引き起こす。ドーパミン作動性ニューロンは、主にモータが2,7を基に化学物質の影響を作り、ロテノン毒性になりやすいです。彼らは遺伝的に扱いやすいですのでハエでパーキンソン病症状を誘導することによって、私たちはより良い病気を理解し、その症状6,8-11を解決することができます。 ショウジョウバエは維持し、迅速なライフサイクルを持ちやすい、この効果を研究するための優れたモデルを提供します。 いくつかの研究は、ロテノン、短期驚愕誘発性の原因となることが示されているショウジョウバエ毎期ハエにおける歩行欠陥がロテノン補充食品で維持されている、彼らは驚愕2-6の後に遅い負走地性の応答を示しています。対照トライアル限り迅速にバイアル装置において上向きに登るため彼らの失敗は驚愕誘発性運動欠陥を示すものである。 長期的にロテノンの効果は、自発的な動きがよくショウジョウバエの活動モニター（のDAM）が正常ショウジョウバエ概日リズムの動きを監視するために使用されている。記載12,13を研究されていない。ハエは、DAMにロードされる個別のチューブ内に配置される。この装置は、ハエが赤外線ビームを遮断回数をカウントする赤外線センサを備えている。これらのカウントは、邪魔されずに移動および活性12,13の尺度として用いることができる。 DAMでハエを確定すると、長期的な運動に対するロテノンの影響を特徴づけることができる。この研究は、MEASへの方法について説明しますより良いロテノン仲介モーターの欠陥の影響を理解するためにURE短期の驚愕誘発性運動と長期自発運動。彼らはこれらの運動欠陥を逆転し得る他の化合物の研究のために可能にするので、パーキンソン病を模倣運動欠乏症の特性が重要である。

<table border="0" cellpadding="0" cellspacing="0" width="628">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Standard narrow vials</td>
			<td style="width:127px;">Genesee Scientific</td>
			<td style="width:119px;">32-120</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Rotenone</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">R8875</td>
			<td>Store in freezer, make fresh for each experiment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td>Solvent for rotenone</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Instant Drosophila medium</td>
			<td style="width:127px;">Carolina Biological&#160;</td>
			<td style="width:119px;">Formula 4-24</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Drosophila activity monitor (DAM)</td>
			<td style="width:127px;">Trikinetics</td>
			<td style="width:119px;">DAM2</td>
			<td>trikinetics.com</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DAM tubes</td>
			<td style="width:127px;">Trikinetics</td>
			<td style="width:119px;">Tubes 5X65 mm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Recipe for Rotenone +food (125 mM dose)</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>Make 62.5 mM rotenone stock solution in DMSO by dissolving 25 mg rotenone in 1 ml DMSO&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>For 125 mM dose, add 10 mM rotenone stock in DMSO to 5 ml water.</td>
		</tr>
	</tbody>
</table>

methods to characterize spontaneous and startle-induced locomotion in a rotenone-induced parkinson's disease model of drosophila

パーキンソン病は、主に黒質に、中枢神経系におけるドーパミン作動性ニューロンの変性に起因する神経変性疾患である。疾患は、ヒトにおける剛性、振戦および痴呆として存在モータの欠陥を引き起こす。ロテノンは、ミトコンドリアにおける電子伝達系の機能を阻害することによって、酸化的損傷を引き起こす殺虫剤である。また、 ショウジョウバエにおけるパーキンソン病をモデル化するために使用される。ハエは、びっくりしている時に上向きに登るためにそれらを強いる固有の負の走地性の応答を持っている。それは、ロテノンは、彼らが下向きにタップされた後に登るハエの能力を混乱の早期死亡率と運動の欠陥の原因となることが確立されている。しかし、自発的な運動にロテノンの効果は十分に文書化されていません。この研究は、内ロテノン誘導性の欠陥を特徴付けるために二つ敏感な再現性、高スループットアッセイを概説ショウジョウバエの短期的な驚愕誘発性運動と長期自発運動。これらのアッセイは、好都合に移動欠陥および治療 ​​薬の有効性の他のショウジョウバエモデルを特徴付けるために適合させることができる。

ショウジョウバエ驚愕誘発性歩行アッセイ 野生型、 カントン-Sは 、ハエは、30秒（ 図1）の後に二重バイアル装置の、それぞれわずか約88％、上部と下部でのハエの5％堅牢な負走地性の反応を示した。 3日間125μMおよび250μMロテノンにさらさハエは、上部セクションと下部セクションのハエの数がわずかに増加中のハエの数がわず?...

Watch this Scientific Journal Video about Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila at JoVE.com

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

パーキンソン病は、運動欠陥を引き起こす中枢神経系におけるドーパミン作動性ニューロンの変性に起因する神経変性疾患である。ロテノンモデルショウジョウバエのパーキンソン病。本論文では、ロテノンによって引き起こされる自発的かつ驚愕誘発性の両方歩行の欠陥を特徴づける2つのアッセイの概要を説明します。

のロテノン誘発性のパーキンソン病モデルにおける自発と驚愕誘発性歩行を特徴づけるための方法<em&gt;ショウジョウバエ</em

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

methods-to-characterize-spontaneous-startle-induced-locomotion

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

10.0K ビュー.  Colby College. パーキンソン病は、運動欠陥を引き起こす中枢神経系におけるドーパミン作動性ニューロンの変性に起因する神経変性疾患である。ロテノンモデルショウジョウバエのパーキンソン病。本論文では、ロテノンによって引き起こされる自発的かつ驚愕誘発性の両方歩行の欠陥を特徴づける2つのアッセイの概要を説明します。 

ビデオ: Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

マウスにおける自発脳内出血をモデル化する自家血注入

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

神経科学

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

アッセイのメソッドショウジョウバエ行動

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

に温痛覚を評価するためのローカルおよびグローバルメソッド<em&gt;ショウジョウバエ</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（<em&gt; Schistocercaアメリカーナ</em&gt;）嗅覚回路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

における自発運動量の測定<em&gt;キイロショウジョウバエ</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

生体内で1 分子追跡ショウジョウバエ運動神経がシナプス前ターミナルで

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

主マウス中脳ニューロンにおける自発的Ca2+ フラックスとその下流効果の定量化

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

パーキンソン病の遺伝的ラットモデルにおける歩行解析のためのラットウォーカーシステムの適用

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

自動画像解析を用いたげっ歯類モデルのサブスタチングニグラにおけるドーパミン作動性ニューロン密度の半定量的決定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

The 6-hydroxydopamine Rat Model of Parkinson's Disease

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and dopaminergic striatal deficit. Animal models have been widely used to simulate human pathology in the laboratory. Rodents are the most used animal models for PD due to their ease of handling and maintenance. Moreover, the anatomy and molecular, cellular, and pharmacological mechanisms of PD are similar in rodents and humans. The infusion of the neurotoxin, 6-hydroxydopamine (6-OHDA), into a medial forebrain bundle (MFB) of rats reproduces the severe destruction of dopaminergic neurons and simulates PD symptoms. This protocol demonstrates how to perform the unilateral microinjection of 6-OHDA in the MFB in a rat model of PD and shows the motor deficits induced by 6-OHDA and predicted dopaminergic lesions through the stepping test. The 6-OHDA causes significant impairment in the number of steps performed with the contralateral forelimb.

The 6-hydroxydopamine (6-OHDA) model has been used for decades to advance the understanding of Parkinson's Disease. In this protocol, we demonstrate how to perform unilateral nigrostriatal lesions in the rat by injecting 6-OHDA in the medial forebrain bundle, assess motor deficits, and predict lesions using the stepping test.

パーキンソン病の6-ヒドロキシドーパミンラットモデル

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in ...

のロテノン誘発性のパーキンソン病モデルにおける自発と驚愕誘発性歩行を特徴づけるための方法

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マウスにおける自発脳内出血をモデル化する自家血注入

アッセイのメソッドショウジョウバエ行動

に温痛覚を評価するためのローカルおよびグローバルメソッド

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（

における自発運動量の測定

生体内で1 分子追跡ショウジョウバエ運動神経がシナプス前ターミナルで

主マウス中脳ニューロンにおける自発的^Ca2+ フラックスとその下流効果の定量化

パーキンソン病の遺伝的ラットモデルにおける歩行解析のためのラットウォーカーシステムの適用

自動画像解析を用いたげっ歯類モデルのサブスタチングニグラにおけるドーパミン作動性ニューロン密度の半定量的決定

パーキンソン病の6-ヒドロキシドーパミンラットモデル