Name: mass histology to quantify neurodegeneration in drosophila
Uploaded: 2016-12-15T12:30:00
Description: Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

This work was supported by grants to D.K. from the Medical Research Foundation of Oregon and from NIH/NINDS (NS047663). E.S. was supported by a training grant from the NIH (T32AG023477).

1.首輪にヘッドを固定し、パラフィンに埋め込み注：固定プロセスの手順はすべて、ドラフト内で行う必要があります。安息香酸メチルは、健康上のリスクをもたらすものではないが、ドラフト内で処理されない場合は圧倒的なことができ非常に明確な臭気を持っています。 <ol><li>ハエを麻酔する前に、（クロロホルム及び酢酸を混在させないでください）99％エタノール30 mLに氷酢酸...

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記載された方法は、 ショウジョウバエの脳における神経変性を定量化する手段を提供します。他の方法は、特定の細胞型を計数するように、神経変性を同定するために使用することができるが、この方法の利点は、より一般的に適用できることです。細胞を計数することは、これらの細胞が確実に特異的な抗体や、常に利用可能でない細胞特異的マーカーの発現、のいずれかを用いて...

平均余命の増加に伴い、アルツハイマー病やパーキンソンなどの神経変性疾患は、一般人口の増大する健康への脅威となっています。国立衛生研究所によると、115万人が世界中で、基礎となる重要な進歩は、それらの多くは、これらの疾患のうちの少なくともいくつかに関与する遺伝子および危険因子を同定することにしたが2050年に認知症により影響を受けると予測されます分子メカニズムはまだ不明かよく理解しています。 線虫（Caenorhabditis elegans）およびキイロショウジョウバエのような単純な無脊椎動物のモデル生物は、短いライフサイクル、子孫の数が多いと、十分に確立し、時にはユニークな遺伝的および分子的方法1の可用性を含む神経変性疾患のメカニズムを研究するための実験種々の利点を提供しています-12。さらに、これらの生物は、公平に適しています神経変性の表現型に対するそれらの悪化または改善効果によって、これらの疾患の要因を特定することができる相互作用の画面。 そのような遺伝的相互作用を分析し、高齢化の影響を評価することは神経変性を検出するために、その重症度を測定するための定量的なプロトコルが必要です。数値性能値13から21を提供するような嗅覚学習、負の走地性、または高速走光性ショウジョウバエにおける行動の側面を、測定するときにこの評価は、比較的容易に行うことができます。ニューロンを計数することによりニューロンの生存に対する効果を決定することも可能です。 PDに影響されるドーパミン作動性ニューロンのように、明確に識別可能である特定の集団に着目した場合ただし、これは可能であり、その後も、結果は22-24議論されています。 ここで説明するプロトコルは、パラフィン連続切片を実行するために、カラー方式を使用して、方法それは、もともとショウジョウバエ 25に解剖学的脳の突然変異体を単離するためにそれを使用しハイゼンベルクとBöhl、によって開発されました。カラー方法の使用は、その後凍結切片、ビブラトーム切片及びプラスチックセクション26-28に含むように適合されています。ここでは、この方法は、次に、神経変性表現型16,21,29-32とハエに開発空胞を測定することができる全体フライヘッドの連続切片を得るために使用されます。これらの測定は、特定の脳領域で行うことができ、または脳全体を覆うことができます。後者のアプローチは、老化中に観察されたように、1つは、弱い変性表現型を識別することができます。最後に、20ハエまで、カラーを使用している場合だけでなく、より少ない時間がかかるだけでなく、原因のわずかな変化にアーティファクトを最小化する、同じ製剤中に対照および実験ハエの分析を可能にする1つの製剤として処理することができます準備。

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	<tbody>
		<tr>
			<td>Name of the Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td></td>
		</tr>
		<tr>
			<td>Collar</td>
			<td>Genesee Scientific</td>
			<td>TS 48-100</td>
			<td>We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. A discription to make collars can be found at http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/fluorescent/index.html&#160;</td>
		</tr>
		<tr>
			<td>Rubber ice cube tray for embedding</td>
			<td>Household store</td>
			<td></td>
			<td>The size can be made to fit by glueing in additional walls&#160;</td>
		</tr>
		<tr>
			<td>Crystallizing dish</td>
			<td>Fisher Scientific company</td>
			<td>08-762-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ether</td>
			<td>Fisher Scientific Company</td>
			<td>E138-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories Inc.</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Choloroform</td>
			<td>Fisher Scientific Company</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher Scientific Company</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylbenzoate</td>
			<td>Fisher Scientific Company</td>
			<td>M205-500</td>
			<td>Distinct Odor</td>
		</tr>
		<tr>
			<td>Use in fume hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Melting Point Paraffin Wax</td>
			<td>Fisher Scientific Company</td>
			<td>T565</td>
			<td>Make sure to keep extra melted in a 65&#176;C waterbath</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems</td>
			<td>Reichert Jung 2040 Autocut</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slide</td>
			<td>Fisher Scientific Company</td>
			<td>12-550D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific Company</td>
			<td>12-545-M</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeClear</td>
			<td>Fisher Scientific Company</td>
			<td>314-629</td>
			<td>Three different containers for washes</td>
		</tr>
		<tr>
			<td>Vertical Staining Jar with Cover</td>
			<td>Ted Pella Inc.&#160;</td>
			<td>432-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific Company</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Solution</td>
			<td>Sigma Life Science</td>
			<td>P8290-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

mass histology to quantify neurodegeneration in drosophila

アルツハイマー病（AD）またはパーキンソン病（PD）などの進行性神経変性疾患は、世界中のヒトの健康に増加脅威です。哺乳動物のモデルは病原性の基礎となるメカニズムに重要な洞察を提供してきたが、一緒に高コストの哺乳動物系の複雑さは、その使用を制限しています。したがって、シンプルだが十分に確立されたショウジョウバエモデル・システムは、これらの疾患に影響される分子経路を調査するための代替手段を提供します。行動障害のほか、神経変性疾患は、神経細胞死と軸索障害などの組織学的表現型によって特徴付けられます。神経変性を定量化するために、それが遺伝的要因と環境要因によって影響される方法を決定するために、我々は大人のハエの脳内の空胞を測定することに基づいている組織学的アプローチを使用しています。系統誤差の影響を最小限に抑えるために、直接制御とEXPからの切片を比較します1つの製剤でerimentalハエは、我々はパラフィン切片のための「カラー」メソッドを使用します。神経変性は、その後、ハエの脳に開発した空胞のサイズ及び/又は数を測定することによって評価されます。これは、いずれかの特定の関心領域に焦点を合わせることによって、または完全長に及ぶ連続切片を得ることにより脳全体を分析することによって行うことができます。通常の老化時に起こるようなので、この方法は、1が厳しい変性だけでなく、いくつかのセクションにのみ検出され、比較的穏やかな表現型だけでなく、を測定することができます。

全体フライヘッドを包含する眼の色素33によって染色された連続切片で説明した方法の結果を使用。この部分は、個々のヘッドのセクションが上から下に表示され、図1Bに示されています。別のハエのセクションは、この例では左から右に見られます。配向およびハエの識別を容易にする、アイレスフライ（正弦oculis）が 3位（矢印、 ...

Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

Mass Histology to Quantify Neurodegeneration in Drosophila

ショウジョウバエは広く神経変性を研究するためのモデル系として使用されます。このプロトコルは、脳内の空胞の形成によって決定されるように、定量化可能な変性方法を記載しています。それはまた、一つのサンプルとして処理し、切片化対照および実験ハエによる実験手順に影響を最小限に抑えることができます。

に神経変性を定量化するために、マス組織学<em&gt;ショウジョウバエ</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Research

JoVE Journal

Neuroscience

Mass Histology to Quantify Neurodegeneration in Drosophila

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

定量シナプス：シナプス数を定量する免疫細胞ベースのアッセイ

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

親油性色素を組み合わせることで、その場でハイブリダイゼーション、免疫組織化学、および組織学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

でアッセイする運動、学習、記憶障害ショウジョウバエ</em神経変性の>モデル

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

In vivo Laser Axotomy in C. elegans

In vivo Laser Axotomy in C. elegans

Cの生体レーザー軸索切断にエレガンス

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Methods to Assay Drosophila Behavior

Methods to Assay Drosophila 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 ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

でProprioceptorsの可視化<em&gt;ショウジョウバエ</em&gt;幼虫および蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

組織学でMRIをマージするために、3D印刷技術を活用：脳セクショニングのためのプロトコルを

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

慢性外傷性脳症モデルの新しい方法<em&gt;ショウジョウバエ</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Modeling Amyloid-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Β42 アミロイド毒性と大人のゼブラフィッシュ脳の神経変性をモデリング

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

ショウジョウバエのニューロン樹状分枝複雑さの定量解析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...