SGは、ニューヨーク州立大学バッファロー校からとJohn R. Oishei財団からの資金によってサポートされています。

1。試薬の調製<ol><li> 、4MMのCaCl 2、4mmののMgCl 2、2mMの塩化カリウム、5mMのHEPESおよび36mmのスクロース、128 mMのNaClを用いて1 ×解離バッファーを準備します。 7.2〜ソリューションと殺菌フィルターPH。 </li><li> 16％ホルムアルデヒドおよび1x解離バッファーの1:1希釈を使用して固定液を準備します。 </li><li> 1Xリン酸を用いて1 × PBTを準備する生理食塩水（PBS）は1:100の比率では7.2とトリトンX - 100のPHでバッファ。 </li><li> 1X PBSでアジ化Naは0.01％（BSA）牛血清アルブミン5％を準備します。 </li></ol> 2。解剖のための準備<ol><li> Sylgardの料理は、解剖のために使用されます。シャーレにSylgard 184シリコーンエラストマーベースと硬化剤の混合物を注ぐことで料理を準備します。混合物が完全に使用する前に固化し、乾燥することができます。 </li><li>解剖する前に、第5位鉗子、ストレートブレードのvannasのはさみのクリーンペア、およびピンのきれいなペアを集める。 </li></ol> 3。第3齢幼虫の解剖<ol><li>ペトリ皿の上に特定の遺伝子型の第3齢幼虫をさまよう収集する。 </li><li>食べ物を取り除くために脱イオン水に幼虫を洗ってください。 </li><li>背側の上で、sylgard皿上に幼虫を置きます。その前部と後部両端の幼虫をピン。 </li><li>ピン幼虫で1 ×解離バッファのドロップを置きます。細かいハサミのニップの後端に近い背側正中線上に幼虫を使用した。はさみを使用して、後端から前端まで幼虫のクチクラをカット。 </li><li>解剖幼虫の上に新たな1 ×解離緩衝液のドロップを置きます。ピンを使用して、幼虫の中央付近にキューティクルの一側縁部を選択し、キューティクルをピン。図に示すように、別のピンを使用して、キューティクルの第二の側縁部を固定。 2本のピンをそれぞれの側面で使用されています。下の図を参照してください。 </li><li>慎重に接続されている分節の神経を持つ2つの光のローブと腹側神経節だけで脳を残して、腸、脂肪体、および気管を取り外します。手順の残りの部分はsylgard皿上で行われます。 </li></ol> 4。ディテールに秘められた幼虫の固定<ol><li> 15分ごとに修正を置き換えることで、30分の修正で切除した幼虫をインキュベートする。 </li><li>固定後、10分ごとにバッファを置き換えることにより、30分間× 1 PBTの幼虫をすすいでください。幼虫は、常にバッファ内でなければならないことに注意することは重要です。 </li></ol> 5。ディテールに秘められた幼虫の抗体染色<ol><li> 1X PBTのNaアジドと、5％BSAで適切な濃度に希釈して一次抗体を準備する。最適な濃度は、種々の抗体に異なります。 </li><li>最後の1X PBTは、準備一次抗体溶液で洗浄し、4℃で一晩湿気チャンバー内でインキュベート交換してください。湿気の多い室は水に浸した金 - ワイプとプラスチック皿を並べることによって構築されています。 </li><li>翌日、10分ごとにバッファを置き換えることにより、30分間× 1 PBTで切除した幼虫をすすいでください。 </li><li> 1X PBTのNaアジドと、5％BSAで適切な濃度に希釈することにより、二次抗体を準備し、時間25℃で二次抗体溶液との幼虫をインキュベートする。二次抗体が敏感な光であるため、光を防止するために箔で湿度の高い室を包みます。 </li><li>インキュベーション後、10分ごとにバッファを置き換えることにより、30分間× 1 PBTの幼虫をすすいでください。 </li></ol> 6。マウントとディテールに秘められた幼虫の可視化<ol><li>スライド上に幼虫をマウントする前に、（図を参照）上に座るためにカバースリップのための十分なスペースと、途中でマニキュアの縦の二重線を分散することでスライドを準備。完全に爪がマニキュア乾かします。 </li><li>スライド上の縦のマニキュアの線（図を参照）との間のスペースに取付バッファのドロップを置きます。スライドは、現在マウントするための準備ができています。 <img src="/files/ftp_upload/2128/2128.6.2.1.jpg" alt="図1ステップ6.2" /> <img src="/files/ftp_upload/2128/2128.6.2.2.jpg" alt="図2ステップ6.2" /></li><li>最後の1X PBTはリンスした後、静かに横方向のピンをはなします。キューティクルを引き裂くしないように注意してください。 </li><li>慎重に残りの2つのピンを削除し、ピンセットで幼虫をピックアップし、プレパラート上に水平に幼虫を置く。幼虫が裏返しと指向腹側が上されていないことを確認します。 </li><li>ゆっくりと上にカバースリップを置き、マニキュア液（図を参照）でエッジを封印。幼虫は蛍光顕微鏡下で可視化するための準備ができています。 <img src="/files/ftp_upload/2128/2128.6.5.jpg" alt="図1ステップ6.5" /></li></ol> 7。代表的な結果<img src="/files/ftp_upload/2128/2128fig1.jpg" alt="図1" /> 図1：野生型larvから幼虫の分節神経の代表画像（CSP、Zinsmaier ら、1994）シナプス小胞のタンパク質のシステインの文字列の蛋白質に対する抗体を用いた。分節性神経がスムーズに染色されることに注意してください。バー=20μmの<img src="/files/ftp_upload/2128/2128fig2.jpg" alt="図2" /> 図2：シナプス小胞のタンパク質のシステインの文字列の蛋白質 （CSP） に対する抗体を用いたモータータンパク質変異体の幼虫から幼虫の分節神経の代表的な画像 。分節性神経は、CSP（矢印）のために明るく染色大規模な集積を示すことに注意してください。

<ol>
	<li>Gunawardena, S., Goldstein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Axonal+Transport+and+Neuronal+Viability+by+Amyoid+Precursor+Protein+Mutations+in+Drosophila.">Disruption of Axonal Transport and Neuronal Viability by Amyoid Precursor Protein Mutations in Drosophila.</a> Neuron. 32 (2), 389-401 (2001).</li><li>Gunawardena, S., Her, L. S., Brusch, R. G., Laymon, R. A., Niesman, I. R., Gordesky-Gold, B., Sintasath, L., Bonini, N. M., Goldstein, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+axonal+transport+by+loss+of+huntingtin+or+expression+of+pathogenic+polyQ+proteins+in+Drosophila.">Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila.</a> Neuron. 40 (1), 1-2 (2003).</li><li>Hurd, D. D., Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+mutations+cause+motor+neuron+disease+phenotypes+by+disrupting+fast+axonal+transport+in+Drosophila.">Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in Drosophila.</a> Genetics. 144 (3), 1075-1085 (1996).</li><li>Zinsmaier, K. E., Eberle, K. K., Buchner, E., Walter, N., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paralysis+and+early+death+in+cysteine+string+protein+mutants+of+Drosophila.">Paralysis and early death in cysteine string protein mutants of Drosophila.</a> Science. 263, 977-980 (1994).</li></ol>

利害の衝突は宣言されません。

ショウジョウバエ幼虫の分節の神経は軸索輸送のメカニズムを研究する強力なシステムです。第3齢幼虫の解剖が正しく行われている場合、このような筋肉やNMJsなどのCNS、PNS、および関連組織のすべてのコンポーネントは、単一の幼虫の中で調べることができます。我々はハードとサクストン（1996）によって発行されたものから、このプロトコルを適応している。このプロト?...

<table><tbody><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Minutien Pins, Stainless Steel</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Mcpherson-Vannas Straight Blade 8cm Scissors</td><td>Fisher Scientific</td><td>50822236</td><td></td></tr><tr><td>Vectashield, Mounting Medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>Sylgard 184 Silicone elastomer</td><td>Dow Corning</td><td>4026148</td><td></td></tr><tr><td>formaldehyde, 16%</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>dCSP3</td><td>Developmental Studies Hybridoma Bank</td><td></td><td></td></tr><tr><td>Alexa Fluor 568 Goat Anti-Mouse IgG</td><td>Invitrogen</td><td>A-11004</td><td></td></tr></tbody></table>

visualization of larval segmental nerves in 3rd instar drosophila larval preparations

キイロショウジョウバエのため、特に便利な遺伝学と完全に配列ゲノムの神経系の発達と機能を研究するための強力なモデル系として浮上している。さらに、幼虫の神経系は、幼虫の体節神経が体の長さに沿って終点の脳とその神経終末内にあるそれらの細胞体と軸索の束が含まれているように軸索輸送のメカニズムを研究する理想的なモデルシステムです。ここでは、幼虫の分節神経内シナプス小胞タンパク質の可視化のための手順を説明します。正しく行えば、神経系のすべてのコンポーネントは、そのような筋肉やNMJsなどの関連組織と一緒に、、そのまま無傷、および可視化する準備が残ります。様々な変異を有する第3齢幼虫は、解剖固定、シナプス小胞の抗体と共にインキュベートし、可視化と野生型の幼虫と比較されます。この手順は、簡単に幼虫分節神経内で観察することができる様々なタンパク質の分布にいくつかの異なるシナプスや神経細胞の抗体と変化に適応することができます。

Watch this Scientific Journal Video about Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations at JoVE.com

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

キイロショウジョウバエの幼虫は、幼虫の分節神経内軸索輸送のメカニズムを調べるために理想的なモデルシステムを提供しています。この手順を使用して、様々な変異を有する第3齢幼虫は、野生型の幼虫と比較することができます。

3の幼虫分節神経の可視化 RD齢ショウジョウバエ幼虫準備

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

visualization-larval-segmental-nerves-3rd-instar-drosophila-larval

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

10.3K ビュー.  SUNY-University at Buffalo. キイロショウジョウバエの幼虫は、幼虫の分節神経内軸索輸送のメカニズムを調べるために理想的なモデルシステムを提供しています。この手順を使用して、様々な変異を有する第3齢幼虫は、野生型の幼虫と比較することができます。

ビデオ: Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

キイロショウジョウバエの幼虫中枢神経系の解剖

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

生物学

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

ホールマウントの胎児神経系の可視化ショウジョウバエ胚

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

神経科学

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

ショウジョウバエ幼虫分節軸索内ではシナプス小胞の in vivo可視化で

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

の形態素解析ショウジョウバエ</em遺伝モザイクを使用>幼虫末梢感覚ニューロンの樹状突起と軸索

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

における損傷応答のライブイメージングや研究のためのマイクロ流体チップを使用<em&gt;ショウジョウバエ</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

生物工学

Visualization of Proprioceptors in Drosophila Larvae and Pupae

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 insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

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 ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

中枢神経系の修復を調査するために傷害パラダイム<em&gt;ショウジョウバエ</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

のライブイメージング<em&gt;ショウジョウバエ</em&gt;幼虫の神経芽細胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Drosophila melanogaster Third Instar Larvaeのライブ脳外植片のイメージング

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

著者スポットライト:非対称細胞分裂ダイナミクスの調査:ショウジョウバエ幼虫の脳外植片のライブイメージングのためのプロトコル 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

ショウジョウバエメラノガスター第3齢幼虫脳の生細胞イメージング

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

3の幼虫分節神経の可視化^RD齢ショウジョウバエ幼虫準備

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キイロショウジョウバエの幼虫中枢神経系の解剖

ホールマウントの胎児神経系の可視化ショウジョウバエ胚

ショウジョウバエ幼虫分節軸索内ではシナプス小胞の in vivo可視化で

の形態素解析ショウジョウバエ幼虫末梢感覚ニューロンの樹状突起と軸索

における損傷応答のライブイメージングや研究のためのマイクロ流体チップを使用

でProprioceptorsの可視化

中枢神経系の修復を調査するために傷害パラダイム

のライブイメージング

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

ショウジョウバエメラノガスター第3齢幼虫脳の生細胞イメージング