私たちは、優れた技術支援のためにコラーゲンプロトコルとユッタ·フェイとウルスラヒンツを共有するためのUrmas Arumaeに感謝したいと思います。さらに我々は親切にビデオshotingのヘルプのためにクリスチャン·F·アッカーマン、ウルリケ·エンゲルとハイデルベルク大学のニコンイメージングセンターともヨアヒムキルシュに感謝したいと思います。研究の一部はドイツ学術振興（SFB 592）を通じて資金を供給された。

1。コラーゲンゲルの調製<ol><li> 455μlの10倍、MEM、112μlの7.5パーセントのNaHCO 3、50μlのグルタミンおよびNaOHを含む、株式媒体を準備します。濃度とNaOHの量は、ラット尾コラーゲン製剤（1.2参照）、1,000μlのある株式媒体の最終容量に依存します。 </li><li> Ebendal（1）に記載のラット尾部コラーゲンを準備します。 4℃のコラーゲン溶液を保存コラーゲン溶液の劣化を観察することができなかった。新しいバッチを調製した後、株式培地（1.1参照）に必要なNaOH濃度を推定する。 NaOHの量と濃度の滴定のための媒体のpHインジケーターを使用しています。 800μlの酸性ラット尾コラーゲンは、株式の培地210μlの混合赤の影を有効にする必要があります。 NaOHの濃度が低すぎるとコラーゲン溶液は、培地のpH指示薬により、黄色に変わります。突くことなく、メディアとのミックスにコラーゲンを追加気泡をucing。氷の上でこれらの手順を実行します。重合は、コラーゲン溶液と株式媒体を混合した後、2時間以内に発生し、新しい溶液を37℃でインキュベートする。実験を開始する前にこれをテストします。 1.3）（1.2参照）在庫培地210μlでコラーゲン溶液800μlを混合します。 8ウェルチャンバースライドのウェル中にこの溶液100μlを置きます。 37でスライドをインキュベート℃、5％CO 2、および細胞培養インキュベーター中で湿気のある条件。細胞培養ベンチ、無菌条件下でコラーゲンゲルを準備します。コラーゲンは、2時間以内にgelatinizes。 </li></ol> 2。胚SCGsの解剖<ol><li>子宮からの時間妊娠中のマウスの収穫胚（E16-18）し、さらに、必要になるまでそれらをPBS中に入れておきましょう。 </li><li>羊膜から1つの胚を解剖。鎖骨レベルの胚の尾の首を。 &quot;昆虫針&quot;を持つシリコンボトムディッシュ上でヘッドを固定します。頭の腹側はあなたに直面しているあなたは顎下領域を参照してくださいする必要があります。d。固定は簡単に準備できるようになります。 </li><li>左と右の顎下領域およびそれとの皮下結合組織の皮膚を取り除きます。 </li><li>舌、infrahyoidal筋肉や胸鎖乳突筋の上にビューをクリアするために、その場所から顎下唾液腺を削除します。 </li><li>喉頭と気管の上にビューをクリアするために慎重にこれらの筋肉を削除します。気管および喉頭を削除します。頚動脈はその後椎前筋の両側に表示されます。 SCGは、頸動脈の分岐点に位置し、楕円形の形状を有している。慎重に神経を除去し、PBS中に置いてください。彼らはそれらに付着している場合は切開ツールから優しく神経を取り除きます。それは、鉗子と同様SCGを除去するための &quot;昆虫針&quot;が搭載された針ホルダーを使用することは有益である。 </li><li>あなたはenouを解剖するまで一度PBS中、PBS中で室温で神経を保存GH神経節。その後、血管や結合組織から神経をきれいにしてください。層流ベンチ下に置かれた解剖顕微鏡下SCG切開とクリーニングを行ってください。 </li><li>最後に、扱いやすくするために注射器に搭載された注射針の助けを借りて、ゼラチン化したコラーゲンゲルに外植神経を配置します。 </li><li> 5％CO 2の湿潤な条件で、37℃で細胞培養インキュベーター中で糊化コラーゲン上SCG外植片をインキュベートする。 </li></ol> 3。 SCG外植片の治療<ol><li> 1〜2時間のインキュベーション後、B27サプリメント、グルタミンおよび抗生物質を含む100μlNeurobasal細胞培養培地とSCG植含むコラーゲンをカバーしています。今チャンバースライドのウェルは200μl（100μlのゲルと100μlの培地）のボリュームが含まれています。しかし今では軸索の成長を促進するために、NGF（60 ng / mlで）を含む、媒体の別の200μlを添加する。従って最終NGF濃度は30 ng / mlである。 </li><li><ol class="lalpha"><li> SCの移行の発症を調査するために、in vitroで 0（DIV0） で一日に直接追加の要因を追加します。 （2）必要に応じて二重濃度のNGF含有する培地中での要因を溶かす。 </li><li>すでに軸索に沿って移行開始のSCへの影響を分析するために、DIV4（実験の潜在的な停止）までDIV3で付加的な要因を追加します。この目的を達成するために、慎重にDIV3では、溶液の180μlを削除し、NGFとダブル濃度（2）で興味のある付加的な要因を含む200μlの新しいメディアを追加します。 </li></ol></li></ol> 4。タイムラプスイメージング分析用倒立顕微鏡を使用しています。種々の目的は、ビューと倍率のフィールドを定義し、使用することができます。 SCG植片のイメージングは​​スライドガラスとコラーゲンを介して行われるように1つの重要な側面は、しかし、使用目的の作動距離であるゲル。 1/10-30分の録画フレームレートは良好な結果（2）を示した。しかし、この側面は、科学的な疑問に調整する必要があります。通常のCCDカメラは、画像取得のために使用することができます。タイムラプスイメージングのための細胞培養インキュベーションチャンバーは、顕微鏡のステージに取り付けなければならない。 5％CO 2の湿潤な条件で、37℃で撮像中に組織をインキュベートする。撮影開始前培養システム一時間を開始します。これはチャンバースライドを含む顕微鏡部品（ 例えば、対物）の温度に調整することができ、温度ドリフト誘発を防ぐことができます。顕微鏡ソフトのヘルプやSCG外植片に異なる印加条件の同時分析のためのソフトウェア制御の電動ステージ（マルチポジションの設定）に分析の特定の領域を（外植片に内および異なる外植片の間）を定義します。使用することができるタイムラプスイメージング野生型組織だけでなく、トランスジェニック動物からの組織のために（3）：SCS（GFP S100B）をマーキング。イメージング蛍光団のための蛍光光源はmicropscopeセットアップで実装する必要があります。標準のフィルタを使用します。 5。 SCの移動距離の定量化<ol><li>エンドポイント（例えばDIV4）で、3〜4時間のためのチャンバースライドに4％PFAでコラーゲンゲルを固定します。連続してPBSで10分間コラーゲンゲルを5回洗浄する。免疫細胞化学（2）のための標準的なプロトコルを適用します。あなたはSCGsの準備を初めて使用する場合は、チロシンヒドロキシラーゼ（TH）カテコールアミン細胞に共通のマーカーに対する免疫組織化学による交感神経節と節の身元を確認します。免疫組織化学（一次抗体後）の間に、ゲルを洗浄するために有益であるより大きな体積を有する24ウェルプレートにコラーゲンゲルを含む植を転送します。 </li><li>免疫組織化学の後、スライドガラス上にコラーゲンゲルを慎重に置きます。慎重に残りの溶液を除去し、コラーゲンが乾燥/二次元に縮小することができます。これは、距離（2）の容易な測定を可能にする。この後、サンプルをマウントします。 </li><li>最後に、フィジー（NIH）のソフトウェアの助けを借りて移動距離を測定します。は、特別なプラグインはこの目的を達成するために必要ではありません。 μm単位の正確な測定を可能にするには、正しい画像のメタデータとフィジーの下の設定、画像やスケールを確認してください。細胞の定量化のためにもフィジーを使用しています。 </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+expressed+in+mouse+transgenic+lines+mark+subsets+of+glia,+neurons,+macrophages,+and+dendritic+cells+for+vital+examination.">Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.</a> The Journal of Neuroscience. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor+and+its+low-affinity+receptor+promote+Schwann+cell+migration.">Nerve growth factor and its low-affinity receptor promote Schwann cell migration.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (7), 2795-2799 (1994).</li><li>Mahanthappa, N. K., Anton, E. S., Matthew, W. 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特別な利害関係は宣言されません。

末梢神経系の発達は、エキサイティングなプロセスです。開発が完了すると、軸索は、ヒトでは、多くの場合100 cm以上であることができます全体の長さに沿って、SCSによってensheathedされています。この目的を達成するために必要なSCの正しい数はまた、完全な軸索のカバレッジを確保するため、周囲に伸びる軸索に沿って移動しなければならない開発およびSCの間に確立されてきました。こ?...

<table border="1"><tbody><tr><td> 試薬/材料の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> 10xのMEM </td><td>ギブコ</td><td> 21430 </td><td></td></tr><tr><td>炭酸水素ナトリウム（7.5％） </td><td>ギブコ</td><td> 25080 </td><td></td></tr><tr><td>グルタミン</td><td>ギブコ</td><td> 25030 </td><td></td></tr><tr><td> NaOHを</td><td>メルク</td><td> 109137 </td><td></td></tr><tr><td> NGF </td><td>ロッシュ</td><td> 1058231 </td><td> R＆Dの＃556-NG-100 </td></tr><tr><td> Neurobasal培地</td><td>ギブコ</td><td> 21103 </td><td></td></tr><tr><td> B27サプリメント</td><td>ギブコ</td><td> 17504 </td><td></td></tr><tr><td>抗生物質</td><td>ギブコ</td><td> 15640 </td><td></td></tr><tr><td> D-PBS </td><td>ギブコ</td><td> 14040 </td><td></td></tr><tr><td>昆虫針</td><td> FST </td><td> 26002から20 </td><td></td></tr><tr><td>注射針</td><td>ブラウン</td><td></td><td> BD＃300013 </td></tr><tr><td> 8穴のチャンバースライド</td><td>ラボTEK </td><td> 177402 </td><td></td></tr></tbody></table>

analyzing murine schwann cell development along growing axons

末梢神経の開発は魅力的なプロセスです。ニューロンはヒトで100以上の30cm離れニューロンの細胞体からしばしば具体的な目標を、支配する軸索を送る。開発中の神経細胞の生存は、対象由来成長因子でなく、シュワン細胞（SCS）のサポートに依存します。この目的を達成するために神経細胞体の地域（または中枢から末梢神経系への移行）のSC ensheath軸索シナプスや神経筋接合部へ。シュワン細胞は神経堤の誘導体であり、全体の軸索は、SCSで覆われるまで、新興の軸索に沿って前駆体として移行されます。これは、末梢神経系の開発のためのSCの移行の重要性を示しており、このプロセスを調査する必要性を強調している。 SCの発展を分析するために、設定も移行のためのそれらの生理学的基質、軸索を含むSCの隣に必要とされる。子宮内の開発による<emは&gt;生体タイムラプスイメージングでは、しかし、マウス（ ハツカネズミ ）のような胎盤の脊椎動物には不可能である。これを回避するために、我々は上頸神経節（SCG）植技術を適応した。神経成長因子（NGF）を用いた治療に基づいたSCG外植片は神経節から周囲に軸索に沿って移行SCの前駆続いて軸索を伸ばす。このシステムの利点は、SCが内因性SCのプールから、それらが同時に成長している自分自身の生理的な軸索に沿って移行することに由来していることです。軸索に沿ってSCの開発がSCの移行についての洞察を得るために更なる可能性を開いて、タイムラプスイメージングによって解析することができるので、このシステムは、特に興味をそそられる。

軸索の成長は、NGF（4）（映画スキームS1は図1方式）を用いた治療により植片をSCGから促進される。このプロセスは、倒立顕微鏡で簡単に表示され、タイムラプスイメージング（ムービーS2）を続けることができます。科学者がマウス胚からSCGを解剖で新たに追加された場合、我々は強くシンプルな抗チロシンヒドロキシラーゼ（TH）免疫組織化学によって手法の妥当?...

Watch this Scientific Journal Video about Analyzing Murine Schwann Cell Development Along Growing Axons at JoVE.com

Analyzing Murine Schwann Cell Development Along Growing Axons

ここでは、SCが伸びる軸索に沿って開発することができますているシュワン細胞（SC）の遊走アッセイを記述します。

成長中の軸索に沿ってマウスのシュワン細胞の発達を分析する

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

analyzing-murine-schwann-cell-development-along-growing-axons

Research

JoVE Journal

Neuroscience

11.5K ビュー.  University of Freiburg. ここでは、SCが伸びる軸索に沿って開発することができますているシュワン細胞（SC）の遊走アッセイを記述します。

ビデオ: Analyzing Murine Schwann Cell Development Along Growing Axons

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

根切断術後の後根の軸索のライブイメージング

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

神経科学

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

単一セルの開発のプロファイリングと成熟網膜神経細胞

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

胎児マウス神経管から神経堤細胞の単離および培養

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

マウス腸の神経前駆細胞の定量細胞遊走アッセイ

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脳軸索のレーザーNanosurgery<em&gt;インビボ</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

ニコチン誘導性カルシウムシグナリングおよび腹側海馬軸索に沿って神経伝達物質放出のライブイメージング

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

エレクトロポレーションによる齧歯類坐骨神経内のシュワン細胞へのインビボ遺伝子導入で

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

勉強するリニアアガロースチャネルを使用します<em&gt;ショウジョウバエ</em&gt;幼虫クロールの動作

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

列およびレイヤーで樹状形態を分析します

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.