この作品は、S.Ge.にNINDSとアメリカ心臓協会によって資金を供給されたこのアプローチは、もともと設立され、ジョンズホプキンス大学で博士宏俊歌の研究室で著者によって開発されました。我々は、彼の指導と支援のために博士宏君の歌に感謝の意を表します。

1。ウイルスのインジェクション高力価のエンジニアレトロウイルス（1 × 10 9単位/ ml）をウイルス上清の遠心分離に続いて、レトロウイルスベクターの同時トランスフェクションにより産生され、HEK293T細胞にVSVGています。源や生産方法については、優れたJoveのデモ（3）を参照してください。 注：ヤングアダルト（4-6週齢）雌のC57BL / 6マウス（Charles River）は、標準条件下で飼育されています。すべての手順は、ストーニーブルック大学のIACUCによって承認されたプロトコルの下で実験動物の管理と使用のための国立研究評議会のガイドに従ってください。 <ol><li>氷の上に動物あたりの冷凍レトロウイルスの融解2ul。 </li><li> Anaesthetize（100μgのケタミン+グラム体重あたり10μgのキシラジン）と定位フレーム（Steolting）で動物をマウント。頭の毛を削除し、70％エタノールで皮膚を拭いてください。 </li><li>頭蓋骨を公開し、以下の座標に歯科用ドリル（0.6ミリメートルドリルビット）を使用して、4つの浅い穴を開けます。 <ul><li>ブレグマからanterioposterior = -2ミリメートル、横= ± 1.6ミリメートル、腹側= 2.5ミリメートル、ブレグマからanterioposterior = -3ミリメートル、横= ± 2.6ミリメートル、腹側= 3.2 mmである。 </li><li>ブレグマからanterioposterior = -2ミリメートル、横= ± 1.6ミリメートル、腹側= 2.5ミリメートル、ブレグマからanterioposterior = -3ミリメートル、横= ± 2.6ミリメートル、腹側= 3.2 mmである。 </li></ul></li><li> 1μlのハミルトン、フラットチップシリンジをマウントし、歯状回の0.25μL/ minの速度でサイトあたり0.5μlのレトロウイルスを注入する。ゆっくり流体逆流を防ぐために注射器を引き出す前に、各注射後（腹側の深さについては、上記の座標を参照してください。）一時停止2分。注射の間で、簡単に滅菌PBSで注射器の先端部と血液の痕跡を除去するアプリケーターの外表面をすすぐ。 </li><li> 滅菌外科用縫合材料で傷口を閉じて（タイプP - 1;サイズ6-0）と注入後の希望の時間段階まで標準的な住宅の条件に動物を返します。 </li></ol> 2。スライスの準備成体マウスからの高品質の急性スライスを取得するには、我々は（下記参照）スライス標本用に変更された最先端のソリューションを使用してください。 <ol><li> 0〜4℃95％の氷とバブルのC O 2 -5％、少なくとも30分間CO 2へのプレチルカットソリューション。 </li><li> Anaesthetizeとtranscardially氷冷酸素飽和切削液で動物を灌流。 </li><li>迅速濾紙冷たい、酸素切削液であらかじめ湿らしたシャーレに脳を取り除く。小脳をカットオフし、（目に見える）注射の座標には、少なくとも1mmの前方の取り付け面をスライス。接着剤は、脳へvibrotome段階と寒さ、酸素切断溶液で満たされたカッティングチャンバにそれをマウントします。 </li><li>冠状または水平スライス（300 -350μm）を作製し、室温ACSFを含むインキュベーションチャンバーに大口径のピペットでそれらを転送するには、95％O 2 / 5％CO 2をバブリング。 </li><li> 30〜60分間32℃でのスライス、連続的にバブリングし、インキュベートする。記録の期間のために室温にチャンバーを戻す。 </li></ol> 3。電気生理学的実験<ol><li> 34℃ - スライスが連続して32℃で酸素飽和ACSFを灌流され、記録室に転送されます。 </li><li>バイポーラタングステン刺激電極は、低倍率の顕微鏡対物レンズを（10X）を使用して歯状回の分子層に配置されます。 </li><li>サブ粒状ゾーン/顆粒細胞層における新生児DGCsは、GFPまたはdTomatoの表現によって識別されます。全細胞patchclampの記録は、高倍率（40倍）下で新生ニューロンに実行されます。 </li><li>記録された細胞の発火特性は、電流クランプモードの下での電流の一連の注入によって得られる。 </li><li>電気刺激は、録音ソフトウェアによって制御される刺激アイソレータを介して刺激電極によって配信され、新生児DGCsにおける誘発性シナプス後電流が記録されます。 </li></ol> 4。データ解析誘発シナプス後応答の振幅は、成人生まれのニューロンの異なる発達段階で分析されています。 5。代表的な結果上記のプロトコルを使用して、 図1と同様に新生児歯状回の神経細胞でGFPの発現は一般的です。新生ニ​​ューロンが容易に可視化すると樹状突起と軸索の両方が確実にラベル付けされることに注意してください。薄いDGC軸索と小さな棘の発現は、ウイルスの質と力価、使用されるプロモーターおよびインビボ発現の長さによって異なります。私たちの手では、新生児の細胞と副細胞小器官は、通常、注射後数日内に、可視、および脊椎の表現であり、開発の初期段階からたどることができます。 W別の時間段階で新生ニューロンからホールセル記録では、成熟（ 図2b）中に既存の神経回路に独自の新生児の細胞特性、例えば活動電位（ 図2a）、だけでなく、シナプス統合のプロセスの研究を可能にする。 <img src="/files/ftp_upload/2548/2548fig1.jpg" alt="図1" /> 図1成体マウスの水平方向の歯状回のセクションにおける新生児の神経細胞の2光子共焦点画像。緑色の細胞はPUX - GFPレトロウイルスで標識21dpi（日後に注入）GFP発現新生児DGCsです。 <img src="/files/ftp_upload/2548/2548fig2.jpg" alt="図2" /> 図2）アクション21解像度。Bにおける新生児DGCの潜在的な発火特性を）代表は21 dpiで新生児DGCに記録された興奮性シナプス後電流（EPSCs）を誘発。

<ol>
	<li>Zhao, C., Deng, W., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functional+implications+of+adult+neurogenesis.">Mechanisms and functional implications of adult neurogenesis.</a> Cell. 132, 645-660 (2008).</li><li>Ge, S., Goh, E. L., Sailor, K. A., Kitabatake, Y., Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+regulates+synaptic+integration+of+newly+generated+neurons+in+the+adult+brain.">GABA regulates synaptic integration of newly generated neurons in the adult brain.</a> Nature. 439, 589-593 (2006).</li><li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells.">Production of replication-defective retrovirus by transient transfection of 293T cells.</a> J Vis Exp. , (2007).</li></ol>

利害の衝突は宣言されません。

哺乳類成体脳の海馬における連続的な神経新生は、成熟脳におけるニューロンの発達との統合を研究するユニークな実験モデルのシステムを提供します。ここで紹介するプロトコルは、レトロウイルス標識および成体マウスの脳における新生児歯状回顆粒ニューロンの統合を研究するために電気生理学的手法を組み合わせたもの。 あなたの実験が成功していることを確認するために、我々は、手順の重要なステップの間に次のことをお勧めします。 不要な感染症や炎症を避けるために、すべてのツールは、手術前（オートクレーブ、滅菌器の任意のタイプ、または70％エタノールによって）滅菌してください。注入前にシリンジの針の先端を洗浄する滅菌PBSを使用してください。 ウイルスの注射のために、動物はよく定位固定装置にマウントする必要があると不必要な動きの源は、注入中に最小化する必要があります。頭がよく、固定と会社であることを確認してください、との計算注入座標の前にレベルブレグマとラムダにマウスの鼻の位置を調整します。部屋の温度への曝露を最小限に抑えるために迅速にシリンジにウイルスを抽出する - レトロウイルスは、特に温度に敏感です。圧力の被害を最小限に抑えるために推奨される速度で徐々にウイルスを注入する。推奨フラット先端シリンジ（対一般的な斜めになった先端を）使用すると、均等に分散歯状回の感染症の領域を確保します。 スライスを準備する前に、ソリューションを切断し、ACSFをインキュベートは、酸素が ​​ソリューションを飽和させないために、少なくとも30分間95％O 2 -5％CO 2でバブリングされている必要があります。動物は、速やかに灌流と組織は、最高のスライス品質の寒さ、酸素飽和溶液で維持されるべきである。 シナプス後応答があるまでの記録のために、徐々に刺激強度を増加させる。必要に応じて歯状回の分子層内に刺激電極の位置を変更します。 要約すると、推奨されるアプローチは、アクティブな、成熟した回路に、初期段階の統合の間に明確な特性と成人生まれのニューロンの可能な機能的役割を探索する方法を提供しています。

<ul><li> 110塩化コリン、2.5のKCl、1.3 KH 2 PO 4、25.0炭酸水素ナトリウム、0.5 CaCl 2で 、7のMgCl 2、10デキストロース、1.3アスコルビン酸ナトリウム、0.6ピルビン酸ナトリウム、5.0キヌレン酸：解決策を（mMで）切断。 </li><li>人工脳脊髄液（ACSF、mMで）：125.0塩化ナトリウム、25.0 炭酸水素ナトリウム、2.5のKCl、2のCaCl 2、1.3 KH 2 PO 4、1.3のMgCl 2、1.3アスコルビン酸ナトリウム、0.6ピルビン酸ナトリウム、10デキストロース。 </li><li>全細胞patchclampのための内部溶液（mMで）：120カリウム、グルコン酸、15のKCl、4のMgCl 2、0.1 EGTA、10.0 HEPES、4 MgATP、0.3のNa 3 GTP、7クレアチンリン酸（pH7.4の、300 mOsm）。 </li></ul>

studying the integration of adult-born neurons

神経発生は、側脳室の副室下帯（SVZ）にし、生涯を通じて海馬歯状回のサブ粒状のゾーン（SGZ）における成体哺乳動物の脳で発生します。前回のレポートでは、成人の海馬における神経新生はてんかん、統合失調症、うつ病や不安（1）を含む多様な脳疾患、関連付けられていることが示されている。正常と異常な成人生まれのニューロンの統合のプロセスを解読すると、これらの疾患の病因を明らかにし、新しい治療法の開発を通知することができる。 SGZの成人の神経発生は、運命の仕様、マイグレーション、シナプスの統合、そして成熟の段階を含む、胚および出生後の神経発達を反映しています。しかし、完全な統合は、長期、6週間にわたって行われます。成人生まれSGZ歯状回顆粒細胞（DGCs）への最初のシナプス入力は、14日間（2）におけるグルタミン酸作動性入力に続いて、GABA作動性である。歯状回における成人生まれのニューロンの回路形成を制御する特定の要因は現在のところ不明です。 当研究室では、これらの増殖細胞に蛍光タンパク質と仮説調節遺伝子を提供するモロニーマウス白血病ウイルスを基に複製欠損レトロウイルスベクターを使用しています。このウイルスの技術は、細胞の誕生日、系統、形態、及びシナプス形成の解析のための高い特異性と分解能を提供します。 典型的な実験は、しばしばユニークなラベル、導入遺伝子、およびin vivoで 、目的の発達過程の単一細胞解析のためのプロモーター要素を含む二、三ウイルスを採用しています。次のプロトコルは単一の緑色（GFP）や赤色（dTomato）蛍光タンパク質のレトロウイルスとパッチクランプ電気生理学を用いて機能的な新生ニューロンの統合を分析するための方法を説明します。

Watch this Scientific Journal Video about Studying the Integration of Adult-born Neurons at JoVE.com

Studying the Integration of Adult-born Neurons

成熟した動物で新生児歯状回顆粒細胞の統合を研究する方法が記載されている。この手法は、生体機能の統合に決定するために電気生理学的記録に続いて新生ニューロンを、ラベルを付けるために設計されたレトロウイルスを使用しています。

アダルト生まれのニューロンの統合を検討し

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

studying-the-integration-of-adult-born-neurons

Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.8K ビュー.  State University of New York at Stony Brook. 成熟した動物で新生児歯状回顆粒細胞の統合を研究する方法が記載されている。この手法は、生体機能の統合に決定するために電気生理学的記録に続いて新生ニューロンを、ラベルを付けるために設計されたレトロウイルスを使用しています。

ビデオ: Studying the Integration of Adult-born Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

子宮内エレクトロポレーションは、皮質ニューロンのサブセットで、遺伝子機能の研究のために初代神経培養した

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

神経科学

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those leg movements that result from passive forces and interactions with other legs through the common contact with the substrate. If we want to understand, which part of a given movement is produced by nervous system motor output, kinematic analysis of stepping movements, therefore, needs to be complemented with electrophysiological recordings of motor activity. The recording of neuronal or muscular activity in a behaving animal is often limited by the electrophysiological equipment which can constrain the animal in its ability to move with as many degrees of freedom as possible. This can either be avoided by using implantable electrodes and then having the animal move on a long tether (i.e. Clarac et al., 1987; Duch &amp; Pfl&uuml;ger, 1995; B&ouml;hm et al., 1997; Gruhn &amp; Rathmayer, 2002) or by transmitting the data using telemetric devices (Kutsch et al, 1993; Fischer et al., 1996; Tsuchida et al. 2004; Hama et al., 2007; Wang et al., 2008). Both of these elegant methods, which are successfully used in larger arthropods, often prove difficult to apply in smaller walking insects which either easily get entangled in the long tether or are hindered by the weight of the telemetric device and its batteries. In addition, in all these cases, it is still impossible to distinguish between the purely neuronal basis of locomotion and the effects exerted by mechanical coupling between the walking legs through the substrate. One solution for this problem is to conduct the experiments in a tethered animal that is free to walk in place and that is locally suspended, for example over a slippery surface, which effectively removes most ground contact mechanics. This has been used to study escape responses (Camhi and Nolen, 1981; Camhi and Levy, 1988), turning (Tryba and Ritzman, 2000a,b; Gruhn et al., 2009a), backward walking (Graham and Epstein, 1985) or changes in velocity (Gruhn et al., 2009b) and it allows the experimenter easily to combine intra- and extracellular physiology with kinematic analyses (Gruhn et al., 2006).
We use a slippery surface setup to investigate the timing of leg muscles in the behaving stick insect with respect to touch-down and lift-off under different behavioral paradigms such as straight forward and curved walking in intact and reduced preparations.

We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.

昆虫に適応運動行動の神経基盤を研究し

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性のためのアダルト生まれの嗅覚ニューロンの選択的なウイルス形質導入 in vivoで Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

ヒト多能性幹細胞から終脳のグルタミン酸作動性ニューロンの仕様

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

抗精神病薬の作用のメカニズムを研究するための方法<em&gt;線虫（Caenorhabditis elegans）</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

ライブ培養神経細胞の抗体供給した後細胞表面および内在化タンパク質の異なる標識化

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

静脈瘤形成と中枢神経の軸索の回復を研究するための Microbiomechanical システム

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

ヒトニューロンのレンチウイルス媒介性形質導入を用いたタウ凝集を研究するための in Vitro モデル

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

自由に泳ぐ魚における可動テトロデスによるニューロンの無線電気生理学的記録

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...