ベンA.バレス（スタンフォード大学）の研究室で：涙点アナライザプラグインは、イメージのためにJはバリーウォーク（Physionコンサルティング現在のアドレス）によって書かれました。 資金調達; <ul><li>アルフレッドP.スローン財団</li><li>エスターA.とジョセフKlingensteinファンド投資法人</li><li>広範な生物医学研究財団</li><li>ハンチントン病のイニシアティブのために治す</li></ul>

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	<li>Barres, B. A., Silverstein, B. E., Corey, D. P., Chun, L. L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological,+morphological,+and+electrophysiological+variation+among+retinal+ganglion+cells+purified+by+panning.">Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.</a> Neuron. 1, 791-803 (1988).</li><li>Meyer-Franke, A., Kaplan, M. R., Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+signaling+interactions+that+promote+the+survival+and+growth+of+developing+retinal+ganglion+cells+in+culture.">Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture.</a> Neuron. 15, 805-819 (1995).</li><li>Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gabapentin+receptor+alpha2delta-1+is+a+neuronal+thrombospondin+receptor+responsible+for+excitatory+CNS+synaptogenesis.">Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.</a> Cell. 139, 380-392 (2009).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 .</li><li>Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+synapse+number+by+glia.">Control of synapse number by glia.</a> Science. 291, 657-661 (2001).</li><li>Christopherson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondins+are+astrocyte-secreted+proteins+that+promote+CNS+synaptogenesis.">Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.</a> Cell. 120, 421-433 (2005).</li><li>Godement, P., Salaun, J., Imbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+and+postnatal+development+of+retinocollicular+projections+in+the+mouse.">Prenatal and postnatal development of retinocollicular projections in the mouse.</a> J. Comp. Neurol. 230, 552-575 (1985).</li><li>Schmidt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+retinotopic+connections:+selective+stabilization+by+an+activity-dependent+mechanism.">Formation of retinotopic connections: selective stabilization by an activity-dependent mechanism.</a> Cell. Mol. Neurobiol. 5, 65-84 (1985).</li><li>Sachs, G. M., Jacobson, M., Caviness, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+changes+in+arborization+patterns+of+murine+retinocollicular+axons.">Postnatal changes in arborization patterns of murine retinocollicular axons.</a> J. Comp. Neurol. 246, 395-408 (1986).</li><li>Elmariah, S. B., Oh, E. J., Hughes, E. G., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+regulate+inhibitory+synapse+formation+via+Trk-mediated+modulation+of+postsynaptic+GABAA+receptors.">Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors.</a> J. Neurosci. 25, 3638-3650 (2005).</li><li>Hughes, E. G., Elmariah, S. B., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+secreted+proteins+selectively+increase+hippocampal+GABAergic+length,+branching+and+synaptogenesis.">Astrocyte secreted proteins selectively increase hippocampal GABAergic length, branching and synaptogenesis.</a> Moll. Cell. Neurosci. 43, 136-145 (2010).</li></ol>

利害の衝突は宣言されません。

上記のシナプスのアッセイは、我々の実験の目標、ここで我々は文化を精製や脳のセクションのいずれかで、大きくRGCsの興奮性の予測に焦点を当てるのコンテキストに基づいています。私たちは、興奮性シナプスを（表1）のラベルにうまく参照テーブルのリストの抗体を提供している。 このシナプスのアッセイは、任意の神経集団または選択的なプリと後シナプスマー...

<table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 抗原 </td><td> 種 </td><td> ポリクローナル/モノクローナル </td><td> ベンダ </td><td> カタログ番号 </td><td> 希釈 </td><td> 文化の作品 </td><td> セクションの作品 </td></tr><tr><td rowspan="10">シナプス前の</td><td>シナプシン</td><td>ウサギ</td><td>ポリクローナル</td><td>シナプスシステム</td><td> 106004 </td><td> 1:750 </td><td> Y </td><td> ND </td></tr><tr><td>シナプシン</td><td>マウス</td><td>モノクローナル</td><td>シナプスシステム</td><td> 106001 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td>ファゴット</td><td>マウス</td><td>モノクローナル</td><td>アッセイデザイン</td><td> VAM - PS003F </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td>ファゴット</td><td>モルモット</td><td>ポリクローナル</td><td>シナプスシステム</td><td> 141004 </td><td> 1:1000 </td><td> Y </td><td> ND </td></tr><tr><td>シナプトタグミン1 </td><td>ウサギ</td><td>ポリクローナル</td><td>シナプスシステム</td><td> 105002 </td><td> 1:750 </td><td> Y </td><td> N </td></tr><tr><td>シナプトブレビン2 （C1.69.1） </td><td>マウス</td><td>モノクローナル</td><td>シナプスシステム</td><td> 104211 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td>シナプトフィジン （C1.7.2） </td><td>マウス</td><td>モノクローナル</td><td>シナプスシステム</td><td> 101011 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> VGlut1 </td><td>マウス</td><td>モノクローナル</td><td>ミリポア</td><td> MAB5502 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td> VGlut1 </td><td>モルモット</td><td>ポリクローナル</td><td>ミリポア</td><td> AB5905 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td> VGlut2 </td><td>モルモット</td><td>ポリクローナル</td><td>ミリポア</td><td> AB2251 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td rowspan="6">シナプス後の</td><td> PSD - 95 （6G6 - 1C9クローン） </td><td>マウス</td><td>モノクローナル</td><td>親和性バイオ試薬</td><td> MA1 - 045 </td><td> 1:750 </td><td> Y </td><td> N </td></tr><tr><td> PSD - 95 </td><td>ウサギ</td><td>ポリクローナル</td><td> ZYMED </td><td> 51から6900 </td><td> 1:500 </td><td> N </td><td> Y </td></tr><tr><td>ホーマー</td><td>マウス</td><td>モノクローナル</td><td>シナプスシステム</td><td> 160011 </td><td> 1:500 </td><td> Y </td><td> ND </td></tr><tr><td>ホーマー</td><td>ラット</td><td>ポリクローナル</td><td>ミリポア</td><td> AB5875 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Gephyrin </td><td>ウサギ</td><td>ポリクローナル</td><td>シナプスシステム</td><td> 147003 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Gephyrin </td><td>マウス</td><td>モノクローナル</td><td>シナプスシステム</td><td> 147 </td><td> 1:200 </td><td> Y </td><td> Y </td></tr></tbody></table> 表1：我々は成功私たちのシナプスのアッセイで使用されていることを良いプリとシナプス後のマーカーのリストの例。これはすべての利用可能なマーカーの網羅的なリストではないことに注意してください。 Y =はい、N =いいえ、ND =データなし。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> ネコ。いいえ </td></tr><tr><td> PBS </td><td>インビトロジェン</td><td> 20012-027 </td></tr><tr><td>ポリ- D -リジン</td><td>シグマ</td><td> P6407 </td></tr><tr><td>ラミニン</td><td> Cultrex </td><td> 3400-010-01 </td></tr><tr><td>トリトンX - 100 </td><td>ロシュダイアグノスティック徳国</td><td> 9002-93-1 </td></tr><tr><td>正常ヤギ血清</td><td>ギブコ</td><td> 16210 </td></tr><tr><td> DAPIとVectaShield </td><td>ベクターラボラトリーズ</td><td> H - 1200 </td></tr><tr><td> OCT </td><td>組織 - Tek社</td><td> 4583 </td></tr><tr><td>トリス-ベース（50 mM）を</td><td>フィッシャー</td><td> BP152 - 5 </td></tr><tr><td>ウシ血清アルブミン</td><td>シグマ</td><td> A2153 </td></tr><tr><td> L -リジン</td><td>シグマ</td><td> L - 1137 </td></tr><tr><td> 16パーセントPFAのソリューション</td><td>電子顕微鏡学</td><td> 15711 </td></tr><tr><td>粒状のPFA </td><td>電子顕微鏡学</td><td> 19210 </td></tr><tr><td> 24ウェル培養プレート</td><td>ファルコン</td><td> 35から3047 </td></tr><tr><td>ヤギ抗マウスアレクサ標識抗体</td><td>インビトロジェン</td><td> --- </td></tr><tr><td> サプライ </td><td> 会社 </td><td> ネコ。いいえ </td></tr><tr><td> 12mmの0号ガラスのカバースリップ</td><td>カールヘクト徳国</td><td> 1105209 </td></tr><tr><td>第1.5ガラスカバースリップ（スライス用） </td><td> VWRサイエンティフィック</td><td> 48393241 </td></tr><tr><td>スライドガラス</td><td> VWRサイエンティフィック</td><td> 48311-703 </td></tr></tbody></table>

quantifying synapses: an immunocytochemistry-based assay to quantify synapse number

神経科学における最も重要な目標の1つは、シナプス形成の初期段階を指示する分子の合図を理解することです。このように、それはシナプス接続の変化を定量化するための客観的なアプローチを開発するために必要不可欠になっています。サンプルの固定から始まる、このプロトコルの詳細については、どのように解離神経細胞培養中および免疫細胞化学を用いて脳切片の両方シナプス数を定量化する。コンパートメント特異的抗体を用いて、我々は、シナプス前終末だけでなく、シナプス後部の専門のサイトにラベルを付けます。我々は、これらのマーカーによって生成された信号間の共局在のポイントとしてシナプスを定義します。これらcolocalizationsの数は、ImageJの解析ソフトウェアのプラットフォームの下涙点アナライザ（リクエストも承りますバリーウォーク、、c.eroglu @ cellbio.duke.eduによって書かれた）プラグインを使用して定量される。このプロトコルで説明されているシナプスのアッセイは、選択前と後シナプスマーカーを持っている任意の神経組織や文化を準備するために適用することができます。このシナプスのアッセイは、広くシナプス開発の研究に利用することができる貴重なツールです。

Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

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

このプロトコルは、解離神経培養および免疫細胞化学を用いて脳切片の両方シナプス数を定量化する方法を詳しく説明します。コンパートメント特異的抗体を用いて、我々は、シナプス前終末だけでなく、シナプス後部の専門のサイトにラベルを付けます。我々は、これらのマーカーによって生成された信号間の共局在のポイントとしてシナプスを定義します。

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 ...

quantifying-synapses-an-immunocytochemistry-based-assay-to-quantify

Research

JoVE Journal

Neuroscience

35.5K ビュー.  Duke University. このプロトコルは、解離神経培養および免疫細胞化学を用いて脳切片の両方シナプス数を定量化する方法を詳しく説明します。コンパートメント特異的抗体を用いて、我々は、シナプス前終末だけでなく、シナプス後部の専門のサイトにラベルを付けます。我々は、これらのマーカーによって生成された信号間の共局在のポイントとしてシナプスを定義?...

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

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神経コロニー形成細胞アッセイ：神経前駆細胞から善意の神経幹細胞を区別するアッセイ

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

神経科学

Methods to Assay Drosophila Behavior

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

Methods to Assay Drosophila Behavior

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

アッセイのメソッドショウジョウバエ行動

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

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

貪食アッセイ：CNS食細胞と神経細胞間の相互作用を評価するためのプロトコル

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

の記録細胞内の電圧応答のための電気生理学的手法<em&gt;ショウジョウバエ</em&gt;光刺激に感光体と介在ニューロン<em&gt;インビボ</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

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 ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海馬の齧歯動物の脳スライスにおけるシナプス密度の評価

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Sod1 突然変異体蛋白質集計における細胞の高いコンテンツ定量化のための分析法の開発

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

視床皮質軸索分岐とシナプスの可視化脊髄共培養系における形成

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

麻酔下のマウスから脳脊髄液のコレクションのための改良法

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Purification of the Dendritic Filopodia-rich Fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

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Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

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

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