この作品は、CPFに科学研究費オランダ機構（NWO）からビディ承認番号H64.09.016によって賄われた。 CPFは、最終原稿の彼女の批判的コメントと修正のための博士SilvinaさんA. Fratantoniに感謝です。 GMRDL / EMMM NWOの一部であり、オランダの技術振興財団STW（プロジェクト12151および11350）によってサポートされており、その一部は経済省によって資金を供給されています。私たちは、援助と支援のため、ニコンインスツルメンツヨーロッパBVのキャサリンクリストファー·ピーターDrentに感謝します。 HX助成科学研究費オランダ機構（助成820.02.006）によってサポートされていましたロイヤル·ダッチ·芸術科学アカデミー（助成11CDP10）とWTによってサポートされていました。

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著者らは、開示することは何もありません。

この記事では、SIMを使用して、in vitroで培養したラット初代海馬ニューロンからの画像樹状突起棘の作業プロトコルが記述されている。初代海馬神経細胞培養法は、Kaechとバンカー18で説明した独自の方法を最適化したものです。主な違いは、をNeurobasal/B27培養アストログリア細胞フィーダー培養の必要性を排除する媒体、およびグリア細胞の増殖を抑制しつつ、ブリューワー<em...

樹状突起棘は、ニューロン膜の小さな突起である。この特徴的な構造は、典型的には、単一のシナプスからの入力を受信するように特化され、2つのニューロン間の物理的接触面積を表している。最も機能的に成熟した樹状突起棘は、球状チップで構成されて頭と呼ばれ、樹状シャフトにヘッドを接続している細い首。しかし、棘は静的ではなく、積極的に移動しても、成人の脳2で連続的にその形態を変更してください。時間の2週間の期間内に、後半胚または出生後早期の時間に由来したラット初代海馬ニューロン培養は、背骨状の構造3への早期糸状仮足から進化し、多数の膜突起を持つ複雑な樹状アーバーを開発しています。この動的挙動および他の特性に基づいて、樹状突起棘は、メモリ記憶およびシナプス伝達のために、4,5の解剖学的基質を提供すると考えられている。 目の与えられた樹状突起棘の大きさと形状がシナプス機能で、それは正確に寸法を測定することが重要ですしていることをE重要な役割。棘は、長さが約200〜2000ナノメートルまで変化と容易に共焦点レーザー走査型蛍光顕微鏡により同定することができる。しかし、長さ以外の脊椎の寸法は、理論的には200ナノメートル6周りの回折によって固定され、通常は従来の光学システム「解像度を下回っている。これらの解決の力は、脊椎のネックとヘッドの幅など細かい詳細を画像化するには不十分である。多くの研究は、この問題を解決するために専用されており、多くの比較的新しい超解像顕微鏡技術は、実質的な進歩を提供した。特に、広視野、非共焦点顕微鏡7-10に横方向に構造化照明顕微鏡（SIM）を使用して任意の発光光を廃棄することなく、古典的な限界を超えた分解能を達成することができる。非リネアと組み合わせてこの技術を用いてr個の顕微鏡技術は、無制限の因子11によって光学顕微鏡の方位分解能を向上させることが理論的に可能である。しかし、ほとんどの実験状況で、SIMは2 1倍に解像限界を超えることができます。このような誘導放出の枯渇（STED）顕微鏡12と光活性化局在化顕微鏡法（PALM）12のような他の超解像光学顕微鏡技術は、樹状突起棘の撮像に適用されている。例えば、PALM等の局在に基づく方法は、超解像を達成するために、原画像の非常に多数を必要とするので、速度が制限される。一方、STEDは、高い画像形成速度を達成することができ、比較的低い光子カウントとSIM 13には当てはまらないかもしれない視野の小さな分野、ではあるが。 この記事では目的は、 試験管内で培養したラット初代海馬ニューロンからの画像樹状突起棘に努めプロトコルを提供するものである</EM&gt; SIMを使用。ラット初代海馬ニューロン培養の確立、開発、トランスフェクションおよび免疫組織化学からなる最初の1とイメージングをサンプリングするために専用の後期相：プロトコルは、2つの区別可能な段階で構成されています。

<table>
	<tbody>
		<tr>
			<td>Fine forceps</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Big forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Big scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope with illumination</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow cell culture hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-temperature dry-oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator (5% CO2, 37 &#176;C)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
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		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Orbital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon structured illumination microscope setup consisting of:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti research inverted microscope with Perfect Focus System</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon CFI Apo TIRF 100x oil objective lens (N.A. 1.49)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 Coherent Sapphire Lasers (458, 488, 514 and 561 nm exitation wavelength)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SIM Illuminator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Stage Controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MCL Nano-Drive piezo controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Intensilight C-HGFIE mercury lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SIM Microscope Enclosure temperature control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Andor EM-CCD Camera iXon DU897</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PC with Microsoft Windows 7 Home Edition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon&#8217;s NiS Elements 6.14 SIM software package</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon type A immersion oil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy

樹状突起棘は、ニューロンの樹状突起から出た突起であり、脳内の興奮性の入力の主要なシナプス後目標を表すものです。技術の進歩は、ニューロンの接続性とシナプス可塑性において重要な要素として、これらの構造を同定した。光学顕微鏡を用いてスパイン形態の定量分析は、光の固有屈折限界に関連する技術的制限による本質的な問題のまま。樹状突起棘は、容易に共焦点レーザー走査型蛍光顕微鏡により同定することができる。長さ以外の脊椎寸法は、通常、200nmの光学顕微鏡法の理論的な解像限界により固定され、従来の光学分解能よりも小さいので、棘の形状及び大きさの微妙な変化を測定することは困難である。 いくつかの最近開発された超解像技術は、樹状突起棘を含む200nmよりも小さい画像の細胞構造に使用されてきた。 Tヘセ技術は、古典的なファーフィールドの動作に基づいており、したがって、既存の試料調製方法の使用を可能にし、試料の表面を越えて画像にしている。ここで説明するには、初代海馬ニューロン培養中の樹状突起棘の画像に超解像構造化照明顕微鏡（SIM）を適用するための作業プロトコルです。 SIMの可能なアプリケーションでは、共焦点顕微鏡のものと重複する。しかし、2つの技術が異なる適用可能性を提示する。共焦点顕微鏡は、物理的なピンホールの使用にフォーカス光のうちの除去を犠牲にして高解像度化を実現しながら、SIMは、より高い実効横方向の解像度を提供しています。このプロトコルでは、一次ニューロンは、DNAプラスミドは、蛍光タンパク質をコードし、SIMを使用して撮像をトランスフェクトした標準プロトコルを使用して、ガラスカバースリップ上で培養される。樹状突起棘の際に、in vitroで 16〜17日後に画像化されているため、ここに記載さ全体プロトコルは、約2週間かかります樹状開発が最適である。プロトコルの完了後に、樹状突起棘は、特殊なソフトウェアを使用してSIM画像スタックの一連の3Dで再構成することができる。

ここで説明するには、SIMを使用して、in vitroでラット初代海馬ニューロンからの樹状突起棘を画像化するための標準化された作業用のプロトコルです。プロトコルのワークフローとその重要なステップは、 図1に示されている。全体的に、プロトコルは、培養、開発およびラット初代海馬ニューロン及び免疫組織化学のトランスフェクションを含む、試料調製の第一段階で?...

Watch this Scientific Journal Video about Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy at JoVE.com

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

この記事では、構造化照明顕微鏡（SIM）を使用してインビトロで海馬ニューロンからイメージ樹状突起棘にワーキングプロトコルについて説明します。 SIMを使用して超解像顕微鏡法は、改善されたディテール個体樹状突起棘の画像化を可能にする、有意にすべての3つの空間次元における光の回折限界を超えて画像解像度を提供する。

構造化照明顕微鏡を用いてラット初代海馬ニューロンの樹状突起棘を画像化

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

imaging-dendritic-spines-rat-primary-hippocampal-neurons-using

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Neuroscience

15.7K ビュー.  University of Amsterdam. この記事では、構造化照明顕微鏡（SIM）を使用してインビトロで海馬ニューロンからイメージ樹状突起棘にワーキングプロトコルについて説明します。 SIMを使用して超解像顕微鏡法は、改善されたディテール個体樹状突起棘の画像化を可能にする、有意にすべての3つの空間次元における光の回折限界を超えて画像解像度を提供する。 

ビデオ: Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofectionとマウス胎児海馬および皮質ニューロンの初代培養

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

神経科学

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

培養海馬ニューロンの樹状突起棘の蛍光タグ融合タンパク質の光退色後蛍光回復（FRAP）

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

軸索、樹状突起および脳スライスにおける個々のニューロンの樹状突起棘から電位感受性色素記録

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

初代海馬ニューロンのリン酸カルシウムトランスフェクション

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

二光子<em&gt; in vivoで</em&gt;間引き頭蓋骨の準備を使用して、マウス皮質における樹状突起棘のイメージング

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

GLUT4 タンパク質デコンボリューション顕微鏡を用いたマウス一次視床下部ニューロンにおける人身売買のライブ映像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

E14-E16スプラーグドーリーラット胚から分離された混合原発海馬および皮質ニューロンからの神経球の生成

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

シナプス可塑性を用いた樹状脊椎の3Dモデリング

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

カエノラブディティス・エレガンスにおける樹状突起棘のイメージング

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

樹状脊椎研究のためのシリアルブロック顔走査電子顕微鏡(SBEM)

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...