Ce travail a été financé par un H64.09.016 VIDI numéro de subvention de l&#39;Organisation néerlandaise pour la recherche scientifique (NWO) à CPF. CPF est reconnaissante au Dr A. Silvina Fratantoni pour ses commentaires critiques et corrections sur le manuscrit final. GMRDL / Emmm sont pris en charge par la Fondation de la technologie VAP néerlandais (de 12151 de projet et 11350), qui fait partie de la NWO, et qui est en partie financé par le ministère des Affaires économiques. Nous remercions Catherine Kitts-et-Peter Drent de Nikon Instruments Europe BV pour l&#39;aide et le soutien. HX a été soutenue par l&#39;Académie royale néerlandaise des arts et des sciences (subvention 11CDP10) et WT a été pris en charge par la subvention de l&#39;Organisation néerlandaise pour la recherche scientifique (subvention 820.02.006).

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Les auteurs n&#39;ont rien à révéler.

Dans cet article, un protocole de travail à l&#39;image épines dendritiques des neurones de l&#39;hippocampe primaires de rat en culture in vitro en utilisant la carte SIM est décrit. Le procédé de culture des neurones hippocampiques primaires est une adaptation de la méthode originale décrite par Kaech Banker et 18. Les différences principales sont l&#39;utilisation d&#39;un milieu Neurobasal/B27 de culture, ce qui élimine la nécessité de cultures nourricières astrocytaires, et l&#39;add...

Une épine dendritique est une petite saillie de la membrane des neurones. Cette structure caractéristique est spécialisée recevoir généralement des données à partir d&#39;une seule synapse et représente la zone de contact physique entre deux neurones. La plupart des épines dendritiques fonctionnellement matures constitués d&#39;une pointe globulaire, appelé tête, et un cou mince qui relie la tête de l&#39;arbre dendritique. Cependant, les épines sont pas statiques et se déplacent activement et modifier leur morphologie en permanence, même dans le cerveau adulte 2. Dans un délai de 2 semaines de temps, des cultures de neurones de l&#39;hippocampe de rat primaire provenant de la fin du temps postnatale embryonnaire ou au début de développer tonnelles dendritiques complexes avec de nombreuses protubérances membranaires qui évoluent de filipodes tôt pour les structures de la colonne vertébrale comme 3. Sur la base de ce comportement dynamique et d&#39;autres caractéristiques, les épines dendritiques sont pensés pour fournir un substrat anatomique pour le stockage en mémoire et 4,5 de la transmission synaptique. Compte tenu ee rôle critique que la taille des épines dendritiques et la forme ont dans la fonction synaptique, il est important de mesurer leurs dimensions avec précision. Épines varient d&#39;environ 200 à 2000 nanomètres de longueur et peuvent être facilement identifiés par microscopie à fluorescence confocale laser à balayage. Cependant, les dimensions de la colonne vertébrale autres que la longueur sont généralement en dessous de la résolution des systèmes optiques classiques de, théoriquement fixée par diffraction autour de 200 nanomètres 6. Ces pouvoirs de résolution sont insuffisantes pour l&#39;imagerie des détails plus fins, telles que la largeur de cou de la colonne vertébrale et la tête. Beaucoup de travail a été consacrée à résoudre ce problème et de nombreux relativement nouvelles techniques de microscopie de super-résolution ont fourni d&#39;importants progrès. En particulier, il est possible d&#39;obtenir une résolution au-delà de la limite classique sans écarter toute lumière d&#39;émission en utilisant la microscopie à lumière structurée latéralement (SIM) dans un grand champ, d&#39;un microscope confocal non-10.7. En utilisant cette technique en combinaison avec des non-linear techniques de microscopie, il est théoriquement possible d&#39;améliorer la résolution latérale du microscope optique par un facteur 11 illimité. Cependant, dans la plupart des cas expérimentaux, SIM permet de dépasser la limite de résolution d&#39;un facteur de deux 1. D&#39;autres techniques de microscopie optique de super-résolution comme l&#39;épuisement d&#39;émission stimulée (STED) microscopie 12 et photo-activation localisation microscopie (PALM) 12 ont été appliquées à l&#39;imagerie des épines dendritiques. Méthodes basées sur la localisation, telles que PALM nécessitent très grand nombre d&#39;images brutes pour atteindre la super-résolution et sont donc limités à la vitesse. D&#39;autre part, STED peut atteindre une vitesse élevée d&#39;imagerie, bien au nombre de photons relativement faibles et les petits champs de vision, ce qui peut ne pas être le cas pour la carte SIM 13. Dans cet article, l&#39;objectif est de fournir un protocole de travail à l&#39;image épines dendritiques des neurones hippocampiques primaires de rat en culture in vitro </em&gt; en utilisant la carte SIM. Le protocole se compose de deux phases distinctes: une première un composé d&#39;établissement, le développement, la transfection et immunohistochimie des cultures de neurones de l&#39;hippocampe de rat primaires et une phase tardive dédié à l&#39;imagerie de l&#39;échantillon.

<table>
	<tbody>
		<tr>
			<td>Fine forceps</td>
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		<tr>
			<td>Big forceps</td>
			<td></td>
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			<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>
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			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope with illumination</td>
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			<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>
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			<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>
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			<td></td>
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		<tr>
			<td>Microcentrifuge</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>
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		<tr>
			<td>SIM Illuminator</td>
			<td></td>
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			<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>
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			<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

Épines dendritiques sont des saillies issues de la dendrite d&#39;un neurone et représentent les cibles primaires de post-synaptiques excitateurs entrées dans le cerveau. Les progrès technologiques ont permis d&#39;identifier ces structures comme des éléments clés de la connectivité neuronale et la plasticité synaptique. L&#39;analyse quantitative de la morphologie de la colonne vertébrale en utilisant la microscopie optique reste un problème essentiel en raison de limitations techniques liées à la limite de réfraction intrinsèque de la lumière. Épines dendritiques peuvent être facilement identifiés par microscopie à fluorescence confocale laser à balayage. Toutefois, la mesure des changements subtils dans la forme et la taille des épines est difficile en raison des dimensions de la colonne vertébrale autres que la longueur sont généralement plus petits que la résolution optique classique fixé par théorique limite de résolution de la microscopie optique de 200 nm. Plusieurs techniques de super résolution récemment développés ont été utilisés pour les structures cellulaires image plus petits que les 200 nm, y compris les épines dendritiques. Ttechniques es sont basés sur les activités en champ lointain classiques et permettent donc l&#39;utilisation de méthodes existantes de préparation d&#39;échantillons et à l&#39;image au-delà de la surface d&#39;un échantillon. Décrit ici est un protocole de travail pour appliquer la résolution de super structuré éclairage microscopie (SIM) pour l&#39;imagerie des épines dendritiques dans des cultures de neurones hippocampiques primaires. Les applications possibles de la carte SIM se chevauchent avec ceux de la microscopie confocale. Cependant, ces deux techniques présentent l&#39;applicabilité différente. SIM offre une résolution latérale plus efficace, tout en microscopie confocale, en raison de l&#39;utilisation d&#39;un sténopé physique, réalise l&#39;amélioration de la résolution au détriment de l&#39;enlèvement de sortir de la lumière de mise au point. Dans ce protocole, les neurones primaires sont cultivées sur des lamelles de verre en utilisant un protocole standard, transfectées avec des plasmides d&#39;ADN codant pour des protéines fluorescentes et imagée en utilisant la carte SIM. Le tout protocole décrit ici prend environ 2 semaines, parce que les épines dendritiques sont imagés après 16-17 jours in vitro, quanddéveloppement dendritique est optimal. Après l&#39;achèvement du protocole, épines dendritiques peuvent être reconstruites en 3D de la série de la carte SIM piles d&#39;images à l&#39;aide de logiciels spécialisés.

Décrit ici est un protocole de travail normalisé pour l&#39;imagerie épines dendritiques des neurones de l&#39;hippocampe primaires de rat in vitro utilisant la carte SIM. Le flux du protocole et de ses étapes cruciales sont présentés dans la figure 1. Globalement, le protocole prend environ 2 semaines de travaux expérimentaux séparés dans une première phase de préparation de l&#39;échantillon, y compris la culture, le développement et la transfection de neurones hippocampiques pri...

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

Cet article décrit un protocole de travail à l&#39;image épines dendritiques de neurones de l&#39;hippocampe in vitro utilisant structuré Illumination Microscopy (SIM). Microscopie de super-résolution utilisant la carte SIM offre une résolution d&#39;image de façon significative au-delà de la limite de diffraction de la lumière dans les trois dimensions de l&#39;espace, ce qui permet l&#39;imagerie des épines dendritiques individuelles avec l&#39;amélioration de détail.

Imagerie épines dendritiques des neurones de l&#39;hippocampe de rat primaire utilisant structuré Illumination Microscopy

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

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Neuroscience

15.7K vues.  University of Amsterdam.  Cet article décrit un protocole de travail à l'image épines dendritiques de neurones de l'hippocampe in vitro utilisant structuré Illumination Microscopy (SIM). Microscopie de super-résolution utilisant la carte SIM offre une résolution d'image de façon significative au-delà de la limite de diffraction de la lumière dans les trois dimensions de l'espace, ce qui permet l'imagerie des épines dendritiques individuelles avec l'amél...

Vidéo: 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.

Nucléofection et de la culture primaire de neurones embryonnaires de souris hippocampe et cortex

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

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Neurosciences

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.

Récupération de fluorescence après photoblanchiment (FRAP) de fluorescence protéines marquées dans les épines dendritiques des neurones de l&#39;hippocampe de culture

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.

Enregistrement colorant sensible à la tension d&#39;axones, des dendrites et des épines dendritiques des neurones individuels dans des tranches de cerveau

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.

Phosphate de calcium transfection de primaire neurones de l&#39;hippocampe

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.

Deux-Photon<em&gt; In vivo</em&gt; Imagerie des épines dendritiques dans le cortex de souris utilisant un crâne-Diluée Préparation

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.

Images en direct de la protéine GLUT4 traite des neurones hypothalamiques primaire de souris au microscope de déconvolution

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.

Génération de neurosphères à partir de neurones hippocampes et corticales primaires mixtes isolées de l'embryon de rat Sprague Dawley E14-E16

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.

Modélisation 3D des épines dendritiques avec plasticité synaptique

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.

Imagerie des épines dendritiques chez Caenorhabditis elegans

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.

Microscopie électronique à balayage en bloc en série (SBEM) pour l’étude des épines dendritiques

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