Este trabalho foi financiado por uma H64.09.016 VIDI número de concessão da Organização Holandesa para Pesquisa Científica (NWO) para CPF. CPF é grato ao Dr. Silvina A. Fratantoni por seus comentários críticos e correções sobre o manuscrito final. GMRDL / Emmm são suportados pelo holandês Tecnologia Fundação STW (12151 projeto e 11350), que faz parte da NWO, e que é parcialmente financiado pelo Ministério dos Assuntos Económicos. Agradecemos a Catherine Cristóvão e Peter Drent da Nikon Instruments Europe BV para a assistência e apoio. HX foi apoiado pela Royal Dutch Academia de Artes e Ciências (concessão 11CDP10) e WT foi apoiado pela concessão da Organização Holandesa para Pesquisa Científica (concessão 820.02.006).

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Neste artigo um protocolo de trabalho para imagem espinhas dendríticas de neurônios do hipocampo de ratos primários cultivados in vitro utilizando SIM é descrito. O método de cultura de neurônios do hipocampo principal é uma adaptação do método original descrito por Kaech e Banker 18. As principais diferenças são a utilização de meio de cultura Neurobasal/B27, o que elimina a necessidade de culturas astrogliais alimentadoras, e a adição do inibidor mitótico FUDR no dia 3, que promove...

A espinha dendrítica é uma pequena saliência da membrana neuronal. Esta estrutura característica é especializada para receber normalmente a entrada de uma única sinapse e representa a área de contato físico entre os dois neurônios. A maioria das espinhas dendríticas funcionalmente maduros consistem em uma ponta globular, denominado cabeça e um pescoço fino que liga a cabeça ao eixo dendrítica. No entanto, as espinhas não são estáticos e mover e alterar a sua morfologia ainda continuamente no cérebro adulto 2 activamente. Dentro de um período de 2 semanas de tempo, ratos culturas de neurônios do hipocampo primários derivados de tempo pós-natal embrionária ou início de tarde desenvolver árvores dendríticas complexas com inúmeras saliências membrana que evoluem a partir de filipodia cedo para estruturas da coluna-como 3. Com base nesse comportamento dinâmico e outras características, espinhas dendríticas são pensados ​​para proporcionar um substrato anatômico para o armazenamento de memória e 4,5 transmissão sináptica. Dada ªe papel crítico que o tamanho da espinha dendrítica e forma têm em função sináptica, é importante para medir com precisão as suas dimensões. Espinhas variam de cerca de 200 a 2000 nanómetros de comprimento e pode ser facilmente identificado por microscopia de fluorescência confocal de varrimento laser. No entanto, outras dimensões do que o comprimento da coluna vertebral são geralmente abaixo de resolução dos sistemas ópticos convencionais, teoricamente fixo por difração de cerca de 200 nanômetros de 6. Esses poderes são insuficientes para resolver imaginando pequenos detalhes, como a largura da coluna pescoços e cabeças. Muito trabalho foi dedicado a resolver este problema e muitos relativamente novas técnicas de microscopia de super-resolução proporcionaram um progresso substancial. Em particular, é possível obter uma resolução para além do limite clássico sem descartar qualquer emissão de luz por meio de microscopia de iluminação estruturada lateralmente (SIM), numa vasta área, microscópio confocal não 7-10. Usando esta técnica, em combinação com os não-lineatécnicas de microscopia de r, que é teoricamente possível a melhorar a resolução lateral do microscópio óptico de um factor 11 ilimitado. No entanto, na maioria das circunstâncias experimentais, de SIM permite ultrapassar o limite de resolução por um factor de dois 1. Outros super-resolução técnicas de microscopia óptica, tais como esgotamento emissão estimulada (STED) microscopia 12 e microscopia localização foto-ativação (PALM) 12 foram aplicadas à imagem das espinhas dendríticas. Métodos baseados em localização, como PALM requerem um grande número de imagens raw para atingir super-resolução e, portanto, são limitados em velocidade. Por outro lado, STED pode alcançar alta velocidade de imagem, embora a contagem relativamente baixos de fótons e pequenos campos de visão, que podem não ser o caso do SIM 13. Neste artigo, o objetivo é fornecer um protocolo de trabalho para imagem espinhas dendríticas de neurônios do hipocampo de ratos primários cultivados in vitro </em&gt; usando SIM. O protocolo consiste em duas fases distintas: inicial consistindo de estabelecimento, desenvolvimento, transfecção e imunohistoquímica de ratos principais culturas de neurônios do hipocampo e uma fase tardia dedicado a provar imagem.

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

As espinhas dendríticas são saliências emergentes da dendrite de um neurónio e representam os alvos primários pós-sinápticos de entradas excitatórios no cérebro. Os avanços tecnológicos têm identificado estas estruturas como elementos-chave na conectividade neuronal e plasticidade sináptica. A análise quantitativa da morfologia da coluna usando microscopia de luz continua a ser um problema essencial, devido a limitações técnicas associadas limite intrínseco da refração da luz. Espinhas dendriticas podem ser prontamente identificados por microscopia de fluorescência confocal de varrimento laser. No entanto, medindo mudanças sutis na forma e tamanho de espinhos é difícil, porque outros do que o comprimento da coluna dimensões são geralmente menores do que a resolução óptica convencional fixada por limite de resolução teórico de microscopia de luz de 200 nm. Várias técnicas de resolução de super recentemente desenvolvidos têm sido utilizados para imagem estruturas celulares menores do que os 200 nm, incluindo espinhas dendriticas. Tstas técnicas baseiam-se em operações de campo distante clássicos e, por conseguinte, permitir a utilização de métodos de preparação de amostras existentes e para além da imagem da superfície de uma amostra. Descrito aqui é um protocolo de trabalho para aplicar resolução estruturada microscopia de iluminação super (SIM) para a imagem das espinhas dendríticas em culturas de neurônios do hipocampo primários. Possíveis aplicações de SIM coincidir com os de microscopia confocal. No entanto, as duas técnicas apresentam aplicabilidade diferente. SIM oferece resolução lateral superior eficaz, ao passo que a microscopia confocal, devido ao uso de um orifício físico, atinge melhoria resolução à custa da remoção de luz fora de foco. Neste protocolo, neurónios primários são cultivadas em lamelas de vidro, utilizando um protocolo padrão, transfectados com os plasmídeos de DNA que codificam as proteínas fluorescentes e fotografadas usando SIM. Todo o protocolo aqui descrito leva aproximadamente duas semanas, porque espinhas dendríticas são visualizados após 16-17 dias in vitro, quandodesenvolvimento dendrítica é o ideal. Após a conclusão do protocolo, espinhas dendríticas pode ser reconstruído em 3D da série de pilhas de imagens do SIM usando software especializado.

Descrito aqui é um protocolo de trabalho padronizado para imagens espinhas dendríticas de neurônios do hipocampo de ratos primários in vitro utilizando SIM. O fluxo de trabalho de protocolo e os seus passos cruciais são mostrados na Figura 1. Geral, o protocolo de demora cerca de 2 semanas de trabalho experimental separadas numa primeira fase de preparação da amostra, incluindo a cultura, o desenvolvimento e a transfecção de neurónios do hipocampo de rato primários e imuno-histoquím...

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

Este artigo descreve um protocolo de trabalho de imagem espinhas dendríticas dos neurônios do hipocampo in vitro utilizando Structured Illumination Microscopy (SIM). Microscopia Super-resolução usando SIM oferece resolução de imagem significativamente além do limite de difração da luz em todas as três dimensões espaciais, permitindo que a imagem de espinhas dendríticas individuais com maior pormenor.

Imagiologia espinhas dendríticas dos neurônios do hipocampo de rato primária usando Structured 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. ...

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

Research

JoVE Journal

Neuroscience

15.7K visualizações.  University of Amsterdam.  Este artigo descreve um protocolo de trabalho de imagem espinhas dendríticas dos neurônios do hipocampo in vitro utilizando Structured Illumination Microscopy (SIM). Microscopia Super-resolução usando SIM oferece resolução de imagem significativamente além do limite de difração da luz em todas as três dimensões espaciais, permitindo que a imagem de espinhas dendríticas individuais com maior pormenor. 

Vídeo: 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 e Cultura Primária de Neurônios do hipocampo do rato embrionárias e Cortical

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

Neurociência

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.

Recuperação de fluorescência Após Fotodegradação (FRAP) de fluorescência Proteínas Tagged em espinhas dendríticas dos neurônios do hipocampo Cultivadas

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.

Tensão sensível Gravação Dye de axônios, dendritos e espinhas dendríticas de neurônios individuais em fatias de cérebro

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.

Fosfato de cálcio transfecção de primário neurônios do hipocampo

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.

Two-Photon<em&gt; In vivo</em&gt; Imagem de espinhas dendríticas no Rato Cortex Usando um desbaste-crânio Preparação

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.

Imagens ao vivo da proteína GLUT4 tráfico de Mouse de neurônios hipotálamo primário usando microscopia de deconvolução

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.

Geração de neuroesferas do hipocampal preliminar misturado e dos neurônios corticais isolados de E14-E16 embrião do rato de Sprague Dawley

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.

Modelagem 3D de Espinhos Dendríticas com Plasticidade Sináptica

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.

Imagem de espinhas dendríticas em 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.

Microscopia eletrônica de varredura de blocos de bloco serial (SBEM) para o estudo de espinhas dendríticas

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