Name: measuring near plasma membrane and global intracellular calcium dynamics in astrocytes
Uploaded: 2009-04-26T23:27:00
Description: Watch this Scientific Journal Video about Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes at JoVE.com

这项工作是支持日本的上原纪念基金会（ES）以及白厅基金会，国立神经疾病与中风和斯坦 - 奥本海默基金会奖（BSK）。

实验步骤实验过程由两个关键部件，是明智的方式在一个步骤如下所述。 第1部分：准备海马星形胶质细胞的培养简单地说，混合海马星形胶质细胞神经元的文化准备使用一个完善的协议1,2,3。我们优化的过程，产生健康的培养的星形胶质细胞。下面列出的所有程序应在无菌层流罩，如环境进行。 准备...

<ol>
	<li>Richler, E., Chaumont, S., Shigetomi, E., Sagasti, A., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+image+activation+of+transmitter-gated+P2X+receptors+in+vitro+and+in+vivo.">An approach to image activation of transmitter-gated P2X receptors in vitro and in vivo.</a> Nature Methods. 5, 87-93 (2008).</li><li>Granseth, B., Odermatt, B., Royle, S. J., Lagnado, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-mediated+endocytosis+is+the+dominant+mechanism+of+vesicle+retrieval+at+hippocampal+synapses.">Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.</a> Neuron. 51, 773-786 (2006).</li><li>Nunez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+hippocampal+neurons+from+P0+newborn+rats.">Primary culture of hippocampal neurons from P0 newborn rats.</a> J Vis Exp. 19, (2008).</li><li>Steyer, J. A., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+view+of+life+within+100+nm+of+the+plasma+membrane.">A real-time view of life within 100 nm of the plasma membrane.</a> Nat Rev Mol Cell Biol. 2, 268-275 (2001).</li><li>Jaiswal, J. K., Fix, M., Takano, T., Nedergaard, M., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+vesicle+fusion+from+lysis+to+monitor+calcium-triggered+lysosomal+exocytosis+in+astrocytes.">Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes.</a> Proc Natl Acad Sci U S A. 104, 14151-14156 (2007).</li><li>Araque, A., Carmignoto, G., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+signalling+between+astrocytes+and+neurons.">Dynamic signalling between astrocytes and neurons.</a> Annu Rev Physiol. 63, 795-813 (2001).</li><li>Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLIA:+listening+and+talking+to+the+synapse.">GLIA: listening and talking to the synapse.</a> Nat Rev Neurosci. 2, 185-193 (2001).</li><li>Bowser, D. N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+excites+interneurons+and+astrocytes+to+increase+synaptic+inhibition+in+neuronal+networks.">ATP excites interneurons and astrocytes to increase synaptic inhibition in neuronal networks.</a> J Neurosci. 24, 8606-8620 (2004).</li><li>Bowser, D. N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+forms+of+astrocyte+single+vesicle+exocytosis+imaged+with+total+internal+reflection+fluorescence+microscopy.">Two forms of astrocyte single vesicle exocytosis imaged with total internal reflection fluorescence microscopy.</a> Proc Natl Acad Sci U S A. 104, 4212-4217 (2007).</li><li>Bowser, D. N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+ATP+is+the+predominant+cause+of+intercellular+calcium+waves+in+astrocytes.">Vesicular ATP is the predominant cause of intercellular calcium waves in astrocytes.</a> J Gen Physiol. 129, 485-491 (2007).</li><li>Agulhon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+role+of+astrocyte+calcium+in+neurophysiology.">What is the role of astrocyte calcium in neurophysiology.</a> Neuron. 59, 932-946 (2008).</li><li>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=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60, 430-440 (2008).</li><li>Lee, S. Y., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+glutamate+targets+NMDA+receptors.">Astrocytic glutamate targets NMDA receptors.</a> J Physiol. 581, 887-888 (2007).</li><li>Shigetomi, E., Bowser, D. N., Sofroniew, M. V., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+forms+of+astrocyte+calcium+excitability+have+distinct+effects+on+NMDA+receptor-mediated+slow+inward+currents+in+pyramidal+neurons.">Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons.</a> J Neurosci. 28, 6659-6663 (2008).</li><li>Cahoy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptome+database+for+astrocytes,+neurons,+and+oligodendrocytes:+a+new+resource+for+understanding+brain+development+and+function.">A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function.</a> J Neurosci. 28, 264-278 (2008).</li></ol>

它是行之有效的，星形胶质细胞显示细胞内钙升高。这些自发地发生，可引发神经元的活动，或通过受体激动剂的应用程序激活星形胶质细胞表面的受体11。其中一个重要的和有争议的问题是是否星形胶质细胞内钙升高可触发释放信号分子，激活受体对神经元的11，12。因为有支持和反对这种观点的证据， 海顿7，第13和麦卡锡 11实验室在评论强调，这是有争议的。根据我们最近的?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>VWR&#174; Micro Cover Slips, Round, No. 1</td>
<td>Tool</td>
<td>VWR</td>
<td>48380-068</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0899</td>
<td>&nbsp;</td>
<tr>
<td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
<tr>
<td>Earle&#x2019;s Balanced Salt Solution (EBSS) (1X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14155-063</td>
<td>&nbsp;</td>
<tr>
<td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle's salts, but no L-glutamine or phenol red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>51200-038</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>HEPES solution 1 M</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H0887</td>
<td>&nbsp;</td>
<tr>
<td>N-2 Supplement (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>&nbsp;</td>
<tr>
<td>Horse Serum, Heat-Inactivated</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>26050-088</td>
<td>&nbsp;</td>
<tr>
<td>PAPAIN-022</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LK003178</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal&#x2122; Medium (1X) Liquid without Phenol Red</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>12348-017</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum-Free Supplement (50X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine-200 mM (100X), liquid</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25030-149</td>
<td>&nbsp;</td>
<tr>
<td>Cell Strainers</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td>
<td>Tool</td>
<td>BD Biosciences</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>21108</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2 hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M2670</td>
<td>&nbsp;</td>
<tr>
<td>HEPES free acid</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G7528</td>
<td>&nbsp;</td>
<tr>
<td>Fluo-4, AM 1 mM solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14217</td>
<td>&nbsp;</td>
<tr>
<td>Pluronic&#174; F-127 20% solution in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>P-3000MP</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil TYPE DF</td>
<td>Microscope</td>
<td>Cargille</td>
<td>16242</td>
<td>&nbsp;</td>
<tr>
<td>Open chamber for 25 mm round coverslips, 100 &mu;l volume</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0362 (RC-21BDW)</td>
<td>&nbsp;</td>
<tr>
<td>P-2 platform for Series 20 chambers, non-heater</td>
<td>Tool</td>
<td>WARNER Instruments</td>
<td>64-0278 (P-2)</td>
<td>&nbsp;</td>
<tr>
<td>FluoSpheres carboxylate-modified microspheres, 0.1 &mu;m, yellow-green fluorescent (505/515) 2% solids</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F8803</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring near plasma membrane and global intracellular calcium dynamics in astrocytes

大脑含有神经胶质细胞。星形胶质细胞，神经胶质细胞类型，长期被称为神经元提供了一个被动的支撑作用。然而，越来越多的证据表明，星形胶质细胞可能也积极参与脑功能，通过与神经元的功能的相互作用。然而，星形胶质细胞生物学的许多基本方面仍然争议，职责不清和/或实验未开发。其中一个重要的问题是星形胶质细胞内钙瞬变的动态。这是有关的，因为钙是完善作为一项重要的第二信使，因为它已被提出，星形胶质细胞钙升高可以触发从星形胶质细胞释放发射机。然而，还没有任何近质膜钙信号在星形胶质细胞的详细或令人满意的描述。全内反射荧光显微镜（TIRF）是一个强大的工具来分析有关生理信号事件，约100纳米的活细胞质膜内。在这里，我们使用TIRF显微镜和描述如何几乎同时监测到附近的细胞膜和全球细胞内钙的动态。这种方法的进一步完善和系统应用的潜力，以了解星形胶质细胞钙信号的精确细节。一个星形胶质细胞钙动力学的详细了解可能会提供一个基础，了解是否，如何，何时和为什么星形胶质细胞和神经元接受钙依赖性的功能上的相互作用。

Watch this Scientific Journal Video about Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes at JoVE.com

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

我们描述了如何来衡量膜和全球培养的星形胶质细胞，利用全内反射和epifluorescence显微镜细胞内钙离子动力学附近。

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Research

JoVE Journal

Biology

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

科研

生物学

Measuring Fast Calcium Fluxes in Cardiomyocytes

测量心肌细胞的快速钙助焊剂

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging Plasma Membrane Deformations With pTIRFM

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

娜功能分析<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt;细胞内的隔间使用质子打死选择器来表达它们的质膜

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis MLKL 介导的血浆膜破裂特征

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

小鼠胰腺胰岛细胞亚群中的钙动力学成像

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...