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

Este trabajo fue apoyado por la Fundación Uehara Memorial de Japón (a ES), así como la Fundación de Whitehall, el Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares y el Premio Stein-Oppenheimer Fundación (a BSK).

PROCEDIMIENTO EXPERIMENTAL El procedimiento experimental consta de dos partes principales que se describen de una manera paso a paso a continuación. Parte 1: preparación de los cultivos de astrocitos del hipocampo En pocas palabras, mezcla de neuronas del hipocampo astrocitos culturas fueron preparados utilizando un protocolo bien establecido 1,2,3. Hemos optimizado el procedimiento para obtener saludable astrocitos cultivado...

<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>

Es un hecho bien establecido que los astrocitos mostrar elevaciones de calcio intracelular. Esto ocurre de forma espontánea, puede ser desencadenada por la actividad neuronal o por la aplicación de agonistas para activar los receptores en la superficie de los astrocitos 11. Una cuestión importante y polémica es si los astrocitos elevaciones de calcio intracelular puede desencadenar la liberación de moléculas de señalización que activan los receptores en las neuronas 11, 12. Esto es controvertido porque no ha ha...

<!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

El cerebro contiene células gliales. Los astrocitos, un tipo de célula glial, han sabido de largo para proporcionar una función de apoyo pasivo a las neuronas. Sin embargo, la evidencia creciente sugiere que los astrocitos pueden también participar activamente en la función cerebral a través de interacciones funcionales con las neuronas. Sin embargo, muchos aspectos fundamentales de la biología de los astrocitos siguen siendo controvertidas, poco claras y / o explorar experimentalmente. Una cuestión importante es la dinámica de los transitorios de calcio intracelular en los astrocitos. Esto es importante porque el calcio se ha consolidado como un importante segundo mensajero y como se ha propuesto que las elevaciones de los astrocitos de calcio puede desencadenar la liberación de los transmisores de los astrocitos. Sin embargo, no ha habido ninguna descripción detallada o la satisfacción de calcio cerca de la membrana plasmática de señalización en los astrocitos. Total de fluorescencia de reflexión interna (TIRF) microscopía es una herramienta poderosa para analizar los eventos de señalización fisiológicamente relevantes dentro de unos 100 nm de la membrana plasmática de las células vivas. En este sentido, el uso de microscopía TIRF y describir la manera de supervisar cerca de la membrana plasmática y la dinámica global de calcio intracelular casi al mismo tiempo. El perfeccionamiento y la aplicación sistemática de este enfoque tiene el potencial de dar a conocer los detalles precisos de la señalización del calcio astrocitos. Una comprensión detallada de la dinámica del calcio astrocitos puede proveer una base para entender si, cómo, cuándo y por qué someterse a los astrocitos y neuronas dependientes de calcio interacciones funcionales.

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

Se describe la forma de medir cerca de la membrana y la dinámica global de calcio intracelular en los astrocitos cultivados usando la reflexión total interna y microscopía de epifluorescencia.

Cerca de la medición de la membrana plasmática y Global Dynamics calcio intracelular en los astrocitos

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

Medición de proteína de la membrana de plasma tarifas endocítica por biotinilación reversible

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

Investigación

Biología

Measuring Fast Calcium Fluxes in Cardiomyocytes

La medición de flujos rápidos de calcio en los cardiomiocitos

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

Cartografía de la difusión molecular en la membrana plasmática de objetivos múltiples de Búsquedas (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

Microscopia intravital para formar imágenes de estructuras subcelulares en ratones vivos que expresan proteínas fluorescentes

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

Imaging membrana plasmática Deformaciones Con 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

Caracterización funcional de Na<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Intercambiadores de compartimentos intracelular usando selección Protón-matanza para expresarlos a la membrana plasmática

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

Determinación de la membrana plasmática particionado en proteínas asociadas periféricamente

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

Caracterización de la ruptura de la membrana plasmática mediado MLKL en Necroptosis

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

Medidas simultáneas de calcio intracelular y el potencial de membrana en el endotelio Cerebral de ratón recién aislado e intacto

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

Imagen de la dinámica del calcio en subpoblaciones de células de islotes pancreáticos de ratón

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