Ce travail a été soutenu par la Fondation Uehara Memorial du Japon (ES) ainsi que la Fondation de Whitehall, l&#39;Institut national des troubles neurologiques et des maladies et un prix de Stein-Oppenheimer de dotation (au BSK).

Procédures expérimentales La procédure expérimentale se compose de deux parties principales qui sont décrits d&#39;une façon par étapes ci-dessous. Partie 1: PRÉPARATION DES CULTURES hippocampique astrocyte Brièvement, mélangé hippocampique astrocyte-neurone cultures ont été préparés en utilisant un protocole bien établi 1,2,3. Nous avons optimisé la procédure de céder saine astrocytes mis en culture. Toutes les procédures énumérées ci-dessous doit être effectuée dans un environnement stérile, comme une hotte à flux laminaire. Préparation des lamelles <ul><li> 22 lamelles mm (VWR, 48380-068) </li><li> Poly-D-Lysine (PDL, Sigma P0899), des aliquotes (1 mg / ml) </li><li> La laminine aliquotes (20 pg / ml): ajouter 49 ml d&#39;eau stérile à 1 mg / ml solution laminine (Sigma L2020), faire 1,2 aliquotes ml et conserver à -20 ° C </li></ul><ol><li> Dissoudre PDL dans l&#39;eau stérile (50 pg / ml) </li><li> Autoclaver les lamelles, rincez à l&#39;eau stérile et le placer dans PDL (20 ml pour 100 lamelles). Laisser à température ambiante jusqu&#39;au lendemain. </li><li> Remplacer PDL à l&#39;eau stérile (3 lavages extensifs). Puis sécher les lamelles et conserver à 4 ° C. </li><li> Le jour avant l&#39;étalement des lamelles les astrocytes place individuelle dans un puits stérile sur une plaque à 6 puits (Multiwell fond plat Plaques avec couvercles, stériles de BD Bioscience). Mettez 400 pi de la laminine sur chaque lamelle et incuber pendant la nuit, dans l&#39;incubateur pour éviter l&#39;évaporation. </li></ol> Dissection Dissection des médias: <ul><li> 500 ml de Earle solution saline équilibrée (EBSS) (1X), liquide (Invitrogen, 14155-063) </li><li> 5 ml solution HEPES (Sigma, H0887) </li></ul> Les médias hippocampe: <ul><li> 412,5 ml de milieu essentiel minimum (MEM) (1X), liquide contient des sels de Earle, mais pas de rouge de L-glutamine ou du phénol (Invitrogen, 51200-038) </li><li> 10 ml de glucose (Sigma, D-(+)-glucose anhydre SIGMA ULTRA G7528) 1 M dans du MEM </li><li> 5 ml de pénicilline-streptomycine (Invitrogen, 15140-122) </li><li> 5 ml de solution de pyruvate de sodium (Sigma, S8636) </li><li> 12,5 ml d&#39;HEPES solution 1 M (Sigma, H0887) </li><li> 5 ml de N-2 Supplément (100X), liquide (Invitrogen, 17502-048) </li><li> 50 ml de sérum de cheval, inactivé par la chaleur (Invitrogen, 26050-088) </li></ul> Solution de papaïne: <ul><li> Ajouter 5 ml de milieu de dissection dans Worthington PAPAÏNE-022 flacon (LK003178; concentration finale de 20 U / ml) et incuber à 37 ° C pendant 60 min. </li></ul><ol><li> Décapitez ratons âgés de P1 (généralement 2 chiots) suivant les lignes directrices révisées et approuvées par les réglementations nationales et de vos soins locaux des animaux et du Comité institutionnel d&#39;utilisation. </li><li> Enlever la peau et le crâne et le cerveau dans une boîte de Pétri remplie avec les médias de dissection froide. </li><li> Suppression des méninges, disséquer les deux hémisphères et disséquer hippocampes. </li><li> Hacher les hippocampes dans ~ 1X1 pièces mm et digérer en solution papaïne à 37 ° C pendant min 11-13. </li><li> Une fois les morceaux de tissu sont installés, retirez soigneusement la papaïne et ajouter 5 ml de milieu de l&#39;hippocampe pour éliminer toute trace de l&#39;enzyme. Répétez cette étape et remettre dans le milieu de l&#39;hippocampe (2 ml). </li><li> Triturer les cellules ~ 5 fois jusqu&#39;à ce qu&#39;il n&#39;y touffes gauche, avec trois flammes poli pipettes de trous progressivement plus petits (1000 um, ~ ~ 500 um et 300 um). </li><li> Une fois trituré, passer les cellules à travers une passoire de cellules (taille des pores, 70 um, BD Biosciences). Compter les cellules dans 10 pl de suspension. Réglez le volume du milieu de l&#39;hippocampe dans le but d&#39;avoir 400 000 cellules / ml. </li><li> Aspirer le laminine hors les lamelles, et avant le couvre peut sécher, la plaque 200 pl de la suspension cellulaire par lamelle. </li><li> Laissez-les à joindre pendant 60 min dans l&#39;incubateur, puis ajouter 2 ml de milieu de l&#39;hippocampe par puits. </li><li> Le lendemain, aspirer ancienne moyennes hippocampe pour enlever les cellules mortes et les débris et ajouter 2 ml de pré-chauffé moyennes hippocampique frais. </li></ol> Entretien Les médias Neurobasal: <ul><li> 500 ml neurobasal (Invitrogen, 12348-017) </li><li> 10 ml de sérum B27 supplément gratuit (Invitrogen, 17504-044) </li><li> 5 ml de pénicilline-streptomycine (Invitrogen, 15140-122) </li><li> 1,25 ml de L-glutamine (Invitrogen, 25030-149) </li></ul> Nourrissez les cultures astrocytes neurone deux fois par semaine avec milieu neurobasal, à commencer quatre jours après placage. Préincuber les médias à environ 30 minutes dans l&#39;incubateur dans un flacon aéré pour équilibrer la température et le CO 2. Partie 2: imagerie calcique Mémoire tampon d&#39;enregistrement hippocampe: 110 mM NaCl, 5,4 mMKCl, 1,8 mM de CaCl2, 0,8 mM MgCl2, 10 mM D-glucose, 10 mM HEPES (Tous les produits chimiques de Sigma) pH 7,4 (ajusté avec NaOH). Chargement colorant indicateur de calcium dans les astrocytes <ol><li> Placer la culture dans un puits sur une plaque à 6 puits remplis avec 2 ml de tampon d&#39;enregistrement hippocampe contenant 2,5 uM Fluo-4, AM (Invitrogen, F-14217) et 0,05% Pluronic ® solution F-127 de 20% dans le DMSO (Invitrogen, P-3000MP) et incuber à température ambiante pendant 10-30 min. </li><li> Retirer Fluo-4 solution et laver lamelles 3 fois avec un tampon d&#39;enregistrement de l&#39;hippocampe et incuber à température ambiante pendant 30 min. </li><li> Placer la lamelle sur la chambre, propre et de l&#39;autre côté de la lamelle avec un liquide de nettoyage pour lentilles. </li><li> Mettez une petite goutte d&#39;huile d&#39;immersion (DF type à immersion d&#39;huile à partir Cargille) sur l&#39;objectif et de mettre la chambre (chambre ouverte pour 25 mm rond lamelle de Warner Instruments) sur la platine du microscope (Olympus IX71). </li><li> Regardez les cellules en utilisant la lumière de transmission de voir comment les cellules regard, et les amener au point. Puis éclairent les cellules avec 488 nm à partir d&#39;un monochromateur (V polychrome de Till Vision) et de déterminer si le signal de fluorescence du fluo-4 est uniforme et détectable dans les astrocytes. </li><li> Utilisez le PEV et l&#39;éclairage TIRF pour mesurer le calcium dans les astrocytes. </li></ol> Microscopie TIRF En bref, nous utilisons un microscope Olympus IX71 équipé d&#39;une caméra Andor IXON DV887DCS EMCCD. Le contrôle de l&#39;excitation et l&#39;acquisition des images est réalisé en utilisant le logiciel TILLVision. Les poutres de 454/488/515 nm Argon (100 mW) et 442 nm état solide (45 MW) lasers sont combinés et contrôlée avec un laser TILL Polyligne combineur, condenseur à double port et de la FRBR acoustoptical filtre accordable et le contrôleur (AOTF; tous de TILL Photonics) et nourris dans une fibre à large bande pour l&#39;entrée en Kineflex le condenseur FRBR. Nous utilisons un Olympus 60X 1,45 NA pour atteindre l&#39;objectif FRBR. Le gain de la caméra est ajusté pour chaque astrocyte pour fournir le meilleur rapport signal sur bruit des images. Le contexte et les principes de microscopie TIRF ont été récemment revu 4, 5. La plupart des composants optiques que nous utilisons ont été achetés auprès TILL Photonics, qui fait maintenant partie de Agilent Technologies (http://www.till-photonics.com/). La profondeur de pénétration TIR peut être calculé à partir des équations ci-dessous. <img style="float:left;" src="/files/ftp_upload/1142/equation.png" alt="Équation" /> d = profondeur de pénétration Indice de réfraction n1 = de verre n2 = indice de réfraction de la cellule a = angle d&#39;incidence NAi = ouverture numérique de l&#39;incidence Afin d&#39;assurer le laser est aligné de façon optimale pour la FRBR, nous trouvons qu&#39;il est utile d&#39;observer 100 nm bille fluorescente (Invitrogen, F8803). Nous présentons des images fixes et des vidéos de perles avec le PEV et la microscopie TIRF. Lorsque dans les FRBR, on observe une augmentation spectaculaire de signal-bruit et les perles d&#39;affichage diffusion brownienne. Nous estimons qu&#39;il est utile d&#39;observer le comportement de 100 nm avec des perles microscopie TIRF sur une base régulière (~ une fois par semaine) pour être sûr que la FRBR optimale se produit, plutôt que de l&#39;éclairage oblique, compromis qui se produirait si l&#39;angle critique ne sont pas égaux à α (voir fig 1). Application de la protéine G agonistes des récepteurs couplés Les astrocytes expriment une variété de GQ récepteurs couplés aux 6, 7, y compris les récepteurs de glutamate métabotropique et des récepteurs P2Y (agoniste, ATP, ADP). L&#39;activation de ces récepteurs conduit à une augmentation significative du taux de calcium intracellulaire dans les astrocytes. Par exemple, on peut facilement observer des élévations de calcium intracellulaire pendant l&#39;application de l&#39;ATP (30 uM) à 8-10 astrocytes. Nous utilisons une solution rapide de commutation de Warner Instruments appelé le Système VC-77SP perfusion rapide Étape (http://www.warneronline.com/index.cfm). Avec cette méthode peut être des solutions appliquées en moins de ~ 10 ms. <img src="/files/ftp_upload/1142/Fig 1a.jpg" alt="Figure 1" /> <img src="/files/ftp_upload/1142/fig1c.jpg" alt="" /> Cartoon FIGURE 1. Et des photographies de l&#39;imagerie mis en place. A. montre une photographie du microscope monté sur une airtable, alors que (B) montre une photographie de l&#39;assemblage laser, les contrôleurs et les boîtes de faisceau. C montre une photo de la platine du microscope à la chambre de montée pour l&#39;imagerie. Sur la gauche du dispositif de perfusion rapide peut être vu (avec le moteur pas à pas et tubes thêta). Sur la droite du headstage d&#39;un amplificateur Axopatch 200A est vu. La caricature schématise le trajet de la lumière dans la mise en place et comment TFRI est atteint. Le laser est focalisé sur le plan focal arrière de l&#39;objectif 60X 1,45 NA et sa position est ajustée décentré afin qu&#39;elle émerge dans l&#39;huile d&#39;immersion à l&#39;angle critique α. A ce point le faisceau souffre d&#39;une réflexion interne totale et se désintègre avec une λ distance (voir l&#39;équation dans le texte principal) dans le milieu de l&#39;indice de réfraction plus faible. Dans ce cas, il s&#39;agit de la mémoire tampon d&#39;enregistrement entourant les astrocytes et les astrocytes eux-mêmes. Le résultat est une excitation optique (et donc d&#39;imagerie) des molécules dans ~ 100 nm de la membrane plasmique. Dans le dessin animé de la cellule de ceci est montré que le vert &quot;excité&quot; récepteurs membranaires, alors que ceux au sein de la cellule ou sur la surface supérieure de la cellule ne sont pas excités. Un compte rendu complet de la microscopie TIRF a été fournie par Steyer et Almers 4. <img src="/files/ftp_upload/1142/fig2.png" alt="Figure 2" /> FIGURE 2. Images de 100 perles fluorescentes nm acquis avec le PEV et la microscopie TIRF. A. montre des images du PEV d&#39;un champ de vue avec des perles de plusieurs dizaines de nm 100 fluorescentes. Le point rouge flèches aux perles qui se sont installés sur la lamelle de verre, tandis que les flèches bleues au point de perles qui se diffusent dans l&#39;eau. B. montre une image TIRF du même champ de vision, comme indiqué en A. Dans ce point de vue que les perles adhérente représentée par les flèches rouges sont visibles. C&#39;est parce que ces s&#39;étaient installés sur le verre et coverlsip étaient donc dans le domaine de ~ 100 nm évanescente. Les perles montré dans une des flèches bleues ne sont pas dans cette région et sont donc invisibles dans les images FRBR. Les parcelles inférieures montrent un rendu 3D des images. Il est clair qu&#39;une augmentation importante de signal-bruit est produit pour les billes dans le champ évanescent lorsque observées par microscopie TIRF. En fait, pour ces images le signal-bruit pour le PEV a été de 7,1 ± 0,6, tandis que pour la FRBR il était de 20 ± 0,7. <img src="/files/ftp_upload/1142/fig3.png" alt="Figure 3" /> FIGURE 3. Images des astrocytes chargées avec Fluo-4 colorant indicateur de calcium acquis avec le PEV et la microscopie TIRF. A. PEV images d&#39;un champ de vue avec cinq astrocytes. B. Une image TIRF du même champ de vue montré dans la note B. que les images de A et B sont sensiblement différentes. C&#39;est parce qu&#39;avec la FRBR seul éclairage de la membrane plasmique des régions en apposition à proximité de la lamelle de verre sont imagés. Les panneaux inférieurs montrent ATP évoqué transitoires du calcium intracellulaire imagées avec le PEV et la microscopie TIRF.

<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>

Il est bien établi que les astrocytes affichage des élévations de calcium intracellulaire. Ils se produisent spontanément, peut être déclenchée par l&#39;activité neuronale ou par application d&#39;agonistes à activer les récepteurs à la surface des astrocytes 11. Une question importante et controversée est de savoir si astrocytes élévations de calcium intracellulaire peut déclencher la libération de molécules de signalisation qui activent les récepteurs des neurones 11, 12. Ceci est controversée, ca...

<!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

Le cerveau contient des cellules gliales. Les astrocytes, un type de cellules gliales, ont longtemps été connues pour fournir un rôle passif de soutien pour les neurones. Cependant, plus de preuves suggèrent que les astrocytes peuvent aussi participer activement à la fonction cérébrale par des interactions fonctionnelles avec les neurones. Toutefois, de nombreux aspects fondamentaux de la biologie des astrocytes restent controversées, clairs et / ou expérimentalement inexploré. Une question importante est la dynamique des transitoires du calcium intracellulaire dans les astrocytes. Cette question est pertinente parce que le calcium est bien établi comme un messager important secondes et parce qu&#39;il a été proposé que les élévations de calcium astrocytaire peut déclencher la libération de transmetteurs d&#39;astrocytes. Cependant, il n&#39;a pas été toute description détaillée ou satisfaisante du calcium à proximité de la membrane plasmique de signalisation dans les astrocytes. Fluorescence totale de réflexion interne (FRBR) microscopie est un outil puissant pour analyser physiologiquement pertinents événements de signalisation dans environ 100 nm de la membrane plasmique des cellules vivantes. Ici, nous utilisons la microscopie TIRF et décrire la façon de surveiller à proximité de la membrane plasmique et la dynamique globale du calcium intracellulaire presque simultanément. Le raffinement et l&#39;application systématique de cette approche a le potentiel pour informer sur les détails précis de la signalisation calcique astrocytaire. Une compréhension détaillée de la dynamique du calcium dans les astrocytes pourrait fournir une base pour comprendre si, comment, quand et pourquoi les astrocytes et les neurones subissent calcium-dépendante des interactions fonctionnelles.

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

Nous décrivons comment mesurer près de la membrane et la dynamique globale du calcium intracellulaire dans les astrocytes cultivés en utilisant la réflexion interne totale et de la microscopie à épifluorescence.

Mesure Proche membrane plasmique et mondiale dynamique du calcium intracellulaire dans les astrocytes

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

measuring-near-plasma-membrane-global-intracellular-calcium-dynamics

Research

JoVE Journal

Biology

13.0K vues.  David Geffen School of Medicine, University of California, Los Angeles. Nous décrivons comment mesurer près de la membrane et la dynamique globale du calcium intracellulaire dans les astrocytes cultivés en utilisant la réflexion interne totale et de la microscopie à épifluorescence.

Vidéo: Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Mesure du taux plasmatique des protéines membranaires par endocytique Biotinylation réversible

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

Recherche

Biologie

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Mesurer les flux de calcium dans les cardiomyocytes rapide

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)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

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

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

Cartographie Diffusion moléculaire dans la membrane plasmique par multiple-cible de Recherches (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

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, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopie intravitale pour l&#39;imagerie Subcellular ouvrages chez des souris vivantes exprimant des protéines 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

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 the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

Imaging Plasma Membrane déformations Avec 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

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

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

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Caractérisation fonctionnelle de Na<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Echangeurs de intracellulaire Compartiments utilisant la sélection Proton-tuant les exprimer à la membrane plasmique

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 using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

Détermination de la Membrane plasmique partitionnement pour les protéines associées à la périphérie

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 is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

Caractérisation de la Rupture des membranes de Plasma induite par le 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

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Mesure simultanée de Calcium intracellulaire et le potentiel de Membrane dans l’endothélium cérébral de souris fraîchement isolées et intacte

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

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Imagerie de la dynamique du calcium dans les sous-populations de cellules d'islet pancréatique de souris

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