Diese Arbeit wurde von der Uehara Memorial Foundation of Japan (ES) sowie die Whitehall-Stiftung, dem National Institute of Neurological Disorders and Stroke und ein Stein-Oppenheimer Endowment Award (für BSK) unterstützt.

Experimentelle Verfahren Das experimentelle Verfahren besteht aus zwei wichtigen Teilen, die in einem Schritt weise Weise beschrieben werden. Teil 1: Vorbereiten des Hippocampus Astrozyten KULTUREN Kurz gesagt wurden gemischt Hippocampus Astrozyten-Neuron Kulturen hergestellt unter Verwendung eines etablierten Protokoll 1,2,3. Wir optimieren das Verfahren zur gesunden kultivierten Astrozyten zu erhalten. Alle unten aufgeführten Schritte sollten in einer sterilen Umgebung wie einer Sterilbank durchgeführt werden. Vorbereitung Deckgläser <ul><li> 22 mm Deckgläser (VWR, 48380-068) </li><li> Poly-D-Lysin (PDL, Sigma P0899), Aliquots (1 mg / ml) </li><li> Laminin Aliquots (20 ug / ml): add 49 ml sterilem Wasser zu 1 mg / ml Laminin-Lösung (Sigma L2020) machen 1,2 ml aliquotiert und bei -20 ° C </li></ul><ol><li> Lösen PDL in sterilem Wasser (50 ug / ml) </li><li> Autoclave die Deckgläser mit sterilem Wasser und in PDL (20 ml für 100 Deckgläser) spülen. Lassen Sie über Nacht bei Raumtemperatur. </li><li> Ersetzen PDL mit sterilem Wasser (3 umfangreiche Waschgänge). Dann trocknen die Deckgläser und lagern bei 4 ° C. </li><li> Am Tag vor dem Ausplattieren der Astrozyten, statt einzelner Deckgläser in einer sterilen auch auf einer 6-well-Platte (Multiwell Flat-Bottom Platten mit Deckel, steril aus BD Bioscience). Legen Sie 400 ul von Laminin auf jedem Deckglas beimpft und über Nacht, in den Inkubator, um Verdunstung zu vermeiden. </li></ol> Zergliederung Dissection Medien: <ul><li> 500 ml Earle Balanced Salt Solution (EBSS) (1x), flüssig (Invitrogen, 14155-063) </li><li> 5 ml HEPES-Lösung (Sigma, H0887) </li></ul> Hippocampus Medien: <ul><li> 412,5 ml Minimum Essential Medium (MEM) (1x), flüssig Enthält Earle-Salzen, aber kein L-Glutamin oder Phenolrot (Invitrogen, 51200-038) </li><li> 10 ml Glucose (Sigma, D-(+)-Glucose wasserfrei SIGMA ULTRA G7528) 1 M in MEM </li><li> 5 ml Penicillin-Streptomycin (Invitrogen, 15140-122) </li><li> 5 ml Natriumpyruvat-Lösung (Sigma, S8636) </li><li> 12,5 ml HEPES-Lösung 1 M (Sigma, H0887) </li><li> 5 ml N-2 Supplement (100x), flüssig (Invitrogen, 17502-048) </li><li> 50 ml Pferdeserum, hitzeinaktiviertem (Invitrogen, 26050-088) </li></ul> Papain-Lösung: <ul><li> 5 ml Dissektion Medien in Worthington PAPAIN-022 Fläschchen (LK003178; Endkonzentration 20 U / ml) und bei 37 ° C für 60 min. </li></ul><ol><li> Enthaupten Rattenbabys im Alter von P1 (typischerweise 2 Welpen) folgende Richtlinien überprüft und durch nationale Vorschriften und Ihrem lokalen Institutional Animal Care und Verwenden Committee genehmigt. </li><li> Entfernen Sie Haut und Schädel und Gehirn Platz in Petrischale mit kaltem Dissektion Medien gefüllt. </li><li> Entfernt Meningen, sezieren beiden Hemisphären und sezieren hippocampi. </li><li> Hacken Sie die hippocampi in ~ 1X1 mm Stücke und verdauen in Papain-Lösung bei 37 ° C für 11-13 min. </li><li> Sobald die Teile des Gewebes niedergelassen haben, zu entfernen Papain sorgfältig und mit 5 ml Medium Hippocampus, um alle Spuren des Enzyms zu entfernen. Wiederholen Sie diesen Schritt und resuspendieren in Hippocampus-Medium (2 ml). </li><li> Man reibt die Zellen ~ 5 mal, bis keine Klumpen links, mit drei Flammen-polierten Pipetten immer kleiner Bohrungen (1000 mu m, ~ 500 um und ~ 300 Fm). </li><li> Sobald verrieben, pass die Zellen durch eine Zelle Sieb (Porengröße 70 um, BD Bioscience). Zählen Sie die Zellen in 10 ul Suspension. Stellen Sie die Lautstärke des Hippocampus Medium, um bis 400000 Zellen / ml haben. </li><li> Saugen Sie das Laminin aus dem Deckgläschen, und bevor die Abdeckungen können trocken, Platte 200 ul der Zellsuspension pro Deckglas. </li><li> Lassen Sie sie für 60 min in den Inkubator legen, und dann 2 ml Hippocampus Medium pro Vertiefung. </li><li> Am nächsten Tag absaugen alten hippokampalen Medium, um tote Zellen und Ablagerungen zu entfernen und 2 ml vorgewärmten frischen Hippocampus Medium. </li></ol> Wartung Neurobasal Medien: <ul><li> 500 ml Neurobasalmedium (Invitrogen, 12348-017) </li><li> 10 ml Serum B27 kostenlose Beilage (Invitrogen, 17504-044) </li><li> 5 ml Penicillin-Streptomycin (Invitrogen, 15140-122) </li><li> 1,25 ml L-Glutamin (Invitrogen, 25030-149) </li></ul> Feed the Astrozyten-Neuron Kulturen zweimal pro Woche mit Neurobasalmedium ab 4 Tage nach dem Ausplattieren. Vorinkubieren den Medien über 30 min im Inkubator in einem belüfteten Kolben auf die Temperatur und CO 2 ausgleichen. Teil 2: Kalzium-Imaging Hippocampus Aufzeichnungspuffer: 110 mM NaCl, 5,4 mMKCl, 1,8 mM CaCl 2, 0,8 mM MgCl 2, 10 mM D-Glucose, 10 mM HEPES (Alle Chemikalien von Sigma) pH 7,4 (eingestellt mit NaOH). Loading Kalzium-Indikator-Farbstoff in Astrozyten <ol><li> Legen Sie die Kultur in einem Brunnen auf einem 6-well-Platte mit 2 ml Hippocampus Aufnahme-Puffer mit 2,5 pM Fluo-4, AM (Invitrogen, F-14217) und 0,05% Pluronic ® F-127 20% ige Lösung in DMSO gefüllt (Invitrogen, P-3000MP) und bei Raumtemperatur für 10-30 min inkubieren. </li><li> Entfernen Fluo-4-Lösung und waschen Deckgläschen 3 mal mit Hippocampus Aufzeichnungspuffer und Inkubieren bei Raumtemperatur für 30 min. </li><li> Legen Sie das Deckglas auf die Kammer, und reinigen Sie die andere Seite des Deckglases mit Objektiv Reinigungsflüssigkeit. </li><li> Setzen Sie einen kleinen Tropfen Immersionsöl (Immersionsöl TYPE DF aus Cargille) auf dem Ziel und setzte Kammer (Open Kammer für 25 mm runde Deckgläschen von Warner Instruments) auf der Bühne des Mikroskops (Olympus IX71). </li><li> Schauen Sie sich die Zellen mit Hilfe von Durchlicht zu sehen, wie die Zellen aussehen, und sie in den Fokus. Dann leuchten die Zellen mit 488 nm aus einem Monochromator (Polychrome V von TILL Vision) und festzustellen, ob das Fluoreszenzsignal von Fluo-4 ist einheitlich und nachweisbar in Astrozyten. </li><li> Verwenden Sie EPI und TIRF Beleuchtung Kalzium in Astrozyten zu messen. </li></ol> TIRF-Mikroskopie Kurz gesagt, werden wir eine Olympus IX71 Mikroskop mit einem Andor IXON DV887DCS EMCCD-Kamera ausgestattet. Die Kontrolle der Anregungs-und Bildaufnahme erfolgt über TILLVision Software. Die Strahlen 454/488/515 nm Argon (100 mW) und 442 nm Festkörper (45 mW) Laser werden kombiniert und gesteuert mit einem TILL Polyline Laser Kombinierer, TIRF Dual-Port-Kondensator und acoustoptical abstimmbaren Filter und Controller (AOTF, alle aus TILL Photonics) und in einen Kineflex breitbandigen Glasfaser für die Einreise in die TIRF Kondensator. Wir verwenden eine Olympus 60X 1,45 NA Objektiv TIRF erreichen. Die Kamera zu gewinnen ist für jeden Astrozyten angepasst werden, um das beste Signal-Rausch-Bilder liefern. Der Hintergrund und Prinzipien der TIRF-Mikroskopie wurden kürzlich bewertet 4., 5. Die meisten der optischen Komponenten verwenden wir wurden von TILL Photonics, die jetzt Teil der Agilent Technologies (http://www.till-photonics.com/) erworben. Das TIR-Eindringtiefe kann aus den folgenden Gleichungen berechnet werden. <img style="float:left;" src="/files/ftp_upload/1142/equation.png" alt="Gleichung" /> d = Eindringtiefe n1 = Brechungsindex des Glases n2 = Brechungsindex der Zelle a = Einfallswinkel NAi = numerische Apertur von Inzidenz Um sicherzustellen, dass die Laser optimal für TIRF ausgerichtet wir finden es hilfreich, 100 nm fluoreszierende Kügelchen (Invitrogen, F8803) zu beobachten. Wir präsentieren Standbilder und Videos von Perlen mit EPI und TIRF-Mikroskopie. Wenn in TIRF, beobachtet man einen dramatischen Anstieg der Signal-Rausch-und die Perlen Display Brownschen Diffusion. Wir finden es sinnvoll, das Verhalten von 100 nm Perlen mit TIRF-Mikroskopie in regelmäßigen Abständen (~ einmal pro Woche), um sicherzustellen, dass eine optimale TIRF, tritt anstelle des kompromittierten Schrägbeleuchtung dass auftreten würden beobachten, ob der kritische Winkel nicht gleich α (siehe Abb. 1). Anwendung von G-Protein-gekoppelten Rezeptor-Agonisten Astrozyten ausdrückliche einer Vielzahl von Gq-gekoppelte Rezeptoren 6, 7, einschließlich metabotropen Glutamat-Rezeptoren und P2Y-Rezeptoren (Agonist, ATP, ADP). Die Aktivierung dieser Rezeptoren führt zu einem signifikanten Anstieg der intrazellulären Calcium-Spiegel innerhalb von Astrozyten. Zum Beispiel kann man leicht beobachten intrazellulären Calcium-Erhöhungen bei der Anwendung von ATP (30 nM) auf Astrozyten 8-10. Wir verwenden eine schnelle Lösung Switcher von Warner Instruments namens VC-77SP Fast-Step Perfusion System (http://www.warneronline.com/index.cfm). Mit dieser Methode Lösungen können in weniger als ~ 10 ms angewendet werden. <img src="/files/ftp_upload/1142/Fig 1a.jpg" alt="Abbildung 1" /> <img src="/files/ftp_upload/1142/fig1c.jpg" alt="" /> ABBILDUNG 1. Cartoon und Fotografien der Bildgebung einzurichten. A. zeigt ein Foto des Mikroskops auf einem Lufttisch montiert, während (B) zeigt ein Foto des Laser-Montage, Steuerungen und Strahl-Boxen. C. zeigt ein Foto des Mikroskoptisches mit der Kammer für die Bildgebung montiert. Auf der linken Seite der schnellen Perfusion Gerät gesehen werden kann (zusammen mit dem Schrittmotor-und Theta-Rohre) werden. Auf der rechten Seite der headstage eines Axopatch 200A Verstärker zu sehen ist. Die Karikatur schematisiert den Lichtweg in die Einrichtung und wie TIRF erreicht ist. Der Laser ist auf der hinteren Brennebene des 60X 1,45 NA Objektiv und seine Position fokussiert wird aus der Mitte so eingestellt, dass es in den Immersionsöl tritt am kritischen Winkel α. An diesem Punkt des Strahls leidet Totalreflexion und zerfällt mit einer Distanz λ (siehe Gleichung in Haupttext) in das Medium mit niedrigerem Brechungsindex. In diesem Fall ist dies die Aufzeichnungspuffer um den Astrozyten und den Astrozyten sich. Das Ergebnis ist optische Anregung (und damit imaging) von Molekülen innerhalb von ~ 100 nm der Plasmamembran. In der Karikatur der Zelle das ist so grün &quot;excited&quot; Membran-Rezeptoren gezeigt, während die innerhalb der Zelle oder auf der Oberseite der Zelle nicht angeregt werden. Eine vollständige Darstellung der TIRF-Mikroskopie wurde von Steyer und Almers 4 wurde zur Verfügung gestellt. <img src="/files/ftp_upload/1142/fig2.png" alt="Abbildung 2" /> ABBILDUNG 2. Images von 100 nm fluoreszierende Kügelchen mit EPI und TIRF-Mikroskopie erworben. A. Zeigt EPI Bilder ein Sichtfeld mit mehreren Dutzend 100 nm fluoreszierende Kügelchen. Die roten Pfeile zeigen auf Perlen, die auf das Deckglas niedergelassen haben, während die blauen Pfeilspitzen zu Perlen, die Diffusion in Wasser zeigen. B. Zeigt eine TIRF-Bild der gleichen Sichtfeld wie in A. In dieser Ansicht wird nur angezeigt, die Anhänger Perlen durch rote Pfeile dargestellt sichtbar sind. Dies liegt daran, diese auf das Glas coverlsip niedergelassen hatten und waren somit in der ~ 100 nm Evaneszentfeld. Die Perlen in A durch blaue Pfeile angezeigt werden, nicht innerhalb dieser Region und sind somit unsichtbar im TIRF Bilder. Die unteren Diagramme zeigen 3D-Rendering der Bilder. Es ist klar, dass eine starke Zunahme der Signal-Rausch-für Perlen im Evaneszentfeld tritt auf, wenn durch TIRF-Mikroskopie beobachtet. In der Tat für diese Bilder das Signal-Rausch-für EPI betrug 7,1 ± 0,6, während für TIRF es 20 war ± 0,7. <img src="/files/ftp_upload/1142/fig3.png" alt="Abbildung 3" /> ABBILDUNG 3. Images von Astrozyten mit Fluo-4 Calcium-Indikator-Farbstoff mit EPI und TIRF-Mikroskopie erworben geladen. A. EPI Bilder ein Sichtfeld mit fünf Astrozyten. B. A TIRF-Bild der gleichen Sichtfeld in B. Beachten Sie, dass die Bilder in A und B signifikant unterschiedlich dargestellt. Dies liegt daran, mit TIRF Beleuchtung nur der Plasmamembran Regionen in enger Apposition an der Deckglas abgebildet werden. Die unteren Bilder zeigen ATP-evozierte intrazellulären Calcium-Transienten mit EPI und TIRF-Mikroskopie abgebildet.

<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 ist gut etabliert, dass Astrozyten intrazellulären Calcium-Erhebungen angezeigt. Diese spontan auftreten, kann durch neuronale Aktivität oder durch die Anwendung von Agonisten an Rezeptoren auf der Oberfläche 11 Astrozyten aktivieren ausgelöst werden. Eine wichtige und umstrittene Frage ist, ob Astrozyten intrazellulären Calcium-Erhebungen können die Freisetzung von Signalmolekülen, die Rezeptoren aktivieren auf Neuronen 11, 12 auslösen. Dies ist umstritten, weil es Beweise für und gegen diese Ansicht, wie ...

<!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

Das Gehirn enthält Gliazellen. Astrozyten, eine Art von Gliazellen, sind seit langem bekannt, eine passive unterstützende Rolle zu Neuronen liefern. Allerdings schlägt immer deutlicher, dass Astrozyten kann auch aktiv in die Gehirnfunktion zu beteiligen durch funktionelle Interaktionen mit Neuronen. Allerdings bleiben viele grundlegende Aspekte der Astrozyten Biologie umstritten, unklar und / oder experimentell erforscht. Ein wichtiges Thema ist die Dynamik der intrazellulären Calcium-Transienten in Astrozyten. Dies ist relevant, weil Kalzium ist auch eine wichtige second messenger gegründet und weil es wurde vorgeschlagen, dass Astrozyten Kalzium Erhebungen können die Freisetzung von Transmittern aus Astrozyten auslösen. Allerdings gibt es keine detaillierte oder befriedigende Beschreibung der in der Nähe Plasmamembran Calcium-Signalgebung in Astrozyten wurden. Total Internal Reflection Fluorescence (TIRF)-Mikroskopie ist ein leistungsfähiges Werkzeug, um physiologisch relevante Signalwege innerhalb von etwa 100 nm der Plasmamembran von lebenden Zellen zu analysieren. Hier verwenden wir TIRF-Mikroskopie und beschreiben, wie man in der Nähe Plasmamembran und globale intrazellulären Calcium-Dynamik fast gleichzeitig zu überwachen. Die weitere Verfeinerung und systematische Anwendung dieses Ansatzes hat das Potenzial, über die genauen Details der Astrozyten Calcium-Signalgebung zu informieren. Ein detailliertes Verständnis der Astrozyten Calcium-Dynamik kann eine Grundlage zu verstehen, wenn, wie, wann und warum Astrozyten und Neuronen durchlaufen eine Calcium-abhängigen funktionellen Interaktionen.

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

Wir beschreiben, wie man in der Nähe Membran und globalen intrazellulären Calcium-Dynamik in kultivierten Astrozyten mit Totalreflexion und Epifluoreszenzmikroskopie messen.

Messen in der Nähe Plasmamembran und Global intrazellulären Calcium-Dynamics in Astrozyten

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 Aufrufe.  David Geffen School of Medicine, University of California, Los Angeles. Wir beschreiben, wie man in der Nähe Membran und globalen intrazellulären Calcium-Dynamik in kultivierten Astrozyten mit Totalreflexion und Epifluoreszenzmikroskopie messen.

Serie: 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.

Mess-Plasmamembranprotein endocytischen Preise von Reversible Biotinylierung

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

Forschung

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.

Mess-Fast-Flux Calcium in Kardiomyozyten

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.

Mapping molekulare Diffusion in der Plasmamembran durch 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 ...

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.

Intravitalmikroskopie zur Imaging subzellulärer Strukturen in lebenden Mäusen Ausdruck Fluoreszierende Proteine

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 Plasmamembran Verformungen Mit 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.

Funktionelle Charakterisierung von Na<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Wärmetauscher von intrazellulären Kompartimente Mit Proton-Tötung Selection man sie an der Plasmamembran Express

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.

Bestimmung der Plasmamembran Partitionierung für peripher-assoziierte Proteine

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.

Charakterisierung der MLKL-vermittelten Plasmamembran Bruch in 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.

Gleichzeitige Messung von intrazellulären Calcium und Membrane Potential in frisch isolierte und intakte Maus zerebralen Endothel

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.

Bildgebende Calciumdynamik in Subpopulationen von Maus-Pankreas-Islet-Zellen

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