Questo lavoro è stato sostenuto dalla Fondazione Memorial Uehara del Giappone (ES) e la Fondazione Whitehall, del National Institute of Neurological Disorder and Stroke e un Stein-Oppenheimer Premio Endowment (a BSK).

Procedure sperimentali La procedura sperimentale consiste di due parti principali che sono descritti in maniera saggia passo sotto. Parte 1: PREPARAZIONE CULTURE astrociti dell&#39;ippocampo In breve, misto astrociti-neuroni dell&#39;ippocampo culture sono state preparate utilizzando un protocollo ben definito 1,2,3. Abbiamo ottimizzato il procedimento per produrre sano astrociti in coltura. Tutte le procedure di seguito elencati deve essere effettuata in un ambiente sterile come una cappa a flusso laminare. Coprioggetto preparazione <ul><li> Coprioggetto 22 mm (VWR, 48380-068) </li><li> Poli-D-lisina (PDL, Sigma P0899), aliquote (1 mg / ml) </li><li> Laminina aliquote (20 mg / ml): aggiungere 49 ml di acqua sterile a 1 mg / ml soluzione laminina (Sigma L2020), fanno aliquote da 1,2 ml e conservare a -20 ° C </li></ul><ol><li> Sciogliere PDL in acqua sterile (50 mg / ml) </li><li> Autoclave i coprioggetti, sciacquare con acqua sterile e posto in PDL (20 ml per 100 lamelle). Lasciare a temperatura ambiente per una notte. </li><li> Sostituire il PDL con acqua sterile (3 lavaggi estensiva). Poi asciugare i coprioggetti e conservare a 4 ° C. </li><li> Il giorno prima della placcatura astrociti, coprioggetto posto individuale in un pozzo sterile su un 6-pozzetti (pozzetti a fondo piatto Piatti con coperchio, sterile da BD Bioscience). Mettere 400 ml di laminina su ogni coprioggetto e incubare una notte, in incubatrice per evitare l&#39;evaporazione. </li></ol> Dissezione Dissezione dei media: <ul><li> 500 ml di Earle soluzione salina bilanciata (EBSS) (1X), liquido (Invitrogen, 14155-063) </li><li> HEPES 5 ml di soluzione (Sigma, H0887) </li></ul> Mezzi di comunicazione dell&#39;ippocampo: <ul><li> 412,5 ml di media minima essenziale (MEM) (1X), liquido Contiene sali di Earle, ma non rosso L-glutammina o fenolo (Invitrogen, 51200-038) </li><li> 10 ml di glucosio (Sigma, D-(+)-glucosio anidro SIGMA ULTRA G7528) 1 M a MEM </li><li> 5 ml di penicillina-streptomicina (Invitrogen, 15140-122) </li><li> 5 ml di soluzione di sodio piruvato (Sigma, S8636) </li><li> 12,5 ml HEPES soluzione 1 M (Sigma, H0887) </li><li> 5 ml N-2 Supplement (100X), liquido (Invitrogen, 17502-048) </li><li> 50 ml di siero di cavallo, inattivato per calore (Invitrogen, 26050-088) </li></ul> Papaina soluzione: <ul><li> Aggiungere 5 ml di dissezione dei media in Worthington papaina-022 flaconcino (LK003178; concentrazione finale, 20 U / ml) e incubare a 37 ° C per 60 min. </li></ul><ol><li> Decapitare ratti di età compresa tra P1 (in genere 2 cuccioli) le linee guida controllati e approvati dalla normativa nazionale e Cura il tuo locale degli animali e del Comitato Istituzionale Usa. </li><li> Togliere la pelle e il cranio e il cervello posto nella piastra di Petri riempite con i media dissezione a freddo. </li><li> Rimosso meningi, sezionare entrambi gli emisferi e sezionare dell&#39;ippocampo. </li><li> Tritate la dell&#39;ippocampo in ~ 1X1 pezzi mm e digerire in soluzione papaina a 37 ° C per 11-13 minuti. </li><li> Una volta che i pezzi di tessuto sono insediati, rimuovere la papaina con cura e aggiungere 5 ml di terreno dell&#39;ippocampo a rimuovere tutte le tracce di un enzima. Ripetere questo passaggio e risospendere in media dell&#39;ippocampo (2 ml). </li><li> Triturare le cellule ~ 5 volte fino a quando non ci sono grumi sinistra, con tre fiamma lucido pipette di fori progressivamente più piccoli (1000 micron, ~ 500 micron e ~ 300 micron). </li><li> Una volta triturato, le cellule passano attraverso un filtro cella (dimensione dei pori, 70 micron, BD Bioscience). Contare le cellule in 10 ml di sospensione. Regolare il volume di terreno dell&#39;ippocampo in modo da avere 400000 cellule / ml. </li><li> Aspirare il laminina fuori i coprioggetti, e prima che le coperture possono asciutto, piatto 200 microlitri della sospensione cellulare per coprioggetto. </li><li> Lasciateli per attaccare per 60 minuti in incubatrice, e poi aggiungere 2 ml di terreno dell&#39;ippocampo per bene. </li><li> Il giorno successivo, aspirare vecchi media dell&#39;ippocampo per rimuovere le cellule morte e detriti e aggiungere 2 ml di pre-riscaldato terreno fresco dell&#39;ippocampo. </li></ol> Manutenzione Mezzi di comunicazione Neurobasal: <ul><li> 500 ml neurobasal (Invitrogen, 12348-017) </li><li> 10 ml di siero B27 supplemento gratuito (Invitrogen, 17504-044) </li><li> 5 ml di penicillina-streptomicina (Invitrogen, 15140-122) </li><li> 1,25 ml L-glutamina (Invitrogen, 25030-149) </li></ul> Feed the culture astrociti-neuroni due volte alla settimana con il mezzo neurobasal, a partire da quattro giorni dopo la placcatura. Preincubare i media di circa 30 minuti in incubatrice in un pallone ventilato per equilibrare la temperatura e CO 2. Parte 2: IMAGING DI CALCIO Registrazione dell&#39;ippocampo tampone: 110 mM NaCl, 5.4 mMKCl, 1,8 mM CaCl 2, 0,8 mM MgCl 2, 10 mM D-glucosio, 10 mM HEPES (Tutti i prodotti chimici da Sigma), pH 7.4 (corretto con NaOH). Caricamento indicatore colorante calcio in astrociti <ol><li> Posizionare la cultura in un bene su un 6-pozzetti riempiti con 2 ml di buffer di registrazione dell&#39;ippocampo contenente 2,5 mM Fluo-4, AM (Invitrogen, F-14217) e 0,05% Pluronic soluzione ® F-127, il 20% in DMSO (Invitrogen, P-3000MP) e incubare a temperatura ambiente per 10-30 min. </li><li> Rimuovere Fluo-4 soluzione e lavare coprioggetto 3 volte con buffer di registrazione dell&#39;ippocampo e incubare a temperatura ambiente per 30 min. </li><li> Posizionare il coprioggetto sulla camera, e pulire il lato del vetrino con il liquido per pulire le lenti. </li><li> Metti una piccola goccia di olio da immersione (olio DF immersione TIPO da Cargille) l&#39;obiettivo e mettere da camera (camera aperta per 25 coprioggetto tondo mm dalla Warner Instruments) sul palco del microscopio (Olympus IX71). </li><li> Guarda le cellule usando la luce di trasmissione per vedere come le cellule aspetto, e portarli a fuoco. Poi illuminare le cellule con 488 nm da un monocromatore (policroma V da TILL Vision) e determinare se il segnale di fluorescenza di Fluo-4 è uniforme e rilevabile negli astrociti. </li><li> Usa EPI e TIRF illuminazione per misurare il calcio negli astrociti. </li></ol> Microscopia TIRF In breve, usiamo un microscopio Olympus IX71 dotato di una fotocamera IXON Andor DV887DCS EMCCD. Il controllo di eccitazione e di acquisizione delle immagini è ottenuta utilizzando un software TILLVision. Le travi di 454/488/515 nm Argon (100 mW) e 442 nm allo stato solido (45 mW) i laser sono combinati e controllati con una FINO Polilinea laser combinatore, TIRF porta a condensatore a doppio e acoustoptical filtro sintonizzabile e controller (AOTF; tutto da FINO Photonics) e inseriti in una fibra Kineflex a banda larga per l&#39;ingresso nel condensatore TIRF. Usiamo un Olympus 60X 1,45 lente NA per raggiungere TIRF. Il guadagno della telecamera è adeguato, per ogni astrociti per fornire il miglior segnale al rumore delle immagini. Lo sfondo ed i principi della microscopia TIRF sono state recentemente recensione 4, 5. La maggior parte dei componenti ottici che utilizziamo sono stati acquistati da TILL Photonics, che ora fa parte di Agilent Technologies (http://www.till-photonics.com/). La profondità di penetrazione TIR può essere calcolato dalle equazioni di seguito. <img style="float:left;" src="/files/ftp_upload/1142/equation.png" alt="Equazione" /> d = profondità di penetrazione n1 = indice di rifrazione del vetro n2 = indice di rifrazione di cellule un angolo di incidenza = NAi = apertura numerica di incidenza Al fine di garantire il laser è allineato in modo ottimale per TIRF riteniamo utile osservare 100 nm perline fluorescenti (Invitrogen, F8803). Vi presentiamo fotogrammi e video di perline con EPI e microscopia TIRF. Quando nel TIRF, si osserva un aumento drammatico nel rapporto segnale-rumore e le perline visualizzazione diffusione browniano. Per noi è utile osservare il comportamento di 100 nm con perline microscopia TIRF su base regolare (~ una volta a settimana) per essere sicuri che TIRF ottimale si verifica, piuttosto che l&#39;illuminazione compromessa obliqua che si verificherebbe se l&#39;angolo critici non sono stati pari a α (vedi Fig. 1). Applicazione di agonisti accoppiata G-proteina recettore Astrociti esprimono una varietà di Gq recettori accoppiati 6, 7 tra cui i recettori metabotropici del glutammato e recettori P2Y (agonista, ATP, ADP). L&#39;attivazione di questi recettori porta ad un aumento significativo dei livelli intracellulari di calcio in astrociti. Per esempio, si può facilmente osservare prospetti del calcio intracellulare durante l&#39;applicazione di ATP (30 mM) a 8-10 astrociti. Usiamo una soluzione rapida switcher da Warner Instruments chiamato VC-77SP Fast-Passo del sistema di perfusione (http://www.warneronline.com/index.cfm). Con questo metodo le soluzioni possono essere applicate in meno di ~ 10 ms. <img src="/files/ftp_upload/1142/Fig 1a.jpg" alt="Figura 1" /> <img src="/files/ftp_upload/1142/fig1c.jpg" alt="" /> Cartoon FIGURA 1. Fotografie e immagini del set up. A. mostra una fotografia del microscopio montato su un airtable, mentre (B) mostra una fotografia del gruppo laser, controllori e scatole fascio. C. Mostra una fotografia della scena microscopio con la camera montata per l&#39;imaging. A sinistra il dispositivo di perfusione veloce può essere visto (insieme con il motore passo-passo e tubi theta). Sulla destra il headstage di un amplificatore Axopatch 200A è visto. Il fumetto schematizza il percorso della luce nel set up e come TIRF è raggiunto. Il laser viene focalizzato sul piano focale posteriore della lente dell&#39;obiettivo 60X 1,45 NA e la sua posizione viene regolata fuori centro in modo che emerge in olio da immersione al α angolo critico. A questo punto il fascio soffre di riflessione interna totale e decade con una distanza λ (vedi equazione nel testo principale) nel mezzo di indice di rifrazione più basso. In questo caso, questo è il buffer di registrazione che circonda la astrociti e gli astrociti stessi. Il risultato è eccitazione ottica (e quindi immagini) di molecole all&#39;interno di ~ 100 nm della membrana plasmatica. Nel cartone animato della cellula, viene visualizzato come verde &quot;eccitato&quot; recettori di membrana, mentre quelli all&#39;interno della cellula o sulla superficie superiore della cella non sono entusiasti. Un resoconto completo di microscopia TIRF è stata fornita da Steyer e Almers 4. <img src="/files/ftp_upload/1142/fig2.png" alt="Figura 2" /> FIGURA 2. Immagini di 100 nm perline fluorescenti acquisito con EPI e microscopia TIRF. A. mostra le immagini EPI di un campo di vista con diverse decine di 100 nm perline fluorescenti. Il punto rosso frecce per perline che si sono insediati sul vetrino di vetro, mentre le frecce blu indicano che sono perle di diffusione in acqua. B. mostra un&#39;immagine TIRF dello stesso campo di vista, come mostrato in A. In questa visione solo le perle aderente mostrato dalle frecce rosse sono visibili. Questo perché questi si erano stabiliti sulla coverlsip vetro e sono stati pertanto nel campo 100 nm ~ evanescente. Le perle mostrato in A con frecce blu non sono in questa regione e sono quindi invisibili nelle immagini TIRF. I grafici mostrano inferiore rendering 3D delle immagini. E &#39;chiaro che un forte aumento del rapporto segnale-rumore si verifica per perline all&#39;interno del campo evanescente quando osservate al microscopio TIRF. Infatti per queste immagini il rapporto segnale-rumore per EPI è stato 7,1 ± 0,6, mentre per TIRF era 20 ± 0,7. <img src="/files/ftp_upload/1142/fig3.png" alt="Figura 3" /> FIGURA 3. Immagini di astrociti caricato con Fluo-4 colorante indicatore di calcio acquisito con EPI e microscopia TIRF. A. EPI immagini di un campo di vista con cinque astrociti. B. Una immagine TIRF dello stesso campo di vista mostrato in B. Nota che le immagini in A e B sono significativamente differenti. Questo perché con TIRF illuminazione solo le regioni membrana plasmatica in apposizione vicino al coprioggetto di vetro sono ripreso. I pannelli inferiori mostrano ATP-evocate transienti di calcio intracellulare ripreso con EPI e microscopia 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>

E &#39;ben dimostrato che gli astrociti visualizzare prospetti del calcio intracellulare. Questi si verificano spontaneamente, può essere attivato da un&#39;attività neuronale o mediante l&#39;applicazione di agonisti per attivare i recettori sulla superficie astrociti 11. Un tema importante e controverso è se astrociti prospetti del calcio intracellulare può innescare il rilascio di molecole di segnalazione che attivano i recettori sui neuroni 11, 12. Questo è controversa perché c&#39;è stata la prova a favore ...

<table><tbody><tr><td>VWR&amp;#174; Micro Cover Slips, Round, No. 1</td><td>Tool</td><td>VWR international</td><td>48380-068</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P0899</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Reagent</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>Earle&amp;rsquo;s Balanced Salt Solution (EBSS) (1X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>14155-063</td><td></td></tr><tr><td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle&amp;rsquo;s salts, but no L-glutamine or phenol red</td><td>Reagent</td><td>Invitrogen</td><td>51200-038</td><td></td></tr><tr><td>Penicillin-Streptomycin liquid</td><td>Reagent</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Sodium pyruvate solution</td><td>Reagent</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>HEPES solution 1 M</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>N-2 Supplement (100X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>Horse Serum, Heat-Inactivated</td><td>Reagent</td><td>Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>PAPAIN-022</td><td>Reagent</td><td>Worthington Biochemical</td><td>LK003178</td><td></td></tr><tr><td>Neurobasal&amp;#8482; Medium (1X) Liquid without Phenol Red</td><td>Reagent</td><td>Invitrogen</td><td>12348-017</td><td></td></tr><tr><td>B-27 Serum-Free Supplement (50X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>L-Glutamine-200 mM (100X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>25030-149</td><td></td></tr><tr><td>Cell Strainers</td><td>Tool</td><td>BD Biosciences</td><td>352350</td><td></td></tr><tr><td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td><td>Tool</td><td>BD Biosciences</td><td>353046</td><td></td></tr><tr><td>NaCl</td><td>Reagent</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; hexahydrate</td><td>Reagent</td><td>Sigma-Aldrich</td><td>21108</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; hexahydrate</td><td>Reagent</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>HEPES free acid</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Reagent</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>Fluo-4, AM 1 mM solution in DMSO</td><td>Reagent</td><td>Invitrogen</td><td>F-14217</td><td></td></tr><tr><td>Pluronic&amp;#174; F-127 20% solution in DMSO</td><td>Reagent</td><td>Invitrogen</td><td>P-3000MP</td><td></td></tr><tr><td>Immersion Oil TYPE DF</td><td>Microscope</td><td>Cargill Labs</td><td>16242</td><td></td></tr><tr><td>Open chamber for 25 mm round coverslips, 100 &amp;mu;l volume</td><td>Tool</td><td>Warner Instruments</td><td>64-0362 (RC-21BDW)</td><td></td></tr><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></td></tr><tr><td>FluoSpheres carboxylate-modified microspheres, 0.1 &amp;mu;m, yellow-green fluorescent (505/515) 2% solids</td><td>Reagent</td><td>Invitrogen</td><td>F8803</td><td></td></tr></tbody></table>

measuring near plasma membrane and global intracellular calcium dynamics in astrocytes

Il cervello contiene cellule gliali. Astrociti, un tipo di cellule gliali, sono da tempo noti per fornire un ruolo passivo di supporto ai neuroni. Tuttavia, una crescente evidenza suggerisce che gli astrociti possono anche partecipare attivamente nella funzione del cervello attraverso le interazioni funzionali con i neuroni. Tuttavia, molti aspetti fondamentali della biologia astrociti rimangono controversi, chiari e / o sperimentale inesplorati. Una questione importante è la dinamica dei transitori calcio intracellulare negli astrociti. Questo è importante perché il calcio è così affermato come un importante messaggero secondo e perché è stato proposto che elevazioni di calcio astrociti possono innescare il rilascio di trasmettitori da astrociti. Tuttavia, non vi è stata alcuna descrizione dettagliata e soddisfacente di calcio vicino a membrana plasmatica di segnalazione negli astrociti. Fluorescenza di riflessione interna totale (TIRF) microscopia è un potente strumento per analizzare gli eventi di segnalazione fisiologicamente rilevanti all&#39;interno di circa 100 nm della membrana plasmatica delle cellule vive. Qui, usiamo microscopia TIRF e descrivono come monitor vicino membrana plasmatica e globale dinamiche di calcio intracellulare quasi simultaneamente. L&#39;ulteriore perfezionamento e l&#39;applicazione sistematica di questo approccio ha il potenziale per informare sui dettagli precisi della segnalazione calcio astrociti. Una comprensione dettagliata delle dinamiche di calcio astrociti possono fornire una base per capire se, come, quando e perché astrociti e neuroni subiscono interazioni funzionali calcio-dipendente.

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

Noi descriviamo come misurare vicino a membrana e globale dinamiche di calcio intracellulare negli astrociti in coltura utilizzando riflessione interna totale e epifluorescenza.

Misura Vicino membrana plasmatica e Global Dynamics calcio intracellulare negli astrociti

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.1K Views.  David Geffen School of Medicine, University of California, Los Angeles. Noi descriviamo come misurare vicino a membrana e globale dinamiche di calcio intracellulare negli astrociti in coltura utilizzando riflessione interna totale e epifluorescenza.

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

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Due registrazioni elettrofisiologiche di sinapticamente-evocati Risposte astrogliale e neuronale in acute fettine di ippocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Ricerca

Neuroscienze

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Indurre Plasticità di astrociti recettori per manipolazione del neuronali di cottura Prezzi

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Imaging intracellulare Ca<sup&gt; 2+</sup&gt; Segnali di striatale astrociti da topi adulti utilizzando indicatori di calcio geneticamente codificate

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Coltura In Vivo-piace murini astrociti utilizzando il protocollo AWESAM veloce, semplice e poco costoso

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Dissezione del locale Ca2 + segnali in cellule coltivate di targeting per membrana Ca2 + indicatori

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Visualizzazione e analisi del trasporto intracellulare di organelli e di altri carichi in Astrociti

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Imaging Dell'assorbimento mitocondriale di Ca2+ in astrociti e neuroni utilizzando indicatori Ca2+ geneticamente codificati (GECI)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

Colture primarie di astrociti e microglia di ratto e loro utilizzo nello studio della sclerosi laterale amiotrofica

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. Under homeostatic conditions, microglia possess a highly ramified morphology. This facilitates their monitoring of the surrounding environment through the continuous extending and retracting of their processes. During brain injury and inflammation, however, microglia become activated and undergo dramatic morphological changes, retracting their ramified processes and swelling their cell body. This facilitates activities such as migration and phagocytosis, which microglia undertake to navigate the brain environment to a less pathological state.
This close relationship between microglial morphology and changes in their activity have enabled considerable insights into various microglial functions. However, such morphological and activity changes are themselves phenomena that can result from any number of intracellular signaling pathways. Moreover, the time-lag between stimulus and response, as well as the highly compartmentalized morphology of microglia, make it difficult to isolate the causative mechanisms that underpin function. To solve this problem, we developed a genetically modified mouse line in which a highly sensitive fluorescent Ca2+-indicator protein is specifically expressed in microglia.
After describing methods for in vivo microglial Ca2+ imaging, this paper presents a structured analysis approach that classifies this Ca2+ activity to rationally defined subcellular regions, thus ensuring that the spatial and temporal dimensions of the encoded information are meaningfully extracted. We believe that this approach will provide a detailed understanding of the intracellular signaling rules that govern the diverse array of microglial activities associated with both higher brain functions and pathological conditions.

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

In this paper, we describe a protocol for in vivo imaging of microglial Ca2+ activity and subsequent analysis of its spatiotemporal dynamics. This method enables thorough characterization of how microglia respond to changes in the brain environment, appropriately capturing the fine spatiotemporal scales at which such events occur.

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. ...

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.

Misurazioni simultanee di calcio intracellulare e potenziale di membrana nell'endotelio cerebrale Mouse appena isolato ed intatto

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

Misura Vicino membrana plasmatica e Global Dynamics calcio intracellulare negli astrociti

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Capitoli in questo video

Due registrazioni elettrofisiologiche di sinapticamente-evocati Risposte astrogliale e neuronale in acute fettine di ippocampo

Indurre Plasticità di astrociti recettori per manipolazione del neuronali di cottura Prezzi

Imaging intracellulare Ca

Coltura In Vivo-piace murini astrociti utilizzando il protocollo AWESAM veloce, semplice e poco costoso

Dissezione del locale Ca^{2 +} segnali in cellule coltivate di targeting per membrana Ca^{2 +} indicatori

Visualizzazione e analisi del trasporto intracellulare di organelli e di altri carichi in Astrociti

Imaging Dell'assorbimento mitocondriale di Ca²⁺ in astrociti e neuroni utilizzando indicatori Ca²⁺ geneticamente codificati (GECI)

Colture primarie di astrociti e microglia di ratto e loro utilizzo nello studio della sclerosi laterale amiotrofica

Spatiotemporal Analysis of Microglial Ca²⁺ Activity at Single-Cell Resolution

Misurazioni simultanee di calcio intracellulare e potenziale di membrana nell'endotelio cerebrale Mouse appena isolato ed intatto