Name: imaging intracellular ca2+ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators
Uploaded: 2014-11-19T14:00:00.000+00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators at JoVE.com

La maggior parte del lavoro e del personale coinvolto sono stati sostenuti dal NIH concedere NS060677 e in parte dal NIH concede MH099559 e MH104069 (a BSK). Una parte del lavoro è stato anche sostenuto dalla Fondazione CHDI.

Tutti i protocolli di animali erano in accordo con la US National Institutes of Health Guide per la cura e l&#39;uso di animali da laboratorio e sono stati approvati dalla cura e l&#39;uso degli animali Comitato Istituzionale presso la UCLA. 1.1) Preparare Micropipette e AAV2 / 5 Virus Loading <ol><li> Utilizzare sottili micropipette di vetro borosilicato per l&#39;iniezione del virus. Tirare la micropipetta utilizzando un programma che tira in due fasi con un estrattore verticale. Smusso la pipetta con un angolo di 40 ° utilizzando una smerigliatrice pipetta. La pipetta che è ideale per uso avrà un diametro di punta di 20 - 40 micron e una lunghezza gambo 6 - 7 mm. Autoclave la pipetta e strumenti chirurgici. Il trapano è sterilizzato strofinando con etanolo al 70%. </li><li> Conservare il virus AAV2 / 5-GfaABC 1 D.cyto-GCaMP3.SV40 (1,5 x 10 13 gc / ml), a -80 ° C in 10 aliquote microlitri. Poco prima di un intervento chirurgico, prendere le aliquote fuori dal freezer e conservare in ghiaccio fino usato per il riempimento the micropipetta. </li><li> Riempire la micropipetta con il virus utilizzando una pompa a siringa. Collegare strettamente una estremità di un pezzo di tubo, attraverso una compressione tubo di dimensioni appropriate raccordo, ad una siringa di vetro con uno stantuffo rinforzato. Fissare la micropipetta autoclavato all&#39;altra estremità del pezzo di tubo attraverso una dimensione appropriata compressione ago asportabile raccordo. </li><li> Prima di caricare il vettore virale, rimuovere le bolle d&#39;aria dal sistema di pompaggio compresa tubazione, siringa e micropipetta dal riempimento del circuito con olio minerale colorato con Sudan IV rosso (~ 1 mg / 50 ml) in una siringa da 1 ml con un ago (27 G ). </li><li> Posizionare un pezzo di parafilm pulita sotto la micropipetta di vetro, e dispensare 5-10 ml di vettore virale sul parafilm. </li><li> Aspirare il vettore virale spostando lo stantuffo della siringa all&#39;indietro a 0.5 ml / min, e marcare il confine tra il vettore e l&#39;olio sulla pipetta di vetro. </li></ol> 1.2) Anestesia, taglio dei capelli, Head fissaggio e preparazione per craniotomia <ol><li> Mettere un mouse (P49-63, C57BL / 6) nella camera riempita con N 2 O e O 2 e 5% isoflurano. Valutare la profondità dell&#39;anestesia dalla perdita di movimento intenzionale e una frequenza respiratoria rallentato (~ 60-90 / min). Tagli capelli sulla testa quando il mouse è anestetizzato. </li><li> Montare il mouse nel telaio stereotassico, con la testa protetta da orecchio bar smussati e il naso inseriti in un sistema di anestesia e ventilazione. Mantenere isoflurano continuo a 2 - 3%. Applicare lacrime artificiali unguento per entrambi gli occhi in quanto i topi perdono la loro istintiva chiusura delle palpebre in anestesia. </li><li> Somministrare 0,05 ml di buprenorfina (0,1 mg / ml) per via sottocutanea per alleviare il dolore. </li><li> Area chirurgica pulita con il 10% di iodio povidone e il 70% di alcol tre volte, alternando tra le due soluzioni e iniziando con iodio povidone. Inizia dal fronte e pulire verso il retro per rimuovere i peli già tagliati. </li><li> Fare un incisione cutanea sulla parte superiore of testa che inizia tra gli occhi e termina tra le orecchie. </li></ol> 1.3) Craniotomia e microiniezioni <ol><li> Rimuovere il periostio facendo scorrere con un batuffolo di cotone e poi asciugare la superficie del cranio con batuffoli di cotone dentale. Fare un segno intorno al sito di iniezione, e forare intorno al bordo del segno usando una piccola bava acciaio alimentato da un trapano ad alta velocità. </li><li> Rimuovere l&#39;osso e pulire la superficie con soluzione salina. </li><li> Posizionare la pipetta nel dorsale laterale sinistro striatum con le seguenti coordinate (mm): antero-posteriore 0,8, mediale-laterale: +2.0, dorso-ventrale dalla superficie piale: -2.4 13. Si noti che queste coordinate significano che la punta dell&#39;ago microiniezione si trova appena sopra striato dorsolaterale (Figura 1A, B), nel senso che penetra leggermente all&#39;interno di questo nucleo. Evitare di danneggiare i vasi sanguigni. Se la pipetta sembra essere proprio su un vaso sanguigno, leggermente regolare lacoordinate da ~ 0,1 - 0,2 mm. </li><li> Inizia iniezione con velocità di iniezione fissato a 0,2 ml / min, e in genere iniettare 1 a 1,5 ml di virus. </li><li> Lasciare la pipetta in posizione dopo l&#39;iniezione per 10 minuti, e quindi estrarre la pipetta lentamente. </li><li> Termina con la chiusura della ferita chirurgica con sutura continua in nylon esterna sutura. </li></ol> 1.4) Recupero <ol><li> Posizionare il mouse in una gabbia pulita su una piastra elettrica per 24 ore. Non restituire un mouse che ha subito un intervento chirurgico per la compagnia di altri animali fino alla completa guarigione. Non lasciare incustodito il mouse fino a quando non ha ripreso conoscenza sufficiente per mantenere decubito sternale. </li><li> Aggiungere Trimetoprim Sulfamethoxiazole (5 ml per acqua 500 ml, contenenti 40 mg di trimetoprim e 200 mg sulfamethoxiazole) in acqua per una settimana. Somministrare Buprenorfina due volte al giorno per un massimo di 3 giorni dopo l&#39;intervento chirurgico. </li><li> Mantenere il mouse su una luce-buio ciclo di 12 ore, nutriti e abbeverati ogni giorno, unND è ponderato una volta al giorno fino a 3 giorni dopo l&#39;intervento chirurgico. In questi 3 giorni, qualsiasi mouse con una perdita di oltre il 10% del peso corporeo è considerato compromessa, e sarà eutanasia invece di essere sottoposto a sperimentazione. </li><li> Separatamente, cambiare la gabbia del mouse due volte alla settimana, e il monitoraggio per la salute generale, almeno una volta al giorno. In genere, il mouse viene utilizzato entro 2 - 3 settimane dopo le microiniezioni stereotassica. </li></ol> 2. acuta del cervello Fetta Preparazione per confocale Ca 2+ Imaging 2.1) Preparazione di Taglio e Soluzioni di registrazione. <ol><li> Preparare soluzione comprendente (mM) di taglio: 194 saccarosio, 30 NaCl, KCl 4,5, 10 D-glucosio, 1 MgCl2, 1,2 NaH 2 PO 4, e 26 NaHCO3, saturata con 95% O 2 e 5% di CO 2. </li><li> Preparare la soluzione di registrazione che comprende (mm): 124 NaCl, KCl 4,5, 1 MgCl 2, 10 D-glucosio, 2 CaCl 2, 1.2 NaH 2 PO 4 </sub&gt;, e 26 NaHCO 3; pH 7,3-7,4, 290-295 mOsm, saturata con 95% O 2 e 5% di CO 2. Riempire il bicchiere fetta cervello che tiene con soluzione di registrazione, e la tiene a 34 ° C. </li></ol> 2.2) Affettare <ol><li> Due o tre settimane dopo microiniezioni stereotassica, profondamente e terminali anestetizzare il mouse, e poi decapitarlo. </li><li> Estrarre il cervello, e usare una lama per rimuovere il uninjected emisfero destro, e montare il sinistro sul vassoio vibratome con colla super. Riempire il vassoio vibratome con tampone di ghiaccio taglio a freddo, e continuare a saturare con il 95% O 2 e il 5% di CO 2. </li><li> Tagliare fettine di cervello striatali coronali o sagittali a 300 micron di spessore. Di solito, 4-5 coronale, o 6-7 fette striatali sagittali possono essere raccolti. </li><li> Trasferire le fette al bicchiere con fetta riscaldato a 34 ° C, e tenerli lì per 30 minuti prima di riporli a temperatura ambiente per la successiva recording. </li><li> Incubare le sezioni di cervello di buffer di registrazione ossigenata a temperatura ambiente per almeno 30 minuti prima del [Ca 2+] i di imaging. </li></ol> 3. L&#39;immunoistochimica (IHC) <ol><li> Due settimane dopo l&#39;iniezione del virus, defluire in mouse transcardially con PBS seguita da 10% di formalina in PBS. </li><li> Rimuovere, post-correzione durante la notte, e cryoprotect il cervello in tamponata 30% di saccarosio per almeno 2 giorni. </li><li> Preparare 40 micron sezioni utilizzando un microtomo criostato. </li><li> Lavare le sezioni 3 volte con PBS a 10 min di intervallo. </li><li> Incubare le sezioni per 1 ora in PBS con siero normale di capra 10% e 0,5% Triton X-100. </li><li> Incubare sezioni notte a 4 ° C in anticorpo primario preparato in PBS con 0,5% Triton X-100. Gli anticorpi primari utilizzati sono i seguenti: pollo anti-GFP 1: 1.000, topo anti-S100β 1: 400, topo anti-NeuN 1: 600, topo anti-glutammina sintetasi 1: 300. </li><li> Lavare le sezioni 3 volte con PBS a 10 min intervalli. </li><li> Incubare le sezioni con anticorpi secondari preparati in PBS con siero normale di capra 10% per 2 ore a temperatura ambiente. Il secondo anticorpi sono stati i seguenti: capra anti-topo Alexa546 1: 500, capra anti-pollo Alexa488 1: 500. </li><li> Montare le sezioni su vetrini, asciutto ed applicare alcune gocce di mezzo di montaggio antifade, quindi coprire lentamente le sezioni con un coprioggetto di vetro. Conservare i vetrini a 4 ° C. </li><li> Prendete le immagini su un microscopio confocale con un obiettivo ad immersione in olio 40X con una apertura numerica di 1,3. </li></ol> 4. confocale [Ca 2+] i Imaging NOTA: [Ca 2+] i di imaging è stato fatto utilizzando un microscopio confocale con un 40X acqua ad immersione lente dell&#39;obiettivo con una apertura numerica di 0,8. <ol><li> Tra 1 e 5 ore dopo il taglio, fetta posto nella camera di registrazione e superfuse con buffer di registrazione ossigenata a temperatura ambiente con una portata di 1 - 2 ml permin. Poi posto un&#39;arpa platino con corde di nylon sulla parte superiore della fetta di ridurre al minimo il movimento durante l&#39;esperimento. </li><li> Visualizza e individuare striato dorsale con campo chiaro luminescenza con un obiettivo 10X immersione in acqua. </li><li> Passare alla lente obiettivo 40X immersione in acqua, e accendere la linea 488 nm del laser Argon. Modificando il piano focale, trovare cellule situate a circa 20 micron sotto la superficie fetta, la visualizzazione di fluorescenza basale a Soma, rami e ramoscelli. Da segnalare, le cellule compromesse solito mostrano il livello di fluorescenza di sopra della media, che dovrebbe essere evitato. </li><li> Utilizzare la modalità di &#39;scansione intermittente&#39; per avviare la scansione. Generalmente, l&#39;intensità del laser regolata a 0,5 - 5% della potenza massima è sufficiente image [Ca 2+] i con cito-GCaMP3. Per il monitoraggio [Ca 2+] i dinamiche in tutto il territorio degli astrociti, la &#39;scansione intermittente&#39;, con una velocità di scansione di 1 secondo per fotogramma o più veloce è raccnded, ma questo deve essere deciso a seconda dell&#39;esperimento in questione. </li><li> Se necessario, per l&#39;immagine [Ca 2+] i nei processi, usare lo zoom digitale di 2 - 3 volte a monitorare 1 - 2 astrociti in tutto il campo visivo. </li><li> Per evitare i pixel saturi durante la registrazione, utilizzare la modalità di ricerca &#39;HiLo&#39; per PseudoColor le cellule. Iniziare sempre con una &quot;registrazione pilota&quot; per circa 5 minuti, per verificare se il colore rosso appare come l&#39;indicazione di saturazione. In tal caso, abbassare la potenza di uscita del laser, o abbassare il PMT / guadagno / valori di offset e di effettuare un&#39;altra registrazione pilota per testare i valori dei pixel sono nell&#39;intervallo non saturo. In genere, i parametri utilizzati per l&#39;imaging combinate astrociti [Ca 2+] i nello striato sono i seguenti: potenza del laser in uscita 0,5-5% (10 mW), PMT 550-650 Volt, Guadagno 2.0 - 3.5X, e offset 0 - 5%. </li></ol>

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I metodi qui descritti hanno permesso di esprimere cito-GCaMP3 negli astrociti striatali in vivo per la successiva [Ca 2+] i di imaging in situ. Questo metodo presenta vantaggi rispetto utilizzando transgenici o topi knock-in, compreso robusta espressione della proteina bersaglio, rapidità e flessibilità di implementazione sperimentale e specificità anatomica. L&#39;espressione di GCaMP3 utilizzando AAV2 / 5 è risultato essere specifico e robusto. La combinazione di GFAP GfaAB...

Gli astrociti sono cellule gliali onnipresenti e abbondanti del cervello. E &#39;ben noto che gli astrociti servono di sostegno vitale e ruoli omeostatici, tra cui il buffering di K + concentrazione nello spazio extracellulare, l&#39;assorbimento di neurotrasmettitori oltre a fornire le sostanze nutritive. Tuttavia, studi recenti dimostrano che anche la visualizzazione [Ca 2+] i segnali, che si verificano spontaneamente e sono aumentate di attività neuronale 1. L&#39;esistenza di astrociti [Ca 2+] i segnalazione è stata sempre più pensato per innescare la loro comunicazione con i neuroni, e come tale è stato interpretato come una forma di &quot;Ca 2+ eccitabilità&quot; all&#39;interno di astrociti. I dati disponibili nel corso degli ultimi due decenni suggeriscono due ambienti in cui gli astrociti e neuroni possono comunicare, forse in maniera bidirezionale. In primo luogo, gli astrociti spesso rispondono con un aumento della [Ca2 +] i, quando attivato da neurotrasmettitori eneuromodulatori rilasciati dai neuroni 2. In secondo luogo, [Ca 2+] i accresce in astrociti causare il rilascio di molecole di segnalazione da astrociti, che a loro volta possono influenzare i neuroni e vasi sanguigni. L&#39;evidenza suggerisce che molecole rilasciate dagli astrociti portano a cambiamenti nelle funzioni delle sinapsi, circuiti e in ultima analisi il comportamento 3-5 tramite segnalazione astrociti-to-neurone. Tuttavia, questo rimane un settore di ricerca in rapido sviluppo, e si è sostenuto che una migliore e dettagliata comprensione di astrociti [Ca 2+] i è necessaria per risolvere alcune delle attuali incertezze 6. Nel lavoro passato, è stato dimostrato che grosso carico di organici Ca 2 + indicatore coloranti in astrociti non riesce a rilevare in modo affidabile [Ca 2+] i segnali all&#39;interno intere astrociti in coltura e in situ 7-10. Questi risultati sono stati discussi da noi e altri 6,11,12. Il Emerging quadro è che [Ca 2+] i segnali all&#39;interno dei processi di astrociti (ad esempio, rami e ramoscelli), che sono i siti principali per le interazioni con i neuroni e vasi sanguigni, sono stati raramente esplorato in dettaglio. Recentemente, l&#39;uso di indicatori di calcio geneticamente codificati (GECIS) come GCaMP3 citosolica, GCaMP5G e GCaMP6 e membrana plasmatica versioni tethered (ad esempio, Lck-GCaMP3) ha permesso lo studio di [Ca 2+] i segnali in piccoli scomparti di astrociti tali processi sottili, vicino alla membrana plasmatica ed entro interi territori 7,8. Tuttavia, GECIS hanno uno svantaggio negli organici Ca 2+ indicatore coloranti e che è il requisito per i metodi genetici per fornire i geni che codificano selettivamente ai astrociti in vivo per periodi di settimane per il GECIS di essere adeguatamente espresso. Espressione in vivo è tipicamente realizzato utilizzando topi transgenici, knock-in topi o con virus a base di consegna appscarafaggi. Nel presente articolo Giove riportiamo i metodi e le procedure utilizzate per fornire GECIS di astrociti striatali che utilizzano virus adeno-associati. Facciamo il cito-GCaMP3 come esempio, ma la stessa procedura di base funziona per qualsiasi altra GECI o fluorescenti giornalista base di proteine.

<table><tbody><tr><td>Syringe Pump</td><td>Harvard Apparatus</td><td>704506</td><td></td></tr><tr><td>Glass Capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td></td></tr><tr><td>Micropipette puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Micropipette grinder</td><td>Narishige</td><td>EG-40</td><td></td></tr><tr><td>pZac2.1 GfaABC&lt;sub&gt;1&lt;/sub&gt;D.cyto-GCaMP3</td><td>Addgene</td><td>44331</td><td>a plasmid sent to UPenn Vector Core for virus packaging</td></tr><tr><td>1 ml syringe</td><td>BD</td><td>309628</td><td></td></tr><tr><td>syringe needle</td><td>BD</td><td>305109</td><td></td></tr><tr><td>AAV2/5 virus</td><td>UPenn vector core</td><td>NA</td><td></td></tr><tr><td>Sudan red IV</td><td>Sigma-Aldrich</td><td>67386</td><td></td></tr><tr><td>Mineral oil</td><td>CVS Pharmacy</td><td>152355</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050 S</td><td></td></tr><tr><td>Stereotaxic instrument</td><td>David Kopf Instruments</td><td>900LS</td><td></td></tr><tr><td>High Speed Rotary Micromotor Kit</td><td>FOREDOM</td><td>K.1070</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Santa cruz biotechnology</td><td>sc-281692</td><td></td></tr><tr><td>Super Glue</td><td>Krazy&amp;reg;Glue</td><td>KG925</td><td></td></tr><tr><td>Microslicer</td><td>Ted Pella</td><td>DTK-Zero 1</td><td></td></tr><tr><td>Confocal microscopes</td><td>Olympus</td><td>FV300 and FV1000</td><td></td></tr><tr><td>Normal goat serum</td><td>Vector</td><td>S-1000</td><td></td></tr><tr><td>chicken anti-GFP</td><td>Abcam</td><td>ab13970</td><td></td></tr><tr><td>mouse anti-s100&amp;beta;</td><td>Sigma-Aldrich</td><td>S2532</td><td></td></tr><tr><td>mouse anti-NeuN</td><td>Millipore</td><td>MAB377</td><td></td></tr><tr><td>mouse anti-glutamine synthetase</td><td>Millipore</td><td>MAB302</td><td></td></tr><tr><td>goat anti-mouse-Alexa546</td><td>Invitrogen</td><td>A11003</td><td></td></tr><tr><td>goat anti-chicken-Alexa488</td><td>Invitrogen</td><td>A11039</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Cover Glass</td><td>Fisher Scientific</td><td>12-548-5J</td><td></td></tr><tr><td>Mounting Medium</td><td>Vector</td><td>H-1000</td><td></td></tr></tbody></table>

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.

Per astrociti espressione specifica del cito-GCaMP3 nello striato, abbiamo usato il virus adeno-associato (AAV) del sierotipo 5, e la GFAP GfaABC 1 D promotore (Figura 1A), che è stato precedentemente dimostrato da guidare robusta GCaMP3 e gene reporter espressione in ippocampo e astrociti corticali 8,14. Due settimane dopo microiniezione virus nello striato del mouse, il mouse (~ 10 settimane) è stato perfuso e IHC è stata eseguita su sezioni di cervello sottili per valutare l&...

Watch this Scientific Journal Video about Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators at JoVE.com

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 ...

imaging-intracellular-ca2-signals-striatal-astrocytes-from-adult-mice

Research

JoVE Journal

Neuroscience

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

14.8K Views.  University of California Los Angeles. 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. 

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

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

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, ...

Ricerca

Biologia

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 ...

Neuroscienze

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 ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

Ex Vivo Formazione immagine di segnalazione alla sinapsi tripartita del diaframma del Mouse cellula-specifico calcio

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imaging del calcio nell'oliva inferiore del topo

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Calcio dei periciti cerebrali e imaging emodinamico nei topi transgenici in vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

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 ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

In vivo Imaging del calcio a campo largo e a due fotoni da un topo utilizzando una grande finestra cranica

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

In vivo Imaging del calcio di insiemi neuronali in reti di neuroni sensoriali primari nei gangli della radice dorsale intatti

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Imaging in tempo reale per l'attività neuronale nelle cellule del giro dentato dei topi 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

Autore in primo piano: Studio dell'attività neurale delle cellule granulari del giro dentato con il microscopio in miniatura

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

Imaging in vivo del calcio delle cellule dei granuli nel giro dentato dell'ippocampo nei topi 

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

Imaging intracellulare Ca

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