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 border="0" cellpadding="0" cellspacing="0" >
	<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 GfaABC1D.cyto-GCaMP3</td>
			<td >Addgene</td>
			<td>44331</td>
			<td >a plasmid sent to UPenn Vector Core for virus packaging</td>
		</tr>
		<tr >
			<td >I 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&#174;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&#946;</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

14.7K 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

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Le risposte di imaging neuronale in preparazione Fetta di organo vomeronasale Esprimere un sensore di calcio geneticamente codificato

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Ricerca

Neuroscienze

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

La registrazione simultanea dei segnali di calcio da parte dei neuroni identificati e comportamento alimentare di Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modellazione Astrocitoma Patogenesi<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Utilizzo corticale astrociti o cellule staminali neurali da condizionale, topi geneticamente ingegnerizzati

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Imaging potenziale di membrana con due tipi di geneticamente codificati fluorescenti di sensori di tensione

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Controllo ottico di una proteina neuronale Utilizzando un acido Innaturale Amino geneticamente codificata in neuroni

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

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 ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Indicatori di sinapsi singola di Soldamento Release e Uptake in Acute Brain Slices da Topi Normali e Huntington

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

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 ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

Ridurre al minimo l'ipossia nelle fette di ippocampale da topi adulti e anziani

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

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.