La majorité des travaux et le personnel impliqués ont été pris en charge par le NIH subvention NS060677 et en partie par les NIH accorde MH099559 et MH104069 (à BSK). Une partie du travail a également été soutenu par la Fondation CHDI.

Tous les protocoles d&#39;animaux étaient en conformité avec les Instituts nationaux américains de la santé Guide pour le soin et l&#39;utilisation des animaux de laboratoire et ont été approuvés par le soin et l&#39;utilisation des animaux Commission institutionnelle à l&#39;UCLA. 1.1) Préparer Micropipette et AAV2 / 5 Virus de chargement <ol><li> Utilisation de fines micropipettes en verre borosilicaté pour l&#39;injection du virus. Tirez la micropipette en utilisant un programme de traction en deux étapes avec un extracteur vertical. Biseau la pipette à un angle de 40 ° en utilisant un broyeur de pipette. La pipette qui est idéal pour être utilisé aura un diamètre de pointe de 20 à 40 um et une longueur de tige de 6-7 mm. Autoclaver la pipette et instruments chirurgicaux. Le foret est stérilisée par essuyage avec 70% d&#39;éthanol. </li><li> Stocker le virus AAV2 / 5-GfaABC une D.cyto-GCaMP3.SV40 (1,5 x 10 13 gc / ml), à -80 ° C dans des aliquotes de 10 pi. Juste avant la chirurgie, prendre les aliquotes du congélateur et stocker sur la glace jusqu&#39;à son utilisation pour le remplissage ee micropipette. </li><li> Remplir la micropipette avec le virus en utilisant une pompe à seringue. Relier hermétiquement une extrémité d&#39;un morceau de tuyau, à travers un tube de compression raccord de taille appropriée, d&#39;une seringue en verre avec un piston renforcé. Attacher la micropipette autoclave à l&#39;autre extrémité du morceau de tube par l&#39;intermédiaire d&#39;une compression de l&#39;aiguille de taille appropriée raccord amovible. </li><li> Avant de charger le vecteur viral, d&#39;éliminer les bulles d&#39;air du système de pompage comprenant un tube, seringue et micropipette par remplissage de l&#39;installation avec une huile minérale de couleur avec du Soudan IV rouge (~ 1 mg / 50 ml) dans une seringue de 1 ml avec une aiguille (27 G ). </li><li> Placez un morceau de parafilm propre sous la micropipette de verre, et distribuer 5 - 10 pi du vecteur viral sur le parafilm. </li><li> Sucer le vecteur viral en déplaçant le piston de la seringue vers l&#39;arrière à 0,5 pi / min, et marquer la limite entre le vecteur et l&#39;huile sur la pipette de verre. </li></ol> 1.2) Anesthésie, coupe de cheveux, HEAD fixation et préparation pour Craniotomy <ol><li> Mettre une souris (P49-63, C57BL / 6) dans la chambre rempli de N 2 O et O 2, et 5% d&#39;isoflurane. Évaluer la profondeur de l&#39;anesthésie par une perte de mouvement intentionnel et un rythme respiratoire ralenti (~ 60-90 / min). Couper les cheveux sur la tête quand la souris est anesthésiée. </li><li> Monter la souris dans le cadre stéréotaxique, avec sa tête fixé par des barres d&#39;oreille émoussées et son nez placés dans un système d&#39;anesthésie et de ventilation. Maintenir l&#39;isoflurane continu à 2 - 3%. Appliquer des larmes artificielles pommade pour les deux yeux puisque les souris perdent leur réflexe de clignement sous anesthésie. </li><li> Administrer 0,05 ml de buprénorphine (0,1 mg / ml) par injection sous-cutanée pour le soulagement de la douleur. </li><li> Zone chirurgicale pur avec 10% de povidone iode et 70% d&#39;alcool trois fois, en alternant entre les deux solutions de départ et avec de la povidone iode. Commencez par l&#39;avant et vers l&#39;arrière essuyer pour enlever les poils déjà coupées. </li><li> Faire une incision de la peau sur le dessus of la tête qui commence entre les yeux et se termine entre les oreilles. </li></ol> 1.3) Craniotomy et microinjections <ol><li> Retirez le périoste en balayant avec un coton-tige, puis sécher la surface du crâne avec des boules de coton dentaires. Faire une marque sur le site d&#39;injection, et percer sur le pourtour de la marque à l&#39;aide d&#39;une petite fraise en acier alimenté par un forage à grande vitesse. </li><li> Retirer l&#39;os et nettoyer la surface avec une solution saline. </li><li> Placez la pipette dans la dorsale latéral gauche striatum avec les coordonnées suivantes (mm): 0,8 antéro-postérieur, médio-latérale: 2,0, dorso-ventral de la surface piales: -2,4 13. A noter que ces coordonnées signifie que la pointe de l&#39;aiguille de micro-injection se trouve juste au-dessus du striatum dorso-latéral (figure 1A, B), ce qui signifie qu&#39;il ne pénètre légèrement à l&#39;intérieur de ce noyau. Évitez d&#39;endommager les vaisseaux sanguins. Si la pipette se trouve être à droite sur un vaisseau sanguin, légèrement ajuster lales coordonnées de ~ 0,1 à 0,2 mm. </li><li> Lancer injection avec un taux d&#39;injection fixée à 0,2 pi / min, et injecter 1 à 1,5 ul de virus. </li><li> Laisser la pipette en place après l&#39;injection pendant 10 min, puis retirer la pipette lentement. </li><li> Terminez par la fermeture de la plaie chirurgicale avec suture continue nylon externe suture. </li></ol> 1.4) Recouvrement <ol><li> Mettez la souris dans une cage propre sur un coussin chauffant pendant 24 heures. Ne retournez pas une souris qui a subi une intervention chirurgicale à la compagnie d&#39;autres animaux jusqu&#39;à guérison complète. Ne laissez pas la souris sans surveillance tant qu&#39;il a repris conscience suffisante pour maintenir décubitus sternal. </li><li> Ajouter triméthoprime Sulfamethoxiazole (5 ml par 500 de l&#39;eau, contenant 40 mg de triméthoprime et 200 mg sulfamethoxiazole) dans l&#39;eau pendant une semaine. Administrer buprénorphine deux fois par jour pour un maximum de trois jours après la chirurgie. </li><li> Maintenir la souris sur un cycle lumière-obscurité de 12 h, nourris et abreuvés quotidiennement, une est pondéré une fois par jour jusqu&#39;à 3 jours après la chirurgie. Dans ces 3 jours, aucune souris avec une perte de plus de 10% du poids du corps est considérée comme compromise, et sera euthanasiée au lieu d&#39;être soumis à l&#39;expérimentation. </li><li> Par ailleurs, changer la cage de la souris deux fois par semaine, et de surveiller la santé générale au moins une fois par jour. En général, la souris est utilisé dans les 2 - 3 semaines après les micro-injections stéréotaxiques. </li></ol> 2. Trancher aiguë cerveau Préparation pour la Imaging confocale Ca 2.1) Préparation de coupe et Solutions d&#39;enregistrement. <ol><li> Préparer une solution comprenant (mM) de coupe: 194 saccharose, 30 NaCl, 4,5 KCl, 10 le D-glucose, 1 de MgCl2, 1,2 NaH 2 PO 4 et NaHCO 3 26, saturé avec 95% O 2 et 5% CO 2. </li><li> Préparer la solution d&#39;enregistrement comprenant (mM): 124 NaCl, 4,5 KCl, 1 MgCl2, 10 le D-glucose, 2 CaCl2, 1,2 NaH 2 PO 4 </sub&gt;, et 26 NaHCO 3; pH 7.3 à 7.4, de 290 à 295 mOsm, saturé avec 95% O 2 et 5% CO 2. Remplir le bécher tranche de cerveau tenue avec une solution d&#39;enregistrement, et les conserver à 34 ° C. </li></ol> 2.2) tranchage <ol><li> Deux à trois semaines après microinjections stéréotaxiques, profondément et terminale anesthésier la souris, puis décapiter. </li><li> Extraire le cerveau, et utiliser une lame pour enlever l&#39;hémisphère droit non injectée, et monter celle de gauche sur le plateau vibratome utilisant la super glue. Remplissez le bac à glace vibratome avec un tampon de coupe froid et gardez-le saturant avec 95% d&#39;O 2 et 5% de CO 2. </li><li> Couper les tranches de cerveau striatum coronale ou sagittale à 300 um d&#39;épaisseur. Habituellement, 4-5 coronaire, ou 6-7 tranches striatales sagittales peuvent être collectées. </li><li> Transférer les tranches dans le bécher de maintien de tranche réchauffé à 34 ° C, et de les y maintenir pendant 30 min avant de les stocker à température ambiante pendant recordi ultérieureng. </li><li> Incuber les tranches de cerveau dans du tampon d&#39;enregistrement oxygénée à la température ambiante pendant au moins 30 min avant de [Ca2 +] i imagerie. </li></ol> 3. L&#39;immunohistochimie (IHC) <ol><li> Deux semaines après l&#39;injection du virus, la souris perfuser transcardiaque avec du PBS suivi par 10% de formaline dans du PBS. </li><li> Retirer, post-fixer nuit, et cryoprotect le cerveau en tampon saccharose à 30% pendant au moins 2 jours. </li><li> Préparer 40 um sections à l&#39;aide d&#39;un microtome de cryostat. </li><li> Rincer les sections 3 fois avec du PBS à intervalle de 10 min. </li><li> Incuber les coupes pendant 1 h dans du PBS avec 10% de sérum de chèvre normal et avec 0,5% de Triton X-100. </li><li> Incuber les coupes pendant une nuit à 4 ° C dans l&#39;anticorps primaire préparée dans du PBS avec 0,5% de Triton X-100. Les anticorps primaires utilisés étaient les suivants: anti-GFP poulet 1: 1000, de souris anti-S100β 1: 400, de souris anti-NeuN 1: 600, de souris anti-glutamine synthétase 1: 300. </li><li> Rincer les sections 3 fois avec du PBS à 10 mdans les intervalles. </li><li> Incuber les coupes avec des anticorps secondaires préparées dans du PBS avec 10% de sérum de chèvre normal pendant 2 h à température ambiante. Les anticorps secondaires étaient les suivants: chèvre anti-souris Alexa546 1: 500, de chèvre anti-poulet Alexa488 1: 500. </li><li> Monter les coupes sur des lames de verre, sécher et appliquer quelques gouttes de antifade milieu de montage, puis couvrir lentement les sections avec une lamelle de verre. Conserver les lames à 4 ° C. </li><li> Prendre des images sur un microscope confocal avec un objectif à immersion dans l&#39;huile 40X avec une ouverture numérique de 1,3. </li></ol> 4. confocale [Ca 2+] i Imaging REMARQUE: [Ca2 +] i a été effectuée en utilisant l&#39;imagerie de microscope confocal avec un objectif 40X à immersion d&#39;eau avec une ouverture numérique de 0,8. <ol><li> Entre 1 et 5 h après la coupe, tranche lieu dans la chambre d&#39;enregistrement et superfuse avec le tampon d&#39;enregistrement oxygéné à température ambiante avec un débit de 1 - 2 ml parmin. Ensuite, placez une harpe de platine avec des cordes en nylon sur le dessus de la tranche pour minimiser le mouvement pendant l&#39;expérience. </li><li> Visualiser et localiser le striatum dorsal avec le champ lumineux luminescence sous un objectif à immersion d&#39;eau 10X. </li><li> Passez à l&#39;immersion dans l&#39;eau 40X objectif, et tourner sur la ligne 488 nm du laser Argon. En changeant le plan focal, trouver des cellules situées à environ 20 um sous la surface de la tranche, affichant fluorescence basale à Soma, branches et rameaux. Fait à noter, les cellules compromis affichent généralement le niveau de fluorescence dessus de la moyenne, qui devrait être évité. </li><li> Utilisez le mode «balayage d&#39;image» pour lancer la numérisation. En général, l&#39;intensité du laser ajusté à 0,5 à 5% de la puissance maximale est suffisante pour l&#39;image [Ca 2+] i par cyto-GCaMP3. Pour la surveillance de [Ca2 +] i dynamique dans l&#39;ensemble du territoire des astrocytes, le &#39;balayage d&#39;image »avec une vitesse de balayage de 1 s par châssis ou plus rapide est recommeDEMVSO, mais cela doit être décidé en fonction de l&#39;expérience en question. </li><li> Si nécessaire, à l&#39;image de [Ca 2+] i dans les processus, utiliser un grossissement numérique de 2 - 3 fois pour surveiller 1 - 2 astrocytes dans l&#39;ensemble du champ de vue. </li><li> Pour éviter pixels saturés pendant l&#39;enregistrement, utilisez le mode de recherche &#39;HiLo&#39; de pseudo-cellules. Toujours commencer par un &quot;enregistrement de pilote&quot; pendant environ 5 min, à tester si la couleur rouge apparaît comme indication de saturation. Si oui, plus la puissance de sortie de laser, ou abaisser le PMT / Gain / valeurs de décalage et d&#39;effectuer un autre enregistrement de pilote pour tester les valeurs de pixels sont dans la gamme non saturé. En règle générale, les paramètres combinés utilisés pour l&#39;imagerie des astrocytes [Ca 2+] i dans le striatum sont les suivantes: laser de puissance de sortie de 0,5 à 5% (de 10 mW), PMT 550 à 650 Volts, Gain 2.0 - 3.5x, et l&#39;offset 0 - 5%. </li></ol>

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Les méthodes décrites ici nous ont permis d&#39;exprimer cyto-GCaMP3 dans les astrocytes du striatum in vivo pour la suite [Ca 2+] i imagerie in situ. Cette méthode a des avantages sur l&#39;aide transgénique ou souris knock-in, y compris l&#39;expression robuste de la protéine ciblée, la rapidité et la souplesse de mise en œuvre expérimentale et la spécificité anatomique. L&#39;expression de GCaMP3 utilisant VAA2 / 5 a été trouvé pour être plus précis et robuste. ...

Les astrocytes sont des cellules gliales omniprésents et abondantes du cerveau. Il est bien établi que les astrocytes servent soutien vital et rôles homéostatiques y compris tampon de concentration de K + dans l&#39;espace extracellulaire, recaptage des neurotransmetteurs, ainsi que fournir des éléments nutritifs. Cependant, des études récentes montrent qu&#39;ils affichent aussi [Ca2 +] i signaux, qui se produisent spontanément et sont augmentés de l&#39;activité neuronale 1. L&#39;existence d&#39;astrocytes [Ca 2+] i la signalisation a été de plus en plus pensé à déclencher leur communication avec les neurones, et en tant que tel a été interprétée comme une forme de &quot;Ca 2+ excitabilité&quot; dans les astrocytes. Les données disponibles au cours des deux dernières décennies suggèrent deux contextes dans lesquels les astrocytes et les neurones peuvent communiquer, peut-être d&#39;une manière bidirectionnelle. Tout d&#39;abord, les astrocytes réagissent souvent avec une augmentation de la [Ca 2+] i lorsqu&#39;il est activé par les neurotransmetteurs etneuromodulateurs libérés par les neurones 2. Deuxièmement, [Ca 2+] i augmente dans les astrocytes provoquer la libération de molécules de signalisation des astrocytes qui peuvent à leur tour affecter les neurones et les vaisseaux sanguins. Les preuves suggèrent que les molécules libérées par les astrocytes conduisent à des changements dans les fonctions de synapses, les circuits et en fin de compte le comportement de signalisation par l&#39;intermédiaire de 3 à 5 astrocyte-à-neurone. Cependant, cela reste un domaine de recherche en plein développement, et il a été avancé que d&#39;une meilleure compréhension et détaillée des astrocytes [Ca 2+] i est nécessaire pour résoudre certaines des incertitudes actuelles 6. Dans le travail passé, il a été démontré que le chargement en vrac de Ca 2+ organiques colorants indicateurs dans les astrocytes ne parvient pas à détecter de manière fiable [Ca 2+] i signaux dans les astrocytes entiers de la culture et in situ 7-10. Ces résultats ont été discutés par nous et d&#39;autres 6,11,12. Le Emerging image est que [Ca 2+] i signaux au sein des processus d&#39;astrocytes (par exemple, les branches et rameaux), qui sont les premiers sites d&#39;interactions avec les neurones et les vaisseaux sanguins, ont rarement été étudiée en détail. Récemment, l&#39;utilisation d&#39;indicateurs de calcium génétiquement codées (GeCIS) comme GCaMP3 cytosolique, GCaMP5G et GCaMP6 et membrane plasmique versions captif (par exemple, Lck-GCaMP3) a permis l&#39;étude de [Ca 2+] i signaux dans de petits compartiments de astrocytes ces processus minces, près de la membrane plasmique et dans des territoires entiers 7,8. Cependant, GeCIS ont un inconvénient plus organiques Ca 2+ colorants indicateurs et qui est l&#39;exigence des méthodes génétiques pour livrer les gènes codant sélective pour les astrocytes in vivo pour des périodes de plusieurs semaines pour la GeCIS être appropriée exprimé. Expression in vivo est généralement réalisé en utilisant des souris transgéniques, souris knock-in ou par le virus basé livraison appcafards. Dans l&#39;article présent JoVE nous présentons les méthodes et procédures utilisées pour fournir GeCIS à astrocytes striataux utilisant des virus adéno-associés. Nous nous concentrons sur cyto-GCaMP3 comme un exemple, mais la même procédure de base fonctionne pour tout autre GECI ou fluorescent reporter à base de protéines.

<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.

Pour les astrocytes expression spécifique de cyto-GCaMP3 dans le striatum, nous avons utilisé un virus adéno-associé (AAV) du 5 serotype, et la GFAP GfaABC 1 promoteur de D (figure 1A), qui a été précédemment montré pour conduire GCaMP3 robuste et gène rapporteur expression dans l&#39;hippocampe et les astrocytes corticaux 8,14. Deux semaines après le virus de la micro-injection dans le striatum de souris, la souris (~ 10 semaines) a été perfusée et IHC a été réali...

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.

Imagerie intracellulaire de Ca<sup&gt; 2+</sup&gt; Signaux dans Striatale astrocytes de souris adultes utilisant des indicateurs de calcium génétiquement codés

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 vues.  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. 

Vidéo: 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.

Réponses d&#39;imagerie neuronale dans les préparations d&#39;organe voméronasal Slice Exprimant un capteur de calcium codées génétiquement

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

Recherche

Neurosciences

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.

Enregistrement simultané des signaux calciques de neurones identifiés et le comportement alimentaire des 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.

Modélisation astrocytome pathogenèse<em&gt; In Vitro</em&gt; Et<em&gt; In Vivo</em&gt; Utilisation corticale astrocytes ou cellules souches neurales à partir de, souris génétiquement modifiées conditionnelle

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 potentiel de membrane avec deux types de capteurs codés génétiquement tension fluorescentes

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.

Contrôle optique d&#39;une protéine Neuronal L&#39;utilisation d&#39;un acide aminé Unnatural génétiquement codificateur dans Neurones

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.

Dissection des signaux locaux Ca2 + dans les cellules cultivées par axés sur la Membrane Ca2 + indicateurs

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.

Indicateurs synapses uniques de la libération et de l’absorption des glutamates dans les tranches cérébrales aigues des souris normales et huntingtones

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.

Visualisation et analyse du transport intracellulaire d'organites et d'autres cargaisons dans les 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 ...

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.

Minimiser l’hypoxie dans les tranches hippocampales des souris adultes et vieillissantes

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.