We are supported by the University of Wisconsin-Madison Medical School start-up (A.V.G.).

Toutes les expériences ont été menées en conformité avec les National Institutes of Health Guide pour le soin et l&#39;utilisation des animaux de laboratoire (NIH Pub. N ° 80-23). Toutes les méthodes et les protocoles utilisés dans ces études ont été approuvées par l&#39;Université du Wisconsin Animal Care et utilisation Comité du Protocole suivant les directives sur les soins et l&#39;utilisation des animaux de laboratoire publiées par le NIH (publication n ° 85-23, révisée en 1996). Tous les animaux utilisés dans cette étude ont reçu des soins humains en conformité avec le Guide pour le soin et l&#39;utilisation des animaux de laboratoire. 1. Heart Enlèvement et Langendorff Perfusion <ol><li> Avant l&#39;isolement du coeur, une solution de Tyrode chaude à 37 ° C en utilisant un bain d&#39;eau et d&#39;une chemise d&#39;eau. </li><li> Anesthésier la souris avec 5% d&#39; isoflurane / 95% de O 2. </li><li> Veiller à ce niveau approprié de l&#39;anesthésie en vérifiant la perte de réflexe de la douleur. </li><li> Effectuer thoracotomie. Ouvrez le coffre à l&#39;aide de courbes 5.5 &quot;ciseaux Mayo et 5.5&quot; hémostatique KellyCèpes pour faire 1 cm découpés sur le devant du thorax. <ol><li> extraire rapidement le cœur de la poitrine (à moins de 30 secondes). Utilisez 4 &quot;courbes pince Iris pour saisir le tissu pulmonaire et découper le poumon, le thymus et le cœur en même temps que le péricarde en utilisant 4.3&quot; ciseaux Iris. Ne pas saisir directement le cœur. </li><li> Laver en oxygéné (95% O 2/5% CO 2), à température constante (36,8 ± 0,4 ° C) a modifié la solution de Tyrode. Utiliser la composition suivante en solution (en mM): NaCl 128,2, KCl 4,7, 1,19 NaH 2 PO 4, 1,05 MgCl2, 1,3 CaCl2, 20,0 NaHCO 3 et du glucose 11,1 (pH 7,35 ± 0,05). </li></ol></li><li> Tout en baignant dans une même solution, d&#39;identifier les poumons, le thymus et les tissus adipeux. les disséquer avec soin du cœur. Utilisez courbe 3 &quot;ciseaux Vannas-Tübingen et 4.3&quot; # 5 forceps. </li><li> Ensuite, identifier l&#39;aorte et canuler il sur un 21 G canule sur-mesure. Utilisez deux 4.3 vigueur # 5B courbe &quot;ps. </li><li> Après canulation perfuser simultanément (par le biais de la canule à un débit d&#39;environ 5 ml / min, ce qui devrait être ajustée en fonction de la pression aortique, voir ci-dessous) et superfuse (dans le bain à un débit d&#39;environ 50 ml / min) le coeur avec la solution de Tyrode chauffée et filtrée à travers toute l&#39;expérience. Passer la solution de perfusion à travers un filtre en nylon de 11 pm en ligne pour éviter le colmatage de la circulation coronaire. </li><li> À l&#39;aide d&#39;un transducteur de pression (ou un manomètre) relié par l&#39;intermédiaire d&#39;un robinet à 3 voies Luer à la ligne de perfusion, de surveiller la pression aortique et la maintenir entre 60 et 80 mmHg. Ajustez la vitesse de perfusion si nécessaire pour maintenir la pression aortique dans cette plage. </li></ol> 2. Cartographie optique du SAN du Langendorff perfusé Coeur entier <ol><li> Instrumentation <ol><li> Placer horizontalement le coeur et la broche de la pointe du ventricule au fond siliconé de la chambre à l&#39;aide de 0,1 mm de diamètre à goupilles prevent mouvement pendant l&#39;expérience induite par flux. 12 </li><li> Insérer une petite (0,012 &quot;ID x 0,005&quot;), le tube de silicium dans le ventricule gauche (VG) par l&#39;intermédiaire de la veine pulmonaire, l&#39;oreillette gauche (LA) et la valve mitrale (MV). Fixer le tube par une suture de soie (4-0) pour le tissu conjonctif de sondage. </li><li> Positionner le coeur avec son côté postérieur vers le haut (figure 1A, panneau de gauche). </li><li> Broche le bord de l&#39;appendice RA (RAA) au fond de la chambre de silicium en utilisant des broches de diamètre minuties 0,1 mm. Régler son niveau afin de rendre la surface postérieure de la RA plat et lui permettre d&#39;être situé plan focal de la caméra. Cela va permettre des mesures optiques à partir de la surface maximale de l&#39;atrium. </li><li> Noose supérieure (SVC) et inférieure (VCI) de la veine cave par une suture de soie (4-0), étirer et épingler l&#39;autre extrémité de la suture au fond d&#39;une chambre de siliconé (voir la figure 3A). Assurez-vous que les sutures ne bloquent pas lachamp de vue optique. </li><li> Placez l&#39;électrode de stimulation sur mesure sur le bord de la RAA. Pour rendre les électrodes, l&#39;utilisation du silicium revêtu de 0,25 mm de diamètre des fils en argent, avec 0,5 mm de distance inter-électrodes et dépourvue de silicium pour une longueur de 1 mm à l&#39;extrémité de stimulation. Ensuite, placez deux aiguilles mm ECG 12 (29 gauge) électrodes monopolaires près de la base des ventricules droit et gauche. Placer le sol ECG électrode à proximité du sommet des ventricules. </li></ol></li><li> tachant <ol><li> Comme les deux colorants fluorescents et électro-mécanique découplant blebbistatin sont sensibles à la lumière, effectuer toutes les procédures décrites ci-dessous dans une pièce sombre. Préparer d&#39;abord sensible à la tension de colorant RH-237 ou di-4-ANNEPS comme une solution mère, 1,25 mg / ml dans du diméthylsulfoxyde (2,5 mM), aliquote il (30 pi chacun) et stocker les aliquots à -20 ° C. </li><li> Diluer 5-10 pi de solution de colorant disponible dans 1 ml de réchauffé (37 ° C) une solution de Tyrode, puis l&#39;injecter dans la ligne de perfusion coronairesur une période de 5-7 minutes en utilisant un orifice d&#39;injection en ligne Luer. </li><li> Préparer blebbistatin comme une solution mère (2 mg / ml dans le diméthylsulfoxyde, 6,8 mM) à l&#39;avance et le stocker à 4 ° C. </li><li> Après 20 minutes de stabilisation, ajouter 0,5 ml de réchauffé (37 ° C) blebbistatin dans le perfusat et diluer 0,1 ml de blebbistatin dans 1 ml de réchauffé (37 ° C), la solution de Tyrode. Puis injecter dans la ligne de perfusion coronaire pendant une période de 5-7 minutes en utilisant un orifice d&#39;injection en ligne Luer. </li></ol></li></ol> 3. cartographie optique du SAN du Atrial Préparation isolé <ol><li> Instrumentation <ol><li> Pour la préparation auriculaire isolée, isoler et cathétériser le cœur comme décrit ci-dessus dans les étapes 1.1-1.5 pour la préparation entière cœur. </li><li> Disséquer les ventricules loin de la face antérieure. Voir Figure 1A, panneau de droite, coupé # 1 pour plus de détails. </li><li> Ouvrez le RA en coupant à travers le va tricuspideLVE (TV) le long de l&#39;axe TV-SVC. Voir Figure 1A, panneau de droite, coupé # 2 pour plus de détails. </li><li> Couper la branche médiane de la crête terminale pour ouvrir le RAA. Voir Figure 1A, panneau de droite, coupé # 3. </li><li> Ouvrez la paroi libre auriculaire antérieure en effectuant une coupe de la ligne médiane de la coupe précédente n ° 3 sur le bord du coin inférieur droit du RAA, aplatir et épingler la paroi libre auriculaire au fond d&#39;une chambre de silicium revêtu. Voir la direction indiquée par la flèche creuse dans la figure 1A, coupe n ° 4. Préserver un rebord du tissu ventriculaire pour épingler la préparation pour éviter d&#39;endommager les oreillettes. </li><li> De même, ouvert LA en coupant à travers le MV le long du coin MV supérieur de l&#39;appendice LA (LAA). </li><li> Pour ouvrir le LAA, coupé à partir du milieu de l&#39;AAL ouvert, à travers la paroi libre auriculaire antérieure jusqu&#39;à près d&#39;un rebord milieu de la LAA. </li><li> Ouvrez la partie antérieure auriculaire paroi alo libreng la même direction, puis aplatir et épingler au fond d&#39;une chambre Sylgard revêtu. </li><li> Déposer partiellement le tissu septal interauriculaire. Cela permettra de réduire la diffusion du signal optique à partir de tissus qui ne sont pas au point. Par conséquent, à la fois le LA et RA ainsi que le réseau SAN et auriculo-ventriculaire jonction (AVJ) , sont accessibles dans cette préparation (figure 1B). </li><li> Soulever la préparation finale d&#39;environ 0,5 mm à partir du fond de la chambre de silicium revêtu afin de permettre surfusion à partir des deux surfaces épicardique et endocardique. </li><li> Superfuse la préparation avec réchauffé (37 ° C) une solution de Tyrode à une vitesse constante de ~ 12 ml / min pour un débit concentré situé près du SVC et ~ 30 ml / min pour une surfusion de bain. </li><li> Placez le Ag / AgCl 2 électrodes de stimulation sur mesure (0,25 mm de diamètre) sur le bord de la RAA disséqués. Ensuite, placez deux aiguilles ECG 12 mm (29G) des électrodes (monopolaire) près de la RAA et LAA, respectivement. Placez le sol ECG élusmonté près du AVJ. </li></ol></li><li> tachant <ol><li> Effectuer une application directe du colorant sensible à la tension (RH-237 ou di-4-ANNEPS). Pour cela, diluer 1-2 ul de la solution de colorant de stock en 1 mm de réchauffé (37 ° C) une solution de Tyrode et relâchez lentement le colorant dilué sur la surface de la préparation à l&#39;aide d&#39;un 1 mm pipette. </li><li> En variante, effectuer une coloration auriculaire avec le colorant sensible à la tension à travers une perfusion coronaire du coeur perfusé de Langendorff similaire à celle décrite ci-dessus pour la préparation entière cardiaque (étapes 2.2.1-2.2.2). <ol><li> Après la coloration de la perfusion coronaire, isoler la préparation auriculaire comme décrit. Effectuer supplémentaire coloration de la surface en cas de besoin pour atteindre un niveau de fluorescence satisfaisante. l&#39;isolement auriculaire rapide ne pas l&#39;eau de Javel du colorant et n&#39;a aucune incidence sur la qualité de la coloration. </li></ol></li><li> Immobiliser la préparation avec l&#39;électro-mécanique découplant, blebbistatin. Diluer 3-5 pi blebbistatin solution mère dans le réchauffé (37 ° C) une solution de Tyrode et lentement libérer le blebbistatin dilué sur la surface de la préparation et de la solution environnante. Depuis blebbistatin a été montré pour être sensible, 13,14 éviter une exposition prolongée à la lumière la lumière lors de la préparation et la coloration. </li><li> Effectuer application blebbistatin supplémentaire autant que nécessaire pour supprimer la contraction. Normalement jusqu&#39;à 3 applications supplémentaires (3-5 pi par chaque application) peut être utilisé pour supprimer l&#39;artefact de mouvement suffisamment pour reconstituer avec précision SAN et activation auriculaire. </li><li> Ajouter 0,5 ml supplémentaires de blebbistatin réchauffé dans le perfusat. Au cours de cette procédure, mettre en pause la surfusion pendant 30 secondes pour permettre au colorant / blebbistatin pour colorer le tissu auriculaire. </li></ol></li></ol> 4. Cartographie optique Set Up NOTE:. Une description détaillée du système de cartographie optique est prévue ailleurs 12 <ol><li> Utilisez un appareil photo avec un systèmeporal résolution de 2000 images / s ou plus, et une série de lentilles de condenseur pour atteindre une résolution spatiale de 100 um / pixel ou plus. Cela est nécessaire pour reconstruire l&#39;activation SAN et la propagation intranodale. </li><li> Pour réduire l&#39;artefact de mouvement de la solution vibrante, fixer un petit verre de couverture sur la surface de la solution sur le cœur / isolé préparation auriculaire. </li><li> Utiliser la lumière d&#39;excitation (520 ± 45 nm de longueur d&#39;onde) fournie par une lampe à halogène à faible bruit de courant constant. Diriger le faisceau de lumière filtrée sur la préparation à l&#39;aide d&#39;un guide de lumière bifurquée flexible. </li><li> Filtrer la fluorescence émise par un filtre passe-haut (&gt; 715 nm). Collecter, numériser et enregistrer le signal fluorescent acquis sur un logiciel d&#39;ordinateur à l&#39;aide fournie par le fabricant de l&#39;appareil. </li></ol> 5. Traitement des données REMARQUE: Les données de cartographie optique est collectée et stockée sous forme d&#39;une série de matrices d&#39;intensité de fluorescence à différents moments. Chaque pixel represeNTS une mesure de l&#39;intensité de fluorescence recueillie à partir d&#39;un emplacement spécifique sur la préparation de tissu à un point de temps spécifique. la fluorescence d&#39;arrière-plan est automatiquement retiré par pixel afin de permettre une meilleure visualisation des changements de fluorescence produites par les variations de tension de la membrane et vers le bas pour correspondre à des potentiels d&#39;action (PA) mesurés par les systèmes de micro-électrodes. Les détails des différentes étapes du traitement des données d&#39;imagerie optique, y compris la segmentation d&#39; images, filtrage spatial, filtrage temporel, et la dérive retrait de la ligne de base, sont fournis dans la revue ciblée 15. <ol><li> Manuellement ou en utilisant l&#39;algorithme spécial (par exemple, une détection de seuil ou le bord) 15 pour déterminer les limites de tissus, créer un masque binaire de 1 (pixels inclus) et 0 (pixels exclus) et appliquer le masque à chaque trame dans la séquence. Ce processus crée une image binaire qui met en lumière des domaines d&#39;intérêt pour un traitement ultérieur. </li><li> Pour améliorer le rapport signal-bruit de l&#39;optdonnées iques, filtrer les signaux dans les zones d&#39;intérêt sélectionnées. Pour cela, utilisez l&#39; espace (ie la moyenne des pixels fluorescents voisins tels que définis par un bac de convolution désiré ou noyau, par exemple 3 x 3 ou, pour les données bruitées, 5 x 5) et / ou filtrage temporel (par exemple, Butterworth, le type Chebyshev 1, Chebyshev type 2, Elliptic etc.) (Figure 2). Gardez à l&#39;esprit la possibilité d&#39;artefacts filtrés induite par l&#39;interprétation des données. Pour plus de détails, voir la revue 15. </li><li> Retirer la dérive de la ligne de base dans les enregistrements optiques en cas de besoin (en utilisant le filtrage passe-haut ou d&#39;un raccord polynôme du signal original), puis normaliser chaque signal de pixel de 0 (fluorescence minimum) à 1 (fluorescence maximale). </li><li> Sélectionner une fenêtre temporelle qui englobe le temps d&#39;activation de tous les pixels d&#39;une seule propagation AP. Attribuez à chaque pixel de son temps d&#39;activation comme le temps de dérivé upstroke maximale (dF / dt max, où F est fluorescenl&#39;intensité de la CE). En utilisant tous les temps d&#39;activation de pixel, reconstruire la carte d&#39;activation isochrone. Chaque isochrone va donc montrer les pixels activés en même temps. </li><li> Pour reconstituer la carte de répartition durée AP (TTA) pour chaque pixel , calculer la durée entre le temps d&#39;activation et du temps à un niveau spécifique de la repolarisation (par exemple, à 80% de repolarisation, APD 80). En utilisant toutes les valeurs APD de pixels, reconstituer la carte de distribution isochrone APD. Chaque isochrone va donc montrer les pixels avec le même APD. </li><li> Pour calculer la vitesse de conduction pour la propagation AP, adapter à une surface de la préparation des données de temps d&#39;activation calculées en 5.4. Pour cela, utilisez polynôme surface de montage ou local lissage de surface du noyau, puis calculer des vecteurs de vitesse de conduction locale du gradient de la surface équipée. </li><li> Pour créer la carte de repolarisation, attribuer à chaque pixel son temps de repolarisation, définie comme la dérivée seconde maximale (d 2 F / dt2) du signal optique à l&#39;extrémité de la PAO, ou le temps de 90% de repolarisation. </li></ol>

<ol>
	<li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ Res. 95 (1), 21-33 (2004).</li><li>Herron, T. J., Lee, P., Jalife, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+voltage+and+calcium+in+cardiac+cells+&amp;+tissues.">Optical imaging of voltage and calcium in cardiac cells &amp; tissues.</a> Circ Res. 110 (4), 609-623 (2012).</li><li>Fedorov, V. V., Glukhov, A. V., Chang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conduction+barriers+and+pathways+of+the+sino-atrial+pacemaker+complex:+their+role+in+normal+rhythm+and+atrial+arrhythmias.">Conduction barriers and pathways of the sino-atrial pacemaker complex: their role in normal rhythm and atrial arrhythmias.</a> Am J Physiol Heart Circ Physiol. , (2012).</li><li>Boyett, M. R., Honjo, H., Kodama, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sinoatrial+node,+a+heterogeneous+pacemaker+structure.">The sinoatrial node, a heterogeneous pacemaker structure.</a> Cardiovasc Res. 47 (4), 658-687 (2000).</li><li>Glukhov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinoatrial+Node+Reentry+in+a+Canine+Chronic+Left+Ventricular+Infarct+Model:+The+Role+of+Intranodal+Fibrosis+and+Heterogeneity+of+Refractoriness.">Sinoatrial Node Reentry in a Canine Chronic Left Ventricular Infarct Model: The Role of Intranodal Fibrosis and Heterogeneity of Refractoriness.</a> Circ Arrhythm Electrophysiol. , (2013).</li><li>Glukhov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calsequestrin+2+deletion+causes+sinoatrial+node+dysfunction+and+atrial+arrhythmias+associated+with+altered+sarcoplasmic+reticulum+calcium+cycling+and+degenerative+fibrosis+within+the+mouse+atrial+pacemaker+complex.">Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex.</a> Eur Heart J. 36 (11), 686-697 (2015).</li><li>John, R. M., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinus+Node+and+Atrial+Arrhythmias.">Sinus Node and Atrial Arrhythmias.</a> Circulation. 133 (19), 1892-1900 (2016).</li><li>Swaminathan, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidized+CaMKII+causes+cardiac+sinus+node+dysfunction+in+mice.">Oxidized CaMKII causes cardiac sinus node dysfunction in mice.</a> J Clin Invest. 121 (8), 3277-3288 (2011).</li><li>Glukhov, A. V., Fedorov, V. V., Anderson, M. E., Mohler, P. J., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+of+the+murine+sinus+node:+high-resolution+optical+mapping+of+ankyrin-B+heterozygous+mice.">Functional anatomy of the murine sinus node: high-resolution optical mapping of ankyrin-B heterozygous mice.</a> Am J Physiol Heart Circ Physiol. 299 (2), H482-H491 (2010).</li><li>Egom, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+sinoatrial+node+function+and+increased+susceptibility+to+atrial+fibrillation+in+mice+lacking+natriuretic+peptide+receptor+C.">Impaired sinoatrial node function and increased susceptibility to atrial fibrillation in mice lacking natriuretic peptide receptor C.</a> J Physiol. 593 (5), 1127-1146 (2015).</li><li>Torrente, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burst+pacemaker+activity+of+the+sinoatrial+node+in+sodium-calcium+exchanger+knockout+mice.">Burst pacemaker activity of the sinoatrial node in sodium-calcium exchanger knockout mice.</a> Proc Natl Acad Sci U S A. 112 (31), 9769-9774 (2015).</li><li>Lang, D., Sulkin, M., Lou, Q., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+action+potentials+and+calcium+transients+in+the+mouse+heart.">Optical mapping of action potentials and calcium transients in the mouse heart.</a> J Vis Exp. (55), e3275 (2011).</li><li>Fedorov, V. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+blebbistatin+as+an+excitation-contraction+uncoupler+for+electrophysiologic+study+of+rat+and+rabbit+hearts.">Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts.</a> Heart Rhythm. 4 (5), 619-626 (2007).</li><li>Swift, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+blebbistatin+for+cardiac+optical+mapping+and+other+imaging+applications.">Properties of blebbistatin for cardiac optical mapping and other imaging applications.</a> Pflugers Arch. 464 (5), 503-512 (2012).</li><li>Laughner, J. I., Ng, F. S., Sulkin, M. S., Arthur, R. M., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+and+analysis+of+cardiac+optical+mapping+data+obtained+with+potentiometric+dyes.">Processing and analysis of cardiac optical mapping data obtained with potentiometric dyes.</a> Am J Physiol Heart Circ Physiol. 303 (7), H753-H765 (2012).</li><li>Liu, J., Dobrzynski, H., Yanni, J., Boyett, M. R., Lei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organisation+of+the+mouse+sinoatrial+node:+structure+and+expression+of+HCN+channels.">Organisation of the mouse sinoatrial node: structure and expression of HCN channels.</a> Cardiovasc Res. 73 (4), 729-738 (2007).</li><li>Verheijck, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+features+of+the+mouse+sinoatrial+node+in+relation+to+connexin+distribution.">Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution.</a> Cardiovasc Res. 52 (1), 40-50 (2001).</li><li>Krishnaswamy, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+parasympathetic+nervous+system+regulation+of+the+sinoatrial+node+in+Akita+diabetic+mice.">Altered parasympathetic nervous system regulation of the sinoatrial node in Akita diabetic mice.</a> J Mol Cell Cardiol. 82, 125-135 (2015).</li><li>Nygren, A., Lomax, A. E., Giles, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+action+potential+durations+in+isolated+mouse+left+and+right+atria+recorded+using+voltage-sensitive+dye+mapping.">Heterogeneity of action potential durations in isolated mouse left and right atria recorded using voltage-sensitive dye mapping.</a> Am J Physiol Heart Circ Physiol. 287 (6), H2634-H2643 (2004).</li><li>Efimov, I. R., Fedorov, V. V., Joung, B., Lin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+cardiac+pacemaker+circuits:+methodological+puzzles+of+the+sinoatrial+node+optical+mapping.">Mapping cardiac pacemaker circuits: methodological puzzles of the sinoatrial node optical mapping.</a> Circ Res. 106 (2), 255-271 (2010).</li><li>Aschar-Sobbi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+atrial+arrhythmia+susceptibility+induced+by+intense+endurance+exercise+in+mice+requires+TNFalpha.">Increased atrial arrhythmia susceptibility induced by intense endurance exercise in mice requires TNFalpha.</a> Nat Commun. 6, 6018 (2015).</li><li>Hansen, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+fibrillation+driven+by+micro-anatomic+intramural+re-entry+revealed+by+simultaneous+sub-epicardial+and+sub-endocardial+optical+mapping+in+explanted+human+hearts.">Atrial fibrillation driven by micro-anatomic intramural re-entry revealed by simultaneous sub-epicardial and sub-endocardial optical mapping in explanted human hearts.</a> Eur Heart J. 36 (35), 2390-2401 (2015).</li><li>Lang, D., Petrov, V., Lou, Q., Osipov, G., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+heart+rate+in+a+rabbit+heart.">Spatiotemporal control of heart rate in a rabbit heart.</a> J Electrocardiol. 44 (6), 626-634 (2011).</li><li>Kanlop, N., Sakai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+study+of+blebbistatin-induced+chaotic+electrical+activities+in+isolated+rat+atrium+preparations.">Optical mapping study of blebbistatin-induced chaotic electrical activities in isolated rat atrium preparations.</a> J Physiol Sci. 60 (2), 109-117 (2010).</li><li>Glukhov, A. V., Flagg, T. P., Fedorov, V. V., Efimov, I. R., Nichols, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+K(ATP)+channel+pharmacology+in+intact+mouse+heart.">Differential K(ATP) channel pharmacology in intact mouse heart.</a> J Mol Cell Cardiol. 48 (1), 152-160 (2010).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+sinoatrial+node+function+and+intra-atrial+conduction+in+murine+gain-of-function+Scn5a+/DeltaKPQ+hearts+suggest+an+overlap+syndrome.">Altered sinoatrial node function and intra-atrial conduction in murine gain-of-function Scn5a+/DeltaKPQ hearts suggest an overlap syndrome.</a> Am J Physiol Heart Circ Physiol. 302 (7), H1510-H1523 (2012).</li></ol>

No conflicts of interest are declared.

Ici, nous avons présenté deux types de préparations souris SAN: 1) intact SAN dans le coeur entier Langendorff perfusé, et 2) SAN dans l&#39;isolement, ouvert préparation auriculaire. Ces deux types de préparations servent différentes fins expérimentales. Dans la préparation du cœur entier Langendorff perfusé, la structure auriculaire intacte est préservée ce qui le rend possible l&#39;étude des troubles du rythme auriculaire complexes , telles que la fibrillation auriculaire, ainsi que les interactions en...

Novel scientific breakthroughs that lead to leaps in the understanding of human physiology are often preceded by technological advances. Fluorescent optical mapping, for example, enables investigation of multiple physiological parameters in both cells and tissues.1,2 It significantly improved our understanding of how the anatomical structure is associated with electrophysiological functions and dysfunctions. In the heart, the natural pacemaker and conduction systems such as the sinoatrial node (SAN) and the atrioventricular junction consist of nodal myocytes that are insulated by the surrounding atrial myocytes.3-5 Such organization creates complex three-dimensional structures with specialized electrical properties. Both structural and functional remodeling of these pacemaker structures has been recognized to form significant electrophysiological heterogeneities.6,7 Understanding the mechanism underlying how such heterogeneities result in SAN dysfunctions and atrial arrhythmogenesis will dramatically benefit the clinical treatment of these diseases. It requires a technique to visualize the propagation of electrical signals at the tissue level, such as optical mapping. 
Recently, accumulating evidence has proven the advance of optical mapping in studies of atrial electrophysiology and pathology.2 However, novel and rigorous studies are dependent on the accurate interpretation of experimental data, whose validity and stability rely on careful experiment protocols. Genetically modified mice are extensively used for research as animal models of human diseases including sick sinus syndrome, pacemaker abnormalities and atrial arrhythmias.6,8-11 Thus, a combination of fluorescent optical mapping with transgenic mouse models provides a powerful tool to study cardiac electrical abnormalities associated with various pathologies. In this paper, we present a protocol for high-resolution optical mapping of the mouse SAN and atrium. Specifically, we discuss and compare different dye loading approaches, time effects on dye bleaching and heart rate stability during the experiment.

<table border="0" cellpadding="0" cellspacing="0" width="1003">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">water jacket</td>
			<td style="width:200px;">Radnoti</td>
			<td colspan="2" style="width:409px;">1660 Series Tissue Bath for Large Organ or Single Cell Isolation Procedures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">water bath / circulator</td>
			<td>Fisher Scientific</td>
			<td>1016S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pressure amplifier</td>
			<td>AD Instruments</td>
			<td>MLT0670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EMD Millipore Nylon Net Filters</td>
			<td>Fisher Scientific</td>
			<td>NY1102500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pressure transducer</td>
			<td>AD Instruments</td>
			<td>MLT0670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless Steel Minutien Pins - 0.1mm Diameter</td>
			<td>Fine Science Tools</td>
			<td>26002-10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfusion pump</td>
			<td>World Precision Instruments</td>
			<td>PERIPRO-4LS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfusion pump</td>
			<td>World Precision Instruments</td>
			<td>PERIPRO-4HS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Tubingen scissors&#160;</td>
			<td>World Precision Instruments</td>
			<td>503379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps</td>
			<td>World Precision Instruments</td>
			<td>501201, 500085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mayo scissors</td>
			<td>World Precision Instruments</td>
			<td>501750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kelly hemostatic forceps</td>
			<td>World Precision Instruments</td>
			<td>501241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iris forceps</td>
			<td>World Precision Instruments</td>
			<td>15917</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iris scissors</td>
			<td>World Precision Instruments</td>
			<td>501263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECG 12 mm needle (29-gauge) electrodes (monopolar)&#160;</td>
			<td>AD Instruments</td>
			<td>MLA1203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">in-line Luer injection port</td>
			<td>Ibidi</td>
			<td>10820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultima-L CMOS camera&#160;</td>
			<td>SciMedia</td>
			<td>MiCAM-05&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">halogen lamp</td>
			<td>Moritex USA Inc</td>
			<td>MHAB-150W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">NaCl</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">S271-1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">CaCl2 (2H2O)</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">C79-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">KCl</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">S217-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">MgCl2 (6H2O)</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">M33-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">NaH2PO4 (H2O)</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">S369-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">NaHCO3</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">S233-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">D-Glucose</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">D16-1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Blebbistatin</td>
			<td style="width:200px;">Tocris Bioscience</td>
			<td style="width:243px;">1760</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">RH237</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:243px;">S1109</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:395px;">Dimethyl sulphoxide (DMSO)</td>
			<td style="width:200px;">Sigma-Aldrich</td>
			<td style="width:243px;">D2650</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-resolution optical mapping of the mouse sino-atrial node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Cartographie optique du SAN Intact du Coeur de Langendorff perfusé Un exemple typique d&#39;une carte de contour d&#39;activation de la RA reconstruit pour un rythme sinusal spontané est représenté sur la figure 3 pour un coeur de souris Langendorff perfusé. Le point d&#39;activation précoce est situé dans la région intercaval près du SVC où le SAN est anatomiquement défini. 3,16 Deux RA cartes de contours d&#39;activation ac...

Watch this Scientific Journal Video about High-resolution Optical Mapping of the Mouse Sino-atrial Node at JoVE.com

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Ici, nous présentons un protocole de cartographie optique de l&#39;activité électrique de l&#39;oreillette droite de la souris et en particulier le nœud sino-auriculaire, à une résolution spatiale et temporelle.

Haute résolution de cartographie optique de la souris Noeud sino-auriculaire

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...

high-resolution-optical-mapping-of-the-mouse-sino-atrial-node

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.5K vues.  University of Wisconsin-Madison School of Medicine and Public Health.  Ici, nous présentons un protocole de cartographie optique de l'activité électrique de l'oreillette droite de la souris et en particulier le nœud sino-auriculaire, à une résolution spatiale et temporelle. 

Vidéo: High-resolution Optical Mapping of the Mouse Sino-atrial Node

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Haute résolution de mesure du comportement Odor-Driven dans les larves de drosophile

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Recherche

Biologie

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Entretien et nettoyage du microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

Principales composantes d&#39;un microscope optique

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Suivi vidéo haute résolution de la locomotion chez les adultes Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Cartographie optique des coeurs de rats perfusés de Langendorff

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Génération de souris dérivées de cellules souches pluripotentes induites

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

Utilisation à haute résolution tomodensitométrie pour visualiser la structure tridimensionnelle et la fonction des vaisseaux sanguins des végétaux

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Tension et calcium double cartographie de canal optique de Cultured HL-1 auriculaire myocytes monocouche

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Cartographie optique du commerce intra-réticulum sarcoplasmique Ca<sup&gt; 2+</sup&gt; Et potentiel transmembranaire dans le Lapin Coeur Langendorff perfusé

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Haute résolution de cartographie optique de la souris Noeud sino-auriculaire

Résumé

Explorer plus de vidéos

Chapitres dans cette vidéo