Nous tenons à remercier Arumae Urmas pour partager un protocole de collagène et Jutta Fey et Ursula Hinz d&#39;assistance technique excellente. En outre, nous tenons à remercier Christian F. Ackermann, Ulrike Engel et le Nikon Imaging Center à l&#39;Université de Heidelberg et aussi Joachim Kirsch d&#39;avoir bien voulu contribuer à la shoting vidéo. Le travail a été partiellement financé par la Deutsche Forschungsgemeinschaft (SFB 592).

1. Préparation de gels de collagène <ol><li> Préparer le milieu de stock, contenant 455 ul MEM 10x, 112 ul de 7,5% de NaHCO 3, 50 ul glutamine et NaOH. La concentration et la quantité de NaOH dépend de la préparation de collagène de queue de rat (voir 1.2), le volume final du milieu stocks étant 1,000 pi. </li><li> Préparer du collagène de queue de rat selon la Ebendal (1). Conserver la solution de collagène à 4 ° C. Une dégradation de la solution de collagène n&#39;a pas pu être observée. Après la préparation d&#39;un nouveau lot, estimer la concentration de NaOH nécessaire pour le support d&#39;actions (voir 1.1). Utiliser l&#39;indicateur de pH du milieu pour le titrage de la quantité et la concentration de NaOH. 800 ul acide de collagène de queue de rat ont à prendre une teinte rouge lorsqu&#39;il est mélangé avec 210 ul du milieu boursier. Si la concentration de NaOH est trop faible, la solution devient jaune collagène, du fait de l&#39;indicateur de pH du milieu. Ajouter le collagène dans le milieu et mélanger sans produitspremière utilisation de bulles. Exécutez ces étapes sur la glace. La polymérisation a lieu dans les 2 heures après la solution de collagène et le moyen de stock ont ​​été mélangés et la nouvelle solution est incubée à 37 ° C. Testez cela avant de commencer une expérience. 1,3) Mélanger 800 ul de solution de collagène avec 210 de moyenne ul stock (voir 1.2). Placer 100 ul de cette solution dans les puits d&#39;une diapositive 8 chambre bien. Incuber les lames à 37 ° C, 5% de CO 2 et les conditions humides dans un incubateur de culture cellulaire. Préparer les gels de collagène sous un banc de culture cellulaire et des conditions stériles. Le collagène gelatinise dans les 2 heures. </li></ol> 2. Dissection de SCGs embryonnaires <ol><li> Récolte des embryons de souris gravides temps (E16-18) de l&#39;utérus et gardez-les dans du PBS jusqu&#39;à nouvel nécessaire. </li><li> Disséquer un embryon à partir de l&#39;amnios. Décapiter l&#39;embryon caudale du niveau de la clavicule. Fixer la tête sur un plat en bas de silicium avec des insectes &quot;aiguilles&quot;. La face ventrale de la tête doit faire face à vous unD Vous devez voir la région sous-maxillaire. La fixation permet de faciliter la préparation. </li><li> Retirez la peau de la région sous-maxillaire gauche et à droite, et avec elle le tissu conjonctif sous-cutané. </li><li> Retirez les glandes sous-maxillaires salivaires de leur emplacement pour dégager la vue sur l&#39;os hyoïde, les muscles infrahyoidal et le muscle sterno. </li><li> Enlevez ces muscles avec soin pour dégager la vue sur le larynx et la trachée. Retirer la trachée et le larynx. Les artères carotides sont alors visibles des deux côtés sur les muscles prévertébraux. La CTB est situé à la bifurcation de l&#39;artère carotide et a une forme ovale. Retirez délicatement les ganglions et les placer dans du PBS. Retirez délicatement les ganglions des outils de dissection, si elles s&#39;en tiennent à leur disposition. Il est avantageux d&#39;utiliser une pince ainsi qu&#39;un porte-aiguille monté avec une &quot;aiguille papillon&quot; pour le retrait CTB. </li><li> Une fois dans du PBS, stocker les ganglions à la température ambiante dans du PBS jusqu&#39;à ce que vous disséqué enouganglions gh. Ensuite, nettoyer les ganglions des vaisseaux sanguins et des tissus conjonctifs. La dissection CTB et le nettoyage sous un microscope à dissection placé sous un banc à flux laminaire. </li><li> Enfin, placer les noyaux explanté sur les gels de collagène gélatinisé à l&#39;aide d&#39;aiguilles de seringues montées sur une seringue pour une manipulation plus aisée. </li><li> Incuber les explants CTB sur le collagène gélatinisé dans un incubateur de culture cellulaire à 37 ° C, avec des conditions de CO 2 humide et 5%. </li></ol> 3. Traitement des explants SCG <ol><li> Après 1-2 heures d&#39;incubation, couvrir le collagène explant CTB contenant avec 100 pl de milieu de culture contenant Neurobasal cellule B27 supplément, la glutamine et des antibiotiques. Maintenant les puits des diapositives de chambre contenir un volume de 200 pi (100 ul gel et 100 pl de milieu). Ajouter un autre ul 200 de la moyenne, mais maintenant contenant NGF (60 ng / ml) pour faciliter la croissance axonale. En conséquence, la concentration finale est NGF 30 ng / ml. </li><li><ol class="lalpha"><li> Pour étudier l&#39;apparition de la SC migration, ajoutez d&#39;autres facteurs directement au jour in vitro 0 (DIV0). Dissoudre les facteurs du milieu contenant du NGF dans le double concentration nécessaire (2). </li><li> Afin d&#39;analyser l&#39;impact sur la SC, qui ont déjà commencé la migration le long des axones, ajouter des facteurs supplémentaires à DIV3 jusqu&#39;à DIV4 (arrêt potentiel de l&#39;expérience). À cette fin, retirez avec précaution 180 ul de la solution, à DIV3, et ajouter 200 ul NGF milieu neuf contenant et les autres facteurs d&#39;intérêt à la concentration double (2). </li></ol></li></ol> 4. Imagerie time-lapse Utiliser un microscope inversé pour les analyses. Divers objectifs peuvent être utilisés, en définissant le champ de vision et le grossissement. Un aspect important, cependant, est la distance de travail de l&#39;objectif utilisé, comme l&#39;imagerie des explants CTB est effectuée par la lame de verre et le collagènegel. La cadence d&#39;enregistrement des 1/10-30 minutes a montré de bons résultats (2). Cependant, cet aspect doit être ajusté à la question scientifique. Une caméra CCD normale peut être utilisée pour l&#39;acquisition des images. Pour l&#39;imagerie en accéléré une chambre d&#39;incubation de culture cellulaire doit être fixé à la platine du microscope. Incuber le tissu lors de l&#39;imagerie à 37 ° C, avec des conditions de CO 2 humide et 5%. Démarrer le système d&#39;incubation une heure avant le début de la formation d&#39;image. Ceci permet aux parties (par exemple, microscopie objectif), y compris la chambre de coulissement pour régler la température et évite les dérives induites par la température. Définir des domaines spécifiques d&#39;analyses (dans le explant et entre les différents explants) à l&#39;aide du logiciel microscope et une étape contrôlée par logiciel motorisé (réglage multiposition) pour les analyses simultanées de différentes conditions appliquées aux explants SCG. Pour imagerie time-lapse de type sauvage tissu ainsi que des tissus d&#39;animaux transgéniques peuvent être utilisésmarquant le SC (S100B: GFP) (3). Pour fluorophores imagerie d&#39;une source de lumière fluorescente doit être mis en œuvre dans la configuration micropscope. Utiliser les filtres standards. 5. Quantification des distances de migration SC <ol><li> Au point de fin (DIV4 par exemple), fixer les gels de collagène avec 4% de PFA dans la chambre de coulissement pour 3-4 heures. Consécutivement laver les gels de collagène 5 fois pendant 10 minutes avec du PBS. Appliquer des protocoles normalisés pour l&#39;immunocytochimie (2). Si vous êtes nouveau à la préparation du GEC, vérifier l&#39;identité du ganglion en ganglion sympathique par immunohistochimie contre la tyrosine hydroxylase (TH) un marqueur commun pour les cellules catécholaminergiques. Au cours de l&#39;immunohistochimie (après l&#39;anticorps primaire), transférer les gels de collagène contenant des explants de plaques à 24 puits, qui ont un plus grand volume, ce qui est bénéfique pour le lavage des gels. </li><li> Après immunohistochimie, placer soigneusement les gels de collagène sur des lames de verre. Soigneusementenlever le reste de la solution et laisser sécher le collagène / réduire à deux dimensions. Cela permet de mesurer facilement les distances (2). Après cela, montez les échantillons. </li><li> Enfin, mesurer les distances de migration à l&#39;aide de Fidji (NIH) des logiciels. Aucun plugin spéciaux sont nécessaires à cette fin. Afin de permettre des mesures correctes dans um, vérifiez les métadonnées imagerie correcte et les paramètres sous les Fidji, l&#39;image et l&#39;échelle. Utilisez Fidji ont également pour la quantification des cellules. </li></ol>

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	<li>Ebendal, T., Rush, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Use of collagen gels to bioassay nerve growth factor activity. IBRO Handh, (1989).</li><li>Heermann, S., Schm&#195;cker, J., Hinz, U., Rickmann, M., Unterbarnscheidt, T., Schwab, M. H., Krieglstein, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuregulin+1+type+III/ErbB+signaling+is+crucial+for+Schwann+cell+colonization+of+sympathetic+axons.">Neuregulin 1 type III/ErbB signaling is crucial for Schwann cell colonization of sympathetic axons.</a> PloS One. 6 (12), e28692 (2011).</li><li>Zuo, Y., Lubischer, J. L., Kang, H., Tian, L., Mikesh, M., Marks, A., Scofield, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+expressed+in+mouse+transgenic+lines+mark+subsets+of+glia,+neurons,+macrophages,+and+dendritic+cells+for+vital+examination.">Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.</a> The Journal of Neuroscience. The Official Journal of the Society for Neuroscience. 24 (49), 10999-11009 (2004).</li><li>Levi-Montalcini, R., Booker, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EXCESSIVE+GROWTH+OF+THE+SYMPATHETIC+GANGLIA+EVOKED+BY+A+PROTEIN+ISOLATED+FROM+MOUSE+SALIVARY+GLANDS.">EXCESSIVE GROWTH OF THE SYMPATHETIC GANGLIA EVOKED BY A PROTEIN ISOLATED FROM MOUSE SALIVARY GLANDS.</a> Proceedings of the National Academy of Sciences of the United States of America. 46 (3), 373-384 (1960).</li><li>Meintanis, S., Thomaidou, D., Jessen, K. R., Mirsky, R., Matsas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuron-glia+signal+beta-neuregulin+promotes+Schwann+cell+motility+via+the+MAPK+pathway.">The neuron-glia signal beta-neuregulin promotes Schwann cell motility via the MAPK pathway.</a> Glia. 34 (1), 39-51 (2001).</li><li>Yamauchi, J., Miyamoto, Y., Chan, J. R., Tanoue, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB2+directly+activates+the+exchange+factor+Dock7+to+promote+Schwann+cell+migration.">ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration.</a> The Journal of cell biology. 181 (2), 351-365 (2008).</li><li>Anton, E. S., Weskamp, G., Reichardt, L. F., Matthew, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor+and+its+low-affinity+receptor+promote+Schwann+cell+migration.">Nerve growth factor and its low-affinity receptor promote Schwann cell migration.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (7), 2795-2799 (1994).</li><li>Mahanthappa, N. K., Anton, E. S., Matthew, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+growth+factor+2,+a+soluble+neuregulin,+directly+increases+Schwann+cell+motility+and+indirectly+promotes+neurite+outgrowth.">Glial growth factor 2, a soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 16 (15), 4673-4683 (1996).</li><li>Yamauchi, J., Chan, J. R., Shooter, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+regulate+Schwann+cell+migration+by+activating+divergent+signaling+pathways+dependent+on+Rho+GTPases.">Neurotrophins regulate Schwann cell migration by activating divergent signaling pathways dependent on Rho GTPases.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (23), 8774-8779 (2004).</li><li>Gumy, L. F., Bampton, E. T. W., Tolkovsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycaemia+inhibits+Schwann+cell+proliferation+and+migration+and+restricts+regeneration+of+axons+and+Schwann+cells+from+adult+murine+DRG.">Hyperglycaemia inhibits Schwann cell proliferation and migration and restricts regeneration of axons and Schwann cells from adult murine DRG.</a> Molecular and Cellular Neurosciences. 37 (2), 298-311 (2008).</li><li>Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., Kashiwagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+spatiotemporal+dynamics+of+multicellular+cell-cycle+progression.">Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.</a> Cell. 132 (3), 487-498 (2008).</li><li>Park, D., Don, A. S., Massamiri, T., Karwa, A., Warner, B., MacDonald, J., Hemenway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+imaging+of+cell+death+using+an+Hsp90+ligand.">Noninvasive imaging of cell death using an Hsp90 ligand.</a> Journal of the American Chemical Society. 133 (9), 2832-2835 (2011).</li><li>Shcherbo, D., Souslova, E. A., Goedhart, J., Chepurnykh, T. V., Gaintzeva, A., Shemiakina, I. I., Gadella, T. W. J., Lukyanov, S., Chudakov, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+and+reliable+FRET/FLIM+pair+of+fluorescent+proteins.">Practical and reliable FRET/FLIM pair of fluorescent proteins.</a> BMC Biotechnology. 9, 24 (2009).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Le développement du système nerveux périphérique est un processus passionnant. Lorsque le développement est terminé, les axones sont entourées par SC sur toute la longueur, ce qui peut, chez l&#39;homme, souvent plus de 100 cm. À cette fin, le nombre exact des SC requis doit être établi au cours du développement et les SC ont aussi à se déplacer le long des axones s&#39;étendent à la périphérie pour assurer la couverture complète des axones. Cela est vrai pour myélinisées, mais aussi pour les axones ...

<table border="1"><tbody><tr><td> Nom de réactif / Matériel </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> MEM 10x </td><td> Gibco </td><td> 21430 </td><td></td></tr><tr><td> Bicarbonate de sodium (7,5%) </td><td> Gibco </td><td> 25080 </td><td></td></tr><tr><td> Glutamine </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> NaOH </td><td> Merck </td><td> 109137 </td><td></td></tr><tr><td> NGF </td><td> Roche </td><td> 1058231 </td><td> R &amp; D # 556-NG-100 </td></tr><tr><td> Moyen Neurobasal </td><td> Gibco </td><td> 21103 </td><td></td></tr><tr><td> B27 Supplément </td><td> Gibco </td><td> 17504 </td><td></td></tr><tr><td> antibiotiques </td><td> Gibco </td><td> 15640 </td><td></td></tr><tr><td> D-PBS </td><td> Gibco </td><td> 14040 </td><td></td></tr><tr><td> aiguilles d&#39;insectes </td><td> FST </td><td> 26002-20 </td><td></td></tr><tr><td> aiguille de la seringue </td><td> Braun </td><td></td><td> BD # 300013 </td></tr><tr><td> 8 Faites glisser chambre bien </td><td> Lab tek </td><td> 177402 </td><td></td></tr></tbody></table>

analyzing murine schwann cell development along growing axons

Le développement des nerfs périphériques est un processus fascinant. Neurones envoient des axones innervent à des objectifs spécifiques, qui chez l&#39;homme sont souvent de plus de 100 cm du soma du neurone. La survie neuronale au cours du développement dépend de facteurs de croissance dérivés cible, mais aussi sur le soutien des cellules de Schwann (CS). Vers l&#39;extrémité des axones ensheath sc de la région de la soma neuronale (ou la transition de la centrale au système nerveux périphérique) de la synapse ou de la jonction neuromusculaire. Les cellules de Schwann sont des dérivés de la crête neurale et migrent le long des axones en tant que précurseurs émergents jusqu&#39;en l&#39;axone entier est couvert de SC. Cela montre l&#39;importance de la migration SC pour le développement du système nerveux périphérique et souligne la nécessité d&#39;enquêter sur ce processus. Afin d&#39;analyser le développement SC, une configuration qui est nécessaire à côté des castes repose aussi sur le substrat physiologique de la migration, de l&#39;axone. En raison du développement intra-utérin <em&gt; in vivo imagerie time-lapse, cependant, n&#39;est pas réalisable chez les vertébrés placentaires comme la souris (Mus musculus). Pour contourner cela, nous avons adapté la technique supérieure du col explant ganglion (CTB). Lors du traitement avec le facteur de croissance des nerfs (NGF) explants CTB étendre les axones, suivis par des précurseurs SC migrent le long des axones du ganglion vers la périphérie. La beauté de ce système est que le SC sont issus d&#39;un pool de SC endogène et qu&#39;ils migrent le long des axones leurs propres physiologiques qui se développent en même temps. Ce système est particulièrement intéressant parce que le développement SC le long des axones peuvent être analysées par imagerie time-lapse, ouvrant de nouvelles possibilités pour mieux comprendre la migration en SC.

La croissance axonale est facilitée à partir SCG explants par traitement avec NGF (4) (schéma de film S1 Figure 1 schéma). Ce processus est facilement visible par n&#39;importe quel microscope inversé et peut être suivi par imagerie time-lapse (film S2). Si un scientifique est nouveau pour disséquer CTB à partir d&#39;embryons de souris, nous recommandons fortement une validation de la technique par un simple anti-tyrosine hydroxylase (TH) immunohistochimie. TH est un marqueur c...

Watch this Scientific Journal Video about Analyzing Murine Schwann Cell Development Along Growing Axons at JoVE.com

Analyzing Murine Schwann Cell Development Along Growing Axons

Nous décrivons ici une cellule de Schwann (SC) essai de migration dans lequel SC sont en mesure de développer le long des axones s&#39;étendent.

Analyser murin développement des cellules de Schwann le long des axones en croissance

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

analyzing-murine-schwann-cell-development-along-growing-axons

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Neuroscience

11.6K vues.  University of Freiburg. Nous décrivons ici une cellule de Schwann (SC) essai de migration dans lequel SC sont en mesure de développer le long des axones s'étendent.

Vidéo: Analyzing Murine Schwann Cell Development Along Growing Axons

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Analyse morphologique des Drosophile Larvaires périphérique dendrites et des axones des neurones sensoriels utilisant mosaïques génétiques

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

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Neurosciences

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Imagerie en temps réel des axones de la racine dorsale après Rhizotomie

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Une seule cellule de profilage de développer et d&#39;âge mûr neurones de la rétine

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolement et culture de cellules de crêtes neurales de tube neural embryonnaire murin

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Un test migration cellulaire quantitative pour murins de neurones entériques progéniteurs

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Laser nanochirurgie de cervelet axones<em&gt; In Vivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Imagerie en direct de la nicotine induit la signalisation du calcium et de la libération de neurotransmetteurs long ventrales hippocampe axones

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In Vivo Transfert de gènes à des cellules de Schwann dans le nerf sciatique Rongeur par électroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Utilisation Agarose linéaire Canaux pour étudier<em&gt; Drosophila</em&gt; Larvaire Ramper Comportement

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

Analyse dendritique Morphologie dans les colonnes et les couches

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...