Ce travail a été soutenu par les Instituts de recherche en santé du Canada (IRSC) subvention pour ASJK a été partiellement financé par une bourse de l&#39;Université Laval. AS est le titulaire d&#39;une chaire de recherche du Canada en neurogenèse postnatale.

<ol>
	<li>Kriegstein, A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+nature+of+embryonic+and+adult+neural+stem+cells.">The glial nature of embryonic and adult neural stem cells.</a> Annu. Rev. Neurosci. 32, 149-184 (2009).</li><li>Kaneko, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+neurons+clear+the+path+of+astrocytic+processes+for+their+rapid+migration+in+the+adult+brain.">New neurons clear the path of astrocytic processes for their rapid migration in the adult brain.</a> Neuron. 67, 213-223 (2010).</li><li>Lois, C., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-distance+neuronal+migration+in+the+adult+mammalian+brain.">Long-distance neuronal migration in the adult mammalian brain.</a> Science. 264, 1145-1148 (1994).</li><li>Snapyan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculature+guides+migrating+neuronal+precursors+in+the+adult+mammalian+forebrain+via+brain-derived+neurotrophic+factor+signaling.">Vasculature guides migrating neuronal precursors in the adult mammalian forebrain via brain-derived neurotrophic factor signaling.</a> J. Neurosci. 29, 4172-4188 (2009).</li><li>Whitman, M. C., Fan, W., Rela, L., Rodriguez-Gil, D. J., Greer, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+form+a+migratory+scaffold+in+the+rostral+migratory+stream.">Blood vessels form a migratory scaffold in the rostral migratory stream.</a> J. Comp. Neurol. 516, 94-104 (2009).</li><li>Lledo, P. M., Saghatelyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+new+neurons+into+the+adult+olfactory+bulb:+joining+the+network,+life-death+decisions,+and+the+effects+of+sensory+experience.">Integrating new neurons into the adult olfactory bulb: joining the network, life-death decisions, and the effects of sensory experience.</a> Trends Neurosci. 28, 248-254 (2005).</li><li>Bolteus, A. J., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+release+and+uptake+regulate+neuronal+precursor+migration+in+the+postnatal+subventricular+zone.">GABA release and uptake regulate neuronal precursor migration in the postnatal subventricular zone.</a> J. Neurosci. 24, 7623-7631 (2004).</li><li>Martini, F. J., Valdeolmillos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actomyosin+contraction+at+the+cell+rear+drives+nuclear+translocation+in+migrating+cortical+interneurons.">Actomyosin contraction at the cell rear drives nuclear translocation in migrating cortical interneurons.</a> J. Neurosci. 30, 8660-8670 (2010).</li><li>Murase, S., Horwitz, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deleted+in+colorectal+carcinoma+and+differentially+expressed+integrins+mediate+the+directional+migration+of+neural+precursors+in+the+rostral+migratory+stream.">Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream.</a> J. Neurosci. 22, 3568-3579 (2002).</li><li>Wilcock, A. C., Swedlow, J. R., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+orientation+distinguishes+stem+cell+and+terminal+modes+of+neuron+production+in+the+early+spinal+cord.">Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord.</a> Development. 134, 1943-1954 (2007).</li><li>Ono, M., Yanagawa, Y., Koyano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+in+inferior+colliculus+of+the+GAD67-GFP+knock-in+mouse:+electrophysiological+and+morphological+properties.">GABAergic neurons in inferior colliculus of the GAD67-GFP knock-in mouse: electrophysiological and morphological properties.</a> Neurosci. Res. 51, 475-492 (2005).</li><li>Bozoyan, L., Khlghatyan, J., Saghatelyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+Control+the+Development+of+the+Migration-Promoting+Vasculature+Scaffold+in+the+Postnatal+Brain+via+VEGF+Signaling.">Astrocytes Control the Development of the Migration-Promoting Vasculature Scaffold in the Postnatal Brain via VEGF Signaling.</a> J. Neurosci. 32, 1687-1704 (2012).</li><li>Bovetti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+form+a+scaffold+for+neuroblast+migration+in+the+adult+olfactory+bulb.">Blood vessels form a scaffold for neuroblast migration in the adult olfactory bulb.</a> J. Neurosci. 27, 5976-5980 (2007).</li><li>Platel, J. C., Heintz, T., Young, S., Gordon, V., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonic+activation+of+GLUK5+kainate+receptors+decreases+neuroblast+migration+in+whole-mounts+of+the+subventricular+zone.">Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in whole-mounts of the subventricular zone.</a> J. Physiol. 586, 3783-3793 (2008).</li><li>Nam, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+features+of+postnatal+subventricular+zone+cell+motility:+a+two-photon+time-lapse+study.">Dynamic features of postnatal subventricular zone cell motility: a two-photon time-lapse study.</a> J. Comp. Neurol. 505, 190-208 (2007).</li><li>Kim, Y., Comte, I., Szabo, G., Hockberger, P., Szele, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mouse+subventricular+zone+stem+and+progenitor+cells+are+sessile+and+epidermal+growth+factor+receptor+negatively+regulates+neuroblast+migration.">Adult mouse subventricular zone stem and progenitor cells are sessile and epidermal growth factor receptor negatively regulates neuroblast migration.</a> PLoS One. 4, e8122 (2009).</li><li>Bortone, D., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCC2+expression+promotes+the+termination+of+cortical+interneuron+migration+in+a+voltage-sensitive+calcium-dependent+manner.">KCC2 expression promotes the termination of cortical interneuron migration in a voltage-sensitive calcium-dependent manner.</a> Neuron. 62, 53-71 (2009).</li><li>Comte, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galectin-3+maintains+cell+motility+from+the+subventricular+zone+to+the+olfactory+bulb.">Galectin-3 maintains cell motility from the subventricular zone to the olfactory bulb.</a> J. Cell Sci. 124, 2438-2447 (2011).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Le ciblage correct des précurseurs neuronaux pour les régions du cerveau appropriés est un processus fondamental qui sous-tend l&#39;ensemble et le bon fonctionnement des circuits neuronaux. La grande majorité des cellules migrent pendant le développement embryonnaire et dans le cerveau postnatal seulement dans quelques régions, comme l&#39;OB, le gyrus denté et le cervelet, le déplacement neuronale a toujours lieu. Les mécanismes orchestrant la migration des cellules dans le cerveau postnatal reste cependant m...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Saccharose </td><td> Sigma </td><td> S9378 </td></tr><tr><td> Glucose (ACSF) </td><td> EMD </td><td> DX0145-3 </td></tr><tr><td> NaCl </td><td> Sigma </td><td> S9625 </td></tr><tr><td> KCI </td><td> Sigma </td><td> P9541 </td></tr><tr><td> MgCl2 x6H 2 O </td><td> Sigma-Aldrich </td><td> M2670 </td></tr><tr><td> NaHCO 3 </td><td> Sigma </td><td> S5761 </td></tr><tr><td> NaH 2 PO 4 xH 2 O </td><td> EMD </td><td> SX0710-1 </td></tr><tr><td> CaCl 2 x2H 2 O </td><td> Sigma-Aldrich </td><td> C3881 </td></tr><tr><td> Dextran TexasRed </td><td> Invitrogen </td><td> D1864 </td></tr><tr><td> Dextran CascaDeblue </td><td> Invitrogen </td><td> D1976 </td></tr><tr><td> Glucose (solution 40X) </td><td> Sigma </td><td> G8769 </td></tr><tr><td> Pyruvat de sodium </td><td> Gibco </td><td> 11360-070 </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3375 </td></tr><tr><td> HBSS </td><td> Gibco </td><td> 14170-112 </td></tr><tr><td> DNase I </td><td> Sigma </td><td> D-5025 </td></tr><tr><td> Trypsine-EDTA </td><td> Gibco </td><td> 25300-054 </td></tr><tr><td> Milieu Neurobasal </td><td> Gibco </td><td> 21103-049 </td></tr><tr><td> BSA </td><td> EMD </td><td> 2930 </td></tr><tr><td> Pen / Strep </td><td> Life Technologies </td><td> 15140-122 </td></tr><tr><td> La kétamine / xylazine </td><td> CDMV </td><td> 5230 </td></tr><tr><td> Pipette Pasteur </td><td> VWR </td><td> 14672-380 </td></tr><tr><td> 15 ml tube conique </td><td&gt; Sarstedt </td><td> 62.553.205 </td></tr><tr><td> 50 ml tube conique </td><td> Sarstedt </td><td> 62.547.205 </td></tr><tr><td> Capillaires en verre (injection stéréotaxique) </td><td> WPI </td><td> 4878 </td></tr><tr><td> Pétrole </td><td> EMD </td><td> PX0045-3 </td></tr><tr><td> Proviodine </td><td> Rougier </td><td> 65655-1370 </td></tr><tr><td> Suturer </td><td> Stoelting </td><td> 50487 </td></tr><tr><td> Anafen </td><td> CDMV </td><td> 11508 </td></tr><tr><td> Seringue de 20 cc </td><td> VWR </td><td> SS-20L2 </td></tr><tr><td> Une boîte de Pétri </td><td> VWR </td><td> 25384-094 </td></tr><tr><td> Gélose </td><td> Laboratoire Mat </td><td> AP-0108 </td></tr><tr><td> Colle </td><td> Permabond </td><td> 910 </td></tr><tr><td> 95% O 2/5% de CO 2 </td><td> Linde </td><td> 24068835 </td></tr><tr><td> Lame &lt;/ Td&gt; <td> WPI </td><td> 501901 </td></tr><tr><td> Filet de nylon </td><td> Warner Instruments </td><td> 64-0198 </td></tr><tr><td> Centrifuger </td><td> Eppendorf </td><td> 5702 000.019 </td></tr><tr><td> Pipette extracteur </td><td> Instrument Sutter </td><td> P-97 </td></tr><tr><td> Nanolitre injecteur </td><td> WPI </td><td> B203MC4 </td></tr><tr><td> Appareil d&#39;injection stéréotaxique </td><td> WPI </td><td> 502900 </td></tr><tr><td> Système de forage micro </td><td> WPI </td><td> 501819 </td></tr><tr><td> Vibratome </td><td> Thermo Scientific </td><td> 920110 </td></tr><tr><td> Grand champ microscope à fluorescence </td><td> Olympe </td><td> BX61WIF </td></tr><tr><td> Caméra CCD </td><td> Photométrie </td><td> CS-HQ2-D </td></tr><tr><td> Chambre d&#39;imagerie ultra-silencieux </td><td> Harvard Apparatus </td><td> 64-1487 </td></tr><tr><td> PH-1Plate-forme Série de chauffage 20 </td><td> Harvard Apparatus </td><td> 64-0284 </td></tr><tr><td> Système de chauffage </td><td> Warner Instruments </td><td> TC-344B </td></tr><tr><td> 40X objectif immersion dans l&#39;eau </td><td> Olympe </td><td> 1-UM587 </td></tr><tr><td> 10X objectif immersion dans l&#39;eau </td><td> Olympe </td><td> 1-UM583 </td></tr><tr><td> Lambda DG-4 </td><td> Sutter Instruments </td><td> DG-4/OF </td></tr><tr><td> MetaMorph logiciel </td><td> Molecular Devices </td><td> 40000 </td></tr><tr><td> Imaris logiciel </td><td> Bitplane </td><td> BPI-IM70-F1 </td></tr></tbody></table>

time-lapse imaging of neuroblast migration in acute slices of the adult mouse forebrain

Il existe un ensemble considérable de preuves indiquant que les nouveaux neurones fonctionnels sont constitutivement produite à partir d&#39;une piscine endogène des cellules souches neurales dans des zones restreintes du cerveau des mammifères adultes. Neuroblastes nés de la zone sous-ventriculaire (SVZ) migrent le long du courant de migration rostrale (RMS) vers leur destination finale dans le bulbe olfactif (OB) 1. Dans la RMS, neuroblastes migrent tangentiellement dans les chaînes entourées par des processus astrocytaires 2,3 à l&#39;aide des vaisseaux sanguins comme un support structurel et une source de facteurs moléculaires nécessaires à la migration 4,5. Dans l&#39;OB, les neuroblastes se détacher des chaînes et migrent radialement dans les différentes couches bulbaires où ils se différencient en interneurones et à s&#39;intégrer dans le réseau existant 1, 6. Dans ce manuscrit, nous décrivons la procédure de migration des cellules de suivi en tranches aiguës du cerveau des rongeurs. L&#39;utilisation des tranches aiguës permet à l&#39;assessment de la migration cellulaire dans le microenvironnement ressemblant à des conditions in vivo et dans les régions du cerveau qui sont difficiles d&#39;accès pour l&#39;imagerie in vivo. En outre, il évite état de culture autant que dans le cas des cultures organotypiques et de la cellule, qui peut éventuellement modifier les propriétés de migration des cellules. Précurseurs neuronaux en tranches aiguës peuvent être visualisés en utilisant l&#39;optique DIC ou des protéines fluorescentes. Étiquetage virale des précurseurs neuronaux dans la SVZ, la greffe neuroblastes de souris journaliste dans la SVZ de souris de type sauvage, et en utilisant des souris transgéniques qui expriment une protéine fluorescente dans les neuroblastes sont toutes des méthodes appropriées pour la visualisation des neuroblastes et après leur migration. La méthode plus tard, cependant, ne permet pas de cellules individuelles à suivre pendant de longues périodes de temps en raison de la forte densité de cellules marquées. On utilise un grand champ du microscope à fluorescence vertical équipé d&#39;une caméra CCD pour obtenir un intervalle d&#39;acquisition relativement rapide (une image toutes les 15 secondes ou 30) pour identifier de manière fiable les phases stationnaire et migrateurs. Une identification précise de la durée des phases stationnaires et migrateurs est cruciale pour l&#39;interprétation univoque des résultats. Nous avons également réalisé plusieurs acquisitions progressives z pour surveiller la migration des neuroblastes en 3D. Imagerie à grand champ fluorescente a été largement utilisé pour visualiser la migration neuronale 7-10. Ici, nous décrivons le protocole détaillé pour l&#39;étiquetage des neuroblastes, l&#39;exécution en temps réel de vidéo-imagerie de la migration des neuroblastes en tranches aiguës du cerveau antérieur de souris adulte, et l&#39;analyse de la migration cellulaire. Alors que le protocole décrit exemple la migration des neuroblastes dans le RMS adulte, elle peut également être utilisée pour suivre la migration des cellules embryonnaires dans le cerveau et le début postnatal.

Watch this Scientific Journal Video about Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain at JoVE.com

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

Nous décrivons un protocole en temps réel videoimaging de la migration neuronale dans le cerveau antérieur de la souris. La migration de viralement étiquetés ou greffés précurseurs neuronales a été enregistré en direct en utilisant des tranches aiguës imagerie à grand champ fluorescent avec un intervalle d&#39;acquisition relativement rapide pour étudier les différentes phases de la migration cellulaire, y compris les durées des phases stationnaire et la migration et la vitesse de migration.

Imagerie time-lapse de la migration des neuroblastes en tranches aiguës du cerveau antérieur souris adultes

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

time-lapse-imaging-neuroblast-migration-acute-slices-adult-mouse

Research

JoVE Journal

Neuroscience

14.8K vues.  Centre de Recherche Université Laval Robert-Giffard. Nous décrivons un protocole en temps réel videoimaging de la migration neuronale dans le cerveau antérieur de la souris. La migration de viralement étiquetés ou greffés précurseurs neuronales a été enregistré en direct en utilisant des tranches aiguës imagerie à grand champ fluorescent avec un intervalle d'acquisition relativement rapide pour étudier les différentes phases de la migration cellulaire, y compris les durées des phases stationnaire et la mi...

Vidéo: Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Un test organotypique à haute résolution imagerie time-lapse de la migration neuronale dans le cerveau postnatal

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Recherche

Neurosciences

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Imagerie time-lapse en direct de relation clonale cellules progénitrices neurales dans le cerveau antérieur développement Zebrafish

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Préparation des tranches aiguës zone sous-ventriculaire pour l&#39;imagerie calcique

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Deux enregistrements électrophysiologiques des synapses-réponses évoquées astrogliales et neuronales aiguës dans des coupes d&#39;hippocampe

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

In vivo postnatale électroporation et imagerie time-lapse de neuroblaste migrations dans Souris aiguë tranches du cerveau

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

Nucléofection de rongeurs Neuroblastes à étudier neuroblaste migrations<em&gt; In vitro</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

À long terme Accéléré imagerie de souris cochléaires explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

En profondeur Physiological Analyse des populations de cellules définies dans le tissu Tranches aiguë de l&#39;Vomeronasal Organ Souris

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

L&#39;imagerie confocale temporelle des neurones migrateurs dans la culture de tranches organotypiques du cerveau embryonnaire de souris en utilisant<em&gt; Dans Utero</em&gt; Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial dysfunction has been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson&#39;s disease (PD), Huntington&#39;s disease, and Alzheimer&#39;s disease. To study the role of mitochondria in models of these diseases, we can measure mitochondrial respiration via oxygen consumption rate (OCR) as a proxy for mitochondrial function. OCR has already been successfully measured in cell cultures, as well as isolated mitochondria. However, these techniques are less physiologically relevant than measuring OCR in acute brain slices. To overcome this limitation, the authors developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice. The technique is optimized with a focus on the striatum, a brain area involved in PD and Huntington&#39;s disease. The analyzer performs a live cell assay using a 24-well plate, which allows the simultaneous kinetic measurement of 24 samples. The method uses circular-punched pieces of striatal brain slices as samples. We demonstrate the effectiveness of this technique by identifying a lower basal OCR in striatal slices of a mouse model of PD. This method will be of broad interest to researchers working in the field of PD and Huntington&#39;s disease.

Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other methods.