Name: time-lapse imaging of neuroblast migration in acute slices of the adult mouse forebrain
Uploaded: 2012-09-12T13:00:00
Description: Watch this Scientific Journal Video about Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain at JoVE.com

Dit werk werd ondersteund door een Canadese Institutes of Health Research (CIHR) subsidie ​​ASJK werd gedeeltelijk ondersteund door een Universite Laval gemeenschap. AS is de ontvanger van een Canada Research Chair in postnatale neurogenese.

<html> 1. Labeling Neuronale Voorlopers Neuroblasten kunnen worden gevisualiseerd met behulp van transgene muizen die selectief fluorescerende eiwitten tot expressie brengen in neuroblasten (dwz Dcx-GFP, Gad67-GFP), door stereotaxically injecteren virale deeltjes codeert fluorescerende eiwitten in de SVZ of RMS of door het enten van neuronale precursors reporter muizen (dwz , Dcx-GFP, Gad67-GFP) in de SVZ van wild-type muizen. We beschrijven de procedure voor het enten en vira...

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De juiste targeting van neuronale precursors de juiste hersengebieden is een fundamenteel proces achter de juiste assemblage en functie van neurale circuits. De meeste cellen migreren tijdens de embryonale ontwikkeling en de postnatale hersenen slechts enkele regio&#39;s, zoals de OB, dentate gyrus en cerebellum, neuronale verplaatsing nog steeds plaats. De mechanismen orkestreren celmigratie in de postnatale hersenen blijven echter slecht begrepen. Kennis van de mechanismen en moleculaire pathways die betrokken zijn in...

<table border="1"><tbody><tr><td> Naam van het reagens </td><td> Vennootschap </td><td> Catalogusnummer </td></tr><tr><td> Sucrose </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> MgCI 2 x6H 2 O </td><td> Sigma-Aldrich </td><td> M2670 </td></tr><tr><td> NaHCO3 </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> CACI 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 (40X oplossing) </td><td> Sigma </td><td> G8769 </td></tr><tr><td> Natrium pyruvat </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> Neurobasal medium </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> Ketamine / Xylazine </td><td> CDMV </td><td> 5230 </td></tr><tr><td> Pasteur pipet </td><td> VWR </td><td> 14672-380 </td></tr><tr><td> 15 ml conische buis </td><td&gt; Sarstedt </td><td> 62.553.205 </td></tr><tr><td> 50 ml conische buis </td><td> Sarstedt </td><td> 62.547.205 </td></tr><tr><td> Glazen capillairen (stereotactische injectie) </td><td> WPI </td><td> 4878 </td></tr><tr><td> Petroleum </td><td> EMD </td><td> PX0045-3 </td></tr><tr><td> Proviodine </td><td> Rougier </td><td> 65655-1370 </td></tr><tr><td> Hechten </td><td> Stoelting </td><td> 50487 </td></tr><tr><td> Anafen </td><td> CDMV </td><td> 11508 </td></tr><tr><td> 20 cc spuit </td><td> VWR </td><td> SS-20L2 </td></tr><tr><td> Petrischaal </td><td> VWR </td><td> 25384-094 </td></tr><tr><td> Agar-agar </td><td> Laboratoire Mat </td><td> AP-0108 </td></tr><tr><td> Lijm </td><td> Permabond </td><td> 910 </td></tr><tr><td> 95% O2 / 5% CO2 </td><td> Linde </td><td> 24068835 </td></tr><tr><td> Blade &lt;/ Td&gt; <td> WPI </td><td> 501901 </td></tr><tr><td> Nylon mesh </td><td> Warner Instruments </td><td> 64-0.198 </td></tr><tr><td> Centrifugeer </td><td> Eppendorf </td><td> 5702 000.019 </td></tr><tr><td> Pipetteer trekker </td><td> Sutter Instrument </td><td> P-97 </td></tr><tr><td> Nanoliter injector </td><td> WPI </td><td> B203MC4 </td></tr><tr><td> Stereotactische injectie-inrichting </td><td> WPI </td><td> 502900 </td></tr><tr><td> Micro boorsysteem </td><td> WPI </td><td> 501819 </td></tr><tr><td> Vibratome </td><td> Thermo Scientific </td><td> 920110 </td></tr><tr><td> Wide-field fluorescentie microscoop </td><td> Olympus </td><td> BX61WIF </td></tr><tr><td> CCD camera </td><td> Lichttechniek </td><td> CS-HQ2-D </td></tr><tr><td> Uiterst stille beeldkamer </td><td> Harvard Apparatus </td><td> 64-1.487 </td></tr><tr><td> PH-1Series 20-platform verwarming </td><td> Harvard Apparatus </td><td> 64 tot 0284 </td></tr><tr><td> Verwarming </td><td> Warner Instruments </td><td> TC-344b </td></tr><tr><td> 40X water immersie objectief </td><td> Olympus </td><td> 1-UM587 </td></tr><tr><td> 10X water immersie objectief </td><td> Olympus </td><td> 1-UM583 </td></tr><tr><td> Lambda DG-4 </td><td> Sutter Instrumenten </td><td> DG-4/OF </td></tr><tr><td> Metamorph software </td><td> Molecular Devices </td><td> 40000 </td></tr><tr><td> Imaris software </td><td> Bitvlak </td><td> BPI-IM70-F1 </td></tr></tbody></table>

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

Er is een groot aantal cijfers blijkt dat aangeeft dat nieuwe functionele neuronen constitutief zijn gegenereerd op basis van een endogene pool van neurale stamcellen in beperkte gebieden van de volwassen hersenen van zoogdieren. Pasgeboren neuroblasten van de subventriculaire zone (SVZ) migreren langs de rostrale migratie stroom (RMS) naar hun eindbestemming in de bulbus olfactorius (OB) 1. In de RMS, neuroblasten tangentiaal migreren in ketens ensheathed door astrocytische processen met 2,3 bloedvaten als structurele steun en een bron van moleculaire factoren vereist voor migratie 4,5. In de OB, neuroblasten los van de ketens en migreren radiaal in de verschillende bulbaire lagen er te differentiëren in interneuronen en te integreren in het bestaande netwerk 1, 6. In dit manuscript beschrijven we de procedure voor toezicht celmigratie in acute plakken van het knaagdier hersenen. Het gebruik van acute segmenten kan de assessment van celmigratie in de micro-omgeving die nauw lijken op in vivo omstandigheden en in hersengebieden die moeilijk toegankelijk zijn voor in vivo imaging. Bovendien voorkomt lange kweken voorwaarde bij organotypische en celkweken die uiteindelijk kunnen veranderen de migratie-eigenschappen van de cellen. Neuronale voorlopers in acute plakjes kan worden gevisualiseerd met behulp van DIC optiek of fluorescerende eiwitten. Virale labeling van neuronale precursors in de SVZ, neuroblasten enten van reporter muizen in de SVZ van wild-type muizen, en transgene muizen die fluorescent eiwit expressie in neuroblasten zijn geschikte werkwijzen voor het visualiseren neuroblasten en na de migratie. De latere methode echter geen afzonderlijke cellen worden bijgehouden voor lange tijd vanwege de hoge dichtheid van gemerkte cellen. We gebruikten een breed gebied fluorescent rechtop microscoop uitgerust met een CCD camera om een ​​relatief snelle acquisitie interval (een ima verwezenlijkenge elke 15 of 30 sec) om betrouwbaar identificeren stationaire fasen en migratie. Een exacte beschrijving van de duur van de stationaire en trekkende fasen is cruciaal voor de eenduidige interpretatie van de resultaten. We hebben ook uitgevoerd meerdere z-stap overnames om neuroblasten migratie in 3D te volgen. Wide-field fluorescerende beeldvorming is uitgebreid gebruikt om neuronale migratie visualiseren 7-10. We beschrijven gedetailleerd protocol voor het labelen neuroblasten, uitvoeren real-time video-imaging van neuroblast migratie acute plakken van de volwassen muis voorhersenen en analyseren celmigratie. Hoewel de beschreven protocol voorbeeld de migratie van neuroblasten in de volwassen RMS kan ook worden gebruikt om celmigratie volgen embryonale en vroege postnatale hersenen.

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

We beschrijven een protocol voor real-time videoimaging van neuronale migratie in de muis voorhersenen. De migratie van viraal gemerkte of geënte neuronale precursors opgenomen in acute levende plakjes met breedveld fluorescerende beeldvorming met een relatief snelle verwerving interval de verschillende fasen van celmigratie, zoals de duur van de stationaire en migratiefasen en de snelheid van bestuderen migratie.

Time-lapse imaging van neuroblast migratie in Acute Schijfjes van de volwassen muis voorhersenen

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

Research

JoVE Journal

Neuroscience

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

Een organotypische Slice Assay voor High-Resolution Time-Lapse Imaging van neuronale migratie in de postnatale Brain

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

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

Time-lapse Live-Imaging van klonaal verwante neurale progenitorcellen in de ontwikkelingslanden zebravis voorhersenen

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

Voorbereiding van Acute subventriculaire zone Schijfjes voor 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 ...

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

Dubbele Elektrofysiologische Opnames van synaptisch-opgeroepen astrogliale en Neuronale responsen bij acute hippocampus Slices

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

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

In vivo Postnatale Elektroporatie en time-lapse imaging van neuroblast Migratie in Muis 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 ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Nucleofectie van knaagdieren neuroblasts om neuroblast Migratie Studie<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

Langdurige Time Lapse Imaging van Muis Cochlear Explantaten

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

Een grondige fysiologische analyse van Defined Cell Populaties in Acute Tissue Slices van de Muis Vomeronasal

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

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Voorbereiding van de Horizontale Schijfjes van Adult Mouse Retina voor elektrofysiologisch onderzoek

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

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

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

Time-lapse Confocal Imaging van Migreren Neuronen in Organotypische Slice Cultuur van Embryonale Muishersenen Met behulp van<em&gt; In Utero</em&gt; Elektroporatie

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 ...