Name: in vivo postnatal electroporation and time-lapse imaging of neuroblast migration in mouse acute brain slices
Uploaded: 2013-11-25T21:45:00
Description: Watch this Scientific Journal Video about In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices at JoVE.com

MS og YZ støttes av KCL og KCL-Kina Doktorgradsstipend. MO ble finansiert av en Bioteknologi og Biological Sciences Research Council PhD student. Vi takker Masaru Okabe og Jun-ichi Miyazaki for PCX-EGFP plasmid og Alain Chedotal og Athena Ypsilanti for verdifulle råd om elektroporering.

Denne fremgangsmåten er i samsvar med de britiske hjemmekontoret Regulations (Animal vitenskapelige prosedyrer Loven, 1986). Forskere skal følge de retningslinjer som er fastsatt og godkjent av deres institusjonelle og nasjonale dyre regulatoriske organisasjoner. En. Postnatal Electroporation 1.1. Utarbeidelse av Glass Kapillærer, DNA Solution og Electroporator <ol><li> Forbered trukket glass kapillærer (OD: 1,5 mm, ID: 0,86 mm) for DNA-injeksjon. (Veiledende ...

<ol>
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Effektiv migrasjon av nevrale stamceller langs RMS sikrer deres påfølgende modning i funksjonelle nerveceller 10. Prominent strømmer av nevrale stamceller rettet mot OB er synlige i menneskets barndom og vil trolig spille en viktig rolle i tidlig postnatal menneskelige hjernens utvikling 27. Dessuten, disse celler er i stand til å målrette områder av hjernen som påvirkes av skade og neurodegenerering 4,28. Å kunne overvåke i sanntid effekten av genmanipulering på neuroblast dyn...

I pattedyrhjernen, oppstår generering av nye nerveceller (neurogenesis) etter fødselen hovedsakelig i to regioner, subventricular sone (SVZ) av den laterale ventriklene og subgranular sone i dentate gyrus av hippocampus en. Betydelig bevis samlet i de siste årene støtter en avgjørende rolle for postnatal neurogenesis i hippocampus og luktelappen minnefunksjoner 1-3. Viktigere, har postnatal neurogenesis også terapeutisk potensial på grunn av sitt forhold til degenerative nevrologiske lidelser, og evnen til neuroblasts å migrere til skadde områder i hjernen 4-6. Subventricular sone (SVZ) har nylig dukket opp som en avgjørende nevrogen nisje. SVZ-avledet neuroblasts migrere mot luktelappen (OB) via rostral migrasjonsstrømmen (RMS), noe som gjør dette den lengste migreringsprosessen i postnatal hjernen 1,7,8. Pattedyr SVZ / RMS / OB-systemet har blitt ennyttig modell for å studere forskjellige trinnene i neurogenesis, for eksempel proliferasjon, migrering og differensiering 1,8. Mange vekstfaktorer og ekstracellulære signaler regulerer SVZ neurogenesis og migrasjon langs RMS, men den intracellulære molekylære mekanismene er langt fra å være fullt ut forstått 1,9. Riktig migrasjon langs RMS er avgjørende for den videre modning av nyfødte nerveceller 10. I tillegg har noen studier vist at SVZ-avledet neuroblasts kan migrere ut av RMS til hjerneskade sider 4-6,11-13. Således undersøkte signalmekanismer regulerings neuroblast migrasjon er grunnleggende ikke bare å forstå neurogenesis men også for potensielle terapeutiske applikasjoner. Her beskriver vi en detaljert protokoll for å merke SVZ nevrale stamceller ved in vivo postnatal elektroporering og overvåke deres migrasjon langs RMS i akutte hjernen skive kulturer ved hjelp av time-lapse roterende disk confocal mikroskoppy. Electroporation er mye brukt i utviklingsstudier fra embryonale til voksne stadier 14-18. Det er et kraftig verktøy for å målrette og manipulere SVZ nevrale stamceller og representerer et billigere og betydelig raskere alternativ til stereotaktisk injeksjon av virale vektorer eller generering av transgene modeller 1,15,19,20. Det er en relativt enkel fremgangsmåte som ikke krever kirurgi, og har høy overlevelse. Electroporation av shRNA eller CRE rekombinase-uttrykke plasmider i muse genetiske modeller anvender LoxP systemet kan brukes til å målrette gener av interesse eller for å oppnå permanent merking av SVZ stamfedre, og dermed representerer et nyttig verktøy for voksne neurogenesis studier 21,22. Imaging RMS neuroblast migrasjon i intakt hjernen er fortsatt utfordrende på grunn av tekniske begrensinger. Imidlertid kan denne prosessen overvåkes ved hjelp confocal roterende disk time-lapse mikroskopi av akutte hjernen skiver, som gir en passende system tett ligner in vivo tilstanden også mottagelig for farmakologisk manipulasjon 23,24. Coupling in vivo postnatal elektroporering med time-lapse bildebehandling vil lette forståelsen av de molekylære mekanismer som styrer neuroblast motilitet og bidra til utvikling av nye tilnærminger for å fremme hjernen reparasjon.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Millicell</td>
			<td style="width:195px;">Millipore</td>
			<td style="width:149px;">PICM0RG50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm Glass bottom culture dish</td>
			<td>MatTek</td>
			<td style="width:149px;">P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gey&#39;s Balanced media</td>
			<td style="width:195px;">Sigma</td>
			<td>G9779-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose, 45%</td>
			<td style="width:195px;">Sigma</td>
			<td>G8769-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:195px;">Sigma</td>
			<td>H3375-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td style="width:195px;">GIBCO</td>
			<td style="width:149px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FCS</td>
			<td style="width:195px;">GIBCO</td>
			<td style="width:149px;">10109-163</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">B27 supplement</td>
			<td style="width:195px;"><a>Invitrogen Life Technologies</a></td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">L-Glutamine</td>
			<td style="width:195px;"><a>Invitrogen Life Technologies</a></td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (phenol red-free)</td>
			<td style="width:195px;">GIBCO</td>
			<td style="width:149px;">31053-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green</td>
			<td style="width:195px;">Sigma</td>
			<td>F7252-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Glass capillaries for injection</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">30-0057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Aspirator tube</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:149px;">A5177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sutter P-97 capillary puller</td>
			<td style="width:195px;">Sutter Instrument</td>
			<td style="width:149px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">ECM830 Square Wave Electroporator</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">45-0052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Platinum Tweezertrodes 7 mm</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">45-0488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Footswitch Model 1250F</td>
			<td style="width:195px;">Harvard Apparatus</td>
			<td style="width:149px;">45-0211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel for electrodes</td>
			<td style="width:195px;">Cefar Compex</td>
			<td style="width:149px;">6602048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td style="width:195px;">Merial</td>
			<td style="width:149px;">AP/DRUGS/220/96</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td style="width:195px;">Leica</td>
			<td style="width:149px;">VT1000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glue</td>
			<td>Roti coll</td>
			<td>Roti coll 1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraViEW VoX spinning disk system</td>
			<td style="width:195px;">Perkin Elmer</td>
			<td style="width:149px;"></td>
			<td>Customized setup (multiple laser sources can be used) equipped with Hamamatsu ORCA R2 C10600-10B CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Volocity software</td>
			<td style="width:195px;">Perkin Elmer</td>
			<td style="width:149px;"></td>
			<td>Acquisition, Quantitation, Visualization Modules</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Environmental chamber for microscopy</td>
			<td style="width:195px;">Solent Scientific</td>
			<td style="width:149px;"></td>
			<td>Custom-made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:344px;">Ti-E inverted microscope</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:149px;"></td>
			<td>CFI Super Plan Fluor ELWD 20X/0.45 NA objective is recommended for the application described in this paper</td>
		</tr>
	</tbody>
</table>

in vivo postnatal electroporation and time-lapse imaging of neuroblast migration in mouse acute brain slices

Subventricular sone (SVZ) er en av de viktigste nevrogene nisjer i barsel hjernen. Her, nevrale stamceller spre seg og gi opphav til neuroblasts stand til å bevege seg langs rostral migrasjonsstrømmen (RMS) mot luktelappen (OB). Dette langdistanse migrasjon er nødvendig for etterfølgende modning av nyfødte nerveceller i OB, men de molekylære mekanismene som regulerer denne prosessen er fortsatt uklart. Gransker signalveier kontrollerende neuroblast motilitet kan ikke bare bidra til å forstå en grunnleggende trinn i neurogenesis, men også ha terapeutisk regenerativ potensial, får muligheten til disse neuroblasts å målrette hjernen områder rammet av skader, hjerneslag, eller degenerasjon. I dette manuskriptet beskriver vi en detaljert protokoll for in vivo postnatal elektroporering og påfølgende time-lapse avbildning av neuroblast migrasjon i muse RMS. Postnatal elektroporering kan effektivt transfektere SVZ stamfarceller, som i sin tur genererer neuroblasts migrere langs RMS. Bruke konfokalmikroskoper spinne disk time-lapse mikroskopi på akutte hjerne skive kulturer, kan neuroblast migrasjon overvåkes i et miljø tett ligner in vivo tilstand. Videre kan neuroblast motilitet spores og kvantitativt analysert. Som et eksempel, beskriver vi hvordan du bruker in vivo postnatal elektroporering av en GFP-uttrykke plasmid å merke og visualisere neuroblasts trekkende langs RMS. Electroporation av shRNA eller CRE rekombinase-uttrykke plasmider i betingede knockout mus som anvender LoxP systemet kan også brukes til å målrette gener av interesse. Farmakologisk manipulering av akutte hjernen skive kulturer kan utføres for å undersøke hvilken rolle ulike signalmolekyler i neuroblast migrasjon. Ved kopling in vivo electroporation med time-lapse bildebehandling, håper vi å forstå de molekylære mekanismene som styrer neuroblast motilitet og bidra til å utvikleling av nye tilnærminger for å fremme hjernen reparasjon.

Merking av SVZ-avledet trekkende neuroblasts kan observeres langs RMS, vanligvis 4-8 dager etter en vellykket electroporation (Figur 1B). Lengre tidspunkter kan også velges, men færre celler vil bli funnet i RMS siden de fleste av dem vil ha kommet inn i OB. Neuroblasts begynne å anskaffe typiske morfologi og funksjoner av modne granule celler i OB rundt 2-3 uker etter elektroporering (ikke vist). Etter dyrkning for ~ 1 hr, kan hjernen skiver fra electroporated museunger bli pålitelig avbildes i opp...

Watch this Scientific Journal Video about In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices at JoVE.com

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

Neuroblast migrasjon er en grunnleggende hendelse i postnatal neurogenesis. Vi beskriver en protokoll for effektiv merking av neuroblasts ved in vivo postnatal elektroporering og påfølgende visualisering av deres migrasjon ved hjelp av time-lapse avbildning av akutte hjernen skiver. Vi inkluderer en beskrivelse for kvantitativ analyse av neuroblast dynamikk av video sporing.

In vivo Postnatal Electroporation og Time-lapse Imaging av neuroblast Migration in Mouse Akutt 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 ...

Research

JoVE Journal

Neuroscience

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

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 ...

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 ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Funksjonell Bildediagnostiske Bruke Ultrasonic blod-hjerne barrieren avbrudd og mangan forbedret MR

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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

Time-lapse Imaging av Neuroblast Migrasjon i Akutt Skiver av Adult Mouse forebrain

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

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

Dual Elektrofysiologiske Opptak av Synaptically-fremkalt Astroglial og Neuronal Responses i akutt hippocampus Slices

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

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Langsiktig Time Lapse Imaging av Mouse Cochlea eksplantater

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

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Ex vivo avbildning av Postnatal Cerebellar Granule Cell Migration Bruke Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

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 av Migrating Neurons i Organotypic Slice Kultur av Embryonic Mouse Brain Using<em&gt; I Utero</em&gt; Elektroporasjon

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 ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

To-fotonet Imaging Microglial prosesser attraksjon mot ATP eller Serotonin i akutt hjernen skiver

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...