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 understøttes af KCL og KCL Kina ph.d.-stipendier. MO blev finansieret af en bioteknologi og biologiske Sciences Research Council Ph.d. studentship. Vi takker Masaru Okabe og Jun-ichi Miyazaki til PCX-EGFP plasmid og Alain Chedotal og Athena Ypsilanti for værdifuld rådgivning om elektroporation.

Denne procedure er i overensstemmelse med det britiske indenrigsministerium forordninger (Animal Scientific Procedures Act, 1986). Forskerne bør følge de retningslinjer, der er fastlagt og godkendt af deres institutionelle og nationale organisationer dyr. 1.. Fødselsdepression Elektroporation 1.1. Fremstilling af glaskapillarer DNA Solution og elektroporator <ol><li> Forbered trukket glaskapillarer (OD: 1,5 mm, ID: 0,86 mm) til DNA-injektion. (Vejledende indsti...

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Effektiv migration af neurale stamfædre langs RMS sikrer deres efterfølgende modning i funktionelle neuroner 10. Fremtrædende strømme af neurale forfædre rettet mod OB er synlige i menneskets barndom og er tilbøjelige til at spille en vigtig rolle i den tidlige postnatale udvikling menneskelige hjerne 27. Desuden er disse celler i stand til målsteder af hjernen påvirkes af skade og neurodegeneration 4,28. At være i stand til at overvåge i realtid effekten af ​​genmanipulati...

I pattedyrs hjerne optræder dannelsen af nye neuroner (neurogenese) efter fødslen hovedsageligt to områder, den subventrikulære zone (SVZ) af de laterale ventrikler og subgranular zone i den tandede gyrus af hippocampus 1. Betydelige beviser indsamlet i de seneste år understøtter en afgørende rolle for postnatal neurogenese i hippocampus og olfaktoriske pære hukommelsesfunktioner 1-3. Vigtigere er det, postnatal neurogenese rummer også terapeutiske potentiale på grund af sit forhold med degenerative neurologiske lidelser, og evne til neuroblasts at migrere til skadelidte steder i hjernen 4-6. Den subventricular zone (SVZ) har for nylig vist sig som en afgørende neurogen niche. SVZ-afledte neuroblasts migrere til lugtekolben (OB) via rostralt vandrende strøm (RMS), hvilket gør dette den længste migration proces i postnatal hjerne 1,7,8. Pattedyrs SVZ / RMS / OB system er blevet ennyttig model til at studere forskellige trin i neurogenese, såsom proliferation, migrering og differentiering 1,8. Mange vækstfaktorer og ekstracellulære signaler regulerer SVZ neurogenesis og migration langs RMS, men de intracellulære molekylære mekanismer er langt fra at være fuldt forstået 1,9. Korrekt migration langs RMS er afgørende for den efterfølgende modning af nyfødte neuroner 10. Derudover har nogle undersøgelser vist, at SVZ-afledte neuroblasts kan migrere ud af RMS til hjerneskade sites 4-6,11-13. Således undersøger signalering mekanismer, der regulerer neuroblast migration er grundlæggende ikke blot at forstå neurogenesis, men også for potentielle terapeutiske anvendelser. Her beskriver vi en detaljeret protokol til at mærke SVZ neurale progenitorer ved in vivo postnatal elektroporation og overvåge deres vandring langs RMS i akutte hjerne skivekulturer bruge time-lapse spinning disk konfokal microscopy. Elektroporation er meget udbredt i udviklingsmæssige undersøgelser fra embryonale til voksne stadier 14-18. Det er et kraftfuldt værktøj til at målrette og manipulere SVZ neurale stamfædre og repræsenterer en billigere og betydeligt hurtigere alternativ til stereotaktisk injektion af virale vektorer eller generering af transgene modeller 1,15,19,20. Det er en relativt enkel procedure, som ikke behøver operation, og har høje overlevelsesrater. Elektroporation af shRNA eller CRE recombinase-udtrykkende plasmider i mus genetiske modeller, der anvender LoxP systemet kan bruges til at målrette gener af interesse eller for at opnå permanent mærkning af SVZ stamceller repræsenterer således et nyttigt redskab for voksne neurogenese studier 21,22. Imaging RMS neuroblast migration i den intakte hjerne er stadig udfordrende på grund af de nuværende tekniske begrænsninger. Dog kan denne proces overvåges ved hjælp af konfokal spinning disk time-lapse mikroskopi af akutte hjernen skiver, som giver en passende system ligner in vivo tilstand også modtagelig for farmakologisk manipulation 23,24. Kobling in vivo postnatal elektroporation med time-lapse imaging vil lette forståelsen af de molekylære mekanismer, der styrer neuroblast motilitet og bidrage til udviklingen af nye metoder til fremme hjernens reparation.

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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>
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		<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

Den subventricular zone (SVZ) er en af ​​de vigtigste neurogene nicher i den postnatale hjerne. Her neurale progenitorceller formere sig og give anledning til neuroblaster i stand til at bevæge sig langs rostralt vandrende strøm (RMS) mod lugtekolben (OB). Denne lange afstande migration er nødvendig for den efterfølgende modning af nyfødte neuroner i OB, men de molekylære mekanismer, der regulerer denne proces er stadig uklart. Undersøge signalveje, der kontrollerer neuroblast motilitet ikke blot kan hjælpe med at forstå et grundlæggende skridt i neurogenese, men også have terapeutisk regenerative potentiale, da evnen af ​​disse neuroblasts at målrette hjernen ramt af skader, slagtilfælde eller degeneration. I dette manuskript beskriver vi en detaljeret protokol for in vivo postnatal elektroporation og efterfølgende time-lapse billeddannelse af neuroblast migration i mus RMS. Fødselsdepression elektroporation kan effektivt transficere SVZ stamfaderceller, som igen genererer neuroblaster migrerer langs RMS. Brug konfokal spinning disk time-lapse mikroskopi om akutte hjerne skivekulturer kan neuroblast migration skal overvåges i et miljø, der ligner in vivo tilstand. Desuden kan neuroblast motilitet spores og analyseres kvantitativt. Som et eksempel beskriver vi, hvordan du bruger in vivo postnatal elektroporation af en GFP-udtrykkende plasmid til at mærke og visualisere neuroblasts migrerer langs RMS. Elektroporering af shRNA eller CRE recombinase-udtrykkende plasmider i betingede knockout mus med den LoxP Systemet kan også anvendes til at målrette gener af interesse. Farmakologisk manipulation af akut hjerne skivekulturer kan udføres for at undersøge den rolle, som forskellige signalmolekyler i neuroblast migration. Ved at koble in vivo elektroporation med time-lapse imaging, håber vi at forstå de molekylære mekanismer, der styrer neuroblast motilitet og bidrage til at udvikleling af nye tilgange til at fremme hjernens reparation.

Mærkning af SVZ-afledte vandrende neuroblaster kan observeres langs RMS, normalt 4-8 dage efter en vellykket elektroporation (figur 1B). Kan også vælges længere tidspunkter, men i mindre celler findes i RMS, da de fleste af dem vil være trådt OB. Neuroblasts begynde at optjene typisk morfologi og funktioner af modne granulceller i OB omkring 2-3 uger efter elektroporation (ikke vist). Efter dyrkning for ~ 1 time, kan hjernen skiver fra elektroporerede museungers pålideligt filmede i op til 3-4 ti...

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 migration er en grundlæggende begivenhed i postnatal neurogenesis. Vi beskriver en protokol til effektiv mærkning af neuroblasts ved in vivo postnatal elektroporation og efterfølgende visualisering af deres migration ved hjælp af time-lapse billeddannelse af akutte hjernen skiver. Vi inkluderer en beskrivelse af den kvantitative analyse af neuroblast dynamik ved video tracking.

In vivo Fødselsdepression Elektroporation og Time-lapse Imaging af neuroblast Migration i Mouse Akut hjerneskiver

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

Funktionel Neuroimaging Med Ultrasonic blod-hjerne barrieren forstyrrelser og mangan-forstærket MRI

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 af neuroblast Migration i Akutte Skiver af den voksne Mouse forhjerne

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

Dobbelte Elektrofysiologiske Optagelser af synaptisk-evoked astrogliale og Neuronal respons i Akut hippocampusskiver

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

Langsigtet Time Lapse Imaging af Mouse Cochlear 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 Imaging af Postnatal cerebellare Granule Cell Migration Brug Konfokal makroskopi

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 konfokal billeddannelse af migrerende neuroner i organotypisk skive kultur af embryonale mus hjernen ved hjælp af<em&gt; I Utero</em&gt; Elektroporation

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-foton billeddannelse af Microglial processer tiltrækning mod ATP eller Serotonin i akut 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 ...