Name: modified roller tube method for precisely localized and repetitive intermittent imaging during long-term culture of brain slices in an enclosed system
Uploaded: 2017-12-28T13:00:00
Description: Watch this Scientific Journal Video about Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System at JoVE

Brug af dyr følger avlsgodkendt og brug af dyr protokoller, der overholder til Animal Care og bruge retningslinjer fra Colorado State University.
Bemærk: Protokollen nedenfor beskriver metoden forberedelse og kultur for langsigtet inkubation og intermitterende billeddannelse af hippocampus skiver. En enkelt hippocampus skive er knyttet til en specielt forberedt photoetched coverslip ved hjælp af et plasma blodprop, og derefter coverslips er forseglet på den flade side af en boret-out rulle...

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+integration+of+rAAV+vector+into+the+AAVS1+region.">Targeted integration of rAAV vector into the AAVS1 region.</a> Virology. 433 (2), 356-366 (2012).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> J Neurosci. 32 (40), 13819-13840 (2012).</li><li>Kim, J. 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Roller tube metode beskrevet her giver mulighed for langsigtet dyrkning og høj opløsning live imaging af skiveskåret hjernevæv. Et stort problem med skive teknik som anvendt her er i montering og vedligeholdelse af skiver. Coverslip belægninger, der understøtter skive vedhæftning, fremme skive udtynding af styrkelse af udkommet af neurites og migration af celler fra Skive; dermed, vi undgik brug af disse substrater. Indsættelse af amino grupper på glasset ved behandling med 3-aminopropyltriethoxysilane forbedret...

Primære kultur af dissocierede neuroner fra regioner i gnavere hjernen er et vigtigt redskab, som forskere for at iagttage Reaktionerne på patologisk impliceret stimuli. Sådanne undersøgelser har dog Ulempen at anskue neuroner i eneste 2D og uden deres glial støttesystem. Desuden, medmindre dyrket under forhold med meget høj tæthed (640 neuroner/mm2 eller ca. 16% af areal) hvor det bliver umuligt at følge den tilfældige udvækst af en dendrit eller axon for mere end en kort afstand fra sin celle organ, hippocampus neuronal levedygtighed forringes over 4 uger betydeligt1, begrænse brugen af dissocierede kulturer til udvidede undersøgelser af aldersbetingede patologier. Dyrkning af skiver forberedt fra gnaver hjerne er en attraktiv mulighed, der overvinder disse begrænsninger ved at opretholde en organiseret celle arkitektur og bæredygtighed uger eller måneder. Betingelserne for opretholdelse af mange forskellige regioner af gnaver hjernen i Skive kultur er blevet beskrevet2.
To store metoder er almindeligt brugt til langtidskulturer af hjernen skiver: dyrkning på membraner på air-liquid grænseflade3 eller dyrkning på coverslips i forseglet rør lov til at rotere i en rulle inkubator at give beluftning4. Skiver kulturperler på membraner kan direkte afbildet med høj opløsning Fluorescens mikroskopi ved hjælp af en opretstående mikroskop og vand fordybelse mål5. Alternativt er skiver kulturperler på membraner blevet overført til glas bund retter at opnå god opløsning af dendritiske spines bruger en inverteret mikroskop6. Begge metoder af imaging skiver dyrkes på membraner er dog åbne systemer, der kræver mellemlang ændringer og ofte bruge svampedræbende og/eller antibiotika til at forebygge eller mindske forurening5,6. Skiver på en membran på grænsefladen luft-medium opretholde fremragende morfologi og overlevelse, men vender tilbage til præcise steder under gentagne imaging på høj forstørrelse er yderst vanskeligt, medmindre eksperimentet følger kun små grupper af celler udtryk for en fluorescerende markør. Selvom skiver dyrkes på membraner har været brugt med viral-medieret udtryk af transgener5,6, kan biosikkerhed protokoller kræve en lukket kultur system anvendes for visse virale vektorer, der bruges til at udtrykke fluorescently markeret proteiner og journalister i celle fysiologi. Derudover kræver fordybelse mål dekontaminering mellem prøver, der vil blive fulgt op i kultur5. En større anvendelse af membran interface kulturer er en kombination af høj opløsning imaging med Elektrofysiologi på én gang point7.
Metoden roller tube med coverslips inde i plastik røret tillader ikke nogen Elektrofysiologi eller lang-dagsorden tænkelig uden at fjerne coverslip. Således, denne metode har oftest udlignet langtidstest hvor efter fiksering observationer foretaget den8. Beskrevet her er en metode, der udnytter roller tube kultur teknik men på boret ud rør med skiver på coverslips, der kan være afbildet gentagne gange så længe som kulturerne vedligeholdes. Det lukkede system kræver ingen ændring af medium til billedbehandling og udnytter photoetched coverslips for at levere referencepunkter, der tillader imaging på høj forstørrelse, efter dage eller uger, felterne præcise tidligere afbildet.
Vi anvender denne metode til at undersøge ændringer i gnavere hippocampus, en større hjerne region involveret i hukommelse og indlæring. Gnaver hippocampus er ofte studeret som en model for patologisk eller aldersrelaterede ændringer observeret under udvikling af kognitiv svækkelse9, såsom dem, der opstår i Annoncen. Vores metode er særligt velegnet til at studere patologiske ændringer, der udvikler sig inden for en enkelt skive over tid reaktion på miljømæssige ændringer, såsom stigninger i Aβ peptider, som er karakteristisk for AD8. En patologi forbundet med menneskelige og gnaver AD hjernen er tilstedeværelsen af cofilin-actin aggregater og stænger, de sidstnævnte indeholdende bundter af filamenter, hvor cofilin og actin er i en 1:1 molar forholdet10,11, 12. stænger er blevet observeret i faste skiver af rotte hippocampus efter Aβ behandling, samt inden for en levende gnavere hjernen skive at udtrykke cofilin-NGL underkastes hypoxi8, og de kan bidrage til den synaptiske dysfunktion set i AD og slagtilfælde. Her bruger vi denne nye dyrkningsbaserede metode for at observere tidsforløb og distribution inden for skiver af udtrykt eksogene kimære fluorescerende proteiner indført af forskellige vira. Vi derefter udnytte den neuronale specifikt udtryk for en cofilin journalist konstruere at følge udviklingen af cofilin stang og samlede patologi i hippocampus skiver i reaktion på behandling med opløselige Aβ oligomerer (Aβo).

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			<td height="40" style="height:40px;width:280px;">Bottoms from 15 cm culture dishes</td>
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			<td style="width:191px;">25384-326</td>
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			<td>2.5&#34; long and 3/16&#34; in diameter</td>
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			<td height="40" style="height:40px;">Machine Screw Nuts (#10-32)</td>
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			<td>5/16&#34;, thick; 5/8&#34;, hole diameter; 1.125&#34;, OD</td>
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		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Swivel Head Deburring Tool, 5&#34;</td>
			<td>Ace Hardware</td>
			<td>26307</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Adhesive Silicone Sheet (Secure Seal)</td>
			<td style="width:220px;">Grace Bio-Labs</td>
			<td style="width:191px;">666581</td>
			<td>0.5 mm Thickness</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">6 mm hole punch</td>
			<td>Office Max</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">12 mm hole punch</td>
			<td>thepunchbunch.com</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">70% Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phototeched Coverslips, 12 mm diameter</td>
			<td>Bellco Glass, Inc.</td>
			<td>1916-91012</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bunsen Burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Absolute Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nanopure Water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">3-aminopropyltriethoxylane</td>
			<td>Sigma-Aldrich</td>
			<td>A3648</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">#5 Dumont Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">McIlwain Tissue Chopper</td>
			<td>Ted Pella, Inc.</td>
			<td>10180</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Double Edge Razor Blades</td>
			<td>Ted Pella, Inc.</td>
			<td>121-6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Whatman Filter Paper</td>
			<td>VWR</td>
			<td>28450-182</td>
			<td>Cut into 5.8 cm diameter circles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Poly-chloro-trifluoro-ethylene (Aclar)</td>
			<td>Ted Pella, Inc.</td>
			<td>10501-10</td>
			<td>Cut into 5.8 cm diameter circles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">#21 Surgical Blade</td>
			<td>VWR Scientific</td>
			<td>25860-144</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">#5 Dumont Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spatula, stainless with tapered end</td>
			<td>VWR</td>
			<td>82027-518</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gey&#39;s Balanced Salt Solution</td>
			<td>Sigma-Aldrich</td>
			<td>G9779&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#160;Glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>15023-021</td>
			<td>25% (w/v) Solution, 0.2 mm filter sterilized</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chicken Plasma</td>
			<td>Cocalico Biologicals</td>
			<td>30-0300-5L</td>
			<td>Rehydrate in sterile water, centrifuge at 2500 x g 30 min at 4 &#176;C, quick freeze aliquots in liquid nitrogen and store at&#160; -80 &#176;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Thrombin, Bovine</td>
			<td>Fisher</td>
			<td>60-516-01KU</td>
			<td>150 units /ml in GBSS/Glucose, quick freeze aliquots in liquid nitrogen and store at -80 &#176;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell Roller System</td>
			<td>Bellco Biotech</td>
			<td>SciERA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Roller Incubator</td>
			<td>Forma</td>
			<td>Model 3956</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">N21-MAX</td>
			<td>ThermoFisher Scientific</td>
			<td>AR008</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pen/Strep (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">200 mM Glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>15023-021</td>
			<td>25% (w/v) Solution, 0.2 mm filter sterilized</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurobasal A</td>
			<td>ThermoFisher Scientific</td>
			<td>10888-022&#160;</td>
			<td>Complete Medium: 48 mL Neurobasal A, 1 mL N21-MAX, 0.625 mL 200 mM Glutamine, 0.180 mL 25% Glucose, 0.250 mL 100x pen/strep.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Third generation lentivirus packaging</td>
			<td>Life Technologies</td>
			<td>K4975-00</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">159 K cutoff centrifugal filters (Centricon)</td>
			<td>EMD Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lentiviral cloning system (InFusion)</td>
			<td>Clonetech</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Plasmids 30323, 50856, 51279</td>
			<td>Addgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neuronal cell viability dye (NeuO)</td>
			<td>Stemcell technologies</td>
			<td>1801</td>
			<td>Thaw once and quick freeze in 4 &#181;L aliquots. Store at -20 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inverted microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope objectives</td>
			<td>Olympus</td>
			<td>air: 4X, 20; oil: 40X, 60X,</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spinning disc confocal system</td>
			<td>Yokagawa</td>
			<td>CSU22</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope EMCCD camera</td>
			<td>Photometrics</td>
			<td>Cascade II</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Linear encoded (x,y), piezo z flat top stage</td>
			<td>ASI</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope lasers and integration</td>
			<td>Intelligent Imaging Innovations</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEK293T cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human Plasmin</td>
			<td>Sigma Aldrich</td>
			<td>P1867</td>
			<td>0.002 U/mL in 0.1% bovine serum albumin (0.2 mm filter sterilized), quick freeze in liquid nitrogen and store at -80 &#176;C.</td>
		</tr>
	</tbody>
</table>

modified roller tube method for precisely localized and repetitive intermittent imaging during long-term culture of brain slices in an enclosed system

Kulturperler gnaver hjernen udsnit er nyttigt for at studere den cellulære og molekylære opførsel af neuroner og glia i et miljø, der har bevaret mange af deres normale i vivo -interaktioner. Udsnit fra en bred vifte af Transgene mus linjer eller brug af virale vektorer for udtryk af fluorescently mærket proteiner eller journalister i vildtype hjernen skiver mulighed for høj opløsning billeddannelse af Fluorescens mikroskopi. Selv om flere metoder er blevet udviklet til billeddannelse hjerne skiver, kombinerer skive kultur med mulighed for at udføre har gentagne høj opløsning billeddannelse af specifikke celler i live skiver over lange perioder stillet problemer. Dette gælder især, når virale vektorer bruges til udtryk af eksogene proteiner, da dette gøres bedst i et lukket system til at beskytte brugere og forhindre krydskontaminering. Simple ændringer til metoden roller tube hjernen skive kultur, der giver mulighed for gentagen høj opløsning billeddannelse af skiver i mange uger i et lukket system er rapporteret. Dyrkning skiver på photoetched coverslips tillader brug af referencepunkter til hurtigt og præcist flytter scenen for at image feltet identisk med tiden før og efter forskellige behandlinger. Eksempler er vist for brug af denne metode, kombineret med specifikke neuronal farvning og udtryk til at observere ændringer i hippocampus skive arkitektur, viral-medieret neuronal udtryk af fluorescerende proteiner og udviklingen af cofilin patologi, der blev tidligere observeret i hippocampus af Alzheimers sygdom (AD) som svar på skive behandling med oligomerer af amyloid-β (Aβ) peptid.

Til at bestemme, hvordan præcist referencepunkternes kan udnyttes for at reimage de samme celler inden for samme over tid, undersøgte vi skiver dyrkes på photoetched coverslips (fig. 6A). Neuroner var visualiseres ved farvning med et afgørende farvestof (100 nM for 2 h; ikke pletter ikke-neuronale celler), der forsvinder fra neuroner over tid uden at skade celler25. Vi identificerede en fiducial mark i en enkelt gr...

Watch this Scientific Journal Video about Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System at JoVE

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Præsenteret her er en modificeret roller tube metode til dyrkning og intermitterende høj opløsning billeddannelse af hjernen, gnaver skiver i mange uger med præcise repositionering på photoetched coverslips. Neuronal levedygtighed og skive morfologi er godt vedligeholdt. Anvendelser af denne fuldstændig lukket system ved hjælp af virus til celletype specifikke udtryk er fastsat.

Ændrede Roller Tube metode for netop lokaliseret og gentagne intermitterende Imaging under langtidskulturer af hjernen skiver i et lukket System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Research

JoVE Journal

Neuroscience

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

En poleret og forstærket tyndt befolket kranie Window for langsigtet Imaging af Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

En Molekylær Udlæsning af Langsigtet olfaktorisk Adaptation in<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Forbedret forarbejdning og konservering af Hippocampale Mouse Skiver for en meget stabil og reproducerbar optagelse af langfristet Potensering

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

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 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 ...

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 ...

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Regioselektiv Biolistic Målretning i organotypisk Brain Skiver anvendelse af en modificeret genpistol

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Isolation, Kultur og opretholdelse på lang sigt af Primary mesencephaliske dopaminerge neuroner af embryonale Gnavere Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Agarose Microchambers for Langsigtet Calcium Imaging af<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

En forenklet metode til Ultra Low Density, Long-Term Primary Hippocampal Neuron Kultur

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Forberedelse af akut hjernen skiver bruger en optimeret N-Methyl-D-glucamine beskyttende genoprettelsesmetode

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...