Name: whole cell recording from an organotypic slice preparation of neocortex
Uploaded: 2011-06-03T12:00:00
Description: Watch this Scientific Journal Video about Whole Cell Recording from an Organotypic Slice Preparation of Neocortex at JoVE.com

De auteurs willen graag Mayumi Sakuraba en Rebecca Foehring bedanken voor de uitstekende technische ondersteuning. Daarnaast zouden we graag Drs bedanken. Rodrigo Andrade voor hulp bij de uitvoering van de organotypische slice cultuur en biolistische transfectie protocollen en dr. Jeanne Nerbonne voor het aanbieden van ons met cDNA constructen voor transfectie. Dit werk werd ondersteund door NIH subsidie: NS044163 van NINDS (tot RCF).

1. De voorbereidingen voor de Dag van Slicing Wij vinden het efficiënter is om de chirurgische instrumenten autoclaaf en oplossingen voor te bereiden voor de dag van in plakken snijden. <ol><li> Autoclave instrumenten. (De operatie en het snijden worden uitgevoerd onder semi-steriele omstandigheden). Autoclaaf de volgende pakketten, individueel verpakt in een autoclaaf papier: <ul class="none"><li> Chirurgie pakket: spatel, # 22 scalpel hanteren, schaar, pincet </li><li> ...

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We hebben het bestuderen van de expressie en functionele rol van voltage-gated kalium kanalen in piramidale neuronen van de rat neocortex (4, 9-11). Vanwege het gebrek aan specifieke farmacologische middelen voor deze kanalen, gebruiken we een genetische benadering voor het manipuleren van kanaal expressie (1,14,15,17-19). Wij gebruiken een organotypische cultuur voorbereiding (2,3, 5-8, 12,13,15-22). Gewijzigd ten opzichte van de aanpak van Stoppini et al. (16), om de cel morfologie en de laminaire pa...

Chirurgie / transfectie / cultuur: <ol><li> Brain Slicer: Campden Vibroslice # MA572 Wereld precisie-instrumenten, Sarasota, FL, USA </li><li> Gene Gun System: Bio-Rad Helios # 165-2431 (Bio-Rad Laboratories, 1000 Alfred Nobel Drive, Hercules, CA 94547) <ul><li> Inclusief: Gene pistool, helium slang met regulator, slangen prep station (# 165 tot 2.418), spuit kit, Tefzel buizen, buizen snijder, optimalisatie kit (# 165 tot 2424), slangen snijder </li><li> Bio-Rad Helium Regulator (# 165-2413) </li><li> disposable materialen voor Helios van Bio-Rad: <ul><li> 1,6 um Goud microdragers: # 165-2264 </li><li> Tefzel Tubing: # 165-2441 </li></ul></li></ul></li><li> Incubator: Forma wetenschappelijk model # 3110 (Thermo-Scientific: (866) 984 tot 3766). </li></ol> Media: <ol><li> Paard Serum: Hyclone donor paarden # 30074 SH. (Hyclone, 925 West 1800 Zuid, Logan, UT 84321) </li><li> HMEM (Minimal Essential Media plus HBSS en HEPES, geen glutamine: Lonza BioWhittaker Catalog # 12-137f): GIBCO / Invitrogen, (800) 955 tot 6288, optie 1. </li><li> HBSS (GIBCO Hanks gebufferde zoutoplossing, # 24020-117): GIBCO / Invitrogen, (800) 955 tot 6288, optie 1. </li><li> MEM (GIBCO minimaal essentieel medium, # 12360-038), GIBCO / Invitrogen, (800) 955 tot 6288, optie 1. </li><li> 250 ml Millipore 0,2 um filter: # SC6PU02RE </li><li> Plastic Transfer pipetten: Fisher # 13-711-20. </li><li> 50 ml Millipore steriflip 0,22 um filter (# SCGP00525) </li></ol> Items 6-8 verkregen uit: Fisher Scientific, 1241 ambassadeur Blvd, Postbus 14989, St. Louis, MO 63132. Opname: <ol><li> Pipet glas: Harvard GC150TF-10: Harvard Apparatuur, 84 oktober Hill Road, Holliston, Massachusetts 01746 </li><li> Sutter P-87 horizontaal elektrode puller: Sutter Instrument Company, One Digital Drive, Novato, CA 94949 </li><li> Axon Instrumenten Multiclamp 700B versterker: Molecular Devices, Inc 1311 Orleans Drive, Sunnyvale, CA 94.089 tot 1,136 </li><li> PClamp 10 data-acquisitie software: Molecular Devices, Inc, 1311 Orleans Drive, Sunnyvale, CA 94089 tot +1,136 </li><li> lectrode positie wordt geregeld met Sutter ROE-200 manipulatoren en PC-200 controller: Sutter Instrument Company, One Digital Drive, Novato, CA 94949. </li><li> Microscoop: Olympus BX-50WI rechtop microscoop met IR-DIC optiek </li><li> IR-gevoelige camera OLY-150 (Olympus) of dage-MTI (dage-MTI, 01 Noord-Roeske Avenue, Michigan City, IN 46360). </li></ol>

whole cell recording from an organotypic slice preparation of neocortex

We hebben het bestuderen van de expressie en functionele rol van voltage-gated kalium kanalen in piramidale neuronen van de rat neocortex. Vanwege het gebrek aan specifieke farmacologische middelen voor deze kanalen, hebben we een genetische benadering voor het manipuleren van kanaal expressie. We maken gebruik van een organotypische cultuur voorbereiding (16) om cel morfologie en de laminaire patroon van de cortex te behouden. We meestal isoleren acute neocorticale plakken op postnatale dagen 8-10 en onderhouden van de plakjes in de cultuur voor 3-7 dagen. Dit laat ons toe om neuronen studeren op dezelfde leeftijd met die in ons werk met acute plakjes en minimaliseert de ontwikkeling van uitbundige prikkelende verbindingen in de slice. We opnemen van visueel-geïdentificeerde piramidale neuronen in lagen II / III of V met behulp van infrarood verlichting (IR-) en differentieel interferentie contrast microscopie (DIC) met whole cell patch clamp in de huidige-of spanning-klem. Wij gebruiken biolistische (Gene gun) transfectie van wild type of mutant kaliumkanaal DNA om expressie van de kanalen om hun functie studie te manipuleren. De getransfecteerde cellen zijn gemakkelijk te herkennen aan epifluorescentie microscopie na co-transfectie met cDNA voor groen fluorescerend eiwit (GFP). We vergelijken opnames van getransfecteerde cellen naar aangrenzende, ongetransfecteerde neuronen in dezelfde laag van dezelfde slice.

Watch this Scientific Journal Video about Whole Cell Recording from an Organotypic Slice Preparation of Neocortex at JoVE.com

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

Dit is een protocol voor te bereiden en een neocorticale slice voorbereiding handhaven organotypische cultuur voor het maken van elektrische opnames van piramidale neuronen.

Hele Cell Opnemen van een organotypische Slice Voorbereiding van de neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

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Neuroscience

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For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

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Organotypische hippocampus Slice culturen

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

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Gepaarde Whole Cell Opnamen in Organotypische hippocampus Slices

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Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...