Name: imaging calcium responses in gfp-tagged neurons of hypothalamic mouse brain slices
Uploaded: 2012-08-24T13:00:00
Description: Watch this Scientific Journal Video about Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices at JoVE.com

Vi takker vores kolleger, der har deltaget i arbejdet opsummeret her. Dette arbejde blev støttet af tilskud fra Deutsche Forschungsgemeinschaft (SFB 894), DFG Schwerpunktprogramm 1392 &#39;Integrativ analyse af lugtesansen «og ved Volkswagen Foundation (TLZ). TLZ er en Lichtenberg professor i Volkswagen Foundation.

1. Fremstilling af Opløsning og agarosegel <ol><li> Fremstille ekstracellulær opløsning ifølge tabellen med dobbeltdestilleret vand. PH-værdien vil være ~ 7,3 efter 10 min beluftning med carbogen (95% O2 / 5% CO2), osmolariteten 300 mOsm 8. Ønskes højere osmolaritet er påkrævet, kan det justeres ved tilsætning af mere glucose (1 mM lig 1 mOsm). Opløsningen filtreres to gange under anvendelse af en 0,2 um membranfilter for at fjerne støvpartikler og eventuelle bakterielle f...

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Et vigtigt spørgsmål i neurovidenskab er at forstå, hvordan hjernen bearbejder social information. En fremherskende kilde til nødvendige oplysninger til social anerkendelse er kodet af olfaktoriske eller pheromonal signaler. Påvisningen af disse signaler ved neuronale populationer i næsen og anerkendelse af de signaler i hjernen, især hypothalamus, spiller en central rolle i mange sociale processer og indflydelse hormoner og andre neuroendokrine faktorer 13-16. En væsentlig hindring for at analysere n...

There was a typo in the abstract of <a href="http://www.jove.com/video/4213">Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices</a>.

Erratum: Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

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imaging calcium responses in gfp-tagged neurons of hypothalamic mouse brain slices

Trods en enorm stigning i vores viden om mekanismerne bag kodning af information i hjernen, et centralt spørgsmål om de præcise molekylære trin samt aktiviteten af ​​specifikke neuroner i multi-funktionelle kerner af områder i hjernen, såsom hypothalamus tilbage. Dette problem omfatter identifikation af de molekylære komponenter involveret i reguleringen af ​​forskellige Neurohormonet signaltransduktionsbegivenheder kaskader. Stigninger i intracellulær Ca2 + spiller en vigtig rolle i reguleringen af følsomheden af neuroner, både på niveau med signaltransduktion og ved synaptiske steder. Nye værktøjer er opstået for at hjælpe med at identificere neuroner i det utal af hjernens neuroner ved at udtrykke grønt fluorescerende protein (GFP) under kontrol af en bestemt promoter. At overvåge både rumligt og tidsligt stimulus-induceret Ca2 +-reaktionerne i GFP-mærkede neuroner, en ikke-grønt fluorescerende Ca2 + indikatorfarvestof needs der skal anvendes. Desuden er konfokal mikroskopi en foretrukken fremgangsmåde til billeddannelse individuelle neuroner i vævssnit på grund af sin evne til at visualisere neuroner i forskellige planer af dybden inde i vævet, og for at begrænse ud-af-fokus fluorescens. Den ratiometrisk Ca2 +-indikator fura-2 er blevet anvendt i kombination med GFP-mærkede neuroner 1. Imidlertid er farvestoffet exciteres af ultraviolet (UV) lys. Prisen for laseren og den begrænsede optiske indtrængningsdybde UV-lys hindret dets anvendelse i mange laboratorier. Endvidere kan GFP-fluorescens gribe ind i fura-2-signalerne 2. Derfor besluttede vi at bruge et rødt fluorescerende Ca2 +-indikatorfarvestof. Den enorme Strokes forskydning af fura-rød tillader flerfarvet analyse af rød fluorescens i kombination med GFP anvendelse af en enkelt excitationsbølgelængde. Vi havde tidligere gode resultater med fura-rød i kombination med GFP-mærkede olfaktoriske neuroner 3. Protokollerne for olfaktoriske vævssnit syntes at arbejde equally godt i hypothalamus neuroner 4. Fura-rød baseret Ca2 + billeddannelse blev også med held kombineret med GFP-mærkede pankreatiske β-celler og GFP-mærkede receptorer udtrykt i HEK-celler 5,6. En lille særhed af fura-rød er, at dens fluorescensintensitet ved 650 nm aftager, når indikatoren binder calcium 7. Derfor fluorescensen af hvilende neuroner med lavt Ca2 +-koncentrationen har relativt høj intensitet. Det skal bemærkes, at andre røde Ca 2 +-indikatorfarvestoffer eksisterer eller er under opbygning, vil der giver bedre eller forbedrede resultater i forskellige neuroner og områder i hjernen.

Watch this Scientific Journal Video about Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices at JoVE.com

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

I denne protokol, opdatere vi de seneste fremskridt i billedbehandling Ca<sup&gt; 2 +</sup&gt; Signaler GFP-mærkede neuroner i hjernen vævsskiver ved hjælp af en rød fluorescerende Ca<sup&gt; 2 +</sup&gt; Indikatorfarvestof.

Imaging Calcium respons i GFP-mærkede neuroner i hypothalamus musehjerne Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

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