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

Wij danken onze collega&#39;s die hebben deelgenomen aan het werk samengevat hier. Dit werk werd ondersteund door subsidies van de Deutsche Forschungsgemeinschaft (SFB 894), &#39;Integrative analyse van reukzin&#39; de DFG Schwerpunktprogramm 1392 en door de Volkswagen Foundation (TLZ). TLZ is een Lichtenberg hoogleraar van de Volkswagen Stichting.

1. Bereiding van de oplossing en Agarose Gel <ol><li> Bereid extracellulaire oplossing volgens de tabel met tweemaal gedestilleerd water. De pH is 7,3 ~ na 10 min beluchting met carbogen (95% O2 / 5% CO 2), de osmolariteit 300 mOsm 8. Indien een hogere osmolariteit vereist is, kan worden aangepast door meer glucose (1 mM gelijk aan 1 mOsm). De oplossing wordt tweemaal gefilterd met een 0,2 pm membraan filter om stofdeeltjes en mogelijke bacteriële besmettingen voorkomen. </li></ol><ta...

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Een belangrijke vraag in de neurowetenschappen is om te begrijpen hoe de hersenen sociale informatie verwerkt. Een grote bron van informatie die nodig is voor de sociale erkenning wordt gecodeerd door olfactorische of feromooncommunicatie signalen. De detectie van deze signalen door neuronale populaties in de neus en de erkenning van de signalen in de hersenen, vooral de hypothalamus, spelen een belangrijke rol in vele maatschappelijke processen en invloed hormonen en andere factoren neuroendocrine 13-16. Een...

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

<table border="1"><tbody><tr><td> Naam </td><td> Vennootschap </td><td> Cat. N ° </td></tr><tr><td> Agar-agar </td><td> Sigma </td><td> A1296 </td></tr><tr><td> Fura-red/AM </td><td> Invitrogen </td><td> F-3021 </td></tr><tr><td> Pluronic F-127 </td><td> Sigma </td><td> P2443 </td></tr><tr><td> Dimethylsulfoxide </td><td> Fisher Scientific </td><td> BP231 </td></tr><tr><td> Vibrerende-Blade Microtoom Hyrax V 50 </td><td> Zeiss </td><td> 9770170 </td></tr><tr><td> Koeling CU 65 voor Microtoom Hyrax V 50 </td><td> Zeiss </td><td> 9920120 </td></tr><tr><td> O 2 / CO 2-Incubator, CB210-UL </td><td> Binder </td><td> 0019389 </td></tr><tr><td> Super lijm, Loctite 406TM </td><td> Henckel </td><td> 142580 </td></tr><tr><td> Dubbele spatels, lepel vorm </td><td>Bochem </td><td> 3182 </td></tr><tr><td> Microlepel spatels, lepel vorm </td><td> Bochem </td><td> 3344 </td></tr><tr><td> Spring Schaar, Moria-Vannas-Wolff - 7mm Blades </td><td> Fine Science Tools </td><td> 15.370-52 </td></tr><tr><td> Spring Schaar, Vannas - 3mm Blades </td><td> Fine Science Tools </td><td> 15.000-00 </td></tr><tr><td> Wagner Schaar </td><td> Fine Science Tools </td><td> 14.071-12 </td></tr><tr><td> Medische tang, Dumont 7b </td><td> Fine Science Tools </td><td> 11.270-20 </td></tr><tr><td> Grote rechthoekige Open Bath Chamber (RC-27) </td><td> Warner Instruments </td><td> 64-0.238 </td></tr><tr><td> Confocale microscoop BioRad Radiance 2100 </td><td> Zeiss </td><td> na </td></tr></tbody></table>

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

Ondanks de enorme toename van de kennis van de onderliggende mechanismen van het coderen van informatie in de hersenen, een centrale vraag over de precieze moleculaire stappen en de activiteit van bepaalde neuronen in multifunctionele kernen van hersengebieden zoals de hypothalamus blijven. Dit probleem omvat de identificatie van de moleculaire componenten betrokken bij de regulatie van verschillende neurohormoon signaalcascades. Verhogingen van de intracellulaire Ca 2 + spelen een belangrijke rol in het reguleren van de gevoeligheid van neuronen, zowel op het niveau van signaaltransductie en synaptische plaatsen. Nieuwe instrumenten ontstaan ​​om neuronen in de veelheid van hersenneuronen identificeren door expressie green fluorescent protein (GFP) onder de controle van een specifieke promotor. Om zowel ruimtelijk als tijdelijk stimulus-geïnduceerde Ca 2 + volgen responsen in GFP-gelabelde neuronen, een niet-green fluorescent Ca 2 + n indicatorkleurstofeeds worden gebruikt. Bovendien is een confocale microscopie favoriete methode imaging individuele neuronen in weefselcoupes vanwege zijn vermogen om neuronen in verschillende vlakken diepte visualiseren in het weefsel en out-of-focus fluorescentie beperken. De ratiometrische Ca 2 + indicator fura-2 werd gebruikt in combinatie met GFP-gelabelde neuronen 1. Echter wordt de kleurstof geëxciteerd door ultraviolet (UV) licht. De kosten van de laser en de beperkte optische indringdiepte van UV licht belemmerd het gebruik in vele laboratoria. Bovendien kan GFP fluorescentie interfereren met de fura-2 signalen 2. Daarom was een rode fluorescerende Ca2 + indicator kleurstof te gebruiken. De enorme Strokes verschuiving van fura red-maakt multicolor analyse van het rode fluorescentie in combinatie met GFP met een excitatiegolflengte. We hadden eerder goede resultaten met behulp van fura-rood in combinatie met GFP-gemerkte olfactorische neuronen 3. De protocollen voor olfactorische weefselcoupes leek te e werkenqually goed in de hypothalamus neuronen 4. Fura-red gebaseerd Ca 2 + imaging werd eveneens met succes in combinatie met GFP-gelabelde pancreatische β-cellen en GFP-gemerkte receptoren tot expressie gebracht in HEK cellen 5,6. Een beetje gril van fura-rood is dat de fluorescentie-intensiteit bij 650 nm is lager dan de indicator bindt calcium 7. Daarom is de fluorescentie van rustende neuronen met lage Ca2 + concentratie relatief hoge intensiteit. Er zij opgemerkt dat andere rode Ca 2 +-indicatorkleurstoffen bestaan ​​of zijn in ontwikkeling, zou dat een beter of betere resultaten in verschillende neuronen en hersengebieden.

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

In dit protocol, updaten we de recente vooruitgang in de beeldvorming Ca<sup&gt; 2 +</sup&gt; Signalen van GFP-gemerkte neuronen in hersenweefsel plakjes met een rode fluorescerende Ca<sup&gt; 2 +</sup&gt; Indicator kleurstof.

Imaging Calcium Reacties in GFP-gelabelde neuronen van de hypothalamus Mouse Brain 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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