Name: recording electrical activity from identified neurons in the intact brain of transgenic fish
Uploaded: 2013-04-30T13:00:00
Description: Watch this Scientific Journal Video about Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish at JoVE.com

Vi tackar Dr Meng-Chin Lin och Ms Yuan Dong för tekniskt bistånd. Detta arbete stöddes av ett bidrag från National Institutes of Health HD053767 (underleverantör till NLW), samt genom medel från Institutionen för fysiologi och tjänstgörande rektor för forskning, University of California-Los Angeles (NLW).

Ett. Dissektion av Brains från Adult Medaka <ol><li> Söva vuxen man eller kvinna (Figur 1A) genom nedsänkning i 5 ml MS-222 (150 mg / L, pH 7,4), vänta ett par minuter efter gälar rörelser har upphört innan halshugga. Alla förfaranden godkändes av Institutional Animal Care och användning kommittén av University of California-Los Angeles. </li><li> Halshugga fisken i fisk saltlösning vid den kaudala änden av kapseln med en sax i en 60-mm diameter petriskål. </li><li> Överför fisk huvud ...

<ol>
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GnRH 3: GFP transgena fiskar ger unika modeller för att studera neurofysiologiska mekanismer bakom neuronal integration och reglering i central kontroll av beteenden som är både direkt och indirekt involverade i fortplantning 3, 8-10. En av de stora fördelarna med denna modell system är att många GnRH3 nervceller som uttrycker GFP är nära den ventrala ytan av hjärnan, vilket möjliggör relativt enkel tillgång till de nervceller för elektrofysiologiska inspelning utan att störa nervbanor ...

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>BX50W (Upright)</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>Axoclamp 200B </td>
 </tr>
 <tr>
 <td>A-D converter</td>
 <td>Computer Interference Corp.</td>
 <td></td>
 <td>Digidata ITC-18 </td>
 </tr>
 <tr>
 <td>Cooled CCD camera</td>
 <td>PCO Computer Optics </td>
 <td></td>
 <td>Sensicam</td>
 </tr>
 <tr>
 <td>Xenon lamp</td>
 <td>Sutter Instruments Co.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>GFP filter set</td>
 <td>Chroma Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Imaging Software</td>
 <td>Intelligent Imaging Innovations</td>
 <td></td>
 <td>Slidebook software </td>
 </tr>
 <tr>
 <td>Electrophysiology Data Acquisition Software</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>Axograph software </td>
 </tr>
 <tr>
 <td>Electrophysiology Data Acquisition Software</td>
 <td>AD Instruments Inc.</td>
 <td></td>
 <td>PowerLab </td>
 </tr>
 <tr>
 <td>Headstage for electrophysiology</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>CV 203BU </td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Sutter Instrument Co </td>
 <td>MP-285 </td>
 <td></td>
 </tr>
 <tr>
 <td>Recording Chamber Platform</td>
 <td>Warner Instrument Corp.</td>
 <td></td>
 <td>P1 </td>
 </tr>
 <tr>
 <td>Recording Chamber</td>
 <td>Warner Instrument Corp.</td>
 <td></td>
 <td>RC-26G </td>
 </tr>
 <tr>
 <td>Electrode Puller</td>
 <td>Sutter instruments</td>
 <td></td>
 <td>P87 </td>
 </tr>
 <tr>
 <td>Filament for electrode puller</td>
 <td>Sutter Instruments</td>
 <td>FB330B</td>
 <td>3.0 mm wide trough filament</td>
 </tr>
 <tr>
 <td>1.5 mm glass capillaries</td>
 <td>World Precision Instruments</td>
 <td>1B150-4</td>
 <td>Microelectrode for recording</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Becton Dickinson</td>
 <td>309586</td>
 <td>3 ml</td>
 </tr>
 <tr>
 <td>MS-222 </td>
 <td>Sigma</td>
 <td>E10521-10G</td>
 <td>Ethyl 3-aminobenzoate methanesulfonate salt</td>
 </tr>
 <tr>
 <td>Fish saline</td>
 <td></td>
 <td></td>
 <td>mM: 134 NaCl; 2.9 KCl; 2.1 CaCl2; 1.2 MgCl2; 10 HEPES </td>
 </tr>
 <tr>
 <td>Electrode solution (loose-patch) </td>
 <td></td>
 <td></td>
 <td>mM: 150 NaCl; 3.5 KCl; 2.5 CaCl2; 1.3 MgCl2; 10 HEPES; 10 glucose </td>
 </tr>
 <tr>
 <td>Electrode solution (whole-cell patch) </td>
 <td></td>
 <td></td>
 <td>mM: 112.5 K-gluconate; NaCl; 17.5 KCl; 0.5 CaCl2; 1 MgCl2; 5 MgATP; 1 EGTA; 10 HEPES; 1 GTP; 0.1 leupeptin;10 phospho-creatine </td>
 </tr>
 </tbody>
</table>

recording electrical activity from identified neurons in the intact brain of transgenic fish

Förstå cellens fysiologi nervbanor som reglerar komplexa beteenden har ökat avsevärt genom att använda modellsystem där detta arbete kan utföras i en intakt hjärna beredning där neurala kretsar i CNS förblir intakt. Vi använder transgen fisk i vilken gonadotropinfrisättande hormon (GnRH) nervceller är genetiskt märkta med grönt fluorescerande protein för identifiering i den intakta hjärnan. Fisk har flera populationer av GnRH neurons, och deras funktioner är beroende av deras läge i hjärnan och den GnRH-genen att de uttrycker en. Vi har fokuserat vår demonstration på GnRH3 neuroner i terminalen nerver (TN) är förknippade med lukt glödlampor med den intakta hjärnan av transgen medaka fisk (Figur 1B och C). Studier tyder på att medaka TN-GnRH3 nervceller är neuromodulatory, i egenskap av en sändare av information från den yttre omgivningen till det centrala nervsystemet; thej inte spelar en direkt roll i regleringen av hypofys-gonadfunktioner, liksom den välkända hypotalamus GnRH1 nervceller 2, 3. tonic mönster av spontan aktionspotential bränning av TN-GnRH3 neuroner är en inneboende egenskap 4-6, frekvensen varav moduleras genom visuella referenser från artfränder 2 och neuropeptid kisspeptin 1 5. I den här videon, använder vi en stabil linje av transgena medaka där TN-GnRH3 nervceller uttrycka en transgen innehållande promotorregionen av Gnrh3 kopplat till förbättrade grönt fluorescerande protein 7 för att visa dig hur man identifierar neurons och övervaka deras elektriska aktiviteten i hela hjärnan framställning 6.

Ett exempel på bilaterala kluster av GFP-märkta TN-GnRH3 neuroner från den utskurna hjärnan hos medaka fisk visas i figurerna 1B och 1C. Varje kluster innehåller cirka 8-10 GnRH nervceller. De spontana neuronala aktiviteter målet TN-GnRH3 noterades i ström-clamp-läge (I = 0) med typiska bränning hastigheter av 0,5-6 Hz. Mönstret av aktionspotential bränning är vanligtvis en tonic eller slå mönster, med en ganska regelbunden interspike intervall. Exempel på spår visas i <...

Watch this Scientific Journal Video about Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish at JoVE.com

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

I den här videon, kommer vi att visa hur man registrerar elektriska aktiviteten från identifierade enskilda nervceller i hela hjärnan beredning, vilket bevarar komplexa neurala kretsar. Vi använder transgen fisk i vilken gonadotropinfrisättande hormon (GnRH) nervceller är genetiskt märkta med en fluorescerande protein för identifiering i den intakta hjärnan beredning.

Inspelning elektrisk aktivitet från Identifierade nervceller i den intakta hjärnan av transgen fisk

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

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Neuroscience

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