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 takker Dr. Meng-Chin Lin og Ms Yuan Dong for teknisk assistanse. Dette arbeidet ble støttet av et stipend fra National Institutes of Health HD053767 (underentrepriser til NLW), og med midler fra Institutt for fysiologi og kontor Vice Chancellor for Research, University of California-Los Angeles (NLW).

En. Disseksjon av Hjerner fra Adult medaka <ol><li> Anesthetize voksen mann eller kvinne (figur 1A) ved å dyppe i 5 ml MS-222 (150 mg / L, pH 7,4), vent et par minutter etter at gjeller bevegelser har opphørt før decapitating. Alle prosedyrer ble godkjent av Institutional Animal Care og bruk Committee of University of California-Los Angeles. </li><li> Decapitate fisken i fisk fysiologisk saltvann ved den caudale enden av operculum med saks i et 60 mm diameter petriskål. </li><li> Transfer fisk hod...

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	<li>Kah, O., Lethimonier, C., Lareyre, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotrophin-releasing+hormone+(GnRH)+in+the+animal+kingdom.">Gonadotrophin-releasing hormone (GnRH) in the animal kingdom.</a> J. Soc. Biol. 198 (1), 53-60 (2004).</li><li>Ramakrishnan, S., Wayne, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+cues+from+conspecifics+alter+electrical+activity+of+gonadotropin-releasing+hormone+neurons+in+the+terminal+nerve+via+visual+signals.">Social cues from conspecifics alter electrical activity of gonadotropin-releasing hormone neurons in the terminal nerve via visual signals.</a> Am. J. Physiol. Regul. Integr. Comp. 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GnRH 3: GFP transgene fiskene gir unike modeller for å studere de nevrofysiologiske mekanismene bak neuronal integrering og regulering i sentral kontroll av atferd som er både direkte og indirekte involvert i tre reproduksjon, 8-10. En av de betydelige fordelene med dette modellsystemet er at mange GnRH3 nevroner uttrykker GFP seg i nærheten av den ventrale overflaten av hjernen, noe som muliggjør forholdsvis enkel tilgang til nervecellene for elektrofysiologisk registrering uten å forstyrre nev...

<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

Forstå cellefysiologi av nevrale kretser som regulerer komplekse atferd er sterkt forbedret ved hjelp av modellsystemer der dette arbeidet kan utføres i en intakt hjerne forberedelse der nevrale kretser i CNS forblir intakt. Vi bruker transgen fisk som gonadotropin-frigjørende hormon (GnRH) nevroner er genetisk merket med grønt fluorescerende protein for identifikasjon i den intakte hjerne. Fisk har flere populasjoner av GnRH-neuroner, og deres funksjon er avhengig av deres plassering i hjernen og den GnRH-genet som de uttrykker en. Vi har fokusert vår demonstrasjon på GnRH3 nevroner lokalisert i terminalen nerver (TN) forbundet med olfactory pærer bruker intakt hjernen av transgene medaka fisk (Figur 1B og C). Studier tyder på at medaka TN-GnRH3 nevroner er neuromodulatory, fungerer som en formidler av informasjon fra det ytre miljøet til det sentrale nervesystemet, tHei ikke spille en direkte rolle i å regulere hypofyse-/gonadesystemet funksjoner, som gjør den velkjente hypothalamus GnRH1 to nerveceller, tre. Den tonic mønster av spontan handling potensial avfyring av TN-GnRH3 nevroner er en iboende egenskap 4-6, frekvensen som er modulert av visuelle signaler fra conspecifics 2 og neuropeptide kisspeptin en fem. I denne videoen bruker vi en stabil linje av transgene medaka der TN-GnRH3 nevroner uttrykker et transgen inneholder promoter regionen Gnrh3 knyttet til forbedret grønt fluorescerende protein syv vise deg hvordan du kan identifisere nevroner og overvåke deres elektrisk aktivitet i hele hjernen forberedelse seks.

Et eksempel på bilaterale klynger av GFP-merket tn-GnRH3 nerveceller fra utskåret hjerne killifisk av fisk er vist på figurene 1B og 1C. Hver klynge inneholder ca 8-10 GnRH nerveceller. De spontane nevrale målselskapets TN-GnRH3 ble registrert i current-klemme-modus (I = 0) med typiske avfyring priser på 0,5-6 Hz. Mønsteret av handlingen potensial avfyring er vanligvis en tonic eller banke mønster, med en ganske vanlig interspike intervall. Eksempel på spor er vist i fig...

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 denne videoen vil vi demonstrere hvordan du tar opp elektrisk aktivitet fra identifiserte enkelt nevroner i en hel hjerne forberedelse, som bevarer komplekse nevrale kretser. Vi bruker transgen fisk hvori gonadotropinfrigjørende hormon (GnRH)-nevronene er genetisk merket med en fluorescerende protein for identifikasjon i den intakte hjerne preparatet.

Innspilling elektriske aktiviteten fra Identifiserte Nerveceller i Intakt Norgesmester Transgenic Fish

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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Retrograde Fluorescent Merking Tillater målrettet Ekstracellulær Single-enhet Opptak fra Identifiserte Nerveceller<em&gt; In vivo</em

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Spenning-sensitive Dye Opptak fra Axoner, dendritter og dendrittiske spines av enkelte nerveceller i hjernen skiver

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

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The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

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The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

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Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

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A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

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Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...