Name: patch-clamp capacitance measurements and ca2+ imaging at single nerve terminals in retinal slices
Uploaded: 2012-01-19T13:00:00
Description: Watch this Scientific Journal Video about Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices at JoVE.com

Vi takker Dr. Fred Rieke for hans slags forklaring av agar-embedded retinal slice forberedelse da vi begynte å bruke protokollen i vårt laboratorium. Vi takker også Lori Vaskalis for illustrasjon av skjematisk oversikt og Drs. Veeramuthu Balakrishnan og Soyoun Cho for nyttige kommentarer til tekst og video. Dette arbeidet ble støttet av en NEI-NIH RO1 stipend, og var også delvis støttet av en Korea Research Foundation Grant finansiert av den koreanske regjeringen [KRF-2008-357-E00032].

1. Ekstern og intern Solutions <ol><li> Forbered slicing løsning (lav kalsium) fra 10x stamløsning og tilsett 2 MgCl, CaCl 2, og D-glukose daglig. Den endelige 1x Løsningen består av (i mm): 119 NaCl, 2,5 KCl, 3.2 2 MgCl, 0,25 CaCl 2, 12 D-glukose, 0,2 L-askorbinsyre, 12 HEPES. Sett pH til 7,4 (med NaOH), og juster osmolaritet til 260 mOsm (med H 2 O og 10x stamløsningen). </li><li> Vei opp 3% lav gelling temperatur agar (agarose type VII-A, A0701, Sigma, Rie...

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En kritisk og vanskelig skritt i protokoll vårt er overføring av del av netthinnen inn i agar løsningen (protokoll 3.4). Det er nødvendig å forsiktig fjerne glasslegemet og rester slice løsning fra retinal stykke og overføre det uten forvrengning eller bøyd. For å oppnå dette bruker vi en liten slikkepott (21-401-25B, Fisherbrand) med et tips bøyd i en 90 ° vinkel, sammen med vinklet spiss tang (11251-35, Fine Science Tools), å plassere retinal stykke gjennom overføringsprosessen i skive l...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Lav gelling-temperatur agar </td><td> Sigma </td><td> A0701 </td><td> Agarose type VII-A </td></tr><tr><td> Patch pipette </td><td> Verden presisjonsinstrumenter </td><td> 1B150F-4 </td><td> Tykke vegger (1,5 mm ytre diameter) borsilikatglass </td></tr><tr><td> Vertikal puller </td><td> Narishige </td><td> PP830 </td><td></td></tr><tr><td> Dental voks </td><td> Cavex </td><td></td><td></td></tr><tr><td> Spring saks </td><td> Fin Science Verktøy </td><td> 15003-08 </td><td></td></tr><tr><td> 45 ° vinklet fin spiss tang </td><td> Fin Science Verktøy </td><td> 11251-35 </td><td></td></tr><tr><td> Barberblad </td><td> Personna </td><td></td><td> Tveegget, rengjøres med 70% etanol og H 2 O </td></tr><tr><td> Sylindrisk tube </td><td> Fisherbrand </td><td> 03-338-1B </td><td> Polyetylen sample hetteglass 2,5 ml </td></tr><tr><td> Hyaluronidase </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Vibratome slicer </td><td> Leica </td><td> VT1000S eller VT1200S </td><td></td></tr><tr><td> Upright mikroskop </td><td> Olympus </td><td> BX51WI </td><td></td></tr><tr><td> 60x vann-immersion objektiv </td><td> Olympus </td><td> LUMPlanFl </td><td> NA 0,90 </td></tr><tr><td> CCD-kamera </td><td> Sony </td><td> XC-75 </td><td></td></tr><tr><td> Kamera controller </td><td> Hamamatsu </td><td> C2400 </td><td></td></tr><tr><td> Monitor &lt;/ Td&gt; <td> Sony </td><td></td><td> 13 &quot;svart og hvit monitor </td></tr><tr><td> Sprøyte filter </td><td> Nalgene </td><td></td><td> 0,2 mikrometer </td></tr><tr><td> Micromanipulator </td><td> Sutter Instrument </td><td> MPC-200 </td><td></td></tr><tr><td> Lås-forsterker </td><td> Heka </td><td></td><td> EPC-9/10 forsterkere har programvareemulering </td></tr><tr><td> Spinning disk laser confocal mikroskop </td><td> Yokogawa </td><td> CSU-X1 </td><td> Live-cell imaging etter patch clamp helcelle opptak </td></tr><tr><td> Slidebook programvare </td><td> Intelligent Imaging Instruments (3i) </td><td></td><td> Imaging datainnsamling og analyse </td></tr><tr><td> Paraformaldehyde </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> Fosfatbuffer løsning </td><td> GIBCO </td><td> 70013 </td><td></td></tr><tr><td> Superfrost skyv </td><td> Fisher Scientific </td><td></td><td> Slide glass </td></tr><tr><td> Anti-fading agenter </td><td> Biomeda corp. </td><td></td><td></td></tr><tr><td> Confocal laser-scanning mikroskop </td><td> Carl Zeiss </td><td> LSM 710 </td><td> Imaging av fast vev </td></tr><tr><td> Slikkepott </td><td> Fisherbrand </td><td> 21-401-25B </td><td></td></tr><tr><td> Manuel vertikal slicer </td><td> Narishige </td><td> ST-20 </td><td></td></tr><tr><td> Oregon Grønn 488 BAPTA-1 </td><td> Invitrogen </td><td> O-6806 </td><td> Ca 2 + sensitive fluorescerende fargestoff </td></tr><tr><td> Alexa Fluor 555 Hydrazide </td><td> Invitrogen </td><td> A-20501MP </td><td> Fluorescerende fargestoff </td></tr></tbody></table>

patch-clamp capacitance measurements and ca2+ imaging at single nerve terminals in retinal slices

Visuelle stimuli er oppdaget og formidlet over et bredt dynamisk spekter av lys intensitet og frekvens endres med spesialisert nevroner i virveldyr netthinnen. To klasser av netthinnens nerveceller, fotoreseptorene og bipolare celler, oppnår dette ved hjelp av bånd-type aktive soner, som muliggjør vedvarende og høy gjennomstrømning nevrotransmitter utgivelse over lange tidsperioder. ON-typen blandet bipolar celle (Mb) terminaler i gullfisk netthinnen, som depolarize til lys stimuli og motta blandet stang og kjegle fotoreseptoren innspill, er egnet for studiet av bånd-type synapser både på grunn av sin store størrelse (~ 10-12 mikrometer diameter), og deres tallrike lateral og gjensidige synaptiske forbindelser med amacrine celle dendritter. Direkte tilgang til Mb bipolar celle terminaler i gullfisk retinal skiver med patch-clamp teknikken tillater måling av presynaptisk Ca 2 + strømmer, membran kapasitans endringer, og gjensidig synaptisk feedback-hemming mediert av GABA &lt;sub&gt; A og GABA C reseptorer uttrykt på terminalene. Presynaptisk membran kapasitans målinger av eksocytose tillate en å studere den kortsiktige plastisitet av eksitatoriske nevrotransmitter utgivelsen 14,15. I tillegg kan kortsiktige og langsiktige plastisitet av inhibitoriske nevrotransmitter løslatelse fra amacrine celler også bli etterforsket av innspillinger av gjensidige tilbakemeldinger hemming ankommer Mb terminal 21. Over korte perioder av gangen (f.eks ~ 10 s), gjennomgår gabaergic gjensidig feedback-hemming fra amacrine celler parvise puls depresjon via GABA vesicle bassenget uttømming 11. Den synaptiske dynamikk retinal microcircuits i indre plexiform laget av netthinnen kan dermed være direkte studert. Hjernen-slice teknikk ble introdusert mer enn 40 år siden, men er fortsatt svært nyttig for etterforskningen av de elektriske egenskapene til nevroner, både på enkelt celle soma, single dendrite eller axon, og microcircuit synaptiske nivå 19. Vev som er for små til å være limt direkte på slicing kammeret er ofte første innebygd i agar (eller plasseres på et filter papir) og deretter skiver 20, 23, 18, ​​9. I denne videoen, ansetter vi pre-embedding agar teknikk med gullfisk netthinnen. Noen av de gigantiske bipolar celle terminaler i vår skiver av gullfisk netthinnen er axotomized (axon-cut) under kutting prosedyren. Dette gir oss muligheten til å isolere enkelt presynaptisk nerve terminal innganger, fordi opptak fra axotomized terminaler utelukker signalene fra de soma-dendrittiske kupé. Alternativt kan man også ta opp fra intakt Mb bipolare celler, med opptak fra terminaler festet til axoner som ikke har blitt kuttet under kutting prosedyren. Totalt sett vil bruk av denne forsøksprotokollen hjelpemiddel i studier av retinal synaptic fysiologi, microcircuit funksjonell analyse, og synaptisk overføring på bånd synapser.

Watch this Scientific Journal Video about Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices at JoVE.com

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Her beskriver vi en protokoll for utarbeidelse av agar-embedded retinal skiver som er egnet for elektrofysiologi og Ca2 + bildebehandling. Denne metoden gjør det mulig å studere bånd-type synapser i retinal microcircuits bruke direkte patch-clamp opptak av single presynaptiske nerveterminaler.

Patch-clamp Kapasitans Målinger og Ca 2 + Imaging ved Singel nerveterminaler i Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

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Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

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