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 venlige forklaring på agar-embedded retinal skive forberedelse, da vi begyndte at bruge protokollen i vores laboratorium. Vi takker også Lori Vaskalis til illustration af skematisk overblik og Drs. Veeramuthu Balakrishnan og Soyoun Cho for nyttige kommentarer til tekst og video. Dette arbejde blev støttet af en NEI-NIH RO1 tilskud, og var også delvist støttet af en Korea Research Foundation Grant finansieret af den koreanske regering [KRF-2008-357-E00032].

1. Eksterne og interne løsninger <ol><li> Forbered udskæring opløsning (lavt calcium) fra 10x stamopløsningen og tilsæt MgCl 2, CaCl 2, og D-glucose dagligt. Den endelige 1x Løsningen består af (i mm): 119 NaCl, 2,5 KCl, 3,2 MgCl 2, 0,25 CaCl 2, 12 D-glucose, 0,2 L-ascorbinsyre, 12 HEPES. Indstil pH til 7,4 (med NaOH), og juster osmolaritet til 260 mOsm (ved hjælp af H 2 O og 10x stamopløsning). </li><li> Der afvejes 3% lave geldannende temperatur agar (A...

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En kritisk og vanskeligt skridt i vores protokol er overførslen af det stykke af nethinden i agar opløsning (protokol 3.4). Det er nødvendigt omhyggeligt at fjerne glaslegemet og resterende skive løsning fra den retinale brik og overføre det uden forvrængning eller bøjes. For at opnå dette, bruger vi en lille spatel (21-401-25B, Fisherbrand) med en spids bøjet til at danne en 90 ° vinkel, sammen med vinklet spids pincet (11251-35, Fin Science Tools), at placere den retinale brik i hele overfø...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Lav geldannende-temperatur agar </td><td> Sigma </td><td> A0701 </td><td> Agarose type VII-A </td></tr><tr><td> Patch pipette </td><td> Verdens præcisionsinstrumenter </td><td> 1B150F-4 </td><td> Tykvæggede (1,5 mm udvendig diameter) borosilikatglas </td></tr><tr><td> Lodret aftrækker </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 Tools </td><td> 15003-08 </td><td></td></tr><tr><td> 45 ° vinklet fin spids pincet </td><td> Fin Science Tools </td><td> 11251-35 </td><td></td></tr><tr><td> Barberblad </td><td> Personna </td><td></td><td> Tveæggede, renses med 70% ethanol og H 2 O </td></tr><tr><td> Cylindrisk rør </td><td> Fisherbrand </td><td> 03 til 338-1B </td><td> Polyethylen prøve hætteglas 2,5 ml </td></tr><tr><td> Hyaluronidase </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Vibratome pålægsmaskine </td><td> Leica </td><td> VT1000S eller VT1200S </td><td></td></tr><tr><td> Opretstående mikroskop </td><td> Olympus </td><td> BX51WI </td><td></td></tr><tr><td> 60x vand-nedsænkning mål </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;sort og hvid skærm </td></tr><tr><td> Sprøjte-filter </td><td> Nalgene </td><td></td><td> 0,2 μm </td></tr><tr><td> Mikromanipulator </td><td> Sutter Instrument </td><td> MPC-200 </td><td></td></tr><tr><td> Lock-in forstærker </td><td> Heka </td><td></td><td> EPC-9/10 forstærkere har software-emulering </td></tr><tr><td> Spinning disk laser konfokal mikroskop </td><td> Yokogawa </td><td> CSU-X1 </td><td> Live-cell imaging efter patch clamp helcelle optagelse </td></tr><tr><td> Slidebook software </td><td> Intelligent Imaging Instrumenter (3i) </td><td></td><td> Imaging dataopsamling og-analyse </td></tr><tr><td> Paraformaldehyd </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> Fosfatbuffer </td><td> GIBCO </td><td> 70013 </td><td></td></tr><tr><td> SuperFrost slide </td><td> Fisher Scientific </td><td></td><td> Slide glas </td></tr><tr><td> Anti-fading agenter </td><td> Biomeda corp. </td><td></td><td></td></tr><tr><td> Konfokal laser-scanning mikroskop </td><td> Carl Zeiss </td><td> LSM 710 </td><td> Billeddannelse af fikseret væv </td></tr><tr><td> Spatel </td><td> Fisherbrand </td><td> 21 til 401-25B </td><td></td></tr><tr><td> Manuel lodret pålægsmaskine </td><td> Narishige </td><td> ST-20 </td><td></td></tr><tr><td> Oregon Green 488 BAPTA-1 </td><td> Invitrogen </td><td> O-6806 </td><td> Ca 2 + følsomme fluorescerende farvestof </td></tr><tr><td> Alexa Fluor 555 Hydrazide </td><td> Invitrogen </td><td> A-20501MP </td><td> Fluorescerende farvestof </td></tr></tbody></table>

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

Visuelle stimuli opfanges og transporteres over et bredt dynamisk område af lys intensitet og frekvens ændringer af specialiserede neuroner i hvirveldyr nethinden. To klasser af retinale neuroner, fotoreceptorer og bipolære celler, opnå dette ved hjælp af bånd-typen aktive zoner, der gør det muligt vedvarende og high-throughput neurotransmitter release over lange perioder. ON-typen blandet bipolar celle (Mb) terminaler i guldfisk nethinden, som depolarisere for lys stimuli og modtage blandet stang og kegle fotoreceptor input, er egnet til studiet af bånd-typen synapser både på grund af deres store størrelse (~ 10-12 μm diameter) og deres talrige laterale og gensidige synaptiske forbindelser med amacrine celle dendritter. Direkte adgang til Mb bipolar celle terminaler i Goldfish retinal skiver med patch-clamp teknik giver mulighed for måling af præsynaptiske Ca 2 + strømme, membran kapacitans ændringer, og gensidig synaptic feedback hæmning medieret af GABA &lt;sub&gt; A og GABA C receptorer udtrykt på terminalerne. Præsynaptisk membran kapacitans målinger af exocytose tillader en at studere på kort sigt plasticitet excitatoriske neurotransmitter release 14,15. Derudover kan kortsigtede og langsigtede plasticitet hæmmende neurotransmitter frigivelse fra amacrine celler også blive undersøgt af optagelser af gensidige feedback hæmning ankommer til Mb terminalen 21. Over kortere perioder (fx ~ 10 s), undergår GABAergic gensidig feedback hæmning fra amacrine celler parret-puls depression via GABA vesikel pool udtynding 11. Den synaptisk dynamik retinal mikrokredsløb i den indre plexiform lag af nethinden kan således direkte undersøgt. Hjernen-slice teknik blev indført mere end 40 år siden, men er stadig meget nyttigt for undersøgelsen af ​​de elektriske egenskaber af neuroner, både på enkelt celle soma, enkelt dendritceller eller Axon, og microcircuit synaptiske niveau 19. Væv, der er for lille til at være limet direkte på udskæring kammer ofte er de første indlejret i agar (eller placeres i et filter papir) og derefter skåret 20, 23, 18, ​​9. I denne video beskæftiger vi før forankring agar teknik med guldfisk nethinden. Nogle af de gigantiske bipolar celle terminaler i vores skiver af guldfisk nethinden er axotomized (Axon-klip) under udskæring procedure. Dette giver os mulighed for at isolere en enkelt præsynaptiske nerve terminal indgange, fordi optagelse fra axotomized terminaler udelukker signalerne fra soma-dendritiske rum. Alternativt kan man også optage fra intakte Mb bipolare celler, ved at optage fra terminaler knyttet til axoner, som ikke er skåret under udskæring procedure. Samlet set vil anvendelsen af ​​denne forsøgsplan støtte i studier af retinale synaptisk fysiologi, mikrokredsløb funktionel analyse, og synaptisk transmission 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 protokol for forberedelse af agar-embedded retinal skiver, der er egnet til elektrofysiologi og Ca2 + billeddannelse. Denne metode gør det muligt at studere bånd-typen synapser i retina mikrokredsløb med direkte patch-clamp optagelser af en enkelt præsynaptiske nerveender.

Patch-clamp Kapacitans Målinger og Ca 2 + Imaging på Single nerveterminaler i Retinal Skiver

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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Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...