Patch Clamp Opptak av Starburst Amacrine celler i en Flat-mount Utarbeidelse av Deafferentated Mouse Retina
Summary
Denne protokollen viser hvordan du utfører hel-celle patch clamp opptak på netthinnens nerveceller fra en flat-mount forberedelse.
Abstract
Pattedyrnetthinnen er en lagdelt vev består av flere neuronale typer. For å forstå hvordan visuelle signaler behandles innen sine intrikate synaptiske nettverk, er elektrofysiologiske opptak ofte brukt til å studere forbindelser mellom enkeltnerveceller. Vi har optimalisert en flat-mount forberedelse for patch clamp opptak av genetisk merket nevroner i både GCL (ganglion celle laget) og INL (indre kjernefysiske lag) muse netthinne. Opptak INL nevroner i flat-mounts er favoriserte over skiver fordi både vertikale og laterale tilkoblinger er bevart i den tidligere konfigurasjon, slik at retinal kretser med store side komponenter som skal studeres. Vi har brukt denne fremgangsmåten for å sammenligne svarene fra speil samarbeid nevroner i netthinne slik som kolinerge starburst amacrine celler (sekker).
Introduction
As an easily accessible part of the central nervous system, the retina has for decades been a useful model in neuroscience studies. Genetic marking of neurons has allowed detailed characterization of synaptic connections in the retina. With many methodologies available to examine function and morphology of retinal neurons, the patch clamp recording technique has been instrumental in our current understanding of vertically transmitted signals in the retina. These signals are originated from photon absorption in photoreceptors and sent to brain visual centers through spiking of retinal ganglion cells (RGCs). Despite a large body of knowledge accumulated thus far, neural diversity in vascularized mammalian retina remains unsolved and obstructs the full appreciation of retinal circuits that subserve normal vision. This is in part because most recordings were performed on retinal slices to trade lateral circuit integrity for access to more proximal retinal neurons1-3. To gain a comprehensive picture on how retina computes visual signals, it is thus desirable to record neurons in flat-mounts wherein lateral connections, large and small, may be better preserved.
When synaptic transmission from photoreceptors to bipolar cells is interrupted due to a defective metabotropic glutamate receptor 6 (mGluR6) signaling pathway in depolarizing bipolar cells4-6 or simply as the result of photoreceptor loss in degenerated retinas7-10, many RGCs exhibit oscillatory activities. These oscillations originate from multiple sources, however the one involving gap junction coupling between AII amacrine cells (AII-ACs) and depolarizing cone bipolar cells (DCBCs) has received the most attention and hence is best understood1,7,11. We have found another source, which persists under pharmacological blockade of the aforementioned AII-AC/DCBC network and drives oscillation of OFF-type SACs in RhoΔCTA and Nob mice with deafferentated retinas7,8,12. Here we detail our protocol of preparing retinal flat-mounts for INL neuron recording. This approach uses commercial mouse lines (Jax stock no. 006410 and 007905) to mark cholinergic retinal neurons by fluorescent protein (tdTomato) expression that is identifiable under a fluorescent microscope equipped with contrast enhancing optics. Some experimental results acquired through this approach have been previously reported4,5,7,13.
Protocol
Representative Results
Discussion
Mange laboratorier har registrert fra GCL nevroner i flat-mount forberedelse 15-18, men våre prosedyrer tillater opptak fra INL nevroner. Vi vektlegger herved flere trinn som er avgjørende for vellykkede rutine opptak.
Friskhet og planhet av netthinnen er viktig for å trenge det med et opptak pipette. I denne forbindelse, fast feste av netthinnen til stemplet nitrocellulosemembranen er viktig, og blir oppnådd ved forbigående absorpsjon av løsningen, etterfulgt av tidsriktig …
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Joung Jang and Xin Guan for technical assistance. We thank Dr. Rory McQuiston of Virginia Commonwealth University for setting up our first patch clamp rig and advices on experimental procedures. We thank Dr. Samuel Wu for suggestions on voltage clamp recording. The work is supported by NIH grants EY013811, EY022228 and a vision core grant EY002520. C-KC is the Alice R. McPherson Retina Research Foundation Endowed Chair at the Baylor College of Medicine.
Materials
| Fixed-stage fluorescent microscope with DIC | Olympus | BX51-WI | |
| Micromanipulators | Sutter | MP-225 | |
| Patch clamp amplifier | A-M System | AM2400 | |
| AD converter | National Instrument | NI-USB-6221 | |
| Heater controller | Warner Instrument | TC-324B | |
| Inline heater | Warner Instrument | SC-20 | |
| Peristaltic pump | Rainin | Dynamax | |
| pipette puller | Sutter Instrument | P-1000 | |
| Glass tube with filament | King Precision Glass | Customized | |
| Stimulator | A.M.P.I. | Master-8 | |
| Biocytin | Sigma | B4261 | |
| NaCl | Sigma | S6191 | |
| KCl | Sigma | P5405 | |
| NaHCO3 | Fisher | BP328-1 | |
| Na2HPO4 | Sigma | S0876 | |
| NaH2PO4 | Sigma | S5011 | |
| CaCl2 | Sigma | C5670 | |
| MgSO4 | Sigma | M1880 | |
| D-glucose | Sigma | G6152 | |
| K-gluconate | Sigma | G4500 | |
| ATP-Mg | Sigma | A9187 | |
| Li-GTP | Sigma | G5884 | |
| EGTA | Sigma | E0396 | |
| HEPES | Sigma | H4034 | |
| KOH | Sigma | P5958 | |
| Cs-methanesulfonate | Sigma | C1426 | |
| CsOH | Sigma | 232041 | |
| Syringer filter | Nalgene | 171 | |
| 1 ml syring | Rainin | 17013002 | |
| 10 ul pipette tip | Genesee Scientific | 24-130RL | |
| Streptavidin-488 | ThermoFisher | S-11223 | |
| 10X PBS | Lonza | 17-517Q |
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