Summary

Patch-clamp gravação de células Starburst Amácrinas em um monte de Luxo Preparação de Deafferentated mouse Retina

Published: October 13, 2016
doi:

Summary

Este protocolo demonstra como executar de células inteiras de gravação patch-clamp em neurônios da retina a partir de uma preparação flat-mount.

Abstract

A retina dos mamíferos é um tecido em camadas compostas de vários tipos neuronais. Para entender como os sinais visuais são processados ​​dentro de sua rede sináptica intrincado, registros eletrofisiológicos são frequentemente usados ​​para estudar as conexões entre os neurônios individuais. Nós otimizamos uma preparação plana de montagem para a gravação de patch-clamp de neurônios geneticamente marcados em ambos GCL (camada de células ganglionares) e INL (camada nuclear interna) de retinas de ratos. Gravação de neurônios do INL em planos montagens é favorecido sobre fatias porque as conexões verticais e laterais são preservados na configuração anterior, permitindo que os circuitos da retina com grandes componentes laterais a ser estudado. Temos utilizado este procedimento para comparar as respostas dos neurônios-espelho-partnered em retinas tais como as células amácrinas starburst colinérgicos (ZEC).

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

A aprovação ética – procedimentos que envolvem indivíduos animais foram conduzidos de acordo com as regras e regulamentos dos Institutos Nacionais de diretrizes de saúde para animais de pesquisa, conforme aprovado pelo Institutional Animal Care e usar comitê de Baylor College of Medicine. 1. externa e Soluções Internas Usar a solução de Ringer de mamífero durante a dissecção retina e como a solução externa na gravação eletrofisiológico subsequente. Preparar a solução de Ringer de m…

Representative Results

Registos representativos de dentro e fora do tipo SACs de uma retina deafferentated rato são mostrados na Figura 1. As células colinérgicas em ambos GCL e INL podem ser identificados de forma confiável por tdTomato fluorescência e direcionados para de células inteiras de gravação patch-clamp sob DIC (Figura 1A) para revelar oscilação de seus potenciais de membrana (top traços) e as correntes sinápticas que dirigi-lo (traços inferiores, <stro…

Discussion

Muitos laboratórios têm gravado a partir de neurônios GCL na preparação 15-18 plana de montagem, mas os nossos procedimentos de permitir a gravação de neurônios do INL. Vimos por este meio enfatizar várias etapas que são críticas para as gravações de rotina bem sucedidas.

A frescura e achatamento da retina são importantes para penetrar com uma pipeta de gravação. A este respeito, a ligação firme da retina para a membrana de nitrocelulose perfurado é primordial e…

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

References

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Cite This Article
Tu, H., Hsu, C., Chen, Y., Chen, C. Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina. J. Vis. Exp. (116), e54608, doi:10.3791/54608 (2016).

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