Patch Clamp grabación de células amacrinas Starburst en un plano de montaje Preparación de Deafferentated retina del ratón
Summary
Este protocolo muestra cómo realizar la grabación de células enteras patch clamp en neuronas de la retina de una preparación plana de montaje.
Abstract
La retina de mamífero es un tejido en capas compuesta de múltiples tipos neuronales. Para entender cómo las señales visuales se procesa dentro de su red sináptica intrincada, registros electrofisiológicos se utilizan con frecuencia para estudiar las conexiones entre las neuronas individuales. Hemos optimizado un preparado de montaje plana para la grabación de patch clamp de neuronas marcadas genéticamente en tanto GCL (capa de células ganglionares) y INL (capa nuclear interna) de las retinas de ratones. Grabación de las neuronas en INL-montajes planos se ve favorecida sobre las rebanadas porque las conexiones verticales y laterales se conservan en la antigua configuración, permitiendo que los circuitos de la retina con grandes componentes laterales para ser estudiado. Hemos utilizado este procedimiento para comparar las respuestas de las neuronas espejo, mediante asociación en las retinas como las células amacrinas 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
Representative Results
Discussion
Muchos laboratorios han registrado desde las neuronas GCL en la preparación 15-18 plana de montaje, pero nuestros procedimientos permiten la grabación de las neuronas de la INL. Por la presente hacemos hincapié en varios pasos que son críticos para las grabaciones de rutina con éxito.
La frescura y planeidad de la retina son importantes para penetrar en él con una pipeta de grabación. En este sentido, la firma de la unión de la retina a la membrana de nitrocelulosa perfora…
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
- Choi, H., et al. Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina. J Neurophysiol. 112 (6), 1491-1504 (2014).
- Gregg, R. G., et al. Nyctalopin expression in retinal bipolar cells restores visual function in a mouse model of complete X-linked congenital stationary night blindness. J Neurophysiol. 98 (5), 3023-3033 (2007).
- Hellmer, C. B., Ichinose, T. Recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques. J Vis Exp. (96), (2015).
- Tu, H. Y., Bang, A., McQuiston, A. R., Chiao, C. C., Chen, C. K. Increased dendritic branching in direction selective retinal ganglion cells in nob1 mice. Invest Ophthalmol Vis Sci. 55 (13), (2014).
- Tu, H. Y., Chen, Y. J., Chiao, C. C., McQuiston, A. R., Chen, C. K. J. Rhythmic membrane potential fluctuations of cholinergic amacrine cells in mice lacking ERG b-waves. Invest Ophthalmol Vis Sci. 56 (7), (2015).
- Demas, J., et al. Failure to maintain eye-specific segregation in nob, a mutant with abnormally patterned retinal activity. Neuron. 50 (2), 247-259 (2006).
- Tu, H. Y., Chen, Y. J., McQuiston, A. R., Chiao, C. C., Chen, C. K. A Novel Retinal Oscillation Mechanism in an Autosomal Dominant Photoreceptor Degeneration Mouse Model. Front Cell Neurosci. 9, 513 (2015).
- Borowska, J., Trenholm, S., Awatramani, G. B. An intrinsic neural oscillator in the degenerating mouse retina. J Neurosci. 31 (13), 5000-5012 (2011).
- Margolis, D. J., Detwiler, P. B. Cellular origin of spontaneous ganglion cell spike activity in animal models of retinitis pigmentosa. J Ophthalmol. , (2011).
- Stasheff, S. F. Emergence of sustained spontaneous hyperactivity and temporary preservation of OFF responses in ganglion cells of the retinal degeneration (rd1) mouse. J Neurophysiol. 99 (3), 1408-1421 (2008).
- Trenholm, S., Awatramani, G. B. Origins of spontaneous activity in the degenerating retina. Front Cell Neurosci. 9, 277 (2015).
- Trenholm, S., et al. Intrinsic oscillatory activity arising within the electrically coupled AII amacrine-ON cone bipolar cell network is driven by voltage-gated Na+ channels. J Physiol. 590 (Pt 10), 2501-2517 (2012).
- Chen, C. K. J., et al. Cell-autonomous changes in displaced cholinergic amacrine cells lacking Gbeta5. Invest Ophthalmol Vis Sci. 56 (7), (2015).
- O’Brien, B. J., Isayama, T., Richardson, R., Berson, D. M. Intrinsic physiological properties of cat retinal ganglion cells. J Physiol. 538 (Pt 3), 787-802 (2002).
- Lee, S., et al. An Unconventional Glutamatergic Circuit in the Retina Formed by vGluT3 Amacrine Cells. Neuron. 84 (4), 708-715 (2014).
- Hoggarth, A., et al. Specific wiring of distinct amacrine cells in the directionally selective retinal circuit permits independent coding of direction and size. Neuron. 86 (1), 276-291 (2015).
- Margolis, D. J., Gartland, A. J., Singer, J. H., Detwiler, P. B. Network oscillations drive correlated spiking of ON and OFF ganglion cells in the rd1 mouse model of retinal degeneration. PLoS One. 9 (1), e86253 (2014).
- Schmidt, T. M., Kofuji, P. An isolated retinal preparation to record light response from genetically labeled retinal ganglion cells. J Vis Exp. (47), (2011).
- Ivanova, E., Yee, C. W., Sagdullaev, B. T. Increased phosphorylation of Cx36 gap junctions in the AII amacrine cells of RD retina. Front Cell Neurosci. 9, 390 (2015).
- Enoki, R., Koizumi, A. A method of horizontally sliced preparation of the retina. Methods Mol Biol. 935, 201-205 (2013).
- Akrouh, A., Kerschensteiner, D. Morphology and function of three VIP-expressing amacrine cell types in the mouse retina. J Neurophysiol. 114 (4), 2431-2438 (2015).
- Wei, W., Elstrott, J., Feller, M. B. Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina. Nat Protoc. 5 (7), 1347-1352 (2010).
