Hochauflösende Quantitative Immungold-Analyse von Membranrezeptoren auf der Netzhaut Ribbon Synapsen
Summary
The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.
Abstract
Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.
Introduction
Glutamat ist der hauptsächliche exzitatorische Neurotransmitter im Netzhaut 1. Retinalen Ganglienzellen (RGC), glutamatergen synaptischen Input von bipolaren Zellen 2 sind die Ausgangsneuronen der Netzhaut erhalten, die visuelle Information an das Gehirn senden. Physiologische Untersuchungen zeigten, dass die synaptische Erregung von RGCs vermittelt wird postsynaptisch von NMDA-Rezeptoren (NMDARs) und AMPA-Rezeptoren (AMPARs) 3,4,5. Obwohl exzitatorischen postsynaptischen Ströme (EPSCs) in RGCs durch AMPARs und NMDARs 3,5,6,7,8, zeigen spontane Miniatur EPSCs (mEPSCs) auf RGCs vermittelt werden nur eine AMPARs vermittelte Komponente 4,5,9. Allerdings ergab Glutamataufnahme Reduktion eines NMDAR Komponente in spontanen EPSCs 5, was darauf hindeutet, dass NMDARs auf RGC Dendriten können außerhalb von Synapsen befinden. Membranassoziierten Guanylat-Kinasen (MAGUKs) wie PSD-95, dass Cluster Neurotransmitter-Rezeptoren, einschließlich Glutamat-Rezeptoren und Ionenkanals bei synaptischen Websites, auch deutliche subsynaptischen Expressionsmuster 10,11,12,13,14 aufweisen.
In den letzten Jahrzehnten konfokalen Immunhistochemie und Pre-Einbettung Elektronenmikroskop (EM) Immunhistochemie wurden zu studieren Membran-Rezeptor-Expression eingesetzt. Obwohl konfokalen Immunfärbung breites Muster der Rezeptorexpression offenbart, macht seine geringere Auflösung unmöglich subzellulären Ort zu unterscheiden zu können. Pre-Einbettung EM-Studien in Säugernetzhaut zeigen, dass NMDAR Untereinheiten in postsynaptischen Elemente bei Konus Bipolarzelle Band Synapsen 15,16,17 vorhanden sind. Dies ist in der scheinbaren Gegensatz zu physiologischen Beweise. Jedoch Diffusion von Reaktionsprodukt ist ein bekannter Artefakt in der Pre-Einbettungs Immunperoxidase-Verfahren. Daher ist dieser Ansatz nicht in der Regel statistisch verlässliche Daten geben und Unterscheidung zwischen Lokalisierung zu synaptischen Membran gegen extrasynaptischen Membran 18,19,20,21 ausschließen. Aufdie andere Hand, physiologischen und anatomischen Daten sind mit einem synaptischen Lokalisation von AMPARs auf RGCs 3,5,7,9,22 konsistent. Somit Glutamatrezeptoren und MAGUKs bei Netzhautband Synapse nicht nur lokalisiert der postsynaptischen sondern auch auf die perisynaptic oder extrasynaptischen Membran Fächern. Jedoch ist ein hochauflösender quantitative Analyse dieser Membranproteine in einer Netzhautband Synapse wird noch benötigt.
Hier entwickelten wir ein postembedding EM Immunogold Technik die subsynaptischen Lokalisierung von NMDAR-Untereinheiten, AMPAR Untereinheiten und PSD-95, gefolgt von der Schätzung der Anzahl, Dichte und die Variabilität dieser Proteine an Synapsen auf Ratten RGCs untersucht markierten Choleratoxin-Untereinheit B mit (CTB) Rückverfolgung Methoden.
Protocol
Representative Results
Discussion
Wir haben vier beschriebenen Techniken für eine erfolgreiche quantitative Post-Einbettung Immun EM: 1) kurze und schwache Fixierung, 2) Gefriersubstitution, 3) nach dem Einbetten von Immunfärbung und 4) Quantifizierung.
EM Immunogold ermöglicht den Nachweis von spezifischen Proteinen in ultradünne Gewebeschnitten. Markierte Antikörper mit Goldpartikeln kann direkt unter Verwendung von EM visualisiert werden. Während mächtig in die subsynaptischen Lokalisierung eines Membranrezeptors z…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Diese Arbeit wurde durch die Interne Programme des National Institute of Neurological Disorders and Stroke (NINDS) und National Institute on Taubheit und andere Communication Disorders (NIDCD), von den National Institutes of Health (NIH) unterstützt. Wir danken dem NINDS EM Anlage und die NIDCD Advanced Imaging-Kern (Code # ZIC DC 000081-03) um Hilfe.
Materials
| Paraformaldehyde | EMS | 15710 | |
| Glutarldehyde | EMS | 16019 | |
| NaH2PO4 | Sigma | S9638 | |
| Na2HPO4 | Sigma | 7782-85-6 | |
| CaCl2 | Sigma | C-8106 | |
| BSA | Sigma | A-7030 | |
| Triton X-100 | Sigma | T-8787 | |
| NaOH | Sigma | 221465 | |
| NaN3 | JT Baker | V015-05 | |
| Glycerol | Gibco BRL | 15514-011 | |
| Lowicryl HM 20 | Polysciences | 15924-1 | |
| Tris-Base | Fisher | BP151-500 | |
| Tris | Fisher | 04997-100 | |
| Anti-GluN2A | Millipore | AB1555P | Dilution 1/50 |
| Anti-GluN2B | Millipore | AB1557P | Dilution 1/30 |
| Anti-GluA2/3 | Millipore | AB1506 | Dilution 1/30 |
| Anti-PSD-95 | Millipore | MA1–046 | Dilution 1/100 |
| Donkey anti-rabbit IgG-10 nm gold particles | EMS | 25704 | Dilution 1/20 |
| Donkey anti-mouse IgG-10 nm gold particles | EMS | 25814 | Dilution 1/20 |
| Donkey anti-mouse IgG-5 nm gold particles | EMS | 25812 | Dilution 1/20 |
| Donkey anti-goat IgG-18 nm gold particles | Jackson ImmunoResearch | 705-215-147 | Dilution 1/20 |
| Formvar-Carbon coated nickel-slot grids. | EMS | FCF2010-Ni | |
| Uranyl acetate | EMS | 22400-1 | |
| Methanol | EMS | 67-56-1 | |
| Lead citrate | Leica | ||
| Leica EM AFS | Leica | ||
| Leica EM CPC | Leica | ||
| Ultromicrotome | Leica | ||
| JEOL 1200 EM | JEOL | ||
| liquid nitrogen | Roberts Oxygen | ||
| Propane | Roberts Oxygen | ||
| CTB | List Biological Laboratories | 104 | 1-1.2% |
| Anti-CTB | List Biological Laboratories | 703 | Dilution 1/4000 |
References
- Copenhagen, D. R., Jahr, C. E. Release of endogenous excitatory amino acids from turtle photoreceptors. Nature. 341, 536-539 (1989).
- Wässle, H., Boycott, B. B. Functional architecture of the mammalian retina. Physiol. Rev. 71, 447-480 (1991).
- Mittman, S., Taylor, W. R., Copenhagen, D. R. Concomitant activation of two types of glutamate receptor mediates excitation of salamander retinal ganglion cells. J. Physiol. 428, 175-197 (1990).
- Matsui, K., Hosoi, N., Tachibana, M. Excitatory synaptic transmission in the inner retina: paired recordings of bipolar cells and neurons of the ganglion cell layer. J. Neurosci. 18, 4500-4510 (1998).
- Chen, S., Diamond, J. S. Synaptically released glutamate activates extrasynaptic NMDA receptors on cells in the ganglion cell layer of rat retina. J. Neurosci. 22, 2165-2173 (2002).
- Diamond, J. S., Copenhagen, D. R. The contribution of NMDA and non-NMDA receptors to the light-evoked input-output characteristics of retinal ganglion cells. Neuron. 11, 725-738 (1993).
- Lukasiewicz, P. D., Wilson, J. A., Lawrence, J. E. AMPA-preferring receptors mediate excitatory synaptic inputs to retinal ganglion cells. J. Neurophysiol. 77, 57-64 (1997).
- Higgs, M. H., Lukasiewicz, P. D. Glutamate uptake limits synaptic excitation of retinal ganglion cells. J. Neurosci. 19, 3691-3700 (1999).
- Taylor, W. R., Chen, E., Copenhagen, D. R. Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells. J. Physiol. 486, 207-221 (1995).
- Kennedy, M. B. Origin of PDZ (DHR, GLGF) domains. Trends. Biochem. Sci. 20, 350 (1995).
- Kim, E., Sheng, M. PDZ domain proteins of synapses. Nat. Rev. Neurosci. 5, 771-781 (2004).
- Migaud, M., et al. Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature. 396, 433-439 (1998).
- Aoki, C., et al. Electron microscopic immunocytochemical detection of PSD-95, PSD-93, SAP-102, and SAP-97 at postsynaptic, presynaptic, and nonsynaptic sites of adult and neonatal rat visual cortex. Synapse. 40, 239-257 (2001).
- Davies, C., Tingley, D., Kachar, B., Wenthold, R. J., Petralia, R. S. Distribution of members of the PSD-95 family of MAGUK proteins at the synaptic region of inner and outer hair cells of the guinea pig cochlea. Synapse. 40, 258-268 (2001).
- Hartveit, E., Brandstätter, J. H., Sassoè-Pognetto, M., Laurie, D. J., Seeburg, P. H., Wässle, H. Localization and developmental expression of the NMDA receptor subunit NR2A in the mammalian retina. J. Comp. Neurol. 348, 570-582 (1994).
- Fletcher, E. L., Hack, I., Brandstätter, J. H., Wässle, H. Synaptic localization of NMDA receptor subunits in the rat retina. J. Comp. Neurol. 420, 98-112 (2000).
- Pourcho, R. G., Qin, P., Goebel, D. J. Cellular and subcellular distribution of NMDA receptor subunit NR2B in the retina. J. Comp. Neurol. 433, 75-85 (2001).
- Ottersen, O. P., Landsend, A. S. Organization of glutamate receptors at the synapse. Eur. J. Neurosci. 9, 2219-2224 (1997).
- Petralia, R. S., Wenthold, R. J., Ariano, M. A. Glutamate receptor antibodies: Production and immunocytochemistry. Receptor Localization: Laboratory Methods and Procedures. , 46-74 (1998).
- Petralia, R. S., Wenthold, R. J. Immunocytochemistry of NMDA receptors. Methods. Mol. Biol. 128, 73-92 (1999).
- Nusser, Z. AMPA and NMDA receptors: similarities and differences in their synaptic distribution. Curr. Opin. Neurobiol. 10, 337-341 (2000).
- Qin, P., Pourcho, R. G. Localization of AMPA-selective glutamate receptor subunits in the cat retina: a light- and electron-microscopic study. Vis. Neurosci. 16, 169-177 (1999).
- Zhang, J., Wang, H. H., Yang, C. Y. Synaptic organization of GABAergic amacrine cells in salamander retina. Visual. Neurosci. 21, 817-825 (2004).
- Zhang, J., Diamond, J. S. Distinct perisynaptic and synaptic localization of NMDA and AMPA receptors on ganglion cells in rat retina. J. Comp. Neurol. 498, 810-820 (2006).
- Zhang, J., Diamond, J. S. Subunit- and Pathway-Specific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina. J. Neurosci. 29, 4274-4286 (2009).
- Peters, A., Palay, S., Webster, H. . The fine structure of the nervous system: neurons and their supporting cells, 3rd ed. , (1991).
- Baude, A., Nusser, Z., Roberts, J. D. B. The metabotropic glutamate receptor (mGluR1) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction. Neuron. 11, 771-787 (1993).
- Van Lookeren Campagne, M., Oestreicher, A. B., van der Krift, T. P., Gispen, W. H., Verkleij, A. J. Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: Suitability for immunogold ultrastructural localization of neural antigens. J. Hist. Cyt. 39, 1267-1279 (1991).
- Matsubara, A., Laake, J. H., Davanger, S., Usami, S., Ottersen, O. P. Organization of AMPA receptor subunits at a glutamate synapse: A quantitative immunogold analysis of hair cell synapses in the rat organ of Corti. J. Neurosci. 16, 4457-4467 (1996).
- Petralia, R. S., et al. Organization of NMDA receptors at extrasynaptic locations. Neurosciences. 167, 68-87 (2010).
- Merighi, A., Polak, J. M., Priestley, J. V. Post-embedding electron microscopic immunocytochemistry. Electron Microscopic Immunocytochemistry: Principles and Practice. , 51-87 (1992).
- Ottersen, O. P., Takumi, Y., Matsubara, A., Landsend, A. S., Laake, J. H., Usami, S. Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear. Prog. Neurobiol. 54, 127-148 (1998).
- Nusser, Z., Lujan, R., Laube, G., Roberts, J. D., Molnar, E., Somogyi, P. Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus. Neuron. 21, 545-559 (1998).
- Takumi, Y., Ramirez-Leon, V., Laake, P., Rinvik, E., Ottersen, O. P. Different modes of expression of AMPA and NMDA receptors in hippocampal synapses. Nat. Neurosci. 2, 618-624 (1999).
- Grimes, W. N., et al. Complex inhibitory microcircuitry regulates retinal signaling near visual threshold. J. Neurophysiol. 114, 341-353 (2015).
- Sassoe-Pognetto, M., Ottersen, O. P. Organization of ionotropic glutamate receptors at dendrodendritic synapses in the rat olfactory bulb. J. Neurosci. 20, 2192-2201 (2000).
