Høy oppløsning Kvantitativ immungull Analyse av membranreseptorer på retina Ribbon Synapses
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 er den viktigste eksitatoriske neurotransmitter i netthinnen 1. Gangliecelle (RGCs), som fikk glutamatergic synaptic innspill fra bipolare celler 2, er utgangsnerveceller i netthinnen som sender visuell informasjon til hjernen. Fysiologiske studier viste at synaptic eksitasjon av RGCs formidles postsynaptisk av NMDA reseptorer (NMDARs) og AMPA reseptorer (AMPARs) 3,4,5. Selv eksitatoriske postsynaptiske strømmer (EPSCs) i RGCs formidles av AMPARs og NMDARs 3,5,6,7,8, spontane miniatyr EPSCs (mEPSCs) på RGCs utviser bare en AMPARs-mediert komponent 4,5,9. Men å redusere glutamatopptak avslørte en NMDAR komponent i spontane EPSCs 5, noe som tyder på at NMDARs på RGC dendritter kan befinne seg utenfor eksitatoriske synapser. Membran-forbundet guanylate kinaser (MAGUKs) som PSD-95 som klynge nevrotransmitterreseptorer, inkludert glutamat reseptorer og ion-kanalens ved synaptiske områder, også stille tydelige subsynaptic uttrykk mønstre 10,11,12,13,14.
Over de siste tiårene, konfokal immunhistokjemi og pre-embedding elektronmikroskop (EM) immunhistokjemi har blitt ansatt for å studere membran reseptor uttrykk. Selv konfokal farging avslører brede mønstre av reseptor uttrykk, gjør sin lavere oppløsning det umulig å bruke for å skille subcellulære sted. Pre-embedding EM studier i pattedyr netthinnen indikerer at NMDAR subenheter er til stede i postsynaptiske elementer ved kjegle bipolar celle bånd synapser 15,16,17. Dette er i tydelig kontrast til fysiologisk bevis. Imidlertid diffusjon av reaksjonsproduktet er et velkjent gjenstand i den forhånds innstøping immunoperoxidase metode. Derfor betyr denne tilnærmingen vanligvis ikke gi statistisk sikre data og kan ekskludere skillet mellom lokalisering til synaptiske membran versus extrasynaptic membran 18,19,20,21. PåDerimot fysiologiske og anatomiske data er i overensstemmelse med en synaptisk lokalisering av AMPARs på RGCs 3,5,7,9,22. Således blir glutamatreseptorer og MAGUKs på retinal bånd synapse lokalisert ikke bare til den postsynaptiske, men også til de perisynaptic eller extrasynaptic membranlommer. Imidlertid er en høy oppløsning kvantitativ analyse av disse membran proteiner i en retinal bånd synapse fortsatt nødvendig.
Her har vi utviklet en postembedding EM immunogold teknikk for å undersøke subsynaptic lokalisering av NMDAR underenheter, Ampar subenheter og PSD-95 etterfulgt av å estimere antall, tetthet og variasjon av disse proteinene i synapsene ut mot rotte RGCs merkes med koleratoksin subenhet B (CTB) retrograd Tracing metoder.
Protocol
Representative Results
Discussion
Vi har beskrevet fire teknikker for vellykket kvantitativ post-embedding immunogold EM: 1) kort og svak fiksering, 2) fryse substitusjon, 3) post-embedding immunogold flekker, og 4) kvantifisering.
EM en immun tillater påvisning av spesifikke proteiner i ultratynne vevssnitt. Antistoffer merket med gull-partikler kan direkte visualisert ved anvendelse EM. Mens kraftig påvise subsynaptic lokalisering av en membran reseptor, kan EM immunogold være teknisk utfordrende, og krever strengere op…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbeidet ble støttet av egenutført programmer av National Institute of nevrologiske lidelser og hjerneslag (NINDS) og National Institute on døvhet og Other Communication Disorders (NIDCD), av National Institutes of Health (NIH). Vi takker ninds EM anlegget og NIDCD avansert bildebehandling kjerne (kode # ZIC DC 000081-03) for å få hjelp.
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 |
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