High-Resolution Kwantitatieve immunogoud Analyse van membraanreceptoren op het netvlies 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
Glutamaat is de belangrijkste exciterende neurotransmitter in de retina 1. Retinale ganglioncellen (RGC's), het ontvangen glutamaat synaptische input van bipolaire cellen 2, zijn de output vormen van de retina die visuele informatie naar de hersenen sturen. Fysiologische studies toonden aan dat synaptische excitatie van RGC postsynaptisch wordt gemedieerd door NMDA-receptoren (NMDARs) en AMPA receptoren (AMPARs) 3,4,5. Hoewel prikkelende postsynaptische stromingen (EPSCs) in RGC worden gemedieerd door AMPARs en NMDARs 3,5,6,7,8, spontane miniatuur EPSCs (mEPSCs) op RGC vertonen slechts een AMPARs-gemedieerde component 4,5,9. Echter, het verminderen glutamaat opname onthulde een NMDAR component spontane EPSCs 5 suggereert dat NMDARs op RGC dendrieten buitenzijde van exciterende synapsen kunnen bevinden. Membraan-geassocieerde guanylate kinases (MAGUKs), zoals PSD-95 dat cluster neurotransmitter receptoren, met inbegrip van glutamaat receptoren en ionkanaals bij synaptische plaatsen, vertonen ook duidelijke subsynaptic expressiepatronen 10,11,12,13,14.
In de afgelopen decennia, confocale immunohistochemie en pre-inbedding elektronenmicroscoop (EM) immunohistochemie werden gebruikt om het membraan receptor expressie te bestuderen. Hoewel confocale immunokleuring onthult brede patronen van receptor expressie, de lagere resolutie niet mogelijk is om te onderscheiden subcellulaire locatie. Pre-inbedding EM studies in zoogdieren netvlies geven aan dat NMDAR subeenheden aanwezig in postsynaptische elementen op kegel bipolaire cel lint synapsen 15,16,17 zijn. Dit is in duidelijke tegenstelling tot fysiologische gegevens. Echter diffusie van reactieproduct een bekende artefact in de pre-inbedding immunoperoxidase methode. Daarom is deze benadering niet geven meestal statistisch betrouwbare gegevens en kunnen onderscheid tussen lokalisatie uitsluiten synaptische membraan versus extrasynaptic membraan 18,19,20,21. OpAnderzijds fysiologische en anatomische gegevens in overeenstemming met een lokalisatie van synaptische AMPARs op RGC 3,5,7,9,22. Aldus glutamaatreceptoren en MAGUKs bij retinale lint synaps gelokaliseerd niet alleen de postsynaptische maar ook de perisynaptic of extrasynaptic membraancompartimenten. Echter, een hoge-resolutie kwantitatieve analyse van deze membraaneiwitten in een retinale lint synaps nog steeds nodig.
Hier hebben we een postembedding EM immunogold techniek om de subsynaptic lokalisatie van NMDAR subeenheden, Ampar subeenheden en PSD-95, gevolgd door een schatting van het aantal, de dichtheid en de veranderlijkheid van deze eiwitten bij synapsen op rat RGC onderzoeken geëtiketteerd met choleratoxine subunit B (CTB) retrograde tracing methoden.
Protocol
Representative Results
Discussion
We hebben beschreven vier technieken voor een succesvolle kwantitatieve post-inbedding immunogold EM: 1) korte en zwakke fixatie, 2) te bevriezen-substitutie, 3) post-inbedding immunogoudkleuring, en 4) kwantificering.
EM immunogoud maakt de detectie van specifieke eiwitten in ultradunne weefselcoupes. Antilichamen gelabeld met gouddeeltjes kunnen direct worden gevisualiseerd middels EM. Terwijl krachtig in het opsporen van de subsynaptic lokalisatie van een membraan receptor, kan EM immunog…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
Dit werk werd ondersteund door de Intramurale Programma's van het Nationaal Instituut voor Neurologische Aandoeningen en Stroke (NINDS) en het National Institute on Doofheid en andere Communication Disorders (NIDCD), van de National Institutes of Health (NIH). Wij danken de NINDS EM-faciliteit en de NIDCD geavanceerde imaging kern (code # ZIC DC 000081-03) voor hulp.
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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