Name: high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses
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Ce travail a été soutenu par les programmes intra-muros de l&#39;Institut national des troubles neurologiques et des maladies (NINDS) et l&#39;Institut national sur la surdité et d&#39;autres troubles de la communication (NIDCD), des National Institutes of Health (NIH). Nous remercions l&#39;installation NINDS EM et l&#39;avancée noyau NIDCD d&#39;imagerie (Code # ZIC DC 000081-03) pour l&#39;assistance.

Soin et la manipulation des animaux étaient conformes aux NIH de protection des animaux et les lignes directrices utilisation Comité. jour postnatal (P) 15-21 Les rats Sprague-Dawley, injectés avec 1-1,2% CTB bilatéralement par le colliculus supérieur, ont été maintenus sur un 12: la lumière de 12 h: cycle d&#39;obscurité. 1. Fixation de tissu rétinien <ol><li> Assembler les matériaux et outils suivants: Un microscope à dissection, 2 pinces avec des pointes très fines, des cis...

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Nous avons décrit quatre techniques pour la réussite de l&#39;intégration post-immuno EM quantitative: 1) à court et à faible fixation, 2) de gel-substitution, 3) post-intégration immunomarquage, et 4) la quantification. EM immunologique permet la détection de protéines spécifiques dans des coupes de tissus ultraminces. Des anticorps marqués avec des particules d&#39;or peuvent être directement visualisées en utilisant EM. Bien que puissante dans la détection de la localisation ...

Le glutamate est le neurotransmetteur excitateur majeur dans la rétine 1. Ganglionnaires rétiniennes de cellules (CGR), recevoir une entrée synaptique glutamatergique de cellules bipolaires 2, les neurones de la rétine de sortie qui envoient des informations visuelles au cerveau. Des études physiologiques ont montré que l&#39;excitation synaptique du CGR est médiée par postsynaptique récepteurs NMDA (de NMDARs) et les récepteurs AMPA (AMPA) 3,4,5. Bien que les courants post-synaptiques excitateurs (de EPSCs) dans CGR sont médiés par des récepteurs AMPA et NMDARs 3,5,6,7,8, EPSCs miniatures spontanés (mEPSCs) sur CGR présentent seulement une composante AMPA médiation 4,5,9. Toutefois, la réduction glutamate absorption révélé une composante de NMDAR dans EPSCs spontanées 5, ce qui suggère que NMDARs sur dendrites RGC peuvent être situés à l&#39;extérieur des synapses excitateurs. guanylate kinase associée à la membrane (de MAGUKs) tels que PSD-95 que les récepteurs de neurotransmetteurs grappe, y compris les récepteurs de glutamate et de canal ioniques sur les sites synaptiques, présentent également des profils d&#39;expression subsynaptic distinctes 10,11,12,13,14. Au cours des dernières décennies, l&#39;immunohistochimie confocale et-enrobage pré microscope électronique (EM) immunohistochimie ont été employés pour étudier l&#39;expression du récepteur de la membrane. Bien immunocoloration confocal révèle les grandes tendances de l&#39;expression du récepteur, sa résolution inférieure, il est impossible d&#39;utiliser de distinguer localisation subcellulaire. Pré-enrobage des études de Em In rétine des mammifères indiquent que les sous-unités NMDAR sont présents dans les éléments post-synaptiques à cônes bipolaires synapses à ruban de cellules 15,16,17. Ceci est en contraste apparent à l&#39;évidence physiologique. Toutefois, la diffusion du produit de réaction est un artefact bien connue dans la méthode de pré-enrobage immunoperoxydase. Par conséquent, cette approche ne donne généralement pas de données statistiques fiables et peut exclure distinction entre la localisation de membrane synaptique par rapport extrasynaptique 18,19,20,21 de la membrane. SurD&#39;autre part, les données physiologiques et anatomiques sont compatibles avec une localisation synaptique des récepteurs AMPA sur CGR 3,5,7,9,22. Ainsi, les récepteurs du glutamate et à la rétine MAGUKs ruban synapse sont localisés, non seulement pour le post-synaptiques, mais aussi dans les compartiments membranaires ou périsynaptiques extrasynaptiques. Cependant, une analyse quantitative à haute résolution de ces protéines membranaires dans un ruban synapse rétine est encore nécessaire. Ici, nous avons développé une technique postembedding EM immunologique d&#39;examiner la localisation subsynaptic de sous-unités NMDAR, sous-unités AMPAR et PSD-95 puis en estimant le nombre, la densité et la variabilité de ces protéines au niveau des synapses SUR DES CGR de rat marquée en utilisant la sous-unité de la toxine cholérique B (CTB) méthodes de traçage rétrograde.

<table border="0" cellpadding="0" cellspacing="0" width="761">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Paraformaldehyde</td>
			<td style="width:167px;">EMS</td>
			<td style="width:127px;">15710</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutarldehyde</td>
			<td>EMS</td>
			<td>16019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Sigma</td>
			<td style="width:127px;">7782-85-6</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma</td>
			<td>C-8106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma</td>
			<td>A-7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>Sigma</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaN3</td>
			<td>JT Baker</td>
			<td>V015-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Gibco BRL</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lowicryl HM 20</td>
			<td>Polysciences</td>
			<td>15924-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-Base</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Fisher</td>
			<td>04997-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluN2A</td>
			<td>Millipore</td>
			<td>AB1555P</td>
			<td>Dilution 1/50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluN2B</td>
			<td>Millipore</td>
			<td>AB1557P</td>
			<td>Dilution 1/30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluA2/3</td>
			<td>Millipore</td>
			<td>AB1506</td>
			<td>Dilution 1/30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-PSD-95</td>
			<td>Millipore</td>
			<td>MA1&#8211;046</td>
			<td>Dilution 1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-rabbit IgG-10 nm gold particles</td>
			<td>EMS</td>
			<td>25704</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-mouse IgG-10 nm gold particles</td>
			<td>EMS</td>
			<td>25814</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-mouse IgG-5 nm gold particles</td>
			<td>EMS</td>
			<td>25812</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-goat IgG-18 nm gold particles</td>
			<td>Jackson ImmunoResearch</td>
			<td style="width:127px;">705-215-147</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formvar-Carbon coated nickel-slot grids.</td>
			<td>EMS</td>
			<td>FCF2010-Ni</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uranyl acetate</td>
			<td>EMS</td>
			<td>22400-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>EMS</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lead citrate</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica EM AFS</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica EM CPC</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultromicrotome</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JEOL 1200 EM</td>
			<td>JEOL</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">liquid nitrogen&#160;</td>
			<td>Roberts Oxygen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propane</td>
			<td>Roberts Oxygen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CTB</td>
			<td>List Biological Laboratories</td>
			<td>104</td>
			<td>1-1.2%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CTB</td>
			<td>List Biological Laboratories</td>
			<td>703</td>
			<td>Dilution 1/4000</td>
		</tr>
	</tbody>
</table>

high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses

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.

Les résultats présentés ici montrent remarquablement différents modèles de localisation de subsynaptic Glua 3.2 et NMDARs dendrites sur RGC dans la rétine de rat, comme décrit précédemment 24,25. 77% de particules de 2/3 Glua immunomarquage dans RGC profils dendritiques étaient situés au sein de la PSD (figure 1A), similaire à la plupart des synapses centrales. Cependant, NMDARs étaient situées soit synaptique ou extrasynaptically. 83% de particu...

Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

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.

Haute résolution quantitative immunomarquage Analyse des Récepteurs Membranaires au rétiniennes Synapses ruban

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Research

JoVE Journal

Neuroscience

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

Enregistrements postsynaptiques au dendrites afférentes Contacter cochléaires cellules ciliées internes: Communiqué multivésiculaires surveillance à une synapse ruban

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

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Neurosciences

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Dissection d&#39;un oeil de la souris pour un mont entiers de l&#39;épithélium pigmentaire rétinien

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The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

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Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

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The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...