Name: high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses
Uploaded: 2016-02-18T14:00:00
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This work was supported by the Intramural Programs of the National Institute of Neurological Disorders and Stroke (NINDS) and National Institute on Deafness and Other Communication Disorders (NIDCD), of the National Institutes of Health (NIH). We thank the NINDS EM facility and the NIDCD advanced imaging core (code # ZIC DC 000081-03) for assistance.

Care and handling of animals were in accordance with NIH Animal Care and Use Committee Guidelines. Postnatal day (P) 15-21 Sprague-Dawley rats, injected with 1-1.2% CTB bilaterally through the superior colliculus, were maintained on a 12:12-hr light:dark cycle.
1. Retinal Tissue Fixation
<ol>
	<li>Assemble the following materials and tools: A dissecting microscope, 2 forceps with very fine tips, scissors, cellulose filter paper, plastic pipette and a microscope slide.</li>
	<li>Anesthetize t...

<ol>
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We have described four techniques for successful quantitative post-embedding immunogold EM: 1) short and weak fixation, 2) freeze-substitution, 3) post-embedding immunogold staining, and 4) quantification.
EM immunogold allows the detection of specific proteins in ultrathin tissue sections. Antibodies labeled with gold particles can be directly visualized using EM. While powerful in detecting the subsynaptic localization of a membrane receptor, EM immunogold can be technically challenging, and...

Glutamate is the major excitatory neurotransmitter in the retina1. Retinal ganglion cells (RGCs), receiving glutamatergic synaptic input from bipolar cells2, are the output neurons of the retina that send visual information to the brain. Physiological studies showed that synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs) 3,4,5. Although excitatory postsynaptic currents (EPSCs) in RGCs are mediated by AMPARs and NMDARs3,5,6,7,8 , spontaneous miniature EPSCs (mEPSCs) on RGCs exhibit only an AMPARs-mediated component 4,5,9. However, reducing glutamate uptake revealed an NMDAR component in spontaneous EPSCs5, suggesting that NMDARs on RGC dendrites may be located outside of excitatory synapses . Membrane-associated guanylate kinases (MAGUKs) such as PSD-95 that cluster neurotransmitter receptors, including glutamate receptors and ion channels at synaptic sites, also exhibit distinct subsynaptic expression patterns 10,11,12,13,14.
Over recent decades, confocal immunohistochemistry and pre-embedding electron microscope (EM) immunohistochemistry have been employed to study membrane receptor expression. Although confocal immunostaining reveals broad patterns of receptor expression, its lower resolution makes it impossible to use to distinguish subcellular location. Pre-embedding EM studies in mammalian retina indicate that NMDAR subunits are present in postsynaptic elements at cone bipolar cell ribbon synapses 15,16,17. This is in apparent contrast to physiological evidence. However, diffusion of reaction product is a well-known artifact in the pre-embedding immunoperoxidase method. Hence, this approach does not usually give statistically reliable data and may exclude distinction between localization to synaptic membrane versus extrasynaptic membrane 18,19,20,21. On the other hand, physiological and anatomical data are consistent with a synaptic localization of AMPARs on RGCs 3,5,7,9,22. Thus, glutamate receptors and MAGUKs at retinal ribbon synapse are localized not only to the postsynaptic but also to the perisynaptic or extrasynaptic membrane compartments. However, a high-resolution quantitative analysis of these membrane proteins in a retinal ribbon synapse is still needed.
Here, we developed a postembedding EM immunogold technique to examine the subsynaptic localization of NMDAR subunits, AMPAR subunits and PSD-95 followed by estimating the number, density and variability of these proteins at synapses onto rat RGCs labeled using cholera toxin subunit B (CTB) retrograde tracing methods.

<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.

The results presented here demonstrate strikingly different subsynaptic localization patterns of GluA 2/3 and NMDARs on RGC dendrites in rat retina, as described previously 24,25. 77% of GluA 2/3 immunogold particles in RGC dendritic profiles were located within the PSD (Figure 1A), similar to most central synapses. However, NMDARs were located either synaptically or extrasynaptically. 83% of GluN2A immunogold particles were localized in the PSD (Figure...

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.

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

Research

JoVE Journal

Neuroscience

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