Name: high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses
Uploaded: 2016-02-18T14:00:00
Description: Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

इस काम के मस्तिष्क संबंधी विकार और स्ट्रोक के राष्ट्रीय संस्थान (NINDS) और नेशनल इंस्टीट्यूट ऑफ बहरापन और अन्य संचार विकार पर (NIDCD), राष्ट्रीय स्वास्थ्य संस्थान के (एनआईएच) के अंदर का कार्यक्रम द्वारा समर्थित किया गया। हम NINDS ईएम सुविधा और NIDCD उन्नत इमेजिंग कोर (कोड # ZIC डीसी 000,081-03) सहायता के लिए धन्यवाद।

देखभाल और जानवरों की हैंडलिंग एनआईएच पशु की देखभाल और उपयोग समिति के दिशा निर्देशों के अनुसार थे। 12 घंटे की प्रकाश: प्रसव के बाद दिन (पी) 15-21 Sprague-Dawley चूहों, द्विपक्षीय बेहतर colliculus के माध्यम से 1-1.2% CTB इंजेक्शन के ...

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हम सफल मात्रात्मक के बाद embedding immunogold ईएम के लिए चार तकनीकों का वर्णन किया है: 1) छोटी और कमजोर निर्धारण, 2) फ्रीज प्रतिस्थापन, 3) के बाद embedding immunogold धुंधला हो जाना, और 4) मात्रा का ठहराव। ईएम immunogold Ultrathin ऊतक वर्?...

ग्लूटामेट रेटिना 1 में प्रमुख उत्तेजक neurotransmitter है। रेटिना नाड़ीग्रन्थि कोशिकाओं (RGCs), द्विध्रुवी कोशिकाओं 2 से ग्लूटामेटरगिक synaptic इनपुट प्राप्त करने, रेटिना के उत्पादन न्यूरॉन्स कि मस्तिष्क के लिए दृश्य जानकारी भेज रहे हैं। शारीरिक अध्ययन से पता चला है कि RGCs के synaptic उत्तेजना NMDA रिसेप्टर्स (NMDARs) और AMPA रिसेप्टर्स (AMPARs) 3,4,5 द्वारा postsynaptically मध्यस्थता है। हालांकि RGCs में उत्तेजक पोस्टअन्तर्ग्रथनी धाराओं (EPSCs) AMPARs और NMDARs द्वारा मध्यस्थता कर रहे 3,5,6,7,8, सहज लघु EPSCs (mEPSCs) पर RGCs केवल एक AMPARs की मध्यस्थता घटक 4,5,9 दिखा रहे हैं। हालांकि, ग्लूटामेट तेज को कम करने सहज EPSCs 5 में एक NMDAR घटक का पता चला है, सुझाव है कि RGC dendrites पर NMDARs उत्तेजक synapses के बाहर स्थित किया जा सकता है। झिल्ली जुड़े guanylate kinases (MAGUKs) ऐसे PSD-95 के रूप में है कि क्लस्टर न्यूरोट्रांसमीटर रिसेप्टर्स, ग्लूटामेट रिसेप्टर्स और आयन चैनल सहितsynaptic स्थलों पर एस, यह भी अलग sub अन्तर्ग्रथनी अभिव्यक्ति पैटर्न 10,11,12,13,14 दिखा रहे हैं। हाल के दशकों, confocal immunohistochemistry और पूर्व embedding इलेक्ट्रॉन माइक्रोस्कोप (ईएम) immunohistochemistry ओवर झिल्ली रिसेप्टर अभिव्यक्ति का अध्ययन करने के लिए नियोजित किया गया है। हालांकि confocal immunostaining रिसेप्टर अभिव्यक्ति की व्यापक पैटर्न का पता चलता है, इसकी कम संकल्प यह असंभव subcellular स्थान भेद करने के लिए उपयोग करने के लिए बनाता है। स्तनधारी रेटिना में प्री-embedding ईएम अध्ययन से संकेत मिलता है कि सब यूनिटों NMDAR शंकु द्विध्रुवी सेल रिबन synapses 15,16,17 पर पोस्टअन्तर्ग्रथनी तत्वों में मौजूद हैं। यह शारीरिक सबूत के लिए स्पष्ट विपरीत है। हालांकि, प्रतिक्रिया उत्पाद का प्रसार पूर्व embedding immunoperoxidase विधि में एक जाने-माने विरूपण साक्ष्य है। इसलिए, इस दृष्टिकोण आमतौर पर सांख्यिकीय रूप से विश्वसनीय आंकड़े नहीं दे करता है और बनाम extrasynaptic झिल्ली 18,19,20,21 synaptic झिल्ली को स्थानीयकरण के बीच भेद बाहर कर सकते हैं। परदूसरी ओर, शारीरिक और शारीरिक डेटा RGCs 3,5,7,9,22 पर AMPARs की एक synaptic स्थानीयकरण के साथ संगत कर रहे हैं। इस प्रकार, ग्लूटामेट रिसेप्टर्स और MAGUKs रेटिना रिबन अन्तर्ग्रथन पर न केवल पोस्टअन्तर्ग्रथनी करने पर भी perisynaptic या extrasynaptic झिल्ली डिब्बों को स्थानीय कर रहे हैं। हालांकि, एक रेटिना रिबन अन्तर्ग्रथन में इन झिल्ली प्रोटीन की एक उच्च संकल्प मात्रात्मक विश्लेषण अभी भी जरूरत है। यहाँ, हम (CTB) NMDAR सब यूनिटों, Ampar सब यूनिटों और PSD-95 चूहे RGCs पर synapses पर नंबर, घनत्व और इन प्रोटीनों की परिवर्तनशीलता का आकलन करने के बाद की sub अन्तर्ग्रथनी स्थानीयकरण की जांच करने का उपयोग लेबल हैजा विष सबयूनिट बी एक postembedding ईएम immunogold तकनीक विकसित प्रतिगामी ट्रेसिंग तरीकों।

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			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
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			<td height="21" style="height:21px;">NaOH</td>
			<td>Sigma</td>
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			<td height="21" style="height:21px;">Anti-GluN2A</td>
			<td>Millipore</td>
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			<td>Dilution 1/50</td>
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			<td>Millipore</td>
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			<td height="21" style="height:21px;">Anti-GluA2/3</td>
			<td>Millipore</td>
			<td>AB1506</td>
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			<td height="21" style="height:21px;">Anti-PSD-95</td>
			<td>Millipore</td>
			<td>MA1&#8211;046</td>
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			<td height="21" style="height:21px;">Donkey anti-rabbit IgG-10 nm gold particles</td>
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			<td>25704</td>
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			<td>EMS</td>
			<td>25814</td>
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			<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>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">Formvar-Carbon coated nickel-slot grids.</td>
			<td>EMS</td>
			<td>FCF2010-Ni</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Uranyl acetate</td>
			<td>EMS</td>
			<td>22400-1</td>
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			<td height="21" style="height:21px;">Methanol</td>
			<td>EMS</td>
			<td>67-56-1</td>
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			<td height="21" style="height:21px;">Lead citrate</td>
			<td>Leica</td>
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			<td height="21" style="height:21px;">Leica EM AFS</td>
			<td>Leica</td>
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			<td height="21" style="height:21px;">Leica EM CPC</td>
			<td>Leica</td>
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			<td height="21" style="height:21px;">Ultromicrotome</td>
			<td>Leica</td>
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			<td height="21" style="height:21px;">JEOL 1200 EM</td>
			<td>JEOL</td>
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			<td height="21" style="height:21px;">liquid nitrogen&#160;</td>
			<td>Roberts Oxygen</td>
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			<td height="21" style="height:21px;">CTB</td>
			<td>List Biological Laboratories</td>
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			<td>List Biological Laboratories</td>
			<td>703</td>
			<td>Dilution 1/4000</td>
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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.

यहाँ प्रस्तुत परिणाम के रूप में पहले 24,25 वर्णित है, चूहे रेटिना में RGC dendrites पर Glua 2/3 के भिन्न sub अन्तर्ग्रथनी स्थानीयकरण पैटर्न और NMDARs प्रदर्शित करता है। RGC वृक्ष के समान प्रोफाइल Glua 2/3 immunogold कणों ?...

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.

उच्च संकल्प मात्रात्मक immunogold झिल्ली रिसेप्टर्स के रेटिना रिबन synapses पर विश्लेषण

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

The overall goal of this experiment is to develop a new approach to analyze membrane proteins at retinal ribbon synapses quantitatively. This method can help answer key questions in the retinal neurobiology field such as the precise location of proteins at synapses within the circuit. The main advantage of technique is that it can estimate the number, density, and rapidity of multiple proteins at retinal ribbon synapses.Demonstrating the procedure of freeze-substitution and the ultra-thin sectioning we have docs Ron Petralia and Ya-Xian Wang from the NIDCD Advanced Imaging Core. To begin this procedure dissect an eyeball under the microscope. To do so, first remove the cornea.Then remove the lens and vitreous from the inter-retinal surface with forceps. Afterward, peel the sclera until the retina is isolated from the eyecup. Cut the retina immediately into 100 200 micrometer thick strips with a razor.Subsequently, mix the retina strips in 4%paraformaldehyde in 0.1 molar PB.Then, in 4%paraformaldehyde plus 0.01%glutaraldehyde at room temperature. After several washes in PB, with 0.15 millimolar calcium chloride, cryoprotect the retinal strips with glycerol in 0.1 molar PB, prior to freeze-substitution. Prior to placing the frozen tissue into the flat embedding holders, cut a thin circle from a clear plastic sheet and place it on the bottom of each holder to allow easier removal of the polymerized specimen box when finished.Now, set the automatic sequence in the AFS. Fill the AFS with liquid nitrogen and place the flat embedding holders in the AFS. Fill them with 1.5%uranyl acetate in methanol and pre-cool them.To plunge-freeze the retinal strips in liquid propane at 184 degrees Celsius, use a fine brush to place the samples on small pieces of double-stick tape attached to the metal stubs on the end of the plunging rods. After that, wick off extra buffer using the brush. Plunge the rods into the liquid propane for about five seconds.Then, transfer them to the AFS using a small transport chamber that is filled with liquid nitrogen. After placing the frozen sample and instruments, forceps and scalpel, into the AFS, cool the instruments in the chamber for several minutes before touching the tissue. Or, cool them by placing the tips of the instruments for a few seconds into the small transport chamber filled with liquid nitrogen.After that, remove the sample and tape from the stub using a fine scalpel. Next, place two pieces of frozen sections in each of the seven wells in the flat embedding aluminium holder with 1.5%uranyl acetate in methanol. Keep them at 90 degrees Celsius for at least 32 hours in the AFS.Then, increase the temperature stepwise to 45 degrees Celsius in the automatic sequence. After that, wash the samples three times in pre-cooled methanol. Next, infiltrate the samples progressively with low-temperature embedding resins as an embedding medium.On the next day, change the resin again and adjust the level of resin to reach the top edge of the wells. Set up the UV light on the AFS and polymerize the samples with UV light in the AFS until the end of the automatic sequence. Then, remove the samples from the AFS.Typically, sample blocks still show some pinkness and color. Place the samples close to the fluorescent light in the chemical fume hood at room temperature overnight, or until they appear completely clear. In this step, cut 70 nonometer thick ultra-thin sections with an ultramicrotome, and collect them on the formvar carbon coated nickel grids.Wash the grids in distilled water once, followed by three washes of TBS. Then, incubate the grids in 20 microliters of 5%BSA in TBS for 30 minutes, and then in 20 microliters of a mixture containing goat anti-CTB and an antibody to one NMDAR sub-unit overnight at room temperature. After that, wash the grids three times with 20 microliters of TBS each time at ph 7.6.Subsequntly, wash the grids once with TBS at ph 8.2 for five minutes. Incubate the grids for two hours with 20 microliters of a mixture containing donkey anti-rabbit IGG coupled to 10 nanometer gold particles, and donkey anti-goat IGG coupled to 18 nanometer gold particles. After two hours, wash the grids in 20 microliters of TBS, at ph 7.6 three times, followed by a wash in the ultra-pure water and then air-dry the grids.Subsequently, counterstain the grids with 5%uranyl acetate in distilled water for eight minutes, followed by 0.3%lead citrate in distilled water for five minutes in the dark. For triple-labeling experiments, incubate the grids overnight at room temperature with 20 microliters of a mixture of anti-goat CTB, anti-mouse PSD-95, and anti-rabbit UN2A. Then, incubate the grids for two hours with 20 microliters of a mixture of IGGs coupled to 18, 10, and five nanometer gold particles.In this section, first set the scale bar. Then measure the length of the PSD of individual RGC dendrites with ImageJ software. Then, count the gold particles within 10 nanometers of the membrane based on an average thickness of seven to nine nanometers for the plasma membrane.Calculate the particle density as the number of gold particles per linear micrometer. Then, measure the distance between the center of each gold particle and the middle of the PSD for synaptic location, or the edge of the PSD, for extra-synaptic location. This image shows the double immunogold labeling of GluA2/3 and CTB.This is the pre-synaptic ribbon and the small gold particles are clustered in the PSD of the RGC processes. This image shows the double immunogold labeling of GluN2B and CTB. The small gold particles are on the extra-synaptic plasma membrane.And this image shows the double immunogold labeling of GluN2A and CTB. Similar to GluA2/3, small particles are clustered within the PSD. The triple immunogold labeling of GluN2A, PSD-95, and CTB is shown here.GluN2A gold particles and PSD-95 gold particles are co-localized within the the PSD on individual CTB-positive RGC dendrites. While attempting this procedure, it is important to remember to fix the tissue very gently, because some protein antigenicity, such as that of NMDA receptors decreases markedly with strong prolonged fixation. After watching this video, you should have a good understanding of how to detect and analyze membrane proteins with great precision at retinal ribbon synapses.

Research

JoVE Journal

Neuroscience

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Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

विच्छेदन, संस्कृति, और विश्लेषण<em&gt; Xenopus laevis</em&gt; भ्रूण ऊतक रेटिना

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Luciferase गतिविधि के माध्यम से रिसेप्टर सक्रियण के मापन: स्तनधारी घ्राण रिसेप्टर्स की उच्च throughput विश्लेषण

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

ऊतक अर्क के उच्च संकल्प परमाणु चुंबकीय अनुनाद स्पेक्ट्रोस्कोपी द्वारा चूहे के मस्तिष्क के Metabolomic विश्लेषण

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

Peering at Brain Polysomes with Atomic Force Microscopy

परमाणु शक्ति माइक्रोस्कोपी के साथ मस्तिष्क polysomes पर peering

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