Name: high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses
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وأيد هذا العمل من قبل برامج جماعية من المعهد الوطني للاضطرابات العصبية والسكتة الدماغية (NINDS) والمعهد الوطني للاضطرابات التواصل الأخرى الصمم و(NIDCD)، من المعاهد الوطنية للصحة (NIH). نشكر منشأة NINDS EM وNIDCD متقدمة الأساسية التصوير (رمز # شركة زيورخ للتأمين العاصمة 000081-03) للحصول على المساعدة.

الرعاية وكان التعامل مع الحيوانات وفقا المعاهد الوطنية للصحة ورعاية الحيوان والمبادئ التوجيهية للجنة استخدام. واستمرت يوم بعد الولادة (P) 15-21 الفئران سبراغ داولي، حقن 1-1،2٪ CTB ثنائيا من خلال أكيمة متفوقة، على 12: ضوء لمدة 12 ساعة: دورة الظلام. <p class="jove_title" style=";text-align:right;d...

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وصفناها أربع تقنيات لنجاح الكمي بعد تضمين immunogold م: 1) تثبيت القصير والضعيف، 2) تجميد الاستبدال، 3) بعد دمج تلطيخ immunogold، و 4) الكمي. EM immunogold يسمح للكشف عن بروتينات معينة في قطاعات النسيج سامسونج. الأجسام المضادة المسمى مع جزيئات الذهب ...

الغلوتامات هو ناقل عصبي مثير رئيسيا في شبكية العين 1. خلايا الشبكية العقدة (RGCs)، وتلقي مدخلات متشابك glutamatergic من خلايا القطبين 2، هي الخلايا العصبية الناتج من شبكية العين التي ترسل المعلومات البصرية إلى الدماغ. وأظهرت الدراسات الفسيولوجية التي الإثارة متشابك من RGCs وتتوسط postsynaptically بواسطة مستقبلات NMDA (NMDARs) ومستقبلات أمبا (AMPARs) 3،4،5. وعلى الرغم من توسط التيارات بعد المشبكي مثير (EPSCs) في RGCs بواسطة AMPARs وNMDARs 3،5،6،7،8، عفوية EPSCs مصغرة (mEPSCs) على RGCs يحمل فقط عنصرا بوساطة AMPARs-4،5،9. ومع ذلك، والحد من امتصاص الغلوتامات كشف عنصرا NMDAR في EPSCs عفوية 5، مما يشير إلى أن NMDARs على التشعبات RGC قد تكون موجودة خارج نقاط الاشتباك العصبي مثير. تحركات guanylate غشاء المرتبطة (MAGUKs) مثل PSD-95 أن مستقبلات الناقل العصبي العنقودية، بما في ذلك مستقبلات الغلوتامات والقناة الايونيةالصورة في مواقع متشابك، أيضا يحمل متميزة أنماط التعبير تحت المشبك 10،11،12،13،14. وعلى مدى العقود الأخيرة، المناعية مبائر وقبل تضمين المجهر الإلكتروني (EM) المناعية المستخدمة لدراسة التعبير مستقبلات الغشاء. على الرغم من أن المناعية متحد البؤر يكشف أنماط واسعة من التعبير مستقبلات، أقل قرارها يجعل من المستحيل استخدامها لتمييز موقع التحت خلوية. وتشير تضمين مسبقة الدراسات EM في شبكية العين الثدييات أن مفارز NMDAR موجودة في العناصر بعد المشبكي في مخروط القطبين نقاط الاشتباك العصبي خلية الشريط 15،16،17. هذا هو على النقيض الواضح على الأدلة الفسيولوجية. ومع ذلك، نشر ناتج التفاعل هو قطعة أثرية معروفة في طريقة المناعي قبل التضمين. وبالتالي، فإن هذا النهج لا تعطي عادة بيانات موثوقة إحصائيا وقد تستبعد التمييز بين التعريب لغشاء متشابك مقابل 18،19،20،21 غشاء extrasynaptic. علىمن ناحية أخرى، البيانات الفسيولوجية والتشريحية تتفق مع التعريب متشابك من AMPARs على RGCs 3،5،7،9،22. وبالتالي، يتم ترجمة مستقبلات الغلوتامات وMAGUKs في المشبك الشريط شبكية العين ليس فقط على بعد المشبكي ولكن أيضا لالمقصورات غشاء perisynaptic أو extrasynaptic. ومع ذلك، لا تزال هناك حاجة إلى تحليل كمي عالية الدقة من هذه البروتينات الغشاء في المشبك الشريط في شبكية العين. هنا، قمنا بتطوير تقنية postembedding EM immunogold لدراسة توطين تحت المشبك من مفارز NMDAR، مفارز AMPAR وPSD-95 تليها تقدير عدد وكثافة وتنوع هذه البروتينات في نقاط الاشتباك العصبي على RGCs الفئران المعنون باسم باستخدام الكوليرا السم الوحيدات ب (CTB) طرق تتبع الوراء.

<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.

النتائج المعروضة هنا تظهر مختلفة لافت للنظر أنماط توطين تحت المشبك من GluA 2/3 وNMDARs على التشعبات RGC في شبكية العين الفئران، كما هو موضح سابقا 24،25. وتقع 77٪ من GluA 2/3 الجسيمات immunogold في لمحات شجيري RGC في مديرية الأمن العام (الشكل 1A)، على غرار مع?...

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.

عالية الدقة التحليل الكمي Immunogold من غشاء المستقبلات في الشبكية نقاط الاشتباك العصبي الشريط

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

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

بعد المشبكي التسجيلات في وارد الاتصال الانشعابيات القوقعة خلايا الشعر الداخلية : رصد النسخة عديد الحويصلات في الشريط المشبك

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

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

تشريح العين ماوس لجبل الجامعة للصبغات الظهارة الشبكية

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

تحليل كمي متشابك الحويصلة التجديد حمام سباحة في الخلايا العصبية مثقف الحبيبية للدماغ باستخدام الأصباغ FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

التصحيح ، المشبك قياسات السعة والكالسيوم 2 + التصوير في محطات عصب واحد في شرائح الشبكية

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

عالية الدقة التصوير بالرنين المغناطيسي الوظيفي للطرق الدماغ المتوسط ​​الإنسان

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

عالية الدقة التصوير لايف من سلوك خلية في الظهارة العصبية النامية

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 ...

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

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

تشريح والثقافة، وتحليل<em&gt; القيطم المورق</em&gt; الأنسجة الجنينية الشبكية

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

تحليل الإنتاجية العالية من الثدييات شمي المستقبلات: قياس مستقبلات التنشيط عبر luciferase المراسل آخر

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

تحليل Metabolomic من دماغ الفئران بواسطة عالية الدقة النووية الرنين المغناطيسي الطيفي مقتطفات من الأنسجة

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

يطل في الدماغ Polysomes مع مجهر القوة الذرية

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