Bu çalışma Ulusal Sağlık Enstitüleri Sağırlık ve Diğer İletişim Bozuklukları Nörolojik Bozukluklar ve İnme Ulusal Enstitüsü (NINDS) ve Ulusal Enstitüsü (NIDCD), (NIH) İntramural Programları tarafından desteklendi. Biz NINDS EM tesis ve yardım için NIDCD gelişmiş görüntüleme çekirdek (kod # ZIC DC 000081-03) teşekkür ederim.

Bakım ve hayvanların taşıma NIH Hayvan Bakımı ve Kullanımı Komite Kurallarına göre idi. 12 saat ışık: Postnatal gün (P) bilateral üstün colliculus ile 1-1.2% CTB enjekte 15-21 Sprague-Dawley sıçanları, 12 muhafaza edildi karanlık döngüsü. 1. Retina Doku Fixation <ol><li> Bir diseksiyon mikroskobu, çok ince ipuçları, makas, selüloz filtre kağıdı, plastik pipet ve bir mikroskop lamı ile 2 forseps: Aşağıdaki malzemeler ve aletler monte edin. </li><li> 2.0 mi, halotan (bir uçucu anestezik) ile kapalı bir bölme sıçan anestezisi. ayak tutam veya göz kırpma refleksi eksikliği sonra arka pençe çekilmesi eksikliği: bu yöntemlerle yeterli anesthetization belirleyin. Sonra, bir giyotinle birlikte derhal başını kesmek. pH 7.4&#39;te 0.1 M fosfat tampon maddesi (PB) içinde% 4 paraformaldehid içeren bir cam tabak içinde iris makas ve yer ile gözler çıkarın. </li><li> diseksiyon mikroskobu kullanarak, mısır kaldırmakgöz küresinin ön keserek ad. forseps ile iç retina yüzeyinden lens ve vitreus çıkarın. </li><li> Retina Göz yuvası izole edilene kadar iki forseps ile sklera soyun. </li><li> Bir jilet ile 200 mikron kalınlığında şeritler ve pH-shift tespitin tabi - 100 içine hemen retina kesti. </li><li> Oda sıcaklığında 20 dakika için 10 - pH 10.5, 30 dakika ve daha sonra% 4 paraformaldehid artı% 0.01 glutaraldehid - 20 pH 6.0&#39;da 0.1 M PB içinde% 4 paraformaldehit retina şeritler düzeltildi. </li><li> (4 ° C&#39;de pH 7.4), 0.15 mM CaCI2 ile PB pek çok kez yıkamadan sonra, 0.1 (% 30 O / N sonra% 10,% 20,% 30, her 60 dakikada bir) M gliserol ile retina şeritler cryoprotect ikamesi dondurmak için önce PB. </li></ol> 2. Freeze-ikamesi NOT: Bu donma-ikame yöntemi erken yayınlanmış bir protokol 19,20 modifiye edilir. Ayrıca, araçlar (eldiven giymek) çok soğuk olduğu önemlidir; Oaletleri ile dokunulduğunda therwise, doku kısmen çözülme olabilir. Bu adımların hepsi AFS odasının içinde yapılır ve cihazlar odasının kenarına üzerinde hareket etmek asla izin verilmez. Benzer şekilde, AFS kullanılan bütün kimyasal maddelerin uygun soğutma gereklidir. <ol><li> -Dalma donma bir EM kriyoprezervasyon enstrüman (TBM) içinde -184 ° C&#39;de sıvı propan retina şeritler. ince bir fırça kullanarak, (fırça kullanarak ekstra tampon kapalı fitil) dalan çubukların ucunda gitmek metal taslakları bağlı çift sopa bant küçük parçalar üzerinde örnekleri yerleştirin. </li><li> Sıvı propan içine çubuklar dalma ve yaklaşık 5 saniye boyunca orada tutun ve sonra sıvı nitrojen ile doldurulmuş küçük bir taşıma odasını kullanarak otomatik dondurarak ikame enstrüman (AFS) transfer ( &#39;LN 2 cryo transferi konteyner soğutmalı&#39;). </li><li> AFS içine donmuş örnek ve aletleri (forseps ve neşter) yerleştirdikten sonra, se için odasında aletleri serinVeral dakika doku dokunmadan, ya da küçük nakil odasına birkaç saniye için araçların ipuçları koyarak bunları soğutmak önce (sıvı azot ile dolu () &#39;LN 2 cryo transferi konteyner soğutmalı&#39;). </li><li> AFS kez ince bir neşter kullanılarak saplama numune ve bandı çıkarın. Ayrıca, azot gazı akış kontrol altında tutmak, TF bu işlemler sırasında açık (TF &#39;LN 2 buharlaştırıcı için regülatör kontrol&#39; olarak tarif edilmiştir). </li><li> (AFS kurmadan önce, yani) düz gömme sahipleri içine donmuş doku yerleştirmeden önce, şeffaf bir plastik levha ince bir daire kesip ve her iyi alt hattına kadar tutucu dibine yerleştirin. Bittiğinde Bu polimerize örnek blok görece daha kolay çıkarılmasını sağlar. NOT: Daha önce, çift Reichert + jelatin kapsülleri (Petralia ve Wenthold, 1999 bakınız) istihdam düz katıştırma sahiplerine alternatif bir yöntem kullanılabilir, ve &lt;sup&gt; 20. </li><li> = 4 ° C / saat (11.3 saat), T2 ile 32 saat, S1 = artış sıcaklığı için T1 = -90 ° C -45 ° C 50 saat, S2 için: AFS (enstrüman terminolojiyi kullanarak) aşağıdaki otomatik dizisini kullanın = 5 ° C / saat (9 saat), (toplam = 142.3 saat) 40 saat süre ile T3 = 0 ° C artış sıcaklığı. önce zamanlı dizisi başlamadan (-90 ° C&#39;de) AFS örnekleri yerleştirmek için 4 saat - 2 adet sürebilir. </li><li> AFS&gt; 32 saat boyunca -90 ° C&#39;de metanol içinde% 1.5 uranil asetat içinde dondurulmuş bölümler bırakın. Düz gömme alüminyum tutucu yedi kuyu her dokuların (tipik olarak) iki adet yerleştirin (üç AFS uyması). <ol><li> kuyularda uranil asetat / metanol içine doku + bandı yerleştirin ve bant doku kaldırmak çok zor ise, hemen önce infiltrasyon (örneğin Lowicryl HM20 gibi) başlangıç ​​gömme ortamına, daha sonra bandı çıkarın. </li></ol></li><li> Daha sonra sıcaklık adım adım artış, saat başına -45 ° C (+ 4 ° C; Otomatik sırayla). </li><li> Her düz gömme sahibinden eski çözüm kaldırmak için ince uçlu bir plastik pipet kullanarak, ve sonra soğutulmuş taze metanol eklemek için başka bir standart plastik pipet kullanarak, önceden soğutulmuş metanol numuneleri üç kez yıkayın. </li><li> 1 ve 2: Daha sonra, aynı yöntemi kullanarak, örneğin 1 de ortamı (HM20 / metanol gömme kütlesi olarak düşük sıcaklık gömme reçine ile kademeli örnekleri infiltre 2 saat boyunca saf reçine ardından 2 saat süre ile, 1 her biri, ve daha sonra reçine değiştirme ve) O / N tutun. </li><li> Sadece kuyu üst kenarına ulaşmak için reçine seviyesinin ayarlanması, ertesi gün tekrar reçine değiştirin. </li><li> örnekleri polimerize (-45 ° C, otomatik sırayla 0 ° C&#39;ye kadar, saatte ortalama + 5 ° C) 40 saat süre ile, UV ışığı ile. </li><li> Sonra AFS örnekler kaldırın. Tipik olarak, örnek bloklar hala renkli bazı pinkness göstermektedir. RT O kimyasal davlumbaz floresan ışık yakın örnekleri, koyun / N veya daha uzun onlar kadar uygulamaKulak tamamen net. </li></ol> 3. Postembedding EM immunogold İmmünositokimya Not: 23,24,25 tarif edildiği gibi Postembedding immünositokimya gerçekleştirilir. <ol><li> % 1 toluidin mavisi ile 1 mikron Ultramikrotomla bölümleri, leke bölümleri kesmek ve bölüm oryantasyonu için onları incelemek; Kesit optimal enine uçağı elde etmek için doku bloğu yönlendirmek. </li><li> Ultramikrotomla 70 nm kalınlığında ultra ince bölümleri kesmek ve Formvar-karbon kaplı nikel yuvası ızgaraları onları toplamak. </li><li> Yıkama bir Tris-tamponlu tuzlu su, üç kez (TBS, 0.05 M Tris tamponu,% 0.7 NaCl, pH 7.6) yıkama izledi damıtılmış H2O içinde bir kez ağlar. </li><li> ve bir NMDAR alt-birimi (anti-tavşan GluN2A 1:50 ila bir antikor: keçi anti-CTB ihtiva eden bir karışım 20 ul damla (3,000 1) daha sonra, 30 dakika TBS içinde% 5 BSA, 20 ul damlalar ızgaraları inkübe ve , GluN2B 1:30), ya da bir anti-tavşan GluA2 / 3 (1:30)% 2 BSA ile TBS-Triton (TBST,% 0.01 Triton X-100, pH 7.6) ve 0.02 M Nan 3 O / RT N. </li><li> 5 dakika TBS (pH 8.2) ve ardından üç ayrı 10 için TBS damla (20 ul) (pH 7.6), 10, ve 20 dk yıkama ızgaraları. </li><li> TBST içinde 18 nM altın parçacıklarına bağlanmıştır 10 nm altın parçacıkları ve eşek anti-keçi IgG (1:20) bağlı eşek anti-tavşan IgG (1:20) ihtiva eden bir karışım damla 2 saat (20 ul) için ızgaraları inkübe 2% BSA ve 0.02 M NaN 3 (pH 8.2). </li><li> ultra saf su içinde, daha sonra 5, 5 ve 10 dakika süre ile TBS 20 ul damlalar (pH 7.6) &#39;de ızgaraları yıkayın ve kurutun. </li><li> Sırasıyla, karanlık koşullarda 8 ve 5 dakika için damıtılmış H2O içinde% 5 uranil asetat ve% 0.3 kurşun sitrat ile karşı-ızgaralar. </li><li> Paket etiketleme deneyleri için, ızgaralar inkübe O / anti-keçi CTB bir karışımının 20 ul damla (1: 3000) oda ısısında N, anti-fare PSD-95 (1: 100) ve anti-tavşan GluN2A (1 : 50). Ardından 2 2 saat süreyle ızgaraları inkübe2% BSA ve 0.02 M NaN 3 TBST içinde 18, 10, ve 5 nm ve altın parçacıklarına bağlanmıştır IgG&#39;lerin bir karışımı (pH 8.2) içinde 0 ul düşer. Yıkama arasında ve antikor inkübasyondan sonra counterstaining çift etiketleme gibi aynı prosedürleri tutun. </li><li> Kontroller ikincil antikorlar, tek başına uygulandığında edildiği yapılmıştır. </li><li> Bir EM isimli ızgaraları ve digitalize görüntüler. 24 gerekirse, sadece parlaklık ve kontrast için Adobe Photoshop 6.0 ​​final rakamları işleyin. </li></ol> 4. sayısallaştırılması <ol><li> El ile 8,000X büyütmede delik veya çatlak olmadan iç pleksiform tabakasının (IPL) seçkin alanlar, daha sonra rastgele 25,000X büyütmede IPL tam derinliğini fotomontaj. </li><li> bunlar: a) retrograd taşınan CTB sinyali (büyük altın parçacıkları) içerir, b) sergiler iyi tanımlanmış membranlar, yarıklar ve postsinaptik yoğunlukları, c) en az iki küçük altın bölümü içerdiğinde konik bipolar çiftlerinde de RGC dendrit tespitPSD içindeki icles veya extrasynaptic plazma zarı boyunca, birden fazla küçük bir altın parçacığı. </li><li> kantitatif analiz için yukarıdaki kriterlere göre, 55 RGC dendrit - 45 toplayın. </li><li> PSD ve NIH ImageJ yazılımı ile bireysel RGC dendritler extrasynaptic plazma zarı uzunlukları ölçün. Membranın 10 nm olan altın parçacıkları sayısı 7, bir ortalama kalınlığına göre, membran bağlantılı - plazma zarı 26 9 nm. </li><li> Doğrusal mikrometre (altın / um) başına altın parçacıkları sayısı parçacık yoğunluğu hesaplayın. Her altın parçacığının merkezi ve (sinaptik konumu için) PSD ortasında ya da (extrasynaptic konumu için) PSD kenarı arasındaki mesafeyi ölçün. </li><li> yazılım tarafından istatistiksel analiz. karşılaştırmak için iki kuyruklu t-testi kullanın. önemi p &lt;0.05 varıldı. Aksi belirtilmedikçe, rapor değerleri ± SEM 18 demek. </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabotropic+glutamate+receptor+(mGluR1)+is+concentrated+at+perisynaptic+membrane+of+neuronal+subpopulations+as+detected+by+immunogold+reaction.">The metabotropic glutamate receptor (mGluR1) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.</a> Neuron. 11, 771-787 (1993).</li><li>Van Lookeren Campagne, M., Oestreicher, A. B., van der Krift, T. P., Gispen, W. H., Verkleij, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-substitution+and+Lowicryl+HM20+embedding+of+fixed+rat+brain:+Suitability+for+immunogold+ultrastructural+localization+of+neural+antigens.">Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: Suitability for immunogold ultrastructural localization of neural antigens.</a> J. Hist. Cyt. 39, 1267-1279 (1991).</li><li>Matsubara, A., Laake, J. H., Davanger, S., Usami, S., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+AMPA+receptor+subunits+at+a+glutamate+synapse:+A+quantitative+immunogold+analysis+of+hair+cell+synapses+in+the+rat+organ+of+Corti.">Organization of AMPA receptor subunits at a glutamate synapse: A quantitative immunogold analysis of hair cell synapses in the rat organ of Corti.</a> J. Neurosci. 16, 4457-4467 (1996).</li><li>Petralia, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+NMDA+receptors+at+extrasynaptic+locations.">Organization of NMDA receptors at extrasynaptic locations.</a> Neuroscience. 167, 68-87 (2010).</li><li>Merighi, A., Polak, J. M., Priestley, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embedding+electron+microscopic+immunocytochemistry.">Post-embedding electron microscopic immunocytochemistry.</a> Electron Microscopic Immunocytochemistry: Principles and Practice. , 51-87 (1992).</li><li>Ottersen, O. P., Takumi, Y., Matsubara, A., Landsend, A. S., Laake, J. H., Usami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+organization+of+a+type+of+peripheral+glutamate+synapse:+the+afferent+synapses+of+hair+cells+in+the+inner+ear.">Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear.</a> Prog. Neurobiol. 54, 127-148 (1998).</li><li>Nusser, Z., Lujan, R., Laube, G., Roberts, J. D., Molnar, E., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+and+pathway+dependence+of+synaptic+AMPA+receptor+number+and+variability+in+the+hippocampus.">Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus.</a> Neuron. 21, 545-559 (1998).</li><li>Takumi, Y., Ramirez-Leon, V., Laake, P., Rinvik, E., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+modes+of+expression+of+AMPA+and+NMDA+receptors+in+hippocampal+synapses.">Different modes of expression of AMPA and NMDA receptors in hippocampal synapses.</a> Nat. Neurosci. 2, 618-624 (1999).</li><li>Grimes, W. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+inhibitory+microcircuitry+regulates+retinal+signaling+near+visual+threshold.">Complex inhibitory microcircuitry regulates retinal signaling near visual threshold.</a> J. Neurophysiol. 114, 341-353 (2015).</li><li>Sassoe-Pognetto, M., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+ionotropic+glutamate+receptors+at+dendrodendritic+synapses+in+the+rat+olfactory+bulb.">Organization of ionotropic glutamate receptors at dendrodendritic synapses in the rat olfactory bulb.</a> J. Neurosci. 20, 2192-2201 (2000).</li></ol>

The authors have no disclosures.

Başarılı kantitatif sonrası gömme immunogold EM için dört teknikleri tarif etmişlerdir: 1) kısa ve zayıf tespit, 2) donma-ikamesi, 3)-sonrası gömme immunogold boyama ve 4) ölçümü. EM immünolojik ultra ince doku kesitlerinde spesifik proteinlerin belirlenmesini sağlar. altın partikülleri ile etiketlenmiş antikorlar ile doğrudan EM kullanılarak görüntülenebilir. Bir zar reseptörü subsynaptic lokalizasyonu tespit güçlü iken, EM immünolojik teknik açıdan zor ola...

Glutamat retinada 1 majör eksitatör nörotransmiter olduğunu. Bipolar hücreleri 2 glutamaterjik sinaptik girdi alan retina ganglion hücrelerinin (RGCs), beyne görsel bilgi göndermek retinanın çıkış nöronları vardır. Fizyolojik çalışmaları RGCs sinaptik uyarım NMDA reseptörlerinin (NMDARs) ve AMPA reseptörleri (AMPARs) 3,4,5 tarafından postsynaptically aracılık ettiğini göstermiştir. RGCs eksitatuvar postsinaptik akımlar (EPSCs) AMPARs ve NMDARs aracılık ettiği, ancak RGCs ilgili 3,5,6,7,8 spontan minyatür EPSCs (mEPSCs) sadece AMPARs aracılı bileşen 4,5,9 sergiler. Ancak, glutamat alımını azaltarak RGC dendritler üzerinde NMDARs uyarıcı sinaps dışında olabilir düşündüren, spontan EPSCs 5 bir NMDAR bileşeni saptandı. Membran ilişkili guanilat kinazlar PSD-95 olarak (MAGUKs) o glutamat reseptörleri ve iyon kanalının da dahil olmak üzere küme nörotransmitter reseptörleri,sinaptik s, ayrıca ayrı subsynaptic ekspresyonu 10,11,12,13,14 sergiler. Son yıllarda, konfokal immünohistokimya ve ön gömme elektron mikroskobu (EM) immünohistokimya üzerinde zar reseptör ekspresyonunu incelemek için istihdam edilmiştir. konfokal immün reseptör ekspresyonunun geniş desenler ortaya koymaktadır rağmen, onun düşük çözünürlüklü imkansız hücre içi konumu ayırt etmek kullanmayı kolaylaştırır. Memeli retinada ön gömme EM çalışmaları NMDAR alt birimleri koni bipolar hücre şerit sinaps 15,16,17 de postsinaptik elemanları mevcut olduğunu göstermektedir. Bunun sebebi, fizyolojiksel kanıt açıkça aksine. Bununla birlikte, reaksiyon ürünü difüzyon ön gömme immünoperoksidaz yöntemi iyi bilinen bir sonucudur. Bu nedenle, bu yaklaşım genellikle istatistiksel güvenilir veriler vermez ve extrasynaptic membran 18,19,20,21 karşı sinaptik membran lokalizasyonu arasındaki ayrımı hariç tutabilir. üzerindeÖte yandan, fizyolojik ve anatomik veriler RGCs 3,5,7,9,22 üzerine AMPARs bir sinaptik lokalizasyonu ile uyumludur. Böylece, retina şerit sinaps glutamat reseptörleri ve MAGUKs postsinaptik değil, aynı zamanda perisynaptic veya extrasynaptic zar bölmelerine sadece lokalizedir. Ancak, retina şerit sinaps bu zar proteinlerinin yüksek çözünürlüklü kantitatif analiz hala ihtiyaç duyulmaktadır. Burada, kolera toksin altbirim B kullanılarak etiketli (CTB) sıçan RGCs üzerine sinaps bu proteinlerin sayısı, yoğunluğu ve değişkenliği tahmin izledi NMDAR alt birimden, Ampar alt birimlerin ve PSD-95 subsynaptic lokalizasyonu incelemek için bir postembedding EM immunogold tekniği geliştirdi retrograd izleme yöntemleri.

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			<td height="21" style="height:21px;width:300px;">Paraformaldehyde</td>
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			<td style="width:127px;">15710</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Glutarldehyde</td>
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			<td>16019</td>
			<td></td>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma</td>
			<td>C-8106</td>
			<td style="width:167px;"></td>
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		<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>
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			<td height="21" style="height:21px;">Glycerol</td>
			<td>Gibco BRL</td>
			<td>15514-011</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Lowicryl HM 20</td>
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			<td>15924-1</td>
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			<td height="21" style="height:21px;">Tris-Base</td>
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			<td>BP151-500</td>
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			<td height="21" style="height:21px;">Tris</td>
			<td>Fisher</td>
			<td>04997-100</td>
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			<td height="21" style="height:21px;">Anti-GluN2A</td>
			<td>Millipore</td>
			<td>AB1555P</td>
			<td>Dilution 1/50</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluN2B</td>
			<td>Millipore</td>
			<td>AB1557P</td>
			<td>Dilution 1/30</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluA2/3</td>
			<td>Millipore</td>
			<td>AB1506</td>
			<td>Dilution 1/30</td>
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		<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>
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		<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>
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		<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.

Daha önce 24,25 açıklandığı gibi burada sunulan sonuçlar, sıçan retina RGC dendritler üzerinde çarpıcı farklı subsynaptic lokalizasyon GluA 2/3 kalıplarını ve NMDARs göstermektedir. RGC dendritik profillerde GluA 2/3 immünolojik parçacıklarının% 77, merkezi sinapslara benzer PSD (Şekil 1A) içinde yer. Ancak, NMDARs ya sinaptik veya extrasynaptically bulunurdu. GluN2A immünolojik partiküllerin% 83 PSD lokalize edilmiştir (Şe...

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.

Retina Şerit Sinapslar at Membran Reseptör Yüksek Çözünürlüklü Sayısal immunogold Analizi

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

high-resolution-quantitative-immunogold-analysis-membrane-receptors

Research

JoVE Journal

Neuroscience

7.4K Görüntüleme.  National Institutes of Health. 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.

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

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Çoklu Ölçeklerde Sayısallaştırılmış Nöronal Morfoloji Otomatik Sholl Analizi

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Nörobilim

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

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

Koklear İç Saç Hücreleri Ulaşın Afferent Dendritler postsinaptik Kayıtlar: Şerit Synapse Multivesicular Yayın İzleme

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

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Retina pigment epiteli Tüm Mount Fare Göz Diseksiyon

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

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM Boyalar Kültür Serebellar Granül Nöronlar Synaptic veziküllü Havuzu Yenileme Kantitatif Analiz

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

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

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 measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

İnsan orta beyin için yüksek çözünürlüklü Fonksiyonel Manyetik Rezonans Görüntüleme Yöntemleri

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

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 nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Gelişmekte Neuroepithelium Hücre Davranış Yüksek çözünürlüklü Canlı Görüntüleme

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

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 basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

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

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Diseksiyon, Kültür ve Analizi<em&gt; Xenopus laevis</em&gt; Embriyonik Retina Doku

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

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Lusiferaz aktivitesi ile reseptör aktivasyonunun bir ölçümü: Memeli Koku reseptörünün yüksek throughput Analizi

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

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Doku Esansı Yüksek Çözünürlük Nükleer Manyetik Rezonans Spektroskopisi ile Sıçan Beyin metabolomic Analizi

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

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

Atomik Kuvvet Mikroskobu ile Beyin Polisomlar de Peering

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