Bu çalışma HEM NIH hibe DA15169 ve DA035224 tarafından finanse edildi

Tüm hayvan taşıma ve doku hasat onaylanan protokol # A1506 (Melikian, PI) ardından, Massachusetts Medical School Kurumsal Hayvan Bakım Kullanım Komitesi Üniversitesi (IACUC) esaslarına uygun olarak gerçekleştirilmiştir. Gerekli çözümleri Yapay beyin omurilik sıvısı (ACSF) - günlük taze olun 125 mM NaCl, 2.5 mM KCI, 1.2 mM NaH 2 PO 4, 1.2 mM MgCl2, 2.4 mM CaCl2, 26 mM NaHCO 3, ve 11 mM glükoz Not: 10x stok solüsyonu olarak ACSF hazırlayın, NaHCO 3 ve glikoz hariç. Taze NaHCO 3 ve glukoz ile tamamlayan, 10x stok günlük 1x çalışan çözümler olun. Sükroz takviye ACSF (SACSF) - günlük taze olun 250 mM suc, 2.5 mM KCI, 1.2 mM NaH 2 PO 4, 1.2 mM MgCl2, 2.4 mM CaCl2, 26 mM NaHCO 3 ve 11, mM glukoz gül Not: 10x stok solüsyonu olarak SACSF hazırlayın, NaHCO 3 ve glikoz hariç. Taze NaHCO 3 ve glukoz ile tamamlayan, 10x stok günlük 1x çalışan çözümler olun. Sülfo-N-hidroksisukkinil-SS-Biotin (sülfo-NHS-SS-biyotin, Pierce Chemical Company) Stok çözeltileri, DMSO içinde 200 mg / ml olabilir ve birden fazla donma / erime döngüsüne karşı dayanıklı olan gerekir. Alikotlar -20 ° C&#39;de saklanır Sukkinil ester hızla sulu çözelti içinde hidrolize edilir, böylece çalışma çözeltiler önce dilimlerine uygulamadan hemen hazırlanmalıdır. Dilim Quench Çözüm ACSF 100 mM glisin ile desteklenmiş RIPA Liziz Buffer 10 mM Tris, pH 7.4, 150 mM NaCl, 1.0 mM EDTA,% 1 Triton-X-100,% 0.1 SDS,% 1 Na deoksikolat Proteaz inhibitörleri ile RIPA (RIPA / PI) - günlük taze olun RIPA 1 uM löpeptin, 1 uM pepstatin, 1 uM aprotinin ve 1 mM fenilmetil sülfonil fluorür ile takviye edilmiştir. 1.. Beyin Dilimleri hazırlayın <ol><li> Taze 1x SACSF ve 1x ACSF olun </li><li> Küçük bir beher buz üzerinde SACSF Chill. Bu durum, taze hasat edilmiş fare beyin tutmak için kullanılacaktır. </li><li> % 95 /% 5 O 2 / CO 2, buz üzerinde 20 dakika ile köpüren tarafından oksijen ile ACSF ve SACSF doyurun. </li><li> P30-38 farenin en uygun doku yaşam için kullanılmalıdır. Servikal dislokasyon ve başları kesilerek hayvanların Kurban ve hızla önceden soğutulmuş, oksijenli SACSF içine beyinleri kaldırın. </li><li> Bir titreşimli mikrotom kullanılarak, ilgi bölgesinde 300 mikron beyin bölümleri yapmak. </li><li> Eğer istenirse, dilimleri başka özel beyin bölgeleri veya ayrı sağa sırasıyla, kontrol ve deney dilimleri olarak kullanmak için sol hemisfer zenginleştirmek için önce toparlanmaya disseke edilebilir. </li><li> Bir ateş cilalı Pastör pipeti kullanarak, 24-yuvalı plakalar içine yerleştirilmiş odaları tabanlı bir örgü dilimleri aktarın. </li><li> Dilimleri sürekli, yumuşak kabarcıklarla, oksijenli ACSF, 40 dakika boyunca 31 ° C geri izin verin. </li></ol> 2. İlaç Tedavisi (uygunsa) ve Dilim Biyotinilasyonu <ol><li> Düzelmesinin ardından, önceden ısıtılmış (37 ° C), oksijenli ACSF köpüren sürekli% 95 /% 5 O 2 / CO 2 dilim yıkayın 3x. </li><li> Test bileşikleri ekleyin ve sürekli oksijenasyonu ile kuluçkaya yatmaktadır. <ol><li> Kolaylık sağlamak için, 10x konsantre ilacın 1/10 th ses eklemek, ve hafifçe ters çevirerek karıştırın. </li><li> Yavaşça istenen sıcaklıkta bir su banyosu içinde plakalar çalkalayın. </li></ol></li><li> Ardından ilaç tedavisi, hızlabuz soğuk ACSF 3x yıkayarak soğuk dilimleri. </li></ol> 3. Biotinylate Yüzey Proteinleri <ol><li> Hemen önce etiketlenmesi için buz gibi soğuk ACSF hazırlanması 1.0 mg / ml sülfo-NHS-biyotin-SS. </li><li> Dilimlerine sülfo-NHS-SS-biotin 0.75 ml ekleyin ve buz, 45 dakika dilimleri kuluçkaya yatmaktadır. </li><li> Buz üzerinde buz soğuk ACSF 10 dakika boyunca inkübe edilir, daha sonra buz soğukluğunda ACSF ile hızlı bir şekilde dilimleri üç kez yıkayın. </li><li> Buz dilim söndürme tamponu ile dilimleri üç kez yıkayın ve ücretsiz sülfo-NHS-SS-biotin gidermek için 0.75 ml dilim söndürme tamponu ile buz üzerinde iki kez, 25 dakika, kuluçkaya yatmaktadır. </li></ol> 4. Doku hazırlanması lizatları <ol><li> Buz soğuk ACSF dilimleri üç kez yıkayın ve bir yangın cilalı Pasteur pipeti kullanarak bir mikro tüp her dilim aktarmak. </li><li> Yavaşça 200 xg, 1 dakika ve dikkatle aspire kalan ACSF santrifüj edilerek pelet dilim. </li><li> 400 ul buz gibi soğuk RIPA / PI ekleyin ve yukarı pipetleme dokusu kırmak ve aşağı bir kez P200 pip aracılığıylaette. </li><li> Aktarım, yeni bir tüpe dilim / RIPA liziz ayrılmış ve tamamlamak için, döndürme, 30 dak, 4 ° C inkübe edin. </li><li> Santrifüj ile hücre yıkıntıları pelet haline, 18,000 xg, 15 dakika, 4 ° C </li><li> , Bir standart olarak sığır serum albümini (BSA) ile BCA protein deneyi kullanılarak lisatı, protein konsantrasyonları belirler. </li></ol> 5. Biyotinilat Proteinler izole <ol><li> Boncuk / total protein oranını optimize. Not: Bireysel protein ekspresyon düzeyleri farklı beyin bölgeleri arasında yaygın olarak değişebilir. Doku lisat belirli bir miktarda biyotinile protein her yakalanır sürece, doğru bir yüzeyi ifade herhangi bir potansiyel değişiklikleri tespit etmek mümkün değildir. Bu nedenle, ampirik önce yeni bir dilim biotinilasyon çalışmaya girişmeden belirli bir protein / beyin bölge için en uygun boncuk / total protein oranını tespit etmek zorunludur. <ol><li> Artan miktar 25 ul streptavidin agaroz boncuklar inkübe, yıkama ve elüsyon adımları bağlanma için, aşağıda tarif edildiği gibi doku lisatı (yaklaşık 25-200 ug) ve bir s devam edin. </li><li> Ortaya çıkan immünoreaktif bantlar sayısal olarak ve artan ya da protein ya da azalmış yüzey ifadesi de doğru olarak izin verecek bağlayıcı doğrusal aralığı içinde bir boncuk / lizat oranını seçmek. </li></ol></li><li> Streptavidin-agaroz taneleri hazırlanması <ol><li> Bütün numuneler için gerekli toplam boncuk hacim belirler. 25 ul / numune = 100 ul boncuk + ekstra = 125 ul boncuk bir miktarı için yeterli bir boncuk hacmi artı bir ilave (yani 4 örnekleri hazırlayın. </li><li> Vortex Streptavidin boncuk stok ve pipet dışarı hacmi istenen. P200 pipet kullanarak ederse, tıkanıklık veya boncuk zarar görmesini önlemek için ucu ucuna kesti. </li><li> Oda bir öfke, 18.000 xg, 1 dakika santrifüj edilerek koruyucu, her RIPA eklenmesinden sonra vorteks ve yıkamalar arasında toplama boncuk kaldırmak için 0.5-1.0 ml RIPA / PI boncuk 3 kere yıkayınfaktöörlere. </li><li> Bir vakum şişeye bağlı bir cam Pasteur pipet kullanılarak yıkamalar arasında tampon aspire. Aspire cam Pasteur pipet ucuna takılı plastik P200 ucu ince kontrol sağlar. Bu pipet içine aspirasyon boncuk risk gibi, ilk iki yıkama için kesinlikle bütün tampon kaldırmak için gerekli değildir. Son yıkamadan sonra, boncuklar aspire olmadan mümkün olduğu kadar fazla tamponunu çıkarın. </li><li> Ve tekrar süspansiyon aşağı yukarı pipetleme, geri orijinal hacmi boncuk getirmek RIPA / PI ekleyin. Boncuklar boru duvarına sopa gibi, bu noktada vorteks kaçının. </li><li> Ucu ile bir P200 kullanarak mikrosantrifüj tüpler içine kısım boncuk kesti. Boncuk eşit askıya ve tüpler arasında dağınık kalmasını sağlamak için aşağı örnekleme arasında birkaç kez yukarı pipet ve. </li></ol></li><li> Streptavidin boncuk Bağlama biyotinile edilmiş proteinler <ol><li> Agaroz boncuklar içeren tüplere hücre lizatları dağıtın. Deneyleri için whe<html> Birden çok numune karşılaştırılan yeniden doğru karşılaştırmalar için, her numune için protein aynı miktarda kullanmak için emin olun. </li><li> 200 ul minimum hacme örnekleri getirmek ilave RIPA / PI ekleyin. Bu örnekler inkübasyon sırasında yeterince karıştırmak ve aynı zamanda örnekleri arasında, protein konsantrasyonları birleştiren garanti eder. </li><li> Bir boru rotator tüpler koyun ve 4 ° C&#39;de gece boyunca karıştırın </li><li> Ayrı tüpler içinde, her bir numune için kullanılan toplam lizat hacmi bir eşdeğerine dağıtmak. Numune hacimleri yüksek, alternatif olarak, toplam lizat hacmi bir kısmı SDS-PAGE jelleri üzerinde maksimum yük birimleri yerleştirmek için, bunun yerine ayrılmış olabilir. Bunlar toplam protein miktarı yüzey ekspresyonunu normalize etmek için kullanılacaktır. </li><li> Eşit 2X hacim veya 6x, SDS-PAGE örnek tamponu içinde 1/5 hacim ve ya örnekleri analiz edilinceye kadar -20 ° C&#39;de boncuk numuneleri ya da mağaza paralel olarak, 4 ° C de inkübe ya ekleyin. </li></ol></li></ol>ove_title &quot;&gt; 6. Zehir ve örnekleri analiz <ol><li> Oda sıcaklığında, 18,000 xg, 2 dakika santrifüjleme ile boncuk Pelet. </li><li> Aspire yüzenler ve yıkama boncuk 0.75 ml RIPA&#39;da ile üç kez. Boncuk kaybını en aza indirmek için, yıkamalar arasında boncuk yukarıdaki tampon küçük bir baş hacmini bırakın. Son yıkamadan sonra, boncuk pelet bozmadan, mümkün olduğunca RIPA çıkarın. </li><li> Disülfid bağı azaltarak streptavidin biyotinile edilmiş proteinler boncuklardan Zehir. Örnek tampon, vorteks de azaltarak 25 ul 2x SDS-PAGE ekleyin ve numuneleri 30 dakika, oda sıcaklığını döndürün. Not: Birçok zar proteinleri ciddi şekilde zarar elektroforetik hareketlilik, SDS-PAGE numune tampon maddesi içinde kaynatılmış zaman toplamak için bir yüksek eğilimi vardır. Bu araştırılmaktadır protein için durum ise, ısıtma örnekleri engellemek ve bunun yerine, oda sıcaklığında yavaşça elute. Kaynar (paralel değerlendirilen bir başka protein kaynamasını gerektiriyorsa örneğin) kesinlikle gerekli ise, SAMPLe tampon toplanmasını en aza indirmek için 2 M üre (nihai konsantrasyon) ile takviye edilebilir. Bazı durumlarda toplama azaltılmasında etkili olsa da, üre genellikle grup görünüşe uzlaşma. </li><li> Immunoblot&#39;da örnekleri analiz. </li><li> Toplam lizat örnekleri çözülme ve boncuk numuneleri, 30 dakika, oda sıcaklığı ile paralel olarak döner. </li><li> SDS-PAGE jelleri üzerinde ayrı proteinler. </li><li> Immünolojik işaretleme ile ilgilenilen protein (ler) belirlenmesi. <ol><li> Bantları uygun miktar tespiti için lineer aralığında tespit emin olun. </li><li> Biyotinilasyon reaktif hasarlı / tehlikeye hücreler yoluyla hücre içi proteinlerin erişim elde olmadığını temin etmek. Bu en iyi araştırılan hücre tipine özel bir hücre içi protein için paralel olarak immünolojik işaretleme ile gerçekleştirilir. </li><li> Bant yoğunluğu ölçmek, toplam protein ekspresyon seviyesinin bir yüzdesi olarak görece protein yüzey yoğunluğunu hesaplamak. </li></ol></li></ol>

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Yazarlar finansal ilgilendiriyor bu çalışma ile bu çatışma var.

Beyinde kritik etkileri sinaptik sinyal endositik kaçakçılığı, kantitatif yetişkin nöronlarda protein yüzey ifade değişikliklerini ölçmek için zor kanıtlamıştır uzun zamandır bilgimiz rağmen. Bu çalışmada, akut beyin dilimleri yüzey proteini ex vivo etiketlemek için güvenilir bir yaklaşım rapor. Sinaptik bağlantıları ve bunların hazırlanması saat sonra hücre canlılığı muhafaza edilmesi gibi beyin dilim hazırlıkları, elektrofizyolojik kayıtlar için yarar bir süredir d...

Endositik ticareti bütün zar proteinlerinin çeşitli plazma zarı tanıtımı ince ayar her yerde bulunan bir hücresel mekanizmadır. Endositozdan sonra hücre içi ortamı 1 için çok önemlidir besin sağlar ve reseptör aktivasyonu 2 tepki olarak reseptör sinyallemesini desensitizes. Geri plazma zarına dönüşüm endositik ek olarak hücre yüzeyinde de 3 protein ekspresyon seviyelerini arttırarak hücre sinyali artırabilir. Ayrıca, membran kaçakçılığı tedirginlikler protein endositik ticaretini yöneten moleküler mekanizmalarının araştırılması gerektiğini vurgulayarak, çok sayıda hastalık ve patolojik koşullarda 4,5 sorumlu tutulmaktadır. Çok protein, son birkaç yıl içinde kanıt montaj klasik klatrin bağımlı içselleştirme mekanizmaları kullanmak da çok klatrin bağımsız endositik mekanizmalar giderek artan bir dizi potansiyel endositik yöneten gösteriyorproteinler 6,7. Böylece, fizyolojik, ilgili sistemlerin ticareti kolaylaştırıcı endositik mekanizmaları araştırmak için ihtiyacı önemli ölçüde büyüdü. Beyinde, reseptörler, iyon kanalları ve nörotransmitter nakliyecilerin endositik kaçakçılığı sinaptik plastisite 8-11 ve sonuçta nöronal heyecanlanabilirliğini ve sinaptik yanıtları etkileyen kötüye 12-15 ilaçlara yanıt kurulmasında başlıca role sahiptir. Nöronal kaçakçılığı çalışmaların çoğunluğu heterolog ekspresyon sistemleri veya kültür birincil nöronlar ya güveniyor Bugüne kadar, ikisi de güvenilir bir yetişkin nöronların oynayan mekanizmaları yansıtıyor olabilir. Burada, sayısal olarak yetişkin kemirgenlerden türetilen akut beyin dilimleri yüzey proteini seviyelerini ölçmek için yüzey biyotinilasyonunu kullanan bir yaklaşım sunulmuştur. Bu yaklaşımı kullanarak, fare striatal dopamin taşıyıcısı hızla re içselleştirilmesini göstermek veri mevcutforbol ester-kaynaklı protein kinaz C (PKC) aktivasyonu için tepkisi uyarma.

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">sulfo NHS-SS-biotin</td>
			<td style="width:83px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Streptavidin agarose</td>
			<td style="width:83px;">Pierce</td>
			<td>20347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">IgG-free, Protease-free Bovine serum albumin</td>
			<td style="width:83px;">Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vibrating microtome sectioner</td>
			<td style="width:83px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking water bath</td>
			<td style="width:83px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Milli-cell mesh-bottomed inserts (8&#181;m pore size)</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:119px;">PI8P 012 50</td>
			<td>These can be washed by hand and re-used</td>
		</tr>
	</tbody>
</table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

Düzenlenmiş endositik kaçakçılığı dinamik bir dakika zaman ölçeğinde reseptör, iyon kanalı, ve taşıyıcı hücre yüzey sunum kontrol ederek, nöromodülasyonundaki etkinlikleri kolaylaştırmak merkezi mekanizmadır. Tek tek proteinlerin endositik kaçakçılığı kontrol eden mekanizmalarla geniş bir çeşitlilik bulunmaktadır. Ticareti moleküler temellerini araştıran çalışmalar öncelikle niceliksel dışsal uyaranlara ve gen manipülasyonu yanıt membran protein yüzey ifadesindeki değişiklikleri ölçmek için yüzey biyotinlasyonu dayanmıştır. Bununla birlikte, bu yaklaşım esas olarak aynen yetişkin nöronların oynayan fizyolojik olarak ilgili mekanizmaları yansıtmayabilir kültürlenmiş hücreler, sınırlayıcı edilmiştir. Ayrıca, kültürlü hücre yaklaşımlar ticareti mekanizmaları bölgeye özgü farklılıkları hafife olabilir. Burada, biz akut beyin dilim hazırlanması için hücre yüzey biyotinilasyonunu uzanan bir yaklaşım açıklar. BizBu yöntem, yetişkin nöronların yüzey zar proteini seviyelerindeki hızlı değişiklikleri ölçmek için bir yüksek kalitede bir yaklaşım sağladığını gösterir. Bu yaklaşım nöronal endositik ticareti alanında geniş bir kullanım olması muhtemeldir.

Nöronal dopamin taşıyıcı hücre hatları 16-20 PKC aktivasyonuna karşılık olarak yerleştirilebilir. Hücre dizileri ve ifade sistemlerinin çeşitli kaynaklı PKC DAT yüzey kayıp gösteren çok sayıda raporlara rağmen, bu kültüre, dopaminerjik nöronların 21-23, bu bulguyu teyit etmek zor olmuştur. Biz doğrudan DAT yetişkin dopaminerjik nöronlarda PKC aktivasyonuna karşılık olarak içselleştiren olup olmadığını test etmek için fare striatal dilimleri kullanılır. Dilim...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Nöronal membran kaçakçılığı dinamik plazma membran proteini kullanılabilirliği ve önemli ölçüde etkiler sinir iletimini kontrol eder. Bugüne kadar, yetişkin nöronlar nöronal endositik ticaretini ölçmek için zor olmuştur. Burada, akut beyin dilimleri yüzey protein ifade ex vivo hızlı değişiklikleri ölçmek için son derece etkili, nicel bir yöntem açıklanmaktadır.

Beyin Dilim Biyotinilasyonu: An<em&gt; Ex Vivo</emYetişkin Nöronlar Bölge özgü Plazma Membran Protein Ticaretiyle Tedbir&gt; Yaklaşım

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Research

JoVE Journal

Neuroscience

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

12.8K Görüntüleme.  University of Massachusetts Medical School.  Nöronal membran kaçakçılığı dinamik plazma membran proteini kullanılabilirliği ve önemli ölçüde etkiler sinir iletimini kontrol eder. Bugüne kadar, yetişkin nöronlar nöronal endositik ticaretini ölçmek için zor olmuştur. Burada, akut beyin dilimleri yüzey protein ifade ex vivo hızlı değişiklikleri ölçmek için son derece etkili, nicel bir yöntem açıklanmaktadır. 

Video: Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

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 ...

Nörobilim

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Görüntüleme İlköğretim Kültürlü Fare Nöronlar içinde Plazma Membran Reseptör Yerleştirme pHluorin etiketli

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

GABA A reseptör Membran Yerelleşme ve Ticareti Eğitim için bir α-bungarotoksin Bağlama Sitesi Tag Kullanımı

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: Fizyolojik Sıcaklık Akut Yetişkin Beyin Dilimleme

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Bir<em&gt; Ex vivo</em&gt; Lazer kaynaklı Omurilik Felçlileri Modeli Gerçek zamanlı aksonal Dejenerasyon mekanizmaları değerlendirmek için

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Beyin Dilimleri Korku Devre Ex Vivo optogenetic Diseksiyon

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

GLUT4 protein Deconvolution mikroskobu kullanarak fare birincil hipotalamik nöronlarda ticareti canlı görüntüleri

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Ateş Ex Vivo sıçan beyin dilimleri kullanarak birincil olayları incelemek için alternatif bir yaklaşım

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Endokrin Drosophila içinde sinyal beyin yanıt-e doğru görüntülenmesi için kalsiyum görüntüleme

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Kemirgen beyninde protein Ifadesini ölçmek için yakın kızılötesi floresan ve yüksek çözünürlüklü tarama kullanma

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...