The authors thank Dr. Mike Ehlers and his laboratory members for originally demonstrating the protocol for subcellular fractionation of synaptic plasma membranes. We also thank Wendy Horsfall for management of the mouse colonies. This work was supported by operating grants from CIHR (AJR and AS).

Aşağıdaki protokol Hayvan Bakımı Kanada Konseyi yönergelerine uygun ve Toronto Üniversitesi Tıp ve Eczacılık Hayvan Bakım Komitesi Fakültesi tarafından onaylanmıştır. Tablo 1 Açıklandığı gibi 1. Gerekli Reaktifler hazırlayın <ol><li> Tablo 1 &#39;de belirtilen tüm konsantrasyonlarda sakkaroz, tampon maddeler, proteaz ve fosfataz (gerekirse) inhibitörleri eklenir ve GKD 2 O. ÖNEMLİ: Deneyin başlamasından önce, reaktifler ve ekipman her 4 ° C&#39;de tüm adımları gerçekleştirmek ve ön-soğutma. Kalıcı bir kalem ile ultrasantrifüjdeki tüpler de dahil, tüm tüpler Etiket. </li></ol> Uygun Beyin Bölgesi 2. Diseksiyon <ol><li> Servikal dislokasyon veya başları kesilerek hayvan kurban. Ev ve hayvan bakımı üzerine kurumsal ve hükümet politikaları doğrultusunda hayvanları euthanize. </li><li> Hızla th kaldırBir soğutulmuş diseksiyon blokta kafatası ve yerden e beyin. </li><li> Taze dokudan uygun bir beyin bölgesini incelemek ve Adım 3&#39;e geçin. </li></ol> Motorlu Cam-Teflon Homogenizer 3. Doku Homojenizasyon <ol><li> 0.32 M HEPES-tamponlu sukroz çözeltisi 4 ml su ihtiva eden bir işaretli 13 mi polipropilen tüp içinde kesilmiş doku (SPM), 50-100 mg, ya da (PSD) 100-200 mg yerleştirin. 15 ml konik cam-teflon doku taşlama / homojenleştirici için örnek aktarmak, 900 rpm (7 ayar) için motor sürücüsünü ayarlayın ve 30 saniye süren 12 vuruş ile örnek homojenize. Numuneler arasında farklı bir cam-teflon homojenizatör kullanın, ya da numuneler arasındaki soğuk distile su ile Homojenleştiriciyi durulayın ve bir Kimwipe ile kurulayın. Not: doku 4 g kadar ancak uygun fraksiyonlama sağlamak için daha az doku kullanmak tavsiye edilir, 0.32 M HEPES-tamponlu sukroz çözeltisi 4 ml homojenize edilebilir. </li><li> T geri homojenize örnek aktarınaynı 13 ml polipropilen tüp o. Yedek miktar daha sonra da protein ve toplam protein fraksiyonunun Western blot analizi için -80 ° C &#39;de ve mağaza homojenat 100 ul. </li></ol> 4. Düşük Hız Santrifugasyon Nükleer Kesir Kaldır (Getirileri Supernatant S1) <ol><li> Santrifüj, 4 ° C&#39;de 10 dakika boyunca 900 xg&#39;de sabit açılı rotor homojenat. Yeni etmek için yüzer maddeyi (S1) Transfer 13 ml tüp etiketlenmiş ve 0.32 M HEPES-tamponlu sukroz 500 ul fraksiyon nükleer pelet (P1) yeniden süspanse edin. Not: (P1) fraksiyonu -80 ° C&#39;de saklandı ve çekirdek kısmının Western blot analizi için kullanılabilir. </li></ol> Ham sinaptosomal Kesir 5. Zenginleştirme (Getirileri Pelet P2) <ol><li> 4 ° C&#39;de 15 dakika boyunca 10,000 x g&#39;de süpernatan (S1) santrifüjleyin. Bir mikrosantrifüj tüpü içinde 500 ul daha sonra da protein kant boyunca -80 ° C&#39;de saklamak için bir süpernatan (S2) ve rezerv kaldırUS-PS ve sitosolik / hafif membran fraksiyonunun western lekeleme analizi. 0.32 M HEPES-tamponlu sukroz çözeltisi 1 ml geri kalan ham sinoptozomal fraksiyonu pelet (P2) yeniden süspanse edin ve daha sonra başka bir 3 mi 0.32 M HEPES-tamponlu sükroz solüsyonunu ilave edin. </li><li> 4 ° C&#39;de 15 dakika boyunca 10,000 x g&#39;de santrifüjleyin. Daha sonra da protein miktar ve sitosolik / hafif membran fraksiyonunun Western blot analizi için -80 ° C&#39;de saklamak için bir mikrosantrifüj tüpü içinde bir süpernatan (S2 &#39;) ve rezerv 500 ul çıkarın. Polipropilen tüp içinde yıkanmıştır Ham sinaptozomal pelet (P2) kaydedin. </li></ol> Ham sinaptosomal Kesir 6. Parçalama (Hypoosmotic Şok) <ol><li> GKD 2 O, 1 ml olarak yeniden süspanse edilerek polipropilen tüp içinde lize Ham sinaptozomal pelet (P2 &#39;) GKD 2 O başka bir 3 mi, 3 darbelerle elle bir cam-teflon homojenizatör doku transfer ve homojenize ekleyin. NOT: Bu önemlidirçabuk mümkün olduğunca hızlı bu adımı gerçekleştirmek ve 6.2 adıma geçin. </li><li> Tekrar aynı 13 mi polipropilen tüp içine hızla homojenleştirici transfer örnekler 1 M HEPES çözeltisi 16 ul geri 4 mM HEPES numunenin ayarlamak karıştırmak için ters. </li><li> 30 dakika tam yok edilmesi sağlamak için 4 ° C&#39;de örnekleri döndürün. </li></ol> Sinaptosomal Membran Kesir 7. Zenginleştirme (P3) <ol><li> 4 ° C&#39;de 20 dakika boyunca 25,000 x g&#39;de santrifüjleyin. Ham yüzer kısım veziküler (S3) sökülmesi ve daha sonra da protein ölçülmesi ve Western blot analizi için -80 ° C&#39;de saklamak için 5 ml&#39;lik bir tüp içinde tutar. 0.32 M HEPES-tamponlu sukroz çözeltisi 1 ml sinaptosomal membran fraksiyonu (P3) yeniden süspanse edin. </li></ol> Sürekli olmayan, sukroz gradyanlı 8. hazırlanması <ol><li> Pipet tam 3.5 ml 1.2 M HEPES-tamponlu sükroz solüsyonu ve tam 3.0 ml her 1.0 M ve 0.8 M HIPE arasındaAyrı 6 ml S-tamponlu sakaroz çözüm kap tüpleri oturtun. Not: Bu sakroz gradyanı yapmak için kullanılan tamponların hacimleri önceden ölçülmesi için yapılır. Hacimlerinin kesin ölçüm çıkan geçişlerini ultrasantrifiij için dengeli olacak sağlamak için önemlidir. </li><li> Bir cam Pasteur pipet ve ampul kullanarak, 12 ml poliallomer ultrasantrifüjdeki tüp 1.2 M HEPES-tamponlu sakaroz çözeltisini aktarın. Dikkatle 1.2 M HEPES-tamponlu sakaroz çözeltisinin üstünde 1.0 M HEPES-tamponlu sükroz çözeltisi tabaka. Son olarak, 1.0 M HEPES-tamponlu sakaroz çözeltisinin üstünde 0.8 M HEPES-tamponlu sükroz çözeltisi tabaka. Her sakaroz çözüm için taze bir Pastör pipet kullanın ve sakaroz degrade bozmamaya özen gösterin. Bir tezgah girdabı veya mikrosantrifüjden titreşimler degrade bütünlüğünü rahatsız edecektir. </li><li> Pasteur pipeti kullanılarak, hazırlanan süreksiz sukroz gradyanın tepesinde yeniden süspansiyon haline getirilmiş sinaptosomal membran fraksiyonu (P3) katmanı.Geçişlerini sallanan bir kova rotor kova içinde onları tartarak dengeli olduğundan emin olun. Kovalar dengeli değilse, dikkatli rotor kova degrade üstüne 0.32 M HEPES-tamponlu sükroz eklemek ve dengelemek için bir P200 pipetlemeyin kullanın. </li></ol> Synaptic Plazma Membran 9. Fraksiyonu (SPM) <ol><li> 4 ° C&#39;de 2 saat boyunca 150,000 x g hızında bir sallanan kepçeli rotor içinde Ultrasantrifüj. Dikkatle ultrasantrifüjdeki kovalar sakaroz geçişlerini çıkarın. Bir 18 G iğne ve 1 ml&#39;lik bir şırıngayı kullanarak, 1.0 M / 1.2 M HEPES-tamponlu sukroz çözeltisi interfaz altındaki tüp delinme ve sinaptik plazma membran tabakası (SPM) geri. </li><li> Her SPM numune 1 ml şırınga kapladığı hacim not edin. Her örnek ayarlamak olmuştur sonra tam 4 mM HEPES 2.5 hacim 0.32 M&#39;ye 1.2 M den sakaroz konsantrasyonunu ayarlamak eklemek, 3.5 ml kalın duvar ultrasantrifüjdeki tüplerde toplanan SPM katmanı yerleştirinEd, 0.32 M sukroz, 0.32 M HEPES-tamponlu sukroz çözeltisi ile boru denge. </li><li> 4 ° C&#39;de 30 dakika boyunca 200,000 x g&#39;de bir sabit açılı rotor Ultrasantrifüj. Çıkarın ve süpernatant atın. 50 mM HEPES / 2 mM EDTA çözeltisi 300 ul plazma zarı sinaptik pelet (SPM) yeniden süspanse edin. -80 ° C&#39;de saklayın örnekleri batı lekeleme için kullanılana kadar. </li></ol> Yoğunluk postsinaptik Fraksiyon 10. Hazırlama (PSD) <ol><li> Buz üzerinde SPM örnekleri çözülme. </li><li> 50 mM HEPES / 2 mM EDTA çözelti içinde% 0.54 Triton X-100 ihtiva eden bir çözelti yapın. </li><li> 5 ml&#39;lik bir tüp içinde polistiren, Triton X-100 / HEPES / EDTA çözeltisi ve 300 ul fraksiyon SPM 2,7 mi birleştirmek ve 4 ° C&#39;de 15 dakika boyunca örnek döndürmek </li><li> 4 ° C&#39;de 20 dakika boyunca 32,000 x g &#39;de 3.5 ml, kalın duvarlı bir ultra santrifüj tüpleri ve santrifüj örnekleri transfer örnekleri. </li><li> -80 ° C&#39;de bir süpernatan (Triton X-100 çözünür fraksiyon [TS]) ve mağaza ayıracağı; ° C. NOT: Bu aşamada, topak &quot;PSD-1T&quot; bölümünü temsil eder. Deterjan ile bir ikinci işlem bir &quot;PSD-2T&quot; fraksiyonu üretecektir. PSD-1T fraksiyonu isteniyorsa, 10,6 adıma geçin. <ol><li> PSD-2T kısmını üretmek için, 50 mM HEPES / 2 mM EDTA çözeltisi içinde 3 mi% 0.5 Triton X-100 pelet yeniden süspanse edin. 4 ° C&#39;de 15 dakika boyunca numune döndürün. 4 ° C&#39;de 20 dakika boyunca 32.000 x g&#39;de santrifüje örnekleri ve 10,6 adıma devam edin. </li></ol></li><li> Süpernatantı atın ve 50 mM HEPES / 2 mM EDTA çözeltisi postsinaptik (PSD) pelletini. Not: yeniden süspansiyon hacmi topağın boyutuna bağlı olabilir; 50-75 ul bir hacim önerilir. PSD fraksiyon deterjan çözünmeyen fraksiyon olduğu için, proteinin tam olarak yeniden süspansiyon sağlamak için,% 0.5 SDS 1-2 ul ilave edilmesi çoğunlukla gereklidir. -80 ° C&#39;de saklayın örnekleri batı lekeleme için kullanılana kadar. </li></ol>

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G., 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=Nuclear+localization+and+cytosolic+overexpression+of+LASP-1+correlates+with+tumor+size+and+nodal-positivity+of+human+breast+carcinoma.">Nuclear localization and cytosolic overexpression of LASP-1 correlates with tumor size and nodal-positivity of human breast carcinoma.</a> BMC Cancer. 7, 198 (2007).</li><li>Heimann, K., Percival, J. M., Weinberger, R., Gunning, P., Stow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+isoforms+of+actin-binding+proteins+on+distinct+populations+of+Golgi-derived+vesicles.">Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles.</a> J Biol Chem. 274, 10743-10750 (1999).</li><li>Neff, R. A., Gomez-Varela, D., Fernandes, C. C., Berg, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+scaffolds+for+nicotinic+receptors+on+neurons.">Postsynaptic scaffolds for nicotinic receptors on neurons.</a> Acta Pharmacol Sin. 30, 694-701 (2009).</li><li>Cella, N., Cornejo-Uribe, R. R., Montes, G. S., Hynes, N. E., Chammas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lysosomal-associated+membrane+protein+LAMP-1+is+a+novel+differentiation+marker+for+HC11+mouse+mammary+epithelial+cells.">The lysosomal-associated membrane protein LAMP-1 is a novel differentiation marker for HC11 mouse mammary epithelial cells.</a> Differentiation. 61, 113-120 (1996).</li><li>Otera, H., 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=Peroxisomal+targeting+signal+receptor+Pex5p+interacts+with+cargoes+and+import+machinery+components+in+a+spatiotemporally+differentiated+manner:+conserved+Pex5p+WXXXF/Y+motifs+are+critical+for+matrix+protein+import.">Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import.</a> Mol Cell Biol. 22, 1639-1655 (2002).</li><li>Goubaeva, F., 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=Cardiac+mitochondrial+connexin+43+regulates+apoptosis.">Cardiac mitochondrial connexin 43 regulates apoptosis.</a> Biochem Biophys Res Commun. 352, 97-103 (2007).</li><li>Lamers, K. 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The authors have no conflicts of interest to disclose.

Başarılı bir sonuç için kritik olan prosedür birkaç adım vardır. 3. adımda, bu homojenleştirme tutarlı bir derecesi, her bir numune için elde edilmesi önemlidir. Motor tahrikli homojenleştirici ile dokuyu, bir homojenizatör zaman, sabit hız havan eli döndürülmesi için değil, aynı zamanda vuruş sayısı ile de kullanılmaktadır. Hipotonik bir solüsyon içerisinde uzun bir homojen hale getirme ya da mitokondri lize edildi ve inkübasyon SPM örneği kontamine çünkü ozmotik şok, inkübasyon s?...

Nöronlar, sinaps ile iletişim kurmak ve bu haberleşme kalitesi sinaps proteinlerin bileşiminde değişikliklerle büyük ölçüde kontrol edilir. Özel olarak, sinaptik sonrası yoğunluğu bulunan proteinlerin sinyal iletim sistemleri 1 ile iyice iskele nörotransmiter reseptörleri nöronal iletişiminde yer. Ayrıca, sinaptik etkinliğinin gücü kalıcı değişiklikler, sinaptik sonrası yoğunluğu 1-6 de ekleme ya da reseptörleri ayrılmasıyla kontrol edilir. Bu nedenle, sinaptik proteinlerin izolasyon ve miktar nöronlar uyaranlara ve sinaptik etkinliğini 7 değiştirmek yanıt yolları içgörü kazanmak için gerekli ve yararlı bir tekniktir. Bu makale süreksiz sakaroz Yokuşta ultrasantrifugasyon kemirgen beyin dokusundan sinaptik proteinler izole etmek ortak bir teknik anlatılmaktadır. Sinaptik plazma zarı fraksiyonu zenginleştirilmiş olabilir dayanan ve izoleampirik olarak 1.2 M sukroz ile benzer olduğu tespit edilmiştir sukroz yoğunluk,. Biyolojik bir soru ile ilgili olarak, alt-hücresel fraksiyonlar sakaroz veya Percoll ya sürekli ya da süreksiz gradyentlerin ayrılabilir. Sürekli eğimleri çok sayıda kısmın içine proteinlerin ayrıştırılması için; Bu, belirli bir payın 8 içindeki proteinlerin birlikte-lokalizasyonunu göstermek için özellikle yararlı olabilir. Bununla birlikte, sürekli geçişlerini hazırlanması daha zahmetlidir ve birçok uygulama için gerekli değildir. Süreksiz gradyanlar hazırlamak için nispeten kolay olan ve az, genellikle tanımlanmış fraksiyonlar halinde proteinleri ayırmak için kullanılabilir. Artan molaritede üç sükroz katmanlardan oluşan çok süreksiz değişim ölçüleri geniş ölçüde sinaptik plazma zarı (SPM) ile ilişkili proteinler izole etmek için kullanılmıştır. Bu sinaptik plazma membranı fraksiyonu, sinaptik sonrası yoğunluk fraksiyonu yok etmek için işlenebilir;Deterjan-çözünmeyen fraksiyon deterjan muamelesi ile izolasyonu ve n, (PSD). Bu işlem, ilk olarak 1960 de tarif 9,10 zaman, elektron mikroskopi kabaca sinaptik membranı ve post-sinaptik yoğunluğu 9-14 fraksiyonları tanımlamak organel ve membranlar göstermek için kullanılmıştır. Bu çalışmalar öncesi ve post-sinaptik zar ve SPM fraksiyonunda sinaptik veziküllerin dahil olduğunu gösterdi; Deterjan tedavisi öncelikle elektron-yoğun sonra sinaptik sonrası yoğunluğu görünür. Prosedürde, hipotonik şok hücre gövdesi 10 gelen sinaptik süreçleri çimdik için kullanılır. Bu adım, mitokondri ozmotik şoka, daha dayanıklı ve sağlam kalır ve bu nedenle bu yöntem sukroz gradyanı ile (Şekil 1) altındaki çöktürmek gerçeğinden yararlanır. Bu aynı zenginleştirme tekniği kullanarak, SPM ve PSD fraksiyonlar biyokimyasal ilk idipoliakrilamid jel elektroforezi ve ana protein bileşenlerinin 15-17 sekans ile tanımlanmıştır. Daha sonra, western blot analizi (Şekil 2), bu kısımlar daha fazla tespit etmek ve tanımlamak sinaptik proteinlerin seviyelerini ölçmek ve kullanılmıştır. Biz Slc6a3 odağının farelerde 18 yineleniyor meydana dopamin taşıyıcı sinaptik seviyelerindeki değişiklikleri ölçmek için laboratuarlarda bu tekniği kullandık. Ayrıca DISC1 interactome 19 bir parçası olan proteinlerin sinaps özgü azalma ortaya çıkarmak için, NMDA reseptörü eksik olan farelerde, bu tekniği kullanılmıştır. Bu fraksiyonlar SPM sinaptik vezikül zar proteinleri, endozom belirteçler, mitokondriyal protein, zar bağlı sentetik enzimler ve sinyal iletim molekülü olarak post-sinaptik yoğunluk tamamlayıcı bileşenleri ve sinaptik plazma zarlarını içeren 20-23, western blot analizi bellidir. Even PSD kısımlar bol mitokondriyal proteinleri ile kirlenmemiş olması ve bunları 13 ayrılması için bir ikinci meyilli sedimentasyon ya da ek saflaştırma adımları gerçekleştirmek gerekli olabilir. Son zamanlarda, tek başına nicel kütle spektrometrisi, sinaptik-sonrası yoğunluğu 100 proteinlerin bir listesi, aynı zamanda bu bileşenlerin 24,25 nispi bolluk bir gösterge sağlamıştır.

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preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient

Nöronal hücre altı bölme teknikleri ve sinaps kaçırılan proteinlerin ölçülmesine olanak sağlar. Başlangıçta 1960 lerin sonlarında tarif edildiği gibi, sinaptik plazma membranı ile ilgili proteinlerin bir sükroz yoğunluk gradyanında ultrasantrifugasyon sureti ile izole edilebilir. Sinaptik membranlar izole edildikten sonra, sinaptik sonrası yoğunluğu olarak tanımlanır, makromoleküler bir komplekstir, daha sonra nedeniyle deterjan çözülemediği için izole edilebilir. Sinaptik plazma zarları ve post-sinaptik yoğunluklu proteinleri izole etmek için kullanılan teknikler, esas olarak 40 yıl sonra aynı kalır, ve yaygın olarak mevcut nörolojik araştırma kullanılır. Bu makalede, sinaptik plazma membranı ve post-sinaptik yoğunluk kesintili bir sakroz gradyanı kullanılarak, ilişkili proteinlerin fraksiyonasyon göstermektedir. Elde edilen protein preparatları, batı lekeleme ya da 2 boyutlu DIGE analiz için uygundur.

Sükroz yoğunluk gradyanı preparasyonu sukroz (0.8, 1.0 ve 1.2 M sükroz) üç mol çözümler net bir ayrım ile sonuçlanmalıdır. Protein numunesi ilave edilmeden önce, gradyanın bir örnek için bakınız Şekil 3A. Degrade önceden çok hazırlanır, ya da diğer ekipmanların gelen titreşim ile bir tezgah yüzeyi üzerinde hazırlanmış ise, degrade tehlikeye olacak ve uygun ayırma elde edilemez olacaktır. Üç çözümler net bir ayrım görünür değilse, bu protein örneği eklemeden ...

Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Bu makalede, bir sükroz gradyanı üzerinde süreksiz bir ultra-santrifüj ile sinaptik plazma membranı ile bağlantılı proteinlerin zenginleştirme göstermektedir. Post-sinaptik yoğunluk proteinlerin takip eden hazırlama de tarif edilmektedir. Protein preparatları western blotting ya da 2 boyutlu DIGE analiz için uygundur.

Süreksiz Sukroz Degrade&#39;yi kullanma Sinaptik Plazma Membran ve postsinaptik Yoğunluk Proteinlerin Hazırlanması

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

preparation-synaptic-plasma-membrane-postsynaptic-density-proteins

Research

JoVE Journal

Neuroscience

31.0K Görüntüleme.  University of Toronto.  Bu makalede, bir sükroz gradyanı üzerinde süreksiz bir ultra-santrifüj ile sinaptik plazma membranı ile bağlantılı proteinlerin zenginleştirme göstermektedir. Post-sinaptik yoğunluk proteinlerin takip eden hazırlama de tarif edilmektedir. Protein preparatları western blotting ya da 2 boyutlu DIGE analiz için uygundur. 

Video: Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Styryl Boyaların Fotoçevrim kullanarak Synaptic veziküllü Havuzları incelenmesi

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Nörobilim

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

Membran Potansiyelleri, Synaptic Yanıtları, Sinir Devresi, Nöromodülasyon ve Kerevit kullanarak kas Histoloji: Öğrenci Laboratuvarı Egzersizler

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

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 ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Süreksiz Percoll Sakkaroz Yoğunluk Gradient kullanarak Fare Cortex Synaptoneurosomes hazırlanması

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Sıçanlarda Sakkaroz Craving Kuluçka değerlendirilmesi için Genel Bir Yöntem

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Kültür Nöronlar Membran Proteinlerin Yanal Difüzyon ve ekzositoz Floresans Kurtarma ve Floresans kaybı Photobleaching kullanarak Değerlendirilen

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Hipokampal Dilim Kültürleri Synaptic Proteinler Post-gömme immunogold Etiketleme

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Astrositik Transporter Akımlar bir Dekonvolüsyon Analizi ile Glutamat Gümrükleme gün Dersin türetilmesi

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

Nöromusküler Kavşak: Ölçme Synapse Boyut, Konfokal Floresan Mikroskobu kullanılarak sinaptik Protein Yoğunluk Parçalanma ve Değişiklikler

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.