Bu analizin geliştirilmesine yardımcı olduğumuz Dr. Ricardo Dolmetsch&#39;e (Stanford Üniversitesi Tıp Fakültesi, bugünkü üyelik: Novartis Biyomedikal Araştırmalar Enstitüsü) ve makalenin eleştirel okumalarından dolayı Dr. Matthew Wood, Takuma Aihara ve Dongsook Kim&#39;e teşekkür ediyoruz.

Tüm hayvan işlemleri, Tsukuba Üniversitesi Hayvan Bakımı ve Kullanımı Komitesi (IACUC) onayı ile gerçekleştirildi. 1. Diseksiyon <ol><li> Embriyonik 13-14 ICR veya C57BL / 6 fetüslerini hazırlamak için servikal dislokasyonla hamile fareleri euthanize edin. Uterusu çıkarıp Hank&#39;in Dengeli Tuz Çözeltisi (HBSS) ile bir petri tabağına koyun. Kanı temizlemek için iyice durulayın. </li><li> Forseps kullanarak sterilize etmek için rahim% 70 etanol ile yeni bir petri kabına aktarın. </li><li> Uterusu HBSS ile yeni bir petri kabına aktarın. Fetusları sterilize edilmiş diseksiyon forsepsleri ve cerrahi makas kullanarak uterusdan alın. </li><li> Cerrahi makas kullanarak fötusları dekapetleyin. Bunları HBSS ile yeni bir petri kabına koyun. </li><li> Deriyi ve kafatayı çıkarın ve sterilize edilmiş diseksiyon forsepsini kullanarak beyinlerini çıkarın. HBSS ile bir petri tablasına koyun. NOT: Bu adımdan itibaren, işlemi temizlemek için en iyisidirBank. </li><li> Stero mikroskop altında sterilize edilmiş diseksiyon forseps kullanarak meninksleri çıkarın. Forseps kullanarak bazal gangliyon, hipokampus ve serebellumu kesin. Kıvrımlı uçlu forseps kullanarak, korteksleri HBSS ile yeni bir petri kabına aktarın. </li><li> Tek kullanımlık bir damlalık veya 25 mL&#39;lik bir pipet kullanarak tüm korteksleri toplayın. Onları 15 mL&#39;lik bir tüp içine koyun ve daha sonra pipetle HBSS&#39;yi çıkarın. </li><li> Korteksleri içeren tüp üzerine 3 mL serebral kortikal enzim çözeltisi ekleyin. Bu tüp bir su banyosu içine koyun ve 37 ° C&#39;de 5 dakika inkübe edin. NOT:% 0.25 tripsin ve 1 mM EDTA ile Fosfat Tamponlu Tuz (PBS) içeren bir serebral kortikal kültür enzimi hazırlayın. 0.22 μm&#39;lik bir filtreyle sterilize edin. </li><li> Korteks içeren tüp içine serebral kortikal enzim solüsyonu ilave 3 mL ekleyin ve ilave bir 5 dakika boyunca bir su banyosu 37 ° C&#39;de inkübe edin. </li><li> 5 mL&#39;lik bir pipet kullanarak serebral kortikal enzim solüsyonunu çıkarın ve 5 mL Dulbe&#39;yi ekleyin% 10 Fetal Sığır Serumu (FBS) içeren cco&#39;nun Modifiye Kartal Ortamı (DMEM). </li><li> Steril bir P1000 ucu olan 5 mL&#39;lik bir pipet kullanarak hafifçe pipetleme yaparak korteklikleri çözün. P200 ucu olan 5 mL pipet kullanarak tekrar pipetleyin. </li><li> 50 mL&#39;lik bir tüp üzerine 40 μm&#39;lik bir naylon hücre süzgeci koyun ve enkazı çıkarmak için süspansiyonu filtreleyin. </li><li> Oda sıcaklığında 5 dakika boyunca 420 xg&#39;de santrifüjleyin ve daha sonra DMEM ortamını çıkarın. </li><li> Serebral kortikal kültür ortamı 5 mL ekleyin ve yavaşça hücre peletini tekrar süspansiyon haline getirin. NOT: 1x B-27 takviyeleri, 2 mM L-glutamin ve 100 U / mL penisilin streptomis ile desteklenmiş nöron bazal ortamdan oluşan serebral bir kortikal kültür ortamı hazırlayın. </li><li> Hemocytometer kullanarak hücreleri sayın ve serebral kortikal kültür ortamı kullanarak hücre konsantrasyonunu (arzu edildiği gibi) ayarlayın. </li><li> Tabak başına 3.0 x 10 6 cortical nöron, kaplanmış 35 mm cam tabaka kaplarında 2.0 mL&#39;dir. NOT: Kaplamadan önce,0.1 mg / mL Poli-D-Lizin hidrobromid (PDL) veya% 0.01 Poli-L-Ornitin (PLO) ile 35 mm.lik cam tabanlı tabaklar. 3 saat süreyle inkübe edin - O / N, 37 ° C&#39;de ve PBS ile en az 3 kez yıkayın. </li></ol> 2. Görüntüleme Vesikül Hareketi <ol><li> Her vezikül türünü etiketleyecek plazmidleri hazırlayın. Örneğin, erken endozomlar için EGFP-Rab5, geç endozomlar için EGFP-Rab7, lizozomlar için LAMP-EGFP ve otofagozomlar 8 , 13 için EGFP-LC3 kullanın. NOT: Bu plasmidler tsuruta.fuminori.fn@u.tsukuba.ac.jp adresinden talep edilebilir. Tipik olarak, 3 ila 5 gün in vitro (DIV) nöronlar bu plazmidlerle transfeksiyon reaktifleri kullanılarak transfekte edilebilir. </li><li> Pipetleme ile 4.0 μg plazmid 200 μL serum barındırmayan ortam ile karıştırılır. Ayrı bir tüpte, 200 μL serum serbest ortamda 8 μL transfeksiyon reaktifini (bkz . Malzeme Tablosu ) seyreltin . Oda sıcaklığında 5 dakika inkübe edin. </li><li> Plazmid çözeltisini ve transfeksiyon rejent solüsyonunu pipetleme ile adım 2.2&#39;de karıştırın. Oda sıcaklığında 20 dakika inkübe edin. </li><li> Her kaplanmış cam alt tabağa saf olmayan ortam ve adım 2.3&#39;ten 400 μL plazmid çözeltisi ilave edin. Nöronları 37 ° C&#39;de 30 dakika boyunca% 5 CO2 inkübatörde inkübe edin. Ortamı 2 mL serebral kortikal kültür ortamı ile değiştirin. Nöronları 37 ° C&#39;de% 5 CO2 inkübatöre 1-2 gün inkübe edin. </li><li> 1 - 2 d transfeksiyondan sonra, transfekte edilen hücreleri görüntü alımı için seçin. NOT: 7 DIV&#39;den küçük erken sinir hücreleri, dinamik vezikül hareketliliğini gösterme eğilimindedir. Floresans probunu orta derecede ifade eden nöronları seçin, çünkü oldukça ifade eden bir nöron seçildiğinde net görüntüler elde edilemez. Ayrıca, akson ve dendritlerin tanımlanmasını kolaylaştırmak için uzun aksonları ve karmaşık dendritleri olan piramidal nöronlar gibi tipik bir nöronal şekli seçin (bakınız Şekil 1A). </li><li> Bir zaman atlamalı görüntüleme sistemi kullanarak görüntü elde etme: Görüntüleme için, 40X Plan Apo 0.95NA veya 100X Plan Apo VC NA1.4 objektif mercekleri bulunan bir Charge-Coupled Device (CCD) kamera ile donatılmış bir fluoresans mikroskop kullanın. Bir inkübasyon sistemi kullanılarak sıcaklığı 37 ° C&#39;de kontrol edin. Görüntü edinme yazılımı tarafından kontrol edilen 100 saniyelik bir periyot boyunca bir çerçeve / 5 sn&#39;de nöron görüntüleri elde edin. Alternatif olarak, görüntüleri daha kısa aralıklarla ve 300 saniyelik bir süre boyunca 2 s&#39;de bir karede olduğu gibi daha uzun zaman periyotlarında elde edebilirsiniz. NOT: Ardışık resimler çekmek için çeşitli uygulamalar kullanılabilir, bunların çoğu bu yöntem için uygundur. Bu nedenle, özel bir uygulama önerilmemektedir; Bunun yerine, her araştırmacı mevcut olan her hangi bir uygulamayı kullanmalıdır. </li></ol> 3. Görüntü Analizi <ol><li> Manifest İzleme 14 ile ImageJ yazılımındaki ardışık görüntüleri açın. NOT: Bugüne kadar, Manuel TakipDeneyleri 15 , 16 , 17 , 18 , 19&#39;da kullanılan dely. Elle İzleme Dr. Fabrice Cordelières tarafından geliştirildi. Ayrıntılı yazılı talimatlar çevrimiçi 20&#39;de mevcuttur. Analiz için görüntüleme verisinin kalitesi yeterli sayıda görüntü ile geliştirilir. 20&#39;den fazla resim kullanmanız önerilir. </li><li> Sıralı resimleri birleştirmek için &lt;Görüntü&gt; → &lt;Yığınlar&gt; → &lt;Yığın Görüntüler&gt; seçimini yapın. &lt;Tamam&gt; &#39;ı tıklayın. </li><li> &lt;Eklentiler&gt; → &lt;Manuel İzleme&gt; seçimini yapın; Bir izleme penceresi açılacaktır. </li><li> &lt;Göster Parametreleri?&gt; Onay kutusunu tıklayın ve parametreler bölümünde izleme parametrelerini tanımlayın. NOT: Zaman aralığı, x / y kalibrasyonu, z kalibrasyonu, merkez için arama kare boyutu dahil olmak üzere birçok parametre ayarlanabilirIng, nokta boyutu, çizgi genişliği ve yazı tipi boyutu. Bu basitleştirilmiş analizde hem zaman aralığı hem de x / y kalibrasyonu tanımlandı. Diğer deneysel koşullar altında, diğer parametreleri de tanımlamak gerekebilir. Burada kullanılan parametreler aşağıdaki gibidir: &lt;Zaman aralığı&gt; 5 s&#39;dir ve &lt;x / y ayarı&gt; 40X Plan Apo 0.95NA objektif lensi için 0.26642 μm veya 100X Plan Apo VC NA1.4 için 0.10657 μm&#39;dir objektif lens. </li><li> Parametreler tanımlandıktan sonra, yeni bir parça başlatmak için &lt;Parça Ekle&gt; &#39;ye tıklayın. </li><li> İlgili salyangozları belirledikten sonra ( örneğin, Şekil 1A&#39;daki mavi ve kırmızı ok uçlarıyla gösterilen veziküller (sağ panel)), xy koordinatlarını kaydetmek için ardışık görüntülerdeki sinyallerin ortasına tıklayın. Kaydedilen XY koordinatlarının, mesafenin ve hızın sonuçları yeni bir pencerede gösterilir. NOT: Lizozom durumunda, büyük parlak floresan sinyalleri ( ör. Fig.Ür 1A (sağ panel), turuncu ok başı) nöronlarda sıklıkla görülür. Bu sinyaller bazen ya biriken veziküller ya da patolojik varikoziteler sergilediğinden, motiliteyi nicelleştirmek için bu sinyallerden kaçınılmalıdır. </li><li> Tüm ardışık görüntülerden veziküllerin xy koordinatlarını toplamak için 3.6 adımını tekrarlayın; Her görüntü, referans noktası seçildikten sonra otomatik olarak ardışık görüntüye geçer. </li><li> Bu verileri yeni bir tabloya ( örneğin, bir e-tablo) dışa aktarın. Uygun bir grafik oluşturmak için uygun bir yazılım kullanın (bkz . Malzeme Listesi ). NOT: Verileri analiz etmek için &lt;Uzaklık&gt; sütununu seçin, bu sütunun tüm değerlerini özetleyin ve bir hareketlilik mesafesini çubuk grafik olarak gösterin (ör. Şekil 1B) . Ek olarak, bu adımı tekrarlayın, toplam hareketlilik mesafesinin ortalamasını hesaplayın ve Şekil 1C&#39;deki gibi bir çubuk grafiği olarak gösterin. Çubuk grafik ve dalga formu verileri oluşturmak için yazılımMalzeme Tablosu. </li></ol>

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	<li>Nicot, A. S., Laporte, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomal+phosphoinositides+and+human+diseases.">Endosomal phosphoinositides and human diseases.</a> Traffic. 9 (8), 1240-1249 (2008).</li><li>De Matteis, M. A., Luini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mendelian+disorders+of+membrane+trafficking.">Mendelian disorders of membrane trafficking.</a> N Engl J Med. 365 (10), 927-938 (2011).</li><li>Lebrand, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+endosome+motility+depends+on+lipids+via+the+small+GTPase+Rab7.">Late endosome motility depends on lipids via the small GTPase Rab7.</a> EMBO J. 21 (6), 1289-1300 (2002).</li><li>Chen, C. S., Bach, G., Pagano, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+transport+along+the+lysosomal+pathway+in+mucolipidosis,+type+IV+disease.">Abnormal transport along the lysosomal pathway in mucolipidosis, type IV disease.</a> Proc Natl Acad Sci U S A. 95 (11), 6373-6378 (1998).</li><li>Venugopal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurologic,+gastric,+and+opthalmologic+pathologies+in+a+murine+model+of+mucolipidosis+type+IV.">Neurologic, gastric, and opthalmologic pathologies in a murine model of mucolipidosis type IV.</a> Am J Hum Genet. 81 (5), 1070-1083 (2007).</li><li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+mechanism+to+regulate+lysosome+motility+for+lysosome+positioning+and+tubulation.">A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation.</a> Nat Cell Biol. 18 (4), 404-417 (2016).</li><li>Tsuruta, F., Green, E. M., Rousset, M., Dolmetsch, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIKfyve+regulates+CaV1.2+degradation+and+prevents+excitotoxic+cell+death.">PIKfyve regulates CaV1.2 degradation and prevents excitotoxic cell death.</a> J Cell Biol. 187 (2), 279-294 (2009).</li><li>Tsuruta, F., Dolmetsch, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIKfyve+mediates+the+motility+of+late+endosomes+and+lysosomes+in+neuronal+dendrites.">PIKfyve mediates the motility of late endosomes and lysosomes in neuronal dendrites.</a> Neurosci Lett. 605, 18-23 (2015).</li><li>McCartney, A. J., Zhang, Y., Weisman, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol+3,5-bisphosphate:+low+abundance,+high+significance.">Phosphatidylinositol 3,5-bisphosphate: low abundance, high significance.</a> Bioessays. 36 (1), 52-64 (2014).</li><li>Shisheva, A., Sbrissa, D., Ikonomov, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plentiful+PtdIns5P+from+scanty+PtdIns(3,5)P2+or+from+ample+PtdIns?+PIKfyve-dependent+models:+Evidence+and+speculation+(response+to:+DOI+10.1002/bies.201300012).">Plentiful PtdIns5P from scanty PtdIns(3,5)P2 or from ample PtdIns? PIKfyve-dependent models: Evidence and speculation (response to: DOI 10.1002/bies.201300012).</a> Bioessays. 37 (3), 267-277 (2015).</li><li>Chow, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+FIG4+causes+neurodegeneration+in+the+pale+tremor+mouse+and+patients+with+CMT4J.">Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J.</a> Nature. 448 (7149), 68-72 (2007).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Vac14,+a+regulator+of+the+signaling+lipid+phosphatidylinositol+3,5-bisphosphate,+results+in+neurodegeneration+in+mice.">Loss of Vac14, a regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice.</a> Proc Natl Acad Sci U S A. 104 (44), 17518-17523 (2007).</li><li>Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., Ohsumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+autophagy+in+response+to+nutrient+starvation+using+transgenic+mice+expressing+a+fluorescent+autophagosome+marker.">In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.</a> Mol Biol Cell. 15 (3), 1101-1111 (2004).</li><li>. Manual Tracking, a plug-in for ImageJ software [Internet] Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/track/track.html">https://imagej.nih.gov/ij/plugins/track/track.html</a> (2017)</li><li>Hu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exopolysaccharide-independent+social+motility+of+Myxococcus+xanthus.">Exopolysaccharide-independent social motility of Myxococcus xanthus.</a> PLoS One. 6 (1), e16102 (2011).</li><li>Tan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+four+type+IV+pilin+homologues+in+Stigmatella+aurantiaca+DSM17044+by+heterologous+expression+in+Myxococcus+xanthus.">Characterization of four type IV pilin homologues in Stigmatella aurantiaca DSM17044 by heterologous expression in Myxococcus xanthus.</a> PLoS One. 8 (9), e75105 (2013).</li><li>Choi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+variant+of+cortactin+linked+to+acute+lung+injury+impairs+lamellipodia+dynamics+and+endothelial+wound+healing.">A genetic variant of cortactin linked to acute lung injury impairs lamellipodia dynamics and endothelial wound healing.</a> Am J Physiol Lung Cell Mol Physiol. 309 (9), L983-L994 (2015).</li><li>Dahirel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+propensity+and+ability+correlate+with+ecological+specialization+in+European+land+snails:+comparative+analysis+of+a+dispersal+syndrome.">Movement propensity and ability correlate with ecological specialization in European land snails: comparative analysis of a dispersal syndrome.</a> J Anim Ecol. 84 (1), 228-238 (2015).</li><li>Hu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+type+IV+pili+activity+and+exopolysaccharides+secretion+controls+motility+patterns+in+single+cells+of+Myxococcus+xanthus.">Interplay between type IV pili activity and exopolysaccharides secretion controls motility patterns in single cells of Myxococcus xanthus.</a> Sci Rep. 6, 17790 (2016).</li><li>. Manual Tracking [Internet] Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/track/Manual">https://imagej.nih.gov/ij/plugins/track/Manual</a> (2017)</li><li>. ImageJ Tracking plug-in [Internet] Available from: <a target="_blank" href="https://imagej.net/Category:Tracking">https://imagej.net/Category:Tracking</a> (2017)</li><li>Tinevez, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrackMate:+An+open+and+extensible+platform+for+single-particle+tracking.">TrackMate: An open and extensible platform for single-particle tracking.</a> Methods. , (2016).</li><li>Ekvall, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tracking+of+small+aquatic+organisms+using+fluorescent+nanoparticles.">Three-dimensional tracking of small aquatic organisms using fluorescent nanoparticles.</a> PLoS One. 8 (11), e78498 (2013).</li><li>Jeyakumar, M., Dwek, R. A., Butters, T. D., Platt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Storage+solutions:+treating+lysosomal+disorders+of+the+brain.">Storage solutions: treating lysosomal disorders of the brain.</a> Nat Rev Neurosci. 6 (9), 713-725 (2005).</li></ol>

Yazarların açıklayacak bir şeyi yok.

Bu protokol vezikül hareketliliğini ölçmek için prosedür tanıtır. Primer nöronlarda, endozomlar ve lizozomlar, genç nöronlarda yüksek motilite gösterme eğilimindedir (4-6 DIV). Sinirsel süreçleri uzatmak için nöronların öncü kısımlara bazı bileşenler vermesi gerektiği göz önüne alındığında, bu aşamada zar ticareti dinamik bir şekilde gerçekleşmelidir. Bu nedenle, genç nöronları nöronal dendritlerde dinamik motilite gözlemlemek için kullanmak önemlidir. Buna ek olarak, daha bü...

Endozom-lizozom ticaretinin hassas kontrolü, nöronal fonksiyonu düzenlemek için vazgeçilmezdir. Özellikle, bu veziküllerin dinamik hareketi, nöronal morfolojinin, gelişimin ve hayatta kalmanın düzenlenmesinde temel faktördür. Bu sistemdeki kusurlar ciddi nöronal bozukluklara neden olur 1 , 2 . Vezikül ticaretini nöronal hastalıklarla ilişkilendiren moleküler mekanizmalar karmaşık kabul edilir ve birçok grup bu önemi incelemeye çalıştı. Örneğin, tedirgin geç endozom motilitesinin, lizozom defektlerinin yol açtığı kalıtsal bir nörodejeneratif bozukluk olan Niemann-Pick C hastalığı 3 ile önemli derecede ilişkili olduğu bildirilmiştir. Başka bir örnek, lizozomal Ca 2+ kanalındaki trpml 1&#39;deki mutasyon olup, lizozomal hareketliliği bozarak lizozomal depolama hastalıkları 4 , 5 , </Sup&gt; 6 . Grubumuz PtdIns&#39;in (3,5) P 2 devresinin düzenlenmesinin nöronlarda endozom ve lizozom motilitesini baskıladığını ve bunun da stres tepkisine karşı savunmasızlıktan 7 , 8 arttığını bildirmiştir. Çoğunlukla geç endozomlar ve lizozomlarda lokalize olan PtdIns (3,5) P2&#39;nin metabolik düzenlenmesi, vezikül ticareti ve füzyon-fisyon işlemleri gibi çok çeşitli hücresel işlevlerde önemli bir rol oynamaktadır 9,10. Zayıflamış PtdIns (3,5) P 2 ciro şiddetli nörodejenerasyon 11,12&#39;ye neden olduğu için, endozom-lizozom motilitesinin sapması regülasyonu, nörodejenerasyon patogenezini anlamak için önemli bir faktör olabilir. Vesikül hareketliliğinin altında yatan moleküler mekanizmaların araştırılması, bu nedenle, bizim vefizimizi derinleştiren umut verici ipuçları sağlayabilirBirkaç nöronal rahatsızlığın kavranması. Bu yazıda, Manuel İzleme adlı özgür yazılım paketini kullanarak nöronlarda vezikül hareketliliğini ölçmek için değerli bir yöntem sunuyoruz. Amaç, vezikül hareketliliğini analiz etmek için hızlı bir kantifikasyon yöntemi geliştirmekti. Bu niceleme, zaman atlamalı filmin her karesinde bir referans noktasına tıklayarak standart bir yaklaşımla yönlendirilir. Manuel İzleme yazılımının kullanılması, bu yaklaşımı diğer uygulamalardan farklı olarak oldukça basit ve geniş yarar sağlar. Dahası, bu yaklaşım glial hücreler gibi diğer hücrelere de uygulanabilir. Bu yöntem ilkel olmasına rağmen, hücresel motilite ve morfolojik değişim de dahil olmak üzere çeşitli analizlere uygulanabilir. Örneğin, bir görüntü dizisi boyunca bir referans noktası tanımladıktan sonra, referans noktalarının konumları ve her bir konumdaki zamana ilişkin bilgiler, veri analizi ve görüntü işleme yumuşaklığı kullanılarak ardışık görüntülerden çıkarılabilirmaldır. Birlikte ele alındığında, bu yöntem basit ama güçlüdür ve endosome-lizozom fonksiyonunu inceleyenler gibi membran ticaretine dayalı çalışmaların geliştirilmiş etkinliğinin geliştirilmesine katkıda bulunur.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium&#160;</td>
			<td>Wako</td>
			<td>044-29765</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher</td>
			<td>31985070</td>
			<td>Serum free medium</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Thermo Fisher</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin Streptmycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Trypsine</td>
			<td>Thermo Fisher</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-ornithine&#160;</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon cell strainer</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher</td>
			<td>11668027</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Polyethyleneimine &#34;Max&#34;</td>
			<td>polysciences</td>
			<td>24765</td>
			<td>Transfection reagent. As an alternative to Lipofectamine2000, 0.1mg/ml Polyethyleneimine dissolved in sterilized water is available. But it is low efficiency and high toxicity.</td>
		</tr>
		<tr>
			<td>BIOREVO BZ-9000</td>
			<td>Keyence</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation system INUG2-K13</td>
			<td>Tokay Hit</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism version 6.0</td>
			<td>GraphPad Software</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel version 15</td>
			<td>Microsoft</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ verion 1.47</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of endosome and lysosome motilities in cultured neurons using fluorescent probes

Beyinde, zar ticaret sistemi, nöronal morfoloji, sinaptik esneklik, hayatta kalma ve glial iletişim gibi nöronal fonksiyonların düzenlenmesinde önemli roller oynar. Bugüne kadar çok sayıda çalışma, bu sistemlerin kusurlarının çeşitli nöronal hastalıklara neden olduğunu bildirmiştir. Böylece vezikül dinamiklerinin altında yatan mekanizmaları anlamak, çeşitli nöronal bozuklukların tedavisinde yardımcı olabilecek etkili ipuçları sağlayabilir. Burada, hareket noktası mesafesi ve hareket hızı gibi vezikül hareketlerini ImageJ platformu için bir yazılım eklentisi kullanarak ölçmek için bir yöntem açıklıyoruz. Niceleme için görüntü elde etmek için, EGFP ile etiketlenmiş vezikül işaretleyici proteinleri olan nöronal endozom-lizozom yapılarını etiketledik ve veziküllerin bir zaman atlamalı mikroskobi ile hareketi gözlemledik. Bu yöntem son derece yararlıdır ve aksonlar ve dendritler gibi nöritlerin ve hem nöronların hem de glial hücrelerin soma&#39;sındaki vezikül hareketliliğinin ölçülmesini basitleştirir. FurthermoYeniden, bu yöntem fibroblastlar ve endotel hücreleri gibi diğer hücre hatlarına da uygulanabilir. Bu yaklaşım, zar ticareti anlayışımızı değerli bir ilerleme sağlayabilir.

Bu deney, in vitro kültürde vezikül dinamiklerini ölçmek için tasarlandı. Bu tahlil, nöronal morfoloji ve hayatta kalma ile ilişkili vezikül hareketliliğini saptamak için kullanılmıştır. Şekil 1A ve B, nöronlarda lizozom motilitesini gösteren temsili verileri göstermektedir. Kortikal nöronlar, lizozom işaretleyici LAMP-EGFP ile transfekte edildi ve standart bir fluoresans mikroskopu kullanılarak gözlendi. Daha önce, spes...

Watch this Scientific Journal Video about Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes at JoVE.com

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Membran kaçakçılığının araştırılması, nöronal işlevleri anlamada çok önemlidir. Burada, nöronlarda vezikül hareketliliğini ölçmek için bir yöntem sunuyoruz. Bu, sinir sistemindeki zar ticaretinin miktarına uyarlanabilen uygun bir yöntemdir.

Floresan Probları Kullanan Kültürlü Nöronlarda Endozom ve Lizozom Hareketlerinin Nicelendirilmesi

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

quantification-endosome-lysosome-motilities-cultured-neurons-using

Research

JoVE Journal

Neuroscience

8.0K Görüntüleme.  University of Tsukuba.  Membran kaçakçılığının araştırılması, nöronal işlevleri anlamada çok önemlidir. Burada, nöronlarda vezikül hareketliliğini ölçmek için bir yöntem sunuyoruz. Bu, sinir sistemindeki zar ticaretinin miktarına uyarlanabilen uygun bir yöntemdir. 

Video: Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nörobilim

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

Kültür Hipokampal Nöronlar Dendritik Spine Floresan Takip edilen Proteinler Photobleaching sonra Floresan Kurtarma (sıkı bağlamak)

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Kültür MSS Nöronlar Dendritik Omurga Morfoloji Analizi

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

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 ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Kültür sempatik nöronlar bulunan dendritik Büyüme indükleyen

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

Kemirgenler davranmak yılında Hareketli Silikon Problar tarafından Nöronlar Büyük ölçekli Kaydı

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

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 ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Immünopresipitasyon ve Açil-Biotin Borsası (ABE) tarafından Yetiştirilmiş Hipokampal Nöronlar Protein Palmitoylation tespiti

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Dört boyutlu Floresans Görüntüleme ile Sinir Terminalleri Yaşayan endozom Dinamiği Görselleştirme

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...