This work was funded by NIH grants DA013137, DA031604, and HD043680. Partial support was provided by a NIH T32 training grant in Biomedical-Behavioral science.

Tüm hayvan protokolleri gözden geçirilmiş ve Güney Carolina Üniversitesi&#39;nde Hayvan Bakım ve Kullanım Komitesi (güvence numarası: A3049-01) tarafından onaylanmıştır. 1. Düşük yoğunluklu Embriyonik Nöronal Kültür <ol><li> Primer kortikal nöron kültürü için hazırlık <ol><li> Çözümler: <ol><li> 10 ml borat tamponu Poli-L-Lizin 5 mg çözülmesiyle Poli-L-Lisin stok çözelti hazırlayın. </li><li> 49 mi borat tamponu içinde 1 ml poli-L-lizin stok seyreltilmesi ile çalışma çözeltisi hazırlayın. </li><li> 395 mi DH 2 O ile başlayan ve daha sonra tüm maddeler (Boraks 1.24 g borik asit + 1.9 g) eklenerek borat tamponu, pH 8.4 hazırlanır. 1 M NaOH ile 8.4 pH ayarlayın. 400 ml ayarlayın ve kaputun altında 0.2 mikron naylon membran filtre ile filtre. </li><li> 445 ml dH 2 O ile başlayan ve diğer tüm maddeler ekleyerek HBSS çözüm, pH 7.2 hazırlayın (50 ml 10X HBSS Stok + 1.2 g HEPES). 1 N HCI, bri tarafından pH&#39;ı 7.2&#39;ye ayarlayınng 500 ml ve kaputun altında 0.2 mikron naylon membran filtre ile filtre. </li><li> % 10 fetal bovin serumu içeren DMEM / F12: Kaplama orta hazırlayın. kaputun altında 0.2 mikron naylon membran filtre ile filtre. </li><li> tam büyüme ortamı hazırlayın: 50 ml Neurobasal orta için, takviyeleri ekleyin (500 ul GlutaMAX (100X) + 500 ul Glukoz antibiyotik antimikotik çözüm (100X + 1 ml B-27 (50X) + 500 ul) + 100 ul% 7.5 sodyum Bikarbonat. kaputun altında 0.2 mikron naylon membran filtre ile filtre. </li></ol></li><li> İki gün kültür öncesi: <ol><li> 2 Poli-L-Lizin çalışma solüsyonu ve kaput O / N altında tutmak ile kaplayın oniki 35 mm cam alt yemekler. </li></ol></li><li> kültür bir gün önce: <ol><li> GKD 2 O. ile boş kaplama yemekler ajan ve durulama Yemekler kaputun altında bir saat kurumaya bırakın. </li><li> % 10 fetal sığır serumu (toplam çözeltinin% 10) ile kaplanmış ortamı (DMEM / F12 hazırlayın. Pipet 2 mi ortam içineher yemeğin ve 37 o C,% 5 CO 2 O / N kuluçkaya yatmaktadır. Not: kültürlerken plastik kültür kaplarına önlemek için önemlidir. cam alt (ters Mikroskop) yemekleri ya da (dik mikroskoplar) cam lamelleri kullanılması keskin ve net görüntüler verir. Bir plastik alt tabak ile karşılaştırıldığında, cam alt çanak floresan mikroskopi sırasında istenmeyen tonları veya parlamayı önler. </li></ol></li></ol></li><li> Hücre kültürü protokolü: <ol><li> HBSS Buffer (100-150 ml), buzdolabında koymak 100 mm petri, forseps, makas, 50 ml tüpler ve kaputu UV ışık altında 15 ml tüpler. </li><li> kültür odası dışında başlık altında% 5 sevofluran öldürücü inhalasyon ile gebe sıçan Euthanize. </li><li> % 70 EtOH, çadır karın cavity.Open plasentayı maruz makas kullanarak rahim ortasında kuyruk yukarıya kesim sırasında forseps kullanarak alt karın derisi ile sterilize edin. </li><li> 8-10 E18 fetus çıkarın. PlaHBSS çözeltisi ihtiva eden petri çanakları 8-10 fetus ce. </li><li> forseps ile fetusun başının arkasını tutun, sonra vücuttan kafasını koparmaya makas kullanın. HBSS 5-7 ml ile dolu başka bir petri yerleştirin. kaput çanak aktarın. </li><li> Soğuk HBSS ile iki ek 100 mm petri doldurun. </li><li> Peel kenara kafatası ve HBSS ile dolu yeni bir petri içine beyin kepçe. </li><li> sonra bunun soğuk HBSS ile yeni bir petri beyin transferi, beyinden gelen keskin kavisli bir forseps, ayrı beyincik ve beyin sapı kullanın. </li><li> kavisli bir forseps ile hemisfer ayırmak, sabitlemek için cımbız kullanın. meninks çıkarın; İşaretli 15 ml santrifüj tüplerine frontal korteks ve transfer parçaları izole. </li><li> Taze 2 mi HBSS ile 15 ml tüp doldurun ve her biri 15 ml&#39;lik bir tüpe 20 ul tripsin EDTA (% 0.5 tripsin ve 5.3 mM EDTA. 10x konsantre edildi) ekleyin. </li><li> Oda sıcaklığında 10-15 dakika kuluçkalayın. Yavaşça tüpü birkaç dakikada girdap, yerleşme izin vermez. </li><li> After 10-15 dk, kullanım cam pipet eski HBSS kaldırmak ve yeni HBSS ile iki kez durulayın. </li><li> (2 mi HBSS içinde 1 mg / ml, 2 mg tripsin inhibitörü) tripsin inhibitörü daldırın, iyice karıştırın ve 5 dakika bekletin. Taze HBSS ile iki kez yıkayın. </li><li> Yine bir kalibre pipet ve çok yavaş homojen hale gelene kadar azaltılmış ucu çapı ile steril bir ucunu kullanarak bubbles.Triturate önleyecek şekilde çok yavaş çiğnemek beyin parçaları, lastik ampul 10-15 kez cam pipet kullanarak. </li><li> Aktarım istenilen yoğunlukta hücreler çözülmüş kaplanmış kültür kaplarına (50 hücre / mm2) hazırlanmış ön. 37 o C,% 5 CO2 O / N inkübe edin. </li><li> 24 saat sonra, taze tam büyüme serumsuz Neurobasal orta DMEM / F12 ortamı değiştirin. </li><li> Deney için en az 12-14 gün% 5 CO 2/95% odada nemlendirilmiş kuluçka makinesi içinde her zaman hücreleri korumak. Taze Neurobasal orta yaşlı orta yaklaşık% 50 yerine her 5-6 gün hücreleri tamamlar. </li></ol></li></ol> 2. Floresan Etiketleme ve İmmünositokimya Not: Birincil kortikal hücre kültürleri immünofloresan markalama 1 ml&#39;lik bir çalışma hacmine sahip cam alt 35 mm&#39;lik bir hücre kültür tabaklarında gerçekleştirilmiştir. <ol><li> fosfat tamponlu tuzlu su pH 7.4 (PBS) ile iki defa cam alt bulaşık yıkama. </li><li> Oda sıcaklığında 15 dakika süreyle% 4 paraformaldehid hücreleri saptamak. </li><li> PBS ile iki kez yıkanır hücreler, 5 dakika boyunca PBS içinde% 0.1 Triton X-100 ile geçirgenliği. </li><li> F-aktine özel leke ile oda sıcaklığında 20 dakika için hücreleri tedavi, AlexaFluor 488 Phalloidin (1:40, 1 ml PBS içinde Phalloidin 25 ul). </li><li> PBS ile iki kez hücreleri durulanır ve 1-2 saat boyunca oda sıcaklığında% 10 normal keçi serumu ile bloke eder. </li><li> % 2 normal keçi serumu ile PBS içinde 4 O ° C&#39;de O / N inkübe: Tavuk poliklonal anti-MAP2 antikoru (2.500 1) ile seyreltilir </li><li> inkübasyondan sonra, iki kez PBS ile durulayın. </li><li> ikincil antikor (Alexa R sulandırmakEd 594-konjuge keçi anti-tavşan IgG&#39;si (1: 500)% 2 normal keçi serumu ve 2 saat oda sıcaklığında inkübe edin. </li><li> PBS ile durulayın ve 10 ul / Hoescht boya çanak ekleyin. </li><li> Oda sıcaklığında 3 dakika kuluçkalayın ve iki kez PBS ile yıkayın. Not: Adım 3 (F-aktin puncta sayma) için artık etiketli hücreler hazır. </li><li> Bir Lamel Kapatma ajan olarak antifade reaktif 100 ul ile hücre örneği korumak ve uzun süreli depolama için karanlıkta 4 o C&#39;de tutun. Not: Phalloidin antifade Lamel reaktifleri ile korunabilir 1 hafta ve floresan kadar karanlıkta 4 ° C&#39;de PBS içinde nispeten kararlıdır. Phalloidin uzatılmış depolama ile difüze başlayabilir Bununla birlikte, uygun resim boyama sonrası 1-3 gün elde edilir. </li></ol> 3. F-aktin puncta Sayma <ol><li> floresan mikroskop açın ve 20 × amacı geçmek. Açık mikroskop yazılımı ve 1280 × 960 piksel görüntü boyutunda program kurmak, ve 0.11 × zum 7 mikron / piksel görüntü çözünürlüğü. </li><li> Kırmızı yeşil (495 nm) / (613 nm) floresan kanal altında eş etiketli F-aktin / map2 nöronların görüntüler elde. </li><li> 5 Yeşil (F-aktin) / Kırmızı (map2) immunolabeled / Mavi (Hoescht) açıkça tanımlanmış dendritik milleri ile bireysel nöronun floresan görüntüleri seçin. </li><li> Sürekli map2 immünofloresan ile ikinci dereceden dendritik segmentte (uzunluk aralığı 25-75 mikron) F-aktin zengin yapıları belirleyin. </li><li> yatay bir seviyeye görüntüleri seçili bölgeyi döndürün. yeni bir resim olarak Kopyala ve yapıştır. </li><li> Her yemeğin tutarlı ayarlamalar kullanarak, görüntülerin arka planını çıkarın. </li><li> Parlak yeşil F-aktin puncta saymak ve elle eğitimli bağımsız gözlemciler tarafından seçilen dendritik parçasının uzunluğunu iz. Bir tablo dosyasına veri verir. </li><li> Map2 dendrit etiketli uzunluğu (L) toplam F-aktin etiketli puncta (N) bölünmesi ile yoğunluğunu hesaplayınız. F-ac sayısı Express verilerinidendrit 10 mikron başına kalay puncta Not: puncta dendritik mili boyanma ortalama yoğunluğu en az 50% bir tepe şiddetine sahip F-aktin floresans (boyut ≤1.5 um), seçilen her bir dendritik bölümüne dahil edilmiştir. </li></ol>

<ol><li>Heller, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biochemical+anatomy+of+cortical+inhibitory+synapses%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The biochemical anatomy of cortical inhibitory synapses.</a> PLoS One. 7, e39572(2012).</li>
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<li>Matus, A., Ackermann, M., Pehling, G., Byers, H. R., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+actin+concentrations+in+brain+dendritic+spines+and+postsynaptic+densities%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">High actin concentrations in brain dendritic spines and postsynaptic densities.</a> Proc Natl Acad Sci USA. 79, 7590-7594 (1982).</li>
<li>Allison, D. W., Gelfand, V. I., Spector, I., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+actin+in+anchoring+postsynaptic+receptors+in+cultured+hippocampal+neurons%3A+differential+attachment+of+NMDA+versus+AMPA+receptors%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors.</a> J Neurosci. 18, 2423-2436 (1998).</li>
<li>Sekino, Y., Kojima, N., Shirao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+actin+cytoskeleton+in+dendritic+spine+morphogenesis%5BTitle%5D)+AND+%22Neurochem+Int%22%5BJournal%5D)">Role of actin cytoskeleton in dendritic spine morphogenesis.</a> Neurochem Int. 51, 92-104 (2007).</li>
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None of the authors have conflicts of interest to declare.

Bu protokol, bize bireysel nöronların dendrit belirlemenizi sağlar 35 mm cam alt yemekleri düşük yoğunlukta sıçan kortikal nöronların kültürleme açıklar. Sonra, dendritik değişiklikleri tespit etmek faloidin ve map2 boyama kullanın. Sonra, F-aktin puncta değişiklikleri ölçmek için özel yazılım kullanılır. F-aktin puncta bireysel nöronun tüm nöronal ağ açıkça görünür olmalıdır değişiklikleri belirlemek için, bu tek bir nörondan uygun ikinci derecede...

Bu çalışmanın temel amacı, nöronal dendritik ağın sinaptik bütünlüğünün ölçümü (tahmini) güvenilir bir yöntem geliştirmektir. Burada Phalloidin boyama ve uzman (NIS-Elemanları) yazılımı kullanılarak takip eden analizi ile dendritler immunositokimyasal (ICC) taramadan oluşan bir kombinasyon kullanılarak primer fare kültürlenmiş nöronlar F-aktin puncta ölçülmesini açıklar. Etiketli phallotoxins büyük ve küçük ipliklerin (F-aktin) için benzer bir yakınlığa sahip fakat bazı aktin antikor 1 farklı olarak, monomerik küresel aktin (G-aktin) bağlanmaz. Phalloidin bağlayıcı Nonspesifik böylece hücresel görüntüleme sırasında en az bir arka plan sağlayarak, ihmal edilebilir düzeydedir. Phalloidin genellikle Phalloidin F-aktin çok daha yoğun etiketleme sağlar flüoresan mikroskopi için hücresel proteinleri etiketlemek için kullanılacak antikorlar çok daha küçüktür. Böylece, nöronlarda F-aktin lokalizasyonu detaylı görüntüler olabilirişaretlenmiş falloidinle kullanılması yoluyla elde edilmiştir. Phalloidin nöronal dendritler (F-aktin) boyama olgun dikenleri olmayan dikenli sinaps 2 ve olgunlaşmamış dikenler de dahil olmak üzere dendritik yapıların, çeşitli temsil eden ayrık &quot;sıcak noktalar&quot; ya da parlak &quot;puncta&quot; oluşturur. Olgunlaşmamış dikenleri ince filopodia ve yama morfolojisi bazı formları içerir ve spinogenesis 3 başlatılmasını temsil edebilir. Olgunlaşmamış dikenleri olan ve olmayan dikenli yamalar PSD95 4 yoksundur. Sadece dikenler sonraki değişiklikler değil, aynı zamanda ek dendritik yapılara F-aktin kurşun üretiminde değişiklikler, böylece synaptodendritic bütünlüğünü 5-7 araştırmak için önemli bir araç faloidin adrestir. Genel olarak, Phalloidin-pozitif (F-aktin) puncta sayısı bir aktif sinapsların arasında bir denge (eksitatör ve inhibitör), aktin dinamiklerini ve sinaps istikrar 8 yansıtmaktadır. belirli t incelemek önemli olmasına rağmenBir tedavinin hedefi bilinmediğinde sinaps (yani, uyarıcı dikenleri) bir Dosya türleri, ilk dendritik yapıların çeşitli genel bütünlüğünü tahmin etmek için gereklidir. F-aktin önleyici sinaps bir synaptopathy gösterebilir F-aktin puncta bir değiştirilmiş sayısı dahil olmak üzere dendritik dikenler ve diğer yapıların, önemli bir bileşeni olduğu için. Bu synaptopathy daha sonra belirli değişiklikler için daha fazla araştırılması olabilir. Birden fazla sinaptik türleri / yapıların tespiti için bizim miktar yöntemi çeşitli deneysel tedaviler aşağıdaki dendritik sinaptik değişiklikler (artış ve azalışlar) genel bir tahmin verir.

<table><tbody><tr><td>35 mm Glass Bottom Dishes No. 1.5 coverglass</td><td>MatTek Corporation</td><td>P35G-1.5-20-C</td><td></td></tr><tr><td>DMEM/F12 medium</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P9155</td><td></td></tr><tr><td>Boric acid</td><td>Sigma</td><td>B0252</td><td></td></tr><tr><td>Borax</td><td>Sigma</td><td>B9876</td><td></td></tr><tr><td>GlutaMax</td><td>Life Technologies</td><td>35050-061</td><td>100X</td></tr><tr><td>Glucose</td><td>VWR</td><td>101174Y</td><td></td></tr><tr><td>HBSS</td><td>Sigma</td><td>H4641</td><td>10X</td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>B-27 supplement</td><td>Life Technologies</td><td>17504-044</td><td>50X</td></tr><tr><td>Antibiotic-Antimycotic solution</td><td>Cellgro</td><td>30004CI</td><td>100X</td></tr><tr><td>Sodium Bicarbonate</td><td>Life Technologies</td><td>25080</td><td></td></tr><tr><td>Vannas Scissors</td><td>World Precision Instruments</td><td>500086</td><td></td></tr><tr><td>Iris Scissors</td><td>World Precision Instruments</td><td>500216</td><td></td></tr><tr><td>Iris Forceps</td><td>World Precision Instruments</td><td>15914</td><td></td></tr><tr><td>Dumont #7 Forceps</td><td>World Precision Instruments</td><td>14097</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>World Precision Instruments</td><td>14095</td><td></td></tr><tr><td>ProLong Gold</td><td>Life Technologies</td><td>P36930</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Cover glass</td><td>VWR</td><td>631-0137</td><td></td></tr><tr><td>AlexaFluor 488 Phalloidin</td><td>Life Technologies</td><td>A12379</td><td></td></tr><tr><td>Normal horse serum</td><td>Life Technologies</td><td>26050-070</td><td></td></tr><tr><td>Chicken polyclonal anti-MAP2</td><td>abcam</td><td>Ab92434</td><td></td></tr><tr><td>Alexa Red 594-conjugated goat anti-chicken IgG</td><td>Life Technologies</td><td>A11042</td><td></td></tr><tr><td>NucBlue Live cell stain ReadyProbes Reagent Hoescht 33342</td><td>Life Technologies</td><td>R37605</td><td></td></tr><tr><td>NIS-Elements software package</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE2000-E inverted fluorescent computer-controlled microscope&amp;nbsp;</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>0.2 &amp;mu;m Nalgene nylon membrane filter</td><td>Fishersci</td><td>151-4020</td><td></td></tr></tbody></table>

quantification of filamentous actin (f-actin) puncta in rat cortical neurons

(F-aktin) İpliksi aktin proteini spinogenesis, sinaptik plastisite ve sinaptik stabilitesinde önemli bir rol oynar. dendritik F-aktin zengin yapılardaki değişiklikler sinaptik bütünlük ve bağlantı değişiklikleri göstermektedir. Burada F-aktin puncta ve sonraki ölçümü teknikleri için birincil sıçan kortikal nöronların, Phalloidin boyama kültürü için ayrıntılı bir protokol sağlar. İlk olarak, E18 fare embriyonlarının frontal korteks, daha sonra, düşük yoğunluklu, hücre kültürü içine en az 12-14 gün boyunca in vitro yetiştirilen nöronlar ayrışmış. Deneysel tedaviden sonra, kortikal nöronlar AlexaFluor 488 falloidinle boyanır ve mikrotübül-ilişkili protein 2 (dendritik F-aktin puncta etiket) (MAP2; nöronal hücreleri ve dendritik bütünlüğünü doğrulamak için). Son olarak, özel yazılım analiz ve rastgele seçilen nöronal dendrite ölçmek için kullanılır. F-aktin zengin yapılar ikinci mertebe dendritik dalları (uzunluk aralığı 25-75 ve tespit edilir# 181; sürekli map2 immünofloresan ile m). Burada sunulan protokol deneysel tedaviler sonrasında dendritik sinaps yapılarında değişiklikler araştırmak için faydalı bir yöntem olacaktır.

Mevcut yöntemlerle, biz ilk kültür sıçan bizi bireysel nöronların dendrit belirlemenizi sağlar 35 mm cam alt yemekleri düşük yoğunlukta, kortikal nöronlar. Şekil 1 &#39;de, diferansiyel girişim kontrast (DIC) ve görüntüler gün 4, 6, 10, in vitro olarak 14, 21 ve 27 fetal sıçan kortikal nöronlar geliştirmek morfolojik değişiklikleri gösterir. uzunluk ve Dentritlerin sayısı kültürlü sıçan primer nöronlar olgunlaşması ile artış...

Watch this Scientific Journal Video about Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons at JoVE.com

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Rat Kortikal Nöronlar Filamentli Aktin (F-aktin) puncta Niceleme

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

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Research

JoVE Journal

Neuroscience

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

12.0K Görüntüleme.  University of South Carolina. Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Video: Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

Sinapslar miktarının: Synapse Numarası ölçmek için bir İmmünositokimya tabanlı Testi

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Nörobilim

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

Vitro F-aktin üzerinde erken Endosomes polimerizasyon

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

Biyokimya

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

Nöronal Büyüme Konileri permeabilize rodamin-aktin ile F-aktin Dikenli Ends Etiketleme

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

Kortikal Ağları Geliştirme Fonksiyonel Kalsiyum Görüntüleme

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

DetectSyn: Sinaps Yoğunluğundaki Değişiklikleri Tespit Etmek için Hızlı, Tarafsız Bir Floresan Yöntemi

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Hipokampal kemirgen beyin dilimleri dansitesi Synapse değerlendirilmesi

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Beyin Dilimlerinde ve Birincil Hücre Kültüründe Piramidal Nöronların Balistik Etiketlemesi

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

Kemirgen ve İnsan Beyin Dokularında Aktüer Polimerizasyon Durumunu Değerlendirmek için Zaman Verimli Floresan Spektroskopi Tabanlı Bir Tahlil

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM

The present protocol describes a method to visualize and measure actin rings and other components of the membrane periodic skeleton of the axon initial segment using cultured rat hippocampal neurons and 3D-structured illumination microscopy (3D-SIM).

Akson Başlangıç Segmentinin Membran Periyodik İskeletinin 3D Yapılı Aydınlatma Mikroskobu (3D-SIM) Kullanılarak Ölçüm Özellikleri

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...