Biz protokolleri geliştirmeye yardımcı oldu, özellikle de burada özgün yordamı geliştirmiştir Cin Fonte açıklanan Link laboratuarı mevcut ve eski üyeleri teşekkür etmek istiyorum. Bu çalışma CDL NIH (AG12423) ve Alzheimer Derneği (Zenith Ödülü) ödül tarafından desteklenmektedir

1) felç tayini için nematodu Büyüme Medya plakaları hazırlamak. Felç Testler, C için rutin olarak kullanılan standart nematodu Büyüme Medya (NGM) tabaklarda yapılmaktadır elegans yayılma ve genetik (Stiernagle 2006). <ol><li> NaCl, Erlenmeyer şişesinin Agar ve Peptonlu içinde bulunduğu NGM çözüm Otoklav ve steril CaCl 2, Urasil, Kolesterol, 1M MgSO 4 ve 1M KPO 4 uygun miktarda ekleyebilirsiniz . , Her biri 60 mm x 15 mm Petri Dish içine sıvı NGM kısım 10 ml. NGM gece oda sıcaklığında katılaşmasını sağlayın. </li><li> Her tabağa bileşikleri / özleri, kısım özü etkilerini test ve özü plaka yüzeyi üzerinde eşit olarak dağıtmak için bir ayırıcı kullanabilirsiniz. Gece oda sıcaklığında kuru ekstresi izin verin. 1 ml ciltlik kuruması için daha fazla zaman gerektirecektir. </li><li> E. 250 mcL ile her plaka Nokta coli suşu OP5O 0.4-0.6 bir optik yoğunluk için bir gecede LB Medya büyüdü. Bakteriler oda sıcaklığında bir gece kurumaya bırakın. </li></ol> İlgili değişkenlerin Plakaların yaş büyük plakalar kurumaya eğilimi gibi, felç tahlil üzerinde önemli bir etkisi vardır. Kullanım plakaları felç testinin bir hafta içinde dökülür. İdeal sonuçlar için, [(2) aşağıya bakınız] senkron solucan popülasyonlarının başlamadan 3-4 gün önce plakaları dökün. Herhangi bir deneme için, aynı partiden dökülür tabak kullanın. Çim boyutu (yani benekli vs. Yayılma) ve / veya bakteri türü değiştirme solucanlar davranışını değiştirebilir. Felç testleri alternatif E. kullanılarak yapılabilir coli suşlarının bir besin kaynağı (örneğin, RNAi deneyler beslenmesinde kullanılan HT115 zorlanma), fakat bu, böylece uygun iç kontrol gerektiren, felç kinetiği değiştirebilirsiniz . 2) Yaş eşzamanlı Worm Populasyonlarının Hazırlık Gerilme CL4176 (smg-1 (cc546 ts); dvIs27 [myo-3 / Aβ ​​minigene + rol-6 (su1006) işaretleyici gen X en sık Aβ indüklenen felç assaying için kullanılır Bu gerginlik ve diğer transgenik modelleri mevcuttur. Caenorhabditis Genetik Merkezi (http://www.cbs.umn.edu/CGC/) nominal ücret karşılığında akademik araştırmacılar. <ol><li> Yaş senkronize, senkronize bir ebeveyn nesil başlatmak için tercih edilir test nüfusun en üst düzeye çıkarmak için. Bir hafta önce test nüfusun felç tahlil başlatarak, 16 yaşında yatıyordu 20-30 gravid yetişkin 2 saatlik bir yumurta için OP50 yayılmış çok 10 cm NGM plakalar üzerine aktarmak için bir platin solucan toplayıcı ° C (müsamahakar sıcaklık CL4176 .) Gravid yetişkin çıkarın ve döl hangi zaman gravid yetişkin &quot;ikinci gün&quot; olacak, 7 gün boyunca büyümeye izin verir. </li><li> (Gün 1) ikinci gün gravid yetişkin yaşa eşzamanlı test nüfusu hazırlamak için kullanılır. Her deneysel koşul için 50-75 yaş senkron solucanları içeren üç nüsha plakaları oluşturmak için nesne. Platin solucan seçici kullanarak, OP50 ile fark 60 mm (10 ml hacim) NGM plakalar üzerine 2 gün gravid yetişkinlerin 10-12 aktarmak. Solucanların sonra yetişkinler almak ve yumurta kuluçkaya yatmak için izin ve 16 büyümeye, 2 saat boyunca 16 ° C&#39;de yumurtalarını ° C izin verin Bu noktada onlara kod plakaları puanlama olmayacak birisi için en iyi golcü deneysel koşullara kör olacak. </li></ol> İlgili değişkenlerin Yumurtladığı embriyonik gelişim evresi (~ optimal koşullar altında vahşi tip solucanlar için 26-hücreli aşama), yetişkin yaş ve beslenme bir fonksiyonudur. Test nüfusun hazırlanması için kullanılan gravid yetişkin, eski ya da açlıktan iseniz, daha sonraki aşamalarda yumurta, nüfus ve felç kinetiği tekrarlanabilirlik yaş senkronize azaltarak belirtilen olacaktır. Farklı kültürlerin (mikrobik kirleticiler Bu testte ciddi etkiler) mono-axenic (yani, sadece OP50 bakteriler) daha önemlidir ve üç nüsha plakalar solucanlar benzer numaraları varsa tekrarlanabilirliği yüksek. 3) Transgen İndüksiyon ve Felç Testi <ol><li> (Gün 3) 25 ° C senkron yumurta sona ermesinden sonra 48 saat Upshift plakaları yatıyordu; solucanlar üçüncü larva dönemi (L3) olmalıdır. Levhalar, plakalar, aynı zamanda 25 ° C ulaşmak için izin vermek için 25 ° C inkübatör istifleme olmadan düzenlenmelidir. </li><li> (4. Gün) upshift başlandıktan sonra 18-20 saat solucanlar felç (zorlanma CL4176) puanlama başlayın. Bu noktada, nüfusun tüm solucanlar, dördüncü larval (L4) evre ulaşmış olması gerekir. Her plakalar tüm solucanlar felç kadar iki saat aralıklarla puanlama devam edin. Bilinmeyen nedenlerden dolayı, felç, vücut uzunluğu boyunca bile değildir: baş bölgeye hareket durdurma solucanın son parçasıdır. Böylece, wfelç son zamanlarda başlatılan ORMs plaka üzerinde çevirmeyin, ancak böylelikle kendi anterior etrafında bakteri takas ve temizlenir bakteri &quot;halo&quot; bırakarak, başlarını taşıyabilirsiniz. Bu solucanlar değişmez tamamen felç olacak ve bu nedenle felç olarak haleler ile solucanlar kategorize edilir. Bazı solucanlar haleler yok ama spontan hareket göstermez, bu solucan seçici dürtüsüyle tarafından test edilmektedir. Kışkırtılması solucan dürtüsüyle üzerine bir tam vücut dalga yayılımı uğramaz, felç gibi, o da atılırsa. Verimli puanlama için, onlar istemeden bir sonraki puanlama süresi rescored olamaz böylece plaka lekesiz sektörü felç solucanlar taşımak için en kolay yoldur. (Bu aynı zamanda bir skor düzeltilmesine imkân verir, yanlış kategorize solucanlar hareketi göstermek için izin verir). </li><li> Bir felç eğrisi oluşturmak için her zaman bir noktada solucanlar kesir felç edilmiş her bir plaka üzerinde bir yüzdesi dönüştürülür ve ortalama &quot;unparalyzed&quot; yüzde upshift başlatılması zaman karşı çizilmiştir. (Bu şekilde çizmek için gerekçe çıkan eğri bir hayatta kalma eğrisi resmen benzer olduğunu ve bu nedenle standart non-parametrik sağkalım istatistikleri kullanılarak analiz edilebilir.) </li></ol> İlgili değişkenlerin Transgenik myo-3/Aβ solucanlar upshift myo-3 organizatörü gelişimsel düzenlenir başlanmalıdır, ve anlamlı geç L4 veya erişkin solucanlar ifade azalttı felç için orta aşama L4 ulaşmadan önce gerçekleşmesi gerekir. Benzer şekilde, solucanlar hasret kalırsanız bu transgen ifadesi engelleniyorsa, bu yüzden solucanları deney boyunca, iyi beslenmiş olması esastır. 16 ° C&#39;ye kadar downshifted olsa bile, biz her türlü şartlar altında kurtarmak için bir felç solucan gözlenen asla ve 12-16 saat upshift sonra, tüm CL4176 solucanlar felç olacak Suşu CL4176, transgen ifadesi yeterli upregülasyonu neden felç zamanı değişmiş olmasına rağmen paralizi (yani, 20-25 ° C) daha düşük sıcaklıklarda oluşabilir. Felç oranı, kullanılan özel transgenik suşu (CL4176 daha hızlı ve yavaş oranları ile myo-3/Aβ suşları inşa ettik) bağlıdır, bu nedenle alternatif suşları ile skor penceresi ayarlanması gerekiyor olabilir. 4) Temsilcisi Sonuçlar Şekil 1&#39;de gösterildiği gibi tedavi edilmemiş ve 6.27 mM kafein (NGM plaka final konsantrasyon) maruz CL4176 bir felç eğri bir arsa. Bu tedavi, bu modeli Aβ toksisite baskılanması itibariyle gösterge niteliğindeki yorumlamak yaklaşık 4 saat, felç bir çok istatistiksel olarak anlamlı bir gecikme ile sonuçlanır. Şekil 2&#39;de gösterildiği gibi, kafestol, kahve bulunan başka bir bileşik etkileri assaying bağımsız bir deney. Bu arsa Aβ zehirlilik etkisi olmayan tedaviler için tipik bir örneğidir. Iki deneylerde tedavi edilmezse CL4176 popülasyonları yaklaşık yarısı 24 saat felç, felç, 28 saat olduğunu unutmayın. Bu kontrolü CL4176 popülasyonlarının felç için tipik zaman dilimleri. Bu zamanlama önemli sapmalar, değişen çevre koşullarına veya transgen kopya kendiliğinden silme göstergesidir. Sıcaklıklarda suşu (CL4176 myo-3/Aβ transgen birden fazla kopya ile kromozomal entegre transgenik dizi içerir. Bu transgenik dizi normalde oldukça istikrarlı olmasına rağmen, herhangi bir yayılma&gt; 16 ° C bir geçirmiş nadir solucanlar için seçecek Aβ ifade ve yavaş felç sonucu düşer varyantları çıkan transgenlerin kendiliğinden silme.) <img src="/files/ftp_upload/2252/2252fig1.jpg" alt="Şekil 1" /> Şekil 1. CL4176 solucanlar tedavi edilmezse veya 6.27 mM kafein (Sigma) maruz gerginlik Felç eğrisi Belirtilen sıcaklık upshift başlangıcından itibaren vardır. Hata çubukları = SEM <img src="/files/ftp_upload/2252/2252fig2.jpg" alt="Şekil 2" /> Şekil 2. 0.48 mM kafestol (Sigma) maruz CL4176 solucanlar Felç eğrisi. Hata çubukları = SEM

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	<li>Arya, U., Dwivedi, H., Subramaniam, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reserpine+ameliorates+Abeta+toxicity+in+the+Alzheimer's+disease+model+in+Caenorhabditis+elegans.">Reserpine ameliorates Abeta toxicity in the Alzheimer's disease model in Caenorhabditis elegans.</a> Exp Gerontol. 44, 462-466 (2009).</li><li>Cohen, E., Bieschke, J., Perciavalle, R. M., Kelly, J. W., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+activities+protect+against+age-onset+proteotoxicity.">Opposing activities protect against age-onset proteotoxicity.</a> Science. 313, 1604-1610 (2006).</li><li>Dosanjh, L. E., Brown, M. K., Rao, G., Link, C. D., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+of+a+transgenic+C.+elegans+expressing+neuronal+amyloid-beta.">Behavioral phenotyping of a transgenic C. elegans expressing neuronal amyloid-beta.</a> J Alzheimers Dis. , (2009).</li><li>Drake, J., Link, C. D., Butterfield, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+precedes+fibrillar+deposition+of+Alzheimer's+disease+amyloid+beta-peptide+(1-42)+in+a+transgenic+Caenorhabditis+elegans+model.">Oxidative stress precedes fibrillar deposition of Alzheimer's disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model.</a> Neurobiol Aging. 24, 415-4120 (2003).</li><li>Fay, D. S., Fluet, A., Johnson, C. J., Link, C. D. <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+aggregation+of+beta-amyloid+peptide+variants.">In vivo aggregation of beta-amyloid peptide variants.</a> J Neurochem. 71, 1616-1625 (1998).</li><li>Florez-McClure, M. L., Hohsfield, L. A., Fonte, G., Bealor, M. T., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+insulin-receptor+signaling+promotes+the+autophagic+degradation+of+beta-amyloid+peptide+in+C.+elegans.">Decreased insulin-receptor signaling promotes the autophagic degradation of beta-amyloid peptide in C. elegans.</a> Autophagy. 3, 569-580 (2007).</li><li>Fonte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+in+vivo+beta-amyloid+peptide+toxicity+by+overexpression+of+the+HSP-16.2+small+chaperone+protein.">Suppression of in vivo beta-amyloid peptide toxicity by overexpression of the HSP-16.2 small chaperone protein.</a> J Biol Chem. 283, 784-7891 (2008).</li><li>Hassan, W. M., Merin, D. A., Fonte, V., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AIP-1+ameliorates+beta-amyloid+peptide+toxicity+in+a+Caenorhabditis+elegans+Alzheimer's+disease+model.">AIP-1 ameliorates beta-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model.</a> Hum Mol Genet. 18, 2739-2747 (2009).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+beta-amyloid+peptide+in+transgenic+Caenorhabditis+elegans.">Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 92, 9368-9372 (1995).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+in+a+transgenic+Caenorhabditis+elegans+Alzheimer's+disease+model.">Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer's disease model.</a> Neurobiol Aging. 24, 397-413 (2003).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta-induced+pathological+behaviors+are+suppressed+by+Ginkgo+biloba+extract+EGb+761+and+ginkgolides+in+transgenic+Caenorhabditis+elegans.">Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans.</a> J Neurosci. 26, 13102-13113 (2006).</li></ol>

Açıklanan protokol etkili Aβ toksisite bileşiklerin etkilerini tahlil etmek için kullanılabilir. Yayınlandı örnekler Ginkgo biloba ekstresi bileşenlerinin etkileri (Wu ve ark 2006) ve reserpin (Arya ve ark 2009) içerir. Diğer sistemlerde Aβ toksisitesine karşı koruyucu bileşikler de (örneğin, memantin, yayımlanmamış sonuçlar) bu modelde koruyucu olduğu gösterilmiştir. Bu modelin kurulması için önemli bir gerekçe C ile iyi geliştirilmiş genetik ve moleküler araçlar kullanılabilir. elegans Aβ toksisite mekanizmaları araştırmak için kullanılabilir. Böylece, Aβ toksisitesi üzerindeki etkileri özel mutasyonlar (örneğin, daf-2-fonksiyon kaybı mutasyonu, Florez-McClure ve ark, 2007 tanıtan tarafından sinyalizasyon gibi insülin azaltmada etkisi) test ve bu model de olabilir (Fonte ve ark 2008) aşırı ifade transgenlerin etkilerini incelemek için kullanılır. Biz son zamanlarda Aβ tek bir amino asit değiştirmelerin (yayınlanmamış veri) toksisitesi üzerindeki etkilerini incelemek için bu modelin geniş kullanımı yaptık. Transgenik bir model deneyleri burada kas hücre fonksiyonu akut Aβ ifade etkisi nitelendirdi. Felç kas hücreleri ve bunların sarkomer zamanda bozulmamış; felç fenotip kas hücre ölümü sonucu değildir. Ancak, kas bütünlüğünü ve solucanlar nihai ölümünden sonra felç (ilk upshift sonra ~ 48 saat) bir arıza var. Biz bu Aβ ifade aşağı etkisi araştırılmıştır veya bir toksisite belirteci olarak kullanılır. Burada açıklanan indüklenebilir Aβ ifade modeli başına β-amiloid oluşumunu modüle tedavileri araştırmak için uygun değildir. (. Fay ve ark 1998; Link ve ark 2001) kurucu Aβ ifade formu kolaylıkla saptanabilir amiloid mevduat transgenik solucanlar rağmen, indüklenebilir Aβ ifade ile transgenik solucanlar nadiren amiloid mevduat ve felç fenotip amiloid birikimi (Drake ve ark bağımsız görünür. 2003). Çözünen oligomerik Aβ birikimi değil, amiloid birikimi bağlı Aβ ifade sonuçlarının akut toksisite sonuçları görünümü ile uyumlu.

<table border="1"><tbody><tr><td> Reaktifler </td><td> Quantity/2L </td><td> Şirket / Katalog # </td><td> Yorumlar </td></tr><tr><td> NaCl </td><td> 6g </td><td> Sigma-Aldrich / S9888 </td><td></td></tr><tr><td> Agar </td><td> 40g </td><td> Sigma-Aldrich / A7002 </td><td></td></tr><tr><td> Pepton </td><td> 5g </td><td> Sigma-Aldrich / P0556 </td><td></td></tr><tr><td> GKD 2 O </td><td> 2L </td><td></td><td></td></tr><tr><td> 1M CaCl 2 </td><td> 2mL </td><td></td><td> GKD 2 O içinde 147.02mg/mL </td></tr><tr><td> Urasil </td><td> 2mL </td><td></td><td> GKD 2 O içinde 2mg/mL </td></tr><tr><td> Kolesterol </td><td> 2mL </td><td></td><td> Etanol içinde 5mg/mL (otoklav değil) </td></tr><tr><td> 1M MgSO 4 </td><td> 2mL </td><td></td><td> GKD 2 O içinde 246.5mg/mL </td></tr><tr><td> 1M KPO 4 </td><td> 50ml </td><td></td><td> 98mg KH 2 PO 4 mL, 48mg K2HPO4/mL GKD 2 O, pH </td></tr><tr><td> Erlenmeyer Flask </td><td></td><td> VWR </td><td></td></tr></tbody></table>

assaying &beta;-amyloid toxicity using a transgenic c. elegans model

Β-amiloid peptid (Aβ) birikmesi genellikle Alzheimer hastalığının indüksiyon merkezi inanıyordu, ancak ilgili toksisite mekanizma (lar) hala belirsiz. Aβ da İçerme Vücut Miyozit, ciddi bir insan miyopati intramüsküler yatırılır. Yoğun çalışılan nematod solucan Caenorhabditis elegans transgenically insan Aβ ifade etmek için tasarlanmış olabilir. Aβ ifade doku ya da zamanlama olarak, transgenik solucanlar Aβ toksisite okuma olarak hizmet kolaylıkla ölçülebilir fenotipleri olabilir. Cohen ve ark; Örneğin, Pan-nöronal Aβ ifade ile transgenik solucanlar kurucu kas-özel ifadesi ile transgenik solucanlar ilerici, yaşa bağlı felç fenotip (Link, 1995, kusurlar, ilişkisel öğrenme (. Dosanjh ve ark 2009) 2006). Bir özellikle yararlı C. elegans modeli, tekrarlanabilir bir felç fenotip sıcaklık upshift (Link ve ark 2003) üzerine çıkan bir Aβ transgen sıcaklık indüklenebilir kas ifade mühendisi mRNA gözetim sistemi ısıya duyarlı bir mutasyon istihdam. Bu modelde karşı Aβ toksisite [örneğin, bir koruyucu transgen (Hassan ve ark 2009) ifade Ginkgo biloba ekstreleri (Wu ve ark 2006) maruz kalma] tekrarlanabilir bu transgenik sıcaklık upshift tarafından tetiklenen felç oranı değiştiren tedaviler solucanlar. İşte biz bu transgenik C. felç oranı ölçmek için bizim protokol tarif Bu ölçüm etkisi deneysel değişkenlerin özellikle dikkat elegans modeli.

Watch this Scientific Journal Video about Assaying Î²-amyloid Toxicity using a Transgenic C. elegans Model at JoVE.com

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

Yoğun çalışılan bir nematod solucanı<em&gt; Caenorhabditis elegans</em&gt; Transgenically insan β-amiloid peptid (Aβ) ifade etmek için tasarlanmış olabilir. Aβ, İndüklenmiş ifade<em&gt; C. elegans</em&gt; Kas hızlı, tekrarlanabilir felç fenotip Aβ toksisite modüle tedavileri izlemek için kullanılabilecek bir yol açar.

Transgenik kullanarak Assaying β-amiloid Toksisite C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

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Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

25.3K Görüntüleme.  University of Colorado. Yoğun çalışılan bir nematod solucanı Caenorhabditis elegans Transgenically insan β-amiloid peptid (Aβ) ifade etmek için tasarlanmış olabilir. Aβ, İndüklenmiş ifade C. elegans Kas hızlı, tekrarlanabilir felç fenotip Aβ toksisite modüle tedavileri izlemek için kullanılabilecek bir yol açar.

Video: Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Sirkadiyen Rhythms Eğitim ve Parametreler Uyku Lokomotor Aktivitesi Assaying Drosophila</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Nörobilim

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

C in vivo Lazer Axotomy elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Içinde Synaptic florasan Otomatik Sayısallaştırma<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

C. elegans Takip ve Davranış Ölçme

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

Uzun vadeli Olfaktör İntibak A Moleküler Saati<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

Comet Assay kullanma Hipokampal Nöronlar DNA Hasarı assaying

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C. vivo Nöronal Kalsiyum Imaging&#39;de elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Bir<em&gt; Caenorhabditis elegans</emAmylopathy Çalışmaları&gt; Model Sistemi

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Sırasında Neuroblast Sitokinez görselleştirme<em&gt; C. elegans</em&gt; Embriyo

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

C. Dauer-spesifik nöronal Yaptırma Vivo Görüntülemede elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...