R01AA016837 (JCB) ve P20AA017828 (AGD ve JCB): Bu çalışmalar Ulusal Sağlık Enstitüleri, Ulusal Alkolizm ve Alkol Bağımlılığı Enstitüsü hibe ile desteklenmiştir.

Tahlil önce Günü&#39;nde gerçekleştirin 1. Adım <ol><li> Taze nematod büyüme ortamına L4 aşamada solucanlar almak (NGM) plakaları OP50 E. bir çim ekildi 20 CO / N de coli, ve kültür onları. Her deney koşulu 10 solucan gerektirir; solucanlar O / N kaybı sağlamak için solucan fazlalığı almak. <ol><li> İlk gün yetişkin Yalnızca tahlil hayvanlar; Birçok mutantlar vahşi tip daha yavaş bir hızda büyür. Test edilen tüm hayvanların ilk gün yetişkin böylece gelişimsel gecikmelere sahip suşlar için toplama zamanlamasını ayarlayın. </li></ol></li></ol> 2. Adım Tahlil günü gerçekleştir <ol><li> Deney için hazırlanması: <ol><li> Standart giremeyen 60 x 15 mm NGM plakalar üzerinde tahlilleri gerçekleştirin. Plakalar her kapak kapalı olarak, 2 saat süre ile 37 ° C&#39;de kullanılacak kurutun. Her deneysel deneme için, dört kurutulmuş NGM tabak kullanın; Bu 0 mM ve 400 mM etanol tahlil plakaları ve beraberindeki alıştırma plakaları olacaktır. NOT:NGM plakalar kullanımı Burada önemli olan; plakalarının bileşimin farklılıklar özellikle bunların ozmolarite, güçlü bir davranışı üzerinde etanol doz etkisini etkileyebilir, bu hayvanlar 20 biriken miktarda etanol değişiklikler, en azından kısmen, kaynaklanmaktadır. NGM agar 160 mOsm. </li><li> Kurutma işleminden sonra, giremeyen NGM plakalar, her deneyde kullanılan ve ağırlığı dikkat edilmesi tartın. Boş levha ağırlığına göre plakalarda ortamın hacmini belirler. Hacme agar ağırlıkça dönüşümünü tahmin etmek üzere ortam su, eşit hacimde aynı ağırlığa sahip olduğunu varsayalım. Not: En tutarlı sonuçlar, orijinal 10 ml hacim 8,3-8,9 ml arasında bir post-kurutma hacmi yeterince dehidrate olan NGM ortamı ile tespit edilmiştir. 2 saat kuru zaman bir alternatif plakalar kuluçka farklılıkları hesaba bu hacim aralığı ulaşana kadar kurumaya olduğunu. </li><li> 4 bakır halkaları (1 iç çapı eritin.Ve 6 cm) levhaların her birinin yüzeyi, yaklaşık 3 için bir alev farklı genotipler ve worms.Grasp her forseps ile bir halka, ve ısı ve tedavi grupları için corrals (Bunsen brülör güçlü bir alev çalışır) olarak hareket halinde sn. Hala &#39;atlama&#39; önlemek için forseps ile halka tutarak hemen bir plaka üzerinde halka yerleştirin. <ol><li> Halka halka üzerinde birkaç noktada forseps ile hafifçe bastırarak agar yüzeyine karşı düz aittir emin olun. Halka yerleştirirken, ek bir üç yüzük için odayı terk etmek dikkatli olun. </li><li> Dört kameranın görüş alanı içerisinde olacak şekilde olduğu gibi birbirine yakın zamanda yerleştirmek için özen levhanın yüzeyine üç ek bakır halkalar eritin. NOT: agar ile iyi bir sızdırmazlık yapma tahlil sırasında halkalarda solucanlar tutmak esastır. Plaka etrafında atlamak Yüzükler agar ile doğru mühür yapmak olası değildir vesolucanlar yuva sağlar ve solucanlar görselleştirmek müdahale ağar, yara olabilir. </li><li> Her tahlil plaka için kurutulmuş ve dereceye giremeyen ve hiçbir etanol alacaksınız gereken eşlik eden &quot;alıştırma&quot; plaka hazırlamak. Bu plakalar üzerinde dört bakır yüzük yerleştirin. </li></ol></li><li> Halkanın kendisinin görüş alanında yazma için özen, aşağıdaki halka kullanılacak solucan suşu ile her bir halkada plakaların alt etiketleyin. Eşlik eden aklimasyonundan plaka tahlil plaka üzerindeki etiketleri eşleştirin. </li><li> Nihai konsantrasyon 0 mM ya da 400 mM metanol (hacim için ağırlık), böylece her bir deney plakası eklemek için% 100 etanol miktarını hesaplayın. Her deney için (n = 1), bir 0 mM ve bir 400 mM etanol plaka olacaktır; alışma plakaları etanol almıyorsunuz. Plaka yüzeyinde etrafında pipetleme, etanol ekleyin. Plaka sızdırmaz laboratuar filmi kullanın ve 2 saat süre ile, bir tezgah üstünde dengelenmeye gelmeye bırakın. </li><li> 1.5 saat geçtikten sonra, tahlil, adım 2.2 aklimasyonundan adımını başlar. </li></ol></li><li> Hareket deneyi yapın: Deney plakaları <ol><li> Dikkatle aklimasyonundan plaka üzerinde bir bakır halka merkezine her deney grubu 10 solucanlar aktarmak. Solucan çekme hafifçe kazıyarak bu noktada agar üzerinde görünür herhangi bir gıda çıkarın. Aynı suşları çalışmalar boyunca aynı sırayla plakalar koymak değil böylece deney grubu deneysel çalışmalar boyunca plakalar koymak sırayı Vary. Not: hedef alıştırma levha gıda deney plakasına aktarılır ise solucanlar gıda ve hareket yeteneği üzerindeki ilacın etkisini gizlenir etrafında birikir, çünkü yemek az miktarda plakaya hayvanlar transfer edilmesidir. </li><li> Bu solucanlar yuva sağlayacak ve tahlil bozacak gibi, çekme ile agar yüzeyine kırmak için değil dikkatli olun. 30 dakika boyunca oda sıcaklığında solucanlar inkübe edin. </li><li> Her plaka insiyatifleri arasında uygun bir aralık olduğundan emin olun. Deneyimli bir deneyci 40 hayvan, 4 yüzük &lt;1.5 dakika boyunca 10 / halka taşıyabilirsiniz, ancak 2 dakika kadar herhangi bir aralık kabul edilebilir. Standart tahlil 10-12 dakika ve 0 mM ve 400 mM etanol hem de maruz 30-32 dk kayıt filmleri vardır. </li><li> 0 mM maruz kalma aklimasyonundan plaka ve yaklaşık 2 dk 30 sn dışında ikinci filmi başlamalıdır ilk önce film dosyasının tasarrufu sağlamak için 400 mM maruz kalma aklimasyonundan plaka başlatın. </li><li> 30 dakikalık bir alışma süresinden sonra, deney plakalarına alıştırma levhalarından solucanlar aktarın. Her plakanın tamamlama arasındaki zamanlama takip, alıştırma plakaya ilave edildi sırayla deney plakalarına dişli (0 mM ya da 400 mM etanol) aktarın. Buharlaşma etanol kaybını en aza indirmek için laboratuvar film ile plaka Seal. </li><li> Udüzleştirilmiş çekme üstüne solucanlar toplamak için ince kenarlı basık solucan kıracağıyla bir çekiş hareketi se. Bu çekme solucanı yapıştırıcı yardımcı olarak yaygın solucan aktarılması kullanılan bakteri, kullanılmadan analiz plakasına giremeyen alıştırma plaka solucanlar transferini gerçekleştirir. Not: plaka ilk solucanlar plakaya eklenmiştir son solucanlar daha uzun etanol maruz çünkü hayvanlar bu aşamada transfer edildiği hızı çok önemlidir, ve daha önceki zaman noktalarında, bazı zamana bağlı etkilere sahip olur. Bu suşlar, deneysel çoğaltır genelinde ilk plaka üzerine yerleştirilir döndürülmesi için önemli nedenidir. </li></ol></li><li> Hareket Testi gerçekleştirin: Film Çekme <ol><li> Böyle bir 2048x2048 7.4 ile bir 0.5x mikroskop objektif, 0.8x büyütme ve bir kamera gibi görüş alanı (yaklaşık 42x42 mm 2 kare), tüm dört yüzük eşzamanlı görüntüleme için izin veren bir mikroskop / kamera arada kullanın81 m piksel CCD. </li><li> Yoğunluk eşik adımında nesne tanıma yardımcı olan görüş alanı, üzerinde bile aydınlatma kullanarak (aşağı bakınız). Bir 3 &quot;x3&quot; arka iyi çalışıyor. </li><li> Görüntü ışık kaynağı bulaşan geleneksel mikroskop ile karşılaştırıldığında arka kullanarak kaybolur kontrastı üreten bir plaka yerleştirilmiş medya tarafı yukarı solucanlar (kapak aşağı). </li><li> Hareketli solucanlar zaman atlamalı filmleri yakalamak için görüntü analiz yazılımı hazırlayın. 2-min (120 kare) film olarak, 12-bit gri ölçekli görüntüler, her 1 sn yakalamak için yazılım ayarlayın. Hala yeterli görüntü çözünürlüğü korurken, çıktı dosyasının boyutunu azaltmak için, 1024x1024 piksel görüntü yakalamak için bir 2x2 binning modunu kullanın. </li><li> Film kaydetmek. Son solucanlar 10 dakika sonra o plaka yerleştirildi ilk plaka (0 mM etanol) 120 kare film kayıt başlayın. Film dosyayı kaydedin. </li><li> İkinci plaka (400 mM etanol) kaydedin. Için bu işlemi tekrarlayınSon solucanlar işleminden 30 dakika sonra başlayan, her iki levha her teşhir için, 30-32 dakikalık bir zaman noktaları yakalamak için birinci plaka (0 mM etanol) yerleştirildi. NOT: analiz edilecek bir sonraki plaka kaydı başlamadan önce her yakalama seansından sonra görüntü dosyalarını kaydetmek için yeterli zaman tanıyın. </li><li> Arşivlenmiş film geleceğe prova ve 8-bit 256 gri skala TIFF dosyaları için her filmin bir kopyasını dönüştürmek, bu filmler açıldı ve ImageJ 28 gibi diğer kamu alanı, açık kaynak kodlu yazılım programları görüntüleme, analiz edilecek izin vermek. NOT: Burada anlatılan görüntü analiz yazılımı özel bir dosya biçimini kullanır. </li></ol></li><li> ImagePro yazılımını kullanarak film analizi. <ol><li> Yakalanan kez, Image-Pro yazılımı veya eşdeğer Artı kullanarak film analiz. NOT: Diğer nesne izleme yazılımı çeşitli başarıyla birden C izlemek için kullanılır olmuştur ki mevcut tek seferde 29 ve böyle de elegansYazılım makul nesne tanımlama adımlar benzer ilkelere dayalı olarak burada tarif için yerine olabilir. açıklanan yöntem küçük farklılıklar olabilir Resim-Pro Plus yazılımı sürümleri 6,0-6,3 ve 7.0, Resim-Pro Plus yazılımının yeni sürümlerini ilgilidir. </li><li> 2-min (120 kare) segmentlerde filmleri analiz. İlk olarak, arka plan düzleştirmek ve solucan nesnelerin kontrastı artırmak için görüntülere bir filtre uygulamak. Aşağıdaki gibi filtre seçin: Menü&gt; İşlem&gt; Filtreler&gt; Donanım&gt; Flatten: Parametreler: Arka plan = Karanlık; Genişlik = 20 Pix Özellik. </li><li> Filtreleme aşamasından sonra film yeniden kaydetmek ama her zaman kendi filtresiz formda orijinal film korumak. <ol><li> Yerleştirilir ve bakır halka ile üst üste büyüklükte faiz (ROI) bir dairesel bölge ile ayrı ayrı halka hayvanların lokomosyonu analiz. Belirlemek ve Menü&gt; Tedbir ile solucanlar izlemek&gt; Parça ... komutunu Nesneleri; Bu Takibi D getirirata Tablo penceresi. Takip Seçenekleri düğmesi belirli parçalar hariç olmak üzere ve herhangi bir deneysel eserler sınırlamak için olanak sağlar. </li></ol></li><li> Otomatik Takip sekmesinde, aşağıdaki parametreleri kullanın: Parça Parametreleri: Hız sınırını (arama yarıçapı) = 400 mm / çerçeve, İvme sınırı = Otomatik, Minimum toplam hat uzunluğu = 400 mikron, Baskın hareket type = kaotik; Parça parametreleri nesneler: kısmi parçalar = yes, Min uzunluğu = 21 kare, İzleme tahmin derinliği = 1 çerçeve izin verin. </li><li> Izleme sürecini başlatmak için, otomatik olarak tüm parçalar Bul Kont / Boyut seçenekleri iletişim kutusunu ve İzleme iletişim kutusunu getirmek için düğmeye işlev tıklayın. Analiz için bir solucan nesne vurgulamak ve hafif arka plan pikselleri görmezden solucan piksel gri ölçekli yoğunluğunu kullanan önemli eşik adım sağlar Kont / Boyutu iletişim kutusunda, içinde Intensity Range Seçimi Manuel seçeneğini seçin. <ol><li> Yoğunluk eşik SLI ayarlayınders, tüm karanlık nesneleri vurgular kapsayıcı bir dizi oluşturmak için (bir alt sınırını ayarlamak için, bir üst sınırını ayarlamak için). 0-4,095 bir ölçekte 0-1,500 bir dizi ince ayar için iyi bir başlangıç ​​noktasıdır. </li><li> Bakır halka ve plakalar üzerinde herhangi bir enkaz gibi tek bir solucan nesnesi daha büyük ve daha küçük nesneleri, dışlamak için bir boyut filtresini uygulayın. Tedbir ile duruşlar çeşitli bireysel solucanlar için boyutları kapsayan bunu yapmak için iki boyut parametrelerini ayarlayın&gt; Kont / Boyut seçenekleri iletişim kutusunda seçin Ölçümler menü öğesi. (Alan aralığı: 28,000-120,000 mikron 2 ve Çevre aralığı: 600-2,500 um). </li><li> Bir mutant suş analiz edilecek plaka üzerinde önemli ölçüde daha küçük ya da diğer hayvanlara göre daha büyükse ayarları değiştirilebilir olması gerekmez, böylece ilk hayvanları izleme önce suşları farklı boyutlarda uyum nesne tanıma için filtre ayarları aralığını genişletmek orta analizi. </li></ol> &lt;/ Li&gt; <li> Takip iletişim kutusunda Devam tıklayarak izleme işlemini tamamlayın. Görme otomatik filtre ayarlarına göre bunu dışlamak için geçerli bir neden olmadıkça, her solucan temsil sağlamak için filmde her solucanın ilerleme çıkış parçaları karşılaştırın. Manuel boyutu filtre kriterlerini doğruladı olmayan solucan nesnelerin varlığı ile üretilen parçaları silin. </li><li> Her çerçeve arasındaki her solucanın hızını hesaplamak için yazılımını kullanın (mesafe kareler arasında 1 saniyede bir nesnenin sentroidinden için gitti) ve solucanlar nüfus için tüm parçalar arasında her parça için ortalama hız ve ortalama hızı görüntüler bakır halka. Bu son ortalama deneysel çalışmalarda açısından n = 1 olmak düşünün. İstatistiksel analizler ve veri arşivleme için bir elektronik tablo programı veri ihracat. </li><li> Veri plakasında ilk halka kaydedildi edildikten sonra, bir sonraki halkaya yatırım getirisini taşımak veparametrelerden birini değiştirmeden, izleme işlemini tekrarlayın. </li><li> Lokomosyon ile göreceli hız hesaplayın: </li><li> Bağıl hız (%) = işlenmiş işlenmemiş (400 mM) hız / (0 mM) hız x 100. NOT: Farklı genetik manipülasyonlar genellikle hayvanların bazal lokomosyon oranını değiştirmek. Hayvanların hareket hızına etanol etkiyi tespit etmek ve farklı genotipler ve koşullar arasında, bu etkilerini karşılaştırmak mümkün olması için, göreceli hızını hesaplar. </li></ol></li></ol>

<ol>
	<li>Prescott, C. A., Kendler, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+environmental+contributions+to+alcohol+abuse+and+dependence+in+a+population-based+sample+of+male+twins.">Genetic and environmental contributions to alcohol abuse and dependence in a population-based sample of male twins.</a> Am. J. Psychiatry. 156, 34-40 (1999).</li><li>Schuckit, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+the+risk+for+alcoholism.">Genetics of the risk for alcoholism.</a> Am. J. Addict. Article Review. 9, 103-112 (2000).</li><li>Heath, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+differences+in+alcohol+sensitivity+and+the+inheritance+of+alcoholism+risk.">Genetic differences in alcohol sensitivity and the inheritance of alcoholism risk.</a> Psychol. Med. 29, 1069-1081 (1999).</li><li>Rodriguez, L. A., Wilson, J. R., Nagoshi, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+psychomotor+sensitivity+to+alcohol+predict+subsequent+alcohol+use.">Does psychomotor sensitivity to alcohol predict subsequent alcohol use.</a> Alcohol. Clin. Exp. Res. 17, 155-161 (1993).</li><li>Schuckit, M. A., Smith, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+8-year+follow-up+of+450+sons+of+alcoholic+and+control+subjects.">An 8-year follow-up of 450 sons of alcoholic and control subjects.</a> Arch. Gen. Psychiatry. 53, 202-210 (1996).</li><li>Kalu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritability+of+level+of+response+and+association+with+recent+drinking+history+in+nonalcohol-dependent+drinkers.">Heritability of level of response and association with recent drinking history in nonalcohol-dependent drinkers.</a> Alcohol. Clin. Exp. Res. 36, 522-529 (2012).</li><li>Davis, S. J., Scott, L. L., Hu, K., Pierce-Shimomura, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+single+residue+in+the+BK+potassium+channel+required+for+activation+by+alcohol+and+intoxication+in.">Conserved single residue in the BK potassium channel required for activation by alcohol and intoxication in.</a> C. elegans. J. Neurosci. 34, 9562-9573 (2014).</li><li>Raabe, R. C., Mathies, L. D., Davies, A. G., Bettinger, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Omega-3+Fatty+Acid+Eicosapentaenoic+Acid+Is+Required+for+Normal+Alcohol+Response+Behaviors+in+C.+elegans.">The Omega-3 Fatty Acid Eicosapentaenoic Acid Is Required for Normal Alcohol Response Behaviors in C. elegans.</a> PLoS ONE. 9, e105999 (2014).</li><li>Topper, S. M., Aguilar, S. C., Topper, V. Y., Elbel, E., Pierce-Shimomura, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+disinhibition+of+behaviors+in+C.+elegans.">Alcohol disinhibition of behaviors in C. elegans.</a> PLoS ONE. 9, e92965 (2014).</li><li>Bhandari, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+intracellular+channels+modulate+acute+ethanol+behaviors+Drosophila,Caenorhabditis+elegans+and+mice.">Chloride intracellular channels modulate acute ethanol behaviors Drosophila,Caenorhabditis elegans and mice.</a> Genes Brain Behav. 11, 387-397 (2012).</li><li>Davies, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+central+role+of+the+BK+potassium+channel+in+behavioral+responses+to+ethanol+in+C.+elegans.">A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans.</a> Cell. 115, 655-666 (2003).</li><li>Davies, A. G., Bettinger, J. C., Thiele, T. R., Judy, M. E., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+variation+in+the+npr-1+gene+modifies+ethanol+responses+of+wild+strains+of+C.+elegans.">Natural variation in the npr-1 gene modifies ethanol responses of wild strains of C. elegans.</a> Neuron. 42, 731-743 (2004).</li><li>Kapfhamer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+RAB-3/A+in+Caenorhabditis+elegans+and+the+mouse+affects+behavioral+response+to+ethanol.">Loss of RAB-3/A in Caenorhabditis elegans and the mouse affects behavioral response to ethanol.</a> Genes Brain Behav. 7, 669-676 (2008).</li><li>Speca, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+role+of+unc-79+in+ethanol+responses+in+lightweight+mutant+mice.">Conserved role of unc-79 in ethanol responses in lightweight mutant mice.</a> PLoS Genet. 6, e1001057 (2010).</li><li>Thiele, T. E., Badia-Elder, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+neuropeptide+Y+in+alcohol+intake+control:+evidence+from+human+and+animal+research.">A role for neuropeptide Y in alcohol intake control: evidence from human and animal research.</a> Physiol. Behav. 79, 95-101 (2003).</li><li>Treistman, S. N., Martin, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BK+Channels:+mediators+and+models+for+alcohol+tolerance.">BK Channels: mediators and models for alcohol tolerance.</a> Trends Neurosci. 32, 629-637 (2009).</li><li>Han, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+GWASs+and+human+protein+interaction+networks+identifies+a+gene+subnetwork+underlying+alcohol+dependence.">Integrating GWASs and human protein interaction networks identifies a gene subnetwork underlying alcohol dependence.</a> Am. J. Hum. Genet. 93, 1027-1034 (2013).</li><li>Kendler, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomewide+association+analysis+of+symptoms+of+alcohol+dependence+in+the+molecular+genetics+of+schizophrenia+(MGS2)+control+sample.">Genomewide association analysis of symptoms of alcohol dependence in the molecular genetics of schizophrenia (MGS2) control sample.</a> Alcohol. Clin. Exp. Res. 35, 963-975 (2011).</li><li>Schuckit, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autosomal+linkage+analysis+for+the+level+of+response+to+alcohol.">Autosomal linkage analysis for the level of response to alcohol.</a> Alcohol. Clin. Exp. Res. 29, 1976-1982 (2005).</li><li>Alaimo, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+metabolism+and+osmolarity+modify+behavioral+responses+to+ethanol+in+C.+elegans.">Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. elegans.</a> Alcohol. Clin. Exp. Res. 36, 1840-1850 (2012).</li><li>Morgan, P. G., Sedensky, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+sensitivity+to+ethanol+in+the+nematode,+Caenorhabditis+elegans.">Mutations affecting sensitivity to ethanol in the nematode, Caenorhabditis elegans.</a> Alcohol. Clin. Exp. Res. 19, 1423-1429 (1995).</li><li>Mitchell, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+concentration-dependent+effects+of+ethanol+on+Caenorhabditis+elegans+behaviour.">The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour.</a> Pharmacogenomics J. 7, 411-417 (2007).</li><li>Vidal-Gadea, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+selects+distinct+crawling+and+swimming+gaits+via+dopamine+and+serotonin.">Caenorhabditis elegans selects distinct crawling and swimming gaits via dopamine and serotonin.</a> Proc. Natl. Acad. Sci. U S A. 108, 17504-17509 (2011).</li><li>Bettinger, J. C., Leung, K., Bolling, M. H., Goldsmith, A. D., Davies, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+environment+modulates+the+development+of+acute+tolerance+to+ethanol+in+Caenorhabditis+elegans.">Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans.</a> PLoS ONE. 7, e35129 (2012).</li><li>Davies, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+genes+influence+toluene-+and+ethanol-induced+locomotor+impairment+in+C.+elegans.">Different genes influence toluene- and ethanol-induced locomotor impairment in C. elegans.</a> Drug Alcohol Depend. 122, 47-54 (2012).</li><li>Newlin, D., Thomson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+challenge+with+sons+of+alcoholics:+a+critical+review+and+analysis.">Alcohol challenge with sons of alcoholics: a critical review and analysis.</a> Psychol. Bull. 108, 383-402 (1990).</li><li>Ponomarev, I., Crabbe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+assess+initial+sensitivity+and+acute+functional+tolerance+to+hypnotic+effects+of+ethanol.">A novel method to assess initial sensitivity and acute functional tolerance to hypnotic effects of ethanol.</a> J. Pharmacol. Exp. Ther. 302, 257-263 (2002).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook. , (2012).</li></ol>

The authors have nothing to disclose.

C. mevcut basit nörobiyoloji ve genetik araçlar elegans solucan davranışı üzerinde etanol etkilerinin moleküler temellerini incelemek için hangi mükemmel bir model olun. Burada, 8,10,20,24,25 etanol akut davranış tepkisinin farklı moleküler ve çevre mediatörleri tanımlamak için kullanılan bir analiz tanımlamaktadırlar. Bu yöntem, bu arada, memelilerde tepki seviyesine bileşik fenotip modeli, farklılaşması ve iki farklı etanol tepki davranışı fenotipleri ilk duyar...

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. This LR phenotype is influenced by genetics and non-genetic components6. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.
The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals10-16, and have been implicated in abuse liability in humans17-19. 
Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming9,23, as well as a role for the environmental variable osmolarity in ethanol responses20, and have highlighted the importance of describing experimental detail of the various assays.
An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C. elegans strains</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 x15 mm Petri plates, triple vented</td>
			<td>Greiner Bio-One</td>
			<td>628161</td>
			<td>Other plate brands will suffice.</td>
		</tr>
		<tr>
			<td>NGM agar</td>
			<td>Various</td>
			<td></td>
			<td>NaCl (3g/L), agar (17g/L), peptone (2.5g/L), 1 mL cholesterol (5mg/mL in ethanol), 1 mL (1M) MgSO4, 1 mL (1M) CaCl2, 25 mL (1M) KPO4, pH=6, 975 mL H2O</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Various</td>
			<td></td>
			<td>e.g. Fisher Scientific #10300</td>
		</tr>
		<tr>
			<td>37&#176;C Incubator</td>
			<td>Various</td>
			<td></td>
			<td>For drying agar</td>
		</tr>
		<tr>
			<td>Digital balance</td>
			<td>Various</td>
			<td></td>
			<td>For determining plate weights and agar volume</td>
		</tr>
		<tr>
			<td>Copper rings</td>
			<td>Plumbmaster</td>
			<td>STK#35583 (48 cap thread gasket)</td>
			<td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>QImaging</td>
			<td>RET-4000R-F-M-12</td>
			<td>This camera has a large field of view.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with C-mount and 0.5X objective</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td>Discontinued model, M60 is current equivalent.</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Schott</td>
			<td>A08923</td>
			<td>3&#8221;x3&#8221;&#160; backlight for even illumination across the field of view</td>
		</tr>
		<tr>
			<td>Imaging and tracking software</td>
			<td>Media Cybernetics</td>
			<td>ImagePro-Plus v6.0-6.3</td>
			<td>Newer versions of the software have tracking functions.</td>
		</tr>
	</tbody>
</table>

an assay for measuring the effects of ethanol on the locomotion speed of caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

Birkaç farklı genotipleri ve eşleştirilmiş kontrollerden Temsilcisi verileri (Şekil 1) 8,24 sunulmuştur; veri özel tahlil hayvanlarda bu vurgu farklılıkları seçildi. maruziyet 10 dakika sonra etki derecesi Şekil 1B-G sol eksen üzerinde gösterilen bir soyu, ilk duyarlılık olarak kabul edilir. 10 dakika sonra kontrole göre daha büyük bir göreceli hızla mutant suşları, etanol (Şekil 1F, G) karşı dirençli olduğu kabul ederken ...

Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Locomotion Speed ​​Etanol Etkilerinin Ölçülmesi için bir Testi<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

an-assay-for-measuring-effects-ethanol-on-locomotion-speed

Research

JoVE Journal

Neuroscience

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

8.5K Görüntüleme.  Virginia Commonwealth University. C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay. 

Video: An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Gaz kromatografisi ile operant etanol kendi kendine uygulama ve etanol belirleme sırasında etanol mikrodivaliz

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Nörobilim

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

Zebra balığı Embriyonik nöroepitelyumda geçirgenliği için bir Assay

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

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 ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Antipsikotik İlaçların Etki Mekanizmaları incelenmesi için Yöntemleri<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Etanol sedasyon Hassasiyet ve hızlı Hoşgörü Ölçüm Ucuz bir, Ölçeklenebilir Davranış Deneyi<em&gt; Drosophila</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Uzun süreli Kalsiyum Görüntüleme için Agaroz microchambers<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Roman Metrik Nematodu Embriyonik Uzama karakterize etmek<em&gt; Caenorhabditis elegans</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Recording Hücre içi Gerilim Yanıtları için Elektrofizyolojik Yöntem<em&gt; Drosophila</em&gt; Işık Uyaranların için Fotoreseptörler ve internöronlar<em&gt; İn Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

bir<emCaenorhabditis elegans</emBeslenme Durumuna Dayalı Bakır Kaçınma Deneyi

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Ratiometric kalsiyum görüntüleme Caenorhabditis Elegans davranışlar içinde bireysel nöronların

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Locomotion Speed ​​Etanol Etkilerinin Ölçülmesi için bir Testi

Locomotion Speed Etanol Etkilerinin Ölçülmesi için bir Testi