Çoklu Zebra balığı için İlaç İdaresi Bir Roman Yöntemi (<em> Danio rerio</em>) Ve Çekilme Niceleme
Summary
A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.
Abstract
Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed.
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.
Introduction
Zebra balığı (Danio rerio) davranışsal 1 ve tıbbi araştırma 2,3 için yararlı bir model organizmadır Hindistan'da menşeli küçük bir teleost türdür. Balığı, sık davranışı üzerindeki etkilerini karakterize etmek amacıyla, çeşitli farmakolojik maddeler test kullanılır. Çeşitli dozlarda ve ilaç programları gibi uyarıcılar 4, anksiyolotikler 5 ve etanol 6-8 gibi bileşiklerin uygulanmasından sonra zebrafish davranışlarını araştırmak için kullanılmıştır.
Bizim laboratuvar da yaygın scototaxic deneyi olarak adlandırılan iyi valide açık / koyu tahlil 9, 20, zebrafish anksiyete ve lokomosyon etanol idarenin programları farklı etkilerini araştırmıştır. Etanol uygulama yeni bir yöntem uzun bir zaman periyodu (21 gün) üzerinde 6 tekrarlayan, günlük uygulama için verimliliğini artırmak için geliştirilmiştir </s> kadar. Daha önce kullanılan yöntemler ancak, biz ilişkili zaman maliyetleri ile, örgü azaltılmış bir yöntem geliştirmek için çalıştı, ve balık çok sayıda ilgi ilacın eşzamanlı, tam zamanlı yönetimini izin, pratik. Geleneksel araştırma kullanılarak, etanol içindeki, zebra balığı halinde ve etanol ve su 12/10 ve uygun bir karışım ihtiva eden başka bir tanka aktarılır. Bu yöntem yaygın olarak kabul iken, Zebra balığı örgü tanıtmak ve ilaç çözeltisinden balık kaldırmak için alınan zaman içinde değişkenlik artabilir. Bu nedenle, söz konusu bileşik için tam maruz tekrarlanan doz içeren bir deney boyunca değişebilir. Taşıma zamanlarda değişkenlik kaynaklanan hata kaynaklarını azaltan bir yöntem arzu edilir. Bizim yöntemi ile her balık özdeş dozlama süresi sonuçlanan, aynı anda tüm balık hareket edebiliyoruz. (Burada anlatıldığı gibi), etanol maruz kalmasını takiben, zebra balığı bir numbe test edilebilirkaygıyı ölçmek de dahil olmak üzere davranışsal deneyleri, r. Yeni yöntemi kullanarak balık grupları dozaj doğru çoğaltmak ve balık grupları konular arasında ve genelinde dozlama standardize etmek yeteneği ötesinde pratik kullanımları vardır. Araştırmacılar kendi deneylerde tekrarlanabilirliği ve doğruluğunu sağlamak için yöntemler kullanabilir görebilirsiniz seferde birden fazla balık takibi için izin verir, yeni yazılım gelişi. Davranışsal sinirbilim için bir model organizma olarak zebrafish yaygın kullanımı göz önüne alındığında, bu yöntem gelecekte farmakolojik çalışmalarda verimlilik ve pratiklik artacak.
Mevcut paradigmada, tekrarlanan doz çizelgesi yaklaşık insan içme programları yansıttığının istihdam edildi. Kontrolü, günlük-orta, ya da haftalık-alem: Balık rastgele bir üç gruba ayrıldı. Dozlama çizelgesi önemli ölçüde önceki çalışmalarda 7 pozlama süreleri aşması nedeniyle seçilmiş, süresi 21 gün idi. Kontrol balık sıfır Alco aldıhol, günlük-orta balık günde bir kez% 0.2 alkol aldı ve haftalık-tıkınırcasına balık haftada% 1.4 alkol kez aldı. Açık / koyu bir görev geri çekilmesinden 2 gün sonra anksiyete değerlendirmek için kullanıldı. Bu bir tarafta duvar beyaz ve diğer tarafta 9 karanlık olan bir dikdörtgen arena kullanan yönetmek için basit bir testtir. Yetişkin Zebra balığı sağlam kontrol koşulları 6,9,13 altında arena karanlık tarafını tercih ederim. Artan anksiyete operasyonel karanlık bölgede harcanan önemli ölçüde daha fazla zaman olarak tanımlanan ve balık ışık bölgesi harcanan nispeten daha fazla zaman harcıyor zaman anksiyete kabul edilebilir azalmıştır. Hareket-izleme yazılımı ile, diğer bilgilendirici değişkenler de ortalama hız, hareketsizlik, kıvrımlı dahildir, miktarı ve bölge 14 geçişler olabilir.
Laboratuvarda geliştirilen dozajlama metodu suda çözünür bileşikler, bir veya daha fazla zebrafi için tatbik edilen herhangi bir araştırma için geçerli olabilirsh. Bu metodoloji yararlanabilecek diğer birçok farmakolojik ajanlar da zebrabalıkları test ediliyor. Genel olarak test bileşikleri, etanol benzer bir şekilde içinde çözülür nikotin, klordiazepoksid, buspiron ve skopolamin, yer alır; su içine kimyasal uygun bir miktarda karıştırarak. Bu nedenle, bu işlemin genel kapsamı çok daha geniş ve etanol ile sınırlı değildir. Ayrıca, birden fazla gün boyunca ilaç verilmesinden sonra, açık / koyu bir görev kullanılabilirdi birçok davranış testleri sadece bir tanesidir. İlaç uygulamasından sonra veya çekilme sırasında, kullanılabilir diğer popüler deneyler yeni tankı dalış testi 15 ve 16 shoaling gibi sosyal davranış testlerini kapsamaktadır. Aşağıdaki prosedür tekrar tekrar ilgi farmakolojik bileşiği içeren çözümler balık ya da bireysel balık gruplarının nakledilmesi etkili bir yöntem açıklayacağım. Buna ek olarak, ışık / karanlık testi ile test anksiyete süreciAlkol tatbikat uzun programları maruz bırakıldıktan sonra geri çekme olan balık gruplar olarak tarif edilecektir.
Protocol
Representative Results
Discussion
Zebra balığı ilaç verilmesini içeren önceki çalışmalarda sadece ilaç solüsyonu 12,16 içine kendi tankından onları taşımak için balık filelere dayanmıştır. Netleştirme her zaman tutarlı değildir ve genellikle nedeniyle önemli bireysel değişkenlik zebrabalıkları, bir kaçış tepkisi beklenenden daha uzun sürer. Geleneksel transfer yöntemleri, yararlı ise de, balık, su dışında geçirmek toplam süreyi azaltarak, hem de hayvanlar arasındaki aktarım süresi değişkenlik mikt…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
The authors have nothing to disclose.
Materials
| Three Shelf benchtop housing system | Aquatic Habitats | N/A | http://aquatichabitats.com/research-systems/z-hab-system/ |
| 1.5L Spawning tank w/400 micron baffle | Aquatic Habitats | N/A | http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/ |
| Pure Grain Ethanol | Luxco, INC | N/A | http://www.luxco.com/public/brands/brands.asp?brandid=21 |
| Ethovision XT Motion tracking software | Noldus Information Technology | http://www.noldus.com/animal-behavior-research/products/ethovision-xt | |
| Pipette | Eppendorf Canada | http://www.eppendorf.ca/int/index.php?l=211&pb=e396f9e705e754a3&action=products&contentid=1&catalognode=9477. | |
| Light/Dark Arena | Custom | Construct as per procedure description. 9.5cm wide, 9.5cm deep, 55cm long |
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