Department of Neurology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
Sleep. 2013 May 1;36(5):689-698G. doi: 10.5665/sleep.2628.
To develop a method, called Caenorhabditis-in-Drop (CiD), encapsulating single worms in aqueous drops, for parallel analysis of behavioral quiescence in C. elegans nematodes.
We designed, constructed, and tested a device that houses an array of aqueous droplets laden with individual worms. The droplets are separated and covered by immiscible, biocompatible oil. We modeled gas exchange across the aqueous/oil interface and tested the viability of the encapsulated animals. We studied the behavior of wild-type animals; of animals with a loss of function mutation in the cGMP-dependent protein kinase gene egl-4; of animals with a loss of function mutation in the gene kin-2, which encodes a cAMP-dependent protein kinase A regulatory subunit; of animals with a gain-of-function mutation in the gene acy-1, which encodes an adenylate cyclase; and of animals that express high levels of the EGF protein encoded by lin-3.
We used CiD to simultaneously monitor the behavior of 24 worms, a nearly 5-fold improvement over the prior best methodology. In support of our gas exchange models, we found that worms remain viable on the chip for 4 days, past the 12-h period needed for observation, but show reduced longevity to that measured on an agar surface. Measurements of duration of lethargus quiescence and total leth-argus quiescence showed reduced amounts as well as reduced variability relative to prior methods. There was reduced lethargus quiescence in animals that were mutant for kin-2 and for acy-1, supporting a wake-promoting effect of PKA in C. elegans, but no change in lethargus quiescence in egl-4 mutants. There was increased quiescence in animals that expressed kin-2 in the nervous system or over-expressed EGF.
CiD is useful for the analysis of behavioral quiescence during lethargus as well as during the adult stage C. elegans. The method is expandable to parallel simultaneous monitoring of hundreds of animals and for other studies of long-term behavior. Using this method, we were successful in measuring, for the first time, quiescence in kin-2(ce179) and in acy-2(ce2) mutants, which are hyperactive. Our observations also highlight the impact of environmental conditions on quiescent behavior and show that longevity is reduced in CiD in comparison to agar surfaces.
开发一种名为 Caenorhabditis-in-Drop（CiD）的方法，将单个蠕虫封装在水性液滴中，用于平行分析秀丽隐杆线虫的行为静止。
我们设计、构建和测试了一种装置，该装置容纳了载有单个蠕虫的水性液滴阵列。液滴被不混溶的、生物相容的油分离并覆盖。我们模拟了气体在水/油界面上的交换，并测试了封装动物的生存能力。我们研究了野生型动物的行为；研究了 cGMP 依赖性蛋白激酶基因 egl-4 功能丧失突变的动物；研究了编码 cAMP 依赖性蛋白激酶 A 调节亚基的基因 kin-2 功能丧失突变的动物；研究了编码腺苷酸环化酶的基因 acy-1 功能获得性突变的动物；以及研究了表达高水平 lin-3 编码的 EGF 蛋白的动物。
我们使用 CiD 同时监测了 24 条蠕虫的行为，这比以前最好的方法提高了近 5 倍。为了支持我们的气体交换模型，我们发现蠕虫在芯片上的存活时间超过了观察所需的 12 小时，达到了 4 天，但与在琼脂表面上的寿命相比有所缩短。测量惰性休眠的持续时间和总惰性休眠的持续时间表明，与以前的方法相比，惰性休眠的持续时间和变异性都有所降低。kin-2 和 acy-1 突变体动物的惰性休眠减少，支持 PKA 在秀丽隐杆线虫中促进觉醒的作用，但 egl-4 突变体动物的惰性休眠没有变化。在神经系统中表达 kin-2 或过表达 EGF 的动物中，静止增加。
CiD 可用于分析秀丽隐杆线虫休眠期间和成年期的行为静止。该方法可扩展到同时监测数百只动物的并行，并用于其他长期行为研究。使用这种方法，我们首次成功地测量了 kin-2(ce179)和 acy-2(ce2)突变体的静止，这两种突变体是过度活跃的。我们的观察结果还强调了环境条件对静止行为的影响，并表明与琼脂表面相比，CiD 中的寿命缩短。
