A HIGH-THROUGHPUT ASSAY FOR GENETIC STUDIES OF SLEEP

唤醒状态是全球神经系统功能的一个基本方面；然而，由于脑电分析的困难，在基因改变的小鼠中很少进行睡眠和清醒的测量。我们建议开发一种高通量的替代小鼠脑电记录的方法，这将极大地促进睡眠和清醒的测量。我们的系统使用一个简单的压电式换能器，包括动物休息的柔性地板。通过呼吸或其他运动使地板膨胀会产生电信号。在慢波睡眠期间，有节奏的呼吸会产生一致的、规则的电信号模式。快速眼动睡眠由于呼吸更不规律而产生不太一致的信号，而清醒时由于各种运动而产生极不规律的模式。因此，可以准确地确定所有三种警戒状态。我们已经在幼鼠身上验证了这一系统。由于不需要手术，而且信号模式比脑电产生的信号模式更简单，因此高通量进行觉醒状态表型鉴定的技术将相对容易。对我们的系统的直接测试和使用将是调查构成睡眠参数的基因。双胞胎研究、领养研究和对近交系小鼠的分析表明，与睡眠相关的特征的变异在很大程度上是由基因决定的。具体地说，我们建议通过将我们的压电记录与传统的脑电记录进行比较，在常见的近交系小鼠身上验证我们的系统。根据我们之前的脑电数据，我们希望证实AKR/J和DBA/2J近交系之间存在巨大的睡眠差异。随后将对AKD2F1杂交种和AKXD共生自交系进行检测。对这些数据的QTL分析应该确定在这两个品系中导致睡眠特征可变性的候选基因。利用这个高通量系统，我们还将能够从这些或其他菌株之间适当的遗传杂交中检测大量的后代，以确认推测的QTL。该系统还使大规模筛选突变小鼠变得更加可行，我们将在拟议的研究中开始检查这类小鼠。总体而言，我们提出的项目将提供神经系统功能的重要指标，特别是将允许通过基因方法了解睡眠和睡眠障碍，这是现有技术无法实现的。

Arousal states are a fundamental aspect of global nervous system function; yet, measures of sleep and wake are rarely performed in genetically altered mice due to the difficulty of EEG analysis. We propose to develop a high-throughput alternative to EEG recordings in mice that will greatly facilitate the measurement of sleep and wake. Our system utilizes a simple piezoelectric transducer comprising the flexible floor upon which the animal rests. Distension of the floor by respiratory or other movements produces electrical signals. During slow wave sleep, rhythmic breathing results in a consistent, regular pattern of electical signals. REM sleep produces a less consistent signal due to more irregular breathing, while wake produces a dramatically irregular pattern caused by a variety of movements. Therefore, all three vigilance states can be accurately determined. We have already validated this system in young rats. Since no surgery is required and the signal patterns are simpler than those produced by EEG, the technology to perform high-throughput phenotyping of arousal states will be relatively easy. An immediate test and use of our system will be to investigate genes that underlie sleep parameters. Twin studies, adoption studies, and the analysis of inbred strains of mice have demonstrated that variation in sleep related traits are largely determined by genotype. Specifically, we propose to validate our system in common inbred strains of mice by comparing our piezo recordings with traditional EEG recordings. Based on our prior EEG data, we expect to confirm large sleep differences between AKR/J and DBA/2J inbred strains. This will be followed by examination of AKD2F1 hybrids and AKXD recominant inbred lines. QTL analysis of these data should identify candidate loci that underlie the variability of sleep traits in these two strains. With this high throughput system we will also be able to examine large numbers of progeny from appropriate genetic crosses between these or other strains to confirm putative QTLs. This system also makes large scale screening of mutagenized mice far more feasible, and we will begin to examine such mice in the proposed study. In general, the project we propose will provide an important index of nervous system function, and specifically will allow genetic approaches towards understanding sleep and sleep disorders that would not be possible with existing technology.