Dissecting an asymmetric brain area implicated in sleep maintenance

就像大多数人喜欢用右手或左手一样，大多数动物的大脑也不对称，否则它们就会对称。虽然我们知道这些不对称是如何在开发过程中产生的一些细节，但我们仍然发现这些不对称在思考和行为方面的功能重要性。人们对左右不对称的作用知之甚少的一种行为是睡眠。由于睡眠对我们一生中正常的健康和认知功能至关重要，因此了解睡眠调节的潜在组织原则非常重要。在研究斑马鱼的睡眠变化时，有与人类自闭症相关的突变，我们发现了一种新的和意想不到的大脑不对称。具体来说，当正常的斑马鱼开始延长晚上熄灯前的睡眠时间时，缺乏自闭症风险基因chd8的斑马鱼却没有这样做。通过观察斑马鱼幼虫的大脑活动，我们发现缺乏chd8的幼虫只有中脑右侧在晚上有高的大脑活动。这导致在大脑的同一区域进一步发现了一种以前未被报道过的物理不对称。在这个项目中，我们现在计划通过确定它是否遵循与其他不对称大脑区域相同的发展规则来研究这种新的不对称，这种发展如何在斑马鱼自闭症模型中出错，以及这种不对称是否以及如何对睡眠持续时间的调节重要。斑马鱼是研究大脑不对称和睡眠之间联系的一个很好的系统，因为现有的实验工具可以可视化和操纵这两种特性。例如，斑马鱼的突变体和实验操作会导致大脑不对称的改变，例如具有“双左”、“双右”或“反向”大脑结构的动物。我们计划测试这些操作是否也会导致调节睡眠的大脑区域的不对称性的变化，这将为我们提供重要的线索，了解该区域在多大程度上遵循已知的产生不对称的发育规则。我们还将使用斑马鱼昼夜循环的自动视频跟踪，看看不对称的变化是否会影响睡眠持续时间，我们可以通过测量斑马鱼在睡眠状态下保持不活动的时间来评估。利用遗传学，我们还将用荧光蛋白标记这种中脑不对称，以了解该区域如何与大脑的其他部分（如已知的睡眠调节中心）联系起来，观察在该区域选择性表达的分子，并了解缺乏chd8的短睡动物如何破坏该区域的形成。这些实验将告诉我们更多关于这种不对称的结构和分子特性，并将告诉我们该区域如何调节睡眠。最后，我们将通过直接控制这一区域的活动来测试这一不对称区域在睡眠中的作用。例如，我们可以用激光消融有选择地切除这个区域，并询问这个区域是否需要适当的睡眠时间。我们还将使用基因干预，将光或药物诱导的蛋白质，可以驱动神经元兴奋性特异性进入这个不对称区域。这将使我们能够直接控制该区域的活动，使我们能够测试打开或关闭该区域是否能够改变斑马鱼的睡眠。总之，这些实验将使我们对大脑不对称如何调节睡眠时间有新的认识，并使我们了解这种调节在自闭症等人类疾病中是如何改变的。

Just as most humans prefer to use either their right or left hand, asymmetries are built into the brains of most animals that are otherwise symmetrical. While we know some details about how these asymmetries arise during development, we are still discovering the functional importance of these asymmetries on thinking and behavior. One behavior for which little is known about the role of left-right asymmetry is sleep. Since sleep is essential for normal health and cognitive function across our lifespan, understanding the underlying organizing principles of sleep regulation is of great importance.While studying sleep changes of zebrafish that have mutations associated with autism in humans, we discovered a new and unexpected brain asymmetry. Specifically, while normal zebrafish begin to lengthen the time they spend asleep in the evening before lights out, zebrafish lacking the autism risk gene called chd8 fail to do so. Using methods to visualize the brain activity of larval zebrafish, which are optically translucent in early stages, we found larvae that lack chd8 have high brain activity only on the right side of the midbrain during the evening. This led to the further discovery of a previously unreported physical asymmetry in same region of the brain. In this project, we now plan to investigate this new asymmetry by determining whether it follows the same developmental rules as other asymmetric brain regions, how this development may go wrong in zebrafish autism models, and whether and how this asymmetry is important for the regulation of sleep duration. The zebrafish is an excellent system in which to study the links between brain asymmetries and sleep because of existing experimental tools to visualize and manipulate both properties. For example, there are zebrafish mutants and experimental manipulations that lead to altered brain asymmetries, such as animals with "double-left", "double-right" or "reversed" brain structures. We plan to test whether these manipulations also lead to changes in the asymmetry of this sleep regulating brain area, which will give us important clues about the extent to which this area follows known developmental rules for the creation of asymmetries. We will also use automated videotracking of zebrafish across the day-night cycle to see if changes in asymmetry affect sleep durations, which we can assess by measuring how long the zebrafish stay inactive in sleep states.Using genetics, we will also label this midbrain asymmetry with fluorescent proteins to see how this area connects with the rest of the brain such as known sleep-regulating centers, to observe what molecules are selectively expressed in this region, and to understand how the formation of this area is disrupted in the short-sleeping animals that lack chd8. These experiments will tell us more about the structural and molecular properties of this asymmetry and will inform how the area may be regulating sleep.Finally, we will test the role of this asymmetric area in sleep by taking direct control of the activity of this area. For example, we can selectively remove this area using laser ablation and ask whether this area is required for proper sleep durations. We will also use genetic interventions to put light- or drug-inducible proteins that can drive neuronal excitability specifically into this asymmetric area. This will give us direct control over the activity of this region, allowing us to test whether turning the area on or off is capable of altering zebrafish sleep. Together these experiments will give us a new understanding of how brain asymmetries regulate sleep duration and knowledge about how this regulation might be altered in human disorders such as autism.