CAREER: Elucidating the Neuronal Basis of Sexual Dimorphism in the Drosophila Circadian System

昼夜节律系统为动物的生理和行为提供内部时间顺序。昼夜节律紊乱对健康有着重要的影响，越来越多的证据表明，许多物种的昼夜节律系统存在性别差异。果蝇是一个功能强大的模型系统，研究两性异形的神经基础，在计时，由于其良好的特征性昼夜节律钟神经元网络和高度保守的分子昼夜节律钟。该项目的主要目标是了解果蝇大脑中昼夜节律系统的基础架构差异如何成为睡眠/觉醒周期性别差异和计时鲁棒性的基础。了解昼夜节律系统中两性异形的机制基础将使我们了解男性和女性如何受到复杂现代环境挑战的不同影响。该项目的教育计划包括一个夏季研究计划，为本科生进行研究，涉及连接组学，神经遗传学和行为，这将使学生获得生物学，生物信息学和工程接口的研究经验。此外，社区外展工作将与哥伦比亚朱克曼研究所的公共外展计划办公室合作进行。他们将参与星期六科学计划背景下的家庭活动，大脑时钟，以及在突破科学计划背景下与纽约市音乐家对话的公共活动，这是一系列旨在通过艺术创造公众参与科学的跨学科活动。昼夜节律系统为生理和行为的多个方面提供内部时间顺序，从而预测环境的变化。和哺乳动物一样，大多数关于果蝇昼夜节律的研究都集中在雄性身上。这个提议的首要假设是，睡眠/觉醒周期的性别差异是由大脑计时中心的网络特性差异介导的。在目标1中，研究小组将测试以下假设：色素分配因子（PDF）/ PDF受体（PDFR）途径突变的性二态表型是由于PDF信号在时钟神经元网络中的差异效应。该团队将使用时程免疫组织化学来比较时钟蛋白和CRISPR-Cas9的表达节律，以敲除时钟神经元子集中的PDFR。此外，研究小组将使用ATP/P2 X2功能连接方法确定女性中的PDFR免疫阳性神经元是否对PDF介导的Morning细胞输出反应较低。在目标2中，研究小组将通过改变特定细胞类型中分子时钟的速度和破坏男性和女性特定时钟类别中的分子振荡来测试晨振子对男性计时神经元网络的影响。在目标3中，研究小组将描述背神经元1后部（DN 1 p）和Morning振荡器之间结构和功能连接模式的性别差异。研究小组将比较DN 1 p与早晨和晚上细胞的连接强度，并测试DN 1 p对侧钟神经元的抑制作用在男性中比女性更明显的假设，分析对浴缸应用谷氨酸的生理反应。总的来说，这些研究将促进我们对女性和男性之间睡眠/觉醒周期差异的机械基础的理解。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

The circadian system provides internal temporal order for an animal’s physiology and behavior. Circadian disruption has significant implications for health, and a growing body of evidence indicates the existence of sex differences in the circadian systems of many species. Drosophila is a powerful model system to study the neuronal basis of sexual dimorphism in timekeeping due to its well-characterized circadian clock neuron network and the highly conserved molecular circadian clock. The main goal of this project is to understand how differences in the underlying architecture of the circadian system in the Drosophila brain underlie sex differences in sleep/wake cycles and robustness in timekeeping. Understanding the mechanistic basis of sexual dimorphism in the circadian system will inform our understanding of how males and females may be differentially affected by the challenges of complex modern environments. The education plan of the project consists of a summer research program for undergraduate students to conduct research involving connectomics, neurogenetics, and behavior that will allow students to gain research experience in the interface of biology, bioinformatics, and engineering. In addition, community outreach efforts will be conducted in partnership with the Columbia Zuckerman Institute’s public outreach programs office. They will involve participation in an event for families, Brain Clocks, in the context of the Saturday Science program, and a public event focused on conversations with New York City-based musicians in the context of the Breakout Science program, a series of interdisciplinary events that aims to create public engagement in science through the arts.The circadian system provides internal temporal order for multiple aspects of physiology and behavior, allowing the anticipation of environmental changes. As in mammals, most of the work on Drosophila circadian rhythms has focused on males. The overarching hypothesis of this proposal is that sex differences in sleep/wake cycles are mediated by differences in the network properties of the brain’s timekeeping center. In Aim 1, the research team will test the hypothesis that the sexually dimorphic phenotypes of the Pigment Dispensing Factor (PDF) / PDF receptor (PDFR) pathway mutations are due to differential effects of PDF signaling in the clock neuron network. The team will use time-course immunohistochemistry to compare expression rhythms of clock proteins and CRISPR-Cas9 to knockout PDFRs in subsets of clock neurons. In addition, the team will determine if the PDFR-immunopositive neurons in females are less responsive to PDF-mediated Morning cell output using the ATP/P2X2 functional connectivity approach. In Aim 2, the team will test the hypothesis that the Morning oscillator has more influence over the male timekeeping neuronal network by changing the speed of the molecular clock in specific cell types and disrupting molecular oscillations in specific clock classes in males and females. In Aim 3, the team will characterize sex differences in structural and functional connectivity patterns between the Dorsal Neurons 1 posterior (DN1p’s) and the Morning oscillator. The team will compare the strength of the DN1p connections to Morning and Evening cells and test the hypothesis that glutamatergic inhibition of the lateral clock neurons by DN1p’s is more pronounced in males than females analyzing physiological responses to bath-applied glutamate. Collectively, the studies will advance our understanding of the mechanistic basis of differences in sleep/wake cycles between females and males.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.