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了内部的时间顺序。昼夜节律紊乱对健康有重大影响，越来越多的证据表明，许多物种的昼夜节律系统中存在性别差异。果蝇具有良好的生物钟特征、神经元网络和高度保守的分子生物钟，是研究性二态在计时中的神经元基础的强大模型系统。这个项目的主要目标是了解果蝇大脑中昼夜节律系统的基本结构的差异是如何导致睡眠/清醒周期中的性别差异以及守时的健壮性的。了解昼夜节律系统中性别二型性的机制基础将有助于我们理解男性和女性如何在复杂的现代环境中受到不同的影响。该项目的教育计划包括一个面向本科生的暑期研究计划，该计划旨在进行涉及连接学、神经遗传学和行为学的研究，使学生能够在生物学、生物信息学和工程学的界面上获得研究经验。此外，社区外展工作将与哥伦比亚大学扎克曼研究所的公共外展项目办公室合作进行。他们将参加一个家庭活动，在周六科学计划的背景下，大脑时钟，以及一个公共活动，重点是在突破科学计划的背景下与纽约的音乐家对话，这是一系列跨学科活动，旨在通过艺术创造公众对科学的参与。生物钟系统为生理和行为的多个方面提供内部时间秩序，允许对环境变化的预期。与哺乳动物一样，研究果蝇昼夜节律的大部分工作都集中在雄性身上。这一建议的主要假设是，睡眠/清醒周期中的性别差异是由大脑计时中心网络属性的差异所调节的。在目标1中，研究小组将检验这样一种假设，即色素分配因子(PDF)/PDF受体(PDFR)途径突变的性别二型性表型是由于PDF信号在时钟神经元网络中的不同作用所致。该团队将使用时程免疫组织化学来比较时钟蛋白和CRISPR-Cas9的表达节奏，以敲除时钟神经元亚群中的PDFR。此外，该团队将使用ATP/P2X2功能连接方法来确定女性的PDFR免疫阳性神经元是否对PDF介导的晨间细胞输出反应较差。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，扰乱男性和女性特定时钟类别中的分子振荡，来测试晨光振荡器对男性计时神经元网络的更大影响的假设。在目标3中，研究小组将描述背侧神经元1后部(DN1p‘s)和晨光振荡器之间结构和功能连接模式的性别差异。该团队将比较DN1p与晨间和晚间细胞的连接强度，并测试DN1p对侧时钟神经元的谷氨酸能抑制在男性比女性更明显的假设，分析对沐浴谷氨酸的生理反应。总而言之，这些研究将促进我们对女性和男性睡眠/清醒周期差异的机制基础的理解。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.