Circadian Structural Plasticity in Central Pacemakers

中央起搏器的昼夜节律结构可塑性

• 批准号: 10409826
• 负责人: Maria Fernanda Ceriani
• 金额: $ 51.45万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10409826
• 关键词: 3-Dimensional; ARNTL gene; Address; Adhesions; Adult; Animals; Argentina; Astrocytes; Automobile Driving; Behavior; Behavioral; Biological Models; Brain; Calcium; Cell Communication; Cells; Circadian Rhythms; Confocal Microscopy; Country; Coupled; Cues; Drosophila genus; Electron Microscopy; Enterobacteria phage P1 Cre recombinase; Face; Feedback; Fiber; Flavoproteins; Genetic; Genetic Transcription; Hypothalamic structure; Immunohistochemistry; Impairment; Injections; Label; Mammals; Mediating; Molecular; Motor Activity; Mus; Neurites; Neuroglia; Neurons; Neuropeptides; Neurotransmitters; Organism; Pacemakers; Periodicity; Photoperiod; Physiological; Pigments; Postdoctoral Fellow; Principal Investigator; Process; Property; Reporter; Reporting; Research; Scanning Electron Microscopy; Signal Transduction; Sleep Wake Cycle; Slice; Structure; Students; Synapses; Synaptophysin; System; Techniques; Testing; Time; Tissues; Training; Translations; Vasoactive Intestinal Peptide; Viral; Virus; Visualization; circadian; circadian pacemaker; density; ex vivo imaging; experience; experimental study; fly; gene product; immunocytochemistry; knock-down; light microscopy; longitudinal analysis; mouse Cre recombinase; neuronal circuitry; polymerization; postsynaptic; presynaptic; promoter; receptor; reconstitution; recruit; response; suprachiasmatic nucleus; tool; vector

SUMARY Circadian rhythms depend on the molecular transcription/translation negative feedback loop (TTL) operating in clock neurons, and on the network properties of these neurons. Among the properties that could be recruited by the circadian clock are changes in the identity of pre/post synaptic partners and/or strength of the connectivity between clock neurons, a property collectively termed as circadian structural plasticity. Our central hypothesis is that circadian structural plasticity within the central circadian clocks of mammals and Drosophila are part of the time-encoding mechanisms. We will employ mouse and fly genetics combined with state-of-the- art quantitative 3D light and electron microscopy techniques to address the extent of structural plasticity within specific neurons of the mouse suprachiasmatic nucleus (SCN) and the Drosophila circadian network. Specific aim 1 will assess how widespread structural plasticity is in the Drosophila circadian network as well as which are the functional consequences of those structural changes. We will explore the extent of circadian neuronal remodeling of subsets of PDF and non-PDF pacemaker neurons using CM and SBEM (sub-aims 1A i and ii). We will examine time-of-day dependent functional connectivity changes among clock neurons through chemogenetic GCamP6-reporting (sub-aim 1B). Sub-aim 1C will examine the behavioral consequences of impairing structural remodeling; sub-aim 1D will further investigate the molecular mechanisms underlying circadian structural plasticity. Specific aim 2 will examine the degree of circadian structural remodeling in SCN VIPergic neurons, which are an essential component of the timekeeping mechanism, through virally mediated sparse-labeling (CM) (sub- aim 2A), or serial block-face scanning electron microscopy (SBEM) with a marker that enables the analysis of dendritic ultrastructure (sub-aim 2C). Finally, we will assess if circadian oscillations in VIP neuronal processes rely on the TTL by repeating experiments in 1A in VIP-specific Bmal1-/- mice (sub-aim 2B). Specific aim 3 will explore if connectivity of VIPergic neurons changes throughout the 24-h cycle. Using GFP reconstitution across synaptic partners (GRASP), we will investigate if these connections change with circadian time through immunocytochemistry and CM analysis in fixed tissue (sub-aim 3A) as well as ex vivo in SCN slices (sub- aim 3B). We will also determine whether GRASP-detected rhythms depend on the canonical TTL by repeating experiments in 2A and 2B in VIP- or SCN astrocyte-specific Bmal1-/- mice (sub-aim 3C). Our experiments test predictions of the hypothesis that circadian structural plasticity represents a defining feature of central neuronal circadian pacemakers. Support for this hypothesis would provide a critical new perspective to understand how these pacemakers encode time at the network level. Furthermore, the experiments we propose represent a unique opportunity for research capacity building in Argentina, where the foreign principal investigator is located, and where students and postdocs will be trained in techniques that are still not fully developed in that country.

SUMARY