Over the past decade, circadian biology has made extraordinary progress in understanding the molecular mechanisms underlying the mammalian circadian clock. Yet we are only beginning to explore how the biological clock in the intact animal relies on yet another, higher level of organization: the complex interactions among the clocks in different organs. To do so will require new technical and theoretical approaches. Here, we propose the development and initial application of a method to record circadian rhythms of individual organs from intact animals. Our method will benefit from several recent advances. 1) The recent development of a CRE-LOX- based method will allow us to restrict the PER2::LUC signal to target tissues. 2) The ongoing development of an apparatus for the in vivo measurement will allow us to record from multiple animals. 3) Our preliminary experiments show that we can deliver luciferin via drinking water and thereby record circadian signals in vivo without implantation surgery. Our immediate goal is to optimize these methods. We must first explore the kinetics and dose- dependence of the response to luciferin and various modes of delivery. Second, we will test luciferin analogs with higher signal strength and longer half-life, and will determine the optimal wavelength for light emission. We will search for the optimal balance among all these competing factors that will yield the highest signal-to-noise ratios for signals arising from the liver. We will then optimize the technique such that signal from brain cells can be detected, a goal supported by luciferin analogs with improved blood-brain barrier permeability and/or brain-selective emission. As a first application of the technology, we will explore the entrainment dynamics of Albumin-CRE (liver) directed bioluminescence by light cycles. Undergraduate students at the largest US women's college, Smith College, will conduct this research. Through a multifaceted program with documented success, we will recruit 1st-year students from under-represented minority groups and engage them in hands-on scientific research, with engaged mentorship. Student research teams will include majors with strong quantitative training (e.g., statistics and data science, engineering) and results will be shared via our lab site on the Open Science Framework. Students will be involved in all aspects of the research and will present results at conferences and contribute to publications.
This research addresses development of methods for recording bioluminescence from behaving freely moving animals. We will determine if we can reliably measure circadian rhythms from the liver using a transgenic mouse and newly developed substrate molecules and imaging equipment. This research might help us develop better advice for people with jobs or diseases that disrupt daily rhythms, a risk factor for metabolic and cardiovascular diseases.
