Functional Organization of the Circadian Oscillator
Satchidananda Panda, Ph.D.
The Salk Institute, San Diego, CA
David Mahoney Neuroimaging Program
June 2006, for 3 years
Imaging the Neuronal Works in the Brain’s “Master Clock”
Salk Institute investigators will use molecular imaging in transgenic” mice (containing human cells) to learn how subsets of neurons that comprise the brain’s Master Clock” work to synchronize sleep and wake cycles and numerous other functions, such as metabolism and cardiac output. The findings are anticipated to lead to means to improve adjustments to temporary and more serious long-term rhythm disruptions.
An estimated 20,000 neurons constitute the master oscillator in the brain’s hypothalamic suprachiasmatic nucleus (SCN). The SCN, which is entrained to the ambient light-dark cycle, orchestrates circadian rhythms in physiology and behavior at different phases in diverse brain centers and in peripheral organs, such as the heart. The neuronal workings of this clock are composed of heterogeneous subpopulations. These subsets respond to different stimuli, emit different signals, and use different neurotransmitters to communicate. The Salk researchers hypothesize, therefore, that each neuronal subpopulation carries out specific functions, and that the ways in which the subpopulations are coordinated determines the overall organization of the circadian rhythm system.
In transgenic mice that have been bred to have cells that have fluorescent and luminescent markers, the investigators will use bioluminescence imaging to visualize subsets of cells as they pass on timing information to other neural cell subsets. Thereafter, the researchers will generate a map of clock function in individual neurons and their interactions through by making two changes. First, they will reset the molecular oscillator in some neurons and monitor how this change in timing information is transmitted to the rest of the neurons. Next, they will destroy specific groups of neurons and assess the timing effects on remaining neurons. Through this process, the investigators expect to characterize the circadian function of principal SCN subpopulations and to determine their overall role in establishing and maintaining circadian rhythm.
Significance: The cellular and systems level findings of how circadian rhythm is maintained, and disrupted, may lead to enhanced treatment for sleep and metabolic disorders affected by such disturbances.
Functional Organization of the Circadian Oscillator
A master circadian oscillator present in the hypothalamic suprachiasmatic nucleus (SCN) generates daily rhythms in behavior and physiology. Neurons of the SCN entrain to the ambient day:night cycle, maintain a robust ~24hr molecular rhythm and temporally orchestrate a plethora of behavior and physiology. There is growing evidence that the SCN is composed of heterogeneous group of neurons differing in neurotransmitter content, and expression of oscillator genes.. We hypothesize that different sub-populations of SCN neurons play specific roles in entrainment, rhythm generation and output regulation, and that interactions among these sub-populations determine the overall oscillator function.
Three well characterized SCN molecular markers are arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), and vasoactive intestinal peptide 2 receptor (VPAC2r). Among SCN neuron,s VIP and AVP are expressed in almost mutually exclusive manner, and VAPC2r is detected in a small subset of AVP or VIP expressing neurons. We propose to use molecular genetics and imaging technology to assess the role of major SCN cell types in the overall organization of the master oscillator.
Aim 1. Single-cell circadian oscillator function in AVP, VIP, and VPAC2r expressing SCN neurons. Using fluorescent reporters to tag neurons expressing AVP, VIP, or VPAC2r and luminescence reporter to monitor circadian oscillation, we will assess circadian oscillator function in individual SCN neurons. This will produce a comprehensive spatio-temporal map of SCN oscillators.
Aim 2. Functional interaction among SCN subpopulations. Using specific photoactivation of SCN neurons and monitoring circadian luminescence reporter in individual cells we will assess functional interactions among neurons.
Aim 3. Role of specific SCN cell types in functional organization of the master oscillator. We will use genetic technique to ablate specific SCN neuron types in adult animals and assess circadian oscillator function in whole animal as well as in individual SCN neurons. Such perturbations will determine role of SCN cell types in overall organization of the master circadian oscillator.
Circadian rhythms in mammals are regulated by 10-20,000 neurons constituting a master oscillator in the hypothalamic suprachiasmatic nucleus (SCN). SCN is entrained to the ambient light dark cycle, maintains intrinsic oscillation, and orchestrates circadian rhythms in physiology and behavior at different phases in diverse brain centers and in peripheral organs. SCN is composed of heterogeneous subpopulation of neurons differing in afferent input, efferent outputs, neurotransmitter-, and peptide- contents. Cellular heterogeneity of the SCN raises the hypothesis that SCN sub-populations carry out specific functions and coordination among these neurons determines the overall organization of the circadian system.
The goal of the project is to systematically assess circadian function of principal SCN subpopulations and determine their overall role in functional organization of the SCN oscillator. The specific aims of the project are:
Aim 1: To assess single-cell circadian oscillator function in Arginine vasopressin (AVP), vasoactive intestinal peptide (VIP) and VIP receptor 2 (VPAC2r) expressing SCN neurons.
Aim 2: To delineate functional interaction among SCN subpopulations.
Aim 3: To determine the role of specific SCN cell types in functional organization of the master oscillator.
Three different types of transgenic mice will be used to mark various major SCN subpopulations, to monitor circadian oscillator function in individual neurons, and to specifically ablate targeted cell types in the SCN. SCN explants cultures from these mice will be monitored for spatial expression of fluorescence markers and circadian luciferase reporter in individual neurons. Gene expression analysis of various sub-populations will be performed. Such analysis will produce both gene expression and temporal heterogeneity map of the SCN. To assess how different SCN sub-regions interact with each other, we will use photoactivable perturbation agents to perturb oscillator function in individual SCN subpopulation and subsequently assess oscillator function in rest of the SCN. Finally, to determine the role of individual cell types in circadian organization, we will specifically ablate target cell types expressing a toxin receptor and assess overall circadian function in the whole organism, as well as in SCN explants cultures.
Panda S., Nayak S.K., Campo B., Walker J.R., Hogenesch J.B., and Jegla T. Illumination of the melanopsin signaling pathway. Science. 2005 Jan 28;307(5709):600-4 .
Sato T.K., Panda S., Miraglia L.J., Reyes T.M., Rudic R.D., McNamara P., Naik K.A., FitzGerald G.A., Kay S.A., and Hogenesch J.B. A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron. 2004 Aug 19;43(4):527-37 .
Panda S., Provencio I., Tu D.C., Pires S.S., Rollag M.D., Castrucci A.M., Pletcher M.T., Sato T.K., Wiltshire T., Andahazy M., Kay S.S., Van Gelder R.N., and Hogenesch J.B. Melanopsin is required for non-image forming photic responses in blind mice. Science. 2003 Jul 25;301(5632):525-7 .
Panda S., Antoch M.P., Miller B.H., Su A.I., Schook A.B., Straume M., Schultz P.G., Kay S.A., Takahashi J.S., and Hogenesch J.B. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell. 2002 May 3;109(3):307-20.