Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Neurosciences

Language

English (en)

Date of Award

January 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Erik Herzog

Abstract

In mammals, the suprachiasmatic nuclei: SCN) in the ventral hypothalamus function as a circadian pacemaker, controlling daily rhythms in behavior and physiology. Together the SCN contain approximately 20,000 neurons that maintain rhythms in firing rate and gene expression. Previous studies led to the assumption that single SCN neurons are capable of self-sustained circadian rhythms. Whether and which SCN neurons can maintain cell-autonomous daily oscillations has not been extensively tested. We measured PERIOD2::LUCIFERASE expression in isolated SCN neurons over multiple days to determine if all SCN neurons were circadian. We then examined neuropeptide content of the recorded neurons. We found that when isolated physically or with a blocker of cell-cell communication, SCN neurons expressed a range of circadian periods, amplitudes, and abilities to sustain cycling. Surprisingly, most cells were sloppy oscillators, switching from rhythmic to arrhythmic or vice versa throughout their lifetime. We also found no evidence for a class of circadian-pacemaker neurons in the SCN based on neuropeptide expression. We conclude that while all SCN neurons are capable of cell-autonomous rhythms, they are intrinsically sloppy with network interactions dramatically increasing the number of circadian neurons. We next used a mathematical model of the mammalian circadian clock to determine whether rates of gene transcription, protein translation, degradation or phosphorylation might explain the ability of SCN neurons to switch between circadian and arrhythmic behaviors. We found that rhythmicity was more sensitive to the rates of protein translation and degradation. We next tested what effect having neurons with different intrinsic circadian behaviors would have on population synchrony. We simulated cells of known circadian phenotypes: e.g. arrhythmic, damped, or self-sustained) in a pattern defined by small-world network properties and varied the positions and proportions of each oscillator type. We found that increasing the number of damped oscillators or placing them in highly connected locations within the network both augmented the rate at which the network synchronized. We conclude that the SCN likely benefit from a heterogeneous population of oscillators, especially when recovering from an environmental perturbation that causes desynchrony. Finally, we generated and characterized two independent lines of transgenic mice to test the role of vasoactive intestinal polypeptide: VIP) neurons in circadian rhythmicity. These mice express Yellow Fluorescent Protein: YFP) under the control of a fragment of the VIP promoter in VIP neurons of the SCN, neocortex, olfactory bulbs, and enteric nervous system. We crossed these mice to generate a line in which VIP neurons are targeted for deletion using Cre-mediated recombination upon addition of tamoxifen. We observed successful deletion of VIP neurons in cultured SCN explants, but have no evidence to date for deletion of SCN neurons in vivo using a variety of protocols. We conclude that our construct is faithfully expressed in VIP neurons and that in vitro experiments show promising results for further study.

Comments

Permanent URL: http://dx.doi.org/10.7936/K71834J8

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