Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Neurosciences

Language

English (en)

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Erik Herzog

Abstract

In mammalian hypothalamus, the suprachiasmatic nucleus: SCN) generates daily behavioral and physiological rhythms as a circadian pacemaker. The 20,000 SCN neurons synchronize to each other and to the ambient cues to generate coherent daily rhythms. Vasoactive intestinal polypeptide: VIP), a neuropeptide produced by SCN neurons, plays a major role in synchronizing SCN neurons to each other. Whether VIP mediates synchrony to environmental cues and how synchrony within the SCN is achieved has not been examined extensively. We recorded PERIOD::LUCIFERASE: PER2::LUC) expression from SCN explant cultures over multiple days following VIP application at different circadian time points to generate a phase response curve which reliably predicted the phase relationship between the SCN and daily increases in VIP. VIP shifted PER2::LUC rhythms in time- and dose-dependent manner. VIP rapidly increased intracellular cAMP in most SCN neurons and simultaneous antagonism of adenylate cyclase: AC) and phospholipase C: PLC) was required to block the VIP-induced phase shifts of SCN PER2 rhythms. We conclude that VIP entrains circadian timing among SCN neurons through rapid and parallel changes in AC and PLC activities. While performing the experiments mentioned above, we found that a single VIP pulse reliably reduced the PER2::LUC rhythm amplitude in the SCN explants. The amplitude reduction was dose-dependent, but not circadian. We found that the amplitude reduction was primarily explained by reduced synchrony among SCN neurons, with little effect on the amplitude of individual neurons. To test if VIP modulates the amplitude of circadian rhythm in vivo, we compared the effects of light on locomotor rhythms in wild-type and VIP-deficient mice. We found that constant light reduced the amplitude of behavioral rhythms in wild type, but not in Vip-/-, mice. Because, theoretically, reduced synchrony among oscillators can facilitate their entrainment to periodic signals, we tested if VIP accelerates entrainment of animals to an 8-h advanced light-cycle or SCN explants to a 10-h advanced temperature cycle. We found that VIP doubled the speed of circadian entrainment both in vivo and in vitro. We conclude that reduced synchrony by VIP accelerates entrainment. Finally, we characterized the spatiotemporal expression of one of the three major VIP receptors, VPAC2R, in various brain areas and SCN. We characterized the specificity of a new antibody and found moderate to weak levels of VPAC2R in cortex, hippocampus, olfactory bulb, cerebellum, arcuate nucleus in hypothalamus, amygdala and ventrolateral thalamus and high levels in the SCN. VPAC2R expression was observed from rostral to caudal SCN with stronger expression in dorsomedial area. SCN neurons expressing VIP or vasopressin all expressed VPAC2R. We found intracellular VPAC2 expression mainly along cell bodies and dendrites, but not along their axons. We found that VPAC2R levels in the SCN do not oscillate in light-dark cycles or in constant conditions. We conclude that VPAC2R presents broadly in the SCN throughout the day to mediate circadian synchrony in the SCN. Taken together, these experiments suggest that VIP signaling mediate entrainment to the daily light cycle and an altered schedule by jet lag or day night shift work through wide expression of VPAC2R in the SCN.

DOI

https://doi.org/10.7936/K7FF3QCH

Comments

Permanent URL: http://dx.doi.org/10.7936/K7FF3QCH

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