Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC227
Circadian Rhythms of PER2 Protein Expression are Maintained in SCN and Extra-SCN Hypothalamic Oscillators of Mice with Disrupted VIP-VPAC2 Signalling.
A. T. Hughes1, C. Guilding1, H. D. Piggins1
1. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
Biological clocks control all aspects of physiology and behaviour and daily rhythms are coordinated by the brain’s dominant circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. This ‘master’ SCN clock synchronises the activity of a myriad of tissue specific oscillators distributed throughout the body. Vasoactive intestinal polypeptide (VIP) and its receptor, VPAC2, play important roles in the functioning of the SCN pacemaker. Mice lacking VPAC2 receptors (Vipr2-/-) express a continuum of locomotor phenotypes from behaviourally rhythmic with a short period, to multiple low power rhythms and arrhythmicity. Vipr2-/- mice show altered SCN neuronal activity and clock gene expression, and display disrupted synchrony of endogenous clock gene (period1) oscillations in the SCN. Further, these mice show altered circadian rhythms in metabolism and food intake. Within the brain, the SCN is not the only site containing endogenous circadian oscillators, nor is it the only site of VPAC2 receptor expression. Using wild type mice, we have recently demonstrated robust endogenous circadian rhythms at both single cell and tissue levels in the arcuate (Arc) and dorsomedial (DMH) nuclei of the mediobasal hypothalamus1, two areas which also express VPAC2 receptors and have been shown to function in the control of energy homeostasis. Consequently we determined the role of VPAC2 receptor signalling in the maintenance of rhythmic expression of the core clock gene protein PERIOD2 (PER2) in these hypothalamic nuclei. We used wild type and Vipr2-/- mice, both expressing a PER2::luciferase fusion protein, which accurately reports PER2 protein expression (PER2::LUC), to record real time bioluminescence in photomultiplier tube assemblies. Following behavioural characterisation of mice in diurnal and circadian (constant dark) conditions, we assessed PER2::LUC expression in microdissected adult brain slice cultures containing SCN, Arc or DMH. Surprisingly, in tissue from Vipr2-/- mice, all three hypothalamic brain regions maintained circadian rhythms of PER2::LUC expression comparable to those of their wild type counterparts, including cultures of tissue taken from behaviourally arrhythmic animals. We found no significant differences in period, phase or amplitude of oscillations in any of the three nuclei between the oscillations of behaviourally rhythmic and arrhythmic Vipr2-/- mice or between the oscillations of wild type and Vipr2-/- mice (all p>0.05, t-tests). These results demonstrate that VPAC2 receptor signalling is not significantly involved in the maintenance of PER2 protein rhythms in the mediobasal hypothalamus in vitro, and raise the intriguing possibility that the loss of VPAC2 receptor signalling differentially affects clock gene expression in the rodent brain.
Where applicable, experiments conform with Society ethical requirements