The suprachiasmatic nucleus (SCN) of the hypothalamus is the master mammalian pacemaker, co-ordinating the multitude of cell-autonomous circadian oscillators across the body to ensure internal synchrony, as well as maintaining an adaptive phase relationship with the light-dark cycle via projections from the retina. Intercellular communication between SCN clock neurons synchronises their oscillations, resulting in coherent output signals to the periphery. Vasoactive intestinal peptide (VIP), a neuropeptide expressed in the retinorecipient ventrolateral region of the SCN, is vital to this circuit-level co-ordination by signalling to its cognate VPAC2 receptor. In addition, VIP is important for the integration of light input into the SCN oscillation. The aims of the work presented in this thesis were to determine the roles of the VIP and VPAC2 cells in controlling circadian rhythmicity, and to elucidate the mechanisms of VIP signalling that underpin these roles. The first two experimental chapters utilise intersectional genetics and viral transduction to address separable roles for the VIP and VPAC2 cell populations. By diphtheria toxin-mediated cell ablation, or by adjusting cell-autonomous periodicity or rhythmicity specifically in these cell populations, I have identified that the VPAC2 cells are important for period setting and rhythmicity of both the SCN ex vivo and mouse behaviour in vivo, while the VIP cells play a vital role in behavioural rhythmicity and phase coherence across the SCN. The next two chapters use application of VIP to SCN slices to address mechanisms of phase-resetting through pharmacological manipulation and microarray analysis. I find that VIP has long lasting effects on all major circadian parameters of the SCN slice oscillation at both the cellular and circuit levels, and that it achieves this through a diversity of molecular pathways, in particular through cAMP/Ca2+ response elements within gene promoters. The final chapter focuses primarily on DUSP4, a negative regulator of the MAP kinase pathway that I have demonstrated to be upregulated by VIP. Here I demonstrate that DUSP4 affects the steady-state period of SCN slices, as well as influences phase shifting characteristics of both slices and mice. To conclude, the work presented here furthers our knowledge of neuropeptidergic communication in mammalian pacemaking. I have undertaken extensive characterisation of the molecular mechanisms through which the VIP neuropeptide influences SCN oscillators, and I have determined differential roles for the VIP and VPAC2 neurons in circadian timekeeping.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:744248 |
| Date | January 2017 |
| Creators | Hamnett, Ryan |
| Contributors | Hastings, Michael |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/267953 |
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