University of Cambridge
Exploring the role of Gastrin-Releasing Peptide Neurons in Circadian Time-Keeping of the Suprachiasmatic Nucleus
Abstract
dc:description.abstractCircadian (approximately one day) rhythms help organisms predict, and thereby adapt to, solar time, ensuring efficient use of energy and resources. In mammals, circadian rhythms are present in practically every cell of the body and are synchronised by a master clock, the hypothalamic suprachiasmatic nucleus (SCN), which provides internal time cues to create coherent circadian rhythms of physiology and behaviour. Time-keeping in the SCN operates at two levels: the intra-cellular, cell-autonomous transcriptional/translational feedback loop (TTFL) and the intercellular network, in which neuropeptidergic signalling couples cell-autonomous rhythms across the SCN. One of several neuropeptidergic axes of the SCN is that formed by cells that express Gastrin-Releasing Peptide (GRP) and cells that express its cognate receptor (GRPR). Although implicated in retinal signalling into the SCN, its general role in the SCN is unknown. This thesis aims to determine the contributions to SCN time-keeping of not only GRP as a neuropeptide, but also of the neurons that release it and/ or respond to it; Grp- and Grpr-expressing neurons, respectively. First, I re-analysed a published single-cell transcriptomic dataset, as well as applied in situ hybridization of SCN sections and slices to reveal that half of Grp neurons also express Vasoactive Intestinal Peptide (Vip), a key neuropeptide for SCN time-keeping. Despite this overlap, I showed that Grp neurons are transcriptionally distinct from other SCN neurons. Second, using organotypic SCN explants that express the circadian TTFL reporter PER2::Luciferase, I investigated the functional contributions of GRP-signalling to SCN network-level timekeeping via pharmacological approaches. Acute treatment with exogenous GRP in early circadian night, but not at other times of the cycle, led to phase delays of up to 3h. In addition, chronic treatment with two different GRPR antagonists reversibly and dose-dependently lengthened SCN period. Together this shows that GRP signalling determines circadian phase and controls network-level period. The role of Grp neurons was then explored with a novel knock-in mouse line in which Cre-recombinase is expressed from the Grp locus to provide genetic access to Grp cells. This required extensive characterisation to ensure that Cre-expression is faithful, that GRP expression was unaffected by introduction of Cre recombinase to the Grp locus, and that circadian behaviour was unaltered. I then mapped the activity patterns of Grp neurons in SCN slices using a Cre-dependent calcium reporter expressed by an AAV. This revealed robust circadian neuronal activity rhythms, with the Grp neurons split into two antiphasic populations, that had a distinct anatomical location in the SCN. The role of the activity of Grp neurons on SCN rhythms was explored with Cre-dependent optogenetic and chemogenetic activators. With both approaches, direct activation of Grp cells during early circadian night (when they are inactive) phase-delayed the SCN TTFL, recapitulating the effects of exogenous GRP. Finally, I present validation of two new intersectional mouse lines in which Grpr-Flp and Vip-Flp can be combined with the Grp-Cre allele. By using suitable Cre- and Flp-dependent AAV-expressed reporters and effectors, these will facilitate future analyses of the complete Grp-Grpr axis and the functional overlap between Grp and Vip neurons.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Gómez García, Elena del Carmen
- Advisor dc:contributor.advisor
-
- Hastings, Michael
Subjects
dc:subject × 8Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.116757
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/381637