Integration of circadian clocks and metabolism in the hypothalamus

Abstract

Recent evidence implicates circadian disruption in the increasing prevalence of obesity. This disruption is thought to occur partly due to aberrant circadian regulation in brain regions that are critical to feeding. The transcription-translation feedback loop (TTFL) drives suprachiasmatic neurons in the mammalian master clock to depolarise and increase action potential (AP) firing during the day and to hyperpolarise and reduce AP firing at night. Intriguingly, core components of the TTFL such as Period1 (Per1) and Cryptochrome1/2 (Cry1/2) are also rhythmically expressed in other brain regions including the arcuate nuclei of the hypothalamus (ARC). ARC neurons are critical sensors of metabolic information and project to downstream brain regions to influence appetite and feeding homeostasis. However, it is currently unknown whether local oscillations in the molecular clockwork drive daily rhythms in ARC neuronal activity. Using a mouse in which Per1 promoter activity is reported by Venus protein (Per1::Venus), electrophysiological recordings were made from Per1::Venus positive ARC neurons maintained in acute adult brain slices. Per1::Venus positive neurons were significantly more active at night, firing APs at ~4Hz, compared to ~2Hz during the day and received more frequent GABAergic input during the day than at night. Importantly, daily variation in firing rate was only observed in neurons located in lateral areas of the ARC. We postulate that rhythmic inhibitory GABA is responsible for generating daily and circadian changes in latARC electrical output, and that this signal originates from a population of GABAergic neurons within the ARC. To establish whether the day-night change in firing of ARC neurons is dependent on an intact TTFL, we made whole-cell recordings from slices prepared from mice lacking Cry1/2 (Cry1-/-Cry2-/-), and found daily variations in firing rate to be absent. Notably, the activity of Cry1-/-Cry2-/- ARC neurons was constitutively elevated across the day-night cycle, which highlights the role of the circadian molecular clockwork in setting excitability in neurons with an intact clock. To confirm that expression of core clock components locally in ARC is responsible for driving daily rhythms we performed bilateral microinjection of rAAV2-Cre-GFP (or control rAAV2-GFP) viral vectors into the ARC of Bmal1fl/fl mice, which led to a cre-dependent increase in daytime firing. We also employed whole-cell methods to record the firing activity of neuropeptide-Y-expressing neurons (NPY-humanised Renilla Green Fluorescent Protein; NPY-hrGFP), which play an established role as stimulators of appetite. However, NPY-hrGFP neurons fired APs at a consistent rate across the day. Collectively, these results demonstrate that the TTFL drives daily changes in electrical activity in a unique sub-population of ARC neurons that not does not overlap with those that express NPY. This study has key implications for our understanding of how the TTFL influences the excitability of neurons in an extra-SCN oscillator with well-established role in feeding homeostasis. Ultimately, this study begins to elucidate how one critical brain area may contribute to a network of circadian oscillators which are important for normal body weight control.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Hugh Piggins (Supervisor) & David Bechtold (Supervisor)