Synaptic vesicle cycling and inflammatory stimulation in central and peripheral oscillators

The 24-hour or circadian rhythm regulates many aspects of behaviour, metabolism and physiology. These rhythms are orchestrated by the suprachiasmatic nucleus (SCN), however many peripheral tissues have been shown to maintain their own rhythms in vitro without SCN input. Skeletal muscle rhythmically expresses many genes, including clock genes, and this study aimed to establish whether the muscle clock is dependent on the circadian transcription-translation feedback loop, and to determine the circadian behaviour of the Flexor Digitorum Brevis (FDB) and diaphragm. By monitoring PER2 production from these tissues using the mPER2::luc mouse, it was observed that both the FDB and the diaphragm maintained their PER2 rhythms in vitro. These rhythms were similar to those observed in the SCN and other peripheral tissues; however the phase of PER2 production was altered by the time of cull. These tissues also showed similar PER2 production profiles to the SCN in Afh/Afh and Cry1-/-Cry2-/- mutant mice, indicating the diaphragm and FDB act as true peripheral oscillators and may be useful as model tissues for circadian research in muscles.Vesicle cycling plays a vital role in cell-cell communication, and the disruption of this cycle is utilised by drugs such as Dysport to abolish unwanted muscle contractions by blocking exocytosis. Dysport, Dynasore (an endocytosis blocker) and TTX (a Na+ channel blocker) were used to treat SCN and muscle cultures in vitro to assess the effect of blocking the vesicle cycle on PER2 production and determine whether vesicle cycling contributes to the circadian phenotype in muscle and neuronal tissues. Whilst Dysport had no effect on PER2 production, Dynasore abolished PER2 rhythms in the SCN, yet increased PER2 production in the diaphragm. These findings suggest that there is some interplay between vesicle cycling and the circadian rhythm, however Dysport poses no threat to the circadian system when used at clinical doses.The immune system is also under circadian control, as studies have shown that clock genes regulate cytokine production and microglia rhythmically express clock proteins. Little is known about the behaviour of microglia within the SCN throughout the day, or the effect of injury and infection on the clock itself. This study aimed to assess if there is an inflammatory input to clock function, and to determine whether microglial recruitment is under temporal control. Pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs; molecules associated with infection or injury) were used in vitro to mimic an immune challenge. And although no effect of PAMPs on PER2 expression in the SCN was observed, significant reduction of PER2 rhythms and culture survival was observed in the presence of DAMPs. SCN sections were also stained for Iba1 to determine their number within the SCN and surrounding areas. It was observed that microglial recruitment altered throughout the day, with a significant peak during the active phase.The findings within this thesis show that there is complex interaction between the circadian system and both the immune system and skeletal muscles, shedding new light on their behaviours. Dysport has also been shown to have no effect on the circadian rhythm, suggesting no detrimental effects of circadian disruption will be seen whilst using this drug. However, further study is required to establish the role of vesicle cycling within the circadian system and to fully characterise the circadian behaviour of the immune system and skeletal muscles.