Physiology of rest/activity cycles in Drosophila melanogaster

Abstract

Sleep is a vital behavioural state present across the whole animal kingdom. In small organisms, a behavioural definition of sleep is typically used to define sleep. Drosophila melanogaster, the common fruit fly, is an important model organism whose rest is considered sleep-like. A key behavioural feature of sleep is a species-specific posture and place preference; however, this aspect of sleep has not been well characterised in Drosophila. In this thesis, I demonstrate a novel assay, Trumelan, which uses side-on video tracking to monitor fly location, pose, and behaviour.

I first find that behavioural classification in Trumelan is highly accurate and can yield expected patterns of daily fly behaviour in both wild-type and circadian mutants. In addition, I compare the behaviour of Trumelan to the commonly used Drosophila Activity Monitor (DAM) assay for studying rest in flies and show that DAM assays overestimate rest. By analysing rest posture, I find that flies adopt a supported upright position at the beginning of rest but show negligible changes during a rest bout. In addition, resting flies prefer to be near, but facing away from, the food source. This preference remains in all stationary behaviours that occur away from the food port, suggesting stationary flies have a general place preference. Finally, I discovered a novel daily rhythm in y-position place preference. Wild-type flies typically rest on the ground during the day but shift to more rest on the ceiling at night. This place preference rhythm remains in constant-dark conditions but is lost in circadian mutants. These findings are the first quantitative analysis of typical rest posture, place preference, and the discovery of a novel place preference under circadian control.

To supplement the behavioural approach for studying sleep, I explored the efficacy of using in vivo luciferase assays for discovering molecular correlates of sleep. I performed a preliminary luciferase screen of a collection of neuronal populations implicated in sleep-wake regulation by utilising calcium signalling as a proxy for neuronal activity. I show luciferase activity rhythms associated with expression in various neuronal populations. Due to low signal strength with firefly Luciferase, I also created new luciferase-expressing flies based on NanoLuc to improve the signal-to-noise ratio. I found that these generate much stronger signals at the expense of a rapid decline in signal.

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Identifiers

ORCID for Herman Wijnen: orcid.org/0000-0002-8710-5176

ORCID for Mariana Vargas-Caballero: orcid.org/0000-0003-2326-4001

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