UC Irvine

ABSTRACT OF THE DISSERTATION

Molecular and Whole Circuit Dynamics of Drosophila Clockwork and Light Integration

By

Logan Roberts

Doctor of Philosophy in Biomedical Sciences

University of California, Irvine, 2016

Professor Todd C. Holmes, Chair

Virtually all forms of life ranging from single-celled prokaryotes to humans have molecular and cellular clocks that calibrate essential physiological and behavioral patterns of activities. These clocks are fine-tuned by various environmental cues with light being the most powerful. Misalignment of circadian rhythms due to altered photic input by conditions such as jet lag and shift work has been linked to numerous negative health effects including diabetes, depression, Alzheimer’s disease and cancer. However, the fundamental question of how circadian neural networks dynamically integrate photic input in real-time has long remained enigmatic. We address this issue by using a combination of transgenic fruit flies, immunocytochemistry, mathematical modeling and 6-day real-time bioluminescence imaging of cultured Drosophila whole brain explants at single cell resolution. Although desynchrony has long been perceived as a negative feature of circadian disruption, our primary findings suggest that transient circuit-wide desynchrony by light may be a key feature of photoentrainment. We also identify the functional contributions of strong and weak neuronal oscillators that permit Drosophila circadian neural networks to drive robust yet adaptable rhythms. Using patch-clamp recordings, pharmacology, genetic recombination and behavioral assays, we show that light activation of the flavoprotein cryptochrome (CRY) is coupled to rapid membrane depolarization and acute behavioral arousal responses in Drosophila via a redox sensor called Hyperkinetic. In addition to CRY’s recognized role in resetting molecular clocks in response to blue light, we show that CRY may also mediate behaviors such as phototaxis in response to ultraviolet light. We are also investigating the growing problem of social jet lag using a novel photoentrainment protocol called LD strobe in combination with a custom bioluminescence imaging system. Overall, our findings indicate that there are distinct dynamic patterns of activity at the molecular, cell-autonomous and circuit-wide levels involved in circadian photoreception which could be harnessed and amplified to treat conditions such as jet lag, shift work and seasonal affective disorder.

Supplemental Movie 1: Raw time-lapse bioluminescence recordings of adult XLG-Per-Luc Drosophila whole-brain explants cultured for 6 days in darkness (Roberts et al., 2015). A: Whole brain culture maintained in constant darkness throughout recording. B: Whole brain culture exposed to a 15 minute 12.57 W/m2 light pulse at CT 22 of the second day in constant darkness (DD). This movie is further discussed in Chapter 2.

Supplemental Movie 2: The animations show changes in the phase and amplitude of XLG-Per-Luc bioluminescence activity for individual oscillators from all neuronal subgroups in either DD (Left) or in response to a phase advancing light pulse (LP, Right). The angle of the disks represents phase and drift of the disks towards the center of the circle and the size of the disks indicates reduction in amplitude. The disks are colored according to neuronal subgroup for the s-LNvs (red), l-LNvs (yellow), LNds (orange), DN1s (blue) and DN3s (green). This movie is further discussed in Chapter 3.

Supplemental Movie 3: Raw time-lapse bioluminescence recordings of cultured adult XLG-Per-Luc Drosophila whole-brain explants in darkness. A: Whole brain culture maintained in constant darkness throughout 7.5 days of recording. B: An 11-day recording of whole brain cultures exposed to a 15 minute 12.57 W/m2 light pulse at CT 22 of the second day in DD. This movie is further discussed in Chapter 6.

Supplemental Movie 4: The animations show changes in the phase and amplitude of XLG-Per-Luc bioluminescence activity for individual oscillators from all neuronal subgroups in either control conditions (Left, LD strobe with no phase shift) or in response to social jet lag (Right). The angle of the disks represents phase and drift of the disks towards the center of the circle and the size of the disks indicates reduction in amplitude. The disks are colored according to neuronal subgroup for the s-LNvs (red), l-LNvs (yellow), LNds (orange), DN1s (blue) and DN3s (green). This movie is further discussed in Chapter 6.