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.

Mosquito disease vector control relies mostly on toxic insecticides. A more environmentally friendly alternative is to make use of light-based behavioral manipulation to attract pests to traps and repel pests away from human habitation. The present technology is based on the assumption that mosquito UV light detection occurs solely through opsin-based photoreception in the eyes. The Holmes Lab has recently found additional UV and short wavelength photoreceptive elements expressed in central brain neurons that strongly modulate complex insect behavioral responses to light. Therefore, there is a need to incorporate these additional elements in disease vector control designs for improved efficiency. Namely, CRYPTOCHROME (CRY), which is classically associated with its role in circadian clock resetting, activates with blue- and UV- light and increases the electrical excitability of circadian/arousal neurons. I hypothesized that CRY and other photoreceptor systems mediate photic input to the circadian/arousal circuit of flies in order to mediate specific light-based behavioral responses. To test this, I modified and adapted light-evoked electrophysiology protocols to test the photoexcitability of primary arousal neurons in response to different stimuli of light using several photoreceptor mutants that lack one of three photoreceptor systems, as well as transgenic flies expressing mosquito CRY1 from a diurnal and nocturnal species. I also modified and adapted several behavioral assays to determine light attraction/avoidance as well as light-pulse arousal behavioral response to these different mutants. These modifications involved developing a new light emitting apparatus that improved on intensity control and spatiotemporal light exposure with a finer tuned spectral emission for UV, violet, blue, and red light. In this dissertation, I found that CRY coordinates with other photoreceptors to mediate light-induced electrical excitability of neurons, which underlie complex sleep/wake and light arousal behaviors. Furthermore, I found that CRY phototransduction persists across species in an intensity-dependent and species-specific manner, controlling light behavioral phototaxis across diurnal and nocturnal mosquito CRY1 profiles. I conclude with non-image forming as an amalgamation of multiple photoreceptor system contributions over a broad spectral range that affect downstream behavioral processes, including CRY as a primary short-wavelength photoreceptor for flies and mosquitos.

Light is a powerful environmental regulator of insect behavior. My thesis focuses on novel phototransduction mechanisms in insect central brain neurons that directly regulate physiology and behavior, independent of external eyes. I hypothesized that novel short wavelength-activated phototransduction mechanisms modulate complex behaviors such as arousal and decision-making. To test this, I developed and integrated molecular, electrophysiological, imaging, and behavioral approaches. I described mechanisms of a direct neuronal blue- and UV-light sensor, CRYPTOCHROME (CRY), in mediating behavioral responses to short wavelength light, and how CRY couples to membrane voltage-gated K+ β subunits, HYPERKINETIC. I also found that the previously uncharacterized Rhodopsin7 is a direct photoreceptor uniquely expressed in circadian neurons mediating circadian photoentrainment. I developed several assays to measure light-evoked avoidance/attraction behaviors with high spatial and temporal resolution in flies and mosquitoes. I demonstrated circadian regulation of these behaviors in Drosophila at the genetic and neuronal circuitry level. I also show valence control of light-evoked behavioral responses by integration of different light inputs, where nocturnal and diurnal mosquito species have unique, circadian clock-controlled behavioral preference to different light environments. Further, I identified that circadian neural circuits in the same mosquito species show striking anatomical and transcriptional diversities, which likely underlie observed behavioral differences. I conclude that light is a powerful cue that drives complex insect behaviors.

Circadian oscillators are capable of endogenous oscillations in free-running conditions. Many studies show that the coordination of the biological clock with environmental inputs, such as daily solar cycles, is critical for regulating physiological and behavioral activities. Conversely, billions of people worldwide are subjecting themselves to chronic misalignment and disruption of biological clocks with environmental inputs. In this study, we developed an entrainment protocol we call LD Strobe that permits us to obtain real-time, longitudinal bioluminescence imaging of the entire Drosophila circadian neural circuit in adult cultured brains, ex vivo. With the LD Strobe schedule, we can now examine circadian disruptions that could potentially lead to the progression of human-related diseases. In the following proposed projects we aim to (1) capture the Drosophila circuit-wide responses to Weekend Light Shifts, (2) determine Drosophila behavioral outputs generated by circadian-disrupted flies after undergoing a simulated weekend, (3) examine the circuit-wide response of flies in a genetic background with uncharacterized disruptions to circadian-modulated physiology and behavior, and (4) determine the circadian regulation light-evoked attraction and avoidance behaviors in daytime- and nighttime-biting mosquitos. Due to similarities between mammalian and insect circadian molecular and neural circuits, features of the circadian physiological and behavioral outputs from the proposed projects may be broadly observed and applicable to humans.