The Physiological and Behavioral Roles of Cholinergic and GABAergic Signaling to Drosophila Circadian Clock Neurons
Persons, Jenna
2019
Abstract
Circadian clocks allow organisms to track and anticipate rhythms in time-giving environmental cues (Zeitgebers) caused by Earth’s 24-hour rotation. In many organisms, master neuronal clocks are essential to synchronize physiology and sleep-wake behavior with daily light and temperature rhythms. Neuronal timekeepers each possess an endogenous molecular clock causal to circadian sleep-wake behavior. The molecular clock’s rhythms approximate the solar day with near 24-hour transcriptional-translational feedback loops. Their slight deviation from the day’s 24-hour cycle requires that clock neurons be reset daily, and stably “entrain” to Zeitgebers to maintain synchrony with the environment. Understanding entrainment is central to understanding circadian clocks; though the precise neurophysiological and molecular mechanisms through which organisms entrain to their environment to coordinate sleep-wake rhythms remains mysterious. Foundational work in circadian model organisms suggests that communication through neuropeptides and transmitters underlies circadian behavior and timing of sleep-wake rhythms. While the criticality of neural communication to circadian behavior is certain, the functions of only a handful of transmitter types within the network’s repertoire have been explored. Remarkably, clock neuron networks exhibit conservation in form and function across network size and separate evolutionary lineages. The clock network of Drosophila melanogaster is spatially and functionally organized similar to mammalian model networks, but with fewer than 1/100th the neurons. Despite their economical scale, Drosophila clock neurons retain many of the neurochemicals that function in mammalian clock networks, and neural connections between clock neuron populations, input and output centers that produce quantifiable circadian rhythms and sleep-wake behavior. My work leverages the Drosophila model’s unique advantages to define the roles of fast-neurotransmitters in circadian neuron physiology and sleep-wake behavior. Using live-neuronal imaging, I characterized the physiological roles of ionotropic GABAergic and acetylcholinergic communication to a critical clock neuron population. I validated the use of a genetically-encoded voltage sensor to directly study membrane excitability without electrophysiology in the Drosophila clock network. I used classic circadian mutants to determine the physiological roles of the molecular clock and light-mediated inputs in setting daily rhythms in transmitter receptivity. Finally, with behavioral studies I determined the functions of GABA and acetylcholine in coordinating circadian rhythmicity and sleep-wake behavior. My biological findings support that fast-neurotransmitters may represent distinct “day” and “night” physiological and behavioral states. Fast-neurotransmitter signaling in mammalian networks is correlated, and perhaps causal to circadian entrainment. In an effort to develop standardized, quantitative measures to study entrainment behavior in Drosophila, I co-developed a free MATLAB-based program, PHASE, to define Activity, Sleep, and Entrainment behavior in data acquired from the universally used DAM-system (TriKinetics, Waltham MA). I demonstrated that PHASE measures entrainment and classic elements of sleep-wake behavior in wild-type flies and circadian mutants in behavior paradigms with equinox light, long- and short-days, and 23- and 25-hour periods. PHASE, when coupled with Drosophila’s extensive genetic tools, may provide key insight into the molecular and neuronal basis of circadian entrainment.Subjects
circadian behavior neural network neurotransmission
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