| Daily rhythms in behavior and physiology are orchestrated by a network of circadian clock neurons. Neuronal connections within this network produce coherence and robustness in circadian timekeeping that are uncharacteristic of rhythms driven by non-neuronal clocks. Using Drosophila as a model system, my thesis research aims to understand how clock neurons are physiologically connected and how their molecular oscillations are coordinated to produce coherent circadian rhythms.;I have developed an experimental approach to address functional connectivity in the fly brain that combines chemogenetic excitation of neurons of interest with simultaneous monitoring of potential postsynaptic physiology with genetically encoded fluorescent sensors. Using this method, I have mapped connections in the clock network mediated by the critical neuropeptide Pigment-Dispersing Factor. In addition, I have performed ex vivo patch-clamp recordings of the fly clock neurons and provided the first electrophysiological characterization of the dorsal lateral neurons (LNds), the Evening Oscillator of the clock network. I find that the neuronal activity LN ds is modulated by multiple fast neurotransmitters, and that a group of dorsal clock neurons provides inhibitory synaptic input onto the LN ds. Furthermore, I find that while GABAergic inhibition of the clock network promotes sleep at night, glutamatergic inhibition promotes wakefulness during the day.;To study how the molecular rhythms of clock neurons are coordinated, I have genetically sped-up or slowed-down the molecular clock in specific subsets of clock neurons and determined how such manipulations affect the molecular oscillations in un-manipulated clock neuron classes and sleep/activity rhythms. I find that the various groups of clock neurons do not display uniform modes of coupling. Rather, they display unique and complex coupling relationships that vary from group to group. In contrast to the widely accepted "Master Pacemaker" model, my results show that the clock network consists of multiple independent oscillators, each unified by its neuropeptide output. Lastly, I find that robust circadian rhythms require coherence of molecular clocks across a much larger proportion of the clock network than previously thought.;Collectively, my thesis research greatly advances our understanding of how the circadian clock neuron network is wired and how it is organized and coordinated. |
