The fruit fly Drosophila exhibits robust daily behavioral rhythms, which are driven by a network of circadian pacemaker neurons in the fly brain. The Narrow Abdomen (NA) sodium leak channel functions rhythmically in pacemaker neurons, downstream of the molecular circadian clock, to depolarize resting membrane potential and promote neuronal excitability. Loss of NA function (NA-LOF) strongly disrupts behavioral rhythms, and these behavioral phenotypes are consistent with decreased circadian neuronal activity. Yet despite some recent advances, the mechanisms of NA channel function and regulation in the circadian system are still not well understood. To further elucidate the role of the NA channel in the circadian neuronal network, we generated mutated versions of the NA transgene and assessed the effects of transgene expression in Drosophila circadian pacemaker neurons. Expression of a putative gain of function na transgene (na-GOF) in pacemaker neurons generates unique behavioral phenotypes, suggesting novel effects on neuronal excitability or/and the molecular circadian clock. Next, we investigated how NA-LOF and NA-GOF mutations affect circadian neuron activity through optical recording of fluorescent voltage and calcium sensors expressed in these neurons. Using the fluorescent voltage sensor ArcLight, we find that both NA-LOF and NA-GOF manipulations suppress spontaneous membrane activity in clock neurons in the Drosophila brain. This finding was surprising because the behavioral effects of NA-LOF and NA-GOF are quite distinct. However, the information provided from these spontaneous assays may be a combination of neuronal input and output, and in some cases information is combined from multiple cells. To further characterize the neurophysiological effects of NA channel manipulation, we next paired optical recording with pharmacology in brain explants. Here we find that both wild-type and NA-LOF DN1p clock neurons are strongly depolarized by the acetylcholine receptor agonist nicotine, while NA-GOF neurons show little response. This suggests that NA-GOF expression already depolarizes the membrane potential of these neurons. We also assessed intracellular calcium levels in the DN1p clock cells after applying the inhibitory neurotransmitter glutamate at either morning (peak) or evening (trough) timepoints. We find that wild-type DN1p neurons show a strong decrease in calcium at the peak timepoint and a much smaller decrease at the trough. In contrast, NA-GOF DN1p neurons show decreases at both timepoints, indicating that they have elevated calcium levels (and elevated activity) at the trough time. Through immunostaining, we find that NA-GOF expression alters the core clock protein PERIOD levels in sLNv and LNd neurons during early day. Taken together, this study shows that overexpression of NA-GOF ion channel in Drosophila pacemaker neurons induce unique behavioral phenotypes, likely by depolarizing membrane potential and increasing neuronal activity. We propose that these changes in neuronal activity may feedback to alter the oscillation of molecular clocks. While these transgenic studies have been informative, we have also established gene-editing methods in order to distinguish the effects of gene mutation from effects of overexpression. We have used the CRISPR-Cas9 system to target the endogenous na locus. In the initial step, we replaced na exons 1-13 with a fluorescent marker flanked by attP integration sites. Through subsequent integrase-mediated recombination, we hope to generate a series of na mutations of interest, including gain-of-function mutations, for future studies.

Circadian clocks drive the daily patterns of behavior and physiology observed in most organisms. These internal clocks allow organisms to advantageously align their behavior to daily cycles in the environment such as light and temperature. The fruit fly Drosophila displays many robust, daily behavioral rhythms including discrete bouts of locomotor activity at dawn (i.e. morning activity) and dusk (i.e. evening activity). The molecular clocks that drive these daily activity bouts are found in approximately 150 circadian pacemaker neurons in the fly brain. Interestingly, the timing of the molecular clocks is synchronous between all pacemaker neurons, yet different subsets of these neurons appear to make quite different contributions to the regulation of morning vs. evening activity. It remains poorly understood how the molecular circadian clock drives daily rhythms in pacemaker neuron activity or how the activities of different groups of pacemaker neurons combine to produce complex behavioral output. The overall goal of this thesis is to characterize how different subsets of Drosophila pacemaker neurons contribute to daily behavioral regulation both individually and as a network. To examine daily patterns of neuronal activity in different groups of circadian clock neurons, we have established imaging methods using genetically encoded fluorescent sensors. For these sensors, changes in fluorescence levels correspond to changes in neuronal activity, thus allowing us to measure neuronal activity patterns in real-time and throughout the day. Using these tools, I have characterized the daily activity patterns of different groups of the clock neurons that agree with published rhythms in activity as assessed by patch-clamp electrophysiology and calcium imaging We have also used genetic and molecular approaches such as RNA interference (RNAi) to alter gene expression in a tissue-specific manner. These approaches allow us to manipulate the function of different groups of clock neurons and to determine how these manipulations affect rhythmic behavior and neuronal activity patterns. We have silenced different subsets of circadian pacemaker neurons using RNAi knockdown of the NARROW ABDOMEN (NA) sodium leak channel and identified a complex role for a subset of the posterior dorsal neurons 1 (DN1p) in regulating locomotor behavior. The DN1p are known to be involved in promoting morning behavior, and recent studies have shown that a subset of the DN1p regulate midday sleep bouts via downstream sleep regulating neurons. Our data suggest that the DN1p neurons likely suppress midday activity through inhibition of other circadian pacemaker neurons, and that this inhibitory role can be compensated for by light. Finally, we have also examined the intracellular mechanisms regulating circadian neuronal output. Rhythmic activity of the NA leak channel and its mammalian ortholog (NALCN) have been shown to contribute to daily excitability rhythms in circadian pacemaker neurons. We used temporally-restricted expression of RNAi and rescue constructs to identify a developmental requirement for the NA channel complex in Drosophila, and we demonstrate that channel complex proteins are very stable in the Drosophila brain. These data suggest that circadian regulation of the NA channel in adults may involve post-translational mechanisms that control activity and not just expression of the channel complex.