DOI

10.17077/etd.4z7z-iv18

Document Type

Dissertation

Date of Degree

Spring 2019

Access Restrictions

Access restricted until 07/29/2021

Degree Name

PhD (Doctor of Philosophy)

Degree In

Genetics

First Advisor

Lear, Bridget C.

First Committee Member

Prahlad, Veena

Second Committee Member

Kitamoto, Toshihiro

Third Committee Member

Johnson, Wayne

Fourth Committee Member

Kay, Alan

Abstract

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.

Keywords

ArcLight, Circadian, DN1p, Drosophila, LNd, Narrow Abdomen

Pages

xiv, 152 pages

Bibliography

Includes bibliographical references (pages 143-152).

Copyright

Copyright © 2019 Stephanie Jean Haase

Available for download on Thursday, July 29, 2021

Included in

Genetics Commons

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