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«Connectivity, Organization, and Network Coordination of the Drosophila Central Circadian Clock by Zepeng Yao A dissertation submitted in partial ...»

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Physiological connectivity does not ensure molecular clock coupling in the lateral neuron network.

Figure 4.S1.

The hierarchical dual-oscillator model of the Drosophila’s circadian clock neuron network.

Figure 4.S2.

The free-running periods of activity rhythms can be genetically manipulated over a wide temporal range.

Figure 4.S3.

The overexpression of DBTS and DBTL coherently accelerates and decelerates the molecular clocks of the PDF positive s-LNvs.

Figure 4.S4.

The PDF positive neurons coherently set free-running periods only within a narrow temporal range.

Figure 4.S5.

Comparison of rhythmicity, internal desynchronization and rhythmic power between flies overexpressing different forms of DBT or SGG in both PDF positive and negative clock neurons and in PDF positive neurons only.

Figure 4.S6.

PDFR signaling is required for the PDF neuron influence over free-running periods.

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between flies overexpressing different forms of DBT or SGG in both PDF positive and negative clock neurons and in PDF negative clock neurons only.

Figure 4.S8.

In the absence of PDFR signaling, the PDF negative neurons determine free-running periods.

Figure 4.S9.

The PDF positive neurons can coherently drive activity rhythms with very long free-running periods in the absence of functional molecular clocks in PDF negative neurons.

Figure 4.S10.

Comparison of PER expression rhythms in sNPF+ and sNPF- LNds from flies with slow-running PDF positive neurons (PdfDBTL) on DD4.

Figure 4.S11.

A multi-oscillator interpretation of free-running activity rhythms.

Figure 5.1.

Neuronal clock speed determines the phase of activity peaks in LD cycles............ 142 Figure 5.2.

Differential influence on the phase of activity peaks in LD by the LNd/5th s-LNv clocks and the LNv clocks.

Figure 5.3.

A subset of the LNd clocks displays delay-specific coupling to the LNv clocks...... 144 Figure 5.4.

The CRY+ DN1p clocks are tightly phased-coupled to the LNv clocks

Figure 5.5.

The lateral clock neurons are sufficient to set the timing of activity peaks under LD.

Figure 5.6.

Coherent free-running activity rhythms require synchrony in all of the lateral clock neurons as well as the CRY+ DN1ps.

Figure 5.S1.

The DvPdf-GAL4 expressing neurons are not sufficient to fully reset the phases of activity peaks under LD.

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free-running periods.

Figure 5.S3.

Rhythmicity and internal desynchronization of free-running rhythms for each GAL4 manipulation.

Figure 5.S4.

The CRY+ DN1ps and all of the lateral clock neurons together are capable of coherently resetting free-running activity rhythms.

Figure 6.1.

The l-LNvs modulate cAMP levels in the s-LNvs.

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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 isolated neurons or nonneuronal clocks. Using Drosophila as a simple yet conserved 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), which constitute the so-called Evening Oscillator of the clock network. I find that the neuronal activity of LNds is modulated by multiple fast neurotransmitters, and that a group of dorsal clock neurons provides inhibitory synaptic input onto the LNds. Lastly, using genetic and behavioral approaches, I find that while GABAergic inhibition of the clock network functions to promote sleep at night, glutamatergic inhibition of the clock network functions to promote wakefulness during the day.

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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 that had dominated the field for more than a decade, my results show that the clock network consists of multiple independent oscillators, each of which is unified by its neuropeptide output. Finally, 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.

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1.1 Circadian clocks Almost every living organism on this planet has an endogenous timing system, the socalled circadian clock, to help anticipate and adapt to the daily cycle of day and night (MooreEde et al., 1982). This endogenous clock orchestrates daily rhythms in physiology, metabolism, and various behaviors. In many animals, including humans, the master clock resides in the brain and consists of a network of so-called clock neurons, each of which contains a molecular clock that generates oscillations in gene expression with a period of approximately 24 hours (Herzog, 2007). Neuronal connections within this network allow clock neurons to coordinate their molecular clocks and produce coherent and robust circadian rhythms that are uncharacteristic of rhythms driven by isolated clock neurons or non-neuronal clocks (Welsh et al., 2010). A major interest in the field is to understand how clock neurons are physiologically connected and how their molecular oscillations are functionally coordinated.

1.2 Drosophila offers an excellent model for the study of circadian clocks The fruit fly, Drosophila melanogaster, has proved an excellent model for the study of circadian clocks due to its genetic accessibility and relative simplicity. Drosophila displays robust circadian rhythms in activity and rest. Under 12h:12h light:dark cycles, Drosophila displays a characteristic bimodal pattern of activity centered around dawn and dusk, and is relatively inactive in the middle of the day and throughout the night (Fig. 1.1). The rest state of Drosophila shares the core characteristics of mammalian sleep, including increased arousal threshold and the presence of homeostatic regulation among others (Hendricks et al., 2000; Shaw et al., 2000; Huber et al., 2004). Genetic studies in Drosophila have identified many of the molecular clock components and led to a transcription/translation feedback loop model of the molecular clock, which is conserved across a wide spectrum of species (reviewed by Dunlap, 1999). A brief introduction of the molecular clock will be given in Section 1.3. In addition, the clock neurons in the fly central brain have been mapped out. The anatomy and neurochemistry of clock neurons will be introduced in Section 1.4. Current models of the clock network function will be discussed in Section 1.5.

Figure 1.1.

Population average activity profile of wild type Canton-S flies.

A population average activity profile (also known as an “eduction plot”) of wild type Canton-S flies (n=32) under a 12h:12h light:dark cycle. Zeitgeber time (ZT) 0 indicates the time of lightson, and ZT12 indicates the time of lights-off. Note that there is an increase of activity levels before lights-on and before lights-off, which are referred to as morning anticipation and evening anticipation, respectively.

1.3 Molecular clocks Many components of the molecular clock are conserved between flies and mammals (reviewed by Yu and Hardin, 2006). The core components of the Drosophila molecular clock include CLOCK (CLK), CYCLE (CYC), PERIOD (PER), and TIMELESS (TIM), which constitute a transcription/translation feedback loop (Fig. 1.2) (reviewed by Hardin, 2011). In brief, heterodimers of CLK and CYC bind to the E-box elements (canonically 5’-CACGTG-3’) in the per and tim promoters and promote the transcription of per and tim (Hao et al., 1997;

Allada et al., 1998; Darlington et al., 1998; Rutila et al., 1998; McDonald et al., 2001; Wang et al., 2001). PER and TIM proteins accumulate in the cytoplasm, later translocate into the nucleus (Vosshall et al., 1994; Curtin et al., 1995; Saez and Young, 1996; Shafer et al., 2002; Meyer et al., 2006) where they act to suppress CLK/CYC function (Lee et al., 1998, 1999; Bae et al., 2000). The cytoplasmic accumulation of PER is delayed by DOUBLETIME (DBT), which phosphorylates PER and targets PER for degradation, whereas it is facilitated by TIM, which stabilizes PER–DBT complexes and enables the accumulation of DBT–PER–TIM complexes in the cytoplasm (Kloss et al., 1998, 2001; Price et al., 1998). Nuclear translocation of PER and TIM is promoted by phosphorylation of PER by CASEIN KINASE 2 (CK2) (Lin et al., 2002;

Akten et al., 2003) and phosphorylation of TIM by SHAGGY (SGG) (Martinek et al., 2001).

Overall, the negative feedback of PER and TIM on their own transcription results in oscillations in the abundance of their mRNAs and proteins with a period of approximately 24 hours (Hardin et al., 1990; Sehgal et al., 1995).

Figure 1.2.

The core feedback loop of the Drosophila molecular clock.

All the genes, regulatory elements, and proteins are defined in the text. “P” represents phosphorylation site(s). See text for details. This figure is reprinted from Adv. Genet. 74. Hardin, P.E., Molecular genetic analysis of circadian timekeeping in Drosophila. 141–173. Copyright (2011), with permission from Elsevier.

1.4 Anatomy and neurochemistry of the Drosophila clock neuron network The neuroanatomy of the Drosophila clock neuron network has been relatively well characterized. There are approximately 150 clock neurons in the adult fly brain, radically fewer than the tens of thousands of neurons in the mammalian clock centers (Herzog, 2007). Despite its relative simplicity, the fly clock neuron network shares both anatomical and functional similarities with that of mammals (Helfrich-Förster, 2004; Vansteensel et al., 2008). The fly’s clock neurons are divided into nine groups based on their anatomy: (1) four pairs of large ventral lateral neurons (l-LNvs); (2) four pairs of small ventral lateral neurons (s-LNvs); (3) one pair of so-called fifth small ventral lateral neurons (5th s-LNvs); (4) six pairs of dorsal lateral neurons (LNds); (5) two pairs of anterior dorsal neurons group 1 (DN1as); (6) ~15 pairs of posterior dorsal neurons group 1 (DN1ps); (7) two pairs of dorsal neurons group 2 (DN2s); (8) ~40 pairs of dorsal neurons group 3 (DN3s); and (9) three to four pairs of lateral posterior neurons (LPNs) (Fig. 1.3) (Kaneko and Hall, 2000; Shafer et al., 2006). Most of the clock neurons send projections to the dorsal protocerebrum, with a notable exception of the l-LNvs, which send a network of fibers onto the surface of the medulla and also project contralaterally to the opposite brain hemisphere (reviewed by Helfrich-Förster, 2005). The DN1as and subsets of the LNds, DN1ps, and DN3s have additional projections towards the accessory medulla, where the l-LNvs and s-LNvs are located (Kaneko and Hall, 2000; Helfrich-Förster, 2005; Shafer et al., 2006;

Helfrich-Förster et al., 2007). The extensive overlap of their neurites suggests that the various classes of clock neurons may be interconnected. However, the physiological connectivity within the clock neuron network remains largely uncharacterized.

The clock neurons are remarkably heterogeneous in their neurochemistry. Pigmentdispersing factor (PDF), a neuropeptide that is critical for circadian rhythms in locomotor activity, is expressed exclusively by the l-LNvs and the s-LNvs (together called the LNvs) in the central brain (Fig. 1.4) (Helfrich-Förster, 1995; Renn et al., 1999). The receptor for PDF (PDFR) is expressed by about half of the clock neurons, most of which co-express a deep-brain blue light photoreceptor Cryptochrome (CRY) (Fig. 1.4a) (Yoshii et al., 2008; Im and Taghert, 2010; Im et al., 2011). Many neuropeptides are expressed in the clock network in addition to PDF, including neuropeptide F (NFP), short neuropeptide F (sNPF), ion transport peptide (ITP), and IPNamide (IPNa), each of which is expressed by only a small number of clock neurons (Fig. 1.4b) (reviewed by Hermann-Luibl and Helfrich-Förster, 2015). The 5th s-LNv and subsets of the LNds cholinergic (Johard et al., 2009), while some DN1s and DN3s are glutamatergic (Hamasaka et al., 2007) (Fig. 1.4b). This remarkable heterogeneity in neuroanatomy and neurochemistry suggests that the various clock neurons play distinct and diverse roles in the control of circadian rhythms.

Figure 1.3.

A schematic of the clock neurons and their projections in the adult fly brain.

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