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1. The Drosophila circadian clock gene cycle controls the development of clock neurons

   https://doi.org/10.1371/journal.pgen.1011441  

   Biondi, Grace; McCormick, Gina; Fernandez, Maria P (October 2024, PLOS Genetics)

   Ewer, John (Ed.)

   Daily behavioral and physiological rhythms are controlled by the brain’s circadian timekeeping system, a synchronized network of neurons that maintains endogenous molecular oscillations. These oscillations are based on transcriptional feedback loops of clock genes, which inDrosophilainclude the transcriptional activatorsClock (Clk)andcycle (cyc). While the mechanisms underlying this molecular clock are very well characterized, the roles that the core clock genes play in neuronal physiology and development are much less understood. TheDrosophilatimekeeping center is composed of ~150 clock neurons, among which the four small ventral lateral neurons (sLN_vs) are the most dominant pacemakers under constant conditions. Here, we show that downregulating the clock genecycspecifically in thePdf-expressing neurons leads to decreased fasciculation both in larval and adult brains. This effect is due to a developmental role ofcyc, as both knocking downcycor expressing a dominant negative form ofcycexclusively during development lead to defasciculation phenotypes in adult clock neurons.Clkdownregulation also leads to developmental effects on sLNv morphology. Our results reveal a non-circadian role forcyc, shedding light on the additional functions of circadian clock genes in the development of the nervous system. 

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2. Overlapping Central Clock Network Circuitry Regulates Circadian Feeding and Activity Rhythms in Drosophila

   https://doi.org/10.1177/07487304241263734  

   Saurabh, Sumit; Meier, Ruth J; Pireva, Liliya M; Mirza, Rabab A; Cavanaugh, Daniel J (October 2024, Journal of Biological Rhythms)

   The circadian system coordinates multiple behavioral outputs to ensure proper temporal organization. Timing information underlying circadian regulation of behavior depends on a molecular circadian clock that operates within clock neurons in the brain. In Drosophila and other organisms, clock neurons can be divided into several molecularly and functionally discrete subpopulations that form an interconnected central clock network. It is unknown how circadian signals are coherently generated by the clock network and transmitted across output circuits that connect clock cells to downstream neurons that regulate behavior. Here, we have exhaustively investigated the contribution of clock neuron subsets to the control of two prominent behavioral outputs in Drosophila: locomotor activity and feeding. We have used cell-specific manipulations to eliminate molecular clock function or induce electrical silencing either broadly throughout the clock network or in specific subpopulations. We find that clock cell manipulations produce similar changes in locomotor activity and feeding, suggesting that overlapping central clock circuitry regulates these distinct behavioral outputs. Interestingly, the magnitude and nature of the effects depend on the clock subset targeted. Lateral clock neuron manipulations profoundly degrade the rhythmicity of feeding and activity. In contrast, dorsal clock neuron manipulations only subtly affect rhythmicity but produce pronounced changes in the distribution of activity and feeding across the day. These experiments expand our knowledge of clock regulation of activity rhythms and offer the first extensive characterization of central clock control of feeding rhythms. Despite similar effects of central clock cell disruptions on activity and feeding, we find that manipulations that prevent functional signaling in an identified output circuit preferentially degrade locomotor activity rhythms, leaving feeding rhythms relatively intact. This demonstrates that activity and feeding are indeed dissociable behaviors, and furthermore suggests that differential circadian control of these behaviors diverges in output circuits downstream of the clock network. 

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3. Synaptic targets of circadian clock neurons influence core clock parameters

   https://doi.org/10.1126/sciadv.adw4666  

   Scholz-Carlson, Eva; Iyer, Aishwarya R; Nern, Aljoscha; Ewer, John; Fernandez, Maria P (September 2025, Science Advances)

   Neuronal connectivity in the circadian clock network is essential for robust endogenous timekeeping. In the Drosophila circadian clock network, the small ventral lateral neurons (sLNvs) serve as critical pacemakers. Peptidergic communication mediated by the neuropeptide Pigment Dispersing Factor (PDF), released by sLNvs, has been well characterized. In contrast, little is known about the role of the synaptic connections that sLNvs form with downstream neurons. Connectomic analyses revealed that the sLNvs form strong synaptic connections with previously uncharacterized neurons called superior lateral protocerebrum 316 (SLP316). Here, we show that silencing the synaptic output from the SLP316 neurons via tetanus toxin expression shortened the free-running period, whereas hyperexciting them by expressing the bacterial voltage-gated sodium channel resulted in period lengthening. Under light-dark cycles, silencing SLP316 neurons caused lower daytime activity and higher daytime sleep. Our results reveal that the main postsynaptic partners of key Drosophila pacemaker neurons are a nonclock neuronal cell type that regulates the timing of sleep and activity. 

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4. Connectomic analysis of the Drosophila lateral neuron clock cells reveals the synaptic basis of functional pacemaker classes

   https://doi.org/10.7554/eLife.79139  

   Shafer, Orie T; Gutierrez, Gabrielle J; Li, Kimberly; Mildenhall, Amber; Spira, Daphna; Marty, Jonathan; Lazar, Aurel A; Fernandez, Maria de (June 2022, eLife)

   Most organisms on Earth possess an internal timekeeping system which ensures that bodily processes such as sleep, wakefulness or digestion take place at the right time. These precise daily rhythms are kept in check by a master clock in the brain. There, thousands of neurons – some of which carrying an internal ‘molecular clock’ – connect to each other through structures known as synapses. Exactly how the resulting network is organised to support circadian timekeeping remains unclear. To explore this question, Shafer, Gutierrez et al. focused on fruit flies, as recent efforts have systematically mapped every neuron and synaptic connection in the brain of this model organism. Analysing available data from the hemibrain connectome project at Janelia revealed that that the neurons with the most important timekeeping roles were in fact forming the fewest synapses within the network. In addition, neurons without internal molecular clocks mediated strong synaptic connections between those that did, suggesting that ‘clockless’ cells still play an integral role in circadian timekeeping. With this research, Shafer, Gutierrez et al. provide unexpected insights into the organisation of the master body clock. Better understanding the networks that underpin circadian rhythms will help to grasp how and why these are disrupted in obesity, depression and Alzheimer’s disease. 

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5. Neonicotinoids disrupt circadian rhythms and sleep in honey bees

   https://doi.org/10.1038/s41598-020-72041-3  

   Tackenberg, Michael C.; Giannoni-Guzmán, Manuel A.; Sanchez-Perez, Erik; Doll, Caleb A.; Agosto-Rivera, José L.; Broadie, Kendal; Moore, Darrell; McMahon, Douglas G. (December 2020, Scientific Reports)

   Abstract Honey bees are critical pollinators in ecosystems and agriculture, but their numbers have significantly declined. Declines in pollinator populations are thought to be due to multiple factors including habitat loss, climate change, increased vulnerability to disease and parasites, and pesticide use. Neonicotinoid pesticides are agonists of insect nicotinic cholinergic receptors, and sub-lethal exposures are linked to reduced honey bee hive survival. Honey bees are highly dependent on circadian clocks to regulate critical behaviors, such as foraging orientation and navigation, time-memory for food sources, sleep, and learning/memory processes. Because circadian clock neurons in insects receive light input through cholinergic signaling we tested for effects of neonicotinoids on honey bee circadian rhythms and sleep. Neonicotinoid ingestion by feeding over several days results in neonicotinoid accumulation in the bee brain, disrupts circadian rhythmicity in many individual bees, shifts the timing of behavioral circadian rhythms in bees that remain rhythmic, and impairs sleep. Neonicotinoids and light input act synergistically to disrupt bee circadian behavior, and neonicotinoids directly stimulate wake-promoting clock neurons in the fruit fly brain. Neonicotinoids disrupt honey bee circadian rhythms and sleep, likely by aberrant stimulation of clock neurons, to potentially impair honey bee navigation, time-memory, and social communication. 

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