Clock Neurons Research Articles

The Roles of Discrete Populations of Neurons Expressing Short Neuropeptide F in Sleep Induction in Drosophila melanogaster.

Sleep is of vital importance in our lives, yet we are far from understanding the neuronal networks that control the amount and timing of sleep. There is substantial conservation of known sleep-regulating transmitters, allowing for studies in simpler organisms to lead the way in gaining insight into the organization of sleep control circuits. In Drosophila melanogaster, we recently showed that optogenetic activation of neurons that produce the neuropeptide Y (NPY)-related transmitter short neuropeptide F (sNPF) increases time spent asleep. However, sNPF is expressed in several neuronal populations, and thus it is unknown which of those populations play roles in the sleep-promoting effect. In this study, we addressed this issue using a genetic approach to limit optogenetic activation to subsets of sNPF-expressing neurons. We found that sleep promotion was shorter-lived when cryptochrome (CRY)-positive neurons were excluded from being activated. Pigment-dispersing factor (PDF) neurons were not required for sleep promotion, nor were mushroom body (MB) neurons. Acute reactions to a short, 10-s period of optogenetic activation were largely unchanged by excluding activation of the three neuronal populations mentioned above. Together, these results suggest that clock neurons that are CRY-positive and PDF-negative are important contributors to the long-lasting sleep promotion produced by sNPF neuron activation. However, other neurons targeted by the sNPF-GAL4 driver appear to mediate the more rapid behavioral responses. Future studies will seek to identify these additional sNPF neuron populations and to determine how sNPF-expressing clock neurons act in concert with other neuronal circuits to promote sleep.

Clock genes regulate sex pheromone production and male mating ability in Bactrocera dorsalis.

Many animals display physiological and behavioral activities limited to specific times of the day. Certain insects exhibit clear daily rhythms in their mating activities that are regulated by an internal biological clock. However, the specific genetic mechanisms underlying this regulation remain largely unexplored. Mating in the fruit fly Bactrocera dorsalis exhibits a daily rhythm and is dependent on sex pheromones produced in the male rectum. We used transcriptome sequencing and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 techniques to understand whether the daily rhythmicity of mating in B. dorsalis and sex pheromone production in the rectum are regulated by clock genes. The results showed that the production of sex pheromones by B. dorsalis males is rhythmic (low during the day and high at night) and is influenced by clock genes. Knockout of the clock genes cryptochrome 1 (cry1) and timeless (tim) reduced the production of sex pheromones and significantly impaired mating ability in males. In addition, quantitative polymerase chain reaction results from 5 different tissues showed cry1 was highly expressed in the head, whereas tim was highly expressed in both the head and rectum (a key site for male sex pheromone production). Transcriptome analysis confirmed that cry1 (head) and tim (head and rectum) exhibit rhythmic expressions consistent with sex pheromone rhythmicity. These results suggest that cry1 may be related to a central clock neuron (like the suprachiasmatic nucleus), whereas the rhythmic expression of tim in the rectum indicates the potential presence of peripheral oscillators. Our study reveals new targets and ideas for improved control of the fruit fly.

Prokineticin 2 protein is diurnally expressed in PER2-containing clock neurons in the mouse suprachiasmatic nucleus.

Expression of prokineticin 2 (PK2) mRNA in the suprachiasmatic nucleus (SCN), also knownas the brain's clock, exhibits circadian oscillations with peak levels midday, zeitgeber time (ZT) 4, and almost undetectable levels during night. This circadian expression profile has substantially contributed to the suggested role of PK2 as an SCN output molecule involved in transmitting circadian rhythm of behavior and physiology. Due to unreliable specificity of PK2 antibodies, the 81 amino acid protein has primarily been studied at the mRNA level and correlation between circadian oscillating mRNAs and protein products are infrequent. Hence, data on PK2 protein expression in the SCN is lacking. In this study a thorough validation of a commercial PK2 antibody for immunohistochemistry (IHC) was performed followed by fluorescence IHC on SCN mouse brain sections at six consecutive ZTs over a 24-hcycle (12:12 light-dark, ZT0 =light ON whereas ZT12 =light OFF). Data were visualized and processed using confocal microscopy. Results showed that PK2 protein expression diurnally oscillates with calculated peak expression ZT5:40 ± 1:40 h. Opposite than described for PK2 mRNA, PK2 immunoreactivity was detectable at all times during the 24-hcycle. PK2 was primarily located in neurons of the shell compartment and > 80 % of these neurons co-expressed the core clock protein PER2. In conclusion, PK2 protein expression oscillates as the mRNA, supporting the suggested role of PK2 as a SCN molecule involved in circadian rhythm regulation.

Neuropeptide-dependent spike time precision and plasticity in circadian output neurons.

Circadian rhythms influence various physiological and behavioral processes such as sleep-wake cycles, hormone secretion, and metabolism. In Drosophila, an important set of circadian output neurons are called pars intercerebralis (PI) neurons, which receive input from specific clock neurons called DN1. These DN1 neurons can further be subdivided into functionally and anatomically distinctive anterior (DN1a) and posterior (DN1p) clusters. The neuropeptide diuretic hormones 31 (Dh31) and 44 (Dh44) are the insect neuropeptides known to activate PI neurons to control activity rhythms. However, the neurophysiological basis of how Dh31 and Dh44 affect circadian clock neural coding mechanisms underlying sleep in Drosophila is not well understood. Here, we identify Dh31/Dh44-dependent spike time precision and plasticity in PI neurons. We first find that a mixture of Dh31 and Dh44 enhanced the firing of PI neurons, compared to the application of Dh31 alone and Dh44 alone. We next find that the application of synthesized Dh31 and Dh44 affects membrane potential dynamics of PI neurons in the precise timing of the neuronal firing through their synergistic interaction, possibly mediated by calcium-activated potassium channel conductance. Further, we characterize that Dh31/Dh44 enhances postsynaptic potentials in PI neurons. Together, these results suggest multiplexed neuropeptide-dependent spike time precision and plasticity as circadian clock neural coding mechanisms underlying sleep in Drosophila.

Overexpression of α-synuclein in PDF neurons alters sleep-wake pattern by regulating lipid metabolism in Drosophila.

Parkinson's disease (PD) is a complex neurodegenerative disorder, characterized by the aggregation of α-synuclein (α-syn). Current research increasingly indicates the prevalence of sleep-wake disorders in early-stage PD, although the underlying pathogenic mechanisms remain unclear. In this study, transgenic Drosophila models were utilized to observe excessive daytime sleepiness and impaired anticipation in flies overexpressing α-syn in pan-neurons and circadian clock neurons. Additionally, deficits in projection of Pigment Dispersing Factor (PDF) neuron terminals, which are involved in Drosophila sleep and circadian rhythm, were identified. An imbalance in lipid metabolism homeostasis was detected in the brains of α-syn overexpressing mutants. Ultimately, the inhibition of Sterol Regulatory Element-Binding Protein (SREBP) activity led to an improvement in the reduced daytime sleep duration phenotype. Our results suggest that lipid pathways play a role in sleep-wake disorders triggered by α-syn mutation and aggregation, thereby providing valuable insights into potential therapeutic avenues for disrupted sleep patterns associated with PD.

Reassessing the involvement of the CREB pathway in the circadian clock of Drosophila melanogaster

Circadian clocks are ubiquitous in almost all organisms on Earth and many key genes are highly conserved among species. In the mammalian suprachiasmatic nucleus, the cAMP response element binding protein (CREB) pathway is known to play a crucial role in conveying light-input to the transcription of clock genes. The fruit fly Drosophila melanogaster also expresses two Creb proteins, CrebA and CrebB, which have been associated with the circadian clock. For example, Drosophila Creb has been suggested to constitute a molecular link between neuronal excitability and clock gene transcription. In this study we subjected flies with clock cell specific CrebA or CrebB mutations to circadian behavioral and bioluminescence assays. Surprisingly, we found that neither loss of CrebA or CrebB did affect free-running locomotor behavior, rhythmic period oscillations in clock neurons, or light-dependent synchronization. In conclusion our findings question the conserved circadian role of the Creb pathway in Drosophila and encourage further studies to elucidate its potential function within insect circadian clocks.

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

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 in Drosophila include the transcriptional activators Clock (Clk) and cycle (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. The Drosophila timekeeping center is composed of ~150 clock neurons, among which the four small ventral lateral neurons (sLNvs) are the most dominant pacemakers under constant conditions. Here, we show that downregulating the clock gene cyc specifically in the Pdf-expressing neurons leads to decreased fasciculation both in larval and adult brains. This effect is due to a developmental role of cyc, as both knocking down cyc or expressing a dominant negative form of cyc exclusively during development lead to defasciculation phenotypes in adult clock neurons. Clk downregulation also leads to developmental effects on sLNv morphology. Our results reveal a non-circadian role for cyc, shedding light on the additional functions of circadian clock genes in the development of the nervous system.

Suprachiasmatic nucleus VIPergic fibers show a circadian rhythm of expansion and retraction

In animals, overt circadian rhythms of physiology and behavior are centrally regulated by a circadian clock located in specific brain regions. In the fruit fly Drosophila and in mammals, these clocks rely on single-cell oscillators, but critical for their function as central circadian pacemakers are network properties that change dynamically throughout the circadian cycle as well as in response to environmental stimuli.1,2,3 In the fly, this plasticity involves circadian rhythms of expansion and retraction of clock neuron fibers.4,5,6,7,8,9,10,11,12,13,14 Whether these drastic structural changes are a universal property of central neuronal pacemakers is unknown. To address this question, we studied neurons of the mouse suprachiasmatic nucleus (SCN) that express vasoactive intestinal polypeptide (VIP), which are critical for the SCN to function as a central circadian pacemaker. By targeting the expression of the fluorescent protein tdTomato to these neurons and using tissue clearing techniques to visualize all SCN VIPergic neurons and their fibers, we show that, similar to clock neurons in the fly, VIPergic fibers undergo a daily rhythm of expansion and retraction, with maximal branching during the day. This rhythm is circadian, as it persists under constant environmental conditions and is present in both males and females. We propose that circadian structural remodeling of clock neurons represents a key feature of central circadian pacemakers that is likely critical to regulate network properties, the response to environmental stimuli, and the regulation of circadian outputs.

Day-night gene expression reveals circadian gene disco as a candidate for diel-niche evolution in moths.

Temporal ecological niche partitioning is an underappreciated driver of speciation. While insects have long been models for circadian biology, the genes and circuits that allow adaptive changes in diel-niches remain poorly understood. We compared gene expression in closely related day- and night-active non-model wild silk moths, with otherwise similar ecologies. Using an ortholog-based pipeline to compare RNA-Seq patterns across two moth species, we find over 25 pairs of gene orthologs showing differential expression. Notably, the gene disco, involved in circadian control, optic lobe and clock neuron development in Drosophila, shows robust adult circadian mRNA cycling in moth heads. Disco is highly conserved in moths and has additional zinc-finger domains with specific nocturnal and diurnal mutations. We propose disco as a candidate gene for the diversification of temporal diel-niche in moths.

A Detailed Re-Examination of the Period Gene Rescue Experiments Shows That Four to Six Cryptochrome-Positive Posterior Dorsal Clock Neurons (DN1p) of Drosophila melanogaster Can Control Morning and Evening Activity.

Animal circadian clocks play a crucial role in regulating behavioral adaptations to daily environmental changes. The fruit fly Drosophila melanogaster exhibits 2 prominent peaks of activity in the morning and evening, known as morning (M) and evening (E) peaks. These peaks are controlled by 2 distinct circadian oscillators located in separate groups of clock neurons in the brain. To investigate the clock neurons responsible for the M and E peaks, a cell-specific gene expression system, the GAL4-UAS system, has been commonly employed. In this study, we re-examined the two-oscillator model for the M and E peaks of Drosophila by utilizing more than 50 Gal4 lines in conjunction with the UAS-period16 line, which enables the restoration of the clock function in specific cells in the period (per) null mutant background. Previous studies have indicated that the group of small ventrolateral neurons (s-LNv) is responsible for controlling the M peak, while the other group, consisting of the 5th ventrolateral neuron (5th LNv) and the three cryptochrome (CRY)-positive dorsolateral neurons (LNd), is responsible for the E peak. Furthermore, the group of posterior dorsal neurons 1 (DN1p) is thought to also contain M and E oscillators. In this study, we found that Gal4 lines directed at the same clock neuron groups can lead to different results, underscoring the fact that activity patterns are influenced by many factors. Nevertheless, we were able to confirm previous findings that the entire network of circadian clock neurons controls M and E peaks, with the lateral neurons playing a dominant role. In addition, we demonstrate that 4 to 6 CRY-positive DN1p cells are sufficient to generate M and E peaks in light-dark cycles and complex free-running rhythms in constant darkness. Ultimately, our detailed screening could serve as a catalog to choose the best Gal4 lines that can be used to rescue per in specific clock neurons.

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

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.

Spatiotemporal changes in Netrin/Dscam1 signaling dictate axonal projection direction in Drosophila small ventral lateral clock neurons.

Axon projection is a spatial- and temporal-specific process in which the growth cone receives environmental signals guiding axons to their final destination. However, the mechanisms underlying changes in axonal projection direction without well-defined landmarks remain elusive. Here, we present evidence showcasing the dynamic nature of axonal projections in Drosophila's small ventral lateral clock neurons (s-LNvs). Our findings reveal that these axons undergo an initial vertical projection in the early larval stage, followed by a subsequent transition to a horizontal projection in the early-to-mid third instar larvae. The vertical projection of s-LNv axons correlates with mushroom body calyx expansion, while the s-LNv-expressed Down syndrome cell adhesion molecule (Dscam1) interacts with Netrins to regulate the horizontal projection. During a specific temporal window, locally newborn dorsal clock neurons secrete Netrins, facilitating the transition of axonal projection direction in s-LNvs. Our study establishes a compelling in vivo model to probe the mechanisms of axonal projection direction switching in the absence of clear landmarks. These findings underscore the significance of dynamic local microenvironments in the complementary regulation of axonal projection direction transitions.

The CoREST complex regulates multiple histone modifications temporal-specifically in clock neurons.

Epigenetic regulation is important for circadian rhythm. In previous studies, multiple histone modifications were found at the Period (Per) locus. However, most of these studies were not conducted in clock neurons. In our screen, we found that a CoREST mutation resulted in defects in circadian rhythm by affecting Per transcription. Based on previous studies, we hypothesized that CoREST regulates circadian rhythm by regulating multiple histone modifiers at the Per locus. Genetic and physical interaction experiments supported these regulatory relationships. Moreover, through tissue-specific chromatin immunoprecipitation assays in clock neurons, we found that the CoREST mutation led to time-dependent changes in corresponding histone modifications at the Per locus. Finally, we proposed a model indicating the role of the CoREST complex in the regulation of circadian rhythm. This study revealed the dynamic changes of histone modifications at the Per locus specifically in clock neurons. Importantly, it provides insights into the role of epigenetic factors in the regulation of dynamic gene expression changes in circadian rhythm.

The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the Drosophila central nervous system

Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.

The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the Drosophila central nervous system.

Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.

Circadian Vesicle Capture Prepares Clock Neuron Synapses for Daily Phase-Delayed Neuropeptide Release.

Drosophila sLNv clock neurons release the neuropeptide PDF to control circadian rhythms. Strikingly, PDF content in sLNv terminals is rhythmic with a peak in the morning hours prior to the onset of activity-dependent release. Because synaptic PDF accumulation, rather than synaptic release, aligns with the late-night elevations in both sLNv neuron excitability and Ca2+, we explored the dependence of presynaptic neuropeptide accumulation on neuropeptide vesicle transport, electrical activity and the circadian clock. Live imaging reveals that anterograde axonal transport is constant throughout the day and capture of circulating neuropeptide vesicles rhythmically boosts presynaptic neuropeptide content hours prior to release. The late-night surge in vesicle capture, like release, requires electrical activity and results in a large releasable pool of presynaptic vesicles to support the later burst of neuropeptide release. The circadian clock is also required suggesting that it controls the switch from vesicle capture to exocytosis, which are normally coupled activity-dependent processes. This toggling of activity transduction maximizes rhythmic synaptic neuropeptide release needed for robust circadian behavior and resolves the previously puzzling delay in timing of synaptic neuropeptide release relative to changes in sLNv clock neuron physiology.

Contribution of neurons that express fruitless and Clock transcription factors to behavioral rhythms and courtship.

Animals need to integrate information across neuronal networks that direct reproductive behaviors and circadian rhythms. In Drosophila, the master regulatory transcription factors that direct courtship behaviors and circadian rhythms are co-expressed in a small set of neurons. In this study we investigate the role of these neurons in both males and females. We find sex-differences in the number of these fruitless and Clock -expressing neurons ( fru ∩ Clk neurons) that is regulated by male-specific Fru. We assign the fru ∩ Clk neurons to the electron microscopy connectome that provides high resolution structural information. We also discover sex-differences in the number of fru -expressing neurons that are post-synaptic targets of Clk -expressing neurons, with more post-synaptic targets in males. When fru ∩ Clk neurons are activated or silenced, males have a shorter period length. Activation of fru ∩ Clk neurons also changes the rate a courtship behavior is performed. We find that activation and silencing fru ∩ Clk neurons impacts the molecular clock in the sLNv master pacemaker neurons, in a cell-nonautonomous manner. These results reveal how neurons that subserve the two processes, reproduction and circadian rhythms, can impact behavioral outcomes in a sex-specific manner.

Clock-dependent chromatin accessibility rhythms regulate circadian transcription.

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

Tango-seq: overlaying transcriptomics on connectomics to identify neurons downstream of Drosophila clock neurons.

Knowing how neural circuits change with neuronal plasticity and differ between individuals is important to fully understand behavior. Connectomes are typically assembled using electron microscopy, but this is low throughput and impractical for analyzing plasticity or mutations. Here, we modified the trans-Tango genetic circuit-tracing technique to identify neurons synaptically downstream of Drosophila s-LNv clock neurons, which show 24hr plasticity rhythms. s-LNv target neurons were labeled specifically in adult flies using a nuclear reporter gene, which facilitated their purification and then single cell sequencing. We call this Tango-seq, and it allows transcriptomic data - and thus cell identity - to be overlayed on top of anatomical data. We found that s-LNvs preferentially make synaptic connections with a subset of the CNMa+ DN1p clock neurons, and that these are likely plastic connections. We also identified synaptic connections between s-LNvs and mushroom body Kenyon cells. Tango-seq should be a useful addition to the connectomics toolkit.

A subclass of evening cells promotes the switch from arousal to sleep at dusk

Animals exhibit rhythmic patterns of behavior that are shaped by an internal circadian clock and the external environment. Although light intensity varies across the day, there are particularly robust differences at twilight (dawn/dusk). These periods are also associated with major changes in behavioral states, such as the transition from arousal to sleep. However, the neural mechanisms by which time and environmental conditions promote these behavioral transitions are poorly defined. Here, we show that the E1 subclass of Drosophila evening clock neurons promotes the transition from arousal to sleep at dusk. We first demonstrate that the cell-autonomous clocks of E2 neurons primarily drive and adjust the phase of evening anticipation, the canonical behavior associated with "evening" clock neurons. We next show that conditionally silencing E1 neurons causes a significant delay in sleep onset after dusk. However, rather than simply promoting sleep, activating E1 neurons produces time- and light-dependent effects on behavior. Activation of E1 neurons has no effect early in the day but then triggers arousal before dusk and induces sleep after dusk. Strikingly, these activation-induced phenotypes depend on the presence of light during the day. Despite their influence on behavior around dusk, invivo voltage imaging of E1 neurons reveals that their spiking rate and pattern do not significantly change throughout the day. Moreover, E1-specific clock ablation has no effect on arousal or sleep. Thus, we suggest that, rather than specifying "evening" time, E1 neurons act, in concert with other rhythmic neurons, to promote behavioral transitions at dusk.