CLOCK-dependent pathway in a single pair of LNd neurons instruct circadian-independent interval timing behavior.

CLOCK-dependent pathway in a single pair of LNd neurons instruct circadian-independent interval timing behavior. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CLOCK-dependent pathway in a single pair of LNd neurons instruct circadian-independent interval timing behavior. Woo Jae Kim, Hongyu Miao, Zekun Wu, Yanan Wei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7471909/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Interval timing is a cognitive ability essential for behaviors such as mating, foraging, and decision-making, and it is distinct from circadian rhythm regulation. Despite the involvement of circadian clock genes in both interval timing and circadian rhythms, the mechanisms differentiating these functions remain unclear. Using Drosophila as a model, we demonstrate that the CLK/CYC heterodimer, but not PER/TIM, is essential for interval timing. Neuronal CLK/CYC expression is necessary and sufficient for sexual experience-dependent shorter mating duration (SMD) behavior. We identified that CLK/CYC expression in a single pair of ITP-positive LN d neurons is pivotal for SMD. These neurons are glutamatergic with output circuits to central brain regions. CLK variants lacking DNA binding motifs dissociate circadian rhythms from interval timing and sleep behaviors in these neurons. Our study uncovers a specialized circuit for interval timing and highlights the non-circadian functions of circadian clock genes. Biological sciences/Physiology Biological sciences/Cell biology/Circadian rhythms CLK/CYC heterodimer mating duration circadian rhythm interval timing pacemaker sleep circadian-independent timing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Statement of Significance This study in Drosophila demonstrates that the CLK/CYC heterodimer is crucial for interval timing, distinct from circadian rhythm regulation. The research pinpoints a specific pair of neurons that are critical for shortened mating duration behavior, and it suggests that CLK variants can influence interval timing dissociation from circadian rhythm and sleep behaviors. Highlights CLK/CYC heterodimer are essential for interval timing. Specialized mechanism of interval timing behavior revealed. Identified a pair of glutamatergic ITP-LN d neurons that independently regulate both interval timing and sleep. Revealed a novel mechanism by which CLK variants regulate sleep and interval timing. Introduction The precise measurement of time intervals, ranging from seconds to minutes, is a critical cognitive function that is essential for behaviors such as mating, foraging, and navigation 1 – 3 . Interval timing is thought to encompass an internal clock mechanism that depends on the synchronization of pacemaker-accumulator circuits with memory circuits in the brain 4 – 7 . Drosophila melanogaster , with its well-characterized neural circuits and molecular mechanisms, serves as an excellent model organism for the study of interval timing 8 . Interval timing has been identified in the mating behaviors of Drosophila , particularly in the duration of male mating. Males demonstrate the ability to adjust their mating duration based on contextual experiences, suggesting the formation of long-term memory to optimize their sexual investment 9 – 13 . The mating duration of male fruit flies serves as an excellent model for investigating interval timing, as it is influenced by both internal states and environmental contexts. Previous research by our group 9 – 11 , 14 – 16 and others 17 – 19 has established robust frameworks for studying mating duration using advanced genetic tools, enabling the dissection of neural circuits underlying interval timing. Notably, males exhibit prolonged mating duration when exposed to rival environments 9 , 10 , 20 . In contrast, they display shortened mating duration (SMD) behavior under sexually saturated conditions, where they reduce their mating investment 11 . These findings highlight the adaptability of mating behavior in response to social and environmental cues, providing a valuable system for exploring the neural and molecular mechanisms of interval timing. The circadian rhythm in Drosophila melanogaster has been a subject of intense research for several decades, and significant progress has been made in understanding the molecular and genetic underpinnings of this biological clock 21 – 23 . Key genes such as period (per) , timeless (tim) , Clock (Clk) and cycle (cyc) have been identified and characterized, providing insights into the feedback loops that regulate the circadian rhythm at the molecular level 24 . These genes form the core of the molecular clock, with their timed expression, localization, post-transcriptional modification, and function being critical for maintaining the circadian cycle. Various regulators, including phosphatases and kinases, act on different steps of this feedback loop to ensure strong and accurately timed rhythms 25 – 27 . Circadian clock plays a role in regulating sleep by ensuring that it occurs at the appropriate time. However, the quantity and quality of sleep are also influenced by other systems that maintain a balance between sleep and wakefulness. Sleep is regulated by separate genetic and cellular mechanisms that control the need for sleep, adjust to environmental signals, and react to extended periods of alertness. In addition, the regulation of sleep according to the body's internal clock requires multiple groups of cells and molecules that coordinate sleep and waking cycles at precise times of the day, rather than being governed by a single oscillating signal 28 , 29 . Circadian rhythm pertains to the 24-hour cycle that governs biological processes, whereas sleep is a reversible state characterized by diminished activity and responsiveness. Interval timing refers to the measurement of durations and intervals, such as the mating duration or foraging actions 20 , 30 , 31 . The primary distinction lies in the periodicity of circadian rhythms and sleep, which adhere to fixed daily patterns, while interval timing is adaptable and not constrained by a 24-hour cycle, enabling organisms to measure time intervals according to their specific requirements 32 . Although the connection between circadian timing and different physiological processes has been extensively studied, the genetic understanding of the interaction between circadian timing and interval timing remains incomplete. Our previous research demonstrated that circadian clock genes, specifically per and tim , rather than Clk and cyc , modulate the rival-induced longer-mating-duration (LMD) behavior, a distinct form of interval timing critical for maximizing sperm competition 9 , 10 , 20 . In this study, we investigate the role of the Clk / cyc gene complex and associated factors within a single pair of LN d neurons in modulating the sexually experienced-dependent shorter-mating-duration (SMD) behavior, which has been previously shown to be elicited by gustatory and pheromonal cues from females 11 . Results Neuronal expression of CLK/CYC heterodimer is necessary and sufficient to generate sexual experience-mediated reduced mating investment. In the context of core circadian rhythm genes, it was observed that mutations in the per or tim genes, as well as compound mutations in both per and tim , did not disrupt SMD behavior (Fig. 1 A-C). However, mutations in the Clk and cyc genes resulted in the absence of SMD behavior (Fig. 1 E-F). Additionally, mutations in the cryptochrome ( cry ) gene ( cry 03 ) and the sleepless mutant of quiver ( qvr 01 ) 33 did not alter the SMD behavior (Fig. 1 D and Fig. S1A). These findings suggest that the CLK/CYC heterodimer is uniquely involved in interval timing behaviors among the core circadian rhythm gene components. The targeted RNAi-mediated knockdown of Clk or cyc in neurons resulted in the disruption of SMD behavior (Fig. 1 I-J and Fig. S1B-C). Additionally, analysis of the fly RNAseq dataset platform, fly SCope, predicted co-expression of Clk and cyc in specific neuronal populations (Fig. 1 G) 34 . While co-expression of Clk and cyc in glial cells was observed (Fig. 1 H), knockdown of these genes in glial populations did not affect SMD behavior (Fig. 1 K-L), indicating that glial expression of CLK/CYC is not essential for interval timing behavior. Furthermore, knockdown of Clk in epithelial, gut, Malpighian tubules (MTs), muscle, hemocyte, and intestinal stem cells (ISCs), despite their high expression of Clk and cyc , did not alter SMD behavior (Fig. 1 M-V and Fig. S1K-L). Collectively, the genetic control experiments and the testing of independent RNAi lines suggest that only neuronal expression of Clk and cyc is required for the sexual experience-mediated reduction in mating investment (Fig. S1D-J). These findings underscore the specificity of the neuronal CLK/CYC expression in mediating the behavioral changes associated with sexual experience, highlighting the importance of cell-type-specific gene expression in the regulation of complex behaviors. The expression of CLK within NPF-expressing cry-positive circadian neurons in the brain is critical for interval timing behavior. Clk and cyc are robustly expressed in neuronal populations throughout the Drosophila body (Fig. 2 A). However, targeted knockdown of Clk in all neurons except those of the ventral nerve cord (VNC) still resulted in the disruption of SMD behavior (Fig. 2 B), indicating that Clk expression within the VNC is not essential for the generation of SMD behavior. Furthermore, the selective knockdown of Clk or cyc in the brain alone was sufficient to impair SMD behavior (Fig. 2 C-E), suggesting that among the diverse neuronal populations, only CLK/CYC expression within the brain is necessary for the manifestation of SMD behavior. Through a genetic rescue approach targeting the cyc gene, we discovered that neuronal expression of cyc , with the exception of the PDF-expressing lateral ventral neurons (LN v ), was sufficient to restore SMD behavior (Fig. 2 F-H), despite the rescue's inability to restore circadian rhythmicity 35 . Additionally, the knockdown of cyc specifically in PDF neurons did not affect SMD behavior (Fig. 2 I), implying that CLK/CYC expression in core oscillator cells, such as sLN v and lLN v , is not required for the generation of interval timing behavior. Utilizing a suite of GAL4 drivers specific to clock cells in conjunction with Clk-RNAi (Fig. 2 J and Fig. S2A), we conducted a detailed analysis revealing that the expression of Clk and cyc in neurons positive for neuropeptide F (NPF) and cry is essential for the generation of SMD behavior (Fig. 2 K-O and Fig. S2B-C). Knockdown of Clk in neurons that are NPF-positive but cry-negative did not impair SMD behavior (Fig. 2 P), indicating that the expression of Clk in both cry-positive and NPF-positive neurons is critical for the manifestation of SMD behavior. NPF-expressing cry-positive neurons are located in the LN d and DN regions of both male and female brains (Fig. 2 Q). However, a unique male brain phenotype was observed, with NPF-positive and CRY-positive neuronal processes extending near the suboesophageal ganglion (SOG) region (Fig. 2 Q), which is known to be important for processing taste information. Previous research from our laboratory has demonstrated the presence of male-specific CRY-positive and NPF-positive LN d neurons 36 , and the role of these neurons’ sexual dimorphism in mating behavior has been reported 37 , 38 . These findings suggest that the sexual dimorphism in the distribution of NPF-expressing cry-positive neurons may underlie sex-specific differences in the processing of sensory information related to SMD behavior. Our results collectively identify a selective requirement for CLK/CYC within specific clock neurons in the modulation of SMD behavior. Two pair of clock neurons are associated with CLK function to generate interval timing. Utilizing the recently published transcriptomic taxonomy dataset for Drosophila circadian neurons 39 , we conducted a targeted screen to identify the minimal subset of Clk-expressing neurons necessary for the generation of SMD (Table. S1). Our analysis revealed that CLK expression within the GAL4 R54D11 -labeled ITP-LN d and 5th sLN v neurons are pivotal for interval timing behavior (Table S1, Fig. 3 A and S3 A). Knockdown of Clk specifically in adults or excluding of the VNC using the GAL4 R54D11 driver was sufficient to impair SMD (Fig. 3 B-D and Fig. S3B), indicating that adult brain expression of Clk in ITP-LN d and 5th sLN v neurons are required to generate SMD behavior. No sexual dimorphism was observed in the expression pattern of the GAL4 R54D11 driver within the brain (Fig. S3C-D). Furthermore, the disruption of SMD following Clk knockdown with the ITP T2A -GAL4 and LN d drivers ( Mai179-GAL4 , marking ITP-LN d , 5th -sLN v , and 2 sNPF-LN d neurons) confirmed that the GAL4 R54D11 driver-labelled neurons are indeed ITP-LN d and 5th sLN v neurons (Fig. 3 E-F). ITP is a key endocrine regulator of water homeostasis in Drosophila 40 . Within the brain and VNC, only a single pair of neurons expressing ITP is located in the LN d region (Fig. 3 H). Analysis of the Fly SCope dataset suggests the presence of a restricted number of neurons that are triply positive for ITP , NPF , and Clk expression within the brain, but not in the VNC (Fig. S3K-N). Knockdown of cyc , induced apoptosis, hyperexcitation, or inhibition of synaptic transmission in ITP-LN d and 5th sLN v neurons, consistently disrupted SMD behavior (Fig. S3E-I). However, inhibiting the neuronal activity of ITP-LN d and 5th sLN v neurons via voltage-gated potassium channel, KCNJ2 resulted in developmental lethality, indicating that their neuronal function is essential for development as well (Fig. S3G). The feminization of ITP-LN d and 5th sLN v neurons through the expression of UAS-tra F had no effect on interval timing behavior (Fig. S3J), demonstrating that the sexual dimorphism of these LN d neurons does not play a role in regulating interval timing. Notably, knockdown of ITP within ITP-LN d neurons did not affect SMD behavior (Fig. 3 G), suggesting that the role of ITP in ITP-LN d neurons is distinct from its function in interval timing. These findings implicate a specialized subset of ITP-LN d and 5th sLN v neurons as a critical node in the circuitry governing interval timing, while also highlighting the diverse functions of ITP in neuronal physiology. The glutamatergic output circuits originating from ITP-LN d neurons are instrumental in the generation of interval timing. Our findings reveal that the expression of flippase in NPF-positive neurons with a UAS-stop cassette can confine the expression of GAL4 R54D11 to four specific neurons in the brain, including the ITP-LN d and 5th -sLN v neurons (Fig. 4 A). Inhibiting the activity of these neurons disrupts SMD behavior without causing developmental lethality (Fig. 4 B), indicating that the KCNJ2-mediated developmental lethality observed with GAL4 R54D11 is likely due to its expression in the VNC (Fig. S3G and Fig. 4 A). Furthermore, hyperexcitation induced by NaChBac, inhibition of synaptic transmission by TNT, or the knockdown of Clk via Clk-RNAi in these four brain neurons all resulted in the disruption of SMD behavior (Fig. 4 C-E), emphasizing the critical role of CLK function and neuronal activity in the ITP-LN d neurons in the brain for the generation of interval timing. Utilizing the fly SCope RNA seq dataset and the FlyWire connectome dataset platform, we inferred the expression patterns of enzymes responsible for neurotransmitter synthesis (Fig. S4A-B) and identified the output neurons from the ITP-LN d 34 , 41 – 48 . The FlyWire connectome data suggests that ITP-LN d neurons project to central brain circuits (Fig. 4 F) and slightly more to the contralateral brain (Fig. S4C). Although the Fly SCope data analysis was inconclusive (Fig. S4A-B), the FlyWire connectome dataset analysis clearly demonstrated that the output circuits from the ITP-LN d neurons consist of glutamatergic and possibly cholinergic co-transmission (Fig. 4 G). Given that the FlyWire connectome data predict the 5th sLN v to be serotonergic neurons (Fig. 4 G) and that ITP-LN d neurons have been confirmed to be non-cholinergic LN d neurons 49 , we conclude that the single pair of ITP-LN d neurons are glutamatergic. The knockdown of VGlut specifically in these neurons disrupts SMD behavior, and the expression of Clk-RNAi in the non-glutamatergic subset of GAL4 R54D11 neurons rescues the disrupted SMD behavior (Fig. 4 H-I). Moreover, knockdown of Clk only in GAL4 R54D11 -positive and glutamatergic neurons is sufficient to disrupt SMD behavior (Fig. 4 J), indicating the crucial role of Clk expression in a single pair of ITP-LN d neurons. Optogenetic activation of GAL4 R54D11 neurons can induce a reduction in mating duration without prior sexual experience (Fig. 4 K), suggesting that artificial neuronal activation of these neurons can mimic the sexual experience-mediated internal states of pacemaker circuits. Variants of the CLK protein that undergo alternative splicing may dissociate circadian rhythms from interval timing within ITP-LN d and 5th -sLN v neurons. We have previously demonstrated that rival-induced prolonged mating duration (LMD), a distinct form of interval timing behavior in male flies, persists even in arrhythmic conditions such as continuous light exposure for five days 10 (Fig. 5 A). Similarly, SMD behavior remains intact under arrhythmic conditions (Fig. 5 B). In the same condition, the rhythmic activity and sleep of flies was completely disorganized (Fig. S5A-B), indicating that interval timing behaviors are independent of the circadian rhythm. We previously conducted experiments at various times throughout the day with Canton-S flies and observed normal SMD behavior 11 , which is consistent with the reported results. The influence of temperature and feeding on the alternative splicing of clock gene products has been explored 50 – 52 , but the role of CLK mRNA variants in regulating various timing behaviors has not been previously reported. The Clk gene produces five distinct mRNA transcripts, Clk-RA , RD , RF , RG , and RH , with Clk-RG and RH lacking the first three exons and thus unable to bind DNA. Our study revealed that Clk ADF -RNAi specifically targets Clk-RA , RB , and RF transcripts, which contain DNA-binding motifs. Surprisingly, the expression of Clk ADF -RNAi did not disrupt SMD behavior, or the circadian rhythm compared to genetic controls (Fig. 5 D-F and Fig. S5C). This suggests that CLK proteins with DNA-binding motifs are not essential for generating interval timing behaviors in ITP-LN d neurons. We next investigated whether CLK expression in ITP-LN d neurons is linked to sleep behaviors. Knockdown of Clk in ITP-LN d neurons partially reduced the duration of sleep during both day and night (Fig. 5 G-J). Remarkably, when we selectively knocked down Clk transcripts with DNA-binding motifs in ITP-LN d neurons (Fig. 5 C), daytime but not nighttime sleep duration decreased (Fig. 5 K-N). This observation clearly indicates that different variants of CLK proteins have differential effects on sleep behavior. We speculate that CLK proteins lacking the DNA-binding motif may play a specific role in regulating sleep and interval timing, suggesting a novel molecular mechanism for the regulation of these behaviors. The CLK/CYC heterodimer, a transcription factor complex, is specifically linked to interval timing and sleep behaviors. Although circadian clock genes are primarily known for their role in regulating circadian rhythms, they also perform various other functions 53 – 55 . Sleep timing is influenced by the circadian clock, yet the two processes can be dissociated; mutations in core clock genes often lead to fragmented sleep patterns but do not necessarily affect overall sleep duration 28 . The cyc 01 and Clk Jrk mutants displayed distinct sleep pattern abnormalities and life span changes, with the cyc 01 mutation exhibiting sexually dimorphic effects on sleep compensation 56 . The CLK/CYC heterodimer transcription factors play a pivotal role in the regulation of numerous clock gene transcripts. However, the specific functions of these target genes in various timing behaviors are not yet fully elucidated (Fig. 6 A) 23 , 57 . Our findings suggest that the CLK/CYC heterodimer lacking a DNA-binding motif is a critical regulator of interval timing and sleep, but not necessarily of circadian rhythm. Consequently, we selected several essential clock genes known to modulate circadian rhythm and reassessed their roles in interval timing and sleep within ITP-LN d neurons. Knockdown of cwo ( clockwork orange ) and Cipc ( Clock interacting protein circadian ) had no impact on SMD behavior, while vri ( vrille ) and Pdp1 ( PAR-domain protein 1 ) knockdown disrupted SMD (Fig. 6 B-D and Fig. S6A). Notably, PER/TIM-related clock genes, such as dco ( discs overgrown ), jet ( jetlag ), Atx2 ( Ataxin-2 ), and cry , did not affect SMD behavior (Fig. S6B-E). The expression levels of these genes did not correlate with their influence on SMD (Fig. S6F). To further investigate the impact of CLK/CYC function on circadian rhythm and sleep within ITP-LN d neurons, we selected cwo and vri as representative genes. Both genes are closely associated with CLK/CYC function, but only vri is involved in interval timing. Knockdown of either genes did not disrupt circadian rhythm (Fig. 6 E and Fig. S6G). Knockdown of vri in ITP-LN d neurons resulted in a decrease in daytime and nighttime sleep duration (Fig. 6 F and Fig. 6 H-I), while cwo knockdown reduced the duration of sleep similarly (Fig. 6 J and Fig. 6 J-K). These results indicate that CLK-VRI interaction is crucial for modulating interval timing and sleep, but not for circadian rhythm regulation, within ITP-LN d neurons. Overexpression of miRNA bantam , which binds to the 3' UTR of Clk and modulates its translation 58 , reversed SMD behavior, indicating the critical role of CLK protein regulation in generating interval timing (Fig. 6 L). Concurrently, the overexpression of mir.ban in ITP-LN d resulted in significant alterations in sleep behavior (Fig. 6 M). Specifically, this was manifested as a decrease in sleep during the daytime and an increase during the nighttime (Fig. 6 N-O). These findings suggest that mir.ban also plays a pivotal role in the regulation of sleep behavior. The SIFa-SIFaR peptidergic signaling pathway is specifically involved in modulating interval timing distinct from its influence on circadian rhythm or sleep behavior. Circadian clock cells are influenced by environmental factors such as temperature, light, and feeding 53 , 55 , 59 – 61 . To elucidate the input signals that specifically regulate SMD behavior in ITP-LN d neurons, we analyzed the fly SCope dataset, which revealed that SIFaR is highly expressed in these neurons (Fig. 7 A). Neuropeptide SIFa, expressed in four neurons in the pars intercerebralis (PI), plays a pivotal role in modulating interval timing behaviors 12 . Knockdown of SIFaR in ITP-LN d neurons, as well as knockdown of Clk in SIFaR-positive cells, disrupted SMD behavior (Fig. 7 B-C), but did not affect circadian rhythm (Fig. S7A). Concomitantly, the absence of SIFaR led to a reduction of both daytime and nighttime sleep duration (Fig. S7B-E). BAcTrace data demonstrated that GAL4 R54D11 neurons receive strong input signals from PI-located SIFa neurons 62 (Fig. 7 D). These findings suggest that SIFa signals are crucial for interval timing and sleep regulation in ITP-LN d neurons, but not for circadian rhythm. Furthermore, we observed that ITP-LN d neurons form strong synapses with SIFa neurons in the superior medial protocerebrum (SMP) and superior lateral protocerebrum (SLP) regions of the brain (Fig. 7 E). The SLP region is known to be involved in upstream processing of mushroom body (MB) learning centers and is where long-range taste projection neurons project 63 . Although sexual experiences did not significantly alter the accumulated calcium levels in ITP-LN d neurons (Fig. S8A-B) and activation of SIFa neurons also did not lead to elevated calcium levels in ITP-LN d (Fig. S8C), the synapses between SIFa and ITP-LN d neurons were significantly reduced (Fig. 7 F-G and Fig. S8D). These data indicate that sexual experiences reduce the inputs to ITP-LN d neurons from SIFa peptidergic signals without dramatic calcium flux change. Using the FlyWire dataset, we analyzed the input circuits to ITP-LN d neurons and identified that cholinergic, glutamatergic, and GABAergic inputs constitute the main neurotransmitters from input neurons, with half coming from visual and half from central class neurons (Fig. S9A-B). Interestingly, the inputs to ITP-LN d neurons predominantly originate from the left side of the brain, potentially representing the involvement of the asymmetric body (AB) associated with this circuit (Fig. S9C) 64 – 68 . The synaptic connectivity of ITP-LN d neurons has previously been shown to be distinct within the clock network, exhibiting fewer intra-network synapses compared to other groups of clock neurons 69 . Previous single-cell RNA sequencing data analysis of 150 clock neurons identified that neural connectivity molecules are key in defining the diverse functions of heterogeneous clock cells 70 . Among these molecules, voltage-gated potassium channel Shaker (Sh) and its beta-subunit Hyperkinetic (Hk) were found to be highly enriched in ITP-LN d neurons. Knockdown of these channels in ITP-LN d neurons reversed SMD behavior, indicating their critical role in maintaining ITP-LN d as pacemaker neurons for interval timing (Fig. S9D-E). Interestingly, the reversed phenotype of interval timing associated with Sh knockdown is linked to a significant shortening of sleep duration, while Hk knockdown shows no effect on sleep (Fig. S9F-M), suggesting that there might exist divergent mechanistic roles for these channels in regulating interval timing and sleep pathways within ITP-LN d neurons, separable from circadian control. Knockdown of the Ih channel, which encodes a low-threshold, voltage-gated ion channel in ITP-LN d , also disrupted SMD behavior (Fig. S9N), suggesting that voltage-gated channels are important regulators of pacemaker function for interval timing. In contrast, knockdown of serotonin receptor 5-HT1A, octopamine receptor in MB (Oamb), or octopamine b3 receptor (Octb3R), which are known to be enriched in ITP-LN d neurons 70 , did not affect SMD behavior (Fig. S9O-Q), indicating that 5-HT and octopamine (OA) transmission are not essential for ITP-LN d 's role in interval timing. These data collectively underscore the pivotal role of voltage-gated channels in maintaining the pacemaker function of CLK-expressing ITP-LN d neurons for interval timing. Discussion In this study, we investigated the role of the CLK/CYC heterodimer and associated factors within a specific pair of LN d neurons in modulating sexual experience-dependent interval timing (SMD) behavior. We found that mutations in Clk and cyc , but not per and tim , disrupt SMD behavior, indicating a unique role for CLK/CYC in interval timing. Neuronal CLK/CYC expression is necessary and sufficient for SMD, while glial expression is not (Fig. 1 ). Specific CLK/CYC expression in NPF + and cry + neurons in the brain, but not in PDF neurons, is essential for SMD (Fig. 2 ). Detailed analysis identifies CLK expression in ITP-LN d and 5th sLN v neurons as pivotal for SMD. Knockdown of CLK in these neurons impairs SMD, as does inhibiting their activity (Fig. 3 ). ITP-LN d neurons are glutamatergic, with output circuits to central brain. Inhibiting glutamatergic transmission or knocking down CLK in these neurons disrupts SMD. Furthermore, optogenetic activation of these neurons can induce SMD without prior experience (Fig. 4 ). We also explored the molecular mechanisms, finding that CLK variants lacking DNA binding motifs dissociate circadian rhythms from interval timing and sleep behaviors in ITP-LN d neurons (Fig. 5 ). CLK-VRI interaction is crucial for modulating interval timing and sleep, but not circadian rhythm, within ITP-LN d neurons (Fig. 6 ). Finally, we demonstrated that the SIFa-SIFaR peptidergic signaling pathway is specifically involved in modulating interval timing distinct from its influence on circadian rhythm (Fig. 7 ). Our study provides a comprehensive understanding of the role of CLK/CYC heterodimer and associated factors in modulating interval timing behavior in Drosophila , shedding light on the neural circuits and molecular mechanisms underlying this complex behavior (Fig. S9R). Despite the central role of circadian clock genes in regulating circadian rhythms, these genes are also involved in various non-circadian functions. Accumulating evidence suggests that clock genes exhibit pleiotropic effects in regulating diverse physiological processes 53 , 55 , 59 , 60 . In addition to their well-established roles in sleep homeostasis 28 , 53 , 59 , 71 – 73 , clock genes have been implicated in the modulation of addiction, psychiatric disorders, and male fertility 53 , 74 , 75 . Recent studies have expanded our understanding of CLK's function, revealing its role in tissue-specific gene regulation and metabolic processes 76 , with Gart being a notable example that is crucial for maintaining feeding rhythms and food intake 73 . Furthermore, distinct clock neurons have been shown to mediate the circadian rhythm of male sex drive 77 . The surprising diversity of circadian clock cells, akin to their functional heterogeneity, suggests that individual clock neurons possess distinct functions beyond circadian rhythm regulation 78 . In conclusion, the diverse non-circadian functions of clock genes underscore their intricate regulatory roles across various physiological processes. The discovery of specific non-circadian functions of clock genes, such as the role of CLK in modulating interval timing and sleep regulation in ITP-LN d neurons, offers valuable insights into the multifaceted functions of these genes. These findings underscore the complexity and versatility of clock genes in orchestrating biological rhythms and behaviors beyond the circadian clock. In our study, we uncovered that various Clk transcripts exhibit distinct timing functions. Our data indicate that Clk-RA , RD , and RF are essential for circadian rhythmicity and sleep, while Clk-RG and RH are critical for interval timing and sleep regulation. These findings suggest that Clk transcripts regulate a spectrum of timing behaviors through their expression in specific clock cells. Moreover, we identified voltage-gated potassium channels Sh and Hk as pivotal for maintaining the pacemaker function of ITP-LN d neurons in regulating interval timing. Elucidating the relationship between Clk-RG/RH function and Sh/Hk in ITP-LN d neurons may provide insights into the molecular mechanisms governing pacemaker regulation in the fly brain for interval timing behavior (Fig. S9R). The traditional perspective on the rhythmic regulation of CLK/CYC dimer-mediated transcriptional control does not account for the PER/TIM-independent mechanisms or the specific dependence of SMD behavior on alternative splicing forms of Clk , which are crucial for behavioral timekeeping within a 5-20-minute range. Our data suggest that a C-terminally deleted CLK protein can exhibit circadian-independent functions, particularly in relation to male interval timing associated with mating investment behavior. Recent studies have reported that alternative splicing of the Clk transcript mediates the circadian clock's response to temperature changes 79 . Additionally, a non-circadian role for cyc has been identified, which regulates the development of clock neurons 80 . There has long been speculation regarding the non-circadian regulation and function of clock genes in controlling oogenesis in female Drosophila 81 and in influencing non-circadian aspects of mating behavior 55 . Therefore, we propose that CLK/CYC dimers composed of different forms of CLK protein may serve diverse functions beyond circadian rhythm regulation, including metabolic control, feeding behavior, mating behavior, and interval timing. Further investigation into the various forms of CLK proteins and their dimerization with CYC will shed light on how these non-circadian functions have evolved alongside the circadian roles of CLK protein. Declarations Acknowledgments We thank Drs. Yuh Nung Jan and Lily Yeh Jan (UCSF, USA) for helpful comments and support on this paper. We are very appreciative to the colleagues who supplied us with several fly strains: Dr. Alex C. Keene (Texas A&M University), Dr. Justin Blau (New York University), Dr. Amita Sehgal (University of Pennsylvania), Dr. Ravi Allada (University of Michigan), Dr. Lihua Jin (Northeast Forestry University), Dr. Zongzhao Zhai (Hunan Normal University), Dr. Wei Zhang (Tsinghua University), Dr. Donggen Luo (Peking University), Dr. Fang Guo (Zhejiang University), Dr. Yufeng Pan and Dr. Junhai Han (Southeast University) and Drs. Young-Joon Kim and Sung-Eun Yoon (Korea Drosophila Resource Center, KDRC). This research was supported a University of Ottawa Startup grant 602496 to WJK, Startup funds from HIT Center for Life Science to WJK, a University of Ottawa Interdisciplinary Research Group Funding Opportunity (IRGFO stream 1 and 2) grants 148101 and 148747 to WJK, a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant (reference: 211406) to WJK, a University of Ottawa Brain and Mind Research Institute/Center for Neural Dynamics Open call project grant 150950 to WJK, a Mitacs Globalink Research Internship Program grant 17268 to WJK. This research was also supported by the Brain Pool Program of the National Research Foundation in Korea grant ZYM5041911 to WJK, Burroughs Wellcome Fund Collaborative Research Travel Grants (reference: 1017486) to WJK and a NVIDIA Academic Hardware Grant Program to WJK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. HM received salary from the ‘Startup funds from HIT Center for Life Science to WJK’. Author Contributions Conceptualization: Woo Jae Kim. Data curation: Hongyu Miao, Zekun Wu, Yanan Wei, Woo Jae Kim. Formal analysis: Hongyu Miao, Zekun Wu, Woo Jae Kim. Funding acquisition: Woo Jae Kim. Investigation: Woo Jae Kim. Methodology: Woo Jae Kim. Project administration: Woo Jae Kim. Resources: Woo Jae Kim. Supervision: Woo Jae Kim. Validation: Hongyu Miao, Woo Jae Kim. Visualization: Hongyu Miao, Zekun Wu, Woo Jae Kim. Writing – original draft: Woo Jae Kim. Writing – review & editing: Hongyu Miao, Woo Jae Kim. Disclosure Statement The authors declare no competing interests. Declaration of Generative AI and AI-assisted Technologies in the Writing Process During the creation of this work, the author(s) utilized QuillBot to rephrase English sentences, verify English grammar, and detect plagiarism, as none of the authors of this paper are native English speakers. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication. Resource Availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Woo Jae Kim ( [email protected] ). Data and code availability All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. The URL of the codes used in this paper are listed in the key resources table. Any additional information required to reanalyze the data in this paper is available from the lead contact upon request. Methods Fly stocks and husbandry Drosophila melanogaster were raised on cornmeal-yeast medium at similar densities to yield adults with similar body sizes. Flies were kept in 12 h light: 12 h dark cycles (LD) at 25℃ (ZT 0 is the beginning of the light phase, ZT12 beginning of the dark phase) except for some experimental manipulation (experiments with the flies carrying tub-GAL80 ts ). Wild-type flies were Canton-S . To reduce the variation from genetic background, all flies were backcrossed for at least 3 generations to CS strain. All mutants and transgenic lines used here have been described previously. The following lines were obtained from Dr. Alex C. Keene (Texas A&M University) and Dr. Justin Blau (New York University): cry 03 , cry-GAL80 , tim-GAL4 (BDSC7126). The following lines were obtained from Dr. Amita Sehgal (University of Pennsylvania): qvr 1 . The following lines were obtained from Dr. Lihua Jin (Northeast Forestry University): Myo1A-GAL4 , esg-GAL4 , Hml-GAL4 (BDSC30139). The following lines were obtained from Dr. Zongzhao Zhai (Hunan Normal University): uro-GAL4 (BDSC91415). The following lines were obtained from Dr. Wei Zhang (Tsinghua University): tsh-GAL80 (BDSC605556), lexAop-FLP (BDSC55819), VGlut-GAL80 (BDSC58448). The following lines were obtained from Dr. Ravi Allada (University of Michigan): UAS-cyc . The following lines were obtained from Dr. Donggen Luo (Peking University) and Dr. Junhai Han (Southeast University): Clk4.1M-GAL4 (BDSC31316), GAL4 R54D11 (BDSC41279), Mai179-GAL4 . The following lines were obtained from Dr. Yufeng Pan (Southeast University): UAS-jGCaMP7s (BDSC79032), empty-RNAi (BDSC36304). The following lines were obtained from Bloomington Stock Center: Canton-S (64349), Df(1)Exel6234 (7708), per 01 (80928), tim 01 (80930), Clk Jrk (80927), cyc 01 (80929), elav c155 (458), Clk-RNAi HMJ02224 (42566), cyc-RNAi HMJ02219 (42563), repo-GAL4 (7415), grh-GAL4 (65637), Mhc-GAL4 (55133), Clk-RNAi JF01453 (31660), Clk-RNAi JF01454 (31661), cyc-RNAi JF03333 (29400), cyc-RNAi JF02185 (31897), cyc-RNAi GL00387 (35461), tub(FRT.GAL80) (38881), otdFLP (600309), cry-GAL4; Pdf-GAL80 (80940), cry-GAL4 (24514), NPF-GAL4 (25681), NPF-GAL4 (25682), tub-GAL80 ts (7108), UAS-CD4tdGFP (35839), UAS-RedStinger (8546), ITP-RNAi (25799), UAS-hid (65403), UAS-KCNJ2 (6595), UAS-NaChBac (9469), UAS-TNT (28838), UAS-traF (4590), UAS>stop>KCNJ2 (67686), VGlut-RNAi (27538), UAS-CsChrimson (55136), cwo-RNAi (26318), vri-RNAi (40862), Pdp1-RNAi (26212), BacTrace (90826), SIFaR-RNAi (34947), lexAop-nSyb-spGFP 1-10 , UAS-CD4-spGFP 11 (64315), Cipc-RNAi (28774), dco-RNAi (27719), jet-RNAi (31058), Atx2-RNAi (36114), cry-RNAi (43217), UAS-mir.ban.A (60671), lexAop-CD8GFP; UAS-mLexA-VP16-NFAT, lexAop-rCD2-GFP (66542), Sh-RNAi (53347), Hk-RNAi (28330), Oamb-RNAi (31233), UAS-mCD8RFP, LexAop-mCD8GFP, nSyb-MKII::nlsLexADBDo, UAS-p65AD::CaM (61679), empty-GAL4 (36303). The following lines were obtained from Qidong Fungene Biotechnology: ITP-RC T2A -GAL4 (FBA00286), VGlut FLP (FRE00001), SIFa-lexA T2A (FBF00116). The following lines were obtained from Korea Drosophila Resource Center: UAS>stop>mCD8GFP (1119), UAS>stop>NaChBac (1183), UAS>stop>TNT (1191). The following lines were obtained from Vienna Drosophila Resource Center: Clk ADF -RNAi (104507), 5-HT1A-RNAi (106094). The following lines were obtained from TsingHua Fly Center: Ih-RNAi (TH02084.N). The following lines were obtained from NIG-FLY Center: Octβ3R-RNAi (31348R-4). The CS background was selected as the experimental background due to its well-characterized and consistent LMD and SMD behaviors. To ensure that genetic variation did not confound our results, all GAL4, UAS, and RNAi lines employed in our assays were rigorously backcrossed into the CS strain, often exceeding ten generations of backcrossing. This approach was undertaken to isolate the effects of our genetic manipulations from those of genetic background. We assert that the extensive backcrossing to the CS background, in concert with the internal control in LMD and SMD, provides a stable platform for the accurate interpretation of the LMD and SMD phenotypes observed in our experiments. To reduce the variation from genetic background, all flies were backcrossed for at least 10 generations to CS strain. For the generation of outcrosses, all GAL4, UAS, and RNAi lines employed as the virgin female stock were backcrossed to the CS genetic background for a minimum of ten generations. Notably, the majority of these lines, which were utilized for LMD assays, have been maintained in a CS backcrossed state for long-term generations subsequent to the initial outcrossing process, exceeding ten backcrosses. Based on our experimental observations, the genetic background of primary significance is that of the X chromosome inherited from the female parent. Consequently, we consistently utilized these fully outcrossed females as virgins for the execution of experiments pertaining to LMD and SMD behaviors. Contrary to the influence on LMD behaviors, we have previously demonstrated that the genetic background exerts negligible influence on SMD behaviors, as reported in our prior publication 11 . The mutants and transgenic lines utilized in this study have been previously characterized, with the exception of the novel transgenic strains that we generated and describe herein. Mating duration assay The mating duration assay in this study has been reported 9–11 . To enhance the efficiency of the mating duration assay, we utilized the Df(1)Exel6234 (DF here after) genetic modified fly line in this study, which harbors a deletion of a specific genomic region that includes the sex peptide receptor (SPR) 82,83 . Previous studies have demonstrated that virgin females of this line exhibit increased receptivity to males 83 . We conducted a comparative analysis between the virgin females of this line and the CS virgin females and found that both groups induced SMD. Consequently, we have elected to employ virgin females from this modified line in all subsequent studies. For group reared (naïve) males, 40 males from the same strain were placed into a vial with food for 5 days. For single reared males, males of the same strain were collected individually and placed into vials with food for 5 days. For experienced males, 40 males from the same strain were placed into a vial with food for 4 days then 80 DF virgin females were introduced into vials for last 1 day before assay. 40 DF virgin females were collected from bottles and placed into a vial for 5 days. These females provide both sexually experienced partners and mating partners for mating duration assays. At the fifth day after eclosion, males of the appropriate strain and DF virgin females were mildly anaesthetized by CO 2 . After placing a single female in to the mating chamber, we inserted a transparent film then placed a single male to the other side of the film in each chamber. After allowing for 1 h of recovery in the mating chamber in 25℃ incubators, we removed the transparent film and recorded the mating activities. Only those males that succeeded to mate within 1 h were included for analyses. The initiation and completion of copulation were recorded to the nearest second, with a precision of ±10 seconds. The total mating duration for each pair was determined from the moment of successful genital apposition until the separation of the male and female Drosophila . Genetic controls with GAL4/+ or UAS/+ lines were omitted from supplementary figures, as prior data confirm their consistent exhibition of normal LMD and SMD behaviors 9–11,14,16 . Hence, genetic controls for LMD and SMD behaviors were incorporated exclusively when assessing novel fly strains that had not previously been examined. In essence, internal controls were predominantly employed in the experiments, as LMD and SMD behaviors exhibit enhanced statistical significance when internally controlled. Within the LMD assay, both group and single conditions function reciprocally as internal controls. A significant distinction between the naïve and single conditions implies that the experimental manipulation does not affect LMD. Conversely, the lack of a significant discrepancy suggests that the manipulation does influence LMD. In the context of SMD experiments, the naïve condition (equivalent to the group condition in the LMD assay) and sexually experienced males act as mutual internal controls for one another. A statistically significant divergence between naïve and experienced males indicates that the experimental procedure does not alter SMD. Conversely, the absence of a statistically significant difference suggests that the manipulation does impact SMD. Hence, we incorporated supplementary genetic control experiments solely if they deemed indispensable for testing. All assays were performed from noon to 4 PM. We conducted blinded studies for every test. Generation of transgenic flies To generate the UAS>stop>Clk-RNAi line, we selected HMJ02224 (BDSC#42566) as the template shRNA. The shRNA sequences were cloned directly with the following primers TTATCCCATATTCAGCCGCTAGCAGT-AGAGCTAGTTGTAGATCTCAA-TAGTTATATTCAAGC and AACTCCGATGTCTCGCCTGAATTCGC-AGAGCTAGTTGTAGATCTCAA-TATGCTTGAATATAAC. The amplified DNA fragment was inserted into the pJFRC28-10XUAS-FRT-stop-FRT-RNAi vector. This vector, supplied by Qidong Fungene Biotechnology Co., Ltd. (http://www.fungene.tech/), is a derivative of the pJFRC28-10XUAS-IVS-GFP-p10 vector (available at https://www.addgene.org/36431). The insertion was achieved by digesting the fragment and the vector with EcoRI and NheI restriction enzymes to create compatible sticky ends. The genetic construct was inserted into the attp5 site on chromosome II and VK0005 site on chromosome III to generate transgenic flies using established techniques, a service conducted by Qidong Fungene Biotechnology Co., Ltd. Immunostaining After 5 days of eclosion, the Drosophila brain was taken from adult flies and fixed in 4% formaldehyde at room temperature for 30 minutes. The sample was than washed three times (5 minutes each) in 1% PBT and then blocked in 5% normal goat serum for 30 minutes. Subsequently, the sample was incubated overnight at 4℃ with primary antibodies in 1% PBT, followed by the addition of fluorophore-conjugated secondary antibodies for one hour at room temperature. Finally, the brain was mounted on plates with an antifade mounting solution (Solarbio) for imaging purposes. Samples were imaged with Zeiss LSM880. Antibodies were used at the following dilutions: Chicken anti-GFP (1:500, Invitrogen), mouse anti-nc82 (1:50, DSHB), rabbit anti-DsRed (1:500, Rockland Immunochemicals), Alexa-488 donkey anti-chicken (1:200, Jackson ImmunoResearch), Alexa-555 goat anti-rabbit (1:200, Invitrogen), Alexa-647 goat anti-mouse (1:200, Jackson ImmunoResearch). Quantitative analysis of fluorescence intensity To quantify the calcium level and synaptic intensity in microscopic images, we introduced ImageJ software 84 . We initially employed ImageJ’s ‘Measure’ feature to calculate average pixel intensity across the entire image or in user-specified sections, and the ‘Plot Profile’ feature to create intensity profiles across areas. To maximize precision, we converted color images to grayscale before analysis. Thresholding methods were also utilized to produce binary images that accurately outlined areas of interest, with pixel intensities of 255 (white) assigned to regions of interest and 0 (black) to the background. Intensity values from the binary image were then transferred to the corresponding locations in the original grayscale image to obtain precise intensity measurements for each object. The ‘Display Results’ feature provided comprehensive data for each object, including average intensity, size, and other relevant statistics. To normalize for fluorescence differences between ROIs, GFP fluorescence for GRASP was normalized to nc82. All specimens were imaged under identical conditions. Optogenetic experiment A 100 mM stock solution of all-trans-retinal (ATR) powder (Sigma) was prepared by dissolving it in 100% alcohol. For optogenetic experiments, 250 μl of the stock solution was mixed with 30 ml of 5% sucrose and 1% agar medium to prepare food with a final concentration of 400 μM ATR. Flies aged between 3 and 5 days were transferred to ATR food for a minimum of 3 days before performing optogenetic experiments 85 . ATR-fed flies and unfed flies were housed in separate transparent tubes and exposed to a 20s red light: 40s no-light cycle treatment overnight before mating duration assay. GCaMP experiment Fly anesthesia was induced using CO 2 . Dissecting the brain after feeding ATR for at least three days. AHL solution (108 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 4 mM NaHCO 3 , 1 mM NaH 2 PO 4 , 5 mM HEPES, 10 mM Sucrose, 5 mM Trehalose, pH 7.5) was used for both dissection and imaging to maintain neuronal activity. We then used Zeiss LSM880 confocal microscope to record calcium signaling fluctuations in ITP-LN d , 5 th sLN v , and ocelli in parallel with activation of SIFa neurons using 5 seconds of red light. The brains were scanned at 1 Hz sampling rate with the max pinhole. Fiji was used to examine ROIs. ΔF/F 0 = (F t − F 0 )/F 0 × 100%. F 0 was the averaged fluorescence of the baseline. Because laser irradiation causes the fluorescence signal to diminish over time, we used the R package feasts (https://feasts.tidyverts.org) to detrend. Single-fly sleep and circadian rhythm recording 96-well white Microfluor 2 plates (Fishier) with 400 μl of food (5% sucrose and 1% agar) were loaded with adult male flies (aged 3–5 days). Flies were entrained to the 12 h:12 h LD cycles for four days at 25 °C to record sleep behavior, then changed to constant darkness for 5-6 days to record circadian rhythms in the absence of light inputs. The fly movement was monitored using a camera at 10s intervals, and the data were then used by the sleep and circadian analysis program SCAMP to analyze sleep and circadian rhythm 86–88 . It calculates activity by shifting the position of Drosophila every 10 seconds and calculates sleep using the standard definition ( Drosophila is recorded as asleep if it remains motionless for at least 5 minutes). For all sleep experiments in Figures 5, 6, S7, and S9, experimental and control groups were assayed concurrently within the same experimental round to minimize batch effects. Bilateral controls (GAL4 driver alone and UAS effector alone) were included for each experimental genotype to validate specificity. Due to the concurrent nature of these assays, the R54D11-GAL4/+ line was used as a shared control for all experimental groups targeting the GAL4 driver side Sample sizes (at least 6–8 flies per genotype) were determined based on prior studies demonstrating reliable detection of robust phenotypes in bilateral control designs 89,90 . Single-nucleus RNA-sequencing analyses snRNAseq dataset analyzed in this paper is published 91 and available at the Nextflow pipelines (VSN, https://github.com/vib-singlecell-nf), the availability of raw and processed datasets for users to explore, and the development of a crowd-annotation platform with voting, comments, and references through SCope (https://flycellatlas.org/scope), linked to an online analysis platform in ASAP ( https://asap.epfl.ch/fca ).Single-cell RNA sequencing (scRNA-seq) data from the Drosophila melanogaster were obtained from the Fly Cell Atlas website (https://flycellatlas.org/scope). The Seurat (v4.2.2) package (https://satijalab.org/seurat) was utilized for data analysis. Violin plots were generated using the “Vlnplot” function, the cell types are split by FCA. Gene expression analysis Single-cell RNA sequencing (scRNA-seq) data from the Drosophila melanogaster were obtained from the GEO under the accession code GSE157504 92 . The same integration method was applied. Data from six time points and under LD and DD conditions were read and integrated using the integration functions provided by the Seurat 4 (version 4.2.2) package 93 . The UMIs data were retrieved, consisting of a grand total of 4,634 cells. The "NormalizeData" function was utilized for the purpose of automated data normalization. Ultimately, we conducted principal component analysis (PCA) on gene expression vectors that were scaled using z-scores. Subsequently, we limited the data to include just the top 40 PCA components. We employed the ‘FindNeighbors’ and ‘FindClusters’ functions from the Seurat package to cluster the data that had been decreased in dimensions. We utilized t-distributed stochastic neighbor embedding (t-SNE) to generate a two-dimensional map that displays the clusters. One cluster that highly express the ITP gene were extracted, and then the marker genes were calculated using the Seurat 'FindMarkers' function. Connectome analysis Whole brain connectomics data were obtained from FlyWire ( https://codex.flywire.ai/ ) 94–99 . The left ITP-LN d (FlyWire Root ID: 720575940634984800) dataset was used to gather information on the synaptic connections between the presynaptic and the postsynaptic neurons of interest. The connectivity was visualized with Sankey diagram and doughnut diagram by the Plotly R Studio library (https://plotly.com/r/). Statistical tests Statistical analysis of mating duration assay was described previously 9–11 . More than 50 males (naïve, experienced and single) were used for mating duration assay. Our experience suggests that the relative mating duration differences between naïve and experienced condition and singly reared are always consistent; however, both absolute values and the magnitude of the difference in each strain can vary. So, we always include internal controls for each treatment as suggested by previous studies 100 . Therefore, statistical comparisons were made between groups that were naïvely reared, sexually experienced and singly reared within each experiment. As mating duration of males showed normal distribution (Kolmogorov-Smirnov tests, p > 0.05), we used two-sided Student’s t tests. The mean ± standard error (s.e.m) ( **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05 ). All analysis was done in GraphPad (Prism). Individual tests and significance are detailed in figure legends. Besides traditional t -test for statistical analysis, we added estimation statistics for all MD assays and two group comparing graphs. In short, ‘estimation statistics’ is a simple framework that—while avoiding the pitfalls of significance testing—uses familiar statistical concepts: means, mean differences, and error bars. More importantly, it focuses on the effect size of one’s experiment/intervention, as opposed to significance testing 101 . In comparison to typical NHST plots, estimation graphics have the following five significant advantages such as (1) avoid false dichotomy, (2) display all observed values (3) visualize estimate precision (4) show mean difference distribution. And most importantly (5) by focusing attention on an effect size, the difference diagram encourages quantitative reasoning about the system under study 102 . Thus, we conducted a reanalysis of all our two group data sets using both standard t tests and estimate statistics. 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Screen of 150 clock neurons. 2025CLKv2Supportinginformationgeneticcontrols.pdf 2025-CLK-v2-Supporting_information-genetic-controls 2025CLKFig.S1v2.pdf 2025CLKFig.S2v2.pdf 2025CLKFig.S3v2.pdf 2025CLKFig.S4v2.pdf 2025CLKFig.S5v2.pdf 2025CLKFig.S6v2.pdf 2025CLKFig.S7v4.pdf 2025CLKFig.S8v2.pdf 2025CLKFig.S9v4.pdf SupplementalFigureLegends.docx GraphicalAbstract.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7471909","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":514738313,"identity":"0c15bd6e-19f7-4e59-afd0-f0db15e0b7f3","order_by":0,"name":"Woo Jae Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYDACHsYGho8NEiCmAfFaGGc2SEiQooWBgZm3gYEELQZnDrdutt1hUcfA3rxNgqHmDhFazja23c49A3QYz7EyCYZjzwhrMTvPCNTSBtQikWMmwdhwmEgtliAt8m+I1QJyGCPYFh4itdifOdh2s7dNQrKNJ63YIuEYEVoke9Kf3fjZVsfPz354440PNURogQM2EJFAgoZRMApGwSgYBXgAAHEiNoVxc/LTAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2826-4177","institution":"HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Woo","middleName":"Jae","lastName":"Kim","suffix":""},{"id":514738314,"identity":"95e3136b-ffcc-4cfb-a076-636ecff46ace","order_by":1,"name":"Hongyu Miao","email":"","orcid":"","institution":"HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Miao","suffix":""},{"id":514738315,"identity":"f696aa4a-5050-4a88-b333-c717cd1808f5","order_by":2,"name":"Zekun Wu","email":"","orcid":"","institution":"Harbin Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Zekun","middleName":"","lastName":"Wu","suffix":""},{"id":514738316,"identity":"42ab0fcc-b41b-4f83-b19d-85dffbf87cb5","order_by":3,"name":"Yanan Wei","email":"","orcid":"","institution":"Harbin Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Wei","suffix":""}],"badges":[],"createdAt":"2025-08-27 13:05:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7471909/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7471909/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91405567,"identity":"31d20876-dd63-4432-8ffa-3f2dc76b8275","added_by":"auto","created_at":"2025-09-16 07:48:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1079581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInterval timing is regulated by neuronal expression of CLK/CYC heterodimer, not PER/TIM. \u003c/strong\u003e(A-F) SMD assays of core circadian rhythm gene mutations. Light gray dots represent naïve males, and pink dots represent experienced ones. Dot plots represent the M. D (mating duration) of each male fly. The mean value and standard error are labeled within the dot plot (black lines). Asterisks represent significant differences, as revealed by the Student’s t test, and ns represents non-significant differences (\u003cem\u003e*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt; 0.001, ****p\u0026lt; 0.0001\u003c/em\u003e). DBMs refers to the Difference between Means. For detailed methods, see the “Methods” for a detailed description of the mating duration assay used in this study.\u003cstrong\u003e \u003c/strong\u003eIn the framework of our investigation, the routine application of internal controls is employed for the vast majority of experimental procedures, as delineated in the \"\u003cstrong\u003eMating Duration Assay\u003c/strong\u003e\" and \"\u003cstrong\u003eStatistical Tests\u003c/strong\u003e\" subsections of the Methods section. The numerical values in the bar graph denote the number of fruit flies in the respective experimental groups, referred to as “n”. Subsequent estimated statistical graphs will employ the same numerical designations. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(G-H) Fly SCope single-cell RNA sequencing data of cells co-expressing \u003cem\u003eClk/cyc\u003c/em\u003e together with (G)\u003cem\u003e elav \u003c/em\u003ein the ‘Neuron’ population and (H) \u003cem\u003erepo\u003c/em\u003e in the ‘Glial Cell’ population.. Annotations and gene names of all the above data are color-coded using red, green, and blue words. When cells overlap, the color of the dots is either yellow, cyan, or magenta. The Venn diagram indicates the number of cells in each overlap category.\u003cstrong\u003e \u003c/strong\u003e(I-J) SMD assays for \u003cem\u003eelav\u003c/em\u003e\u003csup\u003e\u003cem\u003ec155\u003c/em\u003e\u003c/sup\u003e-mediated neuronal knockdown of \u003cem\u003eClk/cyc\u003c/em\u003e via\u003cem\u003e Clk-RNAi \u003c/em\u003eand \u003cem\u003ecyc-RNAi\u003c/em\u003e. (K-L) SMD assays for \u003cem\u003erepo-GAL4\u003c/em\u003e-mediated glial knockdown of \u003cem\u003eClk/cyc\u003c/em\u003e via\u003cem\u003e Clk-RNAi \u003c/em\u003eand \u003cem\u003ecyc-RNAi\u003c/em\u003e. (M-V) SCope data of \u003cem\u003eClk/cyc\u003c/em\u003e expression in different tissue populations and SMD assays for tissue-specific knockdown of \u003cem\u003eClk\u003c/em\u003e via \u003cem\u003eClk-RNAi \u003c/em\u003eusing (N) \u003cem\u003egrh-GAL4\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e(P) \u003cem\u003eMyo1A-GAL4\u003c/em\u003e, (R) \u003cem\u003euro-GAL4\u003c/em\u003e, (T) \u003cem\u003eMhc-GAL4\u003c/em\u003e and (V) \u003cem\u003eHml-GAL4\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/2f90d896869df15999f48015.jpg"},{"id":91406937,"identity":"7d979ba5-22f0-469b-8e9f-cfe9e6c3151e","added_by":"auto","created_at":"2025-09-16 08:04:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1141477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNPF and CRY co-expressed clock neurons in the brain are critical for the generation of interval timing behavior.\u003c/strong\u003e (A) Fly SCope single-cell RNA sequencing data of cells co-expressing \u003cem\u003eClk/cyc\u003c/em\u003e together with \u003cem\u003eelav\u003c/em\u003e in ‘Body’ population. (B) SMD assay for\u003cem\u003e elav\u003c/em\u003e\u003csup\u003e\u003cem\u003ec155\u003c/em\u003e\u003c/sup\u003e driver mediated knockdown of \u003cem\u003eClk\u003c/em\u003e in tsh-negative neurons via \u003cem\u003eClk-RNAi; tsh-GAL80.\u003c/em\u003e (C) Fly SCope single-cell RNA sequencing data of cells co-expressing \u003cem\u003eClk/cyc\u003c/em\u003e together with \u003cem\u003eelav\u003c/em\u003e in ‘Head’ population. (D-E) SMD assays for\u003cem\u003e elav\u003c/em\u003e\u003csup\u003e\u003cem\u003ec155\u003c/em\u003e\u003c/sup\u003e drivers mediated knockdown of \u003cem\u003eClk/cyc\u003c/em\u003e in head neurons via (D) \u003cem\u003etub(FRT.GAL80); otd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFLP\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e; Clk-RNAi \u003c/em\u003eand (D) \u003cem\u003etub(FRT.GAL80); otd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFLP\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e; cyc-RNAi.\u003c/em\u003e (F-H) Genetic rescue experiments of SMD assays for GAL4 mediated overexpression of CYC via (G) \u003cem\u003eelav\u003c/em\u003e\u003csup\u003e\u003cem\u003ec155\u003c/em\u003e\u003c/sup\u003e (H) \u003cem\u003eelav\u003c/em\u003e\u003csup\u003e\u003cem\u003ec155\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e; Pdf-GAL80\u003c/em\u003e in \u003cem\u003ecyc\u003c/em\u003e mutant background flies. (I) SMD assays for \u003cem\u003ePdf-GAL4\u003c/em\u003e-mediated knockdown of\u003cem\u003e Clk\u003c/em\u003e in core oscillator cells via \u003cem\u003eClk-RNAi.\u003c/em\u003e (J) Diagram of circadian-associated driver-labeled clock cells. Subsets of neurons labeled by GAL4 drivers named in italics are color-coded. (K-P) SMD assays for circadian-related-GAL4 mediated knockdown of \u003cem\u003eClk\u003c/em\u003e via \u003cem\u003eClk-RNAi \u003c/em\u003eusing the (K) \u003cem\u003etim-GAL4\u003c/em\u003e, (L) \u003cem\u003eNPF-GAL4\u003c/em\u003e, (M) \u003cem\u003ecry-GAL4\u003c/em\u003e, (N) \u003cem\u003ePdf-GAL4\u003c/em\u003e, (O) \u003cem\u003ecry-GAL4; Pdf-GAL80\u003c/em\u003e, and (P) \u003cem\u003eNPF-GAL4; Pdf-GAL80\u003c/em\u003e. (Q) Male (left three) and female (right one) flies brain expressing \u003cem\u003enpf-GAL4; UAS-mCD8GFP\u003c/em\u003e together with \u003cem\u003ecry-GAL80\u003c/em\u003e and \u003cem\u003ePdf-GAL80\u003c/em\u003e were immunostained with anti-GFP (green) and nc82 (magenta) antibodies. The white circles denote neurons that express various genes, with the genotypes specified to the right of the circles. The yellow arrow indicates NPF-positive cells, whereas the red arrow represents NPF-positive cells that are absent in this genotype. Scale bars represent 50 μm. For detailed methods, see the “Methods” for a detailed description of the immunostaining procedure used in this study.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/b8c25e987f752a88e097b957.jpg"},{"id":91405568,"identity":"688cf3e1-5269-4338-a3d3-885ea94e7a2d","added_by":"auto","created_at":"2025-09-16 07:48:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":945817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITP-LN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and 5\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eth\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e sLN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003ev \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003egenerate SMD behavior through the CLK function.\u003c/strong\u003e (A) SMD assay for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated knockdown of \u003cem\u003eClk\u003c/em\u003e via \u003cem\u003eClk-RNAi\u003c/em\u003e. (B) Brain of male flies expressing \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e together with \u003cem\u003eUAS-CD4tdGFP\u003c/em\u003e was immunostained with anti-GFP (green), and nc82 (blue) antibodies. Scale bars represent 50 μm. The left panel is presented as a gray scale to clearly show the cellular morphology in the adult brain labeled by \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D1\u003c/em\u003e\u003c/sup\u003e driver. Yellow arrows indicate ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5\u003csup\u003eth\u003c/sup\u003e sLN\u003csub\u003ev\u003c/sub\u003e soma. (C) SMD assay for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated adult-stage knockdown of \u003cem\u003eClk\u003c/em\u003e via \u003cem\u003eClk-RNAi \u003c/em\u003etogether with \u003cem\u003etub-GAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003ets\u003c/em\u003e\u003c/sup\u003e. After hybridization, parentals were placed at 22 °C, and after adults had eclosion, they were transferred to 29 °C and left for 5 days before the experiment. (D) SMD assay for\u003cem\u003e GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated knockdown of \u003cem\u003eClk\u003c/em\u003e in tsh-negative neurons via \u003cem\u003eClk-RNAi; tsh-GAL80\u003c/em\u003e. (E-F) SMD assays for ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5\u003csup\u003eth\u003c/sup\u003e sLN\u003csub\u003ev\u003c/sub\u003e labeled-GAL4 mediated knockdown of \u003cem\u003eClk\u003c/em\u003e via \u003cem\u003eClk-RNAi \u003c/em\u003eusing (E) \u003cem\u003eITP\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e and (F) \u003cem\u003eMai179-GAL4\u003c/em\u003e. (G) SMD assay for\u003cem\u003e GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated knockdown of \u003cem\u003eITP\u003c/em\u003e via \u003cem\u003eITP-RNAi\u003c/em\u003e. (H) Male flies brain and VNC expressing\u003cem\u003e ITP\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e driver together with \u003cem\u003eUAS-CD4tdGFP\u003c/em\u003e and \u003cem\u003eUAS-RedStinger\u003c/em\u003e were immunostained with anti-GFP (green), anti-DsRed (red), and anti-nc82 (blue) antibodies. Scale bars represent 50 μm in brain panels and 50 μm in VNC panels. Boxes indicate the magnified regions of interest presented in the middle panels. The text indicates the name of the \u003cem\u003eITP\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e-labeled brain cells. IPC, ITP-producing cells.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/a946badb9015498fdbe2124f.jpg"},{"id":91408056,"identity":"66078462-7ecc-4e15-a775-da00a429a27b","added_by":"auto","created_at":"2025-09-16 08:12:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":753935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNormal glutamatergic output from the ITP-LN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e is necessary for interval timing.\u003c/strong\u003e (A) Male flies expressing the\u003cem\u003e UAS(FRT.stop)mCD8GFP; GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e together with\u003cem\u003e lexAop-FLP; NPF-lexA\u003c/em\u003e were immunostained with anti-GFP (green) and nc82 (magenta) antibodies. Yellow arrows indicate ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5\u003csup\u003eth\u003c/sup\u003e sLN\u003csub\u003ev\u003c/sub\u003e soma. Yellow dash circles indicate \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e-labeled VNC neurons. Scale bars represent 50 μm. (B-E) SMD assays for the GAL4 system labeled only ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5\u003csup\u003eth\u003c/sup\u003e sLN\u003csub\u003ev\u003c/sub\u003e mediated (B) electrical silencing via \u003cem\u003e(FRT.stop)KCNJ2\u003c/em\u003e, (C) electrical activating via \u003cem\u003e(FRT.stop)NaChBac\u003c/em\u003e, (D) inactivation of synaptic transmission via \u003cem\u003e(FRT.stop)TNT\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand\u003cem\u003e \u003c/em\u003e(E) knockdown of \u003cem\u003eClk\u003c/em\u003e via \u003cem\u003e(FRT.stop)Clk-RNAi\u003c/em\u003e. (F-G) Connectome information of ITP-LN\u003csub\u003ed\u003c/sub\u003e output neurons. (F) displays a donut diagram demonstrating the type of output neurons. (G) displays a Sankey histogram depicting the synaptic connections between ITP-LN\u003csub\u003ed\u003c/sub\u003e and output neurons, the neurotransmitters that the output neurons use are displayed on the right. The color indicates the number of synapses connected. The data is from \u003ca href=\"https://codex.flywire.ai/\"\u003ehttps://codex.flywire.ai/\u003c/a\u003e. The blank space in the upper right corner represents the failure to confirm the types of neurotransmitters used by these neurons. (H) SMD assays for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated knockdown of vesicular glutamate transporter via \u003cem\u003eVGlut-RNAi\u003c/em\u003e. (I) SMD assay for\u003cem\u003e GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated knockdown of \u003cem\u003eClk\u003c/em\u003e in VGlut-negative neurons via \u003cem\u003eClk-RNAi; VGlut-GAL80\u003c/em\u003e. (J) SMD assay for\u003cem\u003e GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated knockdown of \u003cem\u003eClk\u003c/em\u003e in glutamatergic neurons via \u003cem\u003eClk-RNAi; VGlut\u003c/em\u003e\u003csup\u003e\u003cem\u003eFLP\u003c/em\u003e\u003c/sup\u003e. (K) Optogenetic experiment for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated electrical activation via \u003cem\u003eUAS-CsChrimson\u003c/em\u003e. Two groups of flies fed all-trans retinaldehyde (ATR) and unfed were irradiated overnight with red light to simulate cellular activation during SMD behavioral generation. See the “Methods” for a detailed description of the optogenetic experiment in this study.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/59c0c1f7c4379ab93117700e.jpg"},{"id":91405573,"identity":"2c09241c-5321-4f73-848f-87a049ce26c4","added_by":"auto","created_at":"2025-09-16 07:48:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1396188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent CLK protein isoforms in ITP-LN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e govern circadian rhythm and interval timing, respectively.\u003c/strong\u003e (A-B) LMD and SMD assays for \u003cem\u003eCanton-S\u003c/em\u003e males under 5 days of constant light. (C) Structures of the different\u003cem\u003e Clk\u003c/em\u003e transcripts. Grey squares represent the exons, brown squares represent CDS, and arrows indicate short hairpin RNAs (shRNAs) for gene silencing. (D) SMD assay for\u003cem\u003e GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated knockdown of \u003cem\u003eClk-RA\u003c/em\u003e, \u003cem\u003eRD\u003c/em\u003e, and \u003cem\u003eRF\u003c/em\u003e via \u003cem\u003eClk\u003c/em\u003e\u003csup\u003e\u003cem\u003eADF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-RNAi\u003c/em\u003e. (E) Genetic control of the SMD assay, which only has \u003cem\u003eClk\u003c/em\u003e\u003csup\u003e\u003cem\u003eADF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-RNAi\u003c/em\u003e without a GAL4 driver. (F) Actogram and Periodogram of single male flies for (top) genetic control (\u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e with \u003cem\u003eempty-RNAi\u003c/em\u003e) and knockdown of \u003cem\u003eClk-RA\u003c/em\u003e, \u003cem\u003eRD\u003c/em\u003e, and \u003cem\u003eRF\u003c/em\u003e via \u003cem\u003eClk\u003c/em\u003e\u003csup\u003e\u003cem\u003eADF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-RNAi \u003c/em\u003eby \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e (bottom). See the “Methods” for a detailed description of the single circadian rhythm experiment in this study. (G-J) Sleep profiles of single flies for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e mediated knockdown of \u003cem\u003eClk\u003c/em\u003e via \u003cem\u003eClk-RNAi \u003c/em\u003eand the quantification of sleep duration\u003cem\u003e.\u003c/em\u003e The data were derived from the average sleep duration over a 3-5 day period under a 12-hour light:12-hour dark cycle. See the “Methods” for a detailed description of the single activity and sleep experiment in this study.\u003cem\u003e \u003c/em\u003e(K-N) Sleep profiles of single flies for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e mediated knockdown of \u003cem\u003eClk-RA\u003c/em\u003e, \u003cem\u003eRD\u003c/em\u003e, and \u003cem\u003eRF\u003c/em\u003e via \u003cem\u003eClk\u003c/em\u003e\u003csup\u003e\u003cem\u003eADF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-RNAi \u003c/em\u003eand the quantification of sleep duration\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/8e14940a4c8936011dfb0012.jpg"},{"id":91408317,"identity":"7d242dec-1949-4253-8103-d9ee99511a01","added_by":"auto","created_at":"2025-09-16 08:20:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1398095,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent circadian genes produce distinct effects on interval timing and sleep behavior.\u003c/strong\u003e (A) Diagram of the molecular clock. Red ovals represent CLK/CYC-associated proteins, and blue ovals represent PER/TIM-associated proteins. (B-D) SMD assay for\u003cem\u003e GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated knockdown of CLK/CYC-associated genes via (B) \u003cem\u003ecwo-RNAi\u003c/em\u003e, (C) \u003cem\u003evri-RNAi\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand (D) \u003cem\u003ePdp1-RNAi\u003c/em\u003e. (E) Actogram and Periodogram of single male flies for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated knockdown of \u003cem\u003evri\u003c/em\u003e or \u003cem\u003ecwo\u003c/em\u003e via \u003cem\u003ecwo-RNAi \u003c/em\u003e(top) or \u003cem\u003evri-RNAi\u003c/em\u003e (bottom). (F-G) Sleep profiles of flies for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e mediated knockdown of \u003cem\u003evri\u003c/em\u003e or \u003cem\u003ecwo\u003c/em\u003e via (F) \u003cem\u003evri-RNAi\u003c/em\u003e or (G) \u003cem\u003ecwo-RNAi\u003c/em\u003e. (H-K) Quantification of sleep duration for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e mediated knockdown of \u003cem\u003evri\u003c/em\u003e or \u003cem\u003ecwo\u003c/em\u003e via (H-I) \u003cem\u003evri-RNAi\u003c/em\u003e or (J-K) \u003cem\u003ecwo-RNAi\u003c/em\u003e. (G) SMD assay for\u003cem\u003e GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver mediated overexpression of microRNA \u003cem\u003ebantam\u003c/em\u003e via \u003cem\u003eUAS-mir.ban.A\u003c/em\u003e. (M-O) Sleep profiles and quantification of flies for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e mediated overexpression of microRNA \u003cem\u003ebantam\u003c/em\u003e via \u003cem\u003eUAS-mir.ban.A\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/ea097faeb9972e01c6657224.jpg"},{"id":91406939,"identity":"01525cba-68be-474c-b551-ba7ebca7af22","added_by":"auto","created_at":"2025-09-16 08:04:06","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":603131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe SIFa-SIFaR peptidergic signaling pathway regulates the formation of interval timing behavior. \u003c/strong\u003e(A) Expression of neuropeptide (NP) and neuropeptide receptor (NPR) in ITP-LN cells. The average UMI count was calculated for each gene in this population, followed by log10 transformation for data normalization. To focus on cells expressing these genes, only genes exhibiting increased expression of NP and NPR were included. (B) SMD assay for \u003cem\u003eSIFaR\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e driver mediated knockdown of \u003cem\u003eClk\u003c/em\u003e in SIFaR-positive neurons via \u003cem\u003eClk-RNAi\u003c/em\u003e. (C) SMD assay for \u003cem\u003eSIFaR\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e driver mediated knockdown of \u003cem\u003eClk\u003c/em\u003e in SIFaR-positive neurons via \u003cem\u003eClk-RNAi\u003c/em\u003e. (D) Male flies expressing \u003cem\u003eSIFa\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-lexA; GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e together with a retro-grade and transsynaptic labeling system,\u003cem\u003e BAcTrace\u003c/em\u003e, were immunostained with anti-GFP (green), anti-DsRed (red), and nc82 (magenta) antibodies. Left: expression pattern of \u003cem\u003eSIFa\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-lexA\u003c/em\u003e, middle: BAcTrace transsynaptic-labeled presynaptic neurons of GAL4\u003csup\u003eR54D11\u003c/sup\u003e neurons; right: SIFa neurons overlap with the BAcTrace-labeled presynaptic neurons of GAL4\u003csup\u003eR54D11\u003c/sup\u003e neurons. Scale bars represent 50 μm. (E) GRASP assay for \u003cem\u003eSIFa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2A-lexA\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e together with \u003cem\u003elexAop-nsyb-spGFP\u003c/em\u003e\u003csup\u003e\u003cem\u003e1-10\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, UAS-CD4-spGFP\u003c/em\u003e\u003csup\u003e\u003cem\u003e11\u003c/em\u003e\u003c/sup\u003e in the SLP and SMP regions of naïve male flies. Scale bars represent 50 μm. Brains of male fly were immunostained with anti-GFP (green) and anti-nc82 (blue) antibodies. The left and bottom panels are presented as a gray scale to clearly show the synapses connection between \u003cem\u003eSIFa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2A-lexA\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e. Boxes indicate the magnified regions of interest presented in the bottom panels. (F-G) Quantification of relative value for synaptic areas that are formed between \u003cem\u003eSIFa\u003c/em\u003e\u003csup\u003e\u003cem\u003e2A-lexA\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e in (E, S8D) between naïve and single male flies. The same quantification was performed for the relative synaptic area in these brain regions between naïve and experienced male flies. The synaptic interactions were visualized utilizing the GRASP system in naïve and experienced male flies. SMP, superior medial protocerebrum; SLP, superior lateral protocerebrum; CB, central brain; PRW, prow. (G) The small panels are presented as a red scale to show the GFP fluorescence marked by the threshold function of ImageJ. See the “Methods” for a detailed description of the fluorescence intensity analysis used in this study.\u003c/p\u003e","description":"","filename":"fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/13ca6960bb972e512cb07c36.jpg"},{"id":91816930,"identity":"6c565a50-61ad-41fc-9e17-a23c9ce8e313","added_by":"auto","created_at":"2025-09-22 06:53:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9185037,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/7a1a8631-7dbf-4618-a8d0-625345f550a3.pdf"},{"id":91405572,"identity":"721f0fe3-6f3a-462f-9f34-17bdfe881f0d","added_by":"auto","created_at":"2025-09-16 07:48:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":476972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. Screen of 150 clock neurons.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2025CLKv5Table.1screenofclockneurons.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/431affe231a141bff0c7fb7a.pdf"},{"id":91405574,"identity":"15e57aff-5772-4bfc-a760-47147a60ecfc","added_by":"auto","created_at":"2025-09-16 07:48:06","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":534414,"visible":true,"origin":"","legend":"2025-CLK-v2-Supporting_information-genetic-controls","description":"","filename":"2025CLKv2Supportinginformationgeneticcontrols.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/086cbe9ae6dbd19f94b3d3f3.pdf"},{"id":91406526,"identity":"5b7b6041-67ae-47c4-a4d1-5838bcf828d2","added_by":"auto","created_at":"2025-09-16 07:56:06","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":942321,"visible":true,"origin":"","legend":"","description":"","filename":"2025CLKFig.S1v2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/ea807e911a61890c0ffbc7f5.pdf"},{"id":91406523,"identity":"7862ad3d-9dde-4629-ba81-4f685acf6cf5","added_by":"auto","created_at":"2025-09-16 07:56:06","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":578094,"visible":true,"origin":"","legend":"","description":"","filename":"2025CLKFig.S2v2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/f053b01624f357fd53ba627c.pdf"},{"id":91405590,"identity":"99ed79e0-0b77-4d07-8984-569860836978","added_by":"auto","created_at":"2025-09-16 07:48:06","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":6088408,"visible":true,"origin":"","legend":"","description":"","filename":"2025CLKFig.S3v2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/8c172fee8590ec17e1392b3d.pdf"},{"id":91408318,"identity":"6ecf763f-fc48-4b52-a653-aa2e246a85ad","added_by":"auto","created_at":"2025-09-16 08:20:06","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1507247,"visible":true,"origin":"","legend":"","description":"","filename":"2025CLKFig.S4v2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/e2f816d6d450961588848d71.pdf"},{"id":91405582,"identity":"780540ac-bbbb-418e-85c0-2c81415ab782","added_by":"auto","created_at":"2025-09-16 07:48:06","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":547554,"visible":true,"origin":"","legend":"","description":"","filename":"2025CLKFig.S5v2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/a1eb7c7f96e7a044bcda6dcd.pdf"},{"id":91406943,"identity":"c367c882-1d09-4fcc-ae29-ad65ebc087ac","added_by":"auto","created_at":"2025-09-16 08:04:06","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1047424,"visible":true,"origin":"","legend":"","description":"","filename":"2025CLKFig.S6v2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/da5d5daa058d2cf6d708fa81.pdf"},{"id":91406941,"identity":"1b7fb083-0dd5-4bfc-a40c-269557950781","added_by":"auto","created_at":"2025-09-16 08:04:06","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":616019,"visible":true,"origin":"","legend":"","description":"","filename":"2025CLKFig.S7v4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/045ba6845b94f45e7bb3bbff.pdf"},{"id":91405591,"identity":"8c0c9fa3-06cb-41b2-b48c-88b558be203f","added_by":"auto","created_at":"2025-09-16 07:48:07","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":19778986,"visible":true,"origin":"","legend":"","description":"","filename":"2025CLKFig.S8v2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/ea2c427008e2f3ae05bba30b.pdf"},{"id":91406534,"identity":"bdb8ba67-4f99-434f-b419-35f11388e10b","added_by":"auto","created_at":"2025-09-16 07:56:06","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":1614660,"visible":true,"origin":"","legend":"","description":"","filename":"2025CLKFig.S9v4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/d9808c61bcbe75c80f9fe393.pdf"},{"id":91405577,"identity":"4e0744a7-2eb0-46a9-ba8c-d54f37f2d120","added_by":"auto","created_at":"2025-09-16 07:48:06","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":19787,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigureLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/f38de47df12ec616c1ae6ed5.docx"},{"id":91408057,"identity":"c748c0b5-1d9d-471c-8106-a09f77b2f540","added_by":"auto","created_at":"2025-09-16 08:12:06","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":14144,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7471909/v1/868a14e86c99c569e0a9b9bf.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"CLOCK-dependent pathway in a single pair of LNd neurons instruct circadian-independent interval timing behavior.","fulltext":[{"header":"Statement of Significance","content":"\u003cp\u003eThis study in \u003cem\u003eDrosophila\u003c/em\u003e demonstrates that the CLK/CYC heterodimer is crucial for interval timing, distinct from circadian rhythm regulation. The research pinpoints a specific pair of neurons that are critical for shortened mating duration behavior, and it suggests that CLK variants can influence interval timing dissociation from circadian rhythm and sleep behaviors.\u003c/p\u003e"},{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eCLK/CYC heterodimer are essential for interval timing.\u003c/li\u003e\n \u003cli\u003eSpecialized mechanism of interval timing behavior revealed.\u003c/li\u003e\n \u003cli\u003eIdentified a pair of glutamatergic ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons that independently regulate both interval timing and sleep.\u003c/li\u003e\n \u003cli\u003eRevealed a novel mechanism by which CLK variants regulate sleep and interval timing.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe precise measurement of time intervals, ranging from seconds to minutes, is a critical cognitive function that is essential for behaviors such as mating, foraging, and navigation\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Interval timing is thought to encompass an internal clock mechanism that depends on the synchronization of pacemaker-accumulator circuits with memory circuits in the brain\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, with its well-characterized neural circuits and molecular mechanisms, serves as an excellent model organism for the study of interval timing\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Interval timing has been identified in the mating behaviors of \u003cem\u003eDrosophila\u003c/em\u003e, particularly in the duration of male mating. Males demonstrate the ability to adjust their mating duration based on contextual experiences, suggesting the formation of long-term memory to optimize their sexual investment\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe mating duration of male fruit flies serves as an excellent model for investigating interval timing, as it is influenced by both internal states and environmental contexts. Previous research by our group \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and others \u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e has established robust frameworks for studying mating duration using advanced genetic tools, enabling the dissection of neural circuits underlying interval timing. Notably, males exhibit prolonged mating duration when exposed to rival environments \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In contrast, they display shortened mating duration (SMD) behavior under sexually saturated conditions, where they reduce their mating investment \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These findings highlight the adaptability of mating behavior in response to social and environmental cues, providing a valuable system for exploring the neural and molecular mechanisms of interval timing.\u003c/p\u003e\u003cp\u003eThe circadian rhythm in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e has been a subject of intense research for several decades, and significant progress has been made in understanding the molecular and genetic underpinnings of this biological clock\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Key genes such as \u003cem\u003eperiod (per)\u003c/em\u003e, \u003cem\u003etimeless (tim)\u003c/em\u003e, \u003cem\u003eClock (Clk)\u003c/em\u003e and \u003cem\u003ecycle (cyc)\u003c/em\u003e have been identified and characterized, providing insights into the feedback loops that regulate the circadian rhythm at the molecular level\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These genes form the core of the molecular clock, with their timed expression, localization, post-transcriptional modification, and function being critical for maintaining the circadian cycle. Various regulators, including phosphatases and kinases, act on different steps of this feedback loop to ensure strong and accurately timed rhythms\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCircadian clock plays a role in regulating sleep by ensuring that it occurs at the appropriate time. However, the quantity and quality of sleep are also influenced by other systems that maintain a balance between sleep and wakefulness. Sleep is regulated by separate genetic and cellular mechanisms that control the need for sleep, adjust to environmental signals, and react to extended periods of alertness. In addition, the regulation of sleep according to the body's internal clock requires multiple groups of cells and molecules that coordinate sleep and waking cycles at precise times of the day, rather than being governed by a single oscillating signal\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCircadian rhythm pertains to the 24-hour cycle that governs biological processes, whereas sleep is a reversible state characterized by diminished activity and responsiveness. Interval timing refers to the measurement of durations and intervals, such as the mating duration or foraging actions\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The primary distinction lies in the periodicity of circadian rhythms and sleep, which adhere to fixed daily patterns, while interval timing is adaptable and not constrained by a 24-hour cycle, enabling organisms to measure time intervals according to their specific requirements\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough the connection between circadian timing and different physiological processes has been extensively studied, the genetic understanding of the interaction between circadian timing and interval timing remains incomplete. Our previous research demonstrated that circadian clock genes, specifically \u003cem\u003eper\u003c/em\u003e and \u003cem\u003etim\u003c/em\u003e, rather than \u003cem\u003eClk\u003c/em\u003e and \u003cem\u003ecyc\u003c/em\u003e, modulate the rival-induced longer-mating-duration (LMD) behavior, a distinct form of interval timing critical for maximizing sperm competition\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we investigate the role of the \u003cem\u003eClk\u003c/em\u003e/\u003cem\u003ecyc\u003c/em\u003e gene complex and associated factors within a single pair of LN\u003csub\u003ed\u003c/sub\u003e neurons in modulating the sexually experienced-dependent shorter-mating-duration (SMD) behavior, which has been previously shown to be elicited by gustatory and pheromonal cues from females\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eNeuronal expression of CLK/CYC heterodimer is necessary and sufficient to generate sexual experience-mediated reduced mating investment.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the context of core circadian rhythm genes, it was observed that mutations in the \u003cem\u003eper\u003c/em\u003e or \u003cem\u003etim\u003c/em\u003e genes, as well as compound mutations in both \u003cem\u003eper\u003c/em\u003e and \u003cem\u003etim\u003c/em\u003e, did not disrupt SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). However, mutations in the \u003cem\u003eClk\u003c/em\u003e and \u003cem\u003ecyc\u003c/em\u003e genes resulted in the absence of SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). Additionally, mutations in the \u003cem\u003ecryptochrome\u003c/em\u003e (\u003cem\u003ecry\u003c/em\u003e) gene (\u003cem\u003ecry\u003c/em\u003e\u003csup\u003e\u003cem\u003e03\u003c/em\u003e\u003c/sup\u003e) and the sleepless mutant of \u003cem\u003equiver\u003c/em\u003e (\u003cem\u003eqvr\u003c/em\u003e\u003csup\u003e\u003cem\u003e01\u003c/em\u003e\u003c/sup\u003e)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e did not alter the SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Fig. S1A). These findings suggest that the CLK/CYC heterodimer is uniquely involved in interval timing behaviors among the core circadian rhythm gene components.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe targeted RNAi-mediated knockdown of \u003cem\u003eClk\u003c/em\u003e or \u003cem\u003ecyc\u003c/em\u003e in neurons resulted in the disruption of SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J and Fig. S1B-C). Additionally, analysis of the fly RNAseq dataset platform, fly SCope, predicted co-expression of \u003cem\u003eClk\u003c/em\u003e and \u003cem\u003ecyc\u003c/em\u003e in specific neuronal populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. While co-expression of \u003cem\u003eClk\u003c/em\u003e and \u003cem\u003ecyc\u003c/em\u003e in glial cells was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), knockdown of these genes in glial populations did not affect SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L), indicating that glial expression of CLK/CYC is not essential for interval timing behavior. Furthermore, knockdown of \u003cem\u003eClk\u003c/em\u003e in epithelial, gut, Malpighian tubules (MTs), muscle, hemocyte, and intestinal stem cells (ISCs), despite their high expression of \u003cem\u003eClk\u003c/em\u003e and \u003cem\u003ecyc\u003c/em\u003e, did not alter SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM-V and Fig. S1K-L). Collectively, the genetic control experiments and the testing of independent RNAi lines suggest that only neuronal expression of \u003cem\u003eClk\u003c/em\u003e and \u003cem\u003ecyc\u003c/em\u003e is required for the sexual experience-mediated reduction in mating investment (Fig. S1D-J). These findings underscore the specificity of the neuronal CLK/CYC expression in mediating the behavioral changes associated with sexual experience, highlighting the importance of cell-type-specific gene expression in the regulation of complex behaviors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe expression of CLK within NPF-expressing cry-positive circadian neurons in the brain is critical for interval timing behavior.\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eClk\u003c/em\u003e and \u003cem\u003ecyc\u003c/em\u003e are robustly expressed in neuronal populations throughout the \u003cem\u003eDrosophila\u003c/em\u003e body (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, targeted knockdown of \u003cem\u003eClk\u003c/em\u003e in all neurons except those of the ventral nerve cord (VNC) still resulted in the disruption of SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating that \u003cem\u003eClk\u003c/em\u003e expression within the VNC is not essential for the generation of SMD behavior. Furthermore, the selective knockdown of \u003cem\u003eClk\u003c/em\u003e or \u003cem\u003ecyc\u003c/em\u003e in the brain alone was sufficient to impair SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E), suggesting that among the diverse neuronal populations, only CLK/CYC expression within the brain is necessary for the manifestation of SMD behavior.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThrough a genetic rescue approach targeting the \u003cem\u003ecyc\u003c/em\u003e gene, we discovered that neuronal expression of \u003cem\u003ecyc\u003c/em\u003e, with the exception of the PDF-expressing lateral ventral neurons (LN\u003csub\u003ev\u003c/sub\u003e), was sufficient to restore SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H), despite the rescue's inability to restore circadian rhythmicity\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Additionally, the knockdown of \u003cem\u003ecyc\u003c/em\u003e specifically in PDF neurons did not affect SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eI), implying that CLK/CYC expression in core oscillator cells, such as sLN\u003csub\u003ev\u003c/sub\u003e and lLN\u003csub\u003ev\u003c/sub\u003e, is not required for the generation of interval timing behavior.\u003c/p\u003e\u003cp\u003eUtilizing a suite of GAL4 drivers specific to clock cells in conjunction with \u003cem\u003eClk-RNAi\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ and Fig. S2A), we conducted a detailed analysis revealing that the expression of \u003cem\u003eClk\u003c/em\u003e and \u003cem\u003ecyc\u003c/em\u003e in neurons positive for neuropeptide F (NPF) and cry is essential for the generation of SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-O and Fig. S2B-C). Knockdown of \u003cem\u003eClk\u003c/em\u003e in neurons that are NPF-positive but cry-negative did not impair SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eP), indicating that the expression of \u003cem\u003eClk\u003c/em\u003e in both cry-positive and NPF-positive neurons is critical for the manifestation of SMD behavior. NPF-expressing cry-positive neurons are located in the LN\u003csub\u003ed\u003c/sub\u003e and DN regions of both male and female brains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eQ). However, a unique male brain phenotype was observed, with NPF-positive and CRY-positive neuronal processes extending near the suboesophageal ganglion (SOG) region (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eQ), which is known to be important for processing taste information. Previous research from our laboratory has demonstrated the presence of male-specific CRY-positive and NPF-positive LN\u003csub\u003ed\u003c/sub\u003e neurons\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and the role of these neurons\u0026rsquo; sexual dimorphism in mating behavior has been reported\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. These findings suggest that the sexual dimorphism in the distribution of NPF-expressing cry-positive neurons may underlie sex-specific differences in the processing of sensory information related to SMD behavior. Our results collectively identify a selective requirement for CLK/CYC within specific clock neurons in the modulation of SMD behavior.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTwo pair of clock neurons are associated with CLK function to generate interval timing.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUtilizing the recently published transcriptomic taxonomy dataset for \u003cem\u003eDrosophila\u003c/em\u003e circadian neurons\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, we conducted a targeted screen to identify the minimal subset of Clk-expressing neurons necessary for the generation of SMD (Table. S1). Our analysis revealed that CLK expression within the \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e-labeled ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5th sLN\u003csub\u003ev\u003c/sub\u003e neurons are pivotal for interval timing behavior (Table S1, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). Knockdown of \u003cem\u003eClk\u003c/em\u003e specifically in adults or excluding of the VNC using the \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver was sufficient to impair SMD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D and Fig. S3B), indicating that adult brain expression of \u003cem\u003eClk\u003c/em\u003e in ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5th sLN\u003csub\u003ev\u003c/sub\u003e neurons are required to generate SMD behavior. No sexual dimorphism was observed in the expression pattern of the \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver within the brain (Fig. S3C-D). Furthermore, the disruption of SMD following Clk knockdown with the \u003cem\u003eITP\u003c/em\u003e\u003csup\u003e\u003cem\u003eT2A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-GAL4\u003c/em\u003e and LN\u003csub\u003ed\u003c/sub\u003e drivers (\u003cem\u003eMai179-GAL4\u003c/em\u003e, marking ITP-LN\u003csub\u003ed\u003c/sub\u003e, 5th -sLN\u003csub\u003ev\u003c/sub\u003e, and 2 sNPF-LN\u003csub\u003ed\u003c/sub\u003e neurons) confirmed that the \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e driver-labelled neurons are indeed ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5th sLN\u003csub\u003ev\u003c/sub\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eITP is a key endocrine regulator of water homeostasis in \u003cem\u003eDrosophila\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Within the brain and VNC, only a single pair of neurons expressing ITP is located in the LN\u003csub\u003ed\u003c/sub\u003e region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Analysis of the Fly SCope dataset suggests the presence of a restricted number of neurons that are triply positive for \u003cem\u003eITP\u003c/em\u003e, \u003cem\u003eNPF\u003c/em\u003e, and \u003cem\u003eClk\u003c/em\u003e expression within the brain, but not in the VNC (Fig. S3K-N). Knockdown of \u003cem\u003ecyc\u003c/em\u003e, induced apoptosis, hyperexcitation, or inhibition of synaptic transmission in ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5th sLN\u003csub\u003ev\u003c/sub\u003e neurons, consistently disrupted SMD behavior (Fig. S3E-I). However, inhibiting the neuronal activity of ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5th sLN\u003csub\u003ev\u003c/sub\u003e neurons via voltage-gated potassium channel, KCNJ2 resulted in developmental lethality, indicating that their neuronal function is essential for development as well (Fig. S3G). The feminization of ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5th sLN\u003csub\u003ev\u003c/sub\u003e neurons through the expression of \u003cem\u003eUAS-tra\u003c/em\u003e\u003csup\u003e\u003cem\u003eF\u003c/em\u003e\u003c/sup\u003e had no effect on interval timing behavior (Fig. S3J), demonstrating that the sexual dimorphism of these LN\u003csub\u003ed\u003c/sub\u003e neurons does not play a role in regulating interval timing. Notably, knockdown of \u003cem\u003eITP\u003c/em\u003e within ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons did not affect SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), suggesting that the role of ITP in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons is distinct from its function in interval timing. These findings implicate a specialized subset of ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5th sLN\u003csub\u003ev\u003c/sub\u003e neurons as a critical node in the circuitry governing interval timing, while also highlighting the diverse functions of ITP in neuronal physiology.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe glutamatergic output circuits originating from ITP-LN\u003c/b\u003e\u003csub\u003e\u003cb\u003ed\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eneurons are instrumental in the generation of interval timing.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur findings reveal that the expression of flippase in NPF-positive neurons with a UAS-stop cassette can confine the expression of \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e to four specific neurons in the brain, including the ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5th -sLN\u003csub\u003ev\u003c/sub\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Inhibiting the activity of these neurons disrupts SMD behavior without causing developmental lethality (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating that the KCNJ2-mediated developmental lethality observed with \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eR54D11\u003c/em\u003e\u003c/sup\u003e is likely due to its expression in the VNC (Fig. S3G and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Furthermore, hyperexcitation induced by NaChBac, inhibition of synaptic transmission by TNT, or the knockdown of \u003cem\u003eClk\u003c/em\u003e via \u003cem\u003eClk-RNAi\u003c/em\u003e in these four brain neurons all resulted in the disruption of SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E), emphasizing the critical role of CLK function and neuronal activity in the ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons in the brain for the generation of interval timing.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUtilizing the fly SCope RNA seq dataset and the FlyWire connectome dataset platform, we inferred the expression patterns of enzymes responsible for neurotransmitter synthesis (Fig. S4A-B) and identified the output neurons from the ITP-LN\u003csub\u003ed\u003c/sub\u003e\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan additionalcitationids=\"CR42 CR43 CR44 CR45 CR46 CR47\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The FlyWire connectome data suggests that ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons project to central brain circuits (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) and slightly more to the contralateral brain (Fig. S4C). Although the Fly SCope data analysis was inconclusive (Fig. S4A-B), the FlyWire connectome dataset analysis clearly demonstrated that the output circuits from the ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons consist of glutamatergic and possibly cholinergic co-transmission (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Given that the FlyWire connectome data predict the 5th sLN\u003csub\u003ev\u003c/sub\u003e to be serotonergic neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) and that ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons have been confirmed to be non-cholinergic LN\u003csub\u003ed\u003c/sub\u003e neurons\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, we conclude that the single pair of ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons are glutamatergic.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe knockdown of \u003cem\u003eVGlut\u003c/em\u003e specifically in these neurons disrupts SMD behavior, and the expression of \u003cem\u003eClk-RNAi\u003c/em\u003e in the non-glutamatergic subset of GAL4\u003csup\u003eR54D11\u003c/sup\u003e neurons rescues the disrupted SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I). Moreover, knockdown of \u003cem\u003eClk\u003c/em\u003e only in GAL4\u003csup\u003eR54D11\u003c/sup\u003e-positive and glutamatergic neurons is sufficient to disrupt SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ), indicating the crucial role of Clk expression in a single pair of ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons. Optogenetic activation of GAL4\u003csup\u003eR54D11\u003c/sup\u003e neurons can induce a reduction in mating duration without prior sexual experience (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), suggesting that artificial neuronal activation of these neurons can mimic the sexual experience-mediated internal states of pacemaker circuits.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVariants of the CLK protein that undergo alternative splicing may dissociate circadian rhythms from interval timing within ITP-LN\u003c/b\u003e\u003csub\u003e\u003cb\u003ed\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eand 5th -sLN\u003c/b\u003e\u003csub\u003e\u003cb\u003ev\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eneurons.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe have previously demonstrated that rival-induced prolonged mating duration (LMD), a distinct form of interval timing behavior in male flies, persists even in arrhythmic conditions such as continuous light exposure for five days\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similarly, SMD behavior remains intact under arrhythmic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In the same condition, the rhythmic activity and sleep of flies was completely disorganized (Fig. S5A-B), indicating that interval timing behaviors are independent of the circadian rhythm. We previously conducted experiments at various times throughout the day with \u003cem\u003eCanton-S\u003c/em\u003e flies and observed normal SMD behavior\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, which is consistent with the reported results.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe influence of temperature and feeding on the alternative splicing of clock gene products has been explored\u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, but the role of CLK mRNA variants in regulating various timing behaviors has not been previously reported. The \u003cem\u003eClk\u003c/em\u003e gene produces five distinct mRNA transcripts, \u003cem\u003eClk-RA\u003c/em\u003e, \u003cem\u003eRD\u003c/em\u003e, \u003cem\u003eRF\u003c/em\u003e, \u003cem\u003eRG\u003c/em\u003e, and \u003cem\u003eRH\u003c/em\u003e, with \u003cem\u003eClk-RG\u003c/em\u003e and \u003cem\u003eRH\u003c/em\u003e lacking the first three exons and thus unable to bind DNA. Our study revealed that \u003cem\u003eClk\u003c/em\u003e\u003csup\u003e\u003cem\u003eADF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-RNAi\u003c/em\u003e specifically targets \u003cem\u003eClk-RA\u003c/em\u003e, \u003cem\u003eRB\u003c/em\u003e, and \u003cem\u003eRF\u003c/em\u003e transcripts, which contain DNA-binding motifs. Surprisingly, the expression of \u003cem\u003eClk\u003c/em\u003e\u003csup\u003e\u003cem\u003eADF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-RNAi\u003c/em\u003e did not disrupt SMD behavior, or the circadian rhythm compared to genetic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F and Fig. S5C). This suggests that CLK proteins with DNA-binding motifs are not essential for generating interval timing behaviors in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons.\u003c/p\u003e\u003cp\u003eWe next investigated whether CLK expression in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons is linked to sleep behaviors. Knockdown of \u003cem\u003eClk\u003c/em\u003e in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons partially reduced the duration of sleep during both day and night (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J). Remarkably, when we selectively knocked down \u003cem\u003eClk\u003c/em\u003e transcripts with DNA-binding motifs in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), daytime but not nighttime sleep duration decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eK-N). This observation clearly indicates that different variants of CLK proteins have differential effects on sleep behavior. We speculate that CLK proteins lacking the DNA-binding motif may play a specific role in regulating sleep and interval timing, suggesting a novel molecular mechanism for the regulation of these behaviors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe CLK/CYC heterodimer, a transcription factor complex, is specifically linked to interval timing and sleep behaviors.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlthough circadian clock genes are primarily known for their role in regulating circadian rhythms, they also perform various other functions\u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Sleep timing is influenced by the circadian clock, yet the two processes can be dissociated; mutations in core clock genes often lead to fragmented sleep patterns but do not necessarily affect overall sleep duration\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003ecyc\u003c/em\u003e\u003csup\u003e\u003cem\u003e01\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eClk\u003c/em\u003e\u003csup\u003e\u003cem\u003eJrk\u003c/em\u003e\u003c/sup\u003e mutants displayed distinct sleep pattern abnormalities and life span changes, with the \u003cem\u003ecyc\u003c/em\u003e\u003csup\u003e\u003cem\u003e01\u003c/em\u003e\u003c/sup\u003e mutation exhibiting sexually dimorphic effects on sleep compensation\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The CLK/CYC heterodimer transcription factors play a pivotal role in the regulation of numerous clock gene transcripts. However, the specific functions of these target genes in various timing behaviors are not yet fully elucidated (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eA)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOur findings suggest that the CLK/CYC heterodimer lacking a DNA-binding motif is a critical regulator of interval timing and sleep, but not necessarily of circadian rhythm. Consequently, we selected several essential clock genes known to modulate circadian rhythm and reassessed their roles in interval timing and sleep within ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons. Knockdown of \u003cem\u003ecwo\u003c/em\u003e (\u003cem\u003eclockwork orange\u003c/em\u003e) and \u003cem\u003eCipc\u003c/em\u003e (\u003cem\u003eClock interacting protein circadian\u003c/em\u003e) had no impact on SMD behavior, while \u003cem\u003evri\u003c/em\u003e (\u003cem\u003evrille\u003c/em\u003e) and \u003cem\u003ePdp1\u003c/em\u003e (\u003cem\u003ePAR-domain protein 1\u003c/em\u003e) knockdown disrupted SMD (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D and Fig. S6A). Notably, PER/TIM-related clock genes, such as \u003cem\u003edco\u003c/em\u003e (\u003cem\u003ediscs overgrown\u003c/em\u003e), \u003cem\u003ejet\u003c/em\u003e (\u003cem\u003ejetlag\u003c/em\u003e), \u003cem\u003eAtx2\u003c/em\u003e (\u003cem\u003eAtaxin-2\u003c/em\u003e), and \u003cem\u003ecry\u003c/em\u003e, did not affect SMD behavior (Fig. S6B-E). The expression levels of these genes did not correlate with their influence on SMD (Fig. S6F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the impact of CLK/CYC function on circadian rhythm and sleep within ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons, we selected \u003cem\u003ecwo\u003c/em\u003e and \u003cem\u003evri\u003c/em\u003e as representative genes. Both genes are closely associated with CLK/CYC function, but only \u003cem\u003evri\u003c/em\u003e is involved in interval timing. Knockdown of either genes did not disrupt circadian rhythm (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and Fig. S6G). Knockdown of \u003cem\u003evri\u003c/em\u003e in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons resulted in a decrease in daytime and nighttime sleep duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-I), while \u003cem\u003ecwo\u003c/em\u003e knockdown reduced the duration of sleep similarly (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ and Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ-K). These results indicate that CLK-VRI interaction is crucial for modulating interval timing and sleep, but not for circadian rhythm regulation, within ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons. Overexpression of miRNA \u003cem\u003ebantam\u003c/em\u003e, which binds to the 3' UTR of Clk and modulates its translation\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, reversed SMD behavior, indicating the critical role of CLK protein regulation in generating interval timing (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). Concurrently, the overexpression of \u003cem\u003emir.ban\u003c/em\u003e in ITP-LN\u003csub\u003ed\u003c/sub\u003e resulted in significant alterations in sleep behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). Specifically, this was manifested as a decrease in sleep during the daytime and an increase during the nighttime (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eN-O). These findings suggest that \u003cem\u003emir.ban\u003c/em\u003e also plays a pivotal role in the regulation of sleep behavior.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe SIFa-SIFaR peptidergic signaling pathway is specifically involved in modulating interval timing distinct from its influence on circadian rhythm or sleep behavior.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCircadian clock cells are influenced by environmental factors such as temperature, light, and feeding\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. To elucidate the input signals that specifically regulate SMD behavior in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons, we analyzed the fly SCope dataset, which revealed that SIFaR is highly expressed in these neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Neuropeptide SIFa, expressed in four neurons in the \u003cem\u003epars intercerebralis\u003c/em\u003e (PI), plays a pivotal role in modulating interval timing behaviors\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Knockdown of \u003cem\u003eSIFaR\u003c/em\u003e in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons, as well as knockdown of \u003cem\u003eClk\u003c/em\u003e in SIFaR-positive cells, disrupted SMD behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C), but did not affect circadian rhythm (Fig. S7A). Concomitantly, the absence of SIFaR led to a reduction of both daytime and nighttime sleep duration (Fig. S7B-E). BAcTrace data demonstrated that GAL4\u003csup\u003eR54D11\u003c/sup\u003e neurons receive strong input signals from PI-located SIFa neurons\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These findings suggest that SIFa signals are crucial for interval timing and sleep regulation in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons, but not for circadian rhythm.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, we observed that ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons form strong synapses with SIFa neurons in the superior medial protocerebrum (SMP) and superior lateral protocerebrum (SLP) regions of the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). The SLP region is known to be involved in upstream processing of mushroom body (MB) learning centers and is where long-range taste projection neurons project\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Although sexual experiences did not significantly alter the accumulated calcium levels in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons (Fig. S8A-B) and activation of SIFa neurons also did not lead to elevated calcium levels in ITP-LN\u003csub\u003ed\u003c/sub\u003e (Fig. S8C), the synapses between SIFa and ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons were significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-G and Fig. S8D). These data indicate that sexual experiences reduce the inputs to ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons from SIFa peptidergic signals without dramatic calcium flux change.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUsing the FlyWire dataset, we analyzed the input circuits to ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons and identified that cholinergic, glutamatergic, and GABAergic inputs constitute the main neurotransmitters from input neurons, with half coming from visual and half from central class neurons (Fig. S9A-B). Interestingly, the inputs to ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons predominantly originate from the left side of the brain, potentially representing the involvement of the asymmetric body (AB) associated with this circuit (Fig. S9C)\u003csup\u003e\u003cspan additionalcitationids=\"CR65 CR66 CR67\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe synaptic connectivity of ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons has previously been shown to be distinct within the clock network, exhibiting fewer intra-network synapses compared to other groups of clock neurons \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Previous single-cell RNA sequencing data analysis of 150 clock neurons identified that neural connectivity molecules are key in defining the diverse functions of heterogeneous clock cells\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Among these molecules, voltage-gated potassium channel Shaker (Sh) and its beta-subunit Hyperkinetic (Hk) were found to be highly enriched in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons. Knockdown of these channels in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons reversed SMD behavior, indicating their critical role in maintaining ITP-LN\u003csub\u003ed\u003c/sub\u003e as pacemaker neurons for interval timing (Fig. S9D-E). Interestingly, the reversed phenotype of interval timing associated with Sh knockdown is linked to a significant shortening of sleep duration, while Hk knockdown shows no effect on sleep (Fig. S9F-M), suggesting that there might exist divergent mechanistic roles for these channels in regulating interval timing and sleep pathways within ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons, separable from circadian control.\u003c/p\u003e\u003cp\u003eKnockdown of the Ih channel, which encodes a low-threshold, voltage-gated ion channel in ITP-LN\u003csub\u003ed\u003c/sub\u003e, also disrupted SMD behavior (Fig. S9N), suggesting that voltage-gated channels are important regulators of pacemaker function for interval timing. In contrast, knockdown of serotonin receptor 5-HT1A, octopamine receptor in MB (Oamb), or octopamine b3 receptor (Octb3R), which are known to be enriched in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, did not affect SMD behavior (Fig. S9O-Q), indicating that 5-HT and octopamine (OA) transmission are not essential for ITP-LN\u003csub\u003ed\u003c/sub\u003e's role in interval timing. These data collectively underscore the pivotal role of voltage-gated channels in maintaining the pacemaker function of CLK-expressing ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons for interval timing.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the role of the CLK/CYC heterodimer and associated factors within a specific pair of LN\u003csub\u003ed\u003c/sub\u003e neurons in modulating sexual experience-dependent interval timing (SMD) behavior. We found that mutations in \u003cem\u003eClk\u003c/em\u003e and \u003cem\u003ecyc\u003c/em\u003e, but not \u003cem\u003eper\u003c/em\u003e and \u003cem\u003etim\u003c/em\u003e, disrupt SMD behavior, indicating a unique role for CLK/CYC in interval timing. Neuronal CLK/CYC expression is necessary and sufficient for SMD, while glial expression is not (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Specific CLK/CYC expression in NPF\u003csup\u003e+\u003c/sup\u003e and cry\u003csup\u003e+\u003c/sup\u003e neurons in the brain, but not in PDF neurons, is essential for SMD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Detailed analysis identifies CLK expression in ITP-LN\u003csub\u003ed\u003c/sub\u003e and 5th sLN\u003csub\u003ev\u003c/sub\u003e neurons as pivotal for SMD. Knockdown of CLK in these neurons impairs SMD, as does inhibiting their activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons are glutamatergic, with output circuits to central brain. Inhibiting glutamatergic transmission or knocking down CLK in these neurons disrupts SMD. Furthermore, optogenetic activation of these neurons can induce SMD without prior experience (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We also explored the molecular mechanisms, finding that CLK variants lacking DNA binding motifs dissociate circadian rhythms from interval timing and sleep behaviors in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003e). CLK-VRI interaction is crucial for modulating interval timing and sleep, but not circadian rhythm, within ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Finally, we demonstrated that the SIFa-SIFaR peptidergic signaling pathway is specifically involved in modulating interval timing distinct from its influence on circadian rhythm (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Our study provides a comprehensive understanding of the role of CLK/CYC heterodimer and associated factors in modulating interval timing behavior in \u003cem\u003eDrosophila\u003c/em\u003e, shedding light on the neural circuits and molecular mechanisms underlying this complex behavior (Fig. S9R).\u003c/p\u003e\u003cp\u003eDespite the central role of circadian clock genes in regulating circadian rhythms, these genes are also involved in various non-circadian functions. Accumulating evidence suggests that clock genes exhibit pleiotropic effects in regulating diverse physiological processes\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. In addition to their well-established roles in sleep homeostasis\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, clock genes have been implicated in the modulation of addiction, psychiatric disorders, and male fertility\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Recent studies have expanded our understanding of CLK's function, revealing its role in tissue-specific gene regulation and metabolic processes\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, with Gart being a notable example that is crucial for maintaining feeding rhythms and food intake\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Furthermore, distinct clock neurons have been shown to mediate the circadian rhythm of male sex drive\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. The surprising diversity of circadian clock cells, akin to their functional heterogeneity, suggests that individual clock neurons possess distinct functions beyond circadian rhythm regulation\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. In conclusion, the diverse non-circadian functions of clock genes underscore their intricate regulatory roles across various physiological processes. The discovery of specific non-circadian functions of clock genes, such as the role of CLK in modulating interval timing and sleep regulation in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons, offers valuable insights into the multifaceted functions of these genes. These findings underscore the complexity and versatility of clock genes in orchestrating biological rhythms and behaviors beyond the circadian clock.\u003c/p\u003e\u003cp\u003eIn our study, we uncovered that various \u003cem\u003eClk\u003c/em\u003e transcripts exhibit distinct timing functions. Our data indicate that \u003cem\u003eClk-RA\u003c/em\u003e, \u003cem\u003eRD\u003c/em\u003e, and \u003cem\u003eRF\u003c/em\u003e are essential for circadian rhythmicity and sleep, while \u003cem\u003eClk-RG\u003c/em\u003e and \u003cem\u003eRH\u003c/em\u003e are critical for interval timing and sleep regulation. These findings suggest that \u003cem\u003eClk\u003c/em\u003e transcripts regulate a spectrum of timing behaviors through their expression in specific clock cells. Moreover, we identified voltage-gated potassium channels Sh and Hk as pivotal for maintaining the pacemaker function of ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons in regulating interval timing. Elucidating the relationship between Clk-RG/RH function and Sh/Hk in ITP-LN\u003csub\u003ed\u003c/sub\u003e neurons may provide insights into the molecular mechanisms governing pacemaker regulation in the fly brain for interval timing behavior (Fig. S9R).\u003c/p\u003e\u003cp\u003eThe traditional perspective on the rhythmic regulation of CLK/CYC dimer-mediated transcriptional control does not account for the PER/TIM-independent mechanisms or the specific dependence of SMD behavior on alternative splicing forms of \u003cem\u003eClk\u003c/em\u003e, which are crucial for behavioral timekeeping within a 5-20-minute range. Our data suggest that a C-terminally deleted CLK protein can exhibit circadian-independent functions, particularly in relation to male interval timing associated with mating investment behavior. Recent studies have reported that alternative splicing of the \u003cem\u003eClk\u003c/em\u003e transcript mediates the circadian clock's response to temperature changes\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Additionally, a non-circadian role for \u003cem\u003ecyc\u003c/em\u003e has been identified, which regulates the development of clock neurons\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. There has long been speculation regarding the non-circadian regulation and function of clock genes in controlling oogenesis in female \u003cem\u003eDrosophila\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e and in influencing non-circadian aspects of mating behavior\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Therefore, we propose that CLK/CYC dimers composed of different forms of CLK protein may serve diverse functions beyond circadian rhythm regulation, including metabolic control, feeding behavior, mating behavior, and interval timing. Further investigation into the various forms of CLK proteins and their dimerization with CYC will shed light on how these non-circadian functions have evolved alongside the circadian roles of CLK protein.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Drs. Yuh Nung Jan and Lily Yeh Jan (UCSF, USA) for helpful comments and support on this paper. We are very appreciative to the colleagues who supplied us with several fly strains: Dr. Alex C. Keene (Texas A\u0026amp;M University), Dr. Justin Blau (New York University), Dr. Amita Sehgal (University of Pennsylvania), Dr. Ravi Allada (University of Michigan), Dr. Lihua Jin (Northeast Forestry University), Dr. Zongzhao Zhai (Hunan Normal University), Dr. Wei Zhang (Tsinghua University), Dr. Donggen Luo (Peking University), Dr. Fang Guo (Zhejiang University), Dr. Yufeng Pan and Dr. Junhai Han (Southeast University) and Drs. Young-Joon Kim and Sung-Eun Yoon (Korea Drosophila Resource Center, KDRC). This research was supported a University of Ottawa Startup grant 602496 to WJK, Startup funds from HIT Center for Life Science to WJK, a University of Ottawa Interdisciplinary Research Group Funding Opportunity (IRGFO stream 1 and 2) grants 148101 and 148747 to WJK, a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant (reference: 211406) to WJK, a University of Ottawa Brain and Mind Research Institute/Center for Neural Dynamics Open call project grant 150950 to WJK, a Mitacs Globalink Research Internship Program grant 17268 to WJK. This research was also supported by the Brain Pool Program of the National Research Foundation in Korea grant ZYM5041911 to WJK, Burroughs Wellcome Fund Collaborative Research Travel Grants (reference: 1017486) to WJK and a NVIDIA Academic Hardware Grant Program to WJK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. HM received salary from the ‘Startup funds from HIT Center for Life Science to WJK’.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u0026nbsp;\u003c/strong\u003eWoo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData curation:\u003c/strong\u003e Hongyu Miao, Zekun Wu,\u0026nbsp;Yanan Wei,\u0026nbsp;Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFormal analysis:\u003c/strong\u003e Hongyu Miao, Zekun Wu,\u0026nbsp;Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding acquisition:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject administration:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResources:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupervision:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation:\u003c/strong\u003e Hongyu Miao, Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization:\u003c/strong\u003e Hongyu Miao, Zekun Wu, Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting – original draft:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting – review \u0026amp; editing:\u003c/strong\u003e Hongyu Miao, Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Generative AI and AI-assisted Technologies in the Writing Process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the creation of this work, the author(s) utilized QuillBot to rephrase English sentences, verify English grammar, and detect plagiarism, as none of the authors of this paper are native English speakers. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResource Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLead contact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Woo Jae Kim ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and code availability\u003c/strong\u003e\u003c/p\u003e\n\u003cul start=\"50\"\u003e\n \u003cli\u003eAll data reported in this paper will be shared by the lead contact upon request.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThis paper does not report original code. The URL of the codes used in this paper are listed in the key resources table.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAny additional information required to reanalyze the data in this paper is available from the lead contact upon request.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eFly stocks and husbandry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDrosophila melanogaster\u003c/em\u003e were raised on cornmeal-yeast medium at similar densities to yield adults with similar body sizes. Flies were kept in 12 h light: 12 h dark cycles (LD) at 25℃ (ZT 0 is the beginning of the light phase, ZT12 beginning of the dark phase) except for some experimental manipulation (experiments with the flies carrying tub-GAL80\u003csup\u003ets\u003c/sup\u003e). Wild-type flies were \u003cem\u003eCanton-S\u003c/em\u003e. To reduce the variation from genetic background, all flies were backcrossed for at least 3 generations to CS strain.\u0026nbsp;All mutants and transgenic lines used here have been described previously.\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Dr. Alex C. Keene (Texas A\u0026amp;M University) and Dr. Justin Blau (New York University): \u003cem\u003ecry\u003csup\u003e03\u003c/sup\u003e\u003c/em\u003e, \u003cem\u003ecry-GAL80\u003c/em\u003e, \u003cem\u003etim-GAL4\u0026nbsp;\u003c/em\u003e(BDSC7126).\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Dr. Amita Sehgal (University of Pennsylvania): \u003cem\u003eqvr\u003csup\u003e1\u003c/sup\u003e\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Dr. Lihua Jin (Northeast Forestry University): \u003cem\u003eMyo1A-GAL4\u003c/em\u003e, \u003cem\u003eesg-GAL4\u003c/em\u003e, \u003cem\u003eHml-GAL4\u0026nbsp;\u003c/em\u003e(BDSC30139).\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Dr. Zongzhao Zhai (Hunan Normal University): \u003cem\u003euro-GAL4\u0026nbsp;\u003c/em\u003e(BDSC91415).\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Dr. Wei Zhang (Tsinghua University): \u003cem\u003etsh-GAL80\u003c/em\u003e (BDSC605556), \u003cem\u003elexAop-FLP\u003c/em\u003e(BDSC55819), \u003cem\u003eVGlut-GAL80\u003c/em\u003e (BDSC58448).\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Dr. Ravi Allada (University of Michigan): \u003cem\u003eUAS-cyc\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Dr. Donggen Luo (Peking University) and Dr. Junhai Han (Southeast University): \u003cem\u003eClk4.1M-GAL4\u003c/em\u003e (BDSC31316), \u003cem\u003eGAL4\u003csup\u003eR54D11\u003c/sup\u003e\u003c/em\u003e(BDSC41279), \u003cem\u003eMai179-GAL4\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Dr. Yufeng Pan (Southeast University): \u003cem\u003eUAS-jGCaMP7s\u0026nbsp;\u003c/em\u003e(BDSC79032), \u003cem\u003eempty-RNAi\u0026nbsp;\u003c/em\u003e(BDSC36304).\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Bloomington Stock Center: \u003cem\u003eCanton-S\u0026nbsp;\u003c/em\u003e(64349), \u003cem\u003eDf(1)Exel6234\u003c/em\u003e (7708), \u003cem\u003eper\u003csup\u003e01\u003c/sup\u003e\u003c/em\u003e (80928), \u003cem\u003etim\u003csup\u003e01\u003c/sup\u003e\u003c/em\u003e (80930), \u003cem\u003eClk\u003csup\u003eJrk\u003c/sup\u003e\u003c/em\u003e (80927), \u003cem\u003ecyc\u003csup\u003e01\u003c/sup\u003e\u003c/em\u003e (80929), \u003cem\u003eelav\u003csup\u003ec155\u003c/sup\u003e\u003c/em\u003e (458), \u003cem\u003eClk-RNAi\u003csup\u003eHMJ02224\u003c/sup\u003e\u003c/em\u003e (42566), \u003cem\u003ecyc-RNAi\u003csup\u003eHMJ02219\u003c/sup\u003e\u003c/em\u003e (42563), \u003cem\u003erepo-GAL4\u003c/em\u003e (7415), \u003cem\u003egrh-GAL4\u003c/em\u003e (65637), \u003cem\u003eMhc-GAL4\u003c/em\u003e (55133), \u003cem\u003eClk-RNAi\u003csup\u003eJF01453\u003c/sup\u003e\u003c/em\u003e (31660), \u003cem\u003eClk-RNAi\u003csup\u003eJF01454\u003c/sup\u003e\u003c/em\u003e (31661), \u003cem\u003ecyc-RNAi\u003csup\u003eJF03333\u003c/sup\u003e\u003c/em\u003e (29400), \u003cem\u003ecyc-RNAi\u003csup\u003eJF02185\u003c/sup\u003e\u003c/em\u003e (31897), \u003cem\u003ecyc-RNAi\u003csup\u003eGL00387\u003c/sup\u003e\u003c/em\u003e (35461), \u003cem\u003etub(FRT.GAL80)\u003c/em\u003e (38881), \u003cem\u003eotdFLP\u003c/em\u003e (600309), \u003cem\u003ecry-GAL4; Pdf-GAL80\u003c/em\u003e (80940), \u003cem\u003ecry-GAL4\u003c/em\u003e (24514), \u003cem\u003eNPF-GAL4\u003c/em\u003e (25681), \u003cem\u003eNPF-GAL4\u003c/em\u003e (25682), \u003cem\u003etub-GAL80\u003csup\u003ets\u003c/sup\u003e\u003c/em\u003e (7108), \u003cem\u003eUAS-CD4tdGFP\u003c/em\u003e (35839), \u003cem\u003eUAS-RedStinger\u003c/em\u003e (8546), \u003cem\u003eITP-RNAi\u003c/em\u003e (25799), \u003cem\u003eUAS-hid\u003c/em\u003e (65403), \u003cem\u003eUAS-KCNJ2\u003c/em\u003e (6595), \u003cem\u003eUAS-NaChBac\u003c/em\u003e (9469), \u003cem\u003eUAS-TNT\u003c/em\u003e (28838), \u003cem\u003eUAS-traF\u003c/em\u003e (4590), \u003cem\u003eUAS\u0026gt;stop\u0026gt;KCNJ2\u003c/em\u003e (67686), \u003cem\u003eVGlut-RNAi\u003c/em\u003e (27538), \u003cem\u003eUAS-CsChrimson\u003c/em\u003e (55136), \u003cem\u003ecwo-RNAi\u003c/em\u003e (26318), \u003cem\u003evri-RNAi\u003c/em\u003e (40862), \u003cem\u003ePdp1-RNAi\u003c/em\u003e (26212), \u003cem\u003eBacTrace\u003c/em\u003e (90826), \u003cem\u003eSIFaR-RNAi\u003c/em\u003e (34947), \u003cem\u003elexAop-nSyb-spGFP\u003csup\u003e1-10\u003c/sup\u003e, UAS-CD4-spGFP\u003csup\u003e11\u003c/sup\u003e\u003c/em\u003e (64315), \u003cem\u003eCipc-RNAi\u003c/em\u003e (28774), \u003cem\u003edco-RNAi\u003c/em\u003e (27719), \u003cem\u003ejet-RNAi\u003c/em\u003e (31058), \u003cem\u003eAtx2-RNAi\u003c/em\u003e (36114), \u003cem\u003ecry-RNAi\u003c/em\u003e (43217), \u003cem\u003eUAS-mir.ban.A\u003c/em\u003e (60671), \u003cem\u003elexAop-CD8GFP; UAS-mLexA-VP16-NFAT, lexAop-rCD2-GFP\u003c/em\u003e (66542), \u003cem\u003eSh-RNAi\u003c/em\u003e (53347), \u003cem\u003eHk-RNAi\u003c/em\u003e (28330), \u003cem\u003eOamb-RNAi\u003c/em\u003e (31233), \u003cem\u003eUAS-mCD8RFP, LexAop-mCD8GFP, nSyb-MKII::nlsLexADBDo, UAS-p65AD::CaM\u003c/em\u003e (61679), \u003cem\u003eempty-GAL4\u003c/em\u003e (36303).\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Qidong Fungene Biotechnology: \u003cem\u003eITP-RC\u003csup\u003eT2A\u003c/sup\u003e-GAL4\u003c/em\u003e (FBA00286), \u003cem\u003eVGlut\u003csup\u003eFLP\u003c/sup\u003e\u003c/em\u003e (FRE00001), \u003cem\u003eSIFa-lexA\u003csup\u003eT2A\u003c/sup\u003e\u003c/em\u003e (FBF00116).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Korea Drosophila Resource Center: \u003cem\u003eUAS\u0026gt;stop\u0026gt;mCD8GFP\u003c/em\u003e (1119), \u003cem\u003eUAS\u0026gt;stop\u0026gt;NaChBac\u003c/em\u003e (1183), \u003cem\u003eUAS\u0026gt;stop\u0026gt;TNT\u003c/em\u003e (1191).\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from Vienna Drosophila Resource Center: \u003cem\u003eClk\u003csup\u003eADF\u003c/sup\u003e-RNAi\u003c/em\u003e (104507), \u003cem\u003e5-HT1A-RNAi\u003c/em\u003e (106094).\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from TsingHua Fly Center: \u003cem\u003eIh-RNAi\u003c/em\u003e (TH02084.N).\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from NIG-FLY Center: \u003cem\u003eOctβ3R-RNAi\u003c/em\u003e (31348R-4).\u003c/p\u003e\n\u003cp\u003eThe CS background was selected as the experimental background due to its well-characterized and consistent LMD and SMD behaviors. To ensure that genetic variation did not confound our results, all GAL4, UAS, and RNAi lines employed in our assays were rigorously backcrossed into the CS strain, often exceeding ten generations of backcrossing. This approach was undertaken to isolate the effects of our genetic manipulations from those of genetic background. We assert that the extensive backcrossing to the CS background, in concert with the internal control in LMD and SMD, provides a stable platform for the accurate interpretation of the LMD and SMD phenotypes observed in our experiments. To reduce the variation from genetic background, all flies were backcrossed for at least 10 generations to \u003cem\u003eCS\u003c/em\u003e strain. For the generation of outcrosses, all GAL4, UAS, and RNAi lines employed as the virgin female stock were backcrossed to the \u003cem\u003eCS\u003c/em\u003e genetic background for a minimum of ten generations. Notably, the majority of these lines, which were utilized for LMD assays, have been maintained in a \u003cem\u003eCS\u003c/em\u003e backcrossed state for long-term generations subsequent to the initial outcrossing process, exceeding ten backcrosses. Based on our experimental observations, the genetic background of primary significance is that of the X chromosome inherited from the female parent. Consequently, we consistently utilized these fully outcrossed females as virgins for the execution of experiments pertaining to LMD and SMD behaviors. Contrary to the influence on LMD behaviors, we have previously demonstrated that the genetic background exerts negligible influence on SMD behaviors, as reported in our prior publication\u003csup\u003e11\u003c/sup\u003e. The mutants and transgenic lines utilized in this study have been previously characterized, with the exception of the novel transgenic strains that we generated and describe herein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMating duration assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mating duration assay in this study has been reported\u003csup\u003e9–11\u003c/sup\u003e. To enhance the efficiency of the mating duration assay, we utilized the \u003cem\u003eDf(1)Exel6234\u003c/em\u003e (DF here after) genetic modified fly line in this study, which harbors a deletion of a specific genomic region that includes the sex peptide receptor (SPR)\u003csup\u003e82,83\u003c/sup\u003e. Previous studies have demonstrated that virgin females of this line exhibit increased receptivity to males\u003csup\u003e83\u003c/sup\u003e. We conducted a comparative analysis between the virgin females of this line and the CS virgin females and found that both groups induced SMD. Consequently, we have elected to employ virgin females from this modified line in all subsequent studies. For group reared (naïve) males, 40 males from the same strain were placed into a vial with food for 5 days. For single reared males, males of the same strain were collected individually and placed into vials with food for 5 days. For experienced males, 40 males from the same strain were placed into a vial with food for 4 days then 80 DF virgin females were introduced into vials for last 1 day before assay. 40 DF virgin females were collected from bottles and placed into a vial for 5 days. These females provide both sexually experienced partners and mating partners for mating duration assays. At the fifth day after eclosion, males of the appropriate strain and DF virgin females were mildly anaesthetized by CO\u003csub\u003e2\u003c/sub\u003e. After placing a single female in to the mating chamber, we inserted a transparent film then placed a single male to the other side of the film in each chamber. After allowing for 1 h of recovery in the mating chamber in 25℃ incubators, we removed the transparent film and recorded the mating activities.\u0026nbsp;Only those males that succeeded to mate within 1 h were included for analyses. The initiation and completion of copulation were recorded to the nearest second, with a precision of ±10 seconds. The total mating duration for each pair was determined from the moment of successful genital apposition until the separation of the male and female \u003cem\u003eDrosophila\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eGenetic controls with \u003cem\u003eGAL4/+\u003c/em\u003e or \u003cem\u003eUAS/+\u003c/em\u003e lines were omitted from supplementary figures, as prior data confirm their consistent exhibition of normal LMD and SMD behaviors\u003csup\u003e9–11,14,16\u003c/sup\u003e. Hence, genetic controls for LMD and SMD behaviors were incorporated exclusively when assessing novel fly strains that had not previously been examined. In essence, internal controls were predominantly employed in the experiments, as LMD and SMD behaviors exhibit enhanced statistical significance when internally controlled. Within the LMD assay, both group and single conditions function reciprocally as internal controls. A significant distinction between the naïve and single conditions implies that the experimental manipulation does not affect LMD. Conversely, the lack of a significant discrepancy suggests that the manipulation does influence LMD. In the context of SMD experiments, the naïve condition (equivalent to the group condition in the LMD assay) and sexually experienced males act as mutual internal controls for one another. A statistically significant divergence between naïve and experienced males indicates that the experimental procedure does not alter SMD. Conversely, the absence of a statistically significant difference suggests that the manipulation does impact SMD. Hence, we incorporated supplementary genetic control experiments solely if they deemed indispensable for testing. All assays were performed from noon to 4 PM. We conducted blinded studies for every test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of transgenic flies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo generate the \u003cem\u003eUAS\u0026gt;stop\u0026gt;Clk-RNAi\u003c/em\u003e line, we selected HMJ02224 (BDSC#42566) as the template shRNA. The shRNA sequences were cloned directly with the following primers TTATCCCATATTCAGCCGCTAGCAGT-AGAGCTAGTTGTAGATCTCAA-TAGTTATATTCAAGC and AACTCCGATGTCTCGCCTGAATTCGC-AGAGCTAGTTGTAGATCTCAA-TATGCTTGAATATAAC. The amplified DNA fragment was\u0026nbsp;inserted into the pJFRC28-10XUAS-FRT-stop-FRT-RNAi vector.\u0026nbsp;This vector, supplied by Qidong Fungene Biotechnology Co., Ltd. (http://www.fungene.tech/), is a derivative of the pJFRC28-10XUAS-IVS-GFP-p10 vector (available at https://www.addgene.org/36431).\u0026nbsp;The insertion was achieved by digesting the fragment and the vector with EcoRI and NheI restriction enzymes to create compatible sticky ends. The genetic construct was inserted into the \u003cem\u003eattp5\u003c/em\u003e site on chromosome II and \u003cem\u003eVK0005\u003c/em\u003e site on chromosome III to generate transgenic flies using established techniques, a service conducted by Qidong Fungene Biotechnology Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunostaining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 5 days of eclosion, the Drosophila brain was taken from adult flies and fixed in 4% formaldehyde at room temperature for 30 minutes. The sample was than washed three times (5 minutes each) in 1% PBT and then blocked in 5% normal goat serum for 30 minutes. Subsequently, the sample was incubated overnight at 4℃ with primary antibodies in 1% PBT, followed by the addition of fluorophore-conjugated secondary antibodies for one hour at room temperature. Finally, the brain was mounted on plates with an antifade mounting solution (Solarbio) for imaging purposes. Samples were imaged with Zeiss LSM880. Antibodies were used at the following dilutions: Chicken anti-GFP (1:500, Invitrogen), mouse anti-nc82 (1:50, DSHB), rabbit anti-DsRed (1:500, Rockland Immunochemicals), Alexa-488 donkey anti-chicken (1:200, Jackson ImmunoResearch), Alexa-555 goat anti-rabbit (1:200, Invitrogen), Alexa-647 goat anti-mouse (1:200, Jackson ImmunoResearch).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative analysis of fluorescence intensity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify the calcium level and synaptic intensity in microscopic images, we introduced ImageJ software\u003csup\u003e84\u003c/sup\u003e. We initially employed ImageJ’s ‘Measure’ feature to calculate average pixel intensity across the entire image or in user-specified sections, and the ‘Plot Profile’ feature to create intensity profiles across areas. To maximize precision, we converted color images to grayscale before analysis. Thresholding methods were also utilized to produce binary images that accurately outlined areas of interest, with pixel intensities of 255 (white) assigned to regions of interest and 0 (black) to the background. Intensity values from the binary image were then transferred to the corresponding locations in the original grayscale image to obtain precise intensity measurements for each object. The ‘Display Results’ feature provided comprehensive data for each object, including average intensity, size, and other relevant statistics. To normalize for fluorescence differences between ROIs, GFP fluorescence for GRASP was normalized to nc82. All specimens were imaged under identical conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptogenetic experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 100 mM stock solution of all-trans-retinal (ATR) powder (Sigma) was prepared by dissolving it in 100% alcohol. For optogenetic experiments, 250 μl of the stock solution was mixed with 30 ml of 5% sucrose and 1% agar medium to prepare food with a final concentration of 400 μM ATR. Flies aged between 3 and 5 days were transferred to ATR food for a minimum of 3 days before performing optogenetic experiments\u003csup\u003e85\u003c/sup\u003e. ATR-fed flies and unfed flies were housed in separate transparent tubes and exposed to a 20s red light: 40s no-light cycle treatment overnight before mating duration assay.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGCaMP experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFly anesthesia was induced using CO\u003csub\u003e2\u003c/sub\u003e. Dissecting the brain after feeding ATR for at least three days. AHL solution (108 mM NaCl, 5 mM KCl, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 4 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 1 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 5 mM HEPES, 10 mM Sucrose, 5 mM Trehalose, pH 7.5) was used for both dissection and imaging to maintain neuronal activity. We then used Zeiss LSM880 confocal microscope to record calcium signaling fluctuations in ITP-LN\u003csub\u003ed\u003c/sub\u003e, 5\u003csup\u003eth\u003c/sup\u003e sLN\u003csub\u003ev\u003c/sub\u003e, and ocelli in parallel with activation of SIFa neurons using 5 seconds of red light. The brains were scanned at 1 Hz sampling rate with the max pinhole. Fiji was used to examine ROIs. ΔF/F\u003csub\u003e0\u003c/sub\u003e = (F\u003csub\u003et\u003c/sub\u003e − F\u003csub\u003e0\u003c/sub\u003e)/F\u003csub\u003e0\u003c/sub\u003e × 100%. F\u003csub\u003e0\u003c/sub\u003e was the averaged fluorescence of the baseline. Because laser irradiation causes the fluorescence signal to diminish over time, we used the R package feasts (https://feasts.tidyverts.org) to detrend.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-fly sleep and circadian rhythm recording\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e96-well white Microfluor 2 plates (Fishier) with 400 μl of food (5% sucrose and 1% agar) were loaded with adult male flies (aged 3–5 days). Flies were entrained to the 12 h:12 h LD cycles for four days at 25 °C to record sleep behavior, then changed to constant darkness for 5-6 days to record circadian rhythms in the absence of light inputs.\u0026nbsp;The fly movement was monitored using a camera at 10s intervals, and the data were then used by the sleep and circadian analysis program SCAMP to analyze sleep and circadian rhythm\u003csup\u003e86–88\u003c/sup\u003e. It calculates activity by shifting the position of \u003cem\u003eDrosophila\u003c/em\u003e every 10 seconds and calculates sleep using the standard definition (\u003cem\u003eDrosophila\u003c/em\u003e is recorded as asleep if it remains motionless for at least 5 minutes).\u0026nbsp;For all sleep experiments in Figures 5, 6, S7, and S9, experimental and control groups were assayed concurrently within the same experimental round to minimize batch effects.\u0026nbsp;Bilateral controls (GAL4 driver alone and UAS effector alone) were included for each experimental genotype to validate specificity. Due to the concurrent nature of these assays, the R54D11-GAL4/+ line was used as a shared control for all experimental groups targeting the GAL4 driver side Sample sizes (at least 6–8 flies per genotype) were determined based on prior studies demonstrating reliable detection of robust phenotypes in bilateral control designs\u003csup\u003e89,90\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-nucleus RNA-sequencing analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003esnRNAseq dataset analyzed in this paper is published\u003csup\u003e91\u003c/sup\u003e and available at the Nextflow pipelines (VSN, https://github.com/vib-singlecell-nf), the availability of raw and processed datasets for users to explore, and the development of a crowd-annotation platform with voting, comments, and references through SCope (https://flycellatlas.org/scope), linked to an online analysis platform in ASAP (\u003ca href=\"https://asap.epfl.ch/fca\"\u003ehttps://asap.epfl.ch/fca\u003c/a\u003e).Single-cell RNA sequencing (scRNA-seq) data from the \u003cem\u003eDrosophila melanogaster\u003c/em\u003e were obtained from the Fly Cell Atlas website (https://flycellatlas.org/scope). The Seurat (v4.2.2) package (https://satijalab.org/seurat) was utilized for data analysis. Violin plots were generated using the “Vlnplot” function, the cell types are split by FCA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell RNA sequencing (scRNA-seq) data from the \u003cem\u003eDrosophila melanogaster\u003c/em\u003e were obtained from the GEO under the accession code GSE157504\u003csup\u003e92\u003c/sup\u003e. The same integration method was applied. Data from six time points and under LD and DD conditions were read and integrated using the integration functions provided by the Seurat 4 (version 4.2.2) package\u003csup\u003e93\u003c/sup\u003e. The UMIs data were retrieved, consisting of a grand total of 4,634 cells. The \"NormalizeData\" function was utilized for the purpose of automated data normalization. Ultimately, we conducted principal component analysis (PCA) on gene expression vectors that were scaled using z-scores. Subsequently, we limited the data to include just the top 40 PCA components. We employed the ‘FindNeighbors’ and ‘FindClusters’ functions from the Seurat package to cluster the data that had been decreased in dimensions. We utilized t-distributed stochastic neighbor embedding (t-SNE) to generate a two-dimensional map that displays the clusters. One cluster that highly express the ITP gene were extracted, and then the marker genes were calculated using the Seurat 'FindMarkers' function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConnectome analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole brain connectomics data were obtained from FlyWire (\u003ca href=\"https://codex.flywire.ai/\"\u003ehttps://codex.flywire.ai/\u003c/a\u003e)\u003csup\u003e94–99\u003c/sup\u003e. The left ITP-LN\u003csub\u003ed\u003c/sub\u003e (FlyWire Root ID: 720575940634984800) dataset was used to gather information on the synaptic connections between the presynaptic and the postsynaptic neurons of interest. The connectivity was visualized with Sankey diagram and doughnut diagram by the Plotly R Studio library (https://plotly.com/r/).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis of mating duration assay was described previously\u003csup\u003e9–11\u003c/sup\u003e. More than 50 males (naïve, experienced and single) were used for mating duration assay. Our experience suggests that the relative mating duration differences between naïve and experienced condition and singly reared are always consistent; however, both absolute values and the magnitude of the difference in each strain can vary. So, we always include internal controls for each treatment as suggested by previous studies\u003csup\u003e100\u003c/sup\u003e. Therefore, statistical comparisons were made between groups that were naïvely reared, sexually experienced and singly reared within each experiment. As mating duration of males showed normal distribution (Kolmogorov-Smirnov tests, p \u0026gt; 0.05), we used two-sided Student’s t tests. The mean ± standard error (s.e.m) (\u003cem\u003e**** = p \u0026lt; 0.0001, *** = p \u0026lt; 0.001, ** = p \u0026lt; 0.01, * = p \u0026lt; 0.05\u003c/em\u003e). All analysis was done in GraphPad (Prism). Individual tests and significance are detailed in figure legends.\u003c/p\u003e\n\u003cp\u003eBesides traditional \u003cem\u003et\u003c/em\u003e-test for statistical analysis, we added estimation statistics for all MD assays and two group comparing graphs. In short, ‘estimation statistics’ is a simple framework that—while avoiding the pitfalls of significance testing—uses familiar statistical concepts: means, mean differences, and error bars. More importantly, it focuses on the effect size of one’s experiment/intervention, as opposed to significance testing\u003csup\u003e101\u003c/sup\u003e. In comparison to typical NHST plots, estimation graphics have the following five significant advantages such as (1) avoid false dichotomy, (2) display all observed values (3) visualize estimate precision (4) show mean difference distribution. And most importantly (5) by focusing attention on an effect size, the difference diagram encourages quantitative reasoning about the system under study\u003csup\u003e102\u003c/sup\u003e. Thus, we conducted a reanalysis of all our two group data sets using both standard \u003cem\u003et\u003c/em\u003e tests and estimate statistics. In 2019, the Society for Neuroscience journal eNeuro instituted a policy recommending the use of estimation graphics as the preferred method for data presentation\u003csup\u003e103\u003c/sup\u003e. For sleep experiments, all statistical analyses were performed using IBM SPSS and Prism software. Data were first tested for normal distribution with the Wilks-Shapiro test. One-way analysis of variance (ANOVA) followed by Tukey-Kramer HSD post hoc tests were applied for multiple group comparisons. The number of animals (n) for each experiment is provided in the figures. Data are presented as mean behavioral responses with error bars indicating the standard error of the mean (SEM), and group differences were considered statistically significant at p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKacelnik A, Brunner D. 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Estimation Statistics, One Year Later. \u003cem\u003eeNeuro\u003c/em\u003e. 2021;8(2):ENEURO.0091-21.2021. doi:10.1523/eneuro.0091-21.2021\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CLK/CYC heterodimer, mating duration, circadian rhythm, interval timing, pacemaker, sleep, circadian-independent timing","lastPublishedDoi":"10.21203/rs.3.rs-7471909/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7471909/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInterval timing is a cognitive ability essential for behaviors such as mating, foraging, and decision-making, and it is distinct from circadian rhythm regulation. Despite the involvement of circadian clock genes in both interval timing and circadian rhythms, the mechanisms differentiating these functions remain unclear. Using \u003cem\u003eDrosophila\u003c/em\u003e as a model, we demonstrate that the CLK/CYC heterodimer, but not PER/TIM, is essential for interval timing. Neuronal CLK/CYC expression is necessary and sufficient for sexual experience-dependent shorter mating duration (SMD) behavior. We identified that CLK/CYC expression in a single pair of ITP-positive LN\u003csub\u003ed\u003c/sub\u003e neurons is pivotal for SMD. These neurons are glutamatergic with output circuits to central brain regions. CLK variants lacking DNA binding motifs dissociate circadian rhythms from interval timing and sleep behaviors in these neurons. Our study uncovers a specialized circuit for interval timing and highlights the non-circadian functions of circadian clock genes.\u003c/p\u003e","manuscriptTitle":"CLOCK-dependent pathway in a single pair of LNd neurons instruct circadian-independent interval timing behavior.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-16 07:48:01","doi":"10.21203/rs.3.rs-7471909/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be63825c-bca5-4326-b8e5-416c4befcc2c","owner":[],"postedDate":"September 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54679489,"name":"Biological sciences/Physiology"},{"id":54679490,"name":"Biological sciences/Cell biology/Circadian rhythms"}],"tags":[],"updatedAt":"2025-09-16T07:48:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-16 07:48:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7471909","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7471909","identity":"rs-7471909","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}