Long-range neuropeptide relay as a central-peripheral communication mechanism for the context-dependent modulation of interval timing behaviors

  1. Tianmu Zhang
  2. Zekun Wu
  3. Yutong Song
  4. Wenjing Li
  5. Yanying Sun
  6. Xiaoli Zhang
  7. Kyle Wong
  8. Justine Schweizer
  9. Khoi-Nguyen Ha Nguyen
  10. Alex Kwan
  11. Woo Jae Kim

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Abstract

Neuropeptides play crucial roles in regulating context-dependent behaviors, but the underlying mechanisms remain elusive. We investigate the role of the neuropeptide SIFa and its receptor SIFaR in regulating two distinct mating duration behaviors in male Drosophila : Longer-Mating-Duration (LMD) and Shorter-Mating-Duration (SMD). We found that SIFaR expression in specific neurons is required for both LMD and SMD behaviors. Social context and sexual experience lead to synaptic reorganization between SIFa and SIFaR neurons, altering internal states of brain. We revealed that the SIFa-SIFaR/Crz-CrzR neuropeptide relay pathway is essential for generating distinct interval timing behaviors, with Crz neurons being responsive to the activity of SIFa neurons. Additionally, CrzR expression in non-neuronal cells is critical for regulating LMD and SMD behaviors. Our study provides insights into how neuropeptides and their receptors modulate context-dependent behaviors through synaptic plasticity and calcium signaling, with implications for understanding the neural circuitry underlying interval timing and neuropeptidergic system modulation of behavioral adaptations.

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    Reply to the reviewers

    Manuscript number: RC-2024-02546

    Corresponding author: Woo Jae, Kim

    1. General Statements

    This is the second version of revision.

    After thoroughly reviewing the comments provided by the EMBO Journal reviewers, we found their feedback to be highly constructive and valuable for enhancing our manuscript without the need for additional experiments. For example, Reviewer 1 acknowledged that our "data are intriguing and some of the experiments are quite convincing," but suggested that the manuscript contained excessive data that required simplification. This sentiment was echoed by Reviewer 2. In response, we have completely reformatted our manuscript to eliminate unnecessary imaging quantification data and CrzR-related screening data. The reviewers noted the density of our experimental data, which has led us to focus on the SIFa to Crz-CrzR circuit mechanisms related to heart function and interval timing in future projects.

    Reviewer 2's comments were generally more moderate, and we successfully addressed all five of their points with detailed explanations and modifications to our manuscript. They positively remarked that "Overall, this highly interesting study advances our knowledge about the behavioral roles of SIFamide and contributes to an understanding of how motivated behavior such as mating is orchestrated by modulatory peptides." Additionally, Reviewer 3 accepted our manuscript without any further comments.

    In summary, we believe we have effectively addressed all concerns raised by Reviewers 1 and 2, resulting in a clearer manuscript that is more accessible to a broader audience.

    2. Point-by-point description of the revisions

    Reviewer #1

    General Comments:* In this revision of their manuscript, Zhang et al have attempted to address most of the points raised by the reviewers, however, they have not assuaged my most important concerns. The manuscript contains a ton of information, but at times this is to the detriment of the narrative flow. I had a lot of trouble following the rationale of each experiment, and the throughline from one experiment to the next is not always obvious. The data are intriguing, and some of the experiments are quite convincing, but other experiments are either superfluous or have methodological issues. I will summarize the most acute issues below.*

    •   *__Answer:__ Thank you for your thoughtful feedback and for acknowledging our efforts to address your previous comments. We appreciate your recognition of the intriguing nature of our data and the convincing aspects of our experiments. In this second revision, we have taken your concerns regarding the narrative flow and data overload to heart. We have completely reshaped our manuscript, significantly reducing unnecessary data, including the NP5270 data and overlapping quantification results that did not contribute meaningfully to the storytelling. Our goal was to streamline the presentation of our findings to enhance clarity and coherence, ensuring that each experiment clearly supports the overarching narrative. We believe these revisions will not only improve the readability of our manuscript but also allow readers to follow the rationale behind each experiment more easily. We are confident that this refined approach will make our contributions clearer and more impactful. Thank you once again for your constructive insights, which have been invaluable in guiding us toward a more focused and compelling presentation of our work.

    * *

    Comment 1. *The authors argue that genetic controls are unnecessary because they have been conducted in previously published papers. I am concerned with this argument, as it is good practice to repeat controls with each experiment. However, I am overall convinced by the basic phenotype indicating that panneuronal SIFaR knockdown eliminates the changes in mating duration associated with previous experience. As for the more restricted 24F06-GAL4, the phenotype is odd-the flies do actually change their mating duration, just in the opposite direction of controls. Doesn't this imply that these flies are still capable of "interval timing", and of changing their mating strategy following exposure to rivals or following sexual experience? *

    __ Answer:__ We appreciate the reviewer's critical comments regarding genetic control and the intriguing phenotypes we observed in specific genetic combinations. We fully agree with the reviewer and have repeated all genetic control experiments for this revision, confirming that our genetic controls consistently demonstrate intact LMD and SMD behaviors, as previously reported. These genetic control experiments have been included in Supplementary Information 1-2. We are grateful to the reviewer for the opportunity to reaffirm that LMD and SMD represent stable behavioral phenotypes suitable for genetically studying interval timing, supported by reproducible data.

    We acknowledge the reviewer's insightful comments about the exciting phenotype observed when SIFaR is knockdown which shows both singly reared and sexually experienced male show lengthened mating duration in contrast to normal LMD and SMD behaviors. Actually, we have observed such phenotype when specific neural circuits are disrupted such as when sNPF peptidergic signaling is disrupted in restricted neuronal population [4]. We are now investigating such phenotype as hypothesis as disinhibition. We explained this phenotype and about disinhibition in main text as below.

    In the spatial, the targeted reduction of SIFaR expression in the GAL424F06 neuronal subset resulted in a notable alteration of mating behavior. Both singly reared and sexually experienced flies exhibited an extended mating duration relative to naïve flies, contrary to the expected reduction. This observation indicates a deficit in the neural mechanism responsible for modulating mating duration, suggesting a disinhibition-like effect within the neural circuitry governing mating behavior. We have also previously observed a similar phenotype when sNPF peptidergic signaling is inhibited in specific neuronal circuits [62]. Disinhibition, characterized by the alleviation of inhibitory constraints, permits the activation of neural circuits that are ordinarily repressed. This process is instrumental in sculpting behavioral patterns and facilitating the sequential progression of behaviors. Through the orchestrated promotion of select neuronal activation and concurrent inhibition of competing neural routes, disinhibition empowers the brain with the ability to dynamically ascertain and preserve the requisite behavioral state, concurrently smoothing the transition to ensuing behavioral phases [63]. It is known that Drosophila neural circuits also exhibit disinhibition phenotypes in light preference and ethanol sensitization [64,65]. Further investigation is needed to uncover the underlying mechanisms of this disinhibition-like phenotype observed in LMD and SMD behaviors.

    This reversed phenotype strongly suggests a disruption in interval timing, as one would expect that if interval timing were normal and intact, male flies would decrease their mating duration in response to appropriate environmental changes. For instance, research has shown that patients with Parkinson's disease exhibit heterogeneity in temporal processing, leading to disrupted interval timing phenotypes [5]. Therefore, if male flies subjected to social isolation or sexual experience do not show a reduction in mating duration compared to control conditions, it indicates a potential disruption in their interval timing mechanisms. We appreciate the reviewer's encouragement to further explore this intriguing disinhibition-like phenotype, and we plan to investigate this aspect in our future projects.

    Comment 2. *I am glad the see the addition of data assessing the extent of SIFaR and CrzR RNAi knockdown; however, this has not completely addressed my concerns about interpretation of behavioral phenotypes. In both cases, the knockdown was assessed by qPCR using the very strong tub-GAL4 driver. mRNA levels are decreased but not nearly eliminated. Thus, when in line 177-178 the authors assert: "Consequently, we infer that the knockdown of SIFaR using the HMS00299 line nearly completely diminishes the levels of the SIFaR protein," the statement is not supported by the data. The qPCR results showed a knockdown at the mRNA level of ~50%. No assays were conducted to measure protein levels. The conclusions should be tempered to align with the data. Furthermore, it is not clear that knockdown is as successful with other drivers, which means that negative behavioral data must be interpreted with caution. For example, the lack of phenotype with repo-GAL4 driving SIFaR RNAi or elav-GAL4 driving CrzR RNAi could be due to a lack of efficient knockdown. This should be acknowledged. *

       __Answer:__ We appreciate the reviewer's critical observation regarding the efficiency of SIFaR knockdown. We fully agree that it is essential to confirm both for ourselves and our readers that the SIFaR knockdown phenotype is robust and convincing. At the outset of this project, we tested all available SIFaR-RNAi strains following established protocols within the fly community to ensure consistency in our findings. When we employed strong drivers such as tub-GAL4 and nSyb-GAL4 for SIFaR-RNAi knockdown, we observed that the flies failed to eclose and exhibited a lethal phenotype during the larval stage, which closely resembles the homozygous lethal phenotype seen in SIFaR mutants. This suggests that, in most cases, the effects of SIFaR knockdown can effectively mimic those of SIFaR mutations. To share our methodology and reinforce our findings, we have added clarifying statements in the main text as follows:

    "Employment of broad drivers, including the tub-GAL4 and the strong neuronal driver nSyb-GAL4, with HMS00299 line consistently results in 100% embryonic lethality (data not shown). This phenotype mirrors the homozygous lethality observed in the SIFaRB322 mutant."

    Due to the significant lethality phenotype observed, we conducted PCR analyses using a combination of tub-GAL80ts and SIFaR-RNAi. As detailed in Fig. 1E, we reared the flies at 22{degree sign}C to suppress RNAi expression and then shifted the temperature to 29{degree sign}C for just three days prior to performing PCR. While our PCR results indicate a 50% reduction in SIFaR levels, we believe that experiments conducted without the tub-GAL80ts system would likely demonstrate an even greater reduction in SIFaR expression. To clarify this point and provide additional context, we have included the following description in the main text:

    "The silencing of SIFaR mRNA was achieved at approximately 50% using the HMS00299 knockdown line in combination with tub-GAL80ts, with RNAi induction lasting for three days (bottom diagram in Fig. 1E). Notably, the same tub-GAL4 driver, when used without the tub-GAL80ts combination, resulted in embryonic lethality while still reducing SIFaR mRNA levels by 50% after three days of RNAi induction. This finding suggests that SIFaR knockdown using the HMS00299 line with GAL4 drivers is likely sufficient to elicit the observed LMD and SMD behaviors. This rationale underscores the effectiveness of our experimental approach and its potential implications for understanding the role of SIFaR in mating behaviors."

    We also concur with the reviewer that the absence of a behavioral phenotype associated with CrzR-RNAi may be due to inefficient RNAi knockdown. Consequently, we have included a description of this issue in the main text as follows:

    "It is important to consider that the 50% knockdown of SIFaR and CrzR may be sufficient to disrupt LMD and/or SMD behavior. However, the lack of phenotype with repo-GAL4 or elav-GAL4 could be due to a less efficient knockdown. This possibility highlights the need for cautious interpretation of negative behavioral data."

    Comment 3. *Regarding the issue of outcrossing, I am confused by the authors' statement: "To reduce the variation from genetic background, all flies were backcrossed for at least 3 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." It's not clear what this means. Perhaps the authors could definitively state how many times each line was outcrossed. The genetic background is important because of 1) the lack of all controls, and 2) the variability of the behavioral phenotype. Often, the presence or absence of LMD or SMD appears to depend on the behavior of the control flies. When these flies show low mating duration, there is typically not a reduction following sexual experience or group raising. Could these differences derive from genetic background or transgenic insertion effects? *

    Answer: We appreciate the reviewer's concern regarding the potential for confusion stemming from our descriptions of the genetic background. As the reviewer noted, we have published multiple papers on LMD and SMD behaviors, and we have conducted our experiments with careful attention to controlling the genetic background [1-3,6-8]. In response to the reviewer's comments about the importance of genetic control and background, we have completed all necessary genetic control experiments and confirmed that all our flies have been backcrossed for more than ten generations to the Canton-S (CS) strain. We believe that we have adequately addressed the reviewer's concerns regarding potential differences arising from genetic background or transgenic insertion effects. To provide readers with more detailed information about our genetic background, we have added a paragraph in the MATERIALS AND METHODS section as follows:

    "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."

    Comment 4. *I continue to have substantial concerns about the thresholding method used across many experiments to quantify overlap, and then to claim that this indicates that synaptic connections are being made between different neuronal populations. The degree of overlap will depend on factors including the settings during imaging (was care taken to prevent pixel saturation?). It is also not clear to me from the methods whether analysis was done on single confocal images or on projections. The images shown in the figures look like maximum projections of a confocal stack. Overlap would have to be assessed on individual confocal sections-it is possible that this is what was done for analysis but not clear from the description in the methods. Furthermore, a lot of figure space is dedicated to superfluous information. For example, in Figure 1F-J, there is a massive amount of space dedicated to assessing the agree of overlap between red stinger and CD4GFP, each driven from the same SIFaR2A driver, and further assessing what percentage of the CD4GFP signal overlaps with nc82, with the apparent goal of showing that a lot of the SIFaR signal is at active zones. This information does little to drive the narrative forward, and is quite confusing to read. Finally, the confocal images are generally too small to actually assess. *

       __Answer:__ We appreciate the reviewer's concerns regarding our imaging quantification methods. We recognize the importance of providing a clear and transparent methodology for both readers and the broader scientific community. Instead of using maximum projection of confocal images, we employed a projection method that incorporates the standard deviation function available in ImageJ. Based on our experience, this approach yields more reliable quantification results, allowing for a more accurate assessment of our data. To ensure clarity and reproducibility, we have detailed our methods in the MATERIALS AND METHODS section as follows:

    "The quantification of the overlap was performed using confocal images with projection by standard deviation function provided by ImageJ to ensure precise measurements and avoid pixel saturation artifacts."

    We appreciate the reviewer's suggestion regarding the inclusion of image quantification data for overlapping regions, which may not be essential to the logical flow of our narrative and could lead to confusion for readers. In response, we have removed nearly all of the quantification data related to overlapping regions, retaining only those that we consider critical for the paper. Currently, only Fig. S3B-E remains, as it is important for illustrating how SIFa neuronal arborization interacts with SIFaR neurons in the central nervous system.

    Additionally, we fully agree with the reviewer that the overall size of the confocal images was too small for effective assessment. To address this concern, we have enlarged all confocal images and increased the spacing in the figures. We believe these improvements will enhance the clarity of our manuscript and facilitate a better understanding of our findings.

    Comment 5. *In general, the figures are still very cluttered, with panels too close together, and the labels are hard to read. *

    Answer: We thank the reviewer for their valuable feedback regarding the clarity of our figures. In response to their concern, we have enlarged the figures to enhance readability and ensure that the panels are more distinct. We believe these adjustments will significantly improve the viewer's ability to interpret the data. We appreciate the reviewer's attention to detail, which has helped us to refine the presentation of our findings.

    Comment 6. *There are no methodological details on how the VFB was used. The authors have not addressed my concern that they are showing only the neuronal skeleton (rather than the actual site of synapses). They are simply identifying all locations where the neuronal skeleton overlaps an entire brain region, and suggesting that these represent synapses. Many papers use the VFB to denote the actual location of synapses, which should be done in Figures 3B and S4A. *

    Answer: We appreciate the reviewer's constructive comments regarding the methodological details of using VFB data. We fully agree that we cannot draw definitive conclusions about SIFa projections to specific regions based solely on neuronal skeleton data, which do not indicate the actual locations of synapses. To address this concern, we have made it clear to readers that the VFB skeleton data serves only as a preliminary indication of potential SIFa projections to GA, FB, and AL.

    To confirm the presence of actual synapses from SIFa neurons, we conducted a thorough analysis using FlyWire data, which validated our findings from VFB. By integrating insights from VFB with the detailed synaptic mapping provided by FlyWire, we can confidently assert the functional relevance of these connections within the context of SIFa neuronal activity. This comprehensive approach not only bolsters our conclusions but also enhances our understanding of how SIFa neurons interact within the broader neural circuitry. We believe this rationale highlights the significance of our work in elucidating the complex relationships among these neuronal populations. We have detailed our findings in the main text as follows:

    "We utilized the "Virtual Fly Brain (VFB)" platform, an interactive tool designed for exploring neuronal connectivity, to gain insights into the connectivity of SIFa neurons with four other neurons, specifically GA, FB, and AL (Fig. 3B and Fig. S4B) [74]. While VFB provides valuable information, it does not offer precise locations of synapses originating from SIFa neurons. To address this limitation, we incorporated data from the FlyWire connectome, which allowed us to confirm that SIFa projections indeed form actual synapses with GA, AL, FB, and SMP (Fig. S3F and S3G) [75]. This multi-faceted approach enhances the robustness of our findings by integrating different data sources to validate neuronal connections."

    Comment 7. *The changes in GRASP and CaLexA with experience are very interesting, and suggest a substantial rearrangement of synaptic connectivity associated with changes in mating duration following group rearing or female exposure. I am still concerned, however, that the nsyb and tGRASP images look so different. I wouldn't expect them to be identical, but it is puzzling that the nsyb-GRASP data show connections in a few discrete brain areas, while the tGRASP data show connections in a much larger overall brain area, but curiously not in the major regions seen with nsyb-GRASP (ie PI, FB and GA). Shouldn't the tGRASP signal appear in all the places that the nsyb-GRASP does? For CaLexA and GRASP data, the methods should indicate the timing of the dissections and staining relative to the group/sexual experience. *

    Answer: We appreciate the reviewer's constructive comments regarding our GRASP data, which indeed reveal an intriguing neural plasticity phenotype, as the reviewer noted. In our previous response, we suggested that the observed differences may be attributed to the distinct SIFa-GAL4 strains utilized, as described in another manuscript focused on SIFa inputs [9]. In that manuscript, we classified the four SIFa neurons into two groups: SIFaDA (dorsal-lateral) and SIFaVP (ventral-posterior). The SIFa2A-GAL4 specifically labels only the SIFaVP neurons, while the SIFa-PT driver labels all four neurons. We acknowledge that we did not clearly communicate this distinction to the reviewer or our readers, and we apologize for any confusion this may have caused. To rectify this oversight, we have added a detailed explanation of these differences in the main text as follows:

    "The subtle differences in GRASP signals observed in Fig. 3A may stem from the distinct expression patterns of the SIFa2A-lexA and GAL4SIFa.PT drivers. We would like to emphasize that the SIFa2A driver labels only a subset of SIFa neurons in other regions (Kim 2024)."

    We recognize that a clear and transparent methodology is essential for generating reproducible data. In response to the reviewer's suggestion, we have revised our MATERIALS AND METHODS section to include more detailed descriptions of the dissection conditions. This enhancement aims to provide readers with the necessary information to replicate our experiments effectively.

    "To ascertain calcium levels and synaptic intensity from microscopic images, we dissected and imaged five-day-old flies of various social conditions and genotypes under uniform conditions. For group reared (naïve) flies, the flies were reared in group condition and dissect right after 5 days of rearing without any further action. For single reared flies, the flies were reared in single condition and dissect at the same time as group reared flies right after 5 days of rearing without any further action. For sexual experienced flies, the flies were reared in group condition after 4 days of rearing and will be given virgins to give them sexual experience for one day, those flies will also be dissected at the same time as group and single reared flies after one day."

    Comment 8. *The calcium imaging data are odd. In most cases, the experimental flies don't actually show an increase in calcium levels but rather a lack of a decrease that is present in the ATR- controls. Also, in the cases where they argue for an excitatory affect of SIF neuron stimulation, the baseline signal intensity appears higher in ATR- controls compared to ATR+ experimental flies (eg Fig 5L, 6O), while it is significantly higher in ATR+ flies compared to ATR- controls when the activation results in decreased calcium signals. Perhaps more details on how these experiments were conducted and whether data were normalized in some way would help to clarify this. *

    Answer: Thank you for your valuable feedback. We appreciate your careful analysis of our calcium imaging data and have addressed your concerns below:

    In our experiments, we observed that ATR+ flies maintained relatively stable calcium levels, whereas ATR- controls exhibited a gradual decrease. Under confocal imaging, GFP signals typically decrease over time, which we observed in ATR- controls. However, ATR+ flies did not exhibit this decline. To better convey this observation, we have refined the language in the manuscript. Specifically, we now describe this as a tendency to sustain the activity of Crz neurons in the OL and AG regions (Fig. 6K-M, Fig. S6G-I). This is supported by the sustained intracellular calcium activity in ATR+ flies compared to the gradual decline to baseline levels observed in ATR- controls (Fig. 6K-M).

    Baseline signal intensity differences: You correctly noted that in some cases, the baseline signal intensity appears higher in ATR- controls compared to ATR+ flies. These differences are likely due to technical factors, such as variations in the distance between the imaged brain and the objective lens. Even minor positional shifts in the brain (forward or backward) can affect the observed signal intensity.

    Our analyses focus on relative changes in fluorescence intensity within the same sample, which we present as line graphs to highlight trends rather than absolute values. However, we acknowledge that showing the magnitude of relative values instead of absolute values may have caused some confusion. We have revised the images to better align with our conclusions, ensuring that the adjustments do not affect the observed relative changes.

    Normalization and experimental details: The calcium imaging data were normalized to ΔF/F to account for differences in baseline fluorescence intensity. However, we recognize that further clarification of the normalization process and experimental setup is essential. We have expanded the methods section to include detailed descriptions of data acquisition, normalization steps, and statistical analyses.

    As the reviewer correctly noted, calcium signals in ATR+ flies are generally higher than those in ATR- flies. However, it appears that the calcium levels exhibit a maintained response rather than a dramatic increase compared to the control ATR- condition, particularly in the case shown in Fig. 6K, which illustrates SIFa-to-Crz signaling. We believe this observation may reflect the actual physiological conditions under which SIFa influences SIFaR neurons to sustain activity during activation. We have included our interpretation of these findings in the main text as follows:

    "Upon optogenetic stimulation of SIFa neurons, we observed a tendency to maintain the activity of Crz neurons in OL and AG regions (Fig. 6K-M, Fig. S6H-J), evidenced by a sustained activity in intracellular Ca2+ levels that persisted in a high level compared to control ATR- condition which shows gradual declining to baseline levels (Fig. 6K-M). In contrast to the OL and AG regions, the cells in the upper region of the SIP consistently show a decrease in Ca2+ levels following stimulation of the SIFa neurons (Fig. 6N-P)."

    To enhance readers' understanding of our calcium imaging results, we have reformatted our GCaMP data for improved clarity and included additional details in the MATERIALS AND METHODS section regarding the quantification of GCaMP imaging methods. Furthermore, as the reviewer correctly noted, discrepancies in baseline activity were due to our error in presenting the baseline data. We have now corrected this oversight accordingly.

    Comment 9. *The models in Fig 4 J and T show data from Song et al, though I could not find a citation for this. I would omit this part of the model since these data are not discussed at all in the manuscript. *

    Answer: We appreciate the reviewer for correctly identifying our oversight in failing to properly cite Song et al.'s paper. This error occurred partly because the preprint was not available at the time we submitted our manuscript. We now have a preprint for Song et al.'s paper, which discusses the contributions of SIFa neurons to various energy balance behaviors, and we plan to submit this paper back-to-back with our current submission to PLOS Biology. We have briefly cited Song et al.'s work in the manuscript; however, we have removed references to it from Fig. 4J and T to avoid any potential confusion for readers.

    Comment 10. *The graphs for the SCOPE data (eg Figure 8I-L) are still too small to make sense of. *

    Answer: We enlarged the tSNE plot generated from the SCOPE data.

    Comment 11. The rationale behind including the data in Figure 9 is not well explained. I would omit this data to help streamline and focus the manuscript.

    Answer: We fully understand and agree with the reviewer's concerns, and we have removed all previous versions of Figure 9 from the manuscript to prevent any confusion regarding the storyline.

    Comment 12. *The single control group is still being duplicated in two different graphs but with different names in each graph. The authors updated figure caption hints at this but does not make it explicit. At the very least, these should be given the same name across all graphs, as is done, for example, in the CaLexA experiments in Figure 4B-C. *

    Answer: We concur with the reviewer and have changed the label for all "group" conditions to "naïve" in all figures.

    Comment 13. *Lines 640-641: Moreover, the pacemaker function is essential for the generation of interval timing capabilities (Meck et al, 2012; Matell, 2014; Buhusi & Meck, 2005), with the heart being recognized as the primary pacemaker organ within the animal body". This is an intriguing idea, however, I attempted to look at the cited references and don't see any claim about the heart being involved in interval timing. I could not find a paper matching the citation of Matell 2014. Meck et al 2012 is an introduction to a Frontiers in Integrative Neuroscience Research Topic and does not mention the heart, nor does the Buhusi and Meck 2005 paper. Perhaps there is a more suitable reference to make the assertion that the fly's interval timer would be affected by changes in heart rate. My suggestion would be to simplify the manuscript, focusing on the most robust findings-the behavioral effect of SIFaR knockdown, the GRASP and CaLexA data showing differences following group rearing or female exposure, and the effect of Crz knockdown in SIFaR neurons. Other details could be included but would have to be verified with more rigorous experiments. *

    __ Answer:__ We appreciate the reviewer's interest in our exploration of the role of heart function in interval timing. While we found that knocking down CrzR in the heart specifically disrupts LMD behavior, we agree that our manuscript needs to be streamlined for clarity. As a result, we have eliminated all CrzR-RNAi knockdown data except for the oenocyte, neuronal and glial knockdown data presented in Fig. S8C-H. This decision was made to ensure a more focused comparison with the SIFaR knockdown experiments shown in Fig. 1. We are dedicated to further investigating the role of Crz-CrzR in heart function and its influence on interval timing in a future project. This approach allows us to maintain clarity in our current manuscript while laying the groundwork for more comprehensive studies ahead.

    In line with the reviewer's suggestions, we have simplified our manuscript by eliminating unnecessary data, such as overlapping image quantification and CrzR-RNAi screening, allowing us to focus on SIFaR knockdown and GRASP, as well as CaLexA with GCaMP imaging. We are grateful to the reviewer for providing us with the opportunity to delineate the role of CrzR in heart function related to LMD as a significant future project. We believe that our manuscript has been greatly improved by the reviewer's constructive feedback.

    __ __

    Reviewer #2

    General Comments:* The authors investigate mating behavior in male fruit flies, Drosophila melanogaster, and test for a role of the SIFamide receptor (SIFaR) in this type of behavior, in particular mating duration in dependence of social isolation and prior mating experience. The anatomy of SIFamide-releasing neurons in comparison with SIFamide receptor-expressing neurons is characterized in a detail-rich manner. Isolating males or exposing them to mating experience modifies the anatomical organization of SIFamidergic axon termini projecting onto SIFamide receptor-expressing neurons. This structural synaptic plasticity is accompanied by changes in calcium influx. Lastly, it is reported that corazonin-releasing neurons are modulated by SIFamide releasing neurons and impact the duration of mating behavior.

    Overall, this highly interesting study advances our knowledge about the behavioral roles of SIFamide, and contributes to an understanding how motivated behavior such as mating is orchestrated by modulatory peptides. The manuscript has some points that are less convincing.*

    __ Answer:__ We appreciate the reviewer's positive feedback regarding our investigation into the role of the SIFamide receptor (SIFaR) in mating behavior in male Drosophila melanogaster. We are pleased that the detailed characterization of SIFamide-releasing neurons and their anatomical changes in response to social isolation and mating experience has been recognized as a valuable contribution to the understanding of synaptic plasticity and its impact on behavior. We are also grateful that the reviewer described our manuscript as a "highly interesting study" that advances knowledge about the behavioral roles of SIFamide and contributes to the understanding of how motivated behaviors, such as mating, are orchestrated by modulatory peptides. We sincerely thank the reviewer for these encouraging comments about our work.

    We acknowledge the reviewer's concerns about certain aspects of our manuscript that may be less convincing. We are committed to addressing these points thoroughly to strengthen our arguments and enhance the clarity of our findings. In response to the feedback, we have made several revisions throughout the manuscript, including clarifying our methodology, enhancing the presentation of our data, and providing additional context where needed. We believe these changes will improve the overall quality of the manuscript and make our conclusions more compelling. Thank you for your thoughtful review, and we look forward to your further insights.

    * *

    Comment 1. *It remains unclear why the authors link the differentially motivated duration of mating behavior with the psychological concept of interval timing. This distracts from the actually interesting neurobiology and is not necessary to make the study interesting. The study deals with the modulation of mating behavior by SIFamide. The abstraction that SIFamide plays a role in the neuronal calculation of time intervals for the perception of time sequenc es is not convincing in itself. *

    •   *__Answer:__ We appreciate the reviewer's thoughtful comments regarding our conclusion that links SIFamide to interval timing in mating behavior. We recognize that our data primarily indicate that SIFamide is essential for normal mating duration and influences the motivation-dependent aspects of this behavior. We also acknowledge the need for more robust evidence to establish a clearer connection between these findings and interval timing. Recent research by Crickmore et al. has provided valuable insights into how mating duration in *Drosophila *serves as an effective model for examining changes in motivation over time as behavioral goals are achieved. For example, around six minutes into mating, sperm transfer occurs, resulting in a significant shift in the male's nervous system, where he no longer prioritizes continuing the mating at the expense of his own survival. This pivotal change is mediated by four male-specific neurons that release the neuropeptide Corazonin (Crz). When these Crz neurons are inhibited, sperm transfer does not take place, and as a result, the male fails to reduce his motivation, leading to matings that can extend for hours instead of the typical duration of approximately 23 minutes [10].

    Recent research conducted by Crickmore et al. has secured NIH R01 funding (Mechanisms of Interval Timing, 1R01GM134222-01) to investigate mating duration and sperm transfer timing in Drosophila as a genetic model for understanding interval timing. Their study emphasizes how fluctuations in motivation over time can affect mating behavior, particularly noting that significant behavioral changes occur during mating. For instance, around six minutes into the mating process, sperm transfer takes place, which corresponds with a notable decrease in the male's motivation to continue mating [10]. These findings indicate that mating duration serves not only as an endpoint for behavior but may also reflect fundamental mechanisms associated with interval timing.

       We believe that by leveraging the robustness and experimental tractability of these findings, along with our own work on SIFamide's role in mating behavior, we can gain deeper insights into the molecular and circuit mechanisms underlying interval timing. We will revise our manuscript to clarify this relationship and emphasize how SIFamide may interact with other neuropeptides and neuronal circuits involved in motivation and timing.

   In addition to the efforts of Crickmore's group to connect mating duration with a straightforward genetic model for interval timing, we have previously published several papers demonstrating that LMD and SMD can serve as effective genetic models for interval timing within the fly research community. For instance, we have successfully connected SMD to an interval timing model in a recently published paper [3], as detailed below:

    "We hypothesize that SMD can serve as a straightforward genetic model system through which we can investigate "interval timing," the capacity of animals to distinguish between periods ranging from minutes to hours in duration.....

    In summary, we report a novel sensory pathway that controls mating investment related to sexual experiences in Drosophila. Since both LMD and SMD behaviors are involved in controlling male investment by varying the interval of mating, these two behavioral paradigms will provide a new avenue to study how the brain computes the 'interval timing' that allows an animal to subjectively experience the passage of physical time [11-16]."

       Lee, S. G., Sun, D., Miao, H., Wu, Z., Kang, C., Saad, B., ... & Kim, W. J. (2023). Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster. *PLoS Genetics*, *19*(5), e1010753.

   We have also successfully linked LMD behavior to an interval timing model and have published several papers on this topic recently [6-8].

   Sun, Y., Zhang, X., Wu, Z., Li, W., & Kim, W. J. (2024). Genetic Screening Reveals Cone Cell-Specific Factors as Common Genetic Targets Modulating Rival-Induced Prolonged Mating in male Drosophila melanogaster. *G3: Genes, Genomes, Genetics*, jkae255.

   Zhang, T., Zhang, X., Sun, D., & Kim, W. J. (2024). Exploring the Asymmetric Body's Influence on Interval Timing Behaviors of Drosophila melanogaster. *Behavior Genetics*, *54*(5), 416-425.

   Huang, Y., Kwan, A., & Kim, W. J. (2024). Y chromosome genes interplay with interval timing in regulating mating duration of male Drosophila melanogaster. *Gene Reports*, *36*, 101999.

   Finally, in this context, we have outlined in our INTRODUCTION section below how our LMD and SMD models are related to interval timing, aiming to persuade readers of their relevance. We hope that the reviewer and readers are convinced that mating duration and its associated motivational changes such as LMD and SMD provide a compelling model for studying the genetic basis of interval timing in *Drosophila*.

    "The dimension of time is the fundamental basis for an animal's survival. Being able to estimate and control the time between events is crucial for all everyday activities [25]. The perception of time in the seconds-to-hours range, referred to as 'interval timing', is involved in foraging, decision making, and learning via activation of cortico-striatal circuits in mammals [26]. Interval timing requires entirely different neural mechanisms from millisecond or circadian timing [27-29]. There is abundant psychological research on time perception because it is a universal cognitive dimension of experience and behavioral plasticity. Despite decades of research, the genetic and neural substrates of temporal information processing have not been well established except for the molecular bases of circadian timing [30,31]. Thus, a simple genetic model system to study interval timing is required. Considering that the mating duration in fruit flies, which averages approximately 20 minutes, is well within the range addressed by interval timing mechanisms, this behavioral parameter provides a relevant context for examining the neural circuits that modulate the Drosophila's perception of time intervals. Such an investigation necessitates an understanding of the extensive neural and behavioral plasticity underlying interval timing [32-37]."

    We would like to highlight that many researchers are currently working to bridge the gap between interval timing as a purely psychological concept and its neurobiological underpinnings, as illustrated in the following articles [15,17-20]. We appreciate the reviewer's concerns regarding the relationship between mating duration and interval timing. However, we believe that our LMD and SMD model can effectively bridge the gap between psychological concepts and neurobiological mechanisms using a straightforward genetic model organism. By employing Drosophila as our model, we aim to elucidate the underlying neural circuits that govern these behaviors, thereby contributing to a deeper understanding of how interval timing is represented in both psychological and biological contexts.

    Matell, M. S. Neurobiology of Interval Timing. Adv. Exp. Med. Biol. 209-234 (2014) doi:10.1007/978-1-4939-1782-2_12.

    Matell, M. S. & Meck, W. H. Cortico-striatal circuits and interval timing: coincidence detection of oscillatory processes. Cogn. Brain Res. 21, 139-170 (2004).

    Merchant, H. & Lafuente, V. de. Introduction to the neurobiology of interval timing. Adv Exp Med Biol 829, 1-13 (2014).

    Golombek, D. A., Bussi, I. L. & Agostino, P. V. Minutes, days and years: molecular interactions among different scales of biological timing. Philosophical Transactions Royal Soc B Biological Sci 369, 20120465 (2014).

    Balcı, F. & Toda, K. Editorial: Psychological and neurobiological mechanisms of time perception and temporal information processing: insight from novel technical approaches. Front. Behav. Neurosci. 17, 1208794 (2023).

    Comment 2. *For all behavioral experiments, genetic controls should always be conducted. That is, both the heterozygous Gal4-line as well as the heterozygous UAS-line should be used as controls. This is laborious, but important and common standard. The authors often report data only for offspring from genetc crosses in which UAS-lines and Gal4-lines are combined (e.g. figure S1). This is not sufficient. *

    •   *__Answer:__ We are grateful for the reviewer's constructive suggestions regarding the genetic control experiments. In response to similar concerns raised by another reviewer, we have conducted all necessary genetic control experiments and included the results in Supplementary Information 1-2. We hope that this thorough effort will demonstrate to both the reviewer and readers that the LMD and SMD behaviors represent stable and reproducible phenotypes for investigating the genetic components of interval timing.

    Comment 3. *There are quite a lot of citations of preprints, including preprints from the authors's own lab. It seems inappropriate to cite non-peer reviewed preprints in order to present the basic principles of the study (interval timing in flies) as recognized knowledge. In general, it is unclear whether the information presented in these multiple preprints will turn out to be credible and acceptable. *

    •   *__Answer:__ We concur with the reviewer and have removed most of the preprint material, retaining only one preprint that discusses SIFa function, which has been co-submitted with this manuscript.

    Comment 4. *Anatomical images are often very small and not informative. For example, figure S1 O, R, S and U shows small images of fly brains and ventral nerve chords that do not convincingly describe the expression of fluorescent proteins. The choice of a threshold to quantify fluorescence seems arbitrary. It is also not clear what the quantification "83% of brain and 71% of VNC SIFaR+ neurons" actually tells us. This quantification does not rely on counting neurons (such as 83% of neurons), but only shows how fluorescence in these neurons overlaps with an immunostaining of an ubiquitous active zone protein. The same is true for figure S2 or S3: overlapping brain areas do not inform you about numbers of cells, as stated in the text. *

    Answer: We appreciate the reviewer's concerns regarding our imaging quantification methods. In response to similar questions raised by another reviewer, we have thoroughly reformatted our methods section and eliminated much of the overlapping data that appeared unnecessary for this paper. We recognize the importance of providing a clear and transparent methodology for both readers and the broader scientific community. Instead of using maximum projection of confocal images, we employed a projection method that incorporates the standard deviation function available in ImageJ. Based on our experience, this approach yields more reliable quantification results, allowing for a more accurate assessment of our data. To ensure clarity and reproducibility, we have detailed our methods in the MATERIALS AND METHODS section as follows:

    "The quantification of the overlap was performed using confocal images with projection by standard deviation function provided by ImageJ to ensure precise measurements and avoid pixel saturation artifacts."

    We appreciate the reviewer's suggestion regarding the inclusion of image quantification data for overlapping regions, which may not be essential to the logical flow of our narrative and could lead to confusion for readers. In response, we have removed nearly all of the quantification data related to overlapping regions, retaining only those that we consider critical for the paper. Currently, only Fig. S3B-E remains, as it is important for illustrating how SIFa neuronal arborization interacts with SIFaR neurons in the central nervous system.

    Additionally, we fully agree with the reviewer that the overall size of the confocal images was too small for effective assessment. To address this concern, we have enlarged all confocal images and increased the spacing in the figures. We believe these improvements will enhance the clarity of our manuscript and facilitate a better understanding of our findings.

    Comment 5. *The authors have consistently confused the extensive overlap of neuronal processes (dendrites and presynaptic regions) across large brain areas with synaptic connections. One cannot infer functional synaptic connectivity from the overlap of these fluorescent signals. *

    Answer: We appreciate the reviewer's feedback and, in light of similar comments from another reviewer, we have removed most of the DenMark and syt.eGFP data, retaining only Fig. 3A. We are grateful for the constructive suggestions, which have significantly enhanced our manuscript. We believe that these revisions have clarified the narrative for readers, allowing for a more focused exploration of SIFaR's role in synaptic plasticity and neuronal orchestration.

    Reviewer #3

    General Comments:* In this revised manuscript, the authors have fully and satisfactorily addressed my comments on the previous version. I recommend publication of this manuscript.*

    __ Answer:__ We would like to extend our heartfelt thanks for the careful consideration and positive assessment of our revised manuscript. Your insightful feedback has been instrumental in shaping the final version of our work, and we are delighted to hear that our revisions have met your expectations.

    Your dedication to ensuring the quality and rigor of the scientific literature is truly commendable, and we are immensely grateful for the time and effort you have devoted to reviewing our paper. Your support for publication is a significant encouragement to us and validates the hard work we have put into addressing the issues you raised.

    Please accept our sincere appreciation for your professional and constructive approach throughout the review process. We look forward to the possibility of contributing to the scientific community through the dissemination of our research.

    REFERENCES

    1. Kim WJ, Jan LY, Jan YN. Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals. Nat Neurosci. 2012;15: 876-883. doi:10.1038/nn.3104
    2. Kim WJ, Jan LY, Jan YN. A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating. Neuron. 2013;80: 1190-1205. doi:10.1016/j.neuron.2013.09.034
    3. Lee SG, Sun D, Miao H, Wu Z, Kang C, Saad B, et al. Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster. PLOS Genet. 2023;19: e1010753. doi:10.1371/journal.pgen.1010753
    4. Zhang X, Miao H, Kang D, Sun D, Kim WJ. Male-specific sNPF peptidergic circuits control energy balance for mating duration through neuron-glia interactions. bioRxiv. 2024; 2024.10.17.618859. doi:10.1101/2024.10.17.618859
    5. Merchant H, Luciana M, Hooper C, Majestic S, Tuite P. Interval timing and Parkinson's disease: heterogeneity in temporal performance. Exp Brain Res. 2008;184: 233-248. doi:10.1007/s00221-007-1097-7
    6. Sun Y, Zhang X, Wu Z, Li W, Kim WJ. Genetic Screening Reveals Cone Cell-Specific Factors as Common Genetic Targets Modulating Rival-Induced Prolonged Mating in male Drosophila melanogaster. G3: Genes, Genomes, Genet. 2024; jkae255. doi:10.1093/g3journal/jkae255
    7. Zhang T, Zhang X, Sun D, Kim WJ. Exploring the Asymmetric Body's Influence on Interval Timing Behaviors of Drosophila melanogaster. Behav Genet. 2024; 1-10. doi:10.1007/s10519-024-10193-y
    8. Huang Y, Kwan A, Kim WJ. Y chromosome genes interplay with interval timing in regulating mating duration of male Drosophila melanogaster. Gene Rep. 2024; 101999. doi:10.1016/j.genrep.2024.101999
    9. Kim WJ, Song Y, Zhang T, Zhang X, Ryu TH, Wong KC, et al. Peptidergic neurons with extensive branching orchestrate the internal states and energy balance of male Drosophila melanogaster. bioRxiv. 2024; 2024.06.04.597277. doi:10.1101/2024.06.04.597277
    10. Thornquist SC, Langer K, Zhang SX, Rogulja D, Crickmore MA. CaMKII Measures the Passage of Time to Coordinate Behavior and Motivational State. Neuron. 2020;105: 334-345.e9. doi:10.1016/j.neuron.2019.10.018
    11. Buhusi CV, Meck WH. What makes us tick? Functional and neural mechanisms of interval timing. Nat Rev Neurosci. 2005;6: 755-765. doi:10.1038/nrn1764
    12. Merchant H, Harrington DL, Meck WH. Neural Basis of the Perception and Estimation of Time. Annu Rev Neurosci. 2012;36: 313-336. doi:10.1146/annurev-neuro-062012-170349
    13. Allman MJ, Teki S, Griffiths TD, Meck WH. Properties of the Internal Clock: First- and Second-Order Principles of Subjective Time. Annu Rev Psychol. 2013;65: 743-771. doi:10.1146/annurev-psych-010213-115117
    14. Rammsayer TH, Troche SJ. Neurobiology of Interval Timing. Adv Exp Med Biol. 2014; 33-47. doi:10.1007/978-1-4939-1782-2_3
    15. Golombek DA, Bussi IL, Agostino PV. Minutes, days and years: molecular interactions among different scales of biological timing. Philosophical Transactions Royal Soc B Biological Sci. 2014;369: 20120465. doi:10.1098/rstb.2012.0465
    16. Jazayeri M, Shadlen MN. A Neural Mechanism for Sensing and Reproducing a Time Interval. Curr Biol. 2015;25: 2599-2609. doi:10.1016/j.cub.2015.08.038
    17. Balcı F, Toda K. Editorial: Psychological and neurobiological mechanisms of time perception and temporal information processing: insight from novel technical approaches. Front Behav Neurosci. 2023;17: 1208794. doi:10.3389/fnbeh.2023.1208794
    18. Gür E, Duyan YA, Arkan S, Karson A, Balcı F. Interval timing deficits and their neurobiological correlates in aging mice. Neurobiol Aging. 2020;90: 33-42. doi:10.1016/j.neurobiolaging.2020.02.021
    19. Merchant H, Lafuente V de. Introduction to the neurobiology of interval timing. Adv Exp Med Biol. 2014;829: 1-13. doi:10.1007/978-1-4939-1782-2_1
    20. Matell MS. Neurobiology of Interval Timing. Adv Exp Med Biol. 2014; 209-234. doi:10.1007/978-1-4939-1782-2_12
  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    The article investigates the role of the neuropeptide SIFa and its receptor SIFaR in regulating two distinct mating duration behaviors in male Drosophila melanogaster, Longer-Mating-Duration (LMD) and Shorter-Mating-Duration (SMD). The study reveals that SIFaR expression in specific neurons is required for both behaviors. It shows that social context and sexual experience lead to synaptic reorganization between SIFa and SIFaR neurons, altering internal brain states. The SIFa-SIFaR/Crz-CrzR neuropeptide relay pathway is essential for generating these behaviors, with Crz neurons responding to SIFa neuron activity. Furthermore, CrzR expression in non-neuronal cells is critical for regulating LMD and SMD behaviors. The study utilizes neuropeptide RNAi screening, chemoconnectome (CCT) knock-in, and genetic intersectional methods to elucidate these findings.

    Major Comments

    • Are the key conclusions convincing? The key conclusions are intriguing but require more robust data to be fully convincing. While the study presents compelling evidence for the involvement of SIFa and SIFaR in mating behaviors, additional experiments are needed to firmly establish the proposed mechanisms.
    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? The authors should qualify certain claims as preliminary or speculative. Specifically, the proposed SIFa-SIFaR/Crz-CrzR neuropeptide relay pathway is only investigated via imaging approach. More experiments using behavioral tests are needed to confirm that Crz relays the SIFa signaling pathway. For example, Crz-Gal4>UAS-SIFaR RNAi should be done to show that SIFaR+ Crz+ cells are necessary for LMD and SMD.
    • Would additional experiments be essential to support the claims of the paper? Yes, additional experiments are essential. Detailed molecular and imaging studies are needed to support claims about synaptic reorganization. For example:
      • More controls are needed for RNAi and Gal80ts experiments, such as Gal4-only control, RNAi-only control, etc.
      • Using synaptic markers and high-resolution imaging to observe synaptic changes directly.
      • Electrophysiological recordings from neurons expressing SIFa and SIFaR to analyze their functional connectivity and activity patterns.
    • Are the suggested experiments realistic in terms of time and resources? The suggested experiments are realistic but will require considerable time and resources. Detailed molecular interaction studies, imaging synaptic plasticity, and electrophysiological recordings could take several months to over a year, depending on the complexity and availability of necessary equipment and expertise. The cost would be moderate to high, involving expenses for reagents, imaging equipment, and animal husbandry for maintaining Drosophila stocks.
    • Are the data and the methods presented in such a way that they can be reproduced? The methods are generally described in detail, allowing for potential reproducibility. However, more precise documentation of certain experimental conditions, such as the timing and conditions of RNAi induction and temperature controls, is necessary. The methods about imaging analysis are too detailed. The exact steps about how to use ImageJ should be removed.
    • Are the experiments adequately replicated and statistical analysis adequate? Most figures in the manuscript need to be re-plotted. The right y-axis "Difference between means" is not necessary, if not confusing. The image panels are too small to see, while the quantification of overlapping cells are unnecessarily large. The figures are too crowded with labels and texts, which makes it extremely difficult to comprehend the data.

    Minor Comments

    • Specific experimental issues that are easily addressable. Clarify the timing of RNAi induction and provide more detailed figure legends for better understanding and reproducibility.
    • Are prior studies referenced appropriately? Yes.
    • Are the text and figures clear and accurate? The text is generally clear, but the figures need re-work. See comment above.
    • Suggestions to improve the presentation of data and conclusions. Use smaller fonts in the bar plots and make the plots smaller. Enlarge the imaging panels and let the pictures tell the story.

    Significance

    Nature and Significance of the Advance

    This study aims to advance understanding of how neuropeptides modulate context-dependent behaviors in Drosophila. It provides novel insights into the role of SIFa and SIFaR in interval timing behaviors, contributing to the broader field of neuropeptide research and behavioral neuroscience. However, the significance of the findings is limited by the preliminary nature of some claims and the need for additional supporting data.

    Context in Existing Literature

    The work builds on previous studies that identified various roles of neuropeptides in behavior modulation but lacked detailed mechanistic insights. By elucidating the SIFa-SIFaR/Crz-CrzR pathway, this study attempts to fill a gap in the literature, but more robust evidence is required to solidify its contributions.

    Interested Audience

    The findings will interest neuroscientists, behavioral biologists, and researchers studying neuropeptides and their roles in behavior and neural circuitry. Additionally, this research may have implications for understanding neuropeptidergic systems in other organisms, making it relevant to a broader audience in the fields of neurobiology and physiology.

    Field of Expertise

    Keywords: Neuropeptides, Drosophila melanogaster, Behavioral Neuroscience. Areas without sufficient expertise: courtship behavior.

    Recommendation

    I recommend a major revision of this manuscript. The study presents intriguing findings, but several key claims are preliminary and require additional experiments for support. The data is poorly presented and the figures can be significantly improved. Detailed molecular and imaging studies, as well as more rigorous statistical analyses, are necessary to strengthen the conclusions. Addressing these concerns will significantly improve the robustness and impact of the paper.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Zhang et al., "Long-range neuropeptide relay as a central-peripheral communication mechanism for the context-dependent modulation of interval timing behaviors".

    The authors investigate mating behavior in male fruit flies, Drosophila melanogaster, and test for a role of the SIFamide receptor (SIFaR) in this type of behavior, in particular mating duration in dependence of social isolation and prior mating experience. The anatomy of SIFamide-releasing neurons in comparison with SIFamide receptor-expressing neurons is characterized in a detail-rich manner. Isolating males or exposing them to mating experience modifies the anatomical organization of SIFamidergic axon termini projecting onto SIFamide receptor-expressing neurons. This structural synaptic plasticity is accompanied by changes in calcium influx. Lastly, it is shown that corazonin-releasing neurons are modulated by SIFamide releasing neurons and impact the duration of mating behavior.

    Overall, this highly interesting study advances our knowledge about the behavioral roles of SIFamide, and contributes to an understanding how motivated behavior such as mating is orchestrated by modulatory peptides. The approach to take the entire organism, including peripheral tissue, into consideration, is very good and a rather unique point. The manuscript has only some points that are less convincing, and these should be addressed.

    Major concerns:

    • It is highly interesting that the duration of mating behavior is dependent on external and motivational factors. In fact, that provides an elegant way to study which neuronal mechanisms orchestrate these factors. However, it remains elusive why the authors link the differentially motivated durations of mating behavior to the psychological concept of interval timing. This distracts from the actually interesting neurobiology, and is not necessary to make the study interesting.
    • In figure 4 A and 4K, fluorescence microscopy images of brains and ventral nerve chords are shown, one illustrating GRASP experiments, and one showing CaLexA experiments. The extreme difference between the differentially treated flies (bright fluorescence versus almost no fluorescence) is - in its drastic form- surprising. Online access to the original confocal microscopy images (raw data) might help to convince the reader that these illustrations do not reflect the most drastic "representative" examples out of a series of brain stainings.
    • In particular for behavioral experiments, genetic controls should always be conducted. That is, both the heterozygous Gal4-line as well as the heterozygous UAS-line should be used as controls. This is laborious, but important.

    Minor comments:

    • Line 75: word missing ("...including FEEDING-RELATED BEHAVIOR, courtship, ...").
    • Line 120: word missing ("SIFaR expression in adult neurons BUT not glia...").
    • I find the figures often to be quite overloaded, and anatomical details often very small (e.g., figure 7A).

    Significance

    Overall, this highly interesting study advances our knowledge about the behavioral roles of SIFamide, and contributes to an understanding how motivated behavior is orchestrated by modulatory peptides. The approach to take the entire organism, including peripheral tissue, into consideration, is very good and a rather unique point.

    Since decades it has been investigated how sensory stimuli are processed and encoded by the brain, and how behavioral actions are executed. Likewise, principles underlying learning and memory, sleep, orentation, circadian rhythms, etc. are subject to intense investigation. However, how motivational factors (sleep pressure, hunger, sexual drive) are actually "encoded", signaled and finally used to orchstrate behavior and guide decision-making is, to a very large degree, unknown - in any species. The model use here (Drosophila and its peptidergic system wit SIFamide as a central hub) represents actually a ideal entry point to study just this question. In this sense, the manuscript is at the forefront of modern, state-of-the-art neurobiology.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    This manuscript from Zhang et al. primarily investigates the contribution of the SIFa neuropeptide receptor (SIFaR) to mating duration in male fruit flies. Through RNAi-mediated downregulation, they show that SIFaR receptor is necessary for previous experience to alter mating duration. Using cell-specific knockdown and rescue of the SIFaR receptor, they identify a population of ~400 neurons that could underlie this effect. This is still a large number of cells but is narrowed from the ~1,200 total SIFaR-expressing neurons. They then use the GRASP synaptic labeling technique to show that SIFa+ neurons form synapses onto the relevant SIFaR-expressing population, and that the area of synaptic contact is systematically altered depending on the fly's past mating history. Finally, they provide evidence to argue that SIFa neurons act through SIFaR neurons that release the neuropeptide corazonin to regulate mating duration. Overall, the authors have used an impressive array of techniques in their attempt to define the neural circuits and molecules involved in changing internal state to modify the duration of mating.

    Major Comments:

    1. The authors are to be commended for the sheer quantity of data they have generated, but I was often overwhelmed by the figures, which try to pack too much into the space provided. As a result, it is often unclear what components belong to each panel. Providing more space between each panel would really help.
    2. The use of three independent RNAi lines to knock down SIFaR expression is experimentally solid, as the common phenotype observed with all 3 lines supports the conclusion that the SIFaR is important for mating duration choice. However, the authors have not tested whether these lines effectively reduce SIFaR expression, nor whether the GAL80 constructs used to delimit knockdown are able to effectively do so. This makes it hard to make definitive conclusions with these manipulations, especially in the face of negative results. A lack of complete knockdown is suggested by the fact that the F24F06 driver rescues lethality when used to express SIFaR in the B322 mutant background, but does not itself produce lethality when used to express SIFaR RNAi. The authors should either conduct experiments to determine knockdown efficiency or explicitly acknowledge this limitation in drawing conclusions from their experiments. A similar concern relates to the CrzR knockdown experiments (eg Figure 7).
    3. Most of the behavioral experiments lack traditional controls, for example flies that contain either the GAL4 or UAS elements alone. The authors should explain their decision to omit these control experiments and provide an argument for why they are not necessary to correctly interpret the data. In this vein, the authors have stated in the methods that stocks were outcrossed at least 3x to Canton-S background, but 3 outcrosses is insufficient to fully control for genetic background.
    4. Throughout the manuscript, the authors appear to use a single control condition (sexually naïve flies raised in groups) to compare to both males raised singly and males with previous sexual experience. These control conditions are duplicated in two separate graphs, one for long mating duration and one for short mating duration, but they are given different names (group vs naïve) depending on the graph. If these are actually the same flies, then this should be made clear, and they should be given a consistent name across the different "experiments".
    5. The authors have consistently conflated overlap of neuronal processes with synaptic connections. Claims of synaptic connectivity deriving solely from overlap of processes should be tempered and qualified.
      • For example, they say (Lines 201-202) "These findings suggest that SIFa neurons and GAL424F06-positive neurons form more synapses in the VNC than in the brain." This is misleading. Overlap of24F06-LexA>CD8GFP and SIFa-GAL4>CD8RFP tells us nothing about synapse number, or even whether actual synapses are being formed.
      • Lines 210-211: "The overlap of DenMark and syt.EGFP signals was highly enriched in both SOG and ProNm regions, indicating that these regions are where GAL424F06 neurons form interconnected networks". This is misleading. Overlap of DenMark and syt.EGFP does not indicate synapses (especially since these molecules can be expressed outside the expected neuronal compartment if driven at high enough levels).
      • Lines 320-322: "Neurons expressing Crz exhibit robust synaptic connections with SIFaR24F06 neurons located in the PRW region of the SOG in the brain (panels of Brain and SOG in Fig. 5A)". This is again misleading. They are not actually measuring synapses here, but instead looking at area of overlap between neuronal processes of Crz and SIFaR cells.
      • In Figs 3B and S4A, they are claiming that all neuronal processes within a given delineated brain area are synapses. The virtual fly brain and hemibrain resource have a way to actually identify synapses. This should be used in addition to the neuron skeleton. Otherwise, it is misleading to label these as synapses.
      • Furthermore, measuring the area of GRASP signal is not the same as quantifying synapses. We don't know if synapse number changes (eg in lines 240-242).
    6. In general, the first part of the manuscript (implicating SIFaR in mating duration) is much stronger than the second part, which attempts to demonstrate that SIFa acts through Crz-expressing neurons to induce its effects. The proof that SIFa acts through Crz-expressing neurons to modify mating duration is tenuous. The most direct evidence of this, achieved via knockdown on Crz in SIFaR-expressing cells, is relegated to supplemental figures. The calcium response of the Crz neurons to SIFa neuron activation (Fig. 6) is more of a lack of a decrease that is observed in controls. Also, this is only done in the VNC. Why not look in the brain, because the authors previously stated a hypothesis that the "transmission of signals through SIFaR in Crz-expressing neurons is limited to the brain" (lines 381-382)?

    Furthermore, the authors suggest that Crz acts on cells in the heart to regulate mating duration. It would be useful to add a discussion/speculation as to possible mechanisms for heart cells to regulate mating decisions. Is there evidence of CrzR in the heart? The SCope data presented in Fig. 7I-L and S7G-H is hard to read.

    1. In several cases, the effects of being raised single are opposite the effects of sexual experience. For example, in Fig. 4T, calcium activity is increased in the AG following sexual experience, but decreased in flies raised singly. Likewise, Crz-neurons in the OL have increased CaLexA signal in singly-raised flies but reduced signals in flies with previous sexual experience. In some cases, manipulations selectively affect LMD or SMD. It would be useful to discuss these differences and consider the mechanistic implications of these differential changes, when they all result in decreased mating duration. This could help to clarify the big picture of the manuscript.

    Minor Comments:

    1. For CaLexA experiments (eg Fig 7A-D), signal intensity should be quantified in addition to area covered. Increased intensity would indicate greater calcium activity within a particular set of neurons.
    2. In Figure 5K: quantification of cell overlap is missing. In the text they state that there are ~100 neurons that co-express SIFaR24F06 and Crz. How was this determined? Is there a graph or numerical summary of this assertion?
    3. In lines 709-711: "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." I had trouble understanding this section of methods. What is done with the data from the internal controls?
    4. Could the authors comment on why the brain GRASP signal is so different in Figures 3A and 4A? I realize that different versions of GRASP were used in these experiments, but I would expect broad agreement between the different approaches.

    Significance

    This study will be most relevant to researchers interested in understanding neuronal control of behavior. The manuscript offers a conceptual advance in identifying cell types and molecules that influence mating duration decisions. The strength of the manuscript is the number of different assays used; however, there is a sense that this has occurred at the cost of providing a cohesive narrative. The first part of the manuscript (detailing the role of SIFaR in LMD and SMD) is relatively stronger and more conclusive.

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    Reply to the reviewers

    Manuscript number: RC-2024-02546

    Corresponding author: Woo Jae, Kim

    1. General Statements

    The goal of this study is to provide the insights of one specific neuron ‘SIFa’ controls interval timing behavior by its receptor ‘SIFaR’ through neuropeptide relay. Interval timing, or the sense of time in the seconds to hours range, is important in foraging, decision making, and learning in humans via activation of cortico-striatal circuits. Interval timing requires completely distinct brain processes from millisecond or circadian timing. In summary, interval timing allows us to subjectively sense the passage of physical time, allowing us to integrate action sequences, thoughts, and behavior, detect developing trends, and predict future consequences.

    Many researchers have tried to figure out how animals, including humans, can estimate time intervals with such precision. However, most investigations have been conducted in the realm of psychology rather than biology thus far. Because the study of interval timing was limited in its ability to intervene in the human brain, many psychologists concentrated on developing convincing theoretical models to explain the known occurrence of interval timing.

    To overcome the limits of studying interval timing in terms of genetic control, we have reported that the time investment strategy for mating in* Drosophila* males can be a suitable behavioral platform to genetically dissect the principle of brain circuit mechanism for interval timing. For example, we previously reported that males prolong their mating when they have previously been exposed to rivals (Kim, Jan & Jan, "Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals" Nature Neuroscience, 2012) (Kim et al, 2012), and this behavior is regulated by visual stimuli, clock genes, and neuropeptide signaling in a subset of neurons (Kim, Jan & Jan, “A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating” Neuron, 2013) (Kim et al, 2013). And we also reported that the sensory inputs are required for sexual experienced males to shorten their mating time (Lee, Sun, et al, “Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster” PLOS genetics, 2023) (Lee et al, 2023).

    Throughout their lives, all animals must make decisions in order to optimize their utility function. Male reproductive success is determined by how many sperms successfully fertilize an egg with a restricted number of investment resources. To optimize male reproductive fitness, a time investment strategy has been devised. As a consequence, we believe that the flexible responses of mating duration to different environmental contexts in Drosophila males might be an excellent model to investigate neural circuits for interval timing.

    The most well-known features of mammalian modulating energy homeostasis between the gut and the brain is one of the most intensively studied neuro-modulatory circuits via the neuronal relay of neuropeptides. In this article, we report that SIFa controls two alternate interval timing behaviors through neuropeptide relay signaling by SIFaR and other important neuropeptides and transmits the internal states of the male brain into decision making. According to our findings, male Drosophila utilize SIFa-SIFaR signaling modulating LMD and SMD behaviors. During our investigation in this regulation, we found a subset of cells that express SIFaR in SOG and AG region are important for the modulation of interval timing behaviors. Furthermore, we discovered a neuropeptide named Corazonin (Crz) which expressed in SIFaR is important for both LMD and SMD behaviors.

    Our discovery of neuropeptide relay of SIFa-SIFaR-Crz-CrzR in male Drosophila in modulating interval timing behaviors will be a huge step forward in our knowledge of interval timing behavior.

    2. Point-by-point description of the revisions

    Reviewer #1

    Comment 1.* The authors are to be commended for the sheer quantity of data they have generated, but I was often overwhelmed by the figures, which try to pack too much into the space provided. As a result, it is often unclear what components belong to each panel. Providing more space between each panel would really help.*

    __ Answer:__ We are grateful for the insightful feedback regarding the structure of our data presentation. In response to your valuable suggestion, we have made adjustments in this revised version by downsizing the diagram and ensuring the spacing between the panels.

    Comment 2.* The use of three independent RNAi lines to knock down SIFaR expression is experimentally solid, as the common phenotype observed with all 3 lines supports the conclusion that the SIFaR is important for mating duration choice. However, the authors have not tested whether these lines effectively reduce SIFaR expression, nor whether the GAL80 constructs used to delimit knockdown are able to effectively do so. This makes it hard to make definitive conclusions with these manipulations, especially in the face of negative results. A lack of complete knockdown is suggested by the fact that the F24F06 driver rescues lethality when used to express SIFaR in the B322 mutant background, but does not itself produce lethality when used to express SIFaR RNAi. The authors should either conduct experiments to determine knockdown efficiency or explicitly acknowledge this limitation in drawing conclusions from their experiments. A similar concern relates to the CrzR knockdown experiments (eg Figure 7).*

       __Answer:__ We appreciate the reviewer's attention to the details of our experimental design. Indeed, the validation of SIFaR-RNAi efficiency is crucial for interpreting our results accurately. In our initial experiments, we focused on the consistent phenotypic outcomes across the three independent RNAi lines, which collectively suggest the importance of SIFaR in LMD and SMD behaviors. However, we recognize the importance of confirming the effectiveness of our RNAi constructs in reducing SIFaR expression. Initially, we incorporated experiments utilizing *elav-GAL80* to demonstrate that the SIFaR knockdown mediated by the *elavc155* driver is sufficient to eliminate LMD and SMD behaviors. The corresponding results are presented in Figure 1C-D, with a detailed description provided in the manuscript as detailed below.

    "The inclusion of elav-GAL80, which suppresses GAL4 activity in a pan-neuronal context, was found to restore both LMD and SMD behaviors when SIFaR was knocked down by a pan-neuronal elavc155 driver (Fig. 1C-D). This observation suggests that the reduction in SIFaR expression mediated by the elavc155 driver is sufficient to significantly impair LMD and SMD behaviors."

       In response to the comments, we have conducted a thorough reevaluation in our revised manuscript. Specifically, we have confirmed the efficiency of the SIFaR-RNAi line HMS00299, which exhibited the most pronounced phenotype when co-expressed with the tub-GAL4 and nSyb-GAL4 drivers, using quantitative real-time PCR (qRT-PCR). It has come to our attention that we omitted mentioning the embryonic lethality induced by the HMS00299 line when combined with either tub-GAL4 or nSyb-GAL4 drivers, which is consistent with the homozygous lethality observed in the *SIFaRB322* mutant. To address this, we have performed qRT-PCR experiments by crossing the HMS00299 line with tub-GAL4; tub-GAL80ts, allowing for the temporary knockdown of SIFaR specifically during the adult stage. We utilized w-/SIFaR-RNAis as a control in these experiments. The outcomes are illustrated in Figure 1E, and we have made the necessary modifications and additions to the manuscript to accurately reflect the efficiency of the SIFaR-RNAi line as detailed below.

    "To ensure that RNAi did not have an off-target effect, we tested three independent RNAi strains and found that all three RNAi successfully disrupted LMD/SMD when expressed in neuronal populations. (Fig. S1E-J). We chose to use the HMS00299 line as SIFaR-RNAi for all our experiments because it efficiently disrupts LMD/SMD without UAS-dicer expression. Employment of broad drivers, including the tub-GAL4 and the strong neuronal driver nSyb-GAL4, with HMS00299 line consistently results in 100% embryonic lethality (data not shown). This phenotype mirrors the homozygous lethality observed in the SIFaRB322 mutant. The efficiency of HMS00299 SIFaR-RNAi lines was also validated through quantitative PCR analysis (Fig. 1E). Consequently, we infer that the knockdown of SIFaR using the HMS00299 line nearly completely diminishes the levels of the SIFaR protein."

    We also examined the knockdown efficiency of CrzR in the experiments related to Figure 8 (revised version), following a similar approach (Fig. S7K).

    Comment 3.* Most of the behavioral experiments lack traditional controls, for example flies that contain either the GAL4 or UAS elements alone. The authors should explain their decision to omit these control experiments and provide an argument for why they are not necessary to correctly interpret the data. In this vein, the authors have stated in the methods that stocks were outcrossed at least 3x to Canton-S background, but 3 outcrosses is insufficient to fully control for genetic background.*

    •   *__Answer:__ We sincerely thank the reviewer for insightful comments regarding the absence of traditional genetic controls in our study of LMD and SMD behaviors. We acknowledge the importance of such controls and wish to clarify our rationale for not including them in the current investigation. The primary reason for not incorporating all genetic control lines is that we have previously assessed the LMD and SMD behaviors of GAL4/+ and UAS/+ strains in our earlier studies. Our past experiences have consistently shown that 100% of the genetic control flies for both GAL4 and UAS exhibit normal LMD and SMD behaviors. Given these findings, we deemed the inclusion of additional genetic controls to be non-essential for the present study, particularly in the context of extensive screening efforts. Consequently, in accordance with the reviewer's recommendation, we conducted genetic validation experiments on novel genetic crosses, including SIFaR-RNAi/+, CrzR-RNAi/+, and GAL4NP5270/+, and incorporated the results in the supplementary figures (Supplementary information 1). We have made the necessary modifications and additions to the manuscript as below.

    "Given those genetic controls, as evidenced by consistent exhibition of normal LMD and SMD behaviors (Supplemental information 1), the observed reduction in SIFaR expression, driven by elavc155, is deemed sufficient to induce significant disruptions in LMD and SMD behaviors."

    However, we understand the value of providing a clear rationale for our methodology choices. To this end, we have added a detailed explanation in the "MATERIALS AND METHODS" section and the figure legends of Figure 1. This clarification aims to assist readers in understanding our decision to omit traditional controls, as outlined below.

    "__Mating Duration Assays for Successful Copulation__The mating duration assay in this study has been reported (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023). 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) (Parks et al. 2004; Yapici et al. 2008). Previous studies have demonstrated that virgin females of this line exhibit increased receptivity to males (Yapici et al. 2008). 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 CO2. After placing a single female into 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. Initiation and completion of copulation were recorded with an accuracy of 10 sec, and total mating duration was calculated for each couple. 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 (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023; Huang et al. 2024; Zhang et al. 2024). 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."

       We appreciate the reviewer's inquiry regarding the genetic background of our experimental lines. In response to the comments, we would like to clarify the following. All of our GAL4, UAS, or RNAi lines, which were utilized as the virgin female stock for outcrosses, have been backcrossed to the Canton-S (CS) genetic background for over ten generations. The majority of these lines, particularly those employed in LMD assays, have been maintained in a CS backcrossed status for several years, ensuring a consistent genetic background across multiple generations. Our experience has indicated that the genetic background, particularly that of the X chromosome inherited from the female parent, plays a pivotal role in the expression of certain behavioral traits. Therefore, we have consistently employed these fully outcrossed females as virgins for conducting experiments related to LMD and SMD behaviors. It is noteworthy that, in contrast to the significance of genetic background for LMD behaviors, we have previously established in our work (Lee *et al*, 2023) that the genetic background does not significantly influence SMD behaviors. This distinction is important for the interpretation of our findings. To provide a comprehensive understanding of our experimental design, we have detailed the genetic background considerations in the __"Materials and Methods"__ section, specifically in the subsection __"Fly Stocks and Husbandry"__ as outlined below.

    "To reduce the variation from genetic background, all flies were backcrossed for at least 3 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 (Lee et al, 2023). All mutants and transgenic lines used here have been described previously."

    Comment 4.* Throughout the manuscript, the authors appear to use a single control condition (sexually naïve flies raised in groups) to compare to both males raised singly and males with previous sexual experience. These control conditions are duplicated in two separate graphs, one for long mating duration and one for short mating duration, but they are given different names (group vs naïve) depending on the graph. If these are actually the same flies, then this should be made clear, and they should be given a consistent name across the different "experiments".*

    •   __Answer:__ We are grateful to the reviewer for highlighting the potential for confusion among readers regarding the visualization methods used in our figures. In response to this valuable feedback, we have now included a more detailed explanation of the graph visualization techniques in the legends of Figure 1, as detailed below. This additional information should enhance the clarity and understanding of the figure for all readers.

    "In the mating duration (MD) assays, light grey data points denote males that were group-reared (or sexually naïve), whereas blue (or pink) data points signify males that were singly reared (or sexually experienced). The dot plots represent the MD 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 unpaired Student’s t test, and ns represents non-significant differences (*p* *

    Comment 5.* The authors have consistently conflated overlap of neuronal processes with synaptic connections. Claims of synaptic connectivity deriving solely from overlap of processes should be tempered and qualified.

    • For example, they say (Lines 201-202) "These findings suggest that SIFa neurons and GAL424F06-positive neurons form more synapses in the VNC than in the brain." This is misleading. Overlap of 24F06-LexA>CD8GFP and SIFa-GAL4>CD8RFP tells us nothing about synapse number, or even whether actual synapses are being formed.*

    •   *__Answer:__ We sincerely thank the reviewer for their insightful and constructive feedback regarding the interpretation of our data. We acknowledge the important point raised about the limitations of inferring synapse numbers from the overlap of membrane GFP and RFP signals. We fully concur that more specific techniques, such as the GRASP method, are necessary to accurately quantify synapse numbers, as we have demonstrated in subsequent sections of our manuscript. In the section where we describe the SIFa-SIFaR neuronal architecture labeled with membrane GFP and RFP, we recognize the need for caution in not overstating the implications of these findings as indicative of synapse formation. In light of the reviewer's comments, we have revised our discussion to more accurately reflect the nature of the SIFa-SIFaR neuronal arborizing patterns, as detailed below. This revision aims to provide a more nuanced interpretation of our observations and to align with the current scientific understanding of synaptic quantification.

    "As previously reported, SIFa neurons arborize extensively throughout the CNS, but the neuronal processes of GAL424F06-positive neurons are enriched in the optic lobe (OL), sub-esophageal ganglion (SOG), and abdominal ganglion (AG) (GFP signal in Fig. 2F). Neuronal processes that are positive for SIFa and SIFaR strongly overlap in the prow (PRW), prothoracic and metathoracic neuromere (ProNm and MesoNm), and AG regions (yellow signals in Fig. S3A). We quantified these overlapping neuronal processes between SIFa- and SIFaR-positive neurons and found that approximately 18% of SIFa neurons and 52% of GAL424F06-positive neurons overlap in brain (Fig. S3B, C), whereas approximately 48% of SIFa and 54% of GAL424F06-positive neurons overlap in VNC (Fig. S3D, E). These findings suggest that SIFa neurons and GAL424F06-positive neurons form more neuronal processes in the VNC than in the brain."

    Lines 210-211: "The overlap of DenMark and syt.EGFP signals was highly enriched in both SOG and ProNm regions, indicating that these regions are where GAL424F06 neurons form interconnected networks". This is misleading. Overlap of DenMark and syt.EGFP does not indicate synapses (especially since these molecules can be expressed outside the expected neuronal compartment if driven at high enough levels).*

    •   *__Answer:__ We are grateful for the reviewer's critical insights regarding our interpretation of the DenMark and syt.eGFP experiments. We acknowledge the reviewer's point that the overlap of DenMark and syt.eGFP signals does not conclusively indicate synapses and that some of these signals can be expressed outside the expected neuronal compartments, particularly at high levels.

 It is important to note that DenMark and syt.eGFP are markers of synaptic polarity. In the original publication of DenMark, the authors demonstrated that while these two markers are closely apposed, they do not necessarily overlap, as seen in the labeled yellow areas. They concluded that these areas could represent closely apposed regions where "LNv neurons establish presynaptic contacts within the aMe, suggesting that these contacts are on the postsynaptic sites of the LNv neurons themselves. (Nicolaï *et al*, 2010)" The authors also observed that DenMark-enriched structures appear juxtaposed to, rather than coexpressed with, syt.eGFP, indicating a potential for synapse formation between R neurons within the eb. In contrast, projections to the suboesophageal ganglion, which show strong Syt–GFP expression, are devoid of DenMark, suggesting a different interpretation of the signals (Nicolaï *et al*, 2010).

 Building on these findings, we have reanalyzed our data with caution (as shown in Figure S3). In the SOG region, where we observed strong yellow signals, these were not limited to cell bodies but also extended to the middle region filled with neural processes. Upon close examination of the DenMark and syt.eGFP signals, we confirmed that these yellow signals are closely juxtaposed, suggesting the possibility of synapse formation between SIFaR24F06 neurons within the SOG. We emphasize that this interpretation is based on the original findings from the DenMark study. To provide clarity for general readers, we have added further explanations regarding the interpretation of these signals, as detailed below. We believe that our revised analysis and the additional explanations will help to clarify the potential implications of our findings, while also acknowledging the limitations and the need for further investigation.

    "DenMark-enriched structures, localized within the SOG, are observed in close apposition to syt.eGFP signals, as indicated by the white-dashed circles (Fig. S3Fa). This spatial relationship suggests that SIFaR-expressing neurons, identified by GAL424F06 labeling, may form synapses with one another within the SOG. The colocalization of yellow signals resulting from the interaction between DenMark and syt.eGFP has been previously interpreted and validated by other researchers, supporting our observation (Nicolaï 2010,Kennedy 2018). In contrast to the yellow signals observed in the SOG, which are indicative of neural processes, the yellow signals detected in the ProNm appear to be associated with cell bodies rather than neural processes, as DenMark signals are often observed to leak out (as shown in Fig. S3Fb) (Nicolaï 2010,Kennedy 2018). Despite the presence of juxtaposed DenMark and syt.GFP signals in the ProNm, the interpretation of the yellow signals as potential synapses between SIFaR neurons remains an open question (indicated by the question mark in Fig. S3K).

    • Lines 320-322: "Neurons expressing Crz exhibit robust synaptic connections with SIFaR24F06 neurons located in the PRW region of the SOG in the brain (panels of Brain and SOG in Fig. 5A)". This is again misleading. They are not actually measuring synapses here, but instead looking at area of overlap between neuronal processes of Crz and SIFaR cells.*
       __Answer:__ We sincerely appreciate the reviewer's critical feedback regarding our initial data interpretation. We acknowledge the important distinction that overlapping membrane markers do not provide a direct measure of synapse formation. In line with the reviewer's suggestion, we have revised the relevant sentence to more accurately reflect this understanding, as detailed below.

    "Neurons expressing Crz were observed in close proximity to SIFaR24F06-expressing neurons within the PRW-SOG of the brain (panels of Brain and SOG in Fig. 6A)."

    In Figs 3B and S4A, they are claiming that all neuronal processes within a given delineated brain area are synapses. The virtual fly brain and hemibrain resource have a way to actually identify synapses. This should be used in addition to the neuron skeleton. Otherwise, it is misleading to label these as synapses.*

       __Answer:__ We are grateful for the reviewer's insightful comments that highlighted the potential for misleading information in our previous submission. Upon careful reexamination of the virtual fly brain model, we have made the necessary corrections and updated the figures in our revised manuscript (Figure 3B and S4B). This reanalysis has allowed us to further substantiate our findings, confirming that SIFa neurons indeed establish dense synaptic connections with multiple regions of the central brain.

    Furthermore, measuring the area of GRASP signal is not the same as quantifying synapses. We don't know if synapse number changes (eg in lines 240-242).*

    Answer: We sincerely appreciate the reviewer's valuable suggestion regarding our quantification methods for assessing synaptic changes using GRASP signals. We acknowledge the reviewer's accurate observation that GRASP signals alone cannot provide an exact quantification of synapse number changes. In response to this feedback, we have employed the 'Particle analysis' function of ImageJ to infer the number of synapses from GRASP signals, clearly labeling them as 'number of particles' (as exemplified in Figures S4G and S4J). Additionally, we have compared the average size of each particle to enable a more precise comparison of synapse number changes (as shown in Figures S4H and S4K). While it is true that GRASP signals should not be directly equated with synapse counts, the quantification of GRASP signal intensity can still provide insights into the underlying synaptic connectivity, as described in the original GRASP paper (Feinberg et al, 2008a). Following this approach, previous studies have used signal intensity quantifications to draw conclusions about changes in synaptic specificity in various mutants. Since our methods for measuring GRASP intensity are consistent with the original techniques, we have updated our Y-axis labeling to reflect 'normalized GFP intensity (Norm. GFP Int.)', as exemplified in Figure 4. This change aims to provide a clearer and more accurate representation of our data.

    * *

    Comment 6.* In general, the first part of the manuscript (implicating SIFaR in mating duration) is much stronger than the second part, which attempts to demonstrate that SIFa acts through Crz-expressing neurons to induce its effects. The proof that SIFa acts through Crz-expressing neurons to modify mating duration is tenuous. The most direct evidence of this, achieved via knockdown on Crz in SIFaR-expressing cells, is relegated to supplemental figures. The calcium response of the Crz neurons to SIFa neuron activation (Fig. 6) is more of a lack of a decrease that is observed in controls. Also, this is only done in the VNC. Why not look in the brain, because the authors previously stated a hypothesis that the "transmission of signals through SIFaR in Crz-expressing neurons is limited to the brain" (lines 381-382)?

    Furthermore, the authors suggest that Crz acts on cells in the heart to regulate mating duration. It would be useful to add a discussion/speculation as to possible mechanisms for heart cells to regulate mating decisions. Is there evidence of CrzR in the heart? The SCope data presented in Fig. 7I-L and S7G-H is hard to read.*

    •   *__Answer:__ We sincerely appreciate the reviewer's constructive feedback on the section of our manuscript that discusses the role of the SIFa-SIFaR connection in regulating mating duration. We understand that the initial presentation may not have been sufficiently convincing. As we detailed in our previous biorXiv preprint (Wong *et al*, 2019), we conducted a comprehensive screen of numerous neuropeptides and their receptors that mediate SIFa signals through SIFaR and added those data in Supplementary Table S1 and S2. Among these, Crz was identified as a key neuropeptide in this pathway and is also well-documented for its role in mating duration (Tayler *et al*, 2012). Our data clearly demonstrate that Crz neurons are responsive to the activity of SIFa neurons, supporting the validity of this connection. Additionally, in another manuscript focusing on the input signals for SIFa (Kim *et al*, 2024), we established that CrzR does not function in SIFa neurons, confirming the bidirectional nature of SIFa-to-Crz signaling.

 Inadvertently, we had relegated the Crz knockdown results to supplementary figures, under the assumption that our screening results regarding the relationship between SIFaR and neuropeptides were already well-covered (Wong *et al*, 2019). In light of the reviewer's comments, we have now relocated the Crz knockdown results, particularly those involving SIFaR-expressing cells, to the main figures (Figure 6F-G). We have also included a more detailed description of our previous screening results within the manuscript, as outlined below, to provide a more comprehensive understanding of our findings.

    "Furthermore, the Crz peptide and Crz-expressing neurons have been characterized as pivotal relay signals in the SIFa-to-SIFaR pathway, which is essential for modulating interval timing behaviors (Wong 2019)."

       We greatly appreciate the reviewer's critical and constructive feedback regarding the detection of SIFa-to-Crz long-distance signaling, particularly their observation that this signaling is detectable from the brain to the VNC but not between brain regions. In response to the reviewer's suggestions, we have made the following adjustments to our manuscript:

    1. We have relocated our SIFa-Crz GCaMP data pertaining to the VNC region to the Figures 6L-O) to maintain focus on the primary findings within the main text.

    2. Our deeper analysis has led to the identification of two cells in the Super Intermediate Protocerebrum (SIP) regions that coexpress both Crz and SIFaR24F06, as well as OL cells (Figure 6D-E).

    3. We have included GCaMP data from the brain region in the main figure to provide a comprehensive view of the signaling dynamics (Fig. 6P-R and Fig. S6N-P).

    4. Upon examining the SIFa-to-Crz signaling through GCaMP calcium imaging, we observed that the calcium levels in Crz+/SIFaR+ SIP neurons consistently decreased upon SIFa activation (Figure 6P-R). In contrast, the calcium signals in Crz+/SIFaR+ OL neurons increased upon SIFa activation, similar to the pattern observed in Crz+ AG neurons in the VNC (Figure 6M-O and Figure S6N-P).

    5. We have summarized these findings in Figure 6S and provided a detailed description of the results in the manuscript, as outlined below. "To elucidate the direct response of Crz neurons to the activity of SIFa neurons, we conducted live calcium (Ca2+) imaging in the Super Intermediate Protocererbrum (SIP), OL and AG region of the VNC, where Crz neurons are situated (Fig. 6D, Fig. S6M). Upon optogenetic stimulation of SIFa neurons, we observed a significant increase in the activity of Crz in OL and AG region (Fig. 6L-O, Fig. S6N-P), evidenced by a sustained elevation in intracellular Ca2+ levels that persisted in a high level before gradually declining to baseline levels, where the cells in top region of the SIP exhibit consistently drop down after stimulated the SIFa neurons (Fig. 6P-R). These calcium level changes were in contrast to the control group (without all-trans retinal, ATR) (Fig. 6L-R, Fig. S6N-P). These findings confirm that Crz neurons in OL and AG are activated in response to SIFa neuronal activity, but the activity of Crz neurons in SIP are inhibited by the activition of SIFa neuron, reinforcing their role as postsynaptic effectors in the neural circuitry governed by SIFa neurons. Moreover, these results provide empirical support for the hypothesis that SIFa-SIFaR/Crz-CrzR long-range neuropeptide relay underlies the neuronal activity-based measurement of interval timing."

      We are grateful for the reviewer's opportunity to elaborate on the intriguing findings concerning the expression of CrzR in the heart and its potential link to mating duration. In the context of traditional interval timing models (Meck *et al*, 2012; Matell, 2014; Buhusi & Meck, 2005), the role of a pacemaker in generating a temporal flow for measuring time is considered essential. The heart, being a well-known pacemaker organ in animals, provides a compelling framework for our discussion. In response to the reviewer's insightful comments, we have expanded upon our hypotheses in the DISCUSSION section, exploring the possible connections between cardiac function and the regulation of mating duration. Our reflections on this topic are detailed as follows:

    "It has been reported that the interaction between the brain and the heart can influence time perception in humans (Khoshnoud et al, 2024). Heart rate is governed by intrinsic mechanisms, such as the muscle pacemaker, as well as extrinsic factors including neural and hormonal inputs (Andersen et al, 2015). Moreover, the pacemaker function is essential for the generation of interval timing capabilities (Meck et al, 2012; Matell, 2014; Buhusi & Meck, 2005), with the heart being recognized as the primary pacemaker organ within the animal body. Consequently, the CrzR in the fly heart may respond to the Crz signal sent by SIFaR+/Crz+ cells and modulate the heart rate, thereby impacting the perception of time in male flies."

       We appreciate the reviewer's interest in the expression of CrzR in the heart and its potential implications for our study. In response to the reviewer's comments, we have conducted a thorough examination of the fly SCope RNAseq dataset. Our analysis revealed that CrzR is indeed broadly expressed in heart tissue, particularly in areas where the Hand gene is also expressed. This significant finding has been incorporated into our manuscript and is depicted in Figure 8L. As illustrated in Figures 8I-L, which present the SCope tSNE plot for various cell types including neurons, glial cells, muscle systems, and heart, the heart tissue exhibits the most robust expression of CrzR. This observation suggests that the Hand-GAL4 mediated CrzR knockdown experiments may provide insights into the role of CrzR expression in the heart and its influence on the interval timing behavior of male fruit flies. We have expanded upon this interpretation in the relevant sections of our manuscript to ensure a clear and comprehensive understanding of our results.

    Comment 7.* In several cases, the effects of being raised single are opposite the effects of sexual experience. For example, in Fig. 4T, calcium activity is increased in the AG following sexual experience, but decreased in flies raised singly. Likewise, Crz-neurons in the OL have increased CaLexA signal in singly-raised flies but reduced signals in flies with previous sexual experience. In some cases, manipulations selectively affect LMD or SMD. It would be useful to discuss these differences and consider the mechanistic implications of these differential changes, when they all result in decreased mating duration. This could help to clarify the big picture of the manuscript.*

       __Answer:__ We sincerely appreciate the reviewer's insightful suggestions regarding the potential mechanistic underpinnings of how differential calcium activities may modulate LMD and SMD behaviors. In response to this valuable input, we have expanded our discussion to include a hypothesis on how neuropeptide relays could potentially induce context-dependent modulation of synaptic changes and calcium activities within distinct neuronal subsets. This addition aims to provide a more comprehensive understanding of the complex interactions at play, as detailed in the revised manuscript.

    "Employing two distinct yet comparable models of interval timing behavior, LMD and SMD, we demonstrated that differential SIFa to SIFaR signaling is capable of modulating context-dependent behavioral responses. Synaptic strengths between SIFa and SIFaR neurons was notably enhanced in group-reared naive males. However, these synaptic strengths specifically diminished in the OL, CB, and AG when males were singly reared, with a particular decrease in the AG region when males were sexually experienced (Fig. 4A-J). Intriguingly, overall calcium signaling within SIFaR24F06 neurons was significantly reduced in group-reared naive males, yet these signals surged dramatically in the OL with social isolation and in the AG with sexual experience (Fig. 4K-T). These calcium signals, as reported by the transcriptional calcium reporter CaLexA, were corroborated by GCaMP live imaging in both the AG and OL regions (Fig. 6L-O and Fig. S6N-P), indicating a close association between elevated calcium levels and LMD and SMD behaviors. The modulation of context-dependent synaptic plasticity and calcium dynamics by the SIFa neuropeptide through a single SIFaR receptor raises the question of how a single receptor can elicit such diverse responses. Recent neuroscientific studies in Drosophila have shown that individual neurons can produce multiple neurotransmitters and that neuropeptides are often colocalized with small molecule neurotransmitters (Nässel 2018,Deng 2019,Croset 2018,Kondo 2020). Consistent with this, we have previously reported that SIFa neurons utilize a variety of neurotransmitters, including glutamate, dopamine, and tyramine (Kim 2024). Therefore, we propose that the SIFa-SIFaR-Crz-CrzR neuropeptidergic relay circuitry may interact with different neurotransmitters in distinct neuronal subpopulations to regulate context-dependent behaviors. Supporting this hypothesis, glutamate, known to function as an inhibitory neurotransmitter in the olfactory pathway of Drosophila (Liu 2013), may be one such candidate. We speculate that neuropeptide cotransmission could underlie the mechanisms facilitating these complex, context-dependent behavioral patterns. Further research is warranted to elucidate how such cotransmission contributes to the intricate behavioral repertoire of the fly."

    Minor Comments: Comment 8.* For CaLexA experiments (eg Fig 7A-D), signal intensity should be quantified in addition to area covered. Increased intensity would indicate greater calcium activity within a particular set of neurons.*

    •   *__Answer:__ We appreciate the reviewer's insightful comments and acknowledge the importance of using intensity measurements in our analysis of CaLexA signals. We concur that the intensity of these signals is indeed correlated with the area measurements, which is a critical factor to consider. In response to the reviewer's valuable suggestion, we have revised our approach and now present our data based on intensity measurements. These have been incorporated as a primary dataset in all our CaLexA results to provide a more accurate representation of our findings. Additionally, we have updated the labeling of our Y-axis to "Norm. GFP Int.", which stands for "normalized GFP intensity". This change ensures clarity and consistency in the presentation of our data, aligning with the reviewer's recommendations and enhancing the overall quality of our manuscript.

    Comment 9.* In Figure 5K: quantification of cell overlap is missing. In the text they state that there are ~100 neurons that co-express SIFaR24F06 and Crz. How was this determined? Is there a graph or numerical summary of this assertion?*

        __Answer:__ We sincerely thank the reviewer for pointing out the oversight in our initial submission regarding the quantification data. In response to this valuable feedback, we have now included the quantification of neurons co-expressing SIFaR24F06 and Crz in the optic lobe (OL) within Figure 6E. This addition ensures that the figure is complete and provides the necessary numerical support for our observations.

    Comment 10.* In lines 709-711: "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." I had trouble understanding this section of methods. What is done with the data from the internal controls?*

       __Answer:__ We appreciate the reviewer's attention to the methodology of our study, particularly regarding the use of internal controls in our mating duration assays. As referenced in our cited work by Bretman et al. (2011) (Bretman *et al*, 2011), our internal control strategy involves a comparison of mating durations between males that have been presented with specific sensory cues and those that have not. This approach includes assessing both males that have been exposed to signals and those that have not, which serves as an internal control for each experimental setup. The purpose of this design is to isolate the effects of our manipulations from other potential confounding factors. In response to the reviewer's comments, we have provided a more detailed description of our mating duration assay in the Methods section. We have also expanded our explanation to clarify how this internal control mechanism ensures that any observed differences in mating duration are attributable to the experimental manipulations and not to extraneous variables. This additional information should provide a clearer understanding of our methodology and the rationale behind our experimental design.

    Comment 11.* Could the authors comment on why the brain GRASP signal is so different in Figures 3A and 4A? I realize that different versions of GRASP were used in these experiments, but I would expect broad agreement between the different approaches.*

       __Answer: __We appreciate the reviewer’s insight. GRASP and t-GRASP are similar technologies that can clearly show the synaptic connection between neurons. GRASP technology was first generated and performed in *C. elegens* (Feinberg *et al*, 2008b). In 2018, the researchers developed a targeted GFP Reconstitution Across Synaptic Partners method, t-GRASP, which resulted in a strong preferential GRASP signal in synaptic regions.

   In our study, we utilized both techniques because of the limitations of the chromosomes where GAL4 and lexA lines located. We also found that during data processing, our method could clearly distinguish the changes in GRASP and t-GRASP signals across three different conditions (naïve, single, and exp.). Therefore, we do not have a particular preference for one technique over the other; both methods are applicable to our experiment.

   The genotype we used in Figure 3A is *SIFa2A-lexA, GAL424F06; lexAop-nSyb-spGFP1-10, UAS-CD4-spGFP11*, where the synaptic transmission occurs from *SIFa2A-lexA *to* GAL424F06*. In Figure 4A, the genotype we used is *GAL4SIFa.PT, lexASIFaR-2A; lexAop-2-post-t-GRASP, UAS-pre-t-GRASP*, where the synaptic transmission occurs from SIFa. PT to SIFaR-2A.

   In our back-to-back submission paper, “Peptidergic neurons with extensive branching orchestrate the internal states and energy balance of male *Drosophila melanogaster,*” (Kim *et al*, 2024) we identified that *SIFa2A* can label posterior-ventral SIFa neurons (SIFaVP), which can only project to ellipsoid body and fan-shaped body. Combining the GRASP technique, Figure 3A cannot show a strong signal as in Figure 4A. We’ve shown in Figure 1G that *SIFaR-2A *covers almost the whole CNS in *Drosophila*. Thus, the synaptic transmission from SIFa. PT (label 4 SIFa neurons) to SIFaR-2A shows a strong signal under the use of the t-GRASP technique. In this case, the GRASP signals in Figure 3A and Figure 4A are so different because of the usage of different GRASP techniques and different fly lines. We appreciate the reviewer's attention to the clarity of our presentation. In response to the comments, we have taken the opportunity to meticulously revise the figure legends to ensure that the differences are explicitly highlighted and easily understood by the readers.

    __ __

    Reviewer #2

    Major concerns: Comment 1.* It is highly interesting that the duration of mating behavior is dependent on external and motivational factors. In fact, that provides an elegant way to study which neuronal mechanisms orchestrate these factors. However, it remains elusive why the authors link the differentially motivated durations of mating behavior to the psychological concept of interval timing. This distracts from the actually interesting neurobiology, and is not necessary to make the study interesting. *

    •   __Answer:__ We are grateful for the opportunity provided by the reviewer to elaborate on our rationale for utilizing the mating duration of male fruit flies as an exemplary genetic model for studying interval timing. At the outset, we would like to acknowledge that mating duration has gained recognition as a valuable genetic model for interval timing, as evidenced by the NIH-NIGMS R01 grant awarded to Michael Crickmore. This grant, which can be reviewed at the provided link (https://grantome.com/grant/NIH/R01-GM134222-01), underscores the significance of this model. Crickmore and colleagues have described in the grant's abstract that "mating duration in *Drosophila* offers a powerful system for exploring changes in motivation over time as behavioral goals are achieved," and it has the potential to provide "the first mechanistic description of a neuronal interval timing system."

In light of this, we have incorporated our rationale into the INTRODUCTION section of our manuscript, as detailed below. We believe that our argumentation, supported by the grant's emphasis on the topic, will not only address the reviewer's concerns but also demonstrate to the broader scientific community the significance of the fruit fly's mating duration as a model for interval timing. This concept has been a cornerstone in the historical development of neuroscientific understanding of time perception. We hope that our expanded discussion will effectively convey the potential of the fruit fly mating duration as a genetic model to offer profound insights into the neural mechanisms underlying interval timing, a concept of enduring importance in the field of neuroscience.

    "The dimension of time is the fundamental basis for an animal's survival. Being able to estimate and control the time between events is crucial for all everyday activities (RICHELLE & LEJEUNE, 1980). The perception of time in the seconds-to-hours range, referred to as ‘interval timing’, is involved in foraging, decision making, and learning via activation of cortico-striatal circuits in mammals (Golombek et al, 2014). Interval timing requires entirely different neural mechanisms from millisecond or circadian timing (Meck et al, 2012; Merchant et al, 2012; Buhusi & Meck, 2005). There is abundant psychological research on time perception because it is a universal cognitive dimension of experience and behavioral plasticity. Despite decades of research, the genetic and neural substrates of temporal information processing have not been well established except for the molecular bases of circadian timing (Buhusi et al, 2009; Tucci et al, 2014). Thus, a simple genetic model system to study interval timing is required. Considering that the mating duration in fruit flies, which averages approximately 20 minutes, is well within the range addressed by interval timing mechanisms, this behavioral parameter provides a relevant context for examining the neural circuits that modulate the Drosophila's perception of time intervals. Such an investigation necessitates an understanding of the extensive neural and behavioral plasticity underlying interval timing (Thornquist et al, 2020; Gautham et al, 2024; Crickmore & Vosshall, 2013)."

    Comment 2.* In figure 4 A and 4K, fluorescence microscopy images of brains and ventral nerve chords are shown, one illustrating GRASP experiments, and one showing CaLexA experiments. The extreme difference between the differentially treated flies (bright fluorescence versus almost no fluorescence) is - in its drastic form- surprising. Online access to the original confocal microscopy images (raw data) might help to convince the reader that these illustrations do not reflect the most drastic "representative" examples out of a series of brain stainings. *

    •   __Answer:__ We sincerely appreciate the reviewer's thoughtful suggestion to enhance the accessibility of our microscopy images for readers who may be interested. In response to this valuable feedback, we have compiled all of our quantified image files into zip format and included them as Supplementary Information 2 and 3. We believe that this additional material will be beneficial for readers seeking a more in-depth view of our data.

    Comment 3.* In particular for behavioral experiments, genetic controls should always be conducted. That is, both the heterozygous Gal4-line as well as the heterozygous UAS-line should be used as controls. This is laborious, but important.*

       __Answer:__ We sincerely appreciate the reviewer's critical feedback regarding the genetic controls in our study. We acknowledge the importance of this aspect and wish to clarify that we have indeed conducted a substantial number of genetic control experiments for both LMD and SMD behaviors. It is worth noting that much of this data has been previously published in other works. Recognizing the interest from another reviewer on the same topic, we have chosen to reiterate our response here for clarity and convenience. Our comprehensive approach to genetic controls ensures the robustness of our findings, and we believe that the published data further substantiates the reliability of our experimental procedures.

   We sincerely thank the reviewer for insightful comments regarding the absence of traditional genetic controls in our study of LMD and SMD behaviors. We acknowledge the importance of such controls and wish to clarify our rationale for not including them in the current investigation. The primary reason for not incorporating all genetic control lines is that we have previously assessed the LMD and SMD behaviors of GAL4/+ and UAS/+ strains in our earlier studies. Our past experiences have consistently shown that 100% of the genetic control flies for both GAL4 and UAS exhibit normal LMD and SMD behaviors. Given these findings, we deemed the inclusion of additional genetic controls to be non-essential for the present study, particularly in the context of extensive screening efforts. However, in accordance with the reviewer's recommendation, we conducted genetic validation experiments on novel genetic crosses, including SIFaR-RNAi/+, CrzR-RNAi/+, and GAL4NP5270/+, and incorporated the results in the supplementary figures (Supplementary information 1). We have made the necessary modifications and additions to the manuscript as below.

    "Given those genetic controls, as evidenced by consistent exhibition of normal LMD and SMD behaviors (Supplemental information. 1), the observed reduction in SIFaR expression, driven by elavc155, is deemed sufficient to induce significant disruptions in LMD and SMD behaviors."

    We understand the value of providing a clear rationale for our methodology choices. To this end, we have added a detailed explanation in the "MATERIALS AND METHODS" section and the figure legends of Figure 1. This clarification aims to assist readers in understanding our decision to omit traditional controls, as outlined below.

    "__Mating Duration Assays for Successful Copulation__The mating duration assay in this study has been reported (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023). 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) (Parks et al. 2004; Yapici et al. 2008). Previous studies have demonstrated that virgin females of this line exhibit increased receptivity to males (Yapici et al. 2008). 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 CO2. After placing a single female into 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. Initiation and completion of copulation were recorded with an accuracy of 10 sec, and total mating duration was calculated for each couple. 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 (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023; Huang et al. 2024; Zhang et al. 2024). 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."

    Minor comments: Comment 4.* Line 75: word missing ("...including FEEDING-RELATED BEHAVIOR, courtship, ..."). *

       __Answer:__ We appreciate your vigilance in identifying this error. We have made the necessary correction to ensure the accuracy of our manuscript.*

    Comment 5.* Line 120: word missing ("SIFaR expression in adult neurons BUT not glia..."). *

       __Answer:__ We appreciate your careful review and attention to detail. Thank you for bringing this to our notice. We have made the necessary corrections to address the error.*

    Comment 6.* I find the figures often to be quite overloaded, and anatomical details often very small (e.g., figure 7A). *

    •   __Answer:__ We appreciate the constructive critique on the layout of our data presentation. Following your insightful recommendation, we have revised the manuscript to enhance clarity. Specifically, we have resized the diagram to be more compact and have also increased the spacing between the panels for better readability.

    __ __

    Reviewer #3

    Major Comments Comment 1.* Are the key conclusions convincing? The key conclusions are intriguing but require more robust data to be fully convincing. While the study presents compelling evidence for the involvement of SIFa and SIFaR in mating behaviors, additional experiments are needed to firmly establish the proposed mechanisms. *

    •   __Answer:__ We are deeply grateful for the insightful and constructive feedback provided by the reviewer on the SIFa-to-SIFaR signaling pathway. We are particularly encouraged by the reviewer's agreement with our findings that support the role of SIFa and SIFaR in regulating mating duration. We concur with the reviewer's suggestion that additional experiments and mechanistic insights are essential to substantiate our conclusions. To this end, we have conducted and included several new experiments, particularly GCaMP data, in the main figures (Figure 6 and S6). Our focus has been intensified on the SIFa-to-Crz signaling, given Crz's established role in controlling mating duration behavior. Below is a summary of the additional experiments we have incorporated:

    1. We have repositioned the SIFa-Crz GCaMP data related to the VNC to Figures 6L-O to ensure that the main text highlights our primary findings.

    2. Our more detailed analysis has identified two cells in the Super Intermediate Protocerebrum (SIP) regions that co-express Crz and SIFaR24F06, along with OL cells (Figure 6D-E).

    3. To provide a complete view of the signaling dynamics, we have included GCaMP data from the brain region in the main figure (Figure 6P-R and Supplementary Figure S6N-P).

    4. Through GCaMP calcium imaging to assess SIFa-to-Crz signaling, we found that calcium levels in Crz+/SIFaR+ SIP neurons consistently decreased with SIFa activation (Figure 6P-R). Conversely, calcium signals in Crz+/SIFaR+ OL neurons increased with SIFa activation, mirroring the pattern seen in Crz+ AG neurons in the VNC (Figure 6M-O and Supplementary Figure S6N-P).

    5. A synthesis of these results is presented in Figure 6S, and we have elaborated on these findings in the manuscript with a detailed description, as detailed below. "To elucidate the direct response of Crz neurons to the activity of SIFa neurons, we conducted live calcium (Ca2+) imaging in the Super Intermediate Protocererbrum (SIP), OL and AG region of the VNC, where Crz neurons are situated (Fig. 6D, Fig. S6M). Upon optogenetic stimulation of SIFa neurons, we observed a significant increase in the activity of Crz in OL and AG region (Fig. 6L-O, Fig. S6N-P), evidenced by a sustained elevation in intracellular Ca2+ levels that persisted in a high level before gradually declining to baseline levels, where the cells in top region of the SIP exhibit consistently drop down after stimulated the SIFa neurons (Fig. 6P-R). These calcium level changes were in contrast to the control group (without all-trans retinal, ATR) (Fig. 6L-R, Fig. S6N-P). These findings confirm that Crz neurons in OL and AG are activated in response to SIFa neuronal activity, but the activity of Crz neurons in SIP are inhibited by the activition of SIFa neuron, reinforcing their role as postsynaptic effectors in the neural circuitry governed by SIFa neurons. Moreover, these results provide empirical support for the hypothesis that SIFa-SIFaR/Crz-CrzR long-range neuropeptide relay underlies the neuronal activity-based measurement of interval timing."

      We are truly grateful for the reviewer's perceptive recommendations concerning the possible mechanisms of LMD and SMD behaviors. In light of this constructive feedback, we have enhanced our discussion to encompass a theoretical framework on the potential role of neuropeptide relays in mediating context-dependent adjustments of synaptic plasticity and calcium signaling within specific neuronal populations. This supplementary perspective is designed to elucidate the intricate dynamics involved, as further elaborated in the updated manuscript.

    "Employing two distinct yet comparable models of interval timing behavior, LMD and SMD, we demonstrated that differential SIFa to SIFaR signaling is capable of modulating context-dependent behavioral responses. Synaptic strengths between SIFa and SIFaR neurons was notably enhanced in group-reared naive males. However, these synaptic strengths specifically diminished in the OL, CB, and AG when males were singly reared, with a particular decrease in the AG region when males were sexually experienced (Fig. 4A-J). Intriguingly, overall calcium signaling within SIFaR24F06 neurons was significantly reduced in group-reared naive males, yet these signals surged dramatically in the OL with social isolation and in the AG with sexual experience (Fig. 4K-T). These calcium signals, as reported by the transcriptional calcium reporter CaLexA, were corroborated by GCaMP live imaging in both the AG and OL regions (Fig. 6L-O and Fig. S6N-P), indicating a close association between elevated calcium levels and LMD and SMD behaviors. The modulation of context-dependent synaptic plasticity and calcium dynamics by the SIFa neuropeptide through a single SIFaR receptor raises the question of how a single receptor can elicit such diverse responses. Recent neuroscientific studies in Drosophila have shown that individual neurons can produce multiple neurotransmitters and that neuropeptides are often colocalized with small molecule neurotransmitters (Nässel 2018,Deng 2019,Croset 2018,Kondo 2020). Consistent with this, we have previously reported that SIFa neurons utilize a variety of neurotransmitters, including glutamate, dopamine, and tyramine (Kim 2024). Therefore, we propose that the SIFa-SIFaR-Crz-CrzR neuropeptidergic relay circuitry may interact with different neurotransmitters in distinct neuronal subpopulations to regulate context-dependent behaviors. Supporting this hypothesis, glutamate, known to function as an inhibitory neurotransmitter in the olfactory pathway of Drosophila (Liu 2013), may be one such candidate. We speculate that neuropeptide cotransmission could underlie the mechanisms facilitating these complex, context-dependent behavioral patterns. Further research is warranted to elucidate how such cotransmission contributes to the intricate behavioral repertoire of the fly."

    Comment 2.* Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? The authors should qualify certain claims as preliminary or speculative. Specifically, the proposed SIFa-SIFaR/Crz-CrzR neuropeptide relay pathway is only investigated via imaging approach. More experiments using behavioral tests are needed to confirm that Crz relays the SIFa signaling pathway. For example, Crz-Gal4>UAS-SIFaR RNAi should be done to show that SIFaR+ Crz+ cells are necessary for LMD and SMD.*

       __Answer:__ We are grateful for the reviewer's constructive suggestion regarding the need to provide additional behavioral assays using RNAi knockdown to substantiate the SIFa-SIFaR/Crz-CrzR neuropeptide relay. Following the reviewer's advice, we have conducted experiments involving SIFaR24F06/Crz-RNAi and Crz-GAL4/SIFaR-RNAi. The outcomes of these experiments have been detailed and are now presented in a clear and comprehensive manner.

   To further aid in the understanding of our results, we have also included a summary diagram in Figure 6S, which illustrates the key findings from these assays. This visual representation is intended to provide a concise overview of the data and to highlight the significance of the SIFa-SIFaR/Crz-CrzR neuropeptide relay in the context of our study.

    Comment 3.* Would additional experiments be essential to support the claims of the paper? Yes, additional experiments are essential. Detailed molecular and imaging studies are needed to support claims about synaptic reorganization. For example: ○ More controls are needed for RNAi and Gal80ts experiments, such as Gal4-only control, RNAi-only control, etc. *

       __Answer:__ We sincerely appreciate the reviewer's critical feedback regarding the genetic controls in our study. We acknowledge the importance of this aspect and wish to clarify that we have indeed conducted a substantial number of genetic control experiments for both LMD and SMD behaviors. It is worth noting that much of this data has been previously published in other works. Recognizing the interest from another reviewer on the same topic, we have chosen to reiterate our response here for clarity and convenience. Our comprehensive approach to genetic controls ensures the robustness of our findings, and we believe that the published data further substantiates the reliability of our experimental procedures.

   We sincerely thank the reviewer for insightful comments regarding the absence of traditional genetic controls in our study of LMD and SMD behaviors. We acknowledge the importance of such controls and wish to clarify our rationale for not including them in the current investigation. The primary reason for not incorporating all genetic control lines is that we have previously assessed the LMD and SMD behaviors of GAL4/+ and UAS/+ strains in our earlier studies. Our past experiences have consistently shown that 100% of the genetic control flies for both GAL4 and UAS exhibit normal LMD and SMD behaviors. Given these findings, we deemed the inclusion of additional genetic controls to be non-essential for the present study, particularly in the context of extensive screening efforts. However, in accordance with the reviewer's recommendation, we conducted genetic validation experiments on novel genetic crosses, including SIFaR-RNAi/+, CrzR-RNAi/+, and GAL4NP5270/+, and incorporated the results in the supplementary figures (Supplementary information 1). We have made the necessary modifications and additions to the manuscript as below.

    "Given those genetic controls, as evidenced by consistent exhibition of normal LMD and SMD behaviors (Supplemental information 1), the observed reduction in SIFaR expression, driven by elavc155, is deemed sufficient to induce significant disruptions in LMD and SMD behaviors."

    We understand the value of providing a clear rationale for our methodology choices. To this end, we have added a detailed explanation in the "MATERIALS AND METHODS" section and the figure legends of Figure 1. This clarification aims to assist readers in understanding our decision to omit traditional controls, as outlined below.

    "__Mating Duration Assays for Successful Copulation__The mating duration assay in this study has been reported (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023). 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) (Parks et al. 2004; Yapici et al. 2008). Previous studies have demonstrated that virgin females of this line exhibit increased receptivity to males (Yapici et al. 2008). 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 CO2. After placing a single female into 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. Initiation and completion of copulation were recorded with an accuracy of 10 sec, and total mating duration was calculated for each couple. 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 (Kim et al. 2012; Kim et al. 2013; Lee et al. 2023; Huang et al. 2024; Zhang et al. 2024). 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."

    *○ Using synaptic markers and high-resolution imaging to observe synaptic changes directly. *

       __Answer:__ We sincerely appreciate the reviewer's constructive suggestion to provide high-resolution imaging for a more direct observation of synaptic changes. While we have already included high-resolution imaging data showcasing postsynaptic and presynaptic alterations using Denmark and syt.eGFP (Figure S3), GRASP (Figure 3A-D), and tGRASP (Figure 4A-J), we recognize the value of further elucidation. Consequently, we have conducted additional experiments to examine the presynaptic changes in SIFaR24F06 neurons under varying social contexts, as presented in Figure 5A-G. We are confident that the comprehensive dataset we have now provided, which includes these new findings, will not only address the reviewer's concerns but also effectively convey to the readers the dynamic and critical nature of SIFa-SIFaR synaptic changes in modulating interval timing behaviors.

    *○ Electrophysiological recordings from neurons expressing SIFa and SIFaR to analyze their functional connectivity and activity patterns. *

       __Answer:__ We sincerely appreciate the reviewer's constructive suggestions regarding the inclusion of electrophysiological recordings from neurons expressing SIFa and SIFaR to analyze functional connectivity and activity patterns. In response to this valuable feedback, we have conducted *in vivo* calcium imaging using the GCaMP indicator. The results have been incorporated into our manuscript, demonstrating SIFa-SIFaR connectivity and alterations in activity patterns (Figure 5H-L), as well as SIFa-Crz connectivity and changes in activity patterns (Figure 6 and Figure S6). We are confident that these additional data provide compelling evidence supporting the notion that the SIFa-SIFaR/Crz-CrzR neuropeptide relay circuits are robustly interconnected and exhibit activity changes in concert with the observed neuronal modifications.

    Comment 4.* Are the suggested experiments realistic in terms of time and resources? The suggested experiments are realistic but will require considerable time and resources. Detailed molecular interaction studies, imaging synaptic plasticity, and electrophysiological recordings could take several months to over a year, depending on the complexity and availability of necessary equipment and expertise. The cost would be moderate to high, involving expenses for reagents, imaging equipment, and animal husbandry for maintaining Drosophila stocks. *

    •   __Answer:__ We are grateful for the reviewers' understanding and support for our additional analysis in the revision experiments. While we have already conducted a multitude of experiments pertinent to this manuscript, we are well-positioned to provide a comprehensive revision of the data within a relatively short timeframe.

    Comment 5.* Are the data and the methods presented in such a way that they can be reproduced? The methods are generally described in detail, allowing for potential reproducibility. However, more precise documentation of certain experimental conditions, such as the timing and conditions of RNAi induction and temperature controls, is necessary. The methods about imaging analysis are too detailed. The exact steps about how to use ImageJ should be removed.*

    •   __Answer:__ We sincerely appreciate the reviewer's meticulous comments regarding the omission of certain methodological details in our manuscript. In response, we have now included a detailed description of the temperature control procedures for conditional RNAi induction in the "Fly Stocks and Husbandry" section, as detailed below.

    "For temperature-controlled experiments, including those utilizing the temperature-sensitive tub-GAL80ts driver, the flies were initially crossed and maintained at a constant temperature of 22℃ within an incubator. The temperature shift was initiated post-eclosion. Once the flies had emerged, they were transferred to an incubator set at an elevated temperature of 29℃ for a defined period, after which the experimental protocols were carried out. Wild-type flies were Canton-S (CS)."

       We appreciate the reviewer's guidance on refining our manuscript. In response to the suggestion, we have streamlined the image analysis methods section, removing excessive details to present the information in a more concise and clear manner as below.

    "Quantitative analysis of fluorescence intensity

    To ascertain calcium levels and synaptic intensity from microscopic images, we dissected and imaged five-day-old flies of various social conditions and genotypes under uniform conditions. The GFP signal in the brains and VNCs was amplified through immunostaining with chicken anti-GFP primary antibody. Image analysis was conducted using ImageJ software. For the quantification of fluorescence intensities, an investigator, blinded to the fly's genotype, thresholded the sum of all pixel intensities within a sub-stack to optimize the signal-to-noise ratio, following established methods (Feinberg 2008). The total fluorescent area or region of interest (ROI) was then quantified using ImageJ, as previously reported. For CaLexA signal quantification, we adhered to protocols detailed by Kayser et al. (Kayser et al, 2014), which involve measuring the ROI's GFP-labeled area by summing pixel values across the image stack. This method assumes that changes in the GFP-labeled area are indicative of alterations in the CaLexA signal, reflecting synaptic activity. ROI intensities were background-corrected by measuring and subtracting the fluorescent intensity from a non-specific adjacent area, as per Kayser et al. (Kayser et al, 2014). For the analysis of GRASP or tGRASP signals, a sub-stack encompassing all synaptic puncta was thresholded by a genotype-blinded investigator to achieve the optimal signal-to-noise ratio. The fluorescence area or ROI for each region was quantified using ImageJ, employing a similar approach to that used for CaLexA quantification (Feinberg 2008)."

    Comment 6.* Are the experiments adequately replicated and statistical analysis adequate? Most figures in the manuscript need to be re-plotted. The right y-axis "Difference between means" is not necessary, if not confusing. The image panels are too small to see, while the quantification of overlapping cells are unnecessarily large. The figures are too crowded with labels and texts, which makes it extremely difficult to comprehend the data.*

       __Answer:__ We appreciate the reviewer's suggestion to refine our figures, and we have indeed reformatted them to provide clearer presentation and improved readability. Regarding the removal of dot blot membranes (DBMs), we have given this considerable thought. While we understand the recommendation, we have chosen to retain the DBMs in our manuscript. Our decision is based on the fact that our analysis encompasses not only traditional t-tests but also incorporates estimation statistics, which have been demonstrated to be effective for biological data analysis (Claridge-Chang & Assam, 2016). The inclusion of DBMs is essential for the accurate interpretation of these estimation statistics, ensuring a comprehensive representation of our findings.

    Minor Comments Comment 7.* Specific experimental issues that are easily addressable. Clarify the timing of RNAi induction and provide more detailed figure legends for better understanding and reproducibility.*

       __Answer:__ We sincerely appreciate the reviewer's suggestion aimed at enhancing our manuscript. As previously addressed in our response to __*Comment 5*__, we have incorporated additional details regarding the timing of RNAi induction within the Methods section. Furthermore, we have expanded upon the figure legends to provide a clearer understanding of our findings, ensuring that the content is accessible to a broader readership.

    Comment 8. Are prior studies referenced appropriately? Yes. *

    __ Answer:__ We are grateful for the reviewer's acknowledgment that our references have been appropriately included and integrated into the manuscript.

    Comment 9. Are the text and figures clear and accurate? The text is generally clear, but the figures need re-work. See comment above. *

    •   *__Answer:__ We appreciate the feedback from the reviewers regarding the clarity of our figures. In response to other reviewers' concerns about the figures appearing too crowded, we have carefully revised the layout of all figures to ensure they are more spacious and aesthetically improved for better readability and visual appeal.

    Comment 10. Suggestions to improve the presentation of data and conclusions. Use smaller fonts in the bar plots and make the plots smaller. Enlarge the imaging panels and let the pictures tell the story. *

    •   __Answer:__ We sincerely appreciate the reviewer's constructive suggestion. In response, we have revised the figures by enlarging the images and adjusting the font sizes in the bar plots to enhance readability and clarity.

    REFERENCES

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    Bretman A, Westmancoat JD, Gage MJG & Chapman T (2011) Males Use Multiple, Redundant Cues to Detect Mating Rivals. Curr Biol 21: 617–622

    Buhusi CV, Aziz D, Winslow D, Carter RE, Swearingen JE & Buhusi MC (2009) Interval Timing Accuracy and Scalar Timing in C57BL/6 Mice. Behav Neurosci 123: 1102–1113

    Buhusi CV & Meck WH (2005) What makes us tick? Functional and neural mechanisms of interval timing. Nat Rev Neurosci 6: 755–765

    Claridge-Chang A & Assam PN (2016) Estimation statistics should replace significance testing. Nat Methods 13: 108–109

    Crickmore MA & Vosshall LB (2013) Opposing Dopaminergic and GABAergic Neurons Control the Duration and Persistence of Copulation in Drosophila. Cell 155: 881–893

    Feinberg EH, VanHoven MK, Bendesky A, Wang G, Fetter RD, Shen K & Bargmann CI (2008a) GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems. Neuron 57: 353–363

    Feinberg EH, VanHoven MK, Bendesky A, Wang G, Fetter RD, Shen K & Bargmann CI (2008b) GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems. Neuron 57: 353–363

    Gautham AK, Miner LE, Franco MN, Thornquist SC & Crickmore MA (2024) Molecular control of temporal integration matches decision-making to motivational state. bioRxiv: 2024.03.01.582988

    Golombek DA, Bussi IL & Agostino PV (2014) Minutes, days and years: molecular interactions among different scales of biological timing. Philosophical Transactions Royal Soc B Biological Sci 369: 20120465

    Khoshnoud S, Leitritz D, Bozdağ MÇ, Igarzábal FA, Noreika V & Wittmann M (2024) When the heart meets the mind: Exploring the brain-heart interaction during time perception. J Neurosci: e2039232024

    Kim WJ, Jan LY & Jan YN (2012) Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals. Nat Neurosci 15: 876–883

    Kim WJ, Jan LY & Jan YN (2013) A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating. Neuron 80: 1190–1205

    Kim WJ, Song Y, Zhang T, Zhang X, Ryu TH, Wong KC, Wu Z, Wei Y, Schweizer J, Nguyen K-NH, et al (2024) Peptidergic neurons with extensive branching orchestrate the internal states and energy balance of male Drosophila melanogaster. bioRxiv: 2024.06.04.597277

    Lee SG, Sun D, Miao H, Wu Z, Kang C, Saad B, Nguyen K-NH, Guerra-Phalen A, Bui D, Abbas A-H, et al (2023) Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster. PLOS Genet 19: e1010753

    Matell MS (2014) Neurobiology of Interval Timing. Adv Exp Med Biol: 209–234

    Meck WH, Doyère V & Gruart A (2012) Interval Timing and Time-Based Decision Making. Frontiers Integr Neurosci 6: 13

    Merchant H, Harrington DL & Meck WH (2012) Neural Basis of the Perception and Estimation of Time. Annu Rev Neurosci 36: 313–336

    Nicolaï LJJ, Ramaekers A, Raemaekers T, Drozdzecki A, Mauss AS, Yan J, Landgraf M, Annaert W & Hassan BA (2010) Genetically encoded dendritic marker sheds light on neuronal connectivity in Drosophila. Proc National Acad Sci 107: 20553–20558

    RICHELLE M & LEJEUNE H (1980) Time in Animal Behaviour. Part III: Mech: 188–199

    Tayler TD, Pacheco DA, Hergarden AC, Murthy M & Anderson DJ (2012) A neuropeptide circuit that coordinates sperm transfer and copulation duration in Drosophila. Proc National Acad Sci 109: 20697–20702

    Thornquist SC, Langer K, Zhang SX, Rogulja D & Crickmore MA (2020) CaMKII Measures the Passage of Time to Coordinate Behavior and Motivational State. Neuron 105: 334-345.e9

    Tucci V, Buhusi CV, Gallistel R & Meck WH (2014) Towards an integrated understanding of the biology of timing. Philosophical Transactions Royal Soc B Biological Sci 369: 20120470

    Wong K, Schweizer J, Nguyen K-NH, Atieh S & Kim WJ (2019) Neuropeptide relay between SIFa signaling controls the experience-dependent mating duration of male Drosophila. Biorxiv: 819045

  6. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    The article investigates the role of the neuropeptide SIFa and its receptor SIFaR in regulating two distinct mating duration behaviors in male Drosophila melanogaster, Longer-Mating-Duration (LMD) and Shorter-Mating-Duration (SMD). The study reveals that SIFaR expression in specific neurons is required for both behaviors. It shows that social context and sexual experience lead to synaptic reorganization between SIFa and SIFaR neurons, altering internal brain states. The SIFa-SIFaR/Crz-CrzR neuropeptide relay pathway is essential for generating these behaviors, with Crz neurons responding to SIFa neuron activity. Furthermore, CrzR expression in non-neuronal cells is critical for regulating LMD and SMD behaviors. The study utilizes neuropeptide RNAi screening, chemoconnectome (CCT) knock-in, and genetic intersectional methods to elucidate these findings.

    Major Comments

    • Are the key conclusions convincing? The key conclusions are intriguing but require more robust data to be fully convincing. While the study presents compelling evidence for the involvement of SIFa and SIFaR in mating behaviors, additional experiments are needed to firmly establish the proposed mechanisms.
    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? The authors should qualify certain claims as preliminary or speculative. Specifically, the proposed SIFa-SIFaR/Crz-CrzR neuropeptide relay pathway is only investigated via imaging approach. More experiments using behavioral tests are needed to confirm that Crz relays the SIFa signaling pathway. For example, Crz-Gal4>UAS-SIFaR RNAi should be done to show that SIFaR+ Crz+ cells are necessary for LMD and SMD.
    • Would additional experiments be essential to support the claims of the paper? Yes, additional experiments are essential. Detailed molecular and imaging studies are needed to support claims about synaptic reorganization. For example:
      • More controls are needed for RNAi and Gal80ts experiments, such as Gal4-only control, RNAi-only control, etc.
      • Using synaptic markers and high-resolution imaging to observe synaptic changes directly.
      • Electrophysiological recordings from neurons expressing SIFa and SIFaR to analyze their functional connectivity and activity patterns.
    • Are the suggested experiments realistic in terms of time and resources? The suggested experiments are realistic but will require considerable time and resources. Detailed molecular interaction studies, imaging synaptic plasticity, and electrophysiological recordings could take several months to over a year, depending on the complexity and availability of necessary equipment and expertise. The cost would be moderate to high, involving expenses for reagents, imaging equipment, and animal husbandry for maintaining Drosophila stocks.
    • Are the data and the methods presented in such a way that they can be reproduced? The methods are generally described in detail, allowing for potential reproducibility. However, more precise documentation of certain experimental conditions, such as the timing and conditions of RNAi induction and temperature controls, is necessary. The methods about imaging analysis are too detailed. The exact steps about how to use ImageJ should be removed.
    • Are the experiments adequately replicated and statistical analysis adequate? Most figures in the manuscript need to be re-plotted. The right y-axis "Difference between means" is not necessary, if not confusing. The image panels are too small to see, while the quantification of overlapping cells are unnecessarily large. The figures are too crowded with labels and texts, which makes it extremely difficult to comprehend the data.

    Minor Comments

    • Specific experimental issues that are easily addressable. Clarify the timing of RNAi induction and provide more detailed figure legends for better understanding and reproducibility.
    • Are prior studies referenced appropriately? Yes.
    • Are the text and figures clear and accurate? The text is generally clear, but the figures need re-work. See comment above.
    • Suggestions to improve the presentation of data and conclusions. Use smaller fonts in the bar plots and make the plots smaller. Enlarge the imaging panels and let the pictures tell the story.

    Significance

    Nature and Significance of the Advance

    This study aims to advance understanding of how neuropeptides modulate context-dependent behaviors in Drosophila. It provides novel insights into the role of SIFa and SIFaR in interval timing behaviors, contributing to the broader field of neuropeptide research and behavioral neuroscience. However, the significance of the findings is limited by the preliminary nature of some claims and the need for additional supporting data.

    Context in Existing Literature

    The work builds on previous studies that identified various roles of neuropeptides in behavior modulation but lacked detailed mechanistic insights. By elucidating the SIFa-SIFaR/Crz-CrzR pathway, this study attempts to fill a gap in the literature, but more robust evidence is required to solidify its contributions.

    Interested Audience

    The findings will interest neuroscientists, behavioral biologists, and researchers studying neuropeptides and their roles in behavior and neural circuitry. Additionally, this research may have implications for understanding neuropeptidergic systems in other organisms, making it relevant to a broader audience in the fields of neurobiology and physiology.

    Field of Expertise

    Keywords: Neuropeptides, Drosophila melanogaster, Behavioral Neuroscience. Areas without sufficient expertise: courtship behavior.

    Recommendation

    I recommend a major revision of this manuscript. The study presents intriguing findings, but several key claims are preliminary and require additional experiments for support. The data is poorly presented and the figures can be significantly improved. Detailed molecular and imaging studies, as well as more rigorous statistical analyses, are necessary to strengthen the conclusions. Addressing these concerns will significantly improve the robustness and impact of the paper.

  7. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Zhang et al., "Long-range neuropeptide relay as a central-peripheral communication mechanism for the context-dependent modulation of interval timing behaviors".

    The authors investigate mating behavior in male fruit flies, Drosophila melanogaster, and test for a role of the SIFamide receptor (SIFaR) in this type of behavior, in particular mating duration in dependence of social isolation and prior mating experience. The anatomy of SIFamide-releasing neurons in comparison with SIFamide receptor-expressing neurons is characterized in a detail-rich manner. Isolating males or exposing them to mating experience modifies the anatomical organization of SIFamidergic axon termini projecting onto SIFamide receptor-expressing neurons. This structural synaptic plasticity is accompanied by changes in calcium influx. Lastly, it is shown that corazonin-releasing neurons are modulated by SIFamide releasing neurons and impact the duration of mating behavior.

    Overall, this highly interesting study advances our knowledge about the behavioral roles of SIFamide, and contributes to an understanding how motivated behavior such as mating is orchestrated by modulatory peptides. The approach to take the entire organism, including peripheral tissue, into consideration, is very good and a rather unique point. The manuscript has only some points that are less convincing, and these should be addressed.

    Major concerns:

    • It is highly interesting that the duration of mating behavior is dependent on external and motivational factors. In fact, that provides an elegant way to study which neuronal mechanisms orchestrate these factors. However, it remains elusive why the authors link the differentially motivated durations of mating behavior to the psychological concept of interval timing. This distracts from the actually interesting neurobiology, and is not necessary to make the study interesting.
    • In figure 4 A and 4K, fluorescence microscopy images of brains and ventral nerve chords are shown, one illustrating GRASP experiments, and one showing CaLexA experiments. The extreme difference between the differentially treated flies (bright fluorescence versus almost no fluorescence) is - in its drastic form- surprising. Online access to the original confocal microscopy images (raw data) might help to convince the reader that these illustrations do not reflect the most drastic "representative" examples out of a series of brain stainings.
    • In particular for behavioral experiments, genetic controls should always be conducted. That is, both the heterozygous Gal4-line as well as the heterozygous UAS-line should be used as controls. This is laborious, but important.

    Minor comments:

    • Line 75: word missing ("...including FEEDING-RELATED BEHAVIOR, courtship, ...").
    • Line 120: word missing ("SIFaR expression in adult neurons BUT not glia...").
    • I find the figures often to be quite overloaded, and anatomical details often very small (e.g., figure 7A).

    Significance

    Overall, this highly interesting study advances our knowledge about the behavioral roles of SIFamide, and contributes to an understanding how motivated behavior is orchestrated by modulatory peptides. The approach to take the entire organism, including peripheral tissue, into consideration, is very good and a rather unique point.

    Since decades it has been investigated how sensory stimuli are processed and encoded by the brain, and how behavioral actions are executed. Likewise, principles underlying learning and memory, sleep, orentation, circadian rhythms, etc. are subject to intense investigation. However, how motivational factors (sleep pressure, hunger, sexual drive) are actually "encoded", signaled and finally used to orchstrate behavior and guide decision-making is, to a very large degree, unknown - in any species. The model use here (Drosophila and its peptidergic system wit SIFamide as a central hub) represents actually a ideal entry point to study just this question. In this sense, the manuscript is at the forefront of modern, state-of-the-art neurobiology.

  8. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    This manuscript from Zhang et al. primarily investigates the contribution of the SIFa neuropeptide receptor (SIFaR) to mating duration in male fruit flies. Through RNAi-mediated downregulation, they show that SIFaR receptor is necessary for previous experience to alter mating duration. Using cell-specific knockdown and rescue of the SIFaR receptor, they identify a population of ~400 neurons that could underlie this effect. This is still a large number of cells but is narrowed from the ~1,200 total SIFaR-expressing neurons. They then use the GRASP synaptic labeling technique to show that SIFa+ neurons form synapses onto the relevant SIFaR-expressing population, and that the area of synaptic contact is systematically altered depending on the fly's past mating history. Finally, they provide evidence to argue that SIFa neurons act through SIFaR neurons that release the neuropeptide corazonin to regulate mating duration. Overall, the authors have used an impressive array of techniques in their attempt to define the neural circuits and molecules involved in changing internal state to modify the duration of mating.

    Major Comments:

    1. The authors are to be commended for the sheer quantity of data they have generated, but I was often overwhelmed by the figures, which try to pack too much into the space provided. As a result, it is often unclear what components belong to each panel. Providing more space between each panel would really help.
    2. The use of three independent RNAi lines to knock down SIFaR expression is experimentally solid, as the common phenotype observed with all 3 lines supports the conclusion that the SIFaR is important for mating duration choice. However, the authors have not tested whether these lines effectively reduce SIFaR expression, nor whether the GAL80 constructs used to delimit knockdown are able to effectively do so. This makes it hard to make definitive conclusions with these manipulations, especially in the face of negative results. A lack of complete knockdown is suggested by the fact that the F24F06 driver rescues lethality when used to express SIFaR in the B322 mutant background, but does not itself produce lethality when used to express SIFaR RNAi. The authors should either conduct experiments to determine knockdown efficiency or explicitly acknowledge this limitation in drawing conclusions from their experiments. A similar concern relates to the CrzR knockdown experiments (eg Figure 7).
    3. Most of the behavioral experiments lack traditional controls, for example flies that contain either the GAL4 or UAS elements alone. The authors should explain their decision to omit these control experiments and provide an argument for why they are not necessary to correctly interpret the data. In this vein, the authors have stated in the methods that stocks were outcrossed at least 3x to Canton-S background, but 3 outcrosses is insufficient to fully control for genetic background.
    4. Throughout the manuscript, the authors appear to use a single control condition (sexually naïve flies raised in groups) to compare to both males raised singly and males with previous sexual experience. These control conditions are duplicated in two separate graphs, one for long mating duration and one for short mating duration, but they are given different names (group vs naïve) depending on the graph. If these are actually the same flies, then this should be made clear, and they should be given a consistent name across the different "experiments".
    5. The authors have consistently conflated overlap of neuronal processes with synaptic connections. Claims of synaptic connectivity deriving solely from overlap of processes should be tempered and qualified.
      • For example, they say (Lines 201-202) "These findings suggest that SIFa neurons and GAL424F06-positive neurons form more synapses in the VNC than in the brain." This is misleading. Overlap of24F06-LexA>CD8GFP and SIFa-GAL4>CD8RFP tells us nothing about synapse number, or even whether actual synapses are being formed.
      • Lines 210-211: "The overlap of DenMark and syt.EGFP signals was highly enriched in both SOG and ProNm regions, indicating that these regions are where GAL424F06 neurons form interconnected networks". This is misleading. Overlap of DenMark and syt.EGFP does not indicate synapses (especially since these molecules can be expressed outside the expected neuronal compartment if driven at high enough levels).
      • Lines 320-322: "Neurons expressing Crz exhibit robust synaptic connections with SIFaR24F06 neurons located in the PRW region of the SOG in the brain (panels of Brain and SOG in Fig. 5A)". This is again misleading. They are not actually measuring synapses here, but instead looking at area of overlap between neuronal processes of Crz and SIFaR cells.
      • In Figs 3B and S4A, they are claiming that all neuronal processes within a given delineated brain area are synapses. The virtual fly brain and hemibrain resource have a way to actually identify synapses. This should be used in addition to the neuron skeleton. Otherwise, it is misleading to label these as synapses.
      • Furthermore, measuring the area of GRASP signal is not the same as quantifying synapses. We don't know if synapse number changes (eg in lines 240-242).
    6. In general, the first part of the manuscript (implicating SIFaR in mating duration) is much stronger than the second part, which attempts to demonstrate that SIFa acts through Crz-expressing neurons to induce its effects. The proof that SIFa acts through Crz-expressing neurons to modify mating duration is tenuous. The most direct evidence of this, achieved via knockdown on Crz in SIFaR-expressing cells, is relegated to supplemental figures. The calcium response of the Crz neurons to SIFa neuron activation (Fig. 6) is more of a lack of a decrease that is observed in controls. Also, this is only done in the VNC. Why not look in the brain, because the authors previously stated a hypothesis that the "transmission of signals through SIFaR in Crz-expressing neurons is limited to the brain" (lines 381-382)?

    Furthermore, the authors suggest that Crz acts on cells in the heart to regulate mating duration. It would be useful to add a discussion/speculation as to possible mechanisms for heart cells to regulate mating decisions. Is there evidence of CrzR in the heart? The SCope data presented in Fig. 7I-L and S7G-H is hard to read.

    1. In several cases, the effects of being raised single are opposite the effects of sexual experience. For example, in Fig. 4T, calcium activity is increased in the AG following sexual experience, but decreased in flies raised singly. Likewise, Crz-neurons in the OL have increased CaLexA signal in singly-raised flies but reduced signals in flies with previous sexual experience. In some cases, manipulations selectively affect LMD or SMD. It would be useful to discuss these differences and consider the mechanistic implications of these differential changes, when they all result in decreased mating duration. This could help to clarify the big picture of the manuscript.

    Minor Comments:

    1. For CaLexA experiments (eg Fig 7A-D), signal intensity should be quantified in addition to area covered. Increased intensity would indicate greater calcium activity within a particular set of neurons.
    2. In Figure 5K: quantification of cell overlap is missing. In the text they state that there are ~100 neurons that co-express SIFaR24F06 and Crz. How was this determined? Is there a graph or numerical summary of this assertion?
    3. In lines 709-711: "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." I had trouble understanding this section of methods. What is done with the data from the internal controls?
    4. Could the authors comment on why the brain GRASP signal is so different in Figures 3A and 4A? I realize that different versions of GRASP were used in these experiments, but I would expect broad agreement between the different approaches.

    Significance

    This study will be most relevant to researchers interested in understanding neuronal control of behavior. The manuscript offers a conceptual advance in identifying cell types and molecules that influence mating duration decisions. The strength of the manuscript is the number of different assays used; however, there is a sense that this has occurred at the cost of providing a cohesive narrative. The first part of the manuscript (detailing the role of SIFaR in LMD and SMD) is relatively stronger and more conclusive.

  9. Version published to 10.1101/2024.06.03.597273 on bioRxiv