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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".
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* 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."
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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).*
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*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).
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- 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)."
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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.
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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).*
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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.*
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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:
- 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.
- 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).
- 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).
- 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).
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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.
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__
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.
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__
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:
- 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.
- 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).
- 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).
- 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).
-
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.
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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.
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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.
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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. *
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* 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.
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