Molecular and Neural Circuit Mechanisms Underlying Sexual Experience-dependent Long-Term Memory in Drosophila.

  1. Dongyu Sun
  2. Xiaoli Zhang
  3. Hongyu Miao
  4. Tianmu Zhang
  5. Woo Jae Kim

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Abstract

The neural and molecular underpinnings of sexual experience-dependent long-term memory (SELTM) in male Drosophila melanogaster remain poorly understood, despite its significance for reproductive success. Here, we dissect the role of a specific class of neurons, termed 'Yuelao' (YL) neurons, in the formation of SELTM and the associated molecular pathways. We employed a combination of genetic manipulations, immunohistochemistry, and behavioral assays in Drosophila to investigate the function of YL neurons and their molecular interactors in SELTM. Utilizing the RNA sequencing data and transgenic tools, we delineated the neural circuits involved in taste and pheromone processing relevant to SELTM. Our findings reveal that the YL neurons, expressing the Orb2 scaffolding protein, are indispensable for the formation of SELTM following sexual experience. These neurons are regulated by the neuromodulator short neuropeptide F (sNPF) and its receptor (sNPF-R), which modulate glutamate release via NMDAR2. We demonstrate that sexual experience triggers synaptic plasticity in YL neurons, characterized by an increase in dendritic and presynaptic terminal areas, and a decrease in intracellular calcium levels. Furthermore, we show that YL neurons are specialized for the generation of appetitive sexual experience-dependent memory and project to brain regions implicated in memory formation. Our study uncovers the YL neurons as a key neural substrate for SELTM in Drosophila, shedding light on the molecular and circuit mechanisms that mediate the formation of long-term memories following sexual experience. These findings provide novel insights into the neural basis of taste-related memory and have broader implications for understanding the interplay between experience, memory formation, and behavior.

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    Point-by-Point Response to Reviewers for Manuscript #RC-2024-02720

    Manuscript Title: Molecular and Neural Circuit Mechanisms Underlying Sexual Experience-dependent Long-Term Memory in Drosophila.

    Corresponding Author: Woo Jae Kim

    We extend our sincere gratitude to the Managing Editor and both reviewers for their diligent and insightful evaluation of our manuscript. The comprehensive feedback provided has been invaluable, guiding us to significantly strengthen the manuscript's scientific rigor, logical cohesion, and overall impact. We have undertaken a substantial revision, incorporating new experimental evidence, reframing the central narrative, and improving data presentation to address all concerns raised.

    The major revisions include:

    1. New Experimental Evidence: We have performed three new sets of experiments to address key questions raised by the reviewers. First, we used the protein synthesis inhibitor cycloheximide to pharmacologically validate that the observed memory is indeed a form of long-term memory (LTM). Then, we performed genetic intersectional analyses to determine if the identified Yuelao (YL) neurons express the canonical sex-determination transcription factors doublesex (dsx) and fruitless (fru).
    2. Narrative Reframing and Logical Restructuring: We fully agree with the reviewers that the logic of the original manuscript was confusing, particularly regarding the distinction between the broad Mushroom Body (MB) Kenyon Cell (KC) population and the specific YL neurons. The manuscript has been extensively rewritten to present a clear, hypothesis-driven narrative. We now frame the initial KC-related findings as part of a broader screening effort that logically led to the identification and focused investigation of the YL neuron circuit.
    3. Refined Central Claim: Guided by the reviewers' feedback and our new data, we have sharpened our central claim. We now propose that YL neurons constitute a critical circuit for forming attractive taste- and pheromone-based memories derived from Gr5a neuronal inputs. This form of appetitive memory is distinct from the previously characterized internal reward state associated with ejaculation, adding a new layer to our understanding of how male flies remember and evaluate reproductive experiences.
    4. Improved Data Quality and Analysis: In response to valid critiques, all imaging figures have been replaced with high-resolution versions. Furthermore, our methods for fluorescence quantification, particularly for the TRIC calcium imaging experiments, have been corrected to include normalization against an internal reference channel, adhering to established best practices. All requested genetic control experiments have been performed. We are confident that these comprehensive revisions have fully addressed all concerns and have transformed our manuscript into a much stronger, more focused, and logically sound contribution. We thank you again for the opportunity to improve our work and look forward to your evaluation of the revised manuscript.

    Responses to Reviewer #1

    General Comments:* This study explores the molecular and neural circuitry mechanisms underlying sexual experience-dependent long-term memory (SELTM) in male Drosophila. The authors use behavioral, imaging, and bioinformatics approaches to identify YL neurons, a subset of mushroom body (MB) projecting neurons, as crucial for SELTM formation. They propose that YL neurons receive inputs from WG neurons via the sNPF-sNPFR pathway and implicate molecular players such as orb2, fmr1, MDAR2-CaMK, and synaptic plasticity in their function.*

    However, the evidence presented does not adequately support the authors' claims. The data fail to cohesively tell a logical story, and key conclusions appear to be based on assumptions and correlations rather than robust evidence.

    •   *__Answer:__ We are deeply grateful to both reviewers for their thorough and constructive evaluation of our manuscript. Their collective feedback has been instrumental in helping us to clarify the study's rationale, strengthen our interpretations, and significantly improve the overall quality and impact of the work. We appreciate the recognition of our study's potential to advance the understanding of how sexual experience modifies future mating behaviors and to elucidate the neuronal and molecular mechanisms of how memory regulates a key sexual behavior in male *Drosophila*.

    •    *In response to the general comments, we have undertaken a major revision of the manuscript to improve the clarity, logic, and presentation. We have rewritten the Abstract and Introduction to more clearly define "sexual experience-dependent long-term memory" (SELTM) and articulate its significance in the context of adaptive decision-making and interval timing. The entire manuscript has been restructured to present a more logical, hypothesis-driven narrative that clearly distinguishes our initial broad screening from the focused investigation of the YL neuron circuit. We have also incorporated alternative interpretations of our data, particularly regarding the role of the YL circuit in regulating baseline mating duration in naive males, which has added more depth to the study. Finally, all figures have been remade in high resolution, and all requested genetic controls and methodological clarifications have been added to ensure rigor and reproducibility. We are confident that these revisions have addressed the reviewers' concerns and have resulted in a much stronger manuscript.

    Comment 1:* The study identifies the knowledge gap (lines 103-104) but fails to integrate relevant literature, particularly Shohat-Ophir et al., Science (2012), and Zer-Krispil et al., Curr Biol (2018). These studies established that ejaculation induces appetitive memory in male Drosophila via corazonin and NPF neurons. The current study does not provide direct evidence that the "act of mating itself" drives SELTM, as it includes both courtship and copulation.*

    Response: Thank you for highlighting these two landmark studies. We fully agree that Shohat-Ophir et al., Science (2012) and Zer-Krispil et al., Curr Biol (2018) were pivotal in demonstrating that ejaculation—and the accompanying corazonin/NPF signalling—can establish an appetitive memory in males.

    In the revised manuscript we have now integrated both papers on lines 111-118:

    “Previous work has shown that successful copulation is intrinsically rewarding to male Drosophila: a single mating encounter elevates brain neuropeptide F (NPF) levels and suppresses subsequent ethanol preference19. Importantly, Zer-Krispil et al. further demonstrated that ejaculation itself—artificially induced by optogenetic activation of corazonin (Crz) neurons—is sufficient to mimic this reward state, driving appetitive memory formation and up-regulation of NPF. These findings indicate that the act of ejaculation, rather than the entire courtship sequence, is the critical sensory event that gates post-mating reward.”

    Comment 2:* The nature of the observed long-lasting reduced mating duration requires clearer characterization: Is this an associative memory or experience-dependent behavioral plasticity? Can the formation of this long-term memory be blocked by protein synthesis inhibitors, such as cycloheximide?*

    Response: We thank the reviewer for this excellent suggestion to pharmacologically characterize the nature of the memory. To definitively test whether the observed SMD is a form of protein synthesis-dependent long-term memory (LTM), we performed a new experiment as suggested.

    We have now included data in new Figure supplement 1I showing that feeding males the protein synthesis inhibitor cycloheximide (CXM) for 24 hours immediately following the sexual experience completely blocks the formation of the long-lasting SMD phenotype. Control flies fed a vehicle solution exhibited robust SMD. This result provides strong evidence that SELTM is not merely a form of transient behavioral plasticity but is a genuine form of LTM that requires de novo protein synthesis for its consolidation, a hallmark of LTM across species.[1]

    The revised text was put on lines 173-176:

    " To determine whether the persistent reduction in mating duration (SMD) depends on de-novo protein synthesis, we fed males the translational inhibitor cycloheximide (CXM). Under this regimen, CXM completely abolished the SMD phenotype (Fig. 1I)."

    Comment 3: While schematics illustrate the working hypotheses, the text lacks detailed explanations, leaving the reader unclear about the rationale behind certain conclusions.

    __Response: __Thank you very much for this insightful comment. We fully agree that the original manuscript did not provide sufficient textual justification for the conclusions derived from the schematics. In the revised version we have therefore added comprehensive explanations immediately following each figure (or schematic) that explicitly state the underlying rationale, the key observations supporting our hypotheses, and the logical steps leading to each conclusion. We believe these additions now make the reasoning transparent and easy to follow. We appreciate your feedback, which has substantially improved the clarity of our work.

    Comment 4*: The logic to draw certain conclusions was confusing and misleading. - For instance, the role of orb2 in SELTM is examined via knockdown in MB Kenyon cells (KCs) (using ok107>orb2-RNAi), which is irrelevant to the claim that orb2 functions in YL neurons. Additionally, RNAseq analyses (Fig. 1N-S) focusing on orb2 expression in a/b KCs are irrelevant to and cannot support the claim that Orb2 functions in YL neurons. *

    *- Similarly, the claim (lines 302-303) that sNPF-R expression is exclusive to MB KCs conflicts with data showing effects when sNPF-R is knocked down in YL neurons. How can knocking-down a gene, which is exclusively expressed in neural population A, in neural population B affect a phenotype? This inconsistency undermines the interpretation of the results. *

    *- Other examples include lines 223-227 and lines 246-249. It is very confusing how the authors came to the indications. *

    - The authors also kept confusing the readers and themselves by mistakenly referring to MB KC a-lobe and YL a-lobe projection. They may know the difference between the two neural populations but they did not always refer to the right one in the text.

    Response: We agree completely with the reviewer that the logic in the original manuscript was confusing and failed to clearly distinguish between the general MB Kenyon Cell (KC) population and the specific YL projection neurons. This was a major flaw, and we are grateful for the opportunity to correct it. We have undertaken a major revision of the manuscript's narrative and structure to present a clear, logical progression of discovery.

    The new logical flow of the manuscript is as follows:

    1. We first establish that sexual experience induces a robust, long-lasting SMD behavior that is dependent on protein synthesis
    2. We then perform initial experiments to implicate the MB as a key brain region. We show that broad inhibition of MB KCs (using the ok107-GAL4 driver) disrupts SMD behavior.This result establishes the general involvement of the MB but lacks cellular specificity.
    3. The remainder of the manuscript then focuses specifically on dissecting the molecular and cellular properties of these YL neurons. Finally, we have meticulously edited the entire manuscript to ensure that we always use precise terminology, clearly distinguishing between "YL neuron projections to the MB α-lobe" and the "MB KC α-lobe."

    Comment 5*: The imaging figures provided are unfocused and poorly resolved, making it difficult to assess data quality. *

    *- Colocalization analyses of orb2 and YL are unconvincing... Maximum intensity projection images are insufficient... complete image stacks with staining of orb2, YL, and KCs (MB-dsRed) are needed for validation. *

    - Quantification of imaging data appears flawed. For example, claims of orb2 and CaMKII upregulation in MB a-lobe projections (e.g., Fig. S2F-J, Fig. 3M,N) are confounded by widespread increases in intensity across the brain, lacking specificity.

    *- The TRIC experiment analysis should normalize GFP signals to internal reference channel (RFP in the TRIC construct)... *

    - In Fig. 6H-J, methods for counting synapse numbers are not described. How are synapse numbers counted in these low-resolution images?

    Response: We sincerely apologize for the poor quality of the imaging data presented in the original manuscript. We agree with the reviewer's critiques and have taken comprehensive steps to rectify these issues.

    • Image Quality: We apologize for not including the full image data in the original submission. The complete figure is now presented in revised Fig. 2J .
    • Fluorescence Quantification: The fluorescence quantification has been re-analyzed. The Methods section now includes a detailed description of our protocol.
    • TRIC Normalization: We apologize for not stating this explicitly in the previous version. As now described in the revised Methods subsection “Quantitative Analysis of Fluorescence Intensity”, all TRIC images were acquired with identical laser power and exposure settings. The GFP signal was background-corrected and then normalized to the RFP fluorescence encoded by the TRIC construct itself (UAS-mCD8RFP), which serves as an internal reference for construct expression and mounting thickness.
    • Synapse Counting: We agree with the reviewer that the resolution of our images was insufficient for accurate synapse particle counting. We have therefore removed the problematic analysis from the former Fig 6H-J. Our conclusions regarding synaptic plasticity now rest on the more robust and quantifiable data showing a significant increase in the total area of dendritic (DenMark) and presynaptic (syt.eGFP) markers. Comment 6: The study presents data from unrelated learning paradigms (e.g., olfactory associative learning, courtship conditioning; Fig. 7) without justifying how these paradigms relate to SELTM. Particularly, the authors claimed that SELTM is related to Gr5a, which leads to appetitive memories, which involve PAM dopaminergic neurons and MB horizontal lobes. However, the olfactory associative learning with electric shock and courtship conditioning lead to aversive memories, that involve PPL1 dopaminergic neurons and the vertical lobes.

    Response: We thank the reviewer for requesting clarification on the rationale for including these experiments. The purpose of these assays was to test the specificity of the YL neuron circuit. A key question is whether YL neurons represent a general-purpose LTM circuit or one specialized for a particular memory modality.

    The data show that knockdown of Orb2 or Nmdar2 specifically in YL neurons has no effect on the formation of LTM for aversive olfactory conditioning or aversive courtship conditioning. These negative results are critically important, as they demonstrate that the YL circuit is

    not required for all forms of LTM. This finding strongly supports our revised central claim that YL neurons are specialized for processing appetitive memories derived from the specific sensory context of mating (i.e., taste and pheromonal cues from Gr5a neurons).

    To improve the narrative flow of the main text, We rearranged the order of the articles. The relevant description is in lines 398-401:

    “To determine whether YL neurons constitute a general LTM circuit or are dedicated to the appetitive context of mating, we tested two canonical aversive paradigms: electric-shock olfactory conditioning and courtship conditioning. If YL neurons serve as a universal LTM module, their genetic impairment should also impair aversive memory.”

    lines 469-472:

    “The inability of YL perturbation to impair aversive memories (Fig. 7) corroborates that this micro-circuit is dedicated to Gr5a-dependent SELTM rather than acting as a generic LTM hub”

    Minor Issues

    Comment 1: Fig 2F. YL projections are labeled as MBONs. Clarify whether YL neurons are the upstream or downstream (MBON) of KCs.

    __Response: __Thank you for this helpful comment. As Huang et al., 2018[2] (Nat. Commun. 9:872) have mentioned, the MB093C-GAL4 driver is the MBON-α3 mushroom body output neuro. Consequently, YL neurons are positioned downstream of the MBON-α3.

    We have now clarified this point in the revised manuscript lines 217-222:

    “Each of these neurons extends a vertical fiber to the dorsal brain region, where they form dense arbors within the α-lobes of the mushroom body. Because the MB093C-GAL4 driver labels MBON-α3 output neuron[51], these YL arbors are positioned postsynaptically within the α-lobe and relay mushroom-body output to the anterior, middle, and posterior superior-medial protocerebrum.”

    Comment 2: Extensive language polishing is required, as several sentences are unclear (e.g., lines 169-172).

    Response: We apologize for the lack of clarity in the original text. The entire manuscript has undergone extensive revision and professional language editing to improve readability, precision, and grammatical accuracy.

    Responses to Reviewer #2

    Major Comments

    Comment 1: Clearer articulation of the rationale, motivation, and significance of the overall study design and individual experiments can strengthen the manuscript and promote readership. For example, the beginnings of the abstract and introduction should define what authors mean by sexual experience-dependent long-term memory and its significance (including why it is "significant for reproductive success" (lines 46 and 92)). Similarly, employing more concrete language throughout the text will help anchor and contextualize the study. Interpretation is occasionally insufficient or does not follow directly from the data provided.

    Response: We thank the reviewer for this valuable advice. We agree that the motivation and significance of our study were not articulated clearly enough. We have rewritten the Abstract and the beginning of the Introduction to address this. The revised text now explicitly defines SELTM as a protein synthesis-dependent, appetitive memory formed in response to gustatory and pheromonal cues. We explain its significance in the context of adaptive behavior, linking it to interval timing, a process by which male flies strategically adjust their mating investment (i.e., mating duration) based on prior experience to optimize reproductive success and energy expenditure. This framing provides a clearer context for our investigation into its underlying neural and molecular mechanisms.

    Comment 2: Long term memory: I do not work on Drosophila memory, but a cursory search suggests that the field generally considers long term memory in Drosophila to last for 24 hr to days (courtship memory lasts for >24 hr). SMD decays between 12-24 hr after copulation. Could SMD be considered a short-term effect?

    Response: This is an important point of clarification, as described in our response to Reviewer #1 (Major Comment 2), we have performed a new experiment demonstrating that the formation of SMD is blocked by the protein synthesis inhibitor cycloheximide (Figure 1I). This dependence on de novo protein synthesis is a defining characteristic of LTM, distinguishing it from short- and intermediate-term memory forms.[1] where memories lasting 12-24 hours are well-established as forms of LTM.[3] Therefore, based on both its duration and its molecular requirements, SMD represents a bona fide form of LTM.

    The relevant statement is in lines 174-178:

    "To determine whether the persistent reduction in mating duration (SMD) depends on de-novo protein synthesis, we fed males the translational inhibitor cycloheximide (CXM). Under this regimen, CXM completely abolished the SMD phenotype (Fig. 1I). This finding suggests that the reduction in mating investment is contingent upon the formation of LTM."

    Comment 3: Fig 1B-E share the same control (naive) group. If these experiments were performed in the same replicate(s), they should be plotted in the same figure. If not, please provide more details on how experimental blocks were set up and how controls compared between replicates.

    Response: Thank you for this helpful suggestion. We understand that sharing the same naive control across multiple panels (Fig. 1B–E) may raise concerns about data independence. However, we chose to present these panels separately for the following reasons:

    1. Clarity and Readability: Each panel (B–E) represents a distinct temporal condition (0 h, 6 h, 12 h, 24 h post-isolation). Separating them avoids visual clutter and allows readers to focus on one time point at a time, improving interpretability.

    __ Consistency with Internal Controls:__

    Although the naive group is identical across panels, each experimental block (i.e., each isolation time point) was run independently on same days, with internal controls (naive vs. experienced) included in every block. This ensures that statistical comparisons remain valid within each panel, even if the naive data overlap.

    We have now added a clear statement in the figure legend explaining that the naive group is shared across panels and that each time point was tested independently with internal controls. This maintains transparency while preserving the visual clarity of the current layout.

    Comment 4: Serial mating (Fig 1F-H): please provide details on the methods. How much time elapsed between successive matings? Is a paired statistical test used? Sperm depletion also affects mating duration, and without this information the authors' conclusion (lines 155-156) does not automatically follow from the data.

    Response:

    1. __ Interval between successive matings__ We have rewritten the Methods to state explicitly that “as soon as one copulation ended the male was transferred immediately to a fresh virgin female, so the next mating began immediately.”

    we add new method:

    "__ Serial mating ____duration ____assay__

    Serial mating duration assay was identical to the standard procedure except that each male was presented with four DF virgin females in immediate succession: upon termination of the first copulation the male was immediately put into a fresh chamber containing the next virgin, the timer was restarted at first contact, and this step was repeated until four complete matings were recorded or 5 min elapsed without initiation, whichever came first."

    __ Statistical test__

    We apologize for omitting this detail. Unpaired t-test was used: for male the mating duration before (naïve) and after sexual experience was recorded, yielding paired observations. Prism’s unpaired t-test module was therefore applied to evaluate the mean difference.

    The figure legend now states “with error bars representing SEM. Asterisks represent significant differences, as revealed by the Unpaired t test and ns represents non-significant difference (**p __ Mating duration versus sperm depletion__

    We apologize for not having made it clear that these two observations are complementary, not contradictory. Previous work has shown that when male Drosophila copulate repeatedly, mating duration remains stable even though the number of sperm transferred—and thus the number of progeny sired—declines progressively [4]

    The revised text is as follows (lines235-241):

    "Previous work has shown that when male Drosophila copulate repeatedly, mating duration remains stable even though the number of sperm transferred—and thus the number of progeny sired—declines progressively. This dissociation confirms that the constant mating duration we observe in our serial-mating experiment (Fig. 1F–H) is consistent with normal sperm depletion and does not compromise the conclusion that the experience-dependent reduction in mating duration reflects long-term memory."

    Thank you for helping us improve the clarity of our study.

    Comment 5: Mating duration assay: Which isolation interval was chosen for the rest of the SMD experiments? The 12 hr en masse mating setup is relatively uncommon among studies on courtship/copulation/post-copulatory phenotypes, and introduces uncertainty and variability in the number and timing of matings that occurred during the 12 hr-window. This source of variability and its implication in interpreting the data should be acknowledged. Moreover, the 3 studies referenced in the methods all house males in groups of 4, whereas this study uses groups of 40. Could density confound the manifestation of SMD?

    Response: We thank the reviewer for these important methodological questions.

    • Isolation Interval: We have clarified in the Methods that virgin females were introduced into vials for last 1 day before assay.
    • Housing Density: This is an excellent point. To control for any potential effects of housing density itself, we have clarified that our "naive" control males are also housed in groups of 40 for the same duration as the "experienced" males. Therefore, the only difference between the two groups is the presence of females, isolating the effect of sexual experience from the effect of social density. Comment 6: SMD behavior: comparing orb2 mutants and controls (Fig 1M and Fig S1K-L), loss of orb2 actually reduces the mating duration in native males (mean ~15 min) relative to controls (~20 min), and have possibly no effect on experienced males (~15 min). This is inconsistent with the SMD behavior demonstrated in Fig 1B-E. The same pattern is found for mushroom body silencing (Fig 1P, Fig S1M-N), orb2 knockdown in YL neurons (Fig 2D, Fig S2A-B), Fmr1 knockdown in YL neurons (Fig 3D, Fig S2B, S3D) and most other experiments where mating duration is not significantly different between naive and experienced males. This might demonstrate a separate role of YL neurons and its related circuit in regulating mating duration in naive males. Could the authors discuss this interpretation? As an aside, plotting genetic controls next to experimental groups is customary and facilitates comparisons between relevant groups.

    Response: Thank you very much for this insightful observation.

    1. Baseline differences among genotypes We agree that absolute mating duration differs slightly between genotypes (e.g. naive orb2∆/+ about 15 min vs. wild-type CS about 20 min). Such differences are common when mutations or transgenes are introduced into distinct genetic backgrounds, and they do not affect the within-genotype comparison that is the essence of SMD (sexual-experience-dependent shortening of mating duration). Therefore, for every experiment we compared naive vs. experienced males of the identical genotype, keeping all other variables constant.

    Consistency of SMD across figures

    In every manipulation that disrupts SMD memory (orb2∆, MB silencing, orb2-RNAi in YL neurons, Fmr1-RNAi in YL neurons, etc.) the naive–experienced difference disappears, whereas the genetic controls retain a significant ΔMD. This is fully consistent with Fig. 1B–E and demonstrates that the memory trace, not the basal duration, is abolished.

    Figure layout

    Following your suggestion, we have re-ordered all bar graphs so that the relevant genetic controls are placed immediately adjacent to the experimental groups, making within-panel comparisons easier.

    We hope these clarifications and adjustments address your concerns.

    Comment 7: Bitmap figures: unfortunately the bitmap figures are compressed and their resolution makes it difficult to evaluate the visual evidence.

    Response: We apologize for the poor quality of the figures. All figures in the revised manuscript, including the scRNA-seq plots, have been remade as high-resolution vector graphics to ensure clarity and detail. For better understanding, different colored illustrations are also placed next to the scRNA-seq.

    Comment 8: Sexual dimorphism of YL neurons: many neurons involved in sexual behaviors express dsx and/or fru. Do YL neurons express them?

    Response: This is an excellent question. To address it, we performed a new set of experiments using genetic intersectional tools to test for the expression of doublesex (dsx) and fruitless (fru) in YL neurons. Our analysis, presented in figure supplement 2B, reveals that YL neurons are indeed fru-negative and dsx-negative. We therefore conclude that YL neurons do not belong to the canonical fru- or dsx-expressing neuronal classes and are unlikely to be intrinsically sex-specific.

    We add explanation in lines 223-229:

    "Our further analysis confirmed the presence of only three pairs of nuclei near the SOG in male brains, whereas female brains exhibit a greater number of nuclei near the AL (Fig. 2I), suggesting subtle sexual dimorphisms in GAL4MB093C-expressing neurons. Importantly, these neurons do not overlap with either fru- or dsx-expressing cells: co-immunostaining for GFP and Fru or Dsx revealed almost no colocalization in any brain region examined (Fig. S2B), indicating that YL neurons are distinct from the canonical sex-specific fru/dsx circuits."

    Comment 9: Genetic controls for some crucial experiments are not provided, e.g. Fig 2J, Fig S3C, Fig S3E-F Fig 5B-C, F, Q-R, Fig S5A-E.

    Response: We thank the reviewer for their careful attention to detail. We have now performed all the missing genetic control experiments.

    Comment 10: Colocalization experiments: please provide more detail on how fluorescence is normalized for each channel across images, especially when the overall expression of an effector is up- or down-regulated after mating.

    Response: We have updated the Methods section under "Quantitative Analysis of Fluorescence Intensity" and "Colocalization Analysis" to provide a detailed description of our normalization procedure.

    Comment 11: Please resolve this apparent contradiction on the expression of Nmdar1 and 2 in YL neurons. On line 261: "both receptors co-expressing in Orb2-positive MB Kenyon cells"; on line 279-281 "Nmdar1 is not expressed with YL neurons [...] whereas Nmdar2 is expressed in a single pair of YL neurons..."

    Response: We apologize for this contradiction, which arose from the confusing narrative structure of the original manuscript. As detailed in our response to Reviewer #1 (Major Comment 4), we have reframed the manuscript.

    Comment 12: Particle analysis (Fig 6H-J): experienced males seem to have more synapses but trend towards smaller average size. It would be helpful to show number of synapses and average size as paired data, or show that the total particle area is larger in experienced males.

    Response: We agree with the reviewer that this analysis was inconclusive and potentially misleading due to the limitations of image resolution. As noted in our response to Reviewer #1, we have removed this particle analysis (former Fig 6H-J) from the revised manuscript. Our claim for increased synaptic plasticity is now supported by the more robust measurement of the total fluorescence area of the pre- and postsynaptic markers, which shows a significant increase in experienced males.

    Minor Comments

    We thank the reviewer for their meticulous attention to detail. We have addressed all minor comments as follows:

    Comment 1: 1. Some figures (e.g. Fig 3M-R) and experiments (e.g. oenocyte scRNA-seq) are not referenced in the text. dnc data is shown alongside amn and rut but the rationale for its inclusion is not provided.

    __Response: __Original Fig. 3M-R (now Fig,3 M-O) was referenced on line 283. The rationale for including dnc data (as a canonical memory mutant) is now clarified in the text on lines 187-189:

    "To ask whether the same molecular machinery underlies the SMD that follows sexual experience, we tested three classical memory mutants: dunce (dnc), amnesiac (amn), and rutabaga(rut)."

    Comment 2: Some references might not point to the intended article (e.g. ref 123).

    __Response: __The reference list has been checked and corrected.

    Comment 3. Please plot genetic controls next to experimental genotypes as they are a crucial part of the experiment.

    __Response: __All relevant figures now include plots of genetic controls next to experimental genotypes.

    Comment 4. The "estimation statistics" plots are not necessary since the authors show individual data points. To further enhance data transparency, the authors may consider reducing the alpha and/or dot size so the individual data points are more readily visible.

    Response: Thank you for this helpful suggestion! We fully agree that data transparency is essential. After carefully testing lower alpha values and smaller dot sizes, we found that either change markedly obscured the dense regions of the distributions. So we didn't change the size of the point.

    The estimation-statistics overlays are kept for two courteous reasons: (i) they provide an immediate visual estimate of the mean difference and its 95 % confidence interval, which is the key statistic we base our conclusions on, and (ii) they spare readers from having to cross-reference separate tables.

    Comment 5. For accessibility, please avoid using green and red in the same plot.

    __Response: __We fully agree that red–green combinations can be problematic for colour-vision-impaired readers. In the present manuscript, however, the only panel that juxtaposes pure red and pure green is the Fly-SCOPE co-expression data. These scRNA-seq plots are provided only as supportive reference; the actual quantitative conclusions are based on independent genetic and imaging experiments that use magenta, cyan, yellow, and greyscale palettes. Moreover, the scope images are accompanied by detailed text descriptions of the overlapping cell clusters, so no essential information is lost even if the colours are indistinguishable

    Comment 6. Fly Cell Atlas: please show color scales used for each gene as the color thresholds are gene-specific by default.The 3-color overlap on SCope also makes it very difficult to see the expression pattern for each gene. One possibility is outlining the Kenyon cells on the tSNE plots and showing the expression for each gene of interest.

    Response: Thank you for this helpful suggestion. To avoid the ambiguity that arises from RGB blending in the three-colour overlay, we have added a small colour-mixing diagram next to the t-SNE plots (revised Fig. 1). This key shows the exact hues produced by pairwise and three-way overlaps:

    • Red + Green = Yellow

    • Red + Blue = Magenta

    • Green + Blue = Cyan

    • Red + Green + Blue = White

    Thus, yellow, magenta or cyan dots indicate co-expression of two genes, while white dots mark cells where all three genes are detected. this diagram allows readers to interpret overlap colours at a glance without re-entering SCope.

    Comment 7. Please also refer to Fly Cell Atlas as such. SCope is a visualization platform that houses multiple datasets.

    __Response: __The reference to Fly Cell Atlas was added.

    Comment 8. Please introduce acronyms and genetic reagents the first time they are mentioned.

    __Response: __All acronyms and genetic reagents are now defined upon their first use.

    Comment 9. Line 184: please specify "split-GAL4 reagents" instead of "advanced genetic tools".

    __Response: __We have replaced "advanced genetic tools" with the more specific term "Split-GAL4 reagents."

    Comment 10. Line 187: there are a few other lines with p>0.05 or p>0.01, so "uniquely" is inaccurate. Are the p-values in Table 1 corrected for multiple testing?

    __Response: __The term "uniquely" has been revised for accuracy. No correction for multiple testing was applied because each entry in Table 1 represents a single pairwise comparison (naive vs. exp). Thus only one p-value was generated per experiment.

    Comment 11. Some immunofluorescence panels lack scale bars.

    __Response: __Scale bars have been added to all immunofluorescence panels.

    Comment 12. Fig S2G-I: do authors mean "naive" instead of "group"?

    __Response: __The term "group" in Fig S2G-I has been corrected to "naive."

    Comment 13. Movie 1 should be referenced when YL neurons are first introduced.

    __Response: __Movie 1 is now referenced when YL neurons are first introduced in the text.

    Comment 14. Is Fig 4L similar to Fig 6L-N?

    __Response: __This error has been corrected after the article was reformatted

    Comment 15. Fig 7: please plot olfactory conditioning experiment results as either percentages, preference index, or paired numbers. "Number of flies/tube" is not as informative.

    __Response: __Thank you for pointing this out. The bars in Fig. 7 indeed represent paired numbers, but we realise this was not stated explicitly. We apologize for the lack of clarity. In the revised manuscript we explained it in detail in figure legend and method. In the figure, we also marked the percentage of flies that chose to avoid the side of the stimulus with gas, and explained it in the Figure legend.

    Reference

    1. Lagasse F, Devaud J-M, Mery F. A Switch from Cycloheximide-Resistant Consolidated Memory to Cycloheximide-Sensitive Reconsolidation and Extinction in Drosophila. J Neurosci. 2009;29: 2225–2230. doi:10.1523/jneurosci.3789-08.2009
    2. Huang C, Maxey JR, Sinha S, Savall J, Gong Y, Schnitzer MJ. Long-term optical brain imaging in live adult fruit flies. Nat Commun. 2018;9: 872. doi:10.1038/s41467-018-02873-1
    3. Tonoki A, Davis RL. Aging Impairs Protein-Synthesis-Dependent Long-Term Memory in Drosophila. J Neurosci. 2015;35: 1173–1180. doi:10.1523/jneurosci.0978-14.2015
    4. Macartney EL, Zeender V, Meena A, Nardo AND, Bonduriansky R, Lüpold S. Sperm depletion in relation to developmental nutrition and genotype in Drosophila melanogaster. Evol Int J Org Evol. 2021;75: 2830–2841. doi:10.1111/evo.14373
  2. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Sun et al. show that Orb2-expressing, glutamatergic mushroom body neurons (YL neurons) are central to the "shorter mating duration (SMD)" behavior, where males reduce their mating duration up to 12 hours after the initial copulation. The authors use SMD as a model for understanding sexual experience-dependent long-term memory in males. A few genes implicated in long-term memory (Fmr1, CrebB) are required in YL neurons for SMD. The Nmdar-CaMKII signaling pathways is also implicated, and mating attenuates Ca2+ signaling and increases synaptic plasticity in the mushroom body and subesophageal ganglion.

    Major comments:

    1. Clearer articulation of the rationale, motivation, and significance of the overall study design and individual experiments can strengthen the manuscript and promote readership. For example, the beginnings of the abstract and introduction should define what authors mean by sexual experience-dependent long-term memory and its significance (including why it is "significant for reproductive success" (lines 46 and 92)). Similarly, employing more concrete language throughout the text will help anchor and contextualize the study. Interpretation is occasionally insufficient or does not follow directly from the data provided.
    2. Long term memory: I do not work on Drosophila memory, but a cursory search suggests that the field generally considers long term memory in Drosophila to last for 24 hr to days (courtship memory lasts for >24 hr). SMD decays between 12-24 hr after copulation. Could SMD be considered a short-term effect?
    3. Fig 1B-E share the same control (naive) group. If these experiments were performed in the same replicate(s), they should be plotted in the same figure. If not, please provide more details on how experimental blocks were set up and how controls compared between replicates.
    4. Serial mating (Fig 1F-H): please provide details on the methods. How much time elapsed between successive matings? Is a paired statistical test used? Sperm depletion also affects mating duration, and without this information the authors' conclusion (lines 155-156) does not automatically follow from the data.
    5. Mating duration assay: Which isolation interval was chosen for the rest of the SMD experiments? The 12 hr en masse mating setup is relatively uncommon among studies on courtship/copulation/post-copulatory phenotypes, and introduces uncertainty and variability in the number and timing of matings that occurred during the 12 hr-window. This source of variability and its implication in interpreting the data should be acknowledged. Moreover, the 3 studies referenced in the methods all house males in groups of 4, whereas this study uses groups of 40. Could density confound the manifestation of SMD?
    6. SMD behavior: comparing orb2 mutants and controls (Fig 1M and Fig S1K-L), loss of orb2 actually reduces the mating duration in native males (mean ~15 min) relative to controls (~20 min), and have possibly no effect on experienced males (~15 min). This is inconsistent with the SMD behavior demonstrated in Fig 1B-E. The same pattern is found for mushroom body silencing (Fig 1P, Fig S1M-N), orb2 knockdown in YL neurons (Fig 2D, Fig S2A-B), Fmr1 knockdown in YL neurons (Fig 3D, Fig S2B, S3D) and most other experiments where mating duration is not significantly different between naive and experienced males. This might demonstrate a separate role of YL neurons and its related circuit in regulating mating duration in naive males. Could the authors discuss this interpretation? As an aside, plotting genetic controls next to experimental groups is customary and facilitates comparisons between relevant groups.
    7. Bitmap figures: unfortunately the bitmap figures are compressed and their resolution makes it difficult to evaluate the visual evidence.
    8. Sexual dimorphism of YL neurons: many neurons involved in sexual behaviors express dsx and/or fru. Do YL neurons express them? If they do, they might be a subset of characterized and named dsx/fru neurons.
    9. Genetic controls for some crucial experiments are not provided, e.g. Fig 2J, Fig S3C, Fig S3E-F Fig 5B-C, F, Q-R, Fig S5A-E.
    10. Colocalization experiments: please provide more detail on how fluorescence is normalized for each channel across images, especially when the overall expression of an effector is up- or down-regulated after mating.
    11. Please resolve this apparent contradiction on the expression of Nmdar1 and 2 in YL neurons. On line 261: "both receptors co-expressing in Orb2-positive MB Kenyon cells"; on line 279-281 "Nmdar1 is not expressed with YL neurons [...] whereas Nmdar2 is expressed in a single pair of YL neurons in both male and female brains".
    12. Particle analysis (Fig 6H-J): experienced males seem to have more synapses but trend towards smaller average size. It would be helpful to show number of synapses and average size as paired data, or show that the total particle area is larger in experienced males.

    Minor comments:

    1. Some figures (e.g. Fig 3M-R) and experiments (e.g. oenocyte scRNA-seq) are not referenced in the text. dnc data is shown alongside amn and rut but the rationale for its inclusion is not provided.
    2. Some references might not point to the intended article (e.g. ref 123).
    3. Please plot genetic controls next to experimental genotypes as they are a crucial part of the experiment.
    4. The "estimation statistics" plots are not necessary since the authors show individual data points. To further enhance data transparency, the authors may consider reducing the alpha and/or dot size so the individual data points are more readily visible.
    5. For accessibility, please avoid using green and red in the same plot.
    6. Fly Cell Atlas: please show color scales used for each gene as the color thresholds are gene-specific by default.The 3-color overlap on SCope also makes it very difficult to see the expression pattern for each gene. One possibility is outlining the Kenyon cells on the tSNE plots and showing the expression for each gene of interest.
    7. Please also refer to Fly Cell Atlas as such. SCope is a visualization platform that houses multiple datasets.
    8. Please introduce acronyms and genetic reagents the first time they are mentioned.
    9. Line 184: please specify "split-GAL4 reagents" instead of "advanced genetic tools".
    10. Line 187: there are a few other lines with p>0.05 or p>0.01, so "uniquely" is inaccurate. Are the p-values in Table 1 corrected for multiple testing?
    11. Some immunofluorescence panels lack scale bars.
    12. Fig S2G-I: do authors mean "naive" instead of "group"?
    13. Movie 1 should be referenced when YL neurons are first introduced.
    14. Is Fig 4L similar to Fig 6L-N?
    15. Fig 7: please plot olfactory conditioning experiment results as either percentages, preference index, or paired numbers. "Number of flies/tube" is not as informative.

    Significance

    The manuscript describes an extensive and comprehensive set of experiments aimed at elucidating the role of a subset of mushroom body neurons in mediating a male post-mating sexual behavior, which the authors use as a model for sexual experience-dependent long-term memory. Long-term post-mating responses in females have been well characterized in Drosophila and other insects, but post-mating long term memory in males are less well understood despite a few studies reporting their importance in mating success. How males adjust their mating duration based on internal and external cues can reveal insights about decision making and interval timer mechanisms. This study represents a functional advancement in the neuronal and molecular mechanisms of how memory and experience regulates a sexual behavior in male Drosophila. Overall, the manuscript can significantly benefit from general editing on clearer articulation of rationale and more appropriate interpretations of data. Higher resolution versions of bitmap figures is also crucial. The SMD experiments invite an alternative interpretation of data that centers on YL neurons' role on regulating mating duration in naive males, which alongside other roles of the mushroom body demonstrated in this manuscript, could add more depth to the study.

    The findings in this manuscript will be of interest to a specialized audience interested in memory, neural circuits of behavior, and Drosophila sexual behavior. I work on Drosophila sexual behavior and circuits, but lacking experience on memory research, I am not as familiar with the mushroom body and conditioning experiments.

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    Referee #1

    Evidence, reproducibility and clarity

    This study explores the molecular and neural circuitry mechanisms underlying sexual experience-dependent long-term memory (SELTM) in male Drosophila. The authors use behavioral, imaging, and bioinformatics approaches to identify YL neurons, a subset of mushroom body (MB) projecting neurons, as crucial for SELTM formation. They propose that YL neurons receive inputs from WG neurons via the sNPF-sNPFR pathway and implicate molecular players such as orb2, fmr1, MDAR2-CaMK, and synaptic plasticity in their function.

    However, the evidence presented does not adequately support the authors' claims. The data fail to cohesively tell a logical story, and key conclusions appear to be based on assumptions and correlations rather than robust evidence.

    Major comments:

    1. The study identifies the knowledge gap (lines 103-104) but fails to integrate relevant literature, particularly Shohat-Ophir et al., Science (2012), and Zer-Krispil et al., Curr Biol (2018). These studies established that ejaculation induces appetitive memory in male Drosophila via corazonin and NPF neurons. The current study does not provide direct evidence that the "act of mating itself" drives SELTM, as it includes both courtship and copulation.
    2. The nature of the observed long-lasting reduced mating duration requires clearer characterization: Is this an associative memory or experience-dependent behavioral plasticity? Can the formation of this long-term memory be blocked by protein synthesis inhibitors, such as cycloheximide?
    3. While schematics illustrate the working hypotheses, the text lacks detailed explanations, leaving the reader unclear about the rationale behind certain conclusions.
    4. The logic to draw certain conclusions was confusing and misleading.
      • For instance, the role of orb2 in SELTM is examined via knockdown in MB Kenyon cells (KCs) (using ok107>orb2-RNAi), which is irrelevant to the claim that orb2 functions in YL neurons. Additionally, RNAseq analyses (Fig. 1N-S) focusing on orb2 expression in a/b KCs are irrelevant to and cannot support the claim that Orb2 functions in YL neurons.
      • Similarly, the claim (lines 302-303) that sNPF-R expression is exclusive to MB KCs conflicts with data showing effects when sNPF-R is knocked down in YL neurons. How can knocking-down a gene, which is exclusively expressed in neural population A, in neural population B affect a phenotype? This inconsistency undermines the interpretation of the results.
      • Other examples include lines 223-227 and lines 246-249. It is very confusing how the authors came to the indications.
      • The authors also kept confusing the readers and themselves by mistakenly referring to MB KC a-lobe and YL a-lobe projection. They may know the difference between the two neural populations but they did not always refer to the right one in the text.
    5. The imaging figures provided are unfocused and poorly resolved, making it difficult to assess data quality.
      • Colocalization analyses of orb2 and YL are unconvincing, especially given that orb2 is well-documented in literature as expressed in MB a-KCs and YL projection wrapping MB a-lobe. Maximum intensity projection images are insufficient for confirming colocalization; complete image stacks with staining of orb2, YL, and KCs (MB-dsRed) are needed for validation.
      • Quantification of imaging data appears flawed. For example, claims of orb2 and CaMKII upregulation in MB a-lobe projections (e.g., Fig. S2F-J, Fig. 3M,N) are confounded by widespread increases in intensity across the brain, lacking specificity.
      • The TRIC experiment analysis should normalize GFP signals to internal reference channel (RFP in the TRIC construct), as per established protocols in the original paper.
      • In Fig. 6H-J, methods for counting synapse numbers are not described. How are synapse numbers counted in these low-resolution images?
    6. The study presents data from unrelated learning paradigms (e.g., olfactory associative learning, courtship conditioning; Fig. 7) without justifying how these paradigms relate to SELTM. Particularly, the authors claimed that SELTM is related to Gr5a, which leads to appetitive memories, which involve PAM dopaminergic neurons and MB horizontal lobes. However, the olfactory associative learning with electric shock and courtship conditioning lead to aversive memories, that involve PPL1 dopaminergic neurons and the vertical lobes.
    7. Some figures are not referred to in the text. For example, Fig S1 K and L (also, what's the difference between these two figures?) and Fig 3M-R. What is MB-V3 in Fig 4J-K?

    Minor issues

    1. Fig 2F. YL projections are labeled as MBONs. Clarify whether YL neurons are the upstream or downstream (MBON) of KCs.
    2. Extensive language polishing is required, as several sentences are unclear (e.g., lines 169-172).

    Significance

    This study potentially advances our understanding of how sexual experience modifies future mating behaviors. While previous work has shown that mating induces appetitive memory in males, the mechanisms linking this memory to future mating behavior remain poorly understood. This work could provide valuable insights into these mechanisms, pending appropriate revisions.

  4. Version published to 10.1101/2024.09.28.615582 on bioRxiv