Hyperexcitation of Monoaminergic Neurons in the Drosophila Mushroom Body Disrupts Memory for Visually Oriented Rival-induced Prolonged Mating

Hyperexcitation of Monoaminergic Neurons in the Drosophila Mushroom Body Disrupts Memory for Visually Oriented Rival-induced Prolonged Mating | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hyperexcitation of Monoaminergic Neurons in the Drosophila Mushroom Body Disrupts Memory for Visually Oriented Rival-induced Prolonged Mating Xinyue Zhou, Dongyu Sun, Yutong Song, Tianmu Zhang, Woo Jae Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4359931/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Male individuals frequently require a prolongation of their mating duration in order to outcompete their rivals for few reproductive chances. This study looks into the roles of monoaminergic neurons in the Drosophila melanogaster mushroom body (MB) as major regulators of males' rival-induced prolonged mating duration (LMD) behavior. Activation screening experiments revealed that hyperexcitation of monoaminergic neurons in the MB, including serotonergic neurons and dopaminergic neurons, disrupts LMD without affecting copulation latency. The co-expression of MB-specific GAL80 ( GAL80 MB247 ) with the monoaminergic GAL4 drivers rescues LMD, confirming the involvement of monoaminergic neurons in the MB. The hyperexcitation of inhibitory GABAergic neurons disrupts mating, but this effect is alleviated by GAL80 MB247 inhibitors, suggesting that critical GABAergic neurons for LMD reside within the MB. In summary, the activation of monoaminergic neurons in the MB disrupts LMD memory, while the hyperactivation of inhibitory GABAergic neurons in the MB impairs mating success. These findings implicate the MB as a crucial neural circuit for integrating visual and social cues to generate memory for LMD behavior. Drosophila melanogaster Mating duration Interval timing Longer-Mating-Duration LMD Mushroom body GABAergic neurons Monoaminergic neurons Memory Hyperexcitation Figures Figure 1 Figure 2 INTRODUCTION The Drosophila melanogaster neurotransmitter (NT) system is a complex network of neurons and synapses that enable communication between cells in the fly's nervous system. It utilizes various neurotransmitters (NTs), including dopamine (DA), serotonin (5-HT), acetylcholine (ACh), and glutamate (Glu), to transmit signals across neurons. The release and reception of neurotransmitters occur at specialized structures called synapses, which allow for the rapid and efficient transmission of signals. This system plays a crucial role in regulating fly behaviors, such as movement, learning, memory, and courtship. By studying the fly neurotransmitter system, researchers gain valuable insights into the fundamental principles of neural communication and behavior (Kahsai and Winther 2011 ; Deng et al. 2019 ). The neurotransmitter system in fruit flies is intricately linked to the long-term memory (LTM) circuit. Neurotransmitters such as DA, 5-HT, Glu, and Ach are pivotal in the modulation of synaptic plasticity, which underlies the formation and storage of long-term memories (Heisenberg 2003 ; Androschuk et al. 2015 ). Dopaminergic signaling pathways, for example, are known to be involved in the induction of long-term potentiation, a cellular mechanism of memory formation (Kaun and Rothenfluh 2017 ; Sabandal et al. 2021 ). Additionally, the neuromodulator octopamine (OA) has been shown to enhance LTM by potentiating synaptic transmission (Sitaraman et al. 2010 ). The serotonergic system in Drosophila is also a key regulator of LTM, with 5-HT acting as a neurotransmitter and neuromodulator. 5-HT-producing neurons are located in the protocerebral bridge (PB) and the ventral nerve cord (VNC), and they project their axons to various regions of the fly's brain, including the mushroom body (MB), the central complex (CC), and the ventral lateral horn (VLH) (Sitaraman et al. 2008 , 2010 ; Johnson et al. 2011 ; Lee et al. 2011 , 2021 ; Huser et al. 2012 ). The integration of these neurotransmitters within specific neural circuits, such as the MB in the fruit fly brain, facilitates the encoding and retrieval of LTM. Therefore, the precise regulation of neurotransmitter release and reception within these circuits is crucial for the establishment and maintenance of long-term memory in fruit flies. The Drosophila mushroom body, also known as the MB, is a key structure in the fruit fly brain that plays a critical role in learning, memory, and decision-making (Heisenberg 2003 ). Comprising of a pair of large, mushroom-shaped structures located dorsally in the fly's brain, the MB receives input from olfactory sensory neurons, as well as other sensory modalities, and integrates this information to guide appropriate behaviors (Davis 2001 ; Kahsai and Zars 2011 ; Séjourné et al. 2011 ). The MB is composed of Kenyon cells, which serve as the principal neurons, and these cells are organized into three distinct lobes: the α, β, and γ lobes. Each lobe receives input from different sensory systems and is involved in distinct aspects of learning and memory (Farris 2005 ). The Longer-Mating-Duration (LMD) in male Drosophila is characterized by an extended duration of mating that is triggered in response to social cues, particularly the presence of rival males (Kim et al. 2012a , a ). LMD behavior is an adaptive response that allows males to increase their reproductive success in competitive environments (Bretman et al. 2009 ). LMD relies on the integration of visual and social cues into memory circuits for its generation (Bretman et al. 2011a ; Kim et al. 2012a ). The memory circuit for LMD in Drosophila has been highly investigated in a neural network that connects the ellipsoid body (EB) to its visual circuit pathway (Kim et al. 2012a , a ). The EB is a region in the fly's brain that plays a key role in visual processing and memory formation. It receives input from various sensory neurons, including those involved in visual perception, and integrates this information with other sensory cues (Neuser et al. 2008 ; Wang et al. 2008 ; Pan et al. 2009 ; Ofstad et al. 2011 ; Kahsai and Zars 2013 ). Even though the MB has been traditionally considered dispensable for visual learning (Wolf et al. 1998 ), recent studies indicate that it plays a pivotal role in visual memory and other cognitive processes. The MB, which consists of three lobes—α, β, and γ—receives input from sensory neurons, including those involved in visual processing. This input enables the MB to integrate visual information with other sensory cues, forming the basis for visual memory (Li et al. 2020 ; Ganguly et al. 2023 ). The connection between the MB and the EB allows for the integration of visual information with other sensory cues, enabling the fly to form and recall visual memories (Solanki et al. 2015 ; Cheong et al. 2020 ). This collaboration between the EB and MB is crucial for visual memory processing in Drosophila (Barth and Heisenberg 1997 ; Kim et al. 2012b ). RESULTS In the previous study investigating the regulation of LMD in male fruit fly, we have identified a crucial role for the memory circuit in mediating this behavior. Utilizing the potassium channel KCNJ2 to inhibit the function of the memory circuit, particularly in cells expressing EB, MB, and FB, we found that R2/R4m region of EB circuits are crucial to be activated to generate LMD memory (Kim et al. 2012a ). Previous studies have determined that a pair of inhibitory neurons is essential for the maintenance of labile memory within the MB (Pitman et al. 2011 ). Moreover, individual Kenyon cells within the MB display a heightened level of self-inhibition through the anterior paired lateral (APL) pathway relative to their inhibition of other Kenyon cells (Amin et al. 2020 ). Given the pivotal role of the MB in visual memory processing and its integration of visual cues with other sensory information, it is justified to test the function of the MB, EB, and other brain regions for LMD memory by activating the MB using transgenes such as NaChBac . NaChBac is a bacterial sodium channel that has been engineered to function in both mammalian and insect nervous systems (Charalambous and Wallace 2011 ). It is commonly used in neuroscience research to study the effects of altering sodium channel activity on neural function. By expressing NaChBac in specific neurons, researchers can manipulate the excitability of those neurons and observe the effects on behavior, physiology, or other neural processes (Nitabach et al. 2006 ). Prior studies have indicated that inhibiting a subset of sexually dimorphic and GABAergic abdominal ganglion (AG) neurons can significantly extend mating duration in male Drosophila (Tayler et al. 2012 ; Crickmore and Vosshall 2013 ). To assess whether the activation of these neurons would elicit a similar effect, we utilized the GAL4 NP5270 (GABAergic AG neurons) driver to express NaChBac. Our findings revealed that the hyperexcitation of these neurons did not alter LMD behavior (Fig. 1A) or the copulation latency (CL) between group-housed and single-housed males (Fig. 1B). These data indicate that the hyperactivation of GABAergic AG neurons does not impact the extension of mating duration or the generation of LMD behavior. The regulation of mating duration has been postulated to involve a delicate interplay between dopaminergic and GABAergic signaling (Crickmore and Vosshall 2013 ). To elucidate the impact of neuronal hyperexcitation on mating duration, we employed the Ddc-GAL4 (serotonergic and dopaminergic neurons)driver, which labels both serotonergic and dopaminergic neurons. Hyperexcitation of Ddc-GAL4 -labeled neurons disrupted LMD without affecting CL (Fig. 1C-D). Furthermore, the activation with 5-HT1B-GAL4 (serotonergic neurons) (Fig. 1E-F) or with TH-GAL4 (dopaminergic neurons) (Fig. 1G-H) also disrupted LMD without altering CL. Contrary to this, hyperexcitation of the OA did not affect LMD or CL (Fig. 1I-J). Remarkably, males with hyperexcited GABAergic neurons failed to mate despite displaying courtship behavior (Fig. 1K-L), indicating that the hyperactivation of inhibitory GABAergic neurons can disrupt mating behaviors. However, this effect does not appear to be mediated by the sexually dimorphic GABAergic neurons within the AG, as their hyperexcitation did not impede mating success (Fig. 1A-B). Utilizing the fly SCope platform, which houses a recently acquired single-cell RNA sequencing dataset, we identified that the expression of Ddc, Tdc2 , TH , 5-HT1B , and Gad1 overlaps in the mushroom body g-Kenyon cells population (Fig. 1M-P) (Li et al. 2022a ), suggesting their potential involvement in LMD regulation (Crickmore and Vosshall 2013 ; Li et al. 2022). We employed GAL4 ok107 and GAL4 MB247 to hyperactivate MB neurons, which disrupted LMD behavior (Fig. 1Q-R). Conversely, the inactivation of MB neurons using the inward rectifier potassium channel, KCNJ2, did not alter LMD (Kim et al. 2012a ). Furthermore, the hyperactivation of EB neurons using GAL4 c547 or FB neurons using GAL4 14–94 or GAL4 104y also disrupted LMD (Fig. 1S and Fig. S1A-B), suggesting that the balance of neuronal activity in EB and FB neurons is necessary for the generation of LMD memory. Single-cell RNA sequencing (SCope) data revealed that dopaminergic neurons are highly co-expressed with MB g-Kenyon cells as well as Dh31-positive EB neurons (Fig. 1T) or AstA-R1-positive FB neurons (Fig. S1C). In contrast, the hyperactivation of dilp2-positive pars intercerebralis (PI) neurons or ap-positive interneurons in the VNC did not affect LMD behavior, despite their high co-expression with dopaminergic neurons (Fig. S1D-H). These data collectively indicate that monoaminergic neurons within the MB are the primary target neurons that disrupt LMD when hyperactivated. To corroborate that monoaminergic neurons within the MB are the primary targets for disrupting LMD behavior when hyperexcited, we utilized MB-specific GAL80 ( GAL80 MB247 ) to inhibit GAL4 activity in MB neurons (Krashes et al. 2007 ). Remarkably, the co-expression of GAL80 MB247 with TH- , Ddc- , or Tdc2-GAL4 (aminergic neurons) drivers, which induce NaChBac-mediated LMD disruption (Fig. 2A-F). Trh-GAL4 , known to selectively target serotonergic neurons (Alekseyenko et al. 2010 ), was found to be not universally expressed in MB neurons, unlike 5-HT1B neurons (Fig. S1I and Fig. 1M). Consequently, the hyperactivation of Trh-positive serotonergic neurons or Trh-positive non-MB neurons did not affect LMD behavior (Fig. S1J and Fig. 2G), indicating that Trh-positive 5-HT neurons are not integral to MB-related LMD memory processing. However, the hyperactivation of Trh neurons disrupted CL in single-housed flies (Fig. 2H), suggesting that Trh-positive serotonergic neurons control CL rather than LMD. Co-expressing Trh-GAL80 with 5-HT1B-GAL4 did not alter the effect of hyperactivation (Fig. 2I-J), whereas the combination of GAL80 MB247 abolished the LMD disruption mediated by hyperactivation (Fig. 2K-L). Single-cell RNA sequencing (SCope) data indicates that the majority of 5-HT1B neurons located in MB overlap with ab-, a’b’-, and g-Kenyon cells (Fig. 2M-P). These findings collectively suggest that monoaminergic neurons within MB Kenyon cells are crucial for the processing of LMD memory. The impairment in mating phenotype observed upon the hyperactivation of Gad1-GAL4 (GABAergic neurons) was alleviated when co-expressed with GAL80 MB247 (Fig. 2Q-R), indicating that the critical GABAergic neurons responsible for mating success are resident within MB neurons (Fig. 2S-T). DISCUSSION This study identified the neural circuits and monoaminergic neurons in the MB as key regulators of the LMD behavior in male Drosophila . Activation screening experiments reveal the disruption of LMD by hyperactivating monoaminergic neurons in the MB, including serotonergic neurons (Fig. 1E-F) and dopaminergic neurons (Fig. 1G-H), without affecting copulation latency. The co-expression of GAL80 MB247 with the disrupting GAL4 drivers rescues LMD (Fig.2C-D, G-H and K-L), confirming the involvement of monoaminergic neurons in the MB. The hyperactivation of inhibitory GABAergic neurons disrupts mating (Fig. 1K-L), but this effect is alleviated by MB-specific GAL80 inhibitors (Fig. 2Q-R), suggesting that critical inhibitory neurons reside within the MB. In summary, the activation of monoaminergic neurons in the MB disrupts LMD memory, while the hyperactivation of inhibitory GABAergic neurons in the MB impairs mating success. These findings implicate the MB as a crucial neural circuit for integrating visual and social cues to regulate mating duration in male Drosophila . This study identified the monoamines, including DA, 5-HT, and OA play crucial roles in memory formation and regulation in Drosophila (Kemenes et al. 2011) . Dopaminergic signaling pathways are involved in the induction of long-term potentiation, a cellular mechanism of memory formation. DA release in the MB facilitates the encoding and retrieval of long-term memories (Yamagata et al. 2015). The neuromodulator OA enhances LTM by potentiating synaptic transmission. Octopaminergic neurons also project to the MB and other brain regions involved in memory. DA and OA exert differential modulatory effects on memory processing across distinct neural circuits (Schwaerzel et al. 2003). Our findings suggest that the OA system is not essential for the generation of LMD memory in male Drosophila (Fig.1I-J) . The serotonergic system in Drosophila exhibits crucial functions in LTM processing. Serotonin-producing neurons in the protocerebral bridge (PB) and VNC project to multiple brain regions, including the MB. 5-HT serves as both a neurotransmitter and neuromodulator, potentiating synaptic transmission and facilitating the expression of genes involved in LTM formation (Sitaraman et al. 2008, 2012). Notably, our data and previous studies (Johnson et al. 2011; Lee et al. 2021) indicate that 5-HT actions via its receptors, such as 5-HT1B (Fig. 1E), are essential for LTM. Indeed, all 5-HT receptors exhibit robust expression in MB neurons (Fig. S1K-O). Serotonin has been demonstrated to enhance LTM in Drosophila through a range of mechanisms, including its impact on synaptic plasticity and gene expression regulation. Specifically, serotonin has been shown to potentiate synaptic transmission and promote the expression of genes involved in LTM formation within MB neurons (Crocker et al. 2016; Wu et al. 2017). In summary, monoamines released in the Drosophila brain act as neurotransmitters and neuromodulators, potentiating synaptic plasticity and promoting gene expression, thereby regulating the formation, consolidation, and retrieval of LTM for LMD. The precise integration of these monoamines within specific neural circuits, such as the MB g-Kenyon cells, enables the encoding and retrieval of LTM in Drosophila . Our findings align with previous reports suggesting that inhibitory circuits within MB play a pivotal role in the generation of LTM (Aranda et al. 2017; Awata et al. 2019; Feng et al. 2021) (Fig. 2U). METHODS EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Fruit fly rearing Drosophila melanogaster were cultured under standard laboratory conditions at 25℃. Samples were prepared as described in the Methods Details. All fly strains are listed in the key resources table. Mating duration assay The mating duration assay in this study has been reported(Kim et al. 2012, 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 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. At the fifth day after eclosion, males of the appropriate strain and DF virgin females were mildly anaesthetized by CO 2 . After placing a single female in to the mating chamber, we inserted a transparent film then placed a single male to the other side of the film in each chamber. After allowing for 1 h of recovery in the mating chamber in 25℃ incubator, 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. All assays were performed from noon to 4pm. We conducted blinded studies for every test. Single-nucleus RNA-sequencing analyses snRNAseq dataset analyzed in this paper is published(Li et al. 2022b) and available at the Nextflow pipelines (VSN, https://github.com/vib-singlecell-nf), the availability of raw and processed datasets for users to explore, and the development of a crowd-annotation platform with voting, comments, and references through SCope (https://flycellatlas.org/scope), linked to an online analysis platform in ASAP ( https://asap.epfl.ch/fca ).Single-cell RNA sequencing (scRNA-seq) data from the Drosophila melanogaster were obtained from the Fly Cell Atlas website (https://doi.org/10.1126/science.abk2432). Statistical Tests Statistical analysis of mating duration assay was described previously(Kim et al. 2012, 2013; Lee et al. 2023). More than 50 males (group and single) were used for mating duration assay. Our experience suggests that the relative mating duration differences between group and single 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(Bretman et al. 2011b). Therefore, statistical comparisons were made between groups that were grouply reared and singly reared within each experiment. As mating duration of males showed normal distribution (Kolmogorov-Smirnov tests, p > 0.05), we used two-sided Student’s t tests. The mean ± standard error (s.e.m) ( **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05 ). All analysis was done in GraphPad (Prism). Individual tests and significance are detailed in figure legends. Besides traditional t -test for statistical analysis, we added estimation statistics for all MD assays and two group comparing graphs. In short, ‘estimation statistics’ is a simple framework that—while avoiding the pitfalls of significance testing—uses familiar statistical concepts: means, mean differences, and error bars. More importantly, it focuses on the effect size of one’s experiment/intervention, as opposed to significance testing(Claridge-Chang and Assam 2016). In comparison to typical NHST plots, estimation graphics have the following five significant advantages such as (1) avoid false dichotomy, (2) display all observed values, (3) visualize estimate precision, (4) show mean difference distribution. And most importantly (5) by focusing attention on an effect size, the difference diagram encourages quantitative reasoning about the system under study(Ho et al. 2019). Thus, we conducted a reanalysis of all of our two group data sets using both standard t -tests and estimate statistics. In 2019, the Society for Neuroscience journal eNeuro instituted a policy recommending the use of estimation graphics as the preferred method for data presentation(Bernard 2021). REAGENTS Genotypes of flies used for experiments in this study. Figure panel Genotype Figures 1A and 1B GAL4 NP5270 /UAS-NachBac Figures 1C and 1D Ddc-GAL4/UAS-NachBac Figures 1E and 1F 5-HT1B-GAL4/UAS-NachBac Figures 1G and 1H TH-GAL4/UAS-NachBac Figures 1I and 1J Tdc2-GAL4/UAS-NachBac Figures 1K and 1L Gad1-GAL4/UAS-NachBac Figures 1Q GAL4 ok107 /UAS-NachBac Figures 1R GAL4 MB247 /UAS-NachBac Figures 1S GAL4 c547 /UAS-NachBac Figures S1A GAL4 14-94 /UAS-NachBac Figures S1B GAL4 104y /UAS-NachBac Figures S1D Dilp2-GAL4/UAS-NachBac Figures S1F and S1G Ap-GAL4/UAS-NachBac Figures S1J Trh-GAL4/UAS-NachBac Figures 2A and 2B TH-GAL4/GAL80 MB247 /UAS-NachBac Figures 2C and 2D Ddc-GAL4/GAL80 MB247 /UAS-NachBac Figures 2E and 2F Tdc2-GAL4/GAL80 MB247 /UAS-NachBac Figures 2G and 2H Trh-GAL4/GAL80 MB247 /UAS-NachBac Figures 2I and 2J 5-HT1B-GAL4/Trh-GAL80/UAS-NachBac Figures 2K and 2L 5-HT1B-GAL4/GAL80 MB247 /UAS-NachBac Figures 2Q and 2R Gad1-GAL4/GAL80 MB247 /UAS-NachBac The following lines were obtained from Bloomington Stock Center (#stock number): 5-HT1B-GAL4 (#86276), Ap-GAL4 (#25685), Ddc-GAL4 (# 7009 ), Dilp2-GAL4 (#37576), Gad1-GAL4 (#51630), GAL41 04y (#81014), GAL4 c547 (# 44272 ), GAL4 MB247 (#50742 ) , GAL4 ok107 (#854), GAL80 MB247 (#64306), Tdc2-GAL4 (#9313), TH-GAL4 (#51982), Trh-GAL4 (#38388), UAS-NachBac (#9469). The following lines were obtained from kyoto drosophila stock center (#stock number): GAL4 NP5270 (Kyoto # 113657). GAL4 14-19 was kind gift from Dr. Ulrike Heberlein (HHMI Janelia Ressearch Campus) Trh-GAL80 was kind gift from Dr. Amita Sehgal (University of Pennsylvania) Declarations ACKNOWLEDGEMENTS We thank Dr. Ulrike Heberlein (HHMI Janelia Ressearch Campus) for sharing GAL4 14-19 driver, Dr. Amita Sehgal (University of Pennsylvania) for kindly sharing Trh-GAL80 fly strain, Drs. Susan Younger, Yuh Nung Jan and Lily Yeh Jan (UCSF, USA) for helpful comments and support on this paper. Author Contributions Conceptualization: Woo Jae Kim. Data curation: Xinyue Zhou, Dongyu Sun, Woo Jae Kim. Formal analysis: Xinyue Zhou, Dongyu Sun, Woo Jae Kim. Funding acquisition: Woo Jae Kim. Investigation: Woo Jae Kim. Methodology: Woo Jae Kim. Project administration: Woo Jae Kim. Resources: Woo Jae Kim. Supervision: Woo Jae Kim. Validation: Xinyue Zhou, Dongyu Sun, Woo Jae Kim. Visualization: Woo Jae Kim. Writing – original draft: Woo Jae Kim. Writing – review & editing: Xinyue Zhou, Dongyu Sun, Tianmu Zhang,Yutong Song, Woo Jae Kim. Funding This research was supported by Startup funds from HIT Center for Life Science to WJK, the Brain Pool Program of the National Research Foundation in Korea grant ZYM5041911 to WJK, Burroughs Wellcome Fund Collaborative Research Travel Grants (reference: 1017486) to WJK and a NVIDIA Academic Hardware Grant Program to WJK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CONFLICT OF INTEREST: The authors declare no competing interests. DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS During the creation of this work, the author(s) utilized QuillBot to rephrase English sentences, verify English grammar, and detect plagiarism, as none of the authors of this paper are native English speakers. 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Proc Natl Acad Sci 112:578–583. https://doi.org/10.1073/pnas.1421930112 Yapici N, Kim Y-J, Ribeiro C, Dickson BJ (2008) A receptor that mediates the post-mating switch in Drosophila reproductive behaviour. Nature 451:33–37. https://doi.org/10.1038/nature06483 Additional Declarations No competing interests reported. Supplementary Files FigS1.png Fig S1. The interconnections between the medial brain and the occipital brain enable the fusion of visual stimuli with other sensory inputs. (A-B) LMD assays for GAL4 14-94 (FB neurons) and GAL4 104y (FB neurons) mediated hyperexcitation via UAS-NaChBac . (C) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of TH (red), AstA-R1 (green) and γ-Kenyon cells (blue) in neuron clusters. Dashed circles indicate MB clusters. (D) LMD assays for dilp2-GAL4 mediated hyperexcitation via UAS-NaChBac . (E) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of TH (red), dilp2 (green) and γ-Kenyon cells (blue) in neuron clusters. Dashed circles indicate MB clusters. (F-G) LMD and CL assays for ap-GAL4 mediated hyperexcitation via UAS-NaChBac . (H) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of TH (red), apterous (green) and γ-Kenyon cells (blue) in neuron clusters. Dashed circles indicate MB clusters. (I) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of TH (red), γ-Kenyon cells (green) and Trh (blue) in neuron clusters. Dashed circles indicate MB clusters. (J) LMD assays for Trh-GAL4 mediated activation via UAS-NaChBac . (K-O) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of γ-Kenyon cells (green), Trh (blue) and 5-HT1A (red/K), 5-HT2A (red/L), 5-HT2B (red/M), 5-HT7 (red/O) in MB clusters. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4359931","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":299059176,"identity":"67d6e8e9-1ba3-4122-8e2d-665d2ac58dc7","order_by":0,"name":"Xinyue Zhou","email":"","orcid":"","institution":"Harbin Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xinyue","middleName":"","lastName":"Zhou","suffix":""},{"id":299059178,"identity":"6e32f0b8-49be-44d9-aad9-8189a577fe1c","order_by":1,"name":"Dongyu Sun","email":"","orcid":"","institution":"Harbin Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Dongyu","middleName":"","lastName":"Sun","suffix":""},{"id":299059180,"identity":"5dc93d9d-5524-45cf-818e-e1a19095428d","order_by":2,"name":"Yutong Song","email":"","orcid":"","institution":"Harbin Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yutong","middleName":"","lastName":"Song","suffix":""},{"id":299059182,"identity":"b0f5839b-e05f-4428-93ad-d8904f8667a5","order_by":3,"name":"Tianmu Zhang","email":"","orcid":"","institution":"Harbin Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Tianmu","middleName":"","lastName":"Zhang","suffix":""},{"id":299059184,"identity":"6725481f-beea-4fea-bfc3-770345413a6e","order_by":4,"name":"Woo Jae Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYDACHsYGho8NEiCmAfFaGGc2SEiQooWBgZm3gYEELQZnDrdutt1hUcfA3rxNgqHmDhFazja23c49A3QYz7EyCYZjzwhrMTvPCNTSBtQikWMmwdhwmEgtliAt8m+I1QJyGCPYFh4itdifOdh2s7dNQrKNJ63YIuEYEVoke9Kf3fjZVsfPz354440PNURogQM2EJFAgoZRMApGwSgYBXgAAHEiNoVxc/LTAAAAAElFTkSuQmCC","orcid":"","institution":"Harbin Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Woo","middleName":"Jae","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-05-02 15:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4359931/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4359931/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56204568,"identity":"f61d0caf-79e9-4353-a6a4-1fb6ac6ed6d0","added_by":"auto","created_at":"2024-05-09 20:47:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":265280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of serotonergic and dopaminergic neurons dirupt LMD behavior.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-L) LMD and CL assays for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eNP5270\u003c/em\u003e\u003c/sup\u003e (A-B)\u003cem\u003e, Ddc-GAL4 \u003c/em\u003e(C-D)\u003cem\u003e, 5-HT1B-GAL4 \u003c/em\u003e(E-F)\u003cem\u003e, TH-GAL4\u003c/em\u003e (G-H),\u003cem\u003e Tdc2-GAL4\u003c/em\u003e (I-J) and \u003cem\u003eGad1-GAL4\u003c/em\u003e (K-L), mediated hyperexcitation via \u003cem\u003eUAS-NaChBac\u003c/em\u003e. Light grey dots in MD assays represent naïve males and blue dots represent single reared males. Black dots in CL assays represent naïve males and blue dots represent single reared males. 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 Student’s \u003cem\u003et\u003c/em\u003e test and ns represents non-significant difference\u003cem\u003e \u003c/em\u003e(\u003cem\u003e*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt; 0.001, ****p\u0026lt; 0.0001\u003c/em\u003e)\u003cem\u003e. \u003c/em\u003eFor detailed methods, see the METHODS for a detailed description of the mating duration assay and court ship latancy assay used in this study.\u003c/p\u003e\n\u003cp\u003e(M-P) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of \u003cem\u003eTH\u003c/em\u003e (red), \u003cem\u003e5-HT1B\u003c/em\u003e (green) and g-Kenyon cells (blue/M), \u003cem\u003eDdc\u003c/em\u003e (blue/N), \u003cem\u003eGad1\u003c/em\u003e (blue/O), \u003cem\u003eTdc2\u003c/em\u003e (blue/P) in neuron clusters. Dashed circles indicate MB clusters. For detailed methods, see the METHODS for a detailed description of the single-nucleus RNA-sequencing analyses used in this study.\u003c/p\u003e\n\u003cp\u003e(Q-S) LMD assays for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eok107\u003c/em\u003e\u003c/sup\u003e(MB neurons),\u003cem\u003e GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(MB neurons), and \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003ec547\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(FB neurons) mediated hyperexcitation via \u003cem\u003eUAS-NaChBac\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(T) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of \u003cem\u003eTH\u003c/em\u003e (red), \u003cem\u003eDh31\u003c/em\u003e (green) and g-Kenyon cells (blue) in neuron clusters. Dashed circles indicate EB and MB clusters.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4359931/v1/d15a3ad9bb34554d1340b2a4.png"},{"id":56204647,"identity":"2186b15d-1cb4-471d-8077-01218b19a9b3","added_by":"auto","created_at":"2024-05-09 20:47:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMushroom bodies play a crucial role in the encoding and retrieval of long-term memory.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-H) LMD and CL assays for \u003cem\u003eTH-GAL4\u003c/em\u003e, \u003cem\u003eDdc-GAL4\u003c/em\u003e,\u003cem\u003e Tdc2-GAL4\u003c/em\u003e, and \u003cem\u003eTrh-GAL4 \u003c/em\u003e(serotonergic neurons) mediated hyperexcitation via \u003cem\u003eUAS-NaChBac\u003c/em\u003e together with \u003cem\u003eGAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e(I-J) LMD and CL assays for \u003cem\u003e5-HT1B-GAL4\u003c/em\u003e mediated hyperexcitation via \u003cem\u003eUAS-NaChBac\u003c/em\u003e together with \u003cem\u003eTrh-GAL80\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(K-L) LMD and CL assays for \u003cem\u003e5-HT1B-GAL4 \u003c/em\u003emediated hyperexcitation via \u003cem\u003eUAS-NaChBac\u003c/em\u003e together with \u003cem\u003eGAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e(M-P) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of \u003cem\u003e5HT1B \u003c/em\u003e(red), Kenyon cells (green) and \u003cem\u003eTrh\u003c/em\u003e (blue) in MB clusters (M), \u003cem\u003e5HT1B \u003c/em\u003e(red), ab-Kenyon cells (green) and \u003cem\u003eTrh\u003c/em\u003e (blue) in MB clusters (N), \u003cem\u003e5HT1B \u003c/em\u003e(red), a’b’-Kenyon cells (green) and \u003cem\u003eTrh\u003c/em\u003e (blue) in MB clusters (O) and \u003cem\u003e5HT1B \u003c/em\u003e(red), g-Kenyon cells (green) and \u003cem\u003eTrh\u003c/em\u003e (blue) in MB clusters (P).\u003c/p\u003e\n\u003cp\u003e(Q-R) LMD and CL assays for \u003cem\u003eGad1-GAL4\u003c/em\u003e (GABAergic neurons) mediated hyperexcitation via \u003cem\u003eUAS-NaChBac\u003c/em\u003e together with \u003cem\u003eGAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e(S-T) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of \u003cem\u003eGad1 \u003c/em\u003e(red), g-Kenyon cells (green) and \u003cem\u003eTrh\u003c/em\u003e (blue/S), \u003cem\u003eTH\u003c/em\u003e (blue/T) in MB clusters. Dashed circles indicate \u003cem\u003eGABA\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eDOPA\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e neurons in MB clusters\u003c/p\u003e\n\u003cp\u003e(U) Male fruit flies and their social interactions Fruit flies utilize their eyes to receive visual inputs and convey these signals to the EB and MB. The EB neurons are then activated and facilitate the consolidation of memory to regulate LMD behavior. At the same time, the MB neurons are suppressed, causing the flies to forget the memory and disrupt the LMD behavior.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4359931/v1/88651a12f162f4b1c1beec67.png"},{"id":58110863,"identity":"02fec6e5-d7fd-4178-b3a9-28fd9cacf242","added_by":"auto","created_at":"2024-06-11 09:22:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":849986,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4359931/v1/0aaacfbd-627a-4529-95c4-bc0637a89690.pdf"},{"id":56204637,"identity":"a5f7360f-8937-4a6a-8b27-e419e89d24b5","added_by":"auto","created_at":"2024-05-09 20:47:20","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":220017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig S1. The interconnections between the medial brain and the occipital brain enable the fusion of visual stimuli with other sensory inputs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) LMD assays for \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003e14-94\u003c/em\u003e\u003c/sup\u003e (FB neurons)\u003cem\u003e \u003c/em\u003eand\u003cem\u003e GAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003e104y\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(FB neurons) mediated hyperexcitation via \u003cem\u003eUAS-NaChBac\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(C) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of \u003cem\u003eTH\u003c/em\u003e (red), \u003cem\u003eAstA-R1 \u003c/em\u003e(green) and γ-Kenyon cells (blue) in neuron clusters. Dashed circles indicate MB clusters.\u003c/p\u003e\n\u003cp\u003e(D) LMD assays for \u003cem\u003edilp2-GAL4\u003c/em\u003e mediated hyperexcitation via \u003cem\u003eUAS-NaChBac\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(E) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of \u003cem\u003eTH\u003c/em\u003e (red), \u003cem\u003edilp2 \u003c/em\u003e(green) and γ-Kenyon cells (blue) in neuron clusters. Dashed circles indicate MB clusters.\u003c/p\u003e\n\u003cp\u003e(F-G) LMD and CL assays for \u003cem\u003eap-GAL4\u003c/em\u003e mediated hyperexcitation via \u003cem\u003eUAS-NaChBac\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(H) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of \u003cem\u003eTH\u003c/em\u003e (red), \u003cem\u003eapterous\u003c/em\u003e (green) and γ-Kenyon cells (blue) in neuron clusters. Dashed circles indicate MB clusters.\u003c/p\u003e\n\u003cp\u003e(I) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of \u003cem\u003eTH\u003c/em\u003e (red), γ-Kenyon cells (green) and\u003cem\u003e Trh\u003c/em\u003e (blue) in neuron clusters. Dashed circles indicate MB clusters.\u003c/p\u003e\n\u003cp\u003e(J) LMD assays for\u003cem\u003e Trh-GAL4\u003c/em\u003e mediated activation via \u003cem\u003eUAS-NaChBac\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(K-O) Single-cell RNA sequencing (SCOPE scRNA-seq) datasets reveal cell clusters colored by expression of γ-Kenyon cells (green),\u003cem\u003e Trh\u003c/em\u003e (blue) and\u003cem\u003e 5-HT1A\u003c/em\u003e (red/K), \u003cem\u003e5-HT2A \u003c/em\u003e(red/L), \u003cem\u003e5-HT2B \u003c/em\u003e(red/M), \u003cem\u003e5-HT7 \u003c/em\u003e(red/O) in MB clusters.\u003c/p\u003e","description":"","filename":"FigS1.png","url":"https://assets-eu.researchsquare.com/files/rs-4359931/v1/8e1e23dc14d8e0790ce7b6cb.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hyperexcitation of Monoaminergic Neurons in the Drosophila Mushroom Body Disrupts Memory for Visually Oriented Rival-induced Prolonged Mating","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe \u003cem\u003eDrosophila melanogaster\u003c/em\u003e neurotransmitter (NT) system is a complex network of neurons and synapses that enable communication between cells in the fly's nervous system. It utilizes various neurotransmitters (NTs), including dopamine (DA), serotonin (5-HT), acetylcholine (ACh), and glutamate (Glu), to transmit signals across neurons. The release and reception of neurotransmitters occur at specialized structures called synapses, which allow for the rapid and efficient transmission of signals. This system plays a crucial role in regulating fly behaviors, such as movement, learning, memory, and courtship. By studying the fly neurotransmitter system, researchers gain valuable insights into the fundamental principles of neural communication and behavior (Kahsai and Winther \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Deng et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe neurotransmitter system in fruit flies is intricately linked to the long-term memory (LTM) circuit. Neurotransmitters such as DA, 5-HT, Glu, and Ach are pivotal in the modulation of synaptic plasticity, which underlies the formation and storage of long-term memories (Heisenberg \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Androschuk et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Dopaminergic signaling pathways, for example, are known to be involved in the induction of long-term potentiation, a cellular mechanism of memory formation (Kaun and Rothenfluh \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sabandal et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, the neuromodulator octopamine (OA) has been shown to enhance LTM by potentiating synaptic transmission (Sitaraman et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The serotonergic system in \u003cem\u003eDrosophila\u003c/em\u003e is also a key regulator of LTM, with 5-HT acting as a neurotransmitter and neuromodulator. 5-HT-producing neurons are located in the protocerebral bridge (PB) and the ventral nerve cord (VNC), and they project their axons to various regions of the fly's brain, including the mushroom body (MB), the central complex (CC), and the ventral lateral horn (VLH) (Sitaraman et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Johnson et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Huser et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe integration of these neurotransmitters within specific neural circuits, such as the MB in the fruit fly brain, facilitates the encoding and retrieval of LTM. Therefore, the precise regulation of neurotransmitter release and reception within these circuits is crucial for the establishment and maintenance of long-term memory in fruit flies. The \u003cem\u003eDrosophila\u003c/em\u003e mushroom body, also known as the MB, is a key structure in the fruit fly brain that plays a critical role in learning, memory, and decision-making (Heisenberg \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Comprising of a pair of large, mushroom-shaped structures located dorsally in the fly's brain, the MB receives input from olfactory sensory neurons, as well as other sensory modalities, and integrates this information to guide appropriate behaviors (Davis \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Kahsai and Zars \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; S\u0026eacute;journ\u0026eacute; et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The MB is composed of Kenyon cells, which serve as the principal neurons, and these cells are organized into three distinct lobes: the α, β, and γ lobes. Each lobe receives input from different sensory systems and is involved in distinct aspects of learning and memory (Farris \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Longer-Mating-Duration (LMD) in male \u003cem\u003eDrosophila\u003c/em\u003e is characterized by an extended duration of mating that is triggered in response to social cues, particularly the presence of rival males (Kim et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003ea\u003c/span\u003e). LMD behavior is an adaptive response that allows males to increase their reproductive success in competitive environments (Bretman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). LMD relies on the integration of visual and social cues into memory circuits for its generation (Bretman et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e). The memory circuit for LMD in \u003cem\u003eDrosophila\u003c/em\u003e has been highly investigated in a neural network that connects the ellipsoid body (EB) to its visual circuit pathway (Kim et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003ea\u003c/span\u003e). The EB is a region in the fly's brain that plays a key role in visual processing and memory formation. It receives input from various sensory neurons, including those involved in visual perception, and integrates this information with other sensory cues (Neuser et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Pan et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ofstad et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kahsai and Zars \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEven though the MB has been traditionally considered dispensable for visual learning (Wolf et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), recent studies indicate that it plays a pivotal role in visual memory and other cognitive processes. The MB, which consists of three lobes\u0026mdash;α, β, and γ\u0026mdash;receives input from sensory neurons, including those involved in visual processing. This input enables the MB to integrate visual information with other sensory cues, forming the basis for visual memory (Li et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ganguly et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The connection between the MB and the EB allows for the integration of visual information with other sensory cues, enabling the fly to form and recall visual memories (Solanki et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cheong et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This collaboration between the EB and MB is crucial for visual memory processing in Drosophila (Barth and Heisenberg \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012b\u003c/span\u003e).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eIn the previous study investigating the regulation of LMD in male fruit fly, we have identified a crucial role for the memory circuit in mediating this behavior. Utilizing the potassium channel KCNJ2 to inhibit the function of the memory circuit, particularly in cells expressing EB, MB, and FB, we found that R2/R4m region of EB circuits are crucial to be activated to generate LMD memory (Kim et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e). Previous studies have determined that a pair of inhibitory neurons is essential for the maintenance of labile memory within the MB (Pitman et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Moreover, individual Kenyon cells within the MB display a heightened level of self-inhibition through the anterior paired lateral (APL) pathway relative to their inhibition of other Kenyon cells (Amin et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the pivotal role of the MB in visual memory processing and its integration of visual cues with other sensory information, it is justified to test the function of the MB, EB, and other brain regions for LMD memory by activating the MB using transgenes such as \u003cem\u003eNaChBac\u003c/em\u003e. NaChBac is a bacterial sodium channel that has been engineered to function in both mammalian and insect nervous systems (Charalambous and Wallace \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). It is commonly used in neuroscience research to study the effects of altering sodium channel activity on neural function. By expressing NaChBac in specific neurons, researchers can manipulate the excitability of those neurons and observe the effects on behavior, physiology, or other neural processes (Nitabach et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrior studies have indicated that inhibiting a subset of sexually dimorphic and GABAergic abdominal ganglion (AG) neurons can significantly extend mating duration in male \u003cem\u003eDrosophila\u003c/em\u003e (Tayler et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Crickmore and Vosshall \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To assess whether the activation of these neurons would elicit a similar effect, we utilized the \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eNP5270\u003c/em\u003e\u003c/sup\u003e (GABAergic AG neurons) driver to express NaChBac. Our findings revealed that the hyperexcitation of these neurons did not alter LMD behavior (Fig.\u0026nbsp;1A) or the copulation latency (CL) between group-housed and single-housed males (Fig.\u0026nbsp;1B). These data indicate that the hyperactivation of GABAergic AG neurons does not impact the extension of mating duration or the generation of LMD behavior.\u003c/p\u003e \u003cp\u003eThe regulation of mating duration has been postulated to involve a delicate interplay between dopaminergic and GABAergic signaling (Crickmore and Vosshall \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To elucidate the impact of neuronal hyperexcitation on mating duration, we employed the \u003cem\u003eDdc-GAL4\u003c/em\u003e (serotonergic and dopaminergic neurons)driver, which labels both serotonergic and dopaminergic neurons. Hyperexcitation of \u003cem\u003eDdc-GAL4\u003c/em\u003e-labeled neurons disrupted LMD without affecting CL (Fig.\u0026nbsp;1C-D). Furthermore, the activation with \u003cem\u003e5-HT1B-GAL4\u003c/em\u003e (serotonergic neurons) (Fig.\u0026nbsp;1E-F) or with \u003cem\u003eTH-GAL4\u003c/em\u003e (dopaminergic neurons) (Fig.\u0026nbsp;1G-H) also disrupted LMD without altering CL. Contrary to this, hyperexcitation of the OA did not affect LMD or CL (Fig.\u0026nbsp;1I-J). Remarkably, males with hyperexcited GABAergic neurons failed to mate despite displaying courtship behavior (Fig.\u0026nbsp;1K-L), indicating that the hyperactivation of inhibitory GABAergic neurons can disrupt mating behaviors. However, this effect does not appear to be mediated by the sexually dimorphic GABAergic neurons within the AG, as their hyperexcitation did not impede mating success (Fig.\u0026nbsp;1A-B). Utilizing the fly SCope platform, which houses a recently acquired single-cell RNA sequencing dataset, we identified that the expression of \u003cem\u003eDdc, Tdc2\u003c/em\u003e, \u003cem\u003eTH\u003c/em\u003e, \u003cem\u003e5-HT1B\u003c/em\u003e, and \u003cem\u003eGad1\u003c/em\u003e overlaps in the mushroom body g-Kenyon cells population (Fig.\u0026nbsp;1M-P) (Li et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e), suggesting their potential involvement in LMD regulation (Crickmore and Vosshall \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al. 2022).\u003c/p\u003e \u003cp\u003eWe employed \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eok107\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e to hyperactivate MB neurons, which disrupted LMD behavior (Fig.\u0026nbsp;1Q-R). Conversely, the inactivation of MB neurons using the inward rectifier potassium channel, KCNJ2, did not alter LMD (Kim et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e). Furthermore, the hyperactivation of EB neurons using \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003ec547\u003c/em\u003e\u003c/sup\u003e or FB neurons using \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003e14\u0026ndash;94\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eGAL4\u003c/em\u003e\u003csup\u003e\u003cem\u003e104y\u003c/em\u003e\u003c/sup\u003e also disrupted LMD (Fig.\u0026nbsp;1S and Fig. S1A-B), suggesting that the balance of neuronal activity in EB and FB neurons is necessary for the generation of LMD memory. Single-cell RNA sequencing (SCope) data revealed that dopaminergic neurons are highly co-expressed with MB g-Kenyon cells as well as Dh31-positive EB neurons (Fig.\u0026nbsp;1T) or AstA-R1-positive FB neurons (Fig. S1C). In contrast, the hyperactivation of dilp2-positive pars intercerebralis (PI) neurons or ap-positive interneurons in the VNC did not affect LMD behavior, despite their high co-expression with dopaminergic neurons (Fig. S1D-H). These data collectively indicate that monoaminergic neurons within the MB are the primary target neurons that disrupt LMD when hyperactivated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo corroborate that monoaminergic neurons within the MB are the primary targets for disrupting LMD behavior when hyperexcited, we utilized MB-specific GAL80 (\u003cem\u003eGAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e) to inhibit GAL4 activity in MB neurons (Krashes et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Remarkably, the co-expression of \u003cem\u003eGAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e with \u003cem\u003eTH-\u003c/em\u003e, \u003cem\u003eDdc-\u003c/em\u003e, or \u003cem\u003eTdc2-GAL4\u003c/em\u003e (aminergic neurons) drivers, which induce NaChBac-mediated LMD disruption (Fig.\u0026nbsp;2A-F). \u003cem\u003eTrh-GAL4\u003c/em\u003e, known to selectively target serotonergic neurons (Alekseyenko et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), was found to be not universally expressed in MB neurons, unlike 5-HT1B neurons (Fig. S1I and Fig.\u0026nbsp;1M). Consequently, the hyperactivation of Trh-positive serotonergic neurons or Trh-positive non-MB neurons did not affect LMD behavior (Fig. S1J and Fig.\u0026nbsp;2G), indicating that Trh-positive 5-HT neurons are not integral to MB-related LMD memory processing. However, the hyperactivation of Trh neurons disrupted CL in single-housed flies (Fig.\u0026nbsp;2H), suggesting that Trh-positive serotonergic neurons control CL rather than LMD. Co-expressing \u003cem\u003eTrh-GAL80\u003c/em\u003e with \u003cem\u003e5-HT1B-GAL4\u003c/em\u003e did not alter the effect of hyperactivation (Fig.\u0026nbsp;2I-J), whereas the combination of \u003cem\u003eGAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e abolished the LMD disruption mediated by hyperactivation (Fig.\u0026nbsp;2K-L). Single-cell RNA sequencing (SCope) data indicates that the majority of 5-HT1B neurons located in MB overlap with ab-, a\u0026rsquo;b\u0026rsquo;-, and g-Kenyon cells (Fig.\u0026nbsp;2M-P). These findings collectively suggest that monoaminergic neurons within MB Kenyon cells are crucial for the processing of LMD memory.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe impairment in mating phenotype observed upon the hyperactivation of \u003cem\u003eGad1-GAL4\u003c/em\u003e (GABAergic neurons) was alleviated when co-expressed with \u003cem\u003eGAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;2Q-R), indicating that the critical GABAergic neurons responsible for mating success are resident within MB neurons (Fig.\u0026nbsp;2S-T).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study identified the neural circuits and monoaminergic neurons in the MB as key regulators of the LMD behavior in male \u003cem\u003eDrosophila\u003c/em\u003e. Activation screening experiments reveal the disruption of LMD by hyperactivating monoaminergic neurons in the MB, including serotonergic neurons (Fig. 1E-F) and dopaminergic neurons (Fig. 1G-H), without affecting copulation latency. The co-expression of \u003cem\u003eGAL80\u003csup\u003eMB247\u003c/sup\u003e\u003c/em\u003e with the disrupting GAL4 drivers rescues LMD (Fig.2C-D, G-H and K-L), confirming the involvement of monoaminergic neurons in the MB. The hyperactivation of inhibitory GABAergic neurons disrupts mating (Fig. 1K-L), but this effect is alleviated by MB-specific GAL80 inhibitors (Fig. 2Q-R), suggesting that critical inhibitory neurons reside within the MB. In summary, the activation of monoaminergic neurons in the MB disrupts LMD memory, while the hyperactivation of inhibitory GABAergic neurons in the MB impairs mating success. These findings implicate the MB as a crucial neural circuit for integrating visual and social cues to regulate mating duration in male \u003cem\u003eDrosophila\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThis study identified the monoamines, including DA, 5-HT, and OA play crucial roles in memory formation and regulation in \u003cem\u003eDrosophila (Kemenes et al. 2011)\u003c/em\u003e. Dopaminergic signaling pathways are involved in the induction of long-term potentiation, a cellular mechanism of memory formation. DA release in the MB facilitates the encoding and retrieval of long-term memories (Yamagata et al. 2015). The neuromodulator OA enhances LTM by potentiating synaptic transmission. Octopaminergic neurons also project to the MB and other brain regions involved in memory. DA and OA exert differential modulatory effects on memory processing across distinct neural circuits (Schwaerzel et al. 2003). Our findings suggest that the OA system is not essential for the generation of LMD memory in male \u003cem\u003eDrosophila\u003c/em\u003e (Fig.1I-J)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe serotonergic system in \u003cem\u003eDrosophila\u003c/em\u003e exhibits crucial functions in LTM processing. Serotonin-producing neurons in the protocerebral bridge (PB) and VNC project to multiple brain regions, including the MB. 5-HT serves as both a neurotransmitter and neuromodulator, potentiating synaptic transmission and facilitating the expression of genes involved in LTM formation (Sitaraman et al. 2008, 2012). Notably, our data and previous studies (Johnson et al. 2011; Lee et al. 2021) indicate that 5-HT actions via its receptors, such as 5-HT1B (Fig. 1E), are essential for LTM. Indeed, all 5-HT receptors exhibit robust expression in MB neurons (Fig. S1K-O). Serotonin has been demonstrated to enhance LTM in \u003cem\u003eDrosophila\u003c/em\u003e through a range of mechanisms, including its impact on synaptic plasticity and gene expression regulation. Specifically, serotonin has been shown to potentiate synaptic transmission and promote the expression of genes involved in LTM formation within MB neurons (Crocker et al. 2016; Wu et al. 2017).\u003c/p\u003e\n\u003cp\u003eIn summary, monoamines released in the \u003cem\u003eDrosophila\u003c/em\u003e brain act as neurotransmitters and neuromodulators, potentiating synaptic plasticity and promoting gene expression, thereby regulating the formation, consolidation, and retrieval of LTM for LMD. The precise integration of these monoamines within specific neural circuits, such as the MB\u0026nbsp;g-Kenyon cells, enables the encoding and retrieval of LTM in \u003cem\u003eDrosophila\u003c/em\u003e. Our findings align with previous reports suggesting that inhibitory circuits within MB play a pivotal role in the generation of LTM\u0026nbsp;\u003cstrong\u003e(Aranda et al. 2017; Awata et al. 2019; Feng et al. 2021)\u003c/strong\u003e (Fig. 2U).\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eEXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFruit fly rearing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDrosophila melanogaster\u003c/em\u003e were cultured under standard laboratory conditions at 25℃. Samples were prepared as described in the Methods Details. All fly strains are listed in the key resources table.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMating duration assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mating duration assay in this\u0026nbsp;study has been reported(Kim et al. 2012, 2013; Lee et al. 2023).\u0026nbsp;To enhance the efficiency of the mating duration assay, we utilized the \u003cem\u003eDf(1)Exel6234\u003c/em\u003e (DF here after) genetic modified fly line in this study, which harbors a deletion of a specific genomic region that includes the sex peptide receptor (SPR)(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 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.\u0026nbsp;At the fifth day after eclosion, males of the appropriate strain and\u0026nbsp;DF\u0026nbsp;virgin females were mildly anaesthetized by CO\u003csub\u003e2\u003c/sub\u003e. After placing a single female in to the mating chamber, we inserted a transparent film then placed a single male to the other side of the film in each chamber. After allowing for 1 h of recovery in the mating chamber in\u0026nbsp;25℃\u0026nbsp;incubator, 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. All assays were performed from noon to 4pm. We conducted blinded studies for every test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-nucleus RNA-sequencing analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003esnRNAseq dataset analyzed in this paper is published(Li et al. 2022b)\u0026nbsp;and available at the Nextflow pipelines (VSN, https://github.com/vib-singlecell-nf), the availability of raw and processed datasets for users to explore, and the development of a crowd-annotation platform with voting, comments, and references through SCope (https://flycellatlas.org/scope), linked to an online analysis platform in ASAP (\u003ca href=\"https://asap.epfl.ch/fca\"\u003ehttps://asap.epfl.ch/fca\u003c/a\u003e).Single-cell RNA sequencing (scRNA-seq) data from the \u003cem\u003eDrosophila melanogaster\u003c/em\u003e were obtained from the Fly Cell Atlas website (https://doi.org/10.1126/science.abk2432).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis of mating duration assay was described previously(Kim et al. 2012, 2013; Lee et al. 2023). More\u0026nbsp;than 50 males (group and single) were used for mating duration assay. Our experience suggests that the relative mating duration differences between group and single 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(Bretman et al. 2011b). Therefore, statistical comparisons were made between groups that were grouply reared and singly reared within each experiment. As mating duration of males showed normal distribution (Kolmogorov-Smirnov tests, p \u0026gt; 0.05), we used two-sided Student’s t tests. The mean ± standard error (s.e.m) (\u003cem\u003e**** = p \u0026lt; 0.0001, *** = p \u0026lt; 0.001, ** = p \u0026lt; 0.01, * = p \u0026lt; 0.05\u003c/em\u003e). All analysis was done in GraphPad (Prism). Individual tests and significance are detailed in figure legends.\u003c/p\u003e\n\u003cp\u003eBesides traditional \u003cem\u003et\u003c/em\u003e-test for statistical analysis, we added estimation statistics for all MD assays and two group comparing graphs. In short, ‘estimation statistics’ is a simple framework that—while avoiding the pitfalls of significance testing—uses familiar statistical concepts: means, mean differences, and error bars. More importantly, it focuses on the effect size of one’s experiment/intervention, as opposed to significance testing(Claridge-Chang and Assam 2016). In comparison to typical NHST plots, estimation graphics have the following five significant advantages such as (1) avoid false dichotomy, (2) display all observed values,\u0026nbsp;(3) visualize estimate precision,\u0026nbsp;(4) show mean difference distribution. And most importantly (5) by focusing attention on an effect size, the difference diagram encourages quantitative reasoning about the system under study(Ho et al. 2019). Thus, we conducted a reanalysis of all of our two group data sets using both standard \u003cem\u003et\u003c/em\u003e-tests and estimate statistics. In 2019, the Society for Neuroscience journal eNeuro instituted a policy recommending the use of estimation graphics as the preferred method for data presentation(Bernard 2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eREAGENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotypes of flies used for experiments in this study.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFigure panel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 1A and 1B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGAL4\u003csup\u003eNP5270\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 1C and 1D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eDdc-GAL4/UAS-NachBac\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 1E and 1F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e5-HT1B-GAL4/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 1G and 1H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eTH-GAL4/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 1I and 1J\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eTdc2-GAL4/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 1K and 1L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGad1-GAL4/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 1Q\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGAL4\u003csup\u003eok107\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 1R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGAL4\u003csup\u003eMB247\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 1S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGAL4\u003csup\u003ec547\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures S1A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGAL4\u003csup\u003e14-94\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures S1B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGAL4\u003csup\u003e104y\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures S1D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eDilp2-GAL4/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures S1F and S1G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eAp-GAL4/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures S1J\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eTrh-GAL4/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 2A and 2B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eTH-GAL4/GAL80\u003csup\u003eMB247\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 2C and 2D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eDdc-GAL4/GAL80\u003csup\u003eMB247\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 2E\u0026nbsp;and 2F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eTdc2-GAL4/GAL80\u003csup\u003eMB247\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 2G\u0026nbsp;and 2H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eTrh-GAL4/GAL80\u003csup\u003eMB247\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 2I and 2J\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e5-HT1B-GAL4/Trh-GAL80/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 2K and 2L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e5-HT1B-GAL4/GAL80\u003csup\u003eMB247\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.741410488245933%\" valign=\"top\"\u003e\n \u003cp\u003eFigures 2Q and 2R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"69.25858951175407%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGad1-GAL4/GAL80\u003csup\u003eMB247\u003c/sup\u003e/UAS-NachBac\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe following lines were obtained from Bloomington Stock Center (#stock number):\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5-HT1B-GAL4\u003c/em\u003e(#86276),\u003cem\u003e\u0026nbsp;Ap-GAL4\u0026nbsp;\u003c/em\u003e(#25685),\u003cem\u003e\u0026nbsp;Ddc-GAL4\u003c/em\u003e\u003cem\u003e(#\u003c/em\u003e\u003cem\u003e7009\u003c/em\u003e),\u003cem\u003eDilp2-GAL4\u0026nbsp;\u003c/em\u003e(#37576),\u003cem\u003eGad1-GAL4\u003c/em\u003e(#51630),\u003cem\u003eGAL41\u003csup\u003e04y\u003c/sup\u003e\u003c/em\u003e(#81014),\u003cem\u003eGAL4\u003csup\u003ec547\u003c/sup\u003e\u003c/em\u003e (#\u003cem\u003e44272\u003c/em\u003e),\u003cem\u003eGAL4\u003csup\u003eMB247\u003c/sup\u003e\u003c/em\u003e (#50742\u003cu\u003e)\u003c/u\u003e, \u003cem\u003eGAL4\u003csup\u003eok107\u003c/sup\u003e\u003c/em\u003e (#854),\u003cem\u003eGAL80\u003csup\u003eMB247\u003c/sup\u003e\u003c/em\u003e (#64306),\u003cem\u003e\u0026nbsp;Tdc2-GAL4\u003c/em\u003e(#9313),\u0026nbsp;\u003cem\u003eTH-GAL4\u0026nbsp;\u003c/em\u003e(#51982),\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrh-GAL4\u003c/em\u003e (#38388),\u0026nbsp;\u003cem\u003eUAS-NachBac\u003c/em\u003e (#9469).\u003c/p\u003e\n\u003cp\u003eThe following lines were obtained from kyoto drosophila stock center\u0026nbsp;(#stock number):\u0026nbsp;\u003cem\u003e\u0026nbsp;GAL4\u003csup\u003eNP5270\u003c/sup\u003e\u003c/em\u003e (Kyoto # 113657).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGAL4\u003csup\u003e14-19\u003c/sup\u003e\u003c/em\u003e was kind gift from Dr. Ulrike Heberlein (HHMI Janelia Ressearch Campus)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrh-GAL80\u0026nbsp;\u003c/em\u003ewas kind gift from Dr. Amita Sehgal (University of Pennsylvania) \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Ulrike Heberlein (HHMI Janelia Ressearch Campus)\u0026nbsp;for sharing \u003cem\u003eGAL4\u003csup\u003e14-19\u003c/sup\u003e\u003c/em\u003e driver, Dr. Amita Sehgal (University of Pennsylvania)\u0026nbsp;for kindly sharing \u003cem\u003eTrh-GAL80\u003c/em\u003e fly strain, Drs. Susan Younger, Yuh Nung Jan and Lily Yeh Jan (UCSF, USA) for helpful comments and support on this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u0026nbsp;\u003c/strong\u003eWoo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData curation:\u003c/strong\u003e Xinyue Zhou, Dongyu Sun, Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFormal analysis:\u003c/strong\u003e Xinyue Zhou, Dongyu Sun, Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding acquisition:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject administration:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResources:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupervision:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation:\u003c/strong\u003e Xinyue Zhou, Dongyu Sun, Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting – original draft:\u003c/strong\u003e Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting – review \u0026amp; editing:\u003c/strong\u003e Xinyue Zhou, Dongyu Sun, Tianmu Zhang,Yutong Song, Woo Jae Kim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Startup funds from HIT Center for Life Science to WJK, the Brain Pool Program of the National Research Foundation in Korea grant ZYM5041911 to WJK, Burroughs Wellcome Fund Collaborative Research Travel Grants (reference: 1017486) to WJK and a NVIDIA Academic Hardware Grant Program to WJK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the creation of this work, the author(s) utilized QuillBot to rephrase English sentences, verify English grammar, and detect plagiarism, as none of the authors of this paper are native English speakers. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlekseyenko OV, Lee C, Kravitz EA (2010) Targeted Manipulation of Serotonergic Neurotransmission Affects the Escalation of Aggression in Adult Male Drosophila melanogaster. PLoS ONE 5:e10806. https://doi.org/10.1371/journal.pone.0010806\u003c/li\u003e\n\u003cli\u003eAmin H, Apostolopoulou AA, Su\u0026aacute;rez-Grimalt R, et al (2020) Localized inhibition in the Drosophila mushroom body. eLife 9:e56954. https://doi.org/10.7554/elife.56954\u003c/li\u003e\n\u003cli\u003eAndroschuk A, Al-Jabri B, Bolduc FV (2015) From Learning to Memory: What Flies Can Tell Us about Intellectual Disability Treatment. 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Nature 451:33\u0026ndash;37. https://doi.org/10.1038/nature06483\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Drosophila melanogaster, Mating duration, Interval timing, Longer-Mating-Duration, LMD, Mushroom body, GABAergic neurons, Monoaminergic neurons, Memory, Hyperexcitation","lastPublishedDoi":"10.21203/rs.3.rs-4359931/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4359931/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMale individuals frequently require a prolongation of their mating duration in order to outcompete their rivals for few reproductive chances. This study looks into the roles of monoaminergic neurons in the \u003cem\u003eDrosophila melanogaster\u003c/em\u003e mushroom body (MB) as major regulators of males' rival-induced prolonged mating duration (LMD) behavior. Activation screening experiments revealed that hyperexcitation of monoaminergic neurons in the MB, including serotonergic neurons and dopaminergic neurons, disrupts LMD without affecting copulation latency. The co-expression of MB-specific GAL80 (\u003cem\u003eGAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e with the monoaminergic GAL4 drivers rescues LMD, confirming the involvement of monoaminergic neurons in the MB. The hyperexcitation of inhibitory GABAergic neurons disrupts mating, but this effect is alleviated by \u003cem\u003eGAL80\u003c/em\u003e\u003csup\u003e\u003cem\u003eMB247\u003c/em\u003e\u003c/sup\u003e inhibitors, suggesting that critical GABAergic neurons for LMD reside within the MB. In summary, the activation of monoaminergic neurons in the MB disrupts LMD memory, while the hyperactivation of inhibitory GABAergic neurons in the MB impairs mating success. These findings implicate the MB as a crucial neural circuit for integrating visual and social cues to generate memory for LMD behavior.\u003c/p\u003e","manuscriptTitle":"Hyperexcitation of Monoaminergic Neurons in the Drosophila Mushroom Body Disrupts Memory for Visually Oriented Rival-induced Prolonged Mating","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-09 20:45:49","doi":"10.21203/rs.3.rs-4359931/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be63825c-bca5-4326-b8e5-416c4befcc2c","owner":[],"postedDate":"May 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-11T09:22:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-09 20:45:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4359931","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4359931","identity":"rs-4359931","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}