A dopamine-gated learning circuit underpins reproductive state-dependent odor preference in Drosophila females

  1. Ariane C Boehm
  2. Anja B Friedrich
  3. Sydney Hunt
  4. Paul Bandow
  5. KP Siju
  6. Jean Francois De Backer
  7. Julia Claussen
  8. Marie Helen Link
  9. Thomas F Hofmann
  10. Corinna Dawid
  11. Ilona C Grunwald Kadow

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    Evaluation Summary:

    In this manuscript, the authors explore the circuit mechanism underlying mating-induced change of odor preference in Drosophila. Olfactory cues during mating induce a long-lasting increase in attraction to polyamines in female flies. The authors use a combination of neurogenetics, imaging, and behaviour to identify elements of the mushroom body and lateral horn circuitry involved in this behaviour. The importance of mushroom body plasticity in female postmating changes highlights a novel pathway for these changes and reveals the variety of mechanisms by which the brain can encode experience and adapt behavior. This paper will be of interest to scientists within the field of reproductive behaviors and neuroscience of internal states.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

Motherhood induces a drastic, sometimes long-lasting, change in internal state and behavior in many female animals. How a change in reproductive state or the discrete event of mating modulates specific female behaviors is still incompletely understood. Using calcium imaging of the whole brain of Drosophila females, we find that mating does not induce a global change in brain activity. Instead, mating modulates the pheromone response of dopaminergic neurons innervating the fly’s learning and memory center, the mushroom body (MB). Using the mating-induced increased attraction to the odor of important nutrients, polyamines, we show that disruption of the female fly’s ability to smell, for instance the pheromone cVA, during mating leads to a reduction in polyamine preference for days later indicating that the odor environment at mating lastingly influences female perception and choice behavior. Moreover, dopaminergic neurons including innervation of the β’1 compartment are sufficient to induce the lasting behavioral increase in polyamine preference. We further show that MB output neurons (MBON) of the β’1 compartment are activated by pheromone odor and their activity during mating bidirectionally modulates preference behavior in mated and virgin females. Their activity is not required, however, for the expression of polyamine attraction. Instead, inhibition of another type of MBON innervating the β’2 compartment enables expression of high odor attraction. In addition, the response of a lateral horn (LH) neuron, AD1b2, which output is required for the expression of polyamine attraction, shows a modulated polyamine response after mating. Taken together, our data in the fly suggests that mating-related sensory experience regulates female odor perception and expression of choice behavior through a dopamine-gated learning circuit.

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  1. Version published to 10.7554/elife.77643 on eLife
  2. eLife

    Evaluation Summary:

    In this manuscript, the authors explore the circuit mechanism underlying mating-induced change of odor preference in Drosophila. Olfactory cues during mating induce a long-lasting increase in attraction to polyamines in female flies. The authors use a combination of neurogenetics, imaging, and behaviour to identify elements of the mushroom body and lateral horn circuitry involved in this behaviour. The importance of mushroom body plasticity in female postmating changes highlights a novel pathway for these changes and reveals the variety of mechanisms by which the brain can encode experience and adapt behavior. This paper will be of interest to scientists within the field of reproductive behaviors and neuroscience of internal states.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Mating influences many behaviours such as enhanced oviposition, suppressed mating, and a change in dietary preference. In this study, Boehm et al explore the circuit basis of the mated female's enhanced preference for polyamines.

    A previous study from this group had identified a mechanism by which mating reduced sensitivity of the olfactory sensory neurons resulting in a preference for higher concentrations of polyamines after mating. However, the preference for polyamines outlasts this mechanism by many days. So, in this study, the authors explore central brain circuits that might encode this persistent behavioural switch. Briefly, they identify neurons within the mushroom body - intrinsic neurons, output neurons and dopaminergic neurons (DAN) - that are involved in this behaviour. They also identify output neurons of the lateral horn that are involved in it.

    The behaviour itself consists of two phases: 1) the mating experience, and 2) the subsequent expression of the polyamine preference. The authors use behavioural assays and neurogenetics to demonstrate that:

    1. The ability to detect odours via the OR67d neurons at the time of mating is necessary to bring about the behavioural switch.

    2. Activity of the intrinsic neurons of the mushroom body is necessary at both times - during the mating and the expression - to bring about the behavioural switch.

    3. They identify one set of dopaminergic neurons - B1 DANs - that are sufficient but not necessary ***at the time of mating*** to induce the switch in virgin females.

    4. They identify a second set of dopaminergic neurons - B2 DANs - that are necessary to ***at the time of expression*** to demonstrate the increased polyamine preference ******in mated females.

    5. They identify a set of mushroom body output neurons (MBONs) downstream of the B1-DANs and show that output from the B1 region is necessary and sufficient at the time of mating for the expression of polyamine preference.

    6. They identify MBONs downstream of the B2 DANs and find that they play no role at the time of mating, but that they are necessary and sufficient at the time of expression to suppress the polyamine preference.

    7. They identify a set of output neurons of the lateral horn and find that they are necessary at the time of expression of polyamine preference.

    The authors also use functional imaging to show that there are no brain-wide changes upon mating in the encoding of one of the polyamines. They explore how cVA (an odour they believe is relevant at the time of mating) is represented in the neurons they have identified. They find that the B1 DANs show enhanced representation of cVA post mating, however, their MBONs do not alter their response to cVA post mating. The B2-MBONs respond to both putrescine and cVA and show no alteration in their response post mating.

    In summary, the authors have identified a mechanism similar to associative learning that operates across the mushroom body and lateral horn, to 'learn' the experience of mating and express it as an enhanced preference to a nutritionally rich food source.

  4. eLife

    Reviewer #2 (Public Review):

    The authors use the model of polyamine attraction and build on their previous observation that mated Drosophila females show increased attraction to polyamines that is outlasting a short term modulation of olfactory sensory neurons. Females do not require exposure to seminal fluid or sperm and do not need to start to ovulate for this change in preference. This is remarkable since the vast majority of female postmating changes in behavior have been shown to rely on sex peptide in the male seminal fluid. It also sets an exciting starting point for the present work, suggesting new mechanisms of how a female can adjust their behavior to mating state.

    The authors find that females have to be able to smell odors detected by the Or system (but not polyamines) during mating in order to change their preference for polyamine.

    Mushroom body Kenyon cells are required during mating and during choice behavior for the polyamine preference of mated females. Activation cVA responsive PAM-b1 neurons of the mushroom body is sufficient to replace the mating experience and change polyamine preference in virgin flies. Activation of the same neurons during mating abolishes the preference development. Other specific mushroom body neurons are required during the choice behavior to promote attraction in mated females or repress it in virgins. Calcium imaging of different mushroom body neurons does not uncover a clear difference in polyamine response between mated and virgin flies. Connectome mining and genetic silencing further indicates that circuit motifs in the lateral horn are also involved in the response to polyamines and might interact with mushroom body circuits.

    While the exact circuits and mechanisms of plasticity that explain the change in postmating preference of polyamines remain to be discovered, this work makes substantial progress in identifying neurons that have a strong impact on development or expression of the preference. It is an exciting paradigm that invites further research.

    This work explores a very interesting example of state-dependent behavioral change in Drosophila. Previously, state dependent changes in sensory neurons have been demonstrated- here, the authors tackle the experimentally much more challenging task of identifying changes in higher order processing areas. The data suggests that polyamine attraction is encoded by a recurrent network of mushroom body neurons. Although the authors do not demonstrate an exact mechanism by which mating/male exposure reconfigures this polyamine attraction network, they have made a substantial advance for our understanding how odor valence is encoded in a flexible and experience dependent way by identifying and characterizing the neuronal players and their roles in induction and expression of preference behavior.

    Their experimental paradigm is special in that it is not a case of classical odor reward learning (mating could be the rewarding experience, but polyamine odor does not have to be present during mating to induce preference). It is also special in that it is a case of long lasting mating induced behavior change that is not dependent on sex peptide or other male seminal fluid proteins. The paradigm has thus great potential to uncover novel mechanisms of encoding experience and adaptively changing behavior.

  5. eLife

    Reviewer #3 (Public Review):

    Mating changes behavior of female fruit flies. Authors previously reported that putrescine-rich foods increase number of progenies per mated female and mated females detect putrescine with IR76b and IR41a and are attracted to putrescine odor (Hussain, Zhang et al., 2016). In another paper, authors reported that this change of putrescine preference is mediated by sex peptide receptor (SPR) and its ligand, myoinhibiotry peptides (MIPs; Hussain, Ucpunar et al., 2016). In yet another paper, authors reported that two types of dopaminergic neurons (DANs) which innervate alpha prime 3 (a'3) or beta prime 1 (b'1) compartment of the mushroom body (MB) show enhanced response to cVA, the male sex pheromone 11-cis-Vaccenyl acetate (Siju et al., 2020). The present study investigated neural circuits that potentially link these observations.

    The authors first showed that putrescine-attraction in mated females is sustained over 7-days, which cannot be explained by SPR-MIP dependent mechanism that disappears in one week. Then they explored a factor that is transferred from males during copulation and required for putrescine-attraction in mated females. They found that blocking synaptic transmission of cVA-sensitive OR67d olfactory receptor neurons during 24 hour period of pairing with males reduces putrescine-attraction 3-5 days later (Figure 1). On the other hand, experiments with mutant flies lacking ability to generate eggs or sperms indicated that fertilization is not essential for the change in odor preference. In a proposed scenario, cVA transferred to the female during copulation activates DANs projecting to the b'1 and that in turn induces a shift in how the MB regulates the expression of polyamine odor preference, possibly by alternating activity of MB output neurons (MBONs) in the beta prime 2 (b'2) compartment.

    Some data are in line with this scenario. Blocking synaptic transmissions of Kenyon cells during mating or odor preference test reduced attraction to putrescine (Figure 2). Activation of dopaminergic neurons projecting to the beta prime 1, gamma 3 and gamma 4 in virgin females promoted attraction to putrescine when tested 3-5 days later (Figure 3). Flies expressing shibire ts1 in the MBONs in the b'1 compartment showed reduced putrescine preference when females were mated at restrictive temperature (Figure 4). Using calcium imaging and EM connectome, authors also found candidate lateral horn output neurons that may mediate putrescine signals from olfactory projection neurons to the b'1 DANs.

    This study utilized molecular genetic tools, behavioral experiments and calcium imaging to comprehensively investigate neural circuits from sensory neurons for cVA or putrescine to the learning circuits of the MB. Addressing points detailed below will strengthen a causal link between enhanced cVA response in beta prime 1 DANs and enhanced putrescine preference in mated females.

    1. The MB is the center for olfactory associative learning. It is not so surprising that 24-hour long activation of any MB cell types have long-term consequence on fly's odor preference. As authors showed in Hussain et al., 2016 and Figure S1, mated females change preference to polyamines but not ammonium. Therefore, it is important to show odor specificity of the circuit manipulations to claim that phenomenon in mated females are recapitulated by each manipulation. Wang et al., 2003 (DOI:https://doi.org/10.1016/j.cub.2003.10.003) reported that blocking a broad set of Kenyon cells impairs innate odor attraction to fruit odors and diluted odors but not repulsion.
    1. Requirement of PAM-b'1 DANs for putrescine-attraction in mated females should be demonstrated. The authors suggested existence of alternative mechanisms that may mask requirement of PAM-b'1 (Figure 3B). In a previous study, the authors reported SPR-dependent mechanism. I suggest testing the requirement of PAM-b'1 DANs in SPR mutant background or one-week after mating when SPR-dependent effect on sensory neurons disappear.
    1. Activation phenotype of MB188B-split-GAL4/UAS-dTrpA1 cannot be ascribed to activation of PMA-b'1 alone because of additional expression in DANs projecting to gamam3 and gamma4 compartments. Run the same experiment with more PMA-b'1 specific driver line.
    1. Some of EM connections are too low to be considered (e.g. two in Figure S3 and five in Figure 5). Although these connections could be functional, previous EM connectome analysis typically set much higher threshold (e.g. 10 in Hulse et al., 2021 DOI: 10.7554/eLife.66039) to avoid considering artifacts.
    1. Data for Kenyon cells (Figure 2) and LHON (Figure 6) are interesting, but not directly related to other data regarding PAM-b'1 and MBON-b'1. Due to lack of long-term changes in MBOB's odor responses in mated females (Figure 5), it is unclear what information needs to be read out from Kenyon cells and how does it affect processing of putrescine signals potentially carried by LHAD1b2.
  6. eLife

    Author Response

    Reviewer 2

    The authors use the model of polyamine attraction and build on their previous observation that mated Drosophila females show increased attraction to polyamines that is outlasting a short term modulation of olfactory sensory neurons. Females do not require exposure to seminal fluid or sperm and do not need to start to ovulate for this change in preference. This is remarkable since the vast majority of female postmating changes in behavior have been shown to rely on sex peptide in the male seminal fluid. It also sets an exciting starting point for the present work, suggesting new mechanisms of how a female can adjust their behavior to mating state.

    We thank the reviewer very much for their encouraging comments.

    The authors find that females have to be able to smell odors detected by the Or system (but not polyamines) during mating in order to change their preference for polyamine.

    Mushroom body Kenyon cells are required during mating and during choice behavior for the polyamine preference of mated females. Activation cVA responsive PAM-b1 neurons of the mushroom body is sufficient to replace the mating experience and change polyamine preference in virgin flies. Activation of the same neurons during mating abolishes the preference development. Other specific mushroom body neurons are required during the choice behavior to promote attraction in mated females or repress it in virgins. Calcium imaging of different mushroom body neurons does not uncover a clear difference in polyamine response between mated and virgin flies. Connectome mining and genetic silencing further indicates that circuit motifs in the lateral horn are also involved in the response to polyamines and might interact with mushroom body circuits.

    While the exact circuits and mechanisms of plasticity that explain the change in postmating preference of polyamines remain to be discovered, this work makes substantial progress in identifying neurons that have a strong impact on development or expression of the preference. It is an exciting paradigm that invites further research.

    This work explores a very interesting example of state-dependent behavioral change in Drosophila. Previously, state dependent changes in sensory neurons have been demonstrated- here, the authors tackle the experimentally much more challenging task of identifying changes in higher order processing areas. The data suggests that polyamine attraction is encoded by a recurrent network of mushroom body neurons. Although the authors do not demonstrate an exact mechanism by which mating/male exposure reconfigures this polyamine attraction network, they have made a substantial advance for our understanding how odor valence is encoded in a flexible and experience dependent way by identifying and characterizing the neuronal players and their roles in induction and expression of preference behavior.

    Their experimental paradigm is special in that it is not a case of classical odor reward learning (mating could be the rewarding experience, but polyamine odor does not have to be present during mating to induce preference). It is also special in that it is a case of long lasting mating induced behavior change that is not dependent on sex peptide or other male seminal fluid proteins. The paradigm has thus great potential to uncover novel mechanisms of encoding experience and adaptively changing behavior.

    Reviewer 3

    Mating changes behavior of female fruit flies. Authors previously reported that putrescine-rich foods increase number of progenies per mated female and mated females detect putrescine with IR76b and IR41a and are attracted to putrescine odor (Hussain, Zhang et al., 2016). In another paper, authors reported that this change of putrescine preference is mediated by sex peptide receptor (SPR) and its ligand, myoinhibiotry peptides (MIPs; Hussain, Ucpunar et al., 2016). In yet another paper, authors reported that two types of dopaminergic neurons (DANs) which innervate alpha prime 3 (a'3) or beta prime 1 (b'1) compartment of the mushroom body (MB) show enhanced response to cVA, the male sex pheromone 11-cis-Vaccenyl acetate (Siju et al., 2020). The present study investigated neural circuits that potentially link these observations.

    The authors first showed that putrescine-attraction in mated females is sustained over 7-days, which cannot be explained by SPR-MIP dependent mechanism that disappears in one week. Then they explored a factor that is transferred from males during copulation and required for putrescineattraction in mated females. They found that blocking synaptic transmission of cVA-sensitive OR67d olfactory receptor neurons during 24 hour period of pairing with males reduces putrescineattraction 3-5 days later (Figure 1). On the other hand, experiments with mutant flies lacking ability to generate eggs or sperms indicated that fertilization is not essential for the change in odor preference. In a proposed scenario, cVA transferred to the female during copulation activates DANs projecting to the b'1 and that in turn induces a shift in how the MB regulates the expression of polyamine odor preference, possibly by alternating activity of MB output neurons (MBONs) in the beta prime 2 (b'2) compartment.

    Some data are in line with this scenario. Blocking synaptic transmissions of Kenyon cells during mating or odor preference test reduced attraction to putrescine (Figure 2). Activation of dopaminergic neurons projecting to the beta prime 1, gamma 3 and gamma 4 in virgin females promoted attraction to putrescine when tested 3-5 days later (Figure 3). Flies expressing shibire ts1 in the MBONs in the b'1 compartment showed reduced putrescine preference when females were mated at restrictive temperature (Figure 4). Using calcium imaging and EM connectome, authors also found candidate lateral horn output neurons that may mediate putrescine signals from olfactory projection neurons to the b'1 DANs.

    This study utilized molecular genetic tools, behavioral experiments and calcium imaging to comprehensively investigate neural circuits from sensory neurons for cVA or putrescine to the learning circuits of the MB. Addressing points detailed below will strengthen a causal link between enhanced cVA response in beta prime 1 DANs and enhanced putrescine preference in mated females.

    1. The MB is the center for olfactory associative learning. It is not so surprising that 24-hour long activation of any MB cell types have long-term consequence on fly's odor preference. As authors showed in Hussain et al., 2016 and Figure S1, mated females change preference to polyamines but not ammonium. Therefore, it is important to show odor specificity of the circuit manipulations to claim that phenomenon in mated females are recapitulated by each manipulation. Wang et al., 2003 (DOI:https://doi.org/10.1016/j.cub.2003.10.003) reported that blocking a broad set of Kenyon cells impairs innate odor attraction to fruit odors and diluted odors but not repulsion.

    We very much appreciate the thorough comments of this reviewer. We have carried out the experiments suggested in the editor’s summary. Due to time and people limitations encountered by the lab’s move during the week of July 11, we were forced to prioritize the number and type of experiments we carried out for this revision.

    We also agree that the change in odor preference due to manipulation of KCs during test is not a very surprising result. We do, however, strongly believe, that the result we received with the inhibition of KCs during mating is not expected. Previous studies using associative learning paradigms suggested that KCs are not essential during learning but only during test:

    • McGuire, S. E., Le, P. T. & Davis, R. L. The Role of Drosophila Mushroom Body Signaling in Olfactory Memory. Science 293, 1330–1333 (2001).

    • Schwaerzel, M., Heisenberg, M. & Zars, T. Extinction Antagonizes Olfactory Olfactory Memory at the Subcellular Level. Neuron 35, 951–960 (2002).

    • Dubnau, J., Grady, L., Kitamoto, T. & Tully, T. Disruption of neurotransmission in Drosophila mushroom body blocks retrieval but not acquisition of memory. Nature 411, 476–480 (2001).).

    Only a very recent study (currently only on BioRXiv, Pribbenow et al. 2022 (https://www.biorxiv.org/content/10.1101/2021.07.01.450776v2) showed that KC output is required during appetitive training suggesting that postsynaptic plasticity in KCs is needed to establish appetitive memories.

    These new findings are in line with our results given that the KCs are likely providing the odor input to DANs and MBONs. We have included a paragraph in the discussion section.

    1. Requirement of PAM-b'1 DANs for putrescine-attraction in mated females should be demonstrated. The authors suggested existence of alternative mechanisms that may mask requirement of PAM-b'1 (Figure 3B). In a previous study, the authors reported SPR-dependent mechanism. I suggest testing the requirement of PAM-b'1 DANs in SPR mutant background or oneweek after mating when SPR-dependent effect on sensory neurons disappear.

    Please see response above to point 4 of the editorial summary. SPR mutants do not undergo the switch in polyamine odor preference. Therefore, SPR signaling likely presents this compensatory mechanism. Nevertheless, MBON-β’1 is required during mating for the transition from virgin to mated female behavior. In the future, we plan to analyze the relationship between SPR and this MBON in detail.

    1. Activation phenotype of MB188B-split-GAL4/UAS-dTrpA1 cannot be ascribed to activation of PMA-b'1 alone because of additional expression in DANs projecting to gamam3 and gamma4 compartments. Run the same experiment with more PMA-b'1 specific driver line.

    Please see response to point 3 of the editorial summary. We do observe a very high preference in virgins with the genetic background MB025B>TrpA1 even in the absence of temperature-mediated activation. Therefore, the experiment, unfortunately, provided no meaningful result. We have instead adjusted the text to include a possible role of γ3 and γ4.

    1. Some of EM connections are too low to be considered (e.g. two in Figure S3 and five in Figure 5). Although these connections could be functional, previous EM connectome analysis typically set much higher threshold (e.g. 10 in Hulse et al., 2021 DOI: 10.7554/eLife.66039) to avoid considering artifacts.

    We thank the reviewer for pointing this out. We have included this reference and the customary threshold of 10 in the methods section.

    1. Data for Kenyon cells (Figure 2) and LHON (Figure 6) are interesting, but not directly related to other data regarding PAM-b'1 and MBON-b'1. Due to lack of long-term changes in MBON's odor responses in mated females (Figure 5), it is unclear what information needs to be read out from Kenyon cells and how does it affect processing of putrescine signals potentially carried by LHAD1b2.

    We agree. In the revised version of the manuscript, we now show that LHAD1b2 neurons appear to undergo a change upon mating. Please see response to the editor’s summary, point 6.

    Kenyon cell output during mating could be required for odor input and odor (i.e. cVA)-mediated activation of MBONs and DANs involved. This would be in line with our data in Fig. 1D,E where we show that ORCO and OR67d OSNs are required during mating to induce the change in behavior.

  11. Version published to 10.1101/2021.07.24.453623v3 on bioRxiv
  12. Version published to 10.1101/2021.07.24.453623v2 on bioRxiv
  13. Version published to 10.1101/2021.07.24.453623v1 on bioRxiv