Orco regulates the circadian activity of pheromone-sensitive olfactory receptor neurons in hawkmoths

  1. Aditi Vijayan
  2. Mauro Forlino
  3. Yajun Chang
  4. Pablo Rojas
  5. Katrin Schröder
  6. Anna C Schneider
  7. Martin E Garcia
  8. Monika Stengl

  • Curated by eLife

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    eLife Assessment

    This valuable study uses technically challenging long-term in vivo recordings and computational modeling to investigate whether hawkmoth olfactory receptor neurons show circadian modulation of spontaneous firing. The study provides solid evidence that ORNs have a circadian pattern of spontaneous firing that is Orco-dependent although Orco transcript abundance does not itself show circadian rhythmicity, and that cAMP can modulate Orco-dependent activity. Together with the computational work, the study proposes the provocative hypothesis that an Orco-centered post-translational feedback loop generates the circadian rhythm of spontaneous firing. Nevertheless, this mechanistic interpretation will require direct testing in future work.

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Abstract

The mating behavior of nocturnal Manduca sexta hawkmoths is under strict temporal control. It is orchestrated via circadian and ultradian oscillations in sex-pheromone stimuli as social Zeitgeber. The extremely sensitive pheromone-detecting olfactory receptor neurons (ORNs) that innervate the long trichoid sensilla on the male’s antennae are peripheral circadian clocks. They express the transcriptional-translational feedback loop (TTFL) circadian clockwork, best characterized in Drosophila melanogaster. In hawkmoths, it is still unknown whether or how the ORN TTFL clockwork regulates the daily rhythms in pheromone sensitivity and in temporal resolution of ultradian pheromone pulses as prerequisites to the temporal regulation of hawkmoth mating behavior. We hypothesize that, rather than the slow TTFL clock, a more rapidly adaptive post-translational feedback loop (PTFL) clockwork, assembled in a signalosome in the ORN plasma membrane, allows for temporal control of pheromone detection via generation of multiscale endogenous membrane potential oscillations. The potential oscillations of the PTFL clock could rapidly synchronize to oscillations of pheromone stimuli at different time scales, thus enabling the prediction of stimulus patterns as a mechanism for active sensing. With in vivo long-term tip recordings of long trichoid sensilla of male hawkmoths, we analyzed the spontaneous spiking activity indicative of the ORNs’ endogenous membrane potential oscillations. Consistent with our hypothesis of a multiscale PTFL clock in hawkmoth ORNs, spontaneous spiking was modulated on ultradian and circadian time scales, with maximum activity at night. When we blocked the evolutionarily conserved olfactory receptor coreceptor (Orco), the circadian modulation was abolished but the ultradian frequency modulation of the spontaneous activity remained. Consistent with PTFL control, Orco was not under the transcriptional control of the TTFL clock, but its modulation of spontaneous spiking activity was dependent on cAMP. We could replicate the experimental data in a conductance-based computational model of an ORN. In this model, Orco conductance changed as a function of fluctuating 2nd messenger levels. This study demonstrates that a PTFL clock is sufficient to impose a circadian pattern on ORN sensitivity. Future experiments are necessary to further challenge our novel hypothesis.

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  1. Version published to 10.7554/elife.108100.4 on eLife
  2. Version published to 10.7554/elife.108100 on eLife
  3. eLife

    eLife Assessment

    This valuable study uses technically challenging long-term in vivo recordings and computational modeling to investigate whether hawkmoth olfactory receptor neurons show circadian modulation of spontaneous firing. The study provides solid evidence that ORNs have a circadian pattern of spontaneous firing that is Orco-dependent although Orco transcript abundance does not itself show circadian rhythmicity, and that cAMP can modulate Orco-dependent activity. Together with the computational work, the study proposes the provocative hypothesis that an Orco-centered post-translational feedback loop generates the circadian rhythm of spontaneous firing. Nevertheless, this mechanistic interpretation will require direct testing in future work.

  4. eLife

    Joint Public Review:

    [Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

    The revised manuscript is much clearer, and the additional analyses address several of the original concerns. RAIN analyses (Rhythmicity Analysis Incorporating Nonparametric methods) now detects circadian rhythmicity in 7/11 recordings under light-dark conditions and 8/12 recordings in constant darkness, compared with 2/12 following treatment with the Orco antagonist. This supports circadian modulation of spontaneous firing and a role for Orco in its normal expression. The expanded qPCR analysis of Orco also supports the conclusion that Orco transcript abundance is not circadian, and the cAMP experiment shows that cAMP can modulate Orco-dependent activity.

    The remaining issue concerns the mechanistic interpretation. The lack of rhythmic Orco transcript abundance does not distinguish an autonomous post-translational feedback-loop (PTFL) clock from a model in which the canonical transcriptional-translational (TTFL) clock acts upstream through cAMP, calcium, kinases, phosphatases, channel trafficking, or related pathways to regulate Orco.

    Similarly, the new Figure 10 provides a useful representation of the authors' hypothesis, but the proposed delayed feedback and coupling mechanisms are not experimentally demonstrated.

    We do not think any further experiments are necessary for the present study. Instead, we recommend that the manuscript should clearly distinguish between what the data show and what remains proposed. The data support circadian modulation of ORN firing a role for Orco in its normal expression, non-circadian Orco transcript abundance, and cAMP-sensitive modulation of Orco-dependent activity. The proposal that an Orco-centred membrane feedback loop generates the rhythm is intriguing and may remain a hypothesis generated through this study that needs formal testing in the future. This should be explicitly stated. While this has been done in the discussion section, elsewhere, including in the abstract and elsewhere, the original claim remains.

  5. eLife

    Author response:

    The following is the authors’ response to the previous reviews.

    The revised manuscript is much clearer, and the additional analyses address several of the original concerns. RAIN analyses (Rhythmicity Analysis Incorporating Nonparametric methods) now detects circadian rhythmicity in 7/11 recordings under light-dark conditions and 8/12 recordings in constant darkness, compared with 2/12 following treatment with the Orco antagonist. This supports circadian modulation of spontaneous firing and a role for Orco in its normal expression. The expanded qPCR analysis of Orco also supports the conclusion that Orco transcript abundance is not circadian, and the cAMP experiment shows that cAMP can modulate Orco-dependent activity.

    The remaining issue concerns the mechanistic interpretation. The lack of rhythmic Orco transcript abundance does not distinguish an autonomous post-translational feedback-loop (PTFL) clock from a model in which the canonical transcriptional-translational (TTFL) clock acts upstream through cAMP, calcium, kinases, phosphatases, channel trafficking, or related pathways to regulate Orco.

    Similarly, the new Figure 10 provides a useful representation of the authors' hypothesis, but the proposed delayed feedback and coupling mechanisms are not experimentally demonstrated.

    We do not think any further experiments are necessary for the present study. Instead, we recommend that the manuscript should clearly distinguish between what the data show and what remains proposed. The data support circadian modulation of ORN firing a role for Orco in its normal expression, non-circadian Orco transcript abundance, and cAMP-sensitive modulation of Orco-dependent activity. The proposal that an Orco-centred membrane feedback loop generates the rhythm is intriguing and may remain a hypothesis generated through this study that needs formal testing in the future. This should be explicitly stated. While this has been done in the discussion section, elsewhere, including in the abstract and elsewhere, the original claim remains.

    We thank the referees and the editors for their efforts and their positive feedback. As requested, we clarified throughout our manuscript that our proposal of an Orco-centered post-transcriptionally controlled feedback loop in the plasma membrane, which we term PTFL clock, is a novel hypothesis that needs to be tested in future experiments.

  6. Version published to 10.7554/elife.108100.3 on eLife
  7. eLife

    eLife Assessment

    This study uses a technically challenging long-term in vivo recordings and computational modeling to investigate whether hawkmoth olfactory receptor neurons show circadian modulation of spontaneous firing. The study provides solid evidence that ORNs have a circadian pattern of spontaneous firing that is Orco-dependent and yet that Orco transcript abundance does express a circadian rhythmicity and that cAMP can modulate all Orco-dependent activity. Together with computational work, the work is valuable in proposing a provocative hypothesis that an Orco-centred post-translational feedback loop generates the circadian rhythm.

  8. eLife

    Joint Public Review:

    The revised manuscript is much clearer, and the additional analyses address several of the original concerns. RAIN analyses (Rhythmicity Analysis Incorporating Nonparametric methods) now detects circadian rhythmicity in 7/11 recordings under light-dark conditions and 8/12 recordings in constant darkness, compared with 2/12 following treatment with the Orco antagonist. This supports circadian modulation of spontaneous firing and a role for Orco in its normal expression. The expanded qPCR analysis of Orco also supports the conclusion that Orco transcript abundance is not circadian, and the cAMP experiment shows that cAMP can modulate Orco-dependent activity.

    The remaining issue concerns the mechanistic interpretation. The lack of rhythmic Orco transcript abundance does not distinguish an autonomous post-translational feedback-loop (PTFL) clock from a model in which the canonical transcriptional-translational (TTFL) clock acts upstream through cAMP, calcium, kinases, phosphatases, channel trafficking, or related pathways to regulate Orco.

    Similarly, the new Figure 10 provides a useful representation of the authors' hypothesis, but the proposed delayed feedback and coupling mechanisms are not experimentally demonstrated.

    We do not think any further experiments are necessary for the present study. Instead, we recommend that the manuscript should clearly distinguish between what the data show and what remains proposed. The data support circadian modulation of ORN firing a role for Orco in its normal expression, non-circadian Orco transcript abundance, and cAMP-sensitive modulation of Orco-dependent activity. The proposal that an Orco-centred membrane feedback loop generates the rhythm is intriguing and may remain a hypothesis generated through this study that needs formal testing in the future. This should be explicitly stated. While this has been done in the discussion section, elsewhere, including in the abstract and elsewhere, the original claim remains.

  9. eLife

    Author response:

    The following is the authors’ response to the previous reviews

    eLife Assessment

    This valuable study uses technically compelling long-term in vivo recordings and computational modeling to investigate whether hawkmoth olfactory receptor neurons show circadian modulation of spontaneous firing. The authors further propose the provocative model that post-translational mechanisms, rather than the transcriptional-translational processes, may contribute to circadian regulation of neuronal excitability.

    We are pleased that our study is recognized as valuable and that our technically very challenging long-term in vivo recordings and computational modeling are appreciated. We agree that we are proposing a provocative model that opposes the current hypothesis in chronobiology, which suggests that all observed biological circadian rhythms are outputs of a transcriptional-translational feedback loop (TTFL) clock. Instead, we suggest that a cell comprises, in addition to the TTFL clock, other posttranslational feedback loop (PTFL) clocks without the need of daily transcription and daily degradation of its core elements. While the circadian TTFL clock is entrained to the daily light-dark cycle, the circadian PTFL clocks are suggested to be entrained to other daily cues such as to the availability of pheromone, or the daily changing levels of hormones and second messenger levels. Our novel hypothesis proposes that the TTFL and PTFL clocks are coupled and linked, constituting an adaptive, plastic network that can tune and phase-lock to different external and internal Zeitgeber signals. However, we certainly do not claim that the TTFL circadian clock is not at all involved in the circadian control of the ORN’s circadian membrane potential rhythms. We clarified our manuscript accordingly.

    However, the evidence for circadian firing in these neurons […] remains incomplete.

    As requested by the reviewers during the previous round of review, we had provided the results of RAIN analysis (Thaben and Westermark, 2014) of individual animals in our first revision (Fig. 4A), which clearly shows that two-thirds of the population express circadian rhythms in key attributes that we used to characterize the spontaneous spiking activity. In the initial review it was assumed by the reviewers that phase alignment of dispersed rhythms would bias interpretations of rhythmicity. After having established with RAIN that individuals show circadian rhythms, albeit dispersed across the population due to the lack of a zeitgeber in DD conditions, phase-alignment of recordings from DD animals is a valid next step to prepare the data for statistical analysis across the population. Phase-alignment of desynchronized rhythms is a generally accepted and necessary method employed in chronobiology (e.g., for insect ORNs: Gosh et al., 2024). It is proven as prerequisite to find and statistically analyze rhythmicity in complex, desynchronized data.

    As we explained in the previous rebuttal and in our first revision, rhythms in electrical activity of insect ORNs cannot be easily synchronized by the light-dark cycle alone, but appear to require daily cycles of pheromone, as shown in other moth species (Gosh et al., 2024). Please be aware that the animals that we used here have never been exposed to pheromone, as we state in the Methods. Therefore, this lack of pheromone exposure can explain why about one third of our experimental population is not expressing any daily or circadian rhythmicity in spiking attributes. This is an important result of our manuscript, reported for the first time for Manduca sexta, providing evidence for our hypothesis that it is not the LD-entrained circadian TTFL clock that governs electrical activity rhythms in ORNs. As we explained in the first revision, and clarified further here in the second revision, we cannot phase-synchronize our animals with cycles of pheromone application in our experimental paradigm because we are researching circadian rhythms in spontaneous spiking activity and not pheromone responses. We failed to obtain phase-alignment with a single pheromone pulse the night before the experiments started. These data were added as supplementary Figure to Fig. 3 in the first revision. Here, we further revised our manuscript to clarify this important finding.

    Thus, as requested by the reviewers in the initial review, we could successfully confirm our previous results of circadian firing in ORNs and the disruption of these circadian rhythms with Orco antagonist OLC15 with RAIN. In the current review, the reviewers raise no further specific critical points or comments that would doubt our careful rhythm analysis of our long-term recordings. Thus, we conclude that we provided clear evidence for our central, exciting new finding. For the first time we demonstrated an unexpected new task for Orco: Orco controls the circadian firing pattern in the spontaneous activity, and thus, of the ORN’s membrane potential, via its property as leak/pacemaker channel.

    However, the evidence […] for post-translational modification of Orco as the underlying mechanism remains incomplete.

    We agree with the reviewers that there are many more experiments and combined efforts of biochemists, structural biologists, and electrophysiologists required to provide complete evidence for post-translational modification of Orco and to reveal the underlying mechanism of its circadian control. It is beyond the scope of the current manuscript to provide all details of post-translational control of Orco.

    The reviewers asked previously for additional evidence that Orco transcription is not controlled via the TTFL clock. As requested, we provided extended qPCR–based evidence that Orco, in contrast to timeless, is not controlled by the TTFL clock on the transcriptional level (Fig. 6 in Revision 1). Furthermore, we added a new result in Revision 1 to demonstrate cAMP-dependent post-translational modulation of Orco open-time probability (Fig 9 in Revision 1) at a ZT at which antennal cAMP levels are low (Schendzielorz et al., 2015). We already showed in Flecke et al., 2010, that the addition of cAMP at different ZTs increases the spontaneous spiking activity only at specific ZTs. Here, we show that the effect of cAMP depends on Orco. Since Orco’s circadian role is not mediated via TTFL control, it can be concluded that post-translational mechanisms provide daily/circadian temporal control. In this additional Figure we provide statistically significant proof that, in agreement with our model-prediction, Orco’s circadian control of the ORN spontaneous activity could be mediated via the second messenger cAMP. ZT-dependent input for Orco would be provided via daily changes in cAMP levels (Schendzielorz et al., 2015).

    In contrast, the study does provide strong evidence that the application of cyclic nucleotides can modulate Orco-dependent activity at a single time point, and reports that the temporal pattern of Orco transcript abundance is not circadian.

    We appreciate that the reviewer confirms that our revised manuscript with additional experiments now provides strong evidence that cAMP modulates Orco-dependent spontaneous activity of M. sexta ORNs. Since we already published that cAMP levels expresses daily rhythms in M. sexta antennae (Schendzielorz et al., 2015), and in vivo cAMP infusion increases spontaneous activity and sensitizes pheromone detection (Flecke et al., 2010), and our computational model here proves that circadian modulation of open time probability of Orco is sufficient to explain our experimental data, it is sufficient for our conclusions to test just the one specific zeitgeber time when endogenous cAMP levels are low and pharmacological cAMP increase has the strongest impact. To further reveal complete ZT-dependence of cAMP modulation of Orco´s control of spontaneous activity is beyond the scope of the current manuscript and not part of the current research question. The structure of Orco is extraordinarily conserved during evolution, thus, the cited experimental results from other laboratories and other species showing that Orco is a hub for posttranslational modification are very likely generalizable to different insect species. We clarified the manuscript accordingly. In Drosophila, Orco has at least 5 phosphorylation sites for protein kinase C (PKC), is cAMP-dependently sensitized, and has a Ca2+/calmodulin binding site that orchestrates the localization of the OR-Orco heteromer to the cilia. However, in fruit fly and other insects, so far, it can only be speculated how circadian control is provided for Orco, since there are no other publications that examine the circadian regulation of Orco in detail. We clarified our manuscript accordingly.

    To summarize, the logical conclusion based on our newly provided data is that the current hierarchical hypothesis in chronobiology based solely on a circadian TTFL clock that controls Orco transcription does not explain our findings in hawkmoth ORNs. Therefore, we suggest a new systemic hypothesis based upon coupled TTFL and PTFL circadian clocks that can also reconcile otherwise inconsistent data published for insect and mammalian circadian clocks (please see reviewed data in: Stengl and Schneider, 2024). We clarified our manuscript in the second revision and added a new Figure 10 to illustrate our novel hypothesis.

    However, the findings are incomplete to exclude a role for transcriptional-translational mechanisms and their associated multi-layered controls in circadian regulation.

    We certainly do not imply excluding a role for the TTFL clock in (indirectly) affecting circadian control of the membrane potential of ORNs. The new qPCR experiments added in the first revision clearly show that the circadian control mediated via Orco is not an output of the TTFL clock via transcriptional control of Orco. Instead, we predict links between a PTFL membrane clock comprising Orco as hub to integrate posttranslational control and the TTFL nuclear clock. We clarified our manuscript accordingly, adding a new Figure 10 to further illustrate and visualize our hypothesis. The predictions of this systemic hypothesis will be challenged in further experiments that, however, are beyond the scope of the current manuscript.

    Joint Public Review:

    This manuscript puts forward the provocative idea that a posttranslational feedback loop regulates daily and ultradian rhythms in neuronal excitability. The authors used in vivo long-term tip recordings of the long trichoid sensilla of male hawkmoths to analyze spontaneous spiking activity indicative of the ORNs' endogenous membrane potential oscillations. This firing pattern was disrupted by pharmacological blockade of the Orco receptor. They then use these recordings together with computational modeling to predict that Orco receptor neuron (ORN) activity is required for circadian, not ultradian, firing patterns. Orco did not show a circadian expression pattern in a qPCR experiment, and its conductance was proposed to be regulated by cyclic nucleotide levels. This evidence led the authors to conclude that a post-translational feedback loop (PTFL) clockwork, associated with the ORN plasma membrane, allows for temporal control of pheromone detection via the generation of multi-scale endogenous membrane potential oscillations. The findings will interest researchers in neurophysiology, circadian rhythms, and sensory biology. However, the manuscript has limited experimental evidence to support its central hypothesis and is undermined by several assumptions that underlie their data analysis and model builds, as well as insufficient biological data including critical controls to validate and/or fully justify the model the authors are proposing.

    We want to remind our reviewers that we used “ORN” as abbreviation for olfactory receptor neuron (= sensory receptor neurons, a.k.a. olfactory sensory neuron (OSN)) and not for Orco receptor neuron, although we focus on the function of Orco. Accordingly, our central finding is that Orco as ion channel is required for the daily/circadian modulation of spontaneous action potential activity generated by the olfactory receptor neurons in the absence of pheromone stimulation.

    We do not understand the specific basis for the conclusions of the reviewers. Therefore, we ask to please specify what experimental evidence is missing to support our central hypothesis that Orco is not directly TTFL- but PTFL clock-controlled, and to name specifically what the “several assumptions” are that undermine our careful data analysis and model builds. Which specific argument in our previous rebuttal was wrong, was not conclusive? Furthermore, please specify your claim that “critical controls are missing”. Which controls are missing for which experiments? We did add a new figure panel in the first revision to demonstrate that neither the addition of DMSO (the OLC15 solvent) nor the repeated attachment of the recording electrode, which was necessary to obtain paired datasets, altered the spontaneous spiking activity (Fig 1B in Revision 1). Furthermore, we expanded the time series of qPCR data and added tim as positive control to Orco (Fig 7), strengthening our argument that Orco expression is not under TTFL control. Dose-response curves of various Orco agonists and antagonists have been published before (see our references in Revision 1) and are therefore not repeated here.

    Our newly added data confirm what our modeling predicted: cAMP increases spontaneous ORN activity dependent on Orco. Previous publications provide evidence for daily rhythms in cAMP concentrations in hawkmoth antennae (Schendzielorz et al., 2012).

    As is true for any other hypothesis, a hypothesis can only be falsified but not validated and needs to be tested by many experiments from many laboratories over a long time until it will be replaced by the next hypothesis that better explains accumulating contradicting evidence. We are very much looking forward to experimental challenges of our provocative new hypothesis by colleagues in the field of olfaction and of chronobiology. We are convinced that our manuscript will greatly stimulate the field, possibly provoking a paradigm switch in chronobiology and in olfactory research.

    Strengths:

    The authors raise several intriguing model-based hypotheses regarding the mechanisms that underlie the generation of olfactory rhythms. The electrophysiological approach and the long-term recording paradigm are elegant and technically impressive. In the revised version, the authors have added additional qPCR data supporting the lack of rhythmic Orco transcript expression and included a new figure suggesting that cAMP can modulate Orco conductance.

    We thank the reviewers for their acknowledgement of our careful work and hope that our further revisions and clarifications help to argue our case.

    Major weaknesses:

    (1) The cAMP experiment was only conducted at one time-point, which is insufficient to support the central claim that "AMP and cGMP may have ZT-dependent effects on Orco conductivity".

    We agree with the reviewers and revised our discussion accordingly to clarify that in this manuscript it is not our central claim that cAMP and cGMP may have ZT-dependent effects on Orco conductivity. Instead, our data show for the first time that Orco controls circadian rhythms of spontaneous activity of ORNs and that the circadian rhythmicity of spontaneous activity is lost when Orco is blocked. Therefore, we provide novel experimental evidence that Orco is a prerequisite to the circadian rhythmicity of spontaneous activity and thus, to circadian rhythms in the membrane potential of ORNs. Furthermore, as requested by the reviewers we provided clear evidence in the first revision that Orco is not controlled at the transcriptional level by the TTFL clock, in contrast to the TTFL clock protein TIMELESS. Thus, it follows logically that Orco is under post-transcriptional control. Since cAMP levels show circadian oscillations and Orco is gated by cAMP (Fig 9 in Revision 1), we used our computational model to show that a cAMP-dependent increase in Orco conductance alone, via daily oscillating concentrations of cAMP, is sufficient to explain our findings. Therefore, we propose here that daily/circadian oscillations of cAMP modify spontaneous spiking activity via Orco on a posttranscriptional level. But it certainly does not provide all evidence for respective mechanisms of how this cAMP modulation of Orco is obtained, since this is beyond the scope of the current manuscript.

    Since we realized that it is difficult for our readers to visualize a circadian PTFL membrane clock we added a new hypothesis-Figure (Figure 10) and considerably focused and clarified especially the discussion of our manuscript. We pointed out that a membrane-associated signalosome that comprises delayed negative feedback mechanisms, and, thus, constitutes an oscillator, a “membrane clock” that generates oscillations. Based upon our data we propose a membrane-associated signalosome constituting a PTFL circadian clock with Orco as central element. This PTFL membrane clock generates superimposed ultradian and circadian rhythms in its outputs: rhythms in the membrane potential, Ca2+, and cAMP levels. The PTFL clock comprises positive feedforward elements that upregulate its outputs, resulting in more depolarization, higher Ca2+- and higher cAMP levels. Via the clock’s delayed negative feedback mechanisms these outputs are downregulated, again, resulting in hyperpolarization, decreasing Ca2+- and cAMP levels. This signalosome comprises the pacemaker channel Orco as a central hub that is controlled via changes in voltage, Ca2+, and cAMP levels. Nevertheless, we predict coupling between the multiscale PTFL membrane clock and the TTFL circadian clock in the nucleus to obtain stable circadian rhythms. As likely mechanism of coupling we predict that Ca2+- and cAMP-dependent kinases interlink both types of clocks, thereby obtaining robust and at the same time flexible interlinked cellular rhythms.

    We hope to now successfully clarify and to visualize our central hypothesis of our manuscript that Orco is not directly controlled by a TTFL circadian clock but is a central element of a membrane-associated posttranslational feedback loop clock (PTFL) clock that is linked to but not forced by the TTFL clock which is predicted to control intracellular Ca2+ homeostasis in a circadian rhythm.

    (2) The revised manuscript continues to rely heavily on prior publications or defers key mechanistic questions (or important manipulations) to future studies. In its current form, the evidence presented remains insufficient to support the central claim that a PTFL constitutes the primary underlying circadian clock mechanism. The proposed model is intriguing, but the data provided do not yet directly demonstrate the novel mechanism.

    We do not understand why the reviewers considers it to be problematic that we “continue to rely heavily on prior publications”. Certainly, we built upon previous publications of our lab as well as on manifold experimental data published by other laboratories in the field of insect olfaction. Our ample citations demonstrate that we have an overview both of the current state of literature and relevant previous literature, dating back to the very first experiments that pioneered pheromone transduction in insects. Based upon our extensive knowledge and experimental data collected in insect olfaction and based on very careful, critical, rigorous analysis of our data and data published by others, we were able to come up with a novel interpretation of the current literature about insect olfaction that differs considerably from the current main views. We consider this to be our strength and judge it as good scientific practice and not a flaw of our work. However, since we do not focus on OR-Orco heteromers and their function in pheromone/odor transduction in the cilia in the current manuscript, we considerably shortened this part of the discussion, avoiding pointing out that highly sensitive moth pheromone transduction greatly differs from less sensitive general odor transduction in Drosophila. Furthermore, since here we focus on cAMP, but not on cGMP-dependent modulation of Orco, we also deleted/considerably shortened this part of our discussion.

    We certainly agree with the reviewers that, while the data provided in the current manuscript are a logical basis for developing our novel hypothesis, they are not a direct and sufficient demonstration of proof and we are not able yet to directly demonstrate and explain the novel mechanism predicted. We would like to point out that if we provided this final proof, it would not be any more a novel hypothesis, but only a novel finding.

    We agree with the reviewers that our provocative hypothesis requires rigorous testing by many further experiments, hopefully not only by our laboratory, but hopefully stimulating new experimental challenges by other laboratories employing different species. But certainly, these experiments with proof-of-principle will take many years and are beyond the scope of our current research paper.

    As per eLife’s assessment system we would like to ask the reviewers to provide detailed feedback as to which experiments/results within the scope of this manuscript would complete this work, or how they think this study should be framed in the light of the results that we obtained. Nevertheless, we hope that with our current careful review the reviewers will be more convinced by our arguments and experiments as valid basis for our provocative new hypothesis.

  10. Version published to 10.1101/2025.06.17.659282v5 on bioRxiv
  11. Version published to 10.1101/2025.06.17.659282v4 on bioRxiv
  12. Version published to 10.7554/elife.108100.2 on eLife
  13. eLife

    eLife Assessment

    This valuable study uses technically compelling long-term in vivo recordings and computational modeling to investigate whether hawkmoth olfactory receptor neurons show circadian modulation of spontaneous firing. The authors further propose the provocative model that post-translational mechanisms, rather than the transcriptional-translational processes, may contribute to circadian regulation of neuronal excitability. However, the evidence for circadian firing in these neurons, and for post-translational modification of Orco as the underlying mechanism, remains incomplete. In contrast, the study does provide strong evidence that the application of cyclic nucleotides can modulate Orco-dependent activity at a single time point, and reports that the temporal pattern of Orco transcript abundance is not circadian.

  14. eLife

    Joint Public Review:

    This manuscript puts forward the provocative idea that a posttranslational feedback loop regulates daily and ultradian rhythms in neuronal excitability. The authors used in vivo long-term tip recordings of the long trichoid sensilla of male hawkmoths to analyze spontaneous spiking activity indicative of the ORNs' endogenous membrane potential oscillations. This firing pattern was disrupted by pharmacological blockade of the Orco receptor. They then use these recordings together with computational modeling to predict that Orco receptor neuron (ORN) activity is required for circadian, not ultradian, firing patterns. Orco did not show a circadian expression pattern in a qPCR experiment, and its conductance was proposed to be regulated by cyclic nucleotide levels. This evidence led the authors to conclude that a post-translational feedback loop (PTFL) clockwork, associated with the ORN plasma membrane, allows for temporal control of pheromone detection via the generation of multi-scale endogenous membrane potential oscillations. The findings will interest researchers in neurophysiology, circadian rhythms, and sensory biology. However, the manuscript has limited experimental evidence to support its central hypothesis and is undermined by several assumptions that underlie their data analysis and model builds, as well as insufficient biological data including critical controls to validate and/or fully justify the model the authors are proposing.

    Strengths:

    The authors raise several intriguing model-based hypotheses regarding the mechanisms that underlie the generation of olfactory rhythms. The electrophysiological approach and the long-term recording paradigm are elegant and technically impressive. In the revised version, the authors have added additional qPCR data supporting the lack of rhythmic Orco transcript expression and included a new figure suggesting that cAMP can modulate Orco conductance.

    Major weaknesses:

    (1) The cAMP experiment was only conducted at one time-point, which is insufficient to support the central claim that "AMP and cGMP may have ZT-dependent effects on Orco conductivity".

    (2) The revised manuscript continues to rely heavily on prior publications or defers key mechanistic questions (or important manipulations) to future studies. In its current form, the evidence presented remains insufficient to support the central claim that a PTFL constitutes the primary underlying circadian clock mechanism. The proposed model is intriguing, but the data provided do not yet directly demonstrate the novel mechanism.

  15. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Joint Public Review

    This manuscript puts forward the provocative idea that a posttranslational feedback loop regulates daily and ultradian rhythms in neuronal excitability. The authors used in vivo long-term tip recordings of the long trichoid sensilla of male hawkmoths to analyze spontaneous spiking activity indicative of the ORNs' endogenous membrane potential oscillations. This firing pattern was disrupted by pharmacological blockade of the Orco receptor. They then use these recordings together with computational modeling to predict that Orco receptor neuron (ORN) activity is required for circadian, not ultradian, firing patterns. Orco did not show a circadian expression pattern in a qPCR experiment, and its conductance was proposed to be regulated by cyclic nucleotide levels. This evidence led the authors to conclude that a post-translational feedback loop (PTFL) clockwork, associated with the ORN plasma membrane, allows for temporal control of pheromone detection via the generation of multi-scale endogenous membrane potential oscillations. The findings will interest researchers in neurophysiology, circadian rhythms, and sensory biology. However, the manuscript has limited experimental evidence to support its central hypothesis and is undermined by several questionable assumptions that underlie their data analysis and model builds, as well as insufficient biological data, including critical controls to validate and/or fully justify the model the authors are proposing.

    We thank the reviewers for their thorough and thoughtful comments and believe that the manuscript is much stronger now after the revision which incorporates the requested changes. We added results of new experiments and additional analyses. Although these new insights did not change the previous conclusions, we significantly reworked the Discussion and added further references to clarify the conclusions we want to make.

    Please note that we used ORN as acronym for “olfactory receptor neuron” throughout the manuscript. ORNs contain odorant receptors (ORs), and in insects these ORs associate with the olfactory receptor co-receptor (Orco) to be trafficked to the membrane of the cilium of the ORN, where they can be contacted by pheromones and odorants. In Manduca sexta, evidence is accumulating for G-protein coupled metabotropic pheromone transduction and not for OR-Orco dependent ionotropic transduction, as shown for Drosophila melanogaster. In both insect species, besides its chaperone function, Orco can form leaky cation channels, which can regulate the spontaneous spiking activity of ORNs. In this study, we explored this role of Orco.

    Strengths:

    The study is notable for its combination of long-term in vivo tip recordings with computational modeling, which is technically challenging and adds weight to the authors' claims. The link between Orco, cyclic nucleotides, and circadian regulation is potentially important for sensory neuroscience, and the modeling framework itself - a stochastic Hodgkin-Huxley formulation that explicitly incorporates channel noise - is a solid and forward-looking contribution. Together, these elements make the study conceptually bold and of clear interest to circadian and olfactory biologists.

    Major weaknesses:

    At the same time, several limitations temper the conclusions. The pharmacological evidence relies on a single antagonist and concentration, without key controls. The circadian analysis is based on relatively small numbers of neurons, with rhythms detected only in subsets, and the alignment procedure used in constant darkness raises concerns of bias. The molecular evidence is sparse, with only three qPCR timepoints, and the model, while creative, rests on assumptions that are not yet fully supported by in vivo data.

    Please see our responses to the detailed comments.

    Detailed comments are provided below:

    (1) The role for Orco proposed in the authors' model largely stems from the effects seen following the administration of (a single dose) of the Orco antagonist, OLC15. However, this hypothesis is undercut by the lack of adequate pharmacological controls, including a basic multipoint OLC15 dose-response series in addition to the administration of blockers for the other channels that are embedded in their model, but which were ruled out as being involved in the modulation of biological rhythms. In addition, these studies would (ideally) also benefit from the inclusion of the same concentration (series) of an inactive OLC15 analog to better control for off-target effects.

    The Orco agonist VUAA1 (Jones et al., 2011) binds directly to Orco and increases the channel open time probability. In M. sexta hawkmoths, we have already published that VUAA 1 increases the low spontaneous activity of ORNs in a dose-dependent fashion (Nolte et al., 2013). Chen and Luetje (2012) systematically varied the chemical structure of VUAA1 to identify new Orco ligands and discovered 22 Orco ligand candidates (OLCs) that either activated or inhibited Orco. In their heterologous expression system, Orco was most sensitive to inhibition by OLC15. Based on these results, we published a dose-response curve of OLC15 inhibition (1-100 µM) using in vivo tip recordings of pheromone-sensitive long trichoid sensilla of M. sexta (Nolte et al., 2016). There, we also demonstrated that OLC15 dose-dependently antagonizes the VUAA1-dependent activation of Orco.

    Furthermore, we tested other published Orco antagonists, which were characterized in heterologous assays, in primary cell cultures of hawkmoth ORNs, as well as in in vivo assays in intact hawkmoths. We focused on amiloride-derived antagonists, because we previously identified an amiloride-sensitive cation channel in hawkmoth ORNs. We found that, in contrast to OLC15, the amilorides HMA and MIA were not Orco-specific antagonists but instead affected different ion channel targets depending on the time of day (Nolte et al., 2016). Based on those experiments and the dose-response curves we determined that the Orco agonist VUAA1 (Jones et al., 2011) and the Orco antagonist OLC15 (Chen and Luetje, 2012) worked best in hawkmoth ORNs to target Orco pharmacologically. Due to those results and other comparative tests with other published Orco antagonists we settled since then in all further experiments on a dose of 50 µM OLC15 as most adequate to antagonize Orco functions in Manudca. In the current study, we focus on Orco without excluding the possibility that other ion channels in the ORNs contribute to the control of membrane potential rhythms.

    We have clarified the Methods section accordingly.

    (2) The expression pattern of Orco was assessed using qPCR at only three timepoints. Rhythmic transcripts can easily be missed with such sparse sampling (Hughes et al., 2017). A minimum of six evenly spaced timepoints across a 24-hour cycle would be required to confidently rule out circadian transcriptional regulation. In addition, the use of the timeless mRNA control from another study is not acceptable. Furthermore, qPCR analysis measures transcript abundance, not transcription, as the authors repeatedly state. Transcriptional studies would require nuclear run-off or, more recently, can be done with snRNAseq analysis. Taken together, these concerns undermine the authors' desire to rule out TTFL-based control that directly led them to implicate a PTTF-based model.

    We agree with the referees that more time points and a direct comparison between timeless and Orco mRNA levels should be included in this manuscript. We included these additional qPCR experiments and edited the manuscript to make clear that we measure transcript abundance, but we will not perform snRNAseq analysis due to time- and financial constraints.

    (3) The modelling presented is based on Orco as a ZT-dependent conductance tied to the cAMP oscillations that were reported by this group in the cockroach and from the presence and functionality in Manduca of homomeric Orco complexes that are devoid of tuning ORs. While these complexes have been generated in cell culture and other heterologous expression systems, as well as presumably exist in vivo in the Drosophila empty neuron and other tuning OR mutants, there is no evidence that these complexes exist in wild-type Manduca ORNs. While this doesn't necessarily undermine every aspect of their models, the authors should note the presence of Orco/OR complexes rather than Orco homomeric complexes.

    Our ELISAs found circadian oscillations in cAMP levels not only in antennae of the Madeira cockroach (Schendzielorz et al., 2014, 2012), but also in hawkmoth antennae (Schendzielorz et al., 2015). For clarification, we added the 2015 citation to the Modeling chapter in the Methods section.

    We agree with the referees that we cannot distinguish between Orco homo- and heteromers in the different compartments of our hawkmoth ORNs but we know that both are expressed in the pheromone-sensitive ORNs. Thus, as the referee suggests, we added text regarding the presence and localization of OR-Orco heteromers. Consistent data collected across different experiments (heterologous expression systems, primary cell cultures of hawkmoth ORNs, in vivo/in situ studies) support that Orco homomers are present in hawkmoth ORNs. In addition to co-expression of MsexOrco and MsexSNMP-1 with either MsexOr-1 or MsexOr-4 in a heterologous expression system, MsexOrco expression alone was already sufficient to increase intracellular Ca2+ levels spontaneously as a result of its property as leaky, non-specific cation channel, and in response to VUAA1 application (Nolte et al., 2013). Both in developing hawkmoth pupae and differentiating primary cell cultures of hawkmoth ORNs, Orco expression started during a developmental time window where ORNs did not yet express pheromone receptors but where Orco affected spontaneous activity and intracellular Ca2+ levels dependent on VUAA1 (Nolte et al., 2016). In vitro patch clamp studies of differentiating cultured hawkmoth ORNs during this time window of pupal development characterized ion channels/currents with properties of Orco as a leaky, non-specific cation channel/current that depends on protein kinase C and cyclic nucleotides (Dolzer et al., 2021, 2008; Krannich and Stengl, 2008; Stengl, 1993). Thus, Orco homomers are present in developing hawkmoth ORNs during a time window where ORNs already express spontaneous activity but they do not heteromerize with pheromone receptors. However, we do not know whether and in what ratio homo- and heteromers of Orco and ORs are present in the respective sensillum compartments of adult hawkmoths because all OR-specific antibodies tested did not work in immunocytochemical studies of hawkmoth antennae (Nolte et al., 2013; Stengl, 1994; Stengl and Hildebrand, 1990). Our hypothesis of differential distribution of Orco homomers in the some and dendrite compartment, and OR-Orco heteromers in the cilia is based on differential immunocytochemical localization of Drosophila ORs mainly in the cilia compartment (Benton et al., 2006).

    We clarified our manuscript accordingly.

    (4) Some aspects of the authors' models, most notably the decision to phase align/optimize their DD and OLC15 recordings, are likely to bias their interpretations.

    It is consensus that insects display daily and circadian rhythms in pheromone-dependent mating, odor-gated feeding, and egg-laying behavior that phase-locks to environmental rhythms, corresponding with daily/circadian rhythms of sensory neuron physiology (e.g., Merlin et al., 2007; Rymer et al., 2007; Schendzielorz et al., 2015, 2012). However, circadian rhythms can be easily masked by stress, like the disturbances during an experimentally very challenging long-term recording experiment over several days. In addition, we observed over the years in our animal raising facility that in 17:7 light-dark cycles the originally nocturnal hawkmoths M. sexta distribute their activity patterns over the course of the day, finding nocturnal as well as diurnal hawkmoths. Thus, light-dark cycles were not enough to ensure phase-synchronized behavioral rhythms, and it is very likely that the nocturnal hawkmoths, next to stress signals, rely heavily on pheromone/odor dependent synchronization as also found in other moth species (Ghosh et al., 2024). Because we focus on spontaneous activity and not on pheromone-dependent physiology in this study, we used isolated males that were never exposed to the female pheromones, taking phase dispersal into account. Therefore, it became necessary in free-running conditions to first determine the respective behavioral rhythm for each animal, and then to phase-align their activity patterns to allow for statistical analysis. Otherwise, circadian differences would average out in a phase-dispersed free-running population. As requested by the referees in point (7), we added RAIN to test for rhythmicity in each of our recordings and revised the manuscript accordingly.

    Furthermore, in preliminary experiments we briefly exposed hawkmoths to pheromone the night before the start of the experiment. However, we failed to obtain phase-synchronized spiking rhythms. Most likely, a circadian pattern of pheromone exposure would have been necessary as zeitgeber, which could not be used here due to long-term pheromone-dependent effects in spiking activity. These results are added as supplementary figure to Fig 3.

    (5) The tip recordings from long trichoid sensilla are critical aspects of this study. These recordings were carried out on upper sensillar tips located on the distal-most second annulus. Since there are approximately 80 annuli on the Manduca antennae, it is unclear whether the recordings are representative of the antennal response.

    We think the reviewers might have misinterpreted our description of the recording site. In the Methods, we state that we clip off the 20 most distal annuli (leaving a stump of about 60 annuli) and insert the reference electrode into the flagellum up to the second annulus from the cut end, i.e., the recording sites are located at 2/3 – 3/4 of the antenna length as seen from the head of the animal. We clarified this in the Methods section.

    In addition, our lab did show with antibody stainings against Orco that apparently all ORNs that innervate long and short trichoid sensilla along the whole flagellum express the same staining pattern (Nolte et al., 2016). Lee and Strausfeld (1990) mapped all types of antennal sensilla, and together with pheromone-dependent tip-recordings of Kaissling et al. (1989) it was shown that most of the male antennal sensilla are pheromone-sensitive long trichoid sensilla, with one of the two innervating ORNs always responding to bombykal, ensuring high sensitivity to pheromone detection. Furthermore, our patch clamp recordings of primary cell cultures of whole male antennae found largely overlapping ion channel populations across ORNs (review: (Stengl, 2010)). This would indicate that all ORNs, whether they express ORs sensitive to pheromone or general odorants, could potentially share the same Orco-dependent spontaneous activity rhythms. Furthermore, in our lab, different experimenters from different years that recorded from long trichoid sensilla on different annuli did not detect obvious differences in neither the spontaneous activity nor the pheromone responses (c.f., Dolzer et al., 2003; Gawalek and Stengl, 2018; Schneider et al., 2025). Thus, it is very likely that we are reporting a general encoding mechanism that is not locally restricted along the antennal flagellum and is very likely shared by all types of OR-Orco expressing ORNs.

    (6.1) The authors do not provide any data in support of their cAMP/cGMP-based Orco gating…

    There are publications supporting cyclic nucleotide gating of Orco in Drosophila, but only after previous phosphorylation via protein kinase C (PKC; review: (Wicher and Miazzi, 2021)). Since Orco is very conserved among insect species, it is likely that PKC- and cGMP/cAMP-dependent regulations are present for Orco in other insect species. To test this, we are currently characterizing second messenger-dependence of spontaneous spiking activity, which is the focus of a follow-up manuscript. Nevertheless, to provide more evidence for our hypothesis of the current manuscript, we added a new set of tip-recording experiments that demonstrate cAMP-dependent gating of Orco. Because of the addition of this figure, we merged figures 8-10 into Figure 8 and added the cAMP data as Figure 9.

    (6.2) … and the PTTF model proposed is somewhat disappointing.

    For a detailed introduction of our PTFL membrane clock hypothesis please see our opinion paper that we refer to in the manuscript (Stengl and Schneider, 2024). We added clarification of how Orco activation can influence cAMP levels. A more elaborate PTFL clock model including many more of the identified ion channels in hawkmoth ORNs is the focus of another manuscript to come.

    (6.3) The model seems to be influenced by their long-held proposal that insect olfactory signaling has a critical metabotropic component involving cyclic nucleotides, PKC, etc, a view that may be influenced by the use of Orco homomeric complexes generated in HEK cells.

    Indeed, we propose a metabotropic pheromone-transduction cascade, which in moths and cockroaches is based on G-protein-mediated activation of phospholipase C but not on adenylyl cyclase activation. Our hypothesis is not influenced by HEK cell heterologous expression studies of Orco but is supported by our own work comparing in vivo tip recordings of intact hawkmoths with patch clamp experiments on hawkmoth primary cell cultures of olfactory receptor neurons, which are able to respond to their species-specific pheromones in vitro (Schneider et al., 2025; Stengl, 2010; Stengl and Funk, 2013; Wicher and Miazzi, 2021). In addition, a multitude of publications by other laboratories with in vivo and in vitro studies using physiological, genetic, and immunocytochemical assays all support a metabotropic signal transduction cascade in insect olfaction (Stengl, 2010; Stengl and Funk, 2013; Takagi et al., 2025; Wicher and Miazzi, 2021). In contrast, the hypothesis suggesting a solely ionotropic pheromone- and general odor-dependent transduction cascade for all insect species is based on very sparse experimental evidence, based primarily on heterologous expression studies such as HEK cells that lack the insect’s WT molecular surroundings, and thus, cannot predict OR-Orco function in vivo. Furthermore, the ionotropic hypothesis is heavily based upon the argument that an inverse 7TM receptor cannot couple to G-proteins, which lacks careful backup via biochemical and structural studies. In addition, the ionotropic hypothesis lacks support via carefully performed physiological in vivo studies in different insect species that paid attention to analysis of the distinct kinetic components of ORN´s odor/pheromone responses and that employ physiological concentrations and durations of odor/pheromone stimuli (please see our most recent publication by Schneider et al. (2025)). We added references to the possible odor transduction mechanisms to the introduction.

    (6.4) Nevertheless, structural studies on Orco do not support a cyclic nucleotide binding site, although PKC-based phosphorylation has been implicated in the fine-tuning/adaptation of olfactory signaling.

    While structural studies did not find evidence for conserved known cyclic nucleotide binding sites on Orco, this does not exclude the presence of indirect cAMP effects via e.g., Orco subunits complexing with other molecules under direct cAMP control, such as other ion channel subunits. Furthermore, it does not exclude so far unknown binding sites, or via sites that fold out only after a specific sequence of previous phosphorylations of the many phosphorylation sites on Orco. Indeed, physiological studies in Drosophila presented evidence for cyclic nucleotide dependence of Orco after previous PKC-dependent phosphorylation (Getahun et al., 2013). Our ongoing in vivo experiments in hawkmoths further corroborate a zeitgeber time-dependent PKC- and cyclic nucleotide-dependent modulation of Orco. These detailed studies will be published in a follow-up publication. In the revised version of this manuscript, we added tip-recording experiments that indicate cAMP involvement in Orco gating (new Figure 9).

    (7) Because only 5/11 LD and 7/10 DD animals showed daily rhythms, with averages lacking clear daily modulation, the methods are not sufficiently reliable enough to reveal novel underlying mechanisms of circadian rhythm generation. The reported results are therefore not yet reliable or quantifiable. To quantify their results, the authors should apply tests for circadian rhythmicity using methods such as RAIN, JTK CYCLE, MetaCycle, or Echo. The use of FFT and Wavelet is applauded, but these methods do not have tests of significance for rhythms and can be biased when analyzing data in which there could only be 1-3 circadian cycles. Because the conclusions appear to be based on 11-12 neurons that were recorded for 2-4 days, the reader is concerned that the methods are not yet perfected to provide strong evidence for circadian regulation of spontaneous firing of ORNs. The average data (e.g., Figure 3Bii and 3Cii) highlight the apparent lack of daily rhythms. In summary, the results would be more compelling if more than 50% of the recordings had significant circadian amplitudes and with similar periods and phases.

    The long-term tip-recordings of intact hawkmoths are very challenging and take a very long time to accomplish, thus, we are very happy that we succeeded in obtaining so many of them (N=40). We are thankful to the reviewers’ suggestion to use RAIN since this analysis revealed circadian rhythms in 7 of 11 LD recordings, 8 of 12 DD recordings, and 2 of 12 OLC15 recordings. Please see also our response to (4) above, commenting the phase-dispersal of activity rhythms observed in our experiments, as well as in the behavior of hawkmoth males in the mating cage.

    (8) The statement that circadian patterns of ORN firing are lost with the Orco antagonist (OLC15) is not strongly supported. The manuscript should be revised to quantify how Orco changed circadian amplitude in the 12 recorded neurons. Measures of circadian amplitude can avoid confusing/vague statements like Line 394 “low and high frequency bands appeared to merge during the activity phase around ZT 0 in the animals that showed clear circadian rhythms (N = 5 of 11 in LD)”. The conclusion that Orco blocks circadian firing appears to be contradicted by Figure 6, which indicates that ~6 of these neurons had circadian periods detected by wavelet. The manuscript would be strengthened with details about the specificity and reproducibility of the Orco antagonist. The authors quantify the gradual decrease in firing with the slope of a linear fit to estimate how the “effectiveness [of OLC15] increased over time.” They conclude that the drug “obliterated circadian rhythms and attenuated the spontaneous activity in several, but not all experiments (N = 8 of 12).” The report would be greatly strengthened with corroborating data from additional Orco antagonists and additional doses of OLC15 (the authors use only 50 uM OLC15).

    According to the valuable suggestions of the referees, we used RAIN to detect circadian rhythms in the spiking attributes in each individual animal. Since only 2 of 12 animals displayed a circadian rhythm in OLC15, statistical comparison of circadian amplitudes is not possible. We revised the results section accordingly and added to the figure legend to make it clearer that the heat maps in Fig 5 are representative from one animal each and not averages across animals.

    As the reviewer states correctly in (7), wavelet results of circadian rhythmicity must be interpreted carefully because of the low number of circadian cycles in ~3-4 day recordings. Since the heatmaps in Figure 5 visually revealed the presence of ultradian rhythms, the main focus of the wavelet analysis in Figure 6 is in the detection and quantification of ultradian periods up to 20 h.

    We revised the Methods section to include references to previous experiments that characterized the effect of different doses of OLC15 and other Orco antagonists and agonists in M. sexta antennae (Nolte et al., 2016). Please see also our response to (1).

    (9) The manuscript includes several statements that are more speculation than conclusion. For example, there is no evidence for tuning or plasticity in this report. Statements like the following should be removed or addressed with experiments that show changes in odor response specificity or sensitivity: "ORN signalosomes are highly plastic endogenous PTFL clocks comprising receptors for circadian and ultradian Zeitgebers that allow to tune into internal physiological and external environmental rhythms as basis for active sensing." (Discussion Line 622). The paper concludes that (line 380) "mean frequency of spontaneous spiking and the frequency of bursting expressed daily modulation, and are both most likely controlled via a circadian clock that targets the leak channel Orco." This is too bold given the available results.

    We revised the manuscript accordingly and clarified which statements are supported via published evidence and which are predictions based upon our novel hypothesis published in our opinion paper (Stengl and Schneider, 2024).

    (10.1) Because Orco conductance is modulated by cyclic nucleotides, it remains highly plausible that circadian regulation occurs upstream at the level of signaling pathways (e.g., calcium, calcium-binding proteins, GPCRs, cyclases, phosphodiesterases).

    We agree with the referees that it is very likely that there are multiple layers of interconnected feedback cycles that control Orco localization and activity. Our novel hypothesis suggests interlocked TTFL and PTFL control of physiological circadian rhythms, not strictly hierarchical TTFL control, which would require a daily turnover of membrane proteins and transcriptional control via the established TTFL clock in insect ORNs. We are currently searching for TTFL control at all levels of odor/pheromone transduction using ZT-dependent transcriptomics in combination with qPCR and single-nucleus transcriptomics, involving also all the molecules suggested by the referees. These studies are ongoing, are very time- and money-consuming, and are beyond the scope of this manuscript. However, we added a set of experiments to this manuscript in which we demonstrate that the effect of increased cAMP on the spontaneous spiking activity is mediated by Orco (new Figure 9).

    (10.2) The possibility that circadian oscillations of cyclic nucleotides are generated by the canonical TTFL mechanism has not been excluded. In fact, extensive work in Drosophila has demonstrated that the TTFL-based molecular clock proteins are required for circadian rhythms in olfaction.

    Our experiments that test circadian TTFL control at different levels of the cAMP transduction cascade in hawkmoth antennae are on the way and are part of another publication. In section 6.2 we already stated that our experiments do not exclude that Orco is under indirect control of the TTFL. We revised our discussion accordingly.

    The experiments published for TTFL dependent control of Drosophila olfaction that we are aware of (Krishnan et al., 1999; Tanoue et al., 2004) do not exclude interlinked PTFL and TTFL clocks. Krishnan et al. (1999) demonstrated that the TTFL clock in antennal olfactory receptor neurons correlates with circadian rhythms in odor responses measured in electroantennogram (EAG) recordings, not in single sensillum recordings as in our experiments. EAG recordings comprise not only voltage responses of the olfactory sensory neurons but also voltage changes generated in non-neuronal antennal cells such as trichogen and tormogen cells that built the transepithelial potential gradient via vATPases that generates the high K+ concentration in the sensillum lymph (Jain et al., 2024; Klein, 1992; Thurm and Küppers, 1980). In addition, EAG recordings most likely contain responses of afferent neurons originating from somata in the brain that maintain central control of the antennae. Thus, EAG recordings are difficult to interpret.

    (11) A defining feature of circadian oscillators is the feedback mechanism that generates a time delay (e.g., PERIOD/TIMELESS repressing their own transcription). While the authors describe how cyclic nucleotides can regulate Orco conductance, they do not provide a convincing explanation of how Orco activity could, in turn, feed back into the proposed PTFL to sustain oscillations. For these reasons, the authors should consider:

    (a) Providing a broader discussion of non-TTFL models of circadian rhythms (e.g., redox cycles, post-translational modifications).

    We revised the discussion accordingly.

    (b) Reassessing Orco expression using a higher-resolution temporal sampling ({greater than or equal to}6 timepoints per 24 h).

    We added those experiments to the revised version of the manuscript (see our response to (2)).

    (c) Clarifying or revising the PTFL model to explicitly address how feedback would be achieved. Alternatively, the data may be more consistent with Orco conductance rhythms being regulated by post-translational mechanisms downstream of the canonical TTFL oscillator, as suggested by the Drosophila olfactory system literature.

    We added possible negative feedback elements to the Discussion to explain how our proposed PTFL could in principle work independent of TTFL clock.

    Minor weaknesses:

    (1) The authors should compare the firing patterns of ORN neurons to the bursts, clusters, and packets of retinal efferent spikes reported in Liu JS and Passaglia CL (2011; JBR). By comparing measures in moths to measures in Limulus, the authors might be able to address the question: Is the daily firing pattern of ORN neurons likely a conserved feature of circadian control of sensory sensitivity?

    We have revised the discussion accordingly.

    (2) The methods need further details. For example, it is unclear if or how single neuron activity was discriminated and whether the results were compromised by the relatively large environmental fluctuations in temperature (21-27oC), humidity (35-60%), or other cues known to modulate spontaneous firing.

    These large fluctuations stem from doing experiments at different seasons (higher temperature and humidity in the summer months, lower temperature and humidity in winter). Throughout each individual experiment, conditions were stable. We clarified the Methods section accordingly.

    Recommendations for the authors:

    The authors should post the code for their computational model to a repository like GitHub.

    The code for the computational model is now available at https://github.com/a-c-schneider/VijayanForlinoEtAl2025_Model.git

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    Takagi S, Abuin L, Mermet J, Lee D, Benton R. 2025. A GPCR signaling pathway in insect odor detection. DOI: https://doi.org/10.1101/2025.10.03.680299

    Tanoue S, Krishnan P, Krishnan B, Dryer SE, Hardin PE. 2004. Circadian Clocks in Antennal Neurons Are Necessary and Sufficient for Olfaction Rhythms in Drosophila. Current Biology 14:638–649. DOI: https://doi.org/10.1016/j.cub.2004.04.009, PMID: 15084278

    Thurm U, Küppers J. 1980. Epithelial physiology of insect sensilla. In: Locke M, Smith DS (Eds). Insect Biology in the Future. Academic Press. p. 735–763. DOI: https://doi.org/10.1016/B978-0-12-454340-9.50039-2

    Wicher D, Miazzi F. 2021. Functional properties of insect olfactory receptors: ionotropic receptors and odorant receptors. Cell and Tissue Research 383:7–19. DOI: https://doi.org/10.1007/s00441-020-03363-x

  16. Mentioned by @rridrobot.bsky.social on Bluesky

    Authors published a paper in Biorxiv, they used pClamp in the study. Including #RRIDs will make this less ambiguous. SciScore made a table with this resource, see “Automated Services” module (download as csv, xml or #jats) #STMpublishing #reproducibility

  17. Version published to 10.1101/2025.06.17.659282v3 on bioRxiv
  18. Version published to 10.7554/elife.108100.1 on eLife
  19. eLife

    eLife Assessment

    In this manuscript, the authors used in vivo long-term tip recordings of the long trichoid sensilla of male hawkmoths to analyze spontaneous spiking activity indicative of the ORNs' endogenous membrane potential oscillations. The authors combine extracellular electrophysiology of the hawkmoth antennae with computational modeling to predict that Orco receptor neuron (ORN) activity is required for circadian, not ultradian, firing patterns. The work provides valuable support for the hypothesis that a posttranslational feedback loop regulates daily and ultradian rhythms in neuronal excitability. Nevertheless, the evidence reported provides only incomplete support for their conclusions, especially with regard to the biological implications of their assumption-heavy models.

  20. eLife

    Joint Public Review:

    This manuscript puts forward the provocative idea that a posttranslational feedback loop regulates daily and ultradian rhythms in neuronal excitability. The authors used in vivo long-term tip recordings of the long trichoid sensilla of male hawkmoths to analyze spontaneous spiking activity indicative of the ORNs' endogenous membrane potential oscillations. This firing pattern was disrupted by pharmacological blockade of the Orco receptor. They then use these recordings together with computational modeling to predict that Orco receptor neuron (ORN) activity is required for circadian, not ultradian, firing patterns. Orco did not show a circadian expression pattern in a qPCR experiment, and its conductance was proposed to be regulated by cyclic nucleotide levels. This evidence led the authors to conclude that a post-translational feedback loop (PTFL) clockwork, associated with the ORN plasma membrane, allows for temporal control of pheromone detection via the generation of multi-scale endogenous membrane potential oscillations. The findings will interest researchers in neurophysiology, circadian rhythms, and sensory biology. However, the manuscript has limited experimental evidence to support its central hypothesis and is undermined by several questionable assumptions that underlie their data analysis and model builds, as well as insufficient biological data, including critical controls to validate and/or fully justify the model the authors are proposing.

    Strengths:

    The study is notable for its combination of long-term in vivo tip recordings with computational modeling, which is technically challenging and adds weight to the authors' claims. The link between Orco, cyclic nucleotides, and circadian regulation is potentially important for sensory neuroscience, and the modeling framework itself - a stochastic Hodgkin-Huxley formulation that explicitly incorporates channel noise - is a solid and forward-looking contribution. Together, these elements make the study conceptually bold and of clear interest to circadian and olfactory biologists.

    Major weaknesses:

    At the same time, several limitations temper the conclusions. The pharmacological evidence relies on a single antagonist and concentration, without key controls. The circadian analysis is based on relatively small numbers of neurons, with rhythms detected only in subsets, and the alignment procedure used in constant darkness raises concerns of bias. The molecular evidence is sparse, with only three qPCR timepoints, and the model, while creative, rests on assumptions that are not yet fully supported by in vivo data.

    Detailed comments are provided below:

    (1) The role for Orco proposed in the authors' model largely stems from the effects seen following the administration of (a single dose) of the Orco antagonist, OLC15. However, this hypothesis is undercut by the lack of adequate pharmacological controls, including a basic multipoint OLC15 dose-response series in addition to the administration of blockers for the other channels that are embedded in their model, but which were ruled out as being involved in the modulation of biological rhythms. In addition, these studies would (ideally) also benefit from the inclusion of the same concentration (series) of an inactive OLC15 analog to better control for off-target effects.

    (2) The expression pattern of Orco was assessed using qPCR at only three timepoints. Rhythmic transcripts can easily be missed with such sparse sampling (Hughes et al., 2017). A minimum of six evenly spaced timepoints across a 24-hour cycle would be required to confidently rule out circadian transcriptional regulation. In addition, the use of the timeless mRNA control from another study is not acceptable. Furthermore, qPCR analysis measures transcript abundance, not transcription, as the authors repeatedly state. Transcriptional studies would require nuclear run-off or, more recently, can be done with snRNAseq analysis. Taken together, these concerns undermine the authors' desire to rule out TTFL-based control that directly led them to implicate a PTTF-based model.

    (3) The modelling presented is based on Orco as a ZT-dependent conductance tied to the cAMP oscillations that were reported by this group in the cockroach and from the presence and functionality in Manduca of homomeric Orco complexes that are devoid of tuning ORs. While these complexes have been generated in cell culture and other heterologous expression systems, as well as presumably exist in vivo in the Drosophila empty neuron and other tuning OR mutants, there is no evidence that these complexes exist in wild-type Manduca ORNs. While this doesn't necessarily undermine every aspect of their models, the authors should note the presence of Orco/OR complexes rather than Orco homomeric complexes.

    (4) Some aspects of the authors' models, most notably the decision to phase align/optimize their DD and OLC15 recordings, are likely to bias their interpretations.

    (5) The tip recordings from long trichoid sensilla are critical aspects of this study. These recordings were carried out on upper sensillar tips located on the distal-most second annulus. Since there are approximately 80 annuli on the Manduca antennae, it is unclear whether the recordings are representative of the antennal response.

    (6) The authors do not provide any data in support of their cAMP/cGMP-based Orco gating, and the PTTF model proposed is somewhat disappointing. The model seems to be influenced by their long-held proposal that insect olfactory signaling has a critical metabotropic component involving cyclic nucleotides, PKC, etc, a view that may be influenced by the use of Orco homomeric complexes generated in HEK cells. Nevertheless, structural studies on Orco do not support a cyclic nucleotide binding site, although PKC-based phosphorylation has been implicated in the fine-tuning/adaptation of olfactory signaling.

    (7) Because only 5/11 LD and 7/10 DD animals showed daily rhythms, with averages lacking clear daily modulation, the methods are not sufficiently reliable enough to reveal novel underlying mechanisms of circadian rhythm generation. The reported results are therefore not yet reliable or quantifiable. To quantify their results, the authors should apply tests for circadian rhythmicity using methods such as RAIN, JTK CYCLE, MetaCycle, or Echo. The use of FFT and Wavelet is applauded, but these methods do not have tests of significance for rhythms and can be biased when analyzing data in which there could only be 1-3 circadian cycles. Because the conclusions appear to be based on 11-12 neurons that were recorded for 2-4 days, the reader is concerned that the methods are not yet perfected to provide strong evidence for circadian regulation of spontaneous firing of ORNs. The average data (e.g., Figure 3Bii and 3Cii) highlight the apparent lack of daily rhythms. In summary, the results would be more compelling if more than 50% of the recordings had significant circadian amplitudes and with similar periods and phases.

    (8) The statement that circadian patterns of ORN firing are lost with the Orco antagonist (OLC15) is not strongly supported. The manuscript should be revised to quantify how Orco changed circadian amplitude in the 12 recorded neurons. Measures of circadian amplitude can avoid confusing/vague statements like Line 394 "low and high frequency bands appeared to merge during the activity phase around ZT 0 in the animals that showed clear circadian rhythms (N = 5 of 11 in LD)". The conclusion that Orco blocks circadian firing appears to be contradicted by Figure 6, which indicates that ~6 of these neurons had circadian periods detected by wavelet. The manuscript would be strengthened with details about the specificity and reproducibility of the Orco antagonist. The authors quantify the gradual decrease in firing with the slope of a linear fit to estimate how the "effectiveness [of OLC15] increased over time." They conclude that the drug "obliterated circadian rhythms and attenuated the spontaneous activity in several, but not all experiments (N = 8 of 12)." The report would be greatly strengthened with corroborating data from additional Orco antagonists and additional doses of OLC15 (the authors use only 50 uM OLC15).

    (9) The manuscript includes several statements that are more speculation than conclusion. For example, there is no evidence for tuning or plasticity in this report. Statements like the following should be removed or addressed with experiments that show changes in odor response specificity or sensitivity: "ORN signalosomes are highly plastic endogenous PTFL clocks comprising receptors for circadian and ultradian Zeitgebers that allow to tune into internal physiological and external environmental rhythms as basis for active sensing." (Discussion Line 622). The paper concludes that (line 380) "mean frequency of spontaneous spiking and the frequency of bursting expressed daily modulation, and are both most likely controlled via a circadian clock that targets the leak channel Orco." This is too bold given the available results.

    (10) Because Orco conductance is modulated by cyclic nucleotides, it remains highly plausible that circadian regulation occurs upstream at the level of signaling pathways (e.g., calcium, calcium-binding proteins, GPCRs, cyclases, phosphodiesterases). The possibility that circadian oscillations of cyclic nucleotides are generated by the canonical TTFL mechanism has not been excluded. In fact, extensive work in Drosophila has demonstrated that the TTFL-based molecular clock proteins are required for circadian rhythms in olfaction.

    (11) A defining feature of circadian oscillators is the feedback mechanism that generates a time delay (e.g., PERIOD/TIMELESS repressing their own transcription). While the authors describe how cyclic nucleotides can regulate Orco conductance, they do not provide a convincing explanation of how Orco activity could, in turn, feed back into the proposed PTFL to sustain oscillations. For these reasons, the authors should consider:

    (a) Providing a broader discussion of non-TTFL models of circadian rhythms (e.g., redox cycles, post-translational modifications).

    (b) Reassessing Orco expression using a higher-resolution temporal sampling ({greater than or equal to}6 timepoints per 24 h).

    (c) Clarifying or revising the PTFL model to explicitly address how feedback would be achieved. Alternatively, the data may be more consistent with Orco conductance rhythms being regulated by post-translational mechanisms downstream of the canonical TTFL oscillator, as suggested by the Drosophila olfactory system literature.

    Minor weaknesses:

    (1) The authors should compare the firing patterns of ORN neurons to the bursts, clusters, and packets of retinal efferent spikes reported in Liu JS and Passaglia CL (2011; JBR). By comparing measures in moths to measures in Limulus, the authors might be able to address the question: Is the daily firing pattern of ORN neurons likely a conserved feature of circadian control of sensory sensitivity?

    (2) The methods need further details. For example, it is unclear if or how single neuron activity was discriminated and whether the results were compromised by the relatively large environmental fluctuations in temperature (21-27oC), humidity (35-60%), or other cues known to modulate spontaneous firing.

  21. eLife

    Author response:

    Joint Public Review

    This manuscript puts forward the provocative idea that a posttranslational feedback loop regulates daily and ultradian rhythms in neuronal excitability. The authors used in vivo long-term tip recordings of the long trichoid sensilla of male hawkmoths to analyze spontaneous spiking activity indicative of the ORNs' endogenous membrane potential oscillations. This firing pattern was disrupted by pharmacological blockade of the Orco receptor. They then use these recordings together with computational modeling to predict that Orco receptor neuron (ORN) activity is required for circadian, not ultradian, firing patterns. Orco did not show a circadian expression pattern in a qPCR experiment, and its conductance was proposed to be regulated by cyclic nucleotide levels. This evidence led the authors to conclude that a post-translational feedback loop (PTFL) clockwork, associated with the ORN plasma membrane, allows for temporal control of pheromone detection via the generation of multi-scale endogenous membrane potential oscillations. The findings will interest researchers in neurophysiology, circadian rhythms, and sensory biology. However, the manuscript has limited experimental evidence to support its central hypothesis and is undermined by several questionable assumptions that underlie their data analysis and model builds, as well as insufficient biological data, including critical controls to validate and/or fully justify the model the authors are proposing.

    We thank the reviewers for their thorough and thoughtful comments and believe that the manuscript will be much stronger once we incorporate the requested changes.

    Please note that we used ORN as acronym for “olfactory receptor neuron” throughout the manuscript. ORNs contain odorant receptors (ORs), and in insects these ORs have to associate with the olfactory receptor co-receptor (Orco) in the cilium of the neuron to form functional OR-Orco complexes for odorant detection. Besides this chaperone function, Orco can form homomers with the potential to act as ionic pacemaker channels; a role which we explore in this study.

    Strengths:

    The study is notable for its combination of long-term in vivo tip recordings with computational modeling, which is technically challenging and adds weight to the authors' claims. The link between Orco, cyclic nucleotides, and circadian regulation is potentially important for sensory neuroscience, and the modeling framework itself - a stochastic Hodgkin-Huxley formulation that explicitly incorporates channel noise - is a solid and forward-looking contribution. Together, these elements make the study conceptually bold and of clear interest to circadian and olfactory biologists.

    Major weaknesses:

    At the same time, several limitations temper the conclusions. The pharmacological evidence relies on a single antagonist and concentration, without key controls. The circadian analysis is based on relatively small numbers of neurons, with rhythms detected only in subsets, and the alignment procedure used in constant darkness raises concerns of bias. The molecular evidence is sparse, with only three qPCR timepoints, and the model, while creative, rests on assumptions that are not yet fully supported by in vivo data.

    Please see our responses to the detailed comments.

    Detailed comments are provided below:

    (1) The role for Orco proposed in the authors' model largely stems from the effects seen following the administration of (a single dose) of the Orco antagonist, OLC15. However, this hypothesis is undercut by the lack of adequate pharmacological controls, including a basic multipoint OLC15 dose-response series in addition to the administration of blockers for the other channels that are embedded in their model, but which were ruled out as being involved in the modulation of biological rhythms. In addition, these studies would (ideally) also benefit from the inclusion of the same concentration (series) of an inactive OLC15 analog to better control for off-target effects.

    The Orco agonist VUAA1 (Jones et al., 2011) binds directly to Orco and increases the channel open time probability. In M. sexta hawkmoths, we have already published that VUAA 1 increases the low spontaneous activity of ORNs in a dose-dependent fashion (Nolte et al., 2016). Chen and Luetje (2012) systematically varied the chemical structure of VUAA1 to identify new Orco ligands and discovered 22 Orco Ligand Candidates (OLC) that either activated or inhibited Orco. In their heterologous expression system, Orco was most sensitive to inhibition by OLC15. Based on these results, we published a dose-response curve of OLC15 inhibition (1-100 µM) using in vivo tip recordings of pheromone-sensitive long trichoid sensilla of M. sexta (Nolte et al., 2016). In that study, we could also demonstrate that OLC15 antagonizes the VUAA1 activation of Orco.

    Furthermore, we tested other published Orco antagonists in in vivo assays in intact hawkmoths, focusing on amiloride-derived antagonists, because we previously identified an amiloride-sensitive cation channel in hawkmoth ORNs. We found that, in contrast to OLC15, the amilorides HMA and MIA were not Orco-specific but instead affected different targets depending on time-of-day (Nolte et al., 2016). Based on those experiments and the dose-response curves we determined that the Orco agonist VUAA1 (Jones et al., 2011) and the Orco antagonist OLC15 (Chen and Luetje, 2012) worked best in hawkmoth ORNs to target Orco pharmacologically. Based on comparative tests with other published Orco antagonists we settled since then in all further experiments on a dose of 50 µM OLC15.

    We will clarify the Methods section accordingly.

    (2) The expression pattern of Orco was assessed using qPCR at only three timepoints. Rhythmic transcripts can easily be missed with such sparse sampling (Hughes et al., 2017). A minimum of six evenly spaced timepoints across a 24-hour cycle would be required to confidently rule out circadian transcriptional regulation. In addition, the use of the timeless mRNA control from another study is not acceptable. Furthermore, qPCR analysis measures transcript abundance, not transcription, as the authors repeatedly state. Transcriptional studies would require nuclear run-off or, more recently, can be done with snRNAseq analysis. Taken together, these concerns undermine the authors' desire to rule out TTFL-based control that directly led them to implicate a PTTF-based model.

    We agree with the referees that more time points and a direct comparison between timeless and Orco mRNA levels should be included in this manuscript. We will include these additional qPCR experiments and edit the manuscript to make clear that we measure transcript abundance, but we will not perform snRNAseq analysis due to time- and financial constraints. We are currently working on the transcriptional control of Orco, both during ontogeny and throughout the day but this work in progress is beyond the scope of this manuscript.

    (3) The modelling presented is based on Orco as a ZT-dependent conductance tied to the cAMP oscillations that were reported by this group in the cockroach and from the presence and functionality in Manduca of homomeric Orco complexes that are devoid of tuning ORs. While these complexes have been generated in cell culture and other heterologous expression systems, as well as presumably exist in vivo in the Drosophila empty neuron and other tuning OR mutants, there is no evidence that these complexes exist in wild-type Manduca ORNs. While this doesn't necessarily undermine every aspect of their models, the authors should note the presence of Orco/OR complexes rather than Orco homomeric complexes.

    Our ELISAs found circadian oscillations in cAMP levels not only in antennae of the Madeira cockroach (Schendzielorz et al., 2014, 2012), but also in hawkmoth antennae (Schendzielorz et al., 2015). We will add the 2015 citation to the Modeling chapter in the Methods section to clarify this.

    We agree with the referees that we cannot distinguish between Orco homo- and heteromers in the different compartments of our hawkmoth ORNs. Thus, as the referee suggests, we will add text regarding the presence and localization of OR-Orco heteromers. However, we have indications that Orco homomers could indeed be present in the hawkmoth ORNs. In a heterologous expression system, MsexOrco expression alone was sufficient to increase intracellular Ca2+ levels in response to VUAA1 application (Nolte et al., 2013). In differentiating primary cell cultures of hawkmoth antennae, Orco expression started during a developmental time window where ORNs did not yet express pheromone receptors, and Orco affected spontaneous activity (Nolte et al., 2016). Thus, Orco homomers are present in developing hawkmoth ORNs during a time window where ORNs already express spontaneous activity but cannot heteromerize with pheromone receptors. However, we do not know whether and in what ratio homo- and heteromers of Orco and ORs are present in the respective sensillum compartments of adult hawkmoths (Nolte et al., 2013; Stengl, 1994; Stengl and Hildebrand, 1990).

    We will clarify our manuscript accordingly.

    (4) Some aspects of the authors' models, most notably the decision to phase align/optimize their DD and OLC15 recordings, are likely to bias their interpretations.

    It is consensus that insects display daily and circadian rhythms in pheromone-dependent mating, odor-gated feeding, and egg-laying behavior that phase-locks to environmental rhythms, corresponding with daily/circadian rhythms of sensory neuron physiology (e.g., Merlin et al., 2007; Rymer et al., 2007; Schendzielorz et al., 2015, 2012). However, circadian rhythms can be easily masked by stress, like the disturbances during a very challenging long-term recording experiment over several days. In addition, we observed in our animal raising facility that in LD 17:7 light-dark cycles the originally nocturnal hawkmoths M. sexta distribute their activity patterns over the course of the day, finding nocturnal as well as diurnal hawkmoths. Thus, light-dark cycles were not enough to ensure phase-synchronized behavioral rhythms, and it is very likely that the nocturnal hawkmoths rely heavily on pheromone/odor dependent synchronization as also found in other moth species (Ghosh et al., 2024). Here, we used isolated males that were never exposed to the female pheromones so that their circadian activity patterns readily disperse. Therefore, it became necessary in free-running conditions to first determine the respective behavioral rhythm for each animal, and then to phase-align their activity patterns to allow for statistical analysis. Otherwise, circadian differences would average out in a free-running population. As requested by the referees in point (7), we will use additional tests for rhythmicity in each of our recordings and revise the manuscript accordingly.

    Assuming that hawkmoths need pheromone presence as additional Zeitgeber, we are currently working on a new set of experiments where we attempt to improve synchronization by exposure to LD cycles and pheromone before DD and OLC15 recordings. We will add these experiments to the manuscript.

    (5) The tip recordings from long trichoid sensilla are critical aspects of this study. These recordings were carried out on upper sensillar tips located on the distal-most second annulus. Since there are approximately 80 annuli on the Manduca antennae, it is unclear whether the recordings are representative of the antennal response.

    We think the reviewers might have misinterpreted our description of the recording site. In the Methods, we state that we clip off the 20 most distal annuli (leaving a stump of about 60 annuli) and insert the reference electrode into the flagellum up to the second annulus from the cut end, i.e., the recording site is located at 2/3 – 3/4 of the antenna length as seen from the head of the animal. We will make this more clear in the Methods section.

    In addition, our lab did show with antibody stainings against Orco that apparently all ORNs that innervate long and short trichoid sensilla along the whole flagellum express the same staining pattern (Nolte et al., 2016). Furthermore, our patch clamp recordings of primary cell cultures of whole male antennae found largely overlapping ion channel populations across ORNs. This would indicate that all ORNs, whether they express pheromone- or general odorant receptors, could potentially share the same Orco-dependent spontaneous activity rhythms. In our lab, different experimenters from different years that recorded from long trichoid sensilla on different annuli did not detect obvious differences in neither the spontaneous activity nor the pheromone responses (c.f., Dolzer et al., 2003; Gawalek and Stengl, 2018; Schneider et al., 2025). Thus, it is very likely that we are reporting a general encoding mechanism that is not locally restricted along the antennal flagellum.

    (5.1) The authors do not provide any data in support of their cAMP/cGMP-based Orco gating…

    There are publications supporting cyclic nucleotide gating of Orco in Drosophila, but only after previous phosphorylation via protein kinase C (PKC; review: (Wicher and Miazzi, 2021)). Since Orco is very conserved among insect species, it is likely that these PKC and cGMP/cAMP-dependent regulations are present in other insect species. We are currently running thorough tip-recording experiments on the regulation of Orco gating, which are beyond the scope of this manuscript. However, we will add a set of experiments to this manuscript that demonstrates cAMP gating of Orco.

    (5.2)… and the PTTF model proposed is somewhat disappointing.

    For a detailed introduction of our PTFL membrane clock hypothesis please see our opinion paper (Stengl and Schneider, 2024).

    (5.3) The model seems to be influenced by their long-held proposal that insect olfactory signaling has a critical metabotropic component involving cyclic nucleotides, PKC, etc, a view that may be influenced by the use of Orco homomeric complexes generated in HEK cells.

    Indeed, we propose a metabotropic pheromone-transduction cascade, which in moths and cockroaches is based on G-protein-mediated activation of phospholipase C but not on adenylyl cyclase activation. Our hypothesis is not influenced by HEK cell heterologous expression studies of Orco but is supported by our own work comparing in vivo tip recordings of intact hawkmoths with patch clamp experiments on hawkmoth primary cell cultures of olfactory receptor neurons, which are able to respond to their species-specific pheromones in vitro ((Schneider et al., 2025; Stengl, 2010; Stengl and Funk, 2013; Wicher and Miazzi, 2021). In addition, a multitude of publications by other laboratories with in vivo and in vitro studies using physiological, genetic, and immunocytochemical assays all support a metabotropic signal transduction cascade in insect olfaction (reviews: Stengl, 2010; Stengl and Funk, 2013; Wicher and Miazzi, 2021). In contrast, the hypothesis suggesting a solely ionotropic pheromone- and general odor-dependent transduction cascade for all insect species is based on very sparse experimental evidence, based primarily on heterologous expression studies such as HEK cells that lack the insect’s WT molecular surroundings, and thus, cannot predict OR-Orco function in vivo. Furthermore, the ionotropic hypothesis is heavily based upon the argument that an inverse 7TM receptor cannot couple to G-proteins, which lacks careful backup via biochemical and structural studies. In addition, the ionotropic hypothesis lacks support via carefully performed physiological in vivo studies in different insect species that paid attention to analysis of the distinct kinetic components of ORN´s odor/pheromone responses and that employ physiological concentrations and durations of odor/pheromone stimuli (please see our most recent publication by Schneider et al. (2025)).

    (5.4) Nevertheless, structural studies on Orco do not support a cyclic nucleotide binding site, although PKC-based phosphorylation has been implicated in the fine-tuning/adaptation of olfactory signaling.

    While structural studies did not find evidence for conserved known cyclic nucleotide binding sites on Orco, this does not exclude the presence of so far unknown binding sites, or via sites that fold out only after a specific sequence of previous phosphorylations of the many phosphorylation sites on Orco. Indeed, physiological studies in Drosophila presented evidence for cyclic nucleotide dependence of Orco after previous PKC-dependent phosphorylation (Getahun et al., 2013). Our ongoing in vivo experiments in hawkmoths further corroborate a PKC- and cAMP-dependent modulation of Orco. These studies will be published in a follow-up publication.

    (6) Because only 5/11 LD and 7/10 DD animals showed daily rhythms, with averages lacking clear daily modulation, the methods are not sufficiently reliable enough to reveal novel underlying mechanisms of circadian rhythm generation. The reported results are therefore not yet reliable or quantifiable. To quantify their results, the authors should apply tests for circadian rhythmicity using methods such as RAIN, JTK CYCLE, MetaCycle, or Echo. The use of FFT and Wavelet is applauded, but these methods do not have tests of significance for rhythms and can be biased when analyzing data in which there could only be 1-3 circadian cycles. Because the conclusions appear to be based on 11-12 neurons that were recorded for 2-4 days, the reader is concerned that the methods are not yet perfected to provide strong evidence for circadian regulation of spontaneous firing of ORNs. The average data (e.g., Figure 3Bii and 3Cii) highlight the apparent lack of daily rhythms. In summary, the results would be more compelling if more than 50% of the recordings had significant circadian amplitudes and with similar periods and phases.

    The long-term tip-recordings of intact hawkmoths are very challenging and take a very long time to accomplish, thus, we are very happy that we succeeded in obtaining so many of them (N=34). Since 5/11 LD recordings and 7/10 DD recordings revealed daily/circadian rhythmicity and since many other physiological recordings at different ZTs of different members of our laboratory all revealed ZT-dependent pheromone-transduction we can be certain that the physiology of hawkmoth antennae is under strict circadian control. Please see also our response to (4) above commenting the phase-dispersal of activity rhythms observed in our experiments, as well as in the behavior of hawkmoth males in the mating cage.

    Nevertheless, we will follow the advice of the referees to apply additional tests for significance of rhythms in spontaneous activity, and we are thankful for the tests suggested that we were not aware of.

    (7) The statement that circadian patterns of ORN firing are lost with the Orco antagonist (OLC15) is not strongly supported. The manuscript should be revised to quantify how Orco changed circadian amplitude in the 12 recorded neurons. Measures of circadian amplitude can avoid confusing/vague statements like Line 394 “low and high frequency bands appeared to merge during the activity phase around ZT 0 in the animals that showed clear circadian rhythms (N = 5 of 11 in LD)”. The conclusion that Orco blocks circadian firing appears to be contradicted by Figure 6, which indicates that ~6 of these neurons had circadian periods detected by wavelet. The manuscript would be strengthened with details about the specificity and reproducibility of the Orco antagonist. The authors quantify the gradual decrease in firing with the slope of a linear fit to estimate how the “effectiveness [of OLC15] increased over time.” They conclude that the drug “obliterated circadian rhythms and attenuated the spontaneous activity in several, but not all experiments (N = 8 of 12).” The report would be greatly strengthened with corroborating data from additional Orco antagonists and additional doses of OLC15 (the authors use only 50 uM OLC15).

    We will revise our data analysis, according to the valuable suggestions of the referees.

    However, based upon our previous studies with other Orco antagonists and different doses of OLC15 (Nolte et al., 2016) we found that 50 µM OLC15 is the best Orco antagonist dose in M. sexta to target Orco-dependent modulation of spontaneous action potential activity of hawkmoth olfactory receptor neurons. Please see also our response to (1).

    (8) The manuscript includes several statements that are more speculation than conclusion. For example, there is no evidence for tuning or plasticity in this report. Statements like the following should be removed or addressed with experiments that show changes in odor response specificity or sensitivity: "ORN signalosomes are highly plastic endogenous PTFL clocks comprising receptors for circadian and ultradian Zeitgebers that allow to tune into internal physiological and external environmental rhythms as basis for active sensing." (Discussion Line 622). The paper concludes that (line 380) "mean frequency of spontaneous spiking and the frequency of bursting expressed daily modulation, and are both most likely controlled via a circadian clock that targets the leak channel Orco." This is too bold given the available results.

    We will revise the discussion accordingly and clarify which statements are supported via published evidence and which are predictions based upon our novel hypothesis published in our opinion paper (Stengl and Schneider, 2024).

    (9.1) Because Orco conductance is modulated by cyclic nucleotides, it remains highly plausible that circadian regulation occurs upstream at the level of signaling pathways (e.g., calcium, calcium-binding proteins, GPCRs, cyclases, phosphodiesterases).

    We agree with the referees that it is very likely that there are multiple layers of interconnected feedback cycles that control Orco localization and activity. Our novel hypothesis suggests interlocked TTFL and PTFL control of physiological circadian rhythms, not strictly hierarchical TTFL control, which would require a daily turnover of membrane proteins and transcriptional control via the established TTFL clock in insect ORNs. We currently search for TTFL control at all levels of odor/pheromone transduction using ZT-dependent transcriptomics in combination with qPCR and single nuclear transcriptomics, involving also all the molecules suggested by the referees. These studies are ongoing, are very time- and money-consuming, and are beyond the scope of this manuscript.

    (9.2) The possibility that circadian oscillations of cyclic nucleotides are generated by the canonical TTFL mechanism has not been excluded. In fact, extensive work in Drosophila has demonstrated that the TTFL-based molecular clock proteins are required for circadian rhythms in olfaction.

    Our experiments that test circadian TTFL control at different levels of the cAMP transduction cascade in hawkmoth antennae are on the way and are part of another publication. We will revise our discussion accordingly.

    The experiments published for TTFL dependent control of Drosophila olfaction that we are aware of (Krishnan et al., 1999; Tanoue et al., 2004) do not exclude interlinked PTFL and TTFL clocks. Krishnan et al. (1999) demonstrate that the TTFL clock in antennal olfactory receptor neurons correlates with circadian rhythms in odor responses measured in electroantennogram (EAG) recordings, not in single sensillum recordings as in our experiments. EAG recordings comprise not only voltage responses of the olfactory sensory neurons but also voltage changes generated in non-neuronal antennal cells such as trichogen and tormogen cells that built the transepithelial potential gradient via vATPases that generates the high K+ concentration in the sensillum lymph (Jain et al., 2024; Klein, 1992; Thurm and Küppers, 1980). In addition, EAG recordings most likely contain responses of afferent neurons originating from somata in the brain that maintain central control of the antennae. Thus, EAG recordings are difficult to interpret.

    (11) A defining feature of circadian oscillators is the feedback mechanism that generates a time delay (e.g., PERIOD/TIMELESS repressing their own transcription). While the authors describe how cyclic nucleotides can regulate Orco conductance, they do not provide a convincing explanation of how Orco activity could, in turn, feed back into the proposed PTFL to sustain oscillations. For these reasons, the authors should consider:

    a) Providing a broader discussion of non-TTFL models of circadian rhythms (e.g., redox cycles, post-translational modifications).

    We will revise the discussion accordingly.

    b) Reassessing Orco expression using a higher-resolution temporal sampling ({greater than or equal to}6 timepoints per 24 h).

    We will add those experiments to the revised version of the manuscript (see our response to (2)).

    c) Clarifying or revising the PTFL model to explicitly address how feedback would be achieved. Alternatively, the data may be more consistent with Orco conductance rhythms being regulated by post-translational mechanisms downstream of the canonical TTFL oscillator, as suggested by the Drosophila olfactory system literature.

    We will revise the manuscript accordingly.

    Minor weaknesses:

    (1) The authors should compare the firing patterns of ORN neurons to the bursts, clusters, and packets of retinal efferent spikes reported in Liu JS and Passaglia CL (2011; JBR). By comparing measures in moths to measures in Limulus, the authors might be able to address the question: Is the daily firing pattern of ORN neurons likely a conserved feature of circadian control of sensory sensitivity?

    We will revise the discussion accordingly.

    (2) The methods need further details. For example, it is unclear if or how single neuron activity was discriminated and whether the results were compromised by the relatively large environmental fluctuations in temperature (21-27oC), humidity (35-60%), or other cues known to modulate spontaneous firing.

    We will clarify the Methods section.

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  22. Mentioned by @rridrobot.bsky.social on Bluesky

    Authors published a paper in Biorxiv, they used pClamp in the study. Including #RRIDs will make this less ambiguous. SciScore made a table with this resource, see “Automated Services” module (download as csv, xml or #jats) #methodsmatter #OpenScience

  23. Version published to 10.1101/2025.06.17.659282v2 on bioRxiv
  24. Mentioned by @biorxiv-physio.bsky.social on Bluesky

    Orco regulates the circadian activity of pheromone-sensitive olfactory receptor neurons in hawkmoths https://www.biorxiv.org/content/10.1101/2025.06.17.659282v1

  25. Mentioned by @biorxivpreprint.bsky.social on Bluesky

    Orco regulates the circadian activity of pheromone-sensitive olfactory receptor neurons in hawkmoths https://www.biorxiv.org/content/10.1101/2025.06.17.659282v1

  26. Version published to 10.1101/2025.06.17.659282v1 on bioRxiv