Neural substrates of cold nociception in Drosophila larva

Reviewer #1 (Public Review):

Summary. The authors goal was to map the neural circuitry underlying cold sensitive contraction in Drosophila. The circuitry underlying most sensory modalities has been characterized but noxious cold sensory circuitry has not been well studied. The authors achieve their goal and map out sensory and post-sensory neurons involved in this behavior.

Strengths. The manuscript provides convincing evidence for sensory and post sensory neurons involved in noxious cold sensitive behavior. They use both connectivity data and functional data to identify these neurons. This work is a clear advance in our understanding of noxious cold behavior. The experiments are done with a high degree of experimental rigor.

Positive comments

-Campari is nicely done to map cold responsive neurons, although it doesn't give data on individual neurons.

-Chrimson and TNT experiments are nicely done.

-Cold temperature activates basin neurons, it's a solid and convincing result.

Weaknesses. Among the few weaknesses in this manuscript is the failure to trace the circuit from sensory neuron to motor neuron; and to ignore analysis of the muscles driving, cold induced contraction. Authors also need to elaborate more on the novel aspects of their work in the introduction or abstract.

Major comments.

-Class three sensory neuron connectivity is known, and role in cold response is known (turner 16, 18). Need to make it clearer what the novelty of the experiments are.

-Why focus on premotor neurons in mechano nociceptive pathways? Why not focus on PMNs innervating longitudinal muscles, likely involved in longitudinal larval contraction? Especially since chosen premotor neurons have only weak effects on cold induced contraction?

Reviewer #2 (Public Review):

Patel et al perform the analysis of neurons in a somatosensory network involved in responses to noxious cold in Drosophila larvae. Using a combination of behavioral experiments, Calcium imaging, optogenetics, and synaptic connectivity analysis in the Drosophila larval they assess the function of circuit elements in the somatosensory network downstream of multimodal somatosensory neurons involved in innocuous and noxious stimuli sensing and probe their function in noxious cold processing, Consistent with their previous findings they find the multidendritic class III neurons, to be the key cold sensing neurons that are both required and sufficient for the CT behaviors response (shown to evoked by noxious cold). They further investigate the downstream neurons identified based on literature and connectivity from EM at different stages of sensory processing characterize the different phenotypes upon activating/silencing those neurons and monitor their responses to noxious cold. The work reveals diverse phenotypes for the different neurons studied and provides the groundwork for understanding how information is processed in the nervous system from sensory input to motor output and how information from different modalities is processed by neuronal networks. However, at times the writing could be clearer and some results interpretations more rigorous.

Specific comments

1. In Figure 1 -supplement 6D-F (Cho co-activation)

The authors find that Ch neurons are cold sensitive and required for cold nociceptive behavior but do not facilitate behavioral responses induced but CIII neurons

The authors show that coactivating mdIII and cho inhibits the CT (a typically observed cold-induced behavioral response) in the second part of the stimulation period, while Cho was required for cold-induced CT. Different levels of activation of md III and Cho (different light intensities) could bring some insights into the observed phenotypes upon Cho manipulation as different levels activate different downstream networks that could correspond to different stimuli. Also, it would be interesting to activate chordotonal during exposure to cold to determine how a behavioral response to cold is affected by the activation of chordotonal sensory neurons.

1. Throughout the paper the co-activation experiments investigate whether co-activating the different candidate neurons and md III neurons facilitates the md III-induced CT response. However, the cold noxious stimuli will presumably activate different neurons downstream than optogenetic activation of MdIII and thus can reveal more accurately the role of the different candidate neurons in facilitating cold nociception.

2. Use of blue lights in behavioral and imaging experiments

Strong Blue and UV have been shown to activate MDIV neurons (Xiang, Y., Yuan, Q., Vogt, N. et al. Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall. Nature 468, 921-926 (2010). https://doi.org/10.1038/nature09576) and some of the neurons tested receive input from MdIV. In their experiments, the authors used blue light to optogenetically activate CDIII neurons and then monitored Calcium responses in Basin neurons, premotor neurons, and ascending neurons and UV light is necessary for photoconversion in Campari Experiments. Therefore, some of the neurons monitored could be activated by blue light and not cdIII activation. Indeed, responses of Basin-4 neurons can be observed in the no ATR condition (Fig 3HI) and quite strong responses of DnB neurons. (Figure 6E) How do authors discern that the effects they see on the different neurons are indeed due to cold nociception and not the synergy of cold and blue light responses could especially be the case for DNB that could have in facilitating the response to cold in a multisensory context (where mdIV are activated by light). In addition, the silencing of DNB neurons during cold stimulation does not seem to give very robust phenotypes (no significant CT decrease compared to empty GAL4 control).

It would be important to for example show that even in the absence of blue light the DNB facilitates the mdIII activation or cold-induced CT by using red light and Chrimson for example or TrpA activation (for coactivation with md III)

Alternatively, in some other cases, the phenotype upon co-activation could be inhibited by blue light (e.g. chair-1 (Figure 5 H-I))

More generally, given the multimodal nature of stimuli activating mdIV , MdIII (and Cho) and their shared downstream circuitry it is important to either control for using the blue light in these stimuli or take into account the presence of the stimulus in interpreting the results as the coactivation of for example Cho and mdIII using blue lights also could activate mdIV (and downstream neurons, alter the state of the network that could inhibit the md III induced CT responses

Assessing the differences in behavioral phenotypes in the different conditions could give an idea of the influence of combining different modalities in these assays. For example, did the authors observe any other behaviors upon co-activation of MDIII and Cho (at the expense of CT in the second part of the stimulation) or did the larvae resume crawling? Blue light typically induces reorientation behavior. What about when co-activating mdIII and Basin-4?

Using Chrimson and red light or TrpA in some key experiments e.g. with Cho, Basin-4, and DNB would clarify the implication of these neurons in cold nociception

1. Basins
   - Page 17 line 442-3 "Neural silencing of all Basin (1-4) neurons, using two independent driver lines (R72F11GAL4 and R57F07GAL4)
   Did the authors check the expression profile of the R57F07 line that they use to probe "all basins"? The expression profile published previously (Ohyama et al, 2015, extended data) shows one basin neuron (identified as basin-4 ) and some neurons in the brain lobes. Also, the split GAL4 that labels Basin-4 (SS00740) is the intersection between R72F11 and R57F07 neurons. Thus the R57F07 likely labels Basin-4 and if that is the case the data in Figure 2 9 and supplement) and Figure 3 related to this driver line, should be annotated as Basin-4, and the results and their interpretation modified to take into account the different phenotypes for all basins and Basin-4 neurons

Page 19 l. 521-525 I am confused by these sentences as the authors claim that Basin-4 showed reduced Calcium responses upon repetitive activation of CDIII md neurons but then they say they exhibit sensitization. Looking at the plots in FIG 3 F-I the Basin-4 responses upon repeated activation seem indeed to decrease on the second repetition compared to the first. What is the sensitization the authors refer to?

On Page 47-In this section of the discussion, the authors emit an interesting hypothesis that the Basin-1 neuron could modulate the gain of behavioral responses. While this is an interesting idea, I wonder what would be the explanation for the finding that co-activation of Cho and MDIII does not facilitate cold nociceptive responses. Would activation of Basin-1 facilitate the cold response in different contexts (in addition to CH0-mediated stimuli?

Page 48 Thus the implication of the inhibitory network in cold processing should be better contextualized

The authors explain the difference in the lower basin-2 Ca- response to Cold/ mdIII activation (compared to Basin-4) despite stronger connectivity, due a stronger inputs from inhibitory neurons to Basin-2 (compared to Basin-4). The previously described inhibitory neurons that synapse onto Basin-2 receive rather a small fraction of inputs from the class III sensory neurons. The differences in response to cold could be potentially assigned to the activation of the inhibitory neurons by the cold-sensing cho- neurons. However, that cannot explain the differences in responses induced by class III neurons. Do the authors refer to additional inhibitory neurons that would receive significant input from MdIII?

Alternative explanations could exist for this difference in activation: electrical synapses from mdII I onto Basin-4, and by stronger inputs from mdIV (compared to Basin-2 in the case of responses to Cold stimulus (Cold induces responses in md IV sensory neurons). Different subtypes of CD III may differentially respond to cold and the cold-sensing ones could synapse preferentially on basin-4 etc.

1. A00c
   Page 26 Figure 4F-I line While Goro may not be involved in cold nociception the A00c (and A05q) seems to be.
   A00c could convey information to other neurons other than Goro and thus be part of a pathway for cold-induced CT.

2. Page 31 766-768 the conclusion that "premotor function is required for and can facilitate cold nociception" seems odd to stress as one would assume that some premotor neurons would be involved in controlling the behavioral responses to a stimulus. It would be more pertinent in the summary to specify which premotor neurons are involved and what is their function

3. There are several Split GAL4 used in the study (with transgenes inserted in attP40 et attP2 site). A recent study points to a mutation related toattP40 that can have an effect on muscle function: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9750024/. The controls used in behavioral experiments do not contain the attP40 site. It would be important to check a control genotype bearing an attP40 site and characterize the different parameters of the CT behavior to cold and take this into account in interpreting the results of the experiments using the Split-GAL4 lines

Reviewer #3 (Public Review):

Summary:
The authors follow up on prior studies where they have argued for the existence of cold nociception in Drosophila larvae. In the proposed pathway, mechanosensitive Class III multidendritic neurons are the noxious cold responding sensory cells. The current study attempts to explore the potential roles of second and third order neurons, based on information of the Class III neuron synaptic outputs that have been obtained from the larval connectome.

Strengths:

The major strength of the manuscript is the detailed discussion of the second and third order neurons that are downstream of the mechanosensory Class III multidendritic neurons. These will be useful in further studies of gentle touch mechanosensation and mechanonociception both of which rely on sensory input from these cells. Calcium imaging experiments on Class III activation with optogenetics support the wiring diagram.

Weaknesses:

The scientific premise is that a full body contraction in larvae that are exposed to noxious cold is a sensorimotor behavioral pathway. This premise is, to start with, questionable. A common definition of behavior is a set of "orderly movements with recognizable and repeatable patterns of activity produced by members of a species (Baker et al., 2001)." In the case of nociception behaviors, the patterns of movement are typically thought to play a protective role and to protect from potential tissue damage.

Does noxious cold elicit a set of orderly movements with a recognizable and repeatable pattern in larvae? Can the patterns of movement that are stimulated by noxious cold allow the larvae to escape harm? Based on the available evidence, the answer to both questions is seemingly no. In response to noxious cold stimulation many, if not all, of the muscles in the larva, simultaneously contract (Turner et al., 2016), and as a result the larva becomes stationary. In response to cold, the larva is literally "frozen" in place and it is incapable of moving away. This incapacitation by cold is the antithesis of what one might expect from a behavior that protects the animals from harm.

Extensive literature has investigated the physiological responses of insects to cold (reviewed in Overgaard and MacMillan, 2017). In numerous studies of insects across many genera (excluding cold adapted insects such as snow flies), exposure to very cold temperatures quickly incapacitates the animal and induces a state that is known as a chill coma. During a chill coma, the insect becomes immobilized by the cold exposure, but if the exposure to cold is very brief the insect can often be revived without apparent damage. Indeed, it is common practice for many laboratories that use adult Drosophila for studies of behavior to use a brief chilling on ice as a form of anesthesia because chilling is less disruptive to subsequent behaviors than the more commonly used carbon dioxide anesthesia. If flies were to perceive cold as a noxious nociceptive stimulus, then this "chill coma" procedure would likely be disruptive to behavioral studies but is not. Furthermore, there is no evidence to suggest that larval sensation of "noxious cold" is aversive.

The insect chill coma literature has investigated the effects of extreme cold on the physiology of nerves and muscles and the consensus view of the field is that the paralysis that results from cold is due to complex and combined action of direct effects of cold on muscle and on nerves (Overgaard and MacMillan, 2017). Electrophysiological measurements of muscles and neurons find that they are initially depolarized by cold, and after prolonged cold exposure they are unable to maintain potassium homeostasis and this eventually inhibits the firing of action potentials (Overgaard and MacMillan, 2017). The very small thermal capacitance of a Drosophila larva means that its entire neuromuscular system will be quickly exposed to the effect of cold in the behavioral assays under consideration here. It would seem impossible to disentangle the emergent properties of a complex combination of effects on physiology (including neuronal, glial, and muscle homeostasis) on any proposed sensorimotor transformation pathway.

Nevertheless, the manuscript before us makes a courageous attempt at attempting this. A number of GAL4 drivers tested in the paper are found to affect parameters of contraction behavior (CT) in cold exposed larvae in silencing experiments. However, notably absent from all of the silencing experiments are measurements of larval mobility following cold exposure. Thus, it is not known from the study if these manipulations are truly protecting the larvae from paralysis following cold exposure, or if they are simply reducing the magnitude of the initial muscle contraction that occurs immediately following cold (ie reducing CT). The strongest effect of silencing occurs with the 19-12-GAL4 driver which targets Class III neurons (but is not completely specific to these cells).

Optogenetic experiments for Class III neurons relying on the 19-12-GAL4 driver combined with a very strong optogenetic acuator (ChETA) show the CT behavior that was reported in prior studies. It should be noted that this actuator drives very strong activation, and other studies with milder optogenetic stimulation of Class III neurons have shown that these cells produce behavioral responses that resemble gentle touch responses (Tsubouchi et al 2012 and Yan et al 2013). As well, these neurons express mechanoreceptor ion channels such as NompC and Rpk that are required for gentle touch responses. The latter makes the reported Calcium responses to cold difficult to interpret in light of the fact that the strong muscle contractions driven by cold may actually be driving mechanosensory responses in these cells (ie through deformation of the mechanosensitive dendrites). Are the cIII calcium signals still observed in a preparation where cold induced muscle contractions are prevented?

A major weakness of the study is that none of the second or third order neurons (that are downstream of CIII neurons) are found to trigger the CT behavioral responses even when strongly activated with the ChETA actuator (Figure 2 Supplement 2). These findings raise major concerns for this and prior studies and it does not support the hypothesis that the CIII neurons drive the CT behaviors.

Later experiments in the paper that investigate strong CIII activation (with ChETA) in combination with other second and third order neurons does support the idea activating those neurons can facilitate body-wide muscle contractions. But many of the co-activated cells in question are either repeated in each abdominal neuromere or they project to cells that are found all along the ventral nerve cord, so it is therefore unsurprising that their activation would contribute to what appears to be a non-specific body-wide activation of muscles along the AP axis. Also, if these neurons are already downstream of the CIII neurons the logic of this co-activation approach is not particularly clear. A more convincing experiment would be to silence the different classes of cells in the context of the optogenetic activation of CIII neurons to test for a block of the effects, a set of experiments that is notably absent from the study.

The authors argument that the co-activation studies support "a population code" for cold nociception is a very optimistic interpretation of a brute force optogenetics approach that ultimately results in an enhancement of a relatively non-specific body-wide muscle convulsion.

Author Response

We thank the reviewers for their suggestions in improving the manuscript. We are currently working on a formal revision and plan to submit a revised manuscript in the near future. However, we would be remiss, if we did not address concerns regarding the conceptual merits of the paper. Below we speak to major points of note that address select reviewer comments and the eLife assessment of our manuscript.

eLife assessment:

However, the strength of evidence is incomplete due to the concern that larval contraction is a result of chilling the nervous system and muscles, which causes spreading depolarization and mechanical contraction of the body, rather than an active sensorimotor response to cold.

Reviewer #3:

The scientific premise is that a full body contraction in larvae that are exposed to noxious cold is a sensorimotor behavioral pathway. This premise is, to start with, questionable. A common definition of behavior is a set of "orderly movements with recognizable and repeatable patterns of activity produced by members of a species (Baker et al., 2001)." In the case of nociception behaviors, the patterns of movement are typically thought to play a protective role and to protect from potential tissue damage.

Does noxious cold elicit a set of orderly movements with a recognizable and repeatable pattern in larvae? Can the patterns of movement that are stimulated by noxious cold allow the larvae to escape harm? Based on the available evidence, the answer to both questions is seemingly no.

We thank the reviewer for their questions and clarify, here. Exposure to cold temperatures does elicit a recognizable and repeatable pattern of behavior across multiple strains, including both wildtype and genetic control strains (w1118, Oregon R) and numerous control conditions that have been previously published (Himmel et al., 2021, Himmel et al., 2023, Patel et al., 2022, Turner et al., 2016, Turner et al., 2018, Tenedini et al., 2019). Our initial publication on Drosophila cold nociception demonstrated a variety of cold-evoked behavior responses including head and/or tail raising of the larva as well as contraction behavior. These behaviors were repeatedly observed in assays involving either local cold stimulation with a cold probe or global cold stimulation on a cold plate. Head and/or tail raise behaviors are consistent with behavior that displaces the larval body from the cold surface, however, exposure to increasingly colder temperatures leads to an increasing level of cold-evoked contraction (CT) responses which result in a reduction of larval area (Turner et al., 2016). Presumably, increasing the level of CIII md neuron activation leads to greater activation of downstream circuitry. We previously performed optogenetic dose response assays to further clarify the increased prevalence CT response to strong noxious cold stimuli and investigated how CIII md neurons discriminate between innocuous touch and noxious cold stimuli. Here, we found that lower-level activation of CIII md neurons lead to predominantly touch-evoked behaviors whereas high-level activation led predominantly to cold-evoked responses (Turner et al., 2016). These analyses were coupled with stimulus-evoked calcium imaging, which revealed that touch-evoked Ca2+ levels were significantly lower than cold-evoked Ca2+ levels (Turner et al., 2016).

In this manuscript, we confirm our previously published findings that neural silencing of CIII md neurons with either tetanus toxin expression or impairing action potential propagation results impaired cold-evoked CT responses (Turner et al., 2016, Turner et al., 2018). However, neural silencing of CIII md neurons did not eliminate cold-evoked CT responses. We interpret this finding as evidence that some component of cold-evoked CT response may be due to cold-induced muscle contraction. Furthermore, in this manuscript, we implicate the requirement of chordotonal (Ch) neurons in cold-evoked CT and demonstrate cold-evoked Ca2+ increases in Ch neurons. Furthermore, neural silencing of multiple sensory neuron types (CIII + Ch or CIII + CII) resulted in greater deficits in cold-evoked behaviors (Turner et al., 2016). Thus, the noxious cold stimulus is detected by multiple peripheral sensory neurons and inhibiting neural activity in CIII md neurons alone cannot eliminate cold-evoked CT responses.

In this manuscript and in several other publications, studies have shown that optogenetic activation of CIII md neurons, or CIII neurons plus CII neurons or Ch neurons elicits CT-like responses (Hwang et al., 2007, Shearin et al., 2013, Turner et al., 2016). Conversely, optogenetic stimulation of CIII md neurons knocked down for paralytic, the α-subunit of voltage-gated sodium channel, did not elicit blue light-evoked CT responses due to impaired action potential propagation. These analyses collectively indicate that CIII md neuron activation is sufficient for eliciting CT-like responses. Additionally, we have previously published electrophysiological recordings of CIII md neurons under cold exposure. To address potential confounds of cold-induced muscle contraction on cold-induced electrical activity of CIII md neurons, we performed these analyses on de-muscled fillets revealing that CIII neural activity is not dependent upon muscles in response to cold. Exposure to noxious cold stimuli results in temperature-dependent increases in CIII neuron firing pattern consisting of both bursting and tonic firing (Himmel et al., 2021, Himmel et al., 2023, Maksymchuk et al., 2022, Patel et al., 2022, Himmel et al., 2022, Maksymchuk et al., 2023).

Reviewer #3:

Can the patterns of movement that are stimulated by noxious cold allow the larvae to escape harm?

We were similarly curious about the neuroethological and/or protective implications of cold-evoked behaviors. In Drosophila larvae, noxious mechanical stimuli-evoked body rolling allows for lateral escape from predatory wasp (Hwang et al., 2007). Reducing the overall surface area that is exposed to cold (e.g., huddling behavior) serves as a protective strategy in many species (Canals et al., 1997, Contreras, 1984, Gilbert et al., 2006, Vickery and Millar, 1984, Hayes et al., 1992). Low temperatures can be fatal to poikilotherms (e.g., insects), however, many species have evolved the ability to cold acclimate thereby increasing their cold tolerance. To explore the potential evolutionary benefit of CIII-mediated contraction response to cold, we previously published work revealing a neural basis for cold acclimation in Drosophila larvae implicating these neurons (Himmel et al., 2021). We demonstrated that cold-evoked CT behavior is evolutionarily conserved across 11 different drosophilid species and that other cold-induced behaviors (e.g., tail raise) were also observed. Furthermore, drosophilid species adapted to rapid temperature swings were more likely to retain the ability to locomote even at lower temperatures (Himmel et al., 2021). Next, we elucidated the role of CIII md neurons in cold acclimation. Silencing CIII md neurons resulted in the inability to cold acclimate. We additionally investigated roles of Ch or CII md neurons, which alone did not inhibit the ability of larvae to cold acclimate. However, combinatorial silencing of CIII with CII or Ch neurons resulted in an inability to cold acclimate but did not obviously increase baseline cold tolerance. We explored how developmental exposure to noxious cold temperature impacts CIII md neuron cold-evoked firing pattern. Electrophysiological analyses revealed that cold acclimation results in hypersensitization in CIII md neurons (Himmel et al., 2021). Lastly, developmental optogenetic activation of CIII md neurons led to increased cold tolerance. Therefore, CIII md neurons are necessary and sufficient for cold tolerance and our collective evidence demonstrate that CIII-mediated cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation (Himmel et al., 2021).

Reviewer #3:

It should be noted that this actuator drives very strong activation, and other studies with milder optogenetic stimulation of Class III neurons have shown that these cells produce behavioral responses that resemble gentle touch responses (Tsubouchi et al 2012 and Yan et al 2013)…The latter makes the reported Calcium responses to cold difficult to interpret in light of the fact that the strong muscle contractions driven by cold may actually be driving mechanosensory responses in these cells (ie through deformation of the mechanosensitive dendrites)…. Are the cIII calcium signals still observed in a preparation where cold induced muscle contractions are prevented?”

We agree with the reviewer that mild activation of CIII md neurons results in gentle touch-like responses. In this manuscript, and other previously published work, it has been shown that optogenetic activation of CIII neurons, or CIII neurons and other sensory neurons, using a variety of optogenetic actuators (ChR2, ChETA, and CsChrimson) promotes bilateral contraction of the larval body along the anterior-posterior axis (Shearin et al., 2013, Hwang et al., 2007, Meloni et al., 2020, Turner et al., 2016, Patel and Cox, 2017, Patel et al., 2022, Himmel et al., 2023).

As described above, in our initial publication documenting larval cold nociception in Drosophila, we investigated how CIII md neurons discriminate multimodal stimuli to elicit stimulus relevant behavioral responses. We reported that increased activation of CIII md neurons results in cold-evoked behaviors, where lower activation results in touch-evoked behaviors. Subsequent, calcium analyses revealed greater stimulus-evoked calcium response to noxious cold and milder calcium response to gentle touch (Turner et al., 2016).

Though we have not performed cold-evoked Ca2+ imaging of CIII md neurons in larval preparations without muscles, we have recorded electrical responses of CIII md neurons in the absence of muscle contractions using de-muscled larvae fillets to analyze cold-evoked firing patterns of CIII md neurons (Himmel et al., 2021, Himmel et al., 2022, Himmel et al., 2023, Patel et al., 2022, Maksymchuk et al., 2022, Maksymchuk et al., 2023). These studies demonstrate the cold-evoked CIII neural activity is not dependent upon muscles.

Reviewer #3:

A major weakness of the study is that none of the second or third order neurons (that are downstream of CIII neurons) are found to trigger the CT behavioral responses even when strongly activated with the ChETA actuator (Figure 2 Supplement 2). These findings raise major concerns for this and prior studies and it does not support the hypothesis that the CIII neurons drive the CT behaviors.”

We conducted extensive screening of interneuron populations post-synaptically connected to CIII neurons in an effort to identify post-synaptic partners that were sufficient to trigger CT response. Much to our surprise, we were unable to find any individual neuron type or driver line that was sufficient to elicit a CT response. However, we provide substantial supporting evidence for our co-activation experiments including neural silencing, EM connectivity and calcium imaging. We also report necessity for the reported second/third order neurons in cold-evoked behavioral responses, where inhibiting neural activity resulted in reduced cold-evoked behavior. Second/third order neurons also exhibit cold-evoked calcium responses. Lastly, we also report CIII-evoked (using optogenetics) increases in calcium response in downstream post-synaptic neurons.

Previously published literature investigating CIV md neuron circuitry has implicated downstream neurons that are not sufficient to elicit rolling behavior upon activation. In CIV md neuron circuit dissection, select neurons are reported as acting downstream of CIV md neurons that require additional circuit components in order to execute rolling behavior. For example, A00c neuron activation alone does not lead to rolling behavior, however, co-activation of A00c and Basin-4 neurons facilitates rolling response (Ohyama et al., 2015). Similarly, co-activation of Basin-1 and Basin-4 neurons significantly enhance rolling probability relative to Basin-4 alone (Ohyama et al., 2015). Further, DnB neurons require Goro command neuron activity to promote rolling behavior (Burgos et al., 2018). Thus, there is precedent for co-activation requirements to elicit robust behavioral output in sensorimotor circuits and we employed a similar strategy after we discovered that activation of second or third order neurons alone did not elicit CT response.

Reviewer #3:

Later experiments in the paper that investigate strong CIII activation (with ChETA) in combination with other second and third order neurons does support the idea activating those neurons can facilitate body-wide muscle contractions. But many of the co-activated cells in question are either repeated in each abdominal neuromere or they project to cells that are found all along the ventral nerve cord, so it is therefore unsurprising that their activation would contribute to what appears to be a non-specific body-wide activation of muscles along the AP axis. Also, if these neurons are already downstream of the CIII neurons the logic of this co-activation approach is not particularly clear.”

We agree with the reviewer’s comment that various cell-types that were investigated are repeated in every abdominal neuromere, however, only select post-synaptic neurons (Basin 1-4, DnB, mCSI, and Chair neurons) are segmentally repeated in every abdominal segment. Conversely, other projection and ascending neurons we investigated (A09e, A00c, A05q, Goro, TePn04/05, and A08n) are not segmentally repeated in every section. We used connectome evidence to guide our experiments on populations of neurons to explore in cold-evoked behavior and as alluded to above our co-activation approach was driven by the observation that an individual subpopulation of connected interneurons was not found to be sufficient to elicit CT behavior. That said, it does not change the findings that inhibition of neural activity in these subpopulations impairs cold-evoked behavior, nor does it change the observation that connected interneurons exhibit cold-evoked Ca2+ responses that can also be observed with optogenetic activation of CIII neurons. Reviewer #3: “The authors argument that the co-activation studies support "a population code" for cold nociception is a very optimistic interpretation of a brute force optogenetics approach that ultimately results in an enhancement of a relatively non-specific body-wide muscle convulsion.” Many studies exploring circuit bases of behavior have applied large-scale optogenetic, including co-activation strategies, or silencing screens to identify circuit components involved in specific behaviors under investigation. We employed similar methods in our circuit-based dissection and our conclusions are not solely based upon optogenetic analyses.

References: BURGOS, A., HONJO, K., OHYAMA, T., QIAN, C. S., SHIN, G. J.-E., GOHL, D. M., SILIES, M., TRACEY, W. D., ZLATIC, M., CARDONA, A. & GRUEBER, W. B. 2018. Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila. eLife, 7:e26016.

CANALS, M., ROSENMANN, M. & BOZINOVIC, F. 1997. Geometrical aspects of the energetic effectivenes of huddling in small mammals. Acta Theriologica 42(3):321-328..

CONTRERAS, L. C. 1984. Bioenergetics of Huddling: Test of a Psycho-Physiological Hypothesis. Journal of Mammalogy, 65, 256-262.

GILBERT, C., ROBERTSON, G., LE MAHO, Y., NAITO, Y. & ANCEL, A. 2006. Huddling behavior in emperor penguins: Dynamics of huddling. Physiol Behav, 88, 479-88.

HAYES, J. P., SPEAKMAN, J. R. & RACEY, P. A. 1992. The Contributions of Local Heating and Reducing Exposed Surface Area to the Energetic Benefits of Huddling by Short-Tailed Field Voles (Microtus agrestis). Physiological Zoology, 65, 742-762.

HIMMEL, N. J., LETCHER, J. M., SAKURAI, A., GRAY, T. R., BENSON, M. N., DONALDSON, K. J. & COX, D. N. 2021. Identification of a neural basis for cold acclimation in Drosophila larvae. iScience, 24, 102657.

HIMMEL, N. J., SAKURAI, A., DONALDSON, K. J. & COX, D. N. 2022. Protocols for measuring cold-evoked neural activity and cold tolerance in Drosophila larvae following fictive cold acclimation. STAR Protoc, 3, 101510.

HIMMEL, N. J., SAKURAI, A., PATEL, A. A., BHATTACHARJEE, S., LETCHER, J. M., BENSON, M. N., GRAY, T. R., CYMBALYUK, G. S. & COX, D. N. 2023. Chloride-dependent mechanisms of multimodal sensory discrimination and nociceptive sensitization in Drosophila. elife, 12:e76863.

HWANG, R. Y., ZHONG, L., XU, Y., JOHNSON, T., ZHANG, F., DEISSEROTH, K. & TRACEY, W. D. 2007. Nociceptive Neurons Protect Drosophila Larvae from Parasitoid Wasps. Current Biology, 17, 2105-2116.

MAKSYMCHUK, N., SAKURAI, A., COX, D. N. & CYMBALYUK, G. 2022. Transient and Steady-State Properties of Drosophila Sensory Neurons Coding Noxious Cold Temperature. Frontiers in Cellular Neuroscience, 16:831803.

MAKSYMCHUK, N., SAKURAI, A., COX, D. N. & CYMBALYUK, G. S. 2023. Cold-Temperature Coding with Bursting and Spiking Based on TRP Channel Dynamics in Drosophila Larva Sensory Neurons. Int J Mol Sci, 24(19):14638.

MELONI, I., SACHIDANANDAN, D., THUM, A. S., KITTEL, R. J. & MURAWSKI, C. 2020. Controlling the behaviour of Drosophila melanogaster via smartphone optogenetics. Scientific Reports, 10, 17614.

OHYAMA, T., SCHNEIDER-MIZELL, C. M., FETTER, R. D., ALEMAN, J. V., FRANCONVILLE, R., RIVERA-ALBA, M., MENSH, B. D., BRANSON, K. M., SIMPSON, J. H., TRUMAN, J. W., CARDONA, A. & ZLATIC, M. 2015. A multilevel multimodal circuit enhances action selection in Drosophila. Nature, 520, 633-639.

PATEL, A. & COX, D. 2017. Behavioral and Functional Assays for Investigating Mechanisms of Noxious Cold Detection and Multimodal Sensory Processing in Drosophila Larvae. BIO-PROTOCOL, 7(13):e2388.

PATEL, A. A., SAKURAI, A., HIMMEL, N. J. & COX, D. N. 2022. Modality specific roles for metabotropic GABAergic signaling and calcium induced calcium release mechanisms in regulating cold nociception. Front Mol Neurosci 15:942548.

SHEARIN, H. K., DVARISHKIS, A. R., KOZELUH, C. D. & STOWERS, R. S. 2013. Expansion of the Gateway MultiSite Recombination Cloning Toolkit. PLoS ONE, 8, e77724-e77724.

TENEDINI, F. M., SÁEZ GONZÁLEZ, M., HU, C., PEDERSEN, L. H., PETRUZZI, M. M., SPITZWECK, B., WANG, D., RICHTER, M., PETERSEN, M., SZPOTOWICZ, E., SCHWEIZER, M., SIGRIST, S. J., CALDERON DE ANDA, F. & SOBA, P. 2019. Maintenance of cell type-specific connectivity and circuit function requires Tao kinase. Nature Communications, 10, 3506.

TURNER, H. N., ARMENGOL, K., PATEL, A. A., HIMMEL, N. J., SULLIVAN, L., IYER, S. C., BHATTACHARYA, S., IYER, E. P. R., LANDRY, C., GALKO, M. J. & COX, D. N. 2016. The TRP Channels Pkd2, NompC, and Trpm Act in Cold-Sensing Neurons to Mediate Unique Aversive Behaviors to Noxious Cold in Drosophila. Curr Biol, 26, 3116-3128.

TURNER, H. N., PATEL, A. A., COX, D. N. & GALKO, M. J. 2018. Injury-induced cold sensitization in Drosophila larvae involves behavioral shifts that require the TRP channel Brv1. PLoS One, 13, e0209577.

VICKERY, W. L. & MILLAR, J. S. 1984. The Energetics of Huddling by Endotherms. Oikos, 43, 88-93.