Homosensory and heterosensory dishabituation engage distinct circuits in Drosophila

Reviewer #1 (Public review):

Summary:

Charonitakis and co-authors characterize dishabituation in adult flies, where they use olfactory habituation to octanol, then dishabituate the flies with disruptions of electric shock or yeast odors. They systematically investigate the neurotransmitters and neural circuits involved in dishabituation and figure out a lot about how this process works in the brain, as an independent circuit. I like the paper, and I like the very structured approach to figuring out the problem.

Strengths:

The introduction nicely sets the stage for the work presented, bringing in knowledge from other organisms and motivating the study.

The results section lays out a logical set of experiments, using a common set of behavioral assays in many flies exposed to thermogenetic or optogenetic manipulation. The paper systematically figures out the necessity and/or sufficiency of specific brain regions and neurotransmitters, culminating in a new understanding of how the important process of dishabituation works.

I like the bar graph representation for the data throughout, with the helpful icons - if a paper figures are going to be 90% bar graphs, it helps when they are super clear like this! And I like how all the parts build up to the conclusion in the last figure, nicely summarizing the thorough characterization of dishabituation.

Weaknesses:

There are no major concerns, but some material could be added for clarity and to make the work more accessible to a more general scientific audience. A figure clearly showing the habituation protocol and the use of the dishabituators would be a good addition, even if the procedure has been done before and is cited. There can always be readers who are seeing this for the first time.

It would also be nice to comment on other ways dishabituation can happen (for example, when the stimulus is removed for a short time and returns) and what their time scales are.

And more generally, the paper could perhaps improve by making a stronger case for why the results are important not just for flies but for neuroscience in general.

Reviewer #2 (Public review):

This is an interesting study in Drosophila comparing potentially differential requirements for subsets of Kenyon Cells (KCs) and Dopaminergic neurons (DANS) in olfactory dishabituation driven by either a novel odor ("homosensory") or footshock ("heterosensory). The authors measure olfactory aversion to Octanol (OCT) in a T-maze, induce olfactory habituation with a 4-minute prior exposure to OCT, and use either brief yeast odor (YO) or footshock (FS) to achieve dishabituation. The major observation that YO-mediated dishabituation is mediated by reward-activated DANs (PAM cluster), while FS-mediated dishabituation is mediated by punishment-activated PPL-DANs is generally solid and convincing. Also convincing are experiments showing the involvement of KCs in the pathway for YO and FS-induced dishabituation, and the argument that KCs drive DAN activation that causes dishabituation, though not experimentally shown, is more than reasonable. The work is significant because, as the authors take pains to point out, circuits and pathways for dishabituation have been very lightly studied, and clear identification of dopaminergic neuron subsets in dishabituation achieved by different means represents unique and interesting progress.

However, the claim that this represents a fundamental difference between homosensory and heterosensory pathways for dishabituation is overstated. The introductory section does not adequately present current broad models for habituation and dishabituation. There are many different time scales, even for Drosophila olfactory habituation. These, as well as potential underlying mechanistic differences, need to be acknowledged; any claim should be specifically qualified for the time scales being studied here. Additionally, there are several unclear, vague, and inaccurate sections and statements. A more careful, precise, and considered presentation of current views, as well as more measured claims of the impact of the findings, would substantially enhance my enthusiasm.

Reviewer #3 (Public review):

Summary:

In this manuscript, Charonitakis, Pasadaki et al. investigated the neural circuits underlying homosensory/within-modal and heterosensory/cross-modal dishabituation of the olfactory avoidance response in Drosophila. Taking advantage of the accessible and sophisticated gene expression manipulation tools in the flies, this study traced neural pathways underlying response facilitation caused by different types of sensory stimuli and revealed both distinct and convergent neural components underlying these different forms of behavioral plasticity. The study first demonstrated that olfactory habituation of the octanol avoidance response can be facilitated by either a different odor (homosensory stimulus) or a foot shock (heterosensory stimulus). Then, the flies' nervous system was manipulated with gene expression tools to identify key neural components involved in mediating the behavioral facilitation caused by different types of sensory stimuli. It was found that different sensory stimuli are input into different parts of the nervous system, and signals converge in the mushroom bodies to generate response facilitation. It was also found that these facilitatory pathways are different from the olfactory habituation pathway in the lateral horns.

Strengths:

The authors took full advantage of the advanced genetic tools in flies and performed a series of experiments to pinpoint neural components in each pathway.

Weaknesses:

The key issue is that the main concepts of this manuscript appear to be based on a misunderstanding/misinterpretation of the literature. As the authors set out to settle the debate "whether the novel dishabituating stimulus elicits sensitization of the habituated circuits, or it engages distinct neuronal routes to bypass habituation reinstating the naïve response", it seems that the authors based their investigation on the premise that "sensitization" is mediated by a facilitatory process within the S-R pathway, and "dishabituation" by a facilitatory process outside the S-R pathway. This is not the status quo in the field, particularly with the prevailing theory like the Dual-Process Theory.

The original version of Dual-Process Theory (Groves and Thompson 1970, but also see Thompson 2008, Neurobiol Learn Mem) already hypothesized that habituation happens within the specific S-R pathway, and sensitization occurs separately in an "organism-wide" state system that modulates the output of all S-R pathways. Dishabituation is recognized by the Dual-Process Theory as sensitization (organism-wide facilitation) manifested on top of existing habituation (depressed S-R pathway). This notion has been supported by a wide range of studies, including cat spinal cord reflex (e.g. Spencer et al. 1966) and work in Aplysia on heterosynaptic facilitation for both sensitization and dishabituation. Therefore, simply showing that the newly identified facilitatory pathways are outside the S-R habituation pathway is insufficient to demonstrate dishabituation.

As behavioral facilitation of a habituated response can be achieved by dishabituating (specific recovery of the S-R pathway) and/or superimposed sensitizing (organism-wide) processes, dishabituation and sensitization of this olfactory response must be first dissociated; however, the study provided no evidence for the dissociation. Without this piece of evidence, the claim of this paper that the newly identified pathways mediate dishabituation is not fully supported.

The literature review of this manuscript has some discrepancies. In the introduction, the authors wrote "initial studies in Aplysia were consistent with the "dual-process theory" (Groves and Thompson 1979), where response recovery due to dishabituation appeared to result from sensitization superimposed on habituation, thus driving reversal of the attenuated response (Carew, Castellucci et al. 1971, Hochner, Klein et al. 1986, Marcus, Nolen et al. 1988, Ghirardi, Braha et al. 1992, Cohen, Kaplan et al. 1997, Antonov, Kandel et al. 1999, Hawkins, Cohen et al. 2006)." Hochner 1986 and Marcus 1988 in fact indicated otherwise. Hochner 1986 suggests that dishabituation and sensitization involve different molecular processes, while Marcus 1988 showed that dishabituation and sensitization have different behavioral characteristics. Therefore, the authors' statement is not supported by the cited literature.

Author response:

Below, we will address point by point any and all concerns of the reviewers.

Reviewer #1:

There are no major concerns, but some material could be added for clarity and to make the work more accessible to a more general scientific audience.

We will add text for clarity and to make the work more accessible to a general audience per this comment and similar suggestions of the other reviewers.

(1.1) A figure clearly showing the habituation protocol and the use of the dishabituators would be a good addition, even if the procedure has been done before and is cited. There can always be readers who are seeing this for the first time.

We do think this is a good idea as the time scales of the experiment will be clearly marked as well and we plan to generate one in the revised manuscript.

(1.2) It would also be nice to comment on other ways dishabituation can happen (for example, when the stimulus is removed for a short time and returns) and what their time scales are.

If the stimulus is withheld, spontaneous recovery occurs, a process distinct from dishabituation and worth exploring on its own. In a previous publication (Semelidou et al. eLife 2018;7:e39569), we have shown that in this habituation paradigm with 4 min exposure either to the aversive Octanol, or the attractive Ethyl Acetate, spontaneous recovery occurs on or after 6 minutes after the habituated stimulus is withheld. This contrasts the immediate effect of the single dishabituating stimulus, delivered for a few seconds at the end of exposure to the habituator. Granted that per Thomson (Neurobiol Learn Mem. 2009), spontaneous recovery is a characteristic of habituation, we will work this point in the text.

(1.3) And more generally, the paper could perhaps improve by making a stronger case for why the results are important not just for flies but for neuroscience in general.

Thank you for the encouragement. We will try to rationally generalize our findings.

Reviewer #2:

(2.1) However, the claim that this represents a fundamental difference between homosensory and heterosensory pathways for dishabituation is overstated.

We had no intention of stating more than the fact that footshock and yeast odor dishabituators relay these stimuli to the mushroom bodies via distinct dopaminergic neurons, hence differentiating distinct dishabituating stimuli via the mechanosensory (footshock) and olfactory (yeast odor) modalities as they engage the mushroom bodies. As the reviewer suggests we will use more measured and specific language to state the above.

(2.2) The introductory section does not adequately present current broad models for habituation and dishabituation.

This was not done intentionally, but rather because we aimed at a less extended introductory section and ostensibly this resulted in brief and possibly inadequate presentation of current habituation models. We will present a much more detailed introduction and detail of habituation and dishabituation models in the revised manuscript (Also see reply to point 3.5 below).

(2.3) There are many different time scales, even for Drosophila olfactory habituation. These, as well as potential underlying mechanistic differences, need to be acknowledged; any claim should be specifically qualified for the time scales being studied here.

We understand and appreciate the point of the reviewer, as well as its significance and we will address this both in the revised text, but also by the paradigm figure we will add as stated above (point 1.1), where the time scales will be explicitly included and emphasized.

(2.4) Additionally, there are several unclear, vague, and inaccurate sections and statements. A more careful, precise, and considered presentation of current views, as well as more measured claims of the impact of the findings, would substantially enhance my enthusiasm.

We will address these concerns of course, though pointing out the specific offending parts would ascertain addressing them thoroughly. As stated above, we will incorporate current views in the introduction and when discussing our results and their impact.

Reviewer #3:

(3.1) The key issue is that the main concepts of this manuscript appear to be based on a misunderstanding/misinterpretation of the literature. As the authors set out to settle the debate "whether the novel dishabituating stimulus elicits sensitization of the habituated circuits, or it engages distinct neuronal routes to bypass habituation reinstating the naïve response", it seems that the authors based their investigation on the premise that "sensitization" is mediated by a facilitatory process within the S-R pathway, and "dishabituation" by a facilitatory process outside the S-R pathway. This is not the status quo in the field, particularly with the prevailing theory like the Dual-Process Theory.

We appreciate the reviewer’s comment and the opportunity to clarify the conceptual framework of our work. Our intention was in fact to test the Groves and Thomson hypothesis (Neurobiol Learn Mem. 2009), in our olfactory habituation system. As such, dishabituation could have been the result of a facilitatory process within the S-R pathway, or from mechanisms outside of it. Our experimental design allowed to distinguish these possibilities and our results clearly show that dishabituation involves circuitry outside the S-R pathway. We do thank the reviewer for pointing out that we have not articulated clearly this intention and we will take care to communicate this effectively in the revised manuscript.

(3.2) The original version of Dual-Process Theory (Groves and Thompson 1970, but also see Thompson 2008, Neurobiol Learn Mem) already hypothesized that habituation happens within the specific S-R pathway, and sensitization occurs separately in an "organism-wide" state system that modulates the output of all S-R pathways.

As mentioned above, we are aware of the Dual-Process hypothesis. In fact, our data demonstrate that activity outside the olfactory S-R pathway, engaging novel neuronal circuits, mediates dishabituation. Unlike habituation, these circuits mediating dishabituation include at minimum, the mushroom bodies, the dopaminergic system and the APL neurons. In our view this does not support the “organism-wide state” system, but rather particular circuits that in agreement with the Groves and Thomson hypothesis, are outside the S-R pathway and modulate its behavioral output. We will work these concepts in the discussion section of the revised manuscript.

(3.3) Dishabituation is recognized by the Dual-Process Theory as sensitization (organism-wide facilitation) manifested on top of existing habituation (depressed S-R pathway). This notion has been supported by a wide range of studies, including cat spinal cord reflex (e.g. Spencer et al. 1966) and work in Aplysia on heterosynaptic facilitation for both sensitization and dishabituation. Therefore, simply showing that the newly identified facilitatory pathways are outside the S-R habituation pathway is insufficient to demonstrate dishabituation.

We respectfully disagree with the concluding sentence here. In all of our experiments, we observe a clear recovery of olfactory avoidance after exposure to the footshock, or yeast odor dishabituators. Moreover, the dishabituators are emulated by (photo)activation of particular neuronal circuits and the recovery of olfactory avoidance is blocked when these circuits are silenced. Regardless of whether this recovery is classified as dishabituation via sensitization or another facilitatory process, the key point is that the habituated response is reliably reinstated contingent upon the dishabituating stimulus. We believe this meets the established criteria for dishabituation.

(3.4) As behavioral facilitation of a habituated response can be achieved by dishabituating (specific recovery of the S-R pathway) and/or superimposed sensitizing (organism-wide) processes, dishabituation and sensitization of this olfactory response must be first dissociated; however, the study provided no evidence for the dissociation. Without this piece of evidence, the claim of this paper that the newly identified pathways mediate dishabituation is not fully supported.

We agree with the reviewer that we have not provided specific evidence dissociating dishabituation and sensitization of the particular olfactory response beyond the evidence implicating particular circuitry in the outcome of facilitation of the olfactory response.

It should be noted that in photoactivation of the implicated circuitries in naïve flies, we do not observe enhanced octanol avoidance, suggesting that activation of these circuits alone does not induce sensitization. Moreover, our results show that neither footshock nor yeast odor drive an organism-wide sensitization, as silencing specific circuits was sufficient to block dishabituation—something that would not be expected if a global sensitization process was responsible of reinstating the olfactory response.

Nonetheless, we will also attempt to dissociate sensitization from dishabituation using mutants previously reported deficient in sensitization (Duerr and Quinn, PNAS 1982), assuming these mutants retain normal olfactory habituation. We will also try sensitization protocols in the case of within-modal dishabituation to further clarify the underlying mechanisms. In principle, this includes using diluted Octanol as the habituating stimulus and attempt dishabituation with concentrated octanol.

(3.5) The literature review of this manuscript has some discrepancies. In the introduction, the authors wrote "initial studies in Aplysia were consistent with the "dual-process theory" (Groves and Thompson 1979), where response recovery due to dishabituation appeared to result from sensitization superimposed on habituation, thus driving reversal of the attenuated response (Carew, Castellucci et al. 1971, Hochner, Klein et al. 1986, Marcus, Nolen et al. 1988, Ghirardi, Braha et al. 1992, Cohen, Kaplan et al. 1997, Antonov, Kandel et al. 1999, Hawkins, Cohen et al. 2006)." Hochner 1986 and Marcus 1988 in fact indicated otherwise. Hochner 1986 suggests that dishabituation and sensitization involve different molecular processes, while Marcus 1988 showed that dishabituation and sensitization have different behavioral characteristics. Therefore, the authors' statement is not supported by the cited literature.

We are grateful to the reviewer for pointing out these significant discrepancies, consequent of multiple rounds of edits followed by our own oversight. These important publications for this manuscript will be referenced properly in the revised version of the manuscript.