Structural basis of CO 2 valence coding in Drosophila
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eLife Assessment
This study provides a detailed anatomical and functional framework for understanding CO₂ processing and behavioral flexibility in Drosophila. The significance of the work is important, as it identifies how specific neural circuits, such as LN23, modulate innately aversive signals across different contexts. The strength of the evidence is convincing, supported by a robust combination of connectomics, anatomical reconstructions, and targeted behavioral manipulations.
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Abstract
In the olfactory system, glomerular sensory channels of single receptor identity support reliable odor recognition for appropriate approach or avoidance behaviors. For many olfactory stimuli, the assigned sensory value is innate but modulated by the internal state and previous experiences. How context-dependent modulation of innate valence coding supports distinct behavioral responses is poorly understood. Here we show that CO 2 sensory information in Drosophila , intrinsically aversive but modified by attractive food signals, diverges from the canonical glomerular channel already in the antennal lobe and is relayed via the polarized local interneuron LN23. LN23 relays sensory input via an extraglomerular CO 2 pathway and manipulation of LN23 activity revealed a dominant role in CO 2 -induced avoidance behavior. The extraglomerular CO 2 pathway projects to the posterior lateral protocerebrum (PLP) adjacent to the canonical Lateral Horn (LH) olfactory processing center and segregates into anatomically distinct valence channels. Connectome data together with functional characterization showed the convergence of parallel CO 2 channels onto two interconnected third-order neurons. These neurons integrate additional sensory modalities via distinct mechanisms: while the glomerular CO 2 pathway converges with food relay neurons onto separated dendritic domains of the PD5 interneuron in the LH, the extraglomerular pathways integrating CO 2 information with antennal humidity and temperature modalities establish antagonistic inputs onto the PLP interneuron PV9. This early anatomical divergence of a defined olfactory channel followed by separated multi-modal integration provides a structural basis for context-dependent valence coding and appropriate behavioral responses.
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eLife Assessment
This study provides a detailed anatomical and functional framework for understanding CO₂ processing and behavioral flexibility in Drosophila. The significance of the work is important, as it identifies how specific neural circuits, such as LN23, modulate innately aversive signals across different contexts. The strength of the evidence is convincing, supported by a robust combination of connectomics, anatomical reconstructions, and targeted behavioral manipulations.
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Reviewer #1 (Public review):
Summary:
The authors set out to better understand how Drosophila responses to CO2 can be aversive or attractive depending on context (especially presence of food odors, temperature, humidity). While some aspects of this circuit had been previously identified, the authors uncovered additional, critical aspects of the circuit to more fully explain these phenomena. One important discovery was the identification of the LN23 interneuron, which receives input from the V glomerulus. LN23 relays sensory input via an extraglomerular CO2 pathway, and manipulation of LN23 activity revealed a dominant role in CO2-induced avoidance behavior.
Through a careful series of experiments, the authors demonstrate important aspects of these parallel (and sometimes converging) circuits - differential sensitivity to CO2 …
Reviewer #1 (Public review):
Summary:
The authors set out to better understand how Drosophila responses to CO2 can be aversive or attractive depending on context (especially presence of food odors, temperature, humidity). While some aspects of this circuit had been previously identified, the authors uncovered additional, critical aspects of the circuit to more fully explain these phenomena. One important discovery was the identification of the LN23 interneuron, which receives input from the V glomerulus. LN23 relays sensory input via an extraglomerular CO2 pathway, and manipulation of LN23 activity revealed a dominant role in CO2-induced avoidance behavior.
Through a careful series of experiments, the authors demonstrate important aspects of these parallel (and sometimes converging) circuits - differential sensitivity to CO2 concentration changes, synaptic plasticity, circuit connectivity, developmental origins, and the effect of chemo and optogenetic manipulations on behavior. Together, they piece together a complex and interconnected circuit diagram for CO2-dependent behaviors that can be modulated by external factors. This finding will be impactful not only for the fly olfactory/gustatory field but also for many others in the sensory neuroscience community who are very interested in understanding state-dependent modulation of sensory circuits.
Strengths:
The experiments were well described and controlled. The addition of the developmental trajectory of the LN23 neurons was interesting. The inclusion of multiple levels of analysis from synaptic contacts and activity-dependent labeling of synapses, circuit analysis guided by connectomes, and detailed behavior analysis for each part of the circuit were all strengths.
Weaknesses:
The circuit is very complex and interconnected. This is important for its function, but it makes reading through the manuscript a challenge. The diagrams are helpful, but still somewhat confusing, and some of the experimental findings do not completely support the model outlined in the final figure.
The main difficulty is visualizing the "default/predominant aversive" LN23 circuit - in the final diagram, there is no "stop" sign on that side, although it's depicted as an inhibition of a "go".
Also, importantly, the findings shown in Figure 5 demonstrate pretty convincingly that LN23 inhibition reduces CO2 avoidance "almost entirely". Also supporting a central role for LN23 is the opposite effect of silencing LN23, with chronic CO2 inducing attraction. If this is the case, then where is the contribution of the other canonical aversive pathway? How does the silencing of LN23 override the PNvbi/uni pathways to aversion? Incorporating this into the figure more prominently would improve the understanding of this contribution to the circuit.A minor weakness is that CO2 levels were not reduced below ambient air. For the first part of the paper addressing the activation of these circuits, there seemed to be a ceiling effect for the LN23 neurons at ambient CO2 levels. It would be interesting to see if there would be some change to the activity labeling experiments if CO2 were reduced or eliminated from the air.
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Reviewer #2 (Public review):
Summary
The authors investigate how parallel olfactory pathways contribute to CO₂ valence processing in Drosophila. By combining multiple approaches, the study identifies LN23 as a previously unrecognized component of the CO₂ circuit and proposes a model in which distinct downstream pathways contribute to aversive and attractive behavioral responses. More broadly, the work aims to connect circuit organization with context-dependent sensory processing and behavioral valence.
Strengths
A major strength of the study is the integration of multiple complementary approaches spanning anatomy, circuit analysis, and behavior. This combination provides a rich and valuable framework for understanding how CO₂ information may be processed across different levels of the olfactory system. The identification of LN23 as an …
Reviewer #2 (Public review):
Summary
The authors investigate how parallel olfactory pathways contribute to CO₂ valence processing in Drosophila. By combining multiple approaches, the study identifies LN23 as a previously unrecognized component of the CO₂ circuit and proposes a model in which distinct downstream pathways contribute to aversive and attractive behavioral responses. More broadly, the work aims to connect circuit organization with context-dependent sensory processing and behavioral valence.
Strengths
A major strength of the study is the integration of multiple complementary approaches spanning anatomy, circuit analysis, and behavior. This combination provides a rich and valuable framework for understanding how CO₂ information may be processed across different levels of the olfactory system. The identification of LN23 as an important component of the CO₂ pathway is particularly interesting and will likely be useful for future studies investigating olfactory processing, behavioral state modulation, and valence coding. The connectomic and anatomical analyses also provide a valuable resource for the community.
Another strength of the manuscript is its conceptual ambition. The work moves beyond a simple labeled-line view of olfactory processing and proposes that flexible behavioral responses may emerge from interactions between parallel downstream pathways and multimodal integration centers. The behavioral manipulations further support an important role for LN23 in CO₂-related behaviors.
Weaknesses
Several aspects of the conceptual interpretation would benefit from additional clarification or more cautious framing relative to the current experimental evidence. In particular, the distinction between atmospheric versus experimentally elevated CO₂ conditions, as well as the interpretation of chronic exposure in terms of habituation, remains somewhat unclear throughout the manuscript.
Some conclusions regarding valence coding and multimodal integration also appear more inferential than directly demonstrated experimentally, especially when moving from anatomical connectivity to functional interpretation.
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Reviewer #3 (Public review):
Summary:
In this manuscript, Javorski and colleagues investigate how CO2 valence is processed in the Drosophila olfactory system. Although CO2 is classically associated with an aversive labeled‑line pathway, its behavioral significance can be modulated by environmental context, such as the presence of food‑related cues. The circuit‑level mechanisms underlying this flexibility remain incompletely understood. The authors address this gap by examining how CO2 sensory information diverges at early stages of olfactory processing and how distinct neural pathways contribute to opposing behavioral outcomes. By identifying the local interneuron LN23 as a relay for CO2‑induced aversion, the study suggests that CO2 valence processing may begin to diverge at the level of the antennal lobe, prior to synaptic integration …
Reviewer #3 (Public review):
Summary:
In this manuscript, Javorski and colleagues investigate how CO2 valence is processed in the Drosophila olfactory system. Although CO2 is classically associated with an aversive labeled‑line pathway, its behavioral significance can be modulated by environmental context, such as the presence of food‑related cues. The circuit‑level mechanisms underlying this flexibility remain incompletely understood. The authors address this gap by examining how CO2 sensory information diverges at early stages of olfactory processing and how distinct neural pathways contribute to opposing behavioral outcomes. By identifying the local interneuron LN23 as a relay for CO2‑induced aversion, the study suggests that CO2 valence processing may begin to diverge at the level of the antennal lobe, prior to synaptic integration in higher‑order brain regions such as the lateral horn.
Strengths:
A major strength of this study is its comprehensive, multi-level experimental design that effectively links neuronal identity, synaptic organization, and behavior. The authors combine calcium‑based anatomical mapping, activity‑dependent reporters, optogenetic and thermogenetic manipulations, and connectomic analyses with behavioral readouts under genetically defined neuronal activation or silencing conditions. Specifically, the identification of LN23 as a component of the CO2 avoidance pathway is supported by anatomical, genetic, and behavioral evidence. Both silencing and activation experiments indicate that LN23 plays an important role in mediating CO2‑induced aversive responses. In contrast, manipulation of the projection neurons (PNv bi and PNv uni) produces more modest behavioral effects, suggesting a degree of specificity for LN23‑associated circuitry within the avoidance pathway. Moreover, the use of previous reported connectome to identify downstream third‑order neurons strengthens the proposed circuit model and provides anatomical support for early divergence of CO2 valence processing.
Weaknesses:
While the study provides a strong mechanistic framework for CO2 aversion, some aspects of context‑dependent valence modulation are less directly addressed and may benefit from further experimental exploration.
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