Skip to main content
NCBI home page
eLife logoLink to eLife
. 2026 Sep 28;14:RP108236. doi: 10.7554/eLife.108236

Selective lifelong suppression of an odor processing channel in response to critical period experience

Hans C Leier 1,†, Julius Jonaitis 2,†, Alexander J Foden 1,†, Abigail J Wilkov 1, Paola Van der Linden Costello 1, Heather T Broihier 1,✉, Andrew M Dacks 2,✉
Editors: Mani Ramaswami3, Albert Cardona4
PMCID: PMC13619131  PMID: 42803516

Abstract

Sensory circuits undergo experience-dependent plasticity during early-life critical periods, attuning the nervous system to levels of key environmental stimuli. During a critical period in the Drosophila olfactory system, we found that exposure to ethyl butyrate (EB) induces glial phagocytosis of odorant receptor Or42a-positive olfactory sensory neuron (OSN) axon terminals which terminate in the VM7 glomerulus (Leier et al., 2025). Here, we extend these findings by establishing functional significance and circuit selectivity in this critical period paradigm. First, using a combination of two-photon Ca2+ imaging and the genetically-encoded voltage indicator ASAP5, we find that Or42a OSN odor-evoked responses are permanently suppressed in animals with critical period odor exposure. Thus, critical period odor exposure results in long-term changes to odor sensitivity in Or42a OSNs. Second, to establish the selectivity of glial pruning for Or42a axon terminals, we examined projection neurons (PNs) postsynaptic to Or42a OSNs as well as a second population of highly EB-responsive OSNs, called Or43b OSNs. We find that (1) within VM7, glial pruning is selective for Or42a terminals, and (2) while Or43b OSNs appear modestly pruned, they maintain their sensitivity to EB. To elucidate this difference, we turned to the Drosophila connectome. We identify striking differences in the scale of inhibitory connectivity to Or42a and Or43b OSNs, although disruption of GABA A receptor expression in both OSN types enhanced pruning. This study expands our understanding of this critical period plasticity paradigm by demonstrating lifelong suppression of pruned Or42a OSNs and establishing its specificity within and between sensory circuits.

Research organism: D. melanogaster

Introduction

Animals integrate information from multiple sensory processing channels to seek out food, reproduce, and avoid danger. In the olfactory system, sensory channels are defined by populations of first-order olfactory sensory neurons (OSNs) expressing unique repertoires of chemoreceptor proteins. For many species, these parallel odor-processing channels converge at an initial processing stage (the olfactory bulb in mammals and the antennal lobe (AL) in insects) in which all OSNs expressing the same combination of receptors synapse at stereotyped glomeruli onto local interneurons and second-order olfactory neurons that project to other regions of the brain (Schlegel et al., 2021; Wilson, 2013).

Because many odorant receptors (ORs) are tuned to recognize a range of ligands with similar structures (Grabe et al., 2016), and many ethologically relevant odorants can bind multiple classes of ORs with varying affinity (Hallem et al., 2004; Hallem and Carlson, 2006; Knaden et al., 2012; Schubert et al., 2014), olfactory circuits are capable of combinatorially encoding a vast odor space of unique compounds with a comparatively small number of receptors (Si et al., 2019). This remarkable capacity is observed even in relatively simple olfactory systems, such as Drosophila melanogaster’s repertoire of ~60 OR genes expressed across ~2600 OSNs (Benton et al., 2025).

While this method of odor coding far exceeds the discriminatory capacity of other sensory systems (Bushdid et al., 2014), it also presents a major vulnerability: at sufficiently high levels, a so-called ‘public odor’ capable of activating many classes of OSNs could exceed the circuit’s ability to discriminate between similar compounds, rendering large parts of the odor landscape inaccessible (Hallem and Carlson, 2006). To counteract this, the Drosophila AL features multiple adaptations. For instance, local interneurons (LNs) synapse with both OSNs and second-order projection neurons (PNs) and exert gain control over the OSN-to-PN relay, using both lateral excitation and inhibition to maintain odor coding across orders of magnitude of intensity (Barth-Maron et al., 2023; Liu and Wilson, 2013; Olsen et al., 2010; Olsen and Wilson, 2008; Root et al., 2008; Root et al., 2007). Gain control allows recent network activity to impact odor-evoked responses over short time periods, whereas prolonged odorant exposure can induce larger scale circuit remodeling, a phenomenon largely restricted to early-life windows of heightened plasticity (Chodankar et al., 2020; Das et al., 2011; Devaud et al., 2003; Devaud et al., 2001; Golovin et al., 2019; Gugel et al., 2023; Jindal et al., 2023; Mallick et al., 2024; Sachse et al., 2007; Sudhakaran et al., 2012).

Recently, we and others uncovered the cellular mechanism of ethyl butyrate (EB)-induced circuit remodeling in the glomerulus VM7, which is innervated by OSNs expressing the EB-sensitive OR Or42a (Leier et al., 2025; Nelson et al., 2024). EB is an important food cue detected by large numbers of ORs, with Or42a being among the most sensitive (Hallem et al., 2004; Hallem and Carlson, 2006; Semmelhack and Wang, 2009). During a 48 hr critical period following eclosion, we found that EB exposure induces glia to invade VM7 and phagocytose Or42a OSN axon terminals, drastically reducing OSN-PN connectivity even after several days had passed (Leier et al., 2025). From a circuit standpoint, we hypothesized that this may be an alternative method of limiting input from the most active information channel in an over-stimulating environment, preserving odor-coding capacity of other EB-sensitive OSNs (Tadres et al., 2022).

In the current study, we define functional consequences and cellular specificity in this critical period paradigm. We previously found that early odor exposure led to a reduction in spontaneous activity of VM7 PNs (Leier et al., 2025), but we did not probe changes in odor-evoked activity. Here, employing two-photon Ca2+ imaging and a genetically-encoded voltage indicator, we find that following critical period odor exposure, Or42a OSN odor-evoked responses remain suppressed at 25 days post eclosion (DPE). These data indicate that critical period EB exposure permanently alters odor processing.

We next addressed if glial pruning is restricted to Or42a OSN axon terminals or whether other neurons engaged in EB odor processing are likewise pruned. Strikingly, within the Or42a-VM7 PN circuit, pruning is selective for Or42a axon terminals since neither axons nor dendrites of VM7 PNs are refined following critical period odor exposure. We then turned to VM2, the second-most EB-sensitive glomerulus in Drosophila, which is innervated by OSNs expressing Or43b (Olsen et al., 2007). While Or43b OSNs are modestly pruned following early-life EB exposure, their odor-evoked responses are unchanged. The existence of critical period Or43b remodeling supports our working hypothesis that pruning of exquisitely EB-sensitive OSNs dampens activity in these hyperactive information channels to maintain odor coding capability. However, we were surprised that EB-evoked responses of Or43b neurons remain relatively normal, arguing that pruning of Or43b presynaptic terminals does not attenuate odor responses below baseline. To elucidate distinct functional consequences of Or42a and Or43b pruning, we capitalized on the recently completed Drosophila connectome (Dorkenwald et al., 2024; Schlegel et al., 2024; Zheng et al., 2018) and compared connectivity of Or42a and Or43b neurons. Our connectomic analysis revealed large-scale differences in the local inhibitory circuitry interacting with OSNs within VM7 and VM2. Specifically, while there are roughly comparable numbers of Or42a and Or43b OSNs, Or42a neurons make roughly sevenfold more synapses with a specific class of highly connected inhibitory interneurons, suggesting Or42a neurons serve an outsize function in EB odor processing. Together, we propose that glial pruning and synaptic inhibition play complementary roles in an interlocking mechanism to adapt and maintain the odor-coding capacity of the olfactory system.

Results and discussion

Lifelong suppression of Or42a OSNs after critical-period EB exposure

We previously showed using whole-cell patch electrophysiological recordings from PNs (Figure 1A) that spontaneous release from Or42a OSN axon terminals is reduced by ~98% following critical-period exposure to EB and remains similarly depressed after a 4 day recovery period (Leier et al., 2025). While this ex vivo recording strategy validated the loss of presynapses observed by confocal microscopy, it did not address the effects of critical-period exposure on the odor-evoked responses of Or42a OSNs. To address this, we used two-photon microscopy to measure Ca2+ flux in Or42a OSNs expressing GCaMP8f (Zhang et al., 2023), following 0–2 DPE 15% EB exposure (Figure 1B).

Figure 1. Lifelong suppression of Or42a OSNs after critical-period EB exposure.

Figure 1.

Open in a new tab

(A) Schematic of the Drosophila olfactory circuit at the level of the AL. OSN, olfactory sensory neuron, PN, projection neuron, LN, local interneuron, EG, ensheathing glia. (B) Overview of odorant-exposure experiments. Flies are exposed to 15% (v/v) ethyl butyrate (EB) or mineral oil vehicle control during the Or42a OSN critical period from 0 to 2 days post-eclosion (DPE). (C–F) Live imaging of Or42a OSN odor-evoked activity at 2 (C, D) or 25 (E, F) DPE, following exposure to 15% EB or mineral oil from 0 to 2 DPE. Or42a-GAL4>UAS-GCaMP8f flies were stimulated with three 1 s puffs (yellow bars) of 5% EB (red traces and data points) or mineral oil vehicle control (gray traces and data points) spaced 10 s apart. Traces (C, E) show the mean ΔF/F0 values of all trials. Data points (D, F) represent the mean peak ΔF/F0 values for each fly. (G–H) Live imaging of ASAP5 responses in Or42a OSNs in 2 DPE flies following exposure to 15% EB or mineral oil from 0 to 2 DPE. Flies were stimulated with odor puffs as above. Traces (G) represent the mean ΔF/F0 values of all trials. Data points (H) represent the mean peak ΔF/F0 values for each animal. Data in (D, F), and (H) are mean± SD. *p<0.05, **p<0.01, unpaired t-test (D) or Mann–Whitney U-test (F, H). Genotypes, raw values, and detailed statistics are provided in Figure 1—source data 1.

Figure 1—source data 1. Numerical data used to generate Figure 1D and F.

In VM7, odor-evoked OSN responses were sharply reduced in flies that had been exposed to EB, relative to mineral oil-exposed controls (Figure 1C–D). Curious about how long this effect persisted, we aged 0–2 DPE-exposed flies to 25 DPE and repeated our imaging. Remarkably, even at an advanced age where olfactory function has declined considerably (Hussain et al., 2018), odor-evoked activity remained suppressed in flies that had been exposed to EB nearly a month earlier (Figure 1E–F). To our knowledge, this is the longest span over which early-life Drosophila olfactory circuit remodeling has been assessed, and highlights the remarkably permanent effects of glial pruning.

While live calcium imaging using GCaMP has been extensively used to study OSN activity in the Drosophila AL (Das et al., 2011; Hong and Wilson, 2015; Semmelhack and Wang, 2009; Sudhakaran et al., 2012), we considered whether the well-known limitations of the technique (Yang and St-Pierre, 2016) may be exacerbated when attempting to assess circuit activity in axon terminals that are actively being phagocytosed. To address this concern, we turned to the recently developed genetically encoded voltage indicator (GEVI) ASAP5 (Hao et al., 2024), using it for the first time in the Drosophila olfactory system. As with GCaMP, Or42a OSNs expressing ASAP5 displayed reduced odor-evoked activity after 0–2 DPE EB exposure compared to mineral oil controls (Figure 1G–H). While odor-evoked GEVI transients likely reflect both Ca2+ influx and action potentials at the axon terminal, the reduction in odor-evoked activity (from either indicator), combined with the reduction in presynaptic markers in OSN terminals and the reduction in spontaneous EPSPs recorded from PNs (Leier et al., 2025), are all consistent with EB exposure dramatically reducing the input from Or42a OSNs to the AL.

Experience-dependent pruning in VM7 is restricted to the presynaptic compartment

Thus far, our work has focused on the presynaptic side of the OSN-PN relay. There are numerous reports of early-life EB exposure inducing structural remodeling of PN dendrites in multiple glomeruli (Chodankar et al., 2020; Das et al., 2011; Golovin et al., 2019; McCann et al., 2011; Sachse et al., 2007), prompting us to carefully examine whether structural or physiological changes in PNs occur within VM7 during its critical period. We began by measuring the odor-evoked responses of VM7 PN dendrites, taking advantage of a newly characterized split-GAL4 line (Xie et al., 2021) to drive expression of GCaMP8f in just two cell types: VM7 and VM5 PNs. VM7 PN EB-evoked responses were reduced after critical-period EB exposure (Figure 2A–B), with a mean change at 2 DPE similar in magnitude to that of Or42a OSNs (45.4% for PNs vs. 32.9% for OSNs in Figure 1D).

Figure 2. Experience-dependent pruning in VM7 is restricted to the presynaptic compartment.

Figure 2.

Open in a new tab

(A–B) Live imaging of VM7 projection neuron (PN) odor-evoked activity at 2 days post-eclosion (DPE), following exposure to 15% ethyl butyrate (EB) or mineral oil from 0 to 2 DPE. VM7 PN split-GAL4 >UAS-GCaMP8f flies were stimulated with three 1 s puffs (yellow bars) of 5% EB (red traces and data points) or mineral oil vehicle control (gray traces and data points) spaced 10 s apart. Traces (A) show the mean ΔF/F0 values of all trials. Data points (B) represent the mean peak ΔF/F0 values for each fly. (C) Representative dendritic arbors of VM7 PNs endogenously expressing the V5-tagged postsynaptic marker Discs large 1 (Dlg1), from flies exposed to mineral oil or 15% EB from 0 to 2 DPE. (D) Normalized counts of Dlg1 puncta. (E) Representative images of VM7 PNs after exposure to mineral oil or 15% EB from 0 to 2 DPE. Neuropil is visualized with antibody staining for the presynaptic active zone protein Bruchpilot (Brp). PN dendritic arbors occupying VM7 are enclosed in white boxes. (F) Volumetric measurements of VM7 PN dendritic arbors shown in (E). (G) Representative images of VM7 PN axonal arbors after exposure to mineral oil or 15% EB from 0 to 2 DPE. (H) Volumetric measurements of VM7 PN axonal arbors shown in (G). (I) Models of VM7 PN presynapses from (G) after exposure to mineral oil or 15% EB from 0 to 2 DPE, defined as Brp puncta masked by mCD8::GFP-labeled VM7 PNs. (J) Normalized counts of VM7 PN presynapses from (I). Data are mean± SD. ns, p≥0.05, *p<0.05, Mann–Whitney U-test. Genotypes, raw values, and detailed statistics are provided in Figure 2—source data 1.

Figure 2—source data 1. Numerical data used to generate Figure 2B, D, F, H and J.

Was this loss in PN responses simply due to reduced innervation from their pruned presynaptic inputs, or had they been modified in other ways? Postsynaptic modifications play an important role in experience-dependent plasticity (Ehrlich and Malinow, 2004), including in the Drosophila olfactory system (Pribbenow et al., 2022); we, therefore, sought to visualize postsynaptic structures in VM7. To accomplish this, we used a newly developed tool to label excitatory postsynapses in a cell type-specific manner, wherein the endogenous postsynaptic scaffolding protein discs large (Dlg1; homolog of mammalian PSD-95) is tagged with V5 in cells expressing GAL4 (Parisi et al., 2023; Sheng and Kim, 2011). Using the same PN split-GAL4 driver as before, we expressed Dlg1-V5 in VM7 PNs and measured the number of postsynaptic puncta within VM7 (Figure 2C), finding that 15% EB exposure from 0 to 2 DPE did not alter VM7 PN postsynaptic content (Figure 2D). While this result may appear incongruous with the loss in presynapses seen in Or42a OSNs, compensation from other glomeruli may preserve the number of postsynapses in PNs (Olsen et al., 2007).

Also, unlike their presynaptic partners, VM7 PN dendrites did not change in volume after 15% EB exposure from 0 to 2 DPE compared to mineral oil-exposed controls (Figure 2E and F) and they showed a striking absence of the punctate membrane appearance associated with glial phagocytosis of presynaptic axon terminals (Leier et al., 2025). Taken together, these data reinforce our previous finding that the intrinsic biophysical properties of VM7 PNs are unaltered even as they receive less innervation from pruned OSNs, and suggest that experience-dependent pruning is restricted to the presynaptic side of the VM7 OSN-PN relay.

Next, we wondered if VM7 PN axon terminals might be pruned or remodeled where they terminate in the mushroom body (Schlegel et al., 2021). While experience-dependent plasticity at PN axon terminals has been observed in other contexts (Kremer et al., 2010), less is known about their dynamics compared to plasticity of PN dendrites. With our VM7/VM5 PN split-GAL4 driving CD8::GFP, we were able to trace 4–5 PN axons from each AL to a tightly defined axonal arbor in the mushroom bodies (Figure 2G). As with PN dendrites, we found no volumetric changes in the axonal arbors of EB-exposed PNs (Figure 2H) and no changes in their presynaptic content (Figure 2I–J).

Experience-dependent pruning and inhibition are decoupled in Or43b OSNs

Another outstanding question from our study of VM7 was whether early-life exposure has a uniform effect on the odor-evoked responses of OSNs in all glomeruli that respond to a single odor. EB robustly activates several classes of ORs in addition to Or42a (Figure 3A; Münch and Galizia, 2016), making it well-suited to interrogate the extent of pruning in glomeruli throughout the AL. Of particular interest to us was VM2, which is innervated by OSNs expressing the odorant receptor (Or43b) with the second-highest affinity for EB (Münch and Galizia, 2016); indeed, Or43b and Or42a as a pair have significantly higher EB responses than other ORs (Figure 3A). Despite this, 0–2 DPE 15% EB exposure only caused a 12% reduction in Or43b OSN presynapses (Figure 3B) and an 18% reduction in VM2 volume (Figure 3C), far less than the changes seen in VM7. This relatively modest result suggests that, as a potential mechanism for maintaining circuit homeostasis in response to neuronal activity (Lee et al., 2021), glial pruning is tightly regulated in a glomerulus-specific manner.

Figure 3. Experience-dependent pruning and inhibition are decoupled in Or43b olfactory sensory neurons (OSNs).

Figure 3.

Open in a new tab

(A) DoOR 2.0 profile of ethyl butyrate (EB) responses across all Drosophila odorant receptors (ORs). (B–C) Representative images (bottom) and quantification of presynaptic content (B) and volume (C) of VM2 (Or43b OSNs) and VM7 (Or42a OSNs) in flies exposed to mineral oil or 15% EB from 0 to 2 days post-eclosion (DPE). Presynapses were visualized with Bruchpilot (Brp) staining. Data are mean± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, unpaired t-test. (D–E) Two-photon imaging of GCaMP8f responses in Or43b OSNs at 2 DPE following exposure to 15% EB or mineral oil from 0 to 2 DPE. Awake flies were stimulated with three 1 s odor puffs (yellow bars) of 5% EB (red traces and data points) or mineral oil (gray traces and data points), spaced 10 s apart. Traces (D) represent the mean ΔF/F0 values of all trials. Data points (E) represent the mean peak ΔF/F0 values for each animal. Genotypes, raw values, and detailed statistics are provided in Figure 3—source data 1.

Figure 3—source data 1. Numerical data used to generate Figure 3A, B, C and E.

Next, we tested the functional consequences of critical-period EB exposure on Or43b OSNs by keeping Or43b-GAL4>UAS-GCaMP8f flies in 15% EB or mineral oil from 0 to 2 DPE, and repeating the experimental scheme in Figure 1B. Given the inhibition observed in other EB-sensitive glomeruli, such as DM2 and DM5, following early-life EB exposure (Das et al., 2011), and our own observation of life-long Or42a OSN inhibition following glial pruning, we postulated that Or43b OSNs would also be inhibited to some degree. Instead, we observed similar levels of EB-evoked activity in pruned Or43b OSNs relative to flies exposed to mineral oil (Figure 3D–E), suggesting that there is a nonlinear relationship between levels of pruning and OSN activity. It is possible that reduced OSN responses occur in parallel with, but not as a result of, glial pruning, or that some threshold of pruning must be reached in order for odor-evoked responses to decrease.

Differential patterns of LN innervation may contribute to interglomerular differences in experience-dependent glial pruning

What factors drive the differences in structural remodeling and inhibition across different glomeruli during early-life EB exposure? One potential explanation is the tuning of the chemoreceptor proteins expressed by each OSN type; however, Or42a and Or43b have very similar response profiles (Grabe et al., 2016; Münch and Galizia, 2016) yet are differentially impacted by EB exposure (Figure 3). We, therefore, asked whether there are any prominent differences in the network connectivity of OSNs innervating VM2 and VM7, using the FlyWire full-brain connectome dataset (Dorkenwald et al., 2024; Schlegel et al., 2024; Zheng et al., 2018) to compare the upstream and downstream synaptic partners of Or24a (Figure 4A and B) and Or43b (Figure 4A and C) OSNs. Although VM7 and VM2 are innervated by relatively similar numbers of OSNs (33 and 37, respectively) and PNs (2–3 per hemisphere) (Figure 4A), VM7 OSNs far exceeded VM2 in the number of synaptic outputs and inputs as well as total number of synaptic partners, such that any given upstream or downstream partner made roughly twice as many synapses with VM7 OSNs relative to VM2 OSNs (Figure 4B–C). Strikingly, the combined volume of VM7 OSNs was ~2.3 times that of VM2 (4275 µm3 relative to 1870 µm3), which was consistent with the ratio of ~2.6 in our glomerular analysis (1883 µm3 [VM7] relative to 712 µm3 [VM2]) (Figure 3C) and indicated that individual VM7 OSNs have greater synaptic connectivity relative to VM2 OSNs.

Figure 4. Differential patterns of local interneuron (LN) innervation may contribute to interglomerular differences in experience-dependent glial pruning.

Figure 4.

Open in a new tab

(A) Numbers of neurons within the cell types included in (B) and (C). (B–C) Sankey diagrams of the synaptic inputs and outputs of Or42a (B) and Or43b (C) olfactory sensory neurons (OSNs). Inputs were thresholded at 99% of all synapses and outputs were thresholded at 95% of all synapses. Brackets contain the number of synapses and percentage of total synapses for that category. All connectomic analyses are derived from the FlyWire FAFB dataset. Synapse counts are provided in Figure 4—source data 1.

Figure 4—source data 1. Numerical data used to generate Figure 4B and C.

Both VM7 and VM2 OSNs receive the vast majority of their synaptic input from several LN subtypes that serve a variety of computational functions within the AL. For instance, both OSN types receive input from the lLN2Ps which are non-spiking, peptidergic LNs that exert intraglomerular gain control (Barth-Maron et al., 2023; Schenk and Gaudry, 2023; Sizemore et al., 2023). However, the demographics of input from LNs were not identical across Or42a and Or43b OSNs. For instance, only the Or42a OSNs received input from the bilaterally projecting il3LN6s which play a critical role in odor localization due to their asymmetric connectivity (Taisz et al., 2023). However, the most prominent difference in the connectivity of Or42a and Or43b OSNs was in their relationship with the lLN2F_b LNs. Although there are only two lLN2F_b LNs in each AL, they are a significant source of GABAergic input within the AL, collectively synapsing upon thousands of downstream targets. Due to their high degree of reciprocal connectivity within the AL, the lLN2F_bs are ‘rich-club’ neurons (Dorkenwald et al., 2024), and have been shown to play an important role in interglomerular gain control by directly targeting OSNs (Barth-Maron et al., 2023). Strikingly, Or42a OSNs receive 953 synapses from lLN2F_b LNs, whereas Or43b OSNs receive only 133 synapses. Furthermore, while both OSN types primarily synapsed upon uniglomerular PNs, Or42a OSNs provided 583 synapses to the two lLN2F_bs, whereas Or43b OSNs only provided 83 synapses. Thus, on both the upstream and downstream sides, Or42a OSNs make roughly sevenfold more inhibitory synapses with ILN2F_b inhibitory interneurons than do Or43b OSNs.

To compare the importance of GABAergic input to experience-dependent pruning of Or42a and Or43b OSNs, we performed RNAi knockdown of the key ionotropic GABAA receptor Rdl (Das et al., 2011; Sudhakaran et al., 2012) in both cell types during critical-period EB or mineral oil exposure. Intriguingly, rdl knockdown modestly increased glial pruning of both Or42a and Or43b presynaptic terminals to a similar degree (Figure 5A–B). While this result further validates that glial pruning is proportional to EB-evoked activity, it suggests that large differences in GABAergic inhibition between Or42a and Or43b OSNs are not solely responsible for their different levels of glial pruning.

Figure 5. GABAergic inputs restrain glial pruning.

Figure 5.

Open in a new tab

(A–B) Representative images (bottom) and quantification of VM7 (A) and VM2 (B) volume in flies exposed to mineral oil or 15% ethyl butyrate (EB) from 0 to 2 days post-eclosion (DPE). Data are mean ± SD. *p<0.05, **p<0.01, ****p<0.0001, one-way ANOVA. Genotypes, raw values, and detailed statistics are provided in Figure 5—source data 1.

Figure 5—source data 1. Numerical data used to generate Figure 5A and B.

LNs have been demonstrated to regulate experience-dependent plasticity in the AL, with particular insights being gained into the glomerulus-specific dynamics of GABAergic inhibitory LNs (Chodankar et al., 2020; Das et al., 2011; Mallick et al., 2024; Sachse et al., 2007). With the accessibility of detailed connectomes of the AL (Dorkenwald et al., 2024; Schlegel et al., 2024), it is possible to explore how differences in the connectivity of specific LN types to OSNs innervating each glomerulus contribute to critical period plasticity. Although there are roughly equal numbers of Or42a and Or43b OSNs and these OSN types are equally sensitive to EB, distinct patterns of connectivity could explain the differences in susceptibility to critical period pruning. The high degree of connectivity of Or42a OSNs (via the lLN2F_b LNs) could make them a ‘hub’ of network activity during EB responses such that glia are responding to the local excitatory-inhibitory ratio, which is influenced by critical LN subtypes (Figure 4A).

In summary, we report two novel advances from our initial findings and previous studies of experience-dependent plasticity in the Drosophila olfactory circuit: (i) true lifelong suppression of a class of OSNs following glial pruning during a critical period and (ii) glial pruning and loss of odor sensitivity in OSNs may occur independently of each other. Importantly, these mechanisms may be viewed as part of an integrated circuit response to a sensory stressor, e.g., an overactive sensory neuron, with the end result of maintaining the odor-encoding space (Hallem and Carlson, 2006). We propose that during sensory critical periods, glial pruning and GABAergic inhibition synergize to limit network excitation and preserve odor encoding. Our work underscores the remarkable capacity of the young olfactory system to adapt to changing environmental conditions and further positions glia as integral components of neuronal circuits.

Methods

Key resources table.

Reagent type (species) or resource Designation Source or reference Identifiers Additional information
Genetic reagent (D. melanogaster) UAS-GCaMP8f BDSC BDSC Stock #92587; RRID:BDSC_92587; FlyBase ID: FBst0092587 Genotype: w[1118]; PBac{y[+mDint2] w[+mC]=20XUAS-IVS-jGCaMP8f}VK00005
Genetic reagent (D. melanogaster) UAS-ASAP5 BDSC BDSC Stock #605337; RRID:BDSC_605337; FlyBase ID: FBst0605337 Genotype: w[*]; S (1)/CyO; PBac{y[+mDint2] w[+mC]=20XUAS-ASAP5}VK00005/TM6B, Tb(1)
Genetic reagent (D. melanogaster) Or42a-mCD8::GFP Golovin et al., 2019 Broadie lab Genotype: w; Or42a-mCD8::GFP
Genetic reagent (D. melanogaster) Or43b-GAL4 BDSC BDSC Stock #23894; RRID:BDSC_23894; FlyBase ID: FBst0023894 Genotype: w[*]; P{w[+mC]=Or43b-GAL4.C}110t8.1
Genetic reagent (D. melanogaster) Or42a-GAL4 BDSC BDSC Stock #9970; RRID:BDSC_9970; FlyBase ID: FBst0009970 Genotype: w[*]; P{w[+mC]=Or42 a-GAL4.F}48.3B
Genetic reagent (D. melanogaster) dlg1[4 K] Parisi et al., 2023 Mosca lab Genotype: dlg1[4 K], UAS-Flp[8208]
Genetic reagent (D. melanogaster) UAS-mCD8::GFP BDSC BDSC Stock #32186; RRID:BDSC_32186; FlyBase ID: FBst0032186 Genotype: w*; P{10XUAS-IVS-mCD8::GFP}attP40
Genetic reagent (D. melanogaster) VM7 PN split-GAL4 Xie et al., 2021 Janelia FlyLight Split-GAL4 Driver Collection, line SS01265 Genotype: w; VT033006-p65ADZp in attP40; R19G08-ZpGdbd in attP2/TM6B
Genetic reagent (D. melanogaster) UAS-LexA RNAi BDSC BDSC Stock # 67947; RRID:BDSC_67947; FlyBase ID: FBst0067947 Genotype: y(1) sc[*] v(1) sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS05772}attP40
Genetic reagent (D. melanogaster) UAS-rdl RNAi BDSC BDSC Stock # 31286; BDSC_31286; FlyBase ID: FBst0031286 Genotype: y(1) v(1); P{y[+t7.7] v[+t1.8]=TRiP.JF01227}attP2
Antibody Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 Thermo Fisher Scientific Cat #A32728;
RRID:AB_2866490 		Used 1:400 (IHC)
Antibody anti-Bruchpilot (Mouse monoclonal) Developmental Studies Hybridoma Bank Cat #nc82;
RRID:AB_2314866 		Used 1:50 (IHC)
Antibody anti-GFP, Alexa Fluor 488 (Rabbit polyclonal) Thermo Fisher Scientific Cat #A21311; RRID:AB_221477 Used 1:200 (IHC)
Antibody anti-V5 tag, Alexa Fluor 488 (Mouse monoclonal) Thermo Fisher Scientific Cat #37–7500 A488; RRID:AB_2610630 Used 1:100
(IHC)
Software, algorithm Imaris Bitplane RRID:SCR_007370
Software, algorithm FlyWire https://flywire.ai/ RRID:SCR_019205
Software, algorithm fafbseg https://github.com/navis-org/fafbseg-py; Schlegel, 2026; Dorkenwald et al., 2024; Schlegel et al., 2024 RRID:SCR_008394 Python package
Software, algorithm DoOR.data https://github.com/ropensci/DoOR.data; Münch, 2026a RRID:SCR_000432 R package for DoOR data
Software, algorithm DoOR.functions https://github.com/ropensci/DoOR.functions; Münch, 2026b RRID:SCR_000432 R package for DoOR functions
Open in a new tab

Drosophila melanogaster stocks

Flies were reared on standard cornmeal-molasses-agar medium in a 25 °C incubator on a 12 hr/12 hr light/dark cycle, conditions which were maintained throughout odorant exposure. Unless otherwise noted, iso31 flies were used as wild-type controls in experimental crosses. Genotypes and reference information for all transgenic fly lines used in this study are provided in the Key resources table.

Odorant exposure

The odorant exposure protocol was maintained from our previous study (Leier et al., 2025). For critical period exposure (0–2 days post-eclosion), 10 days after beginning crosses in standard bottles of fly food stoppered with cotton plugs, all adults (parents and any progeny that had eclosed) were anesthetized briefly with CO2 and discarded, and the cotton plugs were replaced with a layer of nylon mesh secured with rubber bands. A 1 mL solution of ethyl butyrate (Sigma-Aldrich #E15701) diluted volumetrically in mineral oil (Item Number (DPCI): 245-05-0562), or 1 mL of mineral oil-only control, was prepared in a 1.5 mL microcentrifuge tube, vortexed for 5–10 s, and the open tube was taped to the side of a 3.55 L airtight glass container (Glasslock #MHRB-370). The nylon-covered bottles were placed into the Glasslock containers, which were sealed and returned to the 25 °C incubator. After 24 hr, all adults that eclosed during that time were anesthetized with CO2 and separated by genotype into standard vials of fly food secured with nylon mesh. Vials were returned to the Glasslock containers with fresh odorant solution and placed in the incubator for an additional 48 hr, during which the odorant solution was refreshed after 24 hr.

Immunohistochemistry

Adult flies were anesthetized with CO2, decapitated, then dissected in ice-cold PBS. Once dissected, brains were fixed in ice-cold 4% (v/v) PFA in PBST (1% Triton X-100, 0.01 M phosphate, 0.0027 M KCl, and 0.137 M NaCl) for 20 min. Brains were immediately washed three times with PBST, incubated for 1 hr in blocking buffer (5% normal goat serum, 0.3% Triton X-100 in PBS), then incubated for 24 hr at 4 °C with primary antibodies diluted in blocking buffer. Brains were given three 10 min washes with PBST, then incubated for 24 hr at 4 °C with secondary antibodies diluted in blocking buffer as before. The next day, brains were given three 10 min washes with PBST and kept stationary for 30 min in SlowFade Gold mountant (Thermo Fisher #S36936). Mounting was performed essentially as described (Kelly et al., 2017). Unless otherwise noted, all steps were performed with rocking at room temp. A complete list of antibodies and dilutions is provided in the Key resources table.

Confocal microscopy

Images were acquired with a Zeiss LSM 800 confocal laser scanning microscope using a 100 x/1.4 NA Plan-Apochromat objective. Laser intensity and gain were optimized for each z-stack.

Image analysis

Quantification of glomerulus volume and presynaptic content was done using Imaris 9.7.1 (Bitplane). First, the Surfaces function was used on the GFP channel to model the OSN membrane. The following settings were used: baseline subtraction (threshold of 1000) followed by background subtraction (filter width of 10 µm); smoothing enabled (surfaces detail of 0.1 µm); thresholding based on local contrast (largest sphere diameter set to 10 µm), with automatic thresholding enabled; and filter based on number of voxels (cutoff of 500 voxels). The resulting Surfaces were manually processed by a blinded experimenter who used the scissors tool to remove OSN axons prior to the terminal arbor and used the final Surfaces object to mask the Brp channel. The Spots function was then used on the masked channel to quantify the number of Brp puncta within the glomerulus, using the following settings: deconvolution (robust algorithm, standard parameters) followed by background subtraction (filter width of 10 µm); algorithm set to different Spots sizes (region growing); background subtraction enabled; estimated diameter of 0.4 µm; automated quality filter; Spots regions from absolute intensity, with automatic region thresholding enabled.

Fly preparation for in vivo Ca2+ imaging and odor delivery

All in vivo Ca2+ imaging experiments were performed immediately following odorant exposure using a Bruker Ultima two-photon microscope system (Bruker Corporation, Billerica, MA) equipped with a Ti:Sapphire laser (Coherent Inc, Santa Clara, CA), tuned to 920 nm for imaging and controlled by Prairie View software (Bruker). Fluorescence signals were detected using a gallium arsenide phosphide (GaAsP) photomultiplier tube. Imaging was performed at a frame rate of 29.3 Hz. Both male and female flies were used.

Flies were anesthetized on ice and transferred to a custom recording chamber consisting of a square aluminum foil sheet (10 mm×12 mm) affixed to a plastic Petri dish. A central imaging window (~1 mm×1 mm) was cut into the foil to expose the head. Flies were positioned in the window and secured using a UV-cured plastic welder (BONDIC SK8024, NY). The head was immobilized such that the antennae remained dry during dissection. A small incision was made in the cuticle using 26-gauge needles (BD PrecisionGlide, 305110–26 G), and overlying tissue was removed to expose the dorsal surface of the brain for imaging.

The Or42a-GAL4 and Or43b-GAL4 driver lines (Key Resources Table) were used to visualize olfactory sensory neurons (OSNs) projecting to their respective glomeruli. For Ca²+ imaging experiments, GCaMP8f was expressed in OSNs to measure odor-evoked responses to Ethyl Butyrate. For voltage imaging, ASAP5 was expressed in OR42a OSNs. Imaging was performed in the antennal lobe, with a particular focus on glomerular responses to odor stimulation. The imaging chamber was filled with approximately 3 mL of saline.

For odor stimulation, ethyl butyrate was diluted 5:100 in mineral oil and delivered using a modified version of a previously described method. In brief, 50 µL of diluted odorant (in mineral oil) was pipetted onto a small piece of Whatman filter paper, which was inserted into the wide end of a 5¾-inch glass Pasteur pipette (Chemglass Life Sciences, Cat. No. CG-1700–14, Fisher Scientific). The tapered tip of the pipette was placed into a glass stimulation tube that received a continuous humidified air stream. The Pasteur pipette was positioned such that a lateral opening in the stimulation tube allowed for the integration of the odorized airstream into the main flow path. An air source was connected to the back of the pipette, behind the filter paper, enabling airflow to push volatile odorants through the pipette and out the tip into the stimulation tube. Odorized airflow was regulated via a carbon-filtered compressed air system split into a constant background stream (2.5 L/min; Dwyer VFA-25-BV flowmeter) and an odor stream (0.8 L/min; Dwyer VFA-23-BV flowmeter). A Parker solenoid valve (001-0028-900, Hollis, NH) toggled between the odor pipette and constant air flow. Odor stimuli were delivered for 1 s, three times per trial using Prairie View software.

Raw imaging data were imported into FIJI for ROI selection. Fluorescence signals were extracted and processed using a custom MATLAB script (https://github.com/adacks/Salman-et-al; Dacks, 2025). Signal changes were normalized to the baseline fluorescence (F), calculated as the average over the 3 s preceding the first odor stimulus. ΔF/F was calculated as the percent change from baseline, and traces and summary data were visualized accordingly. Processed data were analyzed statistically using GraphPad Prism.

Connectomic analysis

FlyWire (Dorkenwald et al., 2024; Schlegel et al., 2024) analysis was performed with the fafbseg (Dorkenwald et al., 2024; Schlegel et al., 2023; Zheng et al., 2018) Python package. VM7 and VM2 OSN presynaptic and postsynaptic connectivity was extracted using the get_connectivity and NeuronCriteria functions in the flywire module of fafbseg. The presynaptic and postsynaptic root IDs collected were then matched with their respective cell types from get_heirarchical_annotations in the flywire module of fafbseg. The data were filtered to exclude connections with less than five synapses. We sorted the inputs to VM7 OSNs and VM2 OSNs and thresholded the connectivity at 99% of total synaptic partners. We thresholded VM7 OSN and VM2 OSN outputs at 95% of total partners because they have at least four times as many postsynapses compared to presynapses. ‘Other’ consisted of all cells that were not annotated as lLN2F_b/a, lLN2P_b, lLN2T_b/c/d/e, il2LN6, or PN.

Statistics

Statistical analyses were performed with Prism 10.2.1 (GraphPad). All data are from at least two independent experiments. Sample size was not predetermined. For microscopy experiments, each data point represents one glomerulus; for calcium imaging experiments, each data point represents recordings from one animal. Shapiro–Wilk normality testing showed that our data were not normally distributed, so nonparametric tests were used: the Mann-Whitney test for comparisons of two samples, and the Kruskal-Wallis test with Dunn’s test for multiple comparisons for three or more samples. All tests were two-tailed with an alpha of 0.05, and no outliers were excluded. P-values are represented as follows: ns, not significant (p≥0.05); *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

Acknowledgements

We thank the Princeton FlyWire team and members of the Murthy and Seung labs, as well as members of the Allen Institute for Brain Science, for development and maintenance of FlyWire (supported by BRAIN Initiative grants MH117815 and NS126935 to Murthy and Seung). We also acknowledge members of the Princeton FlyWire team and the FlyWire Consortium for neuron proofreading and annotation. We thank Jaeda Coutinho-Budd, Dan Jindal, and members of the Broihier and Dacks labs for thoughtful feedback and stimulating conversations. We gratefully acknowledge stocks obtained from the Janelia Research Campus and the Bloomington Stock Center (NIH P400D018537). This work was supported by NIH grants F30AG097357 (HCL), R01DC016293 (AMD, JJ, AJW), T32GM152319 (HCL, AJF), and R01NS120689 (HCL, AJF, PVLC, HTB).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Heather T Broihier, Email: htb@case.edu.

Andrew M Dacks, Email: amd358@case.edu.

Mani Ramaswami, Trinity College Dublin, Ireland.

Albert Cardona, University of Cambridge, United Kingdom.

Funding Information

This paper was supported by the following grants:

  • National Institutes of Health F30AG097357 to Hans C Leier.

  • National Institutes of Health R01DC016293 to Andrew M Dacks.

  • National Institutes of Health T32GM152319 to Alexander J Foden, Heather T Broihier.

  • National Institutes of Health R01NS120689 to Heather T Broihier.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Data curation, Investigation, Visualization, Writing – original draft, Writing – review and editing.

Data curation, Formal analysis, Validation, Investigation, Visualization, Writing – review and editing.

Conceptualization, Investigation, Visualization.

Conceptualization, Investigation, Visualization, Writing – review and editing.

Investigation, Writing – review and editing.

Conceptualization, Supervision, Project administration, Writing – review and editing.

Conceptualization, Supervision, Writing – original draft, Project administration, Writing – review and editing.

Additional files

MDAR checklist

Data availability

All reagents will be made available upon request. All data in support of the findings of this study can be accessed at https://doi.org/10.17605/OSF.IO/NFWQB.

The following dataset was generated:

Dacks A, Jonaitis J, Leier H. 2026. Leier eLife advance. Open Science Framework.

References

  1. Barth-Maron A, D’Alessandro I, Wilson RI. Interactions between specialized gain control mechanisms in olfactory processing. Current Biology. 2023;33:5109–5120. doi: 10.1016/j.cub.2023.10.041. [DOI] [PubMed] [Google Scholar]
  2. Benton R, Mermet J, Jang A, Endo K, Cruchet S, Menuz K. An integrated anatomical, functional and evolutionary view of the Drosophila olfactory system. EMBO Reports. 2025;26:3204–3225. doi: 10.1038/s44319-025-00476-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bushdid C, Magnasco MO, Vosshall LB, Keller A. Humans can discriminate more than 1 trillion olfactory stimuli. Science. 2014;343:1370–1372. doi: 10.1126/science.1249168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chodankar A, Sadanandappa MK, VijayRaghavan K, Ramaswami M. Glomerulus-selective regulation of a critical period for interneuron plasticity in the Drosophila antennal lobe. The Journal of Neuroscience. 2020;40:5549–5560. doi: 10.1523/JNEUROSCI.2192-19.2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dacks AM. Salman-et-al. 8691f88GitHub. 2025 https://github.com/adacks/Salman-et-al
  6. Das S, Sadanandappa MK, Dervan A, Larkin A, Lee JA, Sudhakaran IP, Priya R, Heidari R, Holohan EE, Pimentel A, Gandhi A, Ito K, Sanyal S, Wang JW, Rodrigues V, Ramaswami M. Plasticity of local GABAergic interneurons drives olfactory habituation. PNAS. 2011;108:E646–E654. doi: 10.1073/pnas.1106411108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devaud JM, Acebes A, Ferrús A. Odor exposure causes central adaptation and morphological changes in selected olfactory glomeruli in Drosophila. The Journal of Neuroscience. 2001;21:6274–6282. doi: 10.1523/JNEUROSCI.21-16-06274.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Devaud JM, Acebes A, Ramaswami M, Ferrús A. Structural and functional changes in the olfactory pathway of adult Drosophila take place at a critical age. Journal of Neurobiology. 2003;56:13–23. doi: 10.1002/neu.10215. [DOI] [PubMed] [Google Scholar]
  9. Dorkenwald S, Matsliah A, Sterling AR, Schlegel P, Yu SC, McKellar CE, Lin A, Costa M, Eichler K, Yin Y, Silversmith W, Schneider-Mizell C, Jordan CS, Brittain D, Halageri A, Kuehner K, Ogedengbe O, Morey R, Gager J, Kruk K, Perlman E, Yang R, Deutsch D, Bland D, Sorek M, Lu R, Macrina T, Lee K, Bae JA, Mu S, Nehoran B, Mitchell E, Popovych S, Wu J, Jia Z, Castro MA, Kemnitz N, Ih D, Bates AS, Eckstein N, Funke J, Collman F, Bock DD, Jefferis G, Seung HS, Murthy M, FlyWire Consortium Neuronal wiring diagram of an adult brain. Nature. 2024;634:124–138. doi: 10.1038/s41586-024-07558-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ehrlich I, Malinow R. Postsynaptic density 95 controls AMPA receptor incorporation during long-term potentiation and experience-driven synaptic plasticity. The Journal of Neuroscience. 2004;24:916–927. doi: 10.1523/JNEUROSCI.4733-03.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Golovin RM, Vest J, Vita DJ, Broadie K. Activity-dependent remodeling of Drosophila olfactory sensory neuron brain innervation during an early-life critical period. The Journal of Neuroscience. 2019;39:2995–3012. doi: 10.1523/JNEUROSCI.2223-18.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grabe V, Baschwitz A, Dweck HKM, Lavista-Llanos S, Hansson BS, Sachse S. Elucidating the neuronal architecture of olfactory glomeruli in the Drosophila antennal lobe. Cell Reports. 2016;16:3401–3413. doi: 10.1016/j.celrep.2016.08.063. [DOI] [PubMed] [Google Scholar]
  13. Gugel ZV, Maurais EG, Hong EJ. Chronic exposure to odors at naturally occurring concentrations triggers limited plasticity in early stages of Drosophila olfactory processing. eLife. 2023;12:e85443. doi: 10.7554/eLife.85443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hallem EA, Ho MG, Carlson JR. The molecular basis of odor coding in the Drosophila antenna. Cell. 2004;117:965–979. doi: 10.1016/j.cell.2004.05.012. [DOI] [PubMed] [Google Scholar]
  15. Hallem EA, Carlson JR. Coding of odors by a receptor repertoire. Cell. 2006;125:143–160. doi: 10.1016/j.cell.2006.01.050. [DOI] [PubMed] [Google Scholar]
  16. Hao YA, Lee S, Roth RH, Natale S, Gomez L, Taxidis J, O’Neill PS, Villette V, Bradley J, Wang Z, Jiang D, Zhang G, Sheng M, Lu D, Boyden E, Delvendahl I, Golshani P, Wernig M, Feldman DE, Ji N, Ding J, Südhof TC, Clandinin TR, Lin MZ. A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events. Neuron. 2024;112:3680–3696. doi: 10.1016/j.neuron.2024.08.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hong EJ, Wilson RI. Simultaneous encoding of odors by channels with diverse sensitivity to inhibition. Neuron. 2015;85:573–589. doi: 10.1016/j.neuron.2014.12.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hussain A, Pooryasin A, Zhang M, Loschek LF, La Fortezza M, Friedrich AB, Blais C-M, Üçpunar HK, Yépez VA, Lehmann M, Gompel N, Gagneur J, Sigrist SJ, Grunwald Kadow IC. Inhibition of oxidative stress in cholinergic projection neurons fully rescues aging-associated olfactory circuit degeneration in Drosophila. eLife. 2018;7:e32018. doi: 10.7554/eLife.32018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jindal DA, Leier HC, Salazar G, Foden AJ, Seitz EA, Wilkov AJ, Coutinho-Budd JC, Broihier HT. Early Draper-mediated glial refinement of neuropil architecture and synapse number in the Drosophila antennal lobe. Frontiers in Cellular Neuroscience. 2023;17:1166199. doi: 10.3389/fncel.2023.1166199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kelly SM, Elchert A, Kahl M. Dissection and immunofluorescent staining of mushroom body and photoreceptor neurons in adult Drosophila melanogaster brains. Journal of Visualized Experiments. 2017;1:56174. doi: 10.3791/56174-v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Knaden M, Strutz A, Ahsan J, Sachse S, Hansson BS. Spatial representation of odorant valence in an insect brain. Cell Reports. 2012;1:392–399. doi: 10.1016/j.celrep.2012.03.002. [DOI] [PubMed] [Google Scholar]
  22. Kremer MC, Christiansen F, Leiss F, Paehler M, Knapek S, Andlauer TFM, Förstner F, Kloppenburg P, Sigrist SJ, Tavosanis G. Structural long-term changes at mushroom body input synapses. Current Biology. 2010;20:1938–1944. doi: 10.1016/j.cub.2010.09.060. [DOI] [PubMed] [Google Scholar]
  23. Lee JH, Kim JY, Noh S, Lee H, Lee SY, Mun JY, Park H, Chung WS. Astrocytes phagocytose adult hippocampal synapses for circuit homeostasis. Nature. 2021;590:612–617. doi: 10.1038/s41586-020-03060-3. [DOI] [PubMed] [Google Scholar]
  24. Leier HC, Foden AJ, Jindal DA, Wilkov AJ, Van der Linden Costello P, Vanderzalm PJ, Coutinho-Budd J, Tabuchi M, Broihier HT. Glia control experience-dependent plasticity in an olfactory critical period. eLife. 2025;13:RP100989. doi: 10.7554/eLife.100989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Liu WW, Wilson RI. Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system. PNAS. 2013;110:10294–10299. doi: 10.1073/pnas.1220560110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mallick A, Tan HL, Epstein JM, Jing Ng CM, Cook OM, Gaudry Q, Dacks AM. Serotonin acts through multiple cellular targets during an olfactory critical period. iScience. 2024;27:111083. doi: 10.1016/j.isci.2024.111083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McCann C, Holohan EE, Das S, Dervan A, Larkin A, Lee JA, Rodrigues V, Parker R, Ramaswami M. The Ataxin-2 protein is required for microRNA function and synapse-specific long-term olfactory habituation. PNAS. 2011;108:E655–E662. doi: 10.1073/pnas.1107198108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Münch D, Galizia CG. DoOR 2.0--Comprehensive mapping of Drosophila melanogaster odorant responses. Scientific Reports. 2016;6:21841. doi: 10.1038/srep21841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Münch D. DoOR.data. db323a4GitHub. 2026a https://github.com/ropensci/DoOR.data
  30. Münch D. DoOR.functions. 15e415eGitHub. 2026b https://github.com/ropensci/DoOR.functions
  31. Nelson N, Vita DJ, Broadie K. Experience-dependent glial pruning of synaptic glomeruli during the critical period. Scientific Reports. 2024;14:9110. doi: 10.1038/s41598-024-59942-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Olsen SR, Bhandawat V, Wilson RI. Excitatory interactions between olfactory processing channels in the Drosophila antennal lobe. Neuron. 2007;54:89–103. doi: 10.1016/j.neuron.2007.03.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Olsen SR, Wilson RI. Lateral presynaptic inhibition mediates gain control in an olfactory circuit. Nature. 2008;452:956–960. doi: 10.1038/nature06864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Olsen SR, Bhandawat V, Wilson RI. Divisive normalization in olfactory population codes. Neuron. 2010;66:287–299. doi: 10.1016/j.neuron.2010.04.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Parisi MJ, Aimino MA, Mosca TJ. A conditional strategy for cell-type-specific labeling of endogenous excitatory synapses in Drosophila. Cell Reports Methods. 2023;3:100477. doi: 10.1016/j.crmeth.2023.100477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pribbenow C, Chen Y-C, Heim M-M, Laber D, Reubold S, Reynolds E, Balles I, Fernández-D V Alquicira T, Suárez-Grimalt R, Scheunemann L, Rauch C, Matkovic T, Rösner J, Lichtner G, Jagannathan SR, Owald D. Postsynaptic plasticity of cholinergic synapses underlies the induction and expression of appetitive and familiarity memories in Drosophila. eLife. 2022;11:e80445. doi: 10.7554/eLife.80445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Root CM, Semmelhack JL, Wong AM, Flores J, Wang JW. Propagation of olfactory information in Drosophila. PNAS. 2007;104:11826–11831. doi: 10.1073/pnas.0704523104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Root CM, Masuyama K, Green DS, Enell LE, Nässel DR, Lee CH, Wang JW. A presynaptic gain control mechanism fine-tunes olfactory behavior. Neuron. 2008;59:311–321. doi: 10.1016/j.neuron.2008.07.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sachse S, Rueckert E, Keller A, Okada R, Tanaka NK, Ito K, Vosshall LB. Activity-dependent plasticity in an olfactory circuit. Neuron. 2007;56:838–850. doi: 10.1016/j.neuron.2007.10.035. [DOI] [PubMed] [Google Scholar]
  40. Schenk JE, Gaudry Q. Nonspiking interneurons in the Drosophila antennal lobe exhibit spatially restricted activity. eNeuro. 2023;10:2022. doi: 10.1523/ENEURO.0109-22.2022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schlegel P, Bates AS, Stürner T, Jagannathan SR, Drummond N, Hsu J, Serratosa Capdevila L, Javier A, Marin EC, Barth-Maron A, Tamimi IF, Li F, Rubin GM, Plaza SM, Costa M, Jefferis G. Information flow, cell types and stereotypy in a full olfactory connectome. eLife. 2021;10:e66018. doi: 10.7554/eLife.66018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schlegel P, Yin Y, Bates AS, Dorkenwald S, Eichler K. A consensus cell type atlas from multiple connectomes reveals principles of circuit stereotypy and variation. bioRxiv. 2023 doi: 10.1101/2023.06.27.546055. [DOI]
  43. Schlegel P, Yin Y, Bates AS, Dorkenwald S, Eichler K, Brooks P, Han DS, Gkantia M, Dos Santos M, Munnelly EJ, Badalamente G, Serratosa Capdevila L, Sane VA, Fragniere AMC, Kiassat L, Pleijzier MW, Stürner T, Tamimi IFM, Dunne CR, Salgarella I, Javier A, Fang S, Perlman E, Kazimiers T, Jagannathan SR, Matsliah A, Sterling AR, Yu S-C, McKellar CE, Costa M, Seung HS, Murthy M, Hartenstein V, Bock DD, Jefferis GSXE, FlyWire Consortium Whole-brain annotation and multi-connectome cell typing of Drosophila. Nature. 2024;634:139–152. doi: 10.1038/s41586-024-07686-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schlegel P. Fafbseg-py. d0da951GitHub. 2026 https://github.com/navis-org/fafbseg-py
  45. Schubert M, Hansson BS, Sachse S. The banana code-natural blend processing in the olfactory circuitry of Drosophila melanogaster. Frontiers in Physiology. 2014;5:59. doi: 10.3389/fphys.2014.00059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Semmelhack JL, Wang JW. Select Drosophila glomeruli mediate innate olfactory attraction and aversion. Nature. 2009;459:218–223. doi: 10.1038/nature07983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sheng M, Kim E. The postsynaptic organization of synapses. Cold Spring Harbor Perspectives in Biology. 2011;3:a005678. doi: 10.1101/cshperspect.a005678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Si G, Kanwal JK, Hu Y, Tabone CJ, Baron J, Berck M, Vignoud G, Samuel ADT. Structured odorant response patterns across a complete olfactory receptor neuron population. Neuron. 2019;101:950–962. doi: 10.1016/j.neuron.2018.12.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sizemore TR, Jonaitis J, Dacks AM. Heterogeneous receptor expression underlies non-uniform peptidergic modulation of olfaction in Drosophila. Nature Communications. 2023;14:5280. doi: 10.1038/s41467-023-41012-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sudhakaran IP, Holohan EE, Osman S, Rodrigues V, Vijayraghavan K, Ramaswami M. Plasticity of recurrent inhibition in the Drosophila antennal lobe. The Journal of Neuroscience. 2012;32:7225–7231. doi: 10.1523/JNEUROSCI.1099-12.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tadres D, Wong PH, To T, Moehlis J, Louis M. Depolarization block in olfactory sensory neurons expands the dimensionality of odor encoding. Science Advances. 2022;8:eade7209. doi: 10.1126/sciadv.ade7209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Taisz I, Donà E, Münch D, Bailey SN, Morris BJ, Meechan KI, Stevens KM, Varela-Martínez I, Gkantia M, Schlegel P, Ribeiro C, Jefferis GSXE, Galili DS. Generating parallel representations of position and identity in the olfactory system. Cell. 2023;186:2556–2573. doi: 10.1016/j.cell.2023.04.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wilson RI. Early olfactory processing in Drosophila: mechanisms and principles. Annual Review of Neuroscience. 2013;36:217–241. doi: 10.1146/annurev-neuro-062111-150533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Xie Q, Brbic M, Horns F, Kolluru SS, Jones RC, Li J, Reddy AR, Xie A, Kohani S, Li Z, McLaughlin CN, Li T, Xu C, Vacek D, Luginbuhl DJ, Leskovec J, Quake SR, Luo L, Li H. Temporal evolution of single-cell transcriptomes of Drosophila olfactory projection neurons. eLife. 2021;10:e63450. doi: 10.7554/eLife.63450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yang HH, St-Pierre F. Genetically encoded voltage indicators: opportunities and challenges. The Journal of Neuroscience. 2016;36:9977–9989. doi: 10.1523/JNEUROSCI.1095-16.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zhang Y, Rózsa M, Liang Y, Bushey D, Wei Z, Zheng J, Reep D, Broussard GJ, Tsang A, Tsegaye G, Narayan S, Obara CJ, Lim JX, Patel R, Zhang R, Ahrens MB, Turner GC, Wang SSH, Korff WL, Schreiter ER, Svoboda K, Hasseman JP, Kolb I, Looger LL. Fast and sensitive GCaMP calcium indicators for imaging neural populations. Nature. 2023;615:884–891. doi: 10.1038/s41586-023-05828-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zheng Z, Lauritzen JS, Perlman E, Robinson CG, Nichols M, Milkie D, Torrens O, Price J, Fisher CB, Sharifi N, Calle-Schuler SA, Kmecova L, Ali IJ, Karsh B, Trautman ET, Bogovic JA, Hanslovsky P, Jefferis G, Kazhdan M, Khairy K, Saalfeld S, Fetter RD, Bock DD. A complete electron microscopy volume of the brain of adult Drosophila melanogaster. Cell. 2018;174:730–743. doi: 10.1016/j.cell.2018.06.019. [DOI] [PMC free article] [PubMed] [Google Scholar]

eLife Assessment

Mani Ramaswami 1

This study in the Drosophila antennal lobe, which contains multiple non-equivalent sensory channels, provides valuable new insight into how early-life sensory experience can produce lasting, cell-type-specific changes in neural circuit function. The work demonstrates that glial-mediated pruning during a defined developmental window leads to persistent suppression of odor responses in one olfactory neuron type, while sparing another. The evidence is convincing and supported by multiple complementary approaches, although some mechanistic interpretations remain speculative and would benefit from additional functional testing.

Reviewer #1 (Public review):

Anonymous

Summary:

This study builds on earlier work showing that early-life odor exposure can trigger glial-mediated pruning of specific olfactory neuron terminals in Drosophila. Moving from indirect to direct functional imaging, the authors show that pruning during a narrow developmental window leads to long-lasting suppression of odor responses in one neuron type (Or42a) but not another (Or43b). The combination of calcium and voltage imaging with connectomic analysis is a strength, though the voltage imaging results are less straightforward to interpret and may not reflect synaptic output changes alone.

Strengths:

Biologically, one of the main strengths of this work is the direct comparison between two odor-responsive OSN types that differ in their long-term adaptation to early-life odor exposure. While Or42a OSNs undergo pruning and remain persistently suppressed into late adulthood, Or43b OSNs, which also respond to the same odor, show little lasting change. This contrast not only underscores the cell-type specificity of critical-period plasticity but also points to a potential role of inhibitory network architecture in determining susceptibility. The persistence of the Or42a suppression well beyond the developmental window provides compelling evidence that early glia-mediated pruning can imprint a stable, life-long functional state on selected sensory channels. By situating these functional outcomes within the context of detailed connectomic data, the study offers a framework for linking structural connectivity to long-term sensory coding stability or vulnerability.

Comments on revised version:

I thank the authors for their careful revision and thoughtful responses to the reviewers' comments. The revised manuscript addresses my previous concerns in a satisfactory manner, and the interpretation of the findings has been appropriately clarified and balanced. I have no further major comments.

Reviewer #2 (Public review):

Anonymous

Recent work from the authors identified the synaptic changes and glial reaction that occurs during exposure of a Drosophila odorant receptor neuron population to continued exposure of a stimulating odorant. This work markedly advanced our understanding of cellular response to critical periods. This current Advance manuscript carries that work forward and examines the non-autonomous responses to constant odorant exposure. The authors discover that the changes to ORN populations are not accompanied by changes to either PN dendrite or PN axon volume, nor are they concurrent with changes in postsynaptic PN structures. These changes are, however, notable accompanied by changes in Ca2+ and voltage responses in ORNs. Importantly, this set of responses is specific for the Or42a ORNs (that are highly sensitive to the odorant in question, ethyl butyrate) and not the Or43b ORNs (which respond to ethyl butyrate, but not as drastically). Finally, the authors include connectomics analyses showing that Or43b and Or42a ORNs differ in their synaptic input/output relationships.

This is an excellent use of the Advance mechanism for the journal as these are important follow-up findings for the parent story. The non-autonomous effects (or lack thereof) on PNs is an important part of the story as is the functional response of Or42a ORNs and the differing response of similarly (but not identically) sensitive Or43b ORNs. The experiments are well conceived, controlled, and conducted. Where the story falters a bit, though, is with the connectomics analysis. The authors show distinct differences between Or43b and Or42b ORN input output relationships and suggest that those differences may underlie the differences observed in their response to ethyl butyrate exposure during the critical period. This is certainly a possibility, but as it stands now, it is too disconnected to offer significant proof. There would have to be additional experiments to address this. Right now, the inclusion of the connectomics work feels like a distraction at best, and a complete non sequitur at worst. To be clear, the connectomics work is well done and I have no issues with its validity, but is not helpful to the central thesis of the work. I would suggest the authors either remove it entirely or strongly rethink how it fits into the paper.

Comments on revised version:

I appreciate the consideration of my comments and the authors' responses. The additional data on PN synapse number is intriguing (and welcome) as is the text discussing potential postsynaptic compensatory mechanisms. I respect the authors' decision in retaining the connectivity analysis, but despite the textual changes, I still feel that it is peripherally related to the main thesis of the work and would best be omitted from the paper and included in a separate, more relevant study. Ultimately, though, that is their choice.

eLife. 2026 Sep 28;14:RP108236. doi: 10.7554/eLife.108236.3.sa3

Author response

Hans C Leier 1, Julius Jonaitis 2, Alexander J Foden 3, Abigail J Wilkov 4, Paola Van der Linden Costello 5, Heather Broihier 6, Andrew M Dacks 7

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study builds on earlier work showing that early-life odor exposure can trigger glial-mediated pruning of specific olfactory neuron terminals in Drosophila. Moving from indirect to direct functional imaging, the authors show that pruning during a narrow developmental window leads to long-lasting suppression of odor responses in one neuron type (Or42a) but not another (Or43b). The combination of calcium and voltage imaging with connectomic analysis is a strength, though the voltage imaging results are less straightforward to interpret and may not reflect synaptic output changes alone.

Strengths:

Biologically, one of the main strengths of this work is the direct comparison between two odor-responsive OSN types that differ in their long-term adaptation to early-life odor exposure. While Or42a OSNs undergo pruning and remain persistently suppressed into late adulthood, Or43b OSNs, which also respond to the same odor, show little lasting change. This contrast not only underscores the cell-type specificity of critical-period plasticity but also points to a potential role of inhibitory network architecture in determining susceptibility. The persistence of the Or42a suppression well beyond the developmental window provides compelling evidence that early glia-mediated pruning can imprint a stable, life-long functional state on selected sensory channels. By situating these functional outcomes within the context of detailed connectomic data, the study offers a framework for linking structural connectivity to long-term sensory coding stability or vulnerability.

Weaknesses:

The narrative begins with the absence of changes in PN dendrites and axons. While this establishes specificity, it is a relatively weak starting point compared to the novel OSN functional results.

We agree that switching the order of Figures 1 and 2 recontextualizes the negative PN morphology findings to make their significance more clear, especially with the addition of PN odour-evoked activity data (see Figures 2A, B of the revised manuscript).

Calcium imaging with GCaMP, though widely used, is an indirect measure of synaptic function, and reduced signals could reflect changes in non-synaptic calcium influx as well as release probability. The interpretation of the voltage imaging results is also unclear: if suppression were solely due to impaired synaptic release, one might expect action potential-evoked voltage signals to remain unchanged. The reported changes raise the possibility of deficits in action potential initiation or propagation, which would shift the mechanistic explanation.

Although it is true that non-synaptic Ca2+ influx could contribute to odour-evoked signals in OSN axon terminals, it seems likely to be a relatively small contribution when compared to Ca2+ influx via voltage-gated Ca2+ channels at the active zone. Given the observation that synaptic markers are eliminated during this form of critical period plasticity and remain decreased even after OSNs regrow their terminals days later (consistent with our observed continued decrease in odour-evoked responses), the most parsimonious explanation is that we are seeing a reduction in synaptic Ca2+ influx. We cannot dismiss the possibility that there is a decreased voltage signal arising from fewer action potentials being elicited by the odour stimulation. However, the reduction in voltage signal must arise at least in part from the observed reduction in Ca2+ influx. We have therefore provided additional text to this effect in the results section.

The difference between Or42a and Or43b OSNs is attributed to varying inhibitory input densities from connectome data, but this remains speculative without functional tests such as manipulating GABA receptor expression in OSNs. In Or43b, there is essentially no strong phenotype, making it premature to ascribe the absence of suppression solely to inhibitory connectivity.

We have tempered our conclusions to posit additional mechanisms that could explain the more mild pruning that occurs for Or43b OSNs. While the pruning phenotype for Or43b OSNs is not as strong as Or42a, it is not absent. To further explore the contribution of inhibition as a candidate mechanism underlying differences in susceptibility of Or42a and Or43b to this form of critical period plasticity we compared the relative impact of knocking down expression of GABA-A receptor (called “rdl”) in Or42a and Or43b OSNs. Consistent with the degree of pruning being regulated inhibition, knocking down expression of rdl enhanced pruning for both Or42a and Or43b OSNs. However, because the magnitude of the enhancement was similar between both OSN types, we agree with the reviewer that inhibitory connectivity cannot be the sole mechanism that explains the difference and have therefore tempered our language appropriately.

Finally, the study does not connect circuit-level changes to behavioral outcomes; assays of odor-guided attraction or discrimination could place the findings in an organismal context.

We agree that behavioral assays will be a critical component for understanding the functional consequences of this form of critical period plasticity. However, the goal of this study was to extend our prior work to determine the longevity and selectivity of the critical period pruning. Behavioral assays testing the consequences of this form of early life plasticity will be a component of future studies.

Some introduction material overlaps with the authors' 2024 paper, and the novelty of the present study could be signposted more clearly.

We have included text to highlight the novelty of the present study.

Reviewer #2 (Public review):

Recent work from the authors identified the synaptic changes and glial reaction that occur during exposure of a Drosophila odorant receptor neuron population to continued exposure of a stimulating odorant. This work markedly advanced our understanding of cellular response to critical periods. This current Advance manuscript carries that work forward and examines the non-autonomous responses to constant odorant exposure. The authors discover that the changes to ORN populations are not accompanied by changes to either PN dendrite or PN axon volume, nor are they concurrent with changes in postsynaptic PN structures. These changes are, however, notable, accompanied by changes in Ca2+ and voltage responses in ORNs. Importantly, this set of responses is specific to the Or42a ORNs (that are highly sensitive to the odorant in question, ethyl butyrate) and not the Or43b ORNs (which respond to ethyl butyrate, but not as drastically). Finally, the authors include connectomics analyses showing that Or43b and Or42a ORNs differ in their synaptic input/output relationships.

This is an excellent use of the Advance mechanism for the journal, as these are important follow-up findings for the parent story. The non-autonomous effects (or lack thereof) on PNs is an important part of the story, as is the functional response of Or42a ORNs and the differing response of similarly (but not identically) sensitive Or43b ORNs. The experiments are well-conceived, controlled, and conducted. Where the story falters a bit, though, is with the connectomics analysis. The authors show distinct differences between Or43b and Or42b ORN input-output relationships, and suggest that those differences may underlie the differences observed in their response to ethyl butyrate exposure during the critical period. This is certainly a possibility, but as it stands now, it is too disconnected to offer significant proof. There would have to be additional experiments to address this. Right now, the inclusion of the connectomics work feels like a distraction at best, and a complete non sequitur at worst. To be clear, the connectomics work is well done and I have no issues with its validity, but it is not helpful to the central thesis of the work. I would suggest the authors either remove it entirely or strongly rethink how it fits into the paper.

We have tempered our stated interpretations of the connectivity analysis and include new experiments examining the impact of GABA signaling on pruning. We have therefore opted to retain the connectivity analysis as we feel that it has been better integrated into the overall narrative of the paper.

Major Concerns:

(1) The examination of PN axon terminals in the MB and LH is interesting, but it is only one possibility. Oftentimes, the volume of neurons remains constant with perturbation, while the synapse number is affected. Figure 1C and E would be greatly helped by examining synapse number (via Brp or Brp-Short) in the PN axons.

We agree that the counting synapse number would provide greater resolution information about synapse function relative to axon volume and have added this analysis to what is now Figure 2.

(2) The use of dlg1[4K] is a strong use of a new tool, but the result is surprising. The presynaptic ORN synapse number onto the PNs is notably changed, but that is not reflected in a postsynaptic PSD-95 change. That suggests a compensatory mechanism that the authors might explore. A good proportion of PN puncta should be postsynaptic to those ORNs, so why aren't they adjusted?

We agree that this result suggests that a compensatory mechanism may be present. We have therefore added new text to point out this observation and potential explanation.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The interpretation of the voltage imaging results would benefit from clarification. If these signals are reduced because of upstream action potential changes rather than synaptic release, this should be explicitly discussed and illustrated with representative raw traces for both OSN types. The proposed link between inhibitory connectivity and selective vulnerability could be tested more directly, for example, by manipulating GABA receptor function in OSNs.

We have now tested the link between inhibitory connectivity and susceptibility to glial pruning by testing the effects of GABA receptor knockdown in either Or42a or Or43b OSNs (fully described above).

Adding an intermediate post-exposure time point for Or42a responses could help resolve whether suppression is immediate or develops over time.

The suppression of Or42a odour-evoked responses is present immediately after the 2 day exposure period and responses remain suppressed until 25 days post-eclosion, indicating that the suppression is immediate and sustained. We therefore respectfully disagree that another physiological time point will help resolve whether the suppression is immediate or develops over time.

In terms of presentation, the introduction could be tightened to reduce overlap with the 2024 paper, figures should have clear axis labels and consistent terminology for neuron types and glomeruli, and a schematic summarising key inhibitory connections for Or42a vs. Or43b would aid clarity

We have now streamlined the introduction, improved clarity on axis labels and checked for consistency of terminology.

Minor Concerns:

(1) The dlg1[4K] is made with a V5 epitope but the authors have it labeled mCD8::GFP in Figure 1F. This is likely a typo and should be corrected.

This typo has now been corrected.

(2) Can the responses be separated in Figures 2A, C, and E? It is difficult to see the differences in oil and EB exposure. This would make it much more straightforward to tell the difference if both traces were clearly visible.

Overlaying the averaged response traces for in Figure 2C, E and G (now Figures 1C, E and G) enables the reader to make direct visual comparisons between the responses of OSNs from flies in each condition to both mineral oil and ethylbutyrate. Separating the individual traces would make it much more difficult to make these comparisons.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. Dacks A, Jonaitis J, Leier H. 2026. Leier eLife advance. Open Science Framework. [DOI]

    Supplementary Materials

    Figure 1—source data 1. Numerical data used to generate Figure 1D and F.
    elife-108236-fig1-data1.xlsx (12.7KB, xlsx)
    Figure 2—source data 1. Numerical data used to generate Figure 2B, D, F, H and J.
    elife-108236-fig2-data1.xlsx (19.1KB, xlsx)
    Figure 3—source data 1. Numerical data used to generate Figure 3A, B, C and E.
    elife-108236-fig3-data1.xlsx (83.2KB, xlsx)
    Figure 4—source data 1. Numerical data used to generate Figure 4B and C.
    elife-108236-fig4-data1.xlsx (10.8KB, xlsx)
    Figure 5—source data 1. Numerical data used to generate Figure 5A and B.
    elife-108236-fig5-data1.xlsx (140.9KB, xlsx)
    MDAR checklist
    elife-108236-mdarchecklist1.docx (89.9KB, docx)

    Data Availability Statement

    All reagents will be made available upon request. All data in support of the findings of this study can be accessed at https://doi.org/10.17605/OSF.IO/NFWQB.

    The following dataset was generated:

    Dacks A, Jonaitis J, Leier H. 2026. Leier eLife advance. Open Science Framework.

    Articles from eLife are provided here courtesy of eLife Sciences Publications, Ltd

    RESOURCES