Selective life-long suppression of an odor processing channel in response to critical period experience

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. 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.

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

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.

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.

(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?