Early-life exposure to elevated ethyl butyrate erodes Or42a OSN connectivity and function

(A) FlyWire full-brain connectome reconstruction (left) and simplified schematic (right) of the Or42a olfactory circuit. Thirty-three olfactory sensory neurons (OSNs) expressing the ethyl butyrate (EB)-sensitive odorant receptor Or42a synapse with projection neurons (PNs) in a single glomerulus (VM7) of the antennal lobe (AL). VM7 also receives lateral input from local interneurons (LNs). (B) Overview of the Drosophila developmental timeline. OSNs synapse with PNs during the first 48 hours after puparium formation. (C) Schematic of the odorant exposure paradigm used in (D–J). White and black bars represent ethyl butyrate (EB) or mineral oil vehicle control, respectively. DPE, days post-eclosion. (D) Representative maximum intensity projections (MIPs) (bottom) and volume measurements (top) of the Or42a-mCD8::GFP OSN terminal arbor in VM7 in 2 DPE flies exposed to mineral oil or the indicated concentrations of EB throughout adulthood. (E) Representative MIPs (bottom) and number of VM7 presynapses (top) in 2 DPE flies exposed to mineral oil or the indicated concentrations of EB throughout adulthood. Presynapses were visualized with nc82 anti-bruchpilot (Brp) staining. Data are mean ± SD. (F–I) Patch-clamp recordings of VM7 PNs from 2 DPE flies exposed to 15% EB or mineral oil throughout adulthood. (F) Representative PN membrane potential traces, (G) spontaneous mean firing rate (Oil, 1.1±0.61 [mean±SD]; EB, 0.017±0.039), (H) interspike interval (ISI) events (Oil, 751.9±450.6; EB, 1757±1114), (I) spike onset latency (Oil, 0.79±0.21; EB, 0.83±0.038), and (J) afterhyperpolarization (AHP) decay time constant (Oil, 92.7±28.8; EB, 96.1±77.4). ns, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis test with Dunn’s multiple comparisons test (D) or Mann-Whitney U-test (F–I). Genotypes are provided in Supplemental Table 1.

The first two days after eclosion is a critical period for Or42a OSNs

(A–C) Schematic of the odorant exposure paradigms used in (D–I). (D–F) Representative maximum intensity projections (MIPs) (bottom) and volume measurements (top) of the Or42a-mCD8::GFP OSN terminal arbor in VM7 in flies exposed to mineral oil or 15% EB for the time periods indicated in (A–C). (G–I) Representative MIPs (bottom) and number of VM7 presynapses (top) in flies exposed to mineral oil or 15% EB for the time periods indicated in (A–C). Presynapses were visualized with nc82 anti-Brp staining. (J) Activation pattern of all glomeruli mapped in the DoOR 2.0 database to EB. Uncolored glomeruli are unmapped (dark gray) or nonresponsive to EB (light gray). (K) Representative MIPs (bottom) and volume measurements (top) of the Or22a OSN terminal arbor in DM2 in 2 DPE flies exposed to mineral oil or 15% EB throughout adulthood. Data are mean ± SD. ns, not significant, ****p < 0.0001, Mann-Whitney U-test. Genotypes are provided in Supplemental Table 1.

The VM7 OSN-PN circuit does not recover from critical period pruning

(A) Schematic of the odorant exposure paradigm used in (B–H). Flies were exposed to mineral oil or 15% EB during the critical period, then recovered without odorant until dissection at 7-8 DPE. (B) Representative MIPs (bottom) and volume measurements (top) of the Or42a-mCD8::GFP OSN terminal arbor in VM7. (C) Representative MIPs (bottom) and number of VM7 presynapses (top). Presynapses were visualized with nc82 anti-Brp staining. Data are mean ± SD. (D–H) Patch-clamp recordings of VM7 PNs from 7-8 DPE flies treated as in (A). (D) Schematic of patch-clamp recordings (left) and representative PN membrane potential traces (right), (E) spontaneous mean firing rate (Oil, 1.2±0.51; EB, 0.14±0.11), (F) interspike interval (ISI) events (Oil, 812.7±633.4; EB, 3027±1671), (G) spike onset latency (Oil, 0.80±0.084; EB, 0.79±0.16), and (H) afterhyperpolarization (AHP) decay time constant (Oil, 107.5±18.9; EB, 126.8±108.2). ns, not significant, **p < 0.01, ***p < 0.001, Mann-Whitney U-test. Genotypes are provided in Supplemental Table 1.

Glial Draper is required for Or42a activity-dependent pruning during its critical period

(A–B) Schematic (A) and representative MIPs (B) of astrocytes and ensheathing glia (EG), the two glial populations present in the neuropil of the antennal lobe. (C) Schematic of the odorant exposure paradigm used in C, D and Figure 5. (D) Representative MIPs (bottom) and volume measurements (top) of the Or42a-mCD8::GFP OSN terminal arbor in VM7. (E) Representative MIPs (bottom) and number of VM7 presynapses (top). Presynapses were visualized with nc82 anti-Brp staining. Data are mean ± SD. ns, not significant, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis test with Dunn’s multiple comparisons test. Pan-glial driver is repo-GAL4. Genotypes are provided in Supplemental Table 1.

Draper is required in ensheathing glia to eliminate VM7 OSN presynaptic terminals

(A) Representative MIPs (bottom) and volume measurements (top) of the Or42a-mCD8::GFP OSN terminal arbor in VM7 in 2 DPE flies exposed to mineral oil or 15% EB throughout adulthood. (B) Representative MIPs (bottom) and number of VM7 presynapses (top). (C) Representative MIPs (bottom) and volume measurements (top) of the Or42a-mCD8::GFP OSN terminal arbor in VM7 in 2 DPE flies exposed to mineral oil or 15% EB throughout adulthood. (D) Representative MIPs (bottom) and number of VM7 presynapses (top). Presynapses were visualized with nc82 anti-Brp staining. Data are mean ± SD. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis test with Dunn’s multiple comparisons test. Astrocyte driver is alrm-GAL4, EG driver is GMR56F03-GAL4. Genotypes are provided in Supplemental Table 1.

Ensheathing glia extend processes into VM7 to perform critical period pruning in a Draper-dependent manner

(A–C) Representative MIPs (bottom) and quantification (top) of percentage of VM7 volume (outline shown by dashed lines) occupied by ensheathing glial processes in 2 DPE flies exposed to 15% EB or mineral oil throughout adulthood. Data are mean ± SD. ns, not significant, **p < 0.01, Mann-Whitney U-test. EG driver is GMR56F03-GAL4. Genotypes are provided in Supplemental Table 1.

Ensheathing glia upregulate Draper in response to critical period Or42a activity and phagocytose its terminal arbor

(A–C) Representative single confocal planes of Brp staining with Draper (A), quantification of GFP mean fluorescence intensity within VM7 (B), and representative single confocal planes of EG membrane with Draper (C) of 2 DPE draper::GFP flies exposed to 15% EB or mineral oil throughout adulthood. Dashed lines indicate the outline of VM7. (C) (D–F) Representative MIPs (bottom) and quantification of pHluorin/tdTomato fluorescence intensity ratio (top) of 2 DPE flies exposed to 15% EB or mineral oil throughout adulthood. Data are mean ± SD. ns, not significant, **p < 0.01, ****p < 0.0001, Mann-Whitney U-test. EG driver is GMR56F03-LexA. Genotypes are provided in Supplemental Table 1.

Genotypes used in experiments.