Glia control experience-dependent plasticity in an olfactory critical period
- Department of Neurosciences, Case Western Reserve University School of Medicine, United States
- Department of Biology, John Carroll University, United States
- Department of Neuroscience, University of Virginia School of Medicine, United States
Figures
Figure 1
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 hr 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–J) 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, raw values, and detailed statistics are provided in Figure 1—source data 1.
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Figure 1—source data 1
Genotypes, raw values, and statistics for experiments shown in Figure 1.
- https://cdn.elifesciences.org/articles/100989/elife-100989-fig1-data1-v1.xlsx
Figure 2
The first 2 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. Scale bar, 10 μm. Data are mean ± SD. ns, not significant, ****p <0.0001, Mann-Whitney U-test. Genotypes, raw values, and detailed statistics are provided in Figure 2—source data 1.
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Figure 2—source data 1
Genotypes, raw values, and statistics for experiments shown in Figure 2.
- https://cdn.elifesciences.org/articles/100989/elife-100989-fig2-data1-v1.xlsx
Figure 3
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 to 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, raw values, and detailed statistics are provided in Figure 3—source data 1.
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Figure 3—source data 1
Genotypes, raw values, and statistics for experiments shown in Figure 3.
- https://cdn.elifesciences.org/articles/100989/elife-100989-fig3-data1-v1.xlsx
Figure 4
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. (F–J) 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, 3.0±0.93 [mean ± SD]; EB, 2.7±0.98), (H) interspike interval (ISI) events (Oil, 313.0±230.6; EB, 373.6±294.5), (I) spike onset latency (Oil, 1.4±1.0; EB, 1.0±0.05), and (J) afterhyperpolarization (AHP) decay time constant (Oil, 79.5±31.8; EB, 124.5±124.4). 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, raw values, and detailed statistics are provided in Figure 4—source data 1.
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Figure 4—source data 1
Genotypes, raw values, and statistics for experiments shown in Figure 4.
- https://cdn.elifesciences.org/articles/100989/elife-100989-fig4-data1-v1.xlsx
Figure 5 with 1 supplement
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, raw values, and detailed statistics are provided in Figure 5—source data 1.
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Figure 5—source data 1
Genotypes, raw values, and statistics for experiments shown in Figure 5.
- https://cdn.elifesciences.org/articles/100989/elife-100989-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
No selective phagocytosis of presynapses during the Or42a critical period.
(A–C) Density of bruchpilot puncta in the Or42a OSN terminal arbor in 2 DPE flies exposed to mineral oil or 15% EB throughout adulthood. Genotypes are repo-GAL4 (A), alrm-GAL4 (B), or GMR56F03- GAL4 (C) crossed with iso31 (-) or the given UAS-luciferase or RNAi lines. Box-and-whisker plots are the median, quartiles, and 10th-90th percentiles. ns, not significant, *p=0.03, Kruskal-Wallis test. Genotypes, raw values, and detailed statistics are provided in Figure 5—source data 1.
Figure 6
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, raw values, and detailed statistics are provided in Figure 6—source data 1.
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Figure 6—source data 1
Genotypes, raw values, and statistics for experiments shown in Figure 6.
- https://cdn.elifesciences.org/articles/100989/elife-100989-fig6-data1-v1.xlsx
Figure 7
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, raw values, and detailed statistics are provided in Figure 7—source data 1.
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Figure 7—source data 1
Genotypes, raw values, and statistics for experiments shown in Figure 7.
- https://cdn.elifesciences.org/articles/100989/elife-100989-fig7-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | Or42a-mCD8::GFP | Golovin et al., 2019 | Broadie lab | |
| Genetic reagent (D. melanogaster) | GMR56F03-Gal4 | Bloomington Drosophila Stock Center (BDSC) | BDSC Stock #39157; RRID:BDSC_39157; FlyBase ID: FBst0039157 | Genotype: w1118; P{GMR56F03-GAL4}attP2 |
| Genetic reagent (D. melanogaster) | UAS-mCD8::RFP | BDSC | BDSC Stock #27398; RRID:BDSC_27398; FlyBase ID: FBst0027398 | Genotype: y1 w*; P{UAS-mCD8.mRFP.LG}18 a |
| 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) | UAS-luciferase | BDSC | BDSC Stock #35788; RRID:BDSC_35788; FlyBase ID: FBst0035788 | Genotype: y1 v1; P{UAS-LUC.VALIUM10}attP2 |
| Genetic reagent (D. melanogaster) | Alrm-Gal4 | Doherty et al., 2009 | Freeman lab | |
| Genetic reagent (D. melanogaster) | GMR56F03-LexA | BDSC | BDSC Stock #53574; RRID:BDSC_53574; FlyBase ID: FBst0053574 | Genotype: w1118; P{GMR56F03-lexA}attP40 |
| Genetic reagent (D. melanogaster) | Repo-Gal4 | Coutinho-Budd et al., 2017 | Coutinho-Budd lab | |
| Genetic reagent (D. melanogaster) | Alrm-LexA | Coutinho-Budd et al., 2017 | Coutinho-Budd lab | |
| Genetic reagent (D. melanogaster) | LexAop-draper RNAi | Coutinho-Budd et al., 2017 | Coutinho-Budd lab | |
| Genetic reagent (D. melanogaster) | UAS-draper I RNAi | McPhee et al., 2010 | Freeman lab | |
| Genetic reagent (D. melanogaster) | UAS-draper RNAi | BDSC | BDSC Stock #36732; RRID:BDSC_36732; FlyBase ID: FBst0036732 | Genotype: y1 sc* v1 sev21; P{TRiP.HMS01623}attP2 |
| Genetic reagent (D. melanogaster) | Draper::GFP | BDSC | BDSC Stock #63184; RRID:BDSC_63184; FlyBase ID: FBst0063184 | Genotype: y1 w*; Mi{PT-GFSTF.1}drprMI07659-GFSTF.1 |
| Genetic reagent (D. melanogaster) | GMR86E01-Gal4 | BDSC | BDSC Stock #45914; RRID:BDSC_45914; FlyBase ID: FBst0045914 | Genotype: w1118; P{GMR86E01-GAL4}attP2 |
| Genetic reagent (D. melanogaster) | UAS-pHluorinSE | BDSC | BDSC Stock #82176; RRID:BDSC_82176; FlyBase ID: FBst0082176 | Genotype: w*; P{10XUAS-pHluorin.CAAX}attP2 |
| Antibody | anti-RFP (Rat polyclonal) | ChromoTek | Cat# 5f8; RRID:AB_2336064 | Used 1:600 (IHC) |
| Antibody | anti-GFP (Rabbit polyclonal) | Abcam | Cat# ab6556; RRID:AB_305564 | Used 1:600 (IHC) |
| Antibody | anti-Bruchpilot (Mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat #nc82; RRID:AB_2314866 | Used 1:50 (IHC) |
| 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 | Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | Cat #A-11034; RRID:AB_2576217 | Used 1:400 (IHC) |
| Antibody | Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 | Thermo Fisher Scientific | Cat #A-11077; RRID:AB_2534121 | Used 1:400 (IHC) |
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Glia control experience-dependent plasticity in an olfactory critical period
eLife 13:RP100989.
https://doi.org/10.7554/eLife.100989.3
