Early Draper-mediated glial refinement of neuropil architecture and synapse number in the Drosophila antennal lobe

Abstract

Glial phagocytic activity refines connectivity, though molecular mechanisms regulating this exquisitely sensitive process are incompletely defined. We developed the Drosophila antennal lobe as a model for identifying molecular mechanisms underlying glial refinement of neural circuits in the absence of injury. Antennal lobe organization is stereotyped and characterized by individual glomeruli comprised of unique olfactory receptor neuronal (ORN) populations. The antennal lobe interacts extensively with two glial subtypes: ensheathing glia wrap individual glomeruli, while astrocytes ramify considerably within them. Phagocytic roles for glia in the uninjured antennal lobe are largely unknown. Thus, we tested whether Draper regulates ORN terminal arbor size, shape, or presynaptic content in two representative glomeruli: VC1 and VM7. We find that glial Draper limits the size of individual glomeruli and restrains their presynaptic content. Moreover, glial refinement is apparent in young adults, a period of rapid terminal arbor and synapse growth, indicating that synapse addition and elimination occur simultaneously. Draper has been shown to be expressed in ensheathing glia; unexpectedly, we find it expressed at high levels in late pupal antennal lobe astrocytes. Surprisingly, Draper plays differential roles in ensheathing glia and astrocytes in VC1 and VM7. In VC1, ensheathing glial Draper plays a more significant role in shaping glomerular size and presynaptic content; while in VM7, astrocytic Draper plays the larger role. Together, these data indicate that astrocytes and ensheathing glia employ Draper to refine circuitry in the antennal lobe before the terminal arbors reach their mature form and argue for local heterogeneity of neuron-glia interactions.

Keywords: Draper; Drosophila; antennal lobe; critical period; glia; pruning; remodeling; synapse.

FIGURE 1

Loss of Draper in ensheathing glia leads to persistent increases in presynaptic content and terminal arbor size of VC1 ORNs. (A) Immunohistochemistry stain (single slice) followed by (A’–A”’) precise, semi-automated tracing (three sample slices shown) of pb2A ORN terminal arbor membrane in flies expressing Or85e-mCD8::GFP. (B) Brp staining (single slice), (B’) MIP of deconvoluted Brp staining, (B”) center points of recognized Brp puncta (MIP) inside pb2A ORN terminal arbors. (C) Surface of pb2A ORN terminal arbors recognized by Imaris (gray) with MIP of deconvoluted Brp channel (magenta) and recognized puncta (white) inside compared to original overlay of confocal channels (inset). (D) Both Brp and Brp-short puncta have a mean diameter of 0.4 μm (manual measurement). (E) Imaris pipeline detects comparable Brp and Brp-short puncta numbers in VC1 ORNs. (F) MIP overlays of Brp stain (magenta) and recognized puncta (white), (G) single coronal slices of membrane staining (green) and traces (white) of pb2A ORN terminal arbors in control flies and flies with constitutive knockdown of drpr in ensheathing glia at 3 DPE and 12 DPE. Draper in ensheathing glia persistently limits (H) presynapse number, (I) volume, (J) surface area, and (K) persistently promotes sphericity of VC1 ORN terminal arbors. In a coronal MIP through the antennal lobe (3.5 μm), (L) ensheathing glia processes (cyan), (M) Brp staining (magenta), (N) overlay. In the region (inset of L) surrounding VC1, (O) ensheathing glia processes (cyan), (P) Brp staining (magenta), (Q) overlay. (R) MIP of ensheathing glia processes (cyan) in the interior of VC1 ORN terminal arbors and recognized glial surface (gray) in control flies and flies with constitutive knockdown of drpr in ensheathing glia at 3 DPE and 12 DPE. (S) Volume of ensheathing glia inside VC1 ORN terminal arbors normalized to terminal arbor volume. Ensheathing glia are more infiltrative into VC1 at 3 DPE than 12 DPE. ^†Surface area is calculated based on light-level confocal microscopy measurements. ^‡Sphericity is the ratio of the surface area of an equal-volume sphere to the surface area of an object and ranges from 0 to 1 (most spherical). For each condition, n > 16 antennal lobes from 8 brains. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns = not significant. All error bars represent mean ± SEM. See (section 2.4. “Statistical analysis”) for details of statistical tests used. Genotypes: (A,A’–A”’,B,B’,B”,C) + /Or85e-mCD8::GFP; + /MZ0709-Gal4. (D,E) Brp is + /Or85e-mCD8::GFP; + /MZ0709-Gal4. Brp-short is Or85e-Gal4/UAS-Brp-short-mCherry; + /Dr & Or85e-Gal4/UAS-Brp-short-mCherry; + /TM3Sb pooled. (F–K) Control is + /Or85e-mCD8::GFP; + /MZ0709-Gal4. drpr knockdown & drpr RNAi is + /Or85e-mCD8::GFP; UAS-drpr RNAi/MZ0709-Gal4. (L–Q) + /UAS-mCD8::GFP; + /GMR5 6F03-Gal4. (R,S) Control is UAS-mCD8::RFP/Or85e-mCD8::GFP; UAS-Luciferase/MZ0709-Gal4. drpr knockdown & drpr RNAi is UAS-mCD8::RFP/Or85e-mCD8::GFP; UAS-drpr RNAi/MZ0709- Gal4. Scale bar = 2 μm (A,B, inset of C,O–Q), 3 μm (A’–A”’,B’,B”,C,F,G,R), 5 μm (L–N).

FIGURE 2

Loss of Draper in astrocytes does not alter VC1 ORN presynaptic content or terminal arbor size, but leads to transient changes in glomerular shape. (A) MIP overlays of Brp stain (magenta) and recognized puncta (white), (B) single coronal slices of membrane staining (green) and traces (white) of pb2A ORN terminal arbors in control flies and flies with constitutive knockdown of drpr in astrocytes at 3 DPE and 12 DPE. Draper in astrocytes does not affect (C) presynapse number or (D) volume but transiently limits (E) surface area and transiently promotes (F) sphericity of VC1 ORN terminal arbors. In a coronal MIP through the antennal lobe (3.5 μm), (G) astrocyte processes (cyan), (H) Brp staining (magenta), (I) overlay. In the region (inset of G) surrounding VC1, (J) astrocyte processes (cyan), (K) Brp staining (magenta), (L) overlay. (M) MIP of astrocyte processes (cyan) in the interior of VC1 ORN terminal arbors and recognized glial surface (gray) in control flies and flies with constitutive knockdown of drpr in astrocytes at 3 DPE and 12 DPE. (N) Volume of astrocytes inside VC1 ORN terminal arbors normalized to terminal arbor volume. Astrocytes are more infiltrative into VC1 at 3 DPE than 12 DPE. ^†Surface area is calculated based on light-level confocal microscopy measurements. ^‡Sphericity is the ratio of the surface area of an equal-volume sphere to the surface area of an object and ranges from 0 to 1 (most spherical). For each condition, n > 16 antennal lobes from 8 brains. **p < 0.01, ****p < 0.0001, and ns = not significant. All error bars represent mean ± SEM. See (section 2.4. “Statistical analysis”) for details of statistical tests used. Genotypes: (A–F) control is + /Or85e-mCD8::GFP; + /R86E01-Gal4. drpr knockdown & drpr RNAi is + /Or85e- mCD8::GFP; UAS-drpr RNAi/R86E01-Gal4. (G–L) + /UAS-mCD8::GFP; + /GMR25H07-Gal4. (M,N) Control is UAS-mCD8::RFP/Or85e-mCD8::GFP; UAS-Luciferase/R86E01-Gal4. drpr knockdown & drpr RNAi is UAS-mCD8::RFP/Or85e-mCD8::GFP; UAS-drpr RNAi/R86E01-Gal4. Scale bar = 2 μm (J–L), 3 μm (A,B,M), 5 μm (G–I).

FIGURE 3

Loss of Draper in astrocytes results in transient increases in presynaptic content and terminal arbor size of VM7 ORNs. (A) MIP overlays of Brp stain (magenta) and recognized puncta (white), (B) single coronal slices of membrane staining (green) and traces (white) of pb1A ORN terminal arbors in control flies and flies with constitutive knockdown of drpr in astrocytes at 3 DPE and 12 DPE. Draper in astrocytes transiently limits (C) presynapse number, (D) volume, and (F) sphericity but not (E) surface area of VM7 ORN terminal arbors. (G) MIP of astrocyte processes (cyan) in the interior of VM7 ORN terminal arbors and recognized glial surface (gray) in control flies and flies with constitutive knockdown of drpr in astrocytes at 3 DPE and 12 DPE. (H) Volume of astrocytes inside VM7 ORN terminal arbors normalized to terminal arbor volume. Astrocytes are equally infiltrative into VM7 at 3 DPE and 12 DPE. ^†Surface area is calculated based on light-level confocal microscopy measurements. ^‡Sphericity is the ratio of the surface area of an equal-volume sphere to the surface area of an object and ranges from 0 to 1 (most spherical). For each condition, n > 16 antennal lobes from 8 brains. *p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant. All error bars represent mean ± SEM. See (section 2.4. “Statistical analysis”) for details of statistical tests used. Genotypes: (A–F) control is + /Or42a-mCD8::GFP; + /R86E01-Gal4. drpr knockdown & drpr RNAi is + /Or42a-mCD8::GFP; UAS-drpr RNAi/R86E01-Gal4. (G,H) Control is UAS-mCD8::RFP/Or42a-mCD8:GFP; UAS-Luciferase/R86E01-Gal4. drpr knockdown & drpr RNAi is UAS-mCD8::RFP/Or42a-mCD8:GFP; UAS-drpr RNAi/R86E01-Gal4. Scale bar = 3 μm.

FIGURE 4

Time course of Draper expression in antennal lobe ensheathing glia and astrocytes. (A–D) Draper expression was visualized at 90 hAPF, 0 DPE, 3 DPE, and 12 DPE with either rabbit anti-Draper antibody (A,B) or a protein trap cassette insertion containing EGFP that allows for endogenous Draper protein visualization (C,D). Insets from 90 hAPF images depict relative Draper expression in adjacent astrocytes and ensheathing glial cells (B,D). (E,F) Quantification of Draper expression in cell bodies of astrocytes (gray circles) and ensheathing glia (red circles) at 90 hAPF, 0 DPE, 3 DPE, and 12 DPE, normalized to average intensity of astrocytes at 90 hAPF. Data shown as mean intensity per animal from at least 5 cells of each glial subtype. **p < 0.01, ****p < 0.001, and ns = not significant. All error bars represent mean ± SEM. See (section 2.4. “Statistical analysis”) for details of statistical tests used. Genotypes: (A,B) w;GMR56F03-Gal4, UAS-mCD8:GFP/ + with ensheathing glia (anti-GFP pseudocolored red), Draper (anti-Draper antibody, green), and astrocytes (anti-GAT antibody, blue). (C,D) w;GMR56F03-Gal4,UAS-mCD8:mCherry/Mi{PT-GFSTF.1}drpr[MI07659-GFSTF.1] to visualize endogenous Draper-EGFP (green), endogenous mCherry for ensheathing glia (red), and anti-GAT for astrocytes (blue).