Extended Data Fig. 6: Astrocyte ablation and manipulation extends critical period plasticity.

a–i, Astrocytes close the critical period. a–c, Representative 3D projections of brains expressing Chrimson::mCherry in aCC–RP2 motor neurons (RN2-gal4,UAS-Chrimson::mCherry) illustrating the three classes of dendritic arbor morphology at 8 h ALH following 4 h of Chrimson activation: control (a), mildly reduced (b) and strongly reduced (c) dendritic arbor size/complexity. Scale bar, 10 μm. d, Quantification of each phenotypic class. N represents number of larvae. Control larvae show no significant dendritic remodelling after 15 min of activation at this stage (P < .12, one-way ANOVA). By contrast, ablation (abl.) of astrocytes results in a significant shift in the distribution of phenotypic classes away from wild type (no light abl. versus 15 min activation abl., P < 0.03, one-way ANOVA). Loss of astrocytes strongly sensitized these motor neurons to remodelling (P < 0.04, two-way ANOVA). Note that control and 4 h data are also displayed in Fig. 3e. Control genotype: RN2-gal4,UAS-Chrimson::mCherry; alrm-lexA,lexAop-myr::GFP. Ablation genotype: RN2-gal4,UAS-Chrimson::mCherry; alrm-lexA,lexAop-rpr. e–h, Representative 3D projections of aCC–RP2 dendrites at 8 h ALH. Scale bar, 5 μm. e, f, Dark-reared controls with (N = 13) or without (N = 15) astrocyte ablation. g, h, GtACR2 silencing in aCC–RP2 from 7–8 h ALH with (N = 12) or without (N = 12) astrocyte ablation; note that astrocyte ablation prolongs the critical period to allow activity-dependent dendrite extension. N represents number of larvae, volume averaged over 4 independent hemisegments (A1–A2). Genotypes: RN2-gal4,UAS-GtACR2::eYFP; alrm-lexA (control), RN2-gal4,UAS-GtACR2::eYFP; alrm-lexA,lexAop-rpr (ablation). i, Quantification by two-way ANOVA (P < 0.009). j–n, Astrocytes do not dampen critical period plasticity. j–m, Representative 3D projections of aCC–RP2 dendrites at 0 h ALH. Scale bar, 5 μm. j, k, Dark-reared controls with (N = 5) or without (N = 7) astrocyte ablation. l, m, Chrimson activation in aCC–RP2 for 1 h in stage 17 embryo terminating at 0 h ALH, with (N = 7) and without (N = 6) astrocyte ablation; note that astrocyte ablation does not enhance activity-induced dendrite retraction. N represents number of larvae, volume averaged over 4 independent hemisegments (A1–A2). Control genotype: RN2-gal4,UAS-Chrimson::mCherry; alrm-lexA,lexAop-myr::GFP. Ablation genotype: RN2-gal4,UAS-Chrimson::mCherry; alrm-lexA,lexAop-rpr. n, Quantification by two-way ANOVA (P < 0.74). o–p′, Representative images of astrocyte morphology in control (o, o′) or following astrocyte ablation (p, p′). White, astrocyte membranes (Gat+). Anterior to the left, dorsal is up. Scale bar, 10 μm. Control Genotype: RN2-gal4,UAS-Chrimson::mCherry; alrm-lexA,lexAop-myr::GFP. Ablation Genotype: RN2-gal4,UAS-Chrimson::mCherry; alrm-lexA,lexAop-rpr. Motor neuron channel not shown. q–v, MCFO clones showing single astrocyte morphology and volume in control (N = 38/13) or following knockdown of gat (N = 11/7), chpf (N = 12/4), nlg4 (N = 23/10), or nlg2 (N = 23/7) at 8 h ALH. N number of clones/number of larvae. The pan-astrocyte marker Gat was used to assay astrocyte ablation at 8 h ALH. Scale bars, 5 μm. Normalized, mean astrocyte volume at the bottom of each MCFO panel (via Imaris Surface). Statistics (one-way ANOVA) relative to control: gat (P < 0.0001), chpf (P < 0.43), nlg4 (P < 0.007), nlg2 (P < 0.37) denoted by asterisks. Genotype: alrm-gal4,UAS-hsMCFO,UAS-RNAi. w–x′, Representative images showing labelling of all astrocytes by MCFO in control (w, w′) or following astrocyte KD of nlg2 (x, x′). Anterior to the top, dorsal is up. Astrocytes tile the entire the CNS and exhibit normal tiling behaviour, as exhibited by non-overlapping territories in single z-slices). Scale bars, 8 μm. Genotype: alrm-gal4,UAS-hsMCFO,UAS-RNAi. Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant. Diamonds are used to denote significance following two-way ANOVA when both one-way and two-way are displayed together. N values reflect biological replicates from 2 independent experiments.