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Astrocytes close a motor circuit critical period

Abstract

Critical periods—brief intervals during which neural circuits can be modified by activity—are necessary for proper neural circuit assembly. Extended critical periods are associated with neurodevelopmental disorders; however, the mechanisms that ensure timely critical period closure remain poorly understood1,2. Here we define a critical period in a developing Drosophila motor circuit and identify astrocytes as essential for proper critical period termination. During the critical period, changes in activity regulate dendrite length, complexity and connectivity of motor neurons. Astrocytes invaded the neuropil just before critical period closure3, and astrocyte ablation prolonged the critical period. Finally, we used a genetic screen to identify astrocyte–motor neuron signalling pathways that close the critical period, including Neuroligin–Neurexin signalling. Reduced signalling destabilized dendritic microtubules, increased dendrite dynamicity and impaired locomotor behaviour, underscoring the importance of critical period closure. Previous work defined astroglia as regulators of plasticity at individual synapses4; we show here that astrocytes also regulate motor circuit critical period closure to ensure proper locomotor behaviour.

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Fig. 1: A critical period for motor circuit plasticity.
Fig. 2: Activity-dependent scaling of dendrite length and synaptic inputs.
Fig. 3: Astrocytes terminate the critical period.
Fig. 4: Neuroligin–Neurexin signalling stabilizes microtubules and closes the critical period.
Fig. 5: Critical period extension alters locomotor behaviour.

Data availability

The raw data files generated during and/or analysed during the current study are available from the corresponding authors on reasonable request. Raw data for any main or supplementary figure (.lsm, .czi or .avi files) can be supplied upon reasonable request.

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Acknowledgements

We thank T. Suzuki, S. Cohen, E. Heckscher, V. Jayaraman and H. Aberle for providing fly stocks; M. Bhat for providing antibodies; K. Monk, J. Skeath, D. Lyons, R. Baines and members of the Doe laboratory for comments on the manuscript. Stocks obtained from the Bloomington Drosophila Stock Center and Shigen National Institute of Genetics (NIH P40OD018537) were used in this study. Funding was provided by HHMI (C.Q.D.), R01 HD27056 (C.Q.D.), R01 NS059991 (M.R.F.) and NIH F32NS098690 (S.D.A.). S.D.A. is a Milton Safenowitz Post-doctoral fellow of the ALSA.

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S.D.A. conceived of the project; N.A.P.-C. performed and analysed all live-imaging experiments; S.D.A. performed and analysed all remaining experiments; M.R.F. and C.Q.D. provided feedback during the project; S.D.A., N.A.P.-C. and C.Q.D. wrote the paper and prepared the figures. All authors commented and approved of the manuscript.

Corresponding authors

Correspondence to Sarah D. Ackerman or Chris Q. Doe.

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The authors declare no competing interests.

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Peer review information Nature thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Activity-dependent remodelling of motor neuron dendrites during a motor circuit critical period.

a–e, Tonic activation of motor neurons during embryogenesis induces dendrite retraction. a, Schematic of the activation paradigm used in this study. For activation of aCC–RP2 motor neurons, RN2-gal4/lexA drove expression of UAS-CsChrimson::mCherry or lexAop/UAS-CsChrimson::mVenus. Crosses were established on day 0 and fed exclusively on yeast paste supplemented with 0.5 mM ATR (required for maximum Chrimson activity) and changed daily for a minimum of 3 days. Timed embryo collections were performed on day 3 for a duration of 1.5 h. Sustained light activation (10,550 lx) was followed by immediate dissection. Optogenetic silencing experiments using UAS-GtACR2::eYFP followed the same scheme. be, Activation of aCC–RP2 motor neurons by Chrimson channelrhodopsin induces dendrite retraction. bd, Representative 3D projections of brains expressing Chrimson::mCherry in aCC–RP2 motor neurons at 0 h ALH following activation during embryonic stage 17 (st17). After activation, brains were categorized qualitatively as control (b), mildly reduced (c) or strongly reduced (d) based on the extent of aCC–RP2 dendritic elaboration (dashed white boxes). Scale bar, 5 μm. e, Distribution of each phenotypic class in control, dark-reared animals versus animals whose aCC–RP2 motor neurons were Chrimson-activated for 15 min, 1 h or 4 h. Dark-reared controls were used throughout as aCC–RP2 motor neurons show sensitivity to Chrimson in the absence of ATR after 15 min and 4 h of Chrimson activation. N (in histogram) represents number of larvae. fi, Complementary assays to define the motor circuit critical period. f, g, Silencing of aCC–RP2 motor neurons for 1 h by GtACR2 (400 ms pulses of 488 nm light per second) (f) or expression of the temperature sensitive shibirets to block synaptic transmission (active at 30 °C) (g), resulted in significant dendrite extension at 0 h ALH, but had no effect at 8 h ALH. N represents number of larvae. 0 h GtACR2: control (N = 11), 1 h silencing (N = 12). 8 h GtACR2: N = 10 per condition. GtACR2 statistics within group (one-way ANOVA): 0 h (P < 0.0001), 8 h (P < 0.76). GtACR2 statistics across groups (two-way ANOVA): P < 0.003. 0 h shibirets: control (N = 7), 1 h silencing (N = 6). 8 h shibirets: control (N = 6), 1 h silencing (N = 7). shibirets statistics within group (one-way ANOVA): 0 h (P < 0.0002), 8 h (P < 0.86). shibirets statistics across groups (two-way ANOVA): P < 0.003. GtACR2 genetics: RN2-gal4, UAS-GtACR2::eYFP. shibirets genetics: RN2-gal4, UAS-shibirets, UAS-myr::GFP. h, i, Activation of aCC–RP2 motor neurons for 1 h by Chrimson (600 ms pulses of 561 nm light per second) (h) or expression of the thermogenetic activator TrpA1 (inactive at 22 °C, fires at ~30 Hz at 27 °C) (i), resulted in significant dendrite retraction at 0 h ALH, but had no effect at 8 h ALH. N represents number of larvae. 0 h Chrimson: control (N = 12), 1 h activation (N = 14). 8 h Chrimson: control (N = 12), 1 h activation (N = 10). Chrimson statistics within group (one-way ANOVA): 0 h (P < 0.0001), 8 h (P < 0.6). Chrimson statistics across groups (two-way ANOVA): P < 0.001. 0 h TrpA1: control (N = 6), 1 h activation (N = 11). 8 h TrpA1: control (N = 5), 1 h activation (N = 6). TrpA1 statistics within group (one-way ANOVA): 0 h (P < 0.0001), 8 h (P < 0.25). TrpA1 statistics across groups (two-way ANOVA): P < 0.0001. Chrimson genetics: RN2-gal4, UAS-Chrimson::mCherry. TrpA1 genetics: RN2-gal4, UAS-TrpA1, UAS-myr::GFP. Data are mean ± s.d. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. Diamonds denote significance following two-way ANOVA when one-way and two-way ANOVA are displayed together. N values reflect biological replicates from 2 independent experiments.

Extended Data Fig. 2 Changes to motor dendrite length and complexity following minutes of altered neuronal activity.

ah, Silencing of RP2 by shibirets induces dendrite extension. ad, MCFO single-neuron labelling at 0 h ALH to visualize the morphology of RP2 motor neuron dendrites at 25 °C in shibirets control (N = 43 neurons, N = 24 brains) (a) compared to neurons silenced with shibirets to block synaptic transmission (active at 30 °C) for 15 min (N = 18/N = 15) (b), 1 h (N = 7/N = 6) (c) or 3 h (N = 29/N = 18) (d). Panel labels with a prime show reconstructions of RP2 dendritic arbors (performed using the Imaris Filaments tool). Blue dots show the seed positions for each filament. Scale bar, 5 μm. Genetics: RN2-gal4,UAS-shibirets,UAS-hsMCFO. eh, Quantification of total dendrite length (e), longest branch length (a measure of distal dendrite extension) (f), number of dendritic branch points (g) and the distribution of dendrite lengths per reconstructed neuron (percentage of all processes) (h) after silencing by shibirets. Statistics (one-way ANOVA) by increasing length of silencing: P < 0.05, P < 0.0001, P < 0.0001 (e); P < 0.007, P < 0.02, P < 0.0001 (f); P < 0.54, P < 0.14, P < 0.8 (g). h, The percentage of long processes (>2 μm) was significantly increased after 1 h (P < 0.0001) and 3 h (P < 0.0001) of silencing (subtle decreases after 15 min, P < 0.04). il, Remodelling of dynamic distal processes within 12 min of aCC–RP2 activation. i, Schematic of a larval brain at 0 h ALH with aCC–RP2 motor neurons in purple. Two hemisegments were imaged per experiment (box). j, k, Motor neuron Chrimson activation results in dendrite retraction within minutes. j, 3D projection of a control isolated CNS at 0 h ALH, time 0 (RN2-gal4,UAS-myr::GFP + ATR). Yellow box highlights intersegmental region used for reconstruction of individual dendrites. Scale bar, 5 μm. j′, j′′, 3D projections from representative time points over a 15-min acquisition period. Top, myr::GFP signal alone. Yellow arrowheads mark the tip of a single reconstructed process. Bottom, green Imaris Filament reconstruction of indicated process. Scale bars, 1 μm. k, 3D projection of an isolated CNS at 0 h ALH for Chrimson-activation, time 0 (RN2-gal4,UAS-CsChrimson::mVenus + ATR). Yellow box highlights intersegmental region used for reconstruction of individual dendrites. Scale bar, 4 μm. k′, k′′, 3D projections from representative time points over a 15-min acquisition period. Top, Chrimson::mVenus signal alone. Yellow arrowheads mark the tip of a single reconstructed process. Bottom, green Imaris Filament reconstruction of indicated process. Scale bars, 1 μm. l, Quantification (one-way ANOVA) of normalized dendrite length over time in myr::GFP controls versus brains that were Chrimson-activated for 3 min (P < 0.99), 8 min (P < 0.06), 12 min (P < 0.05) or 15 min (P < 0.02). N = 10 processes each from N = 4 brains per condition, with processes binned by length into 10 categories. Control length remained stable over the 15-min acquisition period. Chrimson activation results in progressive retraction of motor neuron dendrites. Control box plot specifications (minimum, maximum, centre, upper box bound (75%), lower box bound (25%), minus whisker, plus whisker): 0 min (−0.89, 1.22, 0.51, 0.65, −0.13, −0.76, 0.58), 3 min (−0.96, 1.78, 0.40, 0.95, −0.09, −0.87, 0.82), 8 min (−1.57, 1.33, 0.23, 0.92, −0.77, −0.80, 0.42), 12 min (−1.92, 1.48, 0.30, 1.05, −0.40, −1.51, 0.43), 15 min (−0.89, 1.80, 0.18, 0.97, −0.62, −0.27, 0.84). Chrimson box plot specifications: 0 min (−0.89, 1.22, 0.51, 0.65, −0.13, −0.76, 0.58), 3 min (−0.46, 1.74, 0.10, 0.95, −0.29, −0.17, 0.79), 8 min (–1.25, 1.14, −0.01, 0.88, −0.83, −0.42, 0.26), 12 min (−1.52, 1.01, −0.67, 0.52, −1.35, −0.18, 0.49), 15 min (−1.79, 0.99, −0.95, 0.34, −1.45, −0.34, 0.65). Data are mean ± s.d. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. N values reflect biological replicates from 2 independent experiments.

Extended Data Fig. 3 Quantification of the number of synaptic connections between the GABAergic A23a or cholinergic A18b interneurons and aCC–RP2 motor neurons.

a, TEM reconstruction of the A23a premotor neuron in a first instar larval brain at 4 h ALH; pre-synapses are primarily localized to the contralateral branch (arrow). b, Light microscopy image of a single A23a premotor neuron at 4 h ALH (78F07-lexA) with cyan membranes (lexAop-myr::GFP) and magenta pre-synapses (lexAop-brp-short::Cherry). Most synapses with aCC–RP2 motor neurons are at the contralateral process (arrow). Note the morphological similarity between light and electron microscopy images of A23a. Asterisks, sparse off-target expression not in A23a. Scale bar, 2 μm. c, d, A23a is GABAergic. Scale, 3 μm. Genotype: 78F07-lexA lexAop-myr::GFP. e, Representative image of A23a (A1L) forming 14 synapses (white dots) with aCC (A1R) in the TEM reconstruction. Dorsal view, midline to left. Quantification of A23a–aCC synapses from the TEM reconstruction: 21 in A1R and 13 in A1L; A23a–RP2 synapses: 2 in A1R and 3 in A1L. f, Representative image of A18b (A1L) forming 11 synapses (white dots) with aCC (A1R) in the TEM reconstruction. Dorsal view, midline to left. Quantification of A18b-aCC synapses from the TEM reconstruction: 18 in A1R and 10 in A1L; A18b-RP2 synapses: 7 in A1R and 5 in A1L. g–g′′′′, Quantification of putative synapses between A23a and aCC–RP2 by light microscopy at 4 h ALH. Scale bars, 2 μm. g, Representative 3D projection of aCC–RP2 dendrite membrane (Chrimson::mVenus+; magenta) and A23a Brp-short puncta (white). Genotype: RN2-gal4,UAS-Chrimson::mVenus x 78F07-lexA,lexAop-brp-short::Cherry. g′, aCC–RP2 dendrite membrane (Chrimson::mVenus+). g′′, Imaris surface rendering of g′. g′′′, A23a pre-synapses (Brp-short::Cherry+). g′′′′, Imaris spots measurement of Brp-short puncta within 90 nm of dendritic membrane (red dots, 19 putative direct synapses).

Extended Data Fig. 4 Remodelling of motor neuron synapses during and after critical period closure.

af′, Remodelling of pre-synapses during the critical period. ac, Imaris Surface from control (a) or post-Chrimson activation for 1 h (b) or 4 h (c), terminating at 4 h ALH (critical period open). Magenta, dendrite marker. White, presynaptic Brp-short::Cherry puncta from the excitatory A18b neuron. a′c′, Imaris Spots, presynaptic Brp puncta within 90 nm of dendritic surface. Scale bar, 2 μm. Genotype: RN2-gal4,UAS-Chrimson::mVenus; 94E10-lexA,lexAop-brp-short::Cherry. df, Imaris Surface from control (d) or post-Chrimson activation for 1 h (e) or 4 h (f), terminating at 4 h ALH (critical period open). Magenta, dendrite marker. White, presynaptic Brp-short::Cherry puncta from the inhibitory A23a neuron. d′f′, Imaris Spots, presynaptic Brp puncta within 90 nm of dendritic surface. Scale bar, 2 μm. Genotype: RN2-gal4,UAS-GtACR2::eYFP; 78F07-lexA,lexAop-brp-short::Cherry. Activation caused decreased numbers of excitatory, but not inhibitory, synapses (quantified in Fig. 2t). Overall, we observed Brp puncta numbers matching synapse numbers by TEM in stage-matched control brains (4 h ALH; A18b: 19.5 ± 4.9 Brp+ puncta vs 20 ± 2.5 TEM synapses per hemisegment; A23a: 16.9 ± 4.1 vs 19.5 ± 3.5; number of samples and experiments in Fig. 2 legend). gl, Stability of pre-synapses after critical period closure. g, h, Imaris Surface from control (g) or post-Chrimson activation from 7-8 h ALH (h) (critical period closed; magenta, dendrite marker) with presynaptic Brp-short::Cherry puncta (white) from the excitatory A18b neuron. g′h′, Imaris Spots, presynaptic Brp puncta within 90 nm of dendritic surface. Scale bar, 2 μm. Genotype: RN2-gal4,UAS-Chrimson::mVenus; 94E10-lexA,lexAop-brp-short::Cherry. i, j, Imaris Surface from control (i) or post-GtACR2 silencing (j) from 7–8 h ALH (critical period closed; magenta, dendrite marker) with presynaptic Brp-short::Cherry puncta (white) from the inhibitory A23a neuron. i′j′, Imaris Spots, presynaptic Brp puncta within 90 nm of dendritic surface. Scale bar, 2 μm. Genotype: RN2-gal4,UAS-GtACR2::eYFP; 78F07-lexA,lexAop-brp-short::Cherry. k, l, Quantification (one-way ANOVA) of synapse number following motor neuron excitation (k) or inhibition (l). N represents number of hemisegments/number of larvae: A18b Chrimson N = 10/8 (control); 15/9 (1 h activation, P < 0.57). A18b GtACR2 N = 28/10 (control); 22/9 (1 h silencing, P < 0.94). A23a Chrimson N = 24/14 (control); 20/14 (1 h activation, P < 0.63). A23a GtACR2 N = 22/10 (control); 25/10 (1 h silencing, P < 0.52). mt, Remodelling of excitatory post-synaptic densities during and after the critical period. mo, Representative 3D projection showing dendrite membranes (magenta) and post-synaptic densities (green) in control (m) or following 1 h (n) or 4 h (o) motor neuron activation terminating at 4 h ALH. m′o′, Imaris Spots, post-synaptic puncta within 70 nm of dendritic surface. Scale bar, 2 μm. Genotype: RN2-lexA,lexAop-Chrimson::tdTomato, lexAop-drep-2::GFP. pr, Representative 3D projection showing dendrite membranes (magenta) and post-synaptic densities (green) in control (p) or following 1 h (q) or 4 h (r) motor neuron silencing terminating at 4 h ALH. p′r′, Imaris Spots, post-synaptic puncta within 70 nm of dendritic surface. Scale, 2 μm. Genotype: RN2-gal4,UAS-GtACR2::eYFP, UAS-drep-2::mStrawberry. s, t, N represents number of larvae, number of synapses averaged across 4 hemisegments (A1–A2). N = 10 per all conditions and controls. s, Quantification (one-way ANOVA) of excitatory post-synapse number following motor neuron excitation for 1 h (P < 0.0002) or 4 h (P < 0.0001) relative to control, and following inhibition for 1 h (P < 0.4) or 4 h (P < 0.005) relative to control at 4 h ALH. t, Quantification (one-way ANOVA) of excitatory post-synapse number following motor neuron excitation (P < 0.9) or silencing (P < 0.49) for 1 h relative to control at 8 h ALH. Data are normalized mean (0) ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < .0001. N values reflect biological replicates from 2 independent experiments.

Extended Data Fig. 5 Progressive ensheathment of motor synapses by astrocytes across critical period closure.

ae, Time course of astrocyte–motor neuron synapse association from embryonic stage 17 (a), 0 h ALH (b), 4 h ALH (c), 8 h ALH (d) and 22 h ALH (e). Astrocytes, cyan (Gat+). Motor neuron membranes, green (myr::GFP+). Motor neuron post-synapses, magenta (mStrawberry+). a′e′, Astrocytes and post-synapses alone. Synapses ≤ 90 nm from astrocyte membranes were counted as ensheathed. Scale bar, 5 μm. Genotype: RN2-gal4, UAS-myr::GFP, UAS-drep-2::mStrawberry. f, Quantification of astrocyte-associated post-synapses (percentage of total) revealed a significant interaction between developmental stage and per cent ensheathment (two-way ANOVA, P < 0.0001). N = 6 brains per time point, per cent ensheathment averaged over N ≥ 2 segments (A1–A2). Data are mean ± s.d. ****P < 0.0001. N values reflect biological replicates from 2 independent experiments.

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

ai, Astrocytes close the critical period. ac, 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. eh, 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). jn, Astrocytes do not dampen critical period plasticity. jm, 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). op′, 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. qv, 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. wx′, 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.

Extended Data Fig. 7 Expression of Nrx-1 in motor dendrites during the critical period.

ab′′′, Localization of Nrx-1 (magenta) relative to motor neuron dendritic membranes (orange, myr::GFP+) and pre-synapses (green, Brp+) in control (N = 15) and following motor neuron-specific knockdown of nrx-1 at 0 h ALH (N = 20). N represents number of larvae. aa′′′, Nrx-1 colocalized with motor dendrite membranes (white circle in inset) and synapses (Brp+) in control. bb′′′, Nrx-1 colocalized with synapses, but was absent from dendritic membranes in knockdown brains, as evidenced by increased clarity of Nrx-1+ synaptic puncta (white circle in inset). Scale bar, 2 μm. Insets, 1.5× zoom. Genotype: RN2-gal4, UAS-myr::GFP, UAS-RNAi or control. c, c′, Representative image showing localization of a Nrx-1::GFP fusion (magenta) relative to motor neuron dendrites (green, Denmark::Cherry+). Nrx-1::GFP is present within motor dendrites (white circle, colocalization channel in c′′′). N = 15 animals. Scale bar, 2 μm. Genotype: RN2-gal4, UAS-Nrx-1::GFP, UAS-Denmark::Cherry. N values reflect biological replicates from 2 independent experiments.

Extended Data Fig. 8 Expression of Nlg2 in astrocytes during the critical period is not required for proper excitatory–inhibitory synapse balance.

ab′′′, Localization of Nlg2 (magenta) relative to astrocyte membranes (green, mcd8-GFP+) and pre-synapses (blue, Brp+) in control (N = 15) and following astrocyte-specific knockdown of nlg2 at 0 h ALH (N = 18). N represents number of larvae. aa′′′, Nlg2 colocalized with astrocyte membranes (mcd8-GFP+) and synapses (Brp+) in control. bb′′′, Nlg2 colocalized with synapses (inset), but was absent from astrocyte membranes in knockdown brains, as evidenced by increased clarity of Nlg2+ synaptic puncta. Scale bar, 2 μm. Insets, 1.5× zoom. Genotype: alrm-gal4, UAS-mcd8::GFP, UAS-RNAi or control. cd′′′, Representative images showing aCC–RP2 dendrites (magenta) and excitatory post-synapses (cyan, Drep-2::GFP+) relative to all presynapses (orange, Brp+) in control (N = 7) (cc′′′) versus astrocyte-specific knockdown of nlg2 (N = 9) (dd′′′). c′′′, d′′′, Note close apposition of pre- and post-synapses (arrowheads). N represents number of larvae, with synapses averaged over 4 hemisegments per brain (A1–A2). Scale bar, 3 μm. Genotype: RN2-lexA, lexAop-myr::tdTomato, lexAop-drep-2::GFP; alrm-gal4, UAS-RNAi or control. e, f, Quantification of normalized total synapse number (P < 0.11) (e) and the ratio of excitatory synapses to total synapses (P < 0.96) (f) revealed no significant differences (Mann–Whitney test, two-sided). Data are mean ± s.d. NS, not significant. N values reflect biological replicates from 2 independent experiments.

Extended Data Fig. 9 Tubulin stability correlates with dendrite retention during activity-induced remodelling.

ae, Dendrites with stable microtubules are resistant to activity-induced remodelling. Representative 3D projections of brains expressing Chrimson (green) and the microtubule reporter Zeus (a tagged microtubule binding protein, magenta) in aCC–RP2 motor neurons (RN2-gal4,UAS-Cherry::Zeus,UAS-CsChrimson::mVenus) at 0 h ALH. Brains were preserved with cold fixative to visualize stable microtubule populations in controls (a) and after Chrimson-activation for 15min (b), 1 h (c) or 4 h (d) terminating at 0 h ALH. Panel labels with prime show the Cherry:Zeus channel only. Scale bar, 10 μm. Boxed regions represent ROIs that were used for Imaris Surface reconstructions to determine dendrite and microtubule volume. e, Quantification (one-way ANOVA) of the normalized volume of dendrite membranes (Chrimson::mVenus+) and Cherry::Zeus within the same ROI. Microtubule volumes at each time point were calculated relative to the membrane volume for dark-reared controls. N represents number of larvae, with the volume per animal representing the average volume across 4 hemisegments (A1–A2). In dark-reared controls (N = 4), stable microtubule populations reflect 55 ± 8% of the total dendritic volume. Chrimson activation results in a significant decrease in total dendritic volume after 15 min (N = 6, P < 0.05) and 1 h (N = 4, P < 0.0003) of activation. Microtubule volume is unchanged after 15 min (P < 0.26) or 1 h (P < 0.35). After 4 h of activation (N = 6), both membrane volume (P < 0.0001) and microtubule volume (P < 0.0002) are significantly reduced; however, dendrites with stable microtubules are preferentially retained such that membrane volume is nearly equivalent to the Cherry::Zeus volume (#P < 0.02). Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N values reflect biological replicates from 2 independent experiments.

Extended Data Fig. 10 Assaying permanent motor circuit changes following manipulation of critical period activity.

ac, Stability of remodelled synapses following motor neuron activation during the critical period (CP). a, b, Imaris Surface from control (a) or post-Chrimson activation (b) from 0-4 h ALH (critical period open; magenta, dendrite marker) followed by 20 h recovery in dark conditions. Presynaptic Brp-short::Cherry puncta (white) from the excitatory A18b neuron. a′, b′, Imaris Spots, presynaptic Brp puncta within 90 nm of dendritic surface. Scale bar, 2 μm. Genotype: RN2-gal4,UAS-Chrimson::mVenus; 94E10-lexA,lexAop-brp-short::Cherry. Data not shown: inhibitory A23a synapses following critical period activation and recovery. Genotype: RN2-gal4,UAS-Chrimson::mVenus; 78F07-lexA,lexAop-brp-short::Cherry. c, Quantification of excitatory (P < 0.002) and inhibitory (raw images not shown; P < 0.61) synapse numbers following motor neuron excitation by one-way ANOVA. N represents the number of hemisegments/number of larvae: A18b Chrimson N = 19/3 (control); 15/2 (1 h and 4 h activation combined). A23a Chrimson N = 15/2 (control); 31/8 (1 h and 4 h activation combined). Data are normalized mean (0) ± s.e.m. dg′, Validation of genetic tool for conditional knockdown of astrocyte genes to transiently extend the critical period. N = 10 animals per condition. Orthogonal views through the ventral nerve cord showing the extent of astrocyte infiltration (Gat+, green) into the synapse-dense neuropil (Brp+, magenta). Prime panels show Gat signal alone. de′, In control larvae (25H07-gal4 X UAS-myr::GFP), astrocytes progressively infiltrate the neuropil from 8 h ALH through 44 h ALH. f, f′, When reared at 30 °C to 8 h ALH, expression of UAS-htlDN in astrocytes (tubP-gal80ts; 25H07-gal4) suppressed astrocyte infiltration. g, g′, Shifting to 18 °C at 8 h ALH resulted in inhibition of Gal4 by TubP-Gal80ts, reduced expression of htlDN, and rescued astrocyte infiltration at 44 h ALH (25 °C standard, see Methods for details on staging). Scale bar, 20 μm (8 h), 30 μm (44 h). **P < 0.01. N values reflect biological replicates from 2 independent experiments.

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Video 1: Time-lapse imaging of myr::GFP control for Chrimson-activation

Representative time-lapse video showing control aCC/RP2 dendrite dynamics in a fictive brain preparation (isolated CNS) expressing UAS-myr::GFP under the control of RN2-gal4 at 0 h ALH. Animals were supplied with ATR and reared in the dark to mimic conditions for Chrimson-activation. A single z-stack was acquired every 45 seconds (each stack taking 30” to acquire) and brains were imaged for a total of 15 minutes. Dendrites were highly motile, though largely stable in length over the 15-minute period (white arrowheads).

Video 2: Time lapse imaging showing rapid retraction of dendrites during Chrimson-activation

Representative time-lapse video showing aCC/RP2 dendrite dynamics in a fictive brain preparation during Chrimson-activation (RN2-gal4; UAS-Chrimson::mVenus) at 0 h ALH. A single z-stack was acquired every 45 seconds (each stack taking 30” to acquire) and brains were imaged for a total of 15 minutes. Dendrites were highly motile, with many processes retracting and some processes completely collapsing into their parent dendrite within the 15-minute period (white arrowheads).

Video 3: Time-lapse imaging of wildtype aCC/RP2 dendrites at 4 h ALH

Representative time-lapse video showing control aCC/RP2 dendrite dynamics in a fictive brain preparation expressing UAS-myr::GFP under the control of RN2-gal4 at 4 h ALH. A single z-stack was acquired every 45 seconds (each stack taking 30” to acquire) and brains were imaged for a total of 15 minutes. Dendrites were less motile than 0 h ALH, though still highly dynamic (white arrowheads).

Video 4: Time-lapse imaging of wildtype aCC/RP2 dendrites at 8 h ALH

Representative time-lapse video showing control aCC/RP2 dendrite dynamics in a fictive brain preparation expressing UAS-myr::GFP under the control of RN2-gal4 at 8 h ALH. A single z-stack was acquired every 45 seconds (each stack taking 30” to acquire) and brains were imaged for a total of 15 minutes. Dendrites were less motile than 0 h ALH and 4 h ALH, though still dynamic (white arrowheads).

Video 5: Time-lapse imaging of wildtype aCC/RP2 dendrites at 22 h ALH

Representative time-lapse video showing control aCC/RP2 dendrite dynamics in a fictive brain preparation expressing UAS-myr::GFP under the control of RN2-gal4 at 22 h ALH. A single z-stack was acquired every 45 seconds (each stack taking 30” to acquire) and brains were imaged for a total of 15 minutes. Dendrites showed small dendrite extension/retraction events, but were stable overall (white arrowheads).

Video 6: Time-lapse imaging of wildtype aCC/RP2 dendrites at 22 h ALH post-astrocyte ablation

Representative time-lapse video showing control aCC/RP2 dendrite dynamics in a fictive brain preparation expressing UAS-myr::GFP under the control of RN2-gal4 at 22 h ALH in the absence of astrocytes (alrm lexA lexAop-rpr, verified by Gat staining post-acquisition). A single z-stack was acquired every 45 seconds (each stack taking 30” to acquire) and brains were imaged for a total of 15 minutes. Dendrites showed increased motility relative to 22 h ALH controls (white arrowheads).

Video 7: Time lapse imaging showing rapid retraction of dendrites during Chrimson-activation following microtubule instability

Representative time-lapse video showing aCC/RP2 dendrite dynamics in a fictive brain preparation during Chrimson-activation. aCC/RP2 dendrites were co-labeled with RN2-gal4 driving UAS-Chrimson::mVenus (left panel) and UAS-cherry::zeus (right panel) to mark microtubule populations.. A single z-stack was acquired every 45 seconds (each stack taking 30” to acquire) and brains were imaged for a total of 15 minutes. Cherry::Zeus was depleted from the tip of dendrites just prior to process retraction (white arrowheads).

Video 8: Time lapse imaging of control aCC/RP2 dendrites at 4 h ALH

Representative time-lapse video showing control aCC/RP2 dendrite dynamics in a fictive brain preparation expressing UAS-cherry::zeus under the control of RN2-gal4 at 4 h ALH. UAS-myr::GFP control channel not shown. A single z-stack was acquired every 30 seconds (each stack taking 25” to acquire) and brains were imaged for a total of 15 minutes. Dendritic microtubules were highly dynamic (white arrowheads).

Video 9: Time lapse imaging of aCC/RP2 dendrites overexpressing Nrx-1 at 4 h ALH

Representative time-lapse video showing aCC/RP2 dendrite dynamics in a fictive brain preparation expressing UAS-Nrx-1 and UAS-cherry::zeus under the control of RN2-gal4 at 4 h ALH. A single z-stack was acquired every 30 seconds (each stack taking 25” to acquire) and brains were imaged for a total of 15 minutes. Dendritic microtubules showed less dynamicity under Nrx-1 overexpression conditions (white arrowheads).

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Ackerman, S.D., Perez-Catalan, N.A., Freeman, M.R. et al. Astrocytes close a motor circuit critical period. Nature 592, 414–420 (2021). https://doi.org/10.1038/s41586-021-03441-2

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