Astrocytes close a motor circuit critical period

Sarah D Ackerman; Nelson A Perez-Catalan; Marc R Freeman; Chris Q Doe

doi:10.1038/s41586-021-03441-2

. Author manuscript; available in PMC: 2023 Feb 6.

Published in final edited form as: Nature. 2021 Apr 7;592(7854):414–420. doi: 10.1038/s41586-021-03441-2

Astrocytes close a motor circuit critical period

Sarah D Ackerman ^1,^*, Nelson A Perez-Catalan ², Marc R Freeman ³, Chris Q Doe ^1,^*

PMCID: PMC9901311 NIHMSID: NIHMS1858296 PMID: 33828296

Summary

Critical periods – brief intervals where 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 understood ^1,2. 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, motor neurons scale dendrite length, complexity, and connectivity in response to changes in activity. Astrocytes invaded the neuropil just prior to critical period closure³, and astrocyte ablation prolonged the critical period. Finally, we used a genetic screen to identify astrocyte-motor neuron signaling pathways that close the critical period, including Neuroligin-Neurexin signaling. Reduced signaling destabilized dendritic microtubules, increased dendrite dynamicity, and impaired locomotor behavior, underscoring the importance of critical period closure. Previous work defined astroglia as regulators of plasticity at individual synapses⁴; here, we show that astrocytes also regulate critical period closure to ensure proper locomotor behavior.

Critical periods are brief windows where neural circuit activity can modify the morphological properties of neurons, producing permanent changes to circuit structure and function^1,2,5,6. Critical periods integrate multiple forms of plasticity to modify neural circuits¹. Homeostatic plasticity encompasses changes to synapse number, structure, and function across an entire neuron, as well as changes to connectivity¹. While homeostatic plasticity can occur in the adult brain, dramatic activity-dependent remodeling peaks in early development^5–8. Indeed, failure to terminate critical period plasticity is linked to neurodevelopmental disorders such as autism and epilepsy^2,9. Although putative critical period disorders present with motor defects, the field has largely focused on sensory circuits^2,10. To that end, we developed a novel critical period model within a developing Drosophila motor circuit.

A motor circuit critical period

We focused on two well-characterized Drosophila motor neurons (MNs), aCC and RP2^11,12, which are segmentally repeated in the CNS (Fig. 1a). These MNs are susceptible to activity-induced remodeling, though pioneering studies used chronic activity manipulations and did not define an end-point for homeostatic plasticity^12–14. Here, we expressed the anion channelrhodopsin GtACR2¹⁵ specifically in the aCC/RP2 MNs and delivered acute 1 hour (h) windows of silencing terminating at progressively later times in development. We found that silencing MNs for the last hour of embryogenesis (stage 17) increased aCC/RP2 dendritic volume at 0 h after larval hatching (ALH), whereas silencing for 1 h at later stages showed progressively less of an effect, with no remodeling occurring at 8 h ALH or beyond (Fig. 1f–i, n). In contrast, acute windows of activation (1 h) using the channelrhodopsin Chrimson¹⁶ resulted in significant loss of MN dendrites at 0 h ALH (Fig. 1j–k,n and Extended Data Fig. 1); activating at 8 h ALH and beyond had little or no effect (Fig. 1l–n). Activity-induced changes to dendrite length for single cell RP2 clones (MCFO) ¹⁷ showed similar results (Fig 1o–p). Note that our experiments used far shorter periods of tonic activation than past studies ^14,18,19. Furthermore, although we used tonic activation here (Fig. 1) and below, identical results were observed using 600/400 ms activation or silencing pulses, as well as thermogenetics to activate (TrpA1)¹² or silence (shibire^TS)²⁰ MNs (Extended Data Fig. 1 and Methods). Importantly, dendrite loss following acute activation could be rescued by a 22 h period of dark-rearing (Fig. 1q–u), indicating that activity induces dendrite plasticity, and not excitotoxicity. Together, these experiments define a critical period for activity-dependent motor dendrite plasticity, and to our knowledge, represent the first analyses of motor circuit critical period closure within the CNS^21–23.

Figure 1. — (a) Schematic for reader orientation. A, anterior. P, posterior. L, left. R, right. CNS, central nervous system. MNs, motor neurons. (**b-m**) aCC/RP2 dendrites (single hemisegment) from (**b-e**) dark-reared control, and following optogenetic (**f-i**) silencing or (**j-m**) activation for 1 h ending at the indicated stage. Per stage: left, full dendritic arbor; right, RP2 clone. Scales, 5 μm. Genotypes: Control and silencing: *RN2-gal4,UAS-GtACR2::EYFP*; activation: *RN2-gal4*,*UAS-CsChrimson::mCherry*. RP2 clones: + *UAS-hsMCFO.* (n) Quantification, full arbor. N= #animals, ordered by increasing stage. Silencing: controls: N=8, 13, 14, 11; experimentals: N=7, 16, 25, 21. Silencing statistics by increasing stage: within group (one-way ANOVA): *p<.007*, *p<.01, p<.28*, *p<.66*; across groups (two-way ANOVA): *p<.0001*. Activation controls and experimentals: N= 6 per condition/stage. Activation statistics by increasing stage: within group (one-way ANOVA): *p<6.3×10*⁶, *p<.003*, *p<.07, p<.46*; across groups (two-way ANOVA): *p<.0001*. (**o-p**) Quantification, clones. N = #neurons/#animals by increasing stage. Silencing: controls: N=19/15, 13/10; experimentals: N=14/13, 11/10. Silencing statistics (two-way ANOVA): *p<.004.* Activation: controls: N=7/6, 17/14; experimentals: N=17/17, 8/8. Activation statistics (two-way ANOVA): *p<.008.* (**q-t**) aCC/RP2 dendritic arbor following embryonic activation (st17) and recovery by dark-rearing (0 h vs. 22 h ALH). Scale, 5 μm. Genotype: *RN2-gal4*,*UAS-CsChrimson::mCherry*. (u) Quantification. N= # animals, ordered by increasing stage. Controls: N=5, 10. Experimentals: N=6, 9. Statistics (two-way ANOVA): *p<.0001.* Throughout: Error bars, mean ± SD. Significance: **p<.01; ****p<.0001; NS= not significant. ♦ significance following two-way ANOVA. Ns: biological replicates from 3 technical replicates.

Timescale of motor dendrite remodeling

In vertebrates, homeostatic plasticity functions on a slow timescale − hours to days²⁴. To determine the timescale for MN dendrite expansion following GtACR2 silencing, we silenced aCC/RP2 MNs for 15’, 1 h, or 4 h in stage 17 embryos all terminating at 0 h ALH. Larvae were then immediately dissected and dendritic morphology was assessed in single, well-spaced RP2 neurons using MCFO¹⁷. We observed increased dendritic arbor size and complexity following 1 and 4 h of silencing (Fig. 2a–f). We confirmed these results using the temperature sensitive isoform of shibire²⁰ (Extended Data Fig. 2). In contrast, embryonic Chrimson activation resulted in decreased dendrite length and complexity at 0 h ALH after as little as 15’ of activation (Fig. 2g–l). Furthermore, we observed significant dendrite retraction within 12’ of Chrimson activation by live imaging (Extended Data Fig. 2i–l, Supplementary Videos 1–2). The fact that silencing required more time to show an effect is not surprising, as extension requires generation of new membrane^25,26. We conclude that activity-induced remodeling of Drosophila MNs occurs within minutes, much faster than previously documented for homeostatic plasticity in mammals²⁴.

Figure 2. — (**a-f**) GtACR2 silencing (or control) for the indicated times prior to 0 h ALH. RP2 clones: MCFO. N = #neurons/#animals: N=21/15, 12/9, 13/9, 15/9, respectively. Scale, 5 μm. Genotype: *RN2-gal4,UAS-GtACR2::EYFP*,*UAS-hsMCFO*. (**e-f**) Quantification of morphology. Error bars, mean ± SD. Statistics (one-way ANOVA) by increasing length of silencing for (e) *p<.46*, *p<.02*, *p<.005*; (f) *p<.18*, *p<.02*, *p<.008*. (**g-l**) Chrimson activation (or control) for the indicated times prior to 0 h ALH. RP2 clones: MCFO. N=18/15, 7/6, 16/11, 29/19, respectively. Scale, 5 μm. (**a-l**) Genotypes: *RN2-gal4*,*UAS-CsChrimson::mCherry,UAS-hsMCFO*. (**k-l**) Quantification of morphology. Error bars, mean ± SD. Statistics (one-way ANOVA) by increasing length of activation for (k) *p<.002*, *p<.001*, *p<.0001*; (l) *p<.02*, *p<.0001*, *p<.0001*. (**m-o**) Imaris “Surface” rendering of dendritic membranes (magenta) in control or post-GtACR2 silencing terminating at 4 h ALH. White, presynaptic puncta, A23a neuron (inhibitory). Scale, 2 μm. (**m’-o’**) Imaris “Spots” rendering of puncta ≤ 90 nm of dendritic surface. Genotype: *RN2-gal4,UAS-GtACR2::eYFP; 78F07-lexA,lexAop-brp-short::cherry*. (**p-r**) Imaris “Surface” rendering of MN dendrites (magenta) from (p) control or post-GtACR2 silencing terminating at 4 h ALH. White, presynaptic puncta, A18b neuron (excitatory); (**p’-r’**) Imaris “Spots” rendering of puncta ≤ 90 nm of dendritic surface. Scale, 2 μm. Genotype: *RN2-gal4,UAS-GtACR2::eYFP; 94E10-lexA,lexAop-brp-short::cherry*. (**s-t**) Quantification (one-way ANOVA) following MN (s) inhibition or (t) excitation. N = #hemisegments/#animals: A23a GtACR2 N= 71/23 (control); 36/10 (15’ silencing, *p<.72*); 47/17 (1 h silencing, *p<.0001*); 22/10 (4 h silencing, *p<.0001*). A18b GtACR2 N= 44/19 (control); 17/9 (15’ silencing, *p<.46*); 17/8 (1 h silencing, *p<.22*); 17/9 (4 h silencing, *p<.0001*). A23a Chrimson N= 33/11 (control); 30/9 (15’ activation, *p<.38*); 22/5 (1 h activation, *p<.13*); 29/7 (4 h activation, *p<.14*). A18b Chrimson N= 18/6 (control); 19/8 (15’ activation, *p<.1*); 21/6 (1 h activation, *p<.0001*); 23/10 (4 h activation, *p<.0001*). Error bars, normalized mean (0) ± SEM. Throughout: Significance: *p<.05; **p<.01; ****p<.0001; NS= not significant. Ns: biological replicates from 3 technical replicates.

Motor inputs scale with activity

We showed above that MN scale dendrite length according to activity. An important question is whether these morphological changes are accompanied by changes in excitatory or inhibitory (E/I) synaptic inputs. We examined excitatory cholinergic neuron A18b and inhibitory GABAergic neuron A23a, which are synaptically coupled to aCC/RP2 dendrites in a larval TEM reconstruction^16,29,30. To quantitate E/I synapse number by light microscopy, we expressed a functionally-inactive pre-synaptic marker Bruchpilot^short::Cherry (Brp)³¹ in A18b or A23a. We assayed A23a inhibitory GABAergic synapses onto aCC/RP2 dendrites, quantifying cell-type specific Brp puncta overlapping with aCC/RP2 dendritic membrane (putative synapses) using published standards³² (Extended Data Figs. 3–4). All critical period manipulations terminated at 4 h ALH (stage-matched to the TEM data). We found that 1 h of MN silencing, but not activation, reduced the number of inhibitory synapses between A23a and aCC/RP2 dendrites (Fig. 2m–o’, s–t). Silencing for a longer period (4 h) also yielded a significant increase in A18b excitatory synapses (Fig. 2p–t). Thus, decreasing MN activity leads to a compensatory reduction of inhibitory inputs and a corresponding increase in excitatory inputs to rebalance network activity. We next quantified A18b excitatory cholinergic synapse numbers onto aCC/RP2 dendrites after activation or silencing. We found that MN activation, but not silencing, significantly decreased A18b excitatory synapses onto aCC/RP2 dendrites following 1 and 4 h manipulations (Extended Data Fig. 4a–c‘; quantified in Fig. 2s–t). We did not observe a significant increase in inhibitory synapse number following extended MN activation, possibly due to insufficient dendritic membrane after activity-induced dendrite retraction (Extended Data Fig. 4d–f‘; quantified in Fig. 2t). Thus, increasing MN activity leads to a compensatory reduction of excitatory pre-synaptic inputs. Finally, we expressed a functionally-inactive reporter of excitatory post-synaptic densities (Drep2::GFP or Drep2::mstrawberry)³³, specifically in aCC/RP2, and observed scaling of synapses across the entire dendritic arbor in response to altered activity‒ reduced excitatory post-synapses followed MN activation, increased excitatory post-synapses followed MN silencing during the critical period (Extended Data Fig. 4s). Importantly, homeostatic scaling of MN synapses failed to occur after critical period closure (Extended Data Fig. 4g–l,s–t). In sum, MNs scale E/I inputs relative to their level of activity during the critical period.

Astrocytes terminate the critical period

The mechanisms that close critical periods are poorly defined. Drosophila astrocytes infiltrate the neuropil at late embryogenesis³ and progressively envelop MN synapses as the critical period closes (Extended Data Fig. 5). To test whether astrocytes promote critical period closure, we genetically ablated all astrocytes (see Methods) and used optogenetics to assay for extension of critical period plasticity at 8 h ALH. Astrocyte elimination was confirmed by loss of the astrocyte marker Gat (Fig. 3, Extended Data Fig. 6). As expected, controls closed the critical period by 8 h ALH (Fig. 3a,b; quantified in 3e). In contrast, astrocyte ablation extended dendrite plasticity following activation (Fig. 3c,d; quantified in 3e; Extended Data Fig. 6a–d) or silencing (Extended Data Fig. 6e–i) through 8 h ALH. This effect was not observed at earlier stages (Extended Data Fig. 6j–n), indicating that astrocytes do not constitutively dampen plasticity. Additionally, we found that control motor dendrites were less dynamic after critical period closure, but that astrocyte ablation extends dendrite filopodial dynamicity (Fig. 3f–k, Supplementary Videos 3–6). We conclude that astrocytes are required for the transition from dynamic to stable filopodia and concurrent critical period closure.

Figure 3. — (**a-b**) aCC/RP2 dendrites ± astrocyte ablation in controls or (**c-d**) following Chrimson activation (4–8 h ALH). Scale, 5 μm. Genotypes: *RN2-gal4,UAS-CsChrimson::mCherry*; *alrm-lexA* + *lexAop-myr::GFP* (control) or *lexAop-rpr* (ablation). (e) Quantification. Denoted N= #animals. Control statistics (one-way ANOVA): *p<.*72. Activation statistics (one-way ANOVA): *p<.0001*. Statistics across groups (two-way ANOVA): *p<.04.* (**f-h**) Live imaging: dendrite dynamics. 3D projection, one hemisegment of aCC/RP2 dendrites, 0 h ALH. Yellow boxes, reconstructed regions. Scale: 5 μm. (**g’-h’**) Dynamic dendrite filopodia (arrowheads) over time. Scale, 1 μm. Genotypes: Control: *RN2-gal4,UAS-myr::GFP; alrm-lexA*. Ablation: +*lexAop-rpr*. (**i-k**) Quantification. N=50 dendrites from 5 animals per timepoint. (i) Dendrite displacement statistics across stages (two-way ANOVA): *p<.0001.* Statistics (one-way ANOVA) comparing 22 h ablation with 4 h (*p<.71*), 8 h (*p<.16*) and 22 h (*p<.0001*) controls. (j) Statistics (Fisher’s exact test, two-sided) assaying distribution of dendrite dynamics at 4 h (*p<.08*), 8 h (*p<.04*), and 22 h (*p<.0001*) relative to 0 h control, or relative to 22 h ablation (0h: *p<.007*; 4 h: *p<.007*; 8 h: *p<.91*, 22 h: *p<.0001*). (k) Statistics (one-way ANOVA) to assay extension at (4 h: *p<.81*; 8 h: *p<.02*; 22 h: *p<.01*) or retraction (4 h: *p<.99*; 8 h: *p<.76*; 22 h: *p<.01*) relative to 0 h control. Statistics to assay control extension (0 h: *p<.04*; 4 h: *p<.2*; 8 h: *p<.27*; 22 h: *p<.01*) or retraction (0 h: *p<.67*; 4 h: *p<.9*; 8 h: *p<.99*; 22 h: *p<.001*) relative to 22 h ablation. Throughout: Error bars, mean ± SD. Significance: *p<.05; **p<.01; ****p<.0001; NS= not significant. ♦ significance following two-way ANOVA. ψ: significance for Fisher’s exact tests. Ns: biological replicates from 3 technical replicates.

Astrocyte-MN signaling limits plasticity

To determine how astrocytes close the critical period, we used the astrocyte-specific alrm-gal4 to perform a targeted UAS-RNAi³⁶ knock down (KD) screen (see Methods). Animals were assayed for critical period extension following 1 h of activation from 7–8 h ALH. We identified four genes were required in astrocytes for timely critical period closure: gat (regulates E/I balance), chpf (synthesizes chondroitin sulfate proteoglycans, CSPGs), and the Neuroligins (Nlg) 4 and 2 (Fig. 4a–g).

Figure 4. — (**a-k**) Factors in astrocytes (**a-f**) or MNs (**h-j**) required to close the critical period by 8 h ALH. Top row, dark-reared controls; bottom row, experimentals. Scales, 5 μm. Genotypes: (**a,c-f**) *lexAop-CsChrimson::mVenus,RN2-lexA,alrm-gal4 x UAS-RNAi* or control, (b) *RN2-gal4,UAS-Chrimson::mCherry,lexAop-rpr; alrm-lexA* (**h-j**), *RN2-gal4,UAS-CsChrimson::mCherry x UAS-RNAi* or control. (**g,k**) Quantification. Denoted N= #animals. (g) Astrocyte statistics (two-way ANOVA): ablation (*p<.005*), *gat* KD (*p<.05*), *chpf* KD1 (*p<.0001*), *chpf* KD2 (*o<.0001*), *nlg4* KD1 (*p<.002*), *nlg4* KD2 (*p<.0002*), *nlg2* KD1 (*p<.006*), *nlg2* KD2 (*p<.03*). (k) MN statistics (two-way ANOVA): *lar* KD1 (*p<.001*), *lar* KD2 (*p<.002*), *nrx-1* KD1 (*p<.003*), *nrx-1* KD2 (*p=.05*). (**l-o**) Overexpression of (m) Nrx-1 in MNs or (n) Nlg2 in astrocytes closes the critical period, 4 h ALH. Scale, 5 μm. Genotypes: (**l-m**) *RN2-gal4,UAS-CsChrimson::mCherry* x *UAS-myr::GFP* or *UAS-Nrx-1*, (n) *RN2-lexA,lexAop-Chrimson::tdTomato,alrm-gal4 x UAS-Nlg2,* or *UAS-myr::GFP* control (not shown). (o) Quantification. Denoted N= #animals. Statistics within group (one-way ANOVA): MN control (RN2>GFP, *p<.0001*), astrocyte control (alrm>GFP, *p<.0001*), Nrx-1 OE (*p<.14*), Nlg2 OE (*p<.0002*). Statistics across groups (two-way ANOVA): Nrx-1 OE (*p<.005*), Nlg2 OE (*p<.0007*). (p) Live imaging of aCC/RP2: Chrimson::mVenus (green membranes) and Cherry::zeus (stable microtubules, heatmap), 0 h ALH. Dashed line, retraction landmark. Scale, 1 μm. Genotype: *RN2-gal4, UAS-Chrimson::mVenus, UAS-cherry::zeus.* (q) Quantification (two-way ANOVA): *p<.008*. N=3 animals, each with N=5 dendrites assessed. (**r-s**) Dendritic (myr::GFP) distribution of (**r’-s’)** microtubules (Cherry::Zeus) in (**r-r’**) controls and (**s-s’**) post-overexpression of Nrx-1 in MNs, 4 h ALH. (**r-s’**) Scale, 5 μm. Genotypes: (**r-r’**) *RN2-gal4,UAS-myr::GFP x UAS-Cherry::Zeus,UAS-redstingerNLS* (**s-s’**) *RN2-gal4,UAS-myr::GFP x UAS-Cherry::Zeus,UAS-Nrx-1*. (**t-u**) Quantification (one-way ANOVA) of dendrite volume (*p<.009*) or microtubule:dendrite volume (*p<.0001*). Denoted N= #animals. (**v-w**) Live imaging of stable microtubules (Cherry::Zeus+) in aCC/RP2 (v) control or (w) Nrx-1 overexpression dendrites. (**v-w**) Scale, 1 μm. Genotypes: (v) *RN2-gal4,UAS-Cherry::Zeus x UAS-myr::GFP*, (w) *RN2-gal4, UAS-Cherry::Zeus x UAS-Nrx-1*. Pseudocolor: stable (green), extending (pink), or retracting (blue) dendrites. (x) Quantification, Fisher’s Exact Test (two-sided, *p<.04*). Denoted N= #animals, each with N=10 dendrites assessed. (y) Summary. Throughout: Error bars, mean ± SD. Significance: *p<.05; **p<.01; ***p<.001; ****p<.0001; NS= not significant. ♦ used significance following two-way ANOVA when one-way and two-way are displayed together. Ns: biological replicates from 2 technical replicates.

Neuroligins are cell adhesion proteins known to regulate astrocyte morphogenesis³⁵. In Drosophila, astrocyte-specific KD of nlg2 (murine Nlg1) had no effect on astrocyte volume or tiling, suggesting a more specific defect in astrocyte-motor neuron signaling; KD of the remaining critical period regulators had variable effects on astrocyte morphology but all extended the critical period (Extended Data Fig. 6q–v). Neuroligins bind cell adhesion proteins called Neurexins. We used RNAi against nrx-1, known to bind both nlg2/4^37,38, specifically in aCC/RP2 MNs and observed critical period extension; this is consistent with astrocyte Nlg2 and MN Nrx-1 acting in a common pathway to close the critical period. MN-specific RNAi KD of the CSPG receptor lar⁴⁷ also extended critical period plasticity (Fig. 4h–k). Notably, while Nrx-1 is often pre-synaptic, there is evidence for dendritic localization of these receptors^39–41. Furthermore, antibody staining for endogenous Nrx-1 and Nlg2 revealed localization of this receptor-ligand pair on motor dendrites and astrocytes, respectively (Extended Data Figs. 7–8). Finally, cell-type specific overexpression of Nrx-1 and Nlg2⁴² was sufficient to induce precocious critical period closure (assayed by Chrimson activation from 3–4 h ALH, Fig. 4l–o). We conclude that the Nlg2/Nrx-1 ligand/receptor pair are required in astrocytes and MNs (respectively) for timely critical period closure.

Nrx-1 stabilizes dendritic microtubules

How does Nlg2/Nrx-1 signaling close the critical period? The balance of E/I synapses within neural circuits can instruct critical period timing^2,10. Additionally, excitatory synapses are decreased following astrocyte-specific knockout of neuroligins in mouse³⁵. We observed no significant changes in E/I balance following astrocyte KD of nlg2 (Extended Data Fig. 8c–f), suggesting that critical period closure is not dependent on Nlg2-mediated E/I synapse balance.

Alternatively, Nrx-1 can promote motor axon microtubule stability⁴³, suggesting a microtubule-stabilization mechanism for critical period closure. To test this hypothesis, we used Chrimson::mVenus to activate and visualize aCC/RP2 dendrite membranes at 0 h ALH (peak critical period), and Cherry::Zeus to visualize stable microtubules during and after dendritic retraction. In live preparations, dendrites showed a reduction in Cherry::Zeus intensity immediately preceding activity-dependent retraction (Fig. 4p–q, Supplementary Video 7), suggesting that microtubule collapse within distal branches can induce dendrite retraction. In fixed preparations, we found that proximal dendrites with the highest levels of stable microtubules were protected from activity-dependent retraction (Extended Data Fig. 9). Interestingly, overexpression of Nrx-1 was sufficient to increase both stable microtubules and stable dendrites at 4 h ALH (Fig. 4r–x, Supplementary Videos 8–9). We propose that Nlg2 in astrocytes binds Nrx-1 in MNs to stabilize dendritic microtubules and close the critical period (Fig. 4y; see Discussion).

Critical period timing modifies behavior

In mammals, inappropriate critical period extension has long-term effects on nervous system function². Indeed, we observed persistent changes in MN connectivity at least 24 h following acute MN activation at the end of the critical period (Extended Data Fig. 10a–c), leading us to assay for long-term effects on behavior. We transiently extended the critical period until 12 h ALH (4 h beyond control critical period closure), and then assayed behavior 1.5 days later (Fig. 5a; proof of principle: Extended Data 10d–g‘). Control larvae showed persistent linear locomotion; in contrast, larvae with extended critical periods due to transient MN gene knockdown showed excessive turning leading to abnormal spiraling behavior (Fig. 5b–k). Similar but less severe effects were seen in larvae following astrocyte gene knockdown (Fig. 5l–t). We conclude that a modest extension of the critical period can, in some cases, lead to long-lasting alteration in locomotor behavior.

Figure 5. — (a) Experimental paradigm. Animals were reared at 30°C through 12 ± 2 h ALH to extend critical period plasticity, then shifted to 18°C until 44 h ALH (25° staging standard, see Methods). Genotypes: MNs: *RN2-gal4,CQ2-gal4* x UAS-RNAi,TubP-gal80^ts; astrocytes: *TubP-gal80*^ts,25h07-gal4 x *UAS-RNAi* or control. (b) Five behavioral metrics assayed. (**c-f**) FIMTrack traces (imaged 1 minute, 4 Hz) from individual freely behaving larvae from control (N=38) and transient MN KD of *lar* (N=23) or *nrx-1* (RNAi1 and RNAi2 each N=43). N= #animals. (**g-k**) Quantification and statistics (one-way ANOVA): *p<.0001* for all genotypes, all metrics. (**l-o**) FIMTrack traces from individual freely behaving larvae from control (N=75) and transient astrocyte KD of *chpf* (N=83), *nlg4* (N=80), or *nlg2* (N=80). N= #animals. (**p-t**) Quantification and statistics (one-way ANOVA) relative to control ordered as in graph: (p) *p<.13*, *p<.2*, *p<.02*; (q) *p<.55*, *p<.79*, *p<.96*; (r) *p<.02, p<.09*, *p<.007*; (s) *p<.02*, *p<.8*, *p<.86*; (t) *p<.05*, *p<.61*, *p<.99*. KD of *gat* resulted in unrecoverable paralysis. Throughout: Error bars, mean ± SEM. Significance: *p<.05; **p<.01; ****p<.0001. Ns: biological replicates from 2 technical replicates.

In sum, we identified a novel role for astrocyte Neuroligin to MN Neurexin signaling as essential for closure of a motor critical period required for locomotor function.

Methods

Lead Contact and Materials Availability

Additional information and inquiries regarding resources and reagents should be directed to and will be fulfilled upon request by Lead Contacts Sarah D Ackerman (sarah.d.ackerman@gmail.com) and Chris Q Doe (cdoe@uoregon.edu).

Experimental Model and Subject Details

Fly husbandry

All flies were raised at 25°C on standard cornmeal fly food. Animals were staged relative to a 25°C standard. At 25°C, embryos take 21 hours to hatch into larvae⁵¹; larvae hatch and develop 1.25x faster at 30°C, and 2x slower at 18°C.

Transgenes (in order of appearance)

RN2-gal4 ⁵²
20XUAS-CsChrimson::mCherry ⁵³
20XUAS-CsChrimson::mVenus (BDSC# 55136)
hs-FLPG5;; 10xUAS(FRT.stop)myr::smGdP-HA, 10xUAS(FRT.stop)myr::smGdP-V5, 10xUAS-(FRT.stop)myr::smGdP-FLAG (hs-MCFO, BDSC# 64085)
10XUAS-myr::GFP (BDSC# 32198)
UAS-GtACR2::EYFP ⁵⁴
UAS-shibire^ts1 (BDSC# 44222)
UAS-cherry::zeus ⁵⁵
R78F07-lexA ⁵⁶
8xlexAop-2xbrp-short::cherry ⁵⁷
R94E10-lexA ⁵³
UAS-drep-2::mstrawberry ⁵⁸
lexAop-drep-2::GFP ⁵⁹
alrm-gal4 (Chromosome 2)⁶⁰
10XUAS-mcd8::GFP (BDSC# 5137)
alrm-lexA::GAD (Chromosome 3)⁶⁰
lexAop-rpr ⁶¹
13XlexAop2-CsChrimson::mVenus (BDSC# 55137)
RN2-lexA (courtesy of Ellie Heckscher, UChicago)
alrm-gal4 (Chromosome 3)⁶²
1xUAS-Nrx-1::GFP ⁶³
UAS-DenMark::mCherry (BDSC# 33062)
UAS-SAM.dCas9.GS05146 (BDSC# 82741)
UAS-SAM.dCas9.GS04054 (BDSC# 81420)
13lexAop-CsChrimson::tdTomato (courtesy of Vivek Jayaraman, Janelia Research Campus)
R25H07-gal4 (BDSC# 49145)
CQ2-gal4 ⁵²
tubPgal80^ts (BDSC# 7019)
UAS-htl^DN (BDSC# 5366)

Complete list of RNAi lines and other lines used for screening in astrocytes

gat RNAi (BDSC# 29422), chpf RNAi (BDSC# 53990, NIG# HMJ-22218), shot RNAi (BDSC#s 28366, 64041), dally RNAi (BDSC#s 28747, 33952), nlg4 RNAi (BDSC# 58119, NIG# HMJ-22056), nlg2 RNAi (BDSC#s 58128, 28331), kek2 RNAi (BDSC# 31874, NIG# HMJ-21692), eph RNAi (BDSC# 28511), ptp99a RNAi (BDSC# 25840), arr RNAi (BDSC# 53342), nrx-1 RNAi (BDSC# 27502), dpy RNAi (BDSC# 36691), ft RNAi (BDSC# 29566), sparc RNAi (BDSC# 40885), mew RNAi (BDSC# 27543), sema1b RNAi (BDSC# 28588), tsp RNAi (BDSC# 44116), CG17739 RNAi (BDSC# 28770), gbb RNAi (BDSC# 34898), rangap RNAi (BDSC# 29565), daw RNAi (BDSC# 34974), lrch RNAi (BDSC# 31871), mav RNAi (BDSC# 36809), twin RNAi (BDSC# 32490), stat92e RNAi (BDSC#s 35600, 33637), eaat1 RNAi (BDSC# 43287), nlg3 RNAi (BDSC# 38264), myo RNAi (BDSC# 36840), drpr RNAi (BDSC# 36732), dpp RNAi (BDSC#s 33618, 25782), egr RNAi (BDSC#55276), CG16868 RNAi (BDSC# 29617), tkv RNAi (BDSC# 35653), fasII RNAi (BDSC# 34084), inx2 RNAi (BDSC# 42645), lar RNAi (BDSC# 40938), 7b2 RNAi (BDSC# 27989), octalpha2 RNAi (BDSC# 50678), lpr1 RNAi (BDSC# 50737), egfr RNAi (BDSC# 36773), otk RNAi (BDSC# 25790), tsp96f RNAi (BDSC# 40901), galectin RNAi (BDSC# 34880), sfl RNAi (BDSC#s 34601, 33606), sgl RNAi (BDSC# 65348), trol RNAi (BDSC#s 29440, 42783, 38298), tenA RNAi and overexpression (BDSC#s 42018, 41564), hSod1 misexpression (BDSC#s 33606, 33607). BDSC: Bloomington Drosophila Stock Center, USA. NIG: Shigen National Institute of Genetics, Japan. Independent RNAi lines and/or staining for RNAi efficiency was used to validate all hits.

RNAi lines used for screening in aCC/RP2

lar RNAi (BDSC#s 40938, 34965), nrx-1 RNAi (BDSC#s 27502, 32408), kek2 RNAi (BDSC# 31874), kek6 RNAi (BDSC# 61212), kek1 RNAi (BDSC# 57000), dg RNAi (BDSC# 34895), egfr RNAi (BDSC#s 36773, 25781), tkv RNAi (BDSC#s 35166, 33653), dpp RNAi (BDSC#s 33618, 25782), wit RNAi (BDSC# 25949), brp RNAi (BDSC# 25891). Independent RNAi lines and/or staining for RNAi efficiency was used to validate all hits.

Animal collections

For live imaging of wildtype samples:

For optogenetics (without RNAi or overexpression (OE)): Crosses were reared at 25°C in collection bottles fitted with 3.0% agar apple juice caps containing yeast paste that was supplemented with 0.5mM all-trans retinal (+ATR) (Sigma-Aldrich, R2500–100MG). Crosses were supplied fresh yeast paste (+ATR) for a minimum of 72 h prior to embryo collection to ensure maternal transfer of ATR into embryos. Embryos were then collected on 3.0% agar apple juice caps with yeast paste (+ATR) for 1.5 h and aged at 25°C. To prevent premature optogenetic activation, crosses and embryos were dark-reared until the appropriate developmental stage. At 25° C, Drosophila embryos hatch at 21 h after egg laying (AEL). For assessment of remodeling at 0 h ALH, embryos were aged to 17 h or 20 h AEL prior to light-activation/silencing for 4 h or 1 h, respectively. For 15’ manipulations at 0 h after larval hatching (ALH), optogenetic activation/silencing was performed just after hatching. For assessment of activity-induced remodeling at later larval stages, larvae were collected at 0 h ALH and transferred to fresh apple caps (N=20 larvae per cap) containing yeast paste (+ATR), and then manipulated for the designated length of time just preceding the desired larval stage (4, 8, or 22 h ALH). All animals were dissected immediately following activity manipulation.

For optogenetics (with RNAi or overexpression, OE): Crosses were reared at 25°C in collection bottles fitted with 3.0% agar apple juice caps containing yeast paste that was supplemented with 0.5mM all-trans retinal (+ATR). Crosses were supplied fresh yeast paste (+ATR) for a minimum of 72 h prior to embryo collection to ensure maternal transfer of ATR into embryos. Embryos were then collected on 3.0% agar apple juice caps with yeast paste (+ATR) for 1.5 h and aged at 30°C. To prevent premature optogenetic activation, crosses, embryos, and larvae were dark-reared until the appropriate developmental stage. For assessment of activity-induced remodeling post-critical period, larvae were collected at 0 h ALH and transferred to fresh apple caps (N=10 larvae per cap) containing yeast paste (+ATR) and aged 6.5 h (~ 8 h ALH at 25°C standard) prior to dissection for dark-reared controls, or aged 5.5 h followed by 1 h light activation. For assessment of activity-induced remodeling with OE, larvae were collected at 0 h ALH and transferred to fresh apple caps (N=10 larvae per cap) containing yeast paste (+ATR) and aged 3.5 h (~ 4 h ALH at 25°C standard) prior to dissection for dark-reared controls, or aged 2.5 h followed by 1 h light activation. All animals were dissected immediately following activity manipulation.

For thermogenetics via TrpA1:

Crosses were reared at 22°C in collection bottles fitted with 3.0% agar apple juice caps containing plain yeast paste. Embryos were then collected on 3.0% agar apple juice caps with plain yeast paste for 1.5 h and aged at 22° C. Embryos take 24 h to hatch into larva at 22° C. For assessment of remodeling at 0 h ALH, embryos were aged for 23 h AEL prior to thermogenetic activation (1 h) at 27° C, which induces neuronal firing at ≥ 30 Hz ⁶⁴.

For thermogenetics via shibire^ts:

Crosses were reared at 25°C in collection bottles fitted with 3.0% agar apple juice caps containing plain yeast paste. Embryos were then collected on 3.0% agar apple juice caps with plain yeast paste for 1.5 h and aged at 25° C. At 17 h or 19.5 h AEL, shibire^ts embryos were transferred to 30°C to induce silencing for 3 h or 1 h, respectively. shibire^ts controls were maintained at 25° C. All animals were dissected immediately following activity manipulation.

For behavioral analyses:

For conditional KD of genes in astrocytes or MNs, we used TubGal80^ts as a temperature sensitive repressor of Gal4 (active at 18°C, inactive at 30°C). Crosses were reared at 25°C in collection bottles fitted with 3.0% agar apple juice caps containing plain yeast paste. Embryos were then collected on 3.0% agar apple juice caps with plain yeast paste for 1.5 h and aged at 30°C. Larvae were collected at 0 h ALH and transferred to fresh apple caps (N=40 larvae per cap) supplied with plain yeast and maintained at 30°C for 10 h (~12 ± 2 h ALH at 25°C standard) for robust, Gal4-dependent RNAi expression. Animals were then shifted to 18°C for 64 h to suppress Gal4 activity and assayed for locomotor defects at ~44 h ALH (25°C standard).

For astrocyte ablation:

Crosses were reared (alrm-lexA lexAop-rpr, astrocyte ablation is Rpr-dependent) at 25°C in collection bottles fitted with 3.0% agar apple juice caps containing yeast paste that was supplemented with 0.5mM all-trans retinal (+ATR). Crosses were supplied fresh yeast paste (+ATR) for a minimum of 72 h prior to embryo collection to ensure maternal transfer of ATR into embryos. To prevent premature optogenetic activation or silencing, crosses, embryos, and larvae were dark-reared until the appropriate developmental stage. Larvae were collected at 0 h ALH and transferred to fresh apple caps (N=10 larvae per cap) containing yeast paste (+ATR), and then manipulated for the designated length of time just preceding the desired larval stage (8 h ALH). All animals were dissected immediately following activity manipulation.

METHOD DETAILS

Sustained optogenetic activation/silencing

The following strategy was used for optogenetic activation/silencing in all fixed preparation experiments except for Extended Data Fig. 1f–i. All crosses and resulting embryos/larvae were maintained in dark-rearing conditions until the designated stage. At the designated stage, dark-reared embryos/larvae (reared on apple caps with yeast + ATR) were placed beneath a full spectrum light bulb, shining with an average intensity of 10550 lx (determined with two independent photometer software programs developed for Android: Light Meter© and Photometer©). Animals were dissected in low-light conditions (<100 lx) immediately following activity manipulations. Provision of yeast paste motivated larvae to remain on the top of the agar plate throughout the duration of the manipulation. Any larvae found outside of this area were not processed further. For recovery experiments, animals were transferred back into dark-rearing conditions at 25°C until dissected in low-light conditions. We used tonic activating conditions, which occur in vivo in a number of mutant models ^{51, 65}, to drive the homeostatic response.

Optogenetic activation/silencing with light pulses

The following strategy was used for optogenetic activation/silencing specifically for Extended Data Fig. 1f–i. All crosses and resulting embryos/larvae were maintained in dark-rearing conditions until the designated stage. Animals were maintained on 3.0% agar apple juice caps containing minimal yeast paste that was supplemented with 0.5mM all-trans retinal (+ATR) and manipulated using a Zeiss Axio Zoom v16. Chrimson ⁶⁶ is maximally activated at 590 nm and minimally activated with wavelengths ≤ 500 nm. In contrast, and GtACR2 ⁶⁷ is maximally activated at 470 nm, and minimally activated at wavelengths ≥ 500 nm. Caps were placed under the light path, aperture set to 100%, zoom of 30x to focus the light directly on the larvae. Animals were subjected to 600 ms pulses of 561 nm light, alternating with 400 ms of 488 nm light, for a total of 1 h prior to dissection. Provision of yeast paste motivated larvae to remain on the top of the agar plate throughout the duration of the manipulation. Any larvae found outside of this area were not processed further.

MultiColor FlpOut clone generation

At 15 h AEL, embryos were prepared for heatshock to induce FLP-out clones. Apple caps covered in embryos were sliced to a thickness of ×2 mm and then adhered to a 100 × 26 mm petri dish with water to increase heat transfer. The petri dish was then sealed with parafilm and floated on a 37°C water bath for 17 minutes, followed by 15 minutes at 18°C to prevent further FLP-out events. Embryos were then transferred back to 25°C until the designated stage and/or manipulation.

Immunohistochemistry

Larval brains were dissected in sterile-filtered, ice-cold 1X PBS and mounted on 12mm #1 thickness poly-D-lysine coated round coverslips (Corning^® BioCoat^™, 354085). Brains were fixed for either 12 minutes (0–8h ALH samples) or 15 minutes (22h ALH samples) in fresh 4% paraformaldehyde (Electron Microscopy Sciences, 15710) in .3% PBSTriton, and then washed in .3X PBSTriton to remove fixative. Samples were blocked overnight at 4°C in .3% PBSTriton supplemented with 1% BSA (Fisher, BP1600–100), 1% normal donkey serum and 1% normal goat serum (Jackson ImmunoResearch Laboratories, Inc., 017–000-121 and 005–000-121). Brains were then incubated in primary antibody for one-two days at 4°C. The primary was removed, and brains were washed overnight at 4°C with 0.3% PBST. Brains were then incubated in secondary antibodies overnight at 4°C. The secondary antibodies were removed, and brains transferred to .3% PBSTriton overnight prior to DPX mounting. Brains were dehydrated with an ethanol series: 30%, 50%, 70%, 90%, each for 5 minutes, then twice in 100% ethanol for 10 minutes each (Decon Labs, Inc., 2716GEA). Finally, samples were incubated in xylenes (Fisher Chemical, X5–1) for 2 × 10 minutes, were mounted onto slides containing DPX mountant (Millipore Sigma, 06552), and cured for 1–2 days before imaging.

Primary antibodies: Mouse anti-Cherry (1:500), Clontech Cat. 632543, Figures 1- 4; ED 1,5,6,7; Chicken anti-GFP (1:1000), Aves Cat. GFP-1010, Figures 1,4; ED 1,3,5,6,7,8,9; Guinea Pig anti-GFP (1:500), Frontier Institute Cat. GFP-GP-Af1180, Figures 2; ED 3,10; Rabbit anti-V5 (1:500) Cell Signaling Technology Cat. 13202, Figures 1–2; ED 2,6; Rat anti-HA (1:100) Millipore Sigma Cat. 11867423001, Figures 1–2; ED 2,6; Rabbit anti-Cherry (1:500) Novus Biologicals, Cat. NBP2–25157, Figures 2,4; ED 3,4,9,10; Rabbit anti-Gat (1:4000) M. Freeman lab⁶², Figures 3,4, ED 5,6,8,10; Rabbit anti-GABA (1:500) Sigma A2052, Figures ED 3; Rabbit anti-dsred (1:500) Takara Bio Cat. 632496, Figures 4, ED 4,8; Ms anti-Brp/Nc82 (1:100) DSHB nc82, Figures ED 7,8,10; GP anti-Nrx-1 (1:200) Manzoor Bhat⁶⁸ , Figure ED 7; GP anti-Nlg2 (1:200) Manzoor Bhat⁶⁹ , Figure ED 8.

All secondary antibodies were purchased from Jackson ImmunoResearch and used at a working concentration of 1:400. The following antibodies were used: Alexa Fluor^® Rhodamine RedTM-X Donkey-Anti Mouse (715–295-151), Alexa Fluor^® 488 Donkey anti-Guinea Pig (706–545-148), Alexa Fluor^® 488 Donkey anti-Chicken (703–545-155), Alexa Fluor^® Rhodamine RedTM-X Donkey-Anti Rat (712–295-153), Alexa Fluor^® 488 Donkey-Anti Rat (712–545-153), Alexa Fluor^® 647 Donkey-Anti Rb (711–605-152); Alexa Fluor^® 488 Donkey anti-Rabbit (711–545-152), Alexa Fluor^® Rhodamine RedTM-X Donkey Anti-Rabbit (711–295-152).

Light Microscopy

Fixed larval preparations for dendrite and astrocyte morphology analyses were imaged with a Zeiss LSM 700 laser scanning confocal using a 63x/1.4 NA Oil Plan-Apochromat DIC m27 objective lens. Fixed larval preparations for synapse quantifications were imaged on a Zeiss LSM 800 laser scanning confocal fitted with a 63x/1.40 NA Oil Plan-Apochromat DIC m27 objective lens and GaAsP photomultiplier tubes.

Image processing and analyses

Quantitative analyses (Figures 1–4, Extended Data Figures 1–2,4–6,8–10) were performed using Imaris 9.2.0 (Bitplane AG). Visualization and projection of images for phenotypic categorization (Figures 3, Extended Data Figure 1,6) were performed in FIJI (ImageJ 1.50d, https://imagej.net/Fiji).

Time-lapse imaging of fictive preparations

The following assay was used for live imaging of aCC/RP2 dendrites in isolated CNS (Figures 3–4, Extended Data Figure 2). Larvae were dissected at the indicated stage in a hemolymph-like solution (HL3.1); both lobes and the ventral nerve cord were kept intact. Isolated brains were placed on a 12mm #1 thickness poly-D-lysine coated round coverslips (Corning^® BioCoat^™, 354085), a single 18mm × 18mm × 0.16mm cover glass (Fisher, 12–542B) was pressed against the coverslip until brain lobes were slightly compressed, a drop of HL3.1was used to facilitate sealing. For optogenetic experiments, both dissections and mounting were performed under low light conditions (<100 lx) to delay optogenetic activation for Chrimson experiments and controls. For imaging, we utilized Zeiss LSM 800 laser scanning confocal fitted with a 63x/1.40 NA Oil Plan-Apochromat DIC m27 objective lens and GaAsP photomultiplier tubes, and imaged using a 488 nm laser to visualize dendrites concurrent with a 561 nm laser to activate Chrimson. For Figure 3 and Extended Data Figure 4, continuous scans were obtained every 45 seconds, for 15 minutes, with two hemisegments in the field of view. A z-stack of 25 μm (allowing for drift in Z) with 1 μm step size was performed for Chrimson experiments, and .5 μm for time course analyses. For microtubule dynamics during dendrite retraction (Figure 4), a z-stack of 21 μm (allowing for drift in Z) with 0.3 μm step size was imaged continuously with stacks acquired every 10 seconds for 10 minutes, with only one dendritic branch in the field of view.

Behavioral analyses

Larvae were transferred from 3.0% agar caps with yeast to 1.2% agarose plates for half an hour prior to locomotion assays to avoid inadvertent, temperature-dependent changes in locomotion and to allow the larvae to acclimate to the new crawling surface. Larvae were then transferred to a FIM behavior table⁷⁰ fitted with a fresh 1.2% agarose gel and allowed to further acclimate for two minutes prior to imaging. Two or more independent cohorts of larvae (N=15 larvae per cohort) were tested per genotype. Larval crawling was imaged at 4 Hz, 91 pixels/cm for one minute using a Basler acA2040–25gm camera in the Pylon5 Camera Software Suite (Basler). Data were then analyzed using FIMTrack software using standard settings⁷⁰.

Figure preparation

Images in figures were prepared as either 3D projections in Imaris 9.2.1 (Bitplane AG) or 3D projections in FIJI (ImageJ 1.50d) and assembled using Adobe Illustrator or Adobe Photoshop. Schematics were drawn in Microsoft Powerpoint.

QUANTIFICATION AND STATISTIC ANALYSIS

Phenotypic categorization

For classification of post-activation dendrite morphologies as either control, mildly reduced, or strongly reduced (Figures 3, Extended Data Figure 1,6), 3D projections of data were generated in Fiji to standardize sample angle (dorsal up, anterior top) and fluorescence intensity. Projections were then blinded to genotype and phenotype and scored phenotypically on a numerical scale: 3=control, 2=mildly reduced, 1=strongly reduced. Statistical analyses were performed following unblinding.

Volumetric assays

For quantification of dendrite volume for critical period analysis (Figures 1,4, Extended Data Figure 1,6,9), data was acquired with a voxel size of 0.124 × 0.124 × 0.325 μm³. aCC/RP2 dendrites within a single hemisegment (A1-A2) were captured in a standard ROI spanning 100 pixels by 100 pixels in XY and 8.125 μm in Z with the top of the ROI beginning dorsally at the aCC/RP2 axons for 0h, 4h, and 8h ALH analyses. The ROI was adjusted to account for increased brain size at 22h ALH: 100 pixels by 150 pixels in XY and 8.125 μm in Z at 22h ALH. Dendrites were then reconstructed using the Imaris “Surface” module using default thresholding to determine the total dendritic volume within the ROI. Dendrite volume per brain was determined by averaging the dendrite volume of 4 individual hemisegments. Chrimson and GtACR2 data was normalized to time-matched, experiment-matched, dark-reared controls. For quantification of astrocyte volume (Extended Data Figure 6), data was acquired with a voxel size of 0.397 × 0.397 × 0.427 μm³. A ROI was built around each individual astrocyte (varying sizes) and astrocytes were then reconstructed using the Imaris “Surface” module using default thresholding to determine the total astrocyte volume within the ROI.

Single aCC/RP2 reconstructions

For morphometric analyses of individual MN MCFO clones (Figures 1,2, Extended Data Figure 2), data were acquired with a voxel size of .099 × .099 × .318 μm³. Single MN were captured within a ROI and reconstructed using the Imaris “Filaments” function (starting position: base of cell body; largest diameter filament: 3 μm; seed points: .2 μm; thresholds varied with fluorescence intensity). For reconstruction of aCC (Figure 1o–p only), note that only the ipsilateral aCC branch was reconstructed. Reconstructions were adjusted manually when necessary.

Synapse quantification: pre-motor neuron analyses combined with optogenetics

For quantification of pre-motor inputs on aCC/RP2 (Figure 2, Extended Data Figures 3,4,10), data were acquired with a voxel size of .076 × .076 × .27 μm³ and de-convoluted in Imaris. Chrimson::mVenus+ or GtACR2::eYFP+ aCC/RP2 dendrites within a single hemisegment were reconstructed using the Imaris “Surface” module (no smoothing, thresholds varied with fluorescence intensity). A standard ROI spanned 150 × 150 pixels in XY, and 6.75 μm in Z with the top of the ROI beginning dorsally at the aCC/RP2 axons. A distance transformation was then performed on the “Surface”. Brp-Short::Cherry+ presynapses within the ROI were annotated using the Imaris “Spots” functions and then classified as “direct” synapses if they fell within 90 nm of the “Surface” (accounts from chromatic aberration in addition to the size of the synaptic cleft) based on previously validated criteria⁷¹.

Synapse quantification: post-synapse analyses combined with optogenetics

For quantification of post-synapses on aCC/RP2 following optogenetics (Extended Data Figure 4), data were acquired with a voxel size of .066 × .066 × .27 μm³ and de-convoluted in Imaris. Chrimson::TdTomato+ or GtACR2::eYFP+ aCC/RP2 dendrites within a single hemisegment were reconstructed using the Imaris “Surface” module (no smoothing, thresholds varied with fluorescence intensity). A standard ROI spanned 125 × 125 pixels in XY, and 6.75 μm in Z with the top of the ROI beginning dorsally at the aCC/RP2 axons. A distance transformation was then performed on the “Surface”. Drep-2::GFP or Drep-2::mStrawberry+ post-synapses within the ROI were annotated using the Imaris “Spots” functions and then classified as “direct” synapses if they fell within 70 nm of the “Surface” (accounts for chromatic aberration) based on previously validated criteria⁷¹.

Synapse quantification: association of astrocytes with MN post-synapses

For quantification of astrocyte association with post-synapses of aCC/RP2 (Extended Data Figure 5), data were acquired with a voxel size of .053 × .053 × .31 μm³ and de-convoluted in Imaris. First, aCC/RP2 dendrites (myr::GFP+) within a single hemisegment were reconstructed using the Imaris “Surface” module (no smoothing, thresholds varied with fluorescence intensity). For embryonic stage 17 and 0 h ALH samples, a standard ROI spanned 150 × 200 pixels in XY, and 6.2 μm in Z with the top of the ROI beginning dorsally at the aCC/RP2 axons. For 4 h ALH through 22 h ALH samples, A standard ROI spanned 200 × 200 pixels in XY, and 7.8 μm in Z with the top of the ROI beginning dorsally at the aCC/RP2 axons. A distance transformation was then performed on the “Surface”. Second, Drep-2::mStrawberry+ post-synapses within the ROI were annotated using the Imaris “Spots” functions and then classified as verified post-synapses if they fell within 70 nm of the “Surface” (accounts for chromatic aberration) based on previously validated criteria ⁷¹, and were used to build a new channel. Third, astrocyte membranes within the same R01 were reconstructed using the Imaris “Surface” module (no smoothing, thresholds varied with fluorescence intensity). A distance transformation was then performed on the astrocyte “Surface”. Finally, verified postsynaptic “Spots” were classified as astrocyte-associated if they fell within 90 nm of the astrocyte “Surface” (accounts for chromatic aberration).

Analysis of time-lapse imaging samples

Following acquisition of time-lapse samples (Figures 3–4, Extended Data Figure 2), images were corrected for 3D drift using a Fiji 3D correction plug-in from the Imaris interface, as well as corrected for bleaching using the ‘histogram matching’ function in the same software. Data were then imported to Imaris and analyzed using the “Filaments” module. An automatic detection (starting position: base of cell body; largest diameter filament: 2 μm; seed points: ~.15 μm; thresholds varied with fluorescence intensity) was used for the first time point. Reconstruction of subsequent time points was done manually, using the initial automatic reconstruction as a guide to ensure correct 3D position across timepoints (“cone” reconstruction was set to .3 μm). For quantification of dendrite length extension/retraction events, only dendrites present throughout the 15 minutes were analyzed. For Chrimson experiments, post-activation dendrite lengths were normalized to pre-activation dendrite length to assess retraction over time. Lengths were then compartmentalized to 10 normally distributed values using MATLAB (Mathworks) to minimize variations in brain size and process length between WT and Chrimson-induced activation. Both populations were then plotted against each other as a function of time. For dendrite dynamicity studies, average displacement quantification was calculated per brain (N=5 per timepoint), encompassing 10 reconstructed dendritic processes each. An extension/retraction event was given a value of 1, stability at each time frame was given a value of 0. Motility of a dendrite was defined as an “event” when a length difference of 0.50 μm was detected compared to the previous timepoint. “Motile dendrite” was defined as at least one filopodial event over the 15-min experiment. For quantification of microtubule collapse, retracting dendritic filopodia were identified using Imaris 3D viewer. A retraction event was defined as a ≥0.75 μm change in filopodial length within a period of 40 seconds. The lower boundary of the ROI was created in the retracting process using the final filopodial length as a landmark at t=40 seconds. The upper boundary of the ROI spanned from the landmark to the most distal length of the filopodial process at T₀. Channel intensity was calculated using the “intensity sum” value within Imaris’ “surface” function.

Statistics and Reproducibility

All experiments were performed for N≥2 technical replicates (see legends for exact number) with similar results. Statistics were performed using a combination of Microsoft Excel, MATLAB (MathWorks), and Prism (GraphPad) software. Sample sizes followed published standards. One-way ANOVA was used unless otherwise noted. Error bars, Standard Deviation unless otherwise noted. A 95% confidence interval was used to define the level of significance. Significance: *, p<.05; **, p<.01; ***, p<.001; ****, p<.0001, NS= not significant. ♦ used in place of * to denote significance following two-way ANOVA when both one-way and two-way ANOVA data are displayed on a single graph. All other pertinent information, including sample size, statistical test employed, and variance can be found in the figure legends or labeled within the figure.

Data availability

The raw data files generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Extended Data

Extended Data Figure 3. — (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/RN2 MNs are at the contralateral process (arrow). Note the morphological similarity between light and EM images of A23a. Asterisks, sparse off-target expression not in A23a. Scale, 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” A23a-aCC/RP2 synapses by light microscopy at 4 h ALH. Scales, 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 Figure 4. — (**a-f’**) Remodeling of pre-synapses during the critical period. (**a-c**) Imaris “Surface” from (a) control or post-Chrimson activation for (b) 1 h or (c) 4 h, 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, 2 μm. Genotype: *RN2-gal4,UAS-Chrimson::mVenus; 94E10-lexA,lexAop-brp-short::cherry.* (**d-f**) Imaris “Surface” from (d) control or post-Chrimson activation for (e) 1 h or (f) 4 h, terminating at 4 h ALH (critical period open). Magenta, dendrite marker. White, presynaptic Brp-short::Cherry puncta from the excitatory A23a neuron; (**d’-f’**) Imaris “Spots”, presynaptic Brp puncta within 90 nm of dendritic surface. Scale, 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, see Fig. 2 legend for Ns). (**g-l**) Stability of pre-synapses after critical period closure. (**g-h**) Imaris “Surface” from (g) control or (h) post-Chrimson activation from 7–8 h ALH (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, 2 μm. Genotype: *RN2-gal4,UAS-Chrimson::mVenus; 94E10-lexA,lexAop-brp-short::cherry*. (**i-j**) Imaris “Surface” from (i) control or (j) post-GtACR2 silencing 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, 2 μm. Genotype: *RN2-gal4,UAS-GtACR2::eYFP; 78F07-lexA,lexAop-brp-short::cherry*. (**k-l**) Quantification (one-way ANOVA) of synapse number following (k) MN excitation or (l) inhibition. N = #hemisegments/#animals: A18b Chrimson N= 10/8 (control); 15/9 (1 h activation, *p<.57*). A18b GtACR2 N= 28/10 (control); 22/9 (1 h silencing, *p<.94*). A23a Chrimson N= 24/14 (control); 20/14 (1 h activation, *p<.63*). A23a GtACR2 N=22/10 (control); 25/10 (1 h silencing, *p<.52*). (**m-t**) Remodeling of excitatory post-synaptic densities during and after the critical period. (**m-o**) Representative 3D projection showing dendrite membranes (magenta) and post-synaptic densities (green) in (m) control or following (n) 1 h or (o) 4 h MN activation. (**m’-o’**) Imaris “Spots”, post-synaptic puncta within 70 nm of dendritic surface. Scale, 2 μm. Genotype: *RN2-lexA,lexAop-Chrimson::tdTomato, lexAop-drep-2::GFP.* (**p-r**) Representative 3D projection showing dendrite membranes (magenta) and post-synaptic densities (green) in (p) control or following (q) 1 h or (r) 4 h MN silencing. (**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= #animals, synapse # averaged across 4 hemisegments (A1-A2). N=10 per all conditions and controls. (s) Quantification (one-way ANOVA) of excitatory post-synapse number following MN excitation for 1 h (*p<.0002*) or 4 h (*p<.0001*) relative to control, and following inhibition for 1 h (*p<.4*) or 4 h (*p<.005*) relative to control at 4 h ALH. (t) Quantification (one-way ANOVA) of excitatory post-synapse number following MN excitation (*p<.9*) or silencing (*p<.49*) for 1 h relative to control at 8 h ALH. Throughout: Error bars, normalized mean (0) ± SEM. Significance: *p<.05; **p<.01; ***p<.001; ****p<.0001. N values reflect biological replicates from 2 technical replicates.

Extended Data Figure 5. — (**a-e**) Time course of astrocyte-MN synapse association from (a) embryonic stage 17, (b) 0 h ALH, (c) 4 h ALH, (d) 8 h ALH, and (e) 22 h ALH. Astrocytes, cyan (Gat+). MN membranes, green (myr::GFP+). MN post-synapses, magenta (mStrawberry+). (**a’-e’**) Astrocytes and post-synapses alone. Synapses ≤ 90 nm from astrocyte membranes were counted as ensheathed. Scale, 5 μm. Genotype: *RN2-gal4, UAS-myr::GFP, UAS-drep-2::mStrawberry.*

(f) Quantification of astrocyte-associated post-synapses (% of total) revealed a significant interaction between developmental stage and % ensheathment (two-way ANOVA, *p<.0001*). N= 6 brains per timepoint, % ensheathment averaged over N≥2 segments (A1-A2). Error bars, mean ± SD. Significance: ****, p<.0001. N values reflect biological replicates from 2 technical replicates.

Extended Data Figure 6. — (**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: (a) control, (b) mildly reduced, and (c) strongly reduced dendritic arbor size/complexity. Scale, 10 μm. (d) Quantification of each phenotypic class. Denoted N= #animals. Control animals show no significant dendritic remodeling after 15’ of activation at this stage (p<.12, one-way ANOVA). In contrast, ablation (abl.) of astrocytes results in a significant shift in the distribution of phenotypic classes away from wildtype (no light abl. versus 15’ activation abl., p<.03, one-way ANOVA). Loss of astrocytes strongly sensitized these motor neurons to remodeling (p<.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, 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= #animals, 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<.009*). (**j-n**) Astrocytes do not dampen critical period plasticity. (**j-m**) Representative 3D projections of aCC/RP2 dendrites at 0 h ALH. Scale, 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 late 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= # animals, 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<.74*). (**o-p’**) Representative images of astrocyte morphology in (**o-o’**) control or following (**p-p’**) astrocyte ablation. White, astrocyte membranes (Gat+). Anterior to the left, dorsal is up. Scale, 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*. MN channel not shown. (**q-v**) MCFO clones showing single astrocyte morphology and volume in control (N=38/13) or following KD of *gat* (N=11/7), *chpf* (N=12/4), *nlg4* (N=23/10), or *nlg2* (N=23/7) at 8 h ALH. N= #clones/#animals. The pan-astrocyte marker Gat was used to assay astrocyte ablation at 8 h ALH. Scales, 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<.0001*), *chpf* (*p<.43*), *nlg4* (*p<.007*), *nlg2* (*p<.37*) denoted by asterisks. Genotype: *alrm-gal4,UAS-hsMCFP,UAS-RNAi.* (**w-x’**) Representative images showing labeling of all astrocytes by MCFO in (**w-w’**) control or (**x-x’**) following astrocyte KD of *nlg2*. Anterior to the top, dorsal is up. Astrocytes tile the entire the CNS and exhibit normal tiling behavior, as exhibited by non-overlapping territories in single z-slices). Scales, 8 μm. Genotype: *alrm-gal4,UAS-hsMCFP,UAS-RNAi.* Throughout: error bars, mean ± SD. Significance: *p<.05; **p<.01; ***p<.001; ****p<.0001; NS not significant. ♦ used in place of * to denote significance following two-way ANOVA when both one-way and two-way are displayed together. N values reflect biological replicates from 2 technical replicates.

Extended Data Figure 7. — (**a-b’”**) Localization of Nrx-1 (magenta) relative to MN dendritic membranes (orange, myr::GFP+) and pre-synapses (green, Brp+) in control (N=15) and following MN-specific KD of *nrx-1* at 0 h ALH (N=20). N= #animals. (**a-a’”**) Nrx-1 colocalized with motor dendrite membranes (white circle in inset) and synapses (Brp+) in control. (**b-b’”**) Nrx-1 colocalized with synapses, but was absent from dendritic membranes in KD brains, as evidenced by increased clarity in Nrx-1+ synaptic puncta (white circle in inset). Scale, 2 μm. Insets, 1.5X 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 MN dendrites (green, Denmark::cherry+). Nrx-1::GFP is present within motor dendrites (white circle, colocalization channel in **c’”**). N= 15 animals. Scale, 2 μm. Genotype: *RN2-gal4, UAS-Nrx-1::GFP, UAS-Denmark::cherry*. N values reflect biological replicates from 2 technical replicates.

Extended Data Figure 8. — (**a-b’”**) Localization of Nlg2 (magenta) relative to astrocyte membranes (green, mcd8GFP+) and pre-synapses (blue, Brp+) in control (N=15) and following astrocyte-specific KD of *nlg2* at 0 h ALH (N=18). N= #animals. (**a-a’”**) Nlg2 colocalized with astrocyte membranes (mcd8GFP+) and synapses (Brp+) in control. (**b-b’”**) Nlg2 colocalized with synapses (yellow box), but was absent from astrocyte membranes in KD brains, as evidenced by increased clarity in Nlg2+ synaptic puncta. Scale, 2 μm. Insets, 1.5X zoom. Genotype: *alrm-gal4, UAS-mcd8::GFP, UAS-RNAi* or control. (**c-c’”**) 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) versus (**d-d’”**) astrocyte-specific KD of *nlg2* (N=9). (**c’”, d’”**) Note close apposition of pre- and post-synapses (arrowheads). N= #animals, with synapses averaged over 4 hemisegments per brain (A1-A2). Scale, 3 μm. Genotype: *RN2-lexA, lexAop-myr::tdTomato, lexAop-drep-2::GFP; alrm-gal4, UAS-RNAi* or control. (**e-f**) Quantification of (e) total synapse number (*p<.11*) and (f) the ratio of excitatory synapses to total synapses (*p<.96*) revealed no significant differences (Mann-Whitney test, two-sided). Error bars, mean ± SD. NS, not significant. N values reflect biological replicates from 2 technical replicates.

Extended Data Figure 9. — (**a-e**) Dendrites with stable microtubules are resistant to activity-induced remodeling. Representative 3D projections of brains expressing Chrimson (green) and the microtubule reporter Zeus (a tagged microtubule binding protein, magenta) in aCC/RP2 MNs (*RN2-gal4,UAS-Cherry::Zeus,UAS-CsChrimson::mVenus*) at 0 h ALH. Brains were preserved with cold fixative to visualize stable microtubule populations in (a) control and after Chrimson-activation for (b) 15’, (c) 1 h, or (d) 4 h. Prime panels show Cherry:Zeus channel alone. Scale, 10 μm. Boxed in regions represent regions of interest (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= # animals, 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’ (N=6, *p<.05*) and 1 h (N=4, *p<.0003*) of activation. Microtubule volume is unchanged after 15’ (*p<.26*) or 1 h (*p<.35*). After 4 h of activation (N=6), both membrane volume (*p<.0001*) and microtubule volume (*p<.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<.02). Error bars, mean ± SD. Significance: *p<.05; **p<.01; ***p<.001; ****p<.0001. N values reflect biological replicates from 2 technical replicates.

Extended Data 10. — (**a-c**) Stability of remodeled synapses following MN activation during the critical period (CP). (**a-b**) Imaris “Surface” from (a) control or (b) post-Chrimson activation 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, 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<.002*) and inhibitory (raw images not shown; *p<.61*) synapse numbers following MN excitation by one-way ANOVA. N = #hemisegments/#animals: A18b Chrimson N= 19/3 (control); 15/2 (1 and 4 h activation combined). A23a Chrimson N= 15/2 (control); 31/8 (1 and 4 h activation combined). Error bars, normalized mean (0) ± SEM. (**d-g’**) Validation of genetic tool for conditional KD 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. (**d-e’**) In control animals (*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 through 8 h ALH, expression of *UAS-htl*^DN in astrocytes (*tubP-gal80*^ts; 25H07-gal4) suppressed astrocyte infiltration. (**g-g’**) Shifting to 18°C at 8 h ALH resulted in inhibition of Gal4 by TubP-Gal80^ts, reduced expression of *htl*^DN, and rescued astrocyte infiltration at 44 h ALH (25°C standard, see methods for details on staging). 8 h scale, 20 μm. 44 h scale, 30 μm. Significance: **, p<.01. N values reflect biological replicates from 2 technical replicates.

Supplementary Material

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Acknowledgements

We thank Takashi Suzuki, Stephen Cohen, Ellie Heckscher, Vivek Jayaraman, and Hermann Aberle for providing fly stocks. We thank Manzoor Bhat for providing antibodies. We thank Kelly Monk, Jim Skeath, Dave Lyons, Richard Baines, and members of the Doe lab 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 (CQD), R01 HD27056 (CQD), R01 NS059991 (MRF) and NIH F32NS098690 (SDA). SDA is a Milton Safenowitz Post-doctoral fellow of the ALSA.

Footnotes

Competing Interest Statement

The authors declare no competing financial or non-financial interests.

Data Availability Statement

This study did not generate/analyze datasets/code. Raw data for any main or supplemental figure (.lsm, .czi, or .avi files) can be supplied upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Data Availability Statement

The raw data files generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.

This study did not generate/analyze datasets/code. Raw data for any main or supplemental figure (.lsm, .czi, or .avi files) can be supplied upon request.

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PERMALINK

Astrocytes close a motor circuit critical period

Sarah D Ackerman

Nelson A Perez-Catalan

Marc R Freeman

Chris Q Doe

Summary

A motor circuit critical period

Figure 1. A critical period for motor circuit plasticity.

Timescale of motor dendrite remodeling

Figure 2. Activity-dependent scaling of dendrite length and synaptic inputs.

Motor inputs scale with activity

Astrocytes terminate the critical period

Figure 3. Astrocytes terminate the critical period.

Astrocyte-MN signaling limits plasticity

Figure 4. Neuroligin to Neurexin signaling stabilizes microtubules and closes the critical period.

Nrx-1 stabilizes dendritic microtubules

Critical period timing modifies behavior

Figure 5. Critical period extension alters locomotor behavior.

Methods

Lead Contact and Materials Availability

Experimental Model and Subject Details

Fly husbandry

Complete list of RNAi lines and other lines used for screening in astrocytes

RNAi lines used for screening in aCC/RP2

Animal collections

For live imaging of wildtype samples:

For thermogenetics via TrpA1:

For thermogenetics via shibirets:

For behavioral analyses:

For astrocyte ablation:

METHOD DETAILS

Sustained optogenetic activation/silencing

Optogenetic activation/silencing with light pulses

MultiColor FlpOut clone generation

Immunohistochemistry

Light Microscopy

Image processing and analyses

Time-lapse imaging of fictive preparations

Behavioral analyses

Figure preparation

QUANTIFICATION AND STATISTIC ANALYSIS

Phenotypic categorization

Volumetric assays

Single aCC/RP2 reconstructions

Synapse quantification: pre-motor neuron analyses combined with optogenetics

Synapse quantification: post-synapse analyses combined with optogenetics

Synapse quantification: association of astrocytes with MN post-synapses

Analysis of time-lapse imaging samples

Statistics and Reproducibility

Data availability

Extended Data

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

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

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

Extended Data Figure 4. Remodeling of MN synapses during and after critical period closure.

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

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

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

Extended Data Figure 8. Expression of Nlg2 in astrocytes during the critical period is not required for proper E/I synapse balance.

Extended Data Figure 9. Tubulin stability correlates with dendrite retention during activity-induced remodeling.

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

Supplementary Material

Acknowledgements

Footnotes

Data Availability Statement

References

Method citations

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

For thermogenetics via shibire^ts: