Perisynaptic astrocyte ensheathment of DAergic synapses.

(A) Third instar larval CNS with the VNC highlighted (scale bar: 50 µm). Reconstituted GFP (reconst. GFP) reveals astrocyte contact sites (arrowheads and open arrowheads) with DAergic neuronal processes labeled by myristoylated tdTomato (mtdTomato) (scale bars: 5 µm). Open arrowheads identify typical PEAPODs that resemble synaptic boutons on terminal branches of DAergic neurons. Brp: Bruchpilot (presynaptic marker). (B) Single-cell PEAPODs and synaptic labeling of the DAergic neuron in larval ventral nerve cord (n = 11) (scale bars: 10 µm). (C) Inset zoom in (B), arrowheads denote PEAPODs+ Brp.S-mStraw puncta (scale bar: 2 µm). (D) Schematic drawing of the PEAPODs. Note that GFP reconstitutes at the contact site between the astrocyte sheath and DAergic synapse. (E–H) PEAPODs intensity vs. spontaneous DAergic neuronal burst activation (Ca2+, mR-GECO1) (n = 12). RP, SP, and DP: rising, sustaining, and decay periods, respectively. The area under the curve (AUC) was calculated to show the accumulative PEAPODs response over time. max.: Maximum. (I) PEAPODs intensity. Genetically manipulated DAergic neuronal activity (arrows, up or down) as indicated (n = 8–12). th: DAergic neuron driver. mKcnj2, TNT-G inactive controls: mKcnj2.nc, TNT-Q. (F), (G), (H), one-way ANOVA with Tukey’s test. (I), unpaired t-test. **P < 0.01. ns: Not significant, FC: Fold change.

Astrocyte glycolysis deficiency increases PEAPODs abundance.

(A) Biochemical pathway of glycolysis. Dotted arrows indicate the intermediate steps not shown. The double-headed arrow denotes reversible reactions. (B–E) PEAPODs and abundance analysis (scale bars: 50 µm). Gene knockdown (KD) in the astrocyte (astro) using RNAi were driven by the alrm driver. Genotypes as indicated (n = 10–12. In E, 784–2583 individual PEAPODs in total). (F) Pyk (α-Pyk) was enriched in astrocytes (scale bars: 50 µm). (G) pyk KD diminished Pyk expression (scale bars: 10 µm). In (F) and (G), mCD8-GFP labeled astrocytes, and α-Repo stained glial cell nuclei. (H–K) PEAPODs and abundance analysis (scale bars: 25 µm). Genotypes as indicated (n = 10. In K, 88–180 individual PEAPODs in total). (C), (D), one-way ANOVA with Tukey’s test. (I), (J), unpaired t-test. (E), (K), Kolmogorov-Smirnov test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: Not significant, FC: Fold change.

Glycolysis-deficient astrocytes promote DAergic synaptogenesis.

(A) Astrocyte number. Genotypes as indicated (n = 12). (B) Normal neuropile (marked by α-Brp) infiltration by the astrocyte in the pyk11/7 mutant (scale bars: 20 µm). xz confocal sections show the infiltration in the dorsal (d)-ventral (v) axis of the VNC. Genotypes as indicated (n = 12). alrm: Astrocyte driver, mCD8-GFP: Membrane-tethered GFP, Brp: Bruchpilot (presynaptic marker). (C) Mean Brp levels. Genotypes as indicated (n = 12). (D and E) Single astrocyte MARCM clone. Genotypes as indicated (n = 20–26). MARCM: mosaic analysis with a repressible cell marker. (F and G) Endogenous Brp (Brp-GFP) in DAergic neurons (scale bars: 25 µm; 5 µm in the inset zoom). Genotypes as indicated (n = 12). astro: Astrocyte, KD: Knockdown. (H and I) Sections by transmission electron microscopy (scale bars: 500 nm). Color shade: synapses, blue; astrocyte processes, orange. Note that perisynaptic astrocyte process (arrowheads) were more often observed in astro pyk KD. Genotypes as indicated (n = 311–313 frames from 5 larval CNS). Frame size, 4.28 μm (width) × 4.41 μm (length). astro: Astrocyte, KD: Knockdown. Unpaired t-test. **P < 0.01. ns: Not significant, FC: Fold change.

Astrocyte glycolysis deficiency causes biased DAergic wiring.

(A) Schematic protocol for profiling postsynaptic partners (labeled by trans-Tango) wired to DAergic neurons. FACS: Fluorescence-activated cell sorting, th: DAergic neuron driver, trans-Tango: Transsynaptic labeling system. (B) Clustering of nsyb+, para+ mature neurons (ctrl, n = 5589; astrocyte [astro] pyk knockdown [KD], n = 7236). Note that cluster 12 is distant to the others. uMAP: Uniform manifold approximation and projection. (C) Annotated MNs (ctrl, n = 50; astro pyk KD, n = 131) in the UMAP. MNs gathered in cluster 12. (D) Close-up view of cluster 12 MNs. (E) Plotting FCs in the cell number of annotated populations, astro pyk KD vs. ctrl. The MN showed the highest FC and followed the neuropeptidergic (NP) neuron and the DAergic neuron. glu: Glutamatergic, GABA: GABAergic, cho: Cholinergic, ser: Serotoninergic, OA/TA: Octop-/tyr-aminergic, FC: Fold change.

Biased DAergic wiring alters locomotion patterns.

(A) Schematic illustration of Drosophila larval locomotive movements. Reorientation attempts intervene between peristaltic sessions. (B) Recorded larval locomotion trajectories (scale bars: 2 cm). Astrocyte pyk RNAi larvae travelled in paths with dense branches; six larvae per recording. Genotypes as indicated (n = 30). alrm: Astrocyte driver. (C–H) Quantitative analysis of larval locomotion. Genotypes as indicated (n = 30). Pausing rate (C): The number of frames the larva pauses/the total number of frames the larva appears × 100%, sweeping rate (D): The number of frames the larva sweeps/the total number of frames the larva appears × 100%, bending strength (E): Median bending angle, max. velocity (F): Maximum velocity, peristalsis efficiency (G): Distance per peristalsis, peristalsis frequency (H): Peristalses per second). One-way ANOVA with Tukey’s test. **P < 0.01, ****P < 0.0001. ns: Not significant, FC: Fold change.

Astrocyte glycolysis deficiency disables astrocyte-DAergic neuronal Nlg2-Nrx1 signaling.

(A–H) PEAPODs and abundance analysis (scale bars: 50 µm). Genotypes as indicated (n = 10–12; in D, 659–2286; in H, 1756–3975 individual PEAPODs in total). alrm: Astrocyte driver, th: DAergic neuron driver, KD: Knockdown. (I and J) Endogenous Brp (Brp-GFP) in DAergic neurons (scale bars: 25 µm; 5 µm in the inset zoom). Genotypes as indicated (n = 12). Brp: Bruchpilot (presynaptic marker). (K and L) Glycolysis deficiency led to intra-astrocyte Nlg2 bodies (arrowheads) (scale bars: 5 µm; 2 µm in the inset zoom). The dotted line marks the astrocyte soma. Genotypes as indicated (n = 12). mCD8-GFP: Membrane-tethered GFP. (M) The ER reporter GFP-KDEL decorated Nlg2 bodies (arrowheads) (scale bars: 2 µm). (B), (C), (F), (G), (J), (L), unpaired t-test. (D), (H), Kolmogorov-Smirnov test. ***P < 0.001, ****P < 0.0001. ns: Not significant, FC: Fold change.

The summary list of cells identified by sc-RNA seq and cell type annotation.

Motor neuron subclasses connected to Drosophila larval DAergic system.

The DNA sequence assembled for creating lexAop2-pykRNAi construct.

Stock list.

Trans astrocyte-DAergic neuronal GFP reconstitution, and PEAPODs remodeling during spontaneous DAergic neuronal activation.

(A) Trans astrocyte-DAergic neuronal reconstituted GFP (reconst. GFP) (n = 12) (scale bars: 50 µm). α-HRP (horseradish peroxidase) stains neural tissues. The drivers th-Gal4 (th) and alrm-LexA (alrm) were used here. a.u.: Arbitrary unit. (B) Regional variations in reconstituted GFP (reconst. GFP) intensity across the larval ventral nerve cord (VNC; thoracic segments, t1–t3; abdominal segments, a1–a8) and central brain (n = 3). a.u.: Arbitrary unit. (C) Single-cell PEAPODs and synaptic labeling of the DAergic neuron in larval brain (n = 10) (scale bars: 5 µm). (D) Dual-color live imaging of PEAPODs and spontaneous DAergic neuronal activation. Active DAergic neuronal projections (labeled by mR-GECO1) and their associated PEAPODs. Time-series dF (Ft–F0) images show the differential fluorescence intensity at the time point of t second as compared to the baseline (0 s) (scale bars: 10 µm). Imaging time interval, 0.7 s. One-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, ****P < 0.0001. FC: Fold change.

The knockdown of glycolytic genes that stimulate PEAPODs.

(A–D) Three-dimensional segmentation and numbering of individual PEAPODs (scale bars: 25 µm). Ten largest PEAPODs from the indicated genotypes were sorted by volume (vol.), and they are delineated by the orange line (scale bars: 25 µm). astro: Astrocyte, KD: Knockdown.

pyk mutagenesis using CRISPR-Cas9, lexAop2-pykRNAi construct assembly.

(A) Schematics of the pyk gene region, two predicted transcripts (pyk-RA, -RB), short guide RNA (sgRNA) for guiding Cas9 to the pyk locus, and DNA lesions of the two mutated alleles, pyk11 and pyk7. PAM: Protospacer adjacent motif. (B and C) Western blotting using α-Pyk antibody (B), and quantitated relative Pyk protein levels (C). Loading control, α-Tub (tubulin). Genotypes as indicated (n = 3). (D) Schematic illustration of lexAop2-pykRNAi construct assembly. dsRNA-GL00099 is the TRiP RNAi amplicon (used in pyk RNAi line #1) for generating short hairpin RNA (shRNA) targeting pyk. The ftz intron was purposed to facilitate shRNA nuclear export. The illustrated elements (DNA sequence included in Table S3) arranged as such were cloned into the pUASTattB backbone. Note that 5× UAS was replaced by 13 copies of lexAop2 in tandem (13× lexAop2). (E–G) PEAPODs abundance analysis. Astrocyte (astro) pyk knockdown (KD) using LexA/lexAop system. Genotypes as indicated (n = 12; in G, 1970–3519 individual PEAPODs in total). (C), one-way ANOVA with Tukey’s test. (E), (F), unpaired t-test. (G), Kolmogorov-Smirnov test. ***P < 0.001, ****P < 0.0001. ns: Not significant, FC: Fold change.

PEAPODs in DAergic neuron brp knockdown (KD) transsynaptic (DAergic synapse) tracing by trans-Tango.

(A–C) PEAPODs abundance analysis. Genotypes as indicated (n = 11–14; in C, 1267–2876 individual PEAPODs in total). th: DAergic neuron driver. (D and E) mtdTomato intensity. The postsynaptic partners of DAergic neurons express myristoylated tdTomato (mtdTomato) (scale bars: 50 µm). Arrowheads denote the neuron somata. Genotypes as indicated (n = 12). trans-Tango: Transsynaptic labeling system, astro: Astrocyte, KD: Knockdown. (A), (B), one-way ANOVA with Tukey’s test. (C), Kolmogorov-Smirnov test. (E), unpaired t-test. *P < 0.05, ***P < 0.001, ****P < 0.0001. ns: Not significant, FC: Fold change.

scRNA-seq and cell type annotation of postsynaptic partners connected to DAergic neurons.

(A) Integrated clustering of all cells sequenced from ctrl (n = 11815) and astrocyte (astro) pyk knockdown (KD) (n = 13327) in uMAP. uMAP: Uniform manifold approximation and projection. (B and C) Single cells expressing nsyb (B), para (C). Note that the cells showed a tendency to converge in the upper right groups, e.g., clusters 1, 2, 5, 7, 10, 16, and 19. Color scale: Normalized gene expression level. (D) Highlighted nsyb+para+ cells in clusters. (E) Percentages of nsyb-para- cells and nsyb+para+ mature neurons in all sequenced cells. (F) Percentages of (un)annotated neurons in mature neurons. (G) Percentages of defined populations in annotated mature neurons.

Annotated populations defined by unique marker genes.

(A and B) Motor neuron (MN) and marker gene twit (A). Glutamatergic neuron and marker gene vglut (B). Note that MNs gathered in cluster 12, which was also vglut+. (C) GABAergic neuron and marker gene gad1. (D) Cholinergic neuron and marker gene vacht. (E and F) Serotoninergic neuron and marker genes: sert (E), trhn (F). (G and H) DAergic neuron and marker genes: dat (G), ple (H). (I) Octop-/tyr-aminergic neuron and marker gene: tdc2. (J) Neuropeptidergic neuron and marker gene: phm. Integrated clustering contains all annotated cells from the ctrl and astrocyte pyk knockdown. uMAP: Uniform manifold approximation and projection, color scale: Normalized gene expression level.