Article Figures & Data
- Figure 1.
Near-membrane Ca2+ activity in Drosophila astrocytes. A, Single confocal plane of larvae VNC showing the astrocyte-specific expression of myrGCaMP6s under the control of Alrm-Gal4. B, B’, Time-lapse image series of two microdomain astrocyte Ca2+ transients. C, Superimposition of astrocyte Ca2+ traces (in gray) and their average (black line; n = 46 individual traces). D, Sample traces of recurring Ca2+ transients in five different areas indicated in A. Scale bars 20 µm (A) and 5 µm (B and B’).
- Figure 2.
Acute astrocyte Ca2+ influx through TrpA1 channels suppresses neuronal activity. A, All Alrm > TrpA1 flies became paralyzed within 30 s of exposure to 33°C, while control flies (Alrm-Gal4) show no behavioral defects. n > 80 flies per genotype; pa < 0.0001. B, Time course of paralysis in adult flies of the indicated genotypes on exposure to 33°C; repo-Gal80 suppresses the paralysis in Alrm > TrpA1 background, while elav-Gal80 has no effect. n > 70 flies for each genotype. C, Sample traces of CPG recording from larval NMJs. Postsynaptic potentials in Alrm > TrpA1 animals diminish during a temperature ramp from room temperature (22°C) to 34°C, while Alrm-Gal4 larvae maintain normal activity.
- Figure 3.
Suppression of Rab11 function ameliorates astrocyte Ca2+ influx-induced behavioral defects and impairs GAT trafficking. A, Quantification of paralyzed flies when exposed to 30°C, showing rab11-knockdown suppressed the paralysis in Alrm > TrpA1 flies. n > 100 flies per genotype; pb < 0.00001. B, Quantification of paralyzed flies when Alrm > TrpA1 and Alrm > TrpA1, Rab11-DN flies were transferred to 30°C. n > 45 flies per genotype; pc = 0.0095. C, Single optical section showing astrocyte cell body and neuropil in the VNCs of Alrm > TrpA1-myc (control) and Alrm > TrpA1-myc, Rab11-RNAi (Rab11-RNAi) larvae stained with antibodies against Myc (green) and Brp (magenta). Note the apposition between synapses and astrocyte processes in the neuropil. D, Quantification of relative TrpA1-Myc level, normalized to control. n = 8 individual optical planes for each groups; p d = 0.5761. E, Optical sections showing GAT localization in astrocytes of larval VNCs stained with antibodies against GAT (green) and Repo (magenta) in control and when Rab11-RNAi or Rab11-DN is expressed in astrocytes. F, Western blotting of adult heads showing the effect of Rab11-RNAi expression in astrocytes on GAT level. G, Quantification of F. n = 6 experiments; p e = 0.3125. Scale bars in C and E, 5 µm.
- Figure 4.
Astrocyte Ca2+ influx leads to reduced membrane GAT. A, Confocal images of larval VNC showing astrocyte membrane GAT level in control and Alrm > TrpA1 animals at room temperature (22°C) or 34°C. B, Quantification of relative GAT level shown in A, normalized to Alrm-Gal4 animals at each temperature. n = 8-10 individual optical planes for all groups; p f = 0.4974 and p g < 0.0001, respectively. C, C’, Confocal images with longer exposure showing GAT-containing vesicles (arrowheads) forming in the cytoplasm of Alrm > TrpA1 larvae exposed to 34°C. Scale bars in A and C, 5 µm.
- Figure 5.
Excessive GABA leads to suppression of neuronal activity. A, Quantification of paralyzed flies on exposure to 30°C. Ectopic activation of GABAergic neurons via GAD1-Gal4-induced expression of TrpA1 leads to acute suppression of neuronal activity and paralysis. B, Distinct impact of cortex glia and astrocyte Ca2+ signals on neuronal activity. Ca2+ influx in cortex glia leads to increased neural activity and seizure-like behavior, whereas enhanced astrocyte Ca2+ signal causes suppression of neuronal activity and paralysis. Together, they constitute a Ca2+-dependent glial mechanism to fine-tune neuronal function. C-C”, Model of how astrocyte Ca2+ signaling regulates neuronal activity. Astrocyte Ca2+ influx leads to acute endocytosis of membrane GAT, reduced GABA uptake, and suppression of neuronal activity. Inhibition of Rab11 function reduces the removal of membrane GAT and sustains GABA uptake, hence ameliorating the paralysis caused by astrocyte Ca2+ influx.
Data structure Type of test Observed power (α = 0.05) a (Fig. 2A) Non-normal distribution Mann-Whitney test 1 b (Fig. 3A) Normal distribution Student’s t test 1 c (Fig. 3B) Non-normal distribution Mann-Whitney test 1 d (Fig. 3D) Normal distribution Student’s t test 0.0838271 e (Fig. 3G) Non-normal distribution Wilcoxon signed-rank test 0.2684 f (Fig. 4B) Normal distribution Student’s t test 0.0975257 g (Fig. 4B) Normal distribution Student’s t test 0.999942 Gene product CG no. Effect Function RNAi stock no. Esyt2 CG6643 Enhance ER-PM tethering v28418, v28419 SCAMP CG9195 Enhance SV trafficking v9130 Nep4 CG4058 Enhance Metalloendopeptidase v16669, v100189 Syt4 CG10047 Suppress Vesicle trafficking v33317, BL26730 Syx1A CG31136 Suppress SNARE; Vesicle trafficking v33113 Syt12 CG10617 Suppress Vesicle trafficking v47503 α-SNAP CG6625 Suppress SNARE disassembly v101341, BL29587 Rab11 CG5771 Suppress Endosome trafficking v22198, BL27730
- Movie 1.
Drosophila astrocyte near-membrane Ca2+ activity detected with myrGCaMP6s. Ca2+ transients were recorded from the VNC of undissected 2nd instar larvae. Video speed, 2× real time. Scale bar, 20 µm.
- Movie 2.
Astrocyte Ca2+ influx leads to suppression of neuronal activity in adult flies. Acute paralysis was induced in Alrm > TrpA1 flies when transferred to preheated water bath held at 33°C; control flies did not show obvious behavioral deficit. Video speed is real time.
- Movie 3.
Acute astrocyte Ca2+ influx leads to paralysis in Drosophila larvae. Third instar Alrm > TrpA1 larvae rapidly lose voluntary muscle contraction and became paralyzed following transfer to a preheated Petri dish held at 34°C. Control larvae show enhanced locomotion activity. Video speed is real time.
- Movie 4.
Ca2+ influx in adult ventral ganglion astrocytes causes paralysis. Headless adult Alrm > TrpA1 flies show paralysis when transfer to a preheated Petri dish held at 33°C. The video started 30 s after the flies were transferred to the Petri dish. Video speed is real time.
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