Figure 2. Astrocyte microdomain Ca²⁺ transients are genetically distinct from soma transients and require TrpML.

(A) Responses of microdomain Ca²⁺ transients (frequency) to glutamate (Glu), acetylcholine (Ach), γ-aminobutyric acid (Gaba), tyramine (Tyr), octopamine (Oct) in presence of tetrodotoxin. (B) Effect of tyramine on Ca²⁺ transient frequency in genotypes indicated. Oct-TyrR mutants, and astrocyte-specific (alrm>) expression of RNAis to TyrR or TyrRII (in A and B, n = 6, mean ± SEM, paired t-test). (C) Quantification of microdomain Ca²⁺ transients in Tdc2^RO54, Tβh^nM18 or Oct-TyrR^hono mutants (n = 6, mean ± SEM, one-way ANOVA). (D) Maximally projected astrocyte microdomain Ca²⁺ transients during 6 min in control, trpml¹ mutants, and astrocyte-specific trpml^RNAi (in pseudocolor, grayscale values ranging from 0 to 255. scale bar, 20 µm). Quantification of microdomain Ca²⁺ transients in mutants (loss-of-function mutations or astrocyte-specific RNAi driven by alrm-Gal4) of genes encoding TRP family ion channels (n = 6, mean ± SEM, one-way ANOVA). (E) trpml-myc expression in astrocytes rescues microdomain Ca²⁺ transients in trpml¹ mutants (n = 6, mean ± SEM, one-way ANOVA). (F) Effect of tyramine treatment on controls and trpml¹ mutants (n = 6, mean ± SEM, one-way ANOVA. within groups, paired t-test). Right panel, increase in transients by tyramine (subtracting the basal included) in control and trpml¹ mutants. (G) Application of the antioxidant N-acetyl cysteine (NAC) or H₂O₂ to the larval CNS. (for F and G n = 6, mean ± SEM, across groups, one-way ANOVA; within groups, paired t-test; -, + indicate pre-, post-delivery). (H) H₂O₂ application to control and trpml mutants shows that H₂O₂-dependent increases require TrpML. See source data Figure 2—source data 1.

Figure 2—figure supplement 1. Astrocyte microdomain Ca²⁺ transients require TrpML.

(A) Quantification of microdomain Ca²⁺ transients in intact control and trpml¹ mutant larvae at 1^st instar stage (n = 6, mean ± SEM, t-test). (B) Average amplitude of microdomain Ca²⁺ transients in genotypes as indicated (n = 6, mean ± SEM, one-way ANOVA). (C) Tyramine- induced soma Ca²⁺ rise measured in GCaMP6s intensity (n = 70–73 cells from six larval CNS in each genotype, mean ± SEM, t-test). (D) Gross astrocyte morphology in control and trpml¹. (E) trpml¹ mutant larvae with trpml-myc expression in astrocytes survived to adulthood (n = 3, each n contains 20–40 larvae, mean ± SEM, t-test). (F) Double-labeling TrpML-MYC with a marker for cell membrane (mCD8-GFP) or lysosomes (GFP-Lamp1). Dash lines, cell bodies. Arrows, TrpML-MYC/GFP-Lamp1 colocalization. See source data Figure 2—source data 2.

Figure 2—video 1. Microdomain Ca²⁺ transients in acutely dissected CNS preparations from trpml mutant 3^rd instar (L3) larva.

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Figure 2—video 2. Microdomain Ca²⁺ transients pre/post H₂O₂ treatment in wild type 3^rd instar (L3) larva.

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Figure 2—video 3. Microdomain Ca²⁺ transients pre/post H₂O₂ treatment in trpml¹ mutant 3^rd instar (L3) larva.

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