Figure 1. Characterization of microdomain Ca²⁺ transients in Drosophila astrocytes.

(A) Schematic of larval CNS (white area, neuropil; gray, cortex). An imaging area showing membrane tethered myr-GCaMP5a (green) in astrocytes, in which microdomain Ca²⁺ transients during 6 min were maximally projected. Traces of 8 individual ROIs microdomains (right, a–h) are shown over the entire 6 min window. Pseudocolor grayscale of Ca²⁺ signals (bottom left), grayscale values ranging from 0 to 255 (scale bars, 20 µm). Representative time-lapse images (bottom right) of two indicated microdomain Ca²⁺ transients in 2 ROIs (1, 2). (B) Superimposed traces of individual microdomain Ca²⁺ transients and an average with its full width at half maximum (FWHM, mean ± SD). (C) Histogram showing the distribution of recurrent microdomain Ca²⁺ transients at same ROIs. (D) Responses of microdomain Ca²⁺ transients to tetrodotoxin, 0 extracellular Ca²⁺ and LaCl₃ (n = 6, mean ± SEM, one-way ANOVA). (E) 2-color Ca²⁺ imaging in neighboring astrocytes. In presence of the flippase repo-FLP, myr-GCaMP5a expressing astrocytes switch to express myr-R-GECO1, resulting in 3 types of astrocytes: myr-GCaMP5a⁺ (green), myr-R-GECO1⁺ (red), expressing both indicators (orange) (scale bar, 20 µm). Quantification is from Ca²⁺ imaging of myr-GCaMP5a and myr-R-GECO1 that were exclusively expressed in adjacent astrocytes (n = 3, unpaired t-test). Time-lapse images and superimposed traces of representative microdomain Ca²⁺ transients at two juxtaposed ROIs between myr-GCaMP5a and myr-R-GECO1 expressing astrocytes. See source data Figure 1—source data 1.

Figure 1—figure supplement 1. Spontaneous astrocyte microdomain Ca²⁺ transients in larvae.

(A) Astrocytes expressing myr-GCaMP5a in the ventral nerve cord of 1^st instar larvae. Traces of 8 representative microdomain Ca²⁺ transients. (B) Superimposed traces of individual microdomain Ca²⁺ transients and an average showing its full width at half maximum (FWHM, mean ± SD). (C) Schematic of inducing 2-color Ca²⁺ indicators mosaic expression in neighboring astrocytes (green, red, orange). (D) Two representative microdomain Ca²⁺ transients in dual-labeled myr-GCaMP5a/myr-R-GECO1 astrocytes. Note that both myr-GCaMP5a and myr-R-GECO1 exhibit similar dynamics in the same astrocyte.

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

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Figure 1—video 2. Microdomain Ca²⁺ transients in intact CNS preparation from wildtype 1^st instar larva.

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