Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Abstract

Neuronal Ca(2+) signals can affect excitability and neural circuit formation. Ca(2+) signals are modified by Ca(2+) flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca(2+) stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca(2+) store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP(3)R) can partially compensate for their loss of flight. Ca(2+) measurements show that Orai gain-of-function contributes to the quanta of Ca(2+)-release through mutant InsP(3)Rs and elevates store-operated Ca(2+) entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca(2+) in neurons is required for Drosophila flight.

Fig. 1.

Intracellular Ca²⁺ homeostasis in larval neurons is altered on expression of dsdOrai and dsdSTIM. (A) pseudocolor images represent [Ca²⁺]_ER and SOCE in primary cultures of neurons loaded with Fluo-4 from WT larvae and those expressing dsdOrai or dsdSTIM. (Scale bar, 10 μm.) (B) Single cell traces of SOCE by Ca²⁺ add-back after store depletion. Two cells of each genotype are shown. Arrow heads represent peak values of response, which have been plotted as a box chart in C. (C) Box plots of ΔF/F values of SOCE. The bigger boxes represent the data spread, smaller squares represent mean, and the diamonds on either side represent outlier values. (D) Box plot comparison of [Ca²⁺]_ER between neurons of indicated genotypes. (E) Kolmogorov–Smirnov (K-S) plot analyzing the distribution of [Ca²⁺]_i in neurons loaded with Indo-1. The frequency distribution is significantly shifted to the left for cells with dSTIM RNAi (dsdSTIM), indicating a higher frequency of cells with reduced [Ca²⁺]_i (P_K-S<0.05). (F) Box plot representation of [Ca²⁺]_i in neurons with dsdOrai or dsdSTIM. (n ≥ 150 cells; *, P_ANOVA < 0.05; **, P_ANOVA < 0.01).

Fig. 2.

RNAi knockdown of dOrai or dSTIM in subsets of neurons gives rise to flight motor defects. Pan-neuronal knockdown of dOrai and dSTIM induces (A) change in wing posture, (B) flight defects (n ≥ 100 flies), (C) higher levels of spontaneous firing (n ≥ 15 flies), and (D) defects in air-puff-induced flight patterns. (E) Representative traces of spontaneous firing activity from the DLMs of the indicated genotypes. Histograms represent mean ± SE; (*, P < 0.05; Student's t test). Both GAL4 control strains were tested and found to be similar to WT. Data shown are for glutamatergic GAL4 flies.

Fig. 3.

Overexpression of dOrai⁺ in neuronal subsets partially suppresses flight defects in an itpr mutant. (A) Flies overexpressing dOrai⁺ in aminergic, Dilp2, and glutamatergic neurons or ubiquitously under a HS promoter and subjected to HS initiate unsustained (<5 s) flight patterns in response to air puff. (B) Overexpression of dOrai⁺ (2 copies) either ubiquitously (by a leaky HS GAL4 at 25 °C) or in the indicated subneuronal domains suppresses the elevated spontaneous firing of itpr^ku flies. (**, P < 0.01; Student's t test; n ≥ 20 flies).

Fig. 4.

The dOrai⁺ overexpression in itpr^ku neurons restores intracellular Ca²⁺ homeostasis. (A) SOCE measurements in the indicated genotypes (*, P_ANOVA < 0.05, compared with itpr^ku; and **, P < 0.01, compared with WT). (B) [Ca²⁺]_ER in the indicated genotypes (**, P_ANOVA <0.01, compared with WT). (C) K-S plot for [Ca²⁺]_i in neurons of the indicated genotypes (P_K-S < 0.05 for genotypes expressing dOrai⁺ compared with WT). (D) Box plot representation of [Ca²⁺]_i (*, P_ANOVA < 0.05; **, P_ANOVA < 0.01; n ≥ 170 cells).

Fig. 5.

Suppression of flight and related physiological defects by dominant mutants of dOrai and dSERCA. (A) Air-puff-induced flight patterns in the indicated genotypes. (B) Flight defects in itpr^ku are suppressed by the presence of both Kum¹⁷⁰ and dOrai² or dOrai¹, but not with dOrai mutants or Kum^170/+ on their own. (C) Snapshots taken within the first 5 s of air-puff-induced flight initiation in (i) itpr^ku; (ii) Kum¹⁷⁰/dOrai²; itpr^ku (Movie S1). (D) Spontaneous hyperactivity in DLMs of indicated genotypes; n ≥ 15. (E) Wing posture defects induced by dsdSTIM are suppressed by dOrai² (50%) or Kum¹⁷⁰ (10%). Histograms represent mean ± SE; (*, P < 0.05; **, P < 0.01, compared with itpr^ku; Student's t test).

Fig. 6.

Effects of dOrai and dSERCA mutants on different aspects of intracellular Ca²⁺ release. (A) Changes in stimulated Ca²⁺ release through InsP₃R (measured as ΔF/F) (**, P_ANOVA < 0.01, compared with WT; *, P_ANOVA < 0.05, compared with itpr^ku; n ≥ 150 cells). (B) Effect of Kum¹⁷⁰ and dOrai² on perdurance of InsP₃R mediated Ca²⁺-release signals; n ≥ 40 cells, with similar peak response times. (C) Single cell traces of SOCE by Ca²⁺ add-back after store depletion. (D) SOCE measured in cultured neurons of indicated genotypes (**, P_ANOVA < 0.01, compared with WT). SOCE in dOrai^2/+; itpr^ku is significantly higher than itpr^ku (*, P_ANOVA < 0.05), and is normal in cells of Kum¹⁷⁰/dOrai²; itpr^ku (**, P_ANOVA < 0.01, compared with itpr^ku). Heterozygous dOrai^2/+ partially restores SOCE in dsdSTIM expressing neurons (**, P_ANOVA < 0.01). (E) [Ca²⁺]_ER measurements (**, P_ANOVA < 0.01; *, P_ANOVA < 0.05 in Kum^170/+ genotypes, compared with WT); dOrai^2/+ restores [Ca²⁺]_ER in itpr^ku double mutants (*, P_ANOVA < 0.05). Presence of dOrai² restores [Ca²⁺]_ER in neurons expressing dsdSTIM (**, P_ANOVA < 0.01; n ≥ 170 cells).