dSTIM- and Ral/Exocyst-Mediated Synaptic Release from Pupal Dopaminergic Neurons Sustains Drosophila Flight

Abstract

Manifestation of appropriate behavior in adult animals requires developmental mechanisms that help in the formation of correctly wired neural circuits. Flight circuit development in Drosophila requires store-operated calcium entry (SOCE) through the STIM/Orai pathway. SOCE-associated flight deficits in adult Drosophila derive extensively from regulation of gene expression in pupal neurons, and one such SOCE-regulated gene encodes the small GTPase Ral. The cellular mechanism by which Ral helps in maturation of the flight circuit was not understood. Here, we show that knockdown of components of a Ral effector, the exocyst complex, in pupal neurons also leads to reduced flight bout durations, and this phenotype derives primarily from dopaminergic neurons. Importantly, synaptic release from pupal dopaminergic neurons is abrogated upon knockdown of dSTIM, Ral, or exocyst components. Ral overexpression restores the diminished synaptic release of dStim knockdown neurons as well as flight deficits associated with dSTIM knockdown in dopaminergic neurons. These results identify Ral-mediated vesicular release as an effector mechanism of neuronal SOCE in pupal dopaminergic neurons with functional consequences on flight behavior.

Keywords: Exo84; Neural Circuit; SOCE; Synaptic Maturation.

Figure 1.

Exocyst components are required in pupal neurons for maintaining the duration of adult flight bouts (refer to Figs. 1-1 and 1-2 and Table 1-1). A–C, Box plots represent durations of flight bouts in flies from the indicated genotypes measured by the single flight assay. D, Box plots represent durations of flight bouts in flies with knockdown of Exo84 in neurons during the indicated stages of development. In the box plots, horizontal lines represent medians, crosses indicate means, box limits indicate 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, individual data points are represented as open circles, and the numbers below represent the n number for each box. ***, p < 0.001, n.s., not significant at p < 0.05 by two-tailed Student’s t test. For exact p-values, refer to Table 1-1.

Figure 2.

dStim regulates vesicular release in pupal neurons through Ral (refer to Fig. 2-1 and Table 1-1). A, C, E, Traces represent release of ANF::GFP, estimated by reduction in GFP fluorescence, on depolarization with KCl. Lines represent means and the error bars, standard error of means, from cells as mentioned in the box plots on the right. Point of KCl addition is indicated with a line. B, D, F, Amount of release quantified as area under the curve from 300 to 900 s is represented as box plots for the indicated genotypes. Box plots symbols are as described in Fig. 1. Numbers below each box represent number of cells analyzed from at least three different culture dishes. *, p < 0.05, **, p < 0.01, ***, p < 0.001, n.s., not significant at p < 0.05 by two-tailed Student’s t test (for A) or one-way ANOVA followed by post hoc Tukey’s test (for D and F). All comparisons for significance were with the control values except where marked by a horizontal line. For exact p-values, refer to Table 1-1.

Figure 3.

Extended flight bout durations in adults requires Ral and exocyst function in pupal dopaminergic neurons (refer to Fig. 3-1 and Table 1-1). A, C, Box plots represent flight bout durations of flies from the indicated genotypes measured by the single flight assay. B, Box plots represent flight bout durations of flies with expression of a dominant negative mutant of Ral (Ral^DN) in dopaminergic neurons during the indicated stages of development. D, Box plots represent flight bout durations of flies from the indicated genotypes that were grown at 22°C and transferred to 29°C from 0 to 72 h after Puparium formation for activation of dTrpA1. Box plot symbols are as described in Fig. 1. *, p < 0.05, **, p < 0.01, ***, p < 0.001, n.s., not significant at p < 0.05 by two-tailed Student’s t test (for B) or one-way ANOVA followed by post hoc Tukey’s test (for A, C, and D). All comparisons for significance were with the control values except where marked by a horizontal line. For exact p-values, refer to Table 1-1.

Figure 4.

dStim regulates evoked synaptic release from pupal dopaminergic neurons through Ral (Refer to Fig. 4-1 and Table 1-1). A, Schematic representation of the experimental setup used to image vesicular release from pupal brains. B, Representative images of central regions of pupal brains expressing spH in dopaminergic neurons. Boxed images on the right were obtained by zooming into the inset (white box in the images on the left) at the indicated time points of the time series. C, D, Traces represent average (±SEM) change in fluorescence of spH over time from brains of the indicated genotypes. Points of addition of HL3 and KCl are denoted by vertical lines. E, F, Amount of release quantified as area under the curve from 60 to 300 s (E-Saline–induced) and 300 to 600 s (F-KCl–evoked) from traces in C and D is represented as box plots for the indicated genotypes. Box plot symbols are as described in Fig. 1. Numbers below the boxes represent number of ROIs in which fluorescence changes were measured. They were obtained from a minimum of five brains per genotype. *, p < 0.05, **, p < 0.01, ***, p < 0.001, n.s., not significant at p < 0.05 by one-way ANOVA followed by post hoc Tukey’s test. All comparisons for significance were with the control values except where marked by a horizontal line. For exact p-values, refer to Table 1-1.

Figure 5.

Vesicle release regulated by SOCE, Ral, and the exocyst in pupal neurons regulates flight in adults. A, Cartoon representation of a pupal neuron where SOCE regulates Ral expression. Ral is required for tagging secretory vesicles toward the exocyst complex, thus marking them for both somatic and synaptic release. Such regulated vesicular release of dopamine from pupal neurons appears essential for maturation of a neural circuit required in adults for maintaining the duration of flight bouts. B, In conditions of dStim knockdown, when SOCE is attenuated, Ral levels are reduced, and this in turn has a negative impact on vesicular release in dopaminergic neurons. Consequently, flight bout durations in adult flies are also reduced.