Inositol 1,4,5-trisphosphate receptor and dSTIM function in Drosophila insulin-producing neurons regulates systemic intracellular calcium homeostasis and flight

Abstract

Calcium (Ca(2+)) signaling is known to regulate the development, maintenance and modulation of activity in neuronal circuits that underlie organismal behavior. In Drosophila, intracellular Ca(2+) signaling by the inositol 1,4,5-trisphosphate receptor and the store-operated channel (dOrai) regulates the formation and function of neuronal circuits that control flight. Here, we show that restoring InsP(3)R activity in insulin-producing neurons of flightless InsP(3)R mutants (itpr) during pupal development can rescue systemic flight ability. Expression of the store operated Ca(2+) entry (SOCE) regulator dSTIM in insulin-producing neurons also suppresses compromised flight ability of InsP(3)R mutants suggesting that SOCE can compensate for impaired InsP(3)R function. Despite restricted expression of wild-type InsP(3)R and dSTIM in insulin-producing neurons, a global restoration of SOCE and store Ca(2+) is observed in primary neuronal cultures from the itpr mutant. These results suggest that restoring InsP(3)R-mediated Ca(2+) release and SOCE in a limited subset of neuromodulatory cells can influence systemic behaviors such as flight by regulating intracellular Ca(2+) homeostasis in a large population of neurons through a non-cell-autonomous mechanism.

Figure 1.

Flight and associated physiological defects in itpr mutant adults can be rescued by UASitpr⁺ expression in DILP2 neurons. A, UASitpr^+/+;Dilp2GAL4/+;itpr^ka1091/ug3 and UASitpr^+/+;Dilp2GAL4/+;itpr^wc703/ug3 flies exhibit normal wing posture compared with Dilp2GAL4/+;itpr^ka1091/ug3 and Dilp2GAL4/+;itpr^wc703/ug3 flies which have outspread wings. B, Electrophysiological recordings from the DLMs of tethered flies after delivery of an air puff stimulus (arrows). Dilp2GAL4 rescued flies can respond with a rhythmic pattern that is sustained for the entire 30 s duration of the recording similar to control flies of the genotype Dilp2GAL4/+;itpr^ug3/+. A higher proportion of UASitpr^+/+;Dilp2GAL4/+;itpr^ka1091/ug3 adults showed sustained flight pattern (10/16) in comparison with UASitpr^+/+;Dilp2GAL4/+;itpr^wc703/ug3 (2/16). Air puff-induced flight patterns are absent in itpr heteroalleles (n = 8 of 8 for Dilp2GAL4/+;itpr^ka1091/ug3 and n = 10 of 10 for Dilp2GAL4/+; itpr^wc703/ug3 flies). C, Loss of flight assayed by the cylinder drop test seen in itpr^ka1091/ug3 and Dilp2GAL4/+;itpr^wc703/ug3 is significantly rescued by expression of UASitpr⁺ with Dilp2GAL4 (*p < 0.005; Student's t test). Approximately 100 or more flies were tested for each genotype. Results are expressed as mean ± SEM. D, Single frames from movies of air puff-induced flight in single tethered flies of the indicated genotypes (see also supplemental Video 1, available at www.jneurosci.org as supplemental material). Wings are not visible in the rescued and control condition as they are beating. E, Quantification of spontaneously generated action potentials. The number of spikes observed in recordings over 2 min were counted and averaged from flies of the indicated genotypes to obtain individual firing frequencies in Hz (individual data points are represented by open circles). The spontaneous firing rate in UASitpr^+/+;Dilp2GAL4/+;itpr^ka1091/ug3 (n = 17) and UASitpr^+/+;Dilp2GAL4/+;itpr^wc703/ug3 (n = 16) flies is significantly reduced compared with Dilp2GAL4/+;itpr^ka1091/ug3 (n = 7) and Dilp2GAL4/+;itpr^wc703/ug3 (n = 9) (*p < 0.005; Student's t test). Results are expressed as mean ± SEM. F, Representative traces of spontaneously generated action potentials observed in recordings from the DLMs of indicated itpr mutants and in Dilp2GAL4 rescued flies.

Figure 2.

Adult itpr mutant phenotypes can be rescued by expression of UASitpr⁺ in DILP2 neurons during pupal development. A, UASitpr^+/+;Dilp2GAL4/GAL80^ts;itpr^ka1091/ug3 grown at 19°C all through and maintained at 19°C as adults. The adults exhibit defective wing posture and cannot initiate wing beating in response to an air-puff stimulus (0/21 individual flies tested) similar to Dilp2GAL4/GAL80^ts;itpr^ka1091/ug3 flies on the right and unlike wild-type flies on the left. B, UASitpr^+/+;Dilp2GAL4/GAL80^ts;itpr^ka1091/ug3 grown at 30°C throughout and maintained at 30°C as adults. Wing posture is similar to wild-type flies. These flies can initiate wing beating in response to an air-puff stimulus (10/13 individual flies tested), similar to wild-type flies (see also supplemental Video 2, available at www.jneurosci.org as supplemental material). C, UASitpr^+/+;Dilp2GAL4/GAL80^ts;itpr^ka1091/ug3 grown at 19°C during pupation and maintained at 30°C as adults exhibit a defective wing posture and cannot initiate wing beating in response to an air-puff stimulus (0/12 individual flies tested; see also supplemental Video 3, available at www.jneurosci.org as supplemental material). D, UASitpr^+/+;Dilp2GAL4/GAL80^ts;itpr^ka1091/ug3 grown at 30°C during pupation and maintained at 19°C as adults have a normal wing posture and can initiate wing beating in response to an air-puff stimulus (9/10 individual flies tested; see also supplemental Video 4, available at www.jneurosci.org as supplemental material).

Figure 3.

Expression of dSTIM⁺ in the DILP2 neurons suppresses flight defects of itpr^ka1091/ug3. A, Expression of dSTIM⁺ and dOrai⁺ simultaneously with Dilp2GAL4 (Dilp2) or dSTIM⁺ alone with Dilp2GAL4 or DdcGAL4 (Aminergic) suppresses the wing posture defect of itpr^ka1091/ug3. B, Representative traces of flight patterns from DLMs after delivery of an air-puff stimulus (arrow).Targeted expression of either dOrai⁺ and dSTIM⁺ or dSTIM⁺ on its own, with Dilp2GAL4 or DdcGAL4 in itpr^ka1091/ug3 flies, elicits sustained rhythmic action potentials on delivery of an air-puff, in DLM recordings. C, Complete suppression of the flight defect in itpr^ka1091/ug3 is observed upon expression of dSTIM⁺ with Dilp2GAL4 or DdcGAL4 (**p < 0.01; Student's t test), not seen upon expression of dOrai⁺ alone (Venkiteswaran and Hasan, 2009). D, Frequency of spontaneous firing recorded from the DLMs of itpr^ka1091/ug3 is significantly reduced upon expression of UASdOrai⁺ and/or UASdSTIM⁺ with Dilp2GAL4 or DdcGAL4 in itpr^ka1091/ug3 flies (**p < 0.01; Student's t test). E, Representative traces showing suppression of spontaneous hyperactivity by targeted expression of UASdOrai⁺ and/or UASdSTIM⁺ using Dilp2GAL4 or DdcGAL4.

Figure 4.

Ddc-labeled cells and DILP2 neurons do not overlap in pupal brains. A–F, Projections of confocal Z-stacks of a wild-type Drosophila pupal brain (36 h After Pupa Formation) expressing mCD8GFP with Dilp2GAL4 and immunostained with an anti-Ddc antibody (A, D) and anti-GFP antibody (B, E). C and F are a merge of the corresponding images with anti-Ddc labeling in red and anti-GFP labeling in green. Red arrowheads in A indicate Ddc-stained cells near the DILP2 neurons and in the lateral protocerebrum with processes that lie in close proximity to projections from the insulin-producing neurons. Red arrows indicate Ddc-labeled cells in the subesophageal ganglion. Green arrowheads in B mark the DILP2 neurons in the brain lobes. Green arrowheads in E mark one of a pair of anti-GFP-labeled clusters in the ganglia that sends out processes toward segmentally labeled Ddc cells in the abdominal ganglia (red arrowheads in D). Scale bars (A–F), 50 μm.

Figure 5.

Ddc- and serotonin-labeled cells in larval and adult brains do not overlap with the DILP2 neurons. A–L, Projections of confocal Z-stacks of a wild-type Drosophila adult brain expressing mCD8GFP with Dilp2GAL4 and immunostained with anti-serotonin antibody (A, E, I), anti-Ddc antibody (B, F, J) and anti-GFP antibody (C, G, K). D, H, and L are a merge of the corresponding images with anti-serotonin labeling in blue, anti-Ddc labeling in red, and anti-GFP labeling in green. Successive rows are higher-magnification images. Top and bottom blue arrows in E indicate serotonin-positive varicosities near the DILP2 neurons and in the subesophageal ganglia, respectively. Blue arrowheads in I and red arrowheads in F and J mark cells that are Ddc and serotonin positive and send out processes centrally toward the subesophageal ganglia (red arrows in F, J) that seem to intermingle with the processes sent out by the DILP2 neurons (green arrows in C, G, K). Green arrowhead in C and white arrowheads in D, H mark the DILP2 neurons. Scale bars: A–H, 50 μm; I–L, 20 μm.

Figure 6.

Reduced itpr levels in Drosophila neurons by dsRNA results in flight defects and lowered SOCE. A, Pan-neuronal knockdown of itpr induces wing posture defects that are more pronounced in females and are further enhanced with a UASdicer (dicer) transgene in the background. Wing posture defects are not observed on dsitpr expression with DdcGAL4 (Aminergic) or Dilp2GAL4 (Dilp2) with a UASdicer transgene in the background. B, Significant flight defects are induced on pan-neuronal knockdown of itpr (**p < 0.005; Student's t test). C, Rhythmic action potentials in response to an air-puff stimulus are not observed on pan-neuronal knockdown of itpr. Simultaneous expression of dsitpr in DILP2 and aminergic domains (doubleGAL4) results in a reduced ability to maintain flight. D, Frequency of spontaneous firing in recordings from the DLMs is increased significantly on knockdown of itpr pan-neuronally or in the aminergic and DILP2 domains (*p < 0.05; Student's t test). E, Representative traces of spontaneously generated action potentials in recordings from the DLMs on pan-neuronal or tissue-specific knockdown of itpr (n ≥ 12). F, Pseudo-color image representation of measurement of store Ca²⁺ in neurons of the indicated genotypes by store depletion with thapsigargin followed by Ca²⁺ add-back to measure SOCE. Warmer colors represent higher Ca²⁺. G, Single-cell traces of SOCE in individual cells of the indicated genotypes. Peak values of responses have been plotted as a box chart in (H). H, Pan-neuronal expression of dsditpr lowers SOCE. Box plots representing the distribution of store Ca²⁺ and SOCE in neurons from the indicated genotypes. The bigger boxes represent the data spread (from 25 to 75%), the horizontal line in each box is the median, smaller squares represent mean and the diamonds on either side represent outlier values (*P_ANOVA < 0.05 compared with dsitpr controls; n ≥ 150 cells).

Figure 7.

Restoration of store calcium and SOCE in itpr^ka1091/ug3 neurons upon targeted expression of itpr⁺ to subneuronal domains. A, Fluorescent images show store Ca²⁺ in representative neurons of the indicated genotypes measured by store depletion with thapsigargin followed by Ca²⁺ add-back to measure SOCE. B, Single cell traces of store Ca²⁺ and SOCE of the indicated genotypes. Arrowheads represent peak values of response which have been plotted as a box chart in C and D. Targeted expression of itpr⁺ to either aminergic or DILP2 neurons, in itpr^ka1091/ug3 restores levels of store Ca²⁺ and SOCE in all the neurons. C, Box plot representation of the distribution of SOCE in neurons of the indicated genotypes. The bigger boxes represent the data spread as in Figure 6, smaller squares represent the mean and the diamonds on either side represent outlier upvalues. D, Box plot comparison of store Ca²⁺ in larval neurons with pan-neuronal expression of itpr⁺ or those expressing itpr⁺ specifically in the aminergic or DILP-producing cells. (*P_ANOVA < 0.01 compared with itpr^ka1091/ug3; P_ANOVA > 0.05 compared with wild-type, n ≥ 90 cells).

Figure 8.

dSTIM⁺ expression in itpr^ka1091/ug3 neurons restores intracellular Ca²⁺ homeostasis. A, Single cell traces of SOCE by Ca²⁺ add-back after store depletion. Arrowheads represent peak values of response which have been plotted as a box chart in B. Box plots of ΔF/F values of SOCE (B) and store Ca²⁺ (C) in the indicated genotypes. SOCE and store Ca²⁺ is significantly different compared with itpr^ka1091/ug3 in all conditions where UASdOrai⁺ and/or UASdSTIM⁺ is expressed with Dilp2GAL4 (P_ANOVA < 0.05, compared with itpr^ka1091/ug3). However, SOCE and store Ca²⁺ is significantly different from wild-type only in the condition where UASdOrai⁺ is expressed on its own with Dilp2GAL4 (**P_ANOVA < 0.05, compared with wild-type). D, F, Magnitude of Ca²⁺-release through mutant InsP₃Rs in itpr^ka0191/ug3 neurons is significantly higher with pan-neuronal expression of either dOrai⁺ or dSTIM⁺. Larval neurons of the indicated genotypes, with pan-neuronal expression of Drosophila mAChR, were stimulated with 10 μm carbachol and fluorescent images were taken in the time lapse mode. Images were pseudo-colored to represent [Ca²⁺]_i. Warmer colors represent higher Ca²⁺. E, G, Box plots representing the magnitude of stimulated Ca²⁺ release in neurons of the indicated genotypes by 10 and 20 μm carbachol. Expression of dOrai⁺ partially restores stimulated release through InsP₃R such that it is significantly different from itpr^ka1091/ug3, wild-type and controls (**P_ANOVA < 0.05). Expression of dSTIM⁺ restores stimulated release through InsP₃R such that it is significantly different from itpr^ka1091/ug3 (P_ANOVA < 0.05) but not wild-type and controls (P_ANOVA > 0.05) at 20 μm carbachol. At 10 μm carbachol, stimulated release on dSTIM⁺ expression is significantly different from itpr^ka1091/ug3 and wild-type (**P_ANOVA < 0.05) but not control (Elav^c155/UASdSTIM⁺;UASmAChR, P_ANOVA > 0.05). In control cells expressing pan-neuronal dOrai⁺ or dSTIM⁺ expression (in the absence of itpr^ka1091/ug3) Ca²⁺ release through InsP₃R is similar to wild-type (P_ANOVA > 0.05). Measurements from 150 cells or more were obtained for each box plot.