Compensation of inositol 1,4,5-trisphosphate receptor function by altering sarco-endoplasmic reticulum calcium ATPase activity in the Drosophila flight circuit

Abstract

Ionic Ca2+ functions as a second messenger to control several intracellular processes. It also influences intercellular communication. The release of Ca2+ from intracellular stores through the inositol 1,4,5-trisphosphate receptor (InsP3R) occurs in both excitable and nonexcitable cells. In Drosophila, InsP3R activity is required in aminergic interneurons during pupal development for normal flight behavior. By altering intracellular Ca2+ and InsP3 levels through genetic means, we now show that signaling through the InsP3R is required at multiple steps for generating the neural circuit required in air puff-stimulated Drosophila flight. Decreased Ca2+ release in aminergic neurons during development of the flight circuit can be compensated by reducing Ca2+ uptake from the cytosol to intracellular stores. However, this mode of increasing intracellular Ca2+ is insufficient for maintenance of flight patterns over time periods necessary for normal flight. Our study suggests that processes such as maintenance of wing posture and formation of the flight circuit require InsP3 receptor function at a slow timescale and can thus be modulated by altering levels of cytosolic Ca2+ and InsP3. In contrast, maintenance of flight patterns probably requires fast modulation of Ca2+ levels, in which the intrinsic properties of the InsP3R play a pivotal role.

Figure 1.

itpr mutant phenotypes can be suppressed by a dominant mutation, Kum¹⁷⁰, in the Ca-P60A gene. A, Microsomal vesicles derived from itpr^ka1091/ug3 animals (solid line) release lower levels of Ca²⁺ in response to exogenously added InsP₃, compared with microsomal vesicles derived from wild-type animals (dashed line) as shown in the top panel (A, microsomes prepared from adult head; L, microsomes prepared from larvae). In contrast, InsP₃-stimulated Ca²⁺ release from vesicles prepared from itpr^wc703/wc361 animals appeared similar to those prepared from wild-type animals. Western blot experiments performed with vesicles derived from each genotype exhibit equivalent levels of the InsP₃R protein in all three genotypes (data not shown). B, Kum^170/+; itpr^ka1091/ug3 animals grown throughout at 25°C exhibit normal wing posture compared with itpr^ka1091/ug3 animals in which the wings are spread out. This phenotype is not suppressed in Kum^170/+; itpr^ka1091/ug3 animals when grown at 17.5°C. The wing posture of Kum^170/+ animals at 25 and 17.5°C appears like wild type. C, Animals of Kum^170/+; itpr^ka1091/ug3 grown at 17.5°C after egg laying at 25°C have an improved survival profile compared with itpr^ka1091/ug3 animals grown under the same conditions. Kum^170/+ larvae are fully viable at both 25 and 17.5°C (data not shown). Error bars indicate SEM.

Figure 2.

Effect of Kum¹⁷⁰ on cytosolic Ca²⁺ and flight in itpr mutants. A, Kum¹⁷⁰ reduces the rate of Ca²⁺ clearance from the cytosol in primary larval neurons. a, Time lapse images of larval neurons loaded with Fluo-3 from wild-type (WT) and Kum^170/+ animals before (B) and after (see 5, 30, 60 s) addition of 70 mm KCl as a depolarizing stimulus. BF, Bright-field images; scale bar, 10 μm. b, Plots of the ΔF/F values based on the fluorescence signals recorded from individual neurons of the indicated genotypes. c, Fluorescence levels (ΔF/F) normalized to the peak response (100%) of the individual genotypes tested are shown. The solid lines indicate wild-type (filled circles), itpr^ka1091/ug3 (gray triangles), and itpr^wc703/wc361 (open squares). The dashed lines indicate genotypes with one copy of Kum¹⁷⁰ in wild-type, itpr^ka1091/ug3, and itpr^wc703/wc361 organisms. In all six genotypes tested, larval neurons were loaded with Fluo-4. Peak values post-KCl depolarization were obtained from 45 or more individual neurons. The mean peak value (ΔF/F) for all six genotypes was similar (p > 0.01). ΔF/F values of strains with Kum¹⁷⁰ were significantly (p < 0.05) different from the parent strain at all time points after the peak value. B, Flight defects seen in itpr^ka1091/ug3 and itpr^wc703/wc361 are not suppressed by Kum^170/+. Error bars denote SD. All itpr mutants and Kum^170/+; itpr mutants exhibit significant flight defects (p < 0.05) compared with wild-type (WT). Kum^170/+ organisms have normal flight behavior. C, Kum^170/+ suppresses high levels of spontaneously generated action potentials. Calibration, 10 s. D, Quantification of spontaneously generated action potentials. The number of spikes observed in recordings over 2 min were counted and averaged from animals of the indicated genotypes to obtain individual firing frequencies in Hz. Recordings were obtained from at least five animals of each genotype. Data are expressed as mean ± SE. The spontaneous firing rate of Kum^170/+; itpr^ka1091/ug3 organisms is significantly reduced (p < 0.05) when compared with itpr^ka1091/ug3. E, Air puff stimulated flight patterns in itpr^ka1091/ug3 and Kum^170/+; itpr^ka1091/ug3 animals. Single frames (taken within 1–5 s after initiation of flight) from movies of single fly behavior (1, WT; 2, itpr^ka1091/ug3; 3, Kum^170/+; itpr^ka1091/ug3) are shown in the top panel (see also supplemental movie 1, available at www.jneurosci.org as supplemental material), whereas physiological recordings are shown in the bottom panel. On delivery of a gentle air puff, the DLMs of wild-type flies respond with a rhythmic pattern (wings are not visible for fly 1 in top panel because they are beating). This is absent in itpr^ka1091/ug3 flies in 10 of 10 animals tested (see stationary wings for fly 2). Kum^170/+; itpr^ka1091/ug3 animals responded to an air puff by a rhythmic initiation phase (see fly 3, in which wings are invisible because of wing beating). This generally terminates within 5 s of initiation. (Nine of 14 animals tested showed this initial rhythmic response.) This initial phase of response is accompanied by wing beating (see supplemental movie 1, available at www.jneurosci.org as supplemental material). The air puff response of Kum^170/+ flies is normal. The arrows mark the point of air puff delivery. Calibration, 5 s.

Figure 3.

Characterization of mutant alleles for the dgq locus. A, A schematic with the position of the P element (P14073) located in the 5′-UTR region of the dgq gene, which was used for generating the excision allele dgq^221c. Also shown in this diagram is the position of dgq¹⁸⁷⁴⁵ with corresponding exon/intron boundaries of the dgqα₃ splice variant of the dgq gene. F1, F2, and F3 denote different segments in the dgq gene that were selected for PCR amplification. Approximate positions of the primers used to generate UASdgq^1f1 are shown as 5′1f1 and 3′1f1. B, PCRs performed on genomic DNA isolated from the indicated genotypes. The F1 fragment is absent in dgq^221c homozygous animals, whereas the F2 and F3 are present in all the genotypes tested. C, The Dgqα3 protein is greatly reduced in larval homogenates of dgq^221c and dgq¹⁸⁷⁴⁵ homozygous animals. A Western blot performed with homogenates of first-instar larvae of indicated genotypes shows a significant reduction of Dgqα3 (Gq) protein in dgq^221c and dgq¹⁸⁷⁴⁵ homozygous animals. D, No significant reduction in Dgqα3 (Gq) protein levels could be detected in dgq^221c/+ and dgq^18745/+ heterozygous animals compared with wild-type animals. A subtle reduction in Gqα3 protein level was detected after ubiquitous expression of a dsRNA made against the dgq gene (UASdgq^1f1) compared with animals carrying a single copy of UASdgq^1f1 insert.

Figure 4.

A balance of InsP₃ signaling and SERCA activity controls wing posture and flight behavior. A, Maintenance of wing posture. Normal wing posture in itpr^het (itpr^wc361/wc703) animals is lost after introducing a single copy of dgq^221c leading to a mild defect (dgq^221c/+; itpr^wc703/wc361). Defective wing posture is further enhanced in animals with mutations in three genes of the InsP₃ signaling pathway (dgq^221c/plc21c^P319; itpr^wc703/361). Defective wing posture in these animals is suppressed by introduction of the Kum¹⁷⁰ allele. B, Flight behavior. Flies with a single copy of dgq^221c in the background of itpr^het (dgq^221c/+; itpr^wc703/wc361 and dgq^18745/+; itpr^wc703/wc361) exhibit severe flight defects (∼85%) compared with itpr^wc703/wc361 (∼30%) (p < 0.001). This defect was further enhanced by introducing a single copy of plc21c^P319 (dgq^221c/plc21c^P319; itpr^wc703/wc361) (∼98%) (p < 0.001). Enhanced flight defects arising from introduction of dgq^221c, plc21c^P319 mutant alleles (dgq^221c/+; itpr^wc703/wc361 and dgq^221c/plc21c^P319; itpr^wc703/wc361) were suppressed by Kum¹⁷⁰ and returned to the level seen in itpr^wc703/wc361 (p < 0.05). Error bars indicate SEM.

Figure 5.

Gqα and SERCA mutants affect the rate of spontaneous firing in weak InsP₃R mutants but have no effect on flight patterns. A, A low level of spontaneous firing was recorded from DLMs of wild-type and itpr^wc703/wc361 (itpr^het) organisms. The number of spontaneous spikes increased in dgq^221c/+; itpr^wc703/wc361 (dgq^221c/+; itpr^het) flies and in dgq^221c/plc21c^P319; itpr^wc703/wc361 flies (data not shown). This increase in spontaneous firing was suppressed by the introduction of Kum^170/+ (dgq^221c/plc21c^P319-Kum¹⁷⁰; itpr ^wc703/wc361). Spontaneous firing is similar to wild type in dgq^221c/+ and plc21c^P319/+ animals. B, Average numbers of spikes were calculated as described in Figure 2. Error bars indicate SEM. A significantly enhanced rate of firing was observed in dgq^221c/+; itpr^wc703/wc361 animals compared with itpr^wc703/wc361 animals (p < 0.05). Introduction of Kum¹⁷⁰ suppresses the high rate of spontaneous activity (dgq^221c/plc21c^P319-Kum¹⁷⁰; itpr^het). itpr^het denotes itpr^wc703/wc361. C, Increased penetrance of flies with defective flight patterns in dgq,itpr double and dgq,plc21c,itpr triple mutants. Air puff induced flight in wild-type flies (WT). The rhythmic firing of action potentials measured from DLMs of WT continues throughout the duration of flight. A majority of the flight-competent animals of itpr^wc703/wc361 (Fl, selected through the flight column assay) genotype (4 of 5) have flight patterns similar to WT animals. Nonflier animals (NFl) of itpr^wc703/361 genotype exhibit an initial response to the puff of air but usually fail to sustain the response (4 of 7). The penetrance of this defective flight pattern was higher in dgq^221c/+; itpr^wc703/wc361 (7 of 9) dgq^18745/+; itpr^wc703/wc361 (7 of 9), dgq^221c/plc21c^P319; itpr^wc703/wc361 (7 of 9 animals tested) genotypes. The air puff-induced flight patterns were completely restored in the flier population of dgq^221c/plc21c^P319-Kum¹⁷⁰; itpr^wc703/wc361 genotype (3 of 3), whereas in the nonfliers of this genotype flight patterns remained defective (4 of 5). Flight patterns in dgq^221c/+ and plc21c^P319/+ animals are like WT. Calibration, 5 s.

Figure 6.

The cellular focus of Gq-InsP₃R interactions leading to loss of flight and neuronal rhythmicity lies in aminergic interneurons. A, Rescue of wing posture defects in Gq-InsP₃R mutants. Ubiquitous expression of either UASdgqα3⁺ or UASitpr⁺ in dgq,itpr double-mutant animals (dgq^221c/+;itpr^wc703/wc361) showed rescue of wing posture. The rescue was also seen when UASitpr⁺ or UASdgqα3⁺ expression was confined to the aminergic domain. B, Manipulating levels of InsP₃ signaling in aminergic neurons modulates flight behavior. Expression of UASdgqα3⁺ or UASitpr⁺ either in aminergic neurons (DdcGAL4) or ubiquitously (hsGAL4^L) suppresses flight defects seen in dgq^221c/+; itpr^wc703/wc361 animals (p < 0.05). Alternately, expression of a dsRNA construct for dgq (UASdgq^1f1) in aminergic neurons of itpr^wc703/wc361 (DdcGAL4/UASdgq^1f1;itpr^wc703/wc361) animals results in increased flight defects compared with itpr^wc703/wc361 or DdcGAL4/UASdgq^1f1 animals (p < 0.05 for both genotypes). Ubiquitous expression of UASdgqα3⁺ or UASitpr⁺ has no effect on flight behavior. C, Cell type-specific suppression of neuronal hyperactivity. Flies ubiquitously expressing either UASdgqα3⁺ (hsGAL4^L-dgq^221c/UASdgqα3⁺; itpr^wc703/wc361) or UASitpr⁺ (UASitpr^+/+; hsGAL4^L-dgq^221c/+; itpr^wc703/wc361) show low levels of spontaneous activity compared with dgq^221c/+; itpr^wc703/wc361 animals (p < 0.05). Expression of either UASdgqα3⁺ or UASitpr⁺ in aminergic neurons is sufficient to suppress neuronal hyperactivity seen in flies with compromised InsP₃ signaling (dgq^221c/+; itpr^wc703/wc361; p < 0.05). Ubiquitous expression of UASdgqα3⁺ or UASitpr⁺ has no effect on spontaneous firing. Quantification of average spontaneous firing rates was done as described in Figure 2 and Materials and Methods. Error bars indicate SEM. D, Restoration of flight patterns in response to an air puff. Flight-competent flies were selected after flight test experiments presented in B and subjected to an air puff. Normal flight patterns were restored in response to an air puff in the flier population of hsGAL4^L-dgq^221c/UASdgqα3⁺; itpr^wc703/wc361 (8 of 10) and UASitpr^+/+; hsGAL4^L-dgq^221c/+; itpr^wc703/wc361 animals (9 of 10). Flight patterns in response to an air puff were also restored in flies of the genotype DdcGAL4-dgq^221c/UASdgqα3⁺; itpr^wc703/wc361 (5 of 7) and UAS-itpr^+/+; DdcGAL4-dgq^221c/+; itpr^wc703/wc361 (5 of 8). Alternately, a significant proportion of flight-defective flies of the genotype DdcGAL4/UASdgq^1f1;itpr^wc703/wc361 showed air puff response defects (4 of 7). Expression of UASitpr^+/+ (3 of 3) or UASdgqα3⁺ (6 of 6) ubiquitously with the help of hsGAL4^L did not affect air puff response.