Figure 2: Perineurial Knockdown of Store-Operated Ca²⁺ Entry Pathway (SOCE) Components Impairs Locomotor Activity and Increases Seizure Susceptibility.

(A) Histograms summarizing the percent of flies exhibiting HS-induced seizures at 3 minutes (38.5°C). (a’) An array of glial specific Gal4 drivers were used to knock down dStim (see methods). Only knockdown of dStim using perineurial glia (PG) drivers (46F-Gal4 and GMR85G01-Gal4) recapitulated the pan-glial HS-induced seizure phenotype (p<0.0001, Two-way ANOVA), while knockdown with a third PG driver (NP6293) failed to recapitulate the phenotype. (a’’) Inhibiting Gal4 expression of the RNAi in neurons with Gal80 (C155-Gal80) does not alter the seizures observed with GMR85G01 knockdown, indicating the seizure phenotype does not arise from neuronal knockdown of dStim. Co-expressing Dicer2 with dStim RNAi (to enhance the knockdown) using the GMR85G01 driver does not enhance the seizure phenotype (p>0.05, Two-way ANOVA, N=4 groups of >10 flies/genotype, error bars are mean ± SEM).

(B) Time course of heat-shock induced seizures (38.5°C, HS) for repo>dStim^RNAi and PG>dStim^RNAi are shown (p>0.05, Two-way ANOVA, N=4 groups of 20 flies/genotype, error bars are mean ± SEM, Video 1).

(C) Expression pattern of the perineurial glial driver GMR85G01. GMR85G01 expression of a membrane tethered GFP (mCD8::GFP) reveals high expression in PG cells that enclose the entire larval CNS (brain and VNC); 98 μm projection, scale bar= 100 μm (green: mCD8::GFP, cell membranes; red: anti-elav, neuronal nuclei). For complete expression analysis see Figure S2. GMR85G01 hereafter referred to as PG driver.

(D-E) CPG activity in PG>dStim^RNAi showing PG>dStim^RNAi lose normal rhythmic muscle activity under heat-shock (HS) conditions (38°C). (D) Representative voltage traces of spontaneous CPG activity recorded at larval 3^rd instar muscle 6 at 38°C in control and PG>dStim^RNAi animals (n≥5 preparations/genotype). (E) Quantification of precent muscle potential bursting for CPG recordings of PG>dStim^RNAi animals at room temperature and 38°C HS (marked with pink shadings) (p<0.01, Student’s t-test, n ≥ 5 preparations/genotype).

(F, G) Activity level of adults (F) and 3^rd instar larvae (G) expressing dStim RNAi using the PG driver show significant reduction in total locomotor activity (p<0.0001 for adult flies, p<0.05, p<0.01 for larvae, Student’s t-test, N=8 larvae/genotype, N>100 adult flies/genotype, median is presented).

(H, I) Perineurial conditional knockdown of Orai using Gal4/Gal80^ts. Rearing adult flies at the restrictive temperature for Gal80^ts (>30°C, marked with pink shadings) allows expression of Orai^RNAi only in adults (N=4 groups of 20 flies/condition, error bars are mean ± SEM). PG> Orai^RNAi animals that were reared at 18°C to suppress Gal4-driven transgene expression (marked with blue shading), displayed higher survival rate.

(G) Over the course of several days at 31°C, the majority of PG>Orai^RNAi/Gal80^ts flies died (^~80% mortality after 7 days, p<0.0001, Two-way ANOVA). (H) A significant increase in seizures (p<0.0001, Two-way ANOVA) was seen after seven days of rearing flies at the restrictive temperature for Gal80^ts (31°C). Approximately 30% of surviving adults showed seizures and the rest displayed severe locomotor defects. PG> Orai^RNAi animals that were reared at 18°C to suppress gal4-driven transgene expression (marked with blue shading), displayed increased seizure susceptibility.

*=P<0.05, **=P<0.01, ****=P<0.0001.