Figure 5. Cortex glial knock-down of sandman, a K_2P channel, recapitulates zyd phenotypes.

(A-B) Behavioral analysis of HS induced seizures. (A) Knockdown of sandman (sand) in different glial subtypes: pan-glial (repo), cortex glial (NP2222) and in all glia other than cortex glia (repo>sand^RNAi/GMR54H02>gal80 in which gal80 is constitutively inhibiting gal4 activity and sand^RNAiexpression only in cortex glia). Inset shows analysis after 2 minutes of HS (p=0.0006 for NP2222>sand^2xRNAi, p<0.0001 for repo>sand^RNAi/GMR54H02>gal80, N = 4 groups of >10 flies/genotype). (B) Temperature threshold of repo>sand^RNAi (p=0.5185) and NP2222>sand^2xRNAi (p=0.2302) seizures in comparison to zyd (N = 3 groups of 10/temperature/genotype). (C) Representative voltage traces of spontaneous CPG activity at larval 3^rd instar muscle 6 at 38°C in wildtype, zyd and repo>sand^RNAi (n ≥ 5 preparations/genotype). (D) Quantification of average bursting duration for CPG recordings of the indicated genotypes at 38°C. (E) Light avoidance assay reveals no defect in this behavior at 25°C (N = 3 groups of 20 flies/genotype). (F) Behavioral analysis of HS-induced seizures. Seizures in repo>sand^RNAi animals were not suppressed with either CanB2^RNAi#1 or by feeding flies with 1 mM CsA (N = 3 groups of 20 flies/genotype/treatment). (G-H) Ca²⁺ imaging in larval cortex glial cells using myrGCaMP6s. (G) The average rate of microdomain Ca²⁺ events was reduced in repo>sand^RNAi cortex glia relative to wildtype (20.36 ± 5.5 and 69.83 ± 5.3, p<0.0001). Knockdown of sand on the zyd background did not restore zyd Ca²⁺ microdomain events (n ≥ 5 animals/genotype). (H) Average myrGCaMP6s fluorescence in cortex glia at 25°C. Elevated basal fluorescence of GCaMP6s in zyd relative to wildtype cortex glia (p=0.0003) is not altered following sand knockdown (zyd;;repo>sand^RNAi, N = 4 animals/genotype). Error bars are SEM, ***=P < 0.001, ****=P < 0.0001, Student’s t-test.

Figure 5—figure supplement 1. Cortex glial knockdown of sandman, a K_2P channel, reproduces zyd phenotypes.

Related to Figure 5. (A) Quantification of vortex-induced seizures in wildtype, zyd and repo>sand^RNAi adults. (B-C) Behavioral analysis of HS-induced seizures. (B) Different genetic combinations of the zyd mutation and sand^RNAi were analyzed. Combining a single copy of the zyd mutation does not change the partial seizures observed in cortex-glial knockdown of sand (N = 4 groups of >10 flies/genotype). (C) Pan-glial knockdown of other members of the Drosophila K⁺ channel family besides sand do not cause seizures (N = 2 groups of ≥10 flies/genotype). (D) Pan-glial knockdown of members of the Drosophila K_ir family does not enhance zyd seizures (N = 4 groups of >10 flies/genotype).

Figure 5—video 1. The response of repo>sand^RNAi flies to a 38.5°C heat-shock is shown, following by the response of NP2222>sand^2xRNAi flies to the same condition.

Download video file ^{(3.8MB, mp4)}

DOI: 10.7554/eLife.44186.019

Figure 5—video 2. Representative Ca²⁺ imaging in repo>sand^RNAi cortex glia.

Download video file ^{(3.4MB, mp4)}

DOI: 10.7554/eLife.44186.020

GCaMP6s was expressed specifically in cortex glia using the GMR54H02-gal4 driver.