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. 2019 Apr 26;8:e44186. doi: 10.7554/eLife.44186

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© 2019, Weiss et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 2. — (A) Behavioral analysis of HS-induced seizures. Pan-glial knockdown of the CN regulatory subunit, CanB2, with three partially overlapping hairpins (#1, #2 and #3, see Materials and methods) completely rescues the zyd seizure phenotype, while a single copy of CanB2 knockout allele (CanB2^KO/+) rescues ~60% of seizures (N = 4 groups of ≥15 flies/genotype). Inset shows analysis after 2 minutes of HS (p=0.0001). (B) Behavioral analysis of the recovery from vortex-induced seizures. Pan-glial knockdown of CanB2 and CanA-14F rescues zyd vortex-induced seizures (N = 3 groups of 20 flies/genotype). (C) Representative voltage traces of spontaneous CPG activity at larval 3^rd instar muscle 6 at 38°C in wildtype, zyd and zyd;;repo >CanB2^RNAi#1 animals (n ≥ 5 preparations/genotype). (D) Quantification of average bursting duration for CPG recordings of the indicated genotypes at 38°C (n ≥ 5 preparations/genotype). (E) Detailed analysis (see Materials and methods) of HS induced behaviors of zyd/CanB2^RNAi flies. Cortex glial knockdown of CanB2 leads to seizure rescue in ~30% of zyd;NP2222>CanB2^RNAi flies, with the remaining ~70% displaying partial rescue. Cortex glial CanB2 knockdown with two copies of the RNAi (zyd;NP2222>CanB2^2xRNAi) recapitulates the full rescue seen with pan-glial knockdown. Inhibiting gal4 expression of the RNAi in neurons with gal80 (C155-gal80) does not alter the rescue observed with cortex glial knockdown, and astrocyte specific (alrm-gal4) CanB2 knockdown does not rescue zyd seizures (N = 3 groups of >15 flies/genotype, see Figure 2—figure supplement 1D for complete dataset). (F) Cortex glial conditional knockdown of CanB2 using gal4/gal80^ts. Rearing adult flies at the restrictive temperature (>30°C) for gal80^ts allows expression of CanB2^RNAi only at the adult stage. A significant reduction in seizures (p<0.0001) was seen after four days of rearing flies at the restrictive temperature for gal80^ts (31°C), with only ~25% of adults showing seizures. The reduction in seizures was enhanced when adults were incubated at 31°C for longer periods (N = 3 groups of >10 flies/genotype). Inset shows analysis after 4 days of incubation at 31°C (p=0.0001). (G) Pan-glial knockdown of the Drosophila calcineurin (CN) family (CanA1, CanA-14D/Pp2B-14D, CanA-14F, CanB and CanB2) indicate CanB2 knockdown completely rescues zyd seizures, CanA-14D and CanA-14F knockdowns partially reduce seizures (N = 4 groups of >10 flies/genotype). (H) Pan-glial knockdown of Pp2B-14D and CanA-14F partially rescues the zyd HS seizures phenotype (~25% rescue for Pp2B-14D, p=0.0032; and ~60% rescue for CanA14F, p<0.0001). Knocking down the two genes simultaneously rescues zyd seizures, with only ~10% of flies showing seizures (~90% rescue, p<0.0001, N = 3 groups of >10 flies/genotype). (I) Overexpressing a dominant-negative form on Pp2B-14D (CanA^H217Q) rescues ~50% of zyd seizures regardless of the driver used (repo: p<0.0001; NP2222: p=0.0006; GMR54H02- p=0.0004. N = 3 groups of >10 flies/genotype). (J) Larval Ca²⁺ imaging in cortex glia expressing myrGCaMP6s indicates the elevated basal Ca²⁺ fluorescence at 25°C observed in zyd mutants relative to wildtype cortex glia (p=0.0003) is not altered following CanB2 knockdown (zyd;;repo>CanB2^RNAi, p=0.6096. n ≥ 5 animals/genotype). (K) Microdomain Ca²⁺ oscillations observed in wildtype cortex glia expressing myrGCaMP6s are abolished in zyd cortex glia and are not restored following either CanB2 or CanA14F knockdown (n ≥ 5 animals/genotype). Error bars are SEM, **=P < 0.01, ***=P < 0.001, ****=P < 0.0001, Student’s t-test.

Figure 2—figure supplement 1. — (A) Behavioral analysis of HS-induced seizures. Pan-glial knockdown of the CN regulatory subunit, CanB2, with three partially overlapping hairpins (#1, #2 and #3, see Materials and methods) completely rescues the zyd seizure phenotype, while a single copy of CanB2 knockout allele (CanB2^KO/+) rescues ~60% of seizures (N = 4 groups of ≥15 flies/genotype). Inset shows analysis after 2 minutes of HS (p=0.0001). (B) Behavioral analysis of the recovery from vortex-induced seizures. Pan-glial knockdown of CanB2 and CanA-14F rescues zyd vortex-induced seizures (N = 3 groups of 20 flies/genotype). (C) Representative voltage traces of spontaneous CPG activity at larval 3^rd instar muscle 6 at 38°C in wildtype, zyd and zyd;;repo >CanB2^RNAi#1 animals (n ≥ 5 preparations/genotype). (D) Quantification of average bursting duration for CPG recordings of the indicated genotypes at 38°C (n ≥ 5 preparations/genotype). (E) Detailed analysis (see Materials and methods) of HS induced behaviors of zyd/CanB2^RNAi flies. Cortex glial knockdown of CanB2 leads to seizure rescue in ~30% of zyd;NP2222>CanB2^RNAi flies, with the remaining ~70% displaying partial rescue. Cortex glial CanB2 knockdown with two copies of the RNAi (zyd;NP2222>CanB2^2xRNAi) recapitulates the full rescue seen with pan-glial knockdown. Inhibiting gal4 expression of the RNAi in neurons with gal80 (C155-gal80) does not alter the rescue observed with cortex glial knockdown, and astrocyte specific (alrm-gal4) CanB2 knockdown does not rescue zyd seizures (N = 3 groups of >15 flies/genotype, see Figure 2—figure supplement 1D for complete dataset). (F) Cortex glial conditional knockdown of CanB2 using gal4/gal80^ts. Rearing adult flies at the restrictive temperature (>30°C) for gal80^ts allows expression of CanB2^RNAi only at the adult stage. A significant reduction in seizures (p<0.0001) was seen after four days of rearing flies at the restrictive temperature for gal80^ts (31°C), with only ~25% of adults showing seizures. The reduction in seizures was enhanced when adults were incubated at 31°C for longer periods (N = 3 groups of >10 flies/genotype). Inset shows analysis after 4 days of incubation at 31°C (p=0.0001). (G) Pan-glial knockdown of the Drosophila calcineurin (CN) family (CanA1, CanA-14D/Pp2B-14D, CanA-14F, CanB and CanB2) indicate CanB2 knockdown completely rescues zyd seizures, CanA-14D and CanA-14F knockdowns partially reduce seizures (N = 4 groups of >10 flies/genotype). (H) Pan-glial knockdown of Pp2B-14D and CanA-14F partially rescues the zyd HS seizures phenotype (~25% rescue for Pp2B-14D, p=0.0032; and ~60% rescue for CanA14F, p<0.0001). Knocking down the two genes simultaneously rescues zyd seizures, with only ~10% of flies showing seizures (~90% rescue, p<0.0001, N = 3 groups of >10 flies/genotype). (I) Overexpressing a dominant-negative form on Pp2B-14D (CanA^H217Q) rescues ~50% of zyd seizures regardless of the driver used (repo: p<0.0001; NP2222: p=0.0006; GMR54H02- p=0.0004. N = 3 groups of >10 flies/genotype). (J) Larval Ca²⁺ imaging in cortex glia expressing myrGCaMP6s indicates the elevated basal Ca²⁺ fluorescence at 25°C observed in zyd mutants relative to wildtype cortex glia (p=0.0003) is not altered following CanB2 knockdown (zyd;;repo>CanB2^RNAi, p=0.6096. n ≥ 5 animals/genotype). (K) Microdomain Ca²⁺ oscillations observed in wildtype cortex glia expressing myrGCaMP6s are abolished in zyd cortex glia and are not restored following either CanB2 or CanA14F knockdown (n ≥ 5 animals/genotype). Error bars are SEM, **=P < 0.01, ***=P < 0.001, ****=P < 0.0001, Student’s t-test.