Article Figures & Data
Figures
- Figure 1.
Imp knockdown causes higher occurrence and longer duration of seizures with normal gross motor function. A, Seizure behavior reported as average time to recover after vortexing for eGFP RNAi control (VALIUM22-EGFP.shRNAI.1) and Imp RNAi (TRIP.HMC03794) expressed using neural stem cell driver inscuteable-GAL4 (insc-GAL4) or pan-neuronal driver neuronal synaptobrevin-GAL4 (nSyb-GAL4). Individual points represent each fly, whiskers represent 95% confidence intervals, and bar heights equal the mean. Kruskal–Wallis test determined the significance between all conditions. Relevant comparisons reported (p < 0.05, ***p < 0.01, ***p < 0.001,****p < 0.0001, ns = p > 0.05). N, number of total animals. B, Percentage of flies seizing reported in A observed at 5 s intervals across the entire 60 s trial. Ns are the same as in A. Seizing is defined by supine paralysis, spastic movement, and/or inability to walk with excessive tremoring. Behavioral assays were performed on Day 14. C, Maximum distance climbed (cm) in 30 s for eGFP RNAi control and Imp RNAi expressed in nSyb-GAL4. Individual points represent individual flies, whiskers represent 95% confidence intervals, and bar heights equal the mean. p-values are reported above each bar. Mann–Whitney U determined significance (p < 0.05, ***p < 0.01, ***p < 0.001,****p < 0.0001, ns = p > 0.05). N, number of total animals. All animals were female because their seizure phenotypes were more consistent (Extended Data Fig. 1-1).
- Figure 2.
Developmental loss of neuronal Imp is the primary source of seizure behavior. A, Average time to recover for flies exhibiting seizure behavior for neuronal (nSyb-GAL4) eGFP RNAi (control) and neuronal Imp RNAi. High GAL4 activity animals are raised at 29°C and low GAL4 activity animals are raised at 18°C. Results demonstrate that nSyb-GAL4 RNAi is temperature sensitive. Developmental knockdown animals are raised at 29°C and moved to 18°C within 1 d of adulthood. Adult knockdown animals developed at 18°C and shifted to 29°C within 4 h of adulthood. Behavioral assays were performed on Day 14. Individual points represent individual flies, whiskers represent 95% confidence intervals, and bar heights equal the mean. p-values are reported above each bar. Kruskal–Wallis determined significance between all conditions. N, number of total animals. Relevant comparisons reported (p < 0.05, ***p < 0.01, ***p < 0.001,****p < 0.0001, ns = p > 0.05). Developmental knockdown of Imp is sufficient to recapitulate the seizure phenotype. B, Fold change in Imp expression in third-instar larval brains developed at 18 and 29°C. Individual points represent a biological replicate group of 10 larval brains, whiskers represent 95% confidence intervals, and bar heights equal the mean. C, Percentage of flies presented in A, seizing observed at 5 s intervals across the entire 60 s trial.
- Figure 3.
Sdc interacts with Imp molecularly and functionally and is required for normal neuronal function. A, Average time to recover for flies exhibiting seizure behavior for neuronal (nSyb-GAL4) eGFP RNAi (control) and neuronal Sdc RNAi. Individual points represent each fly and whiskers represent 95% confidence intervals. N, number of total animals. Mann–Whitney U determined significance (p < 0.05, ***p < 0.01, ***p < 0.001,****p < 0.0001, ns = p > 0.05). B, Maximum distance climbed (cm) in 30 s for eGFP RNAi control and Sdc RNAi. Mann–Whitney U significance (*p < 0.05, ***p < 0.01, ***p < 0.001,****p < 0.0001, ns = p > 0.05). C, RNA immunoprecipitation of Imp-GFP protein from control (yw) and Imp-GFP animals using nanobody GFP agarose. Imp is immunoprecipitated specifically in Imp-GFP animals. D, PCR analysis of Imp-GFP RNA immunoprecipitation using primers specific to Sdc in control (yw) and Imp-GFP animals. Sdc product is amplified in Imp-GFP RIP but not in control RIP (yw). E–H, Confocal images of a single slice through the central brain of a third-instar larva stained with ElaV (neurons, cyan), Imp (magenta), and endogenous Sdc-GFP (green). Imp and Sdc proteins are both present in neurons. I, Average time to recover after vortexing for flies exhibiting seizure behavior for neuronal expression (nSyb-GAL4) for eGFP RNAi (control), Imp RNAi, Imp RNAi + UAS-luciferase (dilution effect control), and Imp RNAi + UAS-Sdc cDNA. Points represent individual flies, whiskers represent 95% confidence intervals, and bar heights equal the mean. p-values are reported above each bar. N, number of total animals. Kruskal–Wallis determined significance between all conditions, relevant comparisons reported (p < 0.05, ***p < 0.01, ***p < 0.001,****p < 0.0001, ns = p > 0.05). J, Percentage of flies seizing presented in (E) observed at 5 s intervals across the entire 60 s trial. Expression of Sdc cDNA in Imp knockdown animals rescues seizure deficits.
Tables
Experiment Corresponding figure Data structure Statistical test Power Seizure assay cell-specific Imp knockdown Figure 1A Abnormal, unequal variance Kruskal–Wallis 0.9999965 Forced-climbing assay Imp knockdown Figure 1C Abnormal, unequal variance Mann–Whitney U 0.9830928 Seizure assay staging Imp knockdown Figure 2A Abnormal, unequal variance Kruskal–Wallis 1.0000000 Seizure assay Sdc knockdown Figure 3A Abnormal, unequal variance Mann–Whitney U 0.9999187 Forced-climbing assay Sdc knockdown Figure 3B Abnormal, unequal variance Mann–Whitney U 0.8732388 Seizure assay Sdc rescue Figure 3I Abnormal, unequal variance Kruskal–Wallis 1.0000000
Movies
- Movie 1.
Normal baseline recovery from vortexing in neural stem cell knockdown in control animals (eGFP RNAi). [View online]
- Movie 2.
Slight impairment but relatively quick recovery from vortexing for animals when Imp is knocked down in neural stem cells. [View online]
- Movie 3.
Normal baseline recovery from vortexing in pan-neuronal knockdown in control animals (eGFP RNAi). [View online]
- Movie 4.
Impairment in recovery from vortexing for animals with Imp knocked down pan-neuronally, with visible tonic–clonic episodes. [View online]
- Movie 5.
Impairment in recovery from vortexing for animals with Sdc knocked down pan-neuronally, with visible tonic–clonic episodes. [View online]
- Movie 6.
Recovery from vortexing in animals with Sdc cDNA expression in Imp pan-neuronal knockdown background, with fewer seizing animals and relatively quick recovery. [View online]
Figure 1-1
Imp knockdown has a stronger effect on females. Seizure behavior reported as average time to recover after vortexing for eGFP RNAi (VALIUM22-EGFP.shRNAI.1) Control and Imp RNAi (TRIP.HMC03794) expressed using pan-neuronal driver neuronal synaptobrevin-GAL4 (nSyb-GAL4). Individual points represent each fly, whiskers represent 95% confidence intervals, and bar heights equal the mean. Kruskal-Wallis test determined significance between all conditions. Relevant comparisons reported (p<0.05, ***p<0.01, ***p<0.001,****p<0.0001, ns=p>0.05). N= number of total animals. Download Figure 1-1, TIF file.
In this issue
