Article Figures & Data
Figures
- Figure 1.
CRISPR strategy and validation. A, Scheme of the two-step CRIPSR-Cas9-mediated genome editing for targeting the mutations to the Drosophila para gene. B, Mutant Drosophila lines were confirmed by Sanger sequencing. The wild-type codon is CGA. Step 2 repair plasmids carried the silent mutation CGT for R-R controls, TGT for R-C mutants, and CAT for R-H mutants.
- Figure 2.
R-C and R-H mutants display seizure-like behavior and reduced life span. A, Average seizure probability at any point in a 2 min trial for each independent line (3 R-R/FM7, 3 R-C/FM7, 4 R-H/FM7). B, Average seizure probability for each genotype shown at 40°C over time. At both 22°C and 40°C, R-C and R-H mutants have a significantly greater average seizure probability compared with R-R controls. Comparisons between R-C and R-H flies or between lines of one genotype are not significant: *p < 0.05; **p < 0.01; two-way ANOVA, Tukey's post hoc test. n > 12 trials/group. Bi, Average temperature inside vial over time of vial in 40°C bath. C, Kaplan–Meier survival curve estimates of R-R, R-C, and R-H flies over time. Median survival (50%) line is shown. Median percentage of survival analyzed using the Mantel–Cox log-rank test. Life span is significantly longer in R-R flies compared with R-C and R-H flies. *p < 0.05. n = 30 flies/genotype.
- Figure 3.
R-C and R-H mutants display sustained depolarizations during evoked firing, even after stimulus has ended. A–Ci, Representative trains of action potentials evoked by illustrated stimulus protocol at 22°C (A,B,C) and 30°C (Ai,Bi,Ci). Arrow indicates the appearance of poststimulus depolarization. D, E, Percentage of cells in each genotype with sustained depolarizations during stimulus (D) and poststimulus depolarizations (E) at 22°C, at 30°C, and after cooling back to 22°C. R-C and R-H mutants have increased the incidence of SDs and poststimulus SDs at 30°C. *p < 0.05, **p < 0.01, χ2 test. Any differences before or after heating are not significant. F, G, Average spike frequency shown as a function of current input at ambient temperature (F) and elevated temperature (G). At 22°C, R-Cs have generally increased firing frequency. At 30°C, R-Hs have generally decreased firing frequency compared with R-Cs. n = 7 groups/genotype. *p < 0.05; **p < 0.01, two-way ANOVA, with Tukey’s post hoc test.
- Figure 4.
R-C and R-H mutants display spontaneous sustained depolarizations and reduced inhibitory firing frequency at elevated temperatures. A, B, C, Representative continuous recording of neuron subjected to heating protocol. Chamber temperature over heating protocol shown on A. Ai–Aiii, Bi–Biii, Ci–Ciii, The 1.1 s interval within the labeled part of the temperature curve at 22°C preheating (Ai, Bi, Ci), 27°C mid-heating (Aii, Bii, Cii), and 22°C postcooling (Aiii, Biii, Ciii). D–F, Average frequency and duration of sustained depolarizations (D, E) and firing frequency of action potentials (F) during baseline (22°C), heating (26–29°C), and cooling (22°C). n = 8–9 cells/genotype. *p < 0.05, one-way ANOVA, with Tukey’s post hoc test.
- Figure 5.
R-H neurons exhibit hyperpolarized sodium current activation threshold. A–C, Voltage step-elicited currents at 22°C and 30°C. The first voltage step to elicit current shown. Cells exhibit both an initial transient and then a persistent current. D, Average peak INaT amplitude. E, Average activation threshold at 22°C, 30°C, and following cooling (second threshold at 22°C). R-H mutants have an average activation threshold hyperpolarized to R-C and R-R, but no effect by temperature. *p < 0.05, two-way ANOVA, with Tukey’s post hoc test.
- Figure 6.
R-C and R-H neurons exhibit hyperpolarized sodium current deactivation threshold. A–Ci, Voltage-step sodium channel currents for deactivation at room temperature (22°C; A, B, C) and high temperature (30°C; Ai, Bi, Ci), with the current steps in bold. Cells subjected to a multistep protocol, as shown. Selected sweeps shown to illustrate differences in deactivation properties. D, Average deactivation threshold at 22°C, 30°C, and after cooling back to 22°C. R-C and R-H mutants had hyperpolarized deactivation threshold compared with R-Rs. *p < 0.05, two-way ANOVA, with Tukey’s post hoc test.
Tables
R-R/FM7 R-C/FM7 R-H/FM7 Input resistance, GΩ 0.64 ± 0.02 0.59 ± 0.10 0.56 ± 0.05 Cell capacitance, pF 20.32 ± 2.13 17.72 ± 2.01 21.13 ± 4.31 Action potential threshold, mV 22˚C: −52.45 ± 1.99 22˚C: −50.05 ± 3.81 22˚C: −50.68 ± 3.68 30˚C: −42.71 ± 3.63 30˚C: −44.50 ± 5.26 30˚C: −46.40 ± 2.30 22˚C: −50.25 ± 1.94 22˚C: −51.45 ± 4.88 22˚C: −50.13 ± 2.01 Action potential amplitude, mV 22˚C: 38.59 ± 2.70 22˚C: 33.65 ± 3.02 22˚C: 42.99 ± 2.91 30˚C: 27.41 ± 3.58 30˚C: 23.32 ± 3.32 30˚C: 27.08 ± 3.87 22˚C: 33.78 ± 3.40 22˚C: 35.67 ± 3.69 22˚C: 38.76 ± 4.19 Half-width, ms 22˚C: 3.0 ± 0.1 22˚C: 3.2 ± 0.2 22˚C: 2.8 ± 0.1 30˚C: 2.2 ± 0.1 30˚C: 2.7 ± 0.3 30˚C: 2.6 ± 0.2 22˚C: 2.6 ± 0.1 22˚C: 3.3 ± 0.3 22˚C: 3.1 ± 0.2 Max slope 22˚C: 24.78 ± 2.82 22˚C: 22.45 ± 4.17 22˚C: 29.32 ± 4.07 30˚C: 27.97 ± 4.67 30˚C: 23.96 ± 4.88 30˚C: 26.27 ± 3.75 22˚C: 22.18 ± 3.39 22˚C: 23.72 ± 5.91 22˚C: 25.31 ± 3.66 Adaptation ratio (ISIfirst/ISIfinal at half-maximal firing) 22˚C: 0.49 ± 0.07 22˚C: 0.44 ± 0.09 22˚C: 0.56 ± 0.09 30˚C: 0.27 ± 0.05 30˚C: 0.33 ± 0.08 30˚C: 0.33 ± 0.08 22˚C: 0.45 ± 0.06 22˚C: 0.52 ± 0.09 22˚C: 0.64 ± 0.06 Peak instantaneous frequency, Hz 22˚C: 104.7 ± 12.4 22˚C: 111.5 ± 8.3 22˚C: 97.6 ± 9.2 30˚C: 205.3 ± 22.8 30˚C: 237.8 ± 42.4 30˚C: 206.8 ± 35.6 22˚C: 81.7 ± 10.5 22˚C: 90.7 ± 10.2 22˚C: 83.6 ± 10.2 Values are the mean ± SE at 22°C (during the 5 min preheating), at elevated temperature 30°C, and again at 22°C (during the 5 min postcooling). ISI, Interstimulus interval.
R-R/FM7 R-C/FM7 R-H/FM7 Burst frequency, Hz 22˚C: 0.70 ± 0.24 22˚C: 0.90 ± 0.26 22˚C: 0.70 ± 0.24 27˚C: 0.63 ± 0.26 27˚C: 0.57 ± 0.17 27˚C: 0.63 ± 0.26 22˚C: 0.57 ± 0.20 22˚C: 0.37 ± 0.16 22˚C: 0.57 ± 0.20 Action potential threshold, mV 22˚C: −37.77 ± 1.48 22˚C: −37.48 ± 1.83 22˚C: −38.00 ± 2.45 27˚C: −36.74 ± 1.36 27˚C: −37.06 ± 1.77 27˚C: −33.81 ± 3.10 22˚C: −36.79 ± 1.66 22˚C: −35.95 ± 2.08 22˚C: −36.17 ± 2.40 Action potential amplitude, mV 22˚C: 35.34 ± 2.35 22˚C: 32.27 ± 3.83 22˚C: 28.18 ± 5.16 27˚C: 28.29 ± 2.34 27˚C: 23.16 ± 3.19 27˚C: 23.33 ± 3.87 22˚C: 34.27 ± 3.81 22˚C: 28.52 ± 2.57 22˚C: 31.33 ± 4.16 Action potentials per burst 22˚C: 5.27 ± 0.39 22˚C: 5.24 ± 0.56 22˚C: 4.79 ± 0.35 27˚C: 5.46 ± 0.38 27˚C: 5.05 ± 0.56 27˚C: 4.34 ± 0.87 22˚C: 6.17 ± 0.50 22˚C: 5.31 ± 0.39 22˚C: 5.23 ± 0.49 Resting membrane potential, mV 22˚C: −57.1 ± 2.2 22˚C: −54.2 ± 2.0 22˚C: −54.0 ± 2.6 27˚C: −55.9 ± 1.8 27˚C: −52.1 ± 1.3 27˚C: −47.8 ± 3.2* 30˚C: −57.4 ± 2.4 30˚C: −58.3 ± 4.0 30˚C: −47.9 ± 1.9* 22˚C: −54.9 ± 3.2 22˚C: −49.5 ± 2.1 22˚C: −49.8 ± 1.8 Values are the mean ± SE at 22°C (during the 5 min preheating), “27°C” (during the minute of heating from 26°C to 29°C), and again at 22°C (during the 5 min postcooling).
↵* R-H mutants have depolarized RMP at elevated temperatures compared with R-R controls; p < 0.05, two-way ANOVA.
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