Generation of GEFS+ and Control flies by targeted knock-in. A) Schematic diagram of a sodium channel alpha-subunit. Lysine residue altered in the human K1720T GEFS+ mutation is located in the second transmembrane segment in homology domain III (indicated by *). Comparison of the amino acid sequence in this region reveals a high degree of homology between species. Identical amino acids are shaded gray. Location of lysine residue altered in mutants is indicated by the *. B) Diagram of the wild-type para sodium channel locus and the targeting vectors. The GEFS+ targeting vector contained the K to T substitution and the Control targeting vector contained the control substitution K to K. Location of the substitution indicated by *. Homologous recombination events between incoming linear recombinogenic DNA and endogenous para locus are indicated by crossed lines.

GEFS+ flies exhibit a temperature-induced seizure phenotype. Seizures are defined as a brief period of leg twitches, followed by inability to maintain standing posture, with wing flapping, leg twitching, and sometimes abdomen curling. Individual 2-day old flies were put into vials that were immersed in a water bath (40°C, 2 min). The status of each fly (seizing or not seizing) was determined at 5 second intervals and the probability of seizing at each time point was calculated for the population of flies examined in each genotype. The probability of seizing after 2 minutes at 40°C is significantly different between the mutants (GEFS+/Y and GEFS+/GEFS+), heterozygotes (GEFS+/Control) and Control (Control/Y and Control/Control). (p<0.001, ANOVA Bonferroni post hoc test). Symbols/bars represent mean ± SEM from number of flies indicated (n) for all subsequent figures.

Sodium currents in GEFS+ and Control LNs. A) A LN in the dorsal lateral cluster in the antennal lobe, filled with biocytin during recording, in a Control and GEFS+ brain. Confocal images of the brains fixed and double stained with a fluorescently labeled secondary antibody to biocytin and Nc82 (anti-bruchtpilot antibody). Arrows indicate LN cell body (Control) or axon initial segment (GEFS+). The soma of the GEFS+ cell was lost during pipette removal. B) Depolarizing voltage-step elicited sodium currents at room temperature (23°C) and following heating of the recording solution to high temperature (35°C). I_NaP in Control LN decayed to baseline after the pipet potential returned to −75 mV but it remained activated in the GEFS+ LN at 35°C (arrow). Sodium currents could not be clamped in either genotype. C) I_NaT amplitudes are no different in GEFS+ and Control LNs at 23°C or 35°C (independent t-test, 23°C p=0.28, 35°C p=0.07) and the amplitude did not change with temperature in either genotypes (paired t-test, Control p=0.08, GEFS+ p=0.20). D) The voltage step required to elicit the first inward sodium current is significantly more hyperpolarized in GEFS+ than in Control at both 23°C and 35°C (independent t-test, ***p<0.001). The current activation threshold did not change significantly with temperature in either genotype (paired t-test, Control p=0.06, GEFS+ p=0.14). Symbols/bars represent mean ± SEM.

Temperature-induced hyperpolarizing shift in I_NaP deactivation threshold is significantly larger in GEFS+ LNs. A, B) Families of I_NaP associated with the variable test step during the three-step voltage protocol illustrated in a typical Control and GEFS+ LN at 23°C and 35°C. Only one trace during the pre-pulse is shown for clarity. The first test potential that results in decay of the current back to baseline is defined as the current deactivation threshold voltage and is indicated by the arrow in each set of currents. When the temperature is raised from 23 to 35°C, current deactivation voltage shifts from −45 to −55 mV in Control and −55 to −75 mV in GEFS+ LN. Ai, Bi) I–V curves representing I_NaP amplitude measured during last 10 ms of the test pulse for each test potential from the traces shown above. Dashed lines are linear fits used to calculate the conductance of the I_NaP. C) The deactivation voltage is more hyperpolarized at 35°C compared to 23°C in both Control and GEFS+ (paired t-test, **p<0.01, ***p<0.001). However, the deactivation voltage is significantly more hyperpolarized in GEFS+ than in Control at 35°C (independent t-test, *p<0.05). D) Mean I_NaP conductance reversibly increase at 35°C in both genotypes but was not different between GEFS+ and Control at 23°C (independent t-test, p=0.29) or 35°C (p=0.38). E) Average activation and deactivation threshold voltage of sodium currents in Control and GEFS+ at room temperature and elevated temperature. GEFS+ LNs have a wider voltage range over which sodium channels are conductive.

Alterations in evoked firing properties of GEFS+ LNs at elevated temperature. A, B) Representative trains of spikelets recorded from Control and GEFS+ LNs at 23 and 35°C evoked by the stimulus protocol illustrated. Ai, Bi) Spikelet frequency plotted as a function of injected current for LNs shown above. C) The incidence of post-stimulus depolarization is defined as the percentage of traces in which there was a post-stimulus depolarization that lasted for at least 50 msec in each LN. The incidence of GEFS+ LNs showing post-stimulus depolarizations increased significantly at 35°C compared to 23°C (paired t-test, *p<0.05). In contrast, the incidence of post-stimulus depolarization was low in Control LNs and did not increase significantly at elevated temperature (p=0.36). D) Raising the temperature from 23 to 35°C resulted in a significant increase in the maximal firing frequency in Control LNs (paired t-test, *p<0.05) and a significant decrease in GEFS+ LNs (*p<0.05).

Appearance of sustained depolarizations without spikelets in GEFS+ LNs at high temperature. A, B) Continuous recordings of spontaneous activity in Control and GEFS+ LNs as the temperature is raised and lowered as indicated by the overlay of the temperature probe recording. Ai-Biii are traces on expanded time scales from regions indicated. In both genotypes the regular burst firing frequency gradually decreases as the temperature increases. Prior to cessation of firing, activity in GEFS+ LNs at high temperature is characterized by the appearance of sustained depolarizations without spikelets that are not observed in Control LNs. Normal burst firing resumes in both genotypes when the bath is returned to room temperature. C) The frequency of sustained membrane depolarizations in GEFS+ LNs increased at high temperature (paired t-test, *p<0.05). These events were counted at 23°C (5 min prior to heating), at high temperature (30–35°C) and following return to room temperature (0–5 min after return to room temperature). D) The percentage of membrane depolarizations that were sustained increased at 35°C in GEFS+ (paired t-test, *p<0.05). E) Spikelet frequency increased in Control LNs (*p<0.05), but decreased in GEFS+ LNs (*P<0.05) at 35°C compared to 23°C. F) The spikelet threshold is more hyperpolarized in GEFS+ than in Control LNs at both 23 and 35°C (independent t-test, p<0.05). The spikelet threshold did not change as a function of temperature in either GEFS+ (paired t-test, p=0.95) or Control LNs (p=0.23).

PTX increases GEFS+ sensitivity to heat-induced seizures. A) Flies were fed sucrose with indicated concentration of PTX and tested for seizure susceptibility within 30 min. The probability of seizing in GEFS+ flies after 2 minutes at 37°C increases with PTX concentration (p<0.001, one-way ANOVA). Control flies fed the maximal dose of PTX (0.4 mM) did not show seizure behavior at 37°C. B) In GEFS+ flies, the time spent seizing during the 2 minute heat exposure increases with PTX concentration (p<0.001, one-way ANOVA).