Mutations of the Calcium Channel Gene cacophony Suppress Seizures in Drosophila

Abstract

Bang sensitive (BS) Drosophila mutants display characteristic seizure-like phenotypes resembling, in some aspects, those of human seizure disorders such as epilepsy. The BS mutant parabss1, caused by a gain-of-function mutation of the voltage-gated Na+ channel gene, is extremely seizure-sensitive with phenotypes that have proven difficult to ameliorate by anti-epileptic drug feeding or by seizure-suppressor mutation. It has been presented as a model for intractable human epilepsy. Here we show that cacophony (cacTS2), a mutation of the Drosophila presynaptic Ca++ channel α1 subunit gene, is a particularly potent seizure-suppressor mutation, reverting seizure-like phenotypes for parabss1 and other BS mutants. Seizure-like phenotypes for parabss1 may be suppressed by as much as 90% in double mutant combinations with cacTS2. Unexpectedly, we find that parabss1 also reciprocally suppresses cacTS2 seizure-like phenotypes. The cacTS2 mutant displays these seizure-like behaviors and spontaneous high-frequency action potential firing transiently after exposure to high temperature. We find that this seizure-like behavior in cacTS2 is ameliorated by 85% in double mutant combinations with parabss1.

Fig 1. Drosophila cac^TS2 electrophysiology.

A. Electrical recording from a cac^TS2 DLM fiber following delivery of 55 V HFS stimulation (0.5 msec stimuli at 200 Hz for 300 msec) at room temperature. The stimulation evokes seizure-like electrical activity, indicating that the voltage is at or above seizure threshold. B. Recording from a cac^TS2 DLM fiber following delivery of a 30 V HFS stimulus showing that this stimulation voltage fails to evoke seizure-like activity. This stimulation voltage, near the wild-range, is below seizure threshold for cac^TS2. C. Recording from a wild type Canton Special (CS) DLM fiber following delivery of a 25 V HFS stimulus that is effective in evoking seizure-like activity. D. Recording from a cac^TS2 DLM fiber following a temperature shift from permissive room temperature to restrictive 38°C temperature. Spontaneous seizure-like electrical activity is observed in the DLM fiber, indicating that the mutant is seizure-sensitive at restrictive temperatures. Recording shows a representative example of three spontaneous seizure-like discharges. Horizontal calibration is 1.0 sec for A, B, and C; 4 sec for D and 1.5 sec for D (enlarged). Vertical calibration is 20 mV.

Fig 2. Suppression of bang sensitive behavioral phenotypes by cac^TS2.

A. Behavioral paralysis in sda hemizygotes (white bar) is suppressed in hemizygous double mutants cac^TS2/Y;;sda at room temperature (RT) by about a factor of 10 (black bar; n = 147). Following a brief heat shock (HS = 3 min at 30°C), complete suppression of behavioral paralysis is observed in hemizygous double mutants cac^TS2/Y;;sda (striped bar; n = 93). B. Behavioral paralysis in hemizygous eas cac^TS2/Y double mutants at RT (black bar, n = 147) shows a small reduction compared to hemizygous eas/Y (white bar). Following a brief heat shock (HS = 3 min at 30°C), suppression is by about a factor of 2 (striped bar; n = 93). C. Behavioral paralysis in para^bss1 hemizygotes (white bar) is suppressed in hemizygous para^bss1 cac^TS2 double mutants at RT by about a factor of 3 (black bar; 64% suppression; n = 658). Following a brief HS, suppression is increased in the para^bss1 cac^TS2 double mutants (striped bar; 87% suppression; n = 650). D. Following a brief HS, cac^TS2 completely suppresses behavioral paralysis in para^bss1/+ heterozygous females (n = 60). *P < 0.01, **P = 0.001, ***P < 0.0001, chi-square test.

Fig 3. Electrophysiology of cac^TS2 suppression.

A. Electrical recording from a para^bss1 DLM fiber showing seizure-like activity evoked by a 6 V HFS stimulation, showing that the single BS mutant has a low seizure threshold. B. Electrical recording from a para^bss1 cac^TS2 DLM fiber showing that stimulation at 25 V HFS is below threshold and fails to evoke a seizure event in the double mutant indicating suppression by cac^TS2. C. Electrical recording from a para^bss1 cac^TS2 DLM fiber showing that a high voltage stimulation at 53 V HFS is above threshold and evokes a seizure-like event in the double mutant. For the traces depicted in A-C, the HFS stimulus is delivered immediately after HS (3 min at 30°C). D. At room temperature (RT; steady state), seizure threshold for the para^bss1 cac^TS2/Y double mutant (gray bar) is similar to the para^bss1/Y single mutant (white bar). Immediately following a HS, seizure threshold for para^bss1 cac^TS2/Y is transiently high indicating suppression (black bar; n = 7). E. Average seizure threshold is increased by about a factor of 4 in cac^TS2/Y;;sda double mutants following a brief HS (striped bar; 20.5 ± 2.42 V HFS; n = 7) compared to hemizygous sda single mutant (white and gray bars). F. Average seizure threshold is increased by about a factor of 3 in hemizygous double mutant eas cac^TS2/Y following a brief HS (striped bar; 8.7 ± 1.1 V HFS; n = 11) compared to the eas single mutant (white and gray bars). Quantitative data are represented as mean ± s.e.m. *P < 0.01, **P = 0.001, ***P < 0.0001, (D-F) chi-square test. Horizontal calibration: 800 msec for A-C; Vertical calibration: 20 mV for A-C.

Fig 4. Suppression of para^bss1 and eas behavioral phenotypes by cacRNAi at room temperature.

A. Behavioral paralysis in para^bss1 hemizygotes (white bar) is suppressed by cacRNAi when expressed by a pan-neuronal driver (black bar; genotype: para^bss1 elav^c155-GAL4/Y;;UAS-cacRNAi). B. Behavioral paralysis in eas hemizygotes (white bar) is suppressed by cacRNAi when expressed with a pan-neuronal driver (black bar; genotype: eas elav^c155-GAL4/Y;;UAS-cacRNAi). ***P < 0.0001, chi-square test (n = 212 and 74 for panels A and B, respectively).

Fig 5. Suppression of cac^TS2 temperature-sensitive seizure-like activity by para^bss1.

A. Spontaneous seizure-like activity observed in a cac^TS2 single mutant when the temperature is increased from room temperature to 38°C, indicating that the mutant is seizure-sensitive at restrictive temperature. Recording shows a representative example of 13 spontaneous seizure-like discharges during HS. Enlargement (lower trace) shows one of the spontaneous discharges at a higher sweep speed. B. Spontaneous seizure-like activity is decreased in double mutant homozygous para^bss1 cac^TS2 at restrictive temperature. C. Number of spontaneous seizure-like activity evoked due to restrictive temperature and measured by electrophysiology. Compared to cac^TS2, in homozygous para^bss1 cac^TS2 double mutant flies number of TS seizure-like activity is reduced by about a factor of 5 (white bar: 10 ± 1.2 events/3 min HS; n = 10; compared to gray bar: 2.5 ± 0.42 events/3 min HS; n = 20). D. Behavioral seizure-like activity is decreased in homozygous double mutant para^bss1 cac^TS2 flies at restrictive temperature. Compared to cac^TS2 single mutants (white bar), para^bss1 cac^TS2 double mutants (gray bar) exhibit 5 fold decrease in TS seizure-like behaviors. Quantitative data are represented as mean ± s.e.m. ***P < 0.0001, based on unpaired student-t test (C) and chi-square test (D). Horizontal calibration: A. 15 sec, A (enlarged) 1 sec, B. 15 sec, B (enlarged) 1.5 sec. Vertical calibration: 10 mV.