Seizure termination by acidosis depends on ASIC1a

Abstract

Most seizures stop spontaneously; however, the molecular mechanisms that terminate seizures remain unknown. Observations that seizures reduced brain pH and that acidosis inhibited seizures indicate that acidosis halts epileptic activity. Because acid-sensing ion channel 1a (ASIC1a) is exquisitely sensitive to extracellular pH and regulates neuron excitability, we hypothesized that acidosis might activate ASIC1a, which would terminate seizures. Disrupting mouse ASIC1a increased the severity of chemoconvulsant-induced seizures, whereas overexpressing ASIC1a had the opposite effect. ASIC1a did not affect seizure threshold or onset, but shortened seizure duration and prevented seizure progression. CO2 inhalation, long known to lower brain pH and inhibit seizures, required ASIC1a to interrupt tonic-clonic seizures. Acidosis activated inhibitory interneurons through ASIC1a, suggesting that ASIC1a might limit seizures by increasing inhibitory tone. Our results identify ASIC1a as an important element in seizure termination when brain pH falls and suggest both a molecular mechanism for how the brain stops seizures and new therapeutic strategies.

Figure 1

ASIC1a reduces seizure severity. (a) Seizure response over time in ASIC1a^+/+ and ASIC1a^−/−mice following kainate injection (20 mg/kg IP) (+/+, n = 6; −/−, n = 7; age 13–22 weeks). For each ten–minute interval, the highest level of seizure activity was scored using the Racine seizure scale (see methods). There was a significant main effect of time (F(1, 6) = 27.3, p <0.0001) and a significant time × genotype interaction (F(1, 6) = 2.39, p = 0.039) (ANOVA with repeated measures), suggesting that as time passes seizures were likely to become more severe in the ASIC1a^−/− mice. (b) Maximum Racine score during the 60 minute trial (Mann–Whitney U test; *p = 0.004). (c) Incidence of generalized tonic–clonic seizures (GTCS) in +/+ and −/− mice following PTZ injection (50 mg/kg IP) (+/+, n = 12; −/−, n = 8, age 18–22 weeks; Fisher’s exact test; *p = 0.004). (d) Incidence of continuous, tonic–clonic seizures in wild–type mice injected with 5 mL ACSF (ICV) or PcTx1 (9 ng/mL). Treatment with PcTx1 significantly increased the incidence of sustained seizures (ACSF, n = 10; PcTx1, n = 12, age 9–11 weeks; Fisher’s exact test *p = 0.005). (e) Seizure response over time in ASIC1a^+/+ (WT) and ASIC1a–overexpressing–transgenic (Tg+) mice following 30 mg/kg IP kainate (WT, n = 11; Tg+, n = 9, age 31–36 weeks). With time, seizures were less severe in mice overexpressing ASIC1a (ANOVA with repeated measures; F(1, 3.35) = 3.295, p = 0.022). (f) Maximum Racine score during the 60 minute trial (Mann–Whitney U test; *p = 0.041). (g) Incidence of generalized tonic–clonic seizures (GTCS) in WT and Tg+ mice following PTZ injection (65 mg/kg IP) (WT, n = 13; Tg+, n = 13, age 25–41 weeks; Fisher’s exact test; *p = 0.037). (h) Occurrence of maximal electroconvulsive seizures (MES) in ASIC1a^+/+ and ASIC1a^−/− mice in response to electrical stimulation. ASIC1a disruption did not significantly alter MES threshold (+/+, n = 50, CD50 95% confidence interval = 6.24–7.95; −/−, n = 46, CD50 95% confidence interval = 6.71–7.76). The sample sizes and current intensities were: 5 mA, ASIC1a^+/+ (n = 11) vs. ASIC1a^−/− (n = 7); 6 mA, ASIC1a^+/+ (n = 22) vs. ASIC1a^−/− (n = 21); 7 mA, ASIC1a^+/+ (n = 10) vs. ASIC1a^−/− (n = 10); 8 mA, ASIC1a^+/+ (n = 4) vs. ASIC1a^−/− (n = 4); 10 mA, ASIC1a^+/+ (n = 3) vs. ASIC1a^−/− (n = 4), age 8–22 weeks.

Figure 2

ASIC1a disruption increases seizure duration and progression. (a) Representative EEG tracings and quantification of time from PTZ injection (50 mg/kg IP) until first seizure spikes in ASIC1a^+/+ and ASIC1a^−/− mice (+/+, n = 6; −/−, n = 7, age 18–22 weeks; unpaired t–test: t(11) = 0.544, p = 0.597). (b) Representative EEG tracings and total number of seizure spikes per five minute interval in surviving mice (+/+, n = 6; ⁻/⁻, n = 7). Spike number varied significantly with time (Mixed model analysis; F(1, 5) = 23.5, p < 0.001), and there was a significant time x genotype interaction (F(1,6) = 32.9, p < 0.001), suggesting that ASIC1a^−/−mice had prolonged seizure activity as time elapsed. (c) Incidence of generalized tonic–clonic seizures (GTCS) following PTZ injection (50 mg/kg IP) (+/+, n = 6; −/−, n = 7; Fisher’s exact test; p = 0.078). (d) Survival over time (+/+, n = 6; −/−, n = 7; Mantel–Cox Log Rank, p = 0.025).

Figure 3

ASIC1a disruption reduces post–ictal depression. (a) Representative EEG tracings from an ASIC1a^+/+ and ASIC1a^−/− mouse approximately 5 min. following PTZ injection (50 mg/kg IP). Five, 5–second intervals are denoted by blue vertical bars. These are shown below (b) in rows using an expanded time scale. EEG tracings prior to PTZ injection (base), initial spike–wave activity (1), and seizures associated with forelimb clonus (2) were similar in both genotypes. However, immediately following seizures (3), mice entered a period of post–ictal depression (red boxes). Post–ictal depression quickly reverted to seizure activity in ASIC1a^−/− mice (4, 5). (c) Quantification of post–ictal depression as scored using the post–ictal depression scale (see methods) (+/+, n = 6; −/−, n = 7, age 18–22 weeks; Mann–Whitney U test, *p = 0.011).

Figure 4

ASIC1a mediates the antiepileptic effects of acid in hippocampal slices. (a) Representative CA3 extracellular recording from ASIC1a^+/+ and ASIC1a^−/− slices prior to ictal activity caused by hypomagnesemia (nominal Mg²⁺). (b) Representative CA3 recordings from ASIC1a^+/+ and ASIC1a^−/− slices demonstrating ictal activity before, during, and after pH 6.8 application. A seizure–like discharge during pH 6.8 is expanded in the inset. (c) Latency to seizure onset was recorded in CA3 in response to 0 Mg²⁺ (+/+, n = 7; −/− n = 5; t(10) = 0.366, p = 0.722). (d) Total number of seizure spikes over 4.5 min. before induction of ictal activity (control), and then in the presence of nominal Mg²⁺ at pH 7.35 (baseline), pH 6.8, and after return to pH 7.35 (recovery) (+/+, n = 9; −/− n = 8). In ASIC1a^+/+ mice an ANOVA revealed a significant effect of pH (F(2) = 7.124, p = 0.006), however this was not the case in the ASIC1a^−/− mice (F(2) = 0.104, p = 0.902). A within–subjects comparison revealed a significant pH x genotype interaction (F(2)= 3.78, p = 0.034). At pH 6.8, the spike number was significantly greater in the ASIC1a^−/− mice (unpaired t–test: t(15) = −2.88, *p = 0.006), whereas at baseline and during recovery, the ASIC1a^+/+ and ASIC1a^−/− mice did not significantly differ (unpaired t–test, p = 0.238 and 0.581 respectively).

Figure 5

Inhibitory interneurons have prominent ASIC1a currents. (a) Representative traces of acid–evoked current in interneurons from the hippocampus of ASIC1a^+/+ and ASIC1a^−/− mice. Application of pH 7.0 evoked an inward current in +/+, but not −/− neurons. (b) From ASIC1a^+/+ mice, inhibitory neurons (I) had larger acid–evoked current density than excitatory pyramidal neurons (E) at pH 6.8–7.2 (inhibitory, n = 30; excitatory, n = 18; F(1, 42) = 10.5, p < 0.01). (c) Current–clamp recording demonstrating acid–evoked firing in an ASIC1a^+/+ inhibitory neuron. ASIC1a^−/− neurons did not respond to even greater reductions in pH. pH 6.8 stimulated firing in 78% (n = 9) and pH 7 stimulated firing in 80% (n = 5) of ASIC1a^+/+ inhibitory neurons.

Figure 6

ASIC1a mediates the seizure–terminating effects of 10% CO₂. (a) Kaplan–Meier survival analysis of ASIC1a^+/+ and ASIC1a^−/− mice in response to PTZ injection (90 mg/kg IP) while breathing compressed air (+/+, n = 7; −/−, n = 7, age 13–16 weeks). Both +/+ and −/− mice had the same survival rate (Mantel–Cox Log Rank, p = 0.582). (b) In a parallel experiment, 10% CO₂ was administered at the onset of generalized tonic–clonic seizures. The onset latency and time of CO₂ administration was similar between genotypes (inset) (unpaired t–test, t(14)= 0.663, p = 0.518). The chamber was perfused with CO₂ until minute 15, and then switched to compressed air for the duration of the trial (+/+, n = 8; −/−, n = 8, age 13–16 weeks). In CO₂, the likelihood of survival was significantly greater in the ASIC1a^+/+ mice (Mantel–Cox Log Rank, p = 0.002). (c) Representative pH tracings from a +/+ and −/− mouse brain before PTZ injection, during seizure, and during seizure and 10% CO₂ inhalation. (d) ASIC1a expression did not significantly alter brain pH prior to injection (p = 0.784), during seizures (p = 0.627), or during CO₂ inhalation (p = 0.528) (pre–injection: +/+, n = 5; −/−, n = 5, age 13–15 weeks; t(4) = 0.283; seizure: +/+, n = 5; −/−, n = 5; t(4) = 0.505; CO₂: +/+, n = 4; −/−, n = 4; t(3) = 0.670).