Inhibition of TRPC3 channels suppresses seizure susceptibility in the genetically-epilepsy prone rats

Abstract

Transient receptor potential canonical 3 (TRPC3) channels are important in regulating Ca²⁺ homeostasis and have been implicated in the pathophysiology of chemically induced seizures. Inherited seizure susceptibility in genetically epilepsy-prone rats (GEPR-3s) has been linked to increased voltage-gated Ca²⁺ channel currents in the inferior colliculus neurons, which can affect intraneuronal Ca²⁺ homeostasis. However, whether TRPC3 channels also contribute to inherited seizure susceptibility in GEPR-3s is unclear. This study investigated the effects of JW-65, a potent and selective inhibitor of TRPC3 channels, on acoustically evoked seizure susceptibility in adult male and female GEPR-3s. These seizures consisted of wild running seizures (WRSs) that evolved into generalized tonic-clonic seizures (GTCSs). The results showed that acute administration of low doses of JW-65 significantly decreased by 55-89% the occurrence of WRSs and GTCSs and the seizure severity in both male and female GEPR-3s. This antiseizure effect was accompanied by increased seizure latency and decreased seizure duration. Additionally, female GEPR-3s were more responsive to JW-65's antiseizure effects than males. Moreover, JW-65 treatment for five consecutive days completely suppressed acoustically evoked seizures in male and female GEPR-3s. These findings suggest that inhibiting TRPC3 channels could be a promising antiseizure strategy targeting Ca²⁺ signaling mechanisms in inherited generalized tonic-clonic epilepsy.

Keywords: Brainstem seizures; Calcium signaling; Generalized tonic-clonic seizures; Inherited epilepsy; TRPC3 channels.

Figure 1.

Experimental design. (A) A schematic for evaluating the antiseizure effects of acute JW-65 treatment in male and female GEPR-3s. Prior to administering JW-65 or the vehicle, GEPR-3s were subjected to acoustically evoked seizures. GEPR-3s that experienced seizures were used in pharmacological studies and tested at 0.5, 1, 2, 4, and 24 h post-treatment with either JW-65 (5, 10, 20, and 40 mg/kg, i.p) or the vehicle. (B) A schematic approach for five consecutive daily injections of 10 mg/kg JW-65 in males and female GEPR-3s. Before starting the JW-65 injection regimen, male and female GEPR-3s received intraperitoneal injections of the vehicle and were tested for acoustically evoked seizures. GEPR-3s that experienced seizures were subjected to five consecutive daily injections of JW-65. On the fifth day, GEPR-3s were tested for seizures at 0.5, 1, 2, 4, and 24 h post-treatment.

Figure 2 –

Effects of acute JW-65 treatment on the incidence of acoustically evoked WRSs. The putative seizure-suppressing effect of different doses of JW-65, a potent TRPC3 activator, was evaluated at different posttreatment seizure testing time points (0.5, 1, 2, 4, and 24 h) in adult males (n = 9) and females (n = 9) GEPR-3s. The results showed no statistical difference in the incidence of WRSs following the lowest JW-65 treatment, 5 mg/kg (i.p.), in both female (A) and male (B) GEPR-3s. At a higher dose of 10 mg/kg (i.p.), JW-65 significantly decreased the incidence of WRSs in female GEPR-3s at the 4^th h posttreatment time points (C) and in male GEPR-3s at the 2^nd and 4^th h posttreatment time points (D). The dose of 20 mg/kg (i.p.) significantly reduced the incidence of WRSs in female GEPR-3s at the 1^st and 2^nd h posttreatment time points (E) and in males 2^nd h posttreatment seizure testing time points (F). Finally, the highest dose of 40 mg/kg significantly decreased the incidence of WRSs in female GEPR-3s 1^st, 2^nd, and 4^th h posttreatment time points (G) and in male GEPR-3s 2^nd and at the 4^th h posttreatment time points (H). The data were presented as mean percentages (%)and analyzed using Fisher's exact test. The open bar graphs represented the vehicle treatments, while the filled bar graphs represented JW-65 treatments (gray—5 mg/kg, brown—10mg/kg, green—20 mg/kg, and blue—40 mg/kg). The statistical significance was determined at *p< 0.05 and **p< 0.01.

Figure 3 -

Effects of acute JW-65 treatment on the incidence of acoustically evoked GTCSs. The putative seizure-suppressing effects of different doses of JW-65, a potent TRPC3 activator, were evaluated at different posttreatment seizure testing time points (0.5, 1, 2, 4, and 24 h) in adult males (n = 9) and females (n = 9) GEPR-3s. No statistical difference was found in the occurrence of GTCSs following JW-65 at the lowest dose, 5 mg/kg (i.p.), in female (A) and male (B) GEPR-3s. However, treatment with a higher dose of JW-65, 10 mg/kg (i.p.), significantly decreased the incidence of GTCSs at the 2^nd and 4^th h posttreatment time points in females (C) and male (D) GEPR-3s. Furthermore, treatment with another higher dose of JW-65, 20 mg/kg (i.p.), significantly decreased the incidence of GTCSs in female GEPR-3s at the 1^st, 2^nd, and 4^th h posttreatment time points (E) and in male GEPR-3s 2^nd and 4^th h posttreatment time points (F). Finally, the highest dose of JW-65, 40 mg/kg (i.p.), significantly reduced the incidence of GTCS in female GEPR-3s at 0.5, 1^st, 2^nd, 4^th, and 24^th h posttreatment time points (G) and in male GEPR-3s at the 1^st, 2nd, 4th, and 24^th h posttreatment time points (H). The data were presented as mean percentages (%) and analyzed using Fisher's exact test. The open bar graphs represented the vehicle solution, while the filled bar graphs represented JW-65 treatments. The statistical significance was determined at *p< 0.05 and **p< 0.01.

Figure 4 -

Effects of acute JW-65 treatment on the latency of acoustically evoked seizures. The impact of different doses of JW-65 (5, 10, 20, and 40 mg/k, i.p.), a potent TRPC3 activator, on the seizure latency, was evaluated at different posttreatment seizure testing time points (0.5, 1, 2, 4, and 24 h) in adult males (n = 9) and females (n = 9) GEPR - 3s. The results showed that at the lowest JW-65 dose (5 mg/kg, i.p.), there was no statistical difference in seizure latency in female (A) and male (B) GEPR-3s compared to vehicle-treated GEPR-3s. However, a higher JW-65 dose of 10 mg/kg (i.p.) significantly increased the seizure latency in female GEPR-3s at the 2^nd, 4^th, and 24^th h posttreatment time points (C) and in males at the 1^st, 2^nd, and 4^th h posttreatment time points (D), compared to vehicle-treated GEPR-3s. Furthermore, JW-65 treatment with 20 mg/kg (i.p.) significantly increased the seizure latency in female GEPR-3s at the 1^st, 2^nd, and 4^th h posttreatment time points in female GEPR-3s (E) and male GEPR-3s at the 1^st, 2^nd, 4^th, and 24^th posttreatment time points (F). Finally, the highest dose of JW-65 (40 mg/kg, i.p.) significantly delayed the seizure onset in female GEPR-3s from 0.5 to 4^th h posttreatment time point (G) and in male GEPR-3s from the 1^st to 24^th posttreatment time point (H). Data were presented as mean ± SEM and analyzed using two-way repeated measures analysis of variance followed by Tukey's correction. The open bar graphs indicated the vehicle treatments, while the filled bar graphs represented JW-65 treatments. Statistical significance was indicated by *p< 0.05, **p< 0.01, ***p< 0.001. and ****p< 0.0001.

Figure 5 -

Effects of acute JW-65 treatment on duration of acoustically evoked seizures. The effects of different doses (5, 10, 20, and 40 mg/kg; i.p.) of JW-65, a potent TRPC3 activator, on seizure duration were evaluated at different posttreatment seizure testing time points (0.5, 1, 2, 4, and 24 h) in adult males (n = 9) and females (n = 9) GEPR - 3s. JW-65 treatment at the lowest dose, 5 mg/kg (i.p.), significantly decreased and increased the seizure duration in female GEPR-3s at the 1^st, 2^nd, and 24^th posttreatment time points and at the 4^th posttreatment time point, respectively (A). In male GEPR-3s, this dose significantly decreased the seizure duration between 0.5-2^nd h posttreatment time points (B). At a higher dose (10 mg/kg, i.p.), JW-65 significantly decreased the seizure duration in both female (C) and male (D) GEPR-3s at all testing time points (0.5-24^th h). Furthermore, at 20 mg/kg (i.p.), JW-65 decreased the seizure duration in female GEPR-3s (E) at all testing time points (0.5-24^th h) and in male GEPR-3s (F) at 1^st-24^th h posttreatment. At the highest dose of 40 mg/kg (i.p.), JW-65 significantly decreased seizure duration in female GEPR-3s (G) at 0.5-4^th hour posttreatment time points and in male GEPR-3s at 1^st-4^th h posttreatment (H). Data were presented as mean ± SEM and analyzed using two-way repeated measures analysis of variance followed by Tukey's correction. The open bar graphs represented the vehicle treatment, while the filled bar graphs represented JW-65 treatments. Statistical significance was indicated by *p< 0.05, **p< 0.01, ***p< 0.001. and ****p< 0.0001.

Figure 6 -

Effects of acute JW-65 treatment on the severity of acoustically evoked seizures. The effects of different doses (5, 10, 20, and 40 mk/kg; i.p.) of JW-65, a potent TRPC3 activator, were evaluated at various post-treatment seizure testing time points (0.5, 1, 2, 4, and 24 h) in adult males (n = 9) and females (n = 9) GEPR - 3s. The results showed no statistical difference in the seizure severity with the lowest JW-65 dose (5 mg/kg, i.p.) at all posttreatment time points in female (A) and male (B) GEPR-3s. However, JW-65 treatment with a higher dose, 10 mg/kg (i.p.), significantly decreased the seizure severity in female (C) and male (D) GEPR-3s from 2^nd-4^th posttreatment time points, compared to control-treated GEPR-3s. Furthermore, another higher dose (20 mg/kg, i.p.) of JW-65 significantly suppressed or decreased the seizure severity in female GEPR-3s from 1^st to 4^th h posttreatment time points, compared to control-treated GEPR-3s (E). JW-65 also decreased or suppressed the seizure severity in male GEPR-3s 1^st-2^nd h, compared to control-treated GEPR-3s (F). Finally, JW-65 treatment at the highest dose, 40 mg/kg (i.p.), significantly decreased or suppressed the seizure severity between 0.5-4^th h in female GEPR-3s, compared to control-treated GEPR-3s (G). In male GEPR-3s, JW-65 significantly decreased or suppressed seizure severity in male GEPR-3s from the 1^st-4^th posttreatment time points, compared to control-treated GEPR-3s (H). Data were presented as median ± median absolute deviation and analyzed using the Mann-Whitney test: The open bar graphs represent control-vehicle treatments, while the filled bar graphs represented JW-65 treatments. Statistical significance was indicated by *p< 0.05, **p< 0.01, and ***p< 0.001.

Figure 7 –

Effects of five consecutive daily JW-65 treatments on acoustically evoked seizures. The putative antiseizure effects of five consecutive days JW-65 treatment (10 mg/kg, p.o.), a potent TRPC3 activator, were evaluated at various posttreatment seizure testing time points (0.5, 1, 2, 4, and 24 h) in adult males (n = 7) and females (n = 7) GEPR - 3s. The results showed that JW-65 treatment prevented the occurrence of WRSs in both female (A) and male (B) GEPR-3s within the first 4 h posttreatment, compared to vehicle pre-treatment. JW-65 also prevented the occurrence of GTCSs in both female (C) and male (D) GEPR-3s within the first 4 h posttreatment, compared to vehicle pre-treatment. Additionally, JW-65 increased seizure latency in female (E) and male (F) GEPR-3s and completely suppressed the seizure duration in both female (G) and male (H) within the first 4 h posttreatment compared to vehicle pretreatment. Furthermore, JW-65 completely decreased the seizure severity in female (I) and male (H) GEPR-3s within the first 4 h posttreatment, compared to vehicle pretreatment. Data on the occurrence of WRSs and GTCSs were presented as a mean percentage (%), and Fisher's exact test was used for analysis. Data on seizure latency and duration were presented as mean ± SEM and analyzed using one-way RM ANOVA followed by Tukey's correction. Data on Seizure severity were presented as median±median absolute deviation and analyzed using the Wilcoxon signed-rank test. The open bar graphs represented the vehicle pretreatment (pre-JW-65), while the filled ones represented JW-65. Data on the incidences of WRSs and GTCSs were presented as mean percentages (%) and analyzed using Fisher's exact. Data on seizure latency and duration were presented as mean ± SEM and analyzed using one-way repeated measures analysis of variance followed by Tukey's correction. Data on seizure severity were presented as median±median absolute deviation and analyzed using the Wilcoxon signed-rank test. Statistical significance was indicated by *p< 0.05, **p< 0.01, ***p< 0.001. and ****p< 0.0001.