ONO-2506 inhibits spike-wave discharges in a genetic animal model without affecting traditional convulsive tests via gliotransmission regulation

Abstract

Background and purpose: Anticonvulsants have been developed according to the traditional neurotransmission imbalance hypothesis. However, the anticonvulsive pharmacotherapy currently available remains unsatisfactory. To develop new antiepileptic drugs with novel antiepileptic mechanisms, we have tested the antiepileptic actions of ONO-2506, a glial modulating agent, and its effects on tripartite synaptic transmission.

Experimental approach: Dose-dependent effects of ONO-2506 on maximal-electroshock seizure (MES), pentylenetetrazol-induced seizure (PTZ) and epileptic discharge were determined in a genetic model of absence epilepsy in mice (Cacna1a(tm2Nobs/tm2Nobs) strain). Antiepileptic mechanisms of ONO-2506 were analysed by examining the interaction between ONO-2506 and transmission-modulating toxins (tetanus toxin, fluorocitrate, tetrodotoxin) on release of l-glutamate, d-serine, GABA and kynurenic acid in the medial-prefrontal cortex (mPFC) of freely moving rats using microdialysis and primary cultured rat astrocytes.

Key results: ONO-2506 inhibited spontaneous epileptic discharges in Cacna1a(tm2Nobs/tm2Nobs) mice without affecting MES or PTZ. Given systemically, ONO-2506 increased basal release of GABA and kynurenic acid in the mPFC through activation of both neuronal and glial exocytosis, but inhibited depolarization-induced releases of all transmitters. ONO-2506 increased basal glial release of kynurenic acid without affecting those of l-glutamate, d-serine or GABA. However, ONO-2506 inhibited AMPA-induced releases of l-glutamate, d-serine, GABA and kynurenic acid.

Conclusions and implications: ONO-2506 did not affect traditional convulsive tests but markedly inhibited epileptic phenomena in the genetic epilepsy mouse model. ONO-2506 enhanced release of inhibitory neuro- and gliotransmitters during the resting stage and inhibited tripartite transmission during the hyperactive stage. The results suggest that ONO-2506 is a novel potential glial-targeting antiepileptic drug.

Linked article: This article is commented on by Onat, pp. 1086-1087 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12050.

Figure 1

Typical chromatograms of kynurenic acid analysis using NCE-HPLC. The chromatograms were obtained from 10 μL of a standard solution containing 100 fmol·μL⁻¹ of kynurenic acid, prefrontal perfusate in MRS and HKMRS. The quantification limit for kynurenic acid is 0.2 fmol·10 μL⁻¹.

Figure 2

Effects of ONO-2506 on frequency and duration of spike and wave discharges, MES and PTZ in Cacna1a^{tm2Nobs/tm2Nobs} mice (A) Dose-dependent effects of systemic administration of ONO-2506 (0, 25, 50 and 100 mg·kg⁻¹, i.p.) (n = 8·per group) on the frequency and duration of spike and wave discharges in Cacna1a^{tm2Nobs/tm2Nobs} mice. (B) Dose-dependent effects of systemic administration of ethosuximide (0, 100, 200 and 300 mg·kg⁻¹, i.p.) (n = 5·per group) on the frequency and duration of spike and wave discharges in Cacna1a^{tm2Nobs/tm2Nobs} mice. (C) Lack of dose-dependent effects of systemic administration of ONO-2506 (0, 25, 50 and 100 mg·kg⁻¹, i.p.) (n = 6·per group) on the duration of tonic flexor, tonic extensor and clonic convulsion induced by MES. (D) Lack of dose-dependent effects of systemic administration of ONO-2506 (0, 25, 50 and 100 mg·kg⁻¹, i.p.) (n = 6·per group) on the onset latency for clonic seizures induced by PTZ: score II (facial jerking with muzzle or muzzle and eye) or score III (neck jerks) of the revised Racine's scale (Luttjohann et al., 2009). The putative dose-dependent effects of ONO-2506 on convulsions and spike and wave discharges and of ethosuximide on spike and wave discharges were analysed by anova with Tukey's multiple comparison test. **P < 0.01 significantly different from control. (E) Typical EEG of Cacna1a^{tm2Nobs/tm2Nobs} mice without (control) and with 100 mg·kg⁻¹ ONO-2506 (ONO-2506).

Figure 3

Effects of TTX, FLC and TeNT on basal and K⁺-evoked release of GABA and kynurenic acid in the mPFC. Effects of perfusion with 1 μM TTX and microinjection of 1 ng·0.3 μL⁻¹ TeNT on the basal and K⁺-evoked release of (A) GABA and (B) kynurenic acid in the mPFC. Effects of perfusion with 1 mM FLC on the basal and K⁺-evoked release of (C) GABA and (D) kynurenic acid in the mPFC. Data are means ± SEM (n = 6) of extracellular transmitter levels. The effects of microinjection of TeNT and perfusion with TTX and FLC were compared using manova with Tukey's multiple comparison test. *P < 0.05; **P < 0.01 significantly different from control.

Figure 4

Effects of systemic administration of ONO-2506 on basal release of GABA and kynurenic acid in the mPFC. Dose-dependent effects of ONO-2506 (30, 100 and 300 mg·kg⁻¹) on the extracellular levels of (A) GABA and (B) kynurenic acid in the mPFC. Ordinates: extracellular levels (nM or μM, n = 6) of GABA and kynurenic acid in the mPFC, abscissas: time after administration (min) of ONO-2506 (i.p.). The dose-dependent effects of ONO-2506 on extracellular levels of GABA and kynurenic acid in the mPFC were analysed by manova with Tukey's multiple comparison test. *P < 0.05; **P < 0.01 significantly different from pre-treatment period.

Figure 5

Effects of systemic administration of ONO-2506 on K⁺-evoked releases of L-glutamate, GABA and kynurenic acid in the mPFC. Dose-dependent effects of ONO-2506 (30, 100 and 300 mg·kg⁻¹) on K⁺-evoked releases of (A) L-glutamate, (B) GABA and (C) kynurenic acid in the mPFC. Four hours after systemic administration of ONO-2506, the perfusate was switched from MRS to HKMRS for 20 min (K⁺-evoked stimulation). Ordinates: extracellular levels of l-glutamate (μM, n = 6), GABA (μM, n = 6) and kynurenic acid (nM, n = 6) in the mPFC, abscissas: time after K⁺-evoked stimulation (min). The dose-dependent effects of ONO-2506 on K⁺-evoked releases in the mPFC were analysed using manova with Tukey's multiple comparison test. *P < 0.05; **P < 0.01 significantly different from pre-treatment period.

Figure 6

Effects of TTX, TeNT and FLC on ONO-2506-induced releases of GABA and kynurenic acid in the mPFC. Effects of TTX, TeNT and FLC on the AUC values of 300 mg·kg⁻¹ ONO-2506-induced releases of (A) GABA and (B) kynurenic acid in the mPFC. Ordinates: AUC values of GABA (μM, n = 6) and kynurenic acid (nM, n = 6) in the mPFC. ONO-2506-induced release was calculated by subtraction of the mean of basal release from the extracellular level after ONO-2506 administration from 120 to 180 min. Data are means ± SEM (n = 6). *P < 0.05; **P < 0.01 significantly different from control, by Student's t-test.

Figure 7

Effects of TTX, TeNT and FLC on ONO-2506-induced kynurenic acid release from primary cultured astrocytes. Effects of pre-treatment without (control) or with 1 μM TTX, 3 μg·mL⁻¹ TeNT and 1 mM FLC on ONO-2506-induced kynurenic acid release from astrocytes. After incubation with TTX for 2 h, TeNT for 24 h or FLC for 8 h, ONO-2506 was added to the incubation medium at 10, 30 or 100 μM. Ordinates: % control of kynurenic acid levels (%, n = 6), abscissas: ONO-2506 concentration (μM). *P < 0.05; **P < 0.01 significantly different from 0 μM, repeated anova with Tukey's multiple comparison test.

Figure 8

Effects of TTX, TeNT and FLC on AMPA-induced releases of GABA and kynurenic acid from primary cultured astrocytes. Effects of TTX (1 μM for 2 h), TeNT (0.3 ng·mL⁻¹ for 24 h) and FLC (1 mM for 8 h) on 100 μM AMPA-induced releases of (A) GABA and (B) kynurenic acid from astrocytes. Data are means ± SEM of % control values (%) (n = 6). *P < 0.05; **P < 0.01 significantly different from control; Student's t-test.

Figure 9

Concentration-dependent effects of ONO-2506 on AMPA-induced releases of l-glutamate, d-serine, GABA and kynurenic acid from primary cultured astrocytes. Concentration-dependent effects of ONO-2506 on 100 μM AMPA-induced releases of (A) l-glutamate, (B) d-serine, (C) GABA and (D) kynurenic acid from primary cultured astrocytes. Data are means ± SEM of % control values (%) (n = 6). *P < 0.05; **P < 0.01 significantly different from control; one-way anova with Tukey's multiple comparison test.