Neurochemical Effects of 4-(2Chloro-4-Fluorobenzyl)-3-(2-Thienyl)-1,2,4-Oxadiazol-5(4H)-One in the Pentylenetetrazole (PTZ)-Induced Epileptic Seizure Zebrafish Model

Abstract

Epilepsy is one of the most common neurological disorders, and it is characterized by spontaneous seizures. In a previous study, we identified 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) as a novel anti-epileptic agent in chemically- or genetically-induced epileptic zebrafish and mouse models. In this study, we investigated the anti-epileptic effects of GM-90432 through neurochemical profiling-based approach to understand the neuroprotective mechanism in a pentylenetetrazole (PTZ)-induced epileptic seizure zebrafish model. GM-90432 effectively improved PTZ-induced epileptic behaviors via upregulation of 5-hydroxytryptamine, 17-β-estradiol, dihydrotestosterone, progesterone, 5α -dihydroprogesterone, and allopregnanolone levels, and downregulation of normetanephrine, gamma-aminobutyric acid, and cortisol levels in brain tissue. GM-90432 also had a protective effect against PTZ-induced oxidative stress and zebrafish death, suggesting that it exhibits biphasic neuroprotective effects via scavenging of reactive oxygen species and anti-epileptic activities in a zebrafish model. In conclusion, our results suggest that neurochemical profiling study could be used to better understand of anti-epileptic mechanism of GM-90432, potentially leading to new drug discovery and development of anti-seizure agents.

Keywords: epilepsy; metabolic alteration; neurosteroid; neurotransmitter; zebrafish.

Figure 1

Experimental scheme used to determine the protective effect of GM-90432 against PTZ-induced epileptic seizures in zebrafish larvae and adults. Zebrafish were separated into 4 different treatment groups (G): G1: control group, G2: GM–90432 group, G3: PTZ-induced epilepsy model group, and G4: GM-90432 + PTZ group. At the experimental endpoint, zebrafish were sacrificed for analysis of ROS levels (larva only), and neurotransmitter and neurosteroid analyses.

Figure 2

The levels of 5-HT (A), NM (B), GLN (C), and GABA (D), and excitatory/inhibitory ratio (E) in brain samples of zebrafish exposed to GM-90432, PTZ, or PTZ following pretreatment with GM-90432. Values represent means ± standard error of the mean (SEM) of 12 samples. Significance between control and exposure groups is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 3

The levels of E2 (A), DHT (B), C (C), Prog (D), 5α-dihydroProg (E), and Allo-P (F) in brain samples of zebrafish exposed to GM-90432, PTZ, or PTZ following pretreatment with GM-90432. Values represent means ± SEM of 6 samples. The brain samples from 2 individual zebrafish were pooled to form composite samples. Significance between control and exposure groups is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 4

The enzymatic activity by altered metabolic ratio in brain samples of zebrafish. (A) Altered metabolic ratio (5α-reductase) of T to DHT in brain samples of zebrafish exposed to GM-90432, PTZ, or PTZ following pretreatment with GM-90432. (B) Altered metabolic ratio (aromatase) of T to E2 in brain samples of zebrafish exposed to GM-90432, PTZ, or PTZ following pretreatment with GM-90432. (C) The metabolic pathway of T conversion to DHT or E2. Values represent means ± SEM of 6 samples. Brain samples from 2 individual zebrafish were pooled to form composite samples. Significance between control and exposure groups is indicated by * p < 0.05 or ** p < 0.01.

Figure 5

Protective effect of GM-90432 in PTZ-induced ROS generation. (A) The intracellular ROS were stained with DCF-DA dye for 20 min. Fluorescence images of ROS generation in zebrafish larvae in the 4 experimental groups. (B) The relative fluorescence intensities of ROS levels in individual larvae were quantified. Values represent means ± SEM of 10 samples. Significance between control and exposure groups is indicated by *** p < 0.001.