Role of adenosine signaling on pentylenetetrazole-induced seizures in zebrafish

Abstract

Adenosine is a well-known endogenous modulator of neuronal excitability with anticonvulsant properties. Thus, the modulation exerted by adenosine might be an effective tool to control seizures. In this study, we investigated the effects of drugs that are able to modulate adenosinergic signaling on pentylenetetrazole (PTZ)-induced seizures in adult zebrafish. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) decreased the latency to the onset of the tonic-clonic seizure stage. The adenosine A1 receptor agonist cyclopentyladenosine (CPA) increased the latency to reach the tonic-clonic seizure stage. Both the adenosine A2A receptor agonist and antagonist, CGS 21680 and ZM 241385, respectively, did not promote changes in seizure parameters. Pretreatment with the ecto-5'nucleotidase inhibitor adenosine 5'-(α,β-methylene) diphosphate (AMPCP) decreased the latency to the onset of the tonic-clonic seizure stage. However, when pretreated with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), or with the nucleoside transporter (NT) inhibitors, dipyridamole and S-(4-Nitrobenzyl)-6-thioinosine (NBTI), animals showed longer latency to reach the tonic-clonic seizure status. Finally, our molecular analysis of the c-fos gene expression corroborates these behavioral results. Our findings indicate that the activation of adenosine A1 receptors is an important mechanism to control the development of seizures in zebrafish. Furthermore, the actions of ecto-5'-nucleotidase, ADA, and NTs are directly involved in the control of extracellular adenosine levels and have an important role in the development of seizure episodes in zebrafish.

FIG. 1.

Effect of DPCPX (3, 10, 15 mg/kg) and CPA (1, 2, 4 mg/kg) on locomotor activity and progression of the pentylenetetrazole (PTZ)-induced seizures in zebrafish. (A, C) Locomotor activity was measured as distance traveled during the 30 min following drug pretreatments. (B, D) Latency to the first behavioral manifestation of each seizure stage (I, II, and III) during PTZ exposure. The data are expressed as the mean±SD from 8 and 12 animals for DPCPX and CPA groups, respectively, and were analyzed by one-way ANOVA followed by Dunnett's post hoc test. The symbols represent statistical difference when compared with the respective vehicle control group (DMSO 3%). *p<0.05, **p<0.005, ***p<0.0005.

FIG. 2.

Effect of ZM 241385 (1, 2, 5 mg/kg) and CGS 21680 (1, 2, 5 mg/kg) on locomotor activity and progression of the PTZ-induced seizures in zebrafish. (A, C) Locomotor activity was measured as distance traveled during the 30 min following drug pretreatments. (B, D) Latency to the first behavioral manifestation of each seizure stage (I, II, and III) during PTZ exposure. The data are expressed as the mean±SD from 8 and 12 animals for ZM 241385 and CGS 21680 groups, respectively, and were analyzed by one-way ANOVA followed by Dunnett's post hoc test.

FIG. 3.

Effect of AMPCP (100, 150, 200 mg/kg), EHNA (75, 100, 150 mg/kg), dipyridamole (5, 10, 20 mg/kg), and NBTI (5, 10, 15 mg/kg) on locomotor activity and progression of the PTZ-induced seizures in zebrafish. (A, C, E, G) Locomotor activity was measured as distance traveled during the 30 min following drug pretreatments. (B, D, F, H) Latency to the first behavioral manifestation of each seizure stage (I, II, and III) during PTZ exposure. The data are expressed as the mean±SD from 12, 8, 8, and 12 animals for AMPCP, EHNA, dipyridamole, and NBTI, respectively, and were analyzed by one-way ANOVA followed by Dunnett's post hoc test. The symbols represent statistical difference when compared with the respective vehicle control group (DMSO 3% and Saline). *p<0.05, **p<0.005, ***p<0.0005.

FIG. 4.

Effects of the studied compounds on gene expression of c-fos in zebrafish submitted to PTZ-induced seizures. (A–F) Relative mRNA expression at seizure stage III. The data are expressed as the mean±SD from four samples and were analyzed by one-way ANOVA followed by Duncan's post hoc test. The symbol *represents statistical difference when compared with the vehicle control group (animals that were treated with respective vehicle and were not exposed to PTZ). The symbol ^#represents statistical difference when compared with the vehicle+PTZ group (animals that were exposed to PTZ without receive pretreatment). *^,#p<0.05.

FIG. 5.

Schematic representation of modulation of adenosine signaling in controlling seizures. The scheme shows the action pathway and induced effects of each studied compound.