Abstract
Zebrafish (Danio rerio) is becoming an increasingly important model in epilepsy research. Pentylenetetrazole (PTZ) is a convulsant agent that induces epileptic seizure-like state in zebrafish and zebrafish embryos and is most commonly used in antiepileptic drug discovery research to evaluate seizure mechanisms. Classical antiepileptic drugs, such as valproic acid (VPA) reduce PTZ-induced epileptiform activities. Opioid system has been suggested to play a role in epileptogenesis. The aim of our study is to determine the effects of morphine in PTZ-induced epilepsy model in zebrafish embryos by evaluating locomotor activity and parameters related to oxidant-antioxidant status, inflammation, and cholinergic system as well as markers of neuronal activity c-fos, bdnf, and opioid receptors. Zebrafish embryos at 72 hpf were exposed to PTZ (20 mM), VPA (1 mM), and Morphine (MOR) (100 µM). MOR and VPA pretreated groups were treated with either MOR (MOR + PTZ) or VPA (VPA + PTZ) for 20 min before PTZ expoure. Locomotor activity was quantified as total distance moved (mm), average speed (mm/sec) and exploration rate (%) and analyzed using ToxTrac tracking programme. Oxidant-antioxidant system parameters, acetylcholinesterase activity, and sialic acid leves were evaluated using spectrophotometric methods. The expression of c-fos, bdnf, oprm1, and oprd1 were evaluated by RT-PCR. MOR pretreatment ameliorated PTZ-induced locomotor pattern as evidenced by improved average speed, exploration rate and distance traveled. We report the restoration of inflammatory and oxidant-antioxidant system parameters, c-fos, bdnf, and opioid receptor oprm1 as the possible mechanisms involved in the ameliorative effect of MOR against PTZ-induced epileptogenic process in zebrafish embryos.
Graphical Abstract
Ethical approval
As the zebrafish embryos used were no older than 5 days old, no ethical approval was required for the protocols applied as stated by the Council of Europe (1986), Directive 86/609/EEC.
Consent to participate
All the authors have agreed to authorship, read and approved the manuscript, and given consent to participate.
Consent for publication
All the authors have agreed to authorship, read and approved the manuscript, and given consent for publication.
Author contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Fümet Duygu Üstündağ, İsmail Ünal, Ünsal Veli Üstündağ, Derya Cansız, and Merih Beler. The first draft of the manuscript was written by Fümet Duygu Üstündağ and Ebru Emekli-Alturfan in consultation with A. Ata Alturfan and Pınar Mega Tiber. Ebru Emekli-Alturfan supervised the study. All authors read and approved the final manuscript.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
Data will be available on reasonable request.