Pericardial Injection of Kainic Acid Induces a Chronic Epileptic State in Larval Zebrafish

Abstract

Epilepsy is a common disorder of the brain characterized by spontaneous recurrent seizures, which develop gradually during a process called epileptogenesis. The mechanistic processes underlying the changes of brain tissue and networks toward increased seizure susceptibility are not fully understood. In rodents, injection of kainic acid (KA) ultimately leads to the development of spontaneous epileptic seizures, reflecting similar neuropathological characteristics as seen in patients with temporal lobe epilepsy (TLE). Although this model has significantly contributed to increased knowledge of epileptogenesis, it is technically demanding, costly to operate and hence not suitable for high-throughput screening of anti-epileptic drugs (AEDs). Zebrafish, a vertebrate with complementary advantages to rodents, is an established animal model for epilepsy research. Here, we generated a novel KA-induced epilepsy model in zebrafish larvae that we functionally and pharmacologically validated. KA was administered by pericardial injection at an early zebrafish larval stage. The epileptic phenotype induced was examined by quantification of seizure-like behavior using automated video recording, and of epileptiform brain activity measured via local field potential (LFP) recordings. We also assessed GFP-labeled GABAergic and RFP-labeled glutamatergic neurons in double transgenic KA-injected zebrafish larvae, and examined the GABA and glutamate levels in the larval heads by liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). Finally, KA-injected larvae were exposed to five commonly used AEDs by immersion for pharmacological characterization of the model. Shortly after injection, KA induced a massive damage and inflammation in the zebrafish brain and seizure-like locomotor behavior. An abnormal reorganization of brain circuits was observed, a decrease in both GABAergic and glutamatergic neuronal population and their associated neurotransmitters. Importantly, these changes were accompanied by spontaneous and continuous epileptiform brain discharges starting after a short latency period, as seen in KA rodent models and reminiscent of human pathology. Three out of five AEDs tested rescued LFP abnormalities but did not affect the seizure-like behavior. Taken together, for the first time we describe a chemically-induced larval zebrafish epilepsy model offering unique insights into studying epileptogenic processes in vivo and suitable for high-throughput AED screening purposes and rapid genetic investigations.

Zebrafish as an Innovative Tool for Epilepsy Modeling: State of the Art and Potential Future Directions

“…It is worth mentioning that in ZF, larvae KA has been shown to be able to induce reproducibly convulsive activity also after solubilization in fish water [67], even though its behavioral effects were not detailed in the study employing this route of administration. Interestingly, in one previous study in which KA was bath applied to ZF larvae, clear convulsive seizures resembling those described in adult ZF by Alfaro et al were not observed [66], even though a much lower concentration of KA in fish water (50 µM) was able to induce electrographic activity by Kim et al in 2011 [68].…”

“…In larval ZF, a recent interesting protocol consisted of infusing low KA doses in the common cardinal veins in the pericardium at 3 dpf. In the latter model, the behavioral and EEG effects of KA were well detailed as well as the antiepileptic effects of different ASD and the neurochemical effects of seizures [66]; however, this model is quite challenging from a methodological point of view. It is worth mentioning that in ZF, larvae KA has been shown to be able to induce reproducibly convulsive activity also after solubilization in fish water [67], even though its behavioral effects were not detailed in the study employing this route of administration.…”

The Function and the Affecting Factors of the Zebrafish Gut Microbiota

“…First, it is believed that the composition of gut microbiota and its metabolites and derivatives all play a role in the metabolic homeostasis of the host ( Falcinelli et al, 2015 ), but various endogenous and exogenous factors affect the composition and abundance of the microbiota and it is quite difficult to dissect well the gut microbial-host interactions ( Brugman et al, 2009 ; Roeselers et al, 2011 ; Mottaz et al, 2017 ; Yang et al, 2017 ). Second, although zebrafish are an excellent model organism that has been used to study blood diseases ( Haffter et al, 1996 ; Langenau et al, 2003 ; Dooley et al, 2008 ; van Rooijen et al, 2009 ; Taylor and Zon, 2011 ; Santoriello and Zon, 2012 ), cancer ( Amatruda et al, 2002 ; Ignatius and Langenau, 2009 ; Mione and Trede, 2010 ; Ceol et al, 2011 ; Liu and Leach, 2011 ), heart diseases ( Chen et al, 1996 ; Stainier et al, 1996 ; Poss et al, 2002 ; Poss, 2007 ; Tang et al, 2019 ; Mukherjee et al, 2021 ), muscle disorders ( Bassett and Currie, 2003 ; Follo et al, 2013 ), kidney diseases ( Tobin and Beales, 2008 ; Diep et al, 2011 ; Sander and Davidson, 2014 ; Gehrig et al, 2018 ; Brilli et al, 2019 ), central nervous system diseases ( Stewart et al, 2015 ; Heylen et al, 2021 ), and eye diseases ( Ohnesorge et al, 2019 ; Hong and Luo, 2021 ), zebrafish does have certain disadvantages in mimicking human diseases. These disadvantages include the inclusion of many gene duplications in the zebrafish genome; the phenotypic characteristics of diseases caused by direct homologous genes may differ in zebrafish and humans ( Santoriello and Zon, 2012 ).…”

Connectivity Mapping Using a Novel sv2a Loss-of-Function Zebrafish Epilepsy Model as a Powerful Strategy for Anti-epileptic Drug Discovery

“…Electrographic brain activity of 5 and 6 dpf larvae was assessed by non-invasive local field potential (LFP) recordings from the optic tectum as described previously ( Heylen et al, 2021 ). A blunt glass electrode (soda lime glass, Hilgenberg, Germany) pulled with DMZ Universal Puller (Zeitz, Germany) to an opening of approximately 15–20 microns, connected to a high-impedance amplifier, was filled with artificial cerebrospinal fluid (124 mM NaCl, 2 mM KCl, 2 mM MgSO 4 , 2 mM CaCl 2 , 1.25 mM KH 2 PO 4 , 26 mM NaHCO 3 and 10 mM glucose) and positioned on the skin above the optic tectum of a larva embedded in 2% low-melting point agarose (Thermo Fisher Scientific).…”