INTRODUCTION:

Recurrent seizures brought on by aberrant brain electrical activity are a hallmark of epilepsy, a neurological condition¹. Although this neurological condition affects both adults and children, little is known about many of its characteristics. Zebrafish are perfect for researching neurological conditions that may lead to the development of treatments for epilepsy because of their well-preserved neural systems^2,3. In contrast to traditional rodent models, researchers may gather a large amount of data at a reduced cost and in a shorter amount of time using zebrafish, despite their relatively rudimentary brain structure, and gain various insights into the etiology and management of epilepsy. This minimizes ethical concerns and is especially helpful when screening a large number of potential antiseizure medications (ASDs). In controlled research, chemical inducers have proven indispensable for regulating brain activity⁴. As a result, the majority of chemically induced models in aquatic vertebrates employ substances that throw off the animal's brain's equilibrium between inhibition and stimulation. Among the common chemical inducers are pilocarpine, picrotoxin (PTX), kainic acid (KA), pentylenetetrazole (PTZ), and others⁵. By evaluating behavioral patterns and gene expressions as biomarkers of seizure occurrence, this investigation seeks to thoroughly compare and analyze different chemicals utilized in zebrafish models to cause seizures. It also compares the variations between studies on epilepsy using adult and larvae zebrafish models.

Changing neurotransmitter receptors at excitatory or inhibitory synapses, especially those for GABA and glutamate is the main mechanism of these pharmacological inducers. By creating an inhibitory and excitatory imbalance of impulses in the brain, this alteration aids in epileptogenesis. In the zebrafish brain, the ensuing excitement causes metabolic disruption, neuronal death, and an increase in reactive oxygen species (ROS). Chemical induction provides important insights into the neurological functioning of zebrafish by enabling the observation of behaviors such as freezing, loss of posture, and whole-body contractions. To duplicate different kinds of seizures, including temporal lobe epilepsy, status epilepticus and to investigate effects of chemicals inducing seizures on zebrafish development, a number of parameters can be changed. To ensure the successful induction of models, it becomes crucial to monitor expression of gene indicators like c-fos, that reflects brain activity. A grading system is also used for determine the effectiveness of treatment and assess the severity of seizures⁶.

For the first time, Baraban's group used zebrafish as a seizure model in 2005⁷. That pioneering study's authors reported the zebrafish larvae PTZ model along with c-fos expression, behavioral activity and extracellular electrical activity as a gauge of brain activation. This laid the foundation for the subsequent modelling of epilepsy using zebrafish⁸.

ZEBRAFISH AS EPILEPSY MODELS:

Zebrafish General Features Making them Significant Models for Studying Epilepsy:

Zebrafish are members of the South Asian Phylum of chordates, the Subphylum Vertebrates, the class Osteichthyes, the order Cypriniformes, and the family Barbs. The embryo of this little teleost fish develops incredibly quickly, and as early as 24hours after fertilization (hpf), the entire body development plan is established. This quick development is similar to the first three months of a human embryo's intrauterine life⁹.

Zebrafish have been used as vertebrate model organisms in several studies on a variety of human diseases, including cancer, heart disease, and metabolic issues, since the 1980s¹⁰. Application of zebrafish to model and examine neurological disorders, including epilepsy and diseases related to neurodegeneration like Alzheimer’s and Parkinson’s, has grown significantly in the past ten years. Their unique characteristics, including as their short lifespan and quick generation period, genetic resemblance to humans, and less ethical restrictions than mammals (at least when it comes to larval stages), are probably the primary causes of this¹¹. Since they allow for quick assessments of effectiveness and possible toxicity of novel drug compounds, zebrafish larvae and embryo have been popular in the past ten years as substitute preclinical models for screening and drug discovery in addition to being valuable for disease modelling^12–13.

Like all vertebrates, zebrafish have three primary parts to their brains: Hindbrain/spinal cord, midbrain, and forebrain¹⁴. Adult brain regions like the pallium, sub-pallium, thalamus, and cerebellum are formed by the differentiation of cells from the embryonic forebrain, midbrain, and hindbrain¹⁵. As a result, most of the research to far has been on neurogenesis during the embryonic stages. The mature zebrafish brain, however, may also be a useful tool for researching adult neurogenesis, according to recent research¹⁶.

The CNS of zebrafish larvae is easily manipulable in experiments due to its outward development and optical transparency. A unique model organism for studying neurogenesis and epilepsy, the zebrafish integrates genetics, embryology, and cutting-edge optical techniques. It offers numerous benefits over its murine cousin, including quicker findings and cheaper pharmacological testing¹⁷.

Behavioral, genetic, pharmacological, and electrophysiological investigations have shown that zebrafish exhibit seizures that mimic many of the key features of seizures in mammals¹⁸, which makes them a great model organism for epilepsy research. Zebrafish larvae have been found to display a variety of motor behaviors like those observed in individuals with epilepsy, such as changed swimming pattern and body shaking. These behaviors are easily recognized and staged during seizure evaluation. Lastly, as previously said, zebrafish have a quick life cycle, which makes it possible to analyze several generations of seizures in a short amount of time.

Significance of Larvae Model Vs Adult Model of Zebrafish in studying Epilepsy:

All aforementioned properties make zebrafish larvae ideal for studying epilepsy; nevertheless, adult traits are still little understood. Zebrafish adults and larvae differ in several pathophysiological and pharmacological ways, which should be considered while studying epilepsy¹⁹.

First, as previously mentioned, adults have a fully formed brain that includes the blood-brain barrier (BBB) and has a complicated structure²⁰. The peripheral and central neural systems, together with every other organ, undergo maturation during development from larvae to adult stage until they achieve physiological stability²¹.

Like that of mammals, the zebrafish BBB exhibits intricate organization and function²². Tight connections found in its endothelial cells are what give drugs their size-dependent permeability. At 3 dpf, the BBB formation begins, and by 14 dpf, it is nearly finished. Because of structural and functional maturation and changes of BBB of Zebrafish between the larval and juvenile phases, it ought to be considered that the consequences for epilepsy and effects of ASDs may differ between adults and larvae. It is crucial since the impact, cause, and management of seizures are all impacted by disturbance of the blood-brain barrier²³.

As previously mentioned, neurogenesis differs in several ways between larvae and adults. In the former, it occurs in two stages, while in the latter, it is a vigorous process that encompasses every part of the brain. Furthermore, the two stages differ in how neural precursors are expressed. In addition, like animals, PTZ-induced seizures in adults encourage the growth of new neurons in a particular area of the dorsal telencephalon²⁴. It implies that studying seizures in adult zebrafish could aid in comprehending the connection among epileptogenesis and neurogenesis.

Varied uptake and delivery of chemicals or medications from the external media is another distinction between adults and larvae that may be significant in research on epilepsy. Tiny pores in the chorion, a protective coat that envelops the larvae until 48- or 72hours post-fertilization, enable the passive absorption of drugs or treatments into the body. After 72hours after fertilization, the larvae can absorb the chemicals through their stomach, gills, and skin. During drug exposure, several embryos or larvae are treated simultaneously with varying amounts of drugs diluted in an embryo medium in a 24-well plate. While the adults are treated by dispersing drugs in tanks. Adult zebrafish will uptake drugs through their gills, skin, digestive tract. The BBB is completely developed in adults, though, so for the various substances or medications to reach their intended CNS targets, they must pass through this protective barrier²⁵.

Since there are variations between larvae and adult zebrafish, this parameter should be considered throughout various epilepsy experiments, though the impact of gender difference on epilepsy is unclear. Sex hormones affect how ASDs are metabolized, how epilepsy develops, and how susceptible people, rats, and animals are to seizures^26,27,28. It is difficult to identify the sex of zebrafish at larvae stage like 3 dpf because various genes may not be sufficiently expressed. As a result, gender differences can only be considered to exist in adult zebrafish. Gender differences can influence varied neuronal gene expression patterns and various functional or proliferative activity in specific brain areas in both male and female zebrafish^29,30. Yet, when assessing zebrafish epilepsy, gender is a factor that is usually disregarded^31,32.

Chemical Inducers of Epilepsy in Zebrafish

Pentylenetetrazole:

As antagonist of the GABA receptor, pentylenetetrazole (PTZ) is a tetrazole derivative³³. To investigate seizure activity, PTZ, a pro-convulsant medication, is frequently utilized³⁴. The quick inhibitory synaptic transmission known as GABAergic neurotransmission, which is mediated by GABA's communication with metabotropic and ionotropic membrane receptors, is what PTZ works by blocking. Following membrane depolarization, the interaction between PTZ and GABA-A receptors will block the chloride channel and decrease chloride ion influx. Pro-convulsant activity is facilitated by the action potentials produced by this depolarization, which raise neuronal excitability^35,36,37. Additionally, at slightly negative to positive membrane potentials, PTZ encourages voltage-gated K⁺ channels to become inactive. Both the outward K⁺ current and the neurons' capacity to repolarize are diminished by the inactivation. Neuronal excitability, firing properties, and seizure activity production and maintenance may all be impacted by this modulation³⁸. Epileptogenesis and the emergence of a propensity for recurrent seizures may result from these alterations, which could cause disturbance among inhibition and excitation in brain functioning^39,40.

Drug screening, the development of anti-seizure therapies, and the detection of the negative consequences of seizure induction in pharmaceuticals for other illnesses have all benefited from the use of the PTZ zebrafish model^41,42. This chemical kindling paradigm is a popular choice for experimental epilepsy research because of its many advantages, including its low mortality, ease of reproducibility, and lack of electrodes⁴³. Larval zebrafish behavioral seizure activity can also be assessed by mixing PTZ with a regular bathing media. Larval behavioral traits have been extensively documented and applied to a variety of research techniques⁴⁴.

Many genes have been investigated recently in the PTZ-induced zebrafish model. One important gene that is employed in this situation to confirm that seizures were successfully produced is c-fos. It is due to rapid appearance of c-fos, which is mainly located in zebrafish brain, in reaction to PTZ-like stimuli^45,46. Furthermore, PTZ triggers the activation of bdnf-TrkB signalling pathway, which promotes inflammatory responses. The equilibrium between excitatory and inhibitory neurons may be upset by bdnf increase, notwithstanding the contentious link between it and epilepsy^47,48. Due to blood-brain barrier leaking, PTZ-induced inflammation in the brain has been demonstrated to enhance additional inflammatory mediators, including NFκB, COX-2, IL-1β, and leukocyte infiltration^49,50.

Kainic acid:

Kainic acid (KA) produces its neuroexcitatory effect by binding to AMPA or kainate receptors, which are ionotropic glutamate receptors with postsynaptic excitatory and presynaptic modulatory effects. To allow Ca²⁺ to enter the cell, KA binds to kainate receptors, activating the receptor and depolarizing the neuronal membrane. Calcium excess can cause neuronal injury and death by leading to generation of reactive oxygen species (ROS), that break down cell membranes, and disruption of cellular metabolism^51,52. ROS, the oxidative stress mediators necessary for excitotoxic cell damage, were produced in greater quantities after KA induction due to intracellular calcium entry and glutamate receptor overactivation. Additionally, mitochondrial malfunction, glial activation, endoplasmic reticulum (ER) membrane fragmentation and ER stress, neuroinflammation, and neuronal death are all brought on by Ca²⁺ excess^52,53.

Significant alterations in cell division and electrical activity, such as the induction of interictal events and protracted bursting discharges, can be brought on by KA. The developmental stage and length of exposure, however, may affect the effects of KA exposure. According to research by Menezes et al.⁵⁴, early exposure to KA may have an impact on brain development and change a person's eventual sensitivity to seizures. These results emphasize how crucial it is to consider the developmental stage and exposure duration when examining how KA affects zebrafish behavior and brain function⁶⁴. Additionally, KA influences astrocyte-related neurochemical alterations that cause a temporary decrease in GFAP cell and glutamate uptake levels as well as a sluggish swimming pattern in zebrafish^56,57.

According to reports, KA administered directly to the pericardium causes severe inflammation and brain damage in the larval zebrafish model, resulting in seizure-like locomotor activity. Like the last study, brain cell death is indicated by KA-induced tissue opacity and elevated pro-apoptotic markers. Enhanced levels of pro-inflammatory cytokines, including IL-1β, fas, IL-8, TNF-α, IRF1-β, were also linked to the increased cell death, suggesting an acute inflammatory response like that seen in mammalian temporal lobe epilepsy (TLE). KA-induced locomotor abnormalities, which persist for several days after injection, include jerking movements, intermittent loss of posture, convulsion-like twitches, such as tail or fin twitches, and even whole-body waving. After that, the larvae lose their posture, become hypoactive during light-dark cycles, and become insensitive to tactile stimuli⁵⁸.

Picrotoxin:

Animal models of epilepsy have been created using picrotoxin (PTX) which is non-competitive antagonist of the Gama-Amino Butyric Acid A (GABA-A) receptor, which carries chloride ions across cell membranes^59,60. The GABA-C receptors, which are connected to the chloride channels, are also antagonistic to PTX; however, these receptors induce responses that are slow and sustained⁶¹. By lowering the receptor's opening frequency and mean open time, the PTX mechanism reduces the inhibitory impact of GABA⁶². Since PTX dissolves well in saltwater, it can be administered in any manner. As a result, seizures progress more slowly than those brought on by PTZ⁶³.

When zebrafish are exposed to PTX, they display a range of behavioral alterations. Like PTZ, PTX has a dose-dependent effect on zebrafish locomotor activity^{64, 65}. Large dosages of PTX cause zebrafish larvae to become more motile and exhibit thigmotaxis, a symptom of higher anxiety, in dark and light environments, according to a reported study. Compared to PTZ, PTX offers greater mobility in brighter light and reduced mobility in darker light⁶⁶. Additionally, circular swimming, spasms, corkscrew swimming and spending more duration in the upper portion of tank are all signs of seizure-like behavior that increases with PTX exposure⁶⁷.

Like PTZ, PTX also elicited an increase in neuronal excitability, a sign of the initiation of seizures, which led to an elevation of c-fos transcripts in the zebrafish embryo model after 30 minutes of treatment. In addition to c-fos, PTX causes the zebrafish brain to express more gabra1 and gabrg2⁶⁸.

Pilocarpine:

The hippocampus, striatum, and cortex are the main locations for pilocarpine, a non-selective muscarinic ACh receptor agonist⁶⁹. In animal models, especially those for temporal lobe epilepsy (TLE), status epilepticus (SE), it is commonly employed to cause seizures. Action of pilocarpine involves activation of muscarinic M1 receptor. Once a seizure has begun, it is sustained by NMDA receptor activation⁷⁰. Because of high concentration of muscarinic receptors, nucleus accumbens is major site of damage, and ventral forebrain is where seizures begin⁷¹. EEG recordings revealed a significant increase in hippocampus spike activity after pilocarpine treatment. A few hours after the seizure started, the brain damage started, and it got worse in some locations, like the neocortical areas and the hippocampus hilus⁷². However, pilocarpine research for seizures has been limited to rodents for years until recent studies investigated its potential use in adult zebrafish^{73, 74}.

A proposed method for assessing seizures in zebrafish produced by pilocarpine is a five-stage scoring system that mimics the swimming behavior of epileptic zebrafish⁷⁵. A score like PTZ was used to rate zebrafish larvae in a study by Vermoesen et al.⁷⁶. PTZ and pilocarpine-induced seizures in larval zebrafish were compared in Szep et al.'s work⁷¹. The study found that zebrafish larvae induced with pilocarpine exhibit more suppressed epileptiform activity than those induced with PTZ, hence decreasing the zebrafish's susceptibility to light. The mean total distance travelled was significantly smaller than that of the PTZ one⁷¹.

Table 1: Scoring of seizure severity based on behavior in variety of chemical induced models of epilepsy in Zebrafish.

Stage	Adult [PTZ, PTX]	Larvae [PTZ, PTX, Pilocarpine]
O	Short swim	Normal swimming
1	Enhanced swimming, frequency of opercular movement	Unsteady motion, increased swimming activity
2	Irregular movement	Whirl-pool like circling behavior
3	Circular movement	Brief clonus like spasms leading to posture loss
4	Clonic seizure behavior	Irregular burst movement with imbalance of posture
5	Tonic seizure behavior
6	Death

Advantages and Disadvantages of Chemical Induction of Seizures in Zebrafish:

It is possible to replicate wide range of genetic and pharmacological seizures as seen in rodents, using zebrafish⁷⁸. Chemically generated seizures in zebrafish models have several advantages. First, because the duration of the convulsions is determined by the concentration of the chosen convulsant, controlled studies and repeatable results are made possible. Additionally, chemical induction allows for the observation of behaviors like freezing, imbalance in posture, and whole-body contractions, providing crucial insights into the neurological activity of zebrafish. However, there are several significant disadvantages to employing drugs to cause seizures. One difficulty is the uncertainty in behavior interpretation, particularly the inability to distinguish between hyperactivity, abnormal behavior, seizure behavior in zebrafish larvae. One challenge in interpreting behavior in zebrafish larvae is the inability to distinguish clearly between seizure-like behavior, hyperactivity, and abnormal behavior. This challenge highlights the importance of closely examining behavior and accounting for potential confounding factors. Furthermore, only a limited number of chemical seizure models have been examined for non-behavioral seizure biomarkers, which limits the validity of identified effects. The size of adult zebrafish makes high-throughput studies more challenging, and the application of zebrafish for modelling is restricted, primarily to embryos and larvae⁷⁹. Chemical induction has benefits in epilepsy research including animal models, such as PTZ, KA, pilocarpine, and PTX. These models replicate genuine seizure activity, advance our understanding of seizure mechanisms, and serve as valuable tools for upcoming therapeutic research^80-82. There are limitations as well because these models may not fully capture the different pathways that underlie human seizures.

CONCLUSION:

In addition to helping create novel potential treatments for the human condition, experimental research in epilepsy offers the chance to reveal molecular mechanisms underlying excitability at neuronal level as well as provide insights into brain physiology. As a significant tool for processing data in a short span of duration with reduced expenses than traditional rodent models, without sacrificing a lot of potentially significant information, zebrafish are becoming a classic model in epilepsy research. Future studies on epilepsy should consider the distinct traits of zebrafish adults and larvae, as well as the numerous advantages and disadvantages that each stage has to offer. Adults and larvae of zebrafish are excellent models for researching epilepsy. Zebrafish larvae are easy to handle in a variety of experimental setups and are appropriate for high-throughput screening. However, because of their size, adults are more suitable for EEG, medication injection, dissection. There are several options available for chemical inducers employed in the zebrafish seizure model, each having a unique mechanism. Numerous parameters, including inflammation, oxidative damage, electrolyte imbalance, can be used to study the effects of seizures. PTZ is most utilized chemical in seizure research because of its well-described nature, validation by electrophysiological techniques, and availability in a range of doses. More research prospects are presented by broad topic that can be thoroughly studied, with many facets that are yet largely neglected, particularly in the situations of status epilepticus and temporal lobe epilepsy. These opportunities will help us understand the mechanics behind seizures at deeper level.

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Received on 08.08.2025 Revised on 18.09.2025

Accepted on 22.10.2025 Published on 12.02.2026

Available online from February 14, 2026

Res.J. Pharmacology and Pharmacodynamics.2026;18(1):7-14.

DOI: 10.52711/2321-5836.2026.00002

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Strain/Age	Dose/Route	Behavioral biomarker	Non-behavioral biomarker	Reference
Larvae, AB line/4-5dpf	10,20,40mM PTZ immersed in water	increased locomotor activity	-	[45]
Adult, strain wild-type/ 3-4 months old	170 mg/kg i.p PTZ	Reached seizure score 4 within 150-180sec.á total distance travelled	â GABA áGlutamate âACh âbdnf ácreb1	[48]
Larvae, 7dpf	2.5,5,15 mM PTZ Immersion in water	á Distance travelled á Seizure score	á c-fos	[82]
Adult, Strain wild-type	10 mM immersion	áSeizure score ávelocity or swimming speed	á hsp 70 expression âbdnf ácreb	[83]
Larvae, 7dpf	1.25, 2.5, 5, 10, 20, 40, 80 mM PTZ, immersion	á Distance travelled á Corkscrew swimming	ác-fos âbdnf	[84]