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Seizures induced by pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization.

ABSTRACT: Pentylenetetrazole (PTZ) is a common convulsant agent used in animal models to investigate the mechanisms of seizures. Although adult zebrafish have been recently used to study epileptic seizures, a thorough characterization of the PTZ-induced seizures in this animal model is missing. The goal of this study was to perform a detailed temporal behavior profile characterization of PTZ-induced seizure in adult zebrafish. The behavioral profile during 20 min of PTZ immersion (5, 7.5, 10, and 15 mM) was characterized by stages defined as scores: (0) short swim, (1) increased swimming activity and high frequency of opercular movement, (2) erratic movements, (3) circular movements, (4) clonic seizure-like behavior, (5) fall to the bottom of the tank and tonic seizure-like behavior, (6) death. Animals exposed to distinct PTZ concentrations presented different seizure profiles, intensities and latencies to reach all scores. Only animals immersed into 15 mM PTZ showed an increased time to return to the normal behavior (score 0), after exposure. Total mortality rate at 10 and 15 mM were 33% and 50%, respectively. Considering all behavioral parameters, 5, 7.5, 10, and 15 mM PTZ, induced seizures with low, intermediate, and high severity, respectively. Pretreatment with diazepam (DZP) significantly attenuated seizure severity. Finally, the brain PTZ levels in adult zebrafish immersed into the chemoconvulsant solution at 5 and 10 mM were comparable to those described for the rodent model, with a peak after a 20-min of exposure. The PTZ brain levels observed after 2.5-min PTZ exposure and after 60-min removal from exposure were similar. Altogether, our results showed a detailed temporal behavioral characterization of a PTZ epileptic seizure model in adult zebrafish. These behavioral analyses and the simple method for PTZ quantification could be considered as important tools for future investigations and translational research.

SUBMITTER: Mussulini BH

PROVIDER: S-EPMC3549980 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Purpose of the research: Epilepsy is a continuous process of neurodegeneration categorized by an enduring tendency to generate uncontrolled electrical firing known as seizures causing involuntary movement all over the body. Cognitive impairment and behavioral disturbances are among the more alarming co-morbidities of epilepsy. Anti-epileptic drugs (AEDs) were found to be successful in controlling epilepsy but are reported to worsen cognitive status in patients. Embelin (EMB) is a benzoquinone derived from the plant Embelia ribes and is reported to have central nervous system (CNS) activity. This study aims to evaluate the effectiveness of EMB against pentylenetetrazole (PTZ) induced acute seizures and its associated cognitive dysfunction. This was done via docking studies as well as evaluating neurotransmitter and gene expression in the zebrafish brain. The principal results: Behavioral observations showed that EMB reduced epileptic seizures and the T-maze study revealed that EMB improved the cognitive function of the fish. The docking study of EMB showed a higher affinity toward gamma-aminobutyric acid (GABAA) receptor as compared to the standard diazepam, raising the possibility of EMB working via the alpha subunit of the GABA receptor. EMB was found to modulate several genes, neurotransmitters, and also neuronal growth, all of which play an important role in improving cognitive status after epileptic seizures. Healthy zebrafish treated with EMB alone were found to have no behavioral and biochemical interference or side effects. The immunohistochemistry data suggested that EMB also promotes neuronal protection and neuronal migration in zebrafish brains. Major Conclusions: It was perceived that EMB suppresses seizure-like behavior via GABAA receptor pathway and has a positive impact on cognitive functions. The observed effect was supported by docking study, T-maze behavior, neurotransmitter and gene expression levels, and immunohistology study. The apparatus such as the T-maze and seizure scoring behavior tank were found to be a straightforward technique to score seizure and test learning ability after acute epileptic seizures. These research findings suggest that EMB could be a promising molecule for epilepsy induced learning and memory dysfunction.

Project description:Neural stimulation can reduce the frequency of seizures in persons with epilepsy, but rates of seizure-free outcome are low. Vagus nerve stimulation prevents seizures by continuously activating noradrenergic projections from the brainstem to the cortex. Cortical norepinephrine then increases GABAergic transmission and increases seizure threshold. Another approach, responsive nervous stimulation, prevents seizures by reactively shocking the seizure onset zone in precise synchrony with seizure onset. The electrical shocks abort seizures before they can spread and manifest clinically. The goal of this study was to determine whether a hybrid platform in which brainstem activation triggered in response to impending seizure activity could prevent seizures. We chose the zebrafish as a model organism for this study because of its ability to recapitulate human disease, in conjunction with its innate capacity for tightly controlled high-throughput experimentation. We first set out to determine whether electrical stimulation of the zebrafish hindbrain could have an anticonvulsant effect. We found that pulse train electrical stimulation of the hindbrain significantly increased the latency to onset of pentylenetetrazole-induced seizures, and that this apparent anticonvulsant effect was blocked by noradrenergic antagonists, as is also the case with rodents and humans. We also found that the anticonvulsant effect of hindbrain stimulation could be potentiated by reactive triggering of single pulse electrical stimulations in response to impending seizure activity. Finally, we found that the rate of stimulation triggering was directly proportional to pentylenetetrazole concentration and that the stimulation rate was reduced by the anticonvulsant valproic acid and by larger stimulation currents. Taken as a whole, these results show that that the anticonvulsant effect of brainstem activation can be efficiently utilized by reactive triggering, which suggests that alternative stimulation paradigms for vagus nerve stimulation might be useful. Moreover, our results show that the zebrafish epilepsy model can be used to advance our understanding of neural stimulation in the treatment of epilepsy.

Project description:Zebrafish (Danio rerio) assays provide a versatile pharmacological platform to test compounds on a wide range of behaviors in a whole organism. A major challenge lies in the lack of knowledge about the bioavailability and pharmacodynamic effects of bioactive compounds in this model organism. Here, we employed a combined methodology of LC-ESI-MS/MS analytics and targeted metabolomics with behavioral experiments to evaluate the anticonvulsant and potentially toxic effects of the angular dihydropyranocoumarin pteryxin (PTX) in comparison to the antiepileptic drug sodium valproate (VPN) in zebrafish larvae. PTX occurs in different Apiaceae plants traditionally used in Europe to treat epilepsy but has not been investigated so far. To compare potency and efficacy, the uptake of PTX and VPN into zebrafish larvae was quantified as larvae whole-body concentrations together with amino acids and neurotransmitters as proxy pharmacodynamic readout. The convulsant agent pentylenetetrazole (PTZ) acutely reduced the levels of most metabolites, including acetylcholine and serotonin. Conversely, PTX strongly reduced neutral essential amino acids in a LAT1 (SLCA5)-independent manner, but, similarly to VPN specifically increased the levels of serotonin, acetylcholine, and choline, but also ethanolamine. PTX dose and time-dependent manner inhibited PTZ-induced seizure-like movements resulting in a ~70% efficacy after 1 h at 20 µM (the equivalent of 4.28 ± 0.28 µg/g in larvae whole-body). VPN treated for 1 h with 5 mM (the equivalent of 18.17 ± 0.40 µg/g in larvae whole-body) showed a ~80% efficacy. Unexpectedly, PTX (1-20 µM) showed significantly higher bioavailability than VPN (0.1-5 mM) in immersed zebrafish larvae, possibly because VPN in the medium dissociated partially to the readily bioavailable valproic acid. The anticonvulsive effect of PTX was confirmed by local field potential (LFP) recordings. Noteworthy, both substances specifically increased and restored whole-body acetylcholine, choline, and serotonin levels in control and PTZ-treated zebrafish larvae, indicative of vagus nerve stimulation (VNS), which is an adjunctive therapeutic strategy to treat refractory epilepsy in humans. Our study demonstrates the utility of targeted metabolomics in zebrafish assays and shows that VPN and PTX pharmacologically act on the autonomous nervous system by activating parasympathetic neurotransmitters.

Project description:IntroductionEpilepsy is a chronic neurological condition characterized by behavioral, molecular, and neurochemical alterations. Current antiepileptic drugs are associated with various adverse impacts. The main goal of the current study is to investigate the possible anticonvulsant effect of selenium nanoparticles (SeNPs) against pentylenetetrazole (PTZ)-mediated epileptic seizures in mice hippocampus. Sodium valproate (VPA) was used as a standard anti-epileptic drug.MethodsMice were assigned into five groups (n=15): control, SeNPs (5 mg/kg, orally), PTZ (60 mg/kg, intraperitoneally), SeNPs+PTZ and VPA (200 mg/kg)+PTZ. All groups were treated for 10 days.ResultsPTZ injection triggered a state of oxidative stress in the hippocampal tissue as represented by the elevated lipoperoxidation, heat shock protein 70 level, and nitric oxide formation while decreased glutathione level and antioxidant enzymes activity. Additionally, the blotting analysis showed downregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the epileptic mice. A state of neuroinflammation was recorded following the developed seizures represented by the increased pro-inflammatory cytokines. Moreover, neuronal apoptosis was recorded following the development of epileptic convulsions. At the neurochemical level, acetylcholinesterase activity and monoamines content were decreased in the epileptic mice, accompanied by high glutamate and low GABA levels in the hippocampal tissue. However, SeNP supplementation was found to delay the onset and decreased the duration of tonic, myoclonic, and generalized seizures following PTZ injection. Moreover, SeNPs were found to provide neuroprotection through preventing the development of oxidative challenge via the upregulation of Nrf2 and HO-1, inhibiting the inflammatory response and apoptotic cascade. Additionally, SeNPs reversed the changes in the activity and levels of neuromodulators following the development of epileptic seizures.ConclusionThe obtained results suggest that SeNPs could be used as a promising anticonvulsant drug due to its potent antioxidant, anti-inflammatory, and neuromodulatory activities.

Project description:It has been demonstrated that the zebrafish model of pentylenetetrazole (PTZ)-evoked seizures and the well-established rodent models of epilepsy are similar pertaining to behavior, electrographic features, and c-fos expression. Although this zebrafish model is suitable for studying seizures, to date, inflammatory response after seizures has not been investigated using this model. Because a relationship between epilepsy and inflammation has been established, in the present study we investigated the transcript levels of the proinflammatory cytokines interleukin-1 beta (il1b) and cyclooxygenase-2 (cox2a and cox2b) after PTZ-induced seizures in the brain of zebrafish 7 days post fertilization. Furthermore, we exposed the fish to the nonsteroidal anti-inflammatory drug indomethacin prior to PTZ, and we measured its effect on seizure latency, number of seizure behaviors, and mRNA expression of il1b, cox2b, and c-fos. We used quantitative real-time PCR to assess the mRNA expression of il1b, cox2a, cox2b, and c-fos, and visual inspection was used to monitor seizure latency and the number of seizure-like behaviors.We found a short-term upregulation of il1b, and we revealed that cox2b, but not cox2a, was induced after seizures. Indomethacin treatment prior to PTZ-induced seizures downregulated the mRNA expression of il1b, cox2b, and c-fos. Moreover, we observed that in larvae exposed to indomethacin, seizure latency increased and the number of seizure-like behaviors decreased.This is the first study showing that il1b and cox-2 transcripts are upregulated following PTZ-induced seizures in zebrafish. In addition, we demonstrated the anticonvulsant effect of indomethacin based on (1) the inhibition of PTZ-induced c-fos transcription, (2) increase in seizure latency, and (3) decrease in the number of seizure-like behaviors. Furthermore, anti-inflammatory effect of indomethacin is clearly demonstrated by the downregulation of the mRNA expression of il1b and cox2b. Our results are supported by previous evidences suggesting that zebrafish is a suitable alternative for studying inflammation, seizures, and the effect of anti-inflammatory compounds on seizure suppression.

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Seizures induced by pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization.

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