In Silico Analysis, Anticonvulsant Activity, and Toxicity Evaluation of Schisandrin B in Zebrafish Larvae and Mice.

The aim of this study is to evaluate the anticonvulsant potential of schisandrin B, a main ingredient of Schisandra chinensis extracts. Schisandrin B showed anticonvulsant activity in the zebrafish larva pentylenetetrazole acute seizure assay but did not alter seizure thresholds in the intravenous pentylenetetrazole test in mice. Schisandrin B crosses the blood-brain barrier, which we confirmed in our in silico and in vivo analyses; however, the low level of its unbound fraction in the mouse brain tissue may explain the observed lack of anticonvulsant activity. Molecular docking revealed that the anticonvulsant activity of the compound in larval zebrafish might have been due to its binding to a benzodiazepine site within the GABAA receptor and/or the inhibition of the glutamate NMDA receptor. Although schisandrin B showed a beneficial anticonvulsant effect, toxicological studies revealed that it caused serious developmental impairment in zebrafish larvae, underscoring its teratogenic properties. Further detailed studies are needed to precisely identify the properties, pharmacological effects, and safety of schisandrin B.

6-Gingerol, a Major Constituent of Zingiber officinale Rhizoma, Exerts Anticonvulsant Activity in the Pentylenetetrazole-Induced Seizure Model in Larval Zebrafish.

Zingiber officinale is one of the most frequently used medicinal herbs in Asia. Using rodent seizure models, it was previously shown that Zingiber officinale hydroethanolic extract exerts antiseizure activity, but the active constituents responsible for this effect have not been determined. In this paper, we demonstrated that Zingiber officinale methanolic extract exerts anticonvulsant activity in the pentylenetetrazole (PTZ)-induced hyperlocomotion assay in larval zebrafish. Next, we isolated 6-gingerol (6-GIN)-a major constituent of Zingiber officinale rhizoma. We observed that 6-GIN exerted potent dose-dependent anticonvulsant activity in the PTZ-induced hyperlocomotion seizure assay in zebrafish, which was confirmed electroencephalographically. To obtain further insight into the molecular mechanisms of 6-GIN antiseizure activity, we assessed the concentration of two neurotransmitters in zebrafish, i.e., inhibitory γ-aminobutyric acid (GABA) and excitatory glutamic acid (GLU), and their ratio after exposure to acute PTZ dose. Here, 6-GIN decreased GLU level and reduced the GLU/GABA ratio in PTZ-treated fish compared with only PTZ-bathed fish. This activity was associated with the decrease in grin2b, but not gabra1a, grin1a, gria1a, gria2a, and gria3b expression in PTZ-treated fish. Molecular docking to the human NR2B-containing N-methyl-D-aspartate (NMDA) receptor suggests that 6-GIN might act as an inhibitor and interact with the amino terminal domain, the glutamate-binding site, as well as within the ion channel of the NR2B-containing NMDA receptor. In summary, our study reveals, for the first time, the anticonvulsant activity of 6-GIN. We suggest that this effect might at least be partially mediated by restoring the balance between GABA and GLU in the epileptic brain; however, more studies are needed to prove our hypothesis.

Zebrafish Larvae Carrying a Splice Variant Mutation in cacna1d: A New Model for Schizophrenia-Like Behaviours?

Persons with certain single nucleotide polymorphisms (SNPs) in the CACNA1D gene (encoding voltage-gated calcium channel subunit alpha 1-D) have increased risk of developing neuropsychiatric disorders such as bipolar, schizophrenia and autism. The molecular consequences of SNPs on gene expression and protein function are not well understood. Thus, the use of animal models to determine genotype-phenotype correlations is critical to understanding disease pathogenesis. Here, we describe the behavioural changes in larval zebrafish carrying an essential splice site mutation (sa17298) in cacna1da. Heterozygous mutation resulted in 50% reduction of splice variants 201 and 202 (haploinsufficiency), while homozygosity increased transcript levels of variant 201 above wild type (WT; gain-of-function, GOF). Due to low homozygote viability, we focused primarily on performing the phenotypic analysis on heterozygotes. Indeed, cacna1dasa17298/WT larvae displayed hyperlocomotion-a behaviour characterised in zebrafish as a surrogate phenotype for epilepsy, anxiety or psychosis-like behaviour. Follow-up tests ruled out anxiety or seizures, however, as neither thigmotaxis defects nor epileptiform-like discharges in larval brains were observed. We therefore focused on testing for potential "psychosis-like" behaviour by assaying cacna1dasa17298/WT larval locomotor activity under constant light, during light-dark transition and in startle response to dark flashes. Furthermore, exposure of larvae to the antipsychotics, risperidone and haloperidol reversed cacna1da-induced hyperactivity to WT levels while valproate decreased but did not reverse hyperactivity. Together, these findings demonstrate that cacna1da haploinsufficiency induces behaviours in larval zebrafish analogous to those observed in rodent models of psychosis. Future studies on homozygous mutants will determine how cacna1d GOF alters behaviour in this context.

Pharmacological Validation of the Prepulse Inhibition of Startle Response in Larval Zebrafish using a Commercial Automated System and Software.

While there is an abundance of commercial and standardized automated systems and software for performing the prepulse inhibition (PPI) assay in rodents, to the best of our knowledge, all PPI assays performed in the zebrafish have, until now, been done using custom made systems which were only available to individual groups. This has thereby presented challenges, particularly with regard to issues of data reproducibility and standardization. In the present work, we generated a protocol that utilizes commercially available automated systems to pharmacologically validate the PPI assay in larval zebrafish. Consistent with published findings, we were able to replicate the results of apomorphine, haloperidol and ketamine on the PPI response of 6 days post-fertilization zebrafish larvae.

The Influence of Palmatine Isolated from Berberis sibiricaRadix on Pentylenetetrazole-Induced Seizures in Zebrafish.

Palmatine (PALM) and berberine (BERB) are widely identified isoquinoline alkaloids among the representatives of the Berberidaceae botanical family. The antiseizure activity of BERB was shown previously in experimental epilepsy models. We assessed the effect of PALM in a pentylenetetrazole (PTZ)-induced seizure assay in zebrafish, with BERB as an active reference compound. Both alkaloids were isolated from the methanolic root extract of Berberis sibirica by counter-current chromatography, and their ability to cross the blood-brain barrier was determined via quantitative structure-activity relationship assay. PALM exerted antiseizure activity, as confirmed by electroencephalographic analysis, and decreased c-fos and bdnf levels in PTZ-treated larvae. In a behavioral assay, PALM dose-dependently decreased PTZ-induced hyperlocomotion. The combination of PALM and BERB in ED16 doses revealed hyperadditive activity towards PTZ-induced hyperlocomotion. Notably, we have indicated that both alkaloids may exert their anticonvulsant activity through different mechanisms of action. Additionally, the combination of both alkaloids in a 1:2.17 ratio (PALM: BERB) mimicked the activity of the pure extract, which indicates that these two active compounds are responsible for its anticonvulsive activity. In conclusion, our study reveals for the first time the anticonvulsant activity of PALM and suggests the combination of PALM and BERB may have higher therapeutic value than separate usage of these compounds.

A critical review of zebrafish schizophrenia models: Time for validation?

Schizophrenia is a mental disorder that affects 1% of the population worldwide and is manifested as a broad spectrum of symptoms, from hallucinations to memory impairment. It is believed that genetic and/or environmental factors may contribute to the occurrence of this disease. Recently, the zebrafish has emerged as a valuable and attractive model for various neurological disorders including schizophrenia. In this review, we describe current pharmacological models of schizophrenia with special emphasis on providing insights into the pros and cons of using zebrafish as a behavioural model of this disease. Moreover, we highlight the advantages and utility of using zebrafish for elucidating the genetic mechanisms underlying this psychiatric disorder. We believe that the zebrafish has high potential also in the area of precision medicine and may complement the development of therapeutics, especially for pharmacoresistant patients.