Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage development drugs

Journal of Pharmacological and Toxicological Methods


Introduction: Seizure liability is an adverse property of new candidate drugs typically detected only in later stage pre-clinical safety studies.Consequently, pharmaceutical discovery needs small scale (microplate-based), rapid throughput screens to ‘front-load’ such adverse endpoints in order to reduce associated attrition rates. Of the potential methods available, previously published studies have shown that the quantification of seizure-associated locomotion in the larval zebrafish (Danio rerio) offers high potential for development into such a screen. Here, we present methodology and validation data (on 25 compounds) from a larval zebrafish (Zf) convulsant assay, based on the quantification of high speed locomotion after exposure to a range of test compounds.
Methods: All assays were undertaken in 7 days post fertilization (dpf), WIK-strain Zf larvae, at 27±1 °C. The blinded validation test set consisted of 17 positive and 8 negative controls, based on literature evidence for seizure liability. Initially, a Maximum Tolerated Concentration (MTC) assay was undertaken on each compound to identify the maximum concentration not causing general toxicity, sedation or overt neuromuscular effects. Next, the convulsant assay was undertaken on 5 concentrations from the MTC down, plus a dilution water control. Exposed larvae were videotracked for 1 h, using the Viewpoint Videotrack for Zebrafish™ system, and high speed movements, typically associated with seizure-like locomotor activity, were quantified.
Results: According to classification criteria proposed by the European Centre for the Validation of Alternative Methods (ECVAM), the data generated appeared to offer “sufficient” predictivity (72% overall), particularly considering the potential for throughput and likely positioning within a safety pharmacology front-loading screening cascade.
Discussion: Possible reasons for the misclassifications are discussed, and potential improvements to increase sensitivity and specificity outlined. In all, these initial validation data suggest that this assay offers potential as a medium throughput screen aimed at the early drug discovery detection of this complex safety pharmacological endpoint.
© 2008 Elsevier Inc. All rights reserved.





Hypocretin/Orexin Overexpression Induces An Insomnia-Like Phenotype in Zebrafish

The Journal of Neuroscience


Asmany as 10%of humans suffer chronic sleep disturbances, yet the geneticmechanisms that regulate sleep remainessentially unknown.It is therefore crucial to develop simple and cost-effective vertebratemodels to study the genetic regulation of sleep. The best characterized mammalian sleep/wake regulator is hypocretin/orexin (Hcrt), whose loss results in the sleep disorder narcolepsy and that has also been implicated in feeding behavior, energy homeostasis, thermoregulation, reward seeking, addiction, andmaternal behavior.Here we report that the expression pattern and axonal projections of embryonic and larval zebrafish Hcrt neurons are strikingly similar to those inmammals.We show that zebrafish larvae exhibit robust locomotive sleep/wake behaviors as early as the fifth day of development and that Hcrt overexpression promotes and consolidates wakefulness and inhibits rest. Similar to humans with insomnia, Hcrt-overexpressing larvae are hyperaroused and have dramatically reduced abilities to initiate andmaintain rest at night. Remarkably, Hcrt function is modulated by but does not require normal circadian oscillations in locomotor activity. Our zebrafish model of Hcrt overexpression indicates that the ancestral function of Hcrt is to promote locomotion and inhibit rest and will facilitate the discovery of neural circuits, genes, and drugs that regulate Hcrt function and sleep.



Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation




A major obstacle for the discovery of psychoactive drugs is the inability to predict how small molecules will alter complex behaviors. We report the development and application of a high-throughput, quantitative screen for drugs that alter the behavior of larval zebrafish. We found that the multidimensional nature of observed phenotypes enabled the hierarchical clustering of molecules according to shared behaviors. Behavioral profiling revealed conserved functions of psychotropic molecules and predicted the mechanisms of action of poorly characterized compounds. In addition, behavioral profiling implicated new factors such as ether-a-go-go-related gene (ERG) potassium channels and immunomodulators in the control of rest and locomotor activity. These results demonstrate the power of high-throughput behavioral profiling in zebrafish to discover and characterize psychotropic drugs and to dissect the pharmacology of complex behaviors.

Larval zebrafish locomotor activity assay

(A) At four days post fertilization (dpf), an individual zebrafish larva is pipetted into each well of a 96-well plate with small molecules. Automated analysis software tracks the movement of each larva for 3 days. Each compound is tested on 10 larvae. (B) Locomotor activity of a representative larva. The rest and wake dynamics were recorded, including the number and duration of rest bouts (i.e. a continuous minute of inactivity, (7)), the timing of the first rest bout following a light transition (rest latency), the average waking activity (average activity excluding rest bouts), and the average total activity. Together, these measurements generate a behavioral fingerprint for each compound.

Hierarchical clustering reveals the diversity of drug-induced behaviors

(A) Behavioral profiles are hierarchically clustered to link compounds to behaviors. Each square of the clustergram represents the average relative value (in standard deviations; yellow = higher than controls, blue = lower than controls) for a single behavioral measurement. Dark bars indicate specific clusters analyzed in subsequent figures. (B–F) Normalized waking activity and rest graphs are plotted for behavior-altering compounds (red trace; average of 10 larvae) and representative controls (10 blue traces; average of 10 larvae each). Compounds that altered behavior include the mood stabilizer and anti-epileptic drug sodium valproate (B), the psychotomimetic NMDA antagonist L-701324 (C), the sodium channel agonist pesticide DDT (D), the anti-malarial halofantrine (E), and the calcium channel blocker methoxyverapamil (F).

Unexpected regulators of zebrafish rest/wake states

(A) Podocarpatrien-3-one analogs increase rest latency, the time from light transition to the first rest bout, relative to controls. Error bars represent +/− SEM (B) Many wake-promoting anti-inflammatory and immunomodulating compounds co-cluster (blue—NSAIDs; green—glucocorticoids; pink—PDE inhibitors; yellow—miscellaneous; white—no anti-inflammatory annotation). See Figure S17 for an extended list. (C) A cluster of ERG-blocking compounds specifically increases waking activity at night. (D) Rank sorting the data set by correlation to the ERG blocking cluster results in a significant enrichment of ERG blockers in the top ranks [p<10–13 by the Kolmogorov-Smirnov statistic (see methods)]. Black lines indicate known ERG blockers; red indicates high correlation, green indicates low correlation to the ERG cluster. This analysis also detected potential indirect regulators of ERG function, for example the organophosphate coumaphos (marked with an asterisk), which causes long QT through an unknown mechanism (20).

Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation.

Rihel J, Prober DA, Arvanites A, Lam K, Zimmerman S, Jang S, Haggarty SJ, Kokel D, Rubin LL, Peterson RT, Schier AF.

Part of this publication was performed with ZebraLab and ZebraBox

Eriocaulon buergerianum extract protects PC12 cells and neurons in zebrafish against 6-hydroxydopamine-induced damage

Chinses Medicne


Background: Ericaulon buergerianum (Gujingcao) is an ophthalmic, anti-inflammatory and antimicrobial Chinese
medicinal herb. This study aims to investigate the neuroprotective effects of Ericaulon buergerianum ethanol extract
(EBE) and to elucidate its underlying action mechanism.
Methods: The viability of dopaminergic (DA) neuron in zebrafish was examined by anti-tyrosine hydroxylase (TH)
immunostaining. The locomotor activity of zebrafish was assessed with a digital video tracking system. The viability
and cellular damage of the PC12 cells were determined by MTT and LDH assays respectively. The nuclear
morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33342 dye. Intracellular
nitric oxide (NO) was quantified by DAF-FM diacetate staining. The expression of inducible nitric oxide synthase
(iNOS) was determined by Western blot.
Results: EBE inhibited the 6-OHDA-induced decrease in total distance of movement in zebrafish. Pretreatments of
EBE (25, 50, 100 and 200 μg/ml) increased the viability of 6-OHDA-damaged PC12 cells in a dose dependent
manner. Protection against 6-OHDA-induced nuclear fragmentation and accumulation of apoptotic bodies was also
observed in EBE pretreated cells. Anti-oxidative (inhibition of NO production and iNOS expression in PC12 cells in
vitro) activities of EBE are related to its neuroprotective effects in 6-OHDA-induced DA neuron damage.
Conclusion: EBE exhibited significant neuroprotective activities in zebrafish, including recovery of dopaminergic
neuron loss caused by 6-OHDA in a dose-dependent manner in vivo, inhibition of 6-OHDA-induced decrease of
total distance in movement in zebrafish. The iNOS-NO pathway may be involved.






Toxicity assessment of zebrafish following exposure to CdTe QDs

Journal of Hazardous Materials


CdTe  quantum  dots  (QDs)  are  nanocrystals  of  unique  composition  and properties  that  have  found  many new  commercial  applications;  therefore,their  potential  toxicity  to  aquatic  organisms  has  become  a  hot research  topic.  The  lab  study  was  performed  to  determine  the developmental  and  behavioral  toxicities to  zebrafish  under  continuous exposure  to  low  concentrations  of  CdTe  QDs  (1–400  nM)  coated  with thioglycolic  acid  (TGA).  The  results  show:  (1)  the  120  h  LC50 of 185.9  nM,  (2)  the  lower  hatch  rate  and body length, more malformations, and less heart beat and swimming speed of the exposed zebrafish,(3) the brief burst and a higher basal swimming rate of the exposed zebrafish larvae during a rapidtransition from light-to-dark, and (4) the vascular hyperplasia, vascular bifurcation,vascular crossing and  turbulence  of  the  exposed  FLI-1  transgenic  zebrafish  larvae.





Drug Screening to Treat Early-Onset Eye Diseases: Can Zebrafish Expedite the Discovery?



  The molecular basis of many early-onset eye diseases has been uncovered but the number
of available drug treatments for improving deteriorated vision is still scarce. Consequently, there
is a high demand for new drugs to treat these diseases. This review first provides a brief synopsis
of the utility of zebrafish model for screening drugs with vision benefits. In particular, visual-
motor response (VMR), the activity response of larvae to a change in light stimuli, is proposed to
serve as a simple and efficient tool for screening drugs that may improve vision in various
zebrafish visual mutants. The second part of the review discusses the identification of novel drug
candidates, with particular emphasis on naturally-derived chemicals including traditional
Chinese medicines (TCMs) and nutritional therapies on retinal degenerative diseases. Many of
these chemicals have been used in neuroprotection and/or have been consumed by many
populations for good health and vision; thus, the screening of these chemicals with various
zebrafish visual mutants would expedite the development of novel drugs for treating early-onset
eye diseases.