Pharmacological profiling of brain activity in zebrafish
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Background and Purpose
Epilepsy is a neurological condition characterized by recurring seizures and neuronal hyperexcitability. Cell-based high-throughput screening applications have been essential for drug development and discovering novel biological processes. However, cell-based screens do not provide information on how drug-targeted pathways are integrated into a whole animal. Our objective was to develop and evaluate a screening application using zebrafish larvae to identify signalling mechanisms that modulate neural activity.
Experimental Approach
We developed an in vivo automated high-content screening assay using zebrafish larvae expressing the calcium sensor CaMPARI (calcium-modulated photoactivatable ratiometric integrator) in neurons. This assay can quantify neural activity of multiple individual larvae per well in a 96-well format. We quantified neural activity in 8725 individual larvae, in response to 1292 different drugs to identify molecules that protect against convulsant-induced neuronal hyperexcitability.
Key Results
The assay was effective at identifying drugs that target diverse neurotransmitter signalling systems. While some commonly used anti-convulsants (e.g. phenytoin, carbamazepine, valproic acid) had poor activity in the assay, Kv7 potassium channel activators were consistently effective (ICA-069673, ICA-27243, ICA-110381, retigabine, and ML213). Many compounds approved for treatment of other conditions, including amitriptyline (depression), cyclobenzaprine (muscle spasm), clomipramine (obsessive-compulsive disorder) and ganaxolone (seizures), also strongly suppressed excitability in the assay.
Conclusion and Implications
Neuronal CaMPARI expression in zebrafish larvae is a powerful tool for plate-based compound library screening to identify drugs that suppress hyperexcitability in vivo .
Bullet Point Summary
What is already known
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CaMPARI is an integrative Ca 2+ sensor that can be used to identify active neurons.
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Kv7 activators (retigabine, ML213, and ICA-069673) are effective at reducing convulsant-induced (4-AP) neuronal hyperexcitability.
What this study adds
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An automated in vivo high-content drug screening assay to quantify neural activity.
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A series of drug targets that influence convulsant-induced hyperexcitability.
Clinical significance
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Our new tool will help identify novel compounds and signalling mechanisms that could be pursued as therapeutic targets for diseases involving electrical hyperexcitability.
