As many as 9% of epileptic seizures occur as a result of drug toxicity.
Identifying compounds with seizurogenic side effects is imperative for assessing
compound safety during drug development, however, multiple marketed drugs
still have clinical associations with seizures. Moreover, current approaches for
assessing seizurogenicity, namely rodent EEG and behavioural studies, are
highly resource intensive. This being the case, alternative approaches have
been postulated for assessing compound seizurogenicity, including in vitro, in
vivo, and in silico methods.
In this thesis, experimental work is presented supporting the use of larval
zebrafish as a candidate model organism for developing new seizure liability
screening approaches. Larval zebrafish are translucent, meaning they are
highly amenable to imaging approaches while offering a more ethical alternative
to mammalian research. Zebrafish are furthermore highly fecund facilitating
capacity for both high replication and high throughput. The primary goal of this
thesis was to identify biomarkers in larval zebrafish, both behavioural and
physiological, of compounds that increase seizure liability.
The efficacy of this model organism for seizure liability testing was assessed
through exposure of larval zebrafish to a mechanistically diverse array of
compounds, selected for their varying degrees of seizurogenicity. Their central
nervous systems were monitored using a variety of different techniques
including light sheet microscopy, local field potential recordings, and
behavioural monitoring. Data acquired from these measurements were then
analysed using a variety of techniques including frequency domain analysis,
clustering, functional connectivity, regression, and graph theory. Much of this
analysis was exploratory in nature and is reflective of the infancy of the field.
Experimental findings suggest that larval zebrafish are indeed sensitive to a
wide range of pharmacological mechanisms of action and that drug actions are
reflected by behavioural and direct measurements of brain activity. For
example, local field potential recordings revealed electrographic responses akin
to pre-ictal, inter-ictal and ictal events identified in humans. Ca2+ imaging using
light sheet microscopy found global increases in fluorescent intensity and
functional connectivity due to seizurogenic drug administration. In addition,
[2]
further functional connectivity and graph analysis revealed macroscale network
changes correlated with drug seizurogenicity and mechanism of action. Finally,
analysis of swimming behaviour revealed a strong correlation between high speed swimming behaviours and administration of convulsant compounds.
In conclusion, presented herein are data demonstrating the power of functional
brain imaging, LFP recordings, and behavioral monitoring in larval zebrafish for
assessing the action of neuroactive drugs in a highly relevant vertebrate model.
These data help us to understand the relevance of the 4 dpf larval zebrafish for
neuropharmacological studies and reveal that even at this early developmental
stage, these animals are highly responsive to a wide range of neuroactive
compounds across multiple primary mechanisms of action. This represents
compelling evidence of the potential utility of larval zebrafish as a model
organism for seizure liability testing
