Functional brain imaging in larval zebrafish for characterising the effects of seizurogenic compounds acting via a range of pharmacological mechanisms

Abstract

Background and Purpose: Functional brain imaging using genetically encoded Ca 2+ sensors in larval zebrafish is being developed for studying seizures and epilepsy as a more ethical alternative to rodent models. Despite this, few data have been generated on pharmacological mechanisms of action other than GABA A antagonism.Assessing larval responsiveness across multiple mechanisms is vital to test the translational power of this approach, as well as assessing its validity for detecting unwanted drug-induced seizures and testing antiepileptic drug efficacy.Experimental Approach: Using light-sheet imaging, we systematically analysed the responsiveness of 4 days post fertilisation (dpf; which are not considered protected under European animal experiment legislation) transgenic larval zebrafish to treatment with 57 compounds spanning more than 12 drug classes with a link to seizure generation in mammals, alongside eight compounds with no such link. Key Results: We show 4dpf zebrafish are responsive to a wide range of mechanisms implicated in seizure generation, with cerebellar circuitry activated regardless of the initiating pharmacology. Analysis of functional connectivity revealed compounds targeting cholinergic and monoaminergic reuptake, in particular, showed phenotypic

Differential Electrographic Signatures Generated by Mechanistically-Diverse Seizurogenic Compounds in the Larval Zebrafish Brain

“…For this work, 4dpf transgenic zebrafish, of unknown sex, with a pan-neuronal Ca 2+ sensor (elavl3:GCaMP6s) were used. This transgenic line allowed us to compare the electrographic recording data generated here, with functional imaging data obtained from a separate study (Winter et al, 2021). 4dpf larval zebrafish were selected as these are not considered protected vertebrates under European animal legislation and as such are ethically preferable to the use of older, protected, animals.…”

“…Test compounds were selected based upon their known seizurogenic potential in mammals, including humans, as defined by case studies of seizure incidence and assays in model organisms as outlined in Table 1. Appropriate exposure concentration ranges ensuring CNS responsiveness without generalised toxicity were also selected based upon previously published data (Winter et al, 2021).…”

“…Of these models, the larval zebrafish offers considerable potential as a highly genetically tractable alternative for screening epilepsy related genes and for the study of genetic mutation-induced spontaneous seizures (Baraban et al, 2013a;Griffin et al, 2021). To date, zebrafish have been relatively widely used for assessing drug-induced neural activity, and various studies have described the effects of seizurogenic chemicals on their behaviour (Afrikanova et al, 2013;Alfaro et al, 2011;Baraban et al, 2013b;Berghmans et al, 2007;Winter et al, 2021), electrophysiology (Afrikanova et al, 2013;Baraban et al, 2013bBaraban et al, , 2007Cho et al, 2017;Copmans et al, 2019;Griffin et al, 2017;Hortopan et al, 2010;Hunyadi et al, 2017;Kim et al, 2010b;Liu and Baraban, 2019), and functional imaging phenotypes (Burgstaller et al, 2019;Burrows et al, 2020;Ghannad-Rezaie et al, 2019;Winter et al, 2021Winter et al, , 2017. Electrophysiological assessments, in particular, offer extremely high temporal resolution and allow direct comparison of zebrafish-derived data with that obtained from neurophysiological assessments undertaken in mammals.…”

Estrogens regulate early embryonic development of the olfactory sensory system via estrogen-responsive glia

“…Given that the estrogens/EROB cell cascade crucially and selectively regulates olfactory glomerular development, we examined whether alteration of estrogen signalling affects local neuronal activity in the OB in the developing embryos. To do that, we employed a calcium sensor elavl3(huC):GCaMP6s (hereafter elavl3:GCaMP6s) transgenic zebrafish model in combination with light sheet microscopy (LSM) and a GCaMP6s image processing pipeline established previously in our laboratory (details in Materials and Methods) (Winter et al, 2021(Winter et al, , 2017. Using this system, a full brain volume of GCaMP6s images can be captured that allows us to extract region of interest (ROI)-specific neuronal activity data.…”

“…The detailed procedures for sample preparation for GCaMP6s imaging and for imaging acquisition using a custom-built LSM are described in Winter et al (2021Winter et al ( , 2017. A full brain volume of GCaMP6s images for zebrafish embryo larvae was captured in around 1.8 s (10 horizontal plane optical zslices in 24 μm steps).…”