4-dimensional functional profiling in the convulsant-treated larval zebrafish brain

This is the final version of the article. Available from Springer Nature via the DOI in this record.Functional neuroimaging, using genetically-encoded Ca(2+) sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.This work was funded by the Biological and Biotechnology Research Council (CASE studentship BB/L502510/1, with AstraZeneca Safety Health and Environment), and by the University of Exeter and AstraZeneca

A combined human in silico and CRISPR/Cas9-mediated in vivo zebrafish based approach to provide phenotypic data for supporting early target validation

This is the final version. Available on open access from Frontiers Media via the DOI in this record. Data availability statement: The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding authors.The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts as part of the process for validating potential new drug targets. Traditional approaches (e.g., genetically-modified mice) are optimal for assessing small numbers of genes, but less practical when multiple genes are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation, by providing more confidence in target relevance and facilitating gene selection for definitive loss of function studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including changes in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports further investigation of the potential roles of API5, HSPB7, and LMO2 in human cardiovascular disease. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.Royal SocietyAstraZenec

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

This is the final version. Available on open access from Wiley via the DOI in this recordData availability statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.Background and Purpose Functional brain imaging using genetically encoded Ca2+ 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 GABAA 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 consistency broadly mapping onto what is known about neurotransmitter‐specific circuitry in the larval zebrafish brain. Many seizure‐associated compounds also exhibited altered whole brain functional connectivity compared with controls. Conclusions and Implications This work represents a significant step forward in understanding the translational power of 4dpf larval zebrafish for use in neuropharmacological studies and for studying the events driving transition from small‐scale pharmacological activation of local circuits, to the large network‐wide abnormal synchronous activity associated with seizures.Biotechnology and Biological Sciences Research Council (BBSRC)National Centre for the Replacement Refinement and Reduction of Animals in ResearchUniversity of ExeterMedical Research Council (MRC)AstraZenecaEuropean Unio

Sonoelectrochemical detection of copper within industrial effluent: A critical assessment

The utility of combining an ultrasound probe with conventional electrochemical stripping methodology has been assessed for the mercury free determination of copper within industrial effluent. The introduction of an ultrasound field within the cell during the accumulation stage is shown to significantly enhance the sensitivity of the technique. Two compositionally distinct samples were assessed and the results compared with those obtained from in house and independent analysis (ICP). A brief review of the electroanalytical alternatives to mercury electrodes with regard to copper determination has been included and the various merits and limits of the sonoelectrochemical approach compared and appraised