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FP7

FISH Report Summary

Project ID: 628506
Funded under: FP7-PEOPLE
Country: United Kingdom

Final Report Summary - FISH (Fish Indicatiors of Stress and Health (FISH): Validating the use of embryos and yolk sac larval zebrafish as a model for the study of nociception and veterinary drug testing)

The Marie Curie FISH project (Fish Indicators of Stress and Health – Project IEF 628506) aimed to reduce the number of adult zebrafish used in scientific procedures by replacing adults with non-protected pre-feeding larval zebrafish at the University of Liverpool, UK. Zebrafish are currently a model species for a variety of disciplines including nociception or pain research. Nociception, the detection of potentially harmful stimuli, is the basic mechanism underlying the sensation of pain. Recent investigations have demonstrated that teleost fish have nociceptors, receptors to detect potentially painful stimuli, which are very similar to those found in mammals. In addition, previous studies have identified multiple subtypes of nociceptors in zebrafish suggesting a similar organization of molecular nociceptive circuits between mammals and zebrafish even as early as 1-3 days post fertilisation (dpf). Therefore, the main objective of this study was to explore the use of these young unprotected forms as a valid replacement for adult fish through quantifiable behavioural measurements and to test potential analgesics to inform the development of analgesic protocols. We also aimed to establish a high-throughput approach to test analgesic drugs using these young forms of zebrafish, which should ameliorate the behavioural responses. We also sought to characterise the molecular pathways of nociception through a genome-wide analysis of gene expression of the central nervous system using RNASeq on Illumina. We also aimed to produce novel data on brain activity using light sheet fluorescence microscopy in unprotected zebrafish exposed to innocuous and noxious stimuli.
The first experiment focused on the experimental set-up of the stimulatory systems. To observe the behavioural changes resulting from noxious stimulation, a novel behavioural scoring software was developed in collaboration with the Department of Electrical Engineering and Electronics. This software can quantify the movements of 25 larval zebrafish (YSL) simultaneously in multiwell plates and the resulting data was analysed to determine multiple parameters related to larval motion. This tracking software was based on an object automated detection and tracking using a monitoring algorithm. Both the chemical and thermal noxious stimulations were completed, using a range of irritant agents (acetic and citric acids and CO2) and both cold and hot water temperatures (7, 10, 15, 30, 35 and 40 ⁰C). However, the electrical and mechanical systems proved unfeasible to develop due to technical difficulties where it proved impossible to obtain a similar voltage across the wells and the adoption of larger wells for tracking software use meant it was not possible to mechanically stimulate all larvae at one time. YSL demonstrated reduced activity after noxious stimulations and thresholds for chemical and thermal stimulation were identified (e.g. 0.01% acetic acid). Additionally, larvae exposed to the highest concentrations of acetic acid spent less time active compared to controls, whereas those exposed to citric acid showed an increase in the swimming activity. Larvae exposed to high and low temperatures (7, 10, 35 and 40 ⁰C) provoked a significant change in the swimming activity, demonstrating the utility of thermal and chemical noxious stimuli. Using these results, a range of analgesic drugs (aspirin, lidocaine, morphine and flunixin) were screened at different doses to determine their efficacy to ameliorate the responses observed after stimulation. This approach aimed to provide a relatively high-throughput means of analysing behavioural responses to potential analgesics and identified 2.5 mg/l of aspirin, 5 mg/l of lidocaine and 48 mg/l of morphine via immersion as the most effective. The modulation of the nociceptive-like responses of YSL to stress- (air immersion) and fear/anxiety- (alarm pheromone and caffeine) situations was designed to explore if there are differential responses to stress, fear and nociception. Stressful (air immersion) and the fear/anxiety-elicit (alarm pheromone and caffeine) situations reduced the negative behavioural response evoked by the nociceptive stimulus (acetic acid).
Preliminary studies on the genomic responses to nociception in YSL demonstrated there were 141 genes in common with adult brains taken from noxiously stimulated genes. Many of these transcripts that were regulated in response to chemical nociception have known functions in mammalian pain whereas some are novel and may provide avenues for future studies in collaboration with the Centre for Genomic Research, Liverpool. Initial pilot work in collaboration with Marcello in the Centre for Cell Imaging, Liverpool, using light sheet fluorescence was successful using a novel transgenic which expresses fluorescence linked to calcium signalling in the optic tectum. Technically the images were shaded by the eyes so only brain areas in the optic tectum were visible. A new collaboration with Ahrens and Keller, Janelia Farm USA, allowed us to adopt a novel calcium sensor transgenic which fluoresces in the whole brain (CaMPARI line). Using two photon fluorescence microscopy we have identified several brain areas in the forebrain, midbrain and hindbrain responsive to noxious stimulation.
These results suggest that unprotected 5dpf larval zebrafish respond by reducing activity which is also seen in adult zebrafish and other vertebrates after a painful challenge. The changes in activity affected by nociception can be ameliorated by using a range of immersion analgesics. There are global gene expression changes in whole larvae that are comparable with results from adult brains and brain activity in vivo is observed in 5dpf fish. Therefore, this demonstrates zebrafish are potentially a valid replacement of a protected adult fish with a non-protected larval fish in pain and nociception related research. Analgesic protocols in fish are lacking, therefore, the information provided by this project can assist in informing the development of pain-relief protocols which would be an important refinement in zebrafish research. We envisage three manuscripts being published from this project as a marker of success and these research findings can enable the justification of the adoption of 5dpf zebrafish to replace adults in nociceptive testing.
Marie Curie Fellow: Dr Javier Lope Luna, E-mail jlopez@liverpool.ac.uk
Supervisor: Dr Lynne U. Sneddon, E-mail: lsneddon@liverpool.ac.uk
Web Page: http://www.liv.ac.uk/integrative-biology/research/adaptation-to-environmental-change/fish-indicators-stress-health/

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Record Number: 187044 / Last updated on: 2016-07-18