Reduction in activity by noxious chemical stimulation is ameliorated by immersion in analgesic drugs in zebrafish

Abstract

Research has recently demonstrated that larval zebrafish show similar molecular responses to nociception to those of adults. Our study explored whether unprotected larval zebrafish exhibited altered behaviour after exposure to noxious chemicals and screened a range of analgesic drugs to determine their efficacy to reduce these responses. This approach aimed to validate larval zebrafish as a reliable replacement for adults as well as providing a high-throughput means of analysing behavioural responses. Zebrafish at 5 days postfertilization were exposed to known noxious stimuli: acetic acid (0.01%, 0.1% and 0.25%) and citric acid (0.1%, 1% and 5%). The behavioural response of each was recorded and analysed using novel tracking software that measures time spent active in 25 larvae at one time. Subsequently, the efficacy of aspirin, lidocaine, morphine and flunixin as analgesics after exposure to 0.1% acetic acid was tested. Larvae exposed to 0.1% and 0.25% acetic acid spent less time active, whereas those exposed to 0.01% acetic acid and 0.1-5% citric acid showed an increase in swimming activity. Administration of 2.5 mg l −1 aspirin, 5 mg l −1 lidocaine and 48 mg l −1 morphine prevented the behavioural changes induced by acetic acid. These results suggest that larvae respond to a noxious challenge in a similar way to adult zebrafish and other vertebrates and that the effect of nociception on activity can be ameliorated by using analgesics. Therefore, adopting larval zebrafish could represent a direct replacement of a protected adult fish with a non-protected form in pain-and nociception-related research.

Responses of larval zebrafish to low pH immersion assay. Comment on Lopez-Luna et al.

“…In contrast to and , Lopez-Luna et al (2017) considered reduced (not increased) activity as evidence of 'alleged pain behavior' in zebrafish exposed to 0.1-0.25% acetic acid. interpreted increased activity and cyclooxygenase-2 gene expression as evidence of nociception in larval zebrafish immersed in 0.0025-0.025% acetic acid.…”

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Lopez -Luna et al (2017) observed behavioral responses of larval zebrafish (Danio rerio) exposed for 10 min to pH 2.6-3.6 when acetic acid (0.01-0.25%) or citric acid (0.1-5%) was added to the tank water in the presence or absence of aspirin (1-2.5 mg l −1 ), morphine sulfate (1-48 mg l −1 ), lidocaine (1-5 mg l −1 ) and flunixin (8-20 mg l −1 ). Fish exposed to 0.1-0.25% acetic acid were less active than controls while those exposed to citric acid and 0.01% acetic acid were more active.

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“…However, cyclooxygenase-2 expression is a non-specific marker of several physiological processes (Wang et al, 2016), meaning its expression is also insufficient evidence of nociception. A critical observation is that larval zebrafish in the study by Lopez-Luna et al (2017) continued to exhibit increased activity when exposed to pH 2.6 in the 5 mg l −1 citric acid experiment. Because of the strong likelihood of acute pathological damage to gills, eyes and other tissues at such low pH (Daye and Garside, 1976), the absence of 'alleged pain behavior' in the citric acid treatment calls into question whether nociception was occurring at all.…”

“…It is, therefore, premature for Lopez-Luna et al (2017) and others Lopez-Luna et al (2017a) investigated the utility of using larval zebrafish as a replacement for adults in nociceptive testing. Five days post-fertilisation larvae were held in a 25-well plate and monitored using a video tracking system.…”

“…However, they misinterpret the results as they state that activity increased in Measurements were taken from the study by Lopez-Luna et al (2017a), where zebrafish larvae at 5 days post-fertilisation were held in 3 l of normal water from the aquarium facility or exposed to a range of drugs, and also following exposure to acetic acid and citric acid. Alkalinity was measured using Methyl Orange.…”