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

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 post-fertilization 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.

Effects of Pro-Tex on zebrafish (Danio rerio) larvae, adult common carp (Cyprinus carpio) and adult yellowtail kingfish (Seriola lalandi).

Aquaculture practices bring several stressful events to fish. Stressors not only activate the hypothalamus-pituitary-interrenal-axis, but also evoke cellular stress responses. Up-regulation of heat shock proteins (HSPs) is among the best studied mechanisms of the cellular stress response. An extract of the prickly pear cactus (Opuntia ficus indica), Pro-Tex, a soluble variant of TEX-OE(®), may induce expression of HSPs and reduce negative effects of cellular stress. Pro-Tex therefore is used to ameliorate conditions during stressful aquaculture-related practices. We tested Pro-Tex in zebrafish (Danio rerio), common carp (Cyprinus carpio L.) and yellowtail kingfish (Seriola lalandi) exposed to aquaculture-relevant stressors (thermal stress, net confinement, transport) and assessed its effects on stress physiology. Heat shock produced a mild increase in hsp70 mRNA expression in 5-day-old zebrafish larvae. Pro-Tex increased basal hsp70 mRNA expression, but decreased heat-shock-induced expression of hsp70 mRNA. In carp, Pro-Tex increased plasma cortisol and glucose levels, while it did not affect the mild stress response (increased plasma cortisol and glucose) to net confinement. In gills, and proximal and distal intestine, stress increased hsp70 mRNA expression; in the distal intestine, an additive enhancement of hsp70 mRNA expression by Pro-Tex was seen under stress. In yellowtail kingfish, Pro-Tex reduced the negative physiological effects of transport more efficiently than when fish were sedated with AQUI-S(®). Overall, our data indicate that Pro-Tex has protective effects under high levels of stress only. As Pro-Tex has potential for use in aquaculture, its functioning and impact on health and welfare of fish should be further studied.

Impact of stress, fear and anxiety on the nociceptive responses of larval zebrafish.

Both adult and larval zebrafish have been demonstrated to show behavioural responses to noxious stimulation but also to potentially stress- and fear or anxiety- eliciting situations. The pain or nociceptive response can be altered and modulated by these situations in adult fish through a mechanism called stress-induced analgesia. However, this phenomenon has not been described in larval fish yet. Therefore, this study explores the behavioural changes in larval zebrafish after noxious stimulation and exposure to challenges that can trigger a stress, fear or anxiety reaction. Five-day post fertilization zebrafish were exposed to either a stressor (air emersion), a predatory fear cue (alarm substance) or an anxiogenic (caffeine) alone or prior to immersion in acetic acid 0.1%. Pre- and post-stimulation behaviour (swimming velocity and time spent active) was recorded using a novel tracking software in 25 fish at once. Results show that larvae reduced both velocity and activity after exposure to the air emersion and alarm substance challenges and that these changes were attenuated using etomidate and diazepam, respectively. Exposure to acetic acid decreased velocity and activity as well, whereas air emersion and alarm substance inhibited these responses, showing no differences between pre- and post-stimulation. Therefore, we hypothesize that an antinociceptive mechanism, activated by stress and/or fear, occur in 5dpf zebrafish, which could have prevented the larvae to display the characteristic responses to pain.