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

Abstract

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.

Comparative Physiology of Nociception and Pain

“…Evidence for stress-induced analgesia has also been gathered in teleost fishes. In these studies, a stressful event before noxious stimulation prevents the associated behavioral and physiological responses (3,5,10,80,87). The impact of stress can be reversed by the injection of naloxone, an opioid antagonist (10).…”

Ethical considerations in fish research

“…Empirical studies in fishes have also confirmed the presence of nociceptors, demonstrating their properties are very similar to those found in humans and other mammals (Ashley et al ., , ; Mettam et al ., ; Sneddon, , , ). Studies in fishes have demonstrated changes in brain activity during noxious stimuli (Dunlop & Laming, ; Nordgreen et al ., ; Reilly et al ., ; Sneddon, ) and recorded adverse changes in behaviour and physiology during noxious treatments (Dunlop et al ., ; Maximino, ; Mettam et al ., ; Reilly et al ., ; Roques et al ., ; Sneddon, ; Sneddon et al ., , ; White et al ., ) all of which can be prevented by analgesics known to be effective in relieving pain in mammals (Lopez‐Luna et al ., , , , ; Mettam et al ., ; Newby et al ., ; Nordgreen et al ., ; Schroeder & Sneddon, ; Sneddon, , ; Taylor et al ., ). This empirical evidence taken together supports the idea that fishes may experience pain.…”

“…Opioids inhibit neurotransmitter release pre‐synaptically thereby blocking nociceptors but these drugs also block central transmission. Several studies have shown morphine is an effective analgesic in O. mykiss (Baker et al ., ; Jones et al ., ; Sneddon, ) and other species ( e.g ., goldfish Carassius auratus (L. 1758); Nordgreen et al ., ; D. rerio , Lopez‐Luna et al ., ,b,c,d; Magalhaes et al ., ; Schroeder & Sneddon, ; Taylor et al ., ). Morphine has a pharmocokinetically similar action in fishes compared with mammals, however, morphine persists for a prolonged period after administration due to slower excretion rates (half‐life 37 h; Newby et al ., , ).…”

“…Indomethacin injected intraperitoneally (20 μl of 0.2 mg ml −1 ) did reduce pain related responses in adult D. rerio treated with formalin (Magalhaes et al ., ). Aspirin administered via immersion prevented the reduced activity seen in adult and larval D. rerio when subjected to noxious events (Lopez‐Luna et al ., ,b,c,d; Schroeder & Sneddon, ). Future studies need to investigate NSAIDs further to determine the minimum effective dose and explore any unwanted side‐effects since long‐term use of these drugs in mammals, birds and reptiles can result in gastric ulcers or renal disease.…”

“…Lidocaine effectively prevents behavioural and physiological alterations in O. mykiss during subcutaneous injection of acetic acid (Mettam et al ., ). Exposure to lidocaine by emersion also ameliorates substantial changes in the behaviour of adult D. rerio subject to caudal‐fin clipping (Schroeder & Sneddon, ; Deakin et al ., ) and was also effective when used with larval D. rerio exposed to noxious chemical and thermal stimuli (Lopez‐Luna et al ., ,b,c,d). Infiltration of rainbow trout with lidocaine appears to damage skeletal muscle and connective tissue with higher concentrations of (10 mg kg −1 ) but not to cause adverse behavioural side‐effects (20 mg kg −1 ), compared with saline infiltration (Chatigny et al ., 2018a,b).…”