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

Date Published: August 2, 2017

Publisher: Public Library of Science

Author(s): Javier Lopez-Luna, Qussay Al-Jubouri, Waleed Al-Nuaimy, Lynne U. Sneddon, Zongbin Cui.


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.

Partial Text

Nociception is the sensory mechanism used to perceive actual or potential tissue damage. The neurons that mediate nociceptive information are called nociceptors. Fish can perceive and respond to a wide range of stimuli, including mechanical [1], thermal [2], electrical [3] or chemical [4]. Recent investigations have reported the presence of these neurons in fish [1,5], which, along with the findings of nociceptive pathways and brain activity [6,7] and the regulation of novel candidate genes after a nociceptive event [8], suggest that fish are capable of nociception. Moreover, changes in the behaviour have been recorded after exposure to noxious stimulation [9–11] and these have been ameliorated by administering analgesia [5].

The present study has shown for the first time that larval zebrafish are significantly affected by stress, fear, and anxiety treatments that modulate the response to a noxious stimulus. Exposure to acetic acid for 10 minutes elicited significant behavioural changes in 5dpf zebrafish, with fish showing higher median swimming velocity (mm/s) and lower median time spent active (%) compared with the control group. Fish exposed to air emersion and diazepam swam slower and for less time, whereas those administered with caffeine only had a lower median velocity. Animals exposed to air emersion, diazepam and caffeine prior to the acetic acid challenge did not display these changes.

The novel results presented in this study demonstrate that 5dpf zebrafish showed altered behavioural responses after exposure to a potentially noxious stimulus and that these responses were inhibited by stress- and fear-eliciting situations. Therefore, this suggests the presence of a modulation mechanism of nociception in larval zebrafish, which is activated under potentially threatening or aversive situations. We believe that these findings are also relevant to welfare and handling in experiments involving the use of fish. Since these unprotected 5dpf larval zebrafish display similar behavioural responses to both noxious treatment and the various drugs tested in the present study, they could be proposed as a suitable alternative to replace adult zebrafish in nociception and analgesic studies. These results may propose the question as to whether 5dpf zebrafish should be protected if they show similar responses to potentially painful stimuli, however, further evidence is required such as brain processing mechanisms, altered future motivational state and an ability to learn to avoid noxious stimuli (see Sneddon et al. 2014) before this question can be fully answered.




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