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.

Fig 1. Diagram of the experimental procedures and group names (see Table 1).

Assessment of the impact of air emersion (a), alarm pheromone (substance) (b) and caffeine (c) on the nociceptive-like responses of larval zebrafish. Video recording 1 and 2 indicate the pre- and post-stimulation video recordings, respectively. The names indicate the moment when the challenges were applied or when the substances were introduced in the tank water so only those groups in brackets received that particular treatment.

Fig 2. Velocity shown by 5dpf zebrafish during a 10-minute period before (white boxes) and after (grey boxes) exposure to different treatments.

Each box shows the lower and upper quartile values and the central horizontal black line indicates the median value. The error bars indicate the variation for the rest of the data and outliers are indicated as white dots. Significant differences between pre- and post-stimulation behaviour are indicated with an asterisk (Wilcoxon signed-rank test, P<0.05; n = 10 per group).

Fig 3. Time spent active shown by 5dpf zebrafish during a 10-minute period before (white boxes) and after (grey boxes) exposure to different treatments.

Each box shows the lower and upper quartile values and the central horizontal black line indicates the median value. The error bars indicate the variation for the rest of the data and outliers are indicated as white dots. Significant differences between pre- and post-stimulation behaviour are indicated with an asterisk (Wilcoxon signed-rank test, P<0.05; n = 10 per group).

Fig 4. Change in velocity (%) shown by 5dpf zebrafish exposed to different treatments.

Each box shows the lower and upper quartile values and the central horizontal black line indicates the median value. The error bars indicate the variation for the rest of the data and outliers are indicated as white dots. Significant differences between groups and the control groups are indicated with an asterisk (control in the air emersion challenge: CO/AE), a hashtag (control in the alarm pheromone challenge: CO/AP) or a cross (control in the caffeine challenge: CO/CF) (Wilcoxon signed-rank test, P<0.0031).

Fig 5. Change in time spent active (%) shown by 5dpf zebrafish exposed to different treatments.

Each box shows the lower and upper quartile values and the central horizontal black line indicates the median value. The error bars indicate the variation for the rest of the data and outliers are indicated as white dots. Significant differences between groups and the control groups are indicated with an asterisk (control in the air emersion challenge: CO/AE), a hashtag (control in the alarm pheromone challenge: CO/AP) or a cross (control in the caffeine challenge: CO/CF) (Wilcoxon signed-rank test, P<0.0031).