Social buffering of fear in zebrafish–experiment I

In this study we developed a behavioural paradigm to investigate SB in adult zebrafish when exposed to AS. To do so, focal fish (inside of the test tank) were exposed to AS in the absence or presence of shoal cues [olfactory (O) – i.e. shoal water added to the test tank; visual (V) – i.e. sight of shoal in the demonstrator (demo) tank; see Fig. 1 and Methods for details on the protocol] in order to test the hypothesis that the presence of conspecific cues decreases the fear response of the focal fish towards AS, comparatively to being alone. Focal fish behaviour was video recorded (see Fig. 1 and Supplementary Fig. 1A,B) both before (5 min baseline) and during exposure (30 min test) to AS, and stereotypic behavioural responses to AS (i.e. erratic movement and freezing42) were quantified using a custom-made software (xyz2b; see https://github.com/joseaccruz/xyz2b for detailed information and download). The xyz2b validation against a human observer for erratic movement and freezing quantifications showed the better performance of xyz2b in measuring freezing: r = 0.97, p < 0.001 (see Supplementary Fig. 2A,B and Supplementary Information for further details). Since freezing was the most frequent and consistent behaviour expressed throughout the 30 min test (Supplementary Fig. 2C; see Supplementary Information for further details), it was used as our behavioural measure of the AS-evoked fear response in all experiments.

Figure 1: Behavioural paradigm for the study of SB in zebrafish: experimental setup and behavioural protocol schematics. (A) Schematic representation of the SB experimental setup. Test tank (focal fish) and demo tank (with shoal present or absent depending on the treatment) were side-by-side and physically separated. AS or water (depending on treatment) were administered through a PVC tubing with the help of a syringe. Behaviour was video recorded with side and top cameras. (B) Schematic representation of the behavioural protocol. On day 1 focal fish were left to habituate overnight to the experimental setup. On the following day (day 2), behavioural video recording was initiated with 5 min of baseline (Bl), followed by 30 min of test. Full size image

A two-factor 2 × 2 experimental design [social: alone, SB (O+V) (olfactory + visual shoal cues present); threat: exposed to AS, exposed to water] generated four treatments (Fig. 2A): Alone_Ctrl (alone control exposed to water); Alone_AS (alone exposed to AS); SB (O + V)_Ctrl (control with shoal cues and exposed to water); SB (O + V)_AS (with shoal cues and exposed to AS). Shoals of 8 conspecifics (4 females:4 males) were used since they represent medium-sized shoals observed in zebrafish floodplain habitats43,44 – the same holds for experiment II. Focal fish in the SB (O + V)_AS treatment presented significantly lower freezing behaviour than fish that were administered with the AS when alone (Alone_AS) during the whole 30 min exposure [repeated measures ANOVA with “time” as within-subject factor; time: F (2,76) = 4.141, p = 0.020; treatment: F (1,38) = 38.798, p < 0.001; treatment ∗ time: F (2,76) = 0.006, p = 0.994; corrected p values (p’) after sequential Bonferroni correction for multiple comparisons (LSD post hoc) are reported – 0–10 min: p’ < 0.001; 10–20 min: p’ < 0.001; 20–30 min: p’ < 0.001; Fig. 2D]. Furthermore, there were no differences between the two control treatments Alone_Ctrl and SB (O + V)_Ctrl during the baseline (Bl), and the administration of water in the control treatments did not elicit freezing behaviour [repeated measures ANOVA with “time” as within-subject factor; time: F (1.0, 76.0) = 68.418, p < 0.001; treatment: F (3, 76) = 20.157, p < 0.001; treatment ∗ time: F (3.0, 76.0) = 32.714, p < 0.001; *p’ < 0.05; **p’ < 0.01 and ***p’ < 0.001; Fig. 2C]. Importantly, the erratic movement was also significantly lower in the SB (O + V)_AS treatment comparatively to the Alone_AS treatment, but only in the first 5 minutes of the test, which further enhances the freezing parameter as the best behavioural readout to measure SB to AS-elicited responses in zebrafish (Supplementary Fig. 2D,E). These results show that the presence of conspecific cues reduces the fear response to AS, thus demonstrating the occurrence of social buffering of fear response in zebrafish (see Fig. 2D, Supplementary Movie 1 and Supplementary Movie 2).

Figure 2: Experiment I. (A) Schematic representation of the behavioural treatments. From left to right: Alone_Ctrl–alone focal fish (red outline) administered with water; Alone_AS–alone focal fish (red filling) administered with AS; SB (O + V)_Ctrl–focal fish (green outline) administered with water and exposed simultaneously to shoal water and a shoal of 8 conspecifics, and SB (O + V)_AS–focal fish (green filling) administered with AS and exposed simultaneously to shoal water and a shoal of 8 conspecifics. Grey and red drops represent water and AS administration, respectively. (B) 3D plots representative of each behavioural treatment. Each 3D plot represents the first 5 min after AS onset for the focal fish closest to the mean in each treatment. n = 20 per treatment. Total freezing percentages presented (red circles) in each 3D plot are (from left to right): Alone_Ctrl–1.95%; Alone_AS–56.24%; SB (O + V)_Ctrl–0.00% and SB (O + V)_AS–23.32%. (C) Freezing % in baseline (Bl) vs. first 5 min after AS onset (AS). n = 20 per treatment. Mean ± SEM are shown. *p’ < 0.05; **p’ < 0.01 and ***p’ < 0.001. (D) Freezing % over the 30 min test in 10 min bins. n = 20 per treatment. Mean ± SEM are shown. *p’ < 0.05; **p’ < 0.01 and ***p’ < 0.001. Full size image

Effectiveness of visual and olfactory shoal cues at inducing social buffering–experiment II

SB can be mediated by social cues associated with different sensory modalities7,8,9. To our knowledge, research focused on disentangling the contribution of each sensory cue and its lasting effectiveness on the SB process has never been conducted before. Since in our first experiment the combined presentation of olfactory and visual shoal cues was effective in eliciting SB, we decided to investigate the specific role of each of these two sensory channels in this phenomenon. Although other sensory modalities may also modulate SB – tactile stimulation is known to reduce fear in zebrafish45 – we focused on the olfactory and visual channels, as they were easier to control experimentally. Thus, in a second experiment, we separately tested the effectiveness of olfactory (O) and visual (V) shoal cues on the fear response to the AS. For this purpose, zebrafish were exposed to AS in one of four treatments: Alone_AS; SB (O)_AS; SB (V)_AS; and SB (O + V)_AS (Fig. 3A; see Methods for further details). Whereas there were no differences in baseline freezing levels (i.e. before exposure to AS), either olfactory only, visual only, or both olfactory and visual shoal cues together were effective in reducing freezing behaviour after exposure to AS [repeated measures ANOVA with “time” as within-subject factor; time: F (1, 76) = 74.352, p < 0.001; treatment: F (3, 76) = 10.137, p < 0.001; treatment ∗ time: F (3, 76) = 8.016, p < 0.001. *p’ < 0.05; **p’ < 0.01 and ***p’ < 0.001; Fig. 3B]. However, a more detailed analysis over time (30 min exposure) showed that although both olfactory and visual cues were equally effective in the first 10 min of the test, the visual cue was significantly more effective in decreasing the freezing response in the last 20 min [repeated measures ANOVA with “time” as within-subject factor; time: F (1.6,120.625) = 11.723, p < 0.001; treatment: F (3,76) = 10.284, p < 0.001; treatment ∗ time: F (4.8,120.625) = 3.871, p = 0.003; corrected p values (p’) after sequential Bonferroni correction for multiple comparisons (LSD post hoc): O vs. V, 0–10 min: p’ = 0.194; O vs. V, 10–20 min: p’ = 0.021; O vs. V, 20–30 min: p’ = 0.009; Fig. 3C]. Moreover, in the last 20 min the visual cue was as efficient as the visual and olfactory cues combined, corroborating the higher effectiveness of the sight of shoal to the buffering phenomenon in long-lasting exposures to threat [corrected p values (p’) after sequential Bonferroni correction for multiple comparisons (LSD post hoc): V vs. O + V, 0–10 min: p’ = 0.281; V vs. O + V, 10–20 min: p’ = 0.338; V vs. O + V, 20–30 min: p’ = 0.788; O vs. O + V, 0–10 min: p’ = 0.019; O vs. O + V, 10–20 min: p’ = 0.001; O vs. O + V, 20–30 min: p’ = 0.004; Fig. 3C].

Figure 3: Experiment II. (A) Schematic representation of the behavioural treatments. From left to right: Alone_AS–alone focal fish (red filling) administered with AS; SB (O)_AS–focal fish (orange filling) administered with AS and exposed to shoal water of 8 conspecifics; SB (V)_AS–focal fish (blue filling) administered with AS and exposed to a shoal of 8 conspecifics and SB (O + V)_AS–focal fish (green filling) administered with AS and exposed simultaneously to shoal water and a shoal of 8 conspecifics. Red drops represent AS administration. (B) Freezing % in baseline (Bl) vs. first 5 min after AS onset (AS). n = 20 per treatment. Mean ± SEM are shown. *p’ < 0.05; **p’ < 0.01 and ***p’ < 0.001. (C) Freezing % over the 30 min test in 10 min bins. n = 20 per treatment. Mean ± SEM are shown. *p’ < 0.05; **p’ < 0.01 and ***p’ < 0.001. Full size image

Smaller shoals are equally effective in promoting social buffering–experiment III

Given the greater efficacy of the sight of the shoal to the buffering behaviour, on a third experiment we tested if shoal size modulates the effectiveness of the visual cue in reducing the fear response. Shoal size was manipulated (mixed-sex shoals of 2, 4 and 8 conspecifics) such that 8 experimental treatments were generated: Alone_Ctrl; Alone_AS; SB (2)_Ctrl; SB (2)_AS; SB (4)_Ctrl; SB (4)_AS; SB (8)_Ctrl; SB (8)_AS – where controls were exposed to water (Fig. 4A; see Methods for further details). We verified that conspecific number did not influence freezing responses during the entire duration of the test (Fig. 4C), and that a shoal of 2 conspecifics was enough to significantly decrease freezing behaviour in response to the AS [repeated measures ANOVA with “time” as within-subject factor; time: F (1.6,122.349) = 19.234, p < 0.001; treatment: F (3,76) = 9.822, p < 0.001; treatment ∗ time: F (4.8,122.349) = 1.232, p = 0.299; corrected p values (p’) after sequential Bonferroni correction for multiple comparisons (LSD post hoc): alone vs. shoal 2, 0–10 min: p’ < 0.001; alone vs. shoal 2, 10–20 min: p’ = 0.003; alone vs. shoal 2, 20–30 min: p’ = 0.016; Fig. 4C]. Again, we found no differences between control treatments and the administration of water did not elicit freezing behaviour [repeated measures ANOVA with “time” as within-subject factor; time: F (1.0,152.0) = 131.542, p < 0.001; treatment: F (7,152) = 22.454, p < 0.001; treatment * time: F (7.0,152.0) = 22.358, p < 0.001. *p’ < 0.05; **p’ < 0.01 and ***p’ < 0.001; Fig. 4B].

Figure 4: Experiment III. (A) Schematic representation of the behavioural treatments. From left to right (top panel): Alone_Ctrl–alone focal fish (red outline) administered with water; Alone_AS–alone focal fish (red filling) administered with AS; SB (2)_Ctrl–focal fish (dark blue outline) administered with water and exposed to a shoal of 2 conspecifics; SB (2)_AS–focal fish (dark blue filling) administered with AS and exposed to a shoal of 2 conspecifics. From left to right (bottom panel): SB (4)_Ctrl–focal fish (sea blue outline) administered with water and exposed to a shoal of 4 conspecifics; SB (4)_AS–focal fish (sea blue filling) administered with AS and exposed to a shoal of 4 conspecifics; SB (8)_Ctrl–focal fish (light blue outline) administered with water and exposed to a shoal of 8 conspecifics; SB (8)_AS–focal fish (light blue filling) administered with AS and exposed to a shoal of 8 conspecifics. Grey and red drops represent water and AS administration, respectively. (B) Freezing % in baseline (Bl) vs. first 5 min after AS onset (AS). n = 20 per treatment. Mean ± SEM are shown. *p’ < 0.05; **p’ < 0.01 and ***p’ < 0.001. (C) Freezing % over the 30 min test in 10 min bins. n = 20 per treatment. Mean ± SEM are shown. *p’ < 0.05; **p’ < 0.01 and ***p’ < 0.001. (D) Brain networks as measured by c-fos mRNA expression for each treatment. Circle diameters represent the mean c-fos expression for each brain nuclei. Distinct (≠) and similar (=) co-activation patterns of c-fos mRNA expression between treatments are indicated. Lines linking brain nucleus represent the co-activation between them, as revealed by Pearson’s correlation coefficients (r), with line thicknesses proportional to r value and positive/negative correlations indicated by line colour (green and red, respectively); asterisks indicate significant correlations: *p < 0.05; **p < 0.01 and ***p < 0.001. Full size image

Neuromolecular mechanisms underlying social buffering