Novel zebrafish behavioral assay to identify modifiers of the rapid, nongenomic stress response

Abstract

When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response. Rapid changes in heart rate and blood glucose levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic-pituitary-adrenal axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent hypothalamic-pituitary-adrenal axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires hypothalamic-pituitary-interrenal (HPI) axis activation. In teleost fish, interrenal cells are functionally homologous to the adrenocortical layer. We derived eight frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), five in exon 2 or 5 of nr3c1 (glucocorticoid receptor [GR]) and two in exon 2 of nr3c2 (mineralocorticoid receptor [MR]). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show that this locomotor response requires a functioning HPI axis via the action of mc2r and the canonical GR encoded by nr3c1 gene, but not MR (nr3c2). Our rapid behavioral assay paradigm based on HPI axis biology can be used to screen for genetic and environmental modifiers of the hypothalamic-pituitary-adrenal axis and to investigate the effects of corticosteroids and their cognate receptor interactions on behavior.

Keywords: HPA axis; assay development; behavioral genetics; cortisol; custom nuclease; glucocorticoid receptor.

Figure 1

Stress assay paradigms. A, Experimental flow. Embryos are collected from natural spawning on day 0. Dead embryos are cleaned up and fresh embryo media is provided on 0 and 1 dpf. On 3 dpf, morphologically normal larval fish are plated onto 48‐well plates. On 5 dpf, stress assays are performed. B, Dual light assays. Larvae are acclimated for 30 minutes in infrared (IR) light. Baseline locomotor activity is recorded in IR for 15 minutes, followed by 50 seconds of white light stimulation. Posttreatment locomotor activity is recorded in IR for 20 minutes. C, Hyperosmotic stress assays. Larvae are acclimated for 30 minutes in white light. Baseline locomotor activity is recorded for 15 minutes, followed by addition of NaCl or cinnamon oil (noxious stimulant control). Posttreatment locomotor activity is recorded for 30 minutes. Initial 10 minutes were used for statistical analysis for cinnamon oil assays

Figure 2

Wild‐type larval zebrafish response to acute stressors. We examined the acute response of larvae derived from natural crosses of WT zebrafish in several assays. A, Dual light assays with varied lengths of white light illumination (shown as blue bar with times 15, 30, 60, 600 or 1800 seconds). All larvae were 5 dpf. B, Dual light assays with larvae at different developmental stages (3, 4 or 5 dpf) after a 50‐second white light illumination. C, Cinnamon oil control assays on (3, 4 or 5 dpf) larvae. D, Whole‐body cortisol levels in larvae (5 dpf) after a 50‐second white light illumination. E, NaCl assays with varying NaCl concentrations on 5 dpf larvae (5 dpf). Line graphs in A, B, C and E show the rolling mean of the larval distance moved. The locomotor activity at each second is the mean distance fish moved during the preceding 60 seconds (mean ± 95%CI [shading]). Bar graphs in A, B, C and E show the mean distance larvae moved over the time course (mm/min; mean ± 95%CI) (baseline: 5 minutes; posttreatment: 20 minutes [light assays], 30 minutes [NaCl assays] or 10 minutes [cinnamon oil control assays]). The line graph in D shows pg cortisol per mg of total protein over time following a 50‐second white light exposure at time 0. In bar graphs, different letters indicate a significant difference between groups (Tukey's honest significant difference test, P < 0.05). The number of individual larvae measured is shown at the base of each bar graph

Figure 3

mc2r larvae: Locomotor response to acute stressors. We examined the acute response of larvae derived from natural crosses of mc2r ^+/− fish (WT [+/+], heterozygous [+/−] or homozygous [−/−]) in several assays. (A) Dual light assays, (B) NaCl assays, (C) cinnamon oil control assays and (D) treatment with ACTH or cortisol. The larvae in (D) carried a single copy of the SR4G transgene. Line graphs in A, B and C show the rolling mean of the larval distance moved (5 dpf). The locomotor activity at each second is the mean distance fish moved during the preceding 60 seconds (mean ± 95%CI [shading]). Bar graphs in A, B and C show the mean larval distance moved over the time course (mm/min; mean ± 95%CI) (baseline: 5 minutes; posttreatment: 20 minutes [light assays], 30 minutes [NaCl assays] or 10 minutes [cinnamon oil control assays]). Bar graphs in (D) show relative EGFP transcript levels compared with WT treated with vehicle. In bar graphs, different letters indicate a significant difference between groups (Tukey's honest significant difference test, P < 0.05). The number of individual larvae measured is shown at the base of each bar graph. SR4G: Stress responsive 4‐hour half‐life GFP

Figure 4

nr3c1 larvae: Locomotor response to acute stressors. We examined the acute response of larvae derived from natural crosses of nr3c1 ^+/− fish (WT [+/+], heterozygous [+/−] or homozygous [−/−]) in several assays. Dual light assays for (A) nr3c1 ^ex2 and (B) nr3c1 ^ex5. NaCl assays for (C) nr3c1 ^ex2 and (D) nr3c1 ^ex5. Cinnamon oil control assays for (E) nr3c1 ^ex2 and (F) nr3c1 ^ex5. (G) Dual light assays and (H) cinnamon oil assays following incubation with mifepristone, a GR antagonist. Line graphs in (A) to (H) show the rolling mean of the larval distance moved (5 dpf). The locomotor activity at each second is the mean distance fish moved during the preceding 60 seconds (mean ± 95%CI [shading]). Bar graphs in (A) to (H) show the mean distance larvae moved over the time course (mm/min; mean ± 95%CI) (baseline: 5 minutes; posttreatment: 20 minutes [light assays], 30 minutes [NaCl assays] or 10 minutes [cinnamon oil control assays]). In bar graphs, different letters indicate a significant difference between groups (Tukey's honest significant difference test, P < 0.05). The number of individual larvae measured is shown at the base of each bar graph

Figure 5

nr3c2 larvae: Locomotor response to acute stressors. We examined the acute response of larvae derived from natural crosses of nr3c2 ^+/− fish (WT [+/+], heterozygous [+/−] or homozygous [−/−]) or injected with high efficiency, bi‐allelic TALENs targeting GFP sequences or nr3c2 exon 2 in several assays. (A) Dual light assays for nr3c2 larvae. (B) NaCl assays for TALEN‐injected WT larvae. (C) Dual light assays or (D) cinnamon oil assays following incubation with spironolactone, a MR antagonist. Line graphs in (A) to (D) show the rolling mean of the larval distance moved (5 dpf). The locomotor activity at each second is the mean distance fish moved during the preceding 60 seconds (mean ± 95%CI [shading]). Bar graphs in (A) to (D) show the mean distance larvae moved over the time course (mm/min; mean ± 95%CI) (baseline: 5 minutes; posttreatment: 20 minutes [light assays] or 30 minutes [NaCl assays]). In bar graphs, different letters indicate a significant difference between groups (Tukey's honest significant difference test, P < 0.05). The number of individual larvae measured is shown at the base of each bar graph