11β-Hydroxysteroid dehydrogenase type 2 may mediate the stress-specific effects of cortisol on brain cell proliferation in adult zebrafish (Danio rerio)

Abstract

Stress-induced increases in cortisol can stimulate or inhibit brain cell proliferation, but the mechanisms behind these opposing effects are unknown. We tested the hypothesis that 11β-hydroxysteroid dehydrogenase type 2 (Hsd11b2), a glucocorticoid-inactivating enzyme expressed in neurogenic regions of the adult zebrafish brain, mitigates cortisol-induced changes to brain cell proliferation, using one of three stress regimes: a single 1 min air exposure (acute stress), two air exposures spaced 24 h apart (repeat acute stress) or social subordination (chronic stress). Plasma cortisol was significantly elevated 15 min after air exposure and recovered within 24 h after acute and repeat acute stress, whereas subordinate fish exhibited significant and sustained elevations relative to dominant fish for 24 h. Following acute stress, brain hsd11b2 transcript abundance was elevated up to 6 h after a single air exposure but was unchanged by repeat acute stress or social subordination. A sustained increase in brain Hsd11b2 protein levels occurred after acute stress, but not after repeat or chronic stress. Following acute and repeat acute stress, brain pcna transcript abundance (a marker of cell proliferation) exhibited a prolonged elevation, but was unaffected by social subordination. Interestingly, the number of telencephalic BrdU+ cells increased in fish after a single air exposure but was unchanged by repeat acute stress. Following acute and repeat acute stress, fish expressed lower brain glucocorticoid and mineralocorticoid receptor (gr and mr) transcript abundance while subordinate fish exhibited no changes. Taken together, these results demonstrate stressor-specific regulation of Hsd11b2 in the zebrafish brain that could modulate rates of cortisol catabolism contributing to observed differences in brain cell proliferation.

Keywords: Adult neurogenesis; Glucocorticoid regulation; Neuroendocrinology; Neuroplasticity; Regulation of mitosis; Stress biology.

Fig. 1.

The effects of stress exposure on plasma cortisol. (A) Zebrafish were exposed to air for 1 min (stressor) and allowed to recover for up to 48 h (filled blue circles). (B) For a separate group of fish, the stressor was repeated 24 h after the first stressor and fish were allowed to recover for 15 min or 24 h (open blue circles). (C) Differences in the plasma cortisol of dominant (Dom) and subordinate (Sub) zebrafish after 24 h of social interaction; group-housed (GH) fish are included to act as a control (filled purple squares). Data points identified as statistical outliers were removed prior to analysis and are shown as black triangles. Each symbol represents the cortisol concentration (ng ml⁻¹) of an individual fish, with a solid line and whiskers representing the mean±s.e.m. (acute stress n=10–13, repeat acute stress n=12, social stress n=11–13). Differences in plasma cortisol levels were determined by one-way ANOVA and Tukey's post hoc test, except for repeat acute stress, where a Kruskal–Wallis and Dunn's post hoc test was used (P<0.05). Within each panel, groups that do not share a common letter are significantly different from one another.

Fig. 2.

The effects of stress on hsd11b2 mRNA and Hsd11b2 protein abundance in the adult zebrafish brain. Changes in brain hsd11b2 transcript and Hsd11b2 protein abundance following an acute stressor (A,B), a repeat acute stressor (C,D) and a chronic stressor (E,F). Gene expression was quantified either in brain zone 2 (telencephalon and olfactory bulbs removed; A,C) or in whole brain (E). In B, a representative western blot for acute and repeat acute stress is provided (lane 1=0 min, 2=15 min, 3=6 h, 4=24 h and 6=48 h post-acute stress; lane 5=15 min, 7=24 h post-repeat acute stress; lane 8=internal control). The full blots are provided in Fig. S2. In F, a representative western blot for chronic stress is provided [lane 1=group housed (GH), 2=dominant (Dom), 3=subordinate (Sub), 4=internal control from the same blot]. Transcript abundance was normalized to the mean expression of the two housekeeping genes (ef1α and rpl8). Protein expression of Hsd11b2 was normalized to total protein. Data are shown as individual data points with a solid line and whiskers representing the mean±s.e.m. (hsd11b2, acute stress n=6–8, repeat acute stress n=7–8, social stress n=7; Hsd11b2, acute stress n=4–5, repeat acute stress n=4–5, social stress n=5). Data points identified as statistical outliers were removed prior to analysis and are shown as black triangles. Differences in hsd11b2 mRNA and Hsd11b2 protein levels were determined by one-way ANOVA followed by a Tukey's post hoc test, except for Hsd11b2 protein abundance under acute stress (B), where a Kruskal–Wallis followed by a Dunn's post hoc test was used (P<0.05). Within each panel, groups that do not share a common letter are significantly different from one another.

Fig. 3.

The effects of stress on pcna transcript abundance in the adult zebrafish brain. (A) Zebrafish were exposed to air for 1 min and allowed to recover for up to 48 h (filled blue circles). (B) For a separate group of fish, the stressor was repeated 24 h after the first stressor and fish were allowed to recover for 15 min or 24 h (open blue circles). (C) Dominant (Dom) and subordinate (Sub) zebrafish after 24 h of social stress; group-housed (GH) fish are included as a control (purple squares). pcna transcript abundance in brain zone 2 (telencephalon and olfactory bulbs removed; A,B) and whole brain (C) was normalized to the mean expression of the two housekeeping genes (ef1α and rpl8). Data are shown as individual data points with a solid line and whiskers representing the mean±s.e.m., and statistical outliers as black triangles. Differences in pcna transcript abundance were determined by one-way ANOVA with a Tukey's post hoc test, except for acute stress, where a Kruskal–Wallis with a Dunn's post hoc test was used (P<0.05). Within each panel, groups that do not share a common letter are significantly different from one another.

Fig. 4.

The effects of a single and repeat acute stressor on brain cell proliferation in the telencephalon of adult zebrafish brain. (A) A line drawing traced from original images of cross-sections following immunohistochemical detection, where the vertical dashed line represents the midline of the section and circles represent common locations of BrdU+ cells. The approximate corresponding level in the zebrafish brain atlas is the rostral telencephalon level 71 (Wullimann et al., 1996), based on gross anatomical features across the suite of serial sections for each represented fish. The boxed region indicates the area of the representative photomicrographs of cross-sections through the zebrafish telencephalon of (B) time 0 (baseline), (C) 1.5 h recovery following a single 1 min air exposure stressor and (D) 1.5 h recovery from a repeat stressor 24 h after the initial stressor (i.e. 25.5 h from time 0). Scale bars: 100 µm. To estimate cell proliferation in the telencephalon (E), the number of surface BrdU+ cells (green) in each serial 20 µm section was tallied and normalized to cross-sectional area (µm²). Each data point is the sum tally from 12 serial sections per fish, with a solid line and whiskers representing the mean±s.e.m. for each time point, and statistical outliers shown as black triangles. Changes in cell proliferation following stress exposure were determined using a one-way ANOVA with a Tukey's post hoc test (P<0.05). Within each panel, time points that do not share a common letter are significantly different from one another.

Fig. 5.

The effects of stress exposure on gr transcript abundance in the adult zebrafish brain. (A) Zebrafish were exposed to air for 1 min and allowed to recover for up to 48 h (filled blue circles). (B) For a separate group of fish, the stressor was repeated 24 h after the first stressor and fish were allowed to recover for 15 min or 24 h (open blue circles). (C) Dominant (Dom) and subordinate (Sub) zebrafish after 24 h of social stress; group-housed (GH) fish are included as a control (purple squares). gr transcript abundance was normalized to the mean expression of two housekeeping genes (ef1α and rpl8). Data are shown as individual data points with a solid line and whiskers representing the mean±s.e.m., and statistical outliers as black triangles. Changes in gr transcript abundance in brain zone 2 (telencephalon and olfactory bulbs removed; A,B) and the whole brain (C) were determined using a one-way ANOVA followed by a Tukey's post hoc test (P<0.05). Within each panel, groups that do not share a common letter are significantly different from one another.

Fig. 6.

The effects of stress exposure on mr transcript abundance in the adult zebrafish brain. (A) Zebrafish were exposed to air for 1 min and allowed to recover for up to 48 h (filled blue circles). (B) For a separate group of fish, the stressor was repeated 24 h after the first stressor and fish were allowed to recover for 15 min or 24 h (open blue circles). (C) Dominant (Dom) and subordinate (Sub) zebrafish after 24 h of social stress; group-housed (GH) fish are included as a control (purple squares). mr transcript abundance was normalized to the mean expression of two housekeeping genes (ef1α and rpl8). Data are shown as individual data points with a solid line and whiskers representing the mean±s.e.m., and statistical outliers as black triangles. Changes in mr transcript abundance in brain zone 2 (telencephalon and olfactory bulbs removed; A,B) and the whole brain (C) were determined using a one-way ANOVA followed by a Tukey's post hoc test (P<0.05). Within a panel, groups that do not share a common letter are significantly different from one another.