cDNA encoding myc-tagged bPAC from the soil bacterium Beggiatoa bPAC (Stierl et al., 2011) was PCR amplified with a mutated stop-codon and cloned into a vector containing a viral 2A sequence (Tang et al., 2009) and a fluorescent tdTomato marker flanked by I-SceI and Tol2 transposon recognition sites in the pBR322 backbone. This construct was combined with a fragment of the Pomc promoter, which was PCR amplified from a Pomc-GFP construct (Liu et al., 2003). The Pomc:bPAC-2A-tdTomato plasmid was incubated with 100 ng Tol2 transposase RNA for 10 min and injected in the presence of 0.05% phenol red into wild-type embryos (cross of AB and TL strains) in the one-cell stage. For further propagation of the transgenic line, we selected one founder, Tg(Pomc:bPAC-2A-tdTomato)hd10, with specific tdTomato expression in the pituitary and no ectopic expression.

Zebrafish breeding and maintenance was performed under standard conditions (Westerfield, 2000). Embryos were collected in the morning and raised on a 12:12 light/dark cycle in E2 medium (Westerfield, 2000). Tg(Pomc:bPAC-2A-tdTomato)hd10 were crossed with wild-type fish and their progenies selected for the presence of tdTomato expression in the pituitary at 4 or 5 days post fertilization (dpf) using a fluorescent dissecting microscope. To avoid unspecific activation of bPAC prior to the experiments, transgenic embryos were raised in custom-made reflective containers covered by 550 nm long-pass filters (Thorlabs). Zebrafish experimental procedures were performed according to the guidelines of the German animal welfare law and approved by the local government.

50 pg capped bPAC RNA was prepared using a commercial mRNA kit (mMessage T7 Ultra Kit, Ambion) and injected into one-cell-stage wild-type embryos. Embryos were maintained under filtered light (see above) and subjected to blue-light stimulation at 1 dpf using the stimulation protocol described below (light power: 2.8 mW^*cm⁻²). Groups of 27 embryos were collected immediately after the light-offset and homogenized in 0.1 M HCl on ice. After centrifugation, the supernatant was stored at −20°C. cAMP level was measured following the acetylation protocol from a cAMP ELISA kit (Enzo Life Sciences). Samples from light-stimulated bPAC-injected embryos were diluted 15 times in order to obtain values within the standard range.

6 dpf larvae were fixed overnight at 4°C in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Immunohistochemistry was performed as previously described (Ryu et al., 2007), using either polyclonal antibody against human ACTH (National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, 1:500) or rabbit polyclonal antibody against Myc-Tag (Cell Signaling Technology, 1:500) as primary antibodies, and Alexa Fluor 488 anti-rabbit (Invitrogen, 1:1000) as a secondary antibody. Detection of residual tdTomato fluorescence after the fixation did not require immunohistochemistry. Larvae were imaged in 80% glycerol using a Nikon 20x glycerol objective and a Leica SP5 CLSM. Confocal image stacks were subsequently evaluated using Amira 5.4 (Visualization Sciences Group) to create maximum intensity projections.

For cortisol detection, groups of 30 larvae (6 dpf) were immobilized in ice water, frozen in ethanol/dry ice bath, and stored at −20°C. Cortisol from homogenized samples was extracted with ethyl acetate. We employed a home-made cortisol ELISA protocol (C. M. Yeh, M. Glöck, R. J. De Marco, S. Ryu, unpublished data), using cortisol mouse antibody (EastCoast Bio), cortisol standards (Hydrocortisone, Sigma-Aldrich) and cortisol-HRP (EastCoast Bio). The reactions were stopped using 1M sulfuric acid and read at 450 nm in an ELISA reader (Multiskan Ascent, Thermo Scientific). The data were corrected for dilution factor, extraction efficiency, and recovery function. In all experiments, cortisol samples were taken 2 min after the offset of light, unless otherwise stated.

6 dpf larvae were incubated for 2 h in 1 μM Mifepristone (RU-486, Sigma-Aldrich) dissolved in E2-Medium with 0.1% DMSO. This concentration has been shown to abolish a genomic GC response signal (Weger et al., 2012). During light stimulation, larvae were maintained in the Mifepristone solution to avoid further handling.

A custom-made LED ring was placed at a fixed distance above a mutiwell plate (for behavioral testing) or a single container (for cortisol extraction). The incident angle of the LEDs allowed for homogeneous illumination of the samples. We used custom-made drivers, pulse generators and a TTL control box (USB-IO box, Noldus) to control the LEDs. Larvae were exposed for 18 s or 180 s to either blue- or yellow-light of varying power, using single or multiple stimulation protocols. Each light pulse consisted of 100 ms flashes delivered at 5 Hz. Light power was measured using a hand-held light power meter (Newport). For the multiple stimulation protocol, we used three light pulses delivered with an inter-trial interval of 30 min.

To facilitate light stimulation with a higher throughput, we arranged LEDs so as to homogeneously illuminate a six well plate with a light power of 0.6 mW^*cm⁻². At 4 dpf, we exposed the bPAC-positive (bPAC⁺) larvae and their negative siblings (bPAC⁻) to the above described multiple stimulation protocol. Next, the larvae were placed back in the incubator and kept in E2 medium inside the reflective containers covered by the 550 nm long-pass filters. We repeated this procedure 24 h later. At the end of 5 dpf, we screened the larvae for tdTomato expression in the pituitary. At 6 dpf, both the bPAC⁺ and bPAC⁻ larvae subjected to the above protocol were either directly collected for measuring basal cortisol levels or first stimulated with a single 180 s squared pulse of blue-light (0.6 mW^*cm⁻²) and then collected for measuring light-induced cortisol change. Control animals for each group were handled in the same fashion, but omitting the light stimulation at 4 and 5 dpf. Stimulations were performed within a fixed 3 h window during the larvae's day time.

Behavioral tests were performed using wild-type, bPAC⁺ and bPAC⁻ 6 dpf larvae. Experiments were conducted under infrared (IR) light, delivered through an array of IR-LEDs mounted inside a custom-made light-proof enclosure placed on a vibration-free platform (Newport). We used an infrared-sensitive camera (ICD-49E B/W, Ikegami Tsushinki) to image the movements of the swimming larvae at 25 frames s⁻¹. The lens of the camera (TV Lens, Computer VARI FOCAL H3Z4512 CS-IR, CBC) was surrounded by a custom-made LED ring and positioned above a multiwell plate (Greiner-Bio One). We used EthoVision XT software (Noldus Information Technology) to simultaneously track the movements of 30 larvae swimming individually inside the wells in 50 μL of E2 medium. In all experiments, the larvae were allowed to adjust to the test conditions for 15 min prior to the recordings. Experiments were conducted at room temperature. We continuously monitored the temperature inside a reference well using a thermocouple (npi electronics) connected to a temperature control system (PTC 20, npi electronics; Exos-2 V2 liquid cooling system, Koolance). All the experiments were performed in a blind fashion using unscreened larvae to avoid effects of pre-handling and exposure to unfiltered light. Tests were conducted between 9:00 and 18:00 and the different experimental groups intermixed throughout the day.

All data are shown as mean and standard error of the mean (S.E.M.). To facilitate comparison, locomotor activity is expressed either as distance swam per unit of time (Figures 2A–C) or as percentual motion relative to pre-stimulation baseline levels (Figures 5A–C), as these levels did not differ between bPAC⁺ and bPAC⁻ larvae (Mann–Whitney test, p = 0.11). We used Student's t-tests (two-tailed) for two-group comparisons, or Mann–Whitney U-tests if the data did not fulfill the assumptions of the t-test. ANOVAs were used for multiple group comparisons, followed by Bonferroni's post-hoc tests, or their non-parametric equivalents. We also used repeated-measures linear regression analysis (Fitzmaurice et al., 2004). Analyzes were carried out using MS-Excel, Matlab 2009b (MathWorks), Prism 5, (Graphpad Software), Sigma Plot (Systat), R and Virtual Dub (Freeware).

The small transparent bodies of larval zebrafish make them suitable for non-invasive manipulation of neuronal activity using light. Yet, larval zebrafish are highly sensitive to photic stimuli (Burgess and Granato, 2007). While swimming in darkness, for example, they display stable rates of discontinuous motion (Figure 1A) and react to a brief exposure to light with stereotyped changes in locomotor activity (Macphail et al., 2009). First, they show a drastic reduction of locomotion after the light onset, followed by increased locomotion after the light-offset. Afterward, locomotion decreases gradually until it reaches steady-state levels several minutes later. A drastic reduction of locomotion in response to external stimulation is generally thought of as a fear-related response. Many species secrete cortisol in threatening situations associated with greater fear. Hence, as a prerequisite to develop optogenetic approaches for stress research, we set up to examine the effect of illumination change on locomotion and cortisol level. Since optogenetic photo-actuators work upon absorption of a wide range of light wavelengths, we first tested whether the larval stereotyped reactions to illumination change could be similarly evoked by a squared pulse of either blue- or yellow-light. We observed that both light wavelengths elicited similar motion patterns (Figure 1B). Next, we examined the extent to which such a brief exposure to light could act as a stressful event, and observed that 6 dpf larvae reacted to a 180 s squared pulse of either blue- or yellow-light with increased cortisol levels (Figure 1C; Mann–Whitney test, blue-light: p < 0.01, yellow-light: p = 0.03), thereby specifying that a squared pulse of light can act as a stressful input signal. This effect of a fast transition from darkness to light could not be accounted for by “wakefulness” variations as reflected in motion. Larvae kept for such a brief period of time either under constant white illumination or in complete darkness displayed similar levels of locomotor activity [t-test, t₍₂₄₎ = 0.4, p = 0.72].

Since light in itself can stimulate stress networks, we reasoned that the presence of photo-actuators within the HPI axis would allow us to meaningfully alter its light-triggered activation. In particular, we aimed to manipulate the increase of cortisol triggered by light so as to induce greater and controllable rates of cortisol rise in response to otherwise similarly stressful events. Technically speaking, this means that we aimed to increase the gain of the stress axis by amplifying the output (cortisol) of a constant input signal (light). To this end, we chose to target the expression of Beggiatoa bPAC (Ryu et al., 2010; Stierl et al., 2011) specifically to ACTH-producing pituitary corticotroph cells. Stress activates complex intracellular CRH signaling cascades in multiple cell types (Arzt and Holsboer, 2006). In pituitary cells, an increase in cAMP downstream of CRH receptor activation causes ACTH release. We therefore hypothesized that blue-light stimulation of bPAC will lead to increased cAMP levels in pituitary corticotrophs and, consequently, also to enhanced ACTH release (Figure 2A). Enhanced levels of circulating ACTH will then be expected to co-vary with whole-body cortisol (Figure 2B), as the melanocortin receptor type 2 (MC2R) is predominantly expressed in the interrenal gland and not in the zebrafish brain (Agulleiro et al., 2010). We first demonstrated that bPAC is functional in zebrafish larvae. Injecting bPAC mRNA into embryos in the one-cell stage led to a blue-light dependent elevation of whole-body cAMP at 1 dpf (Figure 2C; Mann–Whitney test, p = 0.19 for non-stimulated control vs. bPAC-injected; p = 0.02 for light-stimulated control vs. bPAC-injected). To target bPAC specifically to pituitary corticotrophs, we used a fragment of the proopiomelanocortin (POMC) promoter whose expression pattern is restricted to corticotroph cells (Liu et al., 2003). To aid in visualization of the expression of the construct, the bPAC protein sequence was fused to myc-tag and a fluorescent reporter, tdTomato, via the viral 2A peptide. The transgenic line expressed bPAC specifically in pituitary corticotrophs, as revealed by the co-localization of ACTH with myc and tdTomato signal (Figure 2D).

Consistent with our observations in wild-type larvae (Figures 1A–C), a 180 s squared pulse of blue-light led to increased cortisol levels in both bPAC-positive (bPAC⁺) and bPAC-negative (bPAC⁻) larvae. However, the former showed substantially higher cortisol levels (Figure 3A; Two-Way ANOVA, light power: F_{(3, 82)} = 29.48, p < 0.0001; genotype: F_{(1, 82)} = 23.09, p < 0.0001; light power X genotype: F_{(3, 82)} = 1.77, p = 0.16; followed by Bonferroni post-tests for within light-power pair comparisons). Yellow-light failed to enhance the rise of cortisol in the bPAC⁺ larvae (Figure 3A; One-Way ANOVA, F_{(3, 36)} = 10.73, p < 0.0001; followed by Bonferroni post-tests for bPAC⁺_blue vs. bPAC⁻_blue, bPAC⁺_yellow or bPAC⁻_yellow, and for bPAC⁺_yellow vs. either bPAC⁻_blue or bPAC⁻_yellow), in line with the fact that bPAC activation is blue-light specific due to its BLUF (blue-light receptor using FAD) type light-sensor domain (Ryu et al., 2010; Stierl et al., 2011). Further, already the lowest light-power caused maximum differences between the cortisol levels of the bPAC⁺ and bPAC⁻ larvae (Figure 3A). This latter result led us to examine the effects of a shorter light stimulation. We then observed that the bPAC⁺ larvae showed enhanced cortisol levels in response to a ten times shorter stimulation, i.e., a light pulse lasting less than 20 s (Figure 3B; Two-Way ANOVA, left, length: F_{(1, 40)} = 33.85, p < 0.0001; genotype: F_{(1, 40)} = 19.56, p < 0.0001; length X genotype: F_{(1, 40)} = 0.47, p = 0.50; right, length: F_{(1, 40)} = 10.85, p = 0.002; genotype: F_{(1, 40)} = 20.37, p < 0.0001; length X genotype: F_{(1, 40)} = 1.13, p = 0.29; followed by Bonferroni post-test for pair comparisons), demonstrating that our approach allows for GC alterations with high temporal resolution.

GCs regulate their own production by decreasing CRH and ACTH outputs from the hypothalamus and pituitary, respectively (Dallman and Yates, 1969; Dallman et al., 1994). We then asked whether optogenetic elevation of endogenous GCs could lead to transient hypercortisolic states repeatedly. After the differential rise of cortisol triggered by blue-light, both the bPAC⁺ and bPAC⁻ larvae had similar and significantly reduced cortisol levels 20 min after the light offset (Figure 4A; bPAC⁺, F_{(2, 27)}= 38.74, p < 0.0001; bPAC⁻, F_{(2, 24)} = 17.70, p < 0.0001; t-test for pair comparisons within time points), indicating that cortisol-mediated negative feedback is fully functional in zebrafish larvae. Next, we repeatedly exposed groups of bPAC⁺ and bPAC⁻ larvae to a sequence of three 180 s squared pulses of blue-light. In order to compare cortisol values resulting from the most recent light pulse and not from previously elevated levels, we used a time interval of 30 min in-between light pulses, which assured similarly low levels in both groups at the time of the second and third pulses (Figure 4A). Using this multiple light stimulation protocol, we observed that the bPAC⁺ larvae responded to each of the light pulses with increased cortisol levels, whereas the bPAC⁻ larvae failed to do so after the first pulse (Figure 4B; Two-Way ANOVA, repeated exposure: F_{(2, 50)} = 12.44, p < 0.0001; genotype: F_{(1, 50)} = 18.55, p < 0.0001; repeated exposure X genotype: F_{(2, 50)} = 0.13, p = 0.88; one sample t-tests for comparisons against basal level). These results demonstrated that multiple light stimulations can repeatedly lead to hypercortisolic states in bPAC⁺ larvae, even if the HPI axis has been down-regulated by previously elevated GC levels. To verify the role of the cortisol-mediated negative feedback in this phenomenon, we applied the same stimulation protocol to bPAC⁺ and bPAC⁻ larvae that had been incubated with mifepristone (Mif), an antagonist for the GC-receptor (GR) that is effective in larval zebrafish (Weger et al., 2012). Under these circumstances, both the bPAC⁺ and bPAC⁻ larvae responded to each of the several light pulses with increased cortisol levels. These stress-induced levels were much higher than those from the non-incubated larvae, verifying that our multiple stimulation protocol leads to down-regulated HPI axis activity. Yet, the bPAC⁺ larvae still showed substantially higher cortisol levels than the bPAC⁻ larvae (Figure 4C; Two-Way ANOVA, repeated exposure: F_{(2, 41)} = 16.30, p < 0.0001; genotype: F_{(1, 41)} = 21.88, p < 0.0001; repeated exposure X genotype: F_{(2, 41)} = 0.72, p = 0.49; one sample t-tests for comparisons against basal level). Also, the basal cortisol levels of both groups of larvae were higher in the Mif-incubated larvae as compared to the non-incubated larvae (Figures 4B,C; Mann–Whitney test, p = 0.004). Taken together, these results show that our approach can be used to induce hypercortisolic states repeatedly, making it possible to examine the effect of repeated GC over-exposure on stress axis development and function.

We noticed that blue-light led to higher post-stimulation locomotion in the bPAC⁺ larvae, as compared to their negative siblings (Figure 5A). Hence, we compared the steady-state post-stimulation motion levels of the bPAC⁺ and bPAC⁻ larvae 20 min after a single pulse of either blue- or yellow-light. We then observed that blue- but not yellow-light enhanced locomotion in the bPAC⁺ larvae, whereas neither blue- nor yellow-light enhanced locomotion in the bPAC⁻ larvae (Figure 5B; Mann–Whitney tests, blue-light: p < 0.04, yellow-light: p = 0.68). We also compared the post-stimulation motion levels of both groups using data from the multiple light stimulation protocol (Figure 4B). Once again, locomotion was higher in the bPAC⁺ than in the bPAC⁻ larvae (Figure 5C; Two-Way ANOVA, repeated exposure: F_{(2, 306)} = 3.0, p = 0.0513; genotype: F_{(1, 306)} = 8.26, p = 0.0043; repeated exposure X genotype: F_{(2, 306)} = 0.19, p = 0.83). Noticiably, the motion values from both groups of larvae plotted against the corresponding cortisol levels could be linearly approximated (Figure 5D; repeated-measures linear regression analysis, p < 0.001). These results indicated that a brief exposure to blue-light can cause not only hypercortisolic states in dark-adapted bPAC⁺ larvae, but also tightly correlated deviations from nominal locomotion.

Early GC overexposure can lead to persistent alterations of HPA axis function (Kapoor et al., 2006; Seckl and Holmes, 2007; Seckl, 2008). We asked whether multiple light stimulations at early stages of development could lead to long-term forms of hypercortisolaemia in bPAC⁺ larvae. To answer this question, we applied the multiple light stimulation protocol (Figure 4) to the bPAC⁺ and bPAC⁻ larvae at 4 and 5 dpf. Later, at 6 dpf, we measured the basal and stress-induced cortisol levels of the larvae following a single 180 s squared pulse of blue-light (Figure 6). We then observed that the bPAC⁺ larvae had increased basal cortisol at 6 dpf (Figure 6A; Wilcoxon signed rank test, bPAC⁺: p = 0.03, bPAC⁻: p = 0.84), whereas the basal cortisol levels of the bPAC⁺ and bPAC⁻ larvae that had not been exposed to early blue-light stimulation did not differ from each other [t-test, t₍₂₃₎ = 1.1, p = 0.31]. Also, the bPAC⁺ larvae responded to a light pulse with higher cortisol level, as compared to either the non-exposed bPAC⁺ larvae or the exposed and non-exposed bPAC⁻ larvae (Figure 6B; Two-Way ANOVA, early stimulation: F_{(1, 21)} = 9.8, p < 0.01; genotype: F_{(1, 21)} = 11.9, p < 0.01; early stimulation X genotype: F_{(1, 21)} = 1.0, p = 0.3, followed by Bonferroni post-tests for within genotype pair comparisons). These results demonstrated that early blue-light stimulation causes long-term hypercortisolaemia in bPAC⁺ larvae.

Soojin Ryu and Rodrigo J. De Marco have submitted a patent application for the use of optogenetic transgenic zebrafish lines to find novel regulators of stress response.