Tricane

Adult homozygous Tg(aanat2:EGFP-ΔCLK) fish were raised in a temperature-controlled recirculation water system under 12-hr:12-hr LD cycles, and transferred to DD at the end of the light period prior to sampling. Pineal glands were sampled at 4-hr intervals throughout two daily cycles under DD at 12 time points corresponding to circadian time (CT) 14, 18, 22, 2, 6, 10, 14b, 18b, 22b, 2b, 6b and 10b (Fig 4A), as previously described [42 (link)]. A pool of 16 pineal glands was collected at each time point. In addition, two control pools of 14 pineal glands were collected from Tg(aanat2:EGFP) fish at time points corresponding to CT2 and CT14b. Fish were anesthetized in 1.5 mM Tricane (Sigma-Aldrich) and decapitated. Fluorescent pineal glands were selectively removed under a dissecting microscope (Olympus SZX12) equipped with filters for excitation (460–490 nm) and emission (510–550 nm) of EGFP. Total RNA for mRNA analysis was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen).

Wild-type zebrafish (Danio rerio) were raised and cared for as per standard procedures in accordance with a protocol approved by the Southern Medical University Animal Care and Use Committee. Embryos and larvae were reared in embryo medium at 28 °C to 5 dpf. Zebrafish larvae with completely development from a single cluster at 5dpf were allocated randomly to a serial of different amount of feeding groups: 20 mg/d, 30 mg/d, 60 mg/d, 80 mg/d, 120 mg/d and 180 mg/d. Each group had 100 larvae and started to feed at 7 dpf for 10, 15 and 20 days in 1.5 L tanks after adaptive of deep water environment for one day. They were fed a larval food (Zeigler AP100) three times daily, which was grinded to powder until food can float on the face of water, and then exchanged by half water after fed for 2 h. For caffeine treatment, larvae were overfed with 1, 2.5, 5 and 8 % caffeine for 20 days, which were mixed well with larval food (Zeigler AP100) and grinded to powder. At each time point, all larvae in a tank were counted and harvested and killed by tricane (Sigma) overdose. The length of zebrafish was measured from tip of the nose to the end of body. Larvae were wiped dry and weighed on a Mettler AE 50 balance to the nearest milligram.

Acetonitrile, methanol, ethanol, tricane (Sigma-Aldrich, St. Louis, MO, USA), formic acid, and ammonium acetate (Sigma-Aldrich, Darmstadt, Germany) were HPLC grade. De-ionized water was prepared using a Milli-Q system (Millipore, Billerica, MA, USA). The blank bovine plasma, and the steroid standards, 11-deoxycorticosterone, corticosterone, aldosterone, 11-deoxycortisol, cortisol, androstenedione, testosterone, and tetra-deuterated cortisol, were bought from Sigma-Aldrich (purity > 99%, USA). The caffeine was refined in-house from food supplementary capsules (Nutricost, Vineyard, UT, USA). The chemical structure and purity were confirmed by NMR (Bruker, Rheinstetten, Germany) and HR-MS (Waters, MA, USA) in our laboratory (See Supplementary Materials). The solid-phase extraction cartridges (HLB, 100 mg sorbent per cartridge) were purchased from Waters (Milford, MA, USA).

Briefly, the acute exposure assays followed the protocol as described [15 (link)]. The ethanol was administered acutely by placing individual zebrafish into 500 mL of 1% for 5 min (male n = 10, female n = 10). Caffeine (300 mg/L) was administered in a 1 L pre-treatment beaker for 5 min (male n = 10, female n = 10). The corresponding controls (male n = 10, female n = 10) which did not receive stimuli treatment during this time were housed in otherwise identical conditions. Following exposure testing, the animals were euthanized in 500 mg/L tricane (Sigma–Aldrich, USA) on an ice bath, and immediately dissected for further analysis.

The zebrafish were raised using the techniques outlined in [22 (link)]. For the present study, two variants of zebrafish larvae were used: the transgenic Tg (flk1:GFP) line, which exhibits green fluorescent protein (GFP) expressing endothelial cells [23 (link)]; and the mutant casper line, which is largely devoid of melanophores and iridophores [11 (link)]. All zebrafish strains were housed and maintained under standard husbandry conditions [3 ]. To prevent excess melanin production in the Tg (flk1:GFP) fish, the embryos were treated with 200 μM of PTU [10 (link)] at 10 h post fertilization (hpf). For fish treated with the anti-angiogenic drug indirubin-3′-monoxime (I3M, or IRO), 4 μM was added to the zebrafish egg water at 8 hpf. All zebrafish experiments were conducted in accordance with St. Michael’s Hospital Animal Care Committee approved protocol ACC403.
A glass-bottom petri dish (MatTek, USA) was filled with 300 μL of molten 1.5% low melting point agarose (Sigma, USA) at 40 °C. The agarose was allowed to set in a 4 °C fridge for 15 min. The zebrafish larvae were anesthetized using a 0.003% (w/v) solution of tricane (Sigma, USA), and pipetted onto the agarose filled petri dishes. Excess egg water was aspirated, and the fish was covered with 20 μL of the molten 40 °C agarose. The dishes containing larvae were left to set at room temperature for 30 min prior to imaging.

Wild-type AB stock was purchased from the Zebrafish International Resource Center (Eugene, OR). Eggs were obtained by placing breeding boats at the base of adult zebrafish tanks overnight, then bleached according to protocols described in The Zebrafish Book51 . From 0 d.p.f. larvae were kept in petri dishes containing embryo medium (E2) without methylene blue, prepared as described in The Zebrafish Book and washed daily up to and including 3 d.p.f. Larvae were anaesthetized with 400 μg ml⁻¹ tricane (Sigma-Aldrich) during in vivo imaging.

Pigmented Xenopus laevis females were housed at ∼18°C under 12 hr light/12 hr dark cycles at the Animal Resource Center (ARC) Bio II vivarium at the University of California, Santa Barbara (UCSB). All the procedures, including Xenopus surgery, ovary harvest, and post-surgical recovery, were performed according to a protocol approved by the institutional animal care and use committee at UCSB. Ovaries were surgically removed under anesthesia (0.3% w/v tricane, Sigma), cut into small pieces and then treated with 2 mg/ml collagenase A (Sigma-Aldrich C5138) in OR2 buffer (100 mM NaCl, 2 mM KCl, 1 mM 5 mM HEPES, pH 7.5) at room temperature until complete de-foliculation. The oocytes were recovered at 18°C for 12–24 hours in OR3 culture medium [50% Leibovitz's media L-15 (Sigma L1518), 13 mM HEPES, 90 g/ml gentamicin, 90 g/ml Fungizone (Amphotericin B), 90 g/ml penicillin/streptomycin, pH 7.5].