2 hydroxypropyl cyclodextrin

To investigate the involvement of NR3C1 in the glucocorticoid-induced testosterone decrease during acute stress, RU486 (17(-hydroxy-11(-(4-dimethy-aminophenyl-1)-17)-(1-prop-1-ynyl)-oestra-4, 9-diene-3-one, (Roussel Uclaf S.A., Paris, France) was administered in vivo by intratesticular injection prior to the stress session. The dose (16 µg) of RU486 was selected based on a previous study conducted in rats [31 (link)]. RU486 was first dissolved in absolute ethanol and subsequently diluted with the vehicle, 45% aqueous 2-hydroxypropyl-cyclodextrin (Catalog Number 0926, Sigma), to attain the needed concentrations (the final concentration of ethanol was 0.8%, which did not affect Leydig cell function [31 (link)]). Animals (n = 6 per group) were divided into 4 groups: Non-stress group, non-stress group of rats injected intratesticularly with RU486, stress group and stress group of rats injected with RU486. Rats were subjected to immobilization stress for 6 h, as described above. At the end of the stress period, animals were euthanized by CO₂, testes were taken and interstitial fluids were prepared according to the previously described method [41 (link)]. In brief, several holes were punctured in the rat testis using needles and placed into centrifuge tubules, and the testis was centrifuged at 800× g for 10 min to collect interstitial fluids.

Ibrutinib (S2680, Selleckchem) or acalabrutinib (HY-17600, MedChemExpress) were solubilized in DMSO (0.5 mg/L). Inhibitor solution was then dissolved in a solution of 10% (2-hydroxypropyl)-cyclodextrin (Sigma-Aldrich) in 1x DPBS. Mice were treated either by oral gavage (200 μL) or by injection into hind footpads (25 μL) as indicated. Assuming an average weight of 25 g/mouse, treatment with 1.56–25 μg of Ibrutinib corresponds to approximately (0.062 mg/kg-1 mg/kg). For acalabrutinib treatments, 0.03125–0.25 mg of acalabrutinib corresponds to approximately 0.00125–0.02 mg/kg, respectively. Ibrutinib and acalabrutinb have a reported ED₅₀ of 0.91mg/kg and 0.34 mg/kg, respectively^{42 (link)}.

On test days, rats were injected i.p. with either saline, or the adenosine receptor antagonist caffeine (Sigma-Aldrich, St. Louis, MO; 50 mg/kg) or the partial inverse agonist at the benzodiazepine allosteric site on the GABA_A receptor, FG-7142 (Sigma-Aldrich, St. Louis, MO; 7.5 mg/kg). Both caffeine and FG-7142 have been shown to be anxiogenic in rodents: caffeine, 50 mg/kg [19 (link)–22 (link)] and FG-7142, 7.5 mg/kg [22 (link),23 (link)]. FG-7142 was dissolved in (40%w/v) 2-hydroxypropyl cyclodextrin (Sigma-Aldrich, St. Louis, MO) in 0.05N HCl to increase its solubility as done in previous studies [24 (link),25 (link)]. Each of these drugs (caffeine and FG-7142) have been reported to cause increased vigilance and arousal behaviors in home cage environment of rats from when compared to vehicle-injected controls [25 (link),26 (link)].