Pregnenolone

Selectively bred, but alcohol naïve Alcohol-preferring (P) rats (male, 3–4 months old; 250–550 g; n = 7–9/group) were obtained from the Alcohol Research Center, Indiana University School of Medicine. Male P rats were examined due to innate activation of neuronal TLR4 signaling in brain^{41 (link),42 (link)} that allowed us to examine inhibition of TLR signaling without activation of the peripheral immune response. Animals were double housed in Plexiglas cages containing corn cob bedding and food and water was available ad libitum. The colony room was maintained on a normal 12 hr light-dark cycle (light onset at 0700 hr). Procedures followed National Institutes of Health Guidelines under UNC Institutional Animal Care and Use Committee approved protocols at University of North Carolina School of Medicine. Rats were habituated to handling for 7 days prior to administration of 3α,5α-THP (15 mg/kg, IP), pregnenolone (75 mg/kg, IP), 3α,5α-THDOC (15 mg/kg, IP), or vehicle (45% w/v 2-hydroxypropyl-β-cyclodextrin) and returned to their home cage. Rats were sacrificed after 45 minutes and the brain was removed and frozen at −80 °C until VTA micropunches were collected from 1 mm cryostat brain sections. This time point was selected because 3α,5α-THP is rapidly metabolized in vivo^{53 (link)}, but has behavioral and pharmacological activity at this time point^{98 (link)}.

Steroids were isolated from 1 ml of cell culture supernatant by liquid/liquid extraction using 3 ml of methyl tertiary butyl ether (MTBE) (Sigma-Aldrich) in borosilicate glass tubes (Fisherbrand, Schwerte, Germany). Samples were vortexed twice for 5 seconds at highest speed, spinned down and subsequently frozen at -20°C for at least 1 h and the top organic layer was poured off into a new clean and dry glass tube. The frozen aqueous layer was discarded. Samples were evaporated for 15 min at 55°C and 0.5 bar using a nitrogen evaporator (Gebrüder Liebisch Labortechnik GmbH, Bielefeld, Germany). Samples were reconstituted in 50/50 methanol/water. They were analysed by tandem mass spectrometry on a Waters Xevo Mass Spectrometer with an acquity uPLC liquid chromatography system as described previously [23 (link)]. Steroids were separated using a HSS T3 1.2 x 50 mm column and identified in positive ionisation mode. System set-up is described in Supporting Information of this article (S2 Table). Steroids were quantified by comparison to a calibration series ranging from 0.5 to 1000 ng/ml and set off against total protein content (μg) per well, which represents the cell number. Steroid concentrations were expressed in mMol/g total protein content and each sample was normalised to its native doxycycline-un-induced state giving the percentile change in steroid output as follows: % change = (steroid concentration sample/steroid concentration native doxycycline-un-induced state) *100. All steroids were quantified using a suitable internal standard (S3 Table). A suit of steroids was considered covering the steroid bio-pathway; cortisol, cortisone, 11-deoxycortisol, corticosterone, androstenedione, testosterone, 5α-dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), 17-hydroxyprogesterone (17OHP), deoxycorticosterone (DOC), progesterone, pregnenolone, and 17-hydroxypregnenolone. Two mass transitions were required to positively quantify each steroid.

For the in vivo study, C57BL/6J 8-week old mice were utilized. Animals were housed and maintained in a 12-h light/dark cycle and fed ad libitum. All procedures were performed according to the European Union policy (Directive 86/609/EEC) (carried out in compliance with Greek Government guidelines) and institutionally approved protocols (Veterinary Directorate of Prefecture of Heraklion (Crete) and FORTH ethics committee (License number: EL91-BIOexp-02)). Mice were anesthetized using an intraperitoneal injection of a ketamine (Nerketan 10, 100 mg/mL) and xylazine (Xylapan, 20 mg/mL) cocktail. Once the hind-limp was lost, the mice were fixed in the supine position, 25 µL of each formulation was administered to each mouse in 60 s intervals, via 1–2 µL dose alternatively into each nostril [26 (link)]. Animals were sacrificed 60 and 120 min after the completion of the administration. Brains were quickly dissected out of the cranium; excess blood was wiped off, and the brain was snap frozen until further processing.
After weighing, brain samples were homogenized in 300 µL ice-cold distilled water/methanol solution (25/75 v/v) and sonicated for 20 min at 4 °C. Next, three volumes of ice-cold acetonitrile were added, followed by sonication for 10 min and centrifugation at 14,000× g/15 min/4 °C. After an extra addition of 100 µL ice-cold acetonitrile to the supernatant, a 10 min-sonication and centrifugation at 14,000× g/15 min/4 °C were carried out. The supernatant was vacuum-dried at a SpeedVac without heating. Prior to analysis, samples were stored at −80 °C.
To quantify the BNN27 levels, the detection limit of the enzymatic method used for BNN27 quantification in the formulations (>2 ppm) was not low enough; therefore, a liquid chromatography mass spectrometry (LC-MSn) method was developed using deuteriated pregnenolone (pregnenolone 17,21,21,21-D4) as the internal standard (70 ng/mL). The analysis was performed on an LTQ-Orbitrap Velos mass spectrometer (MS) (Thermo Fisher Scientific, Bremen, Germany) connected to an Accela ultra-high-performance LC (UHPLC) system. An Acquity UPLC BEH C18 VanGuard pre-column (130 Å, 1.7 µm, 2.1 mm × 100 mm) coupled to an Acquity UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 5 mm) was used. Quality control samples were prepared at three concentrations (low, medium, high) to monitor the instruments’ performance and chromatographic integrity over time. Monitoring occurred in positive ion mode. The standard curve concentration range was 1–2000 ng/mL (Y = 0.000546519 + 0.000897604∙X; R2 = 0.9673; W:1/x). The injection volume was set at 5 µL, and the mobile phase flow rate was set at 0.2 mL/min. Mobile phase solvents were A (95% H₂O, 5% methanol, 0.1% formic acid) and B (methanol, 0.1% formic acid). The eluting gradient program was the following: 0–0.1 min (40% A, 60% B), 0.1–0.5 min (20% A, 80% B), 0.6–6.5 min (5% A, 95% B), 6.51–8.0 min (40% A, 60% B). Data were processed with Xcalibur software (version 2.1, Thermo Scientific, Waltham, MA, USA) and data analysis was conducted using the R programming language. In order to calculate the BNN27 levels in brain tissue, expressed as administered dose percent (ID%), the following formula was applied: ID%/g brain = (Xbrain/X in dose) × 100; where: Xbrain = BNN27 (mg) per g of weighted brain tissue, and X in dose = BNN27 (mg) in 25 µL solution for intranasal administration.

Serum samples stored at −80 °C were analyzed to determine cortisol, pregnenolone, and total and free testosterone levels within two months from sampling. Hormone levels were investigated using IBL ELISA enzyme immunoassays kits according to the manufacturer’s instructions (IBL America, Minneapolis, MN, USA). The ELISA assays have a standard range of 0–25 ng/mL for pregnenolone, 0–16 ng/mL for total testosterone, 0–60 pg/mL for free testosterone, and 0–800 ng/mL for cortisol. Optical density at 450 nm with 650 nm as the reference wavelength was determined using an ELISA microplate absorbance reader (Tecan Infinite F200, Tecan Group Ltd., Männedorf, Switzerland). All samples were measured in duplicate. Samples from each subject were assayed in the same batch. The inter- and intraassay variations of these analyses were all <10%.