Allopregnanolone

BPF (cat. no. 51453), BPA-d₁₆ (cat. no. 451835), 3,4-dichloroaniline (3,4-DCA, cat. no. 437778), Dimethyl sulfoxide (DMSO, cat. no. 472301), and tricaine (MS-222) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-Phenyl-2-thiourea (PTU) was purchased from Fluka (CAS No. 79180).
Standards for the three neurosteroids (progesterone, allopregnanolone, and testosterone) and six neurotransmitters (3-methoxytyramine, 3,4-dihydroxy-L-phenylalanine, norepinephrine, glutamic acid, gamma-aminobutyric acid, and dopamine) used in this study were purchased from either Sigma-Aldrich (St. Louis, MO, USA) or Steraloids, Inc. (Newport, RI, USA). The following internal standards were used: dehydroepiandrosterone-2,2,3,4,4,5-d6 for testosterone; pregnenolone-17β,21,21,21-d4 for progesterone and allopregnanolone; and tryptophan-d3 for the aforementioned six neurotransmitters; these standards were purchased from Sigma-Aldrich and C/D/N Isotopes Inc. (Pointe-Claire, QC, Canada). All organic solvents were of analytical or HPLC grade and purchased from Honeywell Burdick and Jackson (Muskegon, MI, USA). Deionized water was prepared using a Milli-Q purification system (Millipore, Burlington, MA, USA).

Epstein-Barr Virus generation, passaging and maintenance for LCLs prior to the experimental ALLO treatments are as described in Rudzinskas et al., 2023 [19 (link)]. For the present study, N = 19 LCLs were utilized, comprising n = 10 derived from women without a history of PPD (i.e., Controls) and n = 9 from women with past PPD (i.e., PPD). Approximately three days prior to experimental treatments, each LCL was seeded at ~1 × 10⁶ cells/mL in two flasks of 15%-knockout serum replacement (KOSR) RPMI-1640 (a phenol red-free [47 (link)], FBS-free [48 (link)] media). Vehicle treatment stock comprised of 100 μL sterile DMSO (Sigma-Aldrich, St. Louis, MO, USA, D2650-5X5ML) in 20 mL of media. For ALLO treatment stock, 5 mg of allopregnanolone (Sigma-Aldrich, USA, P8887-5MG) was dissolved in 1 mL of DMSO, with 100 μL added to 20 mL of media. The experimental timeline thereafter is described and depicted in Figure 1. In summary, LCLs were treated every 20 h over a 60-h period (mimicking the length of brexanolone infusion for PPD) with either an acute 100 nM spike of allopregnanolone (ALLO) or vehicle (DMSO), totaling three spikes [300 nM total] throughout the experimental treatment. Each LCL flask (N = 38) was then pelleted, frozen and stored at −80 °C for subsequent molecular analyses.

Starting at postnatal day 7 (P7) and thenceforth, mice of the combi-group were injected weekly with 2-hydroxypropyl-β-cyclodextrin/allopregnanolone (25 mg/kg allopregnanolone dissolved in 40% 2-hydroxypropyl-β-cyclodextrin in Ringer’s solution, 4000 mg/kg, i.p., all from Sigma-Aldrich, Munich, Germany). Additionally, these mice were daily injected with miglustat, dissolved in 0.9% NaCl solution, 300 mg/kg i.p. (N-butyldeoxynojirimycin, Zavesca; Actelion Pharmaceuticals, San Francisco, CA, USA) from P10 to P23. From P23 onwards until termination of experiments mice were fed standard chow with embedded miglustat resulting in daily intake of 1200 mg/kg miglustat. The miglu-group was treated like the combi-group, but without administration of cyclodextrin/allopregnanolone, instead mice got vehicle. Mice of the sham-group were injected like those of the combi-group at the various time points with the respective volumes of 0.9% NaCl or without volume and were fed with chaw without drugs.