Millicell cm

The hippocampi for preparation of 400-μm thick organotypic slices with the preserved tissue organization were isolated from the brains of 7-day-old Wistar rats (n = 18), according to the protocol described previously [51 (link)]. The procedure was approved by the IV Local Ethics Committee on Animal Care and Use (Ministry of Science and Higher Education). The slices, obtained by cutting the chilled hippocampi by use of a McIlwain apparatus, were placed on Millicell-CM (Millipore) membranes and cultured initially in DMEM medium (Gibco) containing the following supplements: 25% horse serum (Gibco), 25% HBSS (Gibco), 2 mmol/l L glucose (Sigma), 5-mg/ml HEPES (Gibco), B27 supplement (Gibco), and an antibacterial–antimycotic solution (Sigma). On the day following establishing the ex vivo culture, the serum concentration in the culture medium was gradually decreased and therefore from the 5 day in vitro (DIV) onwards, the slices were kept for the following 7 days in serum-free media and in normoxic gas conditions (5% O₂, 5% CO₂).

Entorhino-hippocampal tissue cultures were prepared from mouse brains of C57Bl6/J mice at postnatal days 3–5 according to previously published protocols (Prang et al., 2003 (link); Del Turco and Deller, 2007 (link); Del Turco et al., 2019 (link)). For brain dissection, an ice-cold preparation medium [Minimal essential medium (MEM, Gibco) containing 2 mM Glutamax (Gibco), pH 7.3] was used. Slices (300–350 μm) were cut using a Leica vibratome (VT1200S, Leica). Organotypic tissue cultures were maintained on porous-MEMbrane filter inserts (Millicell-CM, Millipore) and incubated in a humidified atmosphere with 5% CO₂ at 35°C. Medium for cultivation contained 42% MEM, 25% basal medium eagle (Gibco), 25% heat-inactivated normal horse serum (Gibco), 2.5% HEPES buffer solution (Invitrogen), 0.15% bicarbonate (Invitrogen), 0.675% glucose (Sigma–Aldrich), 0.1 mg/ml streptomycin (Sigma–Aldrich), 100 U/ml penicillin (Sigma–Aldrich) and 2 mM Glutamax. The pH was adjusted to 7.3 and the medium was replaced every 2–3 days. Organotypic tissue cultures were incubated for up to 42 days in vitro (DIV).

Organotypic hippocampal slices were prepared from Wistar rats of either sex at postnatal day 5–7. Briefly, dissected hippocampi^{54 (link)} were cut into 400 μm slices with a tissue chopper and placed on a porous membrane (Millicell CM, Millipore). Cultures were maintained at 37 °C, 5% CO₂ in a medium containing (for 500 ml): 394 ml Minimal Essential Medium (Sigma M7278), 100 ml heat inactivated donor horse serum (H1138 Sigma), 1 mM l-glutamine (Gibco 25030-024), 0.01 mg ml⁻¹ insulin (Sigma I6634), 1.45 ml 5 M NaCl (S5150 Sigma), 2 mM MgSO₄ (Fluka 63126), 1.44 mM CaCl₂ (Fluka 21114), 0.00125% ascorbic acid (Fluka 11140), 13 mM d-glucose (Fluka 49152). No antibiotics were added to the culture medium. The medium was partially exchanged (60–70%) twice per week. Wistar rats were housed and bred at the University Medical Center Hamburg-Eppendorf (UKE). All procedures were performed in compliance with German law (Tierschutzgesetz der Bundesrepublik Deutschland, TierSchG) and according to the guidelines of Directive 2010/63/EU. Protocols were approved by the Behörde für Justiz und Verbraucherschutz (BJV) - Lebensmittelsicherheit und Veterinärwesen, Hamburg.

Organotypic hippocampal slice cultures were prepared and grown with modifications to the interface culture method^{50 (link)} from P6–8 Sprague-Dawley rats. Three hundred-µm-thick, isolated hippocampal brain slices were sectioned using a Leica VT1200S vibratome in ice-cold sterile slicing solution consisting (in mM) of Sucrose 105, NaCl 50, KCl 2.5, NaH₂PO₄ 1.25, MgCl₂ 7, CaCl₂ 0.5, Ascorbic acid 1.3, Sodium pyruvate 3, NaHCO₃ 26, and Glucose 10. Following washes in culture media consisting of 50% Minimal Essential Media, 25% Horse Serum, 25% Hanks Balanced Salt solution, 0.5% L-Glutamine, 28 mM Glucose, and the antibiotics penicillin (100 U/ml) and streptomycin (100 µg/ml), three to four slices were transferred onto each 0.4-µm pore membrane insert (Millicell-CM, Millipore, UK), kept at 37 °C in 5% CO₂ and fed by medium exchange every 2–3 days for a maximum of 21 days in vitro (DIV). The slices were transferred to a microscope recording chamber with the recording artificial cerebrospinal fluid solution containing (in mM): NaCl 125, NaHCO₃ 26, KCl 2.5, NaH₂PO₄ 1.25, MgSO₄ 1.3, CaCl₂ 2, and glucose 16 (osmolarity 300–305 mOsm), continuously bubbled with 95% O₂/5% CO₂. Recordings were carried out at 33–35 °C, with addition of 10 μM NBQX and 50 μM AP5 to reduce the potential for plasticity effects influencing synaptic properties during prolonged recordings.

250 µm thick coronal slices of P10 Cit-k KO mouse forebrain and cerebellum were cut using a Tissue Chopper. The slices were placed on Millicell-CM culture plate inserts (Millipore, Billerica, MS, USA) in a medium composed of 50% basal medium with Earle’s salts, 25% Hanks’ buffered salt solution, 25% horse serum, 5 mg/ml glucose, 0.5 nM triiodothyronine (T3; Sigma–Aldrich, Saint Louis, MS, USA), 60 µg/ml NAC. After 7 days in vitro (DIV), slices were either fixed for 30 min at RT with 4% PFA in 0.1 M PB for subsequent anti-MBP immunolabelings (see above) or pooled (3 millicells/sample) and lysed for the subsequent protein extraction.

Ovaries dissected at 17.5 dpc were placed in culture. Ovaries were cultured in 4-well culture plates in drops of media on 0.4 μM floating filters (Millicell- CM; Millipore Corp., Bedford, MA) in 0.4 ml DMEM-Ham’s F-12 media supplemented with penicillin-streptomycin, 5X ITS-X (Life Technologies, Inc., Grand Island, NY), 0.1% BSA, 0.1% albumax, and 0.05 mg/ml L-ascorbic acid. E₂ and P₄ (Sigma Chemical Co., St. Louis, MO) were dissolved in dimethylsulfoxide (DMSO) at a concentration of 0.1 M and then added to culture media to achieve the desired final concentration. DMSO was added to media at the same percent as the chemical to serve as vehicle control. Ovaries were placed in culture and exposed daily to E₂, P₄ or both hormones at 10⁻⁶ M or DMSO alone (n = 5 ovaries per treatment group). Ovaries were divided randomly among the treatment groups. The ovaries from control and treatment were fixed in Bouin’s fixative and histologically processed.