Minocycline

Slices were prepared from P17–P90 male CD1 EAAT4-GFP or EAAT4-GlyT2-GFP mice. This strain was obtained by crossing EAAT4-GFP mice with a line expressing GlyT2-GFP (gift from H.U. Zeilhofer) (Zeilhofer et al., 2005 (link)), in which some GoCs express GFP. Mice were anesthetized by inhalation of isoflurane, and then killed by decapitation. Transverse slices were prepared as previously described (Valera et al., 2012 (link)). The cerebellum was dissected out and placed in cold artificial cerebrospinal fluid (ACSF) bubbled with carbogen (95% O₂, 5% CO₂), containing (in mM): NaCl 120, KCl 3, NaHCO₃ 26, NaH₂PO₄ 1.25, CaCl₂ 2.5, MgCl₂ 2, glucose 10 and minocycline 0.00005 (Sigma-Aldrich, USA). Then 300 µm-thick transverse slices were prepared (Microm HM 650V, Microm, Germany) in potassium-based medium, containing (in mM): K-gluconate 130, KCl 14.6, EGTA 2, HEPES 20, glucose 25, minocycline 0.00005 and D-AP5 0.05 (Sigma-Aldrich). After cutting, slices were soaked in a sucrose-based medium at 34°C, containing (in mM): sucrose 230, KCl 2.5, NaHCO₃ 26, NaH₂PO₄ 1.25, glucose 25, CaCl₂ 0.8, MgCl₂ 8, and minocyclin 0.00005 (Sigma-Aldrich) and maintained in a water bath at 34°C in bubbled ACSF.

Minocycline (#M9511, Sigma) was administered at 120 mg/kg in distilled water through gavage in a dose volume of 10 ml/kg, twice daily, at 12-hour intervals. Previous studies have demonstrated Minocycline effectively attenuated IL-1β expression in the brain [8 (link),18 (link)].
1-MT (#860646, Sigma) was administered through subcutaneous injection at 50 mg/kg in a dose volume of 5 ml/kg. The injections were administered twice daily at 12-hour intervals, in accordance with the methods of Professor Keith W Kelley, who had administered 50 mg/kg of 1-MT to mice twice a day, and the effect was equal to that observed in studies using 5 mg/day pellets [8 (link)]. We prepared 1-MT using 0.1 M NaOH and adjusted the pH to 9.0 using 1 M HCl.

Human THP-1 monocyte cell line (provided by Dr. Ulrike Erben) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal calf serum (FCS) and 1% penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were seeded in 96-well plates at an initial density of 8 × 10⁴ per well and allowed to attach for 24 h.
To assess the inhibition of RA-degradation by minocycline, cells were treated with 1 µM all-trans RA (Sigma-Aldrich, St. Louis, USA) and minocycline (Minocyclinhydrochlorid, Hovione, Loures, Portugal) at concentrations of 0, 10 and 100 µM.
To further investigate the effects of inflammatory activation on RA degradation, and to quantify the impact on minocycline on the latter, cells were treated with 100 ng/mL lipopolysaccharide, a pro-inflammatory stimuli (LPS; Escherichia coli, Sigma-Aldrich). Vehicle or minocycline were added at 10 and 100 µM. After an incubation period of 24 h, all-trans RA was added to a final concentration of 1 µM.

minocycline dosages for systemic and local administration were chosen according to previous studies showing effects on microglia and behavior^{26 (link),31} . Mice received an average oral minocycline (Sigma-Aldrich) dosage of 40 mg/kg/day for 28 d. For local microinjections, minocycline (20 µg/μl) was infused bilaterally into the DG once daily, for 5 d (shorter period) or 11 d (longer period).

For minocycline experiments, CD-1 mice were IP injected once daily with minocycline (Sigma; 50 mg/kg) or vehicle (10 mM Tris-HCL) from P4 to P9. Dosage was determined based on previously published studies [78 (link)].