Pre 084

The newly synthesised σ1R antagonist, S1RA: 4-[2-[[5-methyl-1-(2-naphthalenyl)-1H-pyrazol-3-yl]oxy]ethyl] morpholine), was obtained from Laboratorios Esteve (Barcelona, Spain). BD1047 (#0956), BD1063 (#0883) and PRE084 (#0589) were obtained from Tocris Bioscience (Bristol, UK). Compounds were dissolved in ethanol/Cremophor EL/physiological saline (1:1:18). To facilitate selective and straightforward access to their targets, the compounds were each injected into the lateral ventricles of mice at 4 μL as previously described or via an injection in the tail vein. Groups of 8 to 10 mice received doses of the selected compounds.

Spinal cord organotypic cultures (SCOCs) were prepared from lumbar sections of Sprague–Dawley pups (8–9 days-old) as previously described (Mòdol-Caballero et al., 2017 (link)). After harvesting, the spinal cord was cut in 350 μm thick transverse sections, that were transferred on Millicell-CM nets (0.4 μm, PICM03050, Millipore) and then into a six-well plate with the incubation medium [50% (v/v) minimal essential medium (MEM, M5775, Sigma), 2 mM glutamine, 25 mM HEPES, 25% (v/v) Hank’s Balanced Salt Solution (HBSS−/−, 14,175, Gibco) supplemented with 25.6 mg/ml glucose and 25% (v/v) heat-inactivated horse serum (26,050–088, Gibco), pH = 7.2). After 7 days in vitro (DIV), chronic excitotoxicity was induced by adding DL-threo-β-hydroxyaspartic acid (THA; 100 μM), a selective inhibitor of glutamate transport (Rothstein et al., 1993 (link)). The Sig-1R ligands were simultaneously co-added in the culture medium and renewed at each medium change twice per week. Sig-1R ligands PRE-084, BD1063 and SA4503 (Tocris) were tested at three different concentrations (30, 3 and 0.3 μM). Riluzole (5 μM) was also assayed as positive control. Slices were maintained for 28 DIV and then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). The in vitro experiments have been performed in three independent cultures and resulting in 12 slices for each experimental condition.