Neuronal Plasticity Induction and Regulation

tdTomato and Lifeact-GFP were purchased from ibidi, Inc. Ser3 peptides with the sequence of MAS(p)GVAVSDGVIKVFN were synthesized by GenScript (GenScript). GW4869, desipramine, fumonisin B1, C2-ceramide, and Pyrazolopyrimidine 2 (PP2) were purchased from Sigma-Aldrich (Sigma-Aldrich, USA). Recombinant IL-1β (PeproTech) was dissolved in DMEM and used after one freeze–thaw cycle at 50 ng/ml. After 14–18 days in vitro (DIV), neurons were treated at 37 °C with 50 ng/ml IL-1β, with control neurons receiving equal volumes of vehicle. cLTP was induced as described previously [24 (link), 25 (link)]. Briefly, hippocampal neurons were maintained in normal ACSF (5 mM HEPES [pH 7.3], 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 33 mM glucose). Osmolarity was adjusted to 290 mosmol/l. Chemical LTP was induced by changing the medium to Mg²⁺-free ACSF (5 mM HEPES [pH 7.3], 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 33 mM glucose, 0.2 mM glycine, 0.02 mM bicuculline, and 0.003 mM strychnine) for 10 min. After that, the incubation solution was altered back to control solution without glycine for 20 min before surface GluA1 labeling and for 30 min before fixation for immunohistochemistry to detect changes in F-actin, respectively. Neurons were treated with vehicle or IL-1β before (1 h), during (10 min), and after cLTP stimulation at indicated time points.