Potassium chloride (kcl)

Cultured hippocampal neurons were prepared from embryonic day 18 Sprague-Dawley rat brains. Dissociated neurons on poly-l-lysine coated (1 mg ml⁻¹) coverslips were placed in neurobasal medium supplemented with B27 (Invitrogen), 0.5 mM l-glutamate and penicillin–streptomycin. For activation, cultured neurons at days in vitro (DIV) 18–21 were treated with NMDA (Sigma; 20 µM, 3 min), KCl (Sigma; 50 mM, 5 min) or l-glutamate (Sigma; 50 µM, 30 min). For induction of NMDA receptor-dependent LTD or mGluR-LTD, cultured neurons were treated with NMDA (20 µM, 3 min) and DHPG (Tocris; 50 μM, 30 min), respectively, and returned to normal conditioning medium. For the blockade of NMDA receptors, cultured neurons were pretreated with APV (Tocris; d-2-amino-5-phosphovalerate; 50 µM, 30 min), followed by stimulation with NMDA (20 µM, 3 min) in the presence of APV or with l-glutamate (50 µM, 1 min) or KCl (30 mM, 1 min) in the absence of APV. For chronic neuronal inhibition or activation, neurons were treated with tetrodotoxin (Tocris; TTX; 1 µM, 48 h) or with bicuculline (Tocris; Bic; 10 µM, 36 h). For blockade of metalloproteinase and γ-secretase activity, neurons were pretreated with GM6001 (Enzo Life Sciences; 2.5 and 25 µM, 30 min), DAPT (Sigma; 250 nM, 2 h; 2 µM, 3 h) and L-685,458 (Calbiochem; 1 µM, 30 min) before and during NMDA stimulation (20 µM, 3 min).

NovoRapid^® insulin aspart 100 units/mL (Novo Nordisk, Bagsværd, Denmark), sotrastaurin (Item No. 16726, Cayman Chemical, Ann Arbor, MI, USA) and (R)-(+)-etomoxir sodium salt (Cat. No. 4539, Tocris Bioscience) were diluted before use in sterile filtered PBS (137 mM NaCl, 2.7 mM KCl (cat. P9541, Merck KGaA), 10 mM Na₂HPO₄ (cat. 12695147, ACROS Organics™), 1.8 mM KH₂PO₄ (cat. 205920025, ACROS Organics™), pH 7.4). BVT948 (Item No. 16615, Cayman Chemical) and wortmannin (Cat. No. 1232, Tocris Bioscience) were dissolved in dimethyl sulfoxide (DMSO) (cat. D8418, Merck KGaA). BMOV was synthesized using the method previously described by Caravan el al. [48 (link)]