Organotypic hippocampal slice, neuronal cultures, transfection and treatments

Organotypic hippocampal slice cultures were prepared as previously described (Pellegrini Giampietro et al., 1999). Hippocampal neuronal primary cultures were prepared from embryonic day 18–19 (E18-E19) rat hippocampi as previously described (Piccoli et al., 2007). Neurons were transfected at DIV7 using calcium-phosphate method. For induction of 'Bic' treatment: hippocampal neurons (DIV14) or organotypic hippocampal slices (DIV14) were incubated with Bicuculline (50 μM; Tocris) and 2.5 mM 4-Aminopyridine (4-AP;Tocris) in Neurobasal medium supplemented with B27; 'Syn' treatment: stimulation of synaptic NMDA receptors was obtained by treating hippocampal neurons at DIV14 with 50 μM Bicuculline (Tocris), 2.5 mM 4-AP and 5 μM ifenprodil in Neurobasal medium supplemented with B27; 'Extrasyn#1' treatment: stimulation of extrasynaptic NMDA receptors was obtained by pretreating hippocampal neurons at DIV14 with 50 μM Bicuculline, 2.5 mM 4-AP and 10 μM MK801 for 30 min, washing them with Neurobasal medium supplemented with B27, incubating with 50mM KCl (Carlo Erba) for 10 min and then with 40 μM Glutamate (Sigma); 'ExtraSyn#2' treatment was performed as previously described by Karpova and co-workers (2013). cLTP (Otmakhov et al., 2004; Oh et al., 2006): Primary neuronal cultures at DIV14 were first incubated in ACSF (125 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 33 mM D-glucose and 25 mM HEPES, pH 7.3) for 30 min, followed by 16 min of stimulation with 0.05 mM Forskolin (Sigma Aldrich), 0.1 mM Picrotoxin (Tocris) and 100 nM Rolipram (Calbiochem) in ACSF (no MgCl₂) to induce NMDAR-dependent cLTP. Control was incubated in ACSF. After stimulation, neurons were replaced in regular ACSF for 2 hr. cLTD (Marcello et al., 2013): primary neuronal cultures at DIV14 were first incubated in ACSF, followed by incubation in ACSF (in presence of MgCl₂) and 50 μM NMDA (Sigma Aldrich) for 10 min. Neurons were then replaced in regular ACSF for 1 hr to induce cLTD. For treatment with 'NLS2 peptide' hippocampal neurons (DIV14) were treated with active (NLS2: RKRKRQKQK) or control (TAT: GRKKRRQRRRPQ) peptide for 24 hr and then treated to induce cLTP the presence of the same peptides.