Apoptosis Induction in Cultured Cortical Neurons

Cortical mouse neurons were cultured as described (Papadia et al., 2008 (link)) at a density of between 9-13 × 10⁴ neurons per cm² from E17.5 mice with Neurobasal growth medium supplemented with B27 (Invitrogen). p53-null founder mice were obtained from Dr. Alan Clarke (University of Cardiff, Cardiff, UK) and were extensively crossed into the CD1 background. Puma-null founder mice were obtained from Dr. Andreas Strasser (Villunger et al., 2003 (link)). Stimulations of cultured neurons were done in all cases after a culturing period of 8-10 days during which cortical neurons develop a network of processes, express functional NMDA-type and AMPA/kainate-type glutamate receptors, and form synaptic contacts. Our cultured neurons are 10-15% GABAergic (assessed by immunofluorescence). Bursts of action potential firing were induced by treatment of neurons with 50 μM bicuculline, and burst frequency was enhanced by addition of 250 μM 4-aminopyridine (Hardingham et al., 2001 (link)). MK-801 (used at 10 μM) was from Tocris, TTX (at 2 μM) and 4-aminopyridine from Calbiochem. Neurons were subjected to trophic deprivation by transferring them from growth medium to a medium containing 10% MEM (Invitrogen), 90% Salt-Glucose-Glycine (SGG) medium ((Bading et al., 1993 (link)); SGG: 114 mM NaCl, 0.219 % NaHCO₃, 5.292 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, 1 mM Glycine, 30 mM Glucose, 0.5 mM sodium pyruvate, 0.1 % Phenol Red; osmolarity 325 mosm/l,(Papadia et al., 2005 (link))). For this model, apoptosis was analysed after 72 h. Neurons were fixed and subjected to DAPI staining and cell death quantified by counting (blind) the number of apoptotic nuclei as a percentage of the total. Approximately 1500 cells were counted per treatment, across 4 independent experiments (i.e. performed on separate cultures). Morphologically, neurons subjected to trophic deprivation show typical signs of apoptotic-like cell death (shrunken cell body and large round chromatin clumps). Furthermore, death was blocked by the pan-caspase inhibitor Q-VD-Oph (50 μM, Fig. 1a). Chemical inducers of apoptosis were used as follows: staurosporine (Calbiochem, 100 nM), 9-cis retinoic acid (Sigma, 5 μM), okadaic acid (Calbiochem, 2 nM), hydrogen peroxide (Sigma, 100 μM). Cell death in all cases was blocked by pre-treatment with Q-VD-Oph (50 μM, Fig. 1a and (Papadia et al., 2008 (link))). These inducers were applied to neurons for 24 h after which the percentage of apoptotic neurons was analysed as described above.