, 2008a), confirming their neuronal identity. For studying the effects of expressing wild-type and chimeric receptors based on GluN2A and GluN2B, constructs were cotransfected with peGFP (ratio 1:1) to identify transfected cells. Coexpression at this ratio was confirmed in the case of pRFP (Papadia et al., 2008). After 48 hr, the transfected neurons were then either
subjected to electrophysiological analysis or their fate following an excitotoxic insult was studied. Pictures of GFP-expressing neurons were taken on a Leica AF6000 LX imaging system, with a DFC350 FX digital camera. Using the automated cell-finder function within the Leica AF6000 software, images of transfected neurons were taken both before and 24 hr after a 1 hr treatment with NMDA (20 μM). Cell death was
assessed by counting the number Sirolimus of surviving GFP-positive neurons. In the vast majority of cases, death was easily spotted as an absence of a healthy GFP-expressing cell where one once was. In place of the cell, there was in most cases (>90%) evidence of death in the form of fragmented neurites, fluorescent cell debris, and a pyknotic nucleus (Papadia et al., 2008). This confirmed that the cells were genuinely dying as opposed to more unlikely scenarios, such as quenching of eGFP fluorescence in a subpopulation of neurons. For each condition, 150–200 neurons were studied over several independent experiments. An identical experimental regime was employed for studying the influence of ICER expression
on vulnerability of GluN2B2A(CTR)/2A(CTR) EGFR inhibitors cancer and GluN2B+/+ neurons to NMDA-induced excitotoxicity. Neurons were transfected with vectors encoding eGFP and the inhibitory CREB family member ICER1 (Stehle et al., 1993), or a control vector (encoding β-globin). We have STK38 previously confirmed that ICER1 expression inhibits CRE-mediated gene expression in neurons (Papadia et al., 2005). The fate of transfected neurons following exposure to NMDA was then studied as described previously. To measure extrasynaptic NMDAR currents, synaptically located NMDARs were blocked by quantal activation-mediated blockade by MK-801, as previously described (Martel et al., 2009 and Papadia et al., 2008). Briefly, whole-cell NMDAR currents were recorded (100 μM NMDA, in Mg2+-free and TTX/PTX-containing recording solution), after which the agonist was washed-out the recording chamber for 2 min. Irreversible NMDAR open-channel blocker MK-801 (10 μM; Tocris Bioscience) was then applied for 10 min, effectively antagonizing NMDARs located at the synapse and experiencing the localized, quantal presynaptic glutamate release (Martel et al., 2009 and Nakayama et al., 2005). Following the 10 min incubation period, MK-801 was then washed out (2 min), and the resulting extrasynaptic NMDAR currents were acquired.