As pharmacological approaches commonly used to differentiate the two subunits are limited at best, the authors used genetic manipulations to engender chimeric receptors in which only the CTD from GluN2A Osimertinib in vitro and GluN2B receptor subunits is C-terminal replaced (CTR). Why focus on the CTD? It has been shown that the CTD of NMDAR subunits is the primary area of sequence divergence, and it is the site that primarily
binds scaffolding proteins, providing a strong rationale for examining its role in excitotoxicity. In the first series of experiments, expression of chimeric GluN2B2A(CTR) receptors in transfected hippocampal neurons produced similar currents as wild-type (WT) subunits (GluN2BWT) and did not affect the proportion of synaptic and extrasynaptic receptors, thus preventing potential confounds arising
from receptor location. Interestingly, NMDA-induced cell death was reduced in chimeric GluN2B2A(CTR) compared to GluN2BWT-containing receptors, suggesting that excitotoxicity is better promoted by CTD2B than CTD2A. Similarly, neurons expressing GluN2A2B(CTR) LY294002 datasheet were more susceptible to cell death than neurons expressing GluN2AWT (Figure 1). Using a different approach, a knockin mouse was generated in which the protein-coding region of the C-terminal exon of the GluN2B subunit was exchanged for that of the GluN2A subunit, named GluN2B2A(CTR)/2A(CTR). Cultured cortical neurons from these mice displayed
similar levels of viability, synaptic connectivity, proportion of extrasynaptic NMDARs, sensitivity to ifenprodil, rundown of NMDA currents, and single channel conductance, compared to those from GluN2B+/+ mice. Notwithstanding these similarities, NMDA currents were about 30% lower in GluN2B2A(CTR)/2A(CTR) than in GluN2B+/+ cells. By adjusting exogenous NMDA concentrations to produce similar currents in both types of cells, the authors confirmed that normalized NMDA current produced more death in GluN2B+/+ than in GluN2B2A(CTR)/2A(CTR) cells. In consequence, a switch in the mouse genome from GluN2B CTD for GluN2A reduces NMDA-dependent Ca2+ influx Isotretinoin and excitotoxicity. However, these differences only occurred at moderate (15–50 μM) NMDA concentrations. When NMDA concentration was increased (e.g., 100 μM), the CTD subtype-specific vulnerability disappeared. These results were confirmed in vivo. Thus, excitotoxic lesions induced by stereotaxic injection of a small dose of NMDA into the hippocampus (CA1-CA3 region) induced smaller lesion volumes in GluN2B2A(CTR)/2A(CTR) compared to GluN2B+/+ mice. Which signaling cascades contribute to differential susceptibility of CTDs to excitotoxic insults? One obvious target, based on previous work by Hardingham’s and other groups, is NMDA-dependent activation of CREB.