Western blot analysis during development confirmed the continued absence of GluN2B and the premature expression of GluN2A in 2B→2A cortical neurons this website (see Figure S1 available online). RT-PCR analysis of mRNA harvested from P17 homozygous 2B→2A animals showed the absence of GluN2B transcript and replacement with exogenous rat mRNA encoding GluN2A, in addition to the endogenous mouse transcript (Figure 1E). Together, these data show that the genetic strategy was successful and predict that in homozygous 2B→2A animals, glutamatergic cortical synapses contain only

GluN2A-containing receptors within a GluN2B null background. Genetic deletion of GluN2B leads to perinatal lethality (Kutsuwada et al., http://www.selleckchem.com/products/BAY-73-4506.html 1996). 2B→2A mice also displayed a high rate of perinatal lethality, indicating that premature expression of GluN2A is not sufficient to fully rescue GluN2B loss of function. Genotyping embryos harvested to prepare cortical cultures showed that the transmission frequency of the 2B→2A allele followed a predicted pattern with an approximately 1:2:1 (WT:HET:2A→2B) ratio from matings between heterozygous 2B→2A animals (19:41:21, in ten cultures).

However, only ≈8% of 2A→2B homozygous animals survived past P0: 6 out of 72 predicted animals (24 months following removal of the neo cassette, data from 38 litters). Surviving homozygous 2B→2A pups exhibited no differences in size at P0 but were significantly stunted in for mass when measured as juveniles (P12–P16) ( Figures 1G and 1H). In homozygous 2B→2A pups at P0, we recorded a decrease in the number of rhythmic mouth suckling movements in response

to stimulation with a feeding needle ( Figure 1I), suggestive of a weakened ability to feed. This is consistent with an observed lack of milk in their bellies at this age ( Figure 1G) and is similar to the GluN2B full knockout animals, which show a complete absence of suckling behavior. These observations underscore the importance of NMDAR signaling during development but also suggest a required and specific role for GluN2B-containing NMDARs. To determine whether or not GluN2A-containing receptors were properly expressed and trafficked to the neuronal membrane in 2B→2A mice, we generated neuronal cultures from homozygous embryos as well as WT embryos at E16–E17 and performed immunofluorescence analysis of GluN1. GluN1 is the obligate subunit of the NMDAR, and thus its expression in dendrites will correlate positively with total receptor levels. We measured similar levels of anti-GluN1 staining in 2B→2A dendrites compared to WT controls by costaining with the dendritic marker MAP2 (Figure 2A). To examine surface-localized protein, we used a live-labeling protocol. This analysis showed that the amount of membrane-associated GluN1 protein in 2B→2A neurons was comparable to WT (Figure 2B).