Analysis of GFP-expressing neurons at P18 revealed a significant increase in the number of dendritic spines on CA1 pyramidal neurons in NgRTKO−/− mice relative to their triple heterozygous littermate controls ( Figures 5A and 5B). These findings are

consistent with the idea that the NgR family members function together in vivo to limit the number of excitatory synapses. To extend this analysis using an independent approach, we performed transmission electron microscopy to visualize the ultrastructural features of excitatory synapses. In micrographs from NgRTKO−/− mice, we observed asymmetric synapses of typical morphology, suggesting that the overall structure and vesicle content of Selleckchem Venetoclax excitatory synapses are normal in the absence of NgRs. However, quantification of the number of excitatory synapses in the apical dendritic regions of CA1 revealed that NgRTKO−/− mice had a significant

increase in the density of excitatory synapses relative to heterozygous littermate controls ( Figures 5C and 5D). Furthermore, this effect was not limited to CA1 neurons, since analysis of CA3 neurons also revealed a clear increase in the number of PSDs in NgRTKO−/− animals ( Figure 5E). Thus, analysis by confocal and electron microscopy suggests that the NgR family functions to limit the number of excitatory synapses in vivo. To address whether the observed increase in synapse number reflects an increase Pictilisib research buy in functional synapses, we performed whole-cell patch-clamp electrophysiology on CA1 pyramidal neurons from acute hippocampal slices obtained from NgRTKO−/− mice and control littermates to quantify the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs). This analysis revealed a significant increase in the frequency of mEPSCs in

NgRTKO−/− mice relative to littermate controls ( Figure 5F and S5C), suggesting that the NgR family restricts the development of functional excitatory synapses. Interestingly, there was a small but significant Oxymatrine decrease in the amplitude of mEPSCs ( Figure 5G and S5D), consistent with the immature spine types observed in NgR1 knockouts ( Lee et al., 2008 and Zagrebelsky et al., 2010). Thus, reducing the expression of the NgR family results in an increase in functional synapses that are slightly reduced in strength. The question remained as to how NgRs work at a mechanistic level to restrict excitatory synapse number. One possibility was that NgRs limit the formation of new synapses in part by inhibiting dendritic growth, thereby reducing the possibility of contact between axons and dendrites. Therefore, we asked whether loss of NgR family members affects dendritic branching.