To assess the function of NgR1 during synapse development, we examined the effect of reducing the expression of NgR1 in cultured hippocampal neurons. Two distinct RNAi-based approaches were used to knockdown NgR1 expression, either direct transfection with short interfering RNA duplexes (siNgR1) or a plasmid encoding a short hairpin RNA to NgR1 (shNgR1) that targets a distinct region of NgR1 mRNA. These RNAis were tested in heterologous cells and primary neuronal cultures, where they selectively reduced NgR1 protein

levels while leaving NgR2 and NgR3 expression unaffected (Figures S2A-S2C). To investigate the effect Dinaciclib datasheet of reducing NgR1 expression on synapse number, hippocampal neurons were cultured, transfected at 9 days in vitro with a plasmid encoding green fluorescent protein (GFP) together with an RNAi to NgR1 or a control RNAi, and fixed 5 days later for staining with antibodies that recognize the presynaptic protein synapsin1 (Syn1) and the Y-27632 postsynaptic protein PSD95. To quantify the number of synapses formed

on the transfected neuron, we counted the number of apposed Syn1/PSD95 puncta along dendrites of GFP-expressing neurons (see Experimental Procedures). Using this approach we found that knockdown of NgR1 resulted in a significant increase in excitatory synaptic number (Figures 2A–2C; all data are listed in Table S1). Similar results were obtained using alternative sets of synaptic markers (GluR2/Syt1 or NR2B/Syt1) (Figures 2E, 2F, and S2D). Furthermore, we also observed an increase in the average size and intensity of synaptic puncta after NgR1 knockdown

(Figures S2E and S2F). We verified the Bay 11-7085 specificity of the NgR1 RNAi phenotype by testing the ability of an RNAi-resistant form of NgR1 (ResNgR1) to rescue the increase in synapse density observed upon knockdown of NgR1. ResNgR1 was validated in heterologous cells (Figure S1B) and then cotransfected in culture neurons along with shNgR. We found that ResNgR1was sufficient to reverse the increase in synaptic number observed with knockdown of NgR1 (Figure 2D), suggesting that the increase in synapse number in NgR1 RNAi-treated neurons is due to the specific knockdown of NgR1 by RNAi. NgR1 belongs to a family that includes two highly homologous proteins, NgR2 and NgR3. All three NgRs are expressed at high levels in the dorsal telencephalon during synaptic development (Figure S2G). To investigate whether NgR2 and NgR3 also function as negative regulators of synapse development, we examined the effect of reducing expression of either NgR2 or NgR3 in cultured hippocampal neurons. Short hairpin RNAs to NgR2 (shNgR2) or NgR3 (shNgR3) were validated in heterologous cells (Figure S2H) and then expressed in neurons, where they resulted in a significant increase in excitatory synapse density (Figure S2I). To extend this finding, we acquired knockout mice for NgR1 ( Zheng et al.