This finding suggests that NgR1 requires a coreceptor to inhibit synapse development. Genome-wide RNA sequencing revealed that of the known NgR1 coreceptors,

only Lingo-1 and TROY are expressed at appreciable levels in 7 DIV neuronal neurons (data not shown). Since Lingo-1 is largely expressed on axons (Lee et al., 2008), we focused on TROY as a potential NgR1 coreceptor that might function in dendrites to inhibit synapse development. Immunostaining with protein-specific antisera revealed that TROY is expressed along the dendrites of cultured neurons and overlaps significantly with all NgR family members (Figure S4E). In addition, TROY knockdown (Figures S4E, S4I, S4J, and S8B) caused a significant increase in synapse density in cultured hippocampal neurons (Figure 4H). Together, DAPT datasheet these findings are consistent

with TROY being the coreceptor that mediates the inhibitory effects of NgR1 on synapse development. To determine whether TROY is required for NgR1-dependent suppression of synapse development, WTNgR1 was overexpressed with or without TROY knockdown (shTROY) and synapse density was quantified. TROY knockdown reversed selleck chemicals llc the reduction in synapse number observed with NgR1 overexpression (Figure 4I). An increase in synapse density was observed, similar to that seen upon TROY knockdown alone. Similar epistasis studies with WTNgR2 and WTNgR3 overexpression revealed that TROY is required for the suppression of synapse development by NgR2 and NgR3 (Figure S4K). Moreover, binding experiments using recombinant TROY protein incubated with heterologous cells expressing different NgR family members show that TROY is capable of binding NgR1 and NgR2, but not NgR3 (Figure S4F), suggesting that NgR1 and NgR2 may signal through TROY directly. It remains unclear whether the affinity of the NgR3-TROY interaction falls below the detection limit of this assay or whether NgR3 acts through an alternative coreceptor. Taken together, these findings identify TROY as a potential

coreceptor for the NgR family that mediates their ability to restrict excitatory synapse number. To address whether the NgR family contributes to synaptic development in vivo, we crossed NgR mutant mice with the GFPM line (Feng et al., 2000), in which a small subset of neurons are genetically labeled with the Thy1-GFP Bay 11-7085 allele, thus enabling visualization of dendritic spines from hippocampal pyramidal neurons. Knockout of any one NgR family member alone was not sufficient to affect the density of dendritic spines in vivo (Figure 5B). Given our previous finding that all three NgR family members play a similar role in limiting synapse development in vitro (Figures 2G and S2I), we hypothesized that these family members might functionally compensate for one another in vivo. To address this possibility, we generated triple knockout mice (NgRTKO−/−).