These data indicate that the LRR domain is critical for promoting excitatory synapse formation in vitro and confirm that the lack of rescue we observed in vivo is not taking place due to dominant-negative effects of the domain deletion mutant proteins. To try to further understand the role of the specific interaction with netrin-G2, we obtained a mutant protein that we termed NGL1(NGL2LRR), in which 20 residues of the NGL-1 LRR domain have been swapped for NGL-2 residues (Seiradake et al., 2011). These

mutations cause NGL1(NGL2LRR) to bind to its normal receptor, netrin-G1, with very low affinity and instead to bind netrin-G2 with high affinity (Seiradake et al., 2011). We coelectroporated shNGL2 and NGL1(NGL2LRR) into a subset of CA1 pyramidal cells and analyzed spine density in SR and SLM. We found that NGL1(NGL2LRR) could fully Androgen Receptor Antagonist in vivo rescue the spine density in SR (Figures 5F and 5G) but had no effect on spine density in SLM (Figures 5F and 5H), indicating that the interaction between NGL-2 and netrin-G2

is critical for driving spine formation in SR. The specific effect of NGL-2 manipulations on high throughput screening SR synapses suggested that NGL-2 might be localized to the dendritic domain of CA1 neurons where SR synapses form. To address this possibility, we coelectroporated GFP-tagged NGL-2 with pCAG-tdTomato as a cytosolic marker. At P14, brains were perfused, sectioned, stained for GFP, and segments of dendrites were imaged in SR and SLM (Figure 6A). The GFP signal was visible in spines and in the dendritic shaft in

a pattern that was predominantly restricted to SR (Figure 6A, top), consistent with an earlier report old (Nishimura-Akiyoshi et al., 2007). We quantified the intensity of the GFP signal normalized to the intensity of the tdTomato signal and found more NGL-2 in SR (Figure 6A, bottom). These data indicate that localization of NGL-2 to a restricted domain of CA1 pyramidal cells could account for the synapse-specific effect of NGL-2. To test whether this localization depends on the interaction with netrin-G2, we generated a GFP-tagged LRR domain deletion mutant version of NGL-2 because the LRR domain is required for binding to Netrin-G2 (Seiradake et al., 2011). We coelectroporated this mutant with pCAG-tdTomato and analyzed the pattern of GFP immunofluorescence within CA1 dendrites. NGL2ΔLRR-GFP was expressed in a punctate pattern throughout the entire length of the CA1 pyramidal cell apical dendrites (Figure 6B, top). We quantified GFP expression levels in SR and SLM and found no significant difference between these regions (Figure 6B, bottom).