Since NGL-2 affects synaptic transmission selectively in the SR pathway but does not affect the properties of individual synapses, we sought to determine whether NGL-2 exerts a cell-autonomous and pathway-specific effect on synapse density. We investigated the role of postsynaptic NGL-2 in regulating spine density by knocking down NGL-2 in a subset of CA1 pyramidal

cells. We electroporated the GFP-containing shNGL2 or control plasmids into embryonic day 15 (E15) mouse embryos (Figure 5A). Animals were perfused at P13–P15, the brains were sectioned and immunostained for GFP (Figure 5B), and spine density was analyzed on secondary apical dendrites in the stratum Raf inhibitor radiatum (Figure 5C). Consistent with the electrophysiological experiments, we found that NGL-2 knockdown caused a significant decrease in spine density on CA1 dendrites in stratum radiatum as compared to the GFP control (Figures 5D and 5G). To determine whether

the effect on spine density was selective to the dendritic segment traversing the SR, we also measured CA1 spine density on secondary apical dendrites in the SLM and found that shNGL2 expression did not affect spine density in this domain (Figures INCB018424 molecular weight 5D and 5H). Thus, postsynaptic knockdown of NGL-2 selectively affects spine density in the stratum radiatum without affecting spine density in the SLM, indicating that a major role of NGL-2 is to regulate synapse density in the SR pathway.

To identify the domains of NGL-2 that mediate its synaptic effects, we coelectroporated shNGL2 with an shRNA-insensitive full-length NGL2∗ or domain deletion mutants and quantified spine density in the SR and SLM (Figures 5E–5H). Expressing NGL2∗ rescued spine density back to control levels on dendrites in the SR (Figures 5F and 5G). We observed no change in spine density in the SLM (Figures 5F and 5H), which is consistent with the targeting of NGL-2 to SR synapses. To determine whether the LRR and PDZ-binding domains of NGL-2 contribute to the spine effects of NGL-2, we generated shRNA-resistant deletion mutants NGL2∗ΔLRR and NGL2∗ΔPDZ (Figure 5E). Like the full-length rescue construct, both mutants are insensitive to shNGL2 (Figure S3B) and reach the surface of HEK293T Electron transport chain cells (Figure S3C). Unlike the full-length NGL2∗, neither NGL2∗ΔLRR nor NGL2∗ΔPDZ could rescue the shNGL2-mediated decrease in spine density in SR (Figures 5F and 5G). Furthermore, neither mutant had an effect in SLM (Figures 5F and 5H). Thus, both the LRR and PDZ-binding domains are required for NGL-2-mediated regulation of spine density in CA1. To further explore the roles of the LRR and PDZ-binding domains in excitatory synapse formation, we overexpressed these mutants, full-length NGL2∗ or EGFP control in cultured hippocampal neurons and analyzed excitatory synapse density by staining for excitatory synapse markers PSD-95 and VGlut1 (Figure S3A).