In granule cells, knockdown of LRRTM4 did not affect the strength of glutamatergic transmission (data not shown), which could be due to incomplete knockdown or the expression of other LRRTMs ( Laurén et al., 2003), which may functionally

compensate. We therefore decided to investigate LRRTM4’s role in synapse development in cortical layer 2/3 (L2/3) pyramidal neurons, which do not express LRRTM2 ( Figure 1A). We first tested whether LRRTM4 regulates synapse formation in cultured cortical neurons and found a significant decrease in the density of dendritic spines and of PSD-95-positive spines after LRRTM4 knockdown ( Figures S7A–S7D). Embryonic day 15.5 mouse embryos were electroporated with control or shLRRTM4 plasmids, resulting in the transduction of L2/3 pyramidal neurons in primary somatosensory cortex ( Figure 7A). We verified mTOR inhibitor by in situ hybridization that LRRTM4 is expressed in mouse P15 L2/3 neurons and that GPC4 is expressed in L2/3 and L4 neurons ( Figures S7E and S7F), indicating that GPC4 is presynaptic to the neurons we recorded from. GFP-positive electroporated L2/3 cells were scattered among a majority of nonelectroporated cells and targeted for whole-cell recording ( Figure 7B). We recorded mEPSCs from labeled cells in acute brain slices and compared mEPSCs from control, GFP-expressing neurons

to shLRRTM4-electroporated neurons ( Figure 7C). Knockdown of LRRTM4 did not affect the frequency of mEPSCs ( Figure 7D) but significantly reduced the mean amplitude of mEPSCs ( Figure 7E). XAV-939 mw These results

indicate that LRRTM4 regulates the strength of glutamatergic synaptic transmission in cortical neurons in vivo, most likely by regulating AMPA receptor content at synapses. To determine whether LRRTM4 may regulate synapse density in vivo as it does in cultured hippocampal and cortical neurons, we analyzed the density of dendritic next spines in L2/3 cortical neurons in electroporated mice at P14. LRRTM4 knockdown resulted in a significant, 18% decrease in the density of dendritic protrusions relative to control neurons (Figures 7F and 7G). Together, these results indicate that endogenous LRRTM4 is required for synapse development in vivo. Cell-surface interactions play key roles in establishing functional neural circuits. Here we identify glypican as an LRRTM4 receptor in an unbiased, proteomics-based approach to find the endogenous receptors for LRRTM2 and LRRTM4. Glypican preferentially interacts with LRRTM4, and this interaction is HS dependent. GPC4 and LRRTM4 localize to opposing membranes of glutamatergic synapses. GPC4 and LRRTM4 expressed on the surface of nonneuronal cells induce clustering of their respective binding partners in cocultured neurons, supporting a trans-synaptic interaction of presynaptic glypican and postsynaptic LRRTM4.