Alternatively, LRRTM4 knockdown may predominantly affect immature spines with low AMPAR content (“silent” synapses), resulting in decreased spine density but no effect on mEPSC frequency. Our current image resolution was not sufficient to rigorously analyze the morphology of individual Src inhibitor spines. Another possible explanation for the lack of decrease in mEPSC frequency after

LRRTM4 knockdown might be that LRRTM4 regulates spine development in select dendritic processes, rather than globally affecting spine density. Loss of LRRTM1 affects VGlut1 clustering in select CA1 hippocampal laminae ( Linhoff et al., 2009), suggesting that at least some LRRTMs may have lamina-specific effects on synapse development. The reduction in synaptic strength after LRRTM4 knockdown

in vivo could be mediated by a direct role of LRRTM4 in AMPAR trafficking. Both LRRTM4 and LRRTM3 were identified as components of AMPAR complexes (Schwenk et al., 2012 and Shanks et al., 2012), and LRRTM2 binds GluR1 via its extracellular domain in heterologous cells (de Wit et al., 2009). A similar reduction in synaptic strength has been observed in GPC4 knockout mice, which was attributed to decreased recruitment of the AMPAR subunit GluR1 to synaptic sites ( Allen et al., 2012). These findings suggest that a disruption of the glypican-LRRTM4 interaction may lead to reduced recruitment or stabilization of AMPARs at the synapse, Dolutegravir resulting in a decrease in synaptic strength. Finally, genome-wide association studies have linked GPC1 and GPC6 to ADHD, neuroticism, and schizophrenia (Calboli et al., 2010, Lesch et al., 2008 and Potkin et al., 2009). The association of glypicans with these nervous system disorders indicates that glypicans may be important for proper brain function. The identification of the trans-synaptic glypican-LRRTM4 interaction as a key regulator of excitatory synapse development should provide an avenue for a deeper understanding of these mafosfamide disorders. Hippocampal neurons were cultured from P0 Long-Evans rats (Charles River) and plated on poly-D-lysine-coated

(Millipore) and laminin-coated (Invitrogen) chamber slides (Nalge Nunc International). Neurons were maintained in Neurobasal-A medium (Invitrogen) supplemented with B27, glucose, glutamax, penicillin/streptomycin (Invitrogen), and 25 μM β-mercaptoethanol. Neurons were transfected using calcium phosphate at 7 DIV. For knockdown experiments, neurons were electroporated at time of plating using a Bio-Rad Gene Pulser Xcell. For Fc treatments of neuronal cultures, Fc proteins (10 μg/ml final concentration) were added to the culture media. For 6-day treatments, half the media was replaced after 3 days with fresh feeding media containing the same final concentration Fc protein. Neurons were fixed in 4% paraformaldehyde, 4% sucrose in PBS, washed in PBS, and blocked in 3% BSA, 0.2% Triton X-100 in PBS.