To determine whether knockdown of cadherin-9 selectively affects DG-CA3 synapse formation, we transfected hippocampal neurons with scrambled control or cadherin-9 shRNA, and examined the formation of DG and CA synapses onto different

types of neurons using find more the SPO assay. Knockdown of cadherin-9 led to significant reductions in the total number of DG synapses and the number of extra-large DG synapses onto CA3 neurons but did not affect the number of CA synapses onto the same cells (Figures 6D–6G), indicating that cadherin-9 regulates a specific class of synapses. In addition, DG synapses that remained on knockdown CA3 neurons were significantly reduced in size compared to controls (Figure 6H), suggesting that cadherin-9 might also control the growth of DG-CA3 synapses. These defects were rescued by coexpression

of shRNA-insensitive cadherin-9, which indicates that the observed effects are due to specific loss of cadherin-9 (Figures 6D–6H). As a further test of specificity, we examined the effects of cadherin-9 shRNA on CA1 neurons, which do not express cadherin-9 (Figure 5A). We found no significant effect of cadherin-9 shRNA expression on the formation of synapses onto Selleck GSK126 CA1 neurons, suggesting that the cadherin-9 shRNA does not cause general synaptic defects (Figures 6E–6H). These results indicate that cadherin-9 plays a specific role in regulating DG synapses onto CA3 neurons, and does not regulate non-DG synapses. Because many DG neurons express cadherin-9, and there are some ectopic DG-DG synapses in culture, we determined if the number of DG-DG synapses would be affected by downregulation of cadherin-9. Expression of cadherin-9 shRNA led to a decrease in the number of DG-DG synapses, suggesting that ectopic synapse formation between DG neurons in culture is driven, at least in part, by cadherin-9-mediated interactions (Figures 6E–6G). We also overexpressed cadherin-9 in cultured hippocampal neurons to determine

whether it is sufficient to induce synapses and found that overexpression does not increase DG synapses on any cell type in culture (Figures 6E–6H). This is consistent with previous studies on N-cadherin, which was also shown to be insufficient to induce synapses or spines (Mendez et al., 2010, Scheiffele et al., 2000 and Togashi et al., 2002). Together, these observations indicate that cadherin-9 regulates the formation of synapses when it is expressed both in the pre- and postsynaptic neuron, supporting a mode of action via homophilic binding. To determine if endogenous cadherin-9 is required for the differentiation of DG-CA3 mossy fiber synapses in vivo, we generated lentiviruses that express cadherin-9 shRNA under control of the human H1 promoter and GFP under control of the rat synapsin promoter. Rat DG neurons were infected with control or cadherin-9 shRNA lentivirus at P5 and assessed by immunofluorescence at P16 (Figure 7A).