Figure 9

Sema5B knockdown increases synapse number and size. (a-d) Confocal images of 9 to 10 days in vitro (DIV) hippocampal neurons expressing postsynaptic density (PSD)-95-red fluorescent protein (RFP) plus either green fluorescent protein (GFP) (a) or GFP-short hairpin RNA (shRNA) (b). Cells were immunolabeled with anti-synaptophysin and synapse number determined by the percentage of PSD-95-RFP puncta that had an associated synaptophysin cluster (c-e). Similar to our previous observations, coexpression of a chick Sema5B with a scrambled shRNA resulted in a significant decrease in synapse number (compare (e) to Figure 4j; Student's t test, P < 0.001). In contrast, knockdown of Sema5B resulted in a significant increase in synapse number ((e); Student's t test shRNA1 and 3, P < 0.005). The increase in synapse number with Sema5B shRNA was abrogated by coexpression with an shRNA-insensitive chick Sema5B (e). In addition to an increase in synapse number, there was a twofold increase in the size of PSD-95-RFP puncta in Sema5B knockdown cells (Student's t test shRNA1 and 3, P < 0.001) that was rescued by coexpression of a chick Sema5B ((f); Student's t test, P = 0.462 compared to control). A similar increase in the size of synaptophysin puncta associated with Sema5B knockdown was observed ((g); Student's t test shRNA1 and 3, P < 0.005). N = at least 25 cells per condition from at least 3 separate cultures. Scale bar 10 μm.