Fig. 2. A, Profile of synaptophysin immunoreactivity in 3- and 10-d-old cultures. A shift from diffuse to punctate staining is observed between these two time points, indicating the absence and presence of synapses, respectively. Ph, Phase contrast, Syn, synaptophysin. B, Quantification of agrin-induced pCREB in 3- and 10-d-old cultures. The agrin isoform Ag4,8 and BDNF, but not Ag0,0, induce an increase in the percentage of pCREB-positive neurons both in 3- and 10-d-old cultures (**p < 0.01 as compared with control medium, ANOVA; n = 4). C, Agrin induces a transient increase in pCREB-immunoreactive cells. Incubation with Ag4,8 or BDNF was analyzed at 0, 10, 30, and 120 min, respectively. Both the Ag4,8 and BDNF responses reach a maximum within 10 min and are reduced within 2 hr. Despite the difference in magnitude, the BDNF- and Ag4,8-induced CREB phosphorylation essentially follows similar kinetics. CM, Control medium.