, 2010), whereas Slits (Whitford et al., 2002) and ephrins (Liebl et al., 2003) are highly enriched

at the CP, similar to the expression of Sema3A. Furthermore, extracellular factors may also influence neuronal polarization by modulating the expression and action of other polarizing factors. For example, Wnt4 and TGF-β1 may regulate Sema3A expression (Kettunen et al., 2005), and Semaphorins may control TGF-β and ephrin signaling (Ikegami et al., 2004). The antagonistic effect of Sema3A and BDNF in polarizing axon/dendrite differentiation shown here (Figure 1), mediated by reciprocal cGMP/cAMP signaling in the neuron (Figure 2; BMS 777607 Shelly, et al., 2010), further underscores the possibility that synergistic and antagonistic actions of extracellular factors

may work in concert to polarize neurons in vivo. The involvement of multiple factors in vivo may account for the observations that disruption of the signaling of a single factor results in only subtle polarity defects. Cultures of dissociated hippocampal and cortical neurons were prepared as previously described (Shelly et al., 2007) and as presented in Supplemental Experimental Procedures. Live images for stripe assays were acquired 12 hr following plating, and immunostaining was performed as described in Supplemental Experimental Procedures. For FRET assays, transfections were carried out 2 hr after plating. For analysis of LKB1, GSK-3β, and Akt phosphorylation by immunoblotting, cells were treated with forskolin (20 μM; 20 min) or BDNF (50 ng/ml, 15 min), either alone or selleck kinase inhibitor together with the PKG inhibitor KT5823 (200 nM), the PDE inhibitor IBMX (50 μM), the PDE4 inhibitor rolipram (1 μM), or the sGC inhibitor ODQ (1 μM). To test for the antagonistic

TCL effects of Sema3A or 8-pCPT-cGMP, increasing concentrations of these factors were incubated together with forskolin or BDNF for 20 min. Whole-cell extracts were prepared at 5 DIV for cortical neurons, before subjected to immunoblotting. HEK293T cells were grown in DMEM medium supplemented with 10% FBS and transiently transfected using calcium-phosphate method. The ubiquitination assay and detection of PKA activity using a fluorescent peptide based “PepTag” assay is described in Supplemental Experimental Procedures. Substrates were patterned as previously described (Shelly et al., 2007) and as presented in Supplemental Experimental Procedures. Microfluidic patterning of the following substrates alone or together with fluorescently conjugated BSA (5 μg/ml) as a marker was performed as follows: F-cAMP, F-cGMP, KT5720 or KT5823 (2 nM); NGF or BDNF (0.5 ng/ml), netrin-1 (0.5 and 0.05 ng/ml); and Sema3A (0.5 and 0.05 μg/ml). The method of in utero electroporation was performed as previously described (Shelly et al., 2007) and as presented in detail in Supplemental Experimental Procedures.