To address these issues, we first cultured NCAM−/− and NCAM+/+ commissural neurons and exposed them to control and gdnf application for quantification of Plexin-A1. We observed that NCAM loss prevented gndf-induced increase of Plexin-A1 levels ( Figures 7A and 7B). Second, the Plexin-A1/Nf160kD ratio in FP and PC domains was determined in E12.5 NCAM+/+ and NCAM−/− embryos. This analysis revealed that the ratio was significantly decreased in the PC domain of NCAM−/− sections, compared with the wild-type ones, although in the FP domain, Selleck C646 the ratio was not statistically significant ( Figures 7C and 7D). In this mouse line, a large amount of axons are still present in

the FP at E12.5 due to the genetic background (C57Black6) and have not yet Selleck LY2157299 experienced FP crossing. This might limit the possibility to detect moderate differences. Third, using the t-BOC reporter, we examined the calpain activity in NCAM+/+ and NCAM−/− commissural neurons. We found that the proportion of cells exhibiting high calpain activity (high t-BOC fluorescence) was significantly decreased by gdnf in the NCAM+/+, but

not NCAM−/−, condition ( Figures 7E and 7F). Similarly, application of a GFRα1 function-blocking antibody abolished the gdnf-induced decrease of high t-BOC-labeled neurons ( Figures 7G and 7H). Finally, calpain activity was measured in commissural tissue dissected from NCAM+/+ and NCAM−/− embryos, exposed to acute stimulation with gdnf and control. The analysis revealed that gdnf could decrease the endogenous calpain activity in NCAM+/+, but not in NCAM−/−, tissue ( Figure 7I). Altogether, these experiments provide evidence that NCAM and GFRα1 are required for the gdnf-induced regulation

of Plexin-A1 levels and calpain activity in spinal commissural neurons. We report here that a local source of gdnf in the FP acting through NCAM, but not RET, regulates the responsiveness of commissural axons to the midline repellent Sema3B. Gdnf makes an important contribution to this process but also acts with NrCAM in the FP to switch on the Sema3B repulsive signaling by inhibiting calpain1-mediated processing of the Sema3B coreceptor Plexin-A1. This mechanism prevents axons responding to Sema3B at the precrossing DOK2 stage, thus allowing them to enter the FP, and then switches on sensitivity to Sema3B at the postcrossing stage (Figure 8). The navigation of commissural axons across the FP has been shown to be a complex multistep process. Upon crossing, axons acquire responsiveness to local repellents to which they were not sensitive before crossing, thus gaining the information to move away (Evans and Bashaw, 2010). Sema3B was shown by previous work to be one such repulsive cue, expelling commissural axons that have crossed the midline (Zou et al., 2000; Nawabi et al., 2010).