DCC mediated axon guidance of spinal interneurons is essential for normal locomotor central pattern generator function.

Coordinated limb rhythmic movements take place through organized signaling in local spinal cord neuronal networks. The establishment of these circuitries during development is dependent on the correct guidance of axons to their targets. It has previously been shown that the well-known axon guidance molecule netrin-1 is required for configuring the circuitry that provides left-right alternating coordination in fictive locomotion. The attraction of commissural axons to the midline in response to netrin-1 has been shown to involve the netrin-1 receptor DCC (deleted in Colorectal Cancer). However, the role of DCC for the establishment of CPG coordination has not yet been resolved. We show that mice carrying a null mutation of DCC displayed an uncoordinated left-right activity during fictive locomotion accompanied by a loss of interneuronal subpopulations originating from commissural progenitors. Thus, DCC plays a crucial role in the formation of spinal neuronal circuitry coordinating left-right activities. Together with the previously published results from netrin-1 deficient mice, the data presented in this study suggest a role for the most ventral originating V3 interneurons in synchronous activities over the midline. Further, it provides evidence that axon crossing in the spinal cord is more intricately controlled than in previously suggested models of DCC-netrin-1 interaction.

Internalization studies of chimeric neuropeptide Y receptors Y1 and Y2 suggest complex interactions between cytoplasmic domains.

Agonist stimulation readily internalizes neuropeptide Y receptor Y1 while there are contradictory results for the Y2 receptor. In order to explore putative functional differences between the Y1 and Y2 receptors we generated reciprocal chimeras by swapping the third intracellular loop, the carboxy terminus or both between human Y1 and Y2. Internalization was studied in a quantitative radioligand binding assay with removal of surface-bound ligand in an acidic-wash procedure. The internalization assay revealed a lower degree of internalization as well as slower kinetics for the Y2 receptor. Generally, reciprocal exchange of receptor segments did not convey properties of the donor receptor but tended to enhance internalization. Surprisingly, insertion of the Y2 carboxy terminus into Y1 gave almost complete internalization (92%), rather than reduced internalization, while the insertion of both segments resulted in internalization equal to the native Y1 receptor. These findings were confirmed by fluorescence microscopy of immuno-stained receptors tagged with a C-terminal FLAG epitope. However, after exposure to high agonist concentrations (100 nM) Y2 was internalized. Studies of Y2 and the closely related Y7 receptor confirmed low internalization for Y2 from chicken and teleost fishes as well as Y7 from two teleosts. The conservation across species of low internalization at physiological concentrations suggests that this is an ancient feature and of vital importance for Y2 function. We propose that amino acid motifs in the third intracellular loop as well as the C terminus of both Y1 and Y2 are able to drive agonist-promoted internalization and that there may be constraining motifs in the Y2 receptor.