Axon guidance at the spinal cord midline-A live imaging perspective

During neural circuit formation, axons navigate several choice points to reach their final target. At each one of these intermediate targets, growth cones need to switch responsiveness from attraction to repulsion in order to move on. Molecular mechanisms that allow for the precise timing of surface expression of a new set of receptors that support the switch in responsiveness are difficult to study in vivo. Mostly, mechanisms are inferred from the observation of snapshots of many different growth cones analyzed in different preparations of tissue harvested at distinct time points. However, to really understand the behavior of growth cones at choice points, a single growth cone should be followed arriving at and leaving the intermediate target. Existing ex vivo preparations, like cultures of an "open-book" preparation of the spinal cord have been successfully used to study floor plate entry and exit, but artifacts prevent the analysis of growth cone behavior at the floor plate exit site. Here, we describe a novel spinal cord preparation that allows for live imaging of individual axons during navigation in their intact environment. When comparing growth cone behavior in our ex vivo system with snapshots from in vivo navigation, we do not see any differences. The possibility to observe the dynamics of single growth cones navigating their intermediate target allows for measuring growth speed, changes in morphology, or aberrant behavior, like stalling and wrong turning. Moreover, observation of the intermediate target-the floor plate-revealed its active participation and interaction with commissural axons during midline crossing.