Guidance and Outgrowth Assays for Embryonic Thalamic Axons

The assay described in this chapter allows for a relatively simple and fast screening for effects of guidance/growth promoting cues and modulators on dorsal thalamic axons growth. As mentioned in the introduction, the conditions of optimal axon growth in vitro are not identical to conditions found in vivo. Therefore, this assay like all other in vitro axon outgrowth assays does not intend to “recapitulate” in vivo situations. This is an advantage in a sense that growth conditions can be controlled and modified as desired. The results can ultimately provide a framework to establish testable hypotheses in vivo. With the emergence of less expensive and more practical real-time imaging techniques (e.g., microscope-independent and environmentally controlled culture chambers), this assay is highly adaptable to monitor the outgrowth of thalamic axons and the effect of various modulators in real time. It is possible, for instance, to visualize and measure acute effects of drugs on the response patterns of thalamic axons, on a much shorter timescale than 72 h in vitro. Furthermore, by simply modifying the dissection steps and testing different extracellular matrix substrates, the response of axons originating from any brain region of interest can be monitored. Therefore, this assay will be helpful to rapidly test the possibility that a given class of axons responds to specific guidance cues and to determine the specific pharmacological agents that may influence growth responses.