Many biological processes have to work together seamlessly to allow a properly functioning brain to develop. One crucial step is the guidance of axons to appropriate targets via molecular cues. In many cases these cues provide directional information by being present in concentration gradients. To respond to such gradients, the growth cone at the tip of the developing axon must detect the direction of increase, and then determine whether to respond by attraction or repulsion. Over the past 20 years much progress has been made in identifying the ligands, receptors and intracellular signalling molecules involved in this process. However a more quantitative understanding has been lacking, making it difficult to predict quantitatively how an axon will behave given particular gradient conditions.
Here, I address this issue from several different directions. Chapter 2 considers whether there is a threshold gradient steepness below which axons from rat dorsal root ganglia are unable to respond to gradients of nerve growth factor. Surprisingly, but consistent with the predictions of a recent mathematical model, I show that there is no threshold: the response drops linearly to zero as gradient steepness decreases. Chapter 3 describes the development of a new microfluidics technology for studying axonal response to gradients which is both high-throughput and allows precise control over gradient parameters for steep gradients. Using this I show that chemotactic responses involving both turning and growth rate modulation can occur at the same gradient steepness for rat superior cervical ganglion axons. Chapter 4 tests the predictions of a mathematical model of the effects of calcium and cAMP levels on whether axons choose to be attracted or repelled. Focussing particularly on the normally repulsive response of axons to gradients of MAG (myelin-associated glycoprotein) I confirm several predictions of the model. Surprisingly, while high levels of calcium or cAMP separately normally switch repulsion to attraction, applying high levels of both together blocks this attraction, as predicted by the model. Finally, chapter 5 considers axon guidance in an in vivo system, that of guidance of axons across the corpus callosum, and provides data showing that later-stage callosal axons show no response to Netrin1 compared to early-stage axons. This suggests the possibility that the change in responsiveness of callosal axons to Netrin1 with age could be due to a down- regulation of DCC (deleted in colorectal cancer) receptors during development.
Together these results significantly extend and refine our understanding of how axons detect and respond to molecular gradients during neural development. Such knowledge will ultimately contribute to our understanding of normal neuronal development, as well as helping to explain how cognitive disorders that may result from miswiring could occur, and how to optimally promote axonal rewiring after injury.