Applications of Video Microscopy to Studies of the Developing Nervous System

Abstract

Growing neurites are guided to their correct targets in the developing nervous system by their highly motile tips, nerve growth cones. To understand how the mammalian cerebral cortex establishes appropriate neural circuits, we have used several light microscopic video techniques to observe growth cones as they extend from developing hamster cerebral cortical neurons and interact with their cellular environment. We describe how Nomarski optics can be combined with video and image processing techniques, termed VEC-DIC (video-enhanced contrast-differential interference contrast) microscopy, to observe the behaviors of neuronal growth cones and the intracellular events that accompany these behaviors. Results from our laboratory on dissociated cortical cell cultures and explants suggest that positive and inhibitory cell-cell interactions in which growth cones grow along other cellular processes or collapse and withdraw from them play a central role in the development of cortical circuits. We show how low-light-level video microscopy of living fluorescently labeled neurons can be used to observe developmental events within intact or semi-intact preparations of the nervous system. With these video techniques we have observed behaviors of fluorescently labeled cortical growth cones as they extend within living neonatal brain slices. Dynamic shape changes of callosal growth cones, growing toward their targets in the contralateral cortex, suggest distinct axon guidance cues in different regions of the corpus callosal pathway. Together these advances in video techniques for Nomarski and fluorescence microscopy will be important in observing directly the mechanisms underlying formation of connections in the developing nervous system.