Dynamic imaging of neuronal cytoskeleton.

Growth cones are the motile tips of growing axons. During development growth cones guide axons along specific pathways to appropriate targets by extending toward or retracting away from attractive or inhibitory guidance cues in their environment. Growth cone motility as well as extension, retraction, and turning behaviors are directed by the neuronal cytoskeleton, principally actin filaments and microtubules. Developing neurons, such as those from the mammalian cerebral cortex, also form connections in the central nervous system (CNS) by extending branches interstitially from the axon shaft. The actin and microtubule cytoskeleton directs growth at the axon tip as well as at axon branch points. Thus, an understanding of how the movement and dynamics of actin filaments and microtubules are regulated and how these two cytoskeletal elements interact is essential for understanding axon pathfinding and formation of connections in the CNS. Imaging the dynamic cytoskeleton in living neurons from the mammalian CNS presents a number of technical challenges. In comparison with larger neurons from invertebrates or the vertebrate periphery, neurons from the cerebral cortex are relatively small, which makes them particularly difficult to microinject with fluorescent probes for labeling and visualizing the cytoskeleton. Although changes in the location and organization of microtubules and actin filaments can occur rapidly in cortical growth cones, imaging periods of many hours are necessary to visualize how these events are related to protracted events such as development of axon branches. Imaging cortical neurons over many hours at a resolution necessary to detect single fluorescently labeled microtubules or actin filament bundles is difficult to achieve without compromising the viability of the neuron. Even limited photodamage has deleterious effects on growth cone motility and axon branching. Most previous work on dynamic imaging of the neuronal cytoskeleton has used direct injection into large neurons from invertebrates such as Aplysia, Helisoma, or grasshopper 1-5 or used injection into the blastula to label neurons from the