Axons are guided to appropriate targets in the developing nervous system by nerve growth cones, the highly motile tips of growing neurites. Growth cones display positive advance or negative retraction in response to cues in the cellular environment These behaviors as well as axon branching play an important role in development of appropriate connections. Studies of environmental cues and intracellular events that influence growth cone steering are proposed toward the long-term goal of understanding axon guidance mechanisms in the mammalian central nervous system (CNS). In vitro preparations of developing hamster sensorimotor cortex will be used with fluorescence and high resolution video microscopy to study axon guidance in a complex CNS environment. In the first specific aim the influence of membrane bound cues on cortical axon guidance will be addressed in in vitro assay systems. Explants of newborn sensorimotor cortex will be plated onto substrates of brain membranes from appropriate or inappropriate targets. Conical neurite outgrowth and growth cone behaviors will be studied with fluorescence and video microscopy. Subsequently, biochemical manipulations of the membrane substrates will be carried out to identify classes of membrane bound cues that influence the guidance of cortical neurites into specific regions of the CNS. In the second specific aim the contributions of the cytoskeleton to the guidance of cortical neurites will be addressed by visualizing the disposition of microtubules in growth cones captured during directional changes and extension of axon branches. Microtubules in the neurites and growth cones of dissociated fixed cortical neurons will be fluorescently labeled by immunocytochemistry with antibodies to various forms of tubulin. The rearrangements of microtubules during these behaviors will be studied by correlating the fluorescence images with matching high resolution video enhanced Nomarski images of the same growth cone. In the third specific aim the behaviors of filopodia, the microspikes extending from neuronal growth cones, will be studied with high resolution video microscopy during growth cone interactions with the cellular environment. Comparisons of filopodial morphologies and behaviors in dissociated cortical cell cultures with those in explanted cortical slices will be used to assess the sensory role of filopodia in growth cone steering events. The long range goal of elucidating growth cone guidance mechanisms in the complex environment of the mammalian CNS is important not only for understanding how specific neural circuits are established during development but for promoting restoration of neural circuitry after injury to the mammalian CNS.
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