The overall goals of this project are to identify and characterize the cellular and molecular signals that pattern axons and dendrites in the developing cortex. The project is motivated by the fact that the correct patterning of axons and dendrites of individual cortical neurons is critical for formation of functional neuronal connections in the brain. Although the importance of correct patterning of axons and dendrites for brain development has long been appreciated, little is known about the signals that regulate this process. To begin to identify these signals we have developed a slice overlay assay that can be used to study the influence of extracellular signals on axonal and dendritic development in the cortex. The proposed project encompasses three specific aims. First, axon and dendrite orienting cues would be further characterized with respect to the time course of their presence, their spatial distribution, and whether they are diffusible in nature. Second, the contribution of cell origin or identity on determining responsiveness to orienting cues will be examined. Cortical neurons will be plated onto striatal slices to determine if their pyramidal morphology is recapitulated, and striatal neurons will be plated onto cortical slices to see if they are capable of assuming a pyramidal morphology. In another set of experiments cortical neurons born at various times, destined to populate various cortical layers, and some of which would normally be pyramidal in shape (layers 5,6), and others which would not (layer 4), will be tested separately for their ability to orient on cortical slices. This might help determine which aspects of cell shape are an autonomous property of neurons. A similar analysis would be performed that compares the responsiveness of inhibitory GABAergic neurons to orienting cues as compared to other neurons. Third, the normal distributions of secreted semaphorins and their effects on cortical axons and dendrites would be further characterized. Receptor bodies, antibodies to neuropilins, or antibodies to specific semaphorins would be used in an effort to perturb axon and dendrite orientation on slices. Finally, experiments are proposed in which cultured cortical neurons would be exposed to a gradient of semaphorins at various times. Cortical axons would be briefly exposed to semaphorins just before they initiate neurites to see if their polarity can be determined by the semaphorin gradient, and after process initiation to determine if, for example, dendrites can reorient in response to a semaphorin gradient.
