Sympathetic neurons from perinatal rat pups extend only a single axon when maintained in culture in the absence of glia and serum. Exposure to recombinant osteogenic protein-1 (OP-1) selectively induces the formation of dendrites that correctly segregate and modify cytoskeletal and membrane proteins and form synaptic contacts of appropriate polarity. OP-1 requires nerve growth factor (NGF) as a cofactor, and, in the presence of optimal concentrations of NGF, OP-1-induced dendritic growth from cultured perinatal neurons is comparable to that observed in situ. Sympathetic neuroblasts that had not formed dendrites in situ also responded to OP-1 in culture, indicating that OP-1 can cause de novo formation as well as regeneration of dendrites. These data imply that specific signals can regulate the development of neuronal shape and polarity.

Neurites of superior cervical ganglion neurons from embryonic, perinatal, and adult rats extended at different rates when placed in tissue culture on similar collagen substrata. Using high resolution cinematography and a time-lapse video recording system, we concluded that these differences arise from variations in individual growth cone behavior. Growth cones of embryonic and perinatal neuronal origin exhibited high peak rates of advance and filopodial and lamellipodial excresences. Perinatal cones differed from embryonic ones in that they were somewhat larger, advanced in straighter paths, and retracted less, consequently translocating at 14 to 29 microns/hr compared with 8 to 22 microns/hr for embryonic cones (ranges of 4-hr means). The growth cones of neurons obtained from adult rats had scant cytoplasm and short branched filopodia, lacked definitive lamellipodia, and traversed the terrain at 4 to 13 microns/hr due to lack of high peak rates of advance and more time spent in stationary or minimal advance phases. Periodic pauses lasting 10 to 20 min, occurring every 20 to 90 min, interrupted the forward advance of growth cones of all ages. During pauses or slow forward movement, the growth cone displayed numerous filopodia whereas, during brief episodes when embryonic and perinatal growth cones moved at peak rates of 200 microns/hr or more, the cone periphery was predominantly lamellipodial. We conclude that the predominance of a lamellipodial or filopodial conformation correlates with the rate of growth cone advance and that age-dependent variations in neurite extension rates are related to differences in growth cone form and pattern of translocation. This is the first documentation of differing behavior of single growth cones of neurons of varying developmental ages in culture.

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Many types of glial-neuronal interactions occur during the development of the nervous system. To determine how such interactions might affect the development of autonomic ganglia, we compared the morphology of embryonic rat sympathetic neurons grown in the absence and in the presence of ganglionic nonneuronal cells in serum-free medium. Dye injections, electron microscopy, and immunocytochemistry were used to distinguish axons from dendrites. In cultures without nonneuronal cells, most (greater than 80%) sympathetic neurons extended only a single axonal process, and this unipolar state persisted for at least 8 weeks. Coculture with ganglionic nonneuronal cells caused sympathetic neurons to become multipolar and to extend multiple (range 1-17) dendrites. Morphometric measurements made after 1 month of coculture indicated that the amount of dendritic growth that occurred in vitro (mean number of dendrites/cell = 7.5; total dendritic length = 1,050 micron) was similar to that normally occurring during a comparable period in situ. In contrast to its prominent effects on dendritic growth, coculture did not cause changes in the number of axons/neuron or in the uptake of neurotransmitter. Cultures with ganglionic nonneuronal cells were immunostained for antigens present on the surfaces of fibroblasts (Thy-1.1, fibronectin) and of glia of the peripheral nervous system (laminin). Fewer than 1% of the nonneuronal cells displayed immunoreactivity for fibroblastic antigens; in contrast, greater than or equal to 99% reacted with antibody to laminin. Moreover, reconstitution experiments revealed that purified populations of laminin-positive Schwann cells promoted dendritic growth. Fibroblasts and heart cells lacked this activity. These data indicate that glia selectively promote dendritic development in sympathetic neurons maintained in serum-free medium.