Sympathetic neurons regenerating in culture were studied in order to gain further insight into the intracellular distribution and phosphorylation of GAP-43, a protein that has been suggested to have a role in axonal outgrowth and neuronal plasticity (Willard et al., 1987). Superior cervical ganglion neurons from embryonic rats were highly reactive with a polyclonal antibody against the growth-associated protein GAP-43 soon after they were placed in culture on a laminin substrate. As these neurons extended neurites, the distribution of GAP-43 reactivity changed. The cell body became progressively less reactive, whereas the growth cone at the tip of the growing neurite reacted strongly. The pattern of immunofluorescence was punctate both in the growth cone and the adjacent neurite, but appeared more diffusely distributed in the cell body. The antibody reacted only with cells that had been subjected to treatment that permeabilized the plasma membrane. When antibody was supplied in the medium of growing neurons, it neither bound to the cells nor altered normal neurite initiation or elongation. Of the different types of cells in these cultures, the antibody reacted only with neurons; it did not react with Schwann cells or fibroblasts. The stimulation of protein kinase C in these cultures resulted in a 7-fold stimulation of the phosphorylation of a protein of similar electrophoretic mobility to GAP-43. These observations demonstrate that GAP-43 is neuron-specific, is present throughout the neuron but at higher levels in the growth cone, and is a major substrate of protein kinase C. The high concentration of GAP-43 in the growth cones may necessitate its increased synthesis in neurons with elongating axons. Its location and phosphorylation by kinase C suggest that it could perform a function in the growth cone that is modulated by extracellular signals, such as those used in pathfinding or in the control of axonal elongation.

Despite evidence that glial cell surfaces and components of the extracellular matrix (ECM) support neurite outgrowth in many culture systems, the relative contributions of these factors have rarely been compared directly. Specifically, it remains to be determined which components of peripheral nerve support growth of central nerve fibers. We have directly compared neurite outgrowth from embryonic day 15 rat retinal explants placed onto beds of (1) Schwann cells without ECM, (2) Schwann cells expressing ECM (including a basal lamina), (3) cell-free ECM prepared from neuron-Schwann cell cultures, (4) nonglial cells (fibroblasts), and (5) 2 isolated ECM components, laminin and type I collagen. From the first day in culture, retinal explants extended neurites when placed on Schwann cells without ECM. Outgrowth on Schwann cells expressing ECM was also extensive, but not obviously different form that on Schwann cells alone. Ultrastructural study revealed that 95% of retinal neurites in ECM-containing cultures contacted other neurites and Schwann cell surfaces exclusively. On cell-free ECM prepared from neuron-Schwann cell cultures, neurite extension was poor to nonexistent. No neurite outgrowth occurred on fibroblasts. Retinal explants also failed to extend neurites onto purified laminin and ammoniated type I collagen substrata; however, growth was rapid and extensive on air-dried type I collagen. In cultures containing islands of air-dried type I collagen on a laminin-coated coverslip, retinal explants attached and extended neurites on collagen, but these neurites did not extend off the island onto the laminin substratum. We conclude from these experiments that neurite extension from embryonic rat retina is supported by a factor found on the surface of Schwann cells and that neither organized nor isolated ECM components provide this neurite promotion. These findings are discussed in relation to possible species differences in growth requirements for retinal ganglion cell neurites and to the specificity of response of different CNS neurites to ECM substrata.

Our goal was to elucidate the pathway of newly synthesized phospholipid into the growing neurite. This was accomplished in pulse-chase studies with the phospholipid precursor [3H]glycerol, using sprouting explant cultures of rat superior cervical ganglion as an experimental system. After the pulse with the precursor and various chase periods, we separated perikarya and neurites microsurgically and extracted their phospholipids. The phospholipid extract from the perikarya exhibited a steep rise followed by a rapid decline in specific radioactivity. In contrast, an increase in neuritic specific radioactivity of phospholipid was observed only after a lag period of "--60 min. Nearly quantitative transfer of newly synthesized phospholipid from the perikarya into the neurites could be demonstrated. Both the decline in perikaryal specific radioactivity and the increase in its neuritic counterpart, i.e., the proximodistal transfer, could be blocked with the microtubule drug colchicine and the metabolic uncoupler, 2,4-dinitrophenol. These observations indicate preferential export of newly synthesized phospholipid from the perikaryon (the major or exclusive site of synthesis) into the growing neurites, most likely by rapid axoplasmic transport of formed elements.The developing, sprouting neuron is characterized by vectorial growth of its processes and very rapid net expansion of its plasmalemma, at a rate of ~0.5 #m2/min per neurite (neurons of the mammalian peripheral nervous system; see reference 17). This system offers the interesting possibility of studying the intracellular pathways(s) of newly synthesized membrane components from their site of synthesis to their point of insertion into the plasma membrane. In such a highly polarized system, incorporation into the plasmalemma of proteins and lipids could occur predominantly in a distal region (at the growth cone), it could occur preferentially at the perikaryon, or else it could occur at random. To gain insight into this problem, we used tritiated glycerol as a precursor for membrane phospholipid in pulse-chase experiments with cultured neurons. After chase, neuritic sprouts were separated from neuronal perikarya, and specific radioactivity of phospholipid was analyzed in the two portions of the neuron. Thus, transfer of newly synthesized phospholipid from the perikaryon to the neurite could be measured. A preliminary report on this work has been presented in abstract form (15). MATERIALS AND METHODSTissue Culture: The experiments were carried out on explant cultures of rat superior cervical ganglia, removed just before, or right aRer, birth of the pups. The ganglia were stripped free of connective tissue ensheathment and cut into small pieces before being placed into collagen-coated Aclar wells (33C Aclar, gauge 5, Allied Chemical Co., Morristown, NJ; cf. reference 5). The neurons were grown for 3 to 4 d in vitro in Leibovitz's medium (LI5; Gibco Laboratories, Grand Island, NY) containing 10% human placental serum, 9 mg/ml glucose, 2.5S nerve growth factor ...

Long term (2- to 3-week) cultures of superior cervical ganglia (SCG) were established from rats and rat embryos ranging in age from 15 days of gestation (E15) to 279 days postnatal (P279). Cultures were grown on a collagen substratum and fed a serum-containing medium with added nerve growth factor. Radial outgrowth of neurites was measured as a function of time for up to 2 to 3 weeks. Computer-aided analysis generated estimates of onset time, initial rate, and subsequent changes in the rate of growth of these neurites. The explants from perinatal rats showed the fastest growth onset time (5 to 13 hr), fastest initial rate of growth (370 to 660 microns/d), and a decline in growth rate during the first 2 weeks in culture. The outgrowth from these perinatal explants was composed of many small fascicles. Neurites from the prenatal explants (E15 to E20) began to grow within 22 hr in vitro. Their rate of growth was lower initially (150 to 300 microns/d) but increased to equal the perinatal explant initial rate before again falling to an intermediate level (200 to 300 microns/d). The outgrowth from prenatal explants contained fewer larger fascicles. Postnatal explants had low initial rates of growth (70 to 176 microns/d) but exhibited an increasing growth rate in vitro, again approaching an intermediate rate of 200 to 250 microns/d after 2 to 3 weeks. Neurite outgrowth from the postnatal explants was delayed by an amount roughly correlated with the age of the animal advancing postnatal age but reached an asymptote of about 50 to 150 microns/d at about P30. The outgrowth was initially sparse but became denser with time in culture. Thus, in a culture system in which medium composition and growth substratum are held constant, marked differences can be observed in pattern, latency, initial rate, and subsequent changes in rate of neurite extension among SCG explants from different ages of rats and rat embryos.

Studies on cellular interactions in the developing nervous system are greatly facilitated by the availability of tissue culture preparations that contain single or combined populations of neurons and non-neuronal cells (NNCs). Using superior cervical ganglia (SCG) from early E15 rats on air-dried collagen, we were able to prepare cultures containing neurons along with Schwann cells (SCs) as the only NNC type present without the use of antimitotic treatment and cultures containing only neurons, following brief antimitotic treatment. Light-microscopic observation of E15 outgrowth showed a uniform population of flattened cells, unlike that of E20 cultures, which contained a mixture of spindle-shaped and flattened cells. Autoradiograms following [3H]thymidine administration to E15 cultures revealed a striking gradient of nuclear labeling: Only a few cells were labeled near the explant and nearly all cells were labeled at the growth front. This was in marked contrast to E20 cultures, in which nuclei were labeled throughout the outgrowth. The conclusion that the labeling gradient is explained by the presence of SCs without other NNC types in E15 cultures was confirmed by immunocytochemical studies. Anti-laminin antibodies stain only those extracellular matrix components related to the SC surface, whereas anti-fibronectin antibodies stain fibroblast-related components (Cornbrooks et al., 1983a). Anti-laminin antibodies stained cell surfaces in both E15 and E20 outgrowth. E15 outgrowth did not stain with anti-fibronectin antibodies although marked staining was obtained in E20 cultures. Electron microscopy confirmed the presence of only SCs in E15, and of both SCs and fibroblasts in E20 outgrowth. Thus, it appears that there is a narrow developmental window in which the ganglia contain neurons and SCs but relatively few fibroblast components; cultures prepared from ganglia at this stage form outgrowth containing only neurites and SCs without antimitotic treatment. Surprisingly, neither SC ensheathment nor SC basal lamina formation was normal in E15 and E20 outgrowth. When either E15 or E20 SCG SCs were transplanted onto dorsal root ganglion neurons free of endogenous SCs, however, the sensory neurites were typically ensheathed or myelinated and basal lamina appeared 9 d later, identifying the SCG NNCs as functionally competent SCs.(ABSTRACT TRUNCATED AT 400 WORDS)