Effects of Fasciculation

This report describes the influence of neurite fasciculation on two aspects of nerve growth from chick spinal ganglia in vitro : the inhibition of outgrowth by high concentrations of nerve growth factor (NGF) and the preferential growth of neurites toward a capillary tube containing NGF. These studies involved a comparison of cultures of single cells, cell aggregates, and intact ganglia and the use of antibodies against the nerve cell adhesion molecule (CAM) to perturb fasciculation under a variety of conditions . The inhibition of outgrowth, which was observed with ganglia and aggregates but not with single cells, was correlated with a thickening of neurite fascicles . In accord with this observation, anti-CAM, which diminishes fasciculation by inhibiting side-to-side interactions between individual neurites, also partially reversed the inhibition of neurite outgrowth at high NGF concentrations . On the basis of these and other studies, we consider the possibility that neurite bundling causes an increase in the elastic tension of a fascicle without a compensatory increase in its adhesion to substratum. It is proposed that this imbalance could inhibit neurites from growing out from a ganglion and even result in retraction of preexisting outgrowth . In the analysis of NGF-directed growth, it was found that a capillary source of NGF produced a steep but transient NGF gradient that subsided before most neurites had emerged from the ganglion. Nevertheless, the presence of a single NGF capillary caused a dramatic and persistent asymmetry in the outgrowth of neurites from ganglia or cell aggregates . In contrast, processes of individual cells did not appear to orient themselves toward the capillary . The most revealing finding was that anti-CAM antibodies caused a decrease in the asymmetry of neurite outgrowth . These results suggest that side-to-side interactions among neurites can influence the guidance of nerve bundles by sustaining and amplifying an initial directional signal . The specification of nerve tracts during embryogenesis is not understood in terms of fundamental mechanisms, particularly at the molecular level. During development, nerve processes often elongate along other nerve fibers or in association with them (9, 15, 27, 28) . This fact, together with the results of our previous studies on the mechanism of neural cell adhesion (1, 30, 31, 34), prompted us to reconsider the possible influence of neurite bundling or fasciculation on nerve guidance . In analyzing neurite fasciculation in cultures of spinal ganglia, we have focused on two previously described phenomena that reveal the effects of local environment on the extent and direction of neurite elongation . The first phenomenon is the halolike outgrowth of neurites from spinal ganglia and its inhibition by high concentrations ofnerve growth factor (NGF) (21, 23). In 1968, Levi-Montalcini and Angeletti (24, 25) clearly demonstrated that the inhibitory effect did not reflect an 370 URS RUTISHAUSER and GERALD M. EDELMAN The Rockefeller University, New York 10021 absence of neurite growth but rather the confining of neurites to the ganglion surface to form a dense capsule. Although these authors drew the important conclusion that NGF stimulates production of neurites at all concentrations, they did not consider the mechanism by which nerve processes are prevented from growing out from the body of a ganglion . The second phenomenon is pertinent to previous suggestions that NGF may be a chemotactic agent for neurites of certain types of nerve cells (7, 8, 10, 11, 13, 20) . Neurites from NGFsensitive tissues, usually ganglia, have been reported to elongate preferentially in the direction of an NGF source, such as a capillary tube or micropipette (8, 11, 13) . Moreover, the injection of NGF into embryos or the inactivation of endogenous NGF by injection of anti-NGF antibodies has been shown to alter or eliminate those cells and neuronal tracts that require this molecule (22, 25, 26). THE JOURNAL OF CELL BIOLOGY " VOLUME 87 NOVEMBER 1980 370-378 ©The Rockefeller University Press " 0021-9525/80/11/0370/09 $1 .00 on July 0, 2017 jcb.rress.org D ow nladed fom In the present work, the role of nerve fasciculation in these two systems has been investigated by microscopic and cinematographic studies comparing single cells, cell aggregates, and intact ganglia, by direct inhibition of neurite-neurite interactions using specific antibodies against the neural cell adhesion molecule (CAM) (30, 31, 34) and by observation of cultures containing ganglia from different spinal regions . The response to NGF under these conditions has been recorded in terms of several quantifiable parameters, including the number, length, width, and rate of growth of neuronal processes that emerge from the ganglion surface . Substratum adhesiveness was also included as a variable. Neurite growth toward an NGF source was analyzed by relating the apparent spatiotemporal distribution of NGF to the amount, direction, and morphology of neurite outgrowth . The results suggest that nerve fasciculation is a major determinant in the overall morphology of neurite outgrowth from ganglia. MATERIALS AND METHODS Ganglia, Cells, and Cell Aggregates Dorsal root ganglia were excised from 10-d-old chick embryos . Thoracic ganglia were used in all experiments except where otherwise noted . Suspensions of ganglion cells were obtained by trypsinization (0 .596 trypsin; Difco Laboratories, Detroit, Mich.) of 200 ganglia for 20 min at 37°C in calcium-free medium. The ganglia were washed three times and dispersed into cells by trituration with a pipette. Over 90% of the cells were viable as judged by trypan blue exclusion . To prepare cell aggregates, cells from 100 ganglia were suspended in 1 .5 ml of Dulbecco's Modified Eagle's Essential Medium supplemented with one-tenth volume of fetal calf serum (DMEM ; Microbiological Associates, Walkersville, Md .) . The suspensions were placed in 35-mm plastic petri dishes and incubated under 13% CO, at 37°C for 20 h on a gyratory shaker (50 rpm; Fisher Scientific Co ., Pittsburgh, Pa). The aggregates ranged in diameter from 200 to 1,000 pm . NGF and antibodies were added as indicated in particular experiments . NGF and Anti-CAM Antibodies Purified 2.5S NGF was purchased from Collaborative Research, Cambridge, Mass. Hans Thoenen (Max Institute for Psychiatry, Munich, Germany) and Lloyd Greene (New York University Medical School) also kindly provided samples of the purified growth factor . The biological activity (23) of these NGF varied by as much as a factor of two, but otherwise gave identical results in our studies . High molecular weight (7S) NGF gave equivalent results when used at about five times the concentration of 2 .5S NGF . Theprocedures for purification ofCAM from chickembryo retina, production of antibodies to CAM in rabbits, and preparation of monovalent Fab' fragments have been described previously (1, 34) . Except where noted, experiments with antibody were carried out with 0 .5 mg Fab'/ml of culture medium and included control cultures with Fab' from unimmunized rabbits. At 0.5 mg/ml, anti-CAM Fab' caused an 80-90% decrease in the initial rate of retinal cell aggregation (30). The cultures with nonimmune Fab' were identical to those without antibody.