The first real insight into the function of NGF in the living animal was provided by experiments done in our laboratory at Washington University in 1959. Cohen obtained specific antibodies to NGF by injecting purified mouse NGF into rabbits. Small amounts of rabbit serum containing the NGF antibodies were then injected into newborn mice. A month later the experimental and control mice were sacrificed. In the mice treated with the NGF antibodies the sympathetic ganglia were reduced to such diminutive size that they were barely visible in the dissecting microscope! Nevertheless, the NGF antibodies had no ill effect on any other organs or tissues and for unexplained reasons did not damage the peripherally located sympathetic ganglia that control the sex organs in both sexes. It has therefore been possible to raise to maturity entire colonies of mice entirely lacking in sympathetic nervous function but normal in all other respects. These animals have provided a most valuable model system for studying how the lack of sympathetic innervation interferes with a multiplicity of body functions.
It is not yet known whether the dramatic and selective effects of the NGF antibodies are due to a direct toxic action of the antibodies on immature sympathetic neurons or to an inactivation by the antibodies of circulating molecules of NGF, thereby indirectly causing the death of the sympathetic neurons by depriving them of the NGF they need in order to survive. This second alternative seems the more likely one; convincing evidence in its favor would provide tangible proof that NGF is an absolute requirement for the survival and growth of immature sympathetic neurons in the living animal.
That NGF is essential for the survival of sympathetic neurons in cell culture has been known for some time. In 1963 Pietro Angeletti and one of us (Levi Montalcini), who were then at the Istituto Superiore di Sanità in Rome, dissected sensory and sympathetic ganglia into their cellular components: neurons, glial cells (which support and nourish the neurons) and fibroblasts (embryonic connective-tissue cells). After 24 hours of culture the glial cells and fibroblasts had survived and multiplied but the neurons had undergone a massive degeneration. The daily addition of minute amounts of NGF to the culture medium, however, enabled the neurons to survive for indefinite periods and to form a dense meshwork of nerve fibers that after a few days completely covered the surface of the culture dish. This ability of NGF to sustain sympathetic neurons in culture has since made possible some ingenious and revealing experiments on neuronal differentiation [see "The Chemical Differentiation of Nerve Cells." by Paul H. Patterson, David D. Potter and Edwin J. Furshpan; SCIENTIFIC AMERICAN, July 1978].
The availability of milligram quantities of pure NGF has made it possible to test its effects in the living organism. All mammals respond to NGF the same way, although for practical reasons rodents are the experimental animals of choice. When 10 micrograms of NGF per gram of body weight is injected into newborn rodents for periods of up to three weeks, their sympathetic ganglia become 10 to 12 times larger than those of control animals. This excessive increase in size of the sympathetic ganglia has been traced to three separate processes: (1) an increased rate of differentiation of the sympathetic neurons, (2) an increase in the total number of neurons in the ganglia and (3) an increase in the size of the fully differentiated neurons.
NGF does not increase the number of neurons in the sympathetic ganglia by enhancing their multiplication. Instead, as was first proposed by I. A. Hendry of the Australian National University, the marked increase in the number of neurons is due to the survival of redundant immature neurons that would ordinarily die off in the course of development. Cell death is a common event in the formation of the nervous system: entire populations of immature neurons die off or are sharply reduced in size. In fact, in the early developmental stages of the chick embryo the dead cells in the sensory and sympathetic ganglia often outnumber the live ones. The generally accepted hypothesis is that the immature neurons that do not establish functional connections with their target cells are doomed to die; the redundant number of neurons ensures that all the appropriate connections are made. The effect of experimentally administered NGF is to enable the redundant neurons to survive and differentiate in spite of their failure to establish connections. It is this effect of NGF that gives rise to the substantially increased numbers of neurons in NGF-treated sympathetic ganglia.