Editor's Note: Neurobiologist Rita Levi-Montalcini, a Nobel laureate in physiology or medicine in 1986, died December 30 at the age of 103. We are making this article co-authored by her free online for the next 30 days. This story was originally published in June 1979 issue of Scientific American.

The human nervous system is a vast network of several billion neurons, or nerve cells, endowed with the remarkable ability to receive, store and transmit information. In order to communicate with one another and with non-neuronal cells the neurons rely on the long extensions called axons, which are somewhat analogous to electrically conducting wires. Unlike wires, however, the axons are fluid-filled cylindrical structures that not only transmit electrical signals but also ferry nutrients and other essential substances to and from the cell body. Many basic questions remain to be answered about the mechanisms governing the formation of this intricate cellular network. How do the nerve cells differentiate into thousands of different types? How do their axons establish specific connections (synapses) with other neurons and non-neuronal cells? And what is the nature of the chemical messages neurons send and receive once the synaptic connections are made?

This article will describe some major characteristics and effects of a protein called the nerve-growth factor (NGF), which has made it possible to induce and analyze under highly favorable conditions some crucial steps in the differentiation of neurons, such as the growth and maturation of axons and the synthesis and release of neurotransmitters: the bearers of the chemical messages. The discovery of NGF has also promoted an intensive search for other specific growth factors, leading to the isolation and characterization of a number of proteins with the ability to enhance the growth of different cell lines.

The peripheral nervous system of vertebrate animals includes three kinds of nerve cells: sensory neurons, which transmit impulses from sensory receptor structures to the brain; motor neurons, which innervate the striated, or skeletal, muscles, and autonomic neurons, which regulate the functional activity of the circulatory system, the organs, the glands and the smooth muscles (such as those of the intestine). Autonomic neurons are of two kinds: sympathetic and parasympathetic. The sensory neurons and some of the sympathetic neurons are situated in chains of ganglia flanking the length of the spinal cord. Because these neurons are uniquely accessible to experimental manipulation much of the research on the development of the nervous system at the cellular level has focused on how the nerve fibers projecting from the sensory and sympathetic ganglia make connections with their corresponding target organs.

In the first half of this century the new science of experimental embryology seemed to offer the best way to study the intimate bond that interlocks the growth of the peripheral neurons and their target organs. Ross G. Harrison of Yale University challenged the nervous system of the larva of amphibians to solve problems it would ordinarily never confront, such as providing nerves for limbs and organs grafted from other species. He wanted to see how sensory and sympathetic ganglia, sending out their nerve fibers to these peripheral "fields of innervation," would adjust to the different dimensions and configurations of the foreign organs.

Harrison's results demonstrated that the developing amphibian nervous system is remarkably flexible in adapting to such novel situations, even to the point of accelerating the growth of nerve fibers in a host species to keep pace with the faster-growing limb of a smaller donor species. He concluded that the embryonic nervous system is highly receptive to influences exerted by the peripheral field. Such influences are not species-specific, however, since they can be evoked by organs or rudimentary limbs that have been transplanted from one species to another.

Viktor Hamburger of Washington University extended Harrison's work but chose to study the chick embryo because its nervous system, although more complex than that of an amphibian, lends itself better to experimental analysis: its nerve centers are more clearly delineated and their strong affinity for silver stain enables the experimenter to visually examine the nerve structures more easily. Hamburger grafted limb buds onto chick embryos at very early stages of development and observed how the modified peripheral field was innervated by sensory and sympathetic fibers. Unfortunately the responses were often so complex that they defied interpretation,

In 1948, in an effort to get more straightforward results, Elmer D. Bueker of Georgetown University modified Hamburger's experimental approach. He had the ingenious idea of replacing one limb bud of a chick embryo with a fragment of a bird or mammalian tumor. The tumor cells were all undifferentiated and hence provided a homogeneous peripheral field, in contrast to the cells of the normal limb bud, which were destined to differentiate into many types of tissue. Bueker's experiment was therefore expected to reveal how a homogeneous but fast-growing peripheral field would be innervated.

Of three different tumors implanted in the body wall of three-day-old chick embryos only one, a mouse tumor of connective-tissue cells called sarcoma 180, grew vigorously and was invaded by nerve fibers growing out from adjacent sensory ganglia. In embryos sacrificed after five days the sensory ganglia innervating the tumor were 33 percent larger than those innervating the normal limb bud on the opposite side of the embryo. At first these findings seemed to suggest that the size of a sensory ganglion depends on the size and rate of growth of its field of innervation. According to this hypothesis the rapidly growing tumor provided a more favorable peripheral field for the innervating sensory fibers than the slowly growing limb bud did.

A reexamination of Bueker's findings by our group at Washington University revealed new aspects of the phenomenon that obliged us to revise his conclusions. We found not only that the growth of the sensory ganglia innervating the sarcoma-180 tumors increased but also that the sympathetic ganglia increased enormously in volume, becoming five to six times larger than they were in control animals. This increase in size of the sympathetic ganglia was considerably greater than that exhibited by the sensory ganglia. Together with the sensory fibers the sympathetic fibers branched all over the peripheral field provided by the tumor but did not form any synapses with the tumor cells.

In addition, and indicating an even more striking departure from normality, the viscera of the embryos with the transplanted tumors were flooded with excessive numbers of sympathetic fibers long before the embryos of the control animals were even sparsely innervated. The sympathetic fibers forced their way into large and small veins, impeding and sometimes completely obstructing the flow of blood. These extraordinary effects suggested that the overgrowth of the sympathetic ganglia was more than simply a response to the rapidly growing peripheral field of innervation provided by the tumor, as Bueker had proposed. Rather it seemed the tumor was releasing some chemical factor that was in turn inducing the remarkable growth of the sympathetic ganglia and the exuberant branching of their nerve fibers.

This hypothesis was tested by transplanting sarcoma-180 tumors into the respiratory membranes of the chick egg, which are permeated with blood vessels from the embryo. The tumor and the developing chick embryo therefore shared the same blood supply, although they were not in direct contact. We found that when the tumor was transplanted into the respiratory membranes, it elicited the same growth-promoting effects on the sympathetic ganglia as it did when it was implanted in the embryo itself, providing convincing proof that the tumor was releasing a soluble factor that was carried in the bloodstream to the embryo.

The next challenge was to identify the postulated nerve-growth factor being released by the tumor. For this purpose we needed a system much less complex than the developing embryo. Tissue culture (which in the early 1950s had yet to become the universal biological tool it is today) seemed to offer a useful alternative. We reasoned that if sarcoma 180 was releasing a chemical factor with the ability to enhance nerve growth, the same effect should appear when an isolated sympathetic ganglion was incubated with the tumor in laboratory glassware. In 1953 one of us (Levi-Montalcini) and Hertha      Meyer did this experiment at the Biophysics Institute in Rio de Janeiro. Sensory and sympathetic ganglia were dissected out of eight-day-old chick embryos and cultured in a semisolid medium in proximity to fragments of mouse sarcoma-180 tumors. Within 10 hours of incubation the isolated ganglion gave rise to a dense halo of nerve fibers, radiating out from the ex-plant like rays from the sun. Control ganglia cultured for the same length of time in the absence of the sarcoma-180 cells displayed only a sparse and irregular outgrowth of nerve fibers.

The discovery that the tumor could exert its growth-enhancing effects on an isolated ganglion in tissue culture was the turning point of the investigation. Whereas our earlier experiments in developing chick embryos had required weeks of painstaking work, we could now in a few hours screen a large number of tissues, organic fluids and chemicals to determine if they were sources of the growth-promoting activity. Furthermore, it now became possible to attempt to isolate the nerve-growth factor from our simplified tissue-culture system.

Stanley Cohen, a biochemist, agreed to join our group at Washington University to undertake the task of identifying the active agent. He soon managed to restrict the growth factor to a fraction of the tumor cells containing both protein and nucleic acid. This finding opened the way to a more precise biochemical analysis, but we would not have made much progress if it had not been for a fortuitous event two years later. In order to determine whether the nerve-growth factor was a protein or a nucleic acid, Cohen and one of us (Levi-Montalcini) treated the tumor-cell extract with snake venom containing a high concentration of the enzyme phosphodiesterase, which degrades nucleic acids. To our surprise we found that adding minute amounts of the venom to the active fraction of the sarcoma-180 cells enhanced the growth-promoting effect of the fraction rather than reduced it. That the snake venom itself was the source of the increased activity was readily confirmed. The addition of a small amount of venom to the culture medium in the absence of the extract of sarcoma 180 elicited the growth of the same dense halo of nerve fibers around an isolated sensory or sympathetic ganglion.

In fact, the nerve-growth factor in the snake venom turned out to be much more abundant and potent than that in the sarcoma-180 cells. Cohen was therefore able to purify the factor from the venom and demonstrate that it was a protein. Injections of the purified snake venom NGF into growing embryos resulted in the same excessive sympathetic innervation of the viscera and the blood vessels as that induced by the sarcoma-180 cells.

The discovery that two unrelated sources—mouse sarcoma and snake venom—harbor NGF suggested the possibility that still other tissues might secrete the factor. The search focused on the submaxillary salivary gland of rodents, which is similar in certain respects to the venom gland of snakes. Indeed, Cohen isolated an NGF from mouse salivary glands that was about 10,000 times more active than that purified from mouse sarcoma 180 and about 10 times more active than that purified from snake venom. Over the next two decades smaller amounts of NGF were found to be secreted by a wide variety of normal and neoplastic (cancer) cells.

ganglia of NGF-treated mice
INCREASED SIZE AND NUMBER of neurons in the sympathetic ganglia of NGF-treated mice result in an increase in the volume of the ganglia. These micrographs are of sections through two ganglia, one ganglion from a three-week-old rat injected daily with saline solution only (left) and the other from a littermate mouse injected with NGF (right). Neurons in NGF-treated rat are substantially enlarged. Note also that cells in ganglia of animals treated with NGF exhibit a greater affinity for toluidine blue, the dye used to stain the cells.
Image: Scientific American, June 1979

In 1969 V. Bocchini and Pietro U. Angeletti at the Laboratory of Cell Biology in Rome devised a method for purifying NGF from mouse salivary glands. With the large quantities of NGF made available by their technique Ruth Hogue Angeletti and Ralph A. Bradshaw of the Washington University School of Medicine were able to determine the sequence of amino acids in the protein. Biologically active NGF is a dimer, or complex of two identical polypeptide chains, each of which has a molecular weight of 13.250 daltons. The NGF molecule has a marked predominance of positively charged amino acids and has a net positive charge at neutral pH. In addition the folded polypeptide chain is stabilized by three covalent sulfur-sulfur bridges between units of the amino acid cysteine at different positions along the chain. Such disulfide bonds are common in proteins that are actively secreted from cells, such as insulin and antibodies. The restraint these molecular bridges impose on the conformation of the polypeptide chain is apparently necessary to prevent its denaturation and inactivation in an environment that is much more subject to adverse conditions than the interior of the cell.

A comparison of the sequence of amino acids in NGF with that of several other polypeptides by William A. Frazier of Washington University revealed that NGF and insulin have certain sequences in common. This observation led to the hypothesis that the gene for NGF evolved from an ancestral gene for proinsulin, the large precursor polypeptide that is cleaved to yield the active molecule of insulin. Frazier suggested that the ancestral proinsulin gene had duplicated itself and the two copies had subsequently evolved divergently, giving rise to proinsulin and a postulated precursor polypeptide for NGF. Although this intriguing possibility remains theoretical, the similarities in the amino acid sequences of NGF and insulin are not great enough to result in any similarity of function: the two molecules have completely different target cells and biological activities.

In 1967 Silvio S. Varon and Eric M. Shooter of the Stanford University School of Medicine isolated NGF by a different procedure and found that the NGF dimer was present as a stable complex with two copies each of two other proteins, which they designated the alpha and gamma subunits. This finding was puzzling, since it was known that the NGF dimer by itself was biologically active. Further investigation revealed that the gamma subunit is a specific enzyme that cleaves polypeptide chains only at a point adjacent to the amino acid arginine. The alpha subunit, on the other hand, has no detectable biological activity. This odd association of three disparate proteins demanded an explanation.

Shooter hypothesized that the alpha and gamma subunits serve to activate, store and protect the NGF molecule. According to his scheme the initial gene product in the manufacture of NGF is a large precursor polypeptide called proNGF, two copies of which form a dimer. Two gamma subunits then associate with the proNGF dimer and cleave the precursor chains to generate the active NGF dimer. Unlike the typical complex of an enzyme and its product, which rapidly dissociates, the 'gamma subunits remain bound to the NGF dimer after the cleavage of the proNGF chains. (The two alpha subunits may bind either to the proNGF dimer before cleavage or to the complex of the gamma subunit and the NGF dimer after cleavage.) The association of the gamma and alpha subunits with the NGF dimer apparently serves to protect it from further degradation by other protein-cleaving enzymes in the body fluids. Shooter's hypothesis recently received support when he and Edward A. Berger isolated the proNGF molecule and demonstrated that incubation of this precursor with the gamma subunit resulted in its complete conversion into active NGF.

The biological activity of a protein resides in its three-dimensional structure, which consists of not only its amino acid sequence but also the precise folding pattern of the polypeptide chain (the secondary structure) and the suprafolding of two or more such chains to form a unique globular entity (the tertiary structure). The only reliable way to determine the secondary and tertiary structure of a protein molecule is to crystallize it and mathematically analyze the crystal's X-ray-diffraction pattern. The recent crystallization of NGF by A. Wlodawer. Keith O. Hodgson and Shooter is therefore an important first step in determining the three-dimensional structure of the molecule by x-ray crystallography.

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.

In addition to apparently being essential to the survival of immature sympathetic neurons NGF seems to play a vital role in guiding nerve fibers toward their corresponding target organs. Three basic mechanisms have been proposed over the years to explain the formation of specific neural circuitry: (1) an elaborate predetermined program encoded genetically in each neuron that unfolds according to rigid and unmodifiable rules, (2) a random process of trial and error in which growing nerve fibers that make the right connections are consolidated and those that fail are reabsorbed and (3) a general program of circuit formation that is brought to completion by an interplay between genetic and extrinsic factors.

The first mechanism can be discounted because it would require very large amounts of genetic information—much more than could be encoded in the entire complement of DNA in the cell nucleus of each neuron. The second mechanism is also unlikely, because such a random process of circuit formation would be extremely time-consuming and wasteful of energy and resources. The third mechanism therefore seems the most probable: the circuitry of the nervous system is established through some combination of genetic and extrinsic factors.

The existence of such extrinsic factors was first suggested by the Spanish neurologist Santiago Ramón y Cajal, who visualized them as chemical signals emanating from peripheral tissues that would direct the growing nerve fibers toward their matching target cells. Cajal named the process neurotropism. His hypothesis was neglected for many years because the methodology for detecting such chemical factors in the living embryo was not yet available. The discovery and isolation of NGF, however, has provided an opportunity to reconsider the concept of neurotropism under more favorable experimental conditions.

Convincing evidence for the role of NGF in the formation of neural circuits in the sympathetic nervous system has come from both animal and test-tube experiments. At the Laboratory of Cell Biology in Rome, NGF was injected into the brain of newborn rodents. An unexpected effect of the treatment was that nerve fibers sprouted from the sympathetic ganglia and invaded the brain and spinal cord. Apparently the NGF injected into the brain diffused through the motor and sensory roots of the spinal cord and reached the sympathetic chain ganglia flanking the cord, where it induced the outgrowth of nerve fibers. This finding implies that the tip of a growing sympathetic nerve fiber elongates along a diffusion gradient of NGF released by the target organ. The NGF gradient does not entirely determine the course of the nerve fibers but helps to orient them in the right direction.

Further evidence for a neurotropism mediated by NGF was provided by some elegant experiments done by Robert B. Campenot of the Harvard Medical School. He devised a three-chambered cell-culture system in which the chambers were separated by impermeable barriers of sterile silicon grease, preventing the diffusion of fluid from one chamber to another. Immature sympathetic neurons were seeded into the central chamber in the presence of NGF and allowed to send out growing fibers under the silicon barriers along scratches made on the bottom of the culture dish. Campenot filled one side chamber with a nutrient solution containing NGF and the other chamber with the same nutrient solution devoid of NGF. He observed that the growing nerve fibers migrated from the central chamber only into the side chamber containing NGF. When the NGF-rich solution was withdrawn from that side chamber, the nerve fibers that had extended into it began to degenerate, even though the neuronal cell bodies in the central NGF-containing chamber persisted in good condition.

These experiments resolved the longstanding question of whether extrinsic chemical factors play a role in guiding the growth of nerve fibers. Since NGF is released in minute amounts from peripheral tissues receiving innervation from sympathetic ganglia, it now seems clear that a diffusion gradient of NGF directs the fibers toward their corresponding target tissues. When the growing nerve fiber and the target cell finally come in contact, the provisional adhesion consolidates into the structural and functional organization of the synapse.

neutropic effect of NGF
NEUROTROPIC EFFECT of NGF was demonstrated by injecting the factor into the brain of newborn rats for 10 days; the control animals were injected with saline solution only. In the NGF-treated animals (bottom) the growth factor diffused from the injection site to the spinal cord and reached the adjacent sympathetic ganglia through the motor and sensory roots. The NGF induced the outgrowth of sympathetic nerve fibers (color), which gained access to the spinal cord through the spinal roots and extended as far as the site of injection in the brain. This abnormal projection of sympathetic fibers (which usually innervate peripheral tissues) in response to NGF provided evidence for the hypothesis that a gradient of NGF guides the direction of growing sympathetic fibers in the periphery. In addition injection of NGF into either the brain or the systemic circulation enhanced the peripheral branching of the sympathetic fibers. Experiment was done by M.L. Menesini Chen and J.S. Chen in authors' laboratory.
Image: Scientific American, June 1979

Once the elongating fibers have estab­lished the appropriate synaptic contacts with the target cells, the continued sur­vival of the innervating cells in the gan­glion appears to depend on the availa­bility of NGF. Studies conducted by Hendry at the Australian National Uni­versity and by K. Stockel and H. Thoe­nen at the Basel Institute for Immunol­ogy have demonstrated that NGF is taken up at the terminal nerve endings of the sympathetic fibers and transport­ed back to the neuronal cell body along the axon. This retrograde axonal trans­port of NGF is absolutely essential for the survival of the innervating neurons. When it is experimentally prevented (either by severing the projecting axons, by treating them with the drug vinblastine, which blocks axonal transport, or by ad­ministering 6-hydroxydopamine, which destroys the nerve endings), the inner­vating sympathetic neurons in the gan­glion die off. The lethal effects of block­ing the axonal transport of NGF can be completely overcome, however, by sup­plying the cell bodies with externally administered NGF. In this case the exter­nal NGF makes up for the NGF that would normally be transported back in­side the axon to the cell body from the innervated cells.

Work in several laboratories has shown that the retrograde axonal transport of NGF follows its interac­tion with specific receptor sites on the nerve terminals of the newly established fibers. Receptors are proteins that are usually located on the external surface of the cell membrane; they provide spe­cific recognition sites for messenger substances such as hormones, neuro­transmitters and growth factors. The existence of such specific receptors on the neuronal surface makes it possible for NGF to exert its effects at exceed­ingly low concentrations (about 2.8 micrograms per liter). The binding of NGF to its receptors triggers a chain of biochemical events that leads ultimately to the outgrowth of the nerve fiber.

Immature sympathetic neurons re­spond to NGF with a burst of metabolic activity that provides the material nec­essary for the growth of the nerve fiber and the manufacture of molecules of neurotransmitter. The cells make more proteins and lipids, take up amino acids from the surrounding medium and burn glucose and other energy-rich com­pounds at a faster rate. These effects can be seen as a general response to the primitive signal conveyed by NGF through its specific receptors on the cell surface.

Within a short time after the binding of NGF to its receptors the protein con­stituents of the cytoplasm of the imma­ture sympathetic neuron are profound­ly rearranged. In particular the filamentous structures called microtubules and microfilaments come to fill all the avail­able space between the cell nucleus and the cell membrane. These filaments play a key role in the growth of the nerve fiber by providing a structural frame­work and the propulsive force for its elongation.

How does NGF control the assembly of these filamentous proteins? One pos­sibility is that it acts directly to enhance the polymerization of tubulin and actin, the monomeric proteins that give rise respectively to microtubules and micro­ filaments. Working at the Laboratory of Cell Biology in Rome, we tested the hy­pothesis by measuring the rate of assem­bly of tubulin and actin in the test tube in both the presence and the absence of NGF. In the absence of NGF dilute solutions of the proteins were unable to polymerize into filaments (or did so at a very low rate) because the thermody­namic tendency of the monomers to stay far apart in solution was greater than their tendency to aggregate. The addi­tion of NGF to the solution, however, induced a rapid and massive polym­erization reaction. Subsequent investi­gation revealed that NGF joins togeth­er the minimum number of monomers needed to initiate polymerization, after which the reaction proceeds spontane­ously to completion. On the basis of these test-tube findings we have hypoth­esized that the massive formation of microtubules and microfilaments in the developing sympathetic neuron is triggered by NGF, and that this effect may lead directly to the growth and elongation of the nerve fiber.

An entirely different function of NGF came to light recently with the discovery that certain non-neuronal cells are able to respond to the protein. Lloyd A. Greene and Arthur S. Tischler of the Harvard Medical School demonstrated in 1977 that cells of the line designated PC12 (which are derived from a rat tumor) respond to NGF by acquiring properties characteristic of sympathetic neurons. The NGF-treated cells send out fibers, become electrically excitable and store and release neurotransmitters of the catecholamine type. When NGF is withdrawn from the culture medium, the cells retract their fibers, lose their other neuronal properties and resume the uncontrolled proliferation characteristic of neoplastic cells.

Shortly after this discovery K. Unsicker and his colleagues at Johns Hopkins University found that immature chromaffin cells obtained from the medulla (inner part) of the adrenal gland and cultured in the presence of NGF acquire the biochemical and morphological properties of sympathetic neurons. Subsequent experiments carried out at the Laboratory of Cell Biology in Rome by Luigi Aloe and one of us (Levi-Montalcini) demonstrated that this remarkable transformation can also take place in the intact animal: repeated injections of NGF into rat fetuses, continued for two to three weeks after birth, resulted in the differentiation of chromaffin cells into sympathetic neurons in the core of the adrenal gland! These findings indicate that NGF plays a much broader role in the living organism than had been supposed.

It should be obvious to the reader that the investigation of NGF, which began more than two decades ago, is far from over. Among the many unanswered questions is why NGF is manufactured and secreted by the venom gland of snakes and the salivary gland of rodents, even though neither of these glands is essential for the life of the organism and the sympathetic neurons that depend on NGF for their survival. The molecule is also manufactured and released in minute amounts by a wide range of normal and neoplastic cells.

The discovery of NGF has made it possible to experimentally manipulate sympathetic neurons with considerable ease, both in tissue culture and in the living animal. With the aid of purified NGF and NGF antibodies the neurobiologist is now able to increase the number of sympathetic neurons in an animal tenfold or to eliminate them altogether. Moreover, the catecholamine­secreting neurons in the brain are strikingly similar to the sympathetic neurons in the peripheral ganglia in both their morphological and biochemical properties. Already information gained from the study of sympathetic neurons has proved applicable to the treatment of disorders of the central catecholamine­secreting neurons, such as Parkinson's disease. A. Bjorklung, B. Bierre and U. Stenevi of the University of Lund recently obtained preliminary evidence that the catecholamine-secreting neurons in the brain respond to NGF with a profuse branching of their nerve fibers. If these findings are confirmed, a powerful tool will become available for modulating the function of such brain circuits, which play a crucial role in many kinds of behavior.