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.
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