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whether this hypothetical substance upregulated neu-
rogenesis, or recruited cells to differentiate as neurons,
or prevented differentiated neurons from dying. As
described above, two sets of careful observations
strongly suggested that the hypothetical substance
worked by maintaining the survival of differentiating
neurons (Levi-Montalcini and Levi, 1942; Hamburger
and Levi-Montalcini, 1949).
By modern standards, the next step would be to
harvest the target tissue (e.g., muscle) and try to isolate
a soluble substance that enhances survival. However,
most of the necessary biochemical tools did not yet
exist in the 1950s. The isolation of a neurotrophic factor
took a few decades to achieve, and it began with a
surprising set of observations. In an effort to provide
neurons with an “unlimited” amount of target tissue,
various mouse tumors were implanted into the chick
hind limb (Bueker, 1948; Levi-Montalcini and Ham-
burger, 1951; Levi-Montalcini and Hamburger, 1953).
One tumor, a connective tissue cell line called sarcoma,
grew rapidly and was invaded by nerve fibers. Within
five days of the transplant, there was a dramatic
increase in the survival of sensory and sympathetic
neurons, while motor neurons were unaffected (Figure
7.11). When the tissue was examined in a little more
detail, a key observation was made: ganglia with
no apparent physical connection to the tumor were
also greatly enlarged. This provided the first indica-
tion that cell survival was mediated by a diffusible
Amore direct demonstration came from experi-
ments in which tumor cells were placed on a vascu-
larized respiratory membrane in the chick egg called
the chorioallantois. In this case, the tumor was not in
contact with sympathetic and sensory ganglia, but it
did share the same blood supply. Even though the
tumor was physically isolated from the nervous
system, it was able to elicit a strong growth-promoting
effect (Figure 7.11). Thus, sarcoma tumor cells must
have released a soluble factor that could be trans-
ported to the neurons through the circulatory system.
As a first step toward isolating the putative survival
factor found in mouse sarcoma, an in vitro assay
system was developed. Sympathetic ganglia were
obtained from chick embryos and placed in a tissue
culture dish, either by themselves or next to mouse
sarcoma tumor cells. When grown next to tumor, the
neurons survived and grew a dense halo of axons
within hours, providing a simple and convenient assay
system. Although biochemical isolation was a slow
process, it was possible to obtain a tumor cell fraction
that had only proteins and nucleic acids. In order to
determine whether either of these components con-
tained the growth factor, a biochemical trick was
Wing bud
removal side
Control side
Wing bud
Late differentiating
Healthy cell
Degenerating cell
Early differentiating
FIGURE 7.10 The target influences neuron survival. Following
unilateral limb bud removal in a 3-day chick embryo (left side), there
is an increase in the number of degenerating cells in the DRG ipsi-
lateral to the ablated limb, as compared to the control side (right).
The number of viable DRG neurons is reduced dramatically.
(Adapted from Hamburger and Levi-Montalcini, 1949)
(VaP) motor neurons depends on contact with a tran-
sient population of muscle cells. If these muscle cells
are ablated a few hours before being contacted by VaP,
then the VaP motor neurons live and innervate the
ventral musculature (Eisen and Melancon, 2001).
Neuron survival clearly depends on the presence of
target tissue, but what is being procured? One simple
hypothesis is that the target cells secrete a chemical
that presynaptic neurons require for their survival. In
fact, an extraordinary series of experiments, coupled
with a few strokes of serendipity, led to the first
endogenous neurotrophic substance to be discovered,
the nerve growth factor (NGF). NGF has since been
shown to largely control the survival of sympathetic
neurons and contribute to the survival of sensory DRG
neurons during development. Although NGF turns
out to be the tip of an enormous iceberg of growth and
survival factors (below), we are going to examine its
discovery in some detail because it remains the best
understood system.
How did scientists arrive at the neurotrophic theory
of cell survival? Viktor Hamburger (1934) first sug-
gested that the target produces a factor that is retro-
gradely transported by the innervating neurons and
influences their development. Initially, it was not clear
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