/ . Embryo/, exp. Morph. Vol. 24, 2, pp. 381-392, 1970
381
Printed in Great Britain
The in vitro effect of
the nerve growth factor on chick embryo spinal
ganglia—a light-microscopic evaluation
ByPEDDRICK WEIS1
From the Department of Anatomy, New Jersey College of
Medicine and Dentistry
SUMMARY
The effect of the nerve growth factor (NGF) on chick embryo spinal ganglia was studied
in the hanging-drop bioassay system by comparison with parallel development in vivo.
The well-differentiated ventrolateral neuroblasts, which in vivo increase 1-33 times in size
during the culture period, did not increase in size at all in vitro. Only 65-72% survived to the
end of the culture period regardless of the NGF concentration.
The less-differentiated mediodorsal (M-D) neuroblasts, which in vivo increase 1-31 times
in size during the culture period, were found to increase equally in vitro if sufficient NGF
was present. Such a quantity was greater than that which evoked maximum outgrowth of
neurites.
Survival of M-D neuroblasts was also related to NGF concentration but did not equal the
/// vivo condition even at the highest concentration. The hyperchromatic type of degeneration
prevented by high NGF concentrations is that which results in vivo from insufficient peripheral
field.
From this and other reports it would appear that the response to NGF seen in vitro is due
only to the M-D neuroblasts, and that all biochemical and cytological observations which
have been reported would therefore represent conditions within those cells only.
INTRODUCTION
The control of development of parts of the nervous system has been attributed in part to a protein, the nerve growth factor (NGF) (Levi-Montalcini &
Angeletti, 1968). Many biochemical observations have been made on the action
of NGF, using the in vitro assay system originally devised for detecting the
presence of NGF (Levi-Montalcini, Meyer & Hamburger, 1954). In regard to
this widely used hanging-drop culture method using chick embryo spinal
ganglia, there are some points which are as yet unclear: (1) there is not any
evidence to show if NGF specifically affects the later-differentiating mediodorsal neuroblasts, as has been suggested (Levi-Montalcini & Angeletti, 1968),
or if it affects the ventrolateral cells as well; (2) the curious inhibition of neuritic
1
Author's address: Department of Anatomy, New Jersey College of Medicine and
Dentistry, 100 Bergen Street, Newark, New Jersey, 07103, U.S.A.
382
p. W E I S
outgrowth which occurs in vitro with higher concentrations of NGF (LeviMontalcini & Booker, 1960; Crain, Benitez & Vatter, 1964) has been given only
superficial attention; and (3) it has not been established to what extent the
NGF-stimulated cultures mimic normal development. The following investigation was initiated in an attempt to add to our knowledge concerning these
points, with the hope that some light would be shed on the in vitro activity of
NGF.
MATERIALS AND METHODS
Lumbosacral spinal ganglia were dissected from chick embryos and placed in
hanging-drop cultures, according to Levi-Montalcini et al. (1954), with the
following modification. The embryos were taken at stage 33 (Hamburger &
Hamilton, 1951) (7-|—8 days of incubation); the drops of rooster plasma and of
NGF-containing medium 199 were placed in disposable Falconware Petri
dishes and sealed with melted petroleum jelly. (This modification was originally
adopted in the laboratory of Dr E. D. Bueker, New York University.) The
cultures contained tenfold serial dilutions of NGF to yield the various degrees of
growth and stunting described by Crain et al. (1964). Control cultures contained
no NGF.
Two preparations of NGF were used. One was the highly purified type of
Schenkein, Levy, Bueker & Tokarsky (1968), while the other was obtained
commercially from Burroughs-Wellcome. Both preparations are extracts of
mouse salivary glands.
At the end of the 20 h culture period, the ganglia were fixed for 1 h at 2-4 °C in
a mixture containing 3 % glutaraldehyde, 1 % OsO4, 0-01 M-CaCl2, and 0-1 M
s-Collidine buffer at pH 7-42 (a modification of the procedure of Trump &
Bulger, 1966). After ethanol dehydration the ganglia were embedded in Maraglass
(Erlandson, 1964). These fine-structural methods were used with the intention
that the tissue blocks could also be used later for electron-microscopical
analysis.
For purposes of comparison with the cultured ganglia, additional ganglia
were excised and fixed immediately both at the beginning and at the end of the
culture period, by which time the embryos had reached Hamburger & Hamilton
stage 35 (8|— 9 days). A total often series of cultures were performed, utilizing
an average of eight ganglia from each of forty eggs.
One-micron sections of embedded ganglia were prepared on a Sorvall MT-1
microtome using glass knives, mounted on glass slides, and stained with toluidine
blue. Suitable sections were photographed in a Zeiss Ultraphot on 10-2 x 12-7 cm
sheet film. The final prints, at x 1000, were assembled into montages and used for
cell counts and size determinations. The growth of the ganglia was compared
with parallel development in ovo by two criteria: (1) the percentage of neuroblasts in both mediodorsal (M-D) and ventrolateral (V-L) areas which survived,
and (2) the increase in size, which, for these irregularly shaped cells, was best
In vitro effect of nerve growth factor
2
383
measured by counting the number within (0-1 mm) in both of the two areas of
each ganglion.
The cultures at the end of the 20 h incubation period show various types of
growth according to the amount of NGF present (Figs. 1-4). The halo of
neuritic outgrowth is optimum at the NGF concentration called one biological
unit (B.U.) by Levi-Montalcini & Cohen (1956). At higher concentrations
'stunting' occurs due to premature cessation of the outgrowth. At 5-10 B.U. the
outgrowth is slightly stunted, at 50-100 B.U. the outgrowth is very stunted, and
above 100 B.U. there is a complete inhibition of outgrowth of neurites. Cultures
FIGURES
1-4
Figs. 1-4. Phase micrographs of living ganglia after 20 h of culture.
Fig. 1. Control culture (no NGF), with a few fibroblast processes.
Fig. 2. Culture with optimum outgrowth (one biological unit of NGF).
Fig. 3. Slightly stunted ganglion (5-10 B.U. of NGF).
Fig. 4. Very stunted ganglion (50-100 B.U. of NGF).
An inhibited ganglion (not shown) would appear similar to a control ganglion,
except for fibroblasts, despite the presence of more than 100 B.U. of NGF.
384
P. WEIS
above 1000 B.U. are not feasible since the clots usually lyse. The cultures used
in this study were taken from the following groups: controls, those with optimum
outgrowth, those that were very stunted, and those whose outgrowth was inhibited completely.
Data analysis was aided by the use of a General Electric 235 digital computer.
RESULTS
At stage 33 (7|—8 days in ovo) the chick lumbosacral spinal ganglia clearly
show the well-differentiated V-L neuroblasts and the less-differentiated M-D
cells, as described by Levi-Montalcini & Levi (1943) (Fig. 5). Since these two
groups were affected differently, they will be discussed separately.
Fig. 5. Cross-section of a normal stage 33 spinal ganglion, the starting-point of this
investigation. The ventrolateral area (VL) consists of larger, more-differentiated
neuroblasts, while the mediodorsal area (MD) consists of smaller, less-differentiated
neuroblasts. The arrow points dorsad.
A. Ventrolateral neuroblasts
In the normally developing ganglion, at the beginning of the culture period
(stage 33), there are 56 ± 1-2 V-L neuroblasts per (0-1 mm)2 with a > 99%
survival rate (Figs. 5, 6). By stage 35 (8|—9 days in ovo), the V-L neuroblasts
have grown so that only 42 + 1-0 fill a (0-1 mm)2 area (Fig. 6). Only a small part
of this change is a result of satellite-cell development. The survival rate is again
> 99%. The neuroblasts have grown 1-33 times during this time (Fig. 7).
In the cultured ganglia the V-L neuroblasts have not been so successful. As
shown in Table 1 and Fig. 6, the cell size in each group has not changed signifi-
In vitro effect of nerve growth factor
385
cantly from the time of explantation and has remained significantly different
(P < 0001) from the stage 35 ganglia. Control cultures and those at all concentrations of NGF have, in addition, poor survival rates of 65-73 % for
V-L cells. These survival rates, which are insignificantly different from_fceach
other, are significantly different (P < 0-001) from both stage 33 and stage 35
in vivo ganglia.
Table 1. Data values for Figure 6
Treatment or stage
Cell
density
d = no./
(01 mm)2*
Mean
cell
size
(S = \00/d)
Mean
growth
rate
(S/S 33 )t
Survival
(%)*
Mediodorsal area
Stage 33
//; vivo
In vitro
Control
Optimum outgrowth
Stunted
Inhibited
Stage 35 in vivo
118 ± 4 0
0-848
100
99-6 ±0-2
111 ±2 7
98 ±3-6
94 ±2-6
89 ±1-3
90 ± 3 0
0-901
1020
1 064
1-124
1110
106
1-20
1 26
1-33
1-31
67 ± 4-9
86 ± 1 1
93 ±1-8
92±l-3
99 ±0-4
Ventrolateral area
Stage 33
In vivo
In vitro
Control
Optimum outgrowth
Stunted
Inhibited
Stage 35 in vivo
56 ±1-2
1-785
100
99-7 ±0-2
61 ±2-7
55 ±2-0
58 + 3-5
57 ±4-2
42 ±1-0
1 -640
1 -820
1 -725
1-755
2-380
0-92
102
0-97
0-98
1-33
71 ± 3-6
72 ±4-4
73 ±3-5
65 ±4-2
99-8 ±0-2
* Data = mean ± S.E.
t S33 = size at stage 33.
Those cells which die may have one of two appearances (Fig. 8). They may
show typical chromatolysis with a loss of basophilia which ends in eventual
complete dissolution of the cell, or they may become hyperchromatic with
condensed nuclei and eventual nuclear pyknosis and cytoplasmic condensation.
Neither type of degeneration predominates in the V-L area.
B. Mediodorsal neuroblasts
Mediodorsal cells in the stage 33 embryo are less differentiated than their
V-L counterparts and are correspondingly smaller (Fig. 5). Their Nissl substance is still diffuse rather than granular as in the V-L cells. There are 118 ±
4-0 M-D cells per (0-1 mm)2 with > 99% survival rate. By stage 35 there are
90 ± 3-0 cells per (0-1 mm)2, which is 1-31 times the stage 33 size (Figs. 6, 9).
In the cultured ganglia the M-D neuroblasts show growth increases and
survival rates which correlate with the concentration of NGF. Controls have
386
P. WEIS
2
111+2-7 cells per (0-1 mm) , or x 1-06 increase in size, with a 67% survival
(Fig. 10). Optimum-growth cultures (1 B.U.) have 98 ±3-6 cells per (0-1 mm)2,
or x 1-20 increase, with an 86 % survival (Fig. 11). Very stunted ganglia (50100 B.U.) have 94 ±2-6 cells per (0-1 mm)2, or a x 1-26 increase, with a 9 3 %
survival. Ganglia whose outgrowth was completely inhibited by > 100 B.U.
1-30
•2 1-20
1-10
100
0-90 L Stage 33
in vivo
Control
Optimum
v
Stunted
Inhibited
v
Stage 35
'
,-„ v/vo
In vitro
100
n
90
80
I
70
Stage 33
in vivo
Control
Optimum
Stunted
Inhibited
Stage 35
in vivo
In vitro
Fig. 6. Graphic representation of data in Table 1. (A) Comparison of growth in vivo
and in vitro of ventrolateral and mediodorsal neuroblasts. Stage 33 neuroblasts in
each area = 1-00. (B) Comparison of survival of neuroblasts in vivo and /// vitro
in both ventrolateral and mediodorsal areas. • , Mediodorsal area; • , ventrolateral
area.
In vitro effect of nerve growth factor
387
11
FIGURES 7-11
Figs. 7-1.1. Oil-immersion (N.A. = 1-25) micrographs of toluidine blue-stained 1 /*
sections of ganglia fixed at end of culture or at comparable time in vivo (Stage 35).
In the cultured ganglia, two types of degenerating neuroblasts can be seen, light
chromatolytic cells (L) and dark condensed cells (D).
Fig. 7. Ventrolateral neuroblasts at stage 35 in vivo.
Fig. 8. Ventrolateral neuroblasts in optimum growth culture. The appearance of
ventrolateral neuroblasts is similar in cultures at any level of NGF concentration,
as well as in control cultures (see text).
Fig. 9. Mediodorsal neuroblasts at stage 35 in vivo.
Fig. 10. Mediodorsal neuroblasts in control culture.
Fig. 11. Mediodorsal neuroblasts in optimum growth culture.
25
E M B 24
388
p. WEIS
of NGF have 89 ± 1-7 cells per (0-1 mm)2, or a x 1-33 growth, with a 92%
survival. These data are shown in Table 1 and Fig. 6.
The control cultures did not grow significantly from stage 33. The NGFstimulated cultures did grow significantly from stage 33 and from control
cultures (P < 0001) and were not significantly different from stage 35 ganglia.
Table 2. Significance of data
(N.S. = not significant; significance indicated by P value
derived from Student's t test.)
Control
Control
Optimum outgrowth
Stunted
Inhibited
Stage 35
Control
Optimum
Stunted
Inhibited
Stage 35
Control
Optimum
Stunted
Inhibited
Stage 35
Control
Optimum
Stunted
Inhibited
Stage 35
Stage 33
Control
Optimum
outgrowth
Stunted
A. Ventrolateral area—s;ize relationships
—
—
—
N.S.
N.S.
N.S.
—
—
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
—
N.S.
< 0001
< 0001
< 0001
< 0001
B. Ventrolateral area—viability relationships
—
—
—
< 0001
N.S.
outgrowth < 0001
—
—
N.S.
—
N.S.
< 0001
N.S.
N.S.
N.S.
< 0001
N.S.
< 0001
< 0001
< 0001
C. Mediodorsal area—size relationships
—
—
—
N.S.
< 005
—
—
outgrowth < 0005
N.S.
—
< 0001
< 0001
< 0001
< 0001
< 005
N.S.
N.S.
N.S.
< 0001
< 0001
D . Mediodorsal area—viability relationships
—
—
—
< 0001
—
—
outgrowth < 0001
< 0001
< 0001
< 0001
< 0005
—
< 0001
< 0001
< 0-005
N.S.
N.S.
< 0001
< 0001
< 0001
Inhibited
—
—
—
—
< 0001
—
—
—
—
< 0001
—
—
—
—
N.S.
—
—
—
—
< 0001
The optimum-growth cultures were not significantly different from stunted
ganglia and were barely significant (0-05 > P > 0-02) in relation to inhibited
ganglia. There is, however, a trend in relation to dosage (Table 2C).
The viability relationships were more indicative of a dose-dependent response.
By this parameter, the optimum-growth cultures were significantly different
(0-005 > P > 0-001) from the others cultured with NGF. However, a maximum
in viability is reached in the stunted ganglia, which are not significantly different
from the inhibited ganglia (Table 2D).
The M-D neuroblasts which do not survive in the control ganglia exhibit
predominantly the chromatolytic type of degeneration. At all levels of NGF-
In vitro effect of nerve growth factor
389
stimulation, however, there are only two or three chromatolytic cells per
(0-1 mm)2 while the remaining degenerated cells are all condensed and hyperchromatic. As survival increases with increased NGF concentrations, it is the
number of hyperchromatic cells which correspondingly decrease.
DISCUSSION
This investigation confirms the statement that while NGF does not support
the maintenance of growth of V-L neuroblasts, the M-D cells are definitely
affected, and furthermore shows that the concentration of NGF in the culture
medium determines the degree of maintenance and growth of M-D cells. In
fact, the responses at various concentrations provide the opportunity to dissect
the action of NGF into three components. At low concentrations there is an
evocation of neuritic outgrowth, that which Crain et al. (1964) referred to as
the 'Montalcinian halo'. However, mortality is high. At higher concentrations
the outgrowth of neurites is progressively inhibited, but there is improved
maintenance of vitality. If one wants to obtain cell survival comparable to that
in vivo, 50-100 times as much NGF as will evoke maximum outgrowth is
required. However, even then the M-D cells do not survive as well as in vivo,
even though the cultures are performed at a time when the ganglia are most
responsive to NGF (Goldberg, 1963). There is a less significant trend in size
increase paralleling the improved survival, but this trend does not level off.
It was anticipated that the V-L cell would not respond to NGF in vitro. The
original experiments of Bueker (1948) and Levi-Montalcini & Hamburger
(1951), using NGF-producing sarcoma implants, did not affect the embryos
until after 7 days in ovo, by which time the V-L cells are well developed and
functional. Apparently, V-L cells are not affected by NGF even when they are
at the stage of development comparable to the responsive stage of the M-D
cells.
At the time of culture the V-L cells have well-formed axons and dendrites,
while the M-D cells have not yet begun to develop processes. Thus, when outgrowth is seen in vitro, it would represent regeneration of fibers from V-L cells
as contrasted with initial outgrowth from M-D cells. This type of investigation
cannot reveal which cell type is providing the outgrowth, but in cultures of
dissociated ganglia in which the satellite cells have been completely removed
from the neuroblasts (Macri & Bueker, 1969) it can be demonstrated that NGF
stimulates only the M-D cells to develop processes (J. N. Macri, personal
communication). Thus, NGF can evoke the outgrowth of neurites from M-D
cells 3-4 days before the time at which Levi-Montalcini & Levi (1943) showed
that normal outgrowth occurs in vivo.
If a neuron has its processes severed the initial reaction in the perikaryon is
usually one of chromatolysis prior to axon regeneration, and if the cut is too
close to the perikaryon the chromatolysis proceeds to degeneration. While this
25-2
390
p. W E I S
reaction is seen in the V-L area, an equally common reaction is the hyperchromatic condensation. This process normally occurs in ovo in only a few
neuroblasts per ganglion, especially in cervical and thoracic ganglia, but rarely
in brachial and lumbosacral ganglia, unless the peripheral field is removed
by limb-bud extirpation prior to neuritic outgrowth (Hamburger & LeviMontalcini, 1949). Therefore, it could be said that some V-L neuroblasts, in
attempting to regenerate, react to the absence of a peripheral field. For the M-D
area, on the other hand, the progressive decrease of hyperchromatic degenerating cells with increasing NGF concentrations is analogous to replacement of the
peripheral field.
Presumably, when the development of spinal ganglia is studied in vitro, a
substitute must be made for the peripheral field. The substitute must have the
functions of the peripheral field: it must be able to maintain vitality and support
growth of the perikaryon as well as to evoke the outgrowth of neurites. From
the results of this investigation, it would appear that NGF can be a partially
effective substitute for the peripheral field in the development of M-D neuroblasts, since the functions of neurite evocation and of cell maintenance and
growth stimulation occur at a ~ 100-fold difference in concentration, and
therefore cannot function simultaneously in culture.
There has been considerable speculation about the role which NGF may play
as a control mechanism in normal development. Bueker, Schenkein & Bane
(1960) demonstrated its presence in the axial region of the chick embryo during
the time of maximum responsiveness to NGF, and Levi-Montalcini & Angeletti
(1961 a) found weak fluorescent-antibody staining (using antiserum to NGF)
in the sympathetic ganglia, as well as NGF-like activity in homogenates of
sympathetic ganglia. These findings indicate that there may be an intrinsic
mechanism in the nervous system for the evocation of neurites which will
seek out the peripheral field. On the other hand, Bueker (1948) demonstrated
an apparent attraction of nerve fibers into NGF-producing sarcomas,
and Levi-Montalcini & Angeletti (1961 b) found NGF-activity in induced
granuloma tissue. This suggests that mesenchymous tissues might be able to
attract neurites into the developing peripheral field.
Whatever the manner in which NGF plays its role, it is apparent that it is
operative in the developmental control only of sympathetic neurons and the
already partially differentiated mediodorsal sensory neurons. That which serves
in the same role for the ventrolateral sensory cells, as well as parasympathetic
and somatic motor neurons, remains unknown. It is also apparent that the
results of metabolic and cytologic studies of the effect of NGF on chick sensory
ganglia (Levi-Montalcini & Angeletti, 1968) must be considered to be representative of changes within the M-D neuroblasts only.
In vitro effect of nerve growth factor
391
RESUME
Veffet dufacteur de croissance du nerf'm vitro sur les ganglions spinaux
de Vembryon de poulet—une evaluation par la microscopie photonique
L'effet du facteur de croissance du nerf (NGF) sur les ganglions spinaux de l'embryon de
poulet a ete etudie dans un systeme de goutte-pendante, en comparaison avec le developpement parallele //; vivo.
Les neuroblastes ventrolateraux bien differencies, qui, in vivo, auginentent de 1,33 fois en
taille pendant la periode de culture, n'augmente pas de taille, in vitro. 65 a 72% des cellules,
seulement, survivent jusqu'a lafinde la periode de culture, independamment de la concentration en NGF.
Les neuroblastes Mesiodorxaus (M-D) moins differencies qui, in vivo, augmentent de 1,31
fois en taille pendant la periode de culture, croissent de la meme facon in vitro, en presence
d'une concentration suffisante de NGF. Cette quantite est plus elevee que celle qui provoque
la croissance maxima des neurites.
La survie des M-D neuroblastes depend egalement de la concentration en NGF mais
n'attient pas celle qu'on observe in vivo, meme a la concentration la plus elevee. Le type
de degenerescence hyperchromatique qu'empechent des concentrations elevees en NGF est
celle qu'on observe //; vivo quand le champ peripherique est insuffisant.
De ces resultats, et d'autres travaux, il ressort que la reponse au NGF observee in vitro est
due aux M-D neuroblastes seulement, et que toutes les observations biochimiques et cytologiques qui ont ete rapportees, ne concernent, de ce fait, que ces seules cellules.
I am grateful to Dr Issac Schenkein of New York University for his gift of NGF, to my
wife, Dr Judith S. Weis of Rutgers University, for sharing her supply of NGF and for
assistance with this manuscript, and to Mrs Jean Chou of our Data Processing Department
for the data analysis.
This research was supported by institutional General Research Support funds.
REFERENCES
E. D. (1948). Implantation of tumors in the hind limb field of the embryonic chick
and the developmental response of the lumbosacral nervous system. Anat. Rec. 102,
369-389.
BUEKER, E. D., SCHENKEIN, I. & BANE, J. L. (1960). The problem of distribution of a nerve
growth factor specific for spinal and sympathetic ganglia. Cancer Res. 20, 1220-1228.
CRAIN, S. L., BENITEZ, H. & VATTER, A. E. (1964). Some cytologic effects of salivary nervegrowth factor on tissue cultures of peripheral ganglia. Ann. N.Y. Acad. Sci. 118, 206-231.
ERLANDSON, R. A. (1964). A new maraglass, D.E.R. 732, embedment for electron microscopy. J. Cell Biol. 22, 704-709.
GOLDBERG, B. S. (1963). Spinal Ganglion Explains of Six to Seventeen Day Chick Embryos
and their Response to the Nerve Growth Factor. Thesis, New York University.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
HAMBURGER, V. & LEVI-MONTALCINF, R. (1949). Proliferation, differentiation and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions.
/. exp. Zool. Ill, 457-501.
BUEKER,
LEVI-MONTALCINI, Ft. & ANGELETTF, P. U. (1961 a). Growth control of the sympathetic
system by a specific protein factor. Q. Rev. Biol. 36, 99-108.
R. & ANGELETTI, P. U. (19616). Biological properties of a nerve-growth
promoting protein and its antiserum. In Regional Neurochemistry, Proc. 4th Int. Neurochem.
Symp. (ed. S. S. Kety & J. Elkes), pp. 362-376. New York: Pergamon.
LEVi-MoNTALCiNr, R. & ANGELETTI, P. U. (1968). Nerve growth factor. Physiol. Rev. 48,
534-569.
LEVI-MONTALCINL,
392
p. WEIS
R. & BOOKER, B. (1960). Excessive growth of the sympathetic ganglia
evoked by a protein isolated from mouse salivary glands. Proc. natn. Acad. Sci. U.S.A.
46, 373-384.
LEVI-MONTALCINI, R. & COHEN, S. (1956). In vitro and in vivo effects of a nerve growthstimulating agent isolated from snake venom. Proc. natn. Acad. Sci. U.S.A. 42, 695-699.
LEVI-MONTALCINI, R. & HAMBURGER, V. (1951). Selective growth stimulating effects of
mouse sarcoma on the sensory and sympathetic nervous system of the chick embryo.
/. exp. Zool. 116, 321-362.
LEVI-MONTALCINI, R. & LEVI, G. (1943). Recherches quantitatives sur la marche du processus de differenciation des neurones dans les ganglions spinaux de l'embryon de poulet.
Archs Biol., Paris 54, 189-206.
LEVI-MONTALCINI, R., MEYER, H. & HAMBURGER, V. (1954). In vitro experiments on the
effects of mouse sarcomas 180 and 37 on the spinal and sympathetic ganglia of the chick
embryo. Cancer Res. 14, 49-57.
MACRI, J. N. & BUEKER, E. D. (1969). The effect of nerve growth factor on the dispersed
cellular elements of spinal ganglia grown in vitro. Anat. Rec. 163, 223-224.
SCHENKEIN, I., LEVY, M., BUEKER, E. D. & TOKARSKY, E. (1968). Nerve growth factor of
very high yield and specific activity. Science, N.Y. 159, 640-643.
TRUMP, B. F. & BULGER, R. E. (1966). New ultrastructural characteristics of cells fixed in
a glutaraldehyde-osmium tetroxide mixture. Lab. Invest. 15, 368-379.
LEVI-MONTALCINI,
{Manuscript received 29 October 1969)