/. Embryol. exp. Morph. Vol. 71, pp. 83-95, 1982
Printed in Great Britain © Company of Biologists Limited 1982
Neuronotrophic effects of
skeletal muscle fractions on spinal cord
differentiation
By L. HSU, 1 D. NATYZAK 1 AND G. L. TRUPIN 2
From the Department of Anatomy, New Jersey School of
Osteopathic Medicine and Department of Anatomy,
Rutgers Medical School, Piscataway
SUMMARY
Soluble fractions of homogenized skeletal muscle were found to promote neuronal
migration and neuritic and glial outgrowth from embryonic chick spinal cord explants.
Fractions obtained from skeletal muscle immobilized by prolonged treatment with curare
were significantly more effective than normal muscle in accelerating neuronal and glial
development. Fractions from other tissues such as brain and lung did not enhance neuronal
differentiation, but were effective in stimulating outgrowth of glial cells. Separate measurements of glial and neuronal responses indicate that tissue fractions produce independent
effects on the glial and neuronal components.
INTRODUCTION
Numerous studies support the concept that peripheral target tissues exert
a regulatory and trophic effect upon central neurons. It has been demonstrated
that surgical removal of chick embryo limb buds produces hypoplasia of
ventral horn neurons (Hamburger & Keefe, 1944). Conversely, implantation
of supernumerary limbs reduces natural cell death and enhances survival of
motorneurons (Hollyday & Hamburger, 1976). In vitro studies have shown
that limb explants (Pollack, 1980) and target tissues such as cardiac and
skeletal muscle (Giller et al 1977; Nishi & Berg, 1977; Ebendal, 1981) are
capable of eliciting neuritic outgrowth and synapse formation in cocultured
ganglionic or spinal cord cultures. Conditioned media from the same tissues
are also effective in promoting neuronal differentiation (Collins, 1978; Helfand,
Riopelle & Wessels, 1978; Bennett, Lai & Nurcombe, 1980; Dribin & Barrett,
1980; Obata & Tanaka, 1980; Coughlin, Bloom & Black, 1981; Henderson,
1
Authors' address: Department of Anatomy, New Jersey School of Osteopathic Medicine,
P.O. Box 55, Piscataway, New Jersey 08854, U.S.A.
2
Author's address: Department of Anatomy, Rutgers Medical School, Piscataway, New
Jersey 08854, U.S.A.
83
84
L. HSU, D. NATYZAK AND G. L. TRUPIN
Hachet & Changeux, 1981), suggesting that this trophic regulation is mediated
by diffusible factors. Potential neuronotrophic1 factors (Varon & Bunge, 1978)
have also been identified in homogenized extracts of various tissues and organs.
Trophic factors that promote neuronal differentiation have been found in
fractions of cardiac and skeletal muscle (McLennan & Hendry, 1978; Lindsay
& Tarbit, 1979; Smith & Appel, 1981) brain and gut (Jacobson, Ebendal,
Hedlund & Norrgren, 1980; Riopelle & Cameron, 1981). Biochemical characterization of factors from these different sources is incomplete (Ebendal,
Belew, Jacobson & Porath, 1979; Bonyhady, Hendry, Hill & McLennan,
1980; Coughlin et al. 1981).
The present study has focused on the trophic effects of homogenized skeletal
muscle fractions on differentiation of spinal cord cultures. We have used a
semiquantitative assay system to evaluate the morphological differentiation
of both glial and neuronal components of embryonic chick spinal cord. To
assess the trophic influence of skeletal muscle with impaired functional activity,
neuronal cultures were also grown in soluble fractions from muscle treated
with curare. Our results demonstrate that normal muscle fractions enhance
neuritic and glial outgrowth. Soluble fractions from curarized muscle are
significantly more effective than normal muscle in accelerating neuronal and
glial development. Fractions from other tissues, such as brain and lung were
effective only in stimulating outgrowth of non-neuronal cells.
MATERIALS AND METHODS
Tissue culture
Spinal cords of 8-day chick embryos were dissected under aseptic conditions
and stripped of meninges. The ventral halves of cord segments from thoracic
to lumbar levels were cut into pieces approximately 0-5 mm3, explanted onto
collagen-coated coverslips and fed one to two drops of growth medium. These
lying-drop preparations were sealed in Maximow chambers and incubated at
37 °C. Cultures were refed every 1-2 days for a period of 4-8 days.
Curare treatment
10-day chick embryos were injected with 0-5 ml of 10 mg/ml sterile d-tubocurarine chloride (Sigma) in Ringer's chick saline (Hall, 1975). The solution
was injected through a pinhole made in the shell and shell membrane above
the airspace. After 5-6 days, the curarized embryos were sacrificed and used
for the preparation of muscle homogenates as described below. Curarized
embryos were slightly smaller than normal embryos of the same age and their
limbs often appeared fixed and stiff at the joints. Samples of skeletal muscle
from normal and curarized embryos were assayed for acetylcholinesterase by
1
Neuronotrophic factors are trophic factors from neural or non-neural tissues which
are directed towards neurons and supporting cells.
Neuronotrophic effects of skeletal muscle fractions
85
the colorimetric method of Ellman, Courtney, Andres & Featherstone (1961).
As in previous studies (Hall, 1975; Oppenheim, Pittman, Gray & Maderut,
1978), curarized muscle showed reduced levels (about 80% of normal muscle)
of acetylcholinesterase activity.
Preparation of tissue fractions
Breast and/or thigh muscles from 15- to 16-day normal or curarized chick
embryos were dissected, cleared of grossly visible tendinous and neuronal
components, and minced to a fine pulp. Approximately 0-5 gm of muscle were
homogenized in 5 ml of Basal Medium Eagle's for 2-3 min at 4 °C with a
teflon-coated tissue homogenizer. The homogenate was centrifuged at 20000 g
and at 105000 g, each time for 60 min. Particulate components floating at the
surface were discarded. The resultant supernatant fraction was sterilized through
a 0-2 /im Sterilet (Amicon) filter and stored at - 1 0 °C. Supernatant fractions
of brain and lung of 15- to 16-day embryos were also prepared as described
above. Total protein concentration of the supernatant fractions was determined
by the dye-binding assay of Bradford (1976) with bovine plasma albumin
(BioRad) as the standard.
Composition of growth media
Spinal cord explants were maintained in experimental and control media
as indicated in Table 1. Two controls were used to provide both a baseline
standard (Group A) and a maximal growth standard (Group D). Group D
cultures were grown in a medium enriched with large quantities of serum and
embryo extracts (GIBCO) that was reported to promote extensive differentiation
of spinal cord explants (Fisher & Federoff, 1977). Group A control cultures
were grown in a medium containing moderate proportions of serum and
embryo extract formulated to ensure healthy but not maximal neuronal
differentiation and glial development (Federoff & Hall, 1979). Experimental
Groups B and C were grown in the same defined medium as Group A supplemented with supernatant fractions of normal or curarized muscle. In groups B
and C, the amount of serum and chick embryo extracts was reduced so that
the total protein content approximated that of the baseline standard (see
Table 1). This standardization of protein content ensured that observed
differences in neuronal development were not due solely to generalized nutrient
effects of the protein components.
To establish that observed differences in neuronal development were specific
responses to skeletal muscle components, cultures grown in muscle fractions
were compared with cultures grown in tissue fractions from lung and brain
(Group E).
5%
0
5%
32%
0
Basal medium eagle's
Basal medium eagle's
1%
* All media contained 50 /*g/ml gentamicin.
Medium 199
with 600 mg % glucose
Basal medium eagle's
1%
5%
Basal medium eagle's
Control group A
(baseline control)
Group B
(normal muscle)
Group C
(curarized muscle)
Control group D
(enriched control)
Group E
2%
10%
Defined medium*
Chick
embryo
extract
Culture groups
Heatinactivated
horse serum
Table 1. Composition of growth media
1900
2000
2000
3200 ±100
0
100% normal muscle
100% lung
100% brain
2300 ±30
2200 ±140
2300 ±40
Total
protein content
(/*g/ml)
Curarized muscle
Normal muscle
0
Homogenized
tissue fraction
oo
z
a
p
•H
Z
I—y
N
H
•
HSU
Neuronotrophic effects of skeletal muscle fractions
87
Table 2. Rating system for quantifying growth of spinal cord explants
Growth parameters
Score
Neuronal migration number of neurons
beyond original
explant
0
None
1
1-10 neurons
2
11-20 neurons
3
21 or more neurons
Neuritic outgrowth - number
of fibres, outgrowth beyond
original explant
Glial outgrowth outgrowth beyond
original explant
No fibres
Fibres sparse, outgrowth = £
explant diameter
Fibres moderately dense, outgrowth = explant diameter
Fibres abundant, outgrowth
= 2 or more times explant
diameter
No glial outgrowth
Glial outgrowth =
explant diameter
Glial outgrowth =
explant diameter
Glial outgrowth =
2 or more times
explant diameter
Rating of growth in spinal cord explants
After 4 or 8 days, cultures were fixed in 10% neutral buffered formalin and
stained with silver (Holmes, 1943). Explants were examined with the light
microscope and rated for three growth parameters to provide a measure of
neuronal and glial development (Table 2). For each growth parameter, cultures
were assigned a numerical score of 0-3. The frequency distribution of these
scores served as an index of the degree of growth and differentiation achieved
by different culture groups. Frequency distribution of scores was subjected
to the Chi-square test. The overall significance of difference in the distribution
of scores between groups for each growth parameter was tested. Each group
was checked against other control or experimental groups (e.g. Group A
against B, C, D, respectively) in order to compare and evaluate the growth
profiles and developmental trends produced by the different media.
RESULTS
Group A cultures - baseline control medium (Table 3)
The morphological differentiation of ventral horn cultures grown in baseline
control medium becomes evident by 4 days in vitro (DIV), and shows further
progression by 8 DIV. Soon after attachment to the collagen substrate, nonneuronal cells begin to spread out from the core of the explant, followed by
an outgrowth of short neuritic fibres and multipolar neurons (Fig. 1). Outward
neuronal migration in Group A cultures is relatively slow; at 4 DIV, most
cultures (54%) remain dense with no visible neurons beyond the explant
(score of 0), while at 8 DIV, 31 % of the cultures still remain unspread. In
contrast, neuritic outgrowth displays a sharp increase between 4 and 8 days,
L. HSU, D. NATYZAK AND G. L. TRUPIN
Neuronotrophic effects of skeletal muscle fractions
89
with a much higher frequency of cultures having scores of 3 (1 % at 4 DIV
versus 24 % at 8 DIV). Glial outgrowth shows a similar shift towards higher
scores between 4 and 8 DIV.
Group B cultures - normal muscle fractions {Table 3)
As compared with control Group A, Group B cultures show a marked
acceleration of early glial outgrowth (Fig. 2), a moderate enhancement of
neuritic outgrowth, but no appreciable increase in neuronal migration. Twentytwo percent of the 4-day cultures in Group B display a maximal glial outgrowth (score of 3), while only 1 % of 4-day cultures in Group A show such
development (P < 001). This early spurt of glial outgrowth in Group B does
not continue, however, and there is relatively little change by 8 DIV.
Comparison of neuritic outgrowth reveals almost identical scores for Group
A and B at 4 DIV. By 8 DIV however, 73 % of Group B cultures have long fibre
outgrowths (scores of 2, 3) while only 61 % of Group A baseline control
cultures have comparable growth (P < 005). This enhancement of neuritic
outgrowth produced by normal muscle fractions in Group B media was in
fact equal to the dense outgrowth produced in Group D cultures grown in
enriched media (29 % of scores of 3 in Group B versus 32 % in Group D).
Group B displays a moderate increase in neuronal migration between 4-8
days, but this is not significantly different from trie parallel increase in neuronal
migration occurring in Group A control cultures during the same period.
Group C cultures - curarized muscle fractions (Table 3)
When compared with Groups A and B, Group C cultures showed significant
advancement in both neuronal and glial development by 4 DIV. Within
Group C, only 28 % of the 4-day cultures showed an absence of neuronal
migration (score of 0). This is indicative of a rapid initiation of neuronal
Fig. 1. Spinal cord explant grown in baseline control medium (Group A) after
4 DIV. A few multipolar neurons (arrows) and short neuritic fibres appear at the
edge of the explant (E). Neuronal elements overlie a background of small glial
cells, x 500. Scale bar = 20 /mi.
Fig. 2. Spinal cord culture grown in normal muscle fraction (Group B) after 4 DIV.
The culture shows a marked acceleration of glial outgrowth. A few neurons have
migrated out from the explant which was located at lower left, x 120. Scale
bar = 83 /im.
Fig. 3. Spinal cord explant grown in curarized muscle fraction (Group C) after
8 DIV. A large number of neurons and an extensive neuritic network can be
seen. x25O. Scale bar = 18/mi.
Fig. 4. Spinal cord explant grown in 100% normal muscle fraction (Group E) after
5 DIV. Two neurons are clearly visible (arrows) and other darkly stained neurons
can be seen emerging from the explant (at top). The culture shows extensive
neuritic outgrowth, x 550. Scale bar = 18/tm.
\j VV 111
A
B
C
D
A
B
C
D
Neuritic
outgrowth
Glial
outgrowth
A
B
C
D
Neuronal
migration
parameter
\J1
54
41
28
31
34
27
15
12
0
0
1
3
0
30
46
43
36
31
36
19
24
43
15
9
41
1
13
10
18
12
34
38
53
53
56
63
55
52
2
3
3
11
21
1
2
13
11
1
22
35
4
3
31
25
22
17
12
6
5
5
2
1
1
2
0
46
49
29
47
27
21
5
19
17
15
11
17
1
13
11
18
13
37
44
22
44
64
50
53
64
2
10
16
31
23
24
29
68
32
17
34
35
17
3
% Distribution of cultures grouped
by scores after 8DIVt
* Number of cultures/group: A = 118; B = 185; C = 110; D = 115.
t Number of cultures/group: A = 186; B = 218; C = 88; D == 180.
(baseline control)
(normal muscle)
(curarized muscle)
(enriched control)
Culture groups
% Distribution of cultures grouped
by sscores after 4 DIV*
Table 3. Effect of soluble muscle fractions on differentiation of spinal cord cultures
d
p
p
CM
H
•z
p
d
X
o
Neuronotrophic effects of skeletal muscle fractions
91
Table 4. Effect 0/100% soluble tissue fractions on differentiation of
spinal cord cultures after 5 DIV
% Distribution of culture
grouped by scores
parameter
Group E* cultures
0
1
2
3
Neuronal
migration
100%
100%
100%
100%
100%
100%
100%
100%
100%
45
95
84
30
74
73
1
0
0
38
5
16
37
22
13
31
33
43
11
0
0
27
4
14
51
47
51
6
0
0
6
0
0
17
20
6
Neuritic
outgrowth
Glial
outgrowth
normal muscle
brain
lung
muscle
brain
lung
muscle
brain
lung
* Number of cultures/group: normal muscle = 84; brain = 58>; lung == 37.
migration, since all other 4-day control and experimental groups have a higher
percentage of cultures showing no sign of migration (score of 0). This rapid
neuronal migration is not significantly different from the extensive neuronal
migration in 4-day Group D cultures maintained in enriched medium. By
8 DIV, neuronal migration in Group C (Fig. 3) actually surpasses that of
Group D cultures (P < 001).
With respect to neuritic outgrowth, Group C cultures are not significantly
different from Group D cultures at 4 DIV, but again surpass Group D cultures
by 8 DIV (68 % versus 32% of cultures with scores of 3; P < 0-01).
Glial development in both experimental groups that contain muscle fractions
(Groups B and C) is more advanced than that of baseline and maximal growth
controls (Groups A and D). Between 4 and 8 days, Groups B and C undergo
relatively little change, but continue to show more extensive glial outgrowth
than Groups A and D. Curarized muscle fractions are more effective than
normal muscle fractions in eliciting glial outgrowth at 4 DIV (35% versus
22% with scores of 3; P < 0 05). In control Groups A and D, only 1 and 4%,
respectively, achieved the same scores for glial outgrowth.
Group D cultures - enriched control medium {Table 3)
Group D medium, enriched with large amounts of serum proteins and
embryo extracts, stimulated extensive neuronal differentiation of ventral horn
cultures. Cultures differentiate rapidly and show marked neuronal migration
and neuritic outgrowths by 4 DIV. Twenty-one percent of the cultures have
over 20+ neurons (score of 3), and the majority of cultures show long, dense
neuritic fibres. Glial development is more limited, with only 4 % of the cultures
92
L. HSU, D. NATYZAK AND G. L. TRUPIN
having maximal outgrowth (score of 3). After 8 DIV, moderate increases in
both fibre and glial development are noted, but neuronal migration remains
unchanged. The primary differences between control Groups A and D are
the more extensive neuronal migration and neuritic outgrowth produced by
the enriched Group D medium (P < 001). There is no significant difference
in glial outgrowth in these two control groups.
Group E cultures - lung, brain and normal muscle fractions {Table 4)
Cultures grown in 100% normal muscle fractions produce significantly
higher levels of neuronal migration and neuritic outgrowth (P < 0 0 1 ; Fig. 4)
than cultures maintained in 100% brain or lung fractions. After 5 DIV, 9 5 %
of cultures grown in lung fractions remain dense with no neuronal migration
beyond the core of the explant. In contrast, only 45 % of the cultures grown
in 100% normal muscle fractions show an absence of neuronal migration
(score of 0), and a combined 17% of these latter cultures display at least
11-20+ neurons (scores of 2, 3). Neuritic outgrowth from cultures grown in
100% muscle fractions is moderate, but superior to neuritic outgrowths in
cultures grown in brain or lung fractions (P < 001). By 5 DIV, brain, lung
and muscle fractions all stimulate a moderate glial extension, with no significant
difference in glial response to the three tissue fractions. These cultures showed
no further neuronal and glial differentiation after 5 DIV.
DISCUSSION
The results of this study indicate that soluble fractions from skeletal muscle
tissue promote growth and differentiation of embryonic chick spinal cord
explants. The migration of multipolar neurons from the core of the explant
and the outgrowth of both neuritic fibres and underlying glial cells are enhanced
by fractions of curarized muscle and to a lesser extent by normal muscle
fractions. This neuronotrophic effect cannot be attributed to a general nutritional
response to soluble tissue proteins since fractions of lung or brain did little to
promote neuronal migration or neuritic extension. These observations are in
agreement with reports that conditioned medium from skeletal muscle cultures
promotes neuritic outgrowth (Dribin & Barrett, 1980; Obata & Tanaka, 1980;
Henderson et al. 1981) and enhanced motorneuron survival (Bennett et al.
1980). Similar studies on effects of brain extracts have yielded somewhat
conflicting results. Tissue extracts from brain and heart have been reported
to promote neuritic outgrowth from sensory (Lindsay & Tarbit, 1979) and
ciliary ganglia (Jacobson et al. 1980). Our study indicates, however, that soluble
brain fractions do not significantly affect neuronal migration or fibre outgrowth
in spinal cord explants. These latter results are in agreement with a recent
survey which showed that soluble brain extracts had a minimal effect on neurite
outgrowth in dissociated spinal cord cultures (Riopelle & Cameron, 1981).
Neuronotrophic effects of skeletal muscle fractions
93
Studies on target tissue regulation of spinal cord development have usually
focused on neuronal differentiation, with little emphasis on accompanying glial
development. In our study, we separately evaluated both glial and neuronal
responses to potential trophic factors. Our results suggest that muscle fractions
produce independent effects on the glial and neuronal components. This view
is supported by two observations: 1) both eurarized and normal muscle fractions
stimulate early glial development, but only curarized muscle fractions promote
extensive neuronal migration; 2) tissue fractions from brain, lung and muscle
produce equivalent glial development, but only muscle fractions elicit a significant neuronal response. These observations are consistent with reports
that enhancement of neuritic outgrowth by muscle conditioned medium was
not blocked when glial proliferation was inhibited by 5-fluoro-2'-deoxyuridine,
(Dribin & Barrett, 1980). Furthermore, Obata & Tanaka (1980) observed
reduced glial outgrowth in spinal cord explants at a time when neuritic outgrowth was enhanced by muscle conditioned medium.
Neuronotrophic factors may affect differentiation by promoting substrate
interactions during neuronal migration and axonal elongation (Varon &
Bunge, 1978; Adler & Varon, 1981). Of the many cell types surveyed, only
nerve, glial and skeletal muscle cells have been shown to produce significant
amounts of substrate attachment material (Schubert, 1977). Similar attachment
substances are also found within medium conditioned by cardiac muscle, and
have been shown to be critical in contact guidance and control of directionality
of neuritic growth (Collins, 1978; Collins & Garrett, 1980). Homogenized
muscle fractions may also contain substances that facilitate neuronal and glial
attachment or act to stimulate proliferation of glial cells (Hanson & Partlow,
1978). We are presently investigating whether skeletal muscle fractions may
also have mitogenic effects.
Muscle fractions from embryos treated with curare are significantly more
effective than normal muscle fractions in promoting neuronal and glial development. Curarized muscle fractions were also more effective in inducing differentiation than an enriched medium specifically designed to promote maximal
neuronal development (Fisher & Federoff, 1977). These observations are consistent with recent reports that embryos immobilized with botulinum toxin or
curare showed a distinct increase in motorneuron survival during periods of
normal cell death (Pittman & Oppenheim, 1978). Blocking physiological activity
at the neuromuscular synapse with these drugs produced a delay in motorneuron degeneration, although the mechanism by which peripheral muscle
prevented death of central neurons was not identified. Our results, utilizing
fractions of homogenized muscle, suggest that an actual component within
curarized muscle acts to promote and sustain neuronal differentiation. It remains
to be determined whether curarized muscle fractions contain factors specific to
curarized tissues or have an altered level of a neuronotrophic component
found in normal muscle. Preliminary biochemical analysis of both normal
and curarized muscle fractions is now in progress.
4
EMB 71
94
L. HSU, D. NATYZAK AND G. L. T R U P I N
This investigation was supported by Grant No. RRO9O85-O3 from the NIH, No. 81-06-006
from the National Osteopathic Foundation to L.H. and Grant No. HD-13315-01 from the
NIH to G.L.T.
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{Received 12 October 1981, revised 1 March 1982)
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