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Normal development of the lateral motor column in the brachial cord in Rana pipiens.

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Normal Development of the Lateral Motor Column
in the Brachial Cord in Rana pipiens'
EMANUEL D. POLLACK
Department of Zoology, University of lowa, Iowa City, Iowa
ABSTRACT
Counts of differentiating motor cells over the length of the brachial
lateral motor column (LMC) indicate that a large decrease in cell number takes place
during the larval period. During the same period an increase in nuclear size of the
motor cells occurs with a maximum size attained just following forelimb emergence.
Comparison between development of the LMC at the brachial and lumbo-sacral levels
indicates a slight lag in brachial LMC development. Cell number remains greater in
the brachial LMC than in the lumbo-sacral LMC, but nuclear size is consistently less
in the brachial column. Probably no significant change in cell number occurs after
metamorphosis, though there is an increase in cell size.
The normal development of the brachial
lateral motor column (LMC) in Rana pipiens has not yet been described. Such a
study would serve to complete the picture
of normal LMC development. The lumbosacral LMC has been described in terms
of its normal development in R. pipiens by
Beaudoin ('55). He found that an initial
large LMC cell number per section during
the early larval period was followed by a
large decrease in cell number per section
during the mid-larval stages and a much
smaller subsequent decrease by the completion of metamorphosis. That the total
number of cells follows this pattern is implied by Beaudoin's study and has since
been confirmed by Kollros (personal communication) and Pollack (unpublished).
The decrease in cell number was accompanied by an increase in the size of the
remaining cells. Similar patterns of LMC
development have been found in R. temporariu (Race and Terry, '65), Eleutherodactylus ricordii (Hughes, '59), and Xenopus Zaevis (Kollros, '56; Baird, '57; Hughes,
'61; Prestige, '67). Species differences are
apparently not reflected in the developmental patterns of the LMC, but rather in
the number and size of the motor cells
(Race and Terry, '65; Hughes, '68).
In X . Zaevis, the LMC of the lumbosacral cord appears prior to that of the
brachial region (Kollros, '56), and for a
large portion of the larval period maintains a lead over the brachial region in its
level of differentiation. It would be of inANAT. REC.,163: 111-120.
terest to ascertain if this situation also occurs in R. pipiens, contrary to the generally observed cephalo-caudal gradient in
developmental events. Within the central
nervous system of amphibians there are
several instances of the more cephalic
regions undergoing developmental changes
earlier than the more caudal regions. The
degenerative processes by which RohonBeard cells disappear in R. pipiens during
the larval period proceed in a cephalocaudal direction (Suter, '66). The differentiation and thickening of the layers of the
optic lobes in R. pipiens progress from
cephalic to caudal poles (Kollros, '53).
During the development of the mesencephalic V nucleus in R. pipiens, the cells
alter their concentration from the cephalic
portion of the optic tectum to the more
caudal portion (Kollros and McMurray,
'55). The cells of the cephalic half of the
mesencephalic V nucleus are larger than
those of the caudal half at mid-larval
stages, which may indicate earlier differentiation of the more cephalic cells since
there is no difference in cell size between
the two regions by metamorphic climax.
In Amblystma, locomotor response and
reflex activities occur first in the more
cephalic regions of the body (Coghill, '29).
Knowing whether differential development
occurs in two spatially separate but morReceived July 25, '68. Accepted Oct. 4, '68.
1This investigation was supported in part by reseamh grant AM02202 from the National Institute of
Azthritis and Metaboh Diseases to Dr. Jerry J.
Kollros
111
112
EMANUEL D. POLLACK
phologically and functionally similar LMC camera lucida at a magnification of
systems in the spinal cord of R. pipiens is 1250 X. An average of 170 nuclei per aniworthwhile.
mal were drawn and the cross-sectional
This study was undertaken to augment areas were measured using a polar planiinformation on the development of the meter (Keuffel and Esser Model 620015).
LMC in R. pipiens, to compare the developRESULTS
ment of the brachial and lumbo-sacral lateral motor columns, and to present some
Description of the lateral motor column
information as to the post-metamorphic
in the brachial cord at various
condition of the spinal cord.
stages of development
MATERIALS AND METHODS
Three clutches of R. pipiens eggs were
obtained by artificially induced ovulation
and fertilization after the method of Rugh
('34). Each fertilized clutch represented a
completely different parentage. The animals were raised at 18-20°C in aerated
and dechlorinated water with a supply of
spinach continuously available.
Tadpoles were sacrificed at alternate
stages from larval stage I11 through larval
stage XXV, following the stages of Taylor
and Kollros ('46). They were then fixed
in Bouin's fixative; the spinal cord was removed, dehydrated through an alcohol series, cleared in methyl salicylate and embedded in paraffin. Serial sections of 10 IJ
were prepared of the brachial cord and
stained with Ehrlich's acid hematoxylin
and light green. Three animals, one from
each clutch, at each stage were used for
histological study.
Two immature female frogs of 66 mm
and 70 mm lengths were anesthetized and
fixed in a fixation-decalcification solution
of picric acid, formalin and formic acid
after the method of Lillie (Humason, '62).
The further histological preparation of
these spinal cords was the same as that
for the larval animals.
For all animals, the differentiating nerve
cells of the left and right brachial motor
columns were counted along the entire
length of the column. Every fifth section
was counted, considering only those cells
which exhibited a distinct nucleolus and
which were either within the column mass
or nearly so, and oriented obliquely from
the dorso-ventral axis. The counting of
cells was done at a magnification of 430 X.
For the purpose of nuclear measurement all of the differentiating nuclei in
sections selected over the length of the
column were drawn with the aid of a
Stage III. No definitive LMC can be
distinguished. The mantle is expanded laterally beyond levels seen at earlier stages.
Stage V. Large numbers of closely
packed and overlapping nuclei are present
as a lateral projection into the marginal
layer, presaging a lateral motor column
(fig. 2). The karyoplasm is densely supplied with coarse granules (fig. 3).
Stage VII. The LMC is fairly well defined at this stage (fig. 4). It is distinguished from the rest of the mantle layer
by an intervening narrow area of light
staining material. The column is located
opposite the ventral part of the central
canal but does not extend below the ventral border of the canal. The nuclei are
arranged in several overlapping layers and
have a regular orientation, with their long
axes at about 30" from the dorsal-ventral
axis. Large numbers of nuclei exhibit a
nucleolus, and occasional cytoplasmic caps
can be observed at the tapered ends of the
ovoid nuclei. The average length of the
LMC is 1450 p.
Stage IX. The orientation of the nuclei, in which the long axis of the nucleus
is directed dorsolaterad to ventromediad,
persists during the remainder of development. Nucleoli are more prominent and
larger than earlier. Cytoplasmic caps on
the nuclei are prevalent. The average
length of the LMC is 1580 p.
Stage XI. The degree of nuclear overlap is greatly reduced. Cell processes are
numerous and easily seen. The nuclear
granules are reduced in coarseness and
give a homogeneous appearance to the
karyoplasm .
Stage X I I I . The LMC is slightly more
ventral than previously, now being at
about the level of the ventral border of
the central canal (fig. 5). It appears as a
distinct region of the mantle layer. A rim
113
BRACHIAL LATERAL MOTOR COLUMN
of cytoplasm appears around the entire
nucleus, in contrast to earlier stages. Dispersed Nissl substance appears in some
cells (fig. 6). The average length of the
LMC is 1850 p.
Stage XV. The LMC is located more
ventrally, with most of the nuclei situated
below the level of the ventral border of the
central canal. A persisting feature of the
nuclei is the very prominent and large
nucleolus. Distinct Nissl bodies appear
around the nucleus. The average length
of the LMC is 2000 u.
Stage XVII. The nuclei are vacuolated
and surrounded by increased quantities of
cytoplasm (fig. 7). The average length of
the LMC is 2120 u. The nuclei do not significantly alter their appearance during the
remainder of the larval period other than
with respect to size.
Cell number and nuclear size as a n
indication of the development of
t h e lateral motor column in
the brachial cord
Progressive changes in the numbers of
motor cells and the size of their nuclei
over different intervals are indicative of
development in the lateral motor columns.
The cell counts are considered in terms of
the differentiating cells of both brachial
motor columns. In addition, information is
provided as to the cell density in units of
counts per section per column. This permits a comparison with data previously
provided for cell number in the lumbosacral LMC.
A maximum number of motor cells appears in the brachial columns at stage
XI; however, the differences from stages
VII and IX are not great (table 1 ) . There
is then a decrease in cell number from
stage XI to stage XV of approximately
40%. Relatively rapid decreases in cell
number continue until stage XXI. Thereafter, a plateau is established for the remainder of the larval period, and, in fact,
into the late juvenile phase. The overall
loss of motor column cells during the larval period is 62% of the maximum number present. Degenerating motor cells and
cells with pycnotic nuclei were occasionally observed. Though little concern was
given to this aspect, it probably accounts,
at least in part, for cell loss in the LMC.
The greatest density of cells is apparent
at stage VII. The decrease in cell number
per section throughout larval development
and beyond is attributable to both the decreasing total cell number and a simultaneous increase in the length of the spinal
cord. While a significant reduction in density occurs postmetamorphically, there is
virtually no change in total cell number
(table 1) . The reduction in cell number per
section during larval development is 69%.
TABLE 1
Brachial LMC cell number and nuclear area i n comparison with the lumbo-sacral
LMC in Rana pipiens
Stage
VII
IX
XI
XI11
xv
XVII
XIX
XXI
xxm
xxv
Juvenile
Mean no.
of motor
cells per
animal 1
12,403-C 646
12,235f 776
13,158f513
9,8682 277
7,902-C 225
7,4732352
6,373-C 204
5,2532206
5,2962 178
5,0372110
4,788-C 404
Mean cell counts per
column per section 2
LumboBrachial
sacral 3
42.8% 2.2
38.4% 1.1
36.5f1.0
27.0 1.5
20.0e1.0
17.7% 0.6
17.2 0.9
14.8 f0.6
14.2f0.3
13.2f0.2
6.3f0.7
*
*
42.8 f1.0
38.8% 1.1
17.7f 1.0
8.3f0.4
7.62 0.4
6.62 0.4
7.9 f 0.4
8.3* 0.6
6.92 0.4
6.5-C 0.4
Mean nuclear area
Brachial
59.1
56.5
71.7
82.7
98.3
96.4
115.3
118.2
116.0
106.6
142.7
~~~~~~
(p2)
Mean
nucleolar
diameter
(P)
64
61
105
105
125
131
143
167
136
136
1.1
1.5
1.6
2.3
2.3
2.5
2.8
2.8
2.7
2.8
3.1
1Counts are the sums of left plus right sides with standard error of the mean. If the cell counts were
corrected followin the method of Abercrombie ('46) utilizing nucleolar size, they would be seen to have
been overestimate3 maximally by the follounng: stages VII-XI, 10-14% ; stages XIII-XXV, 19-22% ; juvenile, 24%.
2 With standard error of the mean.
3From the study of Beaudoin ('55).
4Average ocular mlcrometer measurements from a single animal at each stage.
114
EMANUEL D. POLLACK
Fig.
during
In addition to cell number, there are
also changes in nuclear size (table 1).
The brachial LMC nuclei show an increase
in mean nuclear area from a low value at
stage IX to a high at stage XXI of 105%.
The relationship which exists between
changes in cell number and changes in
nuclear size during the development of the
brachial LMC is presented in figure 1.
DISCUSSION
The present results demonstrate the pattern of normal development of the brachial
LMC in R. pipiens. A decrease in the number of motor cells occurs during the larval
period while the nuclear size increases. The
reciprocity between cell number and nuclear size in the brachial LMC is in accord
with the findings of Beaudoin ('55) for
the lumbo-sacral LMC of R. pipiens. Several differences do occur, however. Beaudoin ('55) demonstrated discernible motor
cells in the lumbo-sacral LMC at stage V
which were of greater density per section
than at stage VII, whereas the cells of the
brachial LMC at stage V showed little indication of differentiation. Reynolds ('63),
on the other hand, observed a differentiated lumbo-sacral LMC at stage I V t .
The greatest rate of cell loss for the brachial columns occurs from stage XI to stage
XV, whereas at the lumbo-sacral level the
greatest decline per section was recorded
from stage IX to stage XI11 (table 1). This
change in cell number per section is compounded of the reduction attributable to
lengthening of the LMC portion of the
spinal cord and that attributable to a decrease in total cell number (e.g., 46% increase and 40% decrease, respectively,
over the stage VII-XVII interval, or an
overall change in count of 59% per section). Hughes ('61) also reports that
growth of X. Zaevis during the larval period produces elongation of the lumbosacral ventral horn section of the spinal
cord.
The reported trends in cell number and
nuclear size for the lateral motor columns
of R. pipiens are similar to the observations made in X. Zaevis (Kollros, '56;
Hughes, '61; Prestige, '67), E. ricordii
(Hughes, '59), and R . temporaria (Race
and Terry, ' 6 5 ) . The presence of a greater
number of motor cells in the brachial
LMC than in the lumbo-sacral LMC at the
end of metamorphosis has been reported
for X. Zaevis (Kollros, '56) on the basis of
counts per section only. In addition, E . ricordii exhibits a similar relationship despite the lack of a larval period (Hughes,
'59). This situation is found to persist in
R. pipiens larvae and, indeed, into the mature frog as reported by Silver ('42).
The results which are reported in this
study demonstrate a decrease in cell number by the end of metamorphosis of 62%
of the maximum number present at early
larval stages. The increase in nuclear area
is 105% from a low at stage VII to a maximum at stage XXI, and an overall increase
by the completion of metamorphosis at
BRACHIAL LATERAL MOTOR COLUMN
stage XXV of 90%. Beaudoin ('55) observed an increase of over 100% in nuclear size in the lumbo-sacral LMC. Race
and Terry ('65) reported a loss in cell
number in the lumbo-sacral LMC of R.
temporaria of 80% by the end of metamorphosis, and an increase in nuclear size
of 52%. The lumbo-sacral LMC in X . Zuevis exhibits a decrease in cell number of
50% during metamorphosis according to
Baird ('57). Hughes ('61) and Prestige
('67) report this loss in X. Zaevis to be
closer to 70%. There is an accompanying
increase in nuclear size of 64% at the
completion of metamorphosis, and of as
much as 90% late in metamorphic climax
(Baird, '57). Other anurans which have
been reported to demonstrate similar decreases in cell number in the lumbo-sacral
ventral horn are Hyla punctatissima with
a loss of about 84% (Hughes, '63), Bufo
marinus which has a cell loss of about
55% (Hughes, '68), and E. martinicensis
which loses about 67% of its ventral horn
cell number by metamorphosis (Hughes,
'68).
Degenerating cells were observed only
on occasion, which may indicate that in
accordance with the findings of Beaudoin
('55) and Race ('61) degeneration may
not play a very great role in the reduction
of cell number in R. pipiens. Decker and
Kollros ('69) have shown that tadpoles of
R. pipiens raised in the cold retain a greater number of cells in the lumbo-sacral
LMC for each particular stage. Thus cells
normally destined to degenerate, regress,
or perhaps even undergo a reverse migration, do not do so in phase with limb
development. The role of cell degeneration
in normal ontogeny, which might account
for cell loss during LMC development, has
been discussed extensively by Glucksmann
('55), and more recently by Prestige ('67)
and Hughes ('68).
While some of the cells of the LMC have
been differentiating, they have also been
growing, attaining a maximum nuclear
size after forelimb emergence. Although
the decrease in cell number in the brachial
LMC of R. pipiens does not continue after
metamorphosis, it has been shown that
the nuclei continue to increase in size in
an apparent response to increasing limb
size. That the progressive changes in the
115
development of the brachial LMC tend to
lag slightly behind the lumbo-sacral LMC
in respect to alteration in cell number has
been demonstrated. What relationship
might exist between the extent of limb development in the two regions of the spinal
cord and the differentiation levels attained
by their respective lateral motor columns
is not yet known.
ACKNOWLEDGMENT
The author is grateful to Dr. Jerry J.
Kollros for his critical reading of the manuscript and valuable comments.
LITERATURE CITED
Abercrombie, M. 1946 Estimation of nuclear
population from microtome sections. Anat.
Rec., 94: 239-247.
Baird, J. J. 1957 Normal development of motor nerve cells in the lumbo-sacral cord of anurans and development following partial limb
ablation and subsequent regeneration. Ph.D.
dissertation, State University of Iowa.
Beaudoin, A. R. 1955 T h e development of lateral motor column cells in the lumbo-sacral
cord in Ranu pipiens. I. Normal development
and development following unilateral limb ablation. Anat. Rec., 121: 81-96.
Coghill, G. 1929 Anatomy and the Problem of
Behaviour. The University Press, Cambridge.
Decker, R., and J. Kollros 1969 The effect of
cold on hind limb growth and lateral motor
column development in R u m pipiens. J. Embryol. Exp. Morph., in press.
Gliicksmann, A. 1951 Cell deaths in normal
vertebrate ontogeny. Biol. Rev., 26: 59-86.
Hughes, A. 1959 Studies in embryonic and
larval development of amphibia. I. The embryology of EZeuthero.odactyZus ricmdii with special
reference to the spinal cord. J. Embryol. Exp.
Momh., 7: 22-58.
1961 Cell degeneration in the larval
ventral horn of Xenupus laevis (Daudin). J .
Embryol. Exp. Morph., 9: 269-284.
1963 On the labelling of larval neurones by melanin of ovarian origin in certain
Anura. J. Anat. Lond., 97: 217-224.
1968 Aspects of Neural Ontogeny.
Logos Press Ltd., London.
Humason, G. L. 1962 Animal Tissue Techniques. W. H. Freeman & Co., San Francisco,
p. 28.
Kollros, J. J. 1953 The development of the
optic lobes in the frog. I. The effects of unilateral enucleation in embryonic stages. J. Exp.
ZOO^., 123: 153-183.
1956 The further development of the
spinal cord, ganglia and nerves. In: Chapter
VI, Division VIII of Normal Table of Xenopus
Zuevis (Daudin). P. D. Nieuwkoop and J. Faber,
eds. North-Holland Publishing Co., Amsterdam,
pp. 63-73.
-
116
EMANUEL D. POLLACK
Kollros, J. J., and V. M. McMurray 1955 The
mesencephalic V nucleus in anurans. I. Normal development in Ranu pipiens. J. Comp.
Neur., 102: 47-63.
Prestige, M. C. 1967 The control of cell number in the lumbar ventral horns during the
development of Xenopus Zaevis tadpoles. J.
Embryol. Exp. Morph., 18: 359-387.
Race, J., Jr., and R. J. Terry 1965 Further
studies on the development of the lateral motor
column in anuran larvae. I. Normal development in Rana temporaria. Anat. Rec., 152: 99106.
Reynolds, W.A. 1963 The effects of thyroxine
upon the initial formation of the lateral motor
column and differentiation of motor neurons in
Rana pipiens. J. Exp. Zool., 153: 237-249.
Rugh, R. 1934 Induced ovulation and artificial
fertilization in the frog. Biol. Bull., 66: 22-24.
Silver, M. L. 1942 The motoneurons of the
spinal cord of the frog. J. Comp. Neur., 77:
1-39.
Suter, J. H. B. 1966 Numbers and distribution
of Rohon-Beard cells in selected larval stages
of Rana pipiens. M.S. Thesis, University of
Iowa.
Taylor, A. C., and J. J. Kollros 1946 Stages in
the normal development of Rana pipiens larvae. Anat. Rec., 94: 7-24.
PLATE
-
m
I-
PLATE 1
Stage VII. The LMC is fairly well defined, with nucleoli becoming
readily visible. The LMC is clearly delimited from the rest of the
mantle layer by a region of light staining material. x 375.
Stage XIII. The LMC appears more ventral than before, and the
degree of nuclear overlap is greatly reduced. X 72.
Stage XIII. High power view demonstrating increased cytoplasm
around the nuclei and the presence of dark staining Nissl substance. x 375.
4
5
6
7 Stage XVII. The nuclei rarely overlap, are more rounded, and have
large nucleoli. x 375.
Stage V. High power view showing the heavily granulated nuclei
in the early stages of LMC differentiation. x 375.
3
2 Stage V. The LMC is in early stages of formation, with cells accumulating as a prospective ventrolateral projection. The arrows indicate the dorsal and ventral limits of the LMC. x 100.
ial spinal cord, showing the lateral motor column (LMC) of normal
Rana pipiens.
The following figures represent typical cross sections through the brach-
EXPLANATION OF FIGURES
BRACHIAL LATERAL MOTOR COLUMN
Emanuel D. Pollack
PLATE 1
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