Normal development of the lateral motor column in the brachial cord in Rana pipiens.
код для вставкиСкачатьNormal 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pattern is implied by Beaudoin's study and has since been confirmed by Kollros (personal communication) and Pollack (unpublished).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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