Neuronal Death in the Development of Normal and Hyperplastic Spinal Ganglia HAROLD D.BIBB Department of Zoology, University of Rhode Island, Kingston, Rhode Island 02881
ABSTRACT The increase of the peripheral area available for innervation by the ninth spinal ganglion of larval Rana berlandieri was accomplished by unilaterally removing at stage V two (numbers 8 and 10) of the three (numbers 8, 9, and 10) ganglia that normally provide the bulk of the sensory innervation to the hindlimb. Peripheral increase significantly enhanced nerve cell number, and the change in neuronal number with developmental stage was also significant. The increase in nerve cell numbers in affected ganglia of 86%over control values a t stage XVI compares favorably with the results of workers using different techniques. The effect of peripheral increase on normally occurring neuronal degeneration during the development of the hyperplastic condition through early and middle larval stages was determined. While the data show a trend toward the presence of fewer degenerating neurons in peripherally overloaded than in control ganglia through stage X, the differences in the numbers of dying nerve cells were not significant. This fact and other considerations suggest that a mechanism other than or in addition to a reduction in the level of neuronal degeneration must be operative to account for the increase in the number of neurons present in ninth ganglia with expanded peripheries. Results are discussed in the context of the temporal pattern of the proliferation of cells that differentiate as neurons, the establishment of central-periphera1 connections, and neuronal degeneration. Since the time t h a t degenerating nerve cells were first described in anuran (Barbieri, '05) and chick (Collin, '06) embryos, naturally occurring neuronal degenerations of the histogenetic type (Glucksmann, '51) have been observed during the development of a variety of neural centers (reviews by Hughes, '68; J a cobson, '70; Prestige, '70; Cowan, '73). This widespread occurrence of neuronal death during the development of the nervous system and reports of greater than normal levels of nerve cell degeneration in neural centers whose peripheral fields have been reduced (Hamburger and Levi-Montalcini, '49; Hamburger, '58; Prestige, '67, '76; Kelly and Cowan, '72; Sohal, '76) have led to a general acceptance of the idea that neuronal death regulates nerve cell numbers in response to peripheral demands by eliminating those nerve cells whose fibers fail to establish proper peripheral connections (Hamburger and Levi-Montalcini, '49; Prestige, '67, '76; Cowan, '73; Price, '74; Landmesser and Pilar, J. EXP. ZOOL. (1978) 206: 65-72.
'74; Hamburger, '58, '75; Pollack and Kollros, '75). Furthermore, not only has the possibility been recognized that changes in the level of neuronal death are involved in the hypoplasia of neural centers following peripheral reduction, but also that reductions in the level of neuronal degeneration, and therefore the survival of nerve cells that would normally die, may account for or contribute to the hyperplasia of affected neural centers following peripheral increase (Cowan, '73; Hamburger, '75; Bibb, '77). While the effect of peripheral reduction on neuronal degeneration has been well documented, the effect of peripheral increase on this process has received far less attention. In the only previously published full report on this topic, Hollyday and Hamburger ('76) presented indirect evidence that enlargement of the periphery by grafting a supernumerary limb bud resulted in a reduction of neuronal
' Supported in part by Grant-in-aid 9800-8025 L from the University of Rhode Island Research Committee. 65
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HAROLD D. BIBB
degeneration in the lateral motor column of chick embryos. As has been discussed elsewhere relative to proliferative activity, the chick lateral motor column has been found to respond differently to peripheral change than do such neural centers as chick and anuran dorsal root ganglia (Cowan, '73; Hollyday and Hamburger, '76; Bibb, '77). The extent to which the effect of peripheral increase on neuronal death in the chick lateral motor column may be generalized to other neural systems is therefore open to question. In a recent study of the response of the ninth spinal ganglion of the anuran R a n a pipiens to peripheral increase, it was found that nerve cell numbers in ganglia whose peripheries had been expanded during early larval development increased over control values during early and middle larval stages, and that this difference in nerve cell numbers was maintained throughout the period studied (Bibb, '77). Although proliferative activity in affected ganglia was greater than that in controls, the possibility of a reduction in the level of neuronal degeneration could not be ruled out. Because the hyperplastic condition was seen to develop during early and middle larval stages, were a reduction in neuronal death to act as a mechanism responsible for or contributing to the greater than normal numbers of nerve cells present in ganglia with increased peripheries, this mechanism would have to operate during this period. This study has been carried out to determine whether neuronal degeneration normally occurs in the ninth ganglion during early and middle larval stages, and if so, to test whether the level of neuronal degeneration is altered in response to peripheral increase. MATERIALS A N D METHODS
Because of increasing limitations on the availability of R a n a pipiens (Bagnara and Frost, '771, a congeneric species, R . berlandieri was used in this investigation. Adult R. berlandieri collected in northern Mexico were purchased from a commercial supplier. Larvae were reared from eggs obtained by means of induced ovulation and fertilization in the laboratory following Rugh's ('34) procedures for R . pipiens. Larvae were maintained a t 19"21°C in aerated pond water with spinach continuously available. Hyperplasia of ninth spinal ganglia was induced by the unilateral removal of ganglia 8 and 10 as described earlier (Bibb, '70, '77). Be-
cause the eighth, ninth, and tenth spinal ganglia normally provide the bulk of the sensory innervation to the hindlimb (Taylor, '441, the unilateral removal of ganglia 8 and 10 effectively increases the size of the peripheral field available for innervation by the remaining ninth ganglion. The ninth ganglion on the operated side gives an hyperplastic response, while t h e unaffected contralateral ninth ganglion may be used as a control (Bibb, '77). Although R a n a berlandieri develops more slowly than does R . pipiens a t the temperatures used in this investigation, the pattern of limb development was found to be quite similar to that of R. pipiens; and the Taylor and Kollros ('46)staging system for R . pipiens larvae was used throughout the study. All ganglionic extirpations were carried out with the aid of a dissecting microscope on rapidly developing stage V larvae. Larvae were anesthetized in a 0.1%solution of ethyl m-aminobenzoate methanesulfonate (Eastman) in preparation for the operation, and ganglia were removed while larvae were immersed in half-strength Holtfreter's solution. Following the operation, larvae were allowed to develop until they reached desired developmental stages, a t which time they were terminally anesthetized in 0.1%ethyl maminobenzoate methanesulfonate. The lumbosacral portion of the spinal cord with its associated ganglia was dissected from the animals, checked with the aid of a dissecting microscope for the completeness of ganglionic removal, and fixed for three days in alcoholic Bouin-Dubosco fixative (Humason, '72). The tissue was then dehydrated in a graded series of alcohols, cleared in methyl salicylate, and embedded in paraffin. Serial sections were cut a t 10 pm, mounted on slides and stained with Delafield's hematoxylin and eosin. Larvae were fixed a t stages V , VI, VIII, IX, X, XII, and XVI. At each stage three larvae of similar size were selected for counts of viable and degenerating neurons in hyperplastic and control ninth spinal ganglia. Counts of viable neurons were made a t a magnification of 1,000 diameters. Estimates of the number of viable neurons present in control and hyperplastic ninth ganglia were obtained by first counting, in every fifth section, all nerve cell nuclei that contained a t least one nucleolus. The sum of these counts for a ganglion was then multiplied by five in order to arrive a t the estimates for the total number of nerve cells present. Because only
+
NEURONAL DEATH IN SPINAL GANGLIA
nuclei containing a nucleolus were counted, no corrections of the Abercrombie ('46) type for duplicate scoring were made (Prestige, '76). Since counts were made in the same manner in control and hyperplastic ganglia, and the determination of the levels of hyperplasia is based on the comparison of the numbers of neurons present in ninth spinal ganglia on operated and control sides of the same larvae under the same histological conditions, differences between the absolute number of nerve cells present and the number of nerve cells counted should be of minimal importance. However, for an estimate of the maximum levels of overestimation of cell number based on counts of nucleolus containing nuclei in material of this type, see Pollock ('69). Degenerating nerve cells were identified on the basis of nuclear morphology using the criteria described by Hughes ('611, and complete counts of degenerating neuronal nuclei were made in control and hyperplastic ninth ganglia a t a magnification of 1,000diameters. Portions of the same degenerating nucleus may be present in more than one section. However, because the number of degenerating nerve cell nuclei per section was small, the position of each degenerating nucleus in a section could be noted and compared with the positions of those in neighboring sections in order to avoid double counts. Results obtained through the use of this procedure in repeated counts of selected ganglia were in agreement. Data on the number of viable and degenerating nerve cells were evaluated for the effects of peripheral increase, developmental stage, and the interaction between peripheral increase and developmental stage using the two factor analysis of variance with a probability level of 0.05 (Ostle and Mensing, '75). Data on the number of degenerating neurons a t each stage were also evaluated using the paired-sample t test, again at a probability level of 0.05 (Zar, '74). RESULTS
Nerve cell number After showing progressive increases through the early stages studied, the number of nerve cells present in ninth ganglia on both the control and operated sides reaches a peak at stage XI1 (fig. 1).The periods of greatest increase in neuronal number are between stages VIII and IX when there are mean net additions of 250 nerve cells to ninth ganglia on the control side and of 1,100 nerve cells on the
67
operated side, and between stages X and XI1 when there is an average net increase of 900 nerve cells in control ganglia and 1,400 nerve cells in ganglia with increased peripheries. Following the peak at, stage XII, there is a net loss of some 1,900 neurons from ninth ganglia on both the operated and control sides by the time that stage XVI is reached. This loss represents a mean net reduction from stage XI1 values of 43% in control ninth ganglia and of 28% in ganglia with increased peripheries. The response of ninth ganglia to the increased periphery is evident as early as stage VIII when the mean numbers of nerve cells present in control and hyperplastic ganglia differ by some 700 neurons. This difference in nerve cell number between ninth ganglia on t h e operated and control sides increases through stage XI1 when there are approximately 2,200 more nerve cells present in hyperplastic than in control ganglia. This level of difference in neuronal numbers in ninth ganglia on the two sides is maintained through the period of net reduction in nerve cell numbers, and ninth ganglia on the operated side contain 86% more neurons than those on the control side at stage XVI. Analysis of variance shows that the difference in nerve cell number in ganglia with normal and increased peripheries is significant. The change in neuronal number with developmental stage was also found to be significant, while the interaction between stage and peripheral increase narrowly failed t o show significance. Degenerations
Degenerating neurons and mitotic figures were found in both control and hyperplastic ninth ganglia at each of the stages studied. The stages and morphology of degenerating neurons in the ninth ganglia of R a m berlandieri were observed to resemble closely those described for the lateral motor column (Hughes, '61) of Xenopus laevis. During the early stages of degeneration the nuclear envelope appears to remain intact but shows an accumulation of condensed chromatin along its inner surface (figs. 2, 3). As the degenerative process proceeds there is a continuing accretion of material organized in dropletlike configurations just inside the nuclear envelope (figs. 3,4). These droplets increase in number and move into the cytoplasm as the integrity of the nucleus is lost (figs. 5, 6). On both the operated and control sides of
68
HAROLD D. BInn
7000
6000
5000
4000
3000
2000
I
I
,
Vlll
IX
x
xv I
XI1
STAGE Fig. 1 Developmental changes in mean nerve cell number in ninth ganglia with normal (filled circles) and experimentally increased (open circles) peripheries. The height of the bars indicates one standard error on either side of the mean. TABLE I
Mean numbers of degenerating neurons in ganglia with normal and increased peripheries (& one standard error) Larval atage
Normal periphery Increased periphery
V+
VI
VIII
IX
1.720.9
9.7k0.3
0.3t0.3
4.0t0.6 25.025.5 25.3k9.6
X
XI1
XVI
29.728.4 30.0k11.2 18.322.7 43.0212.3 19.3k4.8 13.0C1.5 50.5225.7 34.7k9.0
NEURONAL DEATH IN SPINAL GANGLIA
69
Figs. 2-6 The progression of nuclear degeneration in dying ninth spinal ganglion neurons. Arrowheads indicate degenerating nuclei. Photographed a t X 1,000. 2 In a relatively early stage of degeneration t h e nucleus shows the presence of basophilic droplets adjacent to t h e inner surface of the nuclear envelope. 3 , 4 The basophilic droplets increase in size and number as degeneration proceeds. 5 The nuclear envelope is lost. 6 Basophilic droplets disperse into cytoplasm. Viable neurons are routinely present in close proximity to those that are dying, and the presence of mitotic figures (m) near degenerating neurons is not unusual
stage V + larvae (5 days after operation a t stage V) ninth ganglia contained very few degenerating neurons, and in some cases none were present (table 1).The number of degenerating nerve cells in both control and hyperplastic ganglia increased markedly following stage VI. The mean number of degenerating neurons was slightly but consistently larger in control than in hyperplastic ganglia through stage X, and the absolute difference in the mean number of degenerating neurons in ninth ganglia on the two sides remained nearly constant during this period. At stages XI1 and XVI, however, the mean number of degenerating nerve cells was greater in hyperplastic than in control ganglia. Although the differences in the number of degenerating neurons between control and hyperplastic ganglia in individual larvae were all in the same direction a t stages VI, X, and
XVI, differences in both directions were found a t stages VIII, IX, and XII. Differences in the mean number of degenerations on the two sides were small at most of the stages studied, and while the change in number of degenerating neurons with developmental stage was found t o be significant using the analysis of variance, differences in the numbers of degenerating neurons in control and hyperplastic ganglia, and the interaction between developmental stage and peripheral increase were not found t o be significant. Tests of the differences in the number of neuronal degenerations in control and hyperplastic ganglia from larvae of the same stage using the paired-sample t test showed significance only on the data from stage VI larvae. DISCUSSION
As has been discussed elsewhere, the level of
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hyperplasia resulting from the use of the technique employed in this study compares favorably with results obtained by earlier workers who increased the size of the peripheral area available for innervation by grafting supernumerary limbs or limb rudiments to larvae or embryos. The increase in nerve cell number of 86% over control values at stage XVI in ganglia with enlarged peripheries also shows a close correspondence t o the 88%hyperplasia of the ninth spinal ganglion of stage XX R a n a pipiens larvae in which the periphery had been expanded in the same manner as in this investigation (Bibb, '77). The change in nerve cell numbers in the ninth ganglion with developmental stage in Rana berlandieri exhibits the same general pattern of increase and subsequent decrease that is apparent in the eighth, ninth, and tenth spinal ganglia in Xenopus laevis larvae (Prestige, '65) and in the ninth spinal ganglion of larval R . pipiens (Bibb, '77). The period of net nerve cell loss is of greater duration in R . berlandieri, like R . pipiens, than in X. laevis; and the proportion of nerve cells remaining in the ninth ganglion toward the end of the larval period relative to the maximum number present is less in R . berlandieri than in X. laeuis. While the period of net nerve cell loss in the ninth ganglion of R. berlandieri is similar to that of the ninth ganglion (Bibb, '77) and the brachial and lumbosacral motor columns of R. pipiens (Beaudoin, '55; Pollack, '691, differences do exist. Although both R. pipiens and R . berlandieri show peak numbers of neurons in the ninth ganglion during middle larval stages, this peak occurs slightly later in R. berlandieri (stage XII) than in R. pipiens (stage XI. Neuronal numbers are also smaller through most of the larval period in the ninth ganglion of R . berlandieri than those at the same stages in R. pipiens. In R . pipiens larvae, however, net neuronal loss in ninth ganglia with normal peripheries slows considerably between stages XIV and XX, and nerve cell numbers nearly reach a plateau during this period. Hence the similarity between the number of nerve cells present in ninth ganglia with normal peripheries in stage XX R. pipiens larvae and the number present in like ganglia in stage XVI R. berlandieri larvae may indicate that ninth ganglia with normal peripheries contain approximately the same number of neurons a t the end of the larval period in both species. The sequence of morphological changes in
degenerating nuclei of dying neurons is common to neuronal degeneration in a variety of species. While some species differences do exist (Hughes, '611, the general pattern of events described for R a n a berlandieri is strikingly similar to that in the larval ventral horn of Xenopus laevis (Hughes, '61) and R . pipiens (Decker, '76) and in the cervical and lumbosacral motor columns of chick embryos (O'Connor and Wyttenbach, '74). The presence of relatively small numbers of degenerating neurons in ganglion 9 during early larval stages and a subsequent increase in the number of dying nerve cells are in general agreement with Prestige's ('65) findings for ganglia 8 through 10 of larval Xenopus laeuis. However, the data presented here indicate that the increase in the number of degenerating nerve cells may begin somewhat earlier in R a n a berlandieri than in X . laeuis. Also in agreement with Prestige's observations is the presence of degenerating neurons in the ninth ganglion during the period of net nerve cell increase. It is apparent that in the ninth ganglion of R. berlandieri, as is true for X. laeuis, a neuronal turnover system is in operation. The possible significance of such turnover systems has been discussed extensively elsewhere (Prestige, '67, '70; Hughes, '61, '68; Pollack and Kollros, '75). Results of the present study clearly demonstrate that neuronal degeneration occurs throughout the development of the hyperplastic condition in ninth spinal ganglia whose peripheries have been increased. Whether there is a depression in normally occurring neuronal death that is sufficient to account for the increased numbers of nerve cells present in ganglia with expanded peripheries becomes the issue of central importance. Statistical analysis of the data indicates that the overall differences in the number of degenerating neurons in normal and hyperplastic ganglia are not significant. Yet, the trend toward the presence of fewer degenerating neurons in ganglia with expanded peripheries than in those with normal peripheries from stage V + through stage X remains suggestive of the possibility that suppression of normally occurring neuronal loss may contribute to the presence of increased numbers of nerve cells in peripherally overloaded ganglia. In this regard it is perhaps important to emphasize that the mean difference in the number of degenerating neurons in control and hyperplastic ganglia remains nearly con-
NEURONAL DEATH IN SPINAL GANGLIA
stant between stages VI and X. This suggests that the increase in nerve cell numbers resulting from the survival of neurons that would die in the absence of an expanded periphery should also be constant. However, in the 21day period between stages VI and VIII the increase over control values is a maximum of 700 neurons in hyperplastic ganglia, while in the 10-day period between stages VIII and X an additional increase of some 1,100 nerve cells over control values takes place so that at stage X hyperplastic ganglia contain an average of 1,800 more nerve cells than do control ganglia. Therefore the rate of increase in nerve cell number over control values in hyperplastic ganglia is not constant. This observation suggests that the apparent, though not statistically significant, depression in the level of neuronal death does not by itself account for the increased number of nerve cells present in ganglia with expanded peripheries. Thus even if a reduction in the level of neuronal death makes some contribution t o the increased number of nerve cells present in ganglia with expanded peripheries, the action of some additional mechanism would be required to explain fully the development of the hyperplastic condition observed in the present investigation. Results from a variety of studies of the effects of peripheral change on associated neural centers have been consistent with the view that information regarding the periphery is made available to the neural center by way of neural connections between center and periphery, and this view has been forwarded either directly or implicitly in many discussions of central-peripheral interactions (Hamburger, '39, '75; Hamburger and Levi-Montalcini, '49; Kollros, '53; Prestige, '67, '70; Pollack and Kollros, '75; Bibb, '77). It follows that a neural center may be able to respond to peripheral increase only after neural connections between center and periphery are made; and therefore the nature of the response may be related to those mechanisms operative in the neural center during and after the time that information regarding the periphery is made available. In a recent investigation of the effects of peripheral increase on the lateral motor column of chick embryos, Hollyday and Hamburger ('76) found that they could account for the excess numbers of neurons in motor columns with enlarged peripheries solely on the basis of a reduction in normally occurring neuronal
71
death. It is of interest that neurotization of the chick leg bud occurs just as proliferation for the lateral motor column is ending and before normally occurring neuronal degeneration begins on a large scale (Hamburger, '75; Hollyday and Hamburger, '76). Therefore it is not surprising that the chick lateral motor column responds to peripheral increase by a reduction in neuronal degeneration. It appears, however, that a different set of circumstances exists for the ninth spinal ganglion of Rana berlandieri. Nerve fibers are present in the limb bud of R. pipiens larvae as early as stage 11, and by stage IV rudiments of the main mixed trunks of the thigh and the shank are present (Taylor, '43). Although information of this type is not available for R . berlandieri, there is no reason to believe that this temporal pattern would be radically different in the two species. It is of interest in this regard that nerve fibers have been reported to be present at a similarly early stage in the limb bud of larval Xenopus Zaevis (Lamb, '74). Furthermore, unlike the chick lateral motor column, net neuronal numbers in the ninth ganglion of R. berlandieri continue to increase well beyond stages at which nerve fibers would be expected t o reach the limb bud, and mitotic figures were present in the ganglion throughout the period studied. Thus while proliferation for the chick lateral motor column may end before it can be affected by peripheral change, it is unlikely that this is the case for the ninth ganglion of R. berlandieri. In neural centers such as chick dorsal root ganglia (Hamburger and Levi-Montalcini, '49), the optic tectum (Kollros, '53; Currie and Cowan, '74) and the ninth spinal ganglion of R. pipiens (Bibb, '77) proliferation continues beyond the stages a t which peripheral contacts are made, and in these systems peripheral changes do affect proliferative activity. Thus the differentiation as neurons of some of the products of increased proliferation could also contribute to the hyperplasia observed in the study reported here. In emphasizing the exclusion of a role for increased proliferation in the response of the lateral motor column to peripheral enlargement, Hollyday and Hamburger ('76) suggested that the term "hyperplasia" be replaced by "hypothanasia." While this usage may be accurate for the chick lateral motor column, results of the present investigation indicate that adoption of this terminology
72
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would be premature in describing the response of the ninth ganglion of larval Rana berlandieri to peripheral expansion. ACKNOWLEDGMENTS
The author is indebted to Professors Jerry J. Kollros and Emanuel D. Pollack for their critical reading of the manuscript, to Professor Robert F. Costantino for assistance with the statistical analyses, to Miss Bernadette Szumilo for technical aid, and to Mr. Eric D. Scott for the photographic work. LITERATURE CITED Abercrombie, M. 1946 Estimation of nuclear population from microtome sections. Anat. Rec., 94: 239-242. Bagnara, J. T., and J. S. Frost 1977 Leopard frog supply. Science, 197: 106-107. Barbieri, C. 1905 Ricerche sul sviluppo del midollo spinale negli Anfibi. Arch. 2001. (ital.) Napoli, 2: 79-106. Beaudoin, A. R. 1955 The development of lateral motor column cells in the lumbo-sacral cord in Rana pipiens. I. Normal development and development following unilateral limb ablation. Anat. Rec., 121: 81-96. Bibb, H. D. 1970 Ganglionic hypertrophy in Rana pipiens larvae. Anat. Rec., 166: 279. 1977 The production of ganglionic hypertrophy in Rana pipiens larvae. J. Exp. Zool., 200: 265-276. Collin, R. 1906 Recherches cytologiques sur le developpement de la cellule nerveuse. Le Nevraxe, 8: 181-307. Cowan, W. M. 1973 Neuronal death 88 a regulative mechanism in the control of cell number in t he nervous system. In: Development and Aging in t he Nervous System. Academic Press, New York. pp. 19-41. Currie, J., and W. M. Cowan 1974 Some observations on t he early development of the optic tectum in t he frog (Rana pipiensl, with special reference to t he effects of early eye removal on mitotic activity in t he larval tectum. J. Comp. Neur., 156: 123-142. Decker, R. S. 1976 Influence of thyroid hormones on neuronal death and differentiation in larval Rana pipiens. Develop. Biol., 49: 101-118. Glucksmann, A. 1951 Cell deaths in normal vertebrate ontogeny. Biol. Rev., 26: 59-86. Hamburger, V. 1939 Motor and sensory hyperplasia following limb bud transplantations in chick embryos. Physiol. Zool., 12: 268-284. 1958 Regression versus peripheral control of differentiation in motor hypoplasia. Am. J. Anat., 102: 365-410. 1975 Cell death in the development of t he lateral motor column of the chick embryo. J. Comp. Neur., 160: 535-546. Hamburger, V., and R. Levi-Montalcini 1949 Proliferation, differentiation and degeneration in t he spinal ganglia of the chick embryo under normal and experimental conditions. J. Exp. Zool., 111: 457-502. Hollyday, M., and V. Hamburger 1976 Reduction of t he
naturally occurring motor neuron loss by enlargement of the periphery. J. Comp. Neur., 170: 311-320. Hughes, A. 1961 Cell degeneration in the larval ventral horn of Xenopus laeuis (Daudin). J. Embryol. Exp. Morph., 9: 269-284. 1968 Aspects of Neural Ontogeny. Logos Press Ltd., London. Humason, G. L. 1972 Animal Tissue Techniques. Third ed. W. H. Freeman and Company, San Francisco. Jacobson, M. 1970 Developmental Neurobiology. Holt, Rinehart and Winston, New York. Kelly, J. P., and W. M. Cowan 1972 Studies on the development of the chick optic tectum. 111. Effects on early eye removal. Brain Res., 42: 263-288. Kollros, J. J. 1953 The development of the optic lobes in the frog. I. The effects of unilateral enucleation in embryonic stages. J. Exp. Zool., 123: 153-187. Lamb, A. H. 1974 The timing of the earliest motor innervation of the hind limb bud in thexenopus tadpole. Brain Res., 67: 527-530. Landmesser, L., and G. Pilar 1974 Synaptic transmission and cell death during normal ganglionic development. J. Physiol., 241: 737-749. OConnor, T. M., and C. R. Wyttenbach 1974 Cell death in the embryonic chick spinal cord. J. Cell Biol., 60: 448-459. Ostle, B., and R. W. Mensing 1975 Statistics in Research. Third ed. Iowa State University Press, Ames, Iowa. Pollack, E. D. 1969 Normal development of the lateral motor column in the brachial cord in Rana pipiens. Anat. Rec., 163: 111-120. Pollack, E. D., and J. J. Kollros 1975 dell migration into the “established lateral motor column in Rana pipiens larvae. 11. Lumbosacral spinal cord and comparative aspects. J. Exp. Zool., 192: 299-306. Prestige, M. C. 1965 Cell turnover in the spinal ganglia of Xenopus laeuis tadpoles. J. Embryol. Exp. Morph., 13: 63-72. 1967 The control of cell number in the lumbar spinal ganglia during the development of Xenopus laeuis tadpoles. J. Embryol. Exp. Morph., 17: 453-471. 1970 Differentiation, degeneration and the role of the periphery: quantitative considerations. In: The Neurosciences: Second Study Program. Rockefeller University Press, New York, pp. 73-82. 1976 Evidence tha t a t least some of the motor nerve cells tha t die during development have first made peripheral connections. J. Comp. Neur., 170: 123-134. Price, D. L. 1974 The influence of the periphery on spinal motor neurons. N. Y. Acad. Sci., 228: 355-363. Rugh, R. 1934 Induced ovulation and artificial fertilization in the frog. Biol. Bull., 66: 22-24. Sohal, G. S. 1976 An experimental study of cell death in the developing trochlear nucleus. Exp. Neur., 51; 684-698. Taylor, A. C. 1943 Development of the innervation pattern in the limb bud of the frog. Anat. Rec., 87: 379-413. 1944 Selectivity of nerve fibers from the dorsal and ventral roots in the development of the frog limb. J. Exp. Zool., 96: 159-185. Taylor, A. C., and J. J. Kollros 1946 Stages in the normal development ofRana pipiens larvae. Anat. Rec., 94: 7-24. Zar, J. H. 1974 Biostatistical Analysis. Prentice-Hall, Inc. Englewood Cliffs, New Jersey.
ABSTRACT The increase of the peripheral area available for innervation by the ninth spinal ganglion of larval Rana berlandieri was accomplished by unilaterally removing at stage V two (numbers 8 and 10) of the three (numbers 8, 9, and 10) ganglia that normally provide the bulk of the sensory innervation to the hindlimb. Peripheral increase significantly enhanced nerve cell number, and the change in neuronal number with developmental stage was also significant. The increase in nerve cell numbers in affected ganglia of 86%over control values a t stage XVI compares favorably with the results of workers using different techniques. The effect of peripheral increase on normally occurring neuronal degeneration during the development of the hyperplastic condition through early and middle larval stages was determined. While the data show a trend toward the presence of fewer degenerating neurons in peripherally overloaded than in control ganglia through stage X, the differences in the numbers of dying nerve cells were not significant. This fact and other considerations suggest that a mechanism other than or in addition to a reduction in the level of neuronal degeneration must be operative to account for the increase in the number of neurons present in ninth ganglia with expanded peripheries. Results are discussed in the context of the temporal pattern of the proliferation of cells that differentiate as neurons, the establishment of central-periphera1 connections, and neuronal degeneration. Since the time t h a t degenerating nerve cells were first described in anuran (Barbieri, '05) and chick (Collin, '06) embryos, naturally occurring neuronal degenerations of the histogenetic type (Glucksmann, '51) have been observed during the development of a variety of neural centers (reviews by Hughes, '68; J a cobson, '70; Prestige, '70; Cowan, '73). This widespread occurrence of neuronal death during the development of the nervous system and reports of greater than normal levels of nerve cell degeneration in neural centers whose peripheral fields have been reduced (Hamburger and Levi-Montalcini, '49; Hamburger, '58; Prestige, '67, '76; Kelly and Cowan, '72; Sohal, '76) have led to a general acceptance of the idea that neuronal death regulates nerve cell numbers in response to peripheral demands by eliminating those nerve cells whose fibers fail to establish proper peripheral connections (Hamburger and Levi-Montalcini, '49; Prestige, '67, '76; Cowan, '73; Price, '74; Landmesser and Pilar, J. EXP. ZOOL. (1978) 206: 65-72.
'74; Hamburger, '58, '75; Pollack and Kollros, '75). Furthermore, not only has the possibility been recognized that changes in the level of neuronal death are involved in the hypoplasia of neural centers following peripheral reduction, but also that reductions in the level of neuronal degeneration, and therefore the survival of nerve cells that would normally die, may account for or contribute to the hyperplasia of affected neural centers following peripheral increase (Cowan, '73; Hamburger, '75; Bibb, '77). While the effect of peripheral reduction on neuronal degeneration has been well documented, the effect of peripheral increase on this process has received far less attention. In the only previously published full report on this topic, Hollyday and Hamburger ('76) presented indirect evidence that enlargement of the periphery by grafting a supernumerary limb bud resulted in a reduction of neuronal
' Supported in part by Grant-in-aid 9800-8025 L from the University of Rhode Island Research Committee. 65
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degeneration in the lateral motor column of chick embryos. As has been discussed elsewhere relative to proliferative activity, the chick lateral motor column has been found to respond differently to peripheral change than do such neural centers as chick and anuran dorsal root ganglia (Cowan, '73; Hollyday and Hamburger, '76; Bibb, '77). The extent to which the effect of peripheral increase on neuronal death in the chick lateral motor column may be generalized to other neural systems is therefore open to question. In a recent study of the response of the ninth spinal ganglion of the anuran R a n a pipiens to peripheral increase, it was found that nerve cell numbers in ganglia whose peripheries had been expanded during early larval development increased over control values during early and middle larval stages, and that this difference in nerve cell numbers was maintained throughout the period studied (Bibb, '77). Although proliferative activity in affected ganglia was greater than that in controls, the possibility of a reduction in the level of neuronal degeneration could not be ruled out. Because the hyperplastic condition was seen to develop during early and middle larval stages, were a reduction in neuronal death to act as a mechanism responsible for or contributing to the greater than normal numbers of nerve cells present in ganglia with increased peripheries, this mechanism would have to operate during this period. This study has been carried out to determine whether neuronal degeneration normally occurs in the ninth ganglion during early and middle larval stages, and if so, to test whether the level of neuronal degeneration is altered in response to peripheral increase. MATERIALS A N D METHODS
Because of increasing limitations on the availability of R a n a pipiens (Bagnara and Frost, '771, a congeneric species, R . berlandieri was used in this investigation. Adult R. berlandieri collected in northern Mexico were purchased from a commercial supplier. Larvae were reared from eggs obtained by means of induced ovulation and fertilization in the laboratory following Rugh's ('34) procedures for R . pipiens. Larvae were maintained a t 19"21°C in aerated pond water with spinach continuously available. Hyperplasia of ninth spinal ganglia was induced by the unilateral removal of ganglia 8 and 10 as described earlier (Bibb, '70, '77). Be-
cause the eighth, ninth, and tenth spinal ganglia normally provide the bulk of the sensory innervation to the hindlimb (Taylor, '441, the unilateral removal of ganglia 8 and 10 effectively increases the size of the peripheral field available for innervation by the remaining ninth ganglion. The ninth ganglion on the operated side gives an hyperplastic response, while t h e unaffected contralateral ninth ganglion may be used as a control (Bibb, '77). Although R a n a berlandieri develops more slowly than does R . pipiens a t the temperatures used in this investigation, the pattern of limb development was found to be quite similar to that of R. pipiens; and the Taylor and Kollros ('46)staging system for R . pipiens larvae was used throughout the study. All ganglionic extirpations were carried out with the aid of a dissecting microscope on rapidly developing stage V larvae. Larvae were anesthetized in a 0.1%solution of ethyl m-aminobenzoate methanesulfonate (Eastman) in preparation for the operation, and ganglia were removed while larvae were immersed in half-strength Holtfreter's solution. Following the operation, larvae were allowed to develop until they reached desired developmental stages, a t which time they were terminally anesthetized in 0.1%ethyl maminobenzoate methanesulfonate. The lumbosacral portion of the spinal cord with its associated ganglia was dissected from the animals, checked with the aid of a dissecting microscope for the completeness of ganglionic removal, and fixed for three days in alcoholic Bouin-Dubosco fixative (Humason, '72). The tissue was then dehydrated in a graded series of alcohols, cleared in methyl salicylate, and embedded in paraffin. Serial sections were cut a t 10 pm, mounted on slides and stained with Delafield's hematoxylin and eosin. Larvae were fixed a t stages V , VI, VIII, IX, X, XII, and XVI. At each stage three larvae of similar size were selected for counts of viable and degenerating neurons in hyperplastic and control ninth spinal ganglia. Counts of viable neurons were made a t a magnification of 1,000 diameters. Estimates of the number of viable neurons present in control and hyperplastic ninth ganglia were obtained by first counting, in every fifth section, all nerve cell nuclei that contained a t least one nucleolus. The sum of these counts for a ganglion was then multiplied by five in order to arrive a t the estimates for the total number of nerve cells present. Because only
+
NEURONAL DEATH IN SPINAL GANGLIA
nuclei containing a nucleolus were counted, no corrections of the Abercrombie ('46) type for duplicate scoring were made (Prestige, '76). Since counts were made in the same manner in control and hyperplastic ganglia, and the determination of the levels of hyperplasia is based on the comparison of the numbers of neurons present in ninth spinal ganglia on operated and control sides of the same larvae under the same histological conditions, differences between the absolute number of nerve cells present and the number of nerve cells counted should be of minimal importance. However, for an estimate of the maximum levels of overestimation of cell number based on counts of nucleolus containing nuclei in material of this type, see Pollock ('69). Degenerating nerve cells were identified on the basis of nuclear morphology using the criteria described by Hughes ('611, and complete counts of degenerating neuronal nuclei were made in control and hyperplastic ninth ganglia a t a magnification of 1,000diameters. Portions of the same degenerating nucleus may be present in more than one section. However, because the number of degenerating nerve cell nuclei per section was small, the position of each degenerating nucleus in a section could be noted and compared with the positions of those in neighboring sections in order to avoid double counts. Results obtained through the use of this procedure in repeated counts of selected ganglia were in agreement. Data on the number of viable and degenerating nerve cells were evaluated for the effects of peripheral increase, developmental stage, and the interaction between peripheral increase and developmental stage using the two factor analysis of variance with a probability level of 0.05 (Ostle and Mensing, '75). Data on the number of degenerating neurons a t each stage were also evaluated using the paired-sample t test, again at a probability level of 0.05 (Zar, '74). RESULTS
Nerve cell number After showing progressive increases through the early stages studied, the number of nerve cells present in ninth ganglia on both the control and operated sides reaches a peak at stage XI1 (fig. 1).The periods of greatest increase in neuronal number are between stages VIII and IX when there are mean net additions of 250 nerve cells to ninth ganglia on the control side and of 1,100 nerve cells on the
67
operated side, and between stages X and XI1 when there is an average net increase of 900 nerve cells in control ganglia and 1,400 nerve cells in ganglia with increased peripheries. Following the peak at, stage XII, there is a net loss of some 1,900 neurons from ninth ganglia on both the operated and control sides by the time that stage XVI is reached. This loss represents a mean net reduction from stage XI1 values of 43% in control ninth ganglia and of 28% in ganglia with increased peripheries. The response of ninth ganglia to the increased periphery is evident as early as stage VIII when the mean numbers of nerve cells present in control and hyperplastic ganglia differ by some 700 neurons. This difference in nerve cell number between ninth ganglia on t h e operated and control sides increases through stage XI1 when there are approximately 2,200 more nerve cells present in hyperplastic than in control ganglia. This level of difference in neuronal numbers in ninth ganglia on the two sides is maintained through the period of net reduction in nerve cell numbers, and ninth ganglia on the operated side contain 86% more neurons than those on the control side at stage XVI. Analysis of variance shows that the difference in nerve cell number in ganglia with normal and increased peripheries is significant. The change in neuronal number with developmental stage was also found to be significant, while the interaction between stage and peripheral increase narrowly failed t o show significance. Degenerations
Degenerating neurons and mitotic figures were found in both control and hyperplastic ninth ganglia at each of the stages studied. The stages and morphology of degenerating neurons in the ninth ganglia of R a m berlandieri were observed to resemble closely those described for the lateral motor column (Hughes, '61) of Xenopus laevis. During the early stages of degeneration the nuclear envelope appears to remain intact but shows an accumulation of condensed chromatin along its inner surface (figs. 2, 3). As the degenerative process proceeds there is a continuing accretion of material organized in dropletlike configurations just inside the nuclear envelope (figs. 3,4). These droplets increase in number and move into the cytoplasm as the integrity of the nucleus is lost (figs. 5, 6). On both the operated and control sides of
68
HAROLD D. BInn
7000
6000
5000
4000
3000
2000
I
I
,
Vlll
IX
x
xv I
XI1
STAGE Fig. 1 Developmental changes in mean nerve cell number in ninth ganglia with normal (filled circles) and experimentally increased (open circles) peripheries. The height of the bars indicates one standard error on either side of the mean. TABLE I
Mean numbers of degenerating neurons in ganglia with normal and increased peripheries (& one standard error) Larval atage
Normal periphery Increased periphery
V+
VI
VIII
IX
1.720.9
9.7k0.3
0.3t0.3
4.0t0.6 25.025.5 25.3k9.6
X
XI1
XVI
29.728.4 30.0k11.2 18.322.7 43.0212.3 19.3k4.8 13.0C1.5 50.5225.7 34.7k9.0
NEURONAL DEATH IN SPINAL GANGLIA
69
Figs. 2-6 The progression of nuclear degeneration in dying ninth spinal ganglion neurons. Arrowheads indicate degenerating nuclei. Photographed a t X 1,000. 2 In a relatively early stage of degeneration t h e nucleus shows the presence of basophilic droplets adjacent to t h e inner surface of the nuclear envelope. 3 , 4 The basophilic droplets increase in size and number as degeneration proceeds. 5 The nuclear envelope is lost. 6 Basophilic droplets disperse into cytoplasm. Viable neurons are routinely present in close proximity to those that are dying, and the presence of mitotic figures (m) near degenerating neurons is not unusual
stage V + larvae (5 days after operation a t stage V) ninth ganglia contained very few degenerating neurons, and in some cases none were present (table 1).The number of degenerating nerve cells in both control and hyperplastic ganglia increased markedly following stage VI. The mean number of degenerating neurons was slightly but consistently larger in control than in hyperplastic ganglia through stage X, and the absolute difference in the mean number of degenerating neurons in ninth ganglia on the two sides remained nearly constant during this period. At stages XI1 and XVI, however, the mean number of degenerating nerve cells was greater in hyperplastic than in control ganglia. Although the differences in the number of degenerating neurons between control and hyperplastic ganglia in individual larvae were all in the same direction a t stages VI, X, and
XVI, differences in both directions were found a t stages VIII, IX, and XII. Differences in the mean number of degenerations on the two sides were small at most of the stages studied, and while the change in number of degenerating neurons with developmental stage was found t o be significant using the analysis of variance, differences in the numbers of degenerating neurons in control and hyperplastic ganglia, and the interaction between developmental stage and peripheral increase were not found t o be significant. Tests of the differences in the number of neuronal degenerations in control and hyperplastic ganglia from larvae of the same stage using the paired-sample t test showed significance only on the data from stage VI larvae. DISCUSSION
As has been discussed elsewhere, the level of
70
HAROLD D. BIBB
hyperplasia resulting from the use of the technique employed in this study compares favorably with results obtained by earlier workers who increased the size of the peripheral area available for innervation by grafting supernumerary limbs or limb rudiments to larvae or embryos. The increase in nerve cell number of 86% over control values at stage XVI in ganglia with enlarged peripheries also shows a close correspondence t o the 88%hyperplasia of the ninth spinal ganglion of stage XX R a n a pipiens larvae in which the periphery had been expanded in the same manner as in this investigation (Bibb, '77). The change in nerve cell numbers in the ninth ganglion with developmental stage in Rana berlandieri exhibits the same general pattern of increase and subsequent decrease that is apparent in the eighth, ninth, and tenth spinal ganglia in Xenopus laevis larvae (Prestige, '65) and in the ninth spinal ganglion of larval R . pipiens (Bibb, '77). The period of net nerve cell loss is of greater duration in R . berlandieri, like R . pipiens, than in X. laevis; and the proportion of nerve cells remaining in the ninth ganglion toward the end of the larval period relative to the maximum number present is less in R . berlandieri than in X. laeuis. While the period of net nerve cell loss in the ninth ganglion of R. berlandieri is similar to that of the ninth ganglion (Bibb, '77) and the brachial and lumbosacral motor columns of R. pipiens (Beaudoin, '55; Pollack, '691, differences do exist. Although both R. pipiens and R . berlandieri show peak numbers of neurons in the ninth ganglion during middle larval stages, this peak occurs slightly later in R. berlandieri (stage XII) than in R. pipiens (stage XI. Neuronal numbers are also smaller through most of the larval period in the ninth ganglion of R . berlandieri than those at the same stages in R. pipiens. In R . pipiens larvae, however, net neuronal loss in ninth ganglia with normal peripheries slows considerably between stages XIV and XX, and nerve cell numbers nearly reach a plateau during this period. Hence the similarity between the number of nerve cells present in ninth ganglia with normal peripheries in stage XX R. pipiens larvae and the number present in like ganglia in stage XVI R. berlandieri larvae may indicate that ninth ganglia with normal peripheries contain approximately the same number of neurons a t the end of the larval period in both species. The sequence of morphological changes in
degenerating nuclei of dying neurons is common to neuronal degeneration in a variety of species. While some species differences do exist (Hughes, '611, the general pattern of events described for R a n a berlandieri is strikingly similar to that in the larval ventral horn of Xenopus laevis (Hughes, '61) and R . pipiens (Decker, '76) and in the cervical and lumbosacral motor columns of chick embryos (O'Connor and Wyttenbach, '74). The presence of relatively small numbers of degenerating neurons in ganglion 9 during early larval stages and a subsequent increase in the number of dying nerve cells are in general agreement with Prestige's ('65) findings for ganglia 8 through 10 of larval Xenopus laeuis. However, the data presented here indicate that the increase in the number of degenerating nerve cells may begin somewhat earlier in R a n a berlandieri than in X . laeuis. Also in agreement with Prestige's observations is the presence of degenerating neurons in the ninth ganglion during the period of net nerve cell increase. It is apparent that in the ninth ganglion of R. berlandieri, as is true for X. laeuis, a neuronal turnover system is in operation. The possible significance of such turnover systems has been discussed extensively elsewhere (Prestige, '67, '70; Hughes, '61, '68; Pollack and Kollros, '75). Results of the present study clearly demonstrate that neuronal degeneration occurs throughout the development of the hyperplastic condition in ninth spinal ganglia whose peripheries have been increased. Whether there is a depression in normally occurring neuronal death that is sufficient to account for the increased numbers of nerve cells present in ganglia with expanded peripheries becomes the issue of central importance. Statistical analysis of the data indicates that the overall differences in the number of degenerating neurons in normal and hyperplastic ganglia are not significant. Yet, the trend toward the presence of fewer degenerating neurons in ganglia with expanded peripheries than in those with normal peripheries from stage V + through stage X remains suggestive of the possibility that suppression of normally occurring neuronal loss may contribute to the presence of increased numbers of nerve cells in peripherally overloaded ganglia. In this regard it is perhaps important to emphasize that the mean difference in the number of degenerating neurons in control and hyperplastic ganglia remains nearly con-
NEURONAL DEATH IN SPINAL GANGLIA
stant between stages VI and X. This suggests that the increase in nerve cell numbers resulting from the survival of neurons that would die in the absence of an expanded periphery should also be constant. However, in the 21day period between stages VI and VIII the increase over control values is a maximum of 700 neurons in hyperplastic ganglia, while in the 10-day period between stages VIII and X an additional increase of some 1,100 nerve cells over control values takes place so that at stage X hyperplastic ganglia contain an average of 1,800 more nerve cells than do control ganglia. Therefore the rate of increase in nerve cell number over control values in hyperplastic ganglia is not constant. This observation suggests that the apparent, though not statistically significant, depression in the level of neuronal death does not by itself account for the increased number of nerve cells present in ganglia with expanded peripheries. Thus even if a reduction in the level of neuronal death makes some contribution t o the increased number of nerve cells present in ganglia with expanded peripheries, the action of some additional mechanism would be required to explain fully the development of the hyperplastic condition observed in the present investigation. Results from a variety of studies of the effects of peripheral change on associated neural centers have been consistent with the view that information regarding the periphery is made available to the neural center by way of neural connections between center and periphery, and this view has been forwarded either directly or implicitly in many discussions of central-peripheral interactions (Hamburger, '39, '75; Hamburger and Levi-Montalcini, '49; Kollros, '53; Prestige, '67, '70; Pollack and Kollros, '75; Bibb, '77). It follows that a neural center may be able to respond to peripheral increase only after neural connections between center and periphery are made; and therefore the nature of the response may be related to those mechanisms operative in the neural center during and after the time that information regarding the periphery is made available. In a recent investigation of the effects of peripheral increase on the lateral motor column of chick embryos, Hollyday and Hamburger ('76) found that they could account for the excess numbers of neurons in motor columns with enlarged peripheries solely on the basis of a reduction in normally occurring neuronal
71
death. It is of interest that neurotization of the chick leg bud occurs just as proliferation for the lateral motor column is ending and before normally occurring neuronal degeneration begins on a large scale (Hamburger, '75; Hollyday and Hamburger, '76). Therefore it is not surprising that the chick lateral motor column responds to peripheral increase by a reduction in neuronal degeneration. It appears, however, that a different set of circumstances exists for the ninth spinal ganglion of Rana berlandieri. Nerve fibers are present in the limb bud of R. pipiens larvae as early as stage 11, and by stage IV rudiments of the main mixed trunks of the thigh and the shank are present (Taylor, '43). Although information of this type is not available for R . berlandieri, there is no reason to believe that this temporal pattern would be radically different in the two species. It is of interest in this regard that nerve fibers have been reported to be present at a similarly early stage in the limb bud of larval Xenopus Zaevis (Lamb, '74). Furthermore, unlike the chick lateral motor column, net neuronal numbers in the ninth ganglion of R. berlandieri continue to increase well beyond stages at which nerve fibers would be expected t o reach the limb bud, and mitotic figures were present in the ganglion throughout the period studied. Thus while proliferation for the chick lateral motor column may end before it can be affected by peripheral change, it is unlikely that this is the case for the ninth ganglion of R. berlandieri. In neural centers such as chick dorsal root ganglia (Hamburger and Levi-Montalcini, '49), the optic tectum (Kollros, '53; Currie and Cowan, '74) and the ninth spinal ganglion of R. pipiens (Bibb, '77) proliferation continues beyond the stages a t which peripheral contacts are made, and in these systems peripheral changes do affect proliferative activity. Thus the differentiation as neurons of some of the products of increased proliferation could also contribute to the hyperplasia observed in the study reported here. In emphasizing the exclusion of a role for increased proliferation in the response of the lateral motor column to peripheral enlargement, Hollyday and Hamburger ('76) suggested that the term "hyperplasia" be replaced by "hypothanasia." While this usage may be accurate for the chick lateral motor column, results of the present investigation indicate that adoption of this terminology
72
HAROLD D. BIBB
would be premature in describing the response of the ninth ganglion of larval Rana berlandieri to peripheral expansion. ACKNOWLEDGMENTS
The author is indebted to Professors Jerry J. Kollros and Emanuel D. Pollack for their critical reading of the manuscript, to Professor Robert F. Costantino for assistance with the statistical analyses, to Miss Bernadette Szumilo for technical aid, and to Mr. Eric D. Scott for the photographic work. LITERATURE CITED Abercrombie, M. 1946 Estimation of nuclear population from microtome sections. Anat. Rec., 94: 239-242. Bagnara, J. T., and J. S. Frost 1977 Leopard frog supply. Science, 197: 106-107. Barbieri, C. 1905 Ricerche sul sviluppo del midollo spinale negli Anfibi. Arch. 2001. (ital.) Napoli, 2: 79-106. Beaudoin, A. R. 1955 The development of lateral motor column cells in the lumbo-sacral cord in Rana pipiens. I. Normal development and development following unilateral limb ablation. Anat. Rec., 121: 81-96. Bibb, H. D. 1970 Ganglionic hypertrophy in Rana pipiens larvae. Anat. Rec., 166: 279. 1977 The production of ganglionic hypertrophy in Rana pipiens larvae. J. Exp. Zool., 200: 265-276. Collin, R. 1906 Recherches cytologiques sur le developpement de la cellule nerveuse. Le Nevraxe, 8: 181-307. Cowan, W. M. 1973 Neuronal death 88 a regulative mechanism in the control of cell number in t he nervous system. In: Development and Aging in t he Nervous System. Academic Press, New York. pp. 19-41. Currie, J., and W. M. Cowan 1974 Some observations on t he early development of the optic tectum in t he frog (Rana pipiensl, with special reference to t he effects of early eye removal on mitotic activity in t he larval tectum. J. Comp. Neur., 156: 123-142. Decker, R. S. 1976 Influence of thyroid hormones on neuronal death and differentiation in larval Rana pipiens. Develop. Biol., 49: 101-118. Glucksmann, A. 1951 Cell deaths in normal vertebrate ontogeny. Biol. Rev., 26: 59-86. Hamburger, V. 1939 Motor and sensory hyperplasia following limb bud transplantations in chick embryos. Physiol. Zool., 12: 268-284. 1958 Regression versus peripheral control of differentiation in motor hypoplasia. Am. J. Anat., 102: 365-410. 1975 Cell death in the development of t he lateral motor column of the chick embryo. J. Comp. Neur., 160: 535-546. Hamburger, V., and R. Levi-Montalcini 1949 Proliferation, differentiation and degeneration in t he spinal ganglia of the chick embryo under normal and experimental conditions. J. Exp. Zool., 111: 457-502. Hollyday, M., and V. Hamburger 1976 Reduction of t he
naturally occurring motor neuron loss by enlargement of the periphery. J. Comp. Neur., 170: 311-320. Hughes, A. 1961 Cell degeneration in the larval ventral horn of Xenopus laeuis (Daudin). J. Embryol. Exp. Morph., 9: 269-284. 1968 Aspects of Neural Ontogeny. Logos Press Ltd., London. Humason, G. L. 1972 Animal Tissue Techniques. Third ed. W. H. Freeman and Company, San Francisco. Jacobson, M. 1970 Developmental Neurobiology. Holt, Rinehart and Winston, New York. Kelly, J. P., and W. M. Cowan 1972 Studies on the development of the chick optic tectum. 111. Effects on early eye removal. Brain Res., 42: 263-288. Kollros, J. J. 1953 The development of the optic lobes in the frog. I. The effects of unilateral enucleation in embryonic stages. J. Exp. Zool., 123: 153-187. Lamb, A. H. 1974 The timing of the earliest motor innervation of the hind limb bud in thexenopus tadpole. Brain Res., 67: 527-530. Landmesser, L., and G. Pilar 1974 Synaptic transmission and cell death during normal ganglionic development. J. Physiol., 241: 737-749. OConnor, T. M., and C. R. Wyttenbach 1974 Cell death in the embryonic chick spinal cord. J. Cell Biol., 60: 448-459. Ostle, B., and R. W. Mensing 1975 Statistics in Research. Third ed. Iowa State University Press, Ames, Iowa. Pollack, E. D. 1969 Normal development of the lateral motor column in the brachial cord in Rana pipiens. Anat. Rec., 163: 111-120. Pollack, E. D., and J. J. Kollros 1975 dell migration into the “established lateral motor column in Rana pipiens larvae. 11. Lumbosacral spinal cord and comparative aspects. J. Exp. Zool., 192: 299-306. Prestige, M. C. 1965 Cell turnover in the spinal ganglia of Xenopus laeuis tadpoles. J. Embryol. Exp. Morph., 13: 63-72. 1967 The control of cell number in the lumbar spinal ganglia during the development of Xenopus laeuis tadpoles. J. Embryol. Exp. Morph., 17: 453-471. 1970 Differentiation, degeneration and the role of the periphery: quantitative considerations. In: The Neurosciences: Second Study Program. Rockefeller University Press, New York, pp. 73-82. 1976 Evidence tha t a t least some of the motor nerve cells tha t die during development have first made peripheral connections. J. Comp. Neur., 170: 123-134. Price, D. L. 1974 The influence of the periphery on spinal motor neurons. N. Y. Acad. Sci., 228: 355-363. Rugh, R. 1934 Induced ovulation and artificial fertilization in the frog. Biol. Bull., 66: 22-24. Sohal, G. S. 1976 An experimental study of cell death in the developing trochlear nucleus. Exp. Neur., 51; 684-698. Taylor, A. C. 1943 Development of the innervation pattern in the limb bud of the frog. Anat. Rec., 87: 379-413. 1944 Selectivity of nerve fibers from the dorsal and ventral roots in the development of the frog limb. J. Exp. Zool., 96: 159-185. Taylor, A. C., and J. J. Kollros 1946 Stages in the normal development ofRana pipiens larvae. Anat. Rec., 94: 7-24. Zar, J. H. 1974 Biostatistical Analysis. Prentice-Hall, Inc. Englewood Cliffs, New Jersey.
