Ultrastructural Studies on Postnatal Differentiation of Neurons in the Substantia Gelatinosa of R a t Cervical Spinal Cord 'r2
RICHARD S. H A N N A H 3 AND EDWARD J. H. NATHANIEL Faculty of Medicine, University of Manitoba, Manitoba, Canada
ABSTRACT Neuroblasts of the substantia gelatinosa at birth were small with large oval nuclei and scanty cytoplasm. The cytoplasm possessed ribosomes and mitochondria. Granular endoplasmic reticulum and Golgi complexes were generally absent or rudimentary. Electron dense bodies were seldom observed. By the end of the first week, the nuclei of several cells demonstrated early nuclear invaginations; cytoplasm exhibited growth cones, a well developed granular endoplasmic reticulum and Golgi complexes. At several points the channels of endoplasmic reticulum became continuous with the perinuclear space. By the end of the second week, differentiation of the neuroblasts was more advanced. More nuclei showed invagination of their contour. The cytoplasm revealed well developed granular endoplasmic reticulum and multiple Golgi complexes. Numerous vesicles and dense bodies were found adjacent to the Golgi complexes. Arrays of agranular endoplasmic reticulum also appeared late in the second week. By the third week, features of neuronal differentiation, such as nuclear invagination, granular endoplasmic reticulum, agranular membrane configurations, multiple Golgi complexes and dense bodies in the cytoplasm became well established.
The posterior horn of the spinal cord of adult animals has been examined by both light and electron microscopy by Rexed ('64), Szentagothai ('64), Ralston ('65), Nathaniel and Nathaniel ('66a), Petras ('68), and Scheibel and Scheibel ('68). However, to our knowledge, no investigation has been done on the development of posterior horn neurons. Neuronogenesis i n other areas of the central nervous system has been described by Karlsson ('66), Caley and Maxwell ('68) and Nosal and Radouco-Thomas ('71) in rats; Meller et al. ('66), Wechsler ('66), and Pannese ('68) i n chickens; Tennyson ('65) in the rabbit, Bodian ('66) and Kornguth et al. ('67) in the monkey and Billings ('72) in Xenopus. The present study was undertaken to examine postnatal development of the neurons in the rat substantia gelatinosa at the ultrastructural level. MATERIALS AND METHODS
Holtzman albino rats were killed at three day intervals from birth to two weeks, then ANAT. REC., 183: 323-338.
at one week intervals to six weeks of age. Several fixatives were tried and the most acceptable results were obtained with a combination of 3 % paraformaldehyde and 0.5% glutaraldehyde in 0.1 M cacodylate buffer. Animals up to three days of age were cooled to 3°C and fixed by immersion while older animals were perfused via the left ventricle of the heart. The cervical enlargement was immediately exposed and slices of dorsal segments made with a razor blade were immersed in fixative for onehalf hour. The tissue was then rinsed in cacodylate buffer, post-fixed in 1% osmium tetroxide i n cacodylate buffer for one hour, dehydrated in a n ascending series of ethanol and embedded in Araldite. Sections, 0.5 thick, were stained with toluidine blue and examined with the optical microscope to determine the position Received Feb. 20, '74. Accepted Apr. 21, "75. 1 This work has been presented at the Annual meeting of the American Association of Anatomists, Dallas 1972. 2 Supported in part by the Medical Research Council of Canada, National Cancer Institute of Canada and University of Manitoba Grant-in-Aid. 3 Present address: Department of Anatomy, Medical College of Georgia, Augusta, Georgia.
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of the substantia gelatinosa or lamina I1 as defined by Rexed ('52). In the neonate and subsequent ages up to two weeks after birth, the problem of defining lamina I1 was difficult. To assure that samples were homogeneous, all tissue was removed from the area near the entry of the dorsal root. I n animals under two weeks postnatal, the marginal cells and lamina I cells were excluded mainly by ultrastructural examination, A n ultrastructurally homogeneous population of cells is identifiable in the region where the substantia gelatinosa is present at maturity. Thin sections were cut from the region of the substantia gelatinosa and stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron microscope. OBSERVATIONS
Neuroblasts (immature neurons) at birth were distinguished from other cellular elements by two main criteria (fig. 1). The nuclear chromatin was relatively homogeneous in appearance and lacked the peripheral condensation of chromatin seen in glial elements. The cytoplasm was paler in appearance than that observed in glial cells. Similar observations were made by Caley and Maxwell ('68) i n developing rat cerebral cortex and by Radouco-Thomas et al. ('71) in the rat cerebellum. The majority of the neuroblasts present at birth possessed a round or ovoid nucleus (fig. 2). The cytoplasm of these primitive neuroblasts was characterized by paucity of cell organelles. Thus, the cytoplasm contained few scattered free ribosomes, short segments of granular endoplasmic reticulum and few mitochondria. The Golgi complex, when present, was rudimentary. Few axo-somatic synapses were present in these animals. During the first postnatal week, morphological changes occurred in both the nucleus and cytoplasm. The nuclei of these cells lost their oval or circular outline and demonstrated onset of nuclear invagination (fig. 3 ) . Nucleoli, when present, were compact in appearance. The nuclear envelope exhibited numerous connections with granular endoplasmic reticulum. Due to multiple communications between the nuclear envelope and the endoplasmic re-
ticulum, circumscribed areas of cytoplasm became entrapped (figs. 3 , 4 ) . The cytoplasmic features at this period comprised the presence of well defined organelles (fig. 3 ) . Segments of granular endoplasmic reticulum close to the cytoplasmic membrane often exhibited localized dilatations. The walls bounding such a dilatation or cistern were asymmetrical in their morphology. The wall adjacent to the cell membrane was devoid of ribosomes, while the opposite wall had ribosomes attached to it (fig. 5). These structures may represent immature subsurface cisternae, which are similar to those observed by Caley and Maxwell ('68) in rat cerebral cortex. Several neurons showed short bulbous cytoplasmic processes containing a large number of clear vesicles often grouped together in a terminal expansion; these resembled growth cones (figs. 6, 7). The proximal part of these processes generally contained microtubules (fig. 7). Structures resembling growth cones were also observed i n the neuropil. A significant feature of the neuroblasts during the second week of postnatal development was the remarkable irregularity of the nuclear outline resulting in a considerable increase of nuclear surface area (fig. 8). The cytoplasm of these neuroblasts exhibited a n increased number of organelles. The granular endoplasmic reticulum was distributed throughout the cytoplasm. Fewer communications between the perinuclear space and granular endoplasmic reticulum were encountered. Mature subsurface cisterns became evident. The well defined Golgi complexes were associated with considerable numbers of smooth and coated vesicles. There also appeared to be an increased number of Golgi complexes. Lysosome-like dense bodies were seen for the first time i n these developing neuroblasts (fig. 9). A singular feature observed late in the second week of postnatal development of these neurons was the appearance of cisternae of endoplasmic reticulum. Basically, a cistern consisted of dilated granular endoplasmic reticulum enclosing channels of agranular reticulum. These cisternal complexes were located well within the cell as
NEURONAL DIFFERENTIATION IN RAT CORD
well as at the peripheral aspect of the cell. The configuration of these complexes was highly variable (figs. lOa,b,c,d,e). In some neurons the cisternae of agranular reticulum were so tightly packed that the membranes of the adjacent processes became closely approximated, with resultant elimination of the enclosed space (figs. lOf,g) and appeared as aligned dense structures. The ribosomal attachment to the cisternal wall stopped abruptly at the point where the agranular processes projected into the lumen of the cistern, demonstrating the continuity between granular and agranular endoplasmic reticulum. By the end of the second postnatal week, a large number of axosomatic synapses were observed (fig. 9). Such synapses appeared morphologically mature and were characterized by an increased thickness of postsynaptic membrane and a bouton containing numerous vesicles. Mitochondria were also commonly observed within the presynaptic endings. Differentiation during the third postnatal week was restricted to two main features. A greater percentage of cells possessed complex nuclear infoldings and resembled the neurons described in the substantia gelatinosa of the adult rat (Nathaniel and Nathaniel, '66a). The cytoplasm of the cells appeared increased in amount and demonstrated greater growth and complexity of smooth endoplasmic reticulum, which was located within the confines of a dilated cistern of granular endoplasmic reticulum. Short segments of granular endoplasmic reticulum were also found here and there in the cell. Golgi complexes, lysosome-like dense bodies and multivesicular bodies were well developed and scattered throughout the cell. DISCUSSION
On of the most prominent features of the differentiating neuroblast of the substantia gelatinosa observed was the progressive increase in nuclear irregularity resulting an a highly convoluted mature nucleus. This increased nuclear surface area might reflect an increase in the nuclear activity of the cell. Similar nuclear irregularities are manifest in other active cells, such as Ehrlich and Yoshida tumor cells (Wessel and Bernard, '57), mammary
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cancer of the rat (Schultz, '57), and the Harding-Passey melanoma of the mouse (Nathaniel et al., '68; Loader and Nathaniel, '72). This is not to say that nuclei of developing cells which do not exhibit irregularities in outline are not active, for they are probably very active. However, the first signs of nuclear convolution in a cell which exhibits this feature in the mature state may possibly represent a morphological sign of nuclear activity and differentiation. In our study, numerous connections were observed between the granular endoplasmic reticulum and the nuclear membrane in neurons one week after birth. The occurrence of these connections suggested a possible role for the nuclear membrane in the biogenesis of the granular endoplasmic reticulum. Endoplasmic reticulum which appeared to develop from the nuclear margin and extended towards the periphery might have been involved in the production of the immature subsurface cisternae present by the end of the first week. Similar connections between granular endoplasmic reticulum and nuclear membrane have been reported in other neuroblasts by Caley and Maxwell ('68), Pannese ('68), Nosal and Radouco-Thomas ('71) and Billings ('72). The suspected immature subsurface cisternae observed during the first week postnatal lack the well defined pentalaminate structure of mature subsurface cisternae as defined by Siegesmund ('68). The proximity of the dilatations of endoplasmic reticulum to the perikaryon suggests the possibility of metabolite transport into the cell. Whether the suspected subsurface cisternae are capable of transport and how they differ physiologically from mature forms remains unknown. It is interesting that at the time when neuroblasts demonstrated increased cytoplasmic volume and produced axons and dendrites, many direct connections, via the granular endoplasmic reticulum, existed between the nuclear membrane and the suspected subsurface cisternae. Early establishment of such a network, capable of transporting possibly large amounts of metabolites into the cell, might be required to sustain growth. Morphologically mature subsurface cisternae appeared early in the second week post-
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natal when connections between the granular endoplasmic reticulum and the nuclear membrane were not as commonly encountered. Structures resembling the growth cones described by Bodian ('66) in monkey spinal cord and Del Cerro and Snider ('68) in rat cerebellum were observed in one week postnatal neuroblasts of this study. These structures were in direct continuation with the neuronal cell bodies during the period of cell process formation. These vesicle-filled expansions bulged from the perikaryon and also from the terminal end of longer processes. Tennyson ('70) described axonal growth cones containing vesicles, mitochondria, and segments of agranular endoplasmic reticulum i n rabbit dorsal root ganglia. She suggested that the reported variations in growth cone appearance might reflect different states of maturation during development. Microtubules observed in the basal part of the growth cones in this study, have been previously reported by Tennyson ('70) and Yamada et al. ('71). Studies by Hinds and Hinds ('72) in neonatal mouse olfactory bulb showed that dendritic growth cones were characterized by the presence of filopodia, filaments, absence of microtubules and paucity of smooth endoplasmic reticulum. Although several axosomatic synapses were observed at birth, not until one week postnatal were synapses encountered with regularity. During the second postnatal week the number of axosomatic synapses observed on any one individual neuron increased substantially. The axosomatic synapses observed in both the neonate and during the first postnatal week differed in structure from the mature axosomatic synapses. Most of the synapses during this period were characterized by relatively thin, symmetrical, pre and postsynaptic thickenings. Presynaptic vesicles were few in number and mitochondria were only rarely encountered. During the second postnatal week, the synapses appeared morphologically mature. The mature axosomatic synapses resembled Gray's Type I1 synapses, confirming the findings of Nathaniel and Nathaniel ('66b). Developmental changes in synaptic appearance were increased thickening of the post-synaptic membrane, a n increase in the
numbers of both synaptic vesicles and mitochondria in the presynaptic terminal. These findings accord with those of many other investigators as reviewed by Bunge et al. ('67). Whether or not the early synapses were physiologically active is unknown. A possible answer to this problem was offered by Bodian ('66), working with developing monkey spinal cord, who suggested that onset of a particular function follows closely upon the minimal synaptic development essential for the purpose. A secondary addition to the endoplasmic reticulum system was the appearance, towards the end of the second week, of the agranular endoplasmic reticulum in direct continuation with the granular endoplasmic reticulum. Similar whorls of smooth membranes were described in neurons of the posterior horn in adult rats by Nathaniel and Nathaniel ('66a). Membrane systems have also been described in cortical neurons of the rat by Rosenbluth ('62), in the lateral geniculate nucleus of the cat by Morales and Duncan ('66), in striatal neurons of the rat by Anzil et al. ('71) and in medium-sized neurons of the rat substantia nigra by Gulley and Wood ('72). An apparent metabolic role has been elucidated for agranular endoplasmic reticulum. Hendelman ('69), utilizing thallium poisoning in cultured neurons, suggested that the agranular endoplasmic reticulum may be involved in fluid transport. Several membrane systems were observed in continuity with subsurface cisternae and may have been involved in storing or concentrating substances entering or leaving the cell. The late appearance of the agranular membrane systems. at a time when the neurons appeared otherwise mature, may represent the final changeover from cell growth to maturity. ACKNOWLEDGMENTS
The authors wish to thank Dr. K. L. Moore, Chairman of the Department for the facilities provided. The technical assistance of Mr. Paul Perumal is appreciated. LITERATURE CIITED Anzil, A. P., K. Blinzinger and A. Matsushima 1971 Dark cisternal fields: Specialized formations of the endoplasmic reticulum in striatial neurons of a rat. Z. Zellforsch., 113: 553-557.
NEURONAL DIFFERENTIATION IN RAT CORD Bodian, D. 1966 Development of fine structure of spinal cord in monkey fetuses. 1. The motoneuron neuropil at the time of onset of reflex activity. Bull. Johns Hopkins Hosp., 119: 129-149. Bunge, M. B., R. P. Bunge and E. R. Peterson 1967 The onset of synapse formation in spinal cord cultures as studied by electron microscopy. Brain Res., 6: 728-749. Billings, S. M. 1972 Development of the Mauthner cell Xenopus Zaeuis: A light and electron microscopic study of the perikaryon. Z. Anat. Entwickl. Gesch., 136: 168-191. Caley, D. W., and D. S. Maxwell 1968 A n electron microscopic study of neurons during postnatal development of the rat cerebral cortex. J. Comp. Neur., 133: 17-44. Del Cerro, M. P., and R. S. Snider 1968 Studies on the developing cerebellum. I. Ultrastructure of the growth cones. J. Comp. Neur., 133: 341362. Gulley, R. L., and R. L. Wood 1971 The fine structure of the neurons in the rat substantia nigra. Tissue and Cell, 3: 675-690. Hendelman, W. 1969 The effect of thallium on peripheral nervous tissue in culture. A light and electron microscopic study. Anat. Rec., 163: 198-199. Hinds, J. W., and P. L. Hinds 1972 Reconstruction of dendritic growth cones in neonatal mouse olfactory bulb. J. Neurocytol., 1: 169187. Karlsson, U. 1966 Three-dimensional studies of neurons in the lateral geniculate nucleus of the rat. 1. Organelle organization in the perikaryon and its proximal branches. J. Ultrastruct. Res., 16: 429-481. Kornguth, S. E., J. W. Anderson and G . Scott 1967 Observations on the ultrastructure of the developing cerebellum of the Macaca mullata. J. Comp. Neur., 130: 1-23. Loader, K. R., and E. J. Nathaniel 1972 Persistence of colchicine induced differentiation in Harding-Passey Melanoma in mouse - an electron microscopic study. Anat. Rec., 172: 356. Meller, K., J. Eschner and P. Glees 1966 The differentiation of the endoplasmic reticulum in developing neurons of the chick spinal cord. 2. Zellforsch., 69: 189-197. Morales, R., and D. Duncan 1966 Multilaminated bodies and other unusual configurations of endoplasmic reticulum in the cerebellum of the cat. An electron microscopic study. J. Ultrastruct. Res., 15: 480-489. Nathaniel, E. J. H., N. B. Friedman and H. Rychuk 1968 Electron microscopic observations on cells of Harding-Passey melanoma following colchicine administration. Cancer Res., 28: 1031-1040. Nathaniel, E. J. H., and D. R. Nathaniel 1966a Fine structure of the neurons of the posterior horn in the rat spinal cord. Anat. Rec., 155: 629-641. 1966b The ultrastructural features of the synapses i n the posterior horn of the spinal
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cord in the rat. J. Ultrastruct. Res., 14: 540555. Nosal, G., and C. Radouco-Thomas 1971 Ultrastructural study o n the differentiation and development of the nerve cell; the “nucleusribosome” system. In: Advances i n Cytopharmacology. Vol. 1: First International Symposium on Cell Biology and Cytopharmacology. F. Clementi and B. Ceccarelli, eds. Raven Press, New York, pp. 433-456. Pannese, E. 1968 Developmental changes of the endoplasmic reticulum and ribosomes i n nerve cells of the spinal ganglia of the domestic fowl J. Comp. Neur., 132: 331-364. Petras, J. M. 1968 The substantia gelatinosa of Rolandi. Experientia, 24: 1045-1047. Radouco-Thomas, C., G. Nosal and S. RadoucoThomas 1971 The nuclear-ribosomal system during neuronal differentiation and development. In: Chemistry and Brain Development. R. Paoletti and A. N. Davison, eds. Plenum Press, New York, pp. 291-310. Ralston, H. J. 1965 The organization of the substantia gelatinosa Rolandi in the cat lumbosacral spinal cord. Z. Zellforsch., 67: 1-23. Rexed, B. 1964 Some aspects of the cytoarchitectonics and synaptology of the spinal cord. In: Progress in Brain Research. Vol. 11. J. C. Eccles and J. P. Schade, eds. Elsevier, Amsterdam, pp. 58-92. Rosenbluth, J. 1962 Subsurface cisterns and their relationship to the neuronal plasma membrane. J. Cell Biol., 13: 405422. Scheibel, M. E., and A. B. Scheibel 1968 Terminal axonal patterns i n cat spinal cord: 11. The dorsal horn. Brain Res., 9: 32-58. Schultz, H. 1957 Elecktronenmikroskopische Untersuchungen eines Mammakarzinoms der Ratte. Oncologia, 10: 307-329. Siegesmund, K. A. 1968 The fine structure of subsurface cisterns. Anat. Rec., 162: 187-196. Szentagothai, J. 1964 Neuronal and synaptic arrangement in the substantia gelatinosa Rolandi. J. Comp. Neur., 122: 219-239. Tennyson, V. M. 1965 Electron microscopic study of the developing neuroblast of the dorsal root ganglion of the rabbit embryo. J. Comp. Neur., 124: 267-318. 1970 The fine structure of the axon and growth cone of the dorsal root neuroblast of the rabbit embryo. J. Cell Biol., 44: 62-79. Wechsler, W. 1966 Elektronenmikroskopischer Beitrag zur Nervenzelldiferenzierung und Histogenese der grauen Substanz des Riickenmarks von Hiihnerembryonen. Z. Zellforsch., 74: 401-422. Wessel, W., and W. Bernhard 1957 Vergleichende elektronenmikroskopische Untersuchung von Ehrlich-und Yoshida Ascitestumorzellen. Z. fur Krebsforsch., 62: 140-162. Yamada, K. M., B. S. Spooner and N. K. Wessels 1971 Ultrastructure and function of growth cones and axons of cultured nerve cells. J. Cell Biol., 49: 614-635.
PLATE 1 EXPLANATION OF FIGURES
1 This micrograph represents the substantia gelatinosa at birth. The majority of neuroblasts ( N ) possess ovoid nuclei with diffuse nuclear chromatin and scanty amounts of cytoplasm. A glioblast ( G ) , characterized by high overall electron density and thin rim of cytoplasm, is also present. Note the large amount of extra-cellular space present. x 6,000. 2
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A neuroblast at birth which demonstrates an ovoid nucleus with a diffuse nuclear chromatin pattern. The thin rim of cytoplasm contains only scattered free ribosomes. x 14,000.
NEURONAL DIFFERENTIATION IN RAT CORD Richard S. Hannah and Edward J. H. Nathaniel
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
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3
A neuroblast at one week of age. The nucleus demonstrates the early signs of nuclear invagination. The granular endoplasmic reticulum is in communication with the nuclear envelope at many sites (arrow). A small Golgi apparatus (G), multivesicular body (Mb) and centriole ( C ) are also present. X 23,000.
4
A part of a 1-week postnatal neuroblast, which demonstrates connections between the granular endoplasmic reticulum and the nuclear envelope (arrows). x 26,000.
5
Part of a 1-week-old neuroblast containing a suspected primitive subsurface cistern. The membrane of the granular endoplasmic reticulum adjacent to the Flasmalemma is devoid of ribosomes (arrow). X 37,000.
NEURONAL DIFFERENTIATION I N RAT CORD Richard S. Hannah and Edward J. H . Nathaniel
PLATE 2
33 1
PLATE 3 EXPLANATION OF FIGURES
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6
A growth cone, (arrows), containing large clear vesicles, may be observed protruding directly from the cell surface in a 1-week old animal. The nucleus ( N ) is present at the bottom of the micrograph. X 48,000.
7
A growth cone (arrow), composed of large, clear vesicles is present on a cytoplasmic process in a 1-week old animal. Microtubules (Mt), short segments of agranular endoplasmic reticulum and a mitochondrion are also present in the growing cytoplasmic process. The nucleus ( N ) is situated in the lower part of the micrograph. x 31,000.
NEURONAL DIFFERENTIATION IN RAT CORD Richard S . Hannah and Edward J. H. Nathaniel
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
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8
Part of a neuroblast from a 2-week old animal which demonstrates elaborate irregularities in nuclear contour. >: 50,000.
9
In a 2-week old animal the cytoplasm of a neuroblast containing well developed Golgi complexes (G), numerous mitochondria and lysosomelike bodies ( B ) . The arrows demonstrate the numerous axosomatic synapses present. Numerous coated vesicles (asterisk) may be observed in relation to the Golgi complex. >: 19,000.
NEURONAL DIFFERENTIATION IN RAT CORD Richard S. Hannah and Edward J. H. Nathaniel
PLATE 4
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PLATE 5 EXPLANATION O F FIGURES
Fig. 10 This series of micrographs illustrates the possible developmental pattern of the agranular membrane systems.
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10A, B, C
These illustrations show sets of 1, 2 and 3 agranular membranes, respectively, within cisterns of the granular reticulum. A: x 36,900; B: x 53,100; C : x 41,400.
10D
Multiple agranular membranes enclosed within a granular cistern. x 32,400.
10E
A whorl of agranular membranes enclosed within a large granular cistern. Note the proximity and direct communication to the subsurface cistern (arrow). X 34,200.
10F
The dense lines formed by the approximation and fusion of adjacent membranes of agranular reticulum are demonstrated in this micrograph. X 58,500.
10G
A whorl of agranular membranes similar to 10E but with dense lines present. x 58,500.
NEURONAL DIFFERENTIATION I N RAT CORD Richard S. Hannah and Edward J. H. Nathaniel
PLATE 5
RICHARD S. H A N N A H 3 AND EDWARD J. H. NATHANIEL Faculty of Medicine, University of Manitoba, Manitoba, Canada
ABSTRACT Neuroblasts of the substantia gelatinosa at birth were small with large oval nuclei and scanty cytoplasm. The cytoplasm possessed ribosomes and mitochondria. Granular endoplasmic reticulum and Golgi complexes were generally absent or rudimentary. Electron dense bodies were seldom observed. By the end of the first week, the nuclei of several cells demonstrated early nuclear invaginations; cytoplasm exhibited growth cones, a well developed granular endoplasmic reticulum and Golgi complexes. At several points the channels of endoplasmic reticulum became continuous with the perinuclear space. By the end of the second week, differentiation of the neuroblasts was more advanced. More nuclei showed invagination of their contour. The cytoplasm revealed well developed granular endoplasmic reticulum and multiple Golgi complexes. Numerous vesicles and dense bodies were found adjacent to the Golgi complexes. Arrays of agranular endoplasmic reticulum also appeared late in the second week. By the third week, features of neuronal differentiation, such as nuclear invagination, granular endoplasmic reticulum, agranular membrane configurations, multiple Golgi complexes and dense bodies in the cytoplasm became well established.
The posterior horn of the spinal cord of adult animals has been examined by both light and electron microscopy by Rexed ('64), Szentagothai ('64), Ralston ('65), Nathaniel and Nathaniel ('66a), Petras ('68), and Scheibel and Scheibel ('68). However, to our knowledge, no investigation has been done on the development of posterior horn neurons. Neuronogenesis i n other areas of the central nervous system has been described by Karlsson ('66), Caley and Maxwell ('68) and Nosal and Radouco-Thomas ('71) in rats; Meller et al. ('66), Wechsler ('66), and Pannese ('68) i n chickens; Tennyson ('65) in the rabbit, Bodian ('66) and Kornguth et al. ('67) in the monkey and Billings ('72) in Xenopus. The present study was undertaken to examine postnatal development of the neurons in the rat substantia gelatinosa at the ultrastructural level. MATERIALS AND METHODS
Holtzman albino rats were killed at three day intervals from birth to two weeks, then ANAT. REC., 183: 323-338.
at one week intervals to six weeks of age. Several fixatives were tried and the most acceptable results were obtained with a combination of 3 % paraformaldehyde and 0.5% glutaraldehyde in 0.1 M cacodylate buffer. Animals up to three days of age were cooled to 3°C and fixed by immersion while older animals were perfused via the left ventricle of the heart. The cervical enlargement was immediately exposed and slices of dorsal segments made with a razor blade were immersed in fixative for onehalf hour. The tissue was then rinsed in cacodylate buffer, post-fixed in 1% osmium tetroxide i n cacodylate buffer for one hour, dehydrated in a n ascending series of ethanol and embedded in Araldite. Sections, 0.5 thick, were stained with toluidine blue and examined with the optical microscope to determine the position Received Feb. 20, '74. Accepted Apr. 21, "75. 1 This work has been presented at the Annual meeting of the American Association of Anatomists, Dallas 1972. 2 Supported in part by the Medical Research Council of Canada, National Cancer Institute of Canada and University of Manitoba Grant-in-Aid. 3 Present address: Department of Anatomy, Medical College of Georgia, Augusta, Georgia.
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of the substantia gelatinosa or lamina I1 as defined by Rexed ('52). In the neonate and subsequent ages up to two weeks after birth, the problem of defining lamina I1 was difficult. To assure that samples were homogeneous, all tissue was removed from the area near the entry of the dorsal root. I n animals under two weeks postnatal, the marginal cells and lamina I cells were excluded mainly by ultrastructural examination, A n ultrastructurally homogeneous population of cells is identifiable in the region where the substantia gelatinosa is present at maturity. Thin sections were cut from the region of the substantia gelatinosa and stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron microscope. OBSERVATIONS
Neuroblasts (immature neurons) at birth were distinguished from other cellular elements by two main criteria (fig. 1). The nuclear chromatin was relatively homogeneous in appearance and lacked the peripheral condensation of chromatin seen in glial elements. The cytoplasm was paler in appearance than that observed in glial cells. Similar observations were made by Caley and Maxwell ('68) i n developing rat cerebral cortex and by Radouco-Thomas et al. ('71) in the rat cerebellum. The majority of the neuroblasts present at birth possessed a round or ovoid nucleus (fig. 2). The cytoplasm of these primitive neuroblasts was characterized by paucity of cell organelles. Thus, the cytoplasm contained few scattered free ribosomes, short segments of granular endoplasmic reticulum and few mitochondria. The Golgi complex, when present, was rudimentary. Few axo-somatic synapses were present in these animals. During the first postnatal week, morphological changes occurred in both the nucleus and cytoplasm. The nuclei of these cells lost their oval or circular outline and demonstrated onset of nuclear invagination (fig. 3 ) . Nucleoli, when present, were compact in appearance. The nuclear envelope exhibited numerous connections with granular endoplasmic reticulum. Due to multiple communications between the nuclear envelope and the endoplasmic re-
ticulum, circumscribed areas of cytoplasm became entrapped (figs. 3 , 4 ) . The cytoplasmic features at this period comprised the presence of well defined organelles (fig. 3 ) . Segments of granular endoplasmic reticulum close to the cytoplasmic membrane often exhibited localized dilatations. The walls bounding such a dilatation or cistern were asymmetrical in their morphology. The wall adjacent to the cell membrane was devoid of ribosomes, while the opposite wall had ribosomes attached to it (fig. 5). These structures may represent immature subsurface cisternae, which are similar to those observed by Caley and Maxwell ('68) in rat cerebral cortex. Several neurons showed short bulbous cytoplasmic processes containing a large number of clear vesicles often grouped together in a terminal expansion; these resembled growth cones (figs. 6, 7). The proximal part of these processes generally contained microtubules (fig. 7). Structures resembling growth cones were also observed i n the neuropil. A significant feature of the neuroblasts during the second week of postnatal development was the remarkable irregularity of the nuclear outline resulting in a considerable increase of nuclear surface area (fig. 8). The cytoplasm of these neuroblasts exhibited a n increased number of organelles. The granular endoplasmic reticulum was distributed throughout the cytoplasm. Fewer communications between the perinuclear space and granular endoplasmic reticulum were encountered. Mature subsurface cisterns became evident. The well defined Golgi complexes were associated with considerable numbers of smooth and coated vesicles. There also appeared to be an increased number of Golgi complexes. Lysosome-like dense bodies were seen for the first time i n these developing neuroblasts (fig. 9). A singular feature observed late in the second week of postnatal development of these neurons was the appearance of cisternae of endoplasmic reticulum. Basically, a cistern consisted of dilated granular endoplasmic reticulum enclosing channels of agranular reticulum. These cisternal complexes were located well within the cell as
NEURONAL DIFFERENTIATION IN RAT CORD
well as at the peripheral aspect of the cell. The configuration of these complexes was highly variable (figs. lOa,b,c,d,e). In some neurons the cisternae of agranular reticulum were so tightly packed that the membranes of the adjacent processes became closely approximated, with resultant elimination of the enclosed space (figs. lOf,g) and appeared as aligned dense structures. The ribosomal attachment to the cisternal wall stopped abruptly at the point where the agranular processes projected into the lumen of the cistern, demonstrating the continuity between granular and agranular endoplasmic reticulum. By the end of the second postnatal week, a large number of axosomatic synapses were observed (fig. 9). Such synapses appeared morphologically mature and were characterized by an increased thickness of postsynaptic membrane and a bouton containing numerous vesicles. Mitochondria were also commonly observed within the presynaptic endings. Differentiation during the third postnatal week was restricted to two main features. A greater percentage of cells possessed complex nuclear infoldings and resembled the neurons described in the substantia gelatinosa of the adult rat (Nathaniel and Nathaniel, '66a). The cytoplasm of the cells appeared increased in amount and demonstrated greater growth and complexity of smooth endoplasmic reticulum, which was located within the confines of a dilated cistern of granular endoplasmic reticulum. Short segments of granular endoplasmic reticulum were also found here and there in the cell. Golgi complexes, lysosome-like dense bodies and multivesicular bodies were well developed and scattered throughout the cell. DISCUSSION
On of the most prominent features of the differentiating neuroblast of the substantia gelatinosa observed was the progressive increase in nuclear irregularity resulting an a highly convoluted mature nucleus. This increased nuclear surface area might reflect an increase in the nuclear activity of the cell. Similar nuclear irregularities are manifest in other active cells, such as Ehrlich and Yoshida tumor cells (Wessel and Bernard, '57), mammary
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cancer of the rat (Schultz, '57), and the Harding-Passey melanoma of the mouse (Nathaniel et al., '68; Loader and Nathaniel, '72). This is not to say that nuclei of developing cells which do not exhibit irregularities in outline are not active, for they are probably very active. However, the first signs of nuclear convolution in a cell which exhibits this feature in the mature state may possibly represent a morphological sign of nuclear activity and differentiation. In our study, numerous connections were observed between the granular endoplasmic reticulum and the nuclear membrane in neurons one week after birth. The occurrence of these connections suggested a possible role for the nuclear membrane in the biogenesis of the granular endoplasmic reticulum. Endoplasmic reticulum which appeared to develop from the nuclear margin and extended towards the periphery might have been involved in the production of the immature subsurface cisternae present by the end of the first week. Similar connections between granular endoplasmic reticulum and nuclear membrane have been reported in other neuroblasts by Caley and Maxwell ('68), Pannese ('68), Nosal and Radouco-Thomas ('71) and Billings ('72). The suspected immature subsurface cisternae observed during the first week postnatal lack the well defined pentalaminate structure of mature subsurface cisternae as defined by Siegesmund ('68). The proximity of the dilatations of endoplasmic reticulum to the perikaryon suggests the possibility of metabolite transport into the cell. Whether the suspected subsurface cisternae are capable of transport and how they differ physiologically from mature forms remains unknown. It is interesting that at the time when neuroblasts demonstrated increased cytoplasmic volume and produced axons and dendrites, many direct connections, via the granular endoplasmic reticulum, existed between the nuclear membrane and the suspected subsurface cisternae. Early establishment of such a network, capable of transporting possibly large amounts of metabolites into the cell, might be required to sustain growth. Morphologically mature subsurface cisternae appeared early in the second week post-
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RICHARD S. HANNAH AND EDWARD J. H. NATHANIEL
natal when connections between the granular endoplasmic reticulum and the nuclear membrane were not as commonly encountered. Structures resembling the growth cones described by Bodian ('66) in monkey spinal cord and Del Cerro and Snider ('68) in rat cerebellum were observed in one week postnatal neuroblasts of this study. These structures were in direct continuation with the neuronal cell bodies during the period of cell process formation. These vesicle-filled expansions bulged from the perikaryon and also from the terminal end of longer processes. Tennyson ('70) described axonal growth cones containing vesicles, mitochondria, and segments of agranular endoplasmic reticulum i n rabbit dorsal root ganglia. She suggested that the reported variations in growth cone appearance might reflect different states of maturation during development. Microtubules observed in the basal part of the growth cones in this study, have been previously reported by Tennyson ('70) and Yamada et al. ('71). Studies by Hinds and Hinds ('72) in neonatal mouse olfactory bulb showed that dendritic growth cones were characterized by the presence of filopodia, filaments, absence of microtubules and paucity of smooth endoplasmic reticulum. Although several axosomatic synapses were observed at birth, not until one week postnatal were synapses encountered with regularity. During the second postnatal week the number of axosomatic synapses observed on any one individual neuron increased substantially. The axosomatic synapses observed in both the neonate and during the first postnatal week differed in structure from the mature axosomatic synapses. Most of the synapses during this period were characterized by relatively thin, symmetrical, pre and postsynaptic thickenings. Presynaptic vesicles were few in number and mitochondria were only rarely encountered. During the second postnatal week, the synapses appeared morphologically mature. The mature axosomatic synapses resembled Gray's Type I1 synapses, confirming the findings of Nathaniel and Nathaniel ('66b). Developmental changes in synaptic appearance were increased thickening of the post-synaptic membrane, a n increase in the
numbers of both synaptic vesicles and mitochondria in the presynaptic terminal. These findings accord with those of many other investigators as reviewed by Bunge et al. ('67). Whether or not the early synapses were physiologically active is unknown. A possible answer to this problem was offered by Bodian ('66), working with developing monkey spinal cord, who suggested that onset of a particular function follows closely upon the minimal synaptic development essential for the purpose. A secondary addition to the endoplasmic reticulum system was the appearance, towards the end of the second week, of the agranular endoplasmic reticulum in direct continuation with the granular endoplasmic reticulum. Similar whorls of smooth membranes were described in neurons of the posterior horn in adult rats by Nathaniel and Nathaniel ('66a). Membrane systems have also been described in cortical neurons of the rat by Rosenbluth ('62), in the lateral geniculate nucleus of the cat by Morales and Duncan ('66), in striatal neurons of the rat by Anzil et al. ('71) and in medium-sized neurons of the rat substantia nigra by Gulley and Wood ('72). An apparent metabolic role has been elucidated for agranular endoplasmic reticulum. Hendelman ('69), utilizing thallium poisoning in cultured neurons, suggested that the agranular endoplasmic reticulum may be involved in fluid transport. Several membrane systems were observed in continuity with subsurface cisternae and may have been involved in storing or concentrating substances entering or leaving the cell. The late appearance of the agranular membrane systems. at a time when the neurons appeared otherwise mature, may represent the final changeover from cell growth to maturity. ACKNOWLEDGMENTS
The authors wish to thank Dr. K. L. Moore, Chairman of the Department for the facilities provided. The technical assistance of Mr. Paul Perumal is appreciated. LITERATURE CIITED Anzil, A. P., K. Blinzinger and A. Matsushima 1971 Dark cisternal fields: Specialized formations of the endoplasmic reticulum in striatial neurons of a rat. Z. Zellforsch., 113: 553-557.
NEURONAL DIFFERENTIATION IN RAT CORD Bodian, D. 1966 Development of fine structure of spinal cord in monkey fetuses. 1. The motoneuron neuropil at the time of onset of reflex activity. Bull. Johns Hopkins Hosp., 119: 129-149. Bunge, M. B., R. P. Bunge and E. R. Peterson 1967 The onset of synapse formation in spinal cord cultures as studied by electron microscopy. Brain Res., 6: 728-749. Billings, S. M. 1972 Development of the Mauthner cell Xenopus Zaeuis: A light and electron microscopic study of the perikaryon. Z. Anat. Entwickl. Gesch., 136: 168-191. Caley, D. W., and D. S. Maxwell 1968 A n electron microscopic study of neurons during postnatal development of the rat cerebral cortex. J. Comp. Neur., 133: 17-44. Del Cerro, M. P., and R. S. Snider 1968 Studies on the developing cerebellum. I. Ultrastructure of the growth cones. J. Comp. Neur., 133: 341362. Gulley, R. L., and R. L. Wood 1971 The fine structure of the neurons in the rat substantia nigra. Tissue and Cell, 3: 675-690. Hendelman, W. 1969 The effect of thallium on peripheral nervous tissue in culture. A light and electron microscopic study. Anat. Rec., 163: 198-199. Hinds, J. W., and P. L. Hinds 1972 Reconstruction of dendritic growth cones in neonatal mouse olfactory bulb. J. Neurocytol., 1: 169187. Karlsson, U. 1966 Three-dimensional studies of neurons in the lateral geniculate nucleus of the rat. 1. Organelle organization in the perikaryon and its proximal branches. J. Ultrastruct. Res., 16: 429-481. Kornguth, S. E., J. W. Anderson and G . Scott 1967 Observations on the ultrastructure of the developing cerebellum of the Macaca mullata. J. Comp. Neur., 130: 1-23. Loader, K. R., and E. J. Nathaniel 1972 Persistence of colchicine induced differentiation in Harding-Passey Melanoma in mouse - an electron microscopic study. Anat. Rec., 172: 356. Meller, K., J. Eschner and P. Glees 1966 The differentiation of the endoplasmic reticulum in developing neurons of the chick spinal cord. 2. Zellforsch., 69: 189-197. Morales, R., and D. Duncan 1966 Multilaminated bodies and other unusual configurations of endoplasmic reticulum in the cerebellum of the cat. An electron microscopic study. J. Ultrastruct. Res., 15: 480-489. Nathaniel, E. J. H., N. B. Friedman and H. Rychuk 1968 Electron microscopic observations on cells of Harding-Passey melanoma following colchicine administration. Cancer Res., 28: 1031-1040. Nathaniel, E. J. H., and D. R. Nathaniel 1966a Fine structure of the neurons of the posterior horn in the rat spinal cord. Anat. Rec., 155: 629-641. 1966b The ultrastructural features of the synapses i n the posterior horn of the spinal
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cord in the rat. J. Ultrastruct. Res., 14: 540555. Nosal, G., and C. Radouco-Thomas 1971 Ultrastructural study o n the differentiation and development of the nerve cell; the “nucleusribosome” system. In: Advances i n Cytopharmacology. Vol. 1: First International Symposium on Cell Biology and Cytopharmacology. F. Clementi and B. Ceccarelli, eds. Raven Press, New York, pp. 433-456. Pannese, E. 1968 Developmental changes of the endoplasmic reticulum and ribosomes i n nerve cells of the spinal ganglia of the domestic fowl J. Comp. Neur., 132: 331-364. Petras, J. M. 1968 The substantia gelatinosa of Rolandi. Experientia, 24: 1045-1047. Radouco-Thomas, C., G. Nosal and S. RadoucoThomas 1971 The nuclear-ribosomal system during neuronal differentiation and development. In: Chemistry and Brain Development. R. Paoletti and A. N. Davison, eds. Plenum Press, New York, pp. 291-310. Ralston, H. J. 1965 The organization of the substantia gelatinosa Rolandi in the cat lumbosacral spinal cord. Z. Zellforsch., 67: 1-23. Rexed, B. 1964 Some aspects of the cytoarchitectonics and synaptology of the spinal cord. In: Progress in Brain Research. Vol. 11. J. C. Eccles and J. P. Schade, eds. Elsevier, Amsterdam, pp. 58-92. Rosenbluth, J. 1962 Subsurface cisterns and their relationship to the neuronal plasma membrane. J. Cell Biol., 13: 405422. Scheibel, M. E., and A. B. Scheibel 1968 Terminal axonal patterns i n cat spinal cord: 11. The dorsal horn. Brain Res., 9: 32-58. Schultz, H. 1957 Elecktronenmikroskopische Untersuchungen eines Mammakarzinoms der Ratte. Oncologia, 10: 307-329. Siegesmund, K. A. 1968 The fine structure of subsurface cisterns. Anat. Rec., 162: 187-196. Szentagothai, J. 1964 Neuronal and synaptic arrangement in the substantia gelatinosa Rolandi. J. Comp. Neur., 122: 219-239. Tennyson, V. M. 1965 Electron microscopic study of the developing neuroblast of the dorsal root ganglion of the rabbit embryo. J. Comp. Neur., 124: 267-318. 1970 The fine structure of the axon and growth cone of the dorsal root neuroblast of the rabbit embryo. J. Cell Biol., 44: 62-79. Wechsler, W. 1966 Elektronenmikroskopischer Beitrag zur Nervenzelldiferenzierung und Histogenese der grauen Substanz des Riickenmarks von Hiihnerembryonen. Z. Zellforsch., 74: 401-422. Wessel, W., and W. Bernhard 1957 Vergleichende elektronenmikroskopische Untersuchung von Ehrlich-und Yoshida Ascitestumorzellen. Z. fur Krebsforsch., 62: 140-162. Yamada, K. M., B. S. Spooner and N. K. Wessels 1971 Ultrastructure and function of growth cones and axons of cultured nerve cells. J. Cell Biol., 49: 614-635.
PLATE 1 EXPLANATION OF FIGURES
1 This micrograph represents the substantia gelatinosa at birth. The majority of neuroblasts ( N ) possess ovoid nuclei with diffuse nuclear chromatin and scanty amounts of cytoplasm. A glioblast ( G ) , characterized by high overall electron density and thin rim of cytoplasm, is also present. Note the large amount of extra-cellular space present. x 6,000. 2
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A neuroblast at birth which demonstrates an ovoid nucleus with a diffuse nuclear chromatin pattern. The thin rim of cytoplasm contains only scattered free ribosomes. x 14,000.
NEURONAL DIFFERENTIATION IN RAT CORD Richard S. Hannah and Edward J. H. Nathaniel
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
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3
A neuroblast at one week of age. The nucleus demonstrates the early signs of nuclear invagination. The granular endoplasmic reticulum is in communication with the nuclear envelope at many sites (arrow). A small Golgi apparatus (G), multivesicular body (Mb) and centriole ( C ) are also present. X 23,000.
4
A part of a 1-week postnatal neuroblast, which demonstrates connections between the granular endoplasmic reticulum and the nuclear envelope (arrows). x 26,000.
5
Part of a 1-week-old neuroblast containing a suspected primitive subsurface cistern. The membrane of the granular endoplasmic reticulum adjacent to the Flasmalemma is devoid of ribosomes (arrow). X 37,000.
NEURONAL DIFFERENTIATION I N RAT CORD Richard S. Hannah and Edward J. H . Nathaniel
PLATE 2
33 1
PLATE 3 EXPLANATION OF FIGURES
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6
A growth cone, (arrows), containing large clear vesicles, may be observed protruding directly from the cell surface in a 1-week old animal. The nucleus ( N ) is present at the bottom of the micrograph. X 48,000.
7
A growth cone (arrow), composed of large, clear vesicles is present on a cytoplasmic process in a 1-week old animal. Microtubules (Mt), short segments of agranular endoplasmic reticulum and a mitochondrion are also present in the growing cytoplasmic process. The nucleus ( N ) is situated in the lower part of the micrograph. x 31,000.
NEURONAL DIFFERENTIATION IN RAT CORD Richard S . Hannah and Edward J. H. Nathaniel
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
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8
Part of a neuroblast from a 2-week old animal which demonstrates elaborate irregularities in nuclear contour. >: 50,000.
9
In a 2-week old animal the cytoplasm of a neuroblast containing well developed Golgi complexes (G), numerous mitochondria and lysosomelike bodies ( B ) . The arrows demonstrate the numerous axosomatic synapses present. Numerous coated vesicles (asterisk) may be observed in relation to the Golgi complex. >: 19,000.
NEURONAL DIFFERENTIATION IN RAT CORD Richard S. Hannah and Edward J. H. Nathaniel
PLATE 4
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PLATE 5 EXPLANATION O F FIGURES
Fig. 10 This series of micrographs illustrates the possible developmental pattern of the agranular membrane systems.
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10A, B, C
These illustrations show sets of 1, 2 and 3 agranular membranes, respectively, within cisterns of the granular reticulum. A: x 36,900; B: x 53,100; C : x 41,400.
10D
Multiple agranular membranes enclosed within a granular cistern. x 32,400.
10E
A whorl of agranular membranes enclosed within a large granular cistern. Note the proximity and direct communication to the subsurface cistern (arrow). X 34,200.
10F
The dense lines formed by the approximation and fusion of adjacent membranes of agranular reticulum are demonstrated in this micrograph. X 58,500.
10G
A whorl of agranular membranes similar to 10E but with dense lines present. x 58,500.
NEURONAL DIFFERENTIATION I N RAT CORD Richard S. Hannah and Edward J. H. Nathaniel
PLATE 5
