EXPERIMENTALNEUROLOGY
107,187-l%
(1990)
Neurogenic Period of Ascending Tract Neurons in the Upper Lumbar Spinal Cord of the Rat KAILAS N. NANDI, JOHN A. BEAL,~ AND DAVID S. Department
of
Cellular Biology and Anatomy, L.ouisiunu State University Medical Center in Shreveport, P.O. Box 33932, Shreveport, Louisiana 71130
has also been shown that spinal neurogenesis proceeds along gradients which extend from rostra1 to caudal, lateral to medial, and ventral to dorsal (1,16). It has been suggested that the pattern of proliferation along each gradient is such that ascending sensory (relay) neurons complete proliferation well before neurons with short projections, i.e., before propriospinal and local circuit neurons (1). There is considerable evidence to support this theory. First, large spinal neurons complete neurogenesis prior to smaller neurons (1,16,18). Second, long axon neurons, demonstrated in Golgi impregnation studies of the developing spinal cord, were found sprouting axons and dendrites, while short axon neurons were still proliferating ($6). Third, autoradiographic studies using tritiated thymidine in the diencephalon (3), cochlear nuclei (21), and olfactory bulb (13) of the mouse, the cerebellum (2) and occipital neocortex (8) of the rat, and the hippocampus of the monkey (19) have shown that long axon projection neurons complete neurogenesis prior to neurons with short axons. Given the above evidence, then, it seems reasonable to conclude that ascending tract (relay) neurons probably complete neurogenesis prior to spinal neurons with shorter projections. Although it is not known when the neurogenesis of relay neurons is completed, judging from the fact that neurons of the ascending tracts make up less than 1.0% of all spinal neurons (9), it would seem reasonable to further conclude that the proliferation of these neurons would be completed early in the neurogenie period. However, tritiated thymidine autoradiographs of adult lumbar spinal cord, available in our laboratory, revealed that some medium size nerve cell bodies in lamina IV and the nucleus dorsalis, suspected of being ascending tract neurons because of their relatively large size, were still taking up tritiated thymidine when injected in the final one-third of the proliferative period as late as Day 15 of gestation. This phenomenon coupled with recent evidence which demonstrates that GABAergic interneurons of the cerebral cortex proliferate at the same time as the cortical projecting neurons (11,14, 17) suggested that a reevaluation of spinal neurogenesis was in order.
Although the neurogenic period for neurons in the lumbar spinal cord has been clearly established (Days 12 through 16 of gestation), it is not known when the neurogenesis of ascending tract neurons is completed within this period. The purpose of the present study was to determine the duration of the neurogenic period for projection neurons of the ascending tracts. To label neurons undergoing mitosis during this period, tritiated thymidine was administered to fetal rats on Embryonic (E) Days El3 through El6 of gestation. Ascending tract neurons of the lumbar cord were later (Postnatal Days 40-50) labeled in each animal with a retrograde tracer, Fluoro-Gold, applied at the site of a hemisection at spinal cord segment C3. Ascending tract neurons which were undergoing mitosis in the upper lumbar cord were double labeled, i.e., labeled with both tritiated thymidine and Fluoro-Gold. On Day E13,8992% of the ascending tract neurons were double labeled; on Day E14, 35-37%; and on Day E15, l-4%. Results showed, then, that some ascending tract neurons were double labeled through Day El5 and were, therefore, proliferating in the final one-third of the neurogenic period. Ascending tract neurons proliferating on Day El5 were confined to laminae III, IV, V, and X and the nucleus dorsalis. Long tract neurons in the superficial dorsal horn (laminae I and II), on the other hand, were found to have completed neurogenesis on Day El4 of gestation. Results of the present study show that spinal neurogenesis of ascending projection neurons continues throughout most of the neurogenic period and does not completely follow the well-established ventral to dorsal gradient. o 1990 Academic PRESS, I~C.
INTRODUCI-ION
Studies on neurogenesis in the rat using tritiated thymidine autoradiography have shown that neurons of the lumbar spinal cord undergo mitosis between the 12th and 16th day of gestation with the majority proliferating between Embryonic (E) Days El3 and El5 (1,16,18). It 1 To whom
correspondence
should
KNIGHT
be addressed. 187
All
0014-4886/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
188
NANDI,
BEAL,
To determine the exact duration of the neurogenic period of ascending tract neurons, the present study combined tritiated thymidine autoradiography with the application of a retrograde fluorescent tracer, Fluoro-Gold, to simultaneously determine neuronal birthdates (date of final cell division) and neuronal projection, respectively. MATERIALS
AND
AND
KNIGHT
rons labeled with both Fluoro-Gold and tritiated thymidine was counted and subtracted from the total number of neurons labeled with Fluoro-Gold. Over 1500 cells were examined and counted at each stage of development. To avoid counting the same cell on consecutive sections, cells were examined only on alternate sections in each series and only when sectioned through the nucleus of the cell.
METHODS RESULTS
Twelve female rats of the Wistar strain between 150 and 300 days of age were mated and checked for sperm each morning with an intravaginal rinse of phosphatebuffered saline (PBS). The first appearance of sperm in the vagina was defined as the date of conception or Embryonic (E) Day 1. Each pregnant rat was anesthetized via metaphane inhalation and then given an intraperitoneal injection of tritiated thymidine. Three rats were injected on each of Days E13-E16. Tritiated thymidine was purchased from ICN Pharmaceuticals, Inc., with a specific activity of 6.7 Ci/mmole with 1.0 mCi dissolved in 1.0 ml of sterile saline. Total dosage was 5 &i/g body wt (18). Pups of the dams were delivered and raised until 40 to 50 days of age. At this stage, two pups from each of the dams were anesthetized with an intramuscular injection of ketamine hydrochloride (44 mg/kg body wt) and xylazine (5 mg/kg). Under aseptic conditions, a laminectomy was performed over cervical segment C3. A small piece of razor blade was cut and shaped to fit the surgical area. The razor was then positioned and held with a fine forceps and a hemisection was performed at spinal segment C3. A 2.0% solution of Fluoro-Gold (Fluorochrome, Inc.) in sterile saline was applied to a small wedge of Gelfoam and inserted in the severed region of the cord. The edges of the dural incision were approximated and the laminectomy was plugged with Gelfoam. Muscle flaps were then sutured and the wound was closed with sterile autoclips. Each animal was maintained on procaine penicillin (30,000 units/g body wt every 48 h) until the time of sacrifice at 10 to 14 days following surgery. At the time of sacrifice, the pups were anesthetized with ketamine hydrochloride (120 mg/kg) and xylazine (5 mg/kg), then intracardially perfused through the left ventricle with 100 ml of warm PBS containing heparin (200 USP units/ml) and 1.0% procaine hydrochloride followed by 500 ml of 4.0% paraformaldehyde in 0.1 A4 phosphate buffer. Spinal cords were removed and the Ll and L2 spinal cord segments were dehydrated, embedded in paraffin, serially sectioned at 7.0 to 10 pm, and deparaffinized. Slides were then dipped in Ilford K5 nuclear emulsion, exposed in the dark for 50 days at 5”C, and then developed with Kodak D-19. Slides were coverslipped with Aqua-Mount (Lerner) and examined under the light and fluorescent microscope. TO establish the proportion of relay neurons proliferating at each stage of development, the number of neu-
Microscopic examination of transverse sections of the Ll and L2 spinal cord segments at each stage of development revealed neuronal nuclei which were labeled with tritiated thymidine. The nuclei displayed discrete clusters of silver granules with little background fog. Examination under the fluorescent microscope revealed large, medium, and small neurons labeled with Fluoro-Gold (Fig. la). Fluoro-Gold was observed in the cell bodies and proximal dendrites of the labeled neurons. Fluoro-Gold-stained neurons were observed on both the side ipsilateral and the side contralateral to the lesion. The location, distribution, and relative number of stained neurons conformed to those described previously for long tract neurons, as reviewed by Brown (7) and Willis and Coggeshall(22). The majority of the projection (relay) neurons was observed in laminae IV, V, VII, and X. A moderate number were observed in laminae I, III, and VIII and the lateral spinal nucleus. Double labeled neurons, i.e., neurons labeled with both Fluoro-Gold and tritiated thymidine, were observed on Days El3 through El5 (Fig. 2). The percentage of double labeled relay cells ranged from 89 to 92% on Day E13,35 to 37% on Day E14, and 1.0 to 4.0% on Day El5 (Fig. 3). None were observed on Day E16. The final group of projection neurons to complete neurogenesis, those on Day E15, was comprised of both small and medium size cells (Figs. lb-ld). Small cells were defined as those with cell body diameters of 15 pm or less, while medium size neurons were those with diameters greater than 15 pm. Small to medium size double labeled cells were found in both laminae III and IV and the nucleus dorsalis on Day E15. Some medium size, double labeled cells in lamina IV measured up to 30 pm in diameter with nuclei measuring 20 pm in diameter. Small double labeled cells were also observed in laminae V and X on Day E15. Those in lamina V were located in the dorsal one-third of that lamina. Except for the nucleus dorsalis and lamina X, most double labeled neurons on Day El5 were observed in the lateral half of the dorsal horn. Also, most double labeled cells on Day El5 were located on the side ipsilateral to the lesion (Fig. 2). Since there can be temporal differences in the rate of development between animals of the same litter (l), the final day of relay cell development described here should be clearly defined not only as a function of time but also
NEUROGENESIS
FIG. 1. Photomicrographs (a) Separate tritium-labeled neurons in laminae IV, III, Day E15.63X oil immersion
OF
ASCENDING
illustrate tritiated thymidine autoradiography nucleus (arrowhead) and Fluoro-Gold-stained and V, respectively, labeled with both tritiated objective. Scale bar = 10 pm.
as a function of the number of remaining cells undergoing mitosis. Thus, on the final day of neurogenesis for relay neurons, Day E15, as described in this paper, the total number of dividing neurons of all types in each of laminae IV, V, and VII was between 10 and 30 cells per section. This numerical range is the same as that observed in several sets of Nissl-stained autoradiographs taken from another group of rats available in our labora-
TRACT
189
NEURONS
combined with retrograde cell body (arrow). Medium thymidine and Fluoro-Gold.
fluorescent labeling using Fluoro-Gold. (b), intermediate (c), and small (d) size Tritiated thymidine administered on
tory which were injected with tritiated thymidine on Day E15. Also, similar patterns have been described in the literature in autoradiographic studies where rats were injected with tritiated thymidine on Day El5 (1,16,18). DISCUSSION
The types of neurons which were retrogradely stained with Fluoro-Gold in the present study are all presumed
190
NANDI,
BEAL,
AND
KNIGHT
and labeled with tritiated thymidine at any one stage, were ascending tract neurons undergoing mitosis. These would include supraspinal projections as well as neurons which project to upper cervical and cervicomedullary levels via the spinocervical (7, 22) and spinothalamic tracts (12) to synapse on other relay neurons that project to more rostra1 levels. The present study demonstrates significant findings in both the duration and the spatiotemporal pattern of neurogenesis for spinal relay neurons. With regard to the duration, it is interesting to note that the ascending tract neurons, which make up less than 1.0% of the nerve cell population of the spinal cord (9), continue to proliferate into the final one-third of the neurogenic period for all spinal neurons. This temporal pattern does not conform with the classical view of neurogenesis, which depicts long axon neurons completing neurogenesis well before those with short axons. Results of the present study are more consistent with recent findings in the cerebral cortex where GAD (11) and GABA (14,17) immunoreactive cells, presumed short axon neurons, were found to follow the same temporal pattern of neurogenesis as cortical projection neurons. It is apparent, however, that the spinal relay neurons of the present study differ from the cortical projection neurons in that they complete neurogenesis 1 day prior to the completion of the remainder of the neuronal population. Still, the trend is the same, i.e., the duration of the neurogenic period for projection neurons in both the spinal cord and the cerebral cortex is longer than previously thought. With regard to the spatiotemporal pattern, it is interesting to note that although a general ventral to dorsal gradient has been clearly established for spinal neurogenesis (1,16), the present study shows that relay neurons do not completely follow this gradient. Rather, relay neurons in the most dorsal laminae, I and II, complete neurogenesis on Day E14, 1 day prior to the completion of neurogenesis for relay neurons in more .-
100, 90
FIG. 2. Diagrams of spinal cord segment (Ll) showing the location (laminae I-X) of neurons labeled with both Fluoro-Gold and tritiated thymidine on the side ipsilateral (Ipsi) and the side contralateral (Contra) to the lesion site in rate injected with tritiated thymidine on Days E13, E14, and E15. Cell counts taken from a total of 10 sections; each section 7-10 pm thick.
to be relay neurons which give rise to the long ascending tracts of the spinal cord. Since the hemisections were performed at a high cervical level (C3), the long propriospinal neurons, involved in coordinating fore- and hindlimb movements, were not likely to have been stained since they only extend rostrally as far as C5 (4, 10, 15). It is reasonable to conclude, then, that the neurons which were double labeled, i.e., stained with Fluoro-Gold
80 70 60
al 3 a -0
50 40 30 20 10
,
0L El3
Days
of
iGq* El5
El4
Gestation
FIG. 3. Bar graph showing percentage of neurons labeled both Fluoro-Gold and tritiated thymidine from rats injected with ated thymidine on Days E13, E14, and E15.
with triti-
NEUROGENESIS
OF ASCENDING
ventral laminae of the dorsal horn. The early completion of neurogenesis in the dorsal laminae may be related to the fact that there are relatively few long tract relay neurons in the superficial dorsal horn compared to deeper laminae. On the other hand, the projection target of different types of relay neurons might also be a major factor involved in determining the spatiotemporal gradient. It is interesting to note, for example, that all of the relay neurons undergoing neurogenesis on Day El5 are located in areas which give rise to major cerebellar projections. For that reason, it is highly probable that a large proportion of these neurons are spinocerebellar relay neurons. Studies are underway to test this hypothesis. ACKNOWLEDGMENTS This research was supported in part by a grant from the National Science Foundation (BNS-8908601) and the Louisiana Education Quality Support Fund.
REFERENCES 1. ALTMAN, J., ANLI S. A. BAYER. 1984. The development of the rat spinal cord. Adv. An&. Embryol. Cell Bill. 85: 1-166. 2. ALTMAN, J., AND S. A, BAYER. 1985. Embryonic development in the rat cerebellum. III. Regional differences in the time of origin, migration, and settling of Purkinje cells. J. Comp. Neural. 231: 42-65. 3. ANGEVINJZ,J. B. 1970. Time of neuron origin in the diencephalon of the mouse. An autoradiographic study. J. Comp. Neural 139: 129-188. 4. BARILARI, M. G., AND H. G. J. M. KUYPERS. 1969. Propriospinal fibers interconnecting the spinal enlargements in the cat. Brain Res. 14:321-330. 5. BEAL, J. A., AND H. R. BICKNELL. 1984. Development and maturation of neurons in the substantia gelatinosa (SG) of the rat spinal cord. Pages 23-30 in W. G. Willis and M. J. Rowe, Eds., Development, Organization and Processing in Somatosensory Pathways. A. R. Liss, New York. 6. BICKNELL, H. R., AND J. A. BEAL. 1984. Axonal and dendritic development of substantia gelatinosa neurons in the lumbosacral spinal cord of the rat. J. Comp. Neural. 226: 508-522. 7. BROWN, A. G. 1981. Organization in the Spinul Cord. SpringerVerlag, New York.
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NEURONS
191
8. CHRONWALL, B., AND J. R. WOLFF. 1980. Prenatal and postnatal development of GABA-accumulating cells in the occipital neocortex of the rat. J. Comp. Neural. 190: 187-208. 9. CHUNG, K., G. A. KEE’ITER, W. D. WILLIS, AND R. E. COGGESHALL. 1984. An estimate of the ratio of propriospinal to long tract neurons in the sacral spinal cord of the rat. Neurosci. L&t. 44: 173-177. 10. ENGLISH, A. W., J. TIGGES, AND P. R. LENNAFZD.1985. Anatomical organization of long ascending propriospinal neurons in the cat spinal cord. J. Comp. Neurol. 240: 349-358. 11. FAIREN, A., A. COBAS, AND M. FONSECA. 1986. Times of generation of glutamic acid decarboxylase immunoreactive neurons in mouse somatosensory cortex. J. Comp. Neurol. 25 1: 67-83. 12. GRANUM, S. L. 1986. The spinothalamic system of the rat. I. Locations of cells of origin. J. Comp. Neural. 247: 159-180. 13. HINDS, J. W. 1968. Autoradiographic study of histogenesis in the mouse olfactory bulb. I. Time of origin of neurons and neuroglia. J. Comp. Neural. 134: 287-304. 14. MILLER, M. W. 1985. Cogeneration of retrogradely labeled cortico-cortical projection and GABA-immunoreactive local circuit neurons in cerebral cortex. Dev. Brain Res. 23: 187-192. 15. MOLENAAR, I., AND H. G. J. M. KWPERS. 1978. Cells of origin of propriospinal fibers and of fibers ascending to supraspinal levels. A HRP study in cat and rhesus monkey. Brain Res. 162: 429450. 16. NORNES, H. O., AND G. D. DAS. 1974. Temporal pattern of neurogenesis in spinal cord of rat. I. An autoradiographic study-Time and sites of origin, migration and settling patterns of neuroblasts. Brain Res. 73: 121-138. neurons l7* PEDUZZI, J. D. 1988. Genesis of GABA-immunoreactive in the ferret visual cortex. J. Neurosci. 8: 920-931. 18. PENDERGRAST, K. R., AND J. A. BEAL. 1986. An autoradiographic and Golgi analysis of the development of neurons in the superflcial dorsal horn (SDH) of the rat. Anat. Rec. 214: 99A. RAKIC, P., AND R. S. NOWAKOWSKI. 1981. The time of origin of 19. neurons in the hippocampal region of the Rhesus monkey. J. Comp. Neural. 196: 99-128. 20. SNYDER, R. L., R. L. M. FAULL, AND W. R. MEHLER. 1978. A comparative study of the neurons of origin of the spinocerebellar afferents in the rat, cat and squirrel monkey based on the retrograde transport of horseradish peroxidase. J. Comp. Neural. 181:833-852. 21. TABER PIERCE, E. 1967. Histogenesis of the dorsal and ventral cochlear nuclei in the mouse. An autoradiographic study. J. Comp. Neural. 131: 27-54. 22. WILLIS, W. D., AND R. E. COGGESHALL. 1978. Sensory Mechanisms of the Spinal Cord. Plenum, New York/London.
107,187-l%
(1990)
Neurogenic Period of Ascending Tract Neurons in the Upper Lumbar Spinal Cord of the Rat KAILAS N. NANDI, JOHN A. BEAL,~ AND DAVID S. Department
of
Cellular Biology and Anatomy, L.ouisiunu State University Medical Center in Shreveport, P.O. Box 33932, Shreveport, Louisiana 71130
has also been shown that spinal neurogenesis proceeds along gradients which extend from rostra1 to caudal, lateral to medial, and ventral to dorsal (1,16). It has been suggested that the pattern of proliferation along each gradient is such that ascending sensory (relay) neurons complete proliferation well before neurons with short projections, i.e., before propriospinal and local circuit neurons (1). There is considerable evidence to support this theory. First, large spinal neurons complete neurogenesis prior to smaller neurons (1,16,18). Second, long axon neurons, demonstrated in Golgi impregnation studies of the developing spinal cord, were found sprouting axons and dendrites, while short axon neurons were still proliferating ($6). Third, autoradiographic studies using tritiated thymidine in the diencephalon (3), cochlear nuclei (21), and olfactory bulb (13) of the mouse, the cerebellum (2) and occipital neocortex (8) of the rat, and the hippocampus of the monkey (19) have shown that long axon projection neurons complete neurogenesis prior to neurons with short axons. Given the above evidence, then, it seems reasonable to conclude that ascending tract (relay) neurons probably complete neurogenesis prior to spinal neurons with shorter projections. Although it is not known when the neurogenesis of relay neurons is completed, judging from the fact that neurons of the ascending tracts make up less than 1.0% of all spinal neurons (9), it would seem reasonable to further conclude that the proliferation of these neurons would be completed early in the neurogenie period. However, tritiated thymidine autoradiographs of adult lumbar spinal cord, available in our laboratory, revealed that some medium size nerve cell bodies in lamina IV and the nucleus dorsalis, suspected of being ascending tract neurons because of their relatively large size, were still taking up tritiated thymidine when injected in the final one-third of the proliferative period as late as Day 15 of gestation. This phenomenon coupled with recent evidence which demonstrates that GABAergic interneurons of the cerebral cortex proliferate at the same time as the cortical projecting neurons (11,14, 17) suggested that a reevaluation of spinal neurogenesis was in order.
Although the neurogenic period for neurons in the lumbar spinal cord has been clearly established (Days 12 through 16 of gestation), it is not known when the neurogenesis of ascending tract neurons is completed within this period. The purpose of the present study was to determine the duration of the neurogenic period for projection neurons of the ascending tracts. To label neurons undergoing mitosis during this period, tritiated thymidine was administered to fetal rats on Embryonic (E) Days El3 through El6 of gestation. Ascending tract neurons of the lumbar cord were later (Postnatal Days 40-50) labeled in each animal with a retrograde tracer, Fluoro-Gold, applied at the site of a hemisection at spinal cord segment C3. Ascending tract neurons which were undergoing mitosis in the upper lumbar cord were double labeled, i.e., labeled with both tritiated thymidine and Fluoro-Gold. On Day E13,8992% of the ascending tract neurons were double labeled; on Day E14, 35-37%; and on Day E15, l-4%. Results showed, then, that some ascending tract neurons were double labeled through Day El5 and were, therefore, proliferating in the final one-third of the neurogenic period. Ascending tract neurons proliferating on Day El5 were confined to laminae III, IV, V, and X and the nucleus dorsalis. Long tract neurons in the superficial dorsal horn (laminae I and II), on the other hand, were found to have completed neurogenesis on Day El4 of gestation. Results of the present study show that spinal neurogenesis of ascending projection neurons continues throughout most of the neurogenic period and does not completely follow the well-established ventral to dorsal gradient. o 1990 Academic PRESS, I~C.
INTRODUCI-ION
Studies on neurogenesis in the rat using tritiated thymidine autoradiography have shown that neurons of the lumbar spinal cord undergo mitosis between the 12th and 16th day of gestation with the majority proliferating between Embryonic (E) Days El3 and El5 (1,16,18). It 1 To whom
correspondence
should
KNIGHT
be addressed. 187
All
0014-4886/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
188
NANDI,
BEAL,
To determine the exact duration of the neurogenic period of ascending tract neurons, the present study combined tritiated thymidine autoradiography with the application of a retrograde fluorescent tracer, Fluoro-Gold, to simultaneously determine neuronal birthdates (date of final cell division) and neuronal projection, respectively. MATERIALS
AND
AND
KNIGHT
rons labeled with both Fluoro-Gold and tritiated thymidine was counted and subtracted from the total number of neurons labeled with Fluoro-Gold. Over 1500 cells were examined and counted at each stage of development. To avoid counting the same cell on consecutive sections, cells were examined only on alternate sections in each series and only when sectioned through the nucleus of the cell.
METHODS RESULTS
Twelve female rats of the Wistar strain between 150 and 300 days of age were mated and checked for sperm each morning with an intravaginal rinse of phosphatebuffered saline (PBS). The first appearance of sperm in the vagina was defined as the date of conception or Embryonic (E) Day 1. Each pregnant rat was anesthetized via metaphane inhalation and then given an intraperitoneal injection of tritiated thymidine. Three rats were injected on each of Days E13-E16. Tritiated thymidine was purchased from ICN Pharmaceuticals, Inc., with a specific activity of 6.7 Ci/mmole with 1.0 mCi dissolved in 1.0 ml of sterile saline. Total dosage was 5 &i/g body wt (18). Pups of the dams were delivered and raised until 40 to 50 days of age. At this stage, two pups from each of the dams were anesthetized with an intramuscular injection of ketamine hydrochloride (44 mg/kg body wt) and xylazine (5 mg/kg). Under aseptic conditions, a laminectomy was performed over cervical segment C3. A small piece of razor blade was cut and shaped to fit the surgical area. The razor was then positioned and held with a fine forceps and a hemisection was performed at spinal segment C3. A 2.0% solution of Fluoro-Gold (Fluorochrome, Inc.) in sterile saline was applied to a small wedge of Gelfoam and inserted in the severed region of the cord. The edges of the dural incision were approximated and the laminectomy was plugged with Gelfoam. Muscle flaps were then sutured and the wound was closed with sterile autoclips. Each animal was maintained on procaine penicillin (30,000 units/g body wt every 48 h) until the time of sacrifice at 10 to 14 days following surgery. At the time of sacrifice, the pups were anesthetized with ketamine hydrochloride (120 mg/kg) and xylazine (5 mg/kg), then intracardially perfused through the left ventricle with 100 ml of warm PBS containing heparin (200 USP units/ml) and 1.0% procaine hydrochloride followed by 500 ml of 4.0% paraformaldehyde in 0.1 A4 phosphate buffer. Spinal cords were removed and the Ll and L2 spinal cord segments were dehydrated, embedded in paraffin, serially sectioned at 7.0 to 10 pm, and deparaffinized. Slides were then dipped in Ilford K5 nuclear emulsion, exposed in the dark for 50 days at 5”C, and then developed with Kodak D-19. Slides were coverslipped with Aqua-Mount (Lerner) and examined under the light and fluorescent microscope. TO establish the proportion of relay neurons proliferating at each stage of development, the number of neu-
Microscopic examination of transverse sections of the Ll and L2 spinal cord segments at each stage of development revealed neuronal nuclei which were labeled with tritiated thymidine. The nuclei displayed discrete clusters of silver granules with little background fog. Examination under the fluorescent microscope revealed large, medium, and small neurons labeled with Fluoro-Gold (Fig. la). Fluoro-Gold was observed in the cell bodies and proximal dendrites of the labeled neurons. Fluoro-Gold-stained neurons were observed on both the side ipsilateral and the side contralateral to the lesion. The location, distribution, and relative number of stained neurons conformed to those described previously for long tract neurons, as reviewed by Brown (7) and Willis and Coggeshall(22). The majority of the projection (relay) neurons was observed in laminae IV, V, VII, and X. A moderate number were observed in laminae I, III, and VIII and the lateral spinal nucleus. Double labeled neurons, i.e., neurons labeled with both Fluoro-Gold and tritiated thymidine, were observed on Days El3 through El5 (Fig. 2). The percentage of double labeled relay cells ranged from 89 to 92% on Day E13,35 to 37% on Day E14, and 1.0 to 4.0% on Day El5 (Fig. 3). None were observed on Day E16. The final group of projection neurons to complete neurogenesis, those on Day E15, was comprised of both small and medium size cells (Figs. lb-ld). Small cells were defined as those with cell body diameters of 15 pm or less, while medium size neurons were those with diameters greater than 15 pm. Small to medium size double labeled cells were found in both laminae III and IV and the nucleus dorsalis on Day E15. Some medium size, double labeled cells in lamina IV measured up to 30 pm in diameter with nuclei measuring 20 pm in diameter. Small double labeled cells were also observed in laminae V and X on Day E15. Those in lamina V were located in the dorsal one-third of that lamina. Except for the nucleus dorsalis and lamina X, most double labeled neurons on Day El5 were observed in the lateral half of the dorsal horn. Also, most double labeled cells on Day El5 were located on the side ipsilateral to the lesion (Fig. 2). Since there can be temporal differences in the rate of development between animals of the same litter (l), the final day of relay cell development described here should be clearly defined not only as a function of time but also
NEUROGENESIS
FIG. 1. Photomicrographs (a) Separate tritium-labeled neurons in laminae IV, III, Day E15.63X oil immersion
OF
ASCENDING
illustrate tritiated thymidine autoradiography nucleus (arrowhead) and Fluoro-Gold-stained and V, respectively, labeled with both tritiated objective. Scale bar = 10 pm.
as a function of the number of remaining cells undergoing mitosis. Thus, on the final day of neurogenesis for relay neurons, Day E15, as described in this paper, the total number of dividing neurons of all types in each of laminae IV, V, and VII was between 10 and 30 cells per section. This numerical range is the same as that observed in several sets of Nissl-stained autoradiographs taken from another group of rats available in our labora-
TRACT
189
NEURONS
combined with retrograde cell body (arrow). Medium thymidine and Fluoro-Gold.
fluorescent labeling using Fluoro-Gold. (b), intermediate (c), and small (d) size Tritiated thymidine administered on
tory which were injected with tritiated thymidine on Day E15. Also, similar patterns have been described in the literature in autoradiographic studies where rats were injected with tritiated thymidine on Day El5 (1,16,18). DISCUSSION
The types of neurons which were retrogradely stained with Fluoro-Gold in the present study are all presumed
190
NANDI,
BEAL,
AND
KNIGHT
and labeled with tritiated thymidine at any one stage, were ascending tract neurons undergoing mitosis. These would include supraspinal projections as well as neurons which project to upper cervical and cervicomedullary levels via the spinocervical (7, 22) and spinothalamic tracts (12) to synapse on other relay neurons that project to more rostra1 levels. The present study demonstrates significant findings in both the duration and the spatiotemporal pattern of neurogenesis for spinal relay neurons. With regard to the duration, it is interesting to note that the ascending tract neurons, which make up less than 1.0% of the nerve cell population of the spinal cord (9), continue to proliferate into the final one-third of the neurogenic period for all spinal neurons. This temporal pattern does not conform with the classical view of neurogenesis, which depicts long axon neurons completing neurogenesis well before those with short axons. Results of the present study are more consistent with recent findings in the cerebral cortex where GAD (11) and GABA (14,17) immunoreactive cells, presumed short axon neurons, were found to follow the same temporal pattern of neurogenesis as cortical projection neurons. It is apparent, however, that the spinal relay neurons of the present study differ from the cortical projection neurons in that they complete neurogenesis 1 day prior to the completion of the remainder of the neuronal population. Still, the trend is the same, i.e., the duration of the neurogenic period for projection neurons in both the spinal cord and the cerebral cortex is longer than previously thought. With regard to the spatiotemporal pattern, it is interesting to note that although a general ventral to dorsal gradient has been clearly established for spinal neurogenesis (1,16), the present study shows that relay neurons do not completely follow this gradient. Rather, relay neurons in the most dorsal laminae, I and II, complete neurogenesis on Day E14, 1 day prior to the completion of neurogenesis for relay neurons in more .-
100, 90
FIG. 2. Diagrams of spinal cord segment (Ll) showing the location (laminae I-X) of neurons labeled with both Fluoro-Gold and tritiated thymidine on the side ipsilateral (Ipsi) and the side contralateral (Contra) to the lesion site in rate injected with tritiated thymidine on Days E13, E14, and E15. Cell counts taken from a total of 10 sections; each section 7-10 pm thick.
to be relay neurons which give rise to the long ascending tracts of the spinal cord. Since the hemisections were performed at a high cervical level (C3), the long propriospinal neurons, involved in coordinating fore- and hindlimb movements, were not likely to have been stained since they only extend rostrally as far as C5 (4, 10, 15). It is reasonable to conclude, then, that the neurons which were double labeled, i.e., stained with Fluoro-Gold
80 70 60
al 3 a -0
50 40 30 20 10
,
0L El3
Days
of
iGq* El5
El4
Gestation
FIG. 3. Bar graph showing percentage of neurons labeled both Fluoro-Gold and tritiated thymidine from rats injected with ated thymidine on Days E13, E14, and E15.
with triti-
NEUROGENESIS
OF ASCENDING
ventral laminae of the dorsal horn. The early completion of neurogenesis in the dorsal laminae may be related to the fact that there are relatively few long tract relay neurons in the superficial dorsal horn compared to deeper laminae. On the other hand, the projection target of different types of relay neurons might also be a major factor involved in determining the spatiotemporal gradient. It is interesting to note, for example, that all of the relay neurons undergoing neurogenesis on Day El5 are located in areas which give rise to major cerebellar projections. For that reason, it is highly probable that a large proportion of these neurons are spinocerebellar relay neurons. Studies are underway to test this hypothesis. ACKNOWLEDGMENTS This research was supported in part by a grant from the National Science Foundation (BNS-8908601) and the Louisiana Education Quality Support Fund.
REFERENCES 1. ALTMAN, J., ANLI S. A. BAYER. 1984. The development of the rat spinal cord. Adv. An&. Embryol. Cell Bill. 85: 1-166. 2. ALTMAN, J., AND S. A, BAYER. 1985. Embryonic development in the rat cerebellum. III. Regional differences in the time of origin, migration, and settling of Purkinje cells. J. Comp. Neural. 231: 42-65. 3. ANGEVINJZ,J. B. 1970. Time of neuron origin in the diencephalon of the mouse. An autoradiographic study. J. Comp. Neural 139: 129-188. 4. BARILARI, M. G., AND H. G. J. M. KUYPERS. 1969. Propriospinal fibers interconnecting the spinal enlargements in the cat. Brain Res. 14:321-330. 5. BEAL, J. A., AND H. R. BICKNELL. 1984. Development and maturation of neurons in the substantia gelatinosa (SG) of the rat spinal cord. Pages 23-30 in W. G. Willis and M. J. Rowe, Eds., Development, Organization and Processing in Somatosensory Pathways. A. R. Liss, New York. 6. BICKNELL, H. R., AND J. A. BEAL. 1984. Axonal and dendritic development of substantia gelatinosa neurons in the lumbosacral spinal cord of the rat. J. Comp. Neural. 226: 508-522. 7. BROWN, A. G. 1981. Organization in the Spinul Cord. SpringerVerlag, New York.
TRACT
NEURONS
191
8. CHRONWALL, B., AND J. R. WOLFF. 1980. Prenatal and postnatal development of GABA-accumulating cells in the occipital neocortex of the rat. J. Comp. Neural. 190: 187-208. 9. CHUNG, K., G. A. KEE’ITER, W. D. WILLIS, AND R. E. COGGESHALL. 1984. An estimate of the ratio of propriospinal to long tract neurons in the sacral spinal cord of the rat. Neurosci. L&t. 44: 173-177. 10. ENGLISH, A. W., J. TIGGES, AND P. R. LENNAFZD.1985. Anatomical organization of long ascending propriospinal neurons in the cat spinal cord. J. Comp. Neurol. 240: 349-358. 11. FAIREN, A., A. COBAS, AND M. FONSECA. 1986. Times of generation of glutamic acid decarboxylase immunoreactive neurons in mouse somatosensory cortex. J. Comp. Neurol. 25 1: 67-83. 12. GRANUM, S. L. 1986. The spinothalamic system of the rat. I. Locations of cells of origin. J. Comp. Neural. 247: 159-180. 13. HINDS, J. W. 1968. Autoradiographic study of histogenesis in the mouse olfactory bulb. I. Time of origin of neurons and neuroglia. J. Comp. Neural. 134: 287-304. 14. MILLER, M. W. 1985. Cogeneration of retrogradely labeled cortico-cortical projection and GABA-immunoreactive local circuit neurons in cerebral cortex. Dev. Brain Res. 23: 187-192. 15. MOLENAAR, I., AND H. G. J. M. KWPERS. 1978. Cells of origin of propriospinal fibers and of fibers ascending to supraspinal levels. A HRP study in cat and rhesus monkey. Brain Res. 162: 429450. 16. NORNES, H. O., AND G. D. DAS. 1974. Temporal pattern of neurogenesis in spinal cord of rat. I. An autoradiographic study-Time and sites of origin, migration and settling patterns of neuroblasts. Brain Res. 73: 121-138. neurons l7* PEDUZZI, J. D. 1988. Genesis of GABA-immunoreactive in the ferret visual cortex. J. Neurosci. 8: 920-931. 18. PENDERGRAST, K. R., AND J. A. BEAL. 1986. An autoradiographic and Golgi analysis of the development of neurons in the superflcial dorsal horn (SDH) of the rat. Anat. Rec. 214: 99A. RAKIC, P., AND R. S. NOWAKOWSKI. 1981. The time of origin of 19. neurons in the hippocampal region of the Rhesus monkey. J. Comp. Neural. 196: 99-128. 20. SNYDER, R. L., R. L. M. FAULL, AND W. R. MEHLER. 1978. A comparative study of the neurons of origin of the spinocerebellar afferents in the rat, cat and squirrel monkey based on the retrograde transport of horseradish peroxidase. J. Comp. Neural. 181:833-852. 21. TABER PIERCE, E. 1967. Histogenesis of the dorsal and ventral cochlear nuclei in the mouse. An autoradiographic study. J. Comp. Neural. 131: 27-54. 22. WILLIS, W. D., AND R. E. COGGESHALL. 1978. Sensory Mechanisms of the Spinal Cord. Plenum, New York/London.
