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Proliferation and migration of undifferentiated precursor cells in the rat during postnatal gliogenesis
EXPERIMENTAL
XEUROLOGY
Proliferation
16, 263-278
and
Precursor
(1966)
Migration Cells
Postnatal
of
in the
Undifferentiated
Rat
During
Gliogenesis
JOSEPH ALTMAN] Psychophysiological
Laboratory, Cambridge,
Massachusetts Massachztsetts,
Received
July
institute 02139
of
Technology,
11, 1966
Rats aged 13 days were injected with thymidine-Ha and were killed 6 hours, 1, 3, 6, 12, 20 and 60 days afterwards. Incorporation of this labeled precursor of chromosomal DNA was studied autoradiographically with reference to the origin of neuroglia cells in the neocortex. A large proportion of the subependymal cells of the lateral ventricle were labeled after brief survival, indicating rapid regional proliferation. In animals with longer survival there was considerable increase in the proportion of labeled cells, combined with label dilution. In spite of this evidence of rapid and continuous cell multiplication, the concentration of subependymal cells declined considerably with age, indicating that the bulk of new cells do not stay here. The rate of regional proliferation was lower in the white matter and corpus callosum, though there was a considerable increase in the proportion of labeled cells with prolonged survival. Significantly, the addition of this large number of new cells did not lead to a net increase in the total cell population. These facts imply that many of the cells found in these fibrous regions are migratory ones, and as new cells arrive here older ones depart. Finally, in the gray matter of the cortex regional cell proliferation was low at this age, but there was a steady gain in the proportion of labeled cells with increased survival time. On the basis of these findings it is concluded that the cells formed around the lateral ventricle, which are destined to reach the cortex, utilize the white matter and corpus callosum as a migratory pathway. Introduction
The recent introduction of thymidine-H3 autoradiography has made it possibleto investigate aspectsof morphogenesisthat could not be adequately studied in the past with the available histological techniques. Thymidine is a specific precursor of chromosomal DNA and it is incorporated selectively into the newly synthesized DNA strands whenever the chromosomecomplement of the nucleus is duplicated. If animals are injected with tritiated thymidine, the cells that are preparing to undergo mitosis will tend to in1 This research is supported by assistance of William J. Anderson, acknowledged.
the United States Atomic Energy Commission. The Gopal D. Das, and Nicole Poussant is gratefully
263
264
ALTMAN
corporate the radioactively-labeled precursor and the cells formed immediately after the injection will therefore become tagged. These new cells can then be easily identified with fine-resolution autoradiography. This is a technique in which a radiation-sensitive emulsion is applied directly to the tissue section, and the location of the radioactive particles with respect to the apposed cellular material is revealed, after a suitable period of photographic exposure and of development of the latent image, by the location of the reduced silver grains. The sites of cell multiplication, and the regional rates of proliferation, can be established in animals at different stages of their development by killing them within a short period (about l-6 hours) after injection. As animals are killed at progressively longer intervals after injection, the fate of the initially labeled cells, such as their migrations, rates of remultiplication, cellular transformations, and life span, can also be determined. Thymidine-H3 autoradiography has been applied recently with considerable success in investigations dealing with the prenatal development of the central nervous system (9-11, 15, 18-20, 23) and its postnatal maturation 2, 3, 6-9, 17, 21, 22). In our laboratory, the results of a series of autoradiographic studies in which rats of different ages from neonates to mature adults were injected with thymidine-H” and were then allowed to survive for different periods after injection, are being analyzed. The origin and migration of neuroblasts in various regions of the brain that give rise to short-axoned neurons or microneurons have been reported (5, 7, 8). The present paper concerns gliogenesis in the same material. Materials
and
Methods
Material obtained from laboratory-bred, Long-Evans hooded rats, aged 13 days, was analyzed in this study. Fourteen animals were injected intraperitoneally with thymidine-H 3, 10 uc/g body weight (the radiochemical was dissolved in isotonic saline, 1.0 me/ml; specific activity was 6.7 c/millimole). Pairs of the injected animals were killed by cardiac perfusion with 105% neutral formalin after the following survival periods: 6 hours; 1, 3, 6, 12, 20 and 60 days. The removed brains were further fixed in formalin, and then were dehydrated and embedded in Paraplast. Serial coronal sections were cut at 6 u, and three consecutive sections (or set of sections) were preserved out of every twenty (in the younger animals) or thirty. Of the preserved sections, two sets were stained with gallocyanin chromalum and one of these was coated in the darkroom with melted NTB-3 nuclear emulsion by the dipping technique. The dried sections were exposed in lightproof boxes supplied with a desiccant in a refrigerator at 5 C for 91 days and developed in the usual manner (4). Three structures were evaluated microscopically: The ependymal and sub-
POSTNATAL
GLIOGENESIS
265
ependymal layers of the lateral wall of the anterior horn of the lateral ventricle; the white matter and corpus callosum of the dorsal neocortex; a.nd the gray matter of the dorsal neocortex. Cell counting and classification, at a magnification of 640 X, were done in the following manner. The total cell population of the lateral wall of the ependymal and subependymal layers surrounding the lateral aspect of the lateral ventricle was determined in all animals at matched coronal levels through the septal nuclei at approximately the coordinate, A6.2 (14). In this region the proportion of labeled to unlabeled (“new” and “old”) was also determined by classifying 1000 cells in each animal. To determine the proportion of labeled and unlabeled cells in the cerebral white matter, four homologous coronal levels were scanned in all animals. These were at the level of the superior colliculus (A1.O) ; the interventricular foramen of Monro (A6.2) ; the septal nuclei (A8.2)) and the anterior pole (A9.8). In all these sections the total number of cells, and the proportion of labeled to unlabeled cells, was determined in homologous portions of the dorsal cortex in square areas of 182 p, with ten replications, in all animals. In addition, the cells in the midline corpus callosum were counted and classified in a similar manner at the level of the habenular nuclei (A3.8). In a similar manner, the total number of cells, and the proportion of labeled cells, was determined for the gray matter of the dorsal neocortex in three coronal levels, at coordinates Al.0, A6.2, and A9.8. As described in greater detail elsewhere (S), alterations in the number of labeled cells and in the degree of label concentration within cells, as functions ct survival time after injection, can be used as reliable indices of regional rates of cell proliferation. For instance, in rapidly and continuously proliferating regions the number of labeled cells rises rapidly for a short period after injection, due to the remultiplication of the labeled cells. Since remultiplication of labeled cells is combined with progressive dilution of label concentration within cells, more and more cells lose their tagging after a time and the number of labeled cells begins to fall as a consequence in such populations. To determine the degree of label concentration within cells, cells with overlying blackened silver grains can be conveniently classified as (a) all opaque, (b) mostly opaque (these represent the “intensely-labeled cells”) ; and as (c) mostly light, and (d) very light (these represent the “lightlylabeled cells”). Results
The Germinal Matrix of the Lateral Ventricle. An appreciable subependyma1 matrix, with many labeled, cells, is present in 13-day-old rats in the lateral wall of the anterior horn of the lateral ventricle. The typical distribution of thymidine-H3-labeled cells in this region is illustrated in Fig. 1. At this stage of development, cell proliferation has practically come to an end in
266
ALTMAN
the medial wall and part of the dorsal roof of the lateral ventricle, where single-cell-thick ependymal layers surround the lumen of the ventricle. These cells, whose nuclei stain lightly, are generally unlabeled. In contrast, a subependymal layer composed of many cells with darkly staining nuclei surround the ependymal wall on the lateral side of the ventricle. In animals with brief (6 hours) survival after injection, the majority of the pale cells of the ependymal layer are not labeled, whereas a considerable proportion
1. Photomicrograph of lateral ventricle, from a rat 24 hours later. Re!atively few Numerous labeled cells are seen band (SB). BG, basal ganglia; Gallocyanin chromalum; x 50. FIG.
the
an autoradiogram in the region of the injected with thymidine-Hs at 13 days of the cells of the ependymal layer in the subependymal layer (SE) and LV, lumen of CC, corpus callosum;
anterior horn of of age and killed (E) are labeled. in the subcallosal lateral ventricle.
of the darkly-staining cell nuclei of the subependymal layer are intensely labeled. Many labeled cells may also be observed in the dorsolateral arm of the subependymal layer, which is composed of a band or stream of cells interposed between the ventral aspect of the corpus callosum and the dorsal aspect of the basal ganglia. These cells apparently represent migratory elements which are moving outward from the subependymal layer, but since many of these cells are labeled even after the shortest survival period (1 hour), it is apparent that they retain their proliferative capacity to a considerable extent.
POSTNATAL
267
GLIOGENESIS
The cell population of the ependymal and subependymal layers of the lateral wall of the anterior horn of the lateral ventricle declines with increasing age. The total number of this cell population in animals aged 13, 14, 16, 19, 25, 33 and 73 days is plotted in Fig. 2, where each point represents the average of counts made on the right and left lateral ventricles in AGE
R,, f
3
OF ANIMALS
6
20
12 SURVIVAL
AFTER
INJECTION
IN
I.
60
DAYS
FIG. 2. The total number of ependymal and subependymal cells in homologous regions of the lateral wall of the anterior horn of the lateral ventricle as a function of age. The percentage of labeled cells as a function of survival time after injection at 1.3 days of age is &so plotted.
pairs of animals. A considerable drop in the cell population is seen in the 16-day-old animals, the number of cells is essentially unchanged between 16 and 25 days, then it drops again in the older animals. In the same graph I also plotted the changes in the percentage of labeled cells in the same region. After 6-hour survival, 30 and 415% of the germinal cells of the lateral ventricle are labeled, indicating a very high rate of cell multiplication at this period. The majority of these cells, as shown in Fig. 3, are intensely labeled. With 24hour survival, the proportion of intensely-labeled cells declines, but the total number of labeled cells increases to 50 and 5270, with a growing proportion of less-intensely labeled cells. The proportion of intensely-labeled
268
ALTMAN
cells is drastically reduced by the third day after injection, but due to continuing rapid cell multiplication of the initially labeled cells, the total proportion of labeled cells increases to 62 and 67%, the great majority with greatly reduced grain concentration. This survival period marked the asymptote level in the concentration of labeled cells. From this date onward, the proportion of labeled cells declines considerably, and reaches the low level %‘ 90
-
LATERAL
VENTRICLE
p/ P : : ,:
80 70
-
,,t’
,o
,#S’
60
:
0 ---I-O v0 ‘-I*--
.JO--- ..___ __-*se*
0
all opaque
lntenrely
00
mostly light mostly light very opaque 1' lightly
. . ..___g, __,,,,..
labeled labeled
50
40
30
20
m-m------
IO
SURVIVAL
FIG.
matrix
3. Percentage of the lateral
AFTER
INJECTION
IN
DAYS
distribution of intenselyand tightly-labeled ventricle as a function of survival time after
cells in the injection.
germinal
of 10% by the sixtieth day after injection; practically all of these are lightly labeled. These two facts, a high rate of cell proliferation, on the one hand, and a simultaneous decrease in the absolute number of cells, on the other, clearly indicate that the cells produced at a rapid rate at this site do not remain in this region but migrate to other areas of the brain. A reasonable assumption is that the migration route is the subcallosal band of cells previously described. This band consists of a large concentration of cells near the subependymal layer which gradually thins out laterally. Accordingly, I have tested the hypothesis that some of the migratory cells located in this band move by way of the adjacent cerebral white matter and corpus callosum to the cortex.
POSTNATAL
Cerebral White Matter
269
GLIOGENESIS
and Corflus Callosum. In every animal, at five co-
ronal levels, cells were counted and classified in homologous regions of the white matter and corpus callosum in the dorsal aspect of the neocortex. In all instances ten adjacent square areas of 182 p were sampled. The total number of cells obtained in the sampled areas, and the proportion of labeled, or “new,” cells are plotted in Figs. 4-8. A comparison of the data shows considerable, though nonsystematic, variability from animal to animal at differF WHITE
900
MATTER
Level, A 1.0 800
l -m
total
A.-.
A number
x 1s..
x
number of cells of labeled
percentage
cells
of lobeled
cells
700
2ii
600
” B 500 z 2 4oc z 3oc
2oc
100
SURVIVAL
FIG.
sampled
4.
AFTER
INJECTION
IN
DAYS
Total number of cells, and the number and percentage of labeled cells, in ten square areas of 182 k in the cerebral white matter at the coronal level of A1.O.
ent coronal levels, and between animals of different ages at identical levels. The averaged cell density from the five levels is plotted in Fig. 9, which indicates that the cell density of the white matter and corpus callosum does not substantially change between 13 and 73 days of age.’ In Figs. 4-9 I also plotted the number of labeled cells encountered in the 2 The variability obtained in these counts may be due to experimental errors. For instance, at the posterior coronal level (A1.O) higher cell density was obtained in the oldest group, whereas at a more anterior level (A8.2) the cell density in the oldest group was lower than in some of the younger groups. Two obvious sources of experimental error could he the variable shrinkage of the embedded tissue, and discrepancies in the matching of coronal levels in the different age groups.
270
ALTMAN
different age groups. The labeled cells represent the new elements of the population, those that were produced after injection of the radiochemical. These new cells may be products of local proliferation of cells or of the invasion of cells from other proliferative sites. The labeled cells seen in the white matter after brief survival following injection (6 hours) can be attributed to local cell proliferation. The rate of local proliferation appeared to be relatively low, ranging from 5 or 6% at the posterior level (A1.O) to
WHITE Level,
MATTER A 6.2 6-0
total
A.-.A
number
number
percentage
8,s;
3
6
of cells
of labeled
80
cells
of labeled
cells
12 SURVIVAL
AFTER
INJECTION
FIG. 5. TotaI number of cells, and the number white matter at the coronal level of A6.2.
IN
DAYS
and percentage
of labeled
celIs, in the
7% at an anterior level (A8.2). This rate contrasts sharply with the 30 and 41% of labeled cells seen after the same survival period around the lateral wall of the lateral ventricle. In spite of this low rate of local cell multiplication, there was a rapid increase in the population of labeled cells with increasing age, reaching an asymptote level in the range of 43 to 57% 12 days after injection, with a mean value of 49% of labeled, or newlyformed, cells 12 days after injection. This considerable increase can partly be attributed to continuing local cell multiplication, but the steep rise in the number of labeled cells as early as 24 hours after injection (which is pro-
POSTNATAL
271
GLIOGENESIS
portionally much higher than that seen in the ventricular germinal zone), and the relatively low rate of label dilution within cells, suggest that a large proportion of the newly acquired cells must come from other regions of the brain. The assumption of a migratory process is further supported by the fact that notwithstanding the addition of such a large percentage of new cells to the sampled areas in the white matter and corpus callosum, the total cell population does not increase. From these findings I conclude that as new
#
900
-
WHITE Level,
800
MATTER A 8.2
FIG.
total
AX--A
number
x.-en
-
SURVIVAL
white
e-e
AFTER
x
number
INJECTION
6. Total number of cells, and the number matter at the coronal level of A8.2.
of cells
of labeled
percentage
90
cells
of labeled
IN
and
% ‘1
cells
80
DAYS
percentage
of labeled
cells, in the
cells are add.ed to the population older ones are departing, that is, this fibrous region of the brain represents a passageway for migratory cells. If this interpretation is correct, the decline in the number and proportion of labeled cells with prolonged survival may be partly due to continued cell multiplication and concomitant label dilution beyond detectability, but, more importantly, it must be a consequence of the sustained migration of cells and, therefore, the replacement of labeled cells with newer, unlabeled ones. Cerebral Gray Matter. The foregoing results indicate, then, that the cells that multiply at a high rate around the lateral wall of the lateral ventricle leave this proliferative zone of the forebrain by way of the adjacent cerebral
272
ALTMAN
white matter. To determine the final destination of these migratory cells, which do not produce a net increase in the local cell population of the white matter, I investigated changes in the cell population, and in the proportion of labeled cells, in animals of different age groups in the gray matter of the cerebral hemispheres. Scanning through all the layers of the cortex, cells were counted and classified at three coronal levels (A1.O, A6.2 and A9.8) in homologous portions of the dorsal neocortex. The results are plotted in Figs 10-12.
WHITE
MATTER l -e
total
A--WA A---A
number
number
of cells
of labeled
cells
x ,_,, x percentage of labeled
cells
- 10
3
6
FIG.
a’hite
20
12 SURVIVAL
AFTER
INJECTION
7. Total number of cells, and the number matter at the coronal level of A9.8.
IN
II
60
DAYi
and percentage
of labeled
cells, in the
The cell density of the dorsal neocortex, making no distinction here between neurons and neuroglia cells, declined sharply between 13 and 16 days of age at level A1.O, less sharply but distinctly at level A9.8, and not at all clearly at level A6.2. This decline in the packing density of cells, in spite of a considerable increase in the cortex in the absolute number of neuroglia cells, can be attributed to the developmental increase in the volume of the neuropil (12). From the age of 19 days onward the cell density of the neocortex remained essentially unchanged at most levels. In the animals that survived for 6 hours after injection very few labeled cells were seen in the cortex, ranging between 1 and 37%. There was little
POSTNATAL
273
GLIOGENESIS
change in this respect after 24-hour survival, the range increasing slightly to 2-4%. These results indicate a relatively low rate of local cell proliferation at 13 and 14 days of age. However, an appreciable increase in labeled cells was evident by the twelfth day after injection, ranging at different levels in the two animals between 13 and 20%. The low local rate of cell multiplication combined with an appreciable subsequent increase in the population of new cells support the idea that the increase in new cells in this region is due
CORPUS Level,
CALLOSUM A 3.8 e-e A L-W x.s-rx
A., f
3
6
FIG.
8. Total number corpus callosum.
number
of cells
of labeled
percentage
cells
of labeled
cells
12 SURVIVAL
midline
total A number
AFTER
INJECTION
of cells, and the number
IN
DAYS
and percentage
of labeled
cells, in the
to invasion of cells from the underlying white matter. Little change (possibly a slight increase) was observed in the number and proportion of labeled cells from the twelfth day after injection, suggesting that by this time (age of animals 25 days) both local cell proliferation and the invasion of labeled cells has slowed down considerably. Discussion
The subependymal layer of the anterior horn of the lateral ventricle has been identified by several investigators as a germinal zone which remains
274
ALTMAN
mitotically active for some time after birth in various mammals (1, 3, 13, 16, 2 1, 22). The earlier investigators could not establish the fate of the cells formed in this region in adulthood, and it was often argued that this mitotic activity is an abortive phenomenon. With the introduction of thymidine-H3 autoradiography it became possible to follow the life history of these cells, and in a pioneering study Smart (21) concluded tentatively that the cells formed in the subependymal layer in neonatal mice represent spongioblasts
WHITE
900 -
MATTER
Average
from
AND five
CORPUS
‘q
CALLOSUM
levels
800 -
-
mean number
of total cell
--
mean number
of labeled
cells
700 -
ti 600 ” b 500 2 - ii
--
t
i
1
i
3
6
12
20
-I-
& z 400Z
Lo I a.,;
SURVIVAL
FIG.
matter error.
9. Mean number of total and corpus callosum, from
AFTER
INJECTION
IN
II II
60
DAYS
cells, and mean number of labeled cells, in the white five coronal levels. Vertical bars represent the mean
and neuroblasts, which migrate into the brain and become differentiated into neuroglia cells and neurons. The results of Smart suggested that the subependymal cells formed in adult mice no longer migrate into the brain. The migration of newly-formed cells from the anterior horn of the lateral ventricle by way of the subcallosal band was also indicated in my autoradiographic study of cell proliferation in adult rats (3). In subsequent studies (7, 8) suggestive evidence was obtained that a fibrous tract, the fimbria of the hippocampus, serves as a passageway for neuroblasts multiplying in the
POSTNATAL
275
CLIOGENESIS
medial wall of the lateral ventricle; these neuroblasts appeared to move past the pyramidal and polymorph cells of the hippocampus and became differentiated into granule cells of the granular layer of the dentate gyrus. This assumption gained quantitative support in a recent study (5) which showed, in line with present results, that in rats injected with thymidine-HFS at 13 days of age there were over 350 labeled cells in the fimbria in specified sampled fields after 12 days survival, but that the addition of this large num-
# ^^_ IYUU
%
DORSAL
NEOCORTEX
Level, A 1.0
. -80 a-m
total
A---
A number
number
of cell.
of labeled
cells
x . . . . x percentage of labeled 600
-
l
\
r
*,
.
l
SURVIVAL
FIG.
cortical
cells
10.
gray
AFTER
I-
INJECTION
Total number of cells, and the number matter at the coronal level of A1.O.
IN
DAYS
and proportion
of labeled
cells, in the
ber of new cells did not appreciably increase the total cell population of the same region. Accordingly, fibrous tracts can serve as passageways for migratory undifferentiated cells, whether spongioblasts or neuroblasts, in the postnatally developing rat brain. Very few neuroglia cells are present in the cerebral cortex of neonatal rats. The number of neuroglia cells increases rapidly after birth, and this change is reflected quantitatively by the rapidly growing ratio of neuroglia to nerve cell (12). Whereas local cell multiplication in the cortex is very low, there is, at the base of the cerebrum, a large source of multiplying cells around
276
ALTMAN
the outer wall of the lateral ventricle; this suggested the possibility of migration of cells from the latter site to the former. The demonstration that a large proportion of the cells found in the white matter are not interfascicular neuroglia cells but, instead, transitory elements, lends strong support for the assumption of the ventricular origin of spongioblasts and it clarifies the migratory route of these precursors of neuroglia cells.
9DD _
DORSAL
NEOCORTEX -90
Level, A 6.2
200
-
100
-
,,I’X’ h _---e A
a.------R,,f
‘3
6
12 SURVIVAL
FIG. cortical
11.
gray
----
AFTER
INJECTION
Total number of cells, and the number matter at the coronal level of A6.2.
A a-----w
20 IN
*
!’
- IO
60
DAYS
and proportion
of labeled
cells, in the
My quantitative results indicate a very high rate of proliferation for the undifferentiated cells in the ependymal and subependymal layer of the lateral ventricle (30-41% labeled cells after 6-hour survival). As the cells move outward through the white matter this mitotic activity declines considerably (to 57% after 6-hour survival), and the proliferative capacity of these cells decreases further as the cells arrive in the cortex (l-370). This decreasing mitotic activity with increasing distance from the ventricle may reflect the importance of the cerebrospinal fluid system for cell multiplication in the prenatal (and also, presumably, in the postnatal) development of brain tissue. But it is also possible that as the cells get nearer their destination they begin
POSTNATAL
GLIOGENESIS
277
to differentiate and concomitantly lose their proliferative capacity, According to this interpretation, the white matter of the cerebral hemispheres contains, in addition to oligodendrocytes, astrocytes and microglia, also a large complement of undifferentiated, migratory cells with some proliferative capacity. The mode of transformation of these precursor cells will be considered in a subsequent paper.
DORSAL
900
NEOCORTEX
Level. A 9.8
800
80 total
700
number
number
of labeled
percentage
SURVIVAL
FIG. cortical
AFTER
INJECTION
12. Total number of cells, and the number gray matter at the coronal level of A9.8.
IN
of cells cells
of labeled
cells
DAYS
and proportion
of labeled
cells, in the
References 1. 2. 3. 4.
5.
ALLEN, E. 1912. The cessation of mitosis in the central nervous system of the albino rat. J. Comp. Neural. 22: 547-568. Are new neurons formed in the brains of adult mammals? ALTMAN, J. 1962. Science 135: 1127-1128. ALTMAN, J. 1963. Autoradiographic investigation of cell proliferation in the brains of rats and cats. Anat. Record 145: 573-591. ALTMAN, J. 1964. The use of fine-resolution autoradiography in neurological and psychobiological research, pp. 336-359. In “Response of the Nervous System to Ionizing Radiation.” T. J, Haley and R. S. Snider reds.]. Little, Brown, Boston, Massachusetts. ALTMAN, J. Autoradiographic and histological studies of postnatal neurogenesis.
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6.
7. 8.
9.
10 11.
12.
13. 14. 1.5. 16.
17. 18
19.
20.
21.
22.
23.
ALTMAN
II. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in infant rats, with special reference to postnatal neurogenesis in some brain regions. J. Camp. Nercrol. in press. ALTMAN, J., and G. D. DAS. 1964. Autoradiographic examination of the effects of enriched environment on the rate of glial multiplication in the adult rat brain. Nature 204: 1161-1163. ALTMAN, J., and G. D. DAS. 1965. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Camp. Neural. 124: 319-335. ALTMAN, J., and G. D. DAS. 1966. Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats with special reference to postnatal neurogenesis in some brain regions. J. Conzp. Neural. 126: 337-389. ANGEVINE, J. B. 1965. Time of neuron origin in the hippocampal region. An autoradiographic study in the mouse. Exptl. Neurol. Suppl. 2: l-70. ANGEVINE, J. B., and R. L. SIDMAN. 1961. Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature 192: 766-768. BERRY, M. A., A. W. ROGERS, and J. T. EAYRS. 1964. The pattern and mechanism of migration of the neuroblasts of the developing cerebral cortex. J. Anat. 96: 291-292. BRIZZEE, K. R., J. VOGT, and X. KHARETCHKO. 1964. Postnatal changes in glial neuron index with a comparison of methods of cell enumeration in the white rat. Progr. Brain Res. 4: 136-149. BRYANS, W. A. 1959. Mitotic activity in the brain of the adult rat. Anat. Record 133: 65-71. “The Rat Forebrain in Stereotaxic Coordinates.” NoordDE GROOT, J. 1959. Hollandische, Amsterdam. FIJJITA, S. 1963. The matrix ceil and cytogenesis in the developing central nervous system. J. Comp. Neurol. 120: 37-42. GLOBUS, J. H., and H. KUHLENBECK. 1944. The subependymal cell plate (matrix) and its relationship to brain tumors of the ependymal type. J. Neuropathol. Exptl. Neural. 3: l-35. MULE, I. L., and R. L. S~MAN. 1961. An autoradiographic analysis of histogenesis in the mouse cerebellum. FxptZ. Neurol. 4: 277-296. PIERCE, E. T. 1966. Histogenesis of the nudei griseum pontis, corporis pontobulbaris and reticularis tegmenti pontis (Bechterew) in the mouse. An autoradiographic study. J. Comp. Neurol. 126: 219-239. SIDMAN, R. L. 1961. Histogenesis of mouse retina studied with thymidine-H3, pp. 487-506. In “Structure of the Eye.” G. Smelser [ed.]. Academic Press, New York. SIDMAN, R. L., I. L. MIALE, and N. FEDER. 1959. Cell proliferation and migration in the primitive ependymal zone; an autoradiographic study of histogenesis in the nervous system. EzptZ. Neural. 1: 322-333. SMART, I. 1961. The subependymal layer of the mouse brain and its cell production as shown by radioautography after thymidine-Ha injection. J. Camp. Neural. 116: 325-347. SMART, I., and C. P. LEBLOND. 1961. Evidence for division and transformations of neuroglia ceils in the mouse brain, as derived from radioautography after injection of thymidine-Ha. J. Camp. Neural. 116: 349-367. UZMAN, L. L. 1960. The histogenesis of the mouse cerebellum as studied by its tritiated thymidine uptake. J. Camp. Neural. 114: 137-159.
XEUROLOGY
Proliferation
16, 263-278
and
Precursor
(1966)
Migration Cells
Postnatal
of
in the
Undifferentiated
Rat
During
Gliogenesis
JOSEPH ALTMAN] Psychophysiological
Laboratory, Cambridge,
Massachusetts Massachztsetts,
Received
July
institute 02139
of
Technology,
11, 1966
Rats aged 13 days were injected with thymidine-Ha and were killed 6 hours, 1, 3, 6, 12, 20 and 60 days afterwards. Incorporation of this labeled precursor of chromosomal DNA was studied autoradiographically with reference to the origin of neuroglia cells in the neocortex. A large proportion of the subependymal cells of the lateral ventricle were labeled after brief survival, indicating rapid regional proliferation. In animals with longer survival there was considerable increase in the proportion of labeled cells, combined with label dilution. In spite of this evidence of rapid and continuous cell multiplication, the concentration of subependymal cells declined considerably with age, indicating that the bulk of new cells do not stay here. The rate of regional proliferation was lower in the white matter and corpus callosum, though there was a considerable increase in the proportion of labeled cells with prolonged survival. Significantly, the addition of this large number of new cells did not lead to a net increase in the total cell population. These facts imply that many of the cells found in these fibrous regions are migratory ones, and as new cells arrive here older ones depart. Finally, in the gray matter of the cortex regional cell proliferation was low at this age, but there was a steady gain in the proportion of labeled cells with increased survival time. On the basis of these findings it is concluded that the cells formed around the lateral ventricle, which are destined to reach the cortex, utilize the white matter and corpus callosum as a migratory pathway. Introduction
The recent introduction of thymidine-H3 autoradiography has made it possibleto investigate aspectsof morphogenesisthat could not be adequately studied in the past with the available histological techniques. Thymidine is a specific precursor of chromosomal DNA and it is incorporated selectively into the newly synthesized DNA strands whenever the chromosomecomplement of the nucleus is duplicated. If animals are injected with tritiated thymidine, the cells that are preparing to undergo mitosis will tend to in1 This research is supported by assistance of William J. Anderson, acknowledged.
the United States Atomic Energy Commission. The Gopal D. Das, and Nicole Poussant is gratefully
263
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ALTMAN
corporate the radioactively-labeled precursor and the cells formed immediately after the injection will therefore become tagged. These new cells can then be easily identified with fine-resolution autoradiography. This is a technique in which a radiation-sensitive emulsion is applied directly to the tissue section, and the location of the radioactive particles with respect to the apposed cellular material is revealed, after a suitable period of photographic exposure and of development of the latent image, by the location of the reduced silver grains. The sites of cell multiplication, and the regional rates of proliferation, can be established in animals at different stages of their development by killing them within a short period (about l-6 hours) after injection. As animals are killed at progressively longer intervals after injection, the fate of the initially labeled cells, such as their migrations, rates of remultiplication, cellular transformations, and life span, can also be determined. Thymidine-H3 autoradiography has been applied recently with considerable success in investigations dealing with the prenatal development of the central nervous system (9-11, 15, 18-20, 23) and its postnatal maturation 2, 3, 6-9, 17, 21, 22). In our laboratory, the results of a series of autoradiographic studies in which rats of different ages from neonates to mature adults were injected with thymidine-H” and were then allowed to survive for different periods after injection, are being analyzed. The origin and migration of neuroblasts in various regions of the brain that give rise to short-axoned neurons or microneurons have been reported (5, 7, 8). The present paper concerns gliogenesis in the same material. Materials
and
Methods
Material obtained from laboratory-bred, Long-Evans hooded rats, aged 13 days, was analyzed in this study. Fourteen animals were injected intraperitoneally with thymidine-H 3, 10 uc/g body weight (the radiochemical was dissolved in isotonic saline, 1.0 me/ml; specific activity was 6.7 c/millimole). Pairs of the injected animals were killed by cardiac perfusion with 105% neutral formalin after the following survival periods: 6 hours; 1, 3, 6, 12, 20 and 60 days. The removed brains were further fixed in formalin, and then were dehydrated and embedded in Paraplast. Serial coronal sections were cut at 6 u, and three consecutive sections (or set of sections) were preserved out of every twenty (in the younger animals) or thirty. Of the preserved sections, two sets were stained with gallocyanin chromalum and one of these was coated in the darkroom with melted NTB-3 nuclear emulsion by the dipping technique. The dried sections were exposed in lightproof boxes supplied with a desiccant in a refrigerator at 5 C for 91 days and developed in the usual manner (4). Three structures were evaluated microscopically: The ependymal and sub-
POSTNATAL
GLIOGENESIS
265
ependymal layers of the lateral wall of the anterior horn of the lateral ventricle; the white matter and corpus callosum of the dorsal neocortex; a.nd the gray matter of the dorsal neocortex. Cell counting and classification, at a magnification of 640 X, were done in the following manner. The total cell population of the lateral wall of the ependymal and subependymal layers surrounding the lateral aspect of the lateral ventricle was determined in all animals at matched coronal levels through the septal nuclei at approximately the coordinate, A6.2 (14). In this region the proportion of labeled to unlabeled (“new” and “old”) was also determined by classifying 1000 cells in each animal. To determine the proportion of labeled and unlabeled cells in the cerebral white matter, four homologous coronal levels were scanned in all animals. These were at the level of the superior colliculus (A1.O) ; the interventricular foramen of Monro (A6.2) ; the septal nuclei (A8.2)) and the anterior pole (A9.8). In all these sections the total number of cells, and the proportion of labeled to unlabeled cells, was determined in homologous portions of the dorsal cortex in square areas of 182 p, with ten replications, in all animals. In addition, the cells in the midline corpus callosum were counted and classified in a similar manner at the level of the habenular nuclei (A3.8). In a similar manner, the total number of cells, and the proportion of labeled cells, was determined for the gray matter of the dorsal neocortex in three coronal levels, at coordinates Al.0, A6.2, and A9.8. As described in greater detail elsewhere (S), alterations in the number of labeled cells and in the degree of label concentration within cells, as functions ct survival time after injection, can be used as reliable indices of regional rates of cell proliferation. For instance, in rapidly and continuously proliferating regions the number of labeled cells rises rapidly for a short period after injection, due to the remultiplication of the labeled cells. Since remultiplication of labeled cells is combined with progressive dilution of label concentration within cells, more and more cells lose their tagging after a time and the number of labeled cells begins to fall as a consequence in such populations. To determine the degree of label concentration within cells, cells with overlying blackened silver grains can be conveniently classified as (a) all opaque, (b) mostly opaque (these represent the “intensely-labeled cells”) ; and as (c) mostly light, and (d) very light (these represent the “lightlylabeled cells”). Results
The Germinal Matrix of the Lateral Ventricle. An appreciable subependyma1 matrix, with many labeled, cells, is present in 13-day-old rats in the lateral wall of the anterior horn of the lateral ventricle. The typical distribution of thymidine-H3-labeled cells in this region is illustrated in Fig. 1. At this stage of development, cell proliferation has practically come to an end in
266
ALTMAN
the medial wall and part of the dorsal roof of the lateral ventricle, where single-cell-thick ependymal layers surround the lumen of the ventricle. These cells, whose nuclei stain lightly, are generally unlabeled. In contrast, a subependymal layer composed of many cells with darkly staining nuclei surround the ependymal wall on the lateral side of the ventricle. In animals with brief (6 hours) survival after injection, the majority of the pale cells of the ependymal layer are not labeled, whereas a considerable proportion
1. Photomicrograph of lateral ventricle, from a rat 24 hours later. Re!atively few Numerous labeled cells are seen band (SB). BG, basal ganglia; Gallocyanin chromalum; x 50. FIG.
the
an autoradiogram in the region of the injected with thymidine-Hs at 13 days of the cells of the ependymal layer in the subependymal layer (SE) and LV, lumen of CC, corpus callosum;
anterior horn of of age and killed (E) are labeled. in the subcallosal lateral ventricle.
of the darkly-staining cell nuclei of the subependymal layer are intensely labeled. Many labeled cells may also be observed in the dorsolateral arm of the subependymal layer, which is composed of a band or stream of cells interposed between the ventral aspect of the corpus callosum and the dorsal aspect of the basal ganglia. These cells apparently represent migratory elements which are moving outward from the subependymal layer, but since many of these cells are labeled even after the shortest survival period (1 hour), it is apparent that they retain their proliferative capacity to a considerable extent.
POSTNATAL
267
GLIOGENESIS
The cell population of the ependymal and subependymal layers of the lateral wall of the anterior horn of the lateral ventricle declines with increasing age. The total number of this cell population in animals aged 13, 14, 16, 19, 25, 33 and 73 days is plotted in Fig. 2, where each point represents the average of counts made on the right and left lateral ventricles in AGE
R,, f
3
OF ANIMALS
6
20
12 SURVIVAL
AFTER
INJECTION
IN
I.
60
DAYS
FIG. 2. The total number of ependymal and subependymal cells in homologous regions of the lateral wall of the anterior horn of the lateral ventricle as a function of age. The percentage of labeled cells as a function of survival time after injection at 1.3 days of age is &so plotted.
pairs of animals. A considerable drop in the cell population is seen in the 16-day-old animals, the number of cells is essentially unchanged between 16 and 25 days, then it drops again in the older animals. In the same graph I also plotted the changes in the percentage of labeled cells in the same region. After 6-hour survival, 30 and 415% of the germinal cells of the lateral ventricle are labeled, indicating a very high rate of cell multiplication at this period. The majority of these cells, as shown in Fig. 3, are intensely labeled. With 24hour survival, the proportion of intensely-labeled cells declines, but the total number of labeled cells increases to 50 and 5270, with a growing proportion of less-intensely labeled cells. The proportion of intensely-labeled
268
ALTMAN
cells is drastically reduced by the third day after injection, but due to continuing rapid cell multiplication of the initially labeled cells, the total proportion of labeled cells increases to 62 and 67%, the great majority with greatly reduced grain concentration. This survival period marked the asymptote level in the concentration of labeled cells. From this date onward, the proportion of labeled cells declines considerably, and reaches the low level %‘ 90
-
LATERAL
VENTRICLE
p/ P : : ,:
80 70
-
,,t’
,o
,#S’
60
:
0 ---I-O v0 ‘-I*--
.JO--- ..___ __-*se*
0
all opaque
lntenrely
00
mostly light mostly light very opaque 1' lightly
. . ..___g, __,,,,..
labeled labeled
50
40
30
20
m-m------
IO
SURVIVAL
FIG.
matrix
3. Percentage of the lateral
AFTER
INJECTION
IN
DAYS
distribution of intenselyand tightly-labeled ventricle as a function of survival time after
cells in the injection.
germinal
of 10% by the sixtieth day after injection; practically all of these are lightly labeled. These two facts, a high rate of cell proliferation, on the one hand, and a simultaneous decrease in the absolute number of cells, on the other, clearly indicate that the cells produced at a rapid rate at this site do not remain in this region but migrate to other areas of the brain. A reasonable assumption is that the migration route is the subcallosal band of cells previously described. This band consists of a large concentration of cells near the subependymal layer which gradually thins out laterally. Accordingly, I have tested the hypothesis that some of the migratory cells located in this band move by way of the adjacent cerebral white matter and corpus callosum to the cortex.
POSTNATAL
Cerebral White Matter
269
GLIOGENESIS
and Corflus Callosum. In every animal, at five co-
ronal levels, cells were counted and classified in homologous regions of the white matter and corpus callosum in the dorsal aspect of the neocortex. In all instances ten adjacent square areas of 182 p were sampled. The total number of cells obtained in the sampled areas, and the proportion of labeled, or “new,” cells are plotted in Figs. 4-8. A comparison of the data shows considerable, though nonsystematic, variability from animal to animal at differF WHITE
900
MATTER
Level, A 1.0 800
l -m
total
A.-.
A number
x 1s..
x
number of cells of labeled
percentage
cells
of lobeled
cells
700
2ii
600
” B 500 z 2 4oc z 3oc
2oc
100
SURVIVAL
FIG.
sampled
4.
AFTER
INJECTION
IN
DAYS
Total number of cells, and the number and percentage of labeled cells, in ten square areas of 182 k in the cerebral white matter at the coronal level of A1.O.
ent coronal levels, and between animals of different ages at identical levels. The averaged cell density from the five levels is plotted in Fig. 9, which indicates that the cell density of the white matter and corpus callosum does not substantially change between 13 and 73 days of age.’ In Figs. 4-9 I also plotted the number of labeled cells encountered in the 2 The variability obtained in these counts may be due to experimental errors. For instance, at the posterior coronal level (A1.O) higher cell density was obtained in the oldest group, whereas at a more anterior level (A8.2) the cell density in the oldest group was lower than in some of the younger groups. Two obvious sources of experimental error could he the variable shrinkage of the embedded tissue, and discrepancies in the matching of coronal levels in the different age groups.
270
ALTMAN
different age groups. The labeled cells represent the new elements of the population, those that were produced after injection of the radiochemical. These new cells may be products of local proliferation of cells or of the invasion of cells from other proliferative sites. The labeled cells seen in the white matter after brief survival following injection (6 hours) can be attributed to local cell proliferation. The rate of local proliferation appeared to be relatively low, ranging from 5 or 6% at the posterior level (A1.O) to
WHITE Level,
MATTER A 6.2 6-0
total
A.-.A
number
number
percentage
8,s;
3
6
of cells
of labeled
80
cells
of labeled
cells
12 SURVIVAL
AFTER
INJECTION
FIG. 5. TotaI number of cells, and the number white matter at the coronal level of A6.2.
IN
DAYS
and percentage
of labeled
celIs, in the
7% at an anterior level (A8.2). This rate contrasts sharply with the 30 and 41% of labeled cells seen after the same survival period around the lateral wall of the lateral ventricle. In spite of this low rate of local cell multiplication, there was a rapid increase in the population of labeled cells with increasing age, reaching an asymptote level in the range of 43 to 57% 12 days after injection, with a mean value of 49% of labeled, or newlyformed, cells 12 days after injection. This considerable increase can partly be attributed to continuing local cell multiplication, but the steep rise in the number of labeled cells as early as 24 hours after injection (which is pro-
POSTNATAL
271
GLIOGENESIS
portionally much higher than that seen in the ventricular germinal zone), and the relatively low rate of label dilution within cells, suggest that a large proportion of the newly acquired cells must come from other regions of the brain. The assumption of a migratory process is further supported by the fact that notwithstanding the addition of such a large percentage of new cells to the sampled areas in the white matter and corpus callosum, the total cell population does not increase. From these findings I conclude that as new
#
900
-
WHITE Level,
800
MATTER A 8.2
FIG.
total
AX--A
number
x.-en
-
SURVIVAL
white
e-e
AFTER
x
number
INJECTION
6. Total number of cells, and the number matter at the coronal level of A8.2.
of cells
of labeled
percentage
90
cells
of labeled
IN
and
% ‘1
cells
80
DAYS
percentage
of labeled
cells, in the
cells are add.ed to the population older ones are departing, that is, this fibrous region of the brain represents a passageway for migratory cells. If this interpretation is correct, the decline in the number and proportion of labeled cells with prolonged survival may be partly due to continued cell multiplication and concomitant label dilution beyond detectability, but, more importantly, it must be a consequence of the sustained migration of cells and, therefore, the replacement of labeled cells with newer, unlabeled ones. Cerebral Gray Matter. The foregoing results indicate, then, that the cells that multiply at a high rate around the lateral wall of the lateral ventricle leave this proliferative zone of the forebrain by way of the adjacent cerebral
272
ALTMAN
white matter. To determine the final destination of these migratory cells, which do not produce a net increase in the local cell population of the white matter, I investigated changes in the cell population, and in the proportion of labeled cells, in animals of different age groups in the gray matter of the cerebral hemispheres. Scanning through all the layers of the cortex, cells were counted and classified at three coronal levels (A1.O, A6.2 and A9.8) in homologous portions of the dorsal neocortex. The results are plotted in Figs 10-12.
WHITE
MATTER l -e
total
A--WA A---A
number
number
of cells
of labeled
cells
x ,_,, x percentage of labeled
cells
- 10
3
6
FIG.
a’hite
20
12 SURVIVAL
AFTER
INJECTION
7. Total number of cells, and the number matter at the coronal level of A9.8.
IN
II
60
DAYi
and percentage
of labeled
cells, in the
The cell density of the dorsal neocortex, making no distinction here between neurons and neuroglia cells, declined sharply between 13 and 16 days of age at level A1.O, less sharply but distinctly at level A9.8, and not at all clearly at level A6.2. This decline in the packing density of cells, in spite of a considerable increase in the cortex in the absolute number of neuroglia cells, can be attributed to the developmental increase in the volume of the neuropil (12). From the age of 19 days onward the cell density of the neocortex remained essentially unchanged at most levels. In the animals that survived for 6 hours after injection very few labeled cells were seen in the cortex, ranging between 1 and 37%. There was little
POSTNATAL
273
GLIOGENESIS
change in this respect after 24-hour survival, the range increasing slightly to 2-4%. These results indicate a relatively low rate of local cell proliferation at 13 and 14 days of age. However, an appreciable increase in labeled cells was evident by the twelfth day after injection, ranging at different levels in the two animals between 13 and 20%. The low local rate of cell multiplication combined with an appreciable subsequent increase in the population of new cells support the idea that the increase in new cells in this region is due
CORPUS Level,
CALLOSUM A 3.8 e-e A L-W x.s-rx
A., f
3
6
FIG.
8. Total number corpus callosum.
number
of cells
of labeled
percentage
cells
of labeled
cells
12 SURVIVAL
midline
total A number
AFTER
INJECTION
of cells, and the number
IN
DAYS
and percentage
of labeled
cells, in the
to invasion of cells from the underlying white matter. Little change (possibly a slight increase) was observed in the number and proportion of labeled cells from the twelfth day after injection, suggesting that by this time (age of animals 25 days) both local cell proliferation and the invasion of labeled cells has slowed down considerably. Discussion
The subependymal layer of the anterior horn of the lateral ventricle has been identified by several investigators as a germinal zone which remains
274
ALTMAN
mitotically active for some time after birth in various mammals (1, 3, 13, 16, 2 1, 22). The earlier investigators could not establish the fate of the cells formed in this region in adulthood, and it was often argued that this mitotic activity is an abortive phenomenon. With the introduction of thymidine-H3 autoradiography it became possible to follow the life history of these cells, and in a pioneering study Smart (21) concluded tentatively that the cells formed in the subependymal layer in neonatal mice represent spongioblasts
WHITE
900 -
MATTER
Average
from
AND five
CORPUS
‘q
CALLOSUM
levels
800 -
-
mean number
of total cell
--
mean number
of labeled
cells
700 -
ti 600 ” b 500 2 - ii
--
t
i
1
i
3
6
12
20
-I-
& z 400Z
Lo I a.,;
SURVIVAL
FIG.
matter error.
9. Mean number of total and corpus callosum, from
AFTER
INJECTION
IN
II II
60
DAYS
cells, and mean number of labeled cells, in the white five coronal levels. Vertical bars represent the mean
and neuroblasts, which migrate into the brain and become differentiated into neuroglia cells and neurons. The results of Smart suggested that the subependymal cells formed in adult mice no longer migrate into the brain. The migration of newly-formed cells from the anterior horn of the lateral ventricle by way of the subcallosal band was also indicated in my autoradiographic study of cell proliferation in adult rats (3). In subsequent studies (7, 8) suggestive evidence was obtained that a fibrous tract, the fimbria of the hippocampus, serves as a passageway for neuroblasts multiplying in the
POSTNATAL
275
CLIOGENESIS
medial wall of the lateral ventricle; these neuroblasts appeared to move past the pyramidal and polymorph cells of the hippocampus and became differentiated into granule cells of the granular layer of the dentate gyrus. This assumption gained quantitative support in a recent study (5) which showed, in line with present results, that in rats injected with thymidine-HFS at 13 days of age there were over 350 labeled cells in the fimbria in specified sampled fields after 12 days survival, but that the addition of this large num-
# ^^_ IYUU
%
DORSAL
NEOCORTEX
Level, A 1.0
. -80 a-m
total
A---
A number
number
of cell.
of labeled
cells
x . . . . x percentage of labeled 600
-
l
\
r
*,
.
l
SURVIVAL
FIG.
cortical
cells
10.
gray
AFTER
I-
INJECTION
Total number of cells, and the number matter at the coronal level of A1.O.
IN
DAYS
and proportion
of labeled
cells, in the
ber of new cells did not appreciably increase the total cell population of the same region. Accordingly, fibrous tracts can serve as passageways for migratory undifferentiated cells, whether spongioblasts or neuroblasts, in the postnatally developing rat brain. Very few neuroglia cells are present in the cerebral cortex of neonatal rats. The number of neuroglia cells increases rapidly after birth, and this change is reflected quantitatively by the rapidly growing ratio of neuroglia to nerve cell (12). Whereas local cell multiplication in the cortex is very low, there is, at the base of the cerebrum, a large source of multiplying cells around
276
ALTMAN
the outer wall of the lateral ventricle; this suggested the possibility of migration of cells from the latter site to the former. The demonstration that a large proportion of the cells found in the white matter are not interfascicular neuroglia cells but, instead, transitory elements, lends strong support for the assumption of the ventricular origin of spongioblasts and it clarifies the migratory route of these precursors of neuroglia cells.
9DD _
DORSAL
NEOCORTEX -90
Level, A 6.2
200
-
100
-
,,I’X’ h _---e A
a.------R,,f
‘3
6
12 SURVIVAL
FIG. cortical
11.
gray
----
AFTER
INJECTION
Total number of cells, and the number matter at the coronal level of A6.2.
A a-----w
20 IN
*
!’
- IO
60
DAYS
and proportion
of labeled
cells, in the
My quantitative results indicate a very high rate of proliferation for the undifferentiated cells in the ependymal and subependymal layer of the lateral ventricle (30-41% labeled cells after 6-hour survival). As the cells move outward through the white matter this mitotic activity declines considerably (to 57% after 6-hour survival), and the proliferative capacity of these cells decreases further as the cells arrive in the cortex (l-370). This decreasing mitotic activity with increasing distance from the ventricle may reflect the importance of the cerebrospinal fluid system for cell multiplication in the prenatal (and also, presumably, in the postnatal) development of brain tissue. But it is also possible that as the cells get nearer their destination they begin
POSTNATAL
GLIOGENESIS
277
to differentiate and concomitantly lose their proliferative capacity, According to this interpretation, the white matter of the cerebral hemispheres contains, in addition to oligodendrocytes, astrocytes and microglia, also a large complement of undifferentiated, migratory cells with some proliferative capacity. The mode of transformation of these precursor cells will be considered in a subsequent paper.
DORSAL
900
NEOCORTEX
Level. A 9.8
800
80 total
700
number
number
of labeled
percentage
SURVIVAL
FIG. cortical
AFTER
INJECTION
12. Total number of cells, and the number gray matter at the coronal level of A9.8.
IN
of cells cells
of labeled
cells
DAYS
and proportion
of labeled
cells, in the
References 1. 2. 3. 4.
5.
ALLEN, E. 1912. The cessation of mitosis in the central nervous system of the albino rat. J. Comp. Neural. 22: 547-568. Are new neurons formed in the brains of adult mammals? ALTMAN, J. 1962. Science 135: 1127-1128. ALTMAN, J. 1963. Autoradiographic investigation of cell proliferation in the brains of rats and cats. Anat. Record 145: 573-591. ALTMAN, J. 1964. The use of fine-resolution autoradiography in neurological and psychobiological research, pp. 336-359. In “Response of the Nervous System to Ionizing Radiation.” T. J, Haley and R. S. Snider reds.]. Little, Brown, Boston, Massachusetts. ALTMAN, J. Autoradiographic and histological studies of postnatal neurogenesis.
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II. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in infant rats, with special reference to postnatal neurogenesis in some brain regions. J. Camp. Nercrol. in press. ALTMAN, J., and G. D. DAS. 1964. Autoradiographic examination of the effects of enriched environment on the rate of glial multiplication in the adult rat brain. Nature 204: 1161-1163. ALTMAN, J., and G. D. DAS. 1965. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Camp. Neural. 124: 319-335. ALTMAN, J., and G. D. DAS. 1966. Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats with special reference to postnatal neurogenesis in some brain regions. J. Conzp. Neural. 126: 337-389. ANGEVINE, J. B. 1965. Time of neuron origin in the hippocampal region. An autoradiographic study in the mouse. Exptl. Neurol. Suppl. 2: l-70. ANGEVINE, J. B., and R. L. SIDMAN. 1961. Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature 192: 766-768. BERRY, M. A., A. W. ROGERS, and J. T. EAYRS. 1964. The pattern and mechanism of migration of the neuroblasts of the developing cerebral cortex. J. Anat. 96: 291-292. BRIZZEE, K. R., J. VOGT, and X. KHARETCHKO. 1964. Postnatal changes in glial neuron index with a comparison of methods of cell enumeration in the white rat. Progr. Brain Res. 4: 136-149. BRYANS, W. A. 1959. Mitotic activity in the brain of the adult rat. Anat. Record 133: 65-71. “The Rat Forebrain in Stereotaxic Coordinates.” NoordDE GROOT, J. 1959. Hollandische, Amsterdam. FIJJITA, S. 1963. The matrix ceil and cytogenesis in the developing central nervous system. J. Comp. Neurol. 120: 37-42. GLOBUS, J. H., and H. KUHLENBECK. 1944. The subependymal cell plate (matrix) and its relationship to brain tumors of the ependymal type. J. Neuropathol. Exptl. Neural. 3: l-35. MULE, I. L., and R. L. S~MAN. 1961. An autoradiographic analysis of histogenesis in the mouse cerebellum. FxptZ. Neurol. 4: 277-296. PIERCE, E. T. 1966. Histogenesis of the nudei griseum pontis, corporis pontobulbaris and reticularis tegmenti pontis (Bechterew) in the mouse. An autoradiographic study. J. Comp. Neurol. 126: 219-239. SIDMAN, R. L. 1961. Histogenesis of mouse retina studied with thymidine-H3, pp. 487-506. In “Structure of the Eye.” G. Smelser [ed.]. Academic Press, New York. SIDMAN, R. L., I. L. MIALE, and N. FEDER. 1959. Cell proliferation and migration in the primitive ependymal zone; an autoradiographic study of histogenesis in the nervous system. EzptZ. Neural. 1: 322-333. SMART, I. 1961. The subependymal layer of the mouse brain and its cell production as shown by radioautography after thymidine-Ha injection. J. Camp. Neural. 116: 325-347. SMART, I., and C. P. LEBLOND. 1961. Evidence for division and transformations of neuroglia ceils in the mouse brain, as derived from radioautography after injection of thymidine-Ha. J. Camp. Neural. 116: 349-367. UZMAN, L. L. 1960. The histogenesis of the mouse cerebellum as studied by its tritiated thymidine uptake. J. Camp. Neural. 114: 137-159.