Advances in Anatomy

Embryology and Cell Biology

Vol. 122

Editors
F. Beck, Leicester W. Hild, Galveston
W. Kriz, Heidelberg IE. Pauly, Little Rock
Y Sano, Kyoto T.H. Schiebler, Wiirzburg
Kyrill Y. Reznikov

Cell Proliferation and

Cytogenesis in the
Mouse Hippocampus

With 30 Figures

Springer-Verlag
Berlin Heidelberg New York
London Paris Tokyo
Hong Kong Barcelona
Budapest
Dr. Kyrill Yurjevich Reznikov
Central Scientific Research Laboratory,
Medical Faculty, Patrice Lumumba Peoples' Friendship University,
Mic1ucho-Mac1aja Str.8, Moscow, 117198, USSR

ISBN-13 : 978-3-540-53689-5 c-ISBN-13 : 978-3-642-76447-9

DOl : 10.1007/978-3-642-76447-9

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Contents

1 Hippocampal Formation in the Mouse and Rat -

Structural Organization and Development: A Review 1
1.1 Structural Organization of the Hippocampus ...
1.1.1 General Ideas on the Hippocampus, Nomenclature,
and Topography . . . . . . . . . . . . . . . .
1.1.2 Structural Organization of Ammon's Horn
and the Dentate Gyrus . . . . . . . . . . . . 3
1.1.3 Afferent, Efferent, and Longer Intrinsic Connections
in the Hippocampus . . . . . . . . . . . . . . . . 4
1.1.4 Functions of the Hippocampus . . . . . . . . . . . . 6

1.2 Development of the Hippocampus . . . . . . . . . . 6

1.2.1 Neurogenesis and Gliogenesis in the Hippocampus 6
1.2.2 Differentiation in the Hippocampus . 9

1.3 Conclusion . . . . . . . . . ....... 11

2 Materials and Methods . . . . . . . .......... 12

2.1 Animals, Histological Procedures,
and Autoradiography . . . . . . . . . . . . 12

2.2 Description of Experiments and Methods

of Their Analysis . . . . . . . . . . . . . . . 13
2.2.1 Mapping and Quantitative Analysis of Mitoses
and Pyknoses in the Developing Hippocampus . 13
2.2.2 Postnatal Cell Genesis and Death in the Mouse
Dentate Gyrus Under Normal Conditions
and Under Experimental Influences . . . . . . . . . 15
2.2.3 Study of Spatiotemporal Sequences of Neurogenesis
in the Hippocampus and Neocortex ......... 17

3 Cell Proliferation and Cell Death

in the Developing Hippocampus of a Mouse . 19
3.1 Some Aspects of the Study of Cell Proliferation
and Cell Death in the Developing Hippocampus . 19

3.2 Results of the Study . . . . . . . . . . . . . . . . . . 20

V
3.2.1 Registration of Arrangement of Mitoses
and Pyknoses . . . . . . . . . . . . . . . . 20
3.2.2 Quantitative Analysis of Cell Proliferation
and Cell Death . . . . . . . . . . . . . . . . 25
3.3 Discussion . . . . . . . . . . . . . . . . . . . . 28
3.3.1 Cell Proliferation in the Developing Hippocampus 28
3.3.2 Cell Death in the Developing Hippocampus 29
3.4 Conclusion. . . . . . . . . . . . . . . . . . 31

4 Postnatal Production and Death of Cells

in the Mouse Dentate Gyrus . . . . . . . 33
4.1 The Dentate Gyrus as an Object for Experimental
and Clinical Investigations . . . . . . . . . . . . .. 33
4.2 Cytological Characteristics of Proliferating Cells in
the Dentate Gyrus During Postnatal Development 33
4.3 Study of Postnatal Cytogenesis in the Dentate Gyrus 39
4.4 Kinetics of Cell Proliferation and Death
in the Subgranular Zone of the Dentate Gyrus
in Normal and Undernourished Mice 44
4.5 Study of the Regenerative Capacity
of the Dentate Gyrus . . . . . . . . . 50
4.6 Conclusion . . . . . . . . . . . . . . . 50

5 Neurogenesis in the Hippocampus and Neocortex as

the Embryonic Basis for Brain Module Formation. 52
5.1 Neural Modules in the Neocortex and Hippocampus 52
5.2 Sequences of Neurogenesis and Formation
of Modules in the Archi- and Neocortex:
Problems of Interrelationship . . . . . . . . 53
5.3 Results of the Study . . . . . . . . . . . . . . . . 54
5.3.1 Vertical Order and Mosaicism of Neurogenesis
in the Neocortex . . . . . . . . . . . . . . . . . . 54
5.3.2 Spatial and Temporal Sequences of Neurogenesis
in Field CAl of Ammon's Horn . . . . . . . . . . 59
5.4 Discussion . . . . . . . . . . . . . . . . . . . . 63
5.5 Conclusion . . . . . . . . . . . . . . . . . . . . 69

6 Summary .. 71

References . . . 75

Subject Index...................... 82

VI
1 Hippocampal Formation in the Mouse and Rat -
Structural Organization and Development: A Review

1.1 Structural Organization of the Hippocampus

1.1.1 General1deas on the Hippocampus, Nomenclature, and Topography

The hippocampus is one of the most intensely studied formations of the brain.
Such interest in the hippocampus is caused both by its functional role, which is
believed to be learning and memory, and by unique peculiarities of the hippocam-
pal organization and development (see reviews in the two-volumed book edited by
Isaacson and Pribram 1975, 1985). Being a relatively simple part of the cerebral
cortex with respect to its internal structure, the hippocampal formation contains
various subregions with different organization and rates of development. In
particular, the hippocampal formation as compared with other brain structures
has the most protracted span of neurogenesis, which gives an opportunity not only
to study certain stages of neurogenesis in detail but to investigate them experi-
mentally using various effects. Finally, the highly ordered structure of the hip-
pocampus and its significant capacity for reinnervation makes the hippocampus
a favorite experimental model for the analysis of operational principles of neuronal
circuitry and for the study of brain plasticity and regenerative capacity.
A distinction can be made between the terms hippocampal region, the hip-
pocampal formation, and the hippocampus. In the mammalian brain the hip-
pocampal region is the central component of the limbic system, which also includes
the olfactory cortex medial to the rhinal fissure, the amygdala, the septum, the
mamillary bodies, the anterior thalamic nuclei, and some other structures
(MacLean 1952; Hamilton 1976). It is believed that the hippocampal region is
subdivided into two major parts: the hippocampal formation and the parahippo-
campal formation, which correspond to the archicortex and periarchicortex of
Filimonov (1974). In turn, the hippocampal formation consists of the hippo-
campus proper or Ammon's horn, the dentate gyrus, and the subiculum (including
the pro subiculum), while the parahippocampal formation is made up of the
presubiculum, the parasubiculum (including area retroplenialis e), and the ento-
rhinal cortex (Lorente de No 1933, 1934; Blackstad 1956; Angevine 1965, 1975;
Isaacson 1987). At the same time, the term "hippocampus" is often used for the
designation of the hippocampal formation, especially of Ammon's horn and the
closely associated dentate gyrus (Isaacson 1987). In the present work such designa-
tion is allowed while the term "Ammon's horn" is only used for the definition of
the hippocampus proper.
The topography of the hippocampus is rather complicated and essentially
differs in various species of mammals (Ariens Kappers et al. 1960; Angevine 1975).
However, only the placental mammals have a well-developed postcallosal hip-
pocampus, which is located under the temporal cortex and is formed by a fold of
the medial hemispheric wall bulging into the hippocampul fissure. From the lateral
side, the hippocampus borders on the mediobasal wall of the posterior horn of the
lateral ventricle. Ammon's horn through the subiculum and parahippocampal
structures represents the continuation of the cerebral cortex, which is here folded
into a roll with a curl, while the dentate gyrus fringes its free end. According to
three-dimensional reconstructions, the hippocampus in rodents is curved along its
longitudinal ("septo-occipital") axis in such a manner that the dorsal (anterior)
portion has a nearly horizontal orientation and the ventral (posterior) portion
descends to the brain foundation (Blackstad et al. 1970; Hjorth-Simonsen 1972).
In brain sections made perpendicularly to the longitudinal axis of the hip-
pocampus, i.e., in frontal sections for its dorsal portion and horizontal sections for
its ventral portion, the hippocampus has an appearance of two arcs, the smaller
one (dentate gyrus) bordering the inferior end of the larger arc (Ammon's horn)
(Fig. 1). The superior part of Ammon's horn merges with the subiculum and
transits into the cerebral cortex. Correspondingly, Ammon's horn is divided into
regio inferior and regio superior. The finer morphological criteria allowed Lorente
de No (1934) to subdivide Ammon's horn into four fields: CAl, CA2, CA3, and
CA4. Field CAl corresponds to the larger part of the regio superior, CA2 is
situated near the bend of the Ammon's horn arc, CA3 occupies the largest part of
the regio inferior, and CA4 is found within the horseshoe-shaped dentate gyrus in
the region of the hilus and is regarded by many authors as its part (see Amaral
1978, Swanson et al. 1978 for references). The dentate gyrus consists of the
V-shaped fascia dentata and hilus (field CA4). The fascia dentata is divided into
two limbs or blades. Usually the limb closest to the hippocampal fissure is called
the suprapyramidal and the other limb the infrapyramidal (Isaacson 1987).

Fig. 1. Schematic representation of a frontal section through the dorsal hippocampus in the l4-day-old
mouse. V, lateral ventricle, the dorsal part of which fuses in the adult; CAl, CA2, and CA3, fields of
Ammon's hom; the pyramidal layer of Ammon's hom is represented by schematic contours of
pyramidal cell bodies; ipl, infrapyramidal layers of Ammon's hom (alveus and stratum oriens); spl,
suprapyramidallayers of Ammon's hom (stratum radiatum, stratum lacunosum, and stratum molecu-
lare); Sch. c., Schaffer collaterals ofaxons of pyramidal cells; Sb, the subiculum; dl, dorsal (supra-
pyramidal) limb of the dentate gyrus; vi, ventral (infrapyramidal) limb of the dentate gyrus; the
granular layer of the dentate gyrus is represented by dots; mf, mossy fibers of granular cells;f, fimbria

2
1.1.2 Structural Organization of Ammon's Horn and the Dentate Gyrus

Structural organization of Ammon's horn and the dentate gyrus reveals a pro-
nounced orderliness with distinct subdivision into cell and neuropil layers. The
intrinsic organization of Ammon's horn and the dentate gyrus in the rat and mouse
has been studied in detail (Ramon y Cajal 1893; Lorente de No 1934; Blackstad
1956; Laatsch and Cowan 1966; Blackstad et al. 1970; Hjorth-Simonsen 1972,
1973; Andersen 1975; Amaral 1978; Gaarskjaer 1986; Laurberg 1979; Stanfield
et al. 1979; Stanfield and Cowan 1979a; Braitenberg and Schiiz 1983). In Ammon's
horn from the lateral ventricle upward, the following layers have been distin-
guished.

1. The ependymal layer consisting of ependymal cells which borderline the ven-
tricle wall.
2. The alveus containing the myelinated axons of hippocampal pyramidal neurons
running in parallel to the stratum pyramidale, the fibers of the alveolar tract
from the entorhinal cortex, and projections from the septum.
3. The stratum oriens formed by basal dendrites of hippocampal pyramidal
neurons. On the proximal part of these dendrites the commissural afferents
from the contralateral hippocampus are terminated.
4. The stratum pyramidale composed of densely packed bodies of pyramidal
neurons. In the stratum pyramidale the basket short-axoned neurons forming
glomerules around the bodies of pyramidal neurons occur.
5. The stratum lucidum which exists only in field CA3 and represents a strip of
mossy fibers from the dentate gyrus running immediately suprajacent to the
stratum pyramidale.
6. The stratum radiatum containing unbranched primary shafts of pyramidal
neurons both in the regio superior and inferior of Ammon's horn. Schaffer
collaterals ofaxons of pyramidal neurons from CA3 terminate on proximal
parts of apical dendrite shafts in CAL The distal portions of apical dendrite
shafts in the stratum radiatum are the field of termination of commissural
projections from CA3 of the contralateral hippocampus and afferents from the
septum.
7. The stratum lacunosum consisting of branched apical dendrites of pyramidal
neurons. This layer is the terminal field of the perforant path from the ento-
rhinal cortex and of the crossed temporoammonic tract from the contralateral
medial entorhinal cortex.
8. The stratum moleculare formed by terminal branches of apical dendrites from
the stratum pyramidale. Here axons of the perforant path and the crossed
temporoammonic tract from the lateral part of the ipsilateral and contralateral
entorhinal cortex terminate. As well as in other hippocampal strata short-
axoned neurons occur in the stratum moleculare.

The dentate gyrus is divided into three strata or layers: stratum moleculare,
stratum granulosum, and the hilus (polymorphic layer and field CA4). The stratum
moleculare contains vertically oriented apical dendrites of neurons from the
stratum granulosum and afferent fibers running in a horizontal direction. Afferent
fibers terminate in the stratum moleculare according to a strict pattern. Afferents

3
of the lateral perforant path terminate in the superficial one-third of the stratum
moleculare, afferents of the medial perforant path are located in the middle
one-third of the stratum, its inner one-third is occupied by associated and commis-
sural fibers from the hilus, and in the deepest part afferents from the septum run in
a narrow band close to the stratum granulosum.
The stratum granulosum consists mainly of densely packed bodies of granular
neurons. Axons of granular neurons: the mossy fibers run to the hilus where they
send collaterals to local neurons, then run in bundles to field CA3 of Ammon's
horn and terminate with gigantic synapses on the spines of pyramidal cell dendrites
in the stratum lucidum.
The hilus or the polymorphic layer of the dentate gyrus is composed of
modified pyramids in the region termed field CA4 by Lorente de No (1934) and of
several other categories of polymorphic neurons predominantly located closer to
the stratum granulosum, i.e., the polymorphic layer proper. The long-axoned
neurons in the hilus, in particular pyramidal neurons, give rise to ipsilateral and
commissural projections to the inner one-third of the stratum moleculare and to
the hilus of the dentate gyrus. Neurons with short axons either form connections
with other neurons in the hilus or terminate on the granular neuron bodies.
Distribution of glial cells (mainly of astrocytes) in the hippocampus has some
peculiarities (Rose et al. 1976; Gall et al. 1979; Zimmer and Sunde 1984; Kosaka
and Rama 1986). The arrangement of astroglial cell bodies and orientation of their
processes correspond to the laminar organization and afferent fiber orientation in
Ammon's horn and the dentate gyrus. Thus, in the dentate gyrus molecular layer
the astrocyte cell bodies tend to be aligned between commissural-associational
afferents in its inner one-third and afferents of the medial perforant path in the
middle one-third of the layer, and less obviously, between these and afferents of the
lateral perforant path in the outer one-third. The other place of pronounced
alignment of astrocytes in the dentate gyrus is the sub granular zone located in the
hilus immediately beneath its granular layer. The subgranular zone contains
astrocytes of the radial type with processes vertically oriented into the granular
layer (Woodhams et al. 1981; Basco and Rajos 1984; Kosaka and Rama 1986;
Rickmann et al. 1987). In Ammon's horn each neuropil layer is characterized by its
own astroglial architectonics in terms of density and orientation of processes
(Zimmer and Sunde 1984).

1.1.3 Afferent, Efferent, and Longer Intrinsic Connections in the Hippocampus

Ammon's horn and the dentate gyrus have a highly complicated system of afferent,
efferent, and longer intrinsic connections. The following afferent pathways to
Ammon's horn and dentate gyrus are known.

I. Massive ipsilateral and partly contralateral projections from the entorhinal

cortex (see Stewart 1976 for references). A large ipsilateral input from the
entorhinal cortex, the so-called perforant path which perforates the subiculum
and terminates in the outer two-thirds of the dentate molecular layer and the
strata lacunosum and moleculare of field CA3. There is a very slight projection
to the contralateral dentate gyrus, but the only truly bilateral input from the

4
 entorhinal cortex is a pathway to the strata lacunosum and moleculare of CAl
(the so-called crossed temporoammonic tract).
2. The input from the prepyriform cortex to the stratum moleculare of CAl
(Hjorth-Simonsen 1972).
3. The common pathways from the medial septal and diagonal band nuclei which
share the semidisperse terminal field in the strata oriens and radiatum of the
regio inferior of Ammon's horn and terminate in a narrow zone in the deepest
part of the dentate molecular layer (see Swanson and Cowan 1979 for refer-
ences.
4. Afferent fibers from the ipsilateral and contralateral hypothalamus, especially
from the supramamillary and retromamillary regions which innervate both
Ammon's horn and the dentate gyrus, in particular the granular and molecular
layers (see Wyss et al. 1979 for references).
5. The noradrenergic input from the locus coeruleus, which terminates rather
diffusely on apical dendrites of pyramidal and granular neurons in Ammon's
horn and the dentate gyrus but forms the densest plexuses of terminal elements
in the hilus along the border of the dentate granular layer and in the stratum
lucidum of field CA3 (Ungerstedt 1971; Pickel et al. 1974; Swanson and
Hartman 1975).
6. The serotoninergic input from the raphe nuclei that has the innervation pattern
similar to that of noradrenergic afferents (Conrad et al. 1974; Moore and
Halaris 1975).
7. The diffusely organized inputs from regions of the brain stem other than the
locus coeruleus and raphe nuclei (from tegmental reticular fields, the central
grey, the dorsal tegmentum nucleus, and nucleus reticularis tegmenti pontis)
(see Wyss et al. 1979 for references).

The hippocampal commissural system projects via the ventral psalterium to

the strata oriens and radiatum of the contralateral Ammon's horn and to the
molecular layer and hilus of the dentate gyrus (see West et al. 1979; Voneida et al.
1981 for references). The origin of hippocampal commissural projections is:
(a) the input from pyramidal cells of contralateral field CAl, (b) projections from
pyramidal neurons of field CA3 upon pyramidal cells of contralateral subfields
CA3a and CA3b and from polymorphic cells of the hilus upon CA3c pyramids,
and (c) the inputs from giant polymorphic cells of the hilus and from pyramids of
CA3c subfield to the hilus and pyramids of CA3c sub field to contralateral dentate
granular cells (Ribak et al. 1985).
The commissural and the longer associational pathways of the hippocampus
have cells of similar origin and mainly the overlapping zones of termination. At the
same time, the associational projections as a rule are invariably more extensive
than the corresponding commissural connections (Swanson et al. 1978). Besides
that, the only longer associational projections from the dentate gyrus, the mossy
fibers, form ipsilateral connections with pyramidal cells of the regio inferior of
Ammon's horn (Blackstad et al. 1970; Hjorth-Simonsen 1973; Gaarskjaer 1986;
Laurberg 1979).
Lastly, the efferent pathways of the hippocampus consist of cortical and
subcortical projections of pyramidal cells of Ammon's horn (Raisman et al. 1966;
Hjorth-Simonsen 1973; Meibach and Siegel 1977; Swanson and Cowan 1977;

5
1979; Jay et al. 1989). The hippocampus gives rise to an extensive series of
intracortical connections. Field CAl sends a massive unilateral output to the
subiculum and gives rise to more limited projections to the presubiculum, para-
subiculum, entorhinal cortex, cingulate cortex, and pre limbic prefrontal cortex.
Fields CA2-3 send projections to the subiculum, para subiculum, and perirhinal
area. The only subcortical projection of pyramidal cells in fields CAI-3 is the
output to the lateral septal nucleus.

1.1.4 Functions of the Hippocampus

For quite a long period of time the hippocampus was conventionally believed to
playa key role in the regulation of behavioral motivations and emotions of an
organism (Papez 1937; MacLean 1952, 1958). However, numerous physiological
and clinical data provided evidence against concepts regarding the hippocampus as
a central link in the "limbic emotional circle" (see Isaacson and Pribram 1975;
Vinogradova 1975 for critical analysis). The hippocampus was also regarded as
a structure maintaining modulation of orientation reactions due to an inhibitory
influence (Douglas 1967; Kimble 1968; Kimble and Kimble 1970). However, the
absence of extinction of orientation reactions during hippocampoectomia may be
interpreted as disruption of the function of comparing current information with
traces stored in memory (Penfield and Milner 1958; Milner 1970).
At present, there is general agreement that the hippocampus is involved in the
amnestic syndrome. The hippocampal memory disabilities predominantly bear on
knowledge about a particular fact, experience, or declaration that could have only
occurred at a unique time and place. Disrupted or spared categories of memory are
described by various theoretical distinctions such as context-dependent/context-
independent, fact/skill, declarative/procedural, short-/long-term, working/refer-
ence (Pribram 1985; Gerbrandt 1987). Thus, Olton et al. (1979) distinguish
between working memory, which holds information pertinent only to a single trial
of experimental procedures, and reference memory, which holds information
pertinent to several trials. According to Olton et al. (1979) the hippocampus is
responsible for working memory. Lastly, O'Keefe and Nadel (1978) in their
cognitive map theory consider the hippocampus (including adjacent structures) as
a neural substrate for place learning, i.e., learning about spatial relationships of
objects and events in the environment. The diversity of theories about the func-
tional role of the hippocampus is quite explainable because the contribution of the
hippocampus to learning and memory processes remains obscure.

1.2 Development of the Hippocampus

1.2.1 Neurogenesis and Gliogenesis in the Hippocampus

The basic data on the sequence of cytogenesis in the hippocampus were obtained in
the auto radiographic experiments with [3HJthymidine administration in develop-
ing animals and subsequent analysis of the distribution of isotope-labeled cells in
the brain of adult animals. These investigations were performed in the mouse

6
(Angevine 1965; Caviness 1973; Stanfield and Cowan 1979b; Reznikov 1979,
1981), rat (Altman and Das 1965; Bayer and Altman 1974, 1975; Hine and Das
1974; Schlessinger et al. 1975; Kaplan and Hinds 1977; Bayer 1980a, 1982), rabbit
(Fernandez 1969; Fernandez and Bravo 1974; Gueneau et al. 1982), guinea pig
(Altman and Das 1967), and monkey (Nowakowski and Rakic 1981; Rakic and
Nowakowski 1981). In all mammalian species studied, the sequence of neuronal
formation was very much the same, and the only difference was in the duration of
neuro genesis.
Neurogenesis in the mouse and rat hippocampus has been studied the most
profoundly. In the mouse hippocampus, neurogenesis begins on embryonic (E)
day E10. It is known that during this period nerve cells are generated that migrate
to all structures of the hippocampus. However, the peak of neurogenesis and its
termination in various structures of the hippocampus are not the same. Thus, in
Ammon's horn, the production of neurons in the suprapyramidal and in-
frapyramidallayers, as well as of pyramidal neurons in field CA2, is completed
earliest (by E 15), while neurogenesis of pyramidal neurons in fields CA 1 and CA3
continues until the moment of birth (until E19). The general sequence of neurogen-
esis in the pyramidal layer is characterized by the "inside-out" gradient, i.e., first,
neurons in its inner zone (bordering on the stratum oriens) are generated, and in
the last turn, neurons in the outermost zone of the pyramidal layer. In the mouse
dentate gyrus, the formation of neurons in the hilus and molecular layer is
completed earliest (by E15). Neurogenesis in the granular layer begins during
embryogenesis (on ElO) and continues at least until postnatal (P) day P20. The
granular layer of the dentate gyrus is characterized by the "outside-in" gradient of
neurogenesis, i.e., first, neurons in its outermost zone (bordering on the stratum
moleculare) are generated, and in the last turn, neurons in the deep zone of the
granular layer.
In the rat hippocampus, neurogenesis begins on E15. Between E15 and E17
large neurons in the supra- and infrapyramidal layers of Ammon's horn are
generated. Pyramidal neurons form between E17 and E19.1t is known that as well
as in the mouse, neurogenesis of pyramidal neurons in the rat is completed earlier
in field CA2 than in fields CAl and CA3, and in the pyramidal layer the sequence
of neurogenesis is characterized by the inside-out gradient. Moreover, the "sand-
wich gradient" of neurogenesis was described for Ammon's horn (Hine and Das
1974; Bayer 1980a). It is manifested in earlier neuronal formation in the supra- and
infrapyramidallayers in comparison with the pyramidal layer. In the rat dentate
gyrus, large neurons in the molecular layer and hilus also originate earliest
(between E 15 and E 19). Neuronal production in the granular layer begins on E 17,
continues until P20, and persists on a reduced scale even in adult animals. In the
rat, as well as in the mouse, neurogenesis in the granular layer proceeds in the
outside-in sequence.
The above presented data indicate that Ammon's horn and the dentate gyrus
differ essentially in the duration of neuronal production in the pyramidal and
granular layers. While pyramidal neurons are generated simultaneously with other
types of neurons of Ammon's horn, granular neurons of the dentate gyrus, having
begun their genesis at the same period of time, continue to be generated even when
neurogenesis of other types of neurons in the hippocampus has been practically
completed. Thus, in the mouse and rat no less than 80% of granular neurons are

7
produced during the first 3 postnatal weeks (Bayer and Altman 1974; Schlessinger
et al. 1975; Bayer 1980a; Reznikov 1979, 1981). It has been demonstrated that
granular neurons continue to be generated in adult rats as well (Kaplan and Hinds
1977; Bayer 1982, 1985; Kaplan and Bell 1984; Crespo et al. 1986; Trice and
Stanfield 1986).
The postnatal genesis of the majority of granular neurons in the dentate gyrus
is characteristic not only of the rat and mouse, but of other mammalian species as
well. It was described for a 2-month-old rabbit (Gueneau et al. 1982), 6-day-old
guinea pig (Altman and Das 1967), and 35-day-old cat (Wyss and Sripanidkulchai
1985). In the rhesus monkey about 20% of granular neurons are generated during
the first 3 postnatal months, whereas in Ammon's horn, neuronal production is
completed by E80, i.e., by the end of the first half of pregnancy, which constitutes
165 days in this primate species (Rakic and Nowakowski 1981). Neurogenesis in
the monkey dentate gyrus is completed during the juvenile period (4-6 months of
age). In postpubertal animals only glial cells are generated in the dentate gyrus
(Rakic 1985; Eckenhoff and Rakic 1988).
Recently, innovative reports appeared devoted to the study of the terms of
neuronal differentiation of hippocampal neurons possessing different mediatory
specificity. By means of autoradiography with labeled thymidine and immunohis-
tochemical detection of glutamate decarboxylase - an enzyme involved in the
synthesis of gamma-amino butyric acid (GABA) (mediator of inhibitory inter-
neurons) - it was established that the majority of GABAergic neuronal precursors
in the rat hippocampus cease their division and commence neuronal differentiation
on E13, and the smaller amount of these cells between E15 and E18 (Amaral and
Kurz 1985; Liibbers et al. 1985). Similar data were obtained in the study on the
time of origin of neurons immunoreactive to somatostatin - a neuropeptide
involved in a wide range of behavioral functions. It was shown that in the rat
hippocampal formation somatostatin-immunoreactive neurons were generated
between E12 and E15. Most of these cells were generated on E14 in fields CA 1 and
CA3, and on E15 in the hilus (Rapp and Amaral 1988).
In the hippocampus gliogenesis was studied far less sufficiently than neurogen-
esis. Thus, the auto radiographic study of gliogenesis in the rat hippocampus was
carried out without precise analysis of the types of cells generated (Bayer and
Altman 1974). According to these data, about 14% of glial cells in the supra- and
infra-pyramidal layers of Ammon's horn are generated during embryogenesis,
more than 70% of these cells are produced during the first 2 postnatal weeks, and
the rest of the glial cells during later periods of postnatal development. In the
stratum moleculare and stratum polymorphe of the dentate gyrus, 90% and 70%
of the glial cells, respectively, are generated during a postnatal period, 60% and
50% of those, respectively, being generated during the first 2 weeks after birth. On
the whole, the data obtained are indicative of similar rates of gliogenesis in
Ammon's horn and the dentate gyrus.
In the study by Reznikov (1981) the types of glial cells generated in the mouse
dentate gyrus were identified. It was shown that in the stratum moleculare and
stratum polymorphe 80% of the astrocytes are generated during a postnatal
period. Cessation of astrocyte proliferation is more pronounced between P5 and
P16. Oligodendrocytes are characterized by longer periods of genesis than

8
astrocytes. Large-scale production of these cells begins only on P9 and continues
even after P20.
Investigations involving immunocytochemical detection of vimentin and glial
fibrillar acid protein (GFAP), specific to radial glia, permitted the analysis of
formation of this type of glial cells in the rat dentate gyrus (Rickmann et al. 1987).
Vimentin- and GFAP-positive radial fibers can be detected on E13 and E17,
respectively. The orientation of processes and arrangement of cell bodies of the
radial glia change so that by the end of the 1st postnatal week processes form an
intricate network and the cell bodies, originally located in the ventricular zone of
the hippocampus, "move closer" to the granular layer into the subgranular layer of
the dentate gyrus. The majority of cells in the radial glia divide mitotically.
Cell sources of neurogenesis and gliogenesis in the hippocampus were studied
mainly in the rat and mouse. It was shown that during embryonic development,
neurogenesis and gliogenesis in the hippocampus proceed due to migration of cells
from the ventricular germinal zone adjacent to lateral ventricles bordering on the
hippocampus (Bayer and Altman 1974; Stanfield and Cowan 1979b; Bayer 1980b).
During the postnatal period, the ventricular germinal zones of the hippocampus
become "exhausted," and the germinal zones of the dentate gyrus, namely, the
subpial zone, the proliferative zone of the hilus, and the subgranular zone, become
the main sources of neuro- and gliogenesis in the hippocampus. It is noteworthy
that adult animals retain the subgranular zone in a reduced form (Bayer and
Altman 1974; Schlessinger et al. 1975; Bayer 1980b; Reznikov 1975, 1981). In
Chaps. 3 and 4 of the present work a more detailed consideration is given to
proliferative sources of cytogenesis in the hippocampus.

1.2.2 Differentiation in the Hippocampus

Microscopic and submicroscopic data on the development of hippocampal struc-

tures indicate a rather late appearance of definitive signs in the hippocampus. The
study of the developing mouse hippocampus demonstrated that the Ammon's
horn and dentate gyrus primordia can be already observed on E14. By E16,
Ammon's horn becomes characteristically curved, while the dentate gyrus is
represented only by the suprapyramidallimb (Stanfield and Cowan 1979b). The
development of pyramidal neurons in the mouse Ammon's horn involves three
stages: (I) insignificant branching of dendrites (before P5), (2) an increase in the
outgrowth of dendrites, the appearance of first dendrite spines, first signs of the
axon myelinogenesis (between P6 and P9), and (3) an increase in the number of
dendrite spines, the appearance ofmyelinized axons in all areas of Ammon;s horn
(between PIO and P28) (Ferres-Torres and Meyer 1975).
Differentiation of the mouse dentate gyrus occurs mainly during postnatal
development. Formation of the infrapyramidal limb of the dentate gyrus is
completed by the end of the 1st postnatal week. Even in 2-week-old mice neurons
in the inner zone of the granular layer remain poorly differentiated, and only by the
end of the 3rd postnatal week does the granular layer acquire definitive signs
(Reznikov 1975, 1981). Late maturation of the mouse dentate gyrus also shows in
the formation of afferent and efferent connections. First afferents going to the

9
molecular layer of the suprapyramidallimb of the dentate gyrus are detected on PI
and become sufficiently pronounced only by P7. In the molecular layer of the
infrapyramidal limb, first afferents appear on P5. First mossy fibers, going from
the granular neurons to the dendrites of the Ammon's horn pyramids, appear on
PI and become pronounced by P7 (Stanfield and Cowan 1979b). Mature synapses
can be observed on granular neurons of the suprapyramidal limb already on P5,
and by the end of the 2nd postnatal week synapses appear between mossy fibers
and pyramidal cells of fields CA3 and CA4 (LaVail and Wolf 1973). On the whole,
the growth of the mouse hippocampus continues until P40, the volume of this
structure increasing nearly six- to fivefold from the moment of birth (Kretschmann
and Wingert 1968).
In the rat, the dorsomedial wall of telencephalon begins to curve into the
lateral ventricle on El4. On EI5-EI6, a cell-sparse zone ofrandomly oriented cells
forms the primordial Ammon's horn. The pyramidal layer of Ammon's horn can
be observed from E21. On EI8-El9, the ball-like subpial cell mass forms the
primordial dentate gyrus. The suprafimbriallimb of the granular layer appears on
E20, and the primordial infrapyramidal limb of the dentate gyrus can be distin-
guished from E21-E22. The most rapid rate of growth of the granular layer occurs
until P7; however, the growth rate of the granular layer remains rather consider-
able until the end of the 3rd week after birth (Bayer 1980b).
To a great extent, cell differentiation in the rat hippocampus reflects the
sequence of cytogenesis but falls to the later period of development. Thus, in
a newborn rat, pyramidal neurons are immature, i.e., many of them have only
apical shafts without dendritic branches. Only at the end of the 1st postnatal week
do apical dendrites of pyramidal cells approach the stratum moleculare and
produce branching of the third order. During the same period of time, basal
dendrites of pyramids in CA3 enter the stratum oriens; in CAl this occurs only
after 1.5 weeks. On the whole, pyramidal cells mature earlier in CA3 than in CAl
(Engelhardt et al. 1967). At the same time, no essential differences in the rates of
differentiation of pyramidal neurons in fields CA3 and CA4 were discovered
(Minkwitz 1976). Final differentiation of pyramidal neurons of the rat Ammon's
horn is completed by the beginning of the 4th postnatal week (Engelhardt et al.
1967; Minkwitz 1976).
Studies of the development of mossy fibers ending in field CA3 of Ammon's
horn demonstrated spatial and temporal dependence of their formation and
projections on the time of origin of granular neurons. In the rat, first axons of
granular neurons appear on E16, but the majority of mossy fibers forms during the
first 3 postnatal weeks. Throughout the whole period of their development, the
fibers from granular cells, forming in the first turn, are longer and more effectively
diverge in the septotemporal direction than the fibers from later generations of
granular neurons (Gaarskjaer 1986).
Characteristic features of differentiation of granular cells in the dentate gyrus
are varicosities along dendrites, growth cones on dendritic tips, transiently occur-
ring basal dendrites, and progressive formation of dendritic spines (Lubbers and
Frotscher 1988). In the rat dentate gyrus, first synaptic contacts in the molecular
layer appear on P4. Active synaptogenesis takes place between P4 and PII, when
the total number of synapses in the molecular layer redoubles daily. By P25, the
total number of synapses in the molecular layer differs insignificantly from that in

10
adult animals. On the whole, the number of synapses in the molecular layer
increases IOO-fold between P4 and P90 (Crain et al. 1973). Although by P25,
the total number of synapses in the molecular layer of the rat dentate gyrus
approaches the value characteristic of adult animals, their development and
complication continues during later periods as well. In particular, further differen-
tiation of the spine apparatus takes place that can be only sometimes detected in
dendrite spines on P25 (Cotman et al. 1973). It should be noted that the hippocam-
pus of pubertal rats is characterized not only by continuing cell differentiation but
by slow prolonged growth as well (Bayer 1982, 1985). Data exist that the hip-
pocampal growth continues for nearly 1 year (Diamond et al. 1975).

1.3 Conclusion

The present review gives a general idea about the structure and functions of the
hippocampus, and a more detailed description of cytogenesis and differentiation in
Ammon's horn and the dentate gyrus in the mouse and rat. In particular, they
allow a conclusion that there is a striking lack of convergence between the terms of
genesis of long-axoned neurons in Ammon's horn and the dentate gyrus. Pyr-
amidal neurons in Ammon's horn are generated during a comparatively short
period of embryonic development, whereas granular neurons of the dentate gyrus
are characterized by one of the longest (in the central nervous system) periods of
cytogenesis: having begun their genesis simultaneously with pyramidal neurons,
they continue to be actively produced during several postnatal weeks and are
generated even in adult animals (rats). Nevertheless, these significant data do not
exhaust all peculiarities of cell production in the developing hippocampus that
require further investigations. The aim of the present work was to study some
obscure problems of cell production and cytogenesis in the mouse hippocampus, in
particular, spatial and temporal sequences of cell proliferation and cell death in the
hippocampus, peculiarities of postnatal neurogenesis and gliogenesis in the dentate
gyrus, and interrelationship of sequences of neurogenesis and module formation in
the hippocampus. Problems to be considered are formulated in introductory
remarks to the corresponding experimental sections.

11
2 Materials and Methods

2.1 Animals, Histological Procedures, and Autoradiography

Animals. CBA strain mice were used in the experiments. Both male and female
animals were used for the study of early periods of development (including P3),
while only male mice were examined during later periods. Mice with dated time of
conception were analyzed, taking the day after conception as the 1st day of
pregnancy (El). The pregnancies were timed by testing vaginal plugs. Mice born
on E20 were used for the study of postnatal development. The 1st day after birth
was designated as Pl. Animals were kept on a standard laboratory diet with free
access to water under 12-h alternating light and dark periods.

Histological Procedures. Mice were sacrificed either by decapitation or by trans-

cardiac perfusion with Karnovsky's fixative (Karnovsky 1964). Pups aged P1-P7
were killed under hypothermia-induced anesthesia, and older animals were sac-
rificed under Nembutal anesthesia.

Paraffin Embedding for Light Microscopy and Light-Microscopic Autoradio-

graphy. The whole brains of embryos and cerebral hemispheres of older mice
killed by decapitation were placed in Carnoy's solution for 14 h at 4°C. After
dehydration in ethanol and saturation with dioxane the brains were embedded in
paraffin. Cerebral hemispheres were sectioned at 6 Ilm frontally at the level of the
dorsal hippocampus. Slides with mounted sections were stained with 0.1 % cresyl
violet according to Nissl or were processed for autoradiography.

Resin Embedding for Light- and Electron - Microscopic Autoradiography.

Glutaraldehyde-iparaformaldehyde Karnovsky's fixative (Karnovsky 1964) was
perfused transcardially to mice aged P1-P35. Cerebral hemispheres were halved in
the midsagittal plane and immersed overnight in Karnovsky's fixative at room
temperature. The halved brains were rinsed in phosphate buffer (pH 7.4), de-
hydrated in graded ethanols and propylene oxide, and embedded in Durcupan
(Fluca) or in Epon-Araldite. If the material was prepared for both light and
electron microscopy, the buffer rinsing was followed by a 2-h postfixation in 1%
phosphate-buffered osmium tetroxide prior to dehydration and embedding. From
each animal the left and right hemispheres were sectioned frontally and sagittally,
respectively. Semithin (1 Ilm) sections were cut serially at lO-f.lm intervals with an
LKB ultramicrotome and mounted on glass slides. The preparations were stained
with 1% toluidine blue containing 2.5% sodium tetraborate or processed for auto-
radiography. In the osmicated samples, territories were selected for the electron-

12
microscopic study on the basis of light-microscopic examination of semithin
stained sections or semi thin autoradiographs. Ultrathin sections of pale yellow
interference color were cut with Reichert's or LKB ultramicrotomes, floated on
distilled water, and either picked up on uncoated copper grids for electron
microscopy or mounted on glass slides for auto radiographic procedures. Grids
were stained with uranyl acetate and lead citrate (Reynolds 1963) and examined
using a Tesla BS 500 electron microscope.

Autoradiography. [3HJThymidine (specific activity 888 GBq/mM, Isotope, the

USSR, and 1073 GBq/mM, Amersham International) was administered intraperi-
toneally to pregnant female and male mice older than P7, and subcutaneously to
pups aged PI-P7, either with a single dose (5-10 IlCi/g body weight, i.e.,
185-370 kBq/g body weight) or with a series of eight [3HJthymidine injections of
2.5 IlCi/g, each at 12-h intervals. The animals were sacrificed at terms varying from
1 h to 1 month after administration of the isotope (see Sect. 2.2 for details). After
histological treatment thick paraffin (6 11m) and semithin plastic (lllm) sections
were covered by dipping into nuclear emulsion type M (NIKFI, Moscow) diluted
with an equal volume of distilled water. After 28 and 49 days of exposure at 4°C for
thick and semi thin sections, respectively, the preparations were developed in
Kodak D19, fixed and stained through the emulsion either with 0.1 % cresyl violet
(thick sections) or with 1% toluidine blue with 2.5% sodium tetraborate (semithin sec-
tions). Cells with five or more silver grains above the nucleus were regarded as labeled.
For electron-microscopic autoradiography labeled cells were chosen in se-
mithin sections under the light microscope and the block was trimmed for electron
microscopy to contain the labeled cell observed under the light microscope. Thin
sections of yellow interference color were cut with a Reichert's ultramicrotome,
floated on distilled water, and mounted on slide glasses covered with a Parlodion
film. These slides were dipped in Ilford L4 nuclear emulsion diluted 1: 4 with
distilled water and stored in the dark for 40 days at 4°C. They were developed in
Microdal-X, fixed in Kodak F-7 fixer, floated on distilled water, picked up on
copper grids, and stained through the emulsion with uranyl acetate and lead
citrate. Electron micrographs were taken with a Tesla BS 500 electron microscope.

2.2 Description of Experiments and Methods of Their Analysis

2.2.1 Mapping and Quantitative Analysis of Mitoses and Pyknoses

in the Developing Hippocampus

Embryos taken at daily intervals from E14 to E20 and animals on postnatal days
PI, P3, P7, P14, P21, and P60 were used. The animals were killed by decapitation,
and cerebral hemispheres were embedded in paraffin. The frontal sections of the
brain were stained with cresyl violet. The dorsal hippocampus was studied at the
level of the posterior commissure of the endbrain. Mapping of mitoses and
pyknoses was performed using a drawing tube attached to a Zeiss research
microscope. All mitotic phases were registered. Registration of pyknotic nuclei was
performed on the basis of criteria of cell pyknotic degeneration in the germinal
zones of the brain (Altman and Nicholson 1971). Brains from three animals were

13
used for the mapping of each period of development. From one object three serial
sections of the left hemisphere of the hippocampus (every second section) were
mapped. The arrangement of mitoses and pyknoses obtained during the mapping
of the three sections was marked on the summary drawing for each animal.
Orientation of the axis of the mitotic spindle of metaphases, anaphases, and
telophases was registered during the mapping of mitoses in the ventricular zone of
the hippocampus.
Besides mapping of mitoses and pyknoses, mitotic and pyknotic indexes in
Ammon's horn and the dentate gyrus were determined. In the mouse Ammon's
horn, the indexes were calculated in the stratum oriens of field CA3, where
numerous proliferating cells are located during embryogenesis and early postnatal
ontogenesis. In the dentate gyrus the pyknotic indexes were calculated in its
germinal zones (Fig. 2). The count was performed in different regions, which
depended on the term of their development. Thus, before P3 the quantitative
analysis was carried out in the area located between the differentiated part of the
dorsal limb of the granular layer and the outermost layer of the hilus facing the

Fig.2a-c. Schematic representation of hippocampal germinal zones in mice. a 18-day postconception

embryo; b 7-day-old mouse; c adult mouse. I, the suprafimbrial zone; II, the prime germinal zone of
the dentate gyrus (subdividing into the proliferative zone of the hilus and the subgranular zone from
P3); Ill, the subgranular zone

14
truncus cerebri. On P7 and P14, the count was performed in the hilus triangle and
the subgranular zone of the granular layer. All calculations were performed using
a Zeiss microscope (100 x objective, 10 x ocular). Five animals were analyzed for
each term of the experiment. To determine the mitotic and pyknotic indexes in
Ammon's horn and the dentate gyrus, cells were counted in the chosen hippocam-
pal area using eight brain sections (every second section). The figures obtained
were processed by standard statistical methods (Plochinskiy 1970). On the basis of
pyknotic and mitotic indexes, the fraction of dividing cell losses was calculated by
the following equation:

PI 0
FCL = 20 MI 100 Yo ,

where FCL is the fraction of cell losses, PI the pyknotic index, and MI the mitotic
index (Lewis 1975).

2.2.2 Postnatal Cell Genesis and Death in the Mouse Dentate Gyrus
Under Normal Conditions and Under Experimental Influences

Cytological AnalysiY''Oj Proliferating Cells in the Developing Dentate Gyrus. Mice

aged P5, P14, and P60 were injected with a single dose (10 J..lCi/g body weight) of
[3HJthymidine and killed after 1 h. Each age group consisted of four animals.
Cerebellar hemispheres were embedded in Epon-Araldite and processed for light-
microscopic autoradiography. For electron-microscopic autoradiography five
pups aged P12 received a single injection of [3HJthymidine (20 J..lCi/g body weight)
and were sacrificed 1 h later. Durcupan-embedded brain samples were processed
for electron-microscopic autoradiography. The study of pulse-labeled cells was
performed under light and electron microscopes.

Investigation oj Postnatal Neurogenesis and Gliogenesis in the Dentate Gyrus. Mice

aged PI, P5, P9, P13, and Pl7 were injected with eight 2.5 J..lCi/g doses of
[3HJthymidine at 12-h intervals, i.e., in the course of 4 days. All the animals were
killed on P35. Brain hemispheres were embedded in EpQn-Araldite and processed
for light-microscopic autoradiography. Four animals were studied for each period
of the isotope administration. The auto radiographs were examined for quantitative
estimation of neuronal and glial cell production in the dentate gyrus.

Neurogenesis. In each animal, the number of labeled neurons per 1000 nerve cells
in the supra- and infrapyramidallimbs of the granular layer and per 100 neurons in
the molecular and polymorphic layers (without subdivision of the latter into limbs)
was calculated. Estimation of portions of neurons generated during the given age
period was based on modification of the progressively delayed comprehensive
labeling procedure described by Bayer and Altman (1974), according to which all
neurons originating in the dentate gyrus between PI and P35 must be labeled,
when the isotope is introduced between PI and P4 and the animals are killed on
P35. In this case, unlabeled neurons represent cells generated during embryogen-
esis. In animals exposed to [3HJthymidine from P5 to P8 only those neurons

15
should be labeled that were generated between P5 and P35, etc. Further calcu-
lations of the number of neurons generated during various periods of the isotope
administration are given in Sect. 4.3.
Gliogenesis. The terms of gliogenesis were determined separately for astrocytes
and oligodendrocytes. (In view of some difficulties in the identification of oligoden-
drocytes and microglial cells in semi thin toluidine-blue-stained sections, a com-
paratively small population of microglial cells was included into a registered
oligodendroglial cell population.) Both for astrocytes and oligodendrocytes the
number of intensely labeled, weakly labeled, and unlabeled cells per 100 cells of the
same glial type was counted in the molecular and polymorphic layers of the dentate
gyrus of each animal. In this study, the intensely labeled cells defined as having at
least half the number of silver grains of the most heavily labeled cells of the same
type in a given specimen roughly correspond to cells undergoing from one to five
last divisions in the course of 4-day exposure to [3HJthymidine. [It follows from
the duration of the mitotic cycle of glial cells in the mouse which is known to be
20 h (Korr 1975) and from calculations demonstrating that the content of
[3HJthymidine in cells that underwent from one to five divisions during a 4-day
course of injections is expressed as a ratio: 1:3/2:7/4: 15/8:31/16.J Weakly labeled
glial cells are those that continue divisions after the end of exposure to the isotope.
Unlabeled cells either cease their divisions prior to [3HJthymidine administration
or, on the contrary, represent lately differentiating cells that have lost a detectable
label as a result of numerous divisions. The number of intensely and weakly
labeled cells was expressed as a percentage. Results were expressed as arithmetic
means±SEM.
Kinetics of Cell Proliferation and Death in the Subgranular Zone of the Dentate
Gyrus. Analysis of parameters of the mitotic cycle and kinetics of cell death in the
sub granular zone of the dentate gyrus and subependymal zone of brain lateral
ventricles was carried out in normal 20-day-old mice subjected to undernutrition
from PIO to P20. This was achieved by halving the normal diet oflactating mother
mice beginning from the 10th day after delivery. Normally fed and undernourished
20-day-old pups were injected with a single dose of [3HJthymidine (5 IlCi/g body
weight) and killed I, 2, 4, 6, 8, 12, 16, and 24 h after exposure to the isotope. In
both groups two to three animals were sacrificed at each term of the experiment.
Brains were embedded in paraffin and cut frontally. Sections were processed for
autoradiography and stained with hematoxylin. The subgranular zone of the
dentate gyrus at the level of the dorsal hippocampus and the subependymal zone
of the anterior tip of the lateral ventricles were examined under a x 100 oil-
immersion objective. In mice sacrificed I h after the exposure to the isotope, the
indexes of labeled nuclei (LI) were calculated by analyzing 4000 cells in the
subgranular and subependymal zones of each animal. At all terms of the experi-
ment the mitotic indexes (MI) were derived from the counts of 4000 cells in both
germinal zones of each animal. By analyzing 75-100 mitoses (metaphases and
anaphases) the percentage oflabeled mitoses was estimated in the subgranular and
sUbependymal zones of each animal. The parameters of the mitotic cycle were
determined graphically (Quastler and Sherman 1959), and the growth fraction
(GF) and the potential doubling time (PDT) of the cell population were calculated
from the following equations:

16
 GF=LIx T/ts and PDT=ts/LI

where T is the time of the mitotic cycle and ts the duration of the S-phase (Korr
1980). In all animals under study the pyknotic indexes (PI) and indexes of labeled
pyknotic nuclei (LPI) were determined in the subgranular and subependymal
zones, respectively. PI and LPI were calculated by analyzing 4000 cells in these
zones in each animal. The fraction of cell losses (FCL) in the population of
dividing cells was determined according to the equation adduced in Sect. 2.2.1.

Study of the Regenerative Capacity of the Dentate Gyrus. Twelve 3- and 5-day-old
mice received a unilateral incision with an ophthalmic scalpel through the parietal
bone and the right parietal cortex to the level of the dorsal hippocampus. The
incisions were made along the septotemporal axis of the hippocampus. For
orientation of disposibon of the hippocampus the atlas of the mouse brain
(Sidman et al. 1971) was used. The animals were sacrificed on P30 and P60. The
brains were fixed in Carnoy's solution, embedded in paraffin, cut serially in the
frontal plane at the level of the-dorsal hippocampus and stained with cresyl violet.
Only the samples with the transected dentate gyrus were chosen for the study.

2.2.3 Study of Spatiofemporai Sequences of Neurogenesis in the Hippocampus

and Neocortex

Pregnant mice received a single injection of pH]thymidine (10 IlCi/g body weight)
on E13-E19. From each litter, two to three l-day-old animals were killed by
perfusion or by decapitation. The cerebral hemispheres of mice exposed to
pH]thymidine from E15 to E19 were fixed in Karnovsky's fixative and embedded
in Durcupan(Fluca). In animals exposed to [3H]thymidine from E13 to E14, the
cerebral hemispheres were fixed in Carnoy's solution and embedded in paraffin.
From each animal the right and left hemispheres were sectioned frontally and
sagittally, respectively. Slides with mounted plastic (111m) or paraffin (6 11m)
sections were processed for autoradiography and stained with toluidine blue or
with cresyl violet, respectively. Plotting of the distribution of centers of nuclei of
intensely labeled neurons in field CA 1 of the dorsal hippocampus and in field 6 of
the frontal cortex (according to the nomenclature of Caviness 1975) was performed
using a Zeiss microscope with a x 100 oil-immersion objective and a projective
screen. Mapping in the hippocampus was carried out using the frontal paraffin and
plastic brain sections of mice exposed to [3H]thymidine from E12 to E17; mapping
in the neocortex was made using the frontal plastic brain sections of animals
exposed to the isotope from E15 to E17. In both brain structures neurons were
registered as intensely labeled if the number of silvcr grains over the cell nucleus
was half or more of the maximum grain count observed in a given section for
neurons of a given brain structure. The maps of fields CAl and 6 from two animals
for each term of the experiment were mathematically processed using a computer
DZ-28 (USSR) to reveal nonrandom groups of intensely labeled neurons. With
this purpose, an approach was applied allowing evaluation of the ordering of
neurogenesis in brain structures arranged according to the principle of right-
angled lattice formed by vertically and horizontally oriented elements. The frontal

17
sections of fields CAl and 6 answer these criteria if the plane of section passes in
parallel to vertically oriented neuronal processes and cell bodies. The rows of
coordinates of projections of intensely labeled neurons on the horizontal axis of
maps were treated by the U(p2)-grouping test allowing separation of the mixed
aggregate into several reliably existing homogeneous groups (Kildishev and Abol-
entzev 1978). The discovered nonrandom groups were analyzed by means of the
chi-squared test. The numerical characteristics of the revealed neuronal groups
were computed for evaluation of their statistical parameters.

18
3 Cell Proliferation and Cell Death
in the Developing Hippocampus of a Mouse

3.1 Some Aspects ofthe Study of Cell Proliferation and Cell Death
in the Developing Hippocampus

Studies on cytogenesis in the hippocampus mainly involve determination of the

terms of genesis of nerve (and partially of glial) cells in the hippocampal formation
(see Sect 1.2.1). Considerably less attention is paid to the analysis of cell sources of
cytogenesis, i.e., to the study of the distribution of proliferating precursor cells that
later differentiate into nerve and glial cells. Investigations in the mouse and rat
demonstrated that during embryogenesis proliferating cells are located in the
ventricular germinal zone of the hippocampus along the medial wall of lateral
ventricles adjacent to the hippocampus. At the moment of birth, the ventricular
zone reduces and proliferation becomes extraventricular: (a) in the form of prolif-
erating cells scattered in the neuropil layers of Ammon's horn; (b) in the form of
comparatively compact germinal zones in the dentate gyrus (Angevine 1965; Bayer
and Altman 1974; Schlessinger et al. 1975; Stanfield and Cowan 1979b; Bayer
1980a).
Postnatal cell proliferation in the dentate gyrus of the mouse and rat hip-
pocampus was studied more profoundly (Bayer and Altman 1974; Schlessinger
et al. 1975; Reznikov 1'975, 1981; Stanfield and Cowan 1979b). It was shown that
during the first postnatal days the proliferativ.e.zone occupies the larger part of the
dentate gyrus below thesuprapyramidallimb ofthe.granular layer. After forma-
tion of the infrapyramidaJ limb ofthezranular layer, proliferation continues in the
hilus triangle and subgranular zone of the granular layer. Beginning from the end
of the 2nd postnatal week, cells proliferate practically only in the sub granular
zone. This germinal zone reduces in the process of aging but does not completely
disappear even in adult mice and rats (Reznikov 1975; Kaplan and Hinds 1977).
The data on the germinal zones of the hippocampus in the mouse and rat were
found to be very close to the findings obtained during the study of cell proliferation
in the developing hippocampus of the rhesus monkey (Nowakowski and Rakic
1981). At the same time, by far not all problems connected with the analysis of cell
production in the developing hippocampus were studied sufficiently. Thus, there is
no detailed description of the localization of proliferative cells in the embryonic
hippocampus. There are no publications devoted to the mapping of the arrange-
ment of proliferating cells in the hippocampus. Practically no quantitative data
exist· on the age-dependent dynamics of proliferative processes in the hippo-
campus.
Processes of cell death in the developing hippocampus were studied even to
a lesser extent. Meanwhile, pyknotic nuclei (pyknoses) and cell death were revealed

19
in some germinal zones of the mammalian brain. These data were obtained for the
subependymal zone of the endbrain (Smart 1961; Privat and Leblond 1972; Lewis
and Lay 1974; Lewis 1975; Sturrock 1979; Korr 1980) and for the external granular
layer of the cerebellum (Lewis 1975). It should be noted that cell death is observed
riot only in the germinal zones of the developing brain. Pyknoses and death of
differentiating neurons (Prestige 1974; Cowan 1979; Sturrock 1979, 1982; Finlay
et a!. 1982; Cowan et a!. 1984) and of proliferating glioblasts (Pannese and
Ferrarini 1967; Sturrock 1979; Korr 1980) were described. However, the mechan-
isms of death of these cells remain unclarified. While the death of differentiating
neurons is often explained by insufficient contacts with their synaptic targets, the
series of events leading to the death of cells in the germinal zones of the brain and
of glioblasts still remains the object of discussion (Lewis 1975; Lewis eta!' 1977,
1977; Korr 1980; Sturrock 1979, 1982; Sturrock and Smart 1980).
The purpose of the present studies wasto fill the gap in the existing data on cell
proliferation and cell death in the developing hippocampus. The aims were: (a)
mapping of the arrangement of mitoses and pyknoses in the mouse hippocampus
during embryogenesis and postnatal ontogenesis and (b) quantitative analysis of
cell proliferation and cell death in the mouse developing hippocampus.

3.2 Results of the Study

3.2.1 Registration of Arrangement of Mitoses and Pyknoses

On E14, the ventricular (neuroepithelial) zone lying along the wall of the lateral
ventricle is well-pronounced in the hippocampal primordium. The ventricular zone
is continuous with the interstitial zone formed by immature neurons migrating to
the cortical plate. Neurons of the cortical plate are located above the interstitial
zone. On E14, it appears difficult to delineate accurately the anlage of Ammon's
horn, the subiculum, and the retrohippocampal cortex. In the ventromedial direc-
tion from the cortical lamina there lies an aggregation of undifferentiated cells that
evidently represents the anlage of the dentate gyrus. Almost all mitoses registered
on E14 are located in the ventricular zone in direct vicinity to the ventricle surface
(Fig. 3a). A small number of mitoses can be found in the anlage of the dentate
gyrus and in the cortical plate in the anlage of the subiculum. No pyknoses are
revealed on E14.
On E16, the main structures of the hippocampal formation are sufficiently
well-pronounced. Ammon's horn becomes characteristically bow-shaped, and
both the regio superior and inferior of the pyramidal layer can be easily distin-
guished in it. The suprapyramidallimb of the granular layer begins to show in the
dentate gyrus. The hippocampal fimbria is formed. The ventricular zone is rather
prominent on E16, and numerous mitoses are registered in it (Fig. 3b).
Other areas of mitotic activity lie in the subventricular (subependymal) zone of
the subiculum, in the ventral part of the hippocampus bordering on the fimbria
and in the anlage of the dentate gyrus adjacent to the truncus cerebri. Here and
below, the proliferative zone of Ammon's horn bordering on the fimbria is termed
the suprafimbrial zone, and the aggregation of proliferating cells in the dentate
gyrus located between its suprapyramidal limb and the surface adjacent to the

20
Fig. 3a, b. The distribution of mitoses
in the mouse hippocampus on EI4
a and EI5 b. Mitoses are designated
with/Wed circles. Scale bars =200 11m

truncus cerebri the prime germinal zone of the dentate gyrus. No pyknoses are
revealed on E 16~
On-E18, the dorsal hippocampus increases in its volume and changes orienta-
tion of its transversal axis, which becomes parallel to the basis cerebri. The
ventricular zone of the hippocampus becomes considerably thinner than on E14
and E16 and contains less mitoses (Fig. 4a). As well as on E16, mitoses are found in
the subventricular zone of the subiculum, in the suprafimbrial germinal zone of
Ammon's horn, and in the prime germinal zone of the dentate gyrus. No pyknoses
are revealed in the hippocampal formation.
On E20, complete reduction of the ventricular zone is observed (Fig. 4b).
At the same time, the suprafimbrial zone of Ammon's horn grows rapidly, and
numerous mitoses are registered in it. As well as at earlier stages of embryogenesis
(E16, E18), the suprafimbrial zone is connected with the prime germinal zone of
the dentate gyrus by a band of undifferentiated mitotically active cells. Mitoses are
also registered in the subventricular zone of the subiculum, in the region of the
joint of the pyramidal layer of Ammon's horn and subiculum, and, in small
numbers, in various hippocampal layers. Pyknoses are first detected on E20. They
are most numerous in the suprafimbrial zone and in the region of the joint of the
Ammon's horn pyramidal layer and subiculum. No pyknoses are detected in the
dentate gyrus.
On PI, the general view of the hippocampal formation and the distribution of
mitoses in it (Fig. Sa) are similar to those on E20. The main differences consist

21
 Fig. 4a, b. The distribution of mitoses
(filled circles) and pyknoses (crosses)
in the mouse hippocampus on El8
a and E20 b. Scale bars = 200 f.Ull

of the following: a certain increase in the number of mitoses in the stratum oriens
of the regio superior of Ammon's horn, the appearance of mitoses in the molecular
layer of the suprapyramidallimb of the dentate gyrus, and the disappearance of
mitoses registered during earlier periods of development in the region of the joint
of Ammon's horn and the subiculum. The majority of pyknoses is detected in the
suprafimbrial zone, which is sufficiently well-pronounced (Figs. 5a and 6a, b), and
in the region of the joint of Ammon's horn and the subiculum. A small number of
mitoses are revealed in the prime germinal zone of the dentate gyrus and also in the
stratum oriens of the regio superior of Ammon's horn. On P3, the hippocampus
considerably increases in volume and becomes more differentiated than on Pl. The
infrapyramidal limb of the granular layer appears in the dentate gyrus. The
suprafimbrial layer is well-pronounced and contains numerous mitoses and pyk-
noses (Fig. 5b). It remains connected with the prime germinal zone of the dentate
gyrus. Mitoses and pyknoses are still numerous in the prime germinal zone of the
dentate gyrus and in its molecular layer, as well as in the reduced subventricular
zone of the subiculum. Only pyknoses are registered in the region of the joint of the
subiculum and the pyramidal layer of Ammon's horn.
On P7 the hippocampal area in frontal sections of the brain increases nearly
twofold in comparison with P3. The infrapyramidallimb of the granular layer has
already formed in the dentate gyrus. The suprafimbrial zone of Ammon's horn is
considerably reduced and is represented by separate aggregations of undifferen-
tiated cells. Pyknoses and mitoses can be detected in these aggregations (Fig. 7a).
The subventricular zone of the subiculum disappears completely. Due to differenti-
ation and consolidation of neurons of the suprapyramidal and infrapyramidal

22
Fig. Sa, b. The distribution of mitoses (filled circles) and pyknoses (crosses) in the mouse hippocampus
on PI a and P3 b. Scale bars = 200 /lm

limbs of the granular layer, two germinal zones form in the dentate gyrus instead of
the prime germinal zone, namely, a cell-sparse proliferative zone of the hilus and
a compact subgranular zone of the granular layer. Numerous mitoses and pyk-
noses are revealed in these zones. A small number of mitoses and pyknoses is also
registered in the molecular layer of the dentate gyrus and in nonpyramidallayers of
Ammon's horn.
On P14, along with the growth and differentiation of the hippocampal struc-
tures, further reduction of its germinal zones is observed. The compact germinal
zone is preserved only in the dentate gyrus in the form of the subgranular zone
(Fig. 7b). Numerous mitoses and pyknoses are revealed in it. A comparatively
large number of mitoses and pyknoses is still detected in the hilus of the dentate
gyrus, and only single mitoses and pyknoses are revealed in the molecular layer of
the dentate gyrus in the region bordering on the fimbria.
The hippocampus becomes mature by P21. The mitotic activity in it is
insignificant (Fig. 8a). Practically all mitoses and pyknoses are localized in the
subgranular zone of the dentate gyrus, which becomes thinner than on P14. On
P21, pyknoses are more frequent in the hippocampus than mitoses.
On P60, the subgranular zone is represented by small aggregations of undif-
ferentiated cells in the region separating the granular and polymorphic layers of

23
Fig. 6a, b. The suprafimbrial germinal zone of the hippocampus in the l-day-old mouse. a General
view of the suprafimbrial zone. CA3, field CA3 of the stratum pyramidale; SFZ, the suprafimbrial zone;
FR, fimbria hippocampi. Cresyl violet, x 300 b Mitosis (arrowhead) and pyknoses (arrows) in the
suprafimbrial zone. Toluidine blue, x 1100

24
 ---

_---=---=-:- x~;~-------'-'"
x. "1'S.
.
~xx __ x
,
'\
I

Fig. 78, b. The distribution of mitoses (filled circles) and pyknoses (crosses) in the mouse hippocampus
on P7 8 and P14 b. Scale bars = 200 Jlm

the dentate gyrus (Fig. 8b). The total number of pyknoses in the subgranular layer
considerably exceeds the number of mitoses.

3.2.2 Quantitative Analysis of Cell Proliferation and Cell Death

The previous section is devoted to the description of the ventricular zone of the
hippocampus, which is well-pronounced on E14 and E16, is markedly reduced on
E18, and completely disappears on E20. No quantitative differences in the orienta-
tion of the axis of the division spindle of ventricular cells were revealed during this
period of development. The number of mitoses with an axis of the mitotic spindle
going in parallel to or at an angle of less than 300 to the surface of the lateral
ventricle wall comprises more than 50% of the total number of metaphases,
anaphases, and telophases of ventricular cells during all experimentally studied
periods of development (Table 1). No pyknoses were revealed in the ventricular
zone. The above-described suprafimbrial zone takes shape in mice on E16 and is
reduced by P7. The analysis of the mitotic index (MI) in the suprafimbrial zone
(Table 2) is indicative of elevation of the mitotic activity in this zone until P3. The

25
 ---------------
-- -,
....................
"' ... ...
"

/"" ---------

Fig. Sa, b. The distribution of mitoses (filled circles) and pyknoses (crosses) in the mouse hippocampus
on P2! a and P60 b.Sca!e bars = 200 11m

Table 1. Correlation of metaphases, anaphases, and telophases in the ventricular zone of the mouse
embryonic hippocampus with an axis of the mitotic spindle running in parallel or at an angle of more
than 30° to the surface of the lateral ventricle wall

Embryonic The number of Position of the mitotic spindle (%)

days registered metaphases,
anaphases, and telophases In parallel to the surface At an angle of
of the lateral ventricle wall more than 30°

E14 203 56 44
E16 130 53 47
El8 54 59 41

MI is still high enough on P7, sharply decreases by Pl4 (more than sixfold), and
continues to diminish reaching very small values on P60.
Pyknoses appear in the suprafimbrial zone on E20 (Table 2). Until P3 the
pyknotic index (PI) rapidly increases, then it falls, and on P14 becomes smaller
than on P20. PI continues gradually decreasing in the process of aging.
Estimation of the fraction of cell losses (FeL) in the suprafimbrial zone (Table
2) indicates that the amount of pyknotically dying cells in comparison with the
total number of mitotically dividing cells increases considerably between E18 and

26
Table 2. The values of the mitotic (M!) and pyknotic (PI) indexes and of the fraction of cell losses
(FeL) in the germinal zones of the mouse Ammon's horn and dentate gyrus

The age MI P value PI P value FCL

of mice (%) (%) (%)

Ammon's horn
El8 3.1O±0.212 0.00 0.0
<0.01
E20 5.40±0.432 2.57 ± 0.446 1.5
N.S. N.S.
PI 5.99±0.651 3.36±0.464 2.8
<0.05 <0.001
P3 8.78±0.S90 13.94 ±O.961 8.0
N.S <0.001
P7 6.63±0.710 3.27±0.296 2.5
<0.001 <0.001
P14 0.98±0.97 1.00±0.230 5.0
N.S. N.S.
P21 O.77±O.lOS 0.80±O.l70 5.4
<0.05 N.S.
P60 0.30±O.l72 0.37±0.241 6.37
Dentate gyrus
E18 2. 15± 0.440 0.00 0.0
<0.01
E20 6.17±0.851 0.14±0.035 0.1
N.S. <0.001
PI 6.84±0.670 l.35±0.251 1.0
<0.01 <0.01
P3 11.05±0.971 3.31 ±0.540 0.7
<0.05 N.S.
P7 7.32±0.819 2.33±0.490 1.5
<0.01 N.S.
Pl4 3.41 ±0.474 2.17±0.41S 3.2
N.S. N.S.
P21 2.93±0.417 3.12±0.489 5.3
<0.05 N.S.
P60 1.48±0.304 1.85±0.373 6.2

The MI and PI are given as mean ± SEM obtained during the study of five animals for each term of
development; Student's test results for the comparison of the MI or PI at two successive terms of
development, e.g., on EIS and E20, on E20 and PI; NS, not significant (P>0.05).

P3, when these values reach the maximal level. On P7 the FCL decreases more than
threefold in comparison with P3. However, it begins to increase once again and is
only insignificantly lower on P60 than on P3.
Calculation of the MI in the germinal zones of the dentate gyrus (Table 2)
demonstrated a nearly fivefold increase in its value between El8 and P3, i.e., its
elevation in the prime germinal zone of the dentate gyrus that exists during this
period. Beginning from P7 its value begins gradually falling down and decreases by
34% and by 69% on P7 and P14, respectively, in comparison with P3. In 21- and
60-day-old mice proliferation is observed only in the subgranular zone. In com-
parison with P14, the MI decreases by 17% and more than twofold on P21 and
P60, respectively.

27
 The analysis of the PI index values in the germinal zones of the dentate gyrus
(Table 2) demonstrated that though first pyknoses appear in this brain structure
on E20, their large number is registered only after birth, namely, on PI. The PI
gradually increases, reaching a maximal value on P3, when it is 2.4 times higher
than on PI. By P60 the PI is 1.6 times lower than on P21.
Evaluation of the FCL in the germinal zones of the dentate gyrus (Table 2)
demonstrated that it increases from P7 to P60, when the FCL of dividing cells is six
times higher than on PI.

3.3 Discussion

3.3.1 Cell Proliferation in the Developing Hippocampus

In many respects, the results of mapping of the arrangement of mitoses in the

developing mouse hippocampus confirmed the already existing data on its ger-
minal zones. Thus, complete disappearance of the ventricular zone on the last
perinatal day coincides with the data by Stanfield and Cowan (l979b) on the
reduction of the ventricular zone in the mouse hippocampus. The number of
mitoses with an axis of the mitotic spindle going in parallel to or at an angle of less
than 30° to the surface of the ventricle wall is approximately the same for all
studied terms of development of the ventricular zone (EI4-EI8) at 53%-59% of
the total number of metaphases, ana phases, and telophases of ventricular cells.
These data combined with the similar results obtained during the study of the
ventricular zone of the mouse neocortex at earlier periods of neurogenesis
(ElO-EI4) (Landrick and Goffinet 1979) contradict Berry's hypothesis (1974)
about the importance of the orientation of the mitotic spindle for initiating the
migration of postmitotic ventricular cells.
It has been demonstrated that the sub ventricular (subependymal) zone in the
mouse developing hippocampus exists only in the upper corner of the lateral
ventricle, a region which belongs not to Ammon's horn, but to the subiculum.
These data agree with the results of investigations of the developing hippocampus
in the mouse (Stanfield and Cowan 1979b) and monkey (Nowakowski and Rakic
1981), but contradict reports by Bayer (1980a,b) on the presence of the weakly
pronounced subventricular zone in the rat Ammon's horn.
It has been described that the prime germinal zone in the dentate gyrus is
pronounced in mice between E14 and P3. This zone is practically identical to the
"proliferative zone of the dentate gyrus" revealed in rats by Schlessinger et al.
(1975) and to the "anlage of the dentate gyrus" in mice described by Stanfield and
Cowan (1979b). The data obtained on changes in MI in this zone indicate that the
intensity of cell proliferation increases until P3, i.e., until the beginning of trans-
formation of the prime germinal zone into the proliferative zone of the hilus and
the subgranular zone of the dentate gyrus. This transformation is conditioned by
the formation of the infrapyramidallimb of the granular layer, which confines the
area of intensive proliferation to the region of the hilus and a zone of proliferating
cells at the base of the granular layer (the sub granular zone). The proliferative zone
of the hilus, pronounced in mice between P7 and P14 corresponds to the rat
"proliferative zone of the hilus" described by Bayer and Altman (1974). The results

28
concerning the existence of cell proliferation in the subgranular zone during the
advanced period of postnatal ontogenesis agree with the previous data obtained in
the rat, mouse, guinea pig, rabbit, cat, and monkey (see Sect. 4.1).
One of the most important results obtained during the study of cell prolifer-
ation in the developing mouse hippocampus is the discovery of a previously
unknown germinal zone, which was termed a suprafimbrial germinal zone of
Ammon's horn. This zone lying above the fimbria in the regio inferior of Ammon's
horn appears in mice on E16, is maximally pronounced between PI and P3, is
considerably reduced on P7, and completely disappears by P14. Until P3, the
suprafimbrial zone is connected with the prime germinal zone of the dentate gyrus
by a band of actively proliferating cells that lie along the surface of the hippocam-
pus facing the truncus cerebri. According to the classification of the types of brain
germinal zones (Reznikov 1981), the suprafimbrial zone belongs to extraventricu-
lar germinal zones, i.e., zones located at a distance from the surface of brain
ventricles.
What is the cytogenetic importance of the suprafimbrial zone? At present, no
experimental evidence exists concerning the direction of cell differentiation in the
suprafimbrial zone. However, some assumptions seem highly probable. It was
already mentioned that the developing hippocampus practically lacks the subven-
tricular (subependymal) zone. If for the regio superior of Ammon's horn the
migration of cells from the neighboring subventricular zone, located in the upper
corner of the lateral ventricle in the region of the subiculum, appears quite
possible, this process seems highly dubious for the regio inferior in view of spatial
remoteness of this zone and the necessity for cells migrating from it to penetrate
twice through the layer of pyramidal neurons.
It should be noted that the suprafimbrial zone of Ammon's horn forms and
exists during the same periods of development as the subventricular zone of the
subiculum. Therefore, it can be assumed that for the regio inferior of Ammon's
horn the suprafimbrial zone fulfills the cytogenetic role of the lacking subventri-
cular zone. Since the suprafimbrial zone is sufficiently well-pronounced until P7
and completely disappears only in 2-week-old mice, it is highly probable that this
zone is involved in the genesis of glial cells in the regio inferior of Ammon's horn.
The role of the suprafimbrial zone in neurogenesis is more dubious. For pyramidal
neurons, as well as for other macro neurons of Ammon's horn, this possibility
appears highly improbable, since the majority of these cells are generated on E16,
before the appearance of the suprafimbrial zone (Angevine 1965; Caviness 1973).
Concerning the involvement of suprafimbrial cells in the genesis of small short-
axoned neurons (microneurons), this possibility cannot be excluded, but further
investigations are required for its verification.

3.3.2 Cell Death in the Developing Hippocampus

In the present work, the mapping and studies of the distribution ofpyknoses in the
developing hippocampus were carried out. It was already mentioned that pyknotic
cell degeneration during normal ontogenesis may be caused either by the death
of differentiating neurons, in which axons have not found target cells, or by the
death of cells in the germinal zones of the brain or of proliferating glial cells

29
(see Sect. 3.1). In this connection, a question arises whether a certain amount of
pyknoses in the developing hippocampus results from the amitotic death of
superfluous neurons.
At present there is no direct evidence for pyknotic degeneration of differenti-
ating neurons in the developing hippocampus. At the same time, it was calculated
and assumed that due to overproduction of granular neurons in the dentate gyrus,
a certain amount of these cells should be inevitably eliminated (Schlessinger et al.
1975; Cowan 1979). Without going into detail concerning the validity of the
calculations performed, I would only like to point out that the present data
indicate that pyknoses registered in the hippocampus are practically always con-
nected with the mitotic death of proliferating cells and not with neuronal degener-
ation. The following findings support this conclusion: (a) localization of pyknoses
in the areas of cell proliferation, i.e., in the germinal zones of the hippocampus and
in those regions where mitoses of glial cells occur, (b) direct evidence for the
mitotic origin of pyknoses in the subgranular zone of 20-day-old mice (see Sect.
4.4), and (c) simultaneous disappearance of mitotic and pyknotic cells during
ontogenetic reduction of the proliferative zones of the hippocampus.
The area of active proliferation in the region of the joint of Ammon's horn and
the subiculum is the only exception in this respect. In this region, mitoses are
registered during late periods of embryogenesis (EI8-E20), and pyknoses are
revealed right until P3. Of course, it cannot be excluded that these pyknotic cells
have a mitotic origin as well, since in this case mitoses were not registered only
because of their shorter lifetime (0.5 h) in comparison with the duration of
pyknotic degeneration (10-12 h) (Lewis 1975; Korr 1980). This assumption pro-
vides indirect evidence for the presence of pairs of pyknotic nuclei in the region
under study in the absence of mitoses. However, should further investigations
demonstrate that degeneration of superfluous neurons actually takes place in the
region of the joint of Ammon's horn and the subiculum, a question can be raised
about the search of areas of neuronal differentiation in the regions of the joint of
brain structures differing in their genesis.
The results of the present study demonstrated that pyknoses are not revealed
in the ventricular zone of the hippocampus. Similar results were obtained by
Sturrock (1979) during the study of the ventricular zone giving rise to the neo-
cortex in the mouse. Evidently, mitotically active cells in the ventricular zones
of various regions of the normally developing brain do not undergo pyknotic de-
generation.
No pyknoses are detected either in the suprafimbrial zone of Ammon's horn
and the prime germinal zone of the dentate gyrus at the early stages of their
formation (EI6-EI8). In this respect, the germinal zones of the hippocampus differ
from the subventricular zone of the mouse neocortex, where pyknoses can be
registered already beginning from E 15 (Sturrock 1979). Later on, differences in the
age-dependent dynamics of the number of pyknoses in the suprafimbrial zone and
the germinal zones of the dentate gyrus become more pronounced (see Sect. 3.2.2).
The hypothesis suggested below connecting the mitotic death of cells with
processes of gliogenesis may probably help to account for differences in the
number of mitotically dying cells in the germinal zones of the brain. This hypo-
thesis proceeds from the idea that the morphogenetic program of neurogenesis is
more strictly controlled genetically than that of gliogenesis (Jacobson 1978;

30
Reznikov 1981) and, accordingly, requires to a smaller degree the correction of
mistakes appearing during the mitotic division of precursor cells. The mitotic
death of glial cells may serve as a mechanism for the regulation of their production
and elimination of their spontaneous mutations. On the one hand, these notions
are supported by a high percentage of death of mitotically dividing glial cells (Korr
1980), and on the other hand, by the prevalence of neoplastic formations of glial
origin among nerve tissue tumors (Rubinstein 1985). Moreover, proliferating cells
of the ventricular zone, the final mitoses of which are practically always connected
with neuronal production, do not reveal features of pyknotic degeneration (see
Sect. 3.2.2. and Sturrock 1979). Finally, neurons obey their own mechanism of the
neurogenetic program correction, i.e., amitotic death of superfluous neurons that
have not found their target cells.
If the above-suggested hypothesis is true, the absence of pyknoses in the
suprafimbrial zone and the germinal zones of the dentate gyrus during early
periods of their development (El6-E18) can be explained by the fact that at this
stage final mitoses of proliferating cells in the hippocampus are practically always
connected with realization of the program of neurogenesis. In the suprafimbrial
zone, the number of mitoses connected with glial differentiation and, accordingly,
the number of mitotically dying cells increase during the later periods of develop-
ment. A sharp increase in the number of pyknoses in this zone with a peak
corresponding to P3 is probably connected with mass genesis of astrocytes, which
are actively generated in the mouse and rat during the first few days after birth
(Privat and Fulcrand 1977; Korr 1980; Reznikov 1981).
In the mouse dentate gyrus processes of neurogenesis are most pronounced
during the 1st postnatal week and then continue at a slower rate until the end of the
3rd postnatal week (Angevine 1965; Stanfield and Cowan 1979b). During the 1st
postnatal week the fraction of cell losses in the dentate gyrus is comparatively
small. The existence of this fraction can be explained by gliogenesis proceeding in
the dentate gyrus simultaneously with the formation of granular neurons (see Sect.
4.3). During later perioids of development, when neurogenesis in the dentate gyrus
becomes less active, the relative number of mitoses connected with glial cell
production increases, which, accordingly, is accompanied by an increase in the
fraction of dying mitotic cells.

3.4 Conclusion

In the present section the results of studies on cell proliferation and cell death in the
germinal zones of the developing hippocampus in mice are considered and dis-
cussed. The mapping of the distribution of mitoses and pyknoses in the developing
Ammon's horn and dentate gyrus was carried out. The results of mapping and
quantitative analysis of mitoses allowed a more precise definition of the disposition
and age reduction of the germinal zones in the mouse hippocampus: the ventricular
zone of the hippocampus and the germinal zones of the dentate gyrus (the prime
germinal zone, the proliferative zone of the hilus, and the subgranular zone).
Moreover, a previously unknown extraventricular germinal zone of Ammon's
horn that is distinct in its regio inferior adjacent to the fimbria between E16 and
P7, termed a suprafimbrial zone, was revealed. It is assumed that the suprafimbrial

31
zone fulfills cytogenetic functions of the lacking subventricular (subependymal)
zone in Ammon's horn, i.e., represents the main source of gliogenesis in Ammon's
horn.
The results of studies on the distribution and quantitative analysis of pyknoses
in the hippocampus demonstrated that they do not occur in the ventricular zone
and in other germinal zones of Ammon's horn and the dentate gyrus nearly
throughout the whole period of embryogenesis. Later on, pyknoses appear both in
the suprafimbrial zone of Ammon's horn and in the germinal zones of the dentate
gyrus. Each germinal zone is characterized by its own age-dependent dynamics
of absolute and relative amounts of dying cells. It is assumed that the mitotic
death of cells in the germinal zones of the brain, as well as the mitotic death of
differentiating glial cells represent the mechanism of correction of the program of
gliogenesis.

32
4 Postnatal Production and Death of Cells
in the Mouse Dentate Gyrus

4.1 The Dentate Gyrus as an Object for Experimental

and Clinical Investigations

The data analyzed in the previous section indicate that the mouse dentate gyrus is
characterized by a high level of proliferative activity throughout the whole period
of postnatal ontogenesis. These findings agree with the results of previous inves-
tigations showing that in the dentate gyrus of both altricial mammals (mouse, rat,
rabbit, cat) and precocial animals (guinea pig, rhesus monkey) a considerable part
of neurons (and the majority of glial cells) appear after birth (Angevine 1965;
Altman and Das 1965, 1967; Caviness 1973; Bayer and Altman 1974; Hine and
Das 1974; Schlessinger et al. 1975; Kaplan and Hinds 1977; Stanfield and Cowan
1979b; Reznikov 1979, 1981; Bayer 1980a, 1982, 1985; Nowakowski and Rakic
1981; Rakic and Nowakowski 1981; Gueneau et al. 1982; Kaplan and Bell 1984;
Wyss and Sripanidkulchai 1985; Crespo et al. 1986; Trice and Stanfield 1986;
Eckenhoff and Rakic 1988). This clearly demonstrates the importance of the
dentate gyrus as an object for experimental neurobiological and clinical investiga-
tions. The fact that most structures of the dentate gyrus appear during postnatal
periods of development essentially simplifies experimental studies of morpho- and
cytogenesis, formation of nerve connections, and the effect of exogenous factors
on the development of this part of the central nervous system. The interest of
clinicians becomes especially apparent in this case, since the dentate gyrus, due to
its late development, represents one of the most vulnerable structures of the brain
during the period when the developing brain of a child is not protected by the
mother's organism from environmental hazards (see Morgane et al. 1978 for
a review).
This section presents the results of our studies on the cytological mechanisms
of histogenesis in the mouse dentate gyrus during normal postnatal development
and under the action of such exogenous factors as undernourishment and mechan-
ical traumatization. The results obtained were previously communicated by
Reznikov (1979,1981) and Nazarevskaya et al. (1982). However, in the generalized
form they are presented here for the first time.

4.2 Cytological Characteristics of Proliferating Cells in the Dentate Gyrus

During Postnatal Development

Numerous studies on cell proliferation in the germinal zones of the dentate gyrus
in the mouse and rat were based on the analysis of autoradiograms of the paraffin

33
sections of the brain. Such autoradiograms do not permit accurate identification of
the types of proliferating cells when ordinary histological methods of staining are
used. This, in particular, concerns my early work (Reznikov 1975), in which the
sub granular germinal zone of the dentate gyrus in the mouse was described.
Moreover, in these studies proliferating cells in the differentiated part of the
granular layer of the dentate gyrus were described, but the origin of these cells was
not elucidated. Thus, Bayer and Altman (1974) and Schlessinger et al. (1975)
reported on the presence of proliferating cells in the hilus of the dentate gyrus
which they believed to be neuronal precursors for the granular layer. This assump-
tion also required additional verification. In the present study, an attempt was
undertaken to solve the above-mentioned problems on the basis of the analysis of
autorjldiograms of semithin and ultrathin brain sections of 5-, 14-, and 35-day-old
mice pulse labeled with [3HJthymidine.
The results obtained demonstrated that in 5-day-old mice, comparatively
differentiated neurons can be found in the molecular layer, in the outer part of the
granular layer, and in the hilus of the dentate gyrus. An area in the hilus and the
inner part of the granular layer (especially in the infrapyramidallimb) are occupied
by dispersely arranged small cells with oval or angular, rather dark nuclei with
comparatively homogeneous background and delicate blocks of heterochromatin.
The cytoplasm of these cells is sparse and very often inperceptible in semithin
sections. Many undifferentiated cells are labeled with [3HJthymidine and divide
mitotically (Fig. 9a). Identical cells but in a smaller amount can be found in the
molecular layer of the dentate gyrus. On the whole, it appears impossible to
distinguish between different types of proliferating undifferentiated cells in the
dentate gyrus using semi thin sections.
In 2-week-old mice, undifferentiated cells similar to those detected in 5-day-
old animals are dispersely arranged in the hilus and form a compact subgranular
zone in the granular layer. The proliferative activity of these cells is still high (Fig.
9b). It should be noted that undifferentiated proliferating cells can be rarely found
among neurons in the differentiated part of the granular layer (Fig. 9c). Undif-
ferentiated and actively proliferating cells can be found also in the hilus and
molecular layer of 2-week-old mice. These cells resemble those of the subgranular
layer but contain more heterochromatin in their nuclei and possess a more
developed cytoplasm (not shown). At the same time, it appears difficult to give
a more detailed description of these cells using semithin sections. In view of this, an
electron-microscopic study of [3HJthymidine-pulse-labeled cells in the subgranu-
lar zone and hilus of 12-day-old CBA-strain mice was carried out (Reznikov et al.
1981). The results obtained demonstrated that in the polymorphic layer proliferat-
ing cells are represented by glioblasts (Fig. lOa, b). [3HJthymidine-labeled cells
have a polygonal or ovoid nucleus with prominent nucleoli and clumps of
chromatin subjacent to the nuclear envelope. The cytoplasm is poor in organelles;
mainly ribosomal rosettes occur. If labeled cells contact each other, desmosome-
like junctions are often observed between them (Fig. 11).
In 35-day-old mice, nerve cells in all layers of the dentate gyrus are differenti-
ated, neurons of the inner rows of the granular layer being considerably smaller
than similar cells in the outer rows of this layer. A characteristic feature of the
astrocyte distribution in the dentate gyrus is their substantial concentration in the
polymorphic layer. The overwhelming majority of glial cells does not incorporate

34
Fig. 9a-c. [3H)Thymidine-pulse-labeled cells (arrowheads) and mitoses (arrows) in the subgranular
zone of the dentate gyrus in 5-day-old a and 14-day-old b mice and a [3H)thymidine·pulse-labeled cell
(arrowhead) in the differentiated (neuronal) part of the dentate gyrus granular layer in the 14-day-old
mouse c. Toluidine blue, x 1100

35
Fig. lOa, b. Electron micrographs of [3H]thymidine-pulse-labeled glioblasts with polygonal a and
ovoid b nuclei in the polymorphic layer of the dentate gyrus in l2-day-old mice. In both cells, the
cytoplasm is poor in organelles; mainly ribosome rosettes are visible. x 25 000

36
Fig. 11. Electron micrograph of [3H]thymidine-pulse-labeled glioblasts in the polymorphic layer of the
dentate gyrus in the 12-day-old mouse. Between labeled cells desmosome-like junctions (arrows) are
present. x 19000. The same contacts (arrowheads) are shown at larger magnification in the inset.
x 50000

[3HJthymidine. Cells of the subgranular zone are heteromorphic (Fig. 13a). Some
of them possess the same characteristics as undifferentiated cells in the dentate
gyrus of 5-day-old mice. These cells are labeled well enough by [3HJthymidine (not

37
Fig. 12. The astrocyte in the differentiated (neuronal) part of the dentate gyrus granular layer in the
14-day-old mouse. x23 000

shown). Besides that, astrocytes can be found in the subgranular zone. They also
sometimes occur in the granular layer proper (Fig. 12). The number of undifferen-
tiated and glial cells in the subgranular layer of 35-day-old mice amounts to
16%-19% of the total amount of cells in the granular layer (Table 3).
Cells with pyknotic nuclei can be often found in the sub granular zone of 5- and
14-day-old adult mice (Fig. 13a). In Sect. 4.4 the data concerning the mitotic origin

38
Table 3. Correlation of various types of cells in the granular layer of 35-day-old mice (%)

Types of cells Suprapyramidal Infrapyramidal

limb limb

Mixed population of cells of the subgranular zone l8.H 1.3 l6.1±1.6

Granular neurons 78.7±3.1 81.4±3.5
Glial cells in the neuronal part of the granular layer 3.1 ±0.7 2.5±0.5

Values are the means (of five animals) ± SEM.

of these cells are presented. Additional electron-microscopic studies demonstrated

that processes of astrocytes that penetrate into the granular layer from the
polymorphic layer phagocytize dead pyknotic cells. It is significant that a similar
phenomenon can be observed in 1 to 3-day-old mice (Fig. 13b).
The cytological analysis of proliferating cells in the mouse dentate gyrus
indicates that during postnatal ontogenesis the sub granular zone does not only
reduce but becomes heteromorphic: in adult mice it contains both undifferentiated
and glial cells. This fact gives ground for an assumption about a polypotent nature
of proliferating cells in the subgranular zone. There is no doubt of course that the
majority of these cells differentiates into granular neurons (see Sect. 4.3). However,
some amount of subgranular cells transforms into glial cells that, together with
preserved undifferentiated cells, form a non-neuronal zone in the granular layer,
i.e., the subgranular zone in adult animals. In the neuronal part of the granular
layer, glial cells evidently also descend from cells of the subgranular zone. In this
case glioblasts continue to proliferate being already separated in the course of
development from the subgranular zone by rows of differentiating (not dividing)
granular neurons. The results of studies on proliferating cells in the polymorphic
layer (hilus) do not support the ideas of Bayer and Altman (1974) and Schlessinger
et al. (1975) who assert that the proliferative zone of the hilus contains neuronal
precursors. At least in 12-day-old mice the proliferating cells in the hilus are
represented by glioblasts.
Cells with pyknotic nuclei that can be found in the subgranular zone of mice of
various age groups are identical to cell pyknoses in other germinal zones of the
developing hippocampus. Their distribution and hypothetic importance were analyzed
in Sect. 3. The data on the presence in the subgranular zone of astroglial processes
phagocytizing dead cells are of interest mostly due to an observation that this process
takes place in very young (1- to 3-day-old) mice. It cannot be excluded that at early
stages of postnatal development the phagocytic functions are fulfilled by processes of
the radial glia that are morphologically very similar to processes of astrocytes
(Peters et al. 1976). It is known that the radial glia is persistent in the mouse
dentate gyrus up to P30 and exists even in adult animals (Basco and Hajos 1984).

4.3 Study of Postnatal Cytogenesis in the Dentate Gyrus

The existing literary data on cytogenesis in the dentate gyrus leave some problems
unsettled. In the first place, it remains unclear whether postnatal neurogenesis

39
Fig. 13a, b. Cell death in the subgranular layer of the dentate gyrus in normal postnatal mice. a A
pyknotic nucleus (arrowhead) in the subgranular zone of the 35-day-old mouse. Toluidine blue, x 1800.
b Electron micrograph of a phagocytizing glial process near the immature granular layer in the
l-day-old mouse. Note glial filaments that are characteristic of processes of astrocytes and radial glial
cells. x 50000

40
takes place in the dentate gyrus outside the granular layer. On the other hand,
there are data indicating that neurons in the molecular and polymorphic layers of
the mouse and rat complete their formation perinatally (Angevine 1965; Caviness
1973; Hine and Das 1974). On the other hand, Bayer and Altman (1974) reported
on the postnatal formation of a substantial part of neurons of the molecular layer
in the rat. Secondly, gliogenesis in the dentate gyrus was studied insufficiently. In
the above-cited work (Bayer and Altman 1974), the authors adduce quantitative
data on the dynamics of postnatal cytogenesis in the molecular and polymorphic
layers of the rat dentate gyrus. However, this study was performed using Nissl-
stained preparations (paraffin sections) that are of little use for identification of
glial cells and small neurons. This impelled us to conduct quantitative studies of
neuro- and gliogenesis in the mouse dentate gyrus using semi thin sections. Mice
were injected with [3HJthymidine at 12-h intervals in the course of 4 days in five
age groups (PI~P4, P5~P8, P9~PI2, P13~PI6, and PI7~P20). All the animals were
killed on P35. The autoradiograms of semi thin brain sections were used for the
count of the percentage of labeled neurons in the granular layer and intensely and
weakly labeled glial cells (astrocytes and oligodendrocytes) in the molecular and
polymorphic layers (see Sect. 2.2.2 for details).
The results of studies on [3HJthymidine incorporation into neurons of the
mouse dentate gyrus demonstrated that only neurons in the granular layer are
labeled during all periods of exposure to the isotope. Neurons in the molecular and
polymorphic layers do not incorporate [3HJthymidine even when it is injected
between PI and P4. In the granular layer of the dentate gyrus, practically all cells
were labeled except neurons in the region bordering on the molecular layer (Fig.
14a). In the experiments on pH]thymidine administration during later periods of
ontogenesis, the number of labeled cells decreased in consecutive order. In this
case, cells located closer to the polymorphic layer of the dentate gyrus were labeled
in the first turn (Fig. 14b). The data on the terms of formation of granular neurons
during postnatal ontogenesis are represented in Table 4.
These figures make it possible to estimate the percentage of granular neurons
generated in both limbs of the dentate gyrus during different periods of develop-
ment. Thus, about 81 % of neurons in the suprapyramidal limb of the granular
layer are labeled after [3HJthymidine administration between PI and P4, and it
can be concluded that 19% of granular neurons in this limb form prenatally.
Fifty-six percent of neurons in the suprapyramidallimb of the granular level are
labeled after [3HJthymidine administration between P5 and P8, and, consequent-
ly, the number of neurons generated between PI and P4 can be calculated
(81 ~56 = 25%). The results of these studies are indicative of thefollowing dynamics
of neuronal production in the syprapyramidal and infrapyramidal limbs of the
granular layer: embryogenesis, 19% and II %; PI~P4, 25% and 26%; P5~P8, 23%
and 29%; P9~PI2, 12% and 14%; P13~PI6, 10% and 11 %; PI7~35, 11% and
16%. On the whole, the dynamics of neurogenesis in the granular layer of the
mouse dentate gyrus observed is close to the results of similar investigations in the
mouse (Stanfield and Cowan 1979b) and rat (Bayer and Altman 1974). This
indicates that in contrast to the embryonic period, which is characterized by later
terms of neuronal genesis in the mouse dentate gyrus than in the rat one (see Sect.
1.2.1), the postnatal dynamics of genesis of granular neurons reveals close
similarity in both species. Concerning higher rates of neurogenesis in the

41
Fig. 14a, b. [3H)Thymidine·labeled neurons in the granular layer of the dentate gyrus in 35·day-old
mice exposed to the isotope from PI until P4 a and from P13 until PI6 b. Toluidine blue, x 1100

suprapyramidallimb of the granular layer in comparison with the infrapyramidal

limb, the data obtained support the results of previous investigations in the rat,
which revealed the same regularity (Schlessinger et al. 1975). Contrary to Bayer

42
Table 4. The percentage of labeled neurons in the granular layer of the dentate gyrus of
35-day-old mice exposed to [3HJthymidine from PI to P20

Age groups Suprapyramidallimb Infrapyramidallimb

(days of [lHJthymidine
administration)

1-4 81 ± 1.9 89±2.2

5-8 56±2.1 63±2.4
9-12 33±2.0 41 ±2.1
13-16 21 ± 1.7 27 ± 1.8
17-20 11 ± 1.5 16± 1.7

Values are the means (of four animals) ± SEM.

and Altman (1974), I did not discover postnatal neurogenesis in the molecular
layer. Of course, one should take into account the importance of specific differ-
ences of the objects studied (the mouse and rat). However, one cannot completely
exclude that in Nissl-stained autoradiographs of paraffin brain sections large
labeled glial cells were taken for small neurons. This assumption agrees with the
data of other studies on embryonic (rather early) formation of neurons in the
molecular layer both in the mouse (Angevine 1965; Caviness 1973) and rat (Hine
and Das 1974).
The study of postnatal gliogenesis based on the cumulative labeling experi-
ment demonstrated that the majority of glial cells in all layers of the dentate gyrus
contained the label when [3H]thymidine was introduced between PI and P8.
When the isotope was injected during later periods of development, the number of
labeled glial cells progressively decreased. Intensely labeled astrocytes can be seen
in all layers of the dentate gyrus, including the granular layer and (what is most
interesting) the subgranular zone beginning from the first postnatal days. Intensely
labeled oligodendrocytes first appear in a somewhat large amount on P5. The
dynamics of changes in the number of labeled astrocytes (Table 5) demonstrated
that both in the molecular and polymorphic layers astrocytes undergo final
divisions during all periods of [3H]thymidine administration, i.e., from PI to P20.
However, the highest amount of astrocytes goes out from the proliferative com-
partment of astroblasts between P9 and P12. Beginning from Pl7 the number of
astrocytes undergoing final divisions substantially decreases. However, astroblasts
continue to proliferate after P20 (when the isotope is introduced between Pl7 and
P20 from 5% to 8% of these cells have a weak label). It is noteworthy that the rates
of cessation of the astroblast proliferation in the molecular and polymorphic layers
are practically the same.
The results of calculations of the number of labeled oligodendrocytes are
represented in Table 5. It can be seen that a large-scale cessation of their final
divisions both in the molecular and polymorphic layers begins on P9. This process
remains sufficiently pronounced until P20. After this the division of oligoden-
drocytes continues, about 17% and 15% of these cells in the molecular and
polymorphic layers, respectively, dividing between P20 and P35. It follows from
the table that the rates of production of oligodendrocytes in the molecular and
polymorphic layers are essentially the same and that astroglial cells accomplish
their genesis and cease final divisions earlier than oligodendrocytes.

43
Table 5. The percentage of labeled astrocytes and oligodendrocytes in the molecular and polymorphic
layers of the dentate gyrus of 35-day-old mice exposed to [3H]thymidine from PI to P20

Age groups Molecular layer Polymorphic layer

(days of
[3H] thymidine All labeled Intensely All labeled Intensely
administration) cells labeled cells cells labeled cells

Astrocytes
1-4 75±4.0 13±20 n±4.l 16± 1.9
5-S SO±4.4 16± 2.5 78±4.3 19±2.2
9-12 63±3.9 24± 2.8 56±3.6 22±2.4
13-16 42±3.0 19± 2.1 37 ±2.7 IS±2.1
17-20 IS±2.5 1O± 1.6 15±1.7 1O± 1.4
Oligodendrocytes
1-4 74±4.2 3± 0.9 67±4.3 2±0.S
5-8 80±4.5 9± 0.9 75±4.3 6± 1.8
9-12 77±4.0 20± 2.6 78±4.6 24±2.9
13-16 68±3.7 28± 3.2 63±3.4 29±3.3
17-20 3H3.l 20± 2.5 3H2.8 IH2.7

Values are the means (of four animals) ± SEM.

The data obtained on the terms of final divisions by astrocytes and oligoden-
drocytes in the molecular and polymorphic layers of the dentate gyrus are very
close to the data on the terms of gliogenesis in the mouse neocortex (Reznikov
1981). Therefore, it can be assumed that in two brain structures that essentially
differ in the terms of neuronal genesis, generation of glial cells takes place at
approximately one and the same time. It is known that neurons are produced
in the mouse neocortex only during embryogenesis (Reznikov 1981), whereas
in the dentate gyrus about 80% of granular neurons appear after birth. Therefore,
the genesis of glial populations in the neocortex and dentate gyrus does not depend
on the rate of neuronal production. Similar data were obtained in the studies on
the terms ofneurogenesis and gliogenesis in the visual cortex and lateral geniculate
body in the rat. Despite the fact that in the lateral geniculate body neurogenesis is
completed much earlier than in the neocortex (Bruckner et al. 1976), the dynamics
of genesis of glial cells in these structures is essentially the same (Biesold et al. 1976;
MareS and Bruckner 1978).

4.4 Kinetics of Cell Proliferation and Death in the Subgranular Zone

of the Dentate Gyrus in Normal and Undernourished Mice

In the present-day literature only one work exists devoted to the study of kinetics
of cell proliferation in the dentate gyrus (Lewis et al. 1979). However, in this study
which was performed in 1-, 6-, and 12-day-old mice, the parameters of cell
proliferation were determined in the proliferative zone of the hilus which differs
from the subgranular zone in the dynamics of reduction during postnatal develop-
ment and gives rise to other types of cells. This study also demonstrated that in

44
undernourished rats (when pregnant or lactating females received insufficient
quantities of food) the duration of the mitotic cycle of proliferating cells in the
dentate gyrus considerably increased.
Similar data were obtained during the study of the kinetics of cell proliferation
in the subependymal zone of the lateral ventricles of the cerebrum and the external
granular layer of the cerebellum in undernourished rats (Lewis et al. 1977; Deo
et al. 1978). It remained unclear whether undernourishment affects cell prolifer-
ation in the germinal zones of the brain in mice. It was reported that contrary to
rats, the undernourishment in mice during embryonic development does not lead
to the reduction of the DNA content in the brain (Nehrich and Stewart 1978).
Lastly, it appeared of interest to elucidate whether alimentary deficiency enhances
cell death in the germinal zones of the mouse dentate gyrus.
It was reported that in rats growing under conditions of food deprivation the
number ofpyknoses increased in the subependymal zone of the endbrain and in the
external granular layer of the cerebellum (Lewis 1975; Lewis et al. 1977). All this
served as a basis for the study of the kinetics of cell proliferation and death in the
subgranular zone of the dentate gyrus and in the subependymal zone of the lateral
brain ventricles in 20-day-old normal mice and animals of the same age subjected
to undernourishment from PlO to P20. (See Sect. 2.2.2 for a detailed description of
the experiment.)
The results of calculation of the mitotic index (MI) and the index of labeled
nuclei (LI) in the subgranular and subependymal zones in mice sacrificed 1 h after
[3H]thymidine injection demonstrated the absence of essential differences in the
indexes in normally fed animals and mice subjected to undernourishment between
PIO and P20 (Table 6). Moreover, the MI and LI were considerably higher in the
subependymal zone than in the sub granular one. The analysis of curves of labeled
mitoses in the subgranular and subependymal zones (Fig. 15a, b) indicated a signi-
ficant elongation of the mitotic cycle in undernourished mice. Determination of
parameters of the mitotic cycle (Table 7) revealed their almost complete coincid-
ence in the subgranular and subependymal zones. Evaluation of the growth
fraction in both germinal zones of undernourished mice did not reveal any
substantial changes in comparison with control animals (Table 6). The time of
duplication of cell populations in the studied brain germinal zones of undernour-
ished and control mice also did not differ.
The pyknotic indexes (PI) in the subgranular and sub ependymal zones of
control mice exposed to [3H]thymidine and of intact animals were essentially the
same, and no increase in PI after administration of the isotope was observed (Table
6, Fig. 16a, b). These findings indicate that the dose of [3H]thymidine used in the
experiment did not enhance cell death in the studied brain germinal zones. The
total amount of cells with pyknotic nuclei in the subgranular and subependymal
zones increased considerably upon undernourishment. The analysis of curves of
[3H]thymidine-Iabeled pyknotic nuclei in the subgranular and subependymal zones
(Fig. 15a, b) demonstrated that first labeled pyknotic nuclei appeared 4 h after
administration of the isotope (at this stage of the experiment no more than
0.5%-0.8% of pyknotic nuclei contained the label). Then their portion increased
reaching a maximal value after 12 hand 24 h in the subgranular zone and after 16 h
and 24 h in the subependymal zone of the control and undernourished mice,
respectively.

45
Table 6. Parameters of cell proliferation and death in the subgranular zone of the
dentate gyrus and in the lateral ventricular subependymal zone of 20-day-old
control mice and animals of the same age subjected to undernourishment from
PIO

Parameters Control mice Undernourished mice

Subgranular zone
MI (%) 0.52±0.06 0.47±0.08
LI (%) 3.5±0.1 4.2±0.4
GF (%) 6.7 8.7
PDT (h) 268 261
PI (%) 0.41 ±0.03 1.38±0.07b
FCL(%) 3.9 14.7
Subependymal zone
MI (%) 0.76±0.06 0.68±0.04
LI (%) 9.81 ±0.80 1O.62±0.25
GF (%) 18.5 18.3
PDT (h) 109 117
PI (%) 0.40±0.09 0.94±0.08'
FCL(%) 2.9 6.9

Significant difference between control and undernourished groups: ·P<O.OI;

bp<O.OOI MI, the mitotic index; LI, the labeling index; GF, the growth fraction;
PDT, potential doubling time; PI, the pyknotic index; FCL, fraction of cell losses.
The PIon 20-day·old mice not exposed to [,H]thymidine are 0.45% ±0.04% and
0.36% ±0.10% in the subgranular and subependymal zones, respectively. Values
are the means (of four animals)±SEM.

Table 7. Parameters of the mitotic cycle in the subgranular zone of the dentate gyrus and the lateral
ventricular subependymal zone of 20-day-old control and undernourished mice (h)

Animals Parameters

T ts tG2 + 1/2tM tG , + III tM

Subgranular zone
Control mice 19.0 9.4 3.4 6.2
Undernourished mice 21.0 11.0 3.9 6.1
Subependymal zone
Control mice 20.2 10.7 3.1 6.4
Undernourished mice 21.5 11.9 3.9 5.7

T, duration of the mitotic cycle; Is, duration of the S·phase; I G " duration of the G ,-phase; I Gz ' duration
of the Gz-phase; 1M, duration of mitosis.

Thus, the above-described study provided data on the kinetics of cell prolifer-
ation in the subgranular zone of the dentate gyrus. The results obtained demon-
strated that in 20-day-old mice cell proliferation in the sub granular zone of the
dentate gyrus is less intensive than in the subependymal zone of the same animals.
These differences may be associated with a higher cell heterogeneity in the sub-

46
 "I.
100

80

60

40 -

20 -

a 12 4 6 8 12 16 24
0/0 Time (h)
100

80

60

1,0
2

20

12 16 24
Time (h)
Fig. 15a, b. Curves fitted to the data of percentage of labeled mitoses and pyknoses in the lateral
ventricular subependymal zone of the forebrain a and the subgranular zone of the dentate gyrus b in
20-day-old control and undernourished mice at different times after injection of [3H)thymidine. 1 and 3,
percentage oflabeled mitoses and pyknoses in control mice; 2 and 4, percentage oflabeled mitoses and
pyknoses in undernourished mice. Each point represents the results obtained in one animal. Abscissa,
percentage oflabeled mitoses and pyknoses; ordinate, time (in h) after administration of pH)thymidine

granular zone, which contains glial and neuronal cells besides multiplying precur-
sor cells (see Sect. 4.2). The duration of the mitotic cycle and its periods for
proliferating cells was found to be essentially the same in the subgranular and
subependymal zones. These data are in good accord with the results of studies on
the kinetics of cell proliferation in the brain germinal zones in the rat and mouse,
which demonstrated that in the postnatal brain the germinal zones revealed close
similarity in the duration of the mitotic cycle (reviewed by Korr 1980; Reznikov
1981).
The present study also demonstrated that in mice subjected to undernourish-
ment from PIO to P20, no essential changes in the duration of the mitotic cycle
in the subgranular and subependymal zones were observed. The data obtained
differed from the results of the analysis of cell proliferation in the subependymal

47
"/0
1.4 o

0
0

I
<i' ' 0
0

--
_-0'-...... -.._
0
0
1.0 c) I ,~-- 0 "'--2
~ ~/ 0 0
0

0.6 • • • 10
• ·--~~3
0.2 • • • •• 0

a 12 4 6 8 12 16 24
OJ. Time(h)
2.2 0

0 0 0

1.8 ,"" - -(5',

o / 0" ,
r-,,
/
1.4 o 0 0 I " 0
I / 0 '
, 0 '~_/ '2
I
1.0 o 0
0

0.6
0
•

0.2
b
12 4 6 8 12 16 24
Time (h)
Fig. 16a, b. Pyknotic indexes (PI) in the lateral ventricular subependymal zone of the forebrain a and
in b the subgranular zone of the dentate gyrus b in 20-day-old control and undernourished mice at
different times after injection of [3H]thymidine. 1 and 2, PI in control and undernourished mice,
respectively; 3, PI in control mice which were not exposed to [3H]thymidine. Each point represents the
results obtained in one animal. Abscissa, PI in percentage; ordinate, time (in h) after administration of
[3 H]thymidine

zone of 20-day-old rats that revealed a considerable lengthening of the mitotic

cycle upon undernourishment (Lewis et al. 1975). This can be evidently due to
differences in experimental conditions: mice were subjected to undernourishment
from PIO to P20, whereas rats were subjected to undernourishment throughout the
whole embryonic period and during the first 3 postnatal weeks. It should be noted
that in the present experiment an insignificant increase (by 10%) in the duration of
the mitotic cycle upon undernourishment was observed.
The data on the kinetics of cell death in the subgranular and subependymal
zones of the control and undernourished mice are indicative of the mitotic origin of
pyknotic nuclei. First labeled pyknoses appear 4 h after administration of [3H]_
thymidine, i.e., after cells that have incorporated the isotope at the end of the
S-period pass the G 2- and M-periods (see the data on tG2 +ttM in Table 7). The
percentage of labeled pyknotic nuclei increases at the late stages of the experiment,
i.e., after the division of cells labeled during the S-period. At the late stages of the

48
 • .•
..
. .

f
,.
~

..
4',

....
..... -; .
,.
-.,
t'

Fig. 17a-c. back of regeneration of the dentate gyrus granular layer 2 months after its transection in
the 3-day-old mouse. a-c A rostrocaudal series oftransveFse sections through the dorsal hippocampus
in the 63-day-old mouse. The distance between sections is 480 11m. Note the disturbance of morpho-
genesis of the infrapyramidallimb of the granular layer which has acquired a spheric-like shape (c).
Cresyl violet. x 120

49
experiment, the appearance of pairs of labeled pyknotic nuclei was observed. In
this case, both nuclei were always labeled. The presence of pairs of labeled
pyknotic nuclei indicates that the pyknotic degeneration of labeled nuclei occurs
not during the S- and G 2 -periods, but after completion of mitosis. The data
obtained correlate with the results of the studies of cell pyknoses in the subepen-
dymal zone and the external granular layer of the cerebellum in normally fed and
undernourished rats (Lewis 1975; Lewis et al. 1977). These studies also demon-
strated that in the germinal zones of the brain the mitotic nuclei are subjected to
pyknosis and that the number of pyknoses in these zones considerably increases
upon undernourishment. At present, it remains unclear what metabolic distur-
bances lead to cell death in the brain germinal zones upon undernourishment. It
can be assumed that as well as in lymphocytes (Sharmanov 1979) the mechanism of
mitosis of dividing brain cells is disrupted and the frequency of chromosomal
abnormalities increases upon undernourishment.

4.5 Study of the Regenerative Capacity of the Dentate Gyrus

The data presented in Sect. 4.3 demonstrate that about 80% of neurons in the
granular layer of the mouse dentate gyrus are generated during postnatal develop-
ment. In view of this, it appeared of interest to test the regenerative capacity of the
granular layer of the dentate gyrus after its transection in young (3- and 5-day-old)
mice. The dentate gyrus was transected sagitally in the dorsal hippocampus. The
animals were sacrificed on P30 and P60. The Nissl-stained serial sections of
the dentate gyrus were analyzed. The results obtained demonstrated that in all
cases the transected granular layer did not restore its integrity (Fig. 17a-c).
Therefore, the availability of postnatal neurogenesis in the granular layer of the
dentate gyrus is not a sufficient condition for post-traumatic regeneration.

4.6 Conclusion

This section is devoted to the analysis of postnatal cytogenesis in the mouse

dentate gyrus. In some respects, this process resembles cytogenesis in such brain
structures as the cerebellar cortex and the olfactory bulbs that are also character-
ized mainly by postnatal production of granular neurons, the latter forming
a compact layer of densely packed cells. At the same time, cytogenesis in the
dentate gyrus is characterized by certain features not revealed in other brain
structures. Thus, the above-described subgranular zone, in contrast to other
germinal zones of the brain, forms a structural complex with differentiated neurons
of the granular layer. In view of this, postnatal neurogenesis in the granular layer is
characterized by the absence of cell migration. Granular neurons differentiate in
successive rows (outside-in) from cells of the subgranular zone, gradually replacing
the latter. Moreover, there is no other structure in the mammalian brain (including
the mouse) that can be compared with the granular layer of the dentate gyrus in the
duration of neurogenesis. The germinal zones of the dentate gyrus are character-
ized by different potencies for differentiation. Thus, the subgranular zone mainly
gives rise to the population of granular neurons and, moreover, is involved in the

50
generation of glial cells in the granular layer. Cells of the proliferative zone of the
hilus represent mainly the precursors of glial cells of the polymorphic layer.
The study of gliogenesis in the dentate gyrus demonstrated that the order and
rate of production (cessation of division) of astrocytes and oligodendrocytes in the
molecular and polymorphic layers are similar to those of glial cells in the mouse
parietal cortex (Reznikov 1981). These data indicate that in different structures of
the mouse brain the proliferative stage of gliogenesis does not practically depend
on the rate of neuronal production, since in the dentate gyrus neurogenesis is
completed considerably earlier than in the neocortex.
Postnatal production of neurons is very sensitive to environmental hazards.
This assertion is supported by the above-presented data on the increase in the
number of pyknoses in the subgranular layer of young mice subjected to under-
nourishment. Moreover, we have previously demonstrated (Nazarevskaya and
Reznikov 1981) that in mice subjected to undernourishment from PIO to P40 the
number of neurons in the infrapyramidallimb of the granular layer of the dentate
gyrus is lower (P<O.05) than in control animals of the same age.
Finally, the experiments on transection of the dentate gyrus in 3- to 5-day-old
mice demonstrated that the availability of postnatal neurogenesis is not a sufficient
condition for post-traumatic regeneration of damaged brain structures in mammals.

51
5 Neurogenesis in the Hippocampus and Neocortex
as the Embryonic Basis for Brain Module Formation

5.1 Neural Modules in the Neocortex and Hippocampus

At present, it becomes ever more evident that neural centers are organized as
reiterating neuronal ensembles or modules. A conception of modular organization
of neural centers in the eNS was suggested by Szentagothai and Arbib (1975) and
was further developed by many investigators (reviewed by Szentagothai 1983).
According to this conception, the modules represent portions of neural nets which,
under given conditions, function as independent integrative units of neurons. In
different brain structures, the modules vary in their spatial organization, the
number and composition of neurons, their connections, and the character of data
processing, but within the limits of one structure they are mostly the same. The
modules were discovered in all large brain structures including the cerebral cortex.
A great number of works is devoted to the study of modular organization of
the neocortex (reviewed by Szentagothai 1978, 1983; Mountcastle 1979; Eccles
1981; Jones 1984). Many investigators believe that the neocortex has two levels of
modular organization: minicolumns and macrocolumns. Minicolumns represent
indivisible small processing units in the neocortex. According to Mountcastle
(1979), a neocortical minicolumn is a vertical cylinder 30 Jlm in diameter contain-
ing about 110 neurons with a great number of vertical connections and a small
number of horizontal ones. Mountcastle believes that minicolumns are formed on
the basis of ontogenetic columns that are distinct in the developing neocortex and
represent chains of nerve cells that have migrated into the cortex along the radial
glia fibers (Rakic 1978; Rakic and Goldman-Rakic 1982). Macrocolumns are
considerably larger structural and functional processing units than minicolumns.
Each of them contains several hundred minicolumns. The diameter or the width of
macrocolumns vary from 200 to 500 Jlm in different areas of the neocortex.
Macrocolumns are organized around corticocortical afferent fibers penetrating
vertically through the entire depth of the cortex.
The module organization of the hippocampus has been studied far less
sufficiently than that of the neocortex. At present, there is practically no direct
evidence for the presence in the hippocampus of micromodules comparable with
the neocortical minicolumns. Indirect evidence for the existence of such micro-
modules is provided by the data on the electron coupling among neurons in the
pyramidal and granular layers which is realized via gap junctions and is accom-
panied by the molecular transfer. Vertical clusters of pyramidal neurons labeled by
administration of Lucifer yellow into one of the coupled cells were revealed
(reviewed by Schwartzkroin 1982). The data on the presence of macro modules in
the hippocampus are more definite. These macromodules are represented by strips

52
or lamellae of the hippocampal tissue oriented perpendicularly to the long axis of
the hippocampus. Electrophysiological studies demonstrated that both Ammon's
horn and the dentate gyrus are organized as a series of functionally relatively
independent lamellae less than 700 11m wide. In this case, excitation of one lamella
is accompanied by lateral inhibition of the neighboring lamellae (see Andersen
1975; Schwartzkroin 1982 for references). Neuroanatomical evidence for laminal
organization of the hippocampus is less obvious (see Amaral and Witter 1989 for
a critical review). At the same time, the main source of cortical afferent projections
to the dentate gyrus and Ammon's horn, the entorhinal cortex, has a modular
columnar structure (Ikeda et al. 1989). Therefore, further studies are required to
elucidate the modular architectonics of the hippocampus.

5.2 Sequences of Neurogenesis and Formation of Modules

in the Archi- and Neocortex: Problems of Interrelationship

The study of neurogenesis in the mammalian cerebral cortex based on the registra-
tion of PH] thymidine-labeled neurons whose precursors underwent final divisions
at the moment of the isotope administration demonstrated the interconnection
between the spatial and temporal sequence of neuronal genesis with the laminar
organization of the hippocampus and neocortex. Thus, the sandwich and the
inside-out gradients of neurogenesis were described for Ammon's horn (see
Sect. 1.2.1). Earlier neuronal formation in the molecular and polymorphic layers
in comparison with neurogenesis in the granular layer was described for the
dentate gyrus. Moreover, its granular layer is characterized by the outside-in
gradient of neurogenesis (see Sect. 1.2.1). In the neocortex as well as in the
hippocampus, the sequence of neurogenesis reveals an interrelationship with its
laminar organization (reviewed by Jacobson 1978).
It should be noted that the overwhelming majority of autoradiographic
investigations on. spatial and temporal sequence of neurogenesis were not aimed at
elucidation of the embryonic basis of module formation except the works by Rakic
(Rakic et al. 1977; Rakic and-Goldman-Rakic 1982) who advanced a hypothesis
about "proliferative units" in the germinal zones of the brain, which was based on
his findings on the ordered migration of young neurons along the radial glia fibers.
According to this hypothesis, a small group of cells in a germinal zone - a prolif-
erative unit - successively produces neurons that form around a radial fiber (or
several radial fibers) an ontogenetic column which serves as a basis for mini column
formation. Rakic's hypothesis was supported by Smart and coauthors (Smart
1982; Smart and McSherry 1982; Smart and Smart 1982) with their conception of
"radial units" in the developing neocortex and hippocampus which were sub-
divided into arbitrary numbers of radially oriented strips. These strips (radial
units) differed in terms of neurogenesis and were larger than neocortical macro-
columns. The present study represents further elucidation of the relationship
between the spatiotemporal sequence of neurogenesis in the hippocampus and
neocortex and module formation in these structures. The study was begun with the
analysis of this relationship in the neocortex, since its module organization was
studied more profoundly than that of the hippocampus. The next step involved the
analysis of the sequence of neurogenesis in the hippocampus on the basis of the

53
data obtained during the study of the neocortex. The results obtained were
published previously (Reznikov and Nazarevskaya 1983; Reznikov et al. 1984,
1987a; Nazarevskaya and Reznikov 1985).
Here, they are presented in the generalized form (for detailed description of
experimental procedures, see Sect. 2.2.2).

5.3 Results of the Study

5.3.1 Vertical Order and Mosaicism of Neurogenesis in the Neocortex

Both in the frontal and sagittal semithin sections of the forebrain of I-day-old mice
the cortical plate was found to consist of well-defined inner and outer laminae
(Fig. 18). The outer lamina of the plate, giving rise to layers II, III, and IV of the
neocortex, comprises less than one-third of the total thickness of the plate and is
characterized by undifferentiated elongated cells densely packed in columns. Cells
of the inner lamina (prospective layers V, VIa, and VIb) are more differentiated
and do not distinctly show the columnar arrangement.
The distribution of [3H)thymidine-Iabeled neurons in the neocortex differs
considerably depending on the embryonic day of the isotope administration. In
mice exposed to [3H]thymidine on E15; intensely labeled neurons preferentially
occur in the middle and lower parts of the outer lamina of the cortical plate
(prospective layers III and IV), though some intensely labeled cells can be found in
the outermost part of the plate (prospective layer II) and in the upper part of the
inner lamina (prospective layer V). They were arranged in vertical rows of 3-11
cells alternating with unlabeled or weakly labeled cell rows. The mosaic arrange-
ment of intensely labeled neurons in clusters is similarly pronounced both in the
frontal and sagittal sections of the neocortex (Fig. 19a, b). Spindle-shaped cells
migrating into the outer lamina (young neurons, glioblasts) were unlabeled or only
weakly labeled. The mapping of distribution of intensely labeled neurons also
revealed that they are arranged in clusters or groups (Fig. 20a).
Injection of the isotope on El6 resulted in intense labeling only in the outer
lamina of the plate. As well as after injection on E15, intensely labeled neurons
are arranged in clusters alternated with unlabeled or weakly labeled neurons
(Fig. 19c, d). These clusters are shorter than those in mice exposed to the isotope
on El5 (Fig. 20b), but the trend to vertical orientation was obvious. The number
of intensely labeled neurons in clusters in the frontal and sagittal sections was
similar. In contrast to mice exposed to [3H)thymidine on E15, intensely labeled
migrating neurons are observed in the inner part of the neocortex of El6-injected
animals.
Upon isotope injection on E17, intensely labeled neurons were still detectable
in the outermost part of the plate, though their number had decreased substan-
tially. As in E15- and El6-injected animals, after isotope administration on E17,
intensely labeled neurons are arranged in clusters (Fig. 21). The total number of
intensely labeled cells migrating through the neocortex was higher in mice exposed
to [3H)thymidine on El7 than in El6-injected animals. These cells are distributed
throughout the whole cortical plate up to the outermost zone of intensely labeled
neurons that have reached their destination.

54
Fig. 18. Photomicrograph of the part of field 6 of the neocortex in the I-day-old mouse. ML, molecular
layer; OL, outer lamina (prospective layers II, III, and IV); fL, inner lamina (prospective layers V, VIa,
and VIb); Wm, white matter. Frontal section. Toluidine blue, x 400

55
Fig. 19a-d. Clusters of intensely labeled neurons in field 6 of the neocortex in I·day·old mice after
injection of [3H]thymidine on EI5 (a and b, frontal and sagittal sections, respectively) and on EI6 (c and
d, frontal and sagittal sections, respectively). Toluidine blue, x 1100

56
 VIal

a
------------
-- --- -- VI6l

---------

ML

SLs

XX

X Vl
X

.. X Vial

--------------- --- Vlln

b
Fig. 20a, b. Schematic drawings of the distribution of intensely labeled neurons in field 6 of the
frontally sectioned neocortex in I-day-old mice after injection of [3H]thymidine on EI5 a and EI6 b.
Intensely labeled neurons are designated by filled circles; lightly labeled and unlabeled ones by open
circles (they are marked only in the upper part of the drawings); migrating intensely labeled cells are
designated by crosses. ML, molecular layer; SLs, superficial layers (layers II, III, and IV); VL, layer V;
VIaL, layer VIa; VIbL, layer VIb or layer VII. Scale bar = 60 11m

In mice exposed to [3H]thymidine on El8 and E19, there are practically no

labeled neurons that have occupied their final positions in the outer lamina of the
cortical plate.
Computer analysis of the maps of arrangement of intensely labeled neurons in
field 6 of mice exposed to [3H]thymidine on E15-E17 revealed nonrandomly

57
 x x
x x
x
x VL
x
x x x x
x
x

VIaL

--
-----~
VIOL

Fig. 21. Distribution of intensely labeled neurons in field 6 of the neocortex in the l-day-old mouse
after [3H]thymidine injection on E17. Designations and abbreviations as in Fig. 20. Scale bar = 60 Ilm

--------
., MOl.L.
--------

., r.i · I I, I
"
,., I I .~

I·' ,'"'I
••
r1
I. I
... r·,
!W"-'
•• 1 r,
~~ I.. -J t".- l ,., I 1 1 I
~l
I I
IT SPl. is.
1~ I I ,I
~\ ·1 ••
L..
I I
II
II
•
:."1II
I •• 1·1'.1 1 ,
I ·1 I 1(;1 L.. I I
I I II I I • I I ---------
II I I II I I:: I I
, I • LJ JI L_ .. J~ : :
1.1 L.! 1·1
lJ DEEP
is.

"" ....,. ,.to • #111-" .., "

w ~~uu UU LJ U
It, ...............
LJL.......JL--...JUU~U
,.#

Fig. 22. Example of computer-treated map with the distribution of intensely labeled neurons in field
6 of the neocortex in the l-day-old mouse after [3H]thymidine injection on E15. Intensely labeled
neurons are designated by filled circles; their non-random groupings by dashed lines. Along the
horizontal axis of the map, the projections of intensely labeled neurons (filled circles) and widths of
their groupings (brackets) are designated. Abbreviations as in Fig. 20. Scale bar = 60 Ilm

existing groups of intensely labeled neurons (Fig. 22). In five of six cases, nonran-
dom differences from the random distribution of intensely labeled neurons were
revealed at the highest level of statistical significance (P < 0.001) and in one of two

58
cases on El7 at P < 0.01. Since not all intensely labeled neurons have time to reach
their destination in the neocortex by PI after isotope administration on E17, the
maps offield 6 obtained from animals exposed to [3H]thymidine on El5 and El6
were used for statistical analysis of the revealed groups of intensely labeled
neurons. The following results were obtained:
Number of neurons in a group: 4.44±0.25
Width of a group: 14.2±0.53 J.lm
Height of a group: 80.8 ±4.23 J.lm on El5
and 30.6±5.1OJ.lm on El6
Width of spaces: 16.4±0.77 J.lm

5.3.2 Spatial and Temporal Sequences of Neurogenesis in Field CAl

of Ammon's Horn

The Ammon's horn of l-day-old mice is distinctly subdivided into suprayramidal

layers (strata radiatum, lacunosum, and moleculare), the pyramidal layer, and
infrapyramidallayers (stratum oriens and alveus) which, however, are immature
(Fig. 23). The regio superior (field CAl) is considerably thinner than in adult mice.
The majority of cells in all layers of Ammon's horn (especially in the infra-
pyramidal layers) is immature. In the infrapyramidallayers many immature cells
are characteristically spindle-shaped, which is typical of all migrating cells.
The distribution and number of [3H]thymidine intensely labeled neurons in
field CAl in l-day-old mice varies substantially depending on the term of the
isotope administration. After isotope administration on E12, an extremely low
number of intensely labeled neurons are found mainly in the suprapyramidallayers
and in the pyramidal layer along its border with the stratum oriens (Fig. 24a). The
number of intensely labeled neurons in CAl increases slightly after isotope admin-
istration on E13 (Fig. 24b). At this stage of the experiment intensely labeled cells
are found throughout the whole diameter of CAl, but the majority of them are
located in the supra pyramidal layers and in the inner zone of the pyramidal layer.
It should be noted that after isotope administration on E12-E13 intensely labeled
neurons do not exhibit any evident tendency to aggregate in clusters or chains.
In mice exposed to [3H]thymidine on E14, numerous intensely labeled neurons
are found in all layers of CAl, especially in the suprapyramidallayers (Fig. 25). In
the pyramidal layer intensely labeled neurons more frequently occur in its inner
zone adjacent to the stratum oriens. At this stage of the experiment, clusters of
intensely labeled neurons alternating with unlabeled or weakly labeled cells are
pronounced sufficiently distinctly (Fig. 26a). Quite frequently they form radially
oriented rows.
Injection of the isotope on El5 resulted in numerous intensely labeled neurons
in all layers of CA I, but the majority of them is located in the stratum pyramidale
(Fig. 25). They can be found throughout the whole diameter of the pyramidal
layer. At this stage of the experiment, the mosaic-like distribution of clusters of
intensely labeled neurons is most distinctly pronounced (Figs. 26b and 27a, b). It
should be noted that after exposure to the isotope on El5 as well as after its
administration at earlier stages of the experiment (EI2-EI4) the migrating cells in
the stratum oriens remained unlabeled or weakly labeled.

59
Fig. 23. Photomicrograph of the part of field CAl of Ammon's horn in the l-day-old mouse. [PL,
infrapyramidal layer (stratum oriens, alveus, and ependymal layer); PL, pyramidal layer (stratum
pyramidale); SPL, suprapyramidallayer (strata radiatum, lacunosum, and moleculare). Frontal sec-
tion. Toluidine blue, x 550

60
 a

o 0 0
0000 0 0000 000 0 0 0 : 000 000°0 o'8"""-------
o 0 000 0 0 0 a 0 0 ago 00 0 0 ~ c:, 00 0 0 a 0
ogooo°'&o~ 000 0 000 0000 ~o o~o
00000000:0°0
0'6 00 00 0 0 0 a 000 00 0 0 O. 0 0 0 a 00 STN. ON.
0 00 0 00.° 0 0 0 00 ° 0 ° 0 ° ° 0 a goo 0 0
0 00 000 0 0 000 000000
00 0 0 00.- 00 0 • 0
0.0°00°00000.000°0000000000.0
00000000 00.>0000°0000000°00°00
000 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 oe o • 000 .0 .0 0 0 • 0 00
o 0 . 0 ° 0 ° 0 ° 0 . 0 00 0 - : 00
o 0 go
000000°0°°0:0000°0 0 0 ·
0 0 0 0 0 0 0 0000 0 0 0 0 000 000 0 0 000 000000 ° STN. PYN.
o O&oogoooooooo 0800000°000°:°00000000.00 a
0 0 00 0 0 0 00 00 0 00 0 0 0 0 0 . gg~ooooooo
.00
o
o
a a

0:0.°
0 0 0
000 0 0 00
o a 00 0 0 a 0

000 gooD
STN. RAD.
o 0
o 0 : 0 0 LAC.-MOL.
ooooeooooo·o
0 0 o 0 0 0

o·
o 0 o 0 a 00 0
o 0
o a 0 a 0 00
00 00 o~oooo 0·. 0 00
o

Fig. 24a, b. Schematic drawing of distribution of intensely labeled neurons in field CAl of the frontally
sectioned hippocampus in l-day-old mice after injection of [lHlthymidine on E12 a and E13 b.
Intensely labeled neurons are designated by filled circles; unlabeled and lightly labeled ones by open
circles. STR. OR., stratum oriens (and alveus); STR. PYR., stratum pyramidale; STR. RAD. LAC.-
MOL., strata radiatum, lacunosum, and moleculare. Scale bar=50 J.lm

Upon [3H]thymidine injection on E16, the total number of intensely labeled

neurons in CAl is sufficiently lower than after isotope administration on El5
(Fig. 25). The intensely labeled neurons are found in all layers of CAl; however,
the majority of them is located in the strata pyramidale and oriens (Fig. 28a). In
the stratum pyramidale, intensely labeled neurons mainly occur in its outer zone
adjacent to the stratum radiatum. In the stratum oriens, a considerable part of
intensely labeled neurons is spindle-shaped and dark, which is typical of migratory
neurons (not shown). At this stage of the experiment, the intensely labeled neurons
still exhibit a tendency to aggregate in clusters (Fig. 27c, d).

61
0/.
Str. or
20

10
,,
--~

20

10

20
--
Str. rod.
10 Lac-mol

~-L _ _L--L__L-.-L__~~~ ___ _

11 12 13 14 15 16 17 18
Days of injection
Fig. 25. Percentage of intensely labeled neurons in stratum oriens (Str. or.), stratum pyramidale (Str.
pyr.), and strata radiatum, lacunosum, and moleculare (Str. rad. lac.-mol) in field CAl of Ammon's
horn in l-day-old mice after injection of [3H]thymidine from E12 to El8

After [3H]thymidine administration on E17, the overwhelming majority of

intensely labeled cells is located in the infrapyramidallayers of CAl (Fig. 28b).
They display all characteristic properties of migrating neurons. A similar phenom-
enon is observed after isotope administration on EI8 (Fig. 29). After exposure to
[3H]thymidine on E19, practically no intensely labeled cells can be found in CAl
with the exception of single labeled cells evidently of glial origin.
The computer analysis of the maps of arrangement of intensely labeled
neurons in field CAl of Ammon's horn of El3-EI6-injected mice revealed nonran-
domly existing groups of intensely labeled neurons (Fig. 30). In seven of eight
cases, nonrandom differences from the random distribution of intensely labeled
neurons were revealed at the highest level of significance (P < 0.00 I), and only in
one oftwo cases on El3 were the differences found to be insignificant. The maps of
CAl in mice exposed to [3H] thymidine on E14-E15 (when CAl did not practically
contain intensely labeled migrating cells) were used for determination of quantita-
tive parameters of the revealed groups of intensely labeled neurons. The following
results were obtained:
Number of neurons in a group: 4.35 ± 0.16
Width of a group: 10.8 ± O. 73 ~m
Height of a group: 108.0 ± 3.36 ~m
Weight of spaces: 11.1 ±0.44 ~m

62
 • 0 o
00 STR. RAJ]
o
00 LAC.-MOL.
00

o
00
o
a

STR. RAJ].
LAC.-MOL.

Fig. 26a, b. Schematic drawing of distribution of intensely labeled neurons in field CAl of frontally
sectioned hippocampus in l·day-old mice after injection of [3H]thymidine on El4 a and El5 b.
Designations and abbreviations as in Fig. 24. Scale bar= 10 11m

5.4 Discussion

In the present work the generally accepted approach was used for the study of
spatial and temporal sequence of neurogenesis based on the registration of ar-
rangement of intensely labeled neurons, i.e., cells, the ventricular precursors of
which undergo their last divisions at the time of [3H) thymidine administration at
various stages of embryogenesis (Sidman 1970). The results obtained supported
numerous observations on the tendency to the laminar sequence of neuronal
production in the archi- and neocortex. At the same time, the present study did not
confirm the data on the presence of the sandwich gradient of production of
pyramidal neurons in field CAl of Ammon's horn, i.e., their later formation in

63
Fig. 27a--d. Clusters of intensely labeled neurons in the pyramidal layer offield CAl of Ammon's horn
in I-day-old mice after injection of [3H]thymidine on El5 (a and b, frontal and sagittal sections,
respectively) and on El6 (c and d, frontal and sagittal sections, respectively). Toluidine blue, x 1100

64
 ---
°00

o
o
o
o
0
0

O.
o

..0
0 00

0
0

0 0

a
0

0
STR. OR.

STR. PYR.

o 0 o o o o
o o o STR. RAD.
o o o o 0 o
o o o 0
o o o 0 o LAC.-MOL.
o 0 0
o o o 0 o
00 0 00
o o o 0 o
o o 00 o
o 0 0 o 0 o o 0 0 0
o o o 0 o o. 0 0
o
o o o 0 00 0 0
00 o
0 0 o o o 0 0 0 0
000°0°0 o 0
o o 0

STR. OR.

o
0 0 ° 00 0 0

STR. PYR.

0
0 0 o o o
0
0
0
0 0 o 0
o STR. RAD.
0 0 00 0
0
0
o 0 0 0 0 0 o
0 0
o o o o LAC.-MOL
00 0 o. 0 00
0
o o
00 0
0 0 0 0 0
o 0
o
0 000 o
0
0
o o o o 0
o o o
o 0<t goo 0 00 00 o 00 0

Fig. 28a, b. Schematic drawing of distribution of intensely labeled neurons in field CAl of the frontally
sectioned hippocampus in l-day-old mice after injection of [3H]thymidine on E16 a and El7 b.
Designations and abbreviations as in Fig. 24. Scale bar = 50 J.lm

comparison with the neurons of supra- and infrapyramidallayers (see Sect. 1.2.1).
The results obtained indicate that neurons in the suprapyramidal layers form
earliest, prior to neurons of pyramidal and infrapyramidallayers.
In the present study, the mosaic-like distribution of radially oriented clusters
of intensely labeled neurons in the neocortex and Ammon's horn was established.
The descriptive data were supported by the results of computer analysis of the
maps of arrangement of intensely labeled neurons in field 6 of the neocortex and
field CAl of Ammon's horn. Both in the neocortex and Ammon's horn, non-

65
 0- 0 0 0

o o o 0- 0 0

o o
o
0
00 OOo~o
0- 0 0 0
STR. RAll.
o 0 D 0 0 0
o
o o o 00 ~ 0 o 0 0 0 LAC-MOL.
o o o o 0
0 o 0 o o 0 o 0 o
0 0
o 0 o 0 o 0 0 00 0
" 0 o 0 o
o 0
o 0

Fig. 29. Schematic drawing of distribution of intensely labeled neurons in field CAl of the frontally
sectioned hippocampus in the l·day·old mouse after injection of [3H]thymidine on E18. Designations
and abbreviations as in Fig. 24. Scale bar= 50 /lm

.. .,
1 I ,..,
1"'
·1 ,., STI? OR.
I I 1 I r"", 1I
1 1 I I 'I I r"I
I I
: I
1-;
1 1
~.
I
1 ,. 1 I 0
I .. ~ r·,.' I
1·1,.
101
I II I
,..~
.,11, " • .,
r
.11 T 1~lol
I 11 0 ' I I II
If··
III
I..J STR. PYR.
I 1 1o.J I I • ~ I I
i •
l.J I I I II I I II I
I I "I ~ 0
1 I I II I t I I I I: I
i-I I~.J I I I I II I I I I I I II I
"i
1 II I I I I.J I I I I I I 10..J I
I II I I I I I I I I 1.1 I STR. RAll.
I II I I I I 10 1 II I lAC.-MOl.
L~ L.J I I ~ I I .J I
I I I I I
I I I I L.J
L..j L.J

• • - ' _ • • 1.
L-.J LJ L......J L-.J
.01. /':.." ..:..
L.....J L.J LJ L...J L......J LJ LJ LJ
,c:'01 .... ~ .. ..1._
L-.J L.J L.J
).I.,.
L.J LJ

Fig. 30. Example of computer-treated map with distribution of intensely labeled neurons in field CAl
of Ammon's horn in the I·day·old mouse after [3H]thymidine injection on E15. Intensely labeled
neurons are designated by filled circles, their nonrandom groupings by dashed lines. Along the
horizontal axis of the map, the projections of intensely labeled neurons (filled circles) and widths of
their groupings (brackets) are designated. Abbreviations as in Fig. 24. Scale bar = 50 /lm

66
random groups of intensely labeled neurons were represented by vertical columns
of cells alternating with unlabeled or weakly labeled cell rows.
The mosaic-like distribution of radial groups of intensely labeled neurons in
the neocortex and hippocampus in mice and rats exposed to [3H]thymidine at
various stages of embryogenesis is also evident in the maps adduced in the works
by other investigators (Angevine 1965; Gracheva 1973; Bruckner et al. 1976), but
this is not commented upon. In the present work, a more detailed analysis of this
phenomenon is given.
Simultaneous formation of neurons located in the same vertical row but at
varying distances from the pial surface becomes an argument against the hypo-
thesis about the importance of the pial surface for the arrest of young neurons
migrating into the cortex (Berry and Rogers 1965; Berry 1974). It should be noted
that simultaneous formation of neurons in the same vertical row but in various
layers of the structure is vividly pronounced in the mammalian and amphi-
bian tectum (Straznicky and Gaze 1972; Mustari et al. 1979; Reznikov and
Maliovanova 1979; Reznikov et al. 1987b). In the mammalian neocortex and
hippocampus, it is "disguised" by a more distinct laminar order of neurogenesis.
The mosaic-like distribution of intensely labeled neurons in the neocortex and
hippocampus is essentially the projection of processes occurring in the ventricular
zone ofthe brain that gives rise to neurons of the cerebral cortex. This may be due
to two reasons:
I. Differences in the timing of synchronized mitotic cycles between the neighbor-
ing groups of ventricular cells
2. The presence of adjacent groups of ventricular cells differing in the time of
origin of neurons migrating to the same layer of the neighboring ontogenetic
columns.
If a relative delay in the commencement of the cycles between the adjacent
groups of the ventricular cells is longer than the S period, then one group will take
up the isotope, while the other one will not. Accordingly, neurons arising from
these groups of cells will be either labeled or unlabeled. At present, there are no
data on the presence of such groups of highly synchronized ventricular cells in the
developing brain (Korr 1980). Our studies with the registration of arrangement of
mitoses in the developing hippocampus (see Sect. 3.2) did not reveal any mosaicism
in the distribution of mitoses in the ventricular zone, which should have been
undoubtfully observed in the presence of highly synchronized ventricular cells.
Later, Johnston and van der Kooy (1989) revealed the mosaicism in the embryonic
ventricular zone using immunocytochemical proto-oncogene markers and
[3H]thymidine autoradiography. However, the results were obtained in rats ex-
posed to the isotope on E18, i.e., when a great part of neurons in Ammon's horn
and the neocortex had already been generated.
The above-stated considerations suggest a more probable explanation for the
formation of groups of intensely labeled neurons, namely, the existence of essential
differences in the time of origin of neurons destined for the same cortical layer.
This assumption is supported by the data indicating that in some layers of the
neocortex and Ammon's horn in the mouse and rat neurons are generated during
several days of embryonal development (Jacobson 1978; see also Sect. 1.2.1).
The analysis of drawings of distribution of intensely labeled neurons in the

67
mouse neocortex, adduced in the work by Gracheva (1973) in which the double
labeling technique was used, i.e., when [14C]thymidine was introduced I day
after [3H]thymidine, revealed close localization of unlabeled, [3H]_ and
[14C]thymidine-Iabeled neurons, which indicates that the delay in their origin was
no less than 2 days. It cannot be excluded that these differences may be even more
pronounced.
Therefore, it can be concluded that the mosaicism of neurogenesis is condi-
tioned by heterochronous production of neurons belonging to the same cortical
layer by local parts of the ventricular zone. An attempt was made to evaluate the
number of cells in these local parts (loci of neurogenesis). The computer analysis of
the maps of arrangement of intensely labeled neurons in frontal (lllm) sections of
field 6 of the neocortex and field CAl of Ammon's horn demonstrated that in both
cortical structures nonrandom groups of intensely labeled neurons consisted of 4.5
cells on an average. Since the groups of intensely labeled neurons display little
difference in frontal and sagittal brain sections, it can be concluded that these cells
are truly organized in cylindrical clusters consisting of four to five cells.
At the same time, intensely labeled neurons observed in these clusters are in
fact only a part of cell population that have undergone their last mitotic cycle at
the time of pulse exposure to [3H]thymidine. Other cells were in pre- and postsyn-
thetic phases of the mitotic cycle and did not incorporate the isotope. Assuming
that in the middle period of cortical neurogenesis (E14-E16 for the mouse) cells of
the ventricular zone represent a steady-state population (only one of the two
daughter cells continues to proliferate after mitosis and the other one leaves the
ventricular zone), it seems possible to calculate the number of neurons generated
by a single ventricular locus for one mitotic cycle according to the equation for
estimation of the growth fraction in a steady-state population: GF = LI x T Its
(Quastler 1960). For a given case, GF is the number of neurons generated by
a single locus for one mitotic cycle, LI is the number of intensely labeled neurons in
a group, i.e., four to five cells, T is the duration of the mitotic cycle, and ts is the
duration of the S-phase for ventricular cells of the lateral wall in the mouse
endbrain on E15 estimated to be 16 hand 7.l h, respectively (Schmahl 1983).
Making these calculations, we find that on E15 (and similarly on E14 and E16,
since at this time the mitotic cycle in the mouse ventricular zone varies insignifi-
cantly; Schmahl 1983) a single ventricular locus produces 9-11 neurons for one
mitotic cycle. Taking into account that in the middle period of cortical neurogen-
esis precursor or stem cells in the ventricular zone divide asymmetrically producing
dissimilar daughters, one of which becomes a neuron and the other remains a stem
cell (Rakic 1988), and that massive glial production has not started yet (Rickmann
and Wolff 1985), it may be accepted that a ventricular locus in the mouse endbrain
on E14-E16 consists of 9-11 stem cells. These figures are closed to the results of
direct count of the cell number within proliferative units of the ventricular zone in
the Rhesus monkey which was three to five cells at early stages and about 12 cells
at stages when proliferative units started producing postmitotic neurons (Rakic 1988).
The mosaic-like distribution of clusters of intensely labeled neurons in
Ammon's horn and the neocortex also suggests that the main units of neurogenesis
in the cerebral cortex are represented not by separate cells but by groups of
neurons. Of course, this fact does not exclude the possibility that separate cells also
start neuronal differentiation. However, such processes, judging by the results of

68
mapping of arrangement of intensely labeled neurons in the hippocampus and
neocortex, are more pronounced either at the beginning or at the end of neurogen-
esis, whereas during production of the majority of neurons in the cortex, they are
generated as cell groups.
According to the hypothesis of Rakic (1978, 1988), proliferative units in the
ventricular zones (or, as we term them, neurogenetic loci) produce in the process of
brain development ontogenetic columns, which in their turn give rise to cortical
modules (minicolumns). The results obtained permit a comparison of the scale of
neurogenesis in separate neurogenetic loci with the size (number of cells) of cortical
modules. According to the above calculations, a single locus of the ventricular
zone generates 9-11 neurons during one mitotic cycle. Proceeding from the data by
Gracheva (1973) indicating that all the multitude of neurons in the mouse neocor-
tex is generated during 12 successive mitotic cycles of ventricular cells, it can be
concluded that during this period a single neurogenetic locus produces a column
consisting of 108-132 cells. This value is very close to the number of neurons in the
minicolumn of mammalian neocortex (including the mouse), which contains about
110 cells (Rockel et al. 1980). One should bear in mind that these calculations give
only an approximate estimate and that practically complete identity of the least
value obtained with the number of cells in a minicolumn is a mere coincidence. At
the same time, it is quite obvious that the mosaic-like distribution of groups of
intensely labeled neurons described characterizes processes of generation of neu-
ronal groups comparable in scale with the formation of neocortical minicolumns.

5.5 Conclusion

The results obtained during the study of the sequence ofneurogenesis in Ammon's
horn and the neocortex of I-day-old mice exposed to [3H]thymidine on E12-E19
have demonstrated that in both cortical structures along with a well-known
laminar sequence of neuronal production processes exist that bear a relation to
the formation of their radial organization. These processes are expressed in
the availability of mosaic-like distribution of radially orientated groups of
[3H]thymidine intensely labeled neurons. The computer analysis revealed with
a high degree of significance the nonrandom character of grouping of intensely
labeled neurons and allowed determination of their parameters in field 6 of the
parietal cortex and field CAl of Ammon's horn, in particular, the average number
of intensely labeled cells in a single group, which was found to be similar in the
hippocampus (4.35) and neocortex (4.44). The presence of such clusters indicates
that the main units ofneurogenesis in the archi- and neocortex are represented not
by separate neurons but by cell groups.
The mosaicism of neurogenesis is also indicative of heterochronous neuronal
production in the neighbouring ontogenetic columns of Ammon's horn and the
neocortex. This heterochronia is evidently connected with the discrete production
of neurons by local parts of the ventricular germinal zone of the cortex, i.e., with
the presence of neurogenetic loci in the ventricular zone. A hypothesis about the
arrangement of neurogenetic loci in the ventricular zone of the embryonic brain as
discrete units giving rise to ontogenetic columns was advanced by Rakic (1977,
1978). Additional evidence supporting this assumption was presented in this study.

69
Calculations based on the quantitative data obtained permitted a demonstration
that during 12 generations of mitotic cycles characteristic of neurogenesis in the
mouse cortex (Gracheva 1973), a single neurogenetic locus produces a column
consisting of 108-l32 neurons. These values are close to the number of cells in the
neocortical minicolumn which contains 110 neurons according to Mountcastle
(1979). The data obtained do not only indicate that the described groups of
intensely labeled neurons characterize processes of neurogenesis comparable in
scale with elementary functional modules of the neocortex, i.e., microcolumns, but
also present an argument in favor of the presence in the hippocampus of micro-
modules comparable with neocortical minicolumns. In any case, in the hippocam-
pus, an embryonic basis exists for the formation of micromodules.

70
6 Summary

In the present work, processes of cell proliferation, cell death, neurogenesis, and
gliogenesis in the mouse hippocampus were studied. The mapping of distribution
of hippocampal mitoses and counting of their number allowed a more precise
definition of the data concerning the disposition and age reduction of proliferative
sites in Ammon's horn and the dentate gyrus in the mouse. As a result, the
following generalized scheme of development and age reduction of the germinal
zones in the mouse hippocampus has been suggested.

1. Ammon's horn
a) The ventricular zone, from the beginning of formation of the hippocampus
(Ell) until E20
b) The suprafimbrial zone, from El6 until P7
2. Dentate gyrus
a) The prime germinal zone ("the anlage of the dentate gyrus" of Stanfield and
CowanI979b), from E15 until P3
b) The proliferative zone of the hilus, from P3 until Pl4
c) The subgranular zone, from P3 until adult age

The adduced scheme needs some comments:

1. In the hippocampus (as well as in other formations of the developing

brain), primary precursors of all types of cells of neuroectodermal origin are
represented by cells of the ventricular zone. They give rise to cells of secondary
germinal zones in the dentate gyrus and Ammon's horn and are direct precursors
of the majority (if not of all) neuronal cells in Ammon's horn, the earliest
originating generations of neurons in the dentate gyrus, hippocampal radial glial
cells, and, evidently, of a considerable part of astroblasts and oligodendroblasts in
Ammon's horn.
2. In contrast to the subiculum, Ammon's horn in the mouse lacks a subven-
tricular (subependymal) zone. These data differ from the results obtained in the
rat, where the subventricular zone is described both in the subiculum and
Ammon's horn (Bayer 1980b) and from the study in the monkey, where the
subiculum and Ammon's horn lack the subventricular zone (Nowakowski and
Rakic 1981). Thus, in the case of the mouse, the subventricular zone can serve as
a cytoarchitectural characteristic allowing detection of a border between the
developing subiculum and Ammon's horn. (Another developmental feature which
also identifies the border between Ammon's horn and the subiculum is a consider-
able number of dying cells in the region of the joint of these structures on E20-P3).

71
 3. In the developing dentate gyrus and Ammon's horn, several extraventricu-
lar (i.e., located at a distance from brain ventricles) secondary germinal zones exist.
Thus, during embryonic and early postnatal periods of the mouse development,
the prime germinal zone occupies a larger part of the presumptive dentate gyrus. In
the course of further development, the prime germinal zone is partially reduced
and divides into two local germinal zones: the proliferative zone of the hilus and
the subgranular zone. The former disappears comparatively rapidly, while the
latter is reduced only partially and can be found even in adult animals. The
germinal zones of the dentate gyrus are the source of the majority of its neural and
the larger part of glial cells. Thus, in the mouse about 80% of granular neurons
and more than 90% of glial cells are generated during the postnatal period.

Along with cell proliferation, the developing hippocampus is characterized by

processes of cell death. The conducted analysis of distribution of pyknoses in
Ammon's horn and the dentate gyrus demonstrated that cell death preferentially
occurs in the secondary germinal zones of these structures. Similar processes take
place in the subependymal zone of the lateral ventricular wall and in the external
granular layer of the cerebellum in the developing mammalian brain (see Korr
1980 for references). The investigations performed also revealed that pyknotic cells
in the subgranular zone of the dentate gyrus (as well as in the subependymal zone
of the lateral ventricle) became pyknotic shortly after mitosis. This type of cell
death in the developing hippocampus is evidently connected with the postmitotic
death of proliferating cells in the germinal zones and of glioblasts in the nonger-
minal sites of the hippocampus, and not with the amitotic death of differentiating
neurons that did not find contacts with target cells. Since the peak of pyknoses in
the developing hippocampus correlates in time with that of glial production, it
seems probable that the postmitotic cell death represents a mechanism of cor-
rection of errors of gliogenesis.
The spatiotemporal sequence of neurogenesis in Ammon's horn and the
dentate gyrus was studied in some detail in rodents and rhesus monkeys (in
particular, in the mouse by Angevine 1965, 1975; Caviness 1973; Stanfield and
Cowan 1979a; Nazarevskaya and Reznikov 1985). These data allow to conclude
that regularities of neurogenesis in the hippocampus do not principally differ from
those in the neocortex. Both Ammon's horn and the dentate gyrus are character-
ized by a laminar sequence of neuronal generation, which is also well-pronounced
in the neocortex. Of course, there are some differences, but they concern not the
common principles of neurogenesis, but peculiarities of neuronal generation in
some layers or inside a particular layer. Thus, cells in the pyramidal layer of
Ammon's horn are generated in the inside-out sequence, whereas neurons in the
granular layer of the dentate gyrus in the outside-in sequence. In the neocortex,
both gradients of the laminar sequence of neurogenesis can be observed (see
Raedler et al. 1980 for references).
Principles of neurogenesis regulating the formation of radial organization of
the archi- and neocortex reveal even closer similarity. Thus, in Ammon's horn and
the neocortex, the radially orientated groups of simultaneously generated neurons
have been detected. In both structures, they are similarly pronounced and even
coincide in the average cell number in a group (four to five cells). The data
available have confirmed a previously postulated hypothesis (Rakic 1978) on

72
discrete arrangement of the ventricular zone in the form of loci of neurogenesis
(proliferative units) which produce ontogenetic columns in the developing cortex.
Additional calculations have revealed that during neurogenesis a single ventricular
locus forms a column consisting of 108-132 neurons, which is close to the number
of neurons in the basic modular unit in the neocortex, a minicolumn estimated
in 110 cells (Mountcastle 1979). Up to now, there is no direct evidence for the
presence in the hippocampus of micromodules comparable with neocortical mini-
columns. The data obtained do not only indicate similar principles of neurogenesis
in the hippocampus and neocortex, but present an argument in favor of the
existence of such micromodules. At least in the hippocampus there is an embryonic
basis for their formation.
In the hippocampus, gliogenesis reveals no really essential differences from the
same process in the neocortex. The data obtained in the present work concerning
the time sequence of formation of astrocytes and oligodendrocytes in the dentate
gyrus do not principally differ from the results of analogous studies in the mouse
neocortex (Reznikov 1981). Taking into account that in the mouse neocortex
neurogenesis ceases by the end of gestation (Reznikov 1981), while in the mouse
dendate gyrus it continues for at least 3 weeks postnatally, one could assume that
the dynamics of gliogenesis in various brain structures is relatively independent
from that of neurogenesis. This assumption is consistent with the previous data of
the comparative study on the schedule of glial formation in the cortical and
subcortical visual centers of the rat brain (Biesold et al. 1976; Mares and Bruckner
1978).
The analysis of results of the present study combined with the literary data
makes it possible to conclude that the key regularities of cell proliferation and
genesis in the hippocampus are similar to those in the neocortex. At the same time,
the hippocampus is characterized by some unique features of proliferative pro-
cesses and cell genesis. They include:
1. the presence of several extraventricular germinal zones and the lack of the
subventricular germinal zone
2. the absence of migration of young neurons in the dentate gyrus after formation
of its secondary germinal zones.
3. a very long time of generation of granular neurons in the dentate gyrus, which
begins practically simultaneously with formation of other types of neurons in
the dentate gyrus and Ammon's horn (in the mouse, on EIO) and continues
postnatally for a sufficiently long period of time (in the mouse, at least until
P20).
The latter phenomenon has attracted attention of many investigators involved in
the study of the developing hippocampus and requires special remarks.
In his well-known early hypothesis, Altman (1966) subdivided neuronal cells
of the mammalian brain into long-axoned macroneurons and short-axoned
microneurons representing local circuit neurons. According to Altman, formation
of a considerable part of microneurons, particularly of granular cells in the dentate
gyrus, is accomplished postnatally under the effect of environmental stimuli and is
connected with gaining experience. This hypothesis does not fit in well with the
data obtained during the study of neurogenesis in brain structures of various
mammalian species. Thus, in the mouse and rat by far not all types of

73
microneurons (e.g., stellate cells in the neocortex) have a postnatal period of
formation (Berry 1974; Mares and Bruckner 1978; Reznikov 1981). Moreover, it
seems unlikely that production of granular neurons in the dentate gyrus in
mammals is directly connected with postnatal experience, since in the rhesus
monkey, whose level of complexity of obtained information is sufficiently higher
than that in the mouse and rat, the majority of granular neurons in the dentate
gyrus originates during embryogenesis (Rakic and Nowakowski 1981).
The idea about the role of postnatal neuronal production in gaining experience
was revived in connection with the discovery of neurogenesis in the adult bird
brain (Nottebohm 1985) and in the dentate gyrus of adult rats (Kaplan and Bell
1984; Kaplan 1985). On the basis of the data obtained, these authors advanced
a hypothesis assuming that addition of new neurons (especially in the hippocam-
pus) during postnatal development and even in adult mammals may play an
important role in learning and memory (Kaplan and Bell 1984; Nottebohm 1985).
This hypothesis was critically considered by Eckenhoff and Rakic (1988) who did
not observe neurogenesis in the dentate gyrus of postpubertal rhesus monkeys.
The above discussed attempts to establish an interrelationship between pro-
cesses of neurogenesis in the hippocampus and its functional importance un-
doubtedly playa stimulating role for the study of cell proliferation and cytogenesis
in the hippocampus. Further accumulation and more precise definition of experi-
mental data including problems of development, organization, and functioning of
the hippocampal modules are required.

Acknowledgment
Some of the investigations described in the present monograph were performed in collaboration with
Drs. Galina Nazarevskaya and Olga Savrova from the Peoples' Friendship University, Moscow;
Vladimir Derjabin from the Institute of Anthropology of the Moscow State University, Moscow;
Ferenc Hajos, Andras Csillag, and Zoltan Fiilop from the Semmelweis University, Budapest; and
Eduardo Basco from the Havana University, Havana, whose contributions are greatly appreciated.

74
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81
Subject Index

Ammon's hom gliogenesis 8,9,28-31,41,43,44, 51, 73

alvenus 3, 59, 60
ependymal layer 3 Hippocampal connections
field CAl 2,7, 10, 17, 18,59-63,65,66 afferents 4, 5, 9, 10, 53
field CA2 2, 7 associational projections 5
field CA3 2, 4, 7, 10, 14 commissural projections 5
field CA4 2, 4, 10 efferents .5, 9
stratum lacunosum 3, 4, 59-62 hippocampal formation
stratum lucidum 3 hippocampus
stratum moleculare 2, 4, 59-62 differentiation 9-11
stratum oriens 3, 5, 7, 10, 14, 59-62 functions 6
stratum pyramidale 3, 7, 24, 59-62
stratum radiatum 3, 5, 59-62 Lateral geniculate body 44
suprafimbrial zone 14, 20-22, 24, 31, 32, 71 lateral ventricle 2,3,9, 19,29,45
astrocytes 4, 8, 16, 34, 37, 38, 43, 44, 51, 73 limbic system I
astroglial processes 4, 39
Macroneurons 29, 73
Cell death 19, 20, 29, 40, 45, 50, 72 MI, mitotic index 14-16,25-27,45,46
cell differentiation 10, II microglial cells 16
cerebellum microneurons 29, 73, 74
external granular layer 20, 45, 50, 72 mitoses 13, 14,21-25,30,31,47, 67, 72
mitotic cycle 16, 45-48
Dentate gyrus mitotic spindle 14, 25, 26, 28
granular layer 4, 10, 19,20,22,23,34,35,38,
39,42-44,50,72 Neocortex 52-58, 65, 67, 69, 70, 72, 73
hilus 4, 5, 7, 34, 39 nerve tissue tumors 31
molecular layer 3, 7, 10, II neural modules
polymorphic layer 4, 23, 34, 39, 41, 43 macrocolumns, neocortex 52, 53
prime germinal zone 14, 21-23, 27-29, 31, macromodules, hippocampus 52
36,71,72 micromodules, hippocampus 52, 70, 73
proliferative zone of the hilus 9, 14, 23, 28, minicolumns 52, 53, 69, 70, 73
39,71, 72 neurogenesis 6-8, 15, 31, 39,41-43, 50, 51, 63,
regenerative capacity 17, 50 67-69,72-74
subgranular zone 4, 9, 14, 16, 19, 23, 27, 28,
31,34,35,37-39,44-47,50,51,71,72 Oligodendrocytes 8, 16,43,44, 51, 73
desmosome-like junctions 34, 37
Parahippocampal formation
Entorhinal cortex I, 6, 53 parasubiculum I, 6
parietal cortex 51
FCL, fraction of cell losses 15, 26, 28, 46 phagocytic functions 39
fimbria 20, 23, 24, 31 PI, pyknotic index 14, 15, 17,27,28,45,46
frontal cortex 17 presubiculum 1,6
pyknoses 19, 20-25, 30-32, 39, 72
Gamma-aminobutiric acid (GABA) 8
GF, growth fraction 16, 17,46,67,68 Radial glia 4, 39, 71
glioblasts 20, 34, 36, 37, 39, 72 radial fibers 4, 9, 39, 53

82
Sinapses 10-11 Tectum 67
S-period (S-phase) 48, 50, 67, 68
subependymal (subventricular) zone 16, 20, 21, Undernourishment 33, 45, 48, 50, 51
28-30, 32, 45-47, 72-74
subiculum 2, 4, 6, 20-22, 30, 71 Ventricular zone 9,19-21,25,28,31,32,67--69,71

83
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