The hippocampal formation is a critical component of the neural system that is required for the initial storage of long-term memory . The undoubted importance of the hippocampus for memory function has been proved by both experimental and human studies . The great importance of this special brain structure, in addition to its selective impairment in many brain diseases encouraged us to study it by following its development from the age of new born up to adulthood in the most commonly used animal model, the rat. In this study, we tried to collect and correlate histological, morphometric, and immunohistochemical data in the same study.
This histological study showed that the general architecture of the hippocampus proper was already present at P0, whereas that of the DG was not distinct at this age. Previous histological studies in the rat reported that neurons of the hippocampal formation were developed from gestational day G15 into adulthood . Granule neurons of the DG were developed either prenatally from the ventricular zone or postnatally from a secondary proliferative zone, the interhilar zone of the DG , similar to the granule neurons of the mouse cerebellar cortex, which developed mainly postnatally . While, pyramidal neurons of the hippocampus proper were generated only prenatally from the ventricular zone .
Thus, maturational changes that occurred at different hippocampal regions might be specified with respect to different regions in the same structure .
With regard to the hippocampus proper, the nearly mature differentiation of its different areas in this study started only to appear at P14, whereas at P90 there was a clear demarcation between different CA areas. Although the migration of neuronal elements to the PL was finished around the time of birth in the rat, the hippocampus proper showed a nearly mature figure in its histology 15 days after birth .
In this study, axons of pyramidal neurons of the hippocampus proper and the subiculum that formed the fimbrial fibers were first detected at P7. It was previously reported that axonal projections in the fimbria of the rat hippocampus started in the late embryonic life and reached the adult pattern at P10 . Moreover, oligodendrocytes actively extended membrane processes and enwrapped axon fibers into the rat fimbria during the second and third postnatal weeks .
In this study, counting of dark neurons revealed that dark neurons started to appear in CA areas at P14 and showed a very gradual increase toward P90. However, this increase appeared more in CA1, slightly less in CA4, small in CA3, and nearly absent in CA2. This indicated different vulnerabilities in different hippocampal areas . CA1 was highly vulnerable to anoxia, ischemia, and some forms of temporal lobe epilepsy. CA2 and CA3 were resistant sectors, whereas CA4 had medium vulnerability .
The dark neurons observed in CA1 in this study were characterized by dark somata and dark irregular dendrites. Similar finding was observed in CA1 of Wistar rats after brain injury . Dark neurons were considered a manifestation of neuronal injury and their number increased with age . They reflected the early histopathological state of neurons after various brain insults. Some of them died and the others survived depending on the extent of damage to the cytoskeleton of their dendrites .
In this study, it was observed that the lower limb of the DG was less distinct than the upper one at P0 and the distinct lamination of the DG was seen at P7. This finding was consistent with other researchers , who observed that the DG underwent continued reorganization and lamination during early postnatal development in the mouse. It was also reported that the structure of neurons in the lower limb of the DG of the Syrian hamster changed during adolescence . This observation was explained by the effect of gonadal steroid hormones, which increased dramatically during this period of life . This structural maturation was likely related to the adolescent development of hippocampal-dependent cognitive functions, such as learning and memory .
Morphometric data in this study showed that there was a very rapid increase in the number of cell layers of the GL of the DG from P0 to P7. It was previously reported that neurogenesis in the mice DG peaked during the early postnatal stages and persisted through adult life . Similarly, in the guinea pig neurogenesis in the DG occurred at a high rate from P1 to P20 with a peak at 3–6 days of age leading to a considerable increase in cell number during this period. Then, it declined slowly from P20 to P30 and continued in adult animals, at a much reduced rate .
In this study, mitotic and apoptotic figures were observed in the deep part of the GL of the DG at P7. Moreover, at P14 a deep dark layer appeared as a blue line at the interface between the GL and the hilus of the DG. This was exactly consistent with the classification of the GL of the DG in the rat by some researchers  into two separate zones of different origins and maturational states. The superficial zone contained the first formed granule cells that were derived directly from the ventricular cells and were formed from G14 to G22; thus, they were already present at birth. Meanwhile, the deep zone contained granule cells that were derived postnatally from the secondary hilar proliferative center. Initially, this zone displayed the highest mitotic activity. Later, it became restricted to a narrow deep subgranular zone that took over the production of new granule cells into adulthood .
Concomitant with granule cell production and maturity was the sprouting of the neuronal processes, which could be seen in this histological study obviously from P7 rats. It was also associated with a very rapid increase in the thickness of the ML of the hippocampus proper and in both the ML and the OL of the DG. This was most probably due to the growth of granule cell axons, the mossy fibers that occupied the inner ML of the hippocampus and terminated on the proximal part of the apical and basal dendrites of the pyramidal cells of the inferior region of the hippocampus proper  as they projected into CA4 and CA3 of the hippocampus proper [28,29].
Consistently, functional studies on the rat DG denoted that nearly all the developmental changes in the dentate ML occurred between P6-8 and P9-11 .
In this study, apoptotic figures were observed in the hilus of the DG at P7. It was reported that apoptosis is crucial for the development of the brain and that a significant percentage of all central neurons produced during early ontogeny died by apoptosis . It was also observed that in the rat, the cerebellum, which is greatly similar to the DG as it is formed mainly of postnatally generated granule cells, displayed a large peak of apoptosis around P10, a small peak around P21, and low apoptotic levels throughout adulthood .
Apoptosis also might explain the reduction in the number of cell layers in the PL and in the number of light pyramidal neurons in the different CA areas, especially between P0 and P7 observed in this study.
In this study, mitotic figures were detected in the deep part of the GL of the DG at P90. Persistent neurogenesis throughout adult life is a well-known and characteristic phenomenon of the granule cells of the DG. In the adult rat, neurogenesis produces a large pool of new granule cells in the DG . Accumulating evidence had suggested that the new neurons in the adult mouse  and rat  DG played an important role in supporting hippocampal-dependent learning and memory.
The rate of neurogenesis within the DG could be altered under various physiological and pathophysiological conditions. It increased in an enriched environment in animals [34,35]. Conversely, social stress decreased the rate of neurogenesis and might lead to hippocampal atrophy .
With regard to animals, the enriched environment consisted of many components such as expanded learning opportunities, increased social interaction, more physical activities, and larger housing . If this occurred in humans as well, then early diagnosis of some diseases such as autism (which is characterized by hypoplasia of the DG and CA4) and the subsequent exposure to enriched learning conditions might promote enhanced growth of this structure in those children . Similarly, brain diseases such as Alzheimer , Parkinsonism and injury such as stroke had been considered to result in permanent loss of neurons with no possibility of cellular regeneration . Recent studies indicated that exposure to an enriched environment in these brain diseases produced a significant increase in hippocampal neurogenesis with improved spatial memory performance .
In our study, it was observed that the lateral ventricle was wide at P0 and it progressively narrowed till it showed the cribriform appearance at P90. The cerebral ventricular system is a good indicator for brain development and a predictor of neurodevelopmental outcome . Early recognition of ventricular dilatation is important, as in many neurodevelopmental disorders retarded brain growth is associated with ventricular dilatation that might lead to brain atrophy .
In this study, astrocytes were studied as they played a critical role in the development of the central nervous system . Astrocytes also performed a variety of tasks from axon guidance and synaptic support to the control of the blood–brain barrier and blood flow . A key indicator of astroglia activation was the increased accumulation of the glial fibrillary acidic protein (GFAP) . GFAP was the main component of the intermediate filaments of the astroglial lineage .
PTAH, a special stain for detection of glial filaments was used in this study. Variability in staining affinity at different ages was observed. At P0, the stain seemed to fill the whole cytoplasm of the small fibroblast-like astrocytes. With the advancement of age, the astrocyte became gradually larger with an obvious reduction in the astrocyte population. At P7 rats, the glial filaments appeared to concentrate at the central part of the cell. They gradually reincreased again to fill the whole cytoplasm and the extending processes of the large stellate-like astrocytes at P90. Other study indicated that rapid maturation of astrocytes, which consisted of a high expression of GFAP, an increase in overall cell size, and expanding arborization occurred from P11 to P30 in the rat, followed by stabilization of these parameters until P90 . In addition, previous investigators observed that GFAP immunoreactivity, in the gray matter of the visual cortex in the monkey, was high at birth, decreased around the age of 3 months, and increased again toward adulthood . They reported that the period of reduced expression of GFAP coincided with the time of prominent synapse remodeling in the visual cortex. Thus, GFAP might represent an indicator for synaptogenesis . This was also consistent with our observation, as reduced astrocyte staining affinity was observed at P7 rats, which was the time of sprouting of most neuronal processes and the possibility of their integration into the hippocampal circuitry by the formation of synapses.
In addition, similar to our observation it was reported that the number of neuroglial cells in the mouse brain decreased dramatically with maturation .
With regard to the shape of astrocytes, it was mentioned that astrocytes might be fibroblast-like or stellate-like, and they had the potential to acquire very different morphologies, depending on their regional location and their functional interactions with other cell types .
In this study, we were also interested in dating the dendritic growth and maturation by studying one of the most important neuronal cytoskeletal proteins, the MAP2. MAP2 stabilized dendritic processes and it is a useful technique for tracing ontogenetic development of the central nervous system [46–48]. It also exhibited a somatodendritic pattern of localization in the rat hippocampus .
In our study, immunoreactivity for MAP2a,b,c Ab-3 in the hippocampus proper was observed in the neuronal perikarya of the PL at P0. At P7, the immunoreactivity was observed on the perikarya and the sprouting dendrites. Thereafter, it was shifted gradually to the progressively elongated dendrites, which became overcrowded, occupying the whole thickness of the underlying ML at P90. Apical dendrites of pyramidal neurons could only be detected at P90 in the overlying OL, which was consistent with the increase in the thickness of the OL detected by our morphometric study. Meanwhile, apical dendrites of the granule neurons started to appear at P14 and became overcrowded at P90 in the overlying ML. The sprouting of the basal dendrites of the pyramidal neurons and the apical dendrites of the granule neurons were concomitant with the increase in the thickness of the ML, which was detected at P14.
Some researchers observed that the expression of the high molecular weight of MAP2 (MAP2a and b) isoform in the rat significantly increased during development, particularly during the second postnatal week in the MLs of the DG, CA1, and CA3 . They suggested that these proteins played a functional role during neuronal development and in nerve cell survival during stress .
The difference in MAP2 immunoreactivity between different neuronal types and different hippocampal areas observed in this study was also observed in the developing human hippocampus . It was observed that there were region-specific differences in composition and function of the neuronal cytoskeleton. These observations had implications for understanding the role of the neuronal cytoskeleton in the developing, mature, and diseased central nervous system .
Many studies described alterations in MAP2 and neurofibrils as markers for both dendritic and axonal damage respectively during experimental brain injury in the rat . In humans, MAP2 staining decreased in postmortem schizophrenic brain  and ischemia .
The most rapid and important sequences of events in the growth and maturation of the hippocampal formation occurred mainly during the first 2 postnatal weeks with more gradual growth thereafter. Thus, this period is a critical period in the growth of the hippocampal formation in the male albino rat. Hence, determination of its corresponding period in the human with the early diagnosis of some neurodevelopmental disorders affecting the hippocampus may improve the prognosis of these disorders greatly.
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Keywords:© 2011 The Egyptian Journal of Histology
astrocytes; dentate gyrus; hippocampus; microtubule-associated protein 2a; b; c Ab-3; morphometric; postnatal development