Abdelrahim, Eman A.a; Eltony, Sohair A.b
The hippocampal formation is a multicomponent region of the medial temporal lobe that is involved in memory processing . The hippocampal formation consists of the hippocampus proper, the dentate gyrus (DG), and the subiculum. The hippocampus proper is the largest of these components. It is a three-layered archicortex formed of polymorphic (OL), pyramidal (PL)), and molecular (ML) layers.
It is further subdivided into fields designated as cornu Ammonis (CA) from CA1 to CA4. CA4 constitutes the hilus of the DG .
The subiculum is the part of the parahippocampal gyrus that is in direct continuity with the hippocampus proper. It consists of the same layers of the hippocampus proper; however, its pyramidal layer (PL) is thicker. Axons of pyramidal neurons of both the hippocampus proper and the subiculum collect to form a fiber bundle of white matter called the fimbria that passes on the hippocampal surface of the lateral ventricle .
The hippocampal formation is one of the brain structures making up the limbic system, which seems to play a role in emotional and sexual behavior, memory, and motivation .
The hippocampal formation is widely studied in part because of its distinct and highly laminar organization as well as its demonstrated role in learning and memory .
The DG is also a three-layered archicortex formed of molecular (ML), granular (GL), and polymorphic (OL) layers. It is the main target for cortical inputs to the hippocampal formation . Moreover, it is one of the brain regions that continually generate new neurons in adulthood . Its relatively simple structure makes it an attractive model for the study of cortical development .
It is clear that the hippocampal formation is important in the storage of long-term memory  and in studying the neuroplasticity phenomenon ‘adult neurogenesis’ . In addition, the hippocampal circuitry is very susceptible to damage from disease, anoxia, and environmental toxins  and is often implicated as the origin of abnormal electric activity ‘epileptogenic foci’ in the brain .
Behavioral studies have suggested that postnatal developmental abnormalities in the hippocampal formation are thought to contribute to neurodevelopmental disorders such as autism, Down syndrome, epilepsy, and schizophrenia. Systematic studies of the structural development and functional maturation of the hippocampal formation will be necessary to gain insight not only into the information processing but also into the specific maturational processes that might be affected in neurodevelopmental disorders .
The aim of this study was to investigate the postnatal development of the hippocampus proper and the DG as the major parts of the hippocampal formation in male albino rats.
Materials and methods
A total number of 20 male albino rats were used in this study at the following postnatal ages: P0 (day of birth), P7 (1 week), P14 (2 weeks), and P90 (3 months, i.e. adult animals) . Five animals from each age were used. The animals were maintained in the animal house of Assiut University under normal day and night cycles and appropriate temperature, fed rat chow ad libitum, and allowed free access of water.
The animals were anesthetized with ether; their hearts were exposed and perfused with saline until the flowing blood was cleared. The perfusion was completed with Bouin's fluid in three animals and with 10% formalin in two animals from each age. After perfusion, the skull was opened and the brain was removed carefully. The right cerebral hemisphere was cut and immersed in the fixative. The specimens were processed for paraffin sectioning. Coronal serial sections (5–7 μm) were performed for studying the right hippocampal formation. Every tenth section was stained by the Harris hematoxylin and eosin method. Some sections were stained with Mallory's phosphotungstic acid hematoxylin (PTAH) method  for demonstration of glial fibrils within astrocytes.
Expression of microtubule-associated protein 2 (MAP2a,b,c), a neuronal cytoskeletal protein, was detected in formalin-fixed paraffin-embedded sections. The primary antibody used in this study was the MAP2a,b,c Ab-3 (Clone AP18) Mouse Monoclonal Antibody (Thermo Fisher Scientific, Fermont, California, USA). This antibody (Ab-3) is specific for the phosphorylated form of MAP2a,b,c and shows no cross reaction with other MAPs, tau, and tubulin. Ab-3 reacts with dendrites and cell bodies of neurons .
Sections were pretreated and incubated with the primary antibody according to the manufacturer's instructions. The reaction was visualized using the UltraVision ONE Detection System, HRP Polymer and DAB Plus Chromogen (Thermo Fisher Scientific). The procedure was performed according to the manufacturer's instructions. After completion of the reaction, counter staining was performed using hematoxylin.
In this study, most of the parameters of the hippocampal formation were measured as follows. (a) The number of both light and dark pyramidal neurons in the PL of different areas of the CA (CA1, CA2, CA3, and CA4) of the hippocampus proper/field is measured using the touch count method. The measurement was performed viewing hematoxylin and eosin-stained sections by ×40 objective lens in five nonoverlapping fields of 20 serial sections/five different animals from each age. Differentiation between CA1 and CA2 at P0 and P7 was made by their relative relation to the DG. The beginning of CA2 was marked by the lateral end of the upper limb of the DG [12,13]. (b) The thickness of both the ML and the OL in micrometers in both the hippocampus proper and the DG is measured using the arbitrary distance method. The measurement was performed viewing each tenth hematoxylin and eosin-stained section by ×10 objective lens/five different animals from each age (on measuring the thickness of each layer, three measurements were taken along the length of each layer/field and the mean value of these measures was taken; the first measure was at the medial end of each layer, the second was at its lateral end, and the third was at the middle). (c) The number of cell layers in both CA1 area of the PL of the hippocampus proper and the GL in the upper limb of the DG/field was measured using the touch count method. The measurement was performed viewing hematoxylin and eosin-stained sections by ×40 objective lens in five nonoverlapping fields in twenty serial sections/five different animals from each age.
These measurements were performed using a computer-assisted image analyzer (soft imaging system – an Olympus Company) in the Histology Department, Faculty of Medicine, Assiut University, Egypt. The collected data for each parameter were statistically analyzed using analysis of variance (ANOVA) test. A P value of < 0.05 was considered significant.
Hematoxylin and Eosin stain: sections at nearly the same level of the hippocampal formation from each age were selected for examination to obtain accurate comparable results. The general structure of the hippocampal formation was already defined at this age. The CA of the hippocampus proper was subdivided into the superior region, which included CA1 and CA2, and the inferior region, which included CA3 and CA4. CA1 was extended medially as the subiculum, which is a part of the parahippocampal gyrus. A wide lateral ventricle was observed above the hippocampal formation. Fimbrial fibers could not be detected at this age in their site at the hippocampal surface of the lateral ventricle. At this age, CA1 and CA2 were hardly defined from each other. CA1 represented the distal superior region of the hippocampus proper. CA3 represented the proximal inferior region of the hippocampus proper. CA4 represented the continuation of CA3 in the concavity of the DG (Fig. 1). The CA of the hippocampus proper was formed of three layers; OL, PL, and ML. The OL lay just beneath the lateral ventricle and appeared as a highly cellular outer layer. The PL, which is the principal cell layer, was formed of many layers of closely packed small-sized cells. The ML was the least cellular layer and lay deep under the pyramidal cell layer (Fig. 2).
CA1, which represents the major part of the superior region of the CA of the hippocampus proper, was selected to examine the PL. Pyramidal cells were regularly arranged in several layers. They were fusiform in shape with a small amount of karyoplasm and a large oval nucleus that might show a prominent nucleolus. Some cells showed extending processes (Fig. 3).
The DG was a C-shaped capping of the free border of CA4 (Fig. 1). The DG consisted of three layers; a ML, which appeared as an outer thin cellular layer, a thin middle GL, and an inner highly cellular OL (Fig. 2). At this age, these layers could be observed at the upper limb of the DG; however at the lower limb, they were ill defined from each other (Fig. 2).
Examination of the granule cells of the GL at the upper limb of the DG revealed that these cells were few and variable in size and in shape from rounded to oval (Fig. 4).
Mallory's phosphotungstic acid hematoxylin stain: in this staining method, the cytoplasm of astrocytes appeared deep blue in color. At this age, Mallory's phosphotungstic acid hematoxylin stain revealed many longitudinally oriented astrocytes throughout different layers of the hippocampal formation (Fig. 5a). They appeared as small-sized elongated (fibroblast-like) cells (Fig. 5b).
The positivity of this reaction was detected as a golden brown color in the neuronal cell body and dendrites. Examination of the PL, which is the principal layer of the hippocampus proper, showed positive reaction for MAP2 a,b,c Ab-3 in the cytoplasm of the cell body of pyramidal neurons (Fig. 6a).
In the GL of the DG, positive reaction was detected mainly in the cytoplasm of the cell body of granule neurons (Fig. 6b).
Hematoxylin and Eosin stain: at this age, the hippocampal formation apparently increased in size associated with apparent narrowing of the lateral ventricle. Fimbrial fibers could be detected clearly as a bundle of white matter just above the lateral ventricle (Fig. 7). CA4 now could be easily delineated from both the upper and lower limbs of the DG (Figs 7 and 8). However, the differentiation between CA1 and CA2 was still indistinct (Fig. 7). There was a relative increase in the thickness of the ML in both the hippocampus proper and the DG. Both the ML and the OL of the hippocampus proper and the DG looked less cellular than the previous age (Fig. 8).
Pyramidal cells within the PL of CA1 became relatively larger in size and rounded in shape compared with those of P0. They had large rounded nuclei with prominent nucleoli and scanty basophilic karyoplasm. Most of them showed extending processes (Fig. 9).
The DG showed well-differentiated upper and lower limbs and a relatively thicker GL, particularly at its concavity (Figs 7 and 8).
Granule cells within the GL of the upper limb of the DG appeared larger and more rounded than those of P0. They had large rounded nuclei and scanty karyoplasm. The deep cells showed both mitotic and apoptotic figures (Fig. 10).
Mallory's phosphotungstic acid hematoxylin stain: an obvious marked reduction of the astrocyte population was observed compared with P0. Most astrocytes appeared longitudinally oriented fibroblast like. They were relatively larger compared with the previous age (Fig. 11a). The stain tended to concentrate centrally with nearly negative staining at the periphery of the cell (Fig. 11b).
In the hippocampus proper, a positive reaction for MAP2a,b,c Ab-3 was detected in the cytoplasm of the cell body of pyramidal neurons and in their processes, which appeared to extend into the proximal parts of the underlying ML for a relatively short distance (Fig. 12a).
In the DG, positive reaction was detected in the cytoplasm of the cell body of granule neurons, especially in the superficial layers (Fig. 12b).
Hematoxylin and Eosin stain: in the PL of the hippocampus proper, the site of transition from the closely packed small pyramidal neurons at CA1 to the larger loosely arranged pyramidal neurons at CA2 could be detected. There was apparent closure of the lateral ventricle in some areas. The GL of the DG showed a thin deep zone of small dark basophilic cells delineating the GL at its interface with the OL at the hilus of the DG, which contained CA4 (Fig. 13).
The OL and ML of both the hippocampus proper and the DG looked less cellular than the previous age (Fig. 14).
Pyramidal cells of the CA1 were more or less similar to those of the previous age, except that they appeared more packed together (Fig. 15).
Granule cells of the DG were more crowded than those of the previous ages and arranged in many layers. The superficial cells appeared larger and lighter than the deep cells (Fig. 16).
Mallory's phosphotungstic acid hematoxylin stain: at this age, astrocytes seemed to be more populated in CA3 and CA4. They were longitudinally oriented and deeply stained (Fig. 17a). The whole cytoplasm was positively stained (Fig. 17b).
In the hippocampus proper, a positive reaction for MAP2a,b,c Ab-3 was detected in the long slender dendrites projecting from pyramidal neurons toward the underlying ML (Fig. 18a).
In the DG, positive reaction was detected in the dendrites of granule neurons, which could be observed in the overlying ML (Fig. 18b).
Hematoxylin and eosin stain: there was an obvious reduction in the thickness of both the PL of the hippocampus proper and the GL of the DG relative to the previous ages (Fig. 19). A relative increase in the thickness of both the OL and the ML was observed in both the hippocampus proper and the DG. These layers appeared less cellular than the previous age (Fig. 20). The ML of the hippocampus proper could be easily differentiated from that of the DG. It appeared more fibrous. The fibers appeared regularly arranged in longitudinal arrays (Fig. 20). There was an obvious demarcation between CA1 and CA2 of the hippocampus proper at the PL. It appeared as a sudden transition from small densely packed pyramidal neurons at CA1 to larger less packed neurons at CA2. Prominent blood vessels could be observed. The lateral ventricle showed a cribriform appearance (Fig. 19).
Some dark cells were observed in CA1 and were characterized by dark somata and dark irregular processes (Fig. 21).
Granule cells of the DG appeared less crowded than the previous age. The small dark granule cells at this age were not restricted to the deep layers but extended into the superficial ones. Mitotic figures were observed in the deep layers facing the hilus of the DG (Fig. 22).
Mallory's phosphotungstic acid hematoxylin stain: astrocytes were more populated in CA3, CA4, and DG (upper limb). They looked larger in size and denser in staining than the previous ages. Most cells appeared triangular or stellate like (Fig. 23a). Some cells revealed extending processes (Fig. 23b).
In the hippocampus proper, a positive reaction for MAP2a,b,c Ab-3 was detected in the long dendrites of the pyramidal neurons that occupied the whole thickness of the ML. They appeared crowded and interlacing. Apical dendrites of pyramidal neurons could also be detected in the overlying OL (Fig. 24a).
In the DG, positive reaction was detected in the apical dendrites of the granule neurons, which appeared to be branching, interlacing, and occupying the whole thickness of the overlying ML (Fig. 24b).
The number of light pyramidal neurons in the PL of different areas of the hippocampus proper showed a progressive decrease from P0 to P90, with an obvious rapid and marked decrease in the first week of life (between P0 and P7) and a gradual decrease thereafter (Tables 1–4 and Histograms 1–4).
Conversely, the number of dark pyramidal neurons showed a progressive increase from P0 to P90. At the first 2 weeks of life (between P0 and P14), the increase was very gradual in all areas of the hippocampus proper and it became rapid between P14 and P90 but with some variability between the different hippocampal areas. The increase was more in CA1, less in CA4, lesser in CA3, and not obvious in CA2 with nearly flat histogram (Tables 1–4 and Histograms 1–4). Statistical analysis of the data collected on the number of both light and dark pyramidal neurons in CA1, CA2, CA3, and CA4 showed highly significant P values (Tables 1–4).
Measurement of the thickness of the OL of the hippocampus proper showed that the OL was thick at P0. It showed a rapid decrease in thickness in the first week of life (between P0 and P7). Between P7 and P14, it showed a slow increase followed by a rapid increase between P14 and P90, reaching a thickness close to that at P0. These data had a statistically significant P value (Table 5 and Histogram 5).
The thickness of the ML of the hippocampus proper showed a very rapid increase between P0 and P7 and a nearly fixed thickness between P7 and P14. Between P14 and P90, there was a gradual increase in the thickness of this layer. These data were also statistically significant (Table 6 and Histogram 6).
Measurement of the thickness of the ML of the DG showed a very rapid and marked increase between P0 and P7, a very slow increase between P7 and P14, and a rapid increase between P14 and P90 in a manner very similar to that occurring in the ML of the hippocampus proper. These data were also statistically significant (Table 7 and Histogram 7). The thickness of the OL of the DG showed also a very rapid increase between P0 and P7, a very slow increase between P7 and P14, and a rapid increase again between P14 and P90. This resembled that occurring in the ML of the same area and the OL of the hippocampus proper. These data were also statistically significant (Table 8 and Histogram 8).
Morphometric study of the number of cell layers in CA1 area of the PL of the hippocampus proper showed a gradual decrease in the number of cell layers from approximately seven layers at P0 to three layers at P90. The decrease was more rapid between P0 and P7. These data were statistically significant (Table 9 and Histogram 9). Conversely, the number of cell layers in the upper limb of the GL of the DG was very small at P0. It was approximately two layers. It increased very rapidly between P0 and P7 reaching approximately five layers. It remained nearly fixed between P7 and P14. A slight decrease occurred thereafter between P14 and P90. These data were also statistically significant (Table 10 and Histogram 10).
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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