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Research Article

Prenatal and Postnatal Development of the Rat Hippocampus (Histological Study)

Maher M. Kamel, Mohamed M. Abdelaleem, Moustafa A. Saad and Sayed F. El-Sheikh Department of Anatomy, Minia Faculty of Medicine

Abstract The differentiation of the hippocampus starts in the subiculum and proceeds towards the Ammon's horn and the dentate gyrus is the last structure to be formed. Lamination of the subiculum starts on E71 and progress to the Ammon's horn on E71; CA7 is formed, while the CA3 is not completely developed. On E71, the CA3 is completed with formation the ectal limb of dentate gyrus while the endal limb is still unformed and appears as a ball-like mass of cells. Between E02-P7, Ammon's horn shows agreat increase in length as acurved C-shape structure. The endal limb of dentate gyrus appears prenatally and develops rapidly to become morphologically similar to the ectal limb by P1. During the first month of development, there are increases in length of Ammon's horn, dentate gyrus size and size of both pyramidal cells and granule cells. In adult, the length of Ammon's horn is markedly large and the hippocampal subfields are prominent. Key Words: Development, Hippocampus, Ammon's horn and dentate gyrus

Introduction constitute the circuit of emotion. In 7180 Pallium (the ) coats the Maclean named this circuit the "limbic surface of cerebral hemispheres. The system"(72). cerebral cortex is divided according the development level, structure and function This study aimed to identify the into six- layered and three- morphological changes of both layered . The hippocampus is the cornuAmmonis and dentate gyrus in best example for allocortex. The consequent ages during embryological and or are used to postnatal development and analyze these mark the transitional zone between the differences in a functional context. It is neocortex and allocrtex(1). necessary to have the anatomical base of this region by simple microscopic study of The hippocampus was first described by the rat hippocampus using rapid and easy Arantius (7811) in his book "De method (H & E).This method can identify HumanoFoetu" the structure in the temporal layers, pyramidal cells of cornuammonis horn of the lateral ventricles that looks and granule cells of dentate gyrus. likehippocampus (sea horse) or bombycinusvermiscandidus (white silk Materials and methods worm),(78). Twenty four pairs of male and female albino rats were used in the present study. In 7177, Ramóny Cajal and lorente de Nó The rats were put in separate cages, each used Gogi's technique in studying the contained one male and one female rat. cellular architecture of hippocampal They were kept for mating for 09 hours. formation(1179). The next day, female rats were examined for vaginal plug of semen. The first day of In 7131, Papez described his circuit and the conception was determined if the vaginal hippocampus was one of the structures plug is positive and considered to be integrated in(70). He suggested that it might E7.Pregnant female rats were kept separate

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in cages and were followed up for development would be following up to pregnancy dating. From E71-E07, two or reach the final appearance.It is formed of more pregnant rats were taken for each day two major parts, the cornuammonis and the of age, and then sacrificed by decapitation. dentate gyrus. Both structures are curved; The abdomen and the uterus were opened the terminal part of cornuammonis is up, and then the embryos were removed, directed into the concavity of dentate gyrus; heads were separated and injected by Bouin (the hilum). fixative through making pores at the margin of skull cap.From postnatal ages P7, P1, The coruammonis appears as a column of P79, P07, P32 and >P12 (adult), three rats less densely packed, randomly oriented were taken for each age and sacrificed by cells lie adjacent to the tightly packed, decapitation. The skulls were opened and radially oriented neuro-epithelium. The the brains were extracted for removal of subfields of cornuammonisare difficult to hippocampus in the following steps: be identified. The boundary between the 7) In order to expose the hippocampus we subiculum and conruammonis is not recog- need to remove the cerebral cortex nizable. The cells of cornuammonis appear covering it. The first incision is at the end small with small deeply pigmented nuclei. of the hemisphere; the incision should be about 2.1mm deep for adult mouse that The dentate gyrus starts to appear as a not to hurt the hippocampus while to collection of young nerve cells migrating expose it (the incision depth is reduced to from the neuro-epithelium near the 2.0 mm in one day old rat). The 0nd outgrowth of the chorid plexus to a subpial incision is about 7.8-0mm in front of the area in the concavity formed by the neuro- first one, this incision we cut into the epithelium of cornuammonis at its ventral lateral ventricle, both of the incisions go end. to the ventral of the brain and meet there. This piece of cortex is free, after pulling it On E71, the coruammonis is seen to make up, we will see the hippocampus. more arching towards the lateral ventricle 0) On the other side of the brain, both the and temporal cortex, the stratum oriens and sides of the cortex are pulled up that stratum radiatum appear to be formed but covering the hippocampus along the they are thin. The cells appear small and ventricle. Now we can see the dorsal part rounded with deeply pigmented nuclei. of the hippocampus. 3) The rest of the hippocampus is The dentate gyrusarea appeared larger than separated from the cortex covering it the previous age. Cells of the dentate gyrus along the surface of the hippocampus are closely packed in a ball- like subpial towards the ventral part of the mass. The limbs are not formed yet. hippocampus. 9) The hippocampus picked out from the On E71, the cornuammonis appears as a brain. small C- shaped structure, bulging laterally, Either the head (E71-E07), or the towards the cavity of lateral ventricle, hippocampus (P7-adult), was kept in making two limbs, the prominent superior Bouin's fixative for 09 hours, and then limb is continuous with the cortex is called transferred to 721 neutral formalin. All CA7 and faint, uncompleted inferior limb specimens were embedded in paraffin. growing towards the hilum of dentate gyrus Serial sections (1 µm) were prepared in is called CA3. The fimbria rises below the the coronal plane for Haematoxylin and point of meeting between the two limbs of Eosin staining. CA. The hippocampal subfields are well apparent, from ventricular side outwards; Results neuro-epithelium, large sub-ependymal 2) Prenatal ages: (Fig. 2- 4) zone, stratum oriens, pyramidal layer and The hippocampus proper starts to appear by stratum radiatum. The thickness of neuro- the 71th day of intra-utrine life and its epithelium is apparently diminished than

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the previous age with appearance of large layers than CA3, nuclei are vesicular. Cells sub-ependimal zone containing radially of CA3 are larger than CA7. The dentate oriented neuroblasts towards the Cornu gyrus appears as larger mass of cells with Ammonis. The sub-ependymal zone is starting formation of ectal limb of the present throughout the length of granular cell layer that appear superficial to hippocampus, larger towards the subiculum the dentate hilus, less dense with deep than towards Ammon's horn. The cells nuclei and their apical dendrites extending acquire the characteristic pyramidal towards the molecular layer. The remaining appearance starting by this age, with apical cells of the dentate gyrus below the hilus dendrites towards the stratum radiatum. are densely packed with deep nuclei. Cells of CA7 are small, forming more

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On E71, the cornuammonisappears larger vesicular with prominent nucleoli. Cells of C- shaped structure. The CA3 part is CA3 are arranged from 3 to 8 layers, larger completed towards the dentate hilus. The in size with thicker apical dendrites. The subfields of CA could be seen with great dentate gyrus is formed of two limbs; the apparent change in thickness, the neuro- supra-pyramidal (ectal) limb, which appears epithelium layer is thin, sub-ependymal more developed; cells are arranged in layers layer is contracted, pyramidal layer is and larger than the newly formed infra- thickened and stratum radiatum is markedly pyramidal (endal) limb which appears as a enlarged. The dominant neurons in the collection of densely packed cells. The hippocampus proprius are the pyramidal chief cells of the dentate gyrus are the cells. The size and the density of these granular neurons and so, the 3 layers of neurons are variable throughout the CA. dentate gyrus are apparent in the supra- pyramidal cells show marked increase in pyramidal limb, the most superficial layer is apparent size than the previous age. Cells of the molecular layer; the next granule cell the pyramidal layer of CA7 arranged from layer in which granule cells are rounded 8 to 1 layers, with prominent apical with pale nuclei, and the polymorphic layer dendrites that extended to stratum radiatum that related to dentate gyrus hilum and and stratum lacunosum. The nuclei are characterized by presence of large Mossy

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cells. The cells of infra-pyramidal limb are granule cells. The granule cells are small, smaller, dispersed and have deeply rounded with little cytoplasm and have pigmented nuclei, the part of CA3 grows deeply pigmented nuclei, while the cells of towards the dentate gyrus hilum, pyramidal infra-pyramidal (endal) limb are smaller in cells appeared to have indistinct outlines of size and dispersed. cells with very pale nuclei (may be a sign of cell division activity). On E 07, the cornuammonisshows more increase in its length that made more On E02, the cornuammonis increased in its separation between two limbs of C- shape. length that made more space between two The cells of the pyramidal layer of CA7 limbs of its C- shape and made more lateral have prominent apical dendrites. The nuclei bulging and another new superior bulge at are vesicular with prominent nucleoli.The CA7. The pyramidal cells of CA7 are small cells of CA3 are arranged in less layering with prominent apical dendrites directed to than in CA7, they are larger in size with stratum radiatum. The nuclei are vesicular thicker apical dendrites. with prominent nucleoli.The cells of CA3 are larger in size with thicker apical The infra-pyramidal limb of dentate gyrus dendrites than CA7 cells. The dentate gyrus shows slight increase in its length. The cells is more developed, the infra-pyramidal limb of supra-pyramidal (ectal) limb appear to of dentate gyrus shows slight increase in its have vesicular nuclei with more prominent length, but it is not fully developed. The apical extensions towards the molecular outer molecular layer of supra-pyramidal layer. The cells of the infra-pyramidal (ectal) limb of dentate gyrus is apparent (endal) limb appear smaller in size with well, and formed mainly by dendrites of the deeply pigmented nuclei.

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2) Postnatal ages: and dendrites directed towards the On the first day of life, the cornuammonis superficial molecular layer of dentate gyrus. shows more increase in its length than the Cells of infra-pyramidal limb appear more age of the last day of intra-uterine life that dispersed, with more vesicular nuclei. made more bulge into the cavity of lateral ventricle. The pyramidal cells of CA7 are During the first week of life, the smaller in size than CA3 cells with cornuammonis shows great increase in its prominent apical dendrites. The nuclei are length and more lateral bulging into the vesicular. The pyramidal cells of CA3 are cavity of lateral ventricle. The pyramidal larger in size with thicker apical dendrites. cells of CA7 show apparent increase in size Theinfra-pyramidal limb of dentate gyrus is than the previous age with more prominent well formed than the previous age. Granule apical dendrites. The nuclei were vesicular cells of supra-pyramidal limb are rounded with prominent nucleoli. The pyramidal with little cytoplasm with rounded nucleus cells of CA3 near to the hilum of dentate

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gyrus show more increase in size. Most of By the second week of life, the cells had large vesicular nuclei with cornuammonis shows continuous increase prominent nucleoli and thicker apical in its length. The pyramidal cells of CA7 extensions. The infra-pyramidal of dentate are apparently increased size than the gyruslimb is well developed. Granule cells previous age and appeared to be more appear crowded. The cells towards the outer spherical in shape. The nuclei are vesicular molecular layer are larger with vesicular with prominent nucleoli. Cells of CA3 are nuclei and double nucleoli appearance, arranged from 3 to 8 layers, show more while the basal cells towards the increase in size with large vesicular nuclei, polymorphic layer appear small in size with prominent nucleoli and thicker apical deeply stained nuclei. Large, fusiform extensions. Most of nuclei of these cells basket cells are present at the polymorphic showed deeply pigmented chromatin islets. layer, which are more numerous at the ectal The dentate gyruscells show an apparent limb than the endal one. increase in size than the previous age, the infra-pyramidal (endal) limb showed more increase in length.

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On the third week of life, the nuclei, prominent nucleoli and chromatin cornuammonisincreases in its length more islets. The dentate gyrus with its both limbs than the previous age that made more show more increase in length. Granular external bulging. The pyramidal cells of cells are apparently larger.

CA7 part are rounded in shape. The nuclei The cornuammonis in Adult ageis seen as a are vesicular with prominent nucleoli. The large and more curved C- shaped structure pyramidal cells of CA3 show apparently with the fimbria close to the most curved more increase in size with large vesicular part near the dentate gyrus. The pyramidal nuclei, prominent nucleoli and chromatin cells of CA7 are not crowded; the islets. The dentate gyrus with its both limbs development of cell dendrites that push showed more increase in length, granule cells away from each other formed from 8 cells of endal limb were apparently smaller to 1 layers, with prominent apical dendrites and more crowded than ectal limb. Number that extending to stratum radiatum and of granule cell layers in endal limb are from stratum lacunosum. The nuclei are vesicular 1 to 72 while in ectal limb are from 9 to 1 with prominent nucleoli. The cells of CA3 layers with appearance of multiple part are formed of 3 to 8 laers, larger in size chromatin islets in their nuclei giving the with thicker apical dendrites. The pyramidal double nucleoli appearance. cells closer to the dentate gyrus hilum are fewer in layers with increasing in their size, By the first month of age, the cornuam- paler cytoplasm and larger nucleus. The monis greatly increase in size. Cells of the two limbs of dentate gyrusshow apparent pyramidal layer of CA7 part are crowded increase in size with slight increase in and rounded in shape. The nuclei were cellular size. vesicular with prominent nucleoli. Pyramidal cells of CA3 show apparently The following diagram summarizes the more increase in size with large vesicular results:

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Discussion are primitive neuroblasts migrating to their The present study of rat hippocampus shows respective location.(1) the development of inter-digitating structure of the C-shaped Ammon's horn and the V- The stratum pyramidale is starting to appear shape dentate gyrus by using simple H&E between E71- E71. The pyramidal cells of method. These two components show a CA7 are formed earlier than CA3.(119) three layered structure containing a single Between E07 and P7, the pyramidal layer principle layer of densely packed cells expands rapidly more in length than in named pyramidal cell layer in the Ammon's depth. CornuAmmonis grows towards the horn and granular cell layer in the dentate fimbria, with further forward linear gyrus, with superficial fiber layers above expansion making a sharp deflection and deep layers below.(244,42) In this experi- towards the ball- like dentate gyrus.(71) ment, better results were noticed by using Pyramidal cells of Ammon's horn are the formalin fixed sections, where the proceeded in its development by large and structure of the developing hippocampus medium sized cells which are formed earlier proprius and dentate gyrus is observable. As between E71- E71 in the strata oriens and previously mentioned, the hippocampus is radiatum.(0420) Although the dentate gyrus is subdivided into various areas. Many studies the last hippocampal structure to appear, the addressed the question of when and how the neuroblast cells migrating towards the developing hippocampus is subdivided into dentate gyrus are noticed during early fields. There is strong evidence that cell fate development (E71 and E71) as a ball-like is pre-programmed at the time of neuro- mass of neuroblastswith deeply stained genesis,(8,77171). nuclei. From E71 on, fimbrial growth forces migrating cells to move across it and Hippocampal development is composed of through the stratum oriens to get to the multiple steps occur at the level of neuroblat dentate gyrus. The dentate gyrus has a rapid cells: cell proliferation, migration, differen- expansion in size between (E71 and tiation and survival.(0719) The neuro- E71),(71) The granular layer first appears on epithelium is prominent as long as active E71- E71; initially, the ectal limb is more neurogenesis occurs throughout the hippo- prominent than the endal limb. The layer campal region. In the subicular complex, thickens throughout its extent postnatally, Ammon's horn and dentate hilus, neurons and by P1, both limbs are homogenous. As originate mainly between E71- E71.(2,) The the granule cells migrate from the hilus, the dorsal neuro-epithelium may produce more hilus itself is pushed into a V-shape by the neurons than the ventral neuro-epithelium rapidly invaginating Ammon's horn on E71- closer to the fimbria.(8) Hippocampal P7,(6) The sharp fold of the granular layer neurons migrate from hippocampal neuro- may simply explain this distortion.(1) There epithelium, which consists of three are two criteria for development of dentate components: Ammonic neuro-epithelium, gyrus; first, neurons of granule cell layer dentate neuro-epithelium and glio-epithe- located in ectal limb are developed earlier lium.(3) The Ammonic neuro-epithelium than those of endal limb; second, neurons in gives rise to pyramidal cells of Ammon's the outer granule cells are developed earlier horn, the dentate neuro-epithelium is the than the inner cells.(73) origin of granule cells of dentate gyrus and glio-epithelium generate the glial cells.(711) References The sub-ependymal zone is a transitional 7. Altman, J. and S. A. Bayer (7112). zone during the prenatal development of "Mosaic organization of the hippo- hippocampal region that present between campal neuro-epithelium and the the layer of neuro-epithelium and stratum multiple germinal sources of dentate oriens of the developing cornuAmmonis. granule cells." Journal of comparative Mitotic figures are always sparse, and neurology 327(3): 308-390. probably most of the densely packed cells 0. Amaral, D. G., N. Ishizuka and B. Claiborne (7112). "Chapter neurons,

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