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In radiograms of rat embryos that received a single dose of [3H]thymidine between days E16 and E20 and were killed 24 hours after the injection, the heavily labeled cells (those that ceased to multiply soon after the injection) form a horizontal layer in the intermediate zone of the hippocampus, called the inferior band. The fate of these heavily labeled cells was traced in radiograms of the dorsal hippocampus in embryos that received [3H]thymidine on day E18 and were killed at different intervals thereafter. Two hours after injection the labeled proliferative cells are located in the Ammonic neuroepithelium. The heavily labeled cells that leave the neuroepithelium and aggregate in the inferior band 1 day after the injection become progressively displaced toward the stratum pyramidale 2-3 days later, and penetrate the stratum pyramidale of the CA1 region on the 4th day. In the stratum pyramidale of the CA3 region, farther removed from the Ammonic neuroepithelium, the heavily labeled cells are still sojourning in the intermediate zone 4 days after labeling. Observations in methacrylate sections suggest that two morphogenetic features of the developing hippocampus may contribute to the long sojourn of young pyramidal cells in the intermediate zone: the way in which the stratum pyramidale forms and the way in which the alveolar channels develop. The stratum pyramidale of the CA1 region forms before that of the CA3 region, which is the reverse of the neurogenetic gradient in the production of pyramidal cells. We hypothesize that this is so because the pyramidal cells destined to settle in the CA3 region, which will be contacted by granule cells axons (the mossy fibers), have to await the formation of the granular layer on days E21-E22. Concordant with this is the observation that the hippocampal intermediate zone, which contains the sojourning young pyramidal cells, greatly enlarges between days E16 and E20, then suddenly diminishes and disappears by day E22. The other factor that may contribute to the prolonged sojourn of pyramidal cells, specifically those destined to settle in the CA1 region, is the pattern of alveolar channel development. This transient extracellular matrix begins to form several days after the onset of pyramidal cell neurogenesis, grows in a direction opposite to the settling of pyramidal cells in the stratum pyramidale, and does not reach the subicular end of Ammon's horn until day E21.(ABSTRACT TRUNCATED AT 400 WORDS)

Methacrylate-embedded sections and short-survival thymidine radiograms of the hippocampal dentate gyrus were examined in perinatal and postnatal rats in order to trace the site of origin and migration of the precursors of granule cells and study the morphogenesis of the granular layer. The densely packed, spindle-shaped cells of the secondary dentate matrix (a derivative of the primary dentate neuroepithelium) stream in a subpial position towards the granular layer of the internal dentate limb during the perinatal and early postnatal periods. By an accretionary process, the crest of the granular layer forms on day E21 and on the subsequent days the granular layer of the internal dentate limb expands progressively in a lateral direction. Granule cells differentiation, as judged by the transformation of polymorph, darkly staining small cells into rounder, lightly staining larger granule cells, follows the same gradient from the external dentate limb to the internal dentate limb. The secondary dentate matrix is in a process of dissolution by day P5. This matrix is the source of what will later become the outer shell of the granular layer composed of early generated granule cells. The thicker inner shell of the granular layer, formed during the infantile and juvenile periods, derives from an intrinsic, tertiary germinal matrix. On day E22, the dentate migration of the secondary dentate matrix becomes partitioned into two components: a) the subpial component of extradentate origin, referred to in this context as the first dentate migration, and b) the second dentate migration. The latter is distributed in the basal polymorph layer throughout the entire dentate gyrus and is henceforth recognized as the tertiary dentate matrix. The tertiary dentate matrix is prominent between days P3 and P10. It is postulated that the great increase in granule cell population during the infantile period is principally due to cells derived from this intrinsic matrix of the dentate gyrus. Between days P20 and P30 the tertiary dentate matrix disappears in the basal polymorph layer and henceforth proliferative cells become largely confined to the subgranular zone at the base of the granular layer. The subgranular zone is the source of granule cells produced during the juvenile and adult periods.

This study deals with the site of origin, migration, and settling of the principal cell constituents of the rat hippocampus during the embryonic period. The results indicate that the hippocampal neuroepithelium consists of three morphogenetically discrete components--the Ammonic neuroepithelium, the primary dentate neuroepithelium, and the fimbrial glioepithelium--and that these are discrete sources of the large neurons of Ammon's horn, the smaller granular neurons of the dentate gyrus, and the glial cells of the fimbria. The putative Ammonic neuroepithelium is marked in short-survival thymidine radiograms by a high level of proliferative activity and evidence of interkinetic nuclear migration from day E16 until day E19. On days E16 and E17 a diffuse band of unlabeled cells forms outside the Ammonic neuroepithelium. These postmitotic cells are considered to be stratum radiatum and stratum oriens neurons, which are produced in large numbers as early as day E15. A cell-dense layer, the incipient stratum pyramidale, begins to form on day E18 and spindle-shaped cells can be traced to it from the Ammonic neuroepithelium. This migratory band increases in size for several days, then declines, and finally disappears by day E22. It is inferred that this migration contains the pyramidal cells of Ammon's horn that are produced mostly on days E17 through E20. The putative primary dentate neuroepithelium is distinguished from the Ammonic neuroepithelium during the early phases of embryonic development by its location, shape, and cellular dynamics. It is located around a ventricular indentation, the dentate notch, contains fewer mitotic cells near the lumen of the ventricle than the Ammonic neuroepithelium, and shows a different labeling pattern both in short-survival and sequential-survival thymidine radiograms. By day E18, the reduced primary dentate neuroepithelium is surrounded by an aggregate of proliferative cells; this is the secondary dentate matrix. On the subsequent days spindle-shaped cells that have retained their proliferative capacity migrate from the progressively receding secondary dentate matrix to the dentate gyrus itself. The latter, representing a tertiary germinal matrix, becomes highly active during the perinatal period. The putative fimbrial glioepithelium is situated between the primary dentate neuroepithelium and the tip of the hippocampal rudiment. Observations in methacrylate sections and thymidine radiograms suggest that the cells of this germinal matrix, unlike typical neuroepithelial cells, do not undergo interkinetic nuclear migration.(ABSTRACT TRUNCATED AT 400 WORDS)

Volumetric estimates of the total number of granule cells in rats 30, 120, 200, and 365 days old increase linearly by approximately 35 to 43 percent between 1 month and 1 year. Total volume of the granular layer also grows linearly during that time. These results demonstrate a numerical increase in a neuronal population during adulthood in the mammalian brain.

Hippocampal slow wave activity was studied in rats which were normal or had been subjected to denate gyrus granule cell agenesis by focal X-irradiation starting at birth (0-day group) or two days (2-day group) of age. X-irradiation reduced adult brain weight, abolished most (2-day) or all (0-day) granule cells in the lower (endal) blade of the dentate gyrus, and reduced granule cell density by up to 70% in the upper (ectal) blade of the dentate gyrus. X-irradiation did not affect pyramidal cells of the hippocampus proper. Tracking with microelectrodes in urethane anesthetized rats given eserine, sensory, or brain stimulation showed two foci of hippocampal rhythmical slow activity (theta or RSA), one in stratum oriens of CA1 and one in stratum moleculare of the dentate gyrus. These were opposite in phase by approximately 180 degrees and separated by a null zone and phase reversal point occurring at stratum radiatum. There were no significant differences in the amplitude, frequency, null, or phase reveral points in the normal or X-irradiated groups. However, the width of the RSA amplitude peak in the ectal blade was reduced, correlated with the reduction in the length of the ectal blade, and the RSA amplitude peak in the lower blade was absent, correlated with the absence of the lower blade. The fast activity recorded in the hilus of normal rats was absent in the X-irradiated groups. RSA recorded during spontaneous movement (walking) had identical amplitude, frequency, anatomical foci, and phase in both the hippocampus proper and dentate gyrus in normal and X-irradiated rats. Antimuscarinic, but not antinicotinic, agents abolished anesthesia-related RSA, but not movement-related RSA, in all groups of rats. The results are discussed in terms of their relevance to the two-generator hypothesis of RSA.

The total number of granule cells in the dentate gyrus was estimated in 17 male rats, four each aged 30, 120, and 200 days, and five aged 365 days. There is a substantial 35-43% linear increase between 1 month and 1 year. Two parameters of the granular layer are involved in the numerical change. First, total granular layer volume grows linearly with age. Second, average volume of a single granule cell nucleus in the ventral dentate gyrus decreases with age. Older rats tend to have a larger granular layer filled with more and smaller cells. In another group of 21 male rats, 3H-thymidine injections were given on four consecutive days during juvenile (30-33, n = 6) and adult life (60-63, n = 5; 120-123, n = 6; 180-183, n = 4). All animals survived to 200 days of age. The proportion of labeled mature granule cells and labeled presumptive granule cell precursors were determined in anatomically-matched slices. With older ages at injection, there is a decline in labeled mature granule cells and a concurrent increase in labeled precursors. These data are compatible with the constant level of granule cell increase determined volumetrically. Most of the late granule cells originate nearly simultaneously along the base of the main bulk of the granular layer; very few are found in the dorsal tip (septal extreme) and ventral tip (temporal extreme). This study is the first demonstration of a net numerical gain in a neuronal population during adulthood in the mammalian brain. Since the granule adulthood in the mammalian brain. Since the granule cells play a pivotal role in hippocampal function, these data suggest that their influence grows with age.