Evidence that the loss of Purkinje cells and deep cerebellar nuclei neurons in homozygous weaver is not related to neurogenetic patterns.

To determine whether the neurogenetic patterns of Purkinje cells and deep cerebellar nuclei neurons were normal in weaver homozygotes and whether the degeneration of those neuronal types was linked to their time of origin, [3H] thymidine autoradiography was applied on sections of homozygous weaver mice and normal controls on postnatal day 90. The experimental animals were the offspring of pregnant dams injected with [3H] thymidine on embryonic days 11-12, 12-13, 13-14 and 14-15. The results show that the onset of neurogenesis, its pattern of peaks and valleys, and its total span were similar between wild type and homozygous weaver in the cerebellar areas analyzed, indicating that the loss of Purkinje cells and deep cerebellar nuclei neurons is not related to neurogenetic patterns. In weaver homozygotes, the loss of Purkinje cells and deep cerebellar nuclei neurons followed a lateral to medial gradient of increasing severity. Thus, the vermis and the fastigial nucleus, which are medially located, presented the most important neuron loss, whereas in the lateral hemisphere and the dentate nucleus, neuron loss was spared.

The weaver gene has no effect on the generation patterns of mesencephalic dopaminergic neurons.

To determine if the weaver gene has action on late-generated neurons in midbrain areas on postnatal day (P) 8 [(3)H] thymidine autoradiography and tyrosine hydroxylase immunohistochemistry were combined in the same tissue section in homozygous weaver mice and normal controls. The experimental animals were the offspring of pregnant dams injected with [3H] thymidine on embryonic days (E)11-12, E12-13, E13-14 and E14-15. Both the span of neurogenesis and the neurogenetic timetables of dopaminergic neurons were similar between wild-type and homozygous weavers in all midbrain areas analyzed. No loss of late-generated dopaminergic neurons was observed. The cytoarchitecture of the midbrain dopaminergic cell groups were also the same in both experimental groups indicating that cell migration, settling, and cytodifferentiation proceeds normally in spite of the presence of the weaver gene.

The weaver gene continues to target late-generated dopaminergic neurons in midbrain areas at P90.

To determine if lethal action of the weaver gene is more intense in late-generated dopaminergic neurons in midbrain areas on postnatal day (P) 90 [3H] thymidine autoradiography and tyrosine hydroxylase immunohistochemistry were combined in the same tissue section in homozygous weaver mice and normal controls. The experimental animals were the offspring of pregnant dams injected with [3H] thymidine on embryonic days (E) 11-12, E12-13, E13-14 and E14-15. Neurogenetic timetables of dopaminergic neurons were different between wild type and homozygous weavers in all midbrain areas analyzed. A substantial number of late-generated neurons in the substantia nigra pars compacta and in the ventral tegmental area are missing at P90, in these dopaminergic areas the loss is greater than at P20 indicating that neuronal loss is progressive. The greatest loss is in the substantia nigra pars compacta, confirming the report of Bayer et al. [Exp. Brain Res. 105 (1995) 200] at P20, while in the retrorubral field and the interfascicular nucleus late-generated neuron loss was less severe. These results furnish more evidence that dopaminergic neuron loss in homozygous weaver midbrain is a phenomenon linked to development.

Glial cell line-derived neurotrophic factor protects midbrain dopamine neurons from the lethal action of the weaver gene: a quantitative immunocytochemical study.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and repair midbrain dopamine neurons in vivo using animal models created with neurotoxins. The weaver mouse (wv/wv) has natural and spontaneous midbrain dopaminergic cell death which gives a unique opportunity to examine the effects of GDNF. The present study was designed to investigate a possible neuroprotective role by GDNF for midbrain dopamine neurons in the wv/wv. Weaver pups were given 1 microl injections on postnatal day 1. The wv/wv placebo group received a single unilateral injection into the right lateral ventricle of phosphate buffered saline (PBS) while the GDNF treated wv/wv mice received either 1.0 microg/microl or 10.0 microg/microl GDNF in PBS. All mice were sacrificed on postnatal day 20 and their brains were processed for tyrosine hydoxylase (TH) immunocytochemistry. When compared to the placebo group, the 1 microg GDNF group showed significantly less cell death on the injection side, but the contralateral side showed no significant sparing of TH neurons. The combined counts from both sides show significantly more TH staining neurons in the 1 microg GDNF group compared to placebo. When compared to placebo-injected controls, the 10 microg GDNF treated group showed significantly more TH staining neurons on the injected side, contralateral side, and combined. The results demonstrate that GDNF does protect weaver dopaminergic midbrain neurons from the lethal action of the weaver gene and the effect is positively correlated to dosage.

Diverse cell death pathways result from a single missense mutation in weaver mouse.

Neuronal death affects selectively granule cell precursors of the cerebellum and the dopaminergic neurons of midbrain in the weaver mutant mouse. The weaver phenotype is associated with a missense mutation in the gene coding for the GIRK2 potassium channel, which results in chronic depolarization. Using DNA gel electrophoresis, electron microscopy (EM), the in situ end-labeling (ISEL) technique at the light and EM level, and immunohistochemistry for apoptosis-related proteins c-Jun and proliferating cell nuclear antigen (PCNA), we have investigated the mechanisms of cell death in cerebellum and substantia nigra. Between postnatal day P1 and P21, in the external germinal layer of the cerebellum, most degenerating granule cell precursors were found to aggregate to form clusters. Degenerating cells exhibited strong nuclear staining for ISEL, c-Jun, and PCNA and had a typical apoptotic morphology by EM. Increased c-Jun and ISEL staining were also occasionally seen in Purkinje cells. Between P14 and P21, when dopaminergic neurons start to degenerate, staining for ISEL, c-Jun, and PCNA in weaver substantia nigra was the same as in controls. By EM, however, we found only in weaver mice numerous dopaminergic cells that showed extensive vacuolar and autophagic changes of cytoplasm, preservation of membrane and organelle integrity, and absence of chromatin condensation or DNA fragmentation by EM-ISEL. The combination of vacuolar and autophagic changes identifies a novel type of non-necrotic, nonapoptotic cell death. After biochemical analysis of DNA, a clear-cut laddering, suggestive of oligonucleosomal fragmentation, was present in samples from weaver cerebellum. Cell death diversity appears to be influenced by specific features of target cells. These findings may be relevant for understanding the mechanisms of cell death in neurodegenerative diseases.

Phenotypic effects of the weaver gene are evident in the embryonic cerebellum but not in the ventral midbrain.

Degeneration of neurons in two structures, the cerebellum and the dopaminergic neurons in the ventral midbrain, is a well characterized action of the weaver gene. In order to see whether the gene has effects prenatally, both the cerebellum and the ventral midbrain were examined in mouse embryos genotyped for the weaver gene (wv, Girk2) on day E19. Anatomically matched sections of the midline cerebellar vermis were quantitatively analyzed 2 h after the dams were given a single injection of [3H]thymidine. A gene-dose effect was seen in the retardation of fissure development. This was more pronounced in homozygotes (wv/wv) and less so in heterozygotes (wv/+) when compared with wild type controls (+/+). Quantitative measures of the following features showed stepwise differences between genotypes so that the wv/wv are most affected and wv/+ are somewhat affected compared with +/+: surface length of the midline vermis, area of the entire midline vermis and the external germinal layer (egl), total number of cells in the egl, [3H]thymidine-labeled and -unlabeled egl cells, cells in the Purkinje cell layer, cells in the region of the deep nuclei, [3H]thymidine-labeled cells in the Purkinje cell layer (presumptive proliferating Bergmann glia), and [3H]thymidine-labeled cells in the region of the deep nuclei. In contrast to the obvious phenotypic effects of wv in the embryonic cerebellum, qualitative immunocytochemical examination of tyrosine hydroxylase staining in the ventral midbrains of the same embryos showed that the position and density of the presumptive dopaminergic neurons was similar in all genotypes.

Detection of apoptosis in weaver cerebellum by electron microscopic in situ end-labeling of fragmented DNA.

Massive degeneration of granule cell precursors occurs perinatally in the cerebellum of weaver mutant mice. We have studied the electron microscopic (EM) features of granule cell death in weaver and control mice, using an in situ end-labeling (ISEL) technique for detecting DNA fragmentation, a hallmark of apoptosis. In all animals, EM-ISEL revealed the pattern of apoptosis, with an enhanced expression in weaver mice. The weaver gene appears to accelerate the death program, most likely through potassium channel-mediated signals.

Selective vulnerability of late-generated dopaminergic neurons of the substantia nigra in weaver mutant mice.

In homozygous weaver (wv/wv) mutant mice, nearly 50% of the dopaminergic substantia nigra neurons degenerate by postnatal day 20. We have now determined that the total number of dopaminergic neurons in the ventral midbrains of a litter of obligatory homozygous weaver pups and a litter of normal wild-type control pups indicates that no significant differences are present between groups at birth. To test the hypothesis that the subsequent degeneration of these neurons is linked to their time of origin, [3H]thymidine autoradiography was combined with tyrosine hydroxylase immunocytochemistry to construct neurogenetic timetables on postnatal day 20 in wild-type mice and weaver homozygotes. Both groups have the same span of neurogenesis but have statistically different proportions of neurons generated on specific days. In wild-type mice, more than half of the dopaminergic neurons originate on or after embryonic day 12. In contrast, over two-thirds of the surviving dopaminergic neurons in homozygous weaver mice originate on or before embryonic day 11. Our data suggest that the weaver gene does not interfere with the generation of dopaminergic neurons, but it preferentially kills late-generated dopaminergic neurons between birth and postnatal day 20.

Time of neuron origin and gradients of neurogenesis in midbrain dopaminergic neurons in the mouse.

Previous [3H]thymidine studies in Nissl-stained sections in rats established that the substantia nigra pars compacta and the ventral tegmental area originate sequentially according to an anterolateral to posteromedial neurogenetic gradient. We investigated whether that same pattern is found in mice in the dopaminergic neurons in each of these structures. Using tyrosine hydroxylase immunostaining combined with [3H]thymidine autoradiography, the time of origin of dopaminergic midbrain neurons in the retrorubral field, the substantia nigra pars compacta, the ventral tegmental area, and the interfascicular nucleus was determined in postnatal day 20 mice. The dams of the experimental animals were injected with [3H]thymidine on embryonic days (E) 11-E12, E12-E13, E13-E14, and E14-E15. The time of origin profiles for each group indicated significant differences between populations. The retrorubral field and the substantia nigra pars compacta arose nearly simultaneously and contained the highest proportion of neurons, 49 to 37%, generated on or before E11. Progressively fewer early-generated neurons were found in the ventral tegmental area (20%), and the interfascicular nucleus (8.5%). In addition, anterior dorsolateral neurons in the substantia nigra and ventral tegmental area were more likely to be generated early than the posterior ventromedial neurons. These findings indicate that mouse and rat brains have nearly identical developmental patterns in the midbrain, and neurogenetic gradients in dopaminergic neurons are similar to those found in Nissl studies in rats.

Systematic differences in time of dopaminergic neuron origin between normal mice and homozygous weaver mutants.

Immunocytochemical labeling for tyrosine hydroxylase and [3H]thymidine autoradiography were combined in wild-type mice and in mice homozygous for the weaver mutant gene (wv) to see whether the neurogenetic patterns of midbrain dopaminergic neurons was normal in the mutants and whether the degeneration of dopaminergic neurons was linked to their time of origin. Dams of wild-type and homozygous weaver mice were injected with [3H]thymidine on embryonic days (E) 11-E12, E12-E13, E13-E14, and E14-E15 to label neurons in the retrorubral field, the substantia nigra pars compacta, the ventral tegmental area, and the interfascicular nucleus as they were being generated. The quantitatively determined time of origin profiles indicated that wv/wv mice have the same time span of neurogenesis as +/+ mice (E10 to E14), but have significant deficits in the proportion of late-generated neurons in each dopaminergic population. In the retrorubral field and substantia nigra, weaver homozygotes had substantial losses of dopaminergic neurons and had a greater deficit in the proportion of neurons generated late while, in the ventral tegmental area and interfascicular nucleus, there were slight losses of dopaminergic neurons and only slight deficits in the proportion of late-generated neurons. These findings lead to the conclusion that the weaver gene is specifically targeting dopaminergic neurons that are generated late, mainly on E13 and E14.

Correlated quantitative studies of the neostriatum, nucleus accumbens, substantia nigra, and ventral tegmental area in normal and weaver mutant mice.

Normal mice (+/+) and homozygous weaver mutant mice (wv/wv) at 1 year of age were used for three-dimensional computer-aided reconstructions of the nucleus accumbens (NA) and neostriatum (ST) and for quantitative estimations of the total number of medium-sized neurons in the NA and ST, and for the total number of tyrosine hydroxylase (TH)-containing neurons in the ventral tegmental area (VTA) and substantia nigra (SN). The three-dimensional reconstructions showed that the weaver NA and ST are smaller than they are in +/+. Quantitative volumetric measurements of the NA and ST showed wv/wv were smaller than +/+ by nonsignificant differences of 14% and 13%, respectively. The wv/wv group showed statistically significant depletion of neurons in all four structures. On average, NA neurons are reduced by 27%, ST neurons by 22%, VTA-TH neurons by 40%, and SN-TH neurons by 79%. In wv/wv animals, there was a high positive correlation (r = 0.836) between the numbers of SN-TH neurons and ST neurons and a moderate positive correlation (r = 0.534) between the numbers of SN-VTA neurons and NA neurons. The nuclei in TH-containing neurons in wv/wv and +/+ had the same diameters, but in all animals, the SN-TH neurons contained larger nuclei than the VTA-TH neurons. Cytoarchitectonic measurements in control and weaver NA and ST were also similar. In all animals, the NA contains more densely packed neurons with smaller nuclei than those in the ST.

Three-dimensional reconstructions of the developing forebrain in rat embryos.

Using a computerized three-dimensional reconstruction technique with serially sectioned rat embryos, changes in the size and form of the forebrain were studied on Embryonic Days (E) 12 (1 day after closure of the neural tube), E15, E18, and E21 (2 days before birth). During this time, the forebrain changes from a relatively simple tubular structure with thin walls surrounding a large ventricular system to a thick-walled brain with a highly convoluted but reduced ventricular system. On E12, the two components of the forebrain, the telencephalon and the diencephalon, cannot be distinguished. Considering the forebrain as a whole (the embryonic prosencephalon), its volume continually increases between E12 and E21 due to the generation, differentiation, and maturation of neurons and glia. Attention was paid to changes in the sizes of the ventricles, the neuroepithelium and the parenchyma. Volumes of the ventricles and the surrounding neuroepithelium rapidly expanded from E12 to E18 and then decreased by E21, while the volume of the parenchyma continually increased. Differential growth of the telencephalon and that of the diencephalon were compared between E15 and E21. The expansion of the telencephalon was much larger than that of the diencephalon. In the telencephalon, the volumes of the lateral ventricles and the surrounding neuroepithelium increased between E15 and E18 and decreased by E21, while in the diencephalon the volumes of the third ventricle and its surrounding neuroepithelium continually declined between E15 and E21. That observation is compatible with previous work showing that the majority of diencephalic structures develop earlier than those in the telencephalon. It is important to note that volume changes in the ventricles and the neuroepithelium are maintained in "lock-step," suggesting a close relationship between the size of the ventricle and the size of the neuroepithelium.

Cell migration in the rat embryonic neocortex.

Three-dimensional reconstructions of the normal rat embryonic (E) neocortex on days E15, E17, E19, and E21, using Skandha (software designed by J. Prothero, University of Washington, Seattle), show that the neocortical ventricular zone shrinks rapidly in the medial direction during cortical morphogenesis. [3H]thymidine autoradiography indicates that the shrinkage of the ventricular zone occurs before neurons in lateral and ventrolateral parts of layers IV-II are generated. Consequently, most of these neurons originate 400-1000 microns medial to their settling sites in the cortical plate. Embryos killed at daily intervals up to E21 after a single injection of [3H]thymidine on either E17 or E18 revealed the presence of a prominent migratory path, the lateral cortical stream, used by neurons migrating to the lateral and ventrolateral cortical plate; neurons migrating to the dorsal cortical plate follow a direct radial path. Arrival times of neurons in the cortical plate depend on the migratory path and are proportional to the overall distance travelled. Neurons that migrate only radially arrive in the dorsal cortical plate in two days (shortest route). Neurons that migrate laterally arrive in the lateral cortical plate in 3 days (longer route) and in the ventrolateral cortical plate in 4 days (longest route). [3H]thymidine autoradiography also shows that cells generated in the neocortical ventricular zone migrate in the lateral cortical stream for 5 or more days and accumulate in a reservoir. Cells leave the reservoir to enter the piriform cortex and destinations (as yet undetermined) in the basal telencephalon. The lateral cortical stream is found wherever the neocortical primordium surrounds the basal ganglia and is absent behind the basal ganglia. A computer analysis of nuclear orientation in anterior and posterior parts of the intermediate zone in the dorsal neocortex between days E17 and E22 shows that horizontally oriented nuclei are more common anteriorly where many cells are migrating laterally than posteriorly where most cells are migrating radially.

Planar differences in nuclear area and orientation in the subventricular and intermediate zones of the rat embryonic neocortex.

Nuclear area and orientation in the subventricular and intermediate zones was studied quantitatively in coronal vs. sagittal sections of the dorsomedial neocortex. Nissl-stained methacrylate-embedded normal rat embryos were studied between embryonic days (E) 13 and E22. The area of nuclear profiles and the degrees their long axes (defined as a straight line through the two most distant points in the nuclear profile) deviated from the horizontal (defined as parallel to the pial membrane) were determined with a computer-graphics program. Because the nucleus is the most clearly outlined structure in embryonic cells, the area and orientation of the nucleus was taken to reflect the overall size and orientation of the cell body. Nuclear area is larger in the coronal plane than it is in the sagittal plane, especially between E17 and E20. Cell body orientation in the subventricular and lower intermediate zones is predominantly horizontal in the coronal plane and predominantly vertical in the sagittal plane. In the upper intermediate zone, cell body orientation is predominantly vertical in both planes, but more so in the sagittal plane. These data indicate that the majority of cell bodies in the subventricular and lower intermediate zones have a horizontally oriented, flattened elliptical shape with their larger diameters lying within the coronal plane and their smaller diameters in the sagittal plane. Because of the flattening, the cell bodies falsely appear to be vertically oriented in the sagittal plane. Qualitative observations in horizontal sections confirmed the quantitative computer analysis. These results are related to other findings with [3H]thymidine autoradiography concerning cell migration and the sojourn of cells in the subventricular and intermediate zones.

Development of the endopiriform nucleus and the claustrum in the rat brain.

Long-survival [3H]thymidine autoradiography was used to quantitatively determine the time of origin of neurons in the endopiriform nucleus and the claustrum in rats killed on postnatal day 60 after their dams received two consecutive daily injections of [3H]thymidine on embryonic day E13 and E14, E14 and E15, ... E21 and E22. The claustrum originates late, on E15 and E16, and has a strong gradient in the longitudinal direction, posterior (older) to anterior (younger). In contrast, the endopiriform nucleus originates early, on E14 and E15, and lacks a longitudinal gradient but has a strong one in the vertical direction, ventral (older) to dorsal (younger). Sequential-survival [3H]thymidine autoradiography was used to qualitatively determine the germinal sources and settling sites of endopiriform and claustral neurons in embryonic rats. The dams received a single injection of [3H]thymidine on either E14 (to heavily label older endopiriform neurons) or E16 (to heavily label younger claustral neurons) and were killed in sequential 24-h intervals. Neurons in the presumptive endopiriform nucleus settle within two to three days after their peak time of neurogenesis while those in the presumptive claustrum take approximately five days to settle after their peak. It is postulated that endopiriform neurons are generated in the palliostriatal ventricular angle, the neuroepithelium that forms a wedge between the primordia of the neocortex and the basal ganglia, and that claustral neurons are generated in the neocortical neuroepithelium. Divergent developmental patterns between the endopiriform nucleus and the claustrum support the anatomical evidence that these nuclei have different connections. Furthermore, neurogenetic gradients in the claustrum correlate with the pattern of anatomical connections between the claustrum and the neocortex.

Timetables of cytogenesis in the rat subfornical organ.

Timetables of neurogenesis and ependymal cell production in the rat subfornical organ (SFO) were determined by examining the offspring of pregnant rats injected with [3H]thymidine on E13-E14, E14-E156, ... E21-E22, respectively. The proportion of postmitotic cells originating each embryonic day was determined by analyzing, in the adult offspring, the progressive reduction in the proportion of labeled precursors from the maximum amount seen in the E13-E14 group. Neurogenesis was found to occur over an extended period of time, beginning on E12 and continuing through E21. Ependymal cells were generated E15 through E21. Both neuron and ependymal cell production occurred in a triphasic pattern and followed an anterior (older) to posterior (younger) gradient. The anterior to posterior production gradient may be related to the morphological variation which exists along this plane. A production gradient intrinsic to a particular levels was found only in the posterior SFO, where peripheral neurons form earlier than core neurons. That neurogenetic gradient may be related to the core-periphery topographical patterns found in other studies, and suggests that the core neurons, since they are among the last to be formed, may be interneurons.

Mosaic organization of the hippocampal neuroepithelium and the multiple germinal sources of dentate granule cells.

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)

Prolonged sojourn of developing pyramidal cells in the intermediate zone of the hippocampus and their settling in the stratum pyramidale.

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)

Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods.

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.