Interactions between growing thalamocortical afferent axons and the neocortical primordium in normal and reeler mutant mice

The interactions between growing thalamocortical afferent axons and the neocortical primordium were examined during neocortical development of the mouse cerebrum, by labeling the afferents with the carbocyanine fluorescent dye, DiI, which was introduced into the dorsal thalamus of the fixed brains of control and reeler mutant mice. In the neocortical primordium of the control mouse, the labeled afferents running tangentially in the intermediate zone formed a dense plexus in the subplate, the layer below the cortical plate, as early as the 16th gestational day (E16). Small numbers of the afferents invaded the lower cortical plate at E16 and increasing numbers of labeled growing axons extended into the cortical plate at E17. At the 4th postnatal day (P4), labeled afferents grew radially up to the upper cortical plate and terminal arborizations of the afferents were evident in the forming layer IV. In contrast, in the E16 cerebrum of the reeler mutant mouse, in which the cortical layers are inverted, the labeled afferents traversed the neocortical primordium directly towards the superplate, the superficial layer above the cortical plate and the equivalent of the subplate in the control mouse. Thick bundles of labeled axons reached the superplate and made contact with the superplate neurons. At P4 in the reeler neocortex, the afferent axons that had reached the superplate began to change their direction of growth and turned towards the deeper layer. Electron-microscopic observations at E16 revealed that immature synapses were formed on the somata of the subplate neurons in the control mouse, and similar immature synapses were also formed on the superplate neurons of the reeler mutant. At E16 in the control, NGF receptor immunoreactivity was expressed in the intermediate zone, subplate and lower cortical plate, and the mode of expression corresponded to the distribution of thalamocortical afferents. At the same stage of the reeler mutant, expression of NGF receptor immunoreactivity was confined to the afferent axons that had grown through the neocortical primordium towards the superplate. In the control at E17, highly polysialylated NCAM (NCAM-H), a homophilic cell adhesion molecule, was expressed in the subplate, marginal zone and afferent axons. In the reeler mutant at the same stage, this adhesion molecule was expressed in both the superplate and the bundles of the afferent axons. These findings suggest that the subplate and the superplate, which are composed of neurons generated at the earliest stage, attract growing thalamocortical afferent axons specifically by a chemotropic mechanism through the expression of NGF receptor. Furthermore, growth cones of the afferent axons may make contact with the subplate or superplate neurons specifically through the homophilic adhesive activity of NCAM-H expressed on them. On the basis of such mechanisms, the subplate or the superplate could play a role as a tentative target for the thalamocortical afferents prior to arrival at their final targets, i.e., the layer IV cortical neurons in the control and the equivalent neurons in the reeler mutant.     

Early prenatal ontogenesis of the cerebral cortex (neocortex) of the cat (Felis domestica). A Golgi study

Summary The neocortex of the cat undergoes a series of fundamental transformations of its fibrillar-neuronal organization during the course of early prenatal cortical ontogenesis. Some of these transformations assume structural chracteristics and neuronal features which resemble those of phylogenetically older cortical organizations. Following the arrival of corticipetal fibers at the marginal zone of the cerebral vesicle a very primitive neocortical organization, the primordial plexiform layer develops. It is characterized by the external location of the white matter with both corticipetal and a few corticofugal fibers and a few immature neurons sandwiched between the fibers. The primitive plexiform layer is present in the cat from the 20th to the 25th day of gestation. The external (superficial) location of the white matter of the primordial plexiform layer of the cat neocortex is reminiscent of the amphibian cortical organization. It also resembles other primitive structures (spinal cord) of the central nervous system. In view of its short duration and because of the immaturity of its fibrillar-neuronal elements, the primordial plexiform layer is considered to be a transient neocortical organization possibly without functional activity in the cat.The appearance of the cortical plate (25th day of gestation) causes the subdivision of the primordial plexiform layer into an outer and an inner zone. The outer zone becomes layer I and the inner zone layer VI of the neocortex. Both of these layers remain as such throughout cortical development. From the 25th to the 45th day of gestation the fibrillarneuronal structure of layers I and VI develop while the cortical plate grows, passively, by the progressive addition of new cells. The progressive fibrillar-neuronal organization of layers I and VI and the development of structural and functional interactions between them constitutes the primordial neocortical organization of the cerebral cortex of the cat. It is characterized by a superficial (layer I) and a deep (layer VI) plexiform layer composed predominantly of collaterals from the corticipetal fibers arriving at the developing cortex and by three basic types of neurons. The horizontal neurons of layer I with descending axons terminating in layer VI, and the Martinotti neurons of layer VI with ascending axons terminating in layer I, are associative neurons. The large stellate neurons of layer VI are projective neurons. The axons of these cells before entering the white matter send ascending recurrent collaterals to layer I. The fibrillar-neuronal organization of the neocortex during this gestational period (primordial neocortical organization) resembles the organization of the reptilian neocortex. It is postulated that the primordial neocortical organization of the cat is functionally active during this gestational period.The arrival of new types of afferent fibers at the lower region of the cortical plate (45th day of gestation) causes the maturation of the pyramidal neurons of this region of the neocortex. These neurons are recognized at this age as the pyramidal neurons of layer V of the neocortex of the cat. The appearance of these afferent fibers and the maturation of the pyramidal neurons of layer V marks the transformation of the neocortex from its primitive reptilian structure into a distinctly mammalian organization. It is postulated that the cortical plate (pyramidal plate) is a recent addition in neocortical phylogeny representing a mammalian transformation. An analogy seems to exists among the pyramid-like neurons of the amphibian cortex, the pyramid-like neurons of the reptilian neocortex and the pyramid-like neurons (stellate) of layer VI of the mammalian neocortex. This analogy differs from the classical one postulated by Cajal which includes the pyramidal neurons of the mammalian neocortex, which are here considered as recent additions to neocortical phylogeny and hence as distinct mammalian neurons.Supported by Grant HD-03298-09, and by General Research Support Grant FR-05392 from the General Research Branch, National Institutes of Health.     

Synthesis of the foetal protein fetuin by early developing neurons in the immature neocortex

Summary The presence of the foetal protein fetuin has previously been demonstrated by immunocytochemistry to be specifically confined to the primordial plexiform layer, the early cortical plate and subplate zone cells in the developing neocortex of a number of species. In order to investigate its origin there, we have appliedin situ hybridization in paraffin sections of Bouin's fixed foetal sheep brain, using a short anti-sense oligonucleotide probe. The distribution of fetuin mRNA has been compared with that of the protein by using anti-fetuin antibodies and immunocytochemistry. This allowed us to confirm that fetuin is synthesised initially in cells of the primordial plexiform layer and subsequently cortical plate and subplate cells. On the other hand, cells in the ventricular zone that are fetuin (protein) positive do not contain detectable fetuin mRNA. The time course of fetuin mRNA expression in the developing neocortex follows closely the previously described pattern of fetuin (protein) distribution in the sheep brain, apart from its absence from the ventricular zone where its origin is probably by uptake from cerebrospinal fluid.     

The distribution of plasma proteins in the neocortex and early allocortex of the developing sheep brain

Summary The histogenesis of the cerebral neocortex and early allocortex of the sheep has been described and, using an immunohistochemical technique, five plasma proteins have been identified in the telencephalic wall and their distribution followed during its differentiation. The development of the neocortex was studied from 18 days gestation, when the neural tube was still open, to 120 days, when the adult structure was established. A primordial plexiform layer was formed above the ventricular zone by 25 days and by 35 days this layer was divided by the differentiating cortical plate into an outer marginal zone and an inner subplate zone. The appearance of the subventricular and intermediate zones by 50 days gestation completed the formation of the neocortical layers. The differentiation of the allocortex was generally less advanced than the neocortex up to 40 days gestation, when the primordium of the pyramidal layer was beginning to develop.The five plasma proteins identified, fetuin, -fetoprotein, albumin, transferrin and ₁-antitrypsin, are quantitatively the most important in the csf and plasma of the sheep fetus. Fetuin was the earliest plasma protein to be detected in the brain and it was also the most widespread; positive staining for this protein was seen in cells and fibres of all layers as they differentiated and could still be identified in some mature neurons at 120 days. -Fetoprotein and albumin had a limited distribution, appearing in cells in the developing cortical plate for a short period early in gestation (35–40 days), but mainly confined to the ventricular zones later and barely detectable by 80 days gestation. Transferrin appeared to have a different distribution, being detected in fibres first in the primordial plexiform layer and then in the marginal and subplate zones, only later being identified in cells of the cortical plate. From their distribution it is suggested that fetuin and transferrin may play an important role in the differentiation of the cortex and the establishment of correct connections between fiber systems and migrating cells at certain stages of development. ₁-Antitrypsin was only found in a few cells during a restricted period of gestation. All five plasma proteins were identified in precipitated csf and plasma at most ages examined, although at 18 days gestation albumin, transferrin and ₁-antitrypsin and at 120 days, -fetoprotein, could not be detected. The pattern of distribution of plasma proteins in the telencephalic wall suggests that they could originate either by uptake from csf and subsequent migration of protein containing cells or by local synthesis within some cells during a limited period of differentiation.     

Total number of cells in the human newborn telencephalic wall

The total cell numbers were estimated in the neocortical part of the human telencephalon in 10 normal brains of newborn babies within four major developmental zones: the cortical plate/marginal zone, the subplate, the intermediate zone and the ventricular/subventricular zone. Furthermore, the total number of neuron and glial cells was estimated in the cortical plate. The gestational ages ranged from 38 + 0-42 + 5 weeks + days of gestation. The mean total cell number was 32.6 x 10(9) (coefficient of error = 0.04) and the total number of neurons in the cortical plate 19.8 x 10(9) (coefficient of error = 0.06). This indicates that the total number of neocortical neurons equals the total number in the adults, which, however, is not the case for the glial cells.     

Dual origin of the mammalian neocortex and evolution of the cortical plate

Summary A Golgi study of the structural organization of the early developmental stages of the cerebral cortex of the cat has been presented. It has been demonstrated that the structural organization of the mammalian neocortex undergoes a series of fundamental transformations in the course of its early embryonic development. A clear understanding of these early structural changes is essential to comprehend the multi-layered nature of the mammalian cerebral cortex. In order of appearance the following basic transformations have been recognized in mammalian cortical ontogenesis. A. The first recognizable change in the undifferentiated neuroepithelial structure of the cerebral vesicle is the arrival and penetration of corticipetal fibers through its superficial region. The penetration of these afferent fibers into the cerebral vesicle forms a clear plexiform region under the pial surface just above the matrix zone. This clear plexiform region corresponds to the classical marginal zone and is composed, at first, of corticipetal fibers and their collaterals. B. The arrival of these corticipetal fibers induces maturation of some neurons. Primitive-looking and still-developing neurons begin to appear scattered among the afferent fibers without forming any distinct lamination. This combination of an external white matter of afferent fibers with scattered neurons among the fibers has been named the primordial plexiform layer of the mammalian cerebral cortex. Its structure represents a primitive type of nervous organization which is reminiscent of the amphibian brain. In mammalian cortical ontogenesis this primitive plexiform layer has a short duration and it is established as a distinct structure prior to the appearance of the cortical plate. C. The appearance and the formation of the cortical plate within the primordial plexiform layer results in the separation of its neurons and fibers into a superficial and a deep plexiform lamination. Structural and functional interrelationships soon start to develop between the neuronal elements of the superficial and the deep plexiform laminations establishing the primordial neocortical organization, which is characterized by specific types of neurons and fibers. Its structural organization resembles somewhat that of the cerebral cortex of some reptiles. It is important to emphasize that the neurons and fibers of this primordial neocortical organization persist and become components of the adult cerebral cortex. The superficial plexiform becomes layer I and the deep lamination becomes layer VII of the adult mammalian cerebral cortex. Therefore, the cortical plate represents the primordium of only layers VI, V, IV, III, and II of the adult cerebral cortex. The cortical plate is considered to be a distinct mammalian structure of a more recent phylogenetic origin. D. The last significant transformation consists of the sequential growth and maturation of the cortical plate which follows an inside-out progression. The maturation of the neurons of the cortical plate and hence the formation of its laminations seems to be due to the sequential and progressive arrival of corticipetal fibers which takes place during the late embryonic stages of development.According to these observations the mammalian cerebral cortex has a double origin and a possible dual nature. A new interpretation of the basic structural organization of the mammalian neocortex based primarily on this dual nature is introduced and analyzed in this communication. It proposes new ideas concerning the origin, the embryonic development, and the phylogenetic evolution of the mammalian cerebral cortex which differ somewhat from the classical conceptions of cortical development.This work has been supported by the National Institute of Child Health and Human Development Grant no. 09274.     

The role of pioneer neurons in the development of mouse visual cortex and corpus callosum

The primordial plexiform neuropil is very critical to neocortical development. The pioneer neurons, mainly Cajal-Retzius cells in the marginal zone, and subplate neurons in the subplate, differentiate from the primordial plexiform neuropil. In this study, the development of corpus callosum, visual cortex, and subcortical pathways has been observed in C57BL/6 mice with various methods, such as DiI labeling in vitro and in vivo, DiI and DiA in vitro double labeling, immunocytochemistry, and in vivo BrdU and Fast Blue labeling. As early as E14, the primordial plexiform neuropil can be found in the telencephalic wall, and it contains many pioneer neurons. On E15 the primordial plexiform neuropil differentiates into the marginal zone and the subplate. Cajal-Retzius cells exist in the marginal zone, and subplate neurons are in the subplate. Either Cajal-Retzius cells or subplate neurons have long projections toward the ganglionic eminence, suggesting that they migrate tangentially from the ganglionic eminence. Cajal-Retzius cells are involved in radial migration, and subplate neurons participate to guide pathfinding of subcortical pathways. This study reveals how the pioneer neurons, through radial and tangential migration, play an important role in neocortical formation and in the pathfinding of the corpus callosum and subcortical pathways. Furthermore, DiI labeling in vivo has demonstrated the presence of pioneer neurons all along the corpus callosum pathway, especially in the midline. This suggests that pioneer neurons may also play a role in guiding the pathfinding of the corpus callosum. Accepted: 31 July 2001     

A novel monoclonal antibody K1 recognises early neurones in the rat cortex

The aim of this study was to produce monoclonal antibodies specific for neurones that are generated earliest in the rat neocortex. One of the established clones, K1, showed a strong immunoreactivity in the marginal zone at the 19th day of gestation (E19). The immunoreactivity of K1 initially appeared in the primordial plexiform layer at E15, in the subplate at E16, and in the marginal zone by E17. It became undetectable in the first postnatal week. The immunoreactivity was not detected in the neocortex of adults or elderly. Western blotting analysis revealed reactive bands at positions corresponding to proteins of 290 and 280 kDa for the neocortical membrane fractions prepared from E16 to E21 embryos. In these stages, smears of bands were also found at positions corresponding to higher molecular weights. A single band of protein of 280 kDa was detected for the soluble fractions prepared from the embryos at E19 and E21. These reactivities were susceptible to protease, but not to enzymatic or chemical destruction of carbohydrate residues. Electron microscopic analysis showed that the K1 immunoreactivity was detected primarily on the cellular membranes of neurites. In the marginal zone at E19, the K1 immunoreactivity was localised where neurites make contact with other neurites or somata. A portion of these contact points had typical features of synapses. In the cortical plate of the same stage, arrays of tiny K1-immunoreactive puncta were observed on a subset of radial processes. These results suggest that monoclonal antibody K1 is a marker recognising neurites of subplate neurones that extend radially and make neuronal contacts in the marginal zone.     

Subplate in the developing cortex of mouse and human

The subplate is a largely transient zone containing precocious neurons involved in several key steps of cortical development. The majority of subplate neurons form a compact layer in mouse, but are dispersed throughout a much larger zone in the human. In rodent, subplate neurons are among the earliest born neocortical cells, whereas in primate, neurons are added to the subplate throughout cortical neurogenesis. Magnetic resonance imaging and histochemical studies show that the human subplate grows in size until the end of the second trimester. Previous microarray experiments in mice have shown several genes that are specifically expressed in the subplate layer of the rodent dorsal cortex. Here we examined the human subplate for some of these markers. In the human dorsal cortex, connective tissue growth factor‐positive neurons can be seen in the ventricular zone at 15–22 postconceptional weeks (PCW) (most at 17 PCW) and are present in the subplate at 22 PCW. The nuclear receptor‐related 1 protein is mostly expressed in the subplate in the dorsal cortex, but also in lower layer 6 in the lateral and perirhinal cortex, and can be detected from 12 PCW. Our results suggest that connective tissue growth factor‐ and nuclear receptor‐related 1‐positive cells are two distinct cell populations of the human subplate. Furthermore, our microarray analysis in rodent suggested that subplate neurons produce plasma proteins. Here we demonstrate that the human subplate also expresses α2zinc‐binding globulin and Alpha‐2‐Heremans‐Schmid glycoprotein/human fetuin. In addition, the established subplate neuron marker neuropeptide Y is expressed superficially, whereas potassium/chloride co‐transporter (KCC2)‐positive neurons are localized in the deep subplate at 16 PCW. These observations imply that the human subplate shares gene expression patterns with rodent, but is more compartmentalized into superficial and deep sublayers. This increased complexity of the human subplate may contribute to differential vulnerability in response to hypoxia/ischaemia across the depth of the cortex. Combining knowledge of cell‐type specific subplate gene expression with modern imaging methods will enable a better understanding of neuropathologies involving the subplate.     

Changing patterns of synaptic input to subplate and cortical plate during development of visual cortex.

1. The development of excitatory activation in the visual cortex was studied in fetal and neonatal cats. During fetal and neonatal life, the immature cerebral cortex (the cortical plate) is sandwiched between two synaptic zones: the marginal zone above, and an area just below the cortical plate, the subplate. The subplate is transient and disappears by approximately 2 mo postnatal. Here we have investigated whether the subplate and the cortical plate receive functional synaptic inputs in the fetus, and when the adultlike pattern of excitatory synaptic input to the cortical plate appears during development. 2. Extracellular field potential recording to electrical stimulation of the optic radiation was performed in slices of cerebral cortex maintained in vitro. Laminar profiles of field potentials were converted by the current-source density (CSD) method to identify the spatial and temporal distribution of neuronal excitation within the subplate and the cortical plate. 3. Between embryonic day 47 (E47) and postnatal day 28 (P28; birth, E65), age-related changes occur in the pattern of synaptic activation of neurons in the cortical plate and the subplate. Early in development, at E47, E57, and P0, short-latency (probably monosynaptic) excitation is most obvious in the subplate, and longer latency (presumably polysynaptic) excitation can be seen in the cortical plate. Synaptic excitation in the subplate is no longer apparent at P21 and P28, a time when cell migration is finally complete and the cortical layers have formed. By contrast, excitation in the cortical plate is prominent in postnatal animals, and the temporal and spatial pattern has changed. 4. The adultlike sequence of synaptic activation in the different cortical layers can be seen by P28. It differs from earlier ages in several respects. First, short-latency (probably monosynaptic) excitation can be detected in cortical layer 4. Second, multisynaptic, long-lasting activation is present in layers 2/3 and 5. 5. Our results show that the subplate zone, known from anatomic studies to be a synaptic neurophil during development, receives functional excitatory inputs from axons that course in the developing white matter. Because the only mature neurons present in this zone are the subplate neurons, we conclude that subplate neurons are the principal, if not the exclusive, recipients of this input. The results suggest further that the excitation in the subplate in turn is relayed to neurons of the cortical plate via axon collaterals of subplate neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prenatal development of the intrinsic neurons of the rat neocortex: a comparative study of the distribution of GABA-immunoreactive cells and the GABAA receptor.

The ontogenesis of cells showing GABA-like immunoreactivity, and the distribution of the immunoreactivity for the GABAA receptor were studied immunocytochemically in the prenatal rat brain. By embryonic day 14, a few GABA-like immunoreactive (GABA-positive) cells scattered at the subpial limit of the marginal zone (primordial plexiform layer) in the lateral part of the developing cortex. GABA-positive cells appeared progressively within the dorsal and medial sectors of the primordial plexiform layer, occupying deeper positions within the layer. The immunoreactivity for the GABAA receptor covered the whole thickness of the primordial plexiform layer. By embryonic day 16, most GABA-positive cells populated three distinct laminar compartments of the developing cortex: the prospective lamina I, the subplate, and the lower part of the intermediate zone. The GABA-positive cells of the lower intermediate zone appeared to be typical of the developing cerebral cortex of the rat: their neuronal nature was assessed immunocytochemically, using monoclonal antibodies against microtubule-associated protein 2, mainly expressed in neuronal somata and dendrites, and against intermediate filament protein vimentin, expressed in glia. The lower intermediate zone contained cells immunoreactive for microtubule-associated protein 2, although the immunostaining was less intense than in the prospective lamina I and the subplate. Preliminary results showed no vimentin-positive cells in the lower intermediate zone. At embryonic day 16, immunoreactivity for the GABAA receptor was present within the prospective lamina 1 and the subplate. Preliminary results showed no vimentin-positive cell in the lower intermediate zone. At embryonic day 16, immunoreactivity for the GABAA receptor was present within the prospective lamina 1 and the subplate, but not in the lower intermediate zone. From embryonic day 18 onwards, the immunostaining for the GABAA receptor labelled, unambiguously, the subplate as a lamina clearly separated from the suprajacent cortical plate. At embryonic day 18, the GABAA receptor started to be expressed within the lower, differentiating part of the cortical plate. Within the cortical plate, the expression of GABA in neural cell perikarya, and the immunostaining for the GABAA receptor, followed a similar spatio-temporal ("inside-out") gradient during pre- and early postnatal stages. Most GABA-positive cells of the lower intermediate zone started to disappear (or stopped the expression of GABA) by embryonic day 20, but some remained until adulthood. A similar time-course was observed for the microtubule-associated protein 2-immunoreactive cell population located at the same level.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of highly polysialylated NCAM in the neocortex and piriform cortex of the developing and the adult rat

Summary The expression of a highly polysialylated form of the neural cell adhesion molecule (NCAM-H) has been investigated in the neocortex and piriform cortex of the developing and the adult rat by using a monoclonal antibody 12E3, which has been found to recognize the polysialic acid portion of NCAM-H. Immunoblot analysis of the cortical homogenates showed that NCAM-H was temporarily expressed during the late embryonic and early postnatal stages. Further, immunohistochemical observations revealed that NCAM-H appeared at embryonic day 13 (E13) in the plexiform primordium in horizontally-oriented cells, probably Cajal-Retzius cells, which are the first neurons to differentiate. During the late embryonic stage, the marginal zone, subplate, and intermediate zone strongly stained, whereas the ventricular zone stained weakly. After birth, the NCAM-H expression was progressively attenuated from a week onwards, and almost vanished in the adult neocortex. In the primordium of the piriform cortex, NCAM-H immunoreactivity also became positive at E13. The time sequences of the NCAM-H expression in these neurons were similar to those of the neurons in the neocortical area. In the piriform cortex, however, the expression remained in a number of neurons in the layer II, which receives a large number of olfactory fibers from the olfactory bulb, where prolonged neurogenesis and construction of neural circuits take place in adulthood. These results suggest that NCAM-H not only plays an important role in the developing rat cortex, but also may be involved in some functions related to reorganization in the adult piriform cortex.     

Postnatal development of the telencephalon of the tammar wallaby (Macropus eugenii). An accessible model of neocortical differentiation

Summary The sequence of development of cell layers in the neocortex of the tammar has been followed from 24 days gestation to 213 days postnatal. The tammar is born at 27 days gestation and the major period of its development occurs during the subsequent 250 days, most of this time being spent within the pouch. Although the pattern of differentiation of the cell layers appears to resemble that described for many Eutherian mammals, the neocortex is at an embryonic 2 layered stage at birth and a cortical plate is not present throughout the telencephalon until 10–15 days postnatal. A transient subplate zone, presenting a characteristic appearance with widely spaced rows of cells aligned parallel to the cortical surface, develops between 20 and 70 days postnatal, but no secondary proliferative region is seen in the subventricular zone of the dorso-lateral wall.Preliminary experiments with (³H)-thymidine injections indicate that the cortical plate follows the inside-out pattern of development described in many Eutherian mammals and that the oldest neurons are found in the parallel cell rows of the subplate zone. The importance of the late differentiation of the neocortex in relation to the time of birth and the resulting usefulness of the tammar as an experimental model of cortical development is discussed.     

Fetuin as a marker of cortical plate cells in the fetal cow neocortex: a comparison of the distribution of fetuin, alpha 2HS-glycoprotein, alpha-fetoprotein and albumin during early development

Summary Fetuin, ₂HS-glycoprotein (₂HS), -fetoprotein (AFP) and albumin have been shown to be present in some regions of the neocortex in two early stages of development of the cow brain using PAP immunocytochemistry. In the pre-cortical plate stage fibres of the primordial plexiform layer stained positively for fetuin. No staining was seen for albumin but plasma and cerebrospinal fluid (CSF) were positive for ₂HS and AFP. In the early cortical plate stage the strongest fetuin positive staining was seen in the earliest formed cells of the plate. ₂HS staining was much less intense but similar in distribution. The possible role of fetuin, or related glycoproteins, in cortical plate differentiation is discussed. Staining for AFP and for albumin was seen mainly in the ventricular zone and marginal zone fibres, and had a similar distribution and intensity for both proteins. Plasma and CSF stained for all four proteins. Tests showed some cross-reactivity between fetuin and anti-₂HS and, to a much lesser extent, between antisera to AFP and albumin and antigens denatured by fixation.     

Dynamical aspects of neocortical histogenesis in the rat

Summary The formation and transformation of embryonic neocortical cell layers has been studied by means of autoradiographic and morphometric methods in the light of the concept of a dual origin of mammalian neocortex (Marin-Padilla, 1978; Raedler and Raedler, 1978).A primordial plexiform layer begins to form on ED 13, when the first postmitotic preneurons enter the area between ventricular layer and pial basement membrane. Before this stage the area was occupied by single fibres and processes of the ventricular cells only. The further development of the primordial plexiform layer is characterized by a linear increase in width, mainly due to the addition of migrating preneurons and ingrowing fibre bundles. The older preneurons of the primordial plexiform layer are situated outermost in the layer, the younger ones at its inner circumference. The differentiation accordingly follows an outside — inside gradient. The preneurons of this layer are designed to become either the Cajal-Retzius cells of Layer I or subcortical neurons (Layer VII) (Rickmann et al., 1977).While the mitotic activity of cells in the ventricular layer decreases only gradually during the course of neurogenesis, the thickness of the ventricular layer (in the lateral part of neocortex) decreases dramatically from ED 16 on. At the same time the cortical plate starts to form. This is brought about by a translocation of ultrastructurally rather undifferentiated ventricular daughter cells from the inner to the outer circumference of the cerebral wall. The width of the cortical plate grows almost exponentially, especially its medial part. Its development is characterized by the addition of preneurons on its outer circumference while maturation takes place at its inner border.The significance of these developmental features with regard to neocortical histogenesis is discussed.     

Developmental profile of a fetuin-like glycoprotein in neocortex, cerebrospinal fluid and plasma of post-natal tammar wallaby (Macropus eugenii).

Summary A fetuin-like glycoprotein (FLG) has been shown to be present in early cortical plate cells in the developing brain of the tammar wallaby (Macropus eugenii). The developmental sequence of the occurrence of glycoprotein-positive fibres and cells in the dorsolateral telencephalic wall from newborn to day 40 is described. The level of FLG in CSF (cerebrospinal fluid) and plasma of the tammar wallaby has also been measured during pouch life. The presence of FLG in early postnatal fibre systems and in some cells in the primordial plexiform layer, as well as in early cortical plate cells of the tammar is similar to that of fetuin in fetal brain in sheep, pig and cow, and ₂HS glycoprotein in human fetal brain. The sequence of appearance of FLG-positive cells during neocortical development in the tammar is strikingly similar to that of a transient population of early cortical plate cells previously described in fetal cat and sheep cortex. During postnatal development, levels of FLG in tammar plasma and CSF follow a pattern different from that of other species. The developmental expression of all three related glycoproteins in their respective species is discussed.     

On the development of non-pyramidal neurons and axons outside the cortical plate: The early marginal zone as a pallial anlage

Summary The development of non-pyramidal neurons was studied in the pallium of albino rats using autoradiography after thymidine labelling (determination of birth dates), Golgi impregnations (differentiation of dendrites and axons) and electron microscopy including 3D-reconstructions (cytoplasmic differentiation and early synaptogenesis).The marginal zone appears between E13 and E14 and contains glial cells, axons and preneurons from the beginning. The latter can be identified by structural criteria (contacts, cytoplasm, nuclei). The first vertically oriented pyramidal neurons (cortical plate) appear within the marginal zone not before E16, separating its contents into a superficial (lamina I) and a deep portion (intermediate and subventricular zone). Since this old neuronal population of lamina I and the subcortical pallial region can be followed until adulthood, it is proposed to call the early marginal zone a pallial anlage. It can be demonstrated that during the whole period of neuron production (until E21) non-pyramidal neurons are added to all parts of the pallial anlage.The structural differentiation of non-plate neurons is described. Neurons form specific, desmosome-like contacts with axonal growth cones already on E14. Typical synapses (vesicle aggregations) have been observed two days later. In lamina I two types of neurons develop: horizontal neurons (Cajal-Retzius cells) and multipolar neurons (small spiny stellate cells). Subcortical pallial neurons retain mostly their clear horizontal orientation. Only neurons situated very close to the lower border of the cortex show dendritic branches extending into lamina VI. Axons appearing early in the neocortex originate not only from subcortical regions, but also from neurons of the paleopallium, the archicortex, the limbic cortex and the neighbouring neocortex. The tangential growth of the neocortex, as estimated from E14 onwards causes a strong dilution of the elements of the pallial anlage until adulthood.The classification of neurons outside the cortical plate and the fate of the total pallial anlage are discussed. As a consequence of these observations some modifications of the terminology of the Boulder Committee are proposed.     

The early development of thalamocortical and corticothalamic projections in the mouse

The initial ingrowth of corticothalamic and thalamocortical projections was examined in mice at embryonic and perinatal stages. Fibers, in fixed brains, were labeled with the carbocyanine dye 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocianine perchlorate (DiI). By E13, the corticofugal fibers had entered the lowest intermediate zone through which they ran, turned over the corpus striatum, and left the cortex. The fibers were arranged in scattered bundles throughout the corpus striatum. At E14 corticofugal axons reached the internal capsule and at E14.5–E15 they established contact within the thalamus. Meanwhile, the thalamocortical afferents reached the neocortex at E13. At this time fibers ran tangentially within the intermediate zone, immediately underneath the cortical plate. By E14, the fibers had started to invade the subplate and, by E15, thalamocortical fibers had begun their radial growth into the cortex. Such radial growth proceeded steadily, invading each cortical layer as it differentiated cytoarchitectonically from the dense cortical plate. The first retrogradely labeled cells were detected at the cortical plate at E15. By the day of birth (E20), thalamocortical fibers had formed a dense branching system within layers VI and V. Our observations indicate that, in mice, the thalamic axons reach the cortex before corticothalamic projections enter the thalamic nuclei. Moreover, the results suggest that the pathway followed by each fiber system is different. By DiI injections into the internal capsule we have also determined that subplate cells are the first to send axons to the thalamus. Accepted: 11 August 1999     

Corticothalamic and thalamocortical pathfinding in the mouse: dependence on intermediate targets and guidance axis

Recently, increasing attention has been paid to the study of intermediate targets and their relay guidance role in long-range pathfinding. In the present study, mechanisms of corticothalamic and thalamocortical pathfinding were investigated in C57BL/6 mice using in vitro DiI labeling and in vivo cholera toxin labeling. Specifically, three important intermediate targets, the subplate, ganglionic eminence, and reticular thalamic nucleus, were studied for their role in corticothalamic and thalamocortical pathfinding. The results show that the neuroepithelium of the ganglionic eminence is a source of pioneer neurons and pioneer fibers. Through radial and tangential migration, these pioneer neurons and fibers can approach the differentiating field of the ganglionic eminence, the subplate and thalamic reticular nucleus to participate in the formation of these three intermediate targets. Furthermore, the subplate, ganglionic eminence and thalamic reticular nucleus are linked by pioneer neurons and fibers to form a guidance axis. The guidance axis and the three important intermediate targets provide an ideal environment of contact guidance and chemical guidance for the corticothalamic and thalamocortical pathfinding. The concept of a "waiting time" in the subplate and the thalamic reticular nucleus is likely due to the expression of a guidance effect, so that the thalamocortical and corticothalamic projections can be deployed spatially and temporally to the subplate and thalamic reticular nucleus before these projections enter their final destinations, the neocortex and thalamus.Abbreviations CLSM confocal laser scanning microscope - CP cortical plate - DF differentiating field - E embryonic day - GE ganglionic eminence - IC internal capsule - IZ intermediate zone - MZ marginal zone - NP neuroepithelium - P postnatal day - PB phosphate buffer - PBS phosphate-buffered saline - Po posterior group nucleus - Pr5 principle sensory trigeminal nucleus - SI somatosensory cortex - SP subplate - SZ subventricular zone - RT reticular thalamic nucleus - V ventricle - VPM ventral posterior medial nucleus - VZ ventricular zone - WGA wheat germ agglutinin - WM white matter     

Identification of molecules preferentially expressed beneath the marginal zone in the developing cerebral cortex

During cerebral cortical development, the majority of excitatory neurons are born near the ventricle and migrate radially toward the marginal zone (MZ). Since the cells invariably stop migrating beneath the MZ, neurons are aligned in an "inside-out" manner in the cortical plate (CP); that is, the early-born and late-born neurons are ultimately positioned in the deep and superficial layers, respectively. Since dramatic morphological changes occur in cells beneath the MZ, several events critical for proper neuronal maturation and layer formation must take place. In this study, we screened for molecules strongly expressed beneath the MZ, and identified 28 genes that are preferentially expressed in the upper half of the mouse CP on both embryonic day (E) 16.5 and E18.5. Expression analyses in reeler and yotari mice, in which neurons terminate migration throughout the CP, suggested that these genes were indeed related to the events beneath the MZ rather than unrelatedly induced by the structures near the brain surface. Pathway analyses suggested calcium signaling to have an important role in cells beneath the MZ. The gene list presented here will be useful for clarifying the molecular mechanisms that control cortical development.