Development of layer I and the subplate in the rat neocortex

Development of layer I and the subplate of the rat neocortex was examined with [3H]thymidine autoradiography. The experimental animals used for neurogenesis were the offspring of pregnant females injected with [3H]thymidine on 2 consecutive days: Embryonic Day (E) 13-E14, E14-E15, . . . E21-E22, respectively. On Postnatal Day 5, the proportion of layer I and subplate cells originating during 24-h periods were quantified at three anteroposterior levels. Presumptive Cajal-Retzius cells (large horizontal cells) are generated mainly on E14 and subplate cells on E14 and E15 ("outside-in" gradient). Both populations are generated earlier than cells in the cortical plate, which has an "inside-out" gradient. The subplate also has a ventrolateral/older to dorsomedial/younger neurogenetic gradient. The small- to medium-sized horizontal cells in layer I have an extensive period of neurogenesis with an "outside-in" gradient. To study morphogenesis, pregnant females were given single injections of [3H]-thymidine during gestation and embryos were removed in successive 24-h intervals (sequential-survival). On E15 and E16, cells accumulate outside the neuroepithelium in the primordial plexiform layer with older presumptive Cajal-Retzius cells superficial and younger presumptive subplate cells deep. The Cajal-Retzius cells permanently settle superficially among a first system of extracellular channels that appears on E14. Before reaching their final settling sites, subplate cells form the incipient cortical plate in the ventrolateral neocortex on E16. On E17, a seocnd system of extracellular channels appears below the cortical plate. On E18 and E19, subplate cells leave the cortical plate and permanently settle among the deep extracellular channels in a separate layer.     

Origin of the cortical layer I in rodents

Using birthdating techniques, we have studied when cells that settle in the marginal zone (future layer 1) of the cortical neuroepithelium are generated in developing rat embryos. The majority of marginal zone cells are generated at embryonic day 12 (E12), E13 and E14, although some cells generated later can incorporate into this stratum after the cortical plate forms. The nature and the origin of the cell populations that colonize the preplate/marginal zone was studied by means of immunohistochemistry using cell markers for gamma-amino butyric acid (GABA), reelin and the calcium binding proteins calretinin and calbindin. At early stages of development, the preplate is formed by Cajal-Retzius cells, subplate cells, subpial granular layer cells, some interneurons and some glial cells. With the arrival of the cortical plate cells, the subplate cells descend to occupy the stratum below. Layer 1 cells are of diverse origin as some of them are generated in the ventricular zone of the cortical neuroepithelium, whereas other cell populations come from extracortical regions such as the olfactory placode or the ganglionic eminences of the basal telencephalon. The predominant cell type in the marginal zone is the Cajal-Retzius cell, which expresses reelin and calretinin, and is probably generated in the cortical neuroepithelium. These cells can be readily distinguished from cells that come from the ganglionic eminences as these later populations mainly express GABA and calbindin. Finally, our results suggest that the cells of the subpial granular layer might be generated in the rostral pole of the lateral ganglionic eminences.     

GABAergic projections from the subplate to Cajal-Retzius cells in the neocortex

Subplate neurons and Cajal-Retzius cells play an important role in the corticogenesis. Despite morphological evidence, the question whether subplate neurons innervate Cajal-Retzius cells has not been studied yet. We report that electrical stimulation in the subplate resulted in evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in Cajal-Retzius cells. The eIPSC latency showed minor variability and amounted to approximately 4 ms, suggesting the monosynaptic connection. During the first postnatal week: (i) eIPSC amplitude increased, (ii) eIPSC kinetics sped up, (iii) the size of readily releasable pool increased, and (iv) γ-aminobutyric acid release probability decreased. We conclude that GABAergic subplate neurons innervate Cajal-Retzius cells. Surprisingly, despite the transient nature of both cell populations, these projections show developmental adjustments typical for many nontransient synaptic connections.     

Organization of the embryonic and early postnatal murine hippocampus. I. Immunocytochemical characterization of neuronal populations in the subplate and marginal zone

Immunocytochemical techniques were used to characterize the neuronal populations in the hippocampal subplate and marginal zone from embryonic day 13 (E13) to postnatal day 5 (P5). Sections were processed for the visualization of microtubule-associated protein 2 (MAP2) and other antigens such as neurotransmitters, neuropeptides, calcium-binding proteins and a synaptic antigen (Mab SMI81). At E13–E14, only the ventricular zone and the primitive plexiform layer were recognized. Some cells in the later stratum displayed MAP2-, γ-aminobutyric acid (GABA)-and calretinin immunoreactivities. From E15 onwards, the hippocampal and dentate plates became visible. Neurons in the plexiform layers were immunoreactive at E15–E16, whereas the hippocampal and dentate plates showed immunostaining two or three days later. Between E15 and E19 the following populations were distinguished in the plexiform layers: the subventricular zone displayed small neurons that reacted with MAP2 and GABA antibodies; the subplate (prospective stratum oriens) was poorly populated by MAP2- and GABA-positive cells; the inner marginal zone (future stratum radiatum) was heavily populated by multipolar GABAergic cells; the outer marginal zone (stratum lacunosum-moleculare) displayed horizontal neurons that showed glutamate- and calretinin immunoreactivities, their morphology being reminiscent of neocortical Cajal-Retzius cells. Thus, each plexiform layer was populated by a characteristic neuronal population whose distribution did not overlap. Similar segregated neuronal populations were also found in the developing dentate gyrus. At perintal stages, small numbers of neurons in the plexiform layers began to express calbindin D-28K and neuropeptides. During early postnatal stages, neurons in the subplate and inner marginal zones were transformed into resident cells of the stratum oriens and radiatum, respectively. In contrast, calretinin-positive neurons in the stratum lacunosum-moleculare disappeared at postnatal stages. At E15–E19, SMI81-immunoreactive fibers were observed in the developing white matter, subplate and outer marginal zone, which suggests that these layers are sites of early synaptogenesis. At PO-P5, SMI81 immunoreactivity became homogeneously distributed within the hippocampal layers. The present results show that neurons in the hippocampal subplate and marginal zones have a more precocious morphological and neurochemical differentiation than the neurons residing in the principal cell layers. It is suggested that these early maturing neurons may have a role in the targeting of hippocampal afferents, as subplate cells do in the developing neocortex. © 1994 Wiley-Liss, Inc.     

Evidence for tangential migration disturbances in human lissencephaly resulting from a defect in LIS1, DCX and ARX genes

During corticogenesis, neurons adopt different migration pathways to reach their final position. The precursors of pyramidal neurons migrate radially, whereas most of the GABA-containing interneurons are generated in the ventral telencephalon and migrate tangentially into the neocortex. Then, they use a radial migration mode to establish themselves in an inside-out manner in the neocortex, similarly to pyramidal neurons. In humans, the most severe defects in radial migration result in lissencephaly. Lately, a few studies suggested that lissencephaly was also associated with tangential neuronal migration deficits. In the present report, we investigated potential anomalies of this migration mode in three agyric/pachygyric syndromes due to defects in the LIS1, DCX and ARX genes. Immunohistochemistry was performed on paraffin-embedded supra- and infratentorial structures using calretinin, calbindin and parvalbumin antisera. The results were compared with age-matched control brain tissue. In the Miller–Dieker syndrome, GABAergic neurons were found both in upper layers of the cortex and in heterotopic positions in the intermediate zone and in ganglionic eminences. In the DCX mutant brain, few interneurons were dispersed in the cortical plate, with a massive accumulation in the intermediate zone and subventricular zone as well as in the ganglionic eminences. In the ARX-mutated brain, the cortical plate contained almost exclusively pyramidal cells and was devoid of interneurons. The ganglionic eminences and basal ganglia were poorly cellular, suggesting an interneuron production and/or differentiation defect. These data argue for different mechanisms of telencephalic tangential migration impairment in these three agyric/pachygyric syndromes.     

Ephrin-A5 acts as a repulsive cue for migrating cortical interneurons

Cortical interneurons are born in the germinative zones of the ganglionic eminences in the subpallium, and migrate tangentially in spatially and temporally well-defined corridors into the neocortex. Because ephrin-A5 is expressed in the ventricular zone (VZ) of the ganglionic eminences at these developmental stages, we examined the possible effects of this molecule on interneuron migration. Double-immunocytochemistry of dissociated neurons from the medial ganglionic eminences (MGE) revealed that calbindin-positive cells express the EphA4-receptor. In situ , EphA4 is strongly expressed in the subventricular zone of the ganglionic eminences. Using different in vitro assays, we found that ephrin-A5 acts as a repellent cue for MGE neurons. We then examined interneuron migration in slice overlay experiments, where MGE-derived explants from enhanced green fluorescent protein-expressing transgenic mice were homotopically grafted into host slices from wild-type littermate embryos. In these in vitro preparations, interneurons recapitulated in vivo cell migration in several respects. However, interneurons in brain slices also migrated in the VZ of the ganglionic eminences, a region that is strictly avoided in vivo . In situ hybridizations revealed that ephrin-A5 became downregulated in the VZ in vitro . When recombinant ephrin-A5-Fc was added to the slices, it preferentially bound to the VZ, and migrating MGE neurons avoided the VZ as in vivo . The restoration of the normal migration pathway in slices required ephrin-A5 clustering and signalling of Src family kinases. Together, these experiments suggest that ephrin-A5 acts as an inhibitory flank that contributes to define the pathway of migrating interneurons.     

Development of the lateral and medial limbic cortices in the rat in relation to cortical phylogeny

[3H]Thymidine autoradiography was used to investigate neurogenesis of the lateral limbic cortex and morphogenesis of the medial and lateral limbic cortices in adult and embryonic rat brains. Ontogenetic patterns in the limbic cortex are unique because some neurogenetic gradients are linked to those in neocortex, others are linked to those in paleocortex. These findings are related to hypotheses of cortical phylogeny. The experimental animals used for neurogenesis were the offspring of pregnant females injected with [3H]thymidine on 2 consecutive days: Embryonic Day (E) 13-E14, E14-E15, ...E21-E22, respectively. On Postnatal Day (P)60, the proportion of neurons originating during 24-h periods were quantified at nine anteroposterior levels and one sagittal level. Similar to neocortex, deep cells are generated earlier than superficial cells throughout the lateral limbic cortex: layer VI mainly on E14-15, layer V on E15-E16, and layers IV-II on E16-E18. There is a ventral/older to dorsal/younger neurogenetic gradient between the ventral agranular insular, dorsal agranular insular, and gustatory cortical areas and between ventral and dorsal orbital areas beneath the frontal pole. Similar to paleocortex below the rhinal sulcus, limbic cortex in the rhinal sulcus has a "sandwich" gradient: the older posterior agranular insular area is sandwiched by anterior and posterior younger areas (ventral agranular insular and perirhinal). To study morphogenesis, pregnant females were given single injections of [3H]thymidine during gestation and embryos were removed in successive 24-h intervals (sequential-survival). Neurogenesis finishes first in ventral limbic areas, later in dorsal limbic areas, and latest in neocortical areas. The cortical plate in the region of the medial and lateral limbic cortices does not have a separate subplate layer as is found in the region of the neocortex. Instead, layer VI in the limbic cortices has unusually older cells that are generated simultaneously with subplate cells.     

Expression and distribution of metabotropic GABA receptor subtypes GABABR1 and GABABR2 during rat neocortical development

To understand the possible contribution of metabotropic gamma-aminobutyric acid receptors (GABABR) in cortical development, we investigated the expression pattern and the cellular and subcellular localization of the GABABR1 and GABABR2 subtypes in the rat neocortex from embryonic day 14 (E14) to adulthood. At the light microscopic level, both GABABR1 and GABABR2 were detected as early as E14. During prenatal development, both subtypes were expressed highly in the cortical plate. Using double immunofluorescence, GABABR1 colocalized with GABABR2 in neurons of the marginal zone and subplate, indicating that these proteins are coexpressed and could be forming functional GABABRs during prenatal development in vivo. In contrast, only GABABR1 but not GABABR2 was detected in the tangentially migratory cells in the lower intermediate zone. During postnatal development, immunoreactivity for GABABR1 and GABABR2 was distributed mainly in pyramidal cells. Discrete GABABR1-immunopositive cell bodies of interneurons were present throughout the neocortex. In addition, GABABR1 but not GABABR2 was found in identified Cajal-Retzius cells in layer I. At the electron microscopic level, immunoreactivity for GABABR1 and GABABR2 was found in dendritic spines and dendritic shafts at extrasynaptic and perisynaptic sites throughout postnatal development. We further demonstrated the presynaptic localization of GABABR1 and GABABR2, as well as the association of the receptors with asymmetrical synaptic junctions. These results indicate potentially important roles for the GABABRs in the regulation of migratory processes during corticogenesis and in the modulation of synaptic transmission during early development of cortical circuitry.     

Microtubule-associated protein 2 (MAP 2) immunoreactivity in human fetal neocortex

We used a monoclonal antibody to study the immunocytochemical distribution of microtubule-associated protein 2 (MAP 2) in human fetal neocortex between the ages of 16 and 22 weeks gestation. The staining pattern was lamina-specific. Neuronal somata and dendrites in all cortical layers and in the intermediate zone were labelled. Cajal-Retzius cells of layer I, large pyramidal neurons in the inner cortical plate and neurons in the subplate were most strongly immunoreactive. Separate from the underlying cortical plate a thin sheet of small neurons in the inner marginal zone was highlighted by MAP 2 immunoreactivity. The morphologic diversity, density and regional distribution of the interstitial neurons in the subplate was emphasized by MAP 2 staining. In general, the intensity of MAP 2 immunoreactivity in cell somata and dendrites correlated with the degree of neuronal differentiation but the pattern of intracellular staining also varied as a function of laminar position, and presumably cell type.     

Cellular physiology of the neonatal rat cerebral cortex: intrinsic membrane properties, sodium and calcium currents

The cellular physiology of the primary somatosensory cortex was studied in postnatal day (P) 0 to P5 rats using whole-cell patch-clamp recordings. Visually identified Cajal-Retzius, subplate, bifurcated pyramidal, and immature, putatively migrating neurons showed resting membrane potentials between -44 and -50 mV and TTX-sensitive action potentials. Immature pyramidal neurons with the smallest surface area ( approximately 1,600 microm(2)) revealed the largest input resistance ( approximately 1.8 GOmega), and subplate cells with the largest surface area ( approximately 6,200 microm(2)) showed an input resistance of approximately 1 GOmega. Ontogenetically older Cajal-Retzius and subplate cells revealed shorter and larger action potentials compared to bifurcated and immature pyramidal neurons. Whereas Cajal-Retzius and subplate cells responded to injection of depolarizing current pulses with a repetitive nonadapting and fast spiking firing pattern, immature pyramidal neurons showed strong adaptation. Subplate cells revealed the fastest action potentials, largest sodium current amplitude (-714 pA), and highest sodium current density (-38 microA/cm(2)), enabling these cells to transmit afferent activity faithfully to postsynaptic neurons. Whereas all cell types expressed a high-voltage-activated (HVA) calcium current, none of them showed a significant low-voltage-activated calcium current. The largest peak (-25.5 microA/cm(2)) and steady-state (-7.6 microA/cm(2)) HVA calcium current density could be observed in immature presumed migrating neurons. In contrast, Cajal-Retzius and subplate neurons showed a significantly lower peak (-4.9 microA/cm(2)) and steady-state (<-3.3 microA/cm(2)) HVA calcium current density. Whereas a large HVA calcium current may promote neuronal migration of immature neurons, low intracellular calcium levels may provoke apoptosis in Cajal-Retzius and subplate cells.     

Prenatal development of neurons in the human prefrontal cortex: I. A qualitative Golgi study

Golgi-Stensaas and rapid-Golgi staining techniques are used to study neuronal differentiation in the developing human prefrontal cortex in fetuses, premature infants, and full-term newborns from 10.5 to 40 weeks of gestation. Horizontal neurons (Cajal-Retzius neurons) above the cortical plate (in the marginal zone) and randomly oriented neurons below the cortical plate (in the primordial subplate) are more differentiated than the immature bipolar cortical plate neurons in the 10.5-week fetus. During 13.5-15 weeks of gestation the fetal subplate zone can be clearly distinguished-between the cortical plate and the intermediate zone. This subplate zone contains more mature neurons than the cortical plate, especially polymorphous neurons. The basic features of the apical and basal dendrites of pyramidal neurons develop between 17 and 25 weeks of gestation, before the thalamocortical fibres invade the cortical plate. Intensive differentiation of the subplate neurons occurs in this period, when various types of afferent fibres reside in the subplate zone. At least five neuronal types can be distinguished in the subplate, i.e., polymorphous, fusiform, multipolar, normal, and inverted pyramidal neurons. The ingrowth of afferent fibres into the cortical plate between 26 and 34 weeks of gestation coincides with intensive dendritic differentiation and the appearance of spines on dendrites of the prospective layer III and V pyramidal neurons as well as with the differentiation of the double bouquet interneurons in the prospective supragranular layers and layer IV. Multipolar nonpyramidal neurons with the dendritic features of basket neurons are observed between 32 and 34 weeks of gestation in future layer V. They are less differentiated than the double bouquet neurons. The neurons of the subplate zone continue their dendritic differentiation after 26/27 weeks of gestation and are still observed in the full-term newborn. The axonal pattern of the subplate neurons suggests a possible functional role for them as either interneurons or projection neurons.     

Development of GABA-immunoreactivity in the neocortex of the mouse.

The prenatal and postnatal development of GABAergic elements in the neocortex of the mouse was analyzed by GABA-immunocytochemistry. Radial distribution of cells and laminar numerical densities were calculated at each developmental stage to substantiate qualitative observations. The first immunoreactive neurons were observed in the cortical anlage at embryonic day 12-embryonic day 13 (E12-E13) in the primitive plexiform layer. At following prenatal stages (E14-E19), most GABA-positive neurons were present in the marginal zone, subplate, and subventricular zone. GABA-immunoreactivity in the cortical plate appeared early (E14), although the complete maturation of its derivatives was achieved postnatally. At prenatal stages we noted a well-developed system of immunopositive fibers in the subplate. As indicated by the direction of growth cones, most of these fibers had an extracortical origin and invaded the cortex laterally through the internal capsule and striatum. In rostral and middle telencephalic levels, fibers originating in the septal region contributed to the cingulate bundle. Presumably corticofugal fibers and callosal axons were also noticed. At postnatal stages the maturation of GABA-immunoreactivity appeared to be a complex, long-lasting process, in which the adult pattern was produced at the same time as the appearance of certain regressive phenomena. Thus, between postnatal day 0 and postnatal day 8 (P0-P8), GABA-positive populations disappeared from the subventricular zone, marginal zone and to a lesser extent from the subplate. At the same ages we noticed the presence of morphologically abnormal, GABA-immunoreactive neurons in the subventricular zone and subplate which are interpreted as correlates of neuronal degeneration. Most GABA-positive subplate fibers also disappeared whereas GABA-immunoreactive axons were seen in the cingulate bundle until the adult stage. In the derivatives of the cortical plate, the maturation of GABA-immunoreactive elements progressed according to the "inside-out" gradient of cortical development, with the important exception of layer IV, which was the last layer to exhibit an adult-like appearance. Within each layer deriving from the cortical plate (layers VIa to II-III), GABA-immunoreactivity showed a protracted maturation in which the first GABA-positive cells were detected a few days after cell birth but substantial numbers of neurons began to express GABA considerably later. The later phase occurred concurrently with the maturation of GABA-positive axonal plexuses. These results suggest that different GABA-positive populations show different developmental regulation of GABA expression during cortical ontogenesis.     

Localization and development of nerve growth factor-sensitive rat basal forebrain neurons and their afferent projections to hippocampus and neocortex

In order to understand further the role of NGF in the development of NGF-sensitive basal forebrain neurons and their afferent connections to the hippocampus and neocortex, we have used monoclonal antibody 192 IgG to detect and localize NGF receptors immunocytochemically in the developing rat brain. NGF receptor immunoreactivity (NGF-RI) is first visible at embryonic day 13 (E 13) in the ventrolateral telencephalic wall and follows a caudal-to-rostral gradient in its initial appearance. NGF-RI neuronal number and neuropil staining undergo substantial increases before birth, and extensive dendritic growth and increases in perikaryal size continue during the first 3 weeks of postnatal life. This growth and cellular differentiation, however, is followed in the fourth postnatal week and later by an apparent decrease in dendritic arborization and 50% shrinkage in the size of perikarya. Initial NGF-RI fiber outgrowth from immature basal forebrain neurons directed toward appropriate target fields is observed as early as E 15. The formation of a laminar pattern by septal axons in the hippocampal terminal fields and invasion of NB afferents into the cortex occur postnatally over a protracted time. In the hippocampus, NGF-RI is initially diffusely distributed, and wide bands of immature granule and pyramidal cells are almost devoid of immunoreactive fibers; however, with maturity, septal axon terminals become concentrated in narrow zones closely associated with the cellular layers. In the neocortex, early-arriving basal forebrain afferents accumulate in the intermediate zone underneath the darkly immunoreactive subplate before they enter The cortex. Dense subplate and transiently present, radially aligned fiber staining completely disappear in later postnatal week and are gradually replaced by specific axonal and terminal staining associated with NB afferents. The expression of NGF receptor in the subplate zone at the time afferents arrive and its subsequent disappearance with the specific terminal formation suggest that NGF receptor and concomitant accumulation of NGF in the subplate may act as a temporary target for the early-arriving basal forebrain afferents; ingrowing afferents may then be guided by radially oriented NGF-RI fibers to proper synaptic sites.     

Aberrant trajectory of thalamocortical axons associated with abnormal localization of neurocan immunoreactivity in the cerebral neocortex of reeler mutant mice

We examined the molecular mechanisms underlying the formation of the thalamocortical pathway in the cerebral neocortex of normal and reeler mutant mice. During normal development of the mouse neocortex, thalamic axons immunoreactive for the neural cell adhesion molecule L1 rarely invaded the cortical plate and ran centered in the subplate which is immunoreactive for neurocan, a brain-specific chondroitin sulfate proteoglycan. On the other hand, in homozygous reeler mutant mice, thalamic axons took an aberrant course to run obliquely through the cortical plate. Injection of bromodeoxyuridine at embryonic day 11 specifically labeled subplate neurons in normal mice, whilst in the reeler neocortex it labeled cells scattered in the cortical plate as well as in the superficial layer (superplate). Neurocan immunoreactivity was associated with the bromodeoxyuridine-positive cells in the superplate, as well as being present in oblique bands within the cortical plate, along which L1-bearing thalamic axons preferentially ran. The present results support our previous hypothesis proposed for normal rats that a heterophilic molecular interaction between L1 and neurocan is involved in determining the thalamocortical pathway within the neocortical anlage [T. Fukuda et al. (1997) Journal of Comparative Neurology, 382, 141-152].     

Ontogenesis of microtubule-associated protein 2 (MAP2) in embryonic mouse cortex

The developing neocortex in mice from embryonic day 13 (E13) until birth (E19) was immunoreacted with a monoclonal antibody for microtubule-associated protein 2 (MAP2) that is highly specific for neuronal somata and dendrites. In E13 neocortex there was no detectable MAP2 immunoreactivity on tissue sections or on gel blots. From E14 to birth the MAP2 immunoreactivity was present in both tissue sections and immunoblots of homogenized cortex. In the neocortex the staining pattern was lamina-specific. The molecular layer and the cortical subplate contained the most dense staining of dendrites and cell somata. The cortical plate showed weak to moderate staining at these ages while the intermediate and ventricular zones were not stained above background control levels. Gel blots correspondingly did not show detectable levels of MAP2 until E14. Ultrastructural data suggest that MAP2 is present in dendrites in each of the laminae. The laminar pattern of MAP2 immunoreactivity may be due to either the higher density of differentiating dendrites in the molecular and subplate layers or to compartmentalization of MAP2 within individual cortical neurons.