Developmental History of the Subplate and Developing White Matter in the Murine Neocortex. Neuronal Organization and Relationship with the Main Afferent Systems at Embryonic and Perinatal Stages

José Antonio Del Río, Albert Martínez, Carme Auladell and Eduardo Soriano

Department of Cell Biology, Faculty of Biology and Neuroscience Research Center, (C.E.R.N.), University of Barcelona, E-08028 Barcelona, Spain


    Abstract
 Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Notes
 References
 
The neuronal diversity of the subplate and developing white matter in the mouse was studied using a variety of neuronal markers. The subplate was first visible in lateral cortical areas at E13, coinciding with the emergence of the cortical plate. During prenatal development, this layer was formed by morphologically heterogeneous neurons, subsets of which were immunoreactive for GABA- and calcium-binding proteins. From E18 onwards, a few subplate cells also contained neuropeptides. Colocalization experiments demonstrated that the percentages of neurons immunoreactive for each antigen were similar to those described in adult neocortex. By E15, subplate cells had received synaptic contacts. Moreover, a second early-neuronal population was conspicuous from E13 in the lower intermediate zone: the intermediate–subventricular population. Unlike subplate cells, these neurons were morphologically uniform, smaller and horizontally oriented. Nevertheless, a few of these cells also appeared within the ventricular zone, with a perpendicular/ oblique orientation. Most of these cells were GABA-positive and showed calbindin immunoreactivity. At the electron microscopic level, no synaptic contacts were found in these neurons. Tracing studies using DiI showed that subplate neurons were the first to send axons outside the neocortex towards the ganglionic eminence at E13. At E14, subplate axons and ingrowing thalamic fibers met in the striate primordium. Subplate cells retained their projection to the thalamus during prenatal development. Thalamocortical axons reached the subplate at E15, and 1 day later began to invade the upper cortical layers. Early callosal axons, in contrast, did not run through the subplate to reach the contralateral hemisphere, nor did subplate cells send out callosal fibers. Callosal axons ran just above the subventricular zone, intermingled with the intermediate–subventricular neuronal population. We conclude that the subplate neuronal population has a chemical heterogeneity reminiscent of that of the adult cortex and is crucial to the establishment of thalamocortical relationships, whereas the intermediate–subventricular neurons constituted a particular GABAergic population, which includes resident cells and tangentially migrating postmitotic neurons spatially related to the development of callosal connections.


    Introduction
 Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Notes
 References
 
During neocortical histogenesis, neuroblasts destined to form cortical layers II–VI leave the proliferative neuroepithelium and are incorporated into the cell-dense cortical plate following an ‘inside-out’ gradient of positioning (Angevine and Sidman, 1961Go; Rakic, 1974Go; Bayer and Altman, 1991Go). The cortical plate emerges and matures sandwiched between the subplate and the marginal zone, both of which contain the earliest-generated cortical neurons (Marín-Padilla, 1970Go, 1971Go, 1984Go, 1988Go). This developmental sequence suggests that the subplate and the marginal zone might have key roles in the development of the cerebral cortex (Marín-Padilla, 1971Go, 1978Go, 1988Go; Kostovic and Molliver, 1974Go; Kostovic and Rakic, 1980Go, 1990Go; Luskin and Shatz, 1985Go; Chun et al., 1987Go; Shatz et al., 1988Go; De Carlos and O'Leary, 1992Go; O'Leary and Koester, 1993Go; Allendoerfer and Shatz, 1994Go; Supèr et al., 1998Go).

The developmental organization, roles and fate of subplate cells of kittens and primates are relatively well understood [for a review see (Allendoerfer and Shatz 1994Go)]. Subplate neurons are the first cortical neurons to send corticofugal projections, thus pioneering the thalamocortical connections (McConnell et al., 1989Go, 1994Go). Furthermore, a large number of cortical afferents ‘wait’ in the subplate and form transient synaptic interactions with subplate cells, which, for thalamic fibers, appear to be crucial for the entry of axons to the overlying cortex and their later segregation into ocular dominance columns (Marín-Padilla, 1988Go; Ghosh et al., 1990Go; Ghosh and Shatz, 1992aGo,bGo, 1993Go). There is general agreement that most subplate cells in kittens and primates disappear during postnatal stages by cell death, a few remaining in the adult white matter as interstitial neurons (Kostovic and Rakic, 1980Go, 1990Go; Luskin and Shatz, 1985Go; Valverde and Facal-Valverde, 1987Go, 1988Go; Shatz et al., 1988Go; Naegele et al., 1991Go; Allendoerfer and Shatz, 1994Go).

Several lines of evidence suggest important differences in the organization of the subplate during cortical evolution [for reviews see (Allendoerfer and Shatz, 1994Go; Supèr et al., 1998Go). For instance, the subplate is much more evident in the developing neocortex of evolved brains than in rodents, and the archicortex displays a thin subplate layer (Kostovic and Rakic, 1990Go; Soriano et al., 1994Go; Supèr et al., 1998Go). In rodents, the developmental history of subplate cells is relatively controversial. For instance, whereas there is agreement about the early generation of the subplate, its fate remains elusive, since the derivative of the subplate (sublayer VIb) is a neuron-rich lamina unique to rodents (Valverde et al., 1989Go, 1995Go; Bayer and Altman, 1991Go; Ferrer et al., 1992Go; Gillies and Price, 1993Go; Price and Lotto, 1996Go; Price et al., 1997Go). Similarly, thalamocortical axons appear to invade the developing cortical plate in rodents soon after reaching the subplate (Catalano et al., 1991Go; Catalano et al., 1996Go; De Carlos and O'Leary, 1992Go), but the exact interaction of subplate cells with ingrowing afferent systems remains unclear. Lastly, in rodents an additional early-maturing neuronal population has been described in the lower half of the developing white matter as revealed by antibodies against {gamma}-aminobutyric acid (GABA), microtubule-associated protein 2 (MAP2) and ß-tubulin class III (TUJ1) (Van Eden et al., 1989Go; Cobas et al., 1991Go; Del Río et al., 1992Go; Ferrer et al., 1992Go; Menezes and Luskin, 1994Go). Recent studies of this neuronal population using TUJ1-antibodies and 1-1-dioctadecyl 3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) tracing report a migratory phenotype of these cells and offer conflicting data on their origin and birthdates (Menezes and Luskin, 1994Go; De Carlos et al., 1996Go; O'Rourke et al., 1997Go; Tamamaki et al., 1997Go) [see also (Pearlman et al., 1998Go)].

In the present study, the neuronal populations of the subplate and developing white matter (intermediate–subventricular zone) in the neocortex of the mouse were characterized by using immunocytochemistry against a variety of neuronal markers. In addition, the spatial links of these early-neuronal populations with the establishment of the major cortical pathways (i.e. thalamic and callosal projections) were studied using DiI as tracer. Identified neurons were examined using an electron microscope to describe their fine structure, input synapses and relationship with the neighboring milieu.


    Material and Methods
 Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Notes
 References
 
Animals

A total of 91 NMRI mice (Iffa Credo, Lyon, France) from 36 litters were used in the present study. Females were mated overnight (12 p.m. to 8 a.m.) and the mating day, ascertained by the presence of a vaginal plug, was considered as embryonic day 0 (E0). The day of birth was considered as postnatal day 0 (P0). The following developmental stages were examined: E12, E13, E14, E15, E16, E17, E18, E19, P0 and P2.

Immunocytochemical Procedures

Pregnant dams were deeply anesthetized with chloral hydrate (35 mg/kg body wt) and the embryos were removed by caesarian section and perfused transcardially. Postnatal animals were anesthetized with ether or chloral hydrate and perfused through the ascending aorta. For the visualization of all antigens except GABA, specimens were perfused with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (pH, 7.3). Brains were dissected out and postfixed overnight in the same solution. Coronal sections, 50 µm thick, were obtained using a Vibratome, and treated with 3% methanol–10% hydrogen peroxide to inhibit endogenous peroxidases. Thereafter, free-floating sections were incubated with a solution containing 0.2 M glycine, 0.2 M lysine and 10% normal serum of the appropriate species to block nonspecific staining. Afterwards, sections were incubated with one of the following primary antibodies overnight at 4°C: mouse monoclonal anti-MAP2 (diluted 1:2000–5000; clone SMI52; Sternberger-Meyer, Jarrettsville, USA), mouse anti-TUJ1 (diluted 1:3000; BABCO, Richmond, USA), rabbit anti-calbindin D-28k (CALB; diluted 1:2000–4000; Swant, Bellinzona, Switzerland), rabbit anticalretinin (CALR; diluted 1:2000–3000; Swant), rabbit anti-cholecystokinin octapeptide (CCK; diluted 1:500–1500; Incstar, Stillwater, USA), rabbit anti-neuropeptide Y (NPY; diluted 1:1000–2000; Cambridge Research Biomedicals, Cambridge, UK) and rabbit anti-somatostatine (SS; diluted 1:1000, Dakopatts, Glostrup, Denmark). Monoclonal antibodies were visualized using rabbit anti-mouse (diluted 1:75) or biotinylated horse anti-mouse antibodies (diluted 1:200) as secondary antibodies, and mouse peroxidase–anti-peroxidase (PAP) complex (diluted 1:150) or the avidin–biotin–peroxidase complex (ABC) (diluted 1:200) as third layer immunoreagents. For rabbit-raised primary antibodies, the PAP sequence was followed, which included goat anti-rabbit antibodies (diluted 1:75) and rabbit PAP complex (diluted 1:150). Immunoreagents were diluted in 0.1 M PBS, containing 0.2% gelatin, 5% normal serum and 0.1–0.2% Triton X-100. Peroxidase activity was developed with 0.03–0.05% diaminobenzidine (DAB) and 0.01% hydrogen peroxide. Sections were mounted onto gelatinized slides and coverslipped with Eukitt (Merck, Darmstadt, Germany).

For the immunocytochemical visualization of GABA, animals were perfused and processed as above except that 0.25–0.5% glutaraldehyde was added to the fixative solution. After blocking, Vibratome sections were incubated overnight with rabbit anti-GABA antibodies (diluted 1:2500; Incstar) and then processed according to the PAP or ABC sequence.

For the simultaneous visualization of GABA/MAP2, GABA/CALR and GABA/CALB, 50-µm-thick coronal brain sections from four embryos aged E17–E18 were blocked with 10% normal goat and swine sera. After rinsing, sets of sections were incubated overnight with the following combinations of primary antibodies: rabbit anti-GABA and mouse anti-MAP2 antibodies; mouse monoclonal anti-GABA and rabbit anti-CALR; and mouse monoclonal anti-GABA and rabbit anti-CALB (diluted 1:750). Next day, sections were incubated in a cocktail containing fluorescein-labeled goat anti-mouse immunoglobulin G (IgG) and rhodamine-tagged swine anti-rabbit IgG. After rinsing, sections were coverslipped with anti-fading mounting medium (MowiolTM) and examined in a fluorescence photomicroscope equipped with rhodamine and fluorescein filter sets, and with a double-excitation filter allowing the simultaneous visualization of both fluorochromes. Sections selected for quantitative analysis corresponded to the rostrocaudal level corresponding to the first emergence of the hippocampus. This level includes the prospective cingulate, motor and parietal cortices. The number of single and double-labeled cells (e.g. CALB/GABA or MAP2/GABA) in the intermediate zone–subventricular border was counted in sections from embryos aged E17–E18. Five or six sections were harvested per animal, and four or five animals from each litter were used for quantification.

Immunoelectron Microscopy

For electron microscopy, mice from E15, E16, E18 and P0 were perfused with 4% paraformaldehyde and 0.1–0.25% glutaraldehyde dissolved in 0.1 M phosphate buffer (pH 7.3). Vibratome sections were processed for the immunocytochemical visualization of GABA, CALR or MAP2 as described above, except that Triton X-100 was omitted from the immunoreagents. After DAB development, sections were postfixed with 2% osmium tetroxide, block-stained with uranyl acetate and flat-embedded in Araldite. After photodocumentation, selected cells were re-embedded and sectioned in an ultratome. Thin sections were collected on Formvarcoated slot grids and stained with lead citrate.

Immunocytochemical Controls

Immunocytochemical controls, which included omission of primary antibodies or their substitution by normal serum followed by the corresponding immunostaining protocol, prevented immunostaining. In some cases very weak, diffuse immunoreaction in the embryonic marginal and subplate zones was observed when anti-mouse secondary antibodies were used; in these cases the diffuse labeling was abolished by adding anti-mouse Fab fragments to the blocking solution (1:100 diluted; Jackson Laboratories, West Grove, USA).

DiI Experiments

For DiI tracing experiments, embryos at E13–E19 and postnatal animals (P0–P2) were perfused with 4% parafomaldehyde dissolved in 0.1 M phosphate buffer (pH 7.2–7.4). The brains were immersed in the same fixative at 4ºC. Some brains were hemisected sagittally and injected with a crystal of DiI (Molecular Probes, Eugene, OR) in the dorsal thalamus and capsula interna (50 cases), using a thin tungsten needle under microscopic control. Another set of animals received a DiI injection in the prospective somatosensory region, the intermediate zone below the cingulate cortex or the corpus callosum itself (30 cases).

After injection, brains were stored in phosphate-buffered 4% paraformaldehyde at room temperature for 3–10 weeks in the dark. Coronal sections (80 µm thick) were collected in phosphate buffer and counterstained with the DNA-specific dye bisbenzimide (Hoescht 33342; Sigma, Poole Dorset, UK; 3 µM, 30 min) to allow the identification of regional and cytoarchitectonic boundaries. Thereafter, sections were mounted onto gelatinized slides, coverslipped with an anti-fading mounting medium and analyzed in a fluorescence microscope. Selected sections were photoconverted with DAB or immunostained with MAP2 antibodies (diluted 1:750) and fluorescein-labeled secondary antibodies.

Nomenclature

To allow the delimitation of cytoarchitectonic boundaries, parallel Vibratome sections from the animal used for immunocytochemistry were Nissl stained. The delimitation and nomenclature of layers follows previous studies (Del Río et al., 1992Go, 1995Go; Ferrer et al., 1992Go). Thus, at E12 (preplate stage) (De Carlos and O'Leary, 1992Go), the ventricular zone and the primordial plexiform layer were recognized. The cortical plate emerges at E13 in the lateralmost cortical regions. From E13 to E19 the following layers were considered: ventricular zone, subventricular zone, intermediate zone, subplate, cortical plate, marginal zone and sublayer VIa (E16–E17 and E19). From birth onwards the marginal zone and the subplate were considered as layers I and VIb, respectively. The intermediate, subventricular zones and the remainder of the ventricular layer were considered together as the developing white matter during the postnatal period.


    Results
 Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Notes
 References
 
MAP2 Immunoreactivity

To study the emergence and general evolution of neurons in the subplate and developing white matter, sections were immunostained against MAP2, a wide neuronal antigen which has been shown to label early-neuronal populations in cortex (Luskin and Shatz, 1985Go; Crandall et al., 1986Go). At E12, the prospective neocortex showed a simple organization in which only the proliferative ventricular zone and the primitive plexiform layer could be recognized, thus corresponding to the primordial neocortical organization (Marín-Padilla, 1988Go) or preplate stage (De Carlos and O'Leary, 1992Go). Weakly stained neurons were seen at this stage in the primitive plexiform layer (not shown). At E13, the cortical plate emerged in the lateral cortex, following a latero-medial gradient (Smart and Smart, 1982Go; Goffinet, 1984Go); 1 day later (E14) the cortical plate reached dorsal cortical regions (Fig. 1B,CGo). At these stages (E13–E14) MAP2 immunoreactivity increased notably. The emergence of the cortical plate split the neurons populating the primitive plexiform layer into the marginal zone and in the subplate (Fig. 1B,CGo). In this layer, most MAP2-immunoreactive cells had ovoid to pyramidal cell bodies, from which an apical dendrite transversing the cortical plate often arose. In addition, a population of weakly stained MAP2immunoreactive neurons was visible at E13–E14 just above the ventricular zone in the intermediate and subventricular zones (Fig. 1A,BGo). However, their distinctive morphology, i.e. small perikarya and a long dendritic process at one pole, was already recognizable. The majority of these cells were directed toward the dorsomedial cortex along the intermediate–subventricular interface. However, a few such cells displayed different orientations (oblique or radial). Moreover, immunoreactive cells were also seen in medial cortical regions, before the emergence of the cortical plate (Fig. 1AGo).



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Figure 1. MAP2 immunoreactivity in the developing neocortex. (A–C) Photomicrographs illustrating the pattern of MAP2 immunoreactivity in the medial (A) and lateral (B) regions of the mouse neocortex at E14. MAP2-immunopositive elements in the medial cortex (A) are mainly located in the primitive plexiform layer and, to a lesser extent, in the intermediate and subventricular zones. In lateral regions (B), where the cortical primitive plexiform layer is divided between the marginal and the subplate, the immunoreactive elements are also distributed in their derivatives. (C) A Nissl-stained section parallel to those presented in (B). (D,E) Pattern of MAP2 immunoreactivity in the cerebral cortex at E16. MAP2 immunostaining is distributed throughout the cortex, but is especially intense in the marginal zone, subplate and cortical plate. Note the presence of a narrow band of immunoractive cells in the subventricular zone (arrows). (E) A Nissl-stained section parallel to (D). (F–H) Details of MAP2-immunoreacted cells in the middle (F,G) and lower (H) parts of the mouse neocortex at E16 (F–H). Arrows in (F) and (H) label immunoreactive cells populating the intermediate zone (F) or the subventricular–ventricular zone (H). Scale bars: A = 75 µm pertains to B and C; D = 200 µm pertains to E; F = 50 µm pertains to G and H.

 
At subsequent stages (E15–E18) the neocortex showed marked development, which was mainly due to the recruitment of newly generated neurons to form the cortical plate and to the thickening of the intermediate zone with early afferent and efferent fibers (Fig. 1D,EGo). At these stages most cortical plate neurons were MAP2-positive, but immunoreactivity was more prominent in the subplate and marginal zones (Fig. 1DGo). The perikarya of subplate neurons were relatively large (10–15 µm main axis), and cells showed a wide range of shapes. Some subplate neurons had ascending dendrites, although most dendrites were horizontal, some of them also invading the underlying intermediate zone (Fig. 1F,GGo). At perinatal stages (E19–P0), the morphological features and organization of subplate neurons did not differ from those observed in previous stages. A few MAP2-positive neurons were also noticed in the intermediate zone at these perinatal stages (Fig. 1FGo).

At E15–E18, MAP2-positive neurons were very conspicuous just above the proliferative epithelium. Resectioning of immunoreacted Vibratome slices into semithin sections (see Fig. 5C,DGo for calbindin immunostaining) showed that these neurons were located primarily in the subventricular zone and, to a lesser extent, in the adjacent aspect of the intermediate zone. These neurons thus belonged to the intermediate–subventricular border population. In addition, a few immunopositive cells were embedded in the ventricular zone. These MAP2-positive neurons had small cell bodies (main axis 5–6 µm) and displayed a variety of shapes and orientations (Fig. 1D,HGo). MAP2-positive neurons in the intermediate–subventricular border became less conspicuous at perinatal stages (E19–P0), and largely disappeared from the cortex during early postnatal days.



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Figure 5. (A,B) Pattern of CALB- (A) and CALR (B) immunostaining in the cerebral cortex of the mouse at E18. CALB-immunoreactive elements are mainly distributed in the subplate and the subventricular zone, whereas CALR-immunoreactive cells are more abundant in the subventricular zone. (C,D) Photomicrographs of two consecutive semithin sections obtained from a CALB-immunostained Vibratome section at E15. The section in (D) has been Nissl-stained to allow laminar definition. Asterisks label blood vessels, and arrows points to examples of immunoreactive cells seen in both semithin sections. Most neurons belonging to the intermediate–subventricular border population are clearly embedded in the subventricular and even in the ventricular zone. Scale bars: A and C = 75 µm pertain to B and D respectively.

 
GABA Immunoreactivity

A detailed description of the development of GABA immunoreactivity in the murine neocortex has already been published (Del Río et al., 1992Go), so only the main ontogenetical changes in the subplate and intermediate–subventricular zone neurons are reported here. Weak GABA immunoreactivity was first observed at E12 in the primordial plexiform layer. At E13–E14, GABApositive neurons were mainly seen in the marginal zone, subplate and intermediate–subventricular border. At subsequent stages (E15–E18), immunoreactivity was prominent in these early-maturing layers at the same time as immunostaining in the cortical plate increased (Fig. 2AGo). Immunoreactive neurons in the subplate were less densely packed than those stained by MAP2 antibodies (Figs 2B and 5GoGo). GABA-positive subplate cells had heterogeneous shapes but pyramidal-shaped, subplate neurons with apical dendrites were not stained with GABA antibodies (Fig. 2BGo). GABA-immunoreactive neurons in the intermediate–subventricular border displaying the characteristic morphological features of this population were abundant at E15–E18, with a density comparable to that observed with MAP2 immunoreactivity (Figs. 2C and 5GoGo).



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Figure 2. GABA immunoreactivity in the developing cortex. (A) Pattern of GABA immunoreactivity at E18. GABA-immunoreactive elements are distributed throughout the cortex but are mainly accumulated in the marginal zone, subplate and cortical plate. Notice the presence of a band of immunoreactivity in the subventricular zone. (B,C) High-power photomicrograph illustrating GABA-immunoreactive elements in the middle (B) and lower aspects (C) of the mouse neocortex at E18. Arrows point to GABA-positive cells in the intermediate zone (B) and the subventricular–ventricular zone (C). (D–I) Pairs of photomicrographs illustrating examples of double immunoreacted cells (arrows) for GABA (E,G,I) and MAP2 (D,F,H) located in the subplate (D,E), intermediate zone (D,E and F,G) or subventricular–ventricular zone (H,I). The arrowhead in (H,I) labels a dendritic process in the ventricular zone. Scale bars: A = 200 µm; B = 50 µm pertains to C; D = 50 µm pertains to E–I.

 
Double-immunofluorescence experiments performed on sections from E17–E18 brains showed that virtually all GABApositive cells in the subplate and intermediate–subventricular border were MAP2-positive (Fig. 2D–IGo). GABA-positive neurons accounted for a small percentage (17%, n = 349 cells) of the subplate population, as identified with MAP2 antibodies (Fig. 2D,EGo). In contrast, 86% (n = 537 cells) of MAP2-positive neurons in the intermediate–subventricular border were also GABAimmunopositive. These data illustrate the distinct neuronal composition of the subplate and the intermediate–subventricular border population.

TUJ1 Immunoreactivity

The onset and evolution of TUJ1 immunoreactivity was very similar to that observed with MAP2 immunoreactivity. At E12, TUJ1 immunoreactivity was present in the primitive plexiform layer and in immunoreactive cells in the subventricular zone (not shown). At E13–E15, TUJ1 immunoreactivity occurred in postmitotic neurons in the cortical plate as well as in the marginal zone and subplate (Fig. 3AGo). In addition, scattered TUJ1positive cells in the ventricular zone and in the intermediate–subventricular border become more intense (Fig. 3A,CGo). The morphology and orientation of TUJ1-immunoreactive cells resembled that of MAP2and GABA-positive cells. This organization was also observed in late prenatal stages (E17–E19). From P0 onwards, there was a marked reduction in the number of TUJ1-immunoreactive cells in the developing white matter. Moreover, immunoreactive cells were very rare from P2 onwards except in the remaining ventricular zone of the lateral ventricle (not shown).



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Figure 3. TUJ1-immunoreactive elements during neocortical development. (A,B) Panoramic view of coronal sections at E15 (A) and E18 (B) illustrating the arrangement of TUJ1 positive elements. At E15, immunoreactive elements are prominent in the marginal zone and more conspicuous in the rest of cortical layers. At E18, TUJ1-immunopositive elements are distributed in all cortical layers. Notice the immunoreacted fibers in the intermediate and subventricular zones. (C,D) High-power photomicrograph through medial cortical regions showing several immunoreacted cells populating the subventricular–ventricular zone at E15 (C) and E18 (D) (arrows). Cells located perpendicular to the ventricular surface can be seen in both stages (arrowheads in C and D). Moreover, TUJ1-immunoreactive axon fascicles in the lower part of the intermediate zone observed at E15 and, more relevantly, at E18 (open arrows) do not invade the subventricular zone. Scale bars: A,B = 200 µm; C = 50 µm pertains to D.

 
One of the most striking features of the evolution of TUJ1 immunoreactivity is the presence of prominent immunoreactive fibers lying parallel to the pial surface in the neocortex. Some immunoreactive fibers were in the lower part of the intermediate zone, just above the intermediate–subventricular border (Fig. 3B–DGo). These fibers formed dense axonal fascicles and were more conspicuous in medial and lateral portions of the cortex. Other fibers ran transversally in the subplate and adjacent part of the intermediate zone, forming a dense network (Fig. 3BGo). This arrangement was seen throughout prenatal development. At perinatal stages (E19–P0) immunoreactive fibers became less conspicuous, and from P2 onwards individual fibers were no longer seen and the developing white matter showed uniform labeling (not shown).

Calcium-binding Protein Immunoreactivity

At E12, weakly stained CALB-positive cells were present in the primordial plexiform layer and just above the ventricular zone, in lateral sectors of the cortical primordium (not shown). At E13–E14, as shown in Figure 4Go, the cortical plate splits CALBpositive neurons populating the primordial plexiform layer into the marginal zone and the subplate (Fig. 4B–DGo). Immunoreactive neurons in the subplate were morphologically heterogeneous, most of them with horizontal orientation (Fig. 4C,DGo). In the intermediate–subventricular border there were numerous immunoreactive neurons, which showed the distinctive morphology typical of this neuronal population (Fig. 4C,DGo). Such neurons were also present in most medial cortical regions, before the emergence of the cortical plate (Fig. 4CGo). At E15– E18 CALB-immunostaining in the subplate and intermediate–subventricular border was prominent, with labeled neurons appearing more mature than at previous stages (Figs 5 and 6GoGo). No pyramidal-shaped neurons in the subplate were also labeled with CALB antibodies, but a few fibers were immunopositive in this layer. The morphology of CALB-immunoreactive neurons in the intermediate–subventricular border is illustrated in Figure 6Go, compared with that observed using MAP2 and GABA-antibodies. Some of such horizontal neurons had a single process. In other cases, bipolar or multipolar shapes or bizarre morphologies were observed. As seen in semithin sections, most neurons were located in the lower–intermediate and subventricular zones, but a few of them were seen in the proliferative epithelium with dendritic branches running close to the ventricular surface (Fig. 5C,DGo).



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Figure 4. CALB and CALR immunoreactivity at early stages of cortical histogenesis. (A,E) Pattern of CALR immunoreactivity in the cerebral cortex at E14. Notice the dense labeling of the primitive plexiform layer and marginal zone. Scattered immunoreactive cells are also present in other cortical layers. (B) Section parallel to that shown in (A), demonstrating the pattern of CALB immunoreactivity in the cerebral cortex at E14. Neurons are abundant in the primitive plexiform layer, marginal zone, subplate and subventricular zone. Boxed areas are shown at higher magnification in (C,D). (C–E) Details of CALB- (C,D) and CALR (E) immunocytochemistry at E14. In medial cortical regions, where the cortical plate has not yet appeared, CALB-positive cells are in the primitive plexiform layer and subventricular zone. After the emergence of the cortical plate in dorsal regions (D) most CALB-positive neurons are in the marginal zone, subplate and subventricular zone. In contrast, most CALR-positive neurons are in the marginal zone. (F–I) Pairs of photomicrographs showing examples of double-immunoreactive cells (arrows) for CALR (F) and GABA (G), and CALB (H) and GABA (I) in the subventricular zone at E15 (arrows). The open arrow points to other GABA-immunoreactive elements non-immunoreacted for calcium binding proteins. Scale bars: A = 200 µm pertains to B; C = 75 µm pertains to D and E; F = 50 µm pertains to G–I.

 


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Figure 6. Camera lucida drawings illustrating the morphology of MAP2-, GABA- and CALB-immunoreactive neurons in the subplate and the intermediate–subventricular border in the presumptive somatosensory cortex at E18. Scale bar: 50 µm.

 
Calretinin immunoreactivity also appeared early in mouse corticogenesis at E12 in the primordial plexiform layer. With the emergence of the cortical plate, most CALR-positive neurons in the primordial plexiform layer were located in the marginal zone, so only a few CALR-positive neurons occurred in the subplate and in the intermediate–subventricular border (Fig. 4A,EGo). Immunoreactive neurons in the marginal zone correspond to the murine Cajal-Retzius cell population (Del Río et al., 1995Go). At E15–E18, the distribution and density of CALR-positive neurons in the subplate was as in previous stages, although labeled neurons had more mature morphology (Fig. 5BGo). Some fiber bundles in the intermediate zone also displayed CALR immunoreactivity (Fig. 4EGo).

Colocalization studies performed on E17–E18 brains showed that, in the subplate, the intermediate zone and the intermediate– subventricular border, virtually every neuron immunoreactive for CALB or CALR was also GABA-positive, with the exception of a few pyramidal subplate cells which displayed weak CALR immunoreactivity (Fig. 4F–IGo). Moreover, 81% of the GABApositive neurons in the intermediate–subventricular border were CALB-immunoreactive and 23% of the GABAergic neurons in the same layer contained CALR. In the subplate, the percentage of GABAergic neurons showing calcium-binding protein immunostaining was similar. These results indicate that most early GABAergic neurons in the murine neocortex were also labeled with antibodies against calcium-binding proteins.

Neuropeptide Immunocytochemistry

Neuropeptide-immunoreactive neurons appeared late during cortical ontogenesis. NPY-labeled elements were first observed in the neocortex at E16 and their number increased between E18 and P2. At these perinatal stages, large NPY-positive neurons with non-pyramidal shapes were present mainly in the subplate and in layer VIa (Fig. 7A,CGo). Moreover, a few immunoreactive neurons with horizontal orientation were stained in the intermediate–subventricular border (Fig. 6DGo). The developmental pattern of SS immunostaining was similar to that of NPY. Thus, at perinatal stages a few SS-immunoreactive neurons were seen in the subplate and in the intermediate–subventricular border (not shown). The CCK antibodies used only labeled fibers at perinatal stages in the neocortex, but not in other brain regions. CCKpositive fibers were first observed at E16 and increased their number late in development. Immunoreactive axons were present in the subplate and intermediate zone, and also in the marginal zone and in the dense cortical plate (Fig. 7BGo). In contrast to other neuropeptides, no vasoactive intestinal polypeptide-positive elements were detected in the murine neocortex before P5.



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Figure 7. Neuropeptide immunoreactivity in perinatal neocortex. (A) Low-power photomicrograph of a section immunostained for NPY at E18. Scattered immunoreactive cells (arrows) are present mainly in the subplate and intermediate zone. (B) Pattern of CCK immunoreactivity in the neocortex of the mouse at P0. CCK-immunoreactive fibers (arrows) are distributed through all cortical layers. (C,D) High-power photomicrographs illustrating examples of NPY-immunoreactive cells in layer VIb (C) and the subventricular zone (D) at P0. Scale bars: A and C = 100 µm pertain to B and D respectively.

 
DiI Experiments

To determine the relationship between early neuronal cortical populations and the formation of cortical connections, a series of experiments were performed using DiI-tracing techniques. At E13–E14 large DiI injections in the dorsal thalamus did not result in retrogradely labeled neurons in the cortex (Fig. 8AGo). In contrast, when the tracer was positioned in the capsula interna, many neurons were labeled in the cortex (Fig. 8DGo). Such neurons were located in the subplate in dorsal and lateral sectors, but no retrogradely labeled neuron was found in medial cortical regions, which remained at the preplate stage (not shown). Most labeled subplate neurons had pyramidal-shaped perikarya with an apical dendrite reaching the marginal zone (Fig. 8DGo). No retrogradely labeled neurons were observed in the marginal zone. Complementary injections of DiI in the dorsal cortex at E13–E14 showed that corticofugal axons terminated in growth cones in the capsula interna and prospective striatum, thus indicating that subplate cell axons do not reach the thalamus at these stages (Fig. 8CGo).



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Figure 8. Development of thalamocortical relationships during the prenatal stage. (A) DiI-labeled axons after a typical DiI injection in the dorsal thalamus at E14. Thalamocortical axons run through the striatal primordium approaching lateral regions of the cortex. (B,C) A bisbenzimide view (B) of the section seen in (C). After a DiI injection in the parietal cortex at E14 (asterisk), corticofugal axons terminate in growth cones in the striatal primordium. (D,E) Retrogradely labeled cells in the neocortex after a DiI injection in the internal capsula at E14. (E) A bisbenzimide staining of the boxed area in (D). Pyramidal-like subplate cells (arrows). In lateral regions, some cortical plate cells (arrowheads) are also retrogradely labeled after the DiI injection. (F–H) Distribution of thalamocortical axons in the neocortex during embryonic development. At E16 (F) most thalamic axons are accumulated in the subplate, but a few branches are seen to enter the deep cortical plate. Two days later (H), thalamic axons invading upper cortical layers were more abundant (layer VIa). (G) A bisbenzimide view of (H). (I,J) High-power magnifications of retrogradely labeled cells in the subplate (arrows) after DiI injection in the dorsal thalamus at E15 (I) or E16 (J). Scale bars: A = 100 µm pertains to B and C; D = 75 µm pertains to E–H; I = 50 µm pertains to J.

 
One day later (E15) and at following embryonic stages, numerous subplate cells were consistently retrogradely labeled after DiI injections in the dorsal thalamus, demonstrating that their axons had reached this target region. Thalamic injections at E16–E19 resulted in almost constant numbers of retrogradely labeled subplate cells. These labeled neurons showed several morphologies, including pyramidal and multipolar shapes (Fig. 8I,JGo). Thus, the present results show that subplate cells project to the dorsal thalamus during murine corticogenesis and that their axons reach this target region by E15.

Retrograde labeling of cortical plate neurons following thalamic injections was observed after a short, but significant, delay with regard to that of subplate cells. Thus, injections in the capsula interna at E14 labeled a few undifferentiated cortical plate neurons, exclusively in the lateralmost cortical regions (Fig. 8DGo). At E15, a few pyramidal-shaped neurons located in dorso-lateral sectors of the cortical plate were labeled after thalamic injections. Their frequency increased at later embryonic stages (E16–E19).

The developmental distribution of anterogradely labeled thalamocortical axons was also analyzed. At E13, thalamocortical afferents did not reach the cortex, but terminated within the capsula interna. One day later, single thalamic fibers were seen entering into the cortical intermediate zone laterally, without reaching the subplate (Fig. 8AGo). This spatio-temporal sequence suggests that thalamocortical fibers and corticothalamic axons from subplate cells meet each other and cross at a certain level in the prospective striatum (Molnár et al., 1998bGo). At E15 many thalamocortical fibers were present in the prospective neocortex. After entering from the striatal primordium, thalamic axons crossed the intermediate zone obliquely and ran through the subplate for a certain distance, often forming fascicles (not shown). At this stage, labeled fibers were confined to the subplate. Single thalamic fibers were seen running through the subplate below different prospective cortical areas, which suggests that the subplate serves not only as a targeting compartment for specific thalamic axons, but also as a permissive milieu for thalamic axons growing towards more medial cortical regions.

At E16, thalamocortical axons started to invade the lower, decondensed portion of the cortical plate (Fig. 8FGo). These ascending collaterals arising from numerous thalamic fibers in the subplate were short and unbranched. At E17–E18, a larger number of axon collaterals innervated the prospective layer VIa, but only very exceptionally did they penetrate into the condensed portion of the cortical plate (Fig. 8HGo). At later embryonic and perinatal stages, the number of thalamic axonal branches ascending from the subplate increased notably, although their general distribution remained essentially the same, below the dense cortical plate.

The relationship of subplate cells and subventricular zone neurons with the formation of callosal connections was also studied. The first cortical axons directed towards the telencephalic midline were detected at E16, especially following injections of DiI in lateral cortical aspects (Fig. 9A,DGo). Such early callosal fibers ran towards the corpus callosum through the inner half of the intermediate zone just above the subventricular zone (Fig. 9A,BGo). Early callosal fibers, therefore, did not grow through the subplate or the upper intermediate zone to the corpus callosum. In addition, a few DiI-labeled neurons, after injections in the lateral or medial cortex, were occasionally found in the intermediate–subventricular border, embedded in the bundles of anterogradely labeled callosal axons, with a morphology reminiscent of neuronal populations in this layer (Fig. 9BGo).



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Figure 9. Development of callosal connections. (A) Result of a typical injection of DiI in a lateral cortical region of the mouse at E17, viewed after photoconvertion of the fluorescent label. From the injection site (right), callosal axons (arrows) reach towards the cingulate area running the through the inner intermediate zone, near the subventricular zone. (B) High-power magnifications of retrogradely labeled cells in the intermediate zone (arrow) after DiI injection in the ipsilateral cortex at E18. (C) Distribution of retrogradely labeled callosal cells after injection of DiI in the contralateral hemisphere at E17. Most retrogradely labeled cells are located in the cortical plate. (D) High-power photomicrograph of a section parallel to that shown in (C), demonstrating the absence of retrogradely labeled callosal cells in the subplate. Pyramidal-like retrogradely labeled cells are located in the dense cortical plate and in the upper part of layer VIa. Scale bar: A = 200 µm pertains to B–D; E = 75 µm.

 
Callosal axons first crossed the corpus callosum at E17. To visualize the first cortical neurons sending out callosal axons, crystals of DiI were injected into the corpus callosum or the contralateral intermediate zone, just below the cingulate cortex, at E17–P0 (Fig. 9D,EGo). These experiments demonstrated that the first callosal neurons were located in infragranular layers V and VIa, but hardly any retrogradely labeled neuron was found in the subplate layer VIb (Fig. 9D,EGo). Similar experiments performed on postnatal animals failed to label layer VIb cells, which indicates that in the mouse subplate cells do not send callosal fibers.

Electron Microscopy

Subplate cells identified with MAP2-, GABA- and CALR immunocytochemistry were subjected to electron microscopic analyses from E15 to E18 (Fig. 10Go). Subplate neurons had a relatively large nucleus, rich in euchromatin and with a prominent nucleolus (Fig. 10AGo). The cytoplasm contained abundant organelles, especially endoplasmic reticulum, polyribosomes and Golgi complex. Their dendrites had numerous microtubules, as well as biosynthetic and vesicular organelles (Fig. 10AGo). These relatively mature features were in contrast with the undifferentiated neurons in the cortical plate. Morphologically identifiable synaptic contacts were observed on both the perikarya and dendritic surfaces of subplate neurons from E15 onwards (Fig. 10B,EGo). Axon terminals were usually small and contained a few synaptic vesicles, although larger boutons were also seen. In some cases, horizontal fibers were seen to establish ‘en passant’ synaptic contacts with subplate cells (Fig. 10CGo).



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Figure 10. Fine structural characteristics of subplate and intermediate–subventricular cells. (A) Electron micrograph of a MAP2-immunoreactive neuron located in the subplate at E16. The nucleus (n) is large and has evenly dispersed chromatin and contains one nucleolus. Cytoplasmic organelles are distributed around the nucleus. (B,C) Examples of asymmetric (B) and symmetric (D) synaptic contacts (arrows) on MAP2-immunoreacted elements in the subplate at E18 (B) and E16 (D). (D) Electron micrograph of a GABA-immunoreactive cell embedded between non-immunoreacted cells. (E) Examples of focal adhesion contacts (arrow) between MAP2-immunoreacted subventricular cells at E16. Scale bars: A and D = 1 µm; B = 0.2 µm pertains to E; C = 0.2 µm.

 
The fine structure of cells in the intermediate zone–subventricular border was markedly different to that of subplate cells (Fig. 10DGo). The nuclei were ovoid, with one or two indentations of the nuclear envelope. Perichromatin and heterochromatin granules were frequent. The cytoplasm was scarce and contained few cisternae of endoplasmic reticulum and polyribosomes, a poorly developed Golgi apparatus, but abundant mitochondria (Fig. 10DGo). Immunolabeled profiles showed similar content of organelles and few microtubules. Most such cells and their processes were embedded between the perikarya of nearby cells (Fig. 10DGo). On occasions, these cells were adjacent to radially oriented thick profiles, most of which corresponded to radial glia. Only in a few cases were such neurons found in direct contact with horizontal axonal bundles in the lower intermediate or subventricular zones, presumably of callosal origin. We were unable to find synaptic contacts between these axons and the perikarya on dendritic processes of immunoreactive neurons in the intermediate–subventricular border.


    Discussion
 Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Notes
 References
 
The main findings of the present study are: (i) the subplate and the intermediate zone–subventricular border are formed by immunocytochemically and morphologically distinct neuronal populations; (ii) the neuronal populations in these layers are heterogeneous; and (iii) they are also differentially related to the establishment of thalamocortical relationships and callosal connections.

The Subplate Displays a Neuronal Complexity Similar to that of the Adult Cortex

Using several neuronal markers, the present study supports the view that the subplate and the marginal zone are the derivatives of the primordial plexiform layer, as shown elsewhere (Marín-Padilla, 1970Go, 1971Go, 1978Go, 1988Go; Rickmann et al., 1977Go; Rickmann and Wolff, 1985Go; Bayer and Altman, 1991Go; De Carlos and O'Leary, 1992Go; O'Leary and Koester, 1993Go) [for a review see also (Supèr et al., 1998Go)]. Neurons populating the subplate and marginal zone exhibit mature features (i.e. immunoreactivity against neuronal antigens and elaborate dendrites) 2 days before the first early-maturing cortical plate neurons, reinforcing the hypothesis of early maturation of the subplate and marginal zone. Our study also shows that the subplate is formed by a heterogeneous neuronal population according to neurotransmitter identity, morphology and content of calcium-binding proteins and neuropeptides. Furthermore, the relative percentages of such subpopulations appear to be similar to those reported in layers deriving from the cortical plate (Hendry et al., 1984Go; Jones and Hendry, 1986Go; Rogers, 1992aGo,bGo; Andressen et al., 1993Go). For instance, only 17% of subplate cells were GABAergic, most of them presenting immunoreactivity for CALB or CALR. These characteristics are very similar to that of the adult cortex (Rogers, 1992aGo; Andressen et al., 1993Go). On the other hand, Valverde and co-workers have also pointed out the morphological diversity of subplate cells, which included spiny neurons as well as smooth neurons (Valverde et al., 1989Go). It can be concluded, therefore, that the subplate contains neuronal populations similar to those of the cortical plate derivatives. This appears to be the case for the marginal zone, which is also formed by a subpopulation of GABAergic neurons and glutamate-immunoreactive Cajal–Retzius cells (Del Río et al., 1995Go). Thus, the derivatives of the primitive plexiform layer in the mouse cortex, i.e. the subplate and marginal zones, contain a mixture of excitatory and inhibitory neurons, which has also been described in kittens (Antonini and Shatz, 1990Go; Finney et al., 1998Go).

The Intermediate Zone–Subventricular Border: a Distinct Neuronal Population Unique to Rodents

With the neuronal markers used here (e.g. CALB, MAP2), the intermediate zone–subventricular border neuronal populations were shown to be spatially segregated from neurons in the primitive plexiform layer even at the preplate stage. It is also worth noting that the intermediate–subventricular populations are identifiable before the primitive plexiform layer becomes split into the marginal and subplate zones. At these stages, the area between the primitive plexiform layer and the ventricular zone is filled by the earliest corticopetal fibers originating from preplate neurons, forming the incipient intermediate zone (De Carlos and O'Leary, 1992Go; O'Leary and Koester, 1993Go). Thus, the present observations taken from the moment when these neurons are first visible with CALB, GABA, MAP2 and TUJ1 antibodies suggest that intermediate–subventricular neurons do not form part of the preplate. It cannot be ruled out, however, that at earlier developmental stages this particular neuronal population could form part of the primitive plexiform layer (De Diego et al., 1994Go).

Our colocalization study demonstrates that most of this neuronal population is GABAergic, as suggested in other studies (Cobas et al., 1991Go; Del Río et al., 1992Go; Ferrer et al., 1992Go; De Diego et al., 1994Go). Furthermore, most of these neurons contain CALB, and a few others show CALR immunoreactivity. Thus, subventricular–intermediate neurons constitute a unique neuronal population in the developing cortex, with highly uniform and distinctive immunohistochemical properties and morphology. This observation again emphasizes the differences between such populations and those originating from the preplate, the subplate and the marginal zone.

Potential Roles of the Subplate in the Rodent Cortex during Corticogenesis

In kittens, subplate cells are the first neurons to send out axons from the cortex and these have been shown to reach the thalamus before thalamocortical axons enter the cortex [for a review see (Allendoerfer and Shatz, 1994Go)]. Furthermore, ablation of subplate cells in the visual cortex of kittens demonstrated that subplate neurons are necessary for the invasion of thalamic fibers into the upper-lying cortex and for their later segregation into ocular-dominance columns (Ghosh et al., 1990Go; Ghosh and Shatz, 1992aGo). Our results support this hypothesis and show that, in rodents, subplate cells are also the first neurons to grow corticofugal axons. However, consistent with the studies of De Carlos and O'Leary or Molnár et al. in rats (De Carlos and O'Leary, 1992Go; Molnár et al., 1998aGo), Molnár et al. in mice (Molnár et al., 1998bGo) and Metin and Godement in hamsters (Metin and Godement, 1996Go), the growth of subplate cell axons and thalamocortical fibers is almost simultaneous, so both fiber systems meet at a certain position in the ganglionic eminence (prospective striatum). This developmental sequence rules out the possibility that subplate cells may direct the initial outgrowth of thalamocortical fibers. Moreover, the axons of subplate cells in our material reach the thalamus from E15 onwards, as shown by the retrograde labeling of subplate neurons after injections in the thalamus. This suggests that the axons of subplate cells lay down corticothalamic fibers. This contrasts with data reported in ferrets, where layer V neurons rather than subplate or layer VI cells are the first cortical neurons to reach thalamic nuclei (Clascá et al., 1995Go). Different stages of evolution could explain these dissimilarities. In addition, other transient neuronal populations in the capsula interna in carnivores and primates open the possibility of coordinated roles directing corticothalamic connections in carnivores and primates (Letinic and Kostovic, 1996Go; Menendez Suso et al., 1997Go; Adams et al., 1997Go).

In the present study subplate cells were not found to project to the contralateral hemisphere via the corpus callosum, in agreement with other studies in rodents (De Carlos and O'Leary, 1992Go; Koester and O'Leary, 1993Go; O'Leary and Koester, 1993Go; Ozaki and Wahlsten, 1998Go). Moreover, rat subplate cells do not project to the superior colliculus (De Carlos and O'Leary, 1992Go; O'Leary and Koester, 1993Go). This is in contrast to kittens or humans, where subplate cell axons innervate the superior colliculus and, more exceptionally, the contralateral cortex, which suggests that the number of target regions of subplate cells has increased during the evolution of the cerebral cortex (McConnell et al., 1989Go, 1994Go; DeAzevedo et al., 1997Go). In addition, recent studies reported on the possibility that transient cells populating the intermediate zone could pioneer the cortical projection to the superior colliculus (Tamamaki, 1995Go; Metin and Godement, 1996Go; Tamamaki et al., 1997Go). Future experiments may identify a cell population responsible for pioneering the cortico-collicular projection in rodents.

The subplate has been regarded as a ‘waiting’ compartment, where a number of different axonal systems ‘wait’ for varying periods depending of the species and the thalamocortical projection (Lund and Mustari, 1977Go; Rakic, 1983Go; Ghosh and Shatz, 1992aGo) [for a review see (O'Leary et al., 1994Go)]. Consistent with recent findings in rats, our results show that thalamocortical axons remain restricted to the subplate for only 2 days (De Carlos and O'Leary, 1992Go; O'Leary and Koester, 1993Go). Further, a study in kittens reduces the waiting period to 15 days (Ghosh and Shatz, 1992bGo). However, such a short delay does not necessarily rule out transient synaptic interaction between afferent fibers and subplate neurons. In fact, our electron microscopic study shows that subplate cells form morphologically identifiable synaptic contacts from E15 onwards, even before afferent systems start to grow into the cortical plate, reinforcing the view that subplate cells may be postsynaptic targets for different axonal systems, including thalamocortical axons or GABAergic neurons, well before the onset of synaptogenesis in the cortical plate (König et al., 1977Go; Kostovic and Rakic, 1980Go; Hermann et al., 1991). The list of afferent systems connecting the subplate may be longer if we take into account that catecholaminergic and serotoninergic fibers run in this layer (Levitt and Moore, 1979Go; Schlumpf et al., 1980Go; Caviness and Korde, 1981Go; Lidov and Molliver, 1982Go; Crandall and Caviness, 1984Go). Taken together, these findings suggest that subplate cells participate in elaborate cortical circuits with several afferent systems, most probably modulating the input of information from subcortical regions to the developing neocortex.

Finally, the subplate may also be viewed as an intracortical pathway. Although most afferent axons run through the intermediate zone, they invade the subplate long before arborizing in their target zone (Ghosh and Shatz, 1992bGo, 1993Go; Allendoerfer and Shatz, 1994Go). In fact, several molecules promoting axonal extension, such as neuronal adhesion molecules, proteoglycans, lectin-binding residues and neurotrophic factors (NT3 or BDNF) and receptors (P75), have been found in the subplate (Steward and Pearlman 1987Go; Allendoerfer et al., 1990Go; Sheppard et al., 1991Go; Gotz et al., 1992Go; Bicknese et al., 1994Go; Emerling and Lander, 1996Go; Fukuda et al., 1997Go; Fukumitsu et al., 1998Go).

Developmental Significance of the Intermediate–Subventricular Border Neuronal Population

Several studies have shown that neurons populating the intermediate–subventricular border may represent tangentially migrating postmitotic neurons that could be further incorporated in the developing neocortex (O'Rourke et al., 1997Go; Pearlman et al., 1998Go). However, this seems unlikely for all neurons of the intermediate–subventricular border, for several reasons: (i) the shape of some labeled cells is not consistent with the definition of tangentially migrating neurons (unipolar, with a long leading process) given by other authors (Menezes and Luskin, 1994Go; O'Rourke et al., 1997Go). This is particularly remarkable from E15 onwards, when dendritic branching or bizarre morphologies are very frequent, especially in the ventricular zone. (ii) Their fine structure, with a poorly developed Golgi complex and unpolarized cytoplasm, is unlike that of migratory neuroblasts. (iii) These neurons express antigens presented by postmigratory neurons, including neuropeptides, and, in addition, these neuronal populations are present during the prenatal (E14–E19) and early postnatal stages in a similar location. Finally, (iv) numerous labeled cells are oriented perpendicularly to the ventricular surface, often with their main dendrites contacting the ventricular surface. Thus, although a large number of these neurons with unipolar morphology and horizontal orientation may be postmitotic cells, some cells may be considered as a resident neuronal population of the developing intermediate–subventricular border different to those migrating neurons, or microglial/endothelial cells present in the ventricular area (Fishell et al., 1993Go; Memberg and Hall, 1995Go; O'Rourke et al., 1997Go). During postnatal development, this neuronal population undergoes massive cell death during the early postnatal stages (Wood et al., 1992Go; Blaschke et al., 1996Go, 1998Go) (J.A. Del Río et al., submitted for publication). It appears that these cells are generated in other regions, migrate tangentially to these locations and reside within the ventricular zone (O'Rourke et al., 1997Go; Tamamaki et al., 1997Go; Pearlman et al., 1998Go).

The role of the cells populating the intermediate–subventricular border during cortical histogenesis is unknown. We can only speculate as to their function. Our preliminary observations suggested that neurons in the intermediate– subventricular zone may constitute a path for callosal axons, since there was a clear overlap of early callosal fibers and the neurons. However, in our electron microscopic observations we were unable to find a single synaptic contact on such cells. This suggests that other mechanisms of axonal guidance could be present. For instance, guide-post cells can use a wide range of recognition molecules or even metabolic coupling to interact with axons and guide them (Tessier-Lavigne and Goodman, 1996Go). Recently, one of these cue molecules, the diffusible glycoprotein netrin-1, has been proposed to play a crucial role in the development of corticothalamic connections as well as in the formation of cerebral commissures (Serafini et al., 1996Go; Metin et al., 1997Go; Richards et al., 1997Go). In addition, members of the semaphorin family of chemorepulsive molecules (Sema F and G) are expressed by ventricular and subventricular cells during prenatal development (Skaliora et al., 1998Go). The expression of these chemorepulsive molecules in conjunction with our present observations reinforces the idea of a possible role of the intermediate–subventricular neurons as an ‘axonal barrier’ for corticothalamic or thalamocortical axons, as initially suggested by Menezes and Luskin (Menezes and Luskin 1994Go). An extensive analysis of netrin-1 and semaphorin expression as well as their receptors during cortical development would elucidate their putative role. Our present findings, showing DiI-labeled cells coupling with callosal axons, suggesting transmembrane labeling and the presence of focal adhesion contacts at the ultrastructural level, may support this ‘guiding’ role for this neuronal population.

The strategic position of these neurons just above or intermingled with the entire ventricular proliferative zone makes them good candidates for other roles, such as the control of proliferation in the ventricular zone. For instance, GABA, the only neurotransmitter reported in these cells, has been shown to be a trophic molecule during early development, as well as one of the putative factors recently proposed to control neuroblast proliferation via the GABAa receptor expressed in progenitor cells just before they become postmitotic (Poulter et al., 1993Go; LoTurco et al., 1995Go; Barker et al., 1998Go; Ma and Barker, 1998Go). Moreover, it is interesting that the emergence of this population in the cerebral cortex around E13–E14 is coincident with the start of the ‘second’ phase of cortical neuronegenesis (producing supragranular neurons), and that their degeneration is also coincidental with the end of neurogenesis [for a review see (Bayer and Altman, 1991Go)].


    Notes
 Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Notes
 References
 
The authors wish to thank Robin Rycroft for linguistic advice. This study was supported by Ministerio de Educación y Cultura, PM95-0102 y PM98-047 (J.A.D.R.) and DIGICYT SAF94-0743-CO2-01 and SAF97-1429-E (E.S.).

Address correspondence to Dr José Antonio del Río, Department of Cell Biology, Faculty of Biology, University of Barcelona, Diagonal 645, E-08028 Barcelona, Spain. Email: jario{at}porthos.bio.ub.es.


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 Introduction
 Material and Methods
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 Discussion
 Notes
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