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.     

The functions of the preplate in development and evolution of the neocortex and hippocampus

Recently, it has been shown that the early developmental organization of the archicortical hippocampus resembles that of the neocortex. In both cortices at embryonic stages, a preplate is present, which is split by the formation of the cortical plate into a marginal zone and a subplate layer. The pioneer neurons of the preplate are believed to form a phylogenetically ancient cortical structure. Neurons in these preplate layers are the first postmitotic neurons and have important roles in the development of the cerebral cortex. Cajal–Retzius cells in the marginal zone regulate the phenotype of radial glial cells and may direct neuronal migration establishing the inside-out gradient of corticogenesis. Furthermore, pioneer neurons form the initial axonal connections with other (sub)cortical structures. A significant difference between the hippocampus and neocortex, however, is that in the hippocampus, most afferents are guided by the pioneer neurons in the prominent marginal zone, while in the neocortex most ingrowing afferent axons enter via the subplate. At later developmental periods, most pioneer neurons disappear by cell death or transform into other neuronal shapes. Here, we review the early developmental organization of the mammalian cerebral cortex (both neocortex and hippocampus) and discuss the functions and fate of pioneer neurons in cortical development, in particular that of Cajal–Retzius cells. Evaluating the developmental properties of the hippocampus and neocortex, we present the hypothesis that the distribution of the main ingrowing afferent systems in the developing neocortex, which differs from the one in the hippocampal region, may have enabled the specific evolution of the neocortex.     

Disabled-1 mRNA and protein expression in developing human cortex

Disabled-1 (Dab1) forms part of the Reelin-Dab1 signalling pathway that controls neuronal positioning during brain development; Dab1 deficiency gives rise to a reeler-like inversion of cortical layers. To establish a timetable of Dab1 expression in developing human brain, Dab1 mRNA and protein expression were studied in prenatal human cortex. The earliest Dab1 signal was detected at 7 gestational weeks (GW), the stage of transition from preplate to cortical plate, suggesting a role of the Reelin-Dab1 signalling pathway in preplate partition. From 12 to 20 GW, the period of maximum cortical migration, Dab1 expression was prominent in the upper tiers of the cortical plate, to decline after midgestation. Radially orientated apical dendrites of Dab1-expressing neurons indicated a predominant pyramidal phenotype. Pyramidal cells in hippocampus and entorhinal cortex displayed a more protracted time of Dab1 expression compared to neocortex. In addition, at later stages (18-25 GW), Dab1 was also expressed in large neurons scattered throughout intermediate zone and subplate. From 14 to 22 GW, particularly high levels of Dab1 mRNA and protein were observed in cells of the ventricular/subventricular zone displaying the morphology of radial glia. The partial colocalization of vimentin and Dab1 in cells of the ventricular zone supported a radial glia phenotype. The concentration of Dab1 protein in ventricular endfeet and initial portions of radial processes of ventricular-zone cells points to a possible involvement of Dab1 in neurogenesis. Furthermore, a subset of Cajal-Retzius cells in the marginal zone colocalized Dab1 and Reelin, and may thus represent a novel target of the Reelin-Dab1 signalling pathway.     

Growth and targeting of subplate axons and establishment of major cortical pathways.

In the developing mammalian neocortex, the first postmitotic neurons form the "preplate" superficial to the neuroepithelium. The preplate is later split into a marginal zone (layer 1) and subplate by cortical plate neurons that form layers 2-6. Cortical efferent axons from layers 5 and 6 and cortical afferent axons from thalamus pass between cortex and subcortical structures through the internal capsule. Here, we identify in rats the axonal populations that establish the internal capsule, and characterize the potential role of subplate axons in the development of cortical efferent and afferent projections. The early growth of cortical efferent and afferent axons was studied using 1-1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) as an anterograde and retrograde tracer in aldehyde-fixed brains of embryonic rats. Cortical axons first enter the nascent internal capsule on embryonic day (E) 14 and originate from lateral and anterior cortex; axons from posterior cortex extend rostrally but do not yet exit cortex. The labeled axons, tipped by growth cones with complex morphologies, take a pathway deep to the preplate. Preplate neurons extend these early cortical efferents, based on the developmental stage of the cortex, and on their location and morphology. Most of these cells later occupy the subplate. Cortical plate neurons extend axons into the internal capsule by E16. En route to the internal capsule, cortical plate axons take the same path as the earlier-growing preplate axons, through the intermediate zone deep to subplate. Subplate axons reach thalamus by E16; the first cortical plate axons enter thalamus about a day later. Thalamic axons enter cortex by E16, prior to other cortical afferents. On E15, both preplate and thalamic axons reach the midpoint of the internal capsule. To determine the subcortical distribution of subplate axons, we used Dil as a retrograde tracer in aldehyde-fixed brains and fast blue and rhodamine-B-isothiocyanate as in vivo retrograde markers in neonatal rats. Tracers were injected into the superior colliculus, the principal midbrain target of layer 5 neurons, at times before, during, and after the arrival of cortical axons, or into the subcortical pathway of primary layer 5 axons at two points, the cerebral peduncle caudal to the internal capsule, and the pyramidal decussation at the junction of the hindbrain and spinal cord, at times shortly after the passing of cortical axons. In every case, the labeled neurons are confined to layer 5; subplate neurons are not labeled.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prenatal development of GABA-ergic neurons in the neocortex of the rat

The present study shows that in the prenatal rat neocortex the GABA immunoreactive neurons are not limited to the marginal, subplate, and intermediate zones, but are also found in all fetal zones of the cerebral anlage. The first GABA-ergic cells are observed on embryonic day 14 in the plexiform primordium. On embryonic day 15, a second population of GABA-ergic cells is observed in the intermediate zone. Beginning on day 16 of gestation and continuing throughout gestation, GABA-ergic neurons are observed in the marginal zone, the subplate zone, the cortical plate, and the ventricular and subventricular zones. Furthermore, while the number of GABA-ergic cells in the cortical plate increases, GABA-ergic neurons in the intermediate zone and subventricular zone decrease in number after embryonic day 19.     

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.     

Fibronectin-like immunoreactivity in the developing cerebral cortex

In the developing cerebral cortex of the mouse, binding of antibodies directed against the extracellular matrix glycoprotein fibronectin occurs with a distinct temporal and spatial pattern. On the 10th embryonic day (E10), when the wall of the telencephalic vesicle is made up of only the proliferating cells of the ventricular zone, antifibronectin (aFN) binding is restricted to the blood vessels and pia-arachnoid. Fibronectin-like immunoreactivity first appears in the neuropil as small points of immunofluorescence among the earliest postmitotic neurons that form the preplate (E11-12). A short time later (E12-13), aFN immunoreactivity becomes more diffuse but continues to be restricted to the preplate. As newly arriving neurons form the cortical plate within the preplate (E13-14), aFN binding is present in the marginal zone above the cortical plate and in the subplate below it. Both the marginal zone and the subplate contain early afferents and the cells that were previously part of the preplate. Binding of aFN is transient; by E18-19 it has diminished to the point where it is no longer detectable except in the blood vessels and pia-arachnoid. The transient appearance of fibronectin-like immunostaining in the zones that contain early cortical afferents suggests that fibronectin plays a role in forming the migratory pathway for the growth cones of these axons. In this role it may be acting in concert with other extracellular matrix components such as hyaluronectin, glycosaminoglycans, and laminin, which have been shown to have similar spatial distributions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the chemokine receptor Cxcr4 mRNA during mouse brain development

The expression of Cxcr4 mRNA that encodes the receptor for the chemokine Sdf1 was studied during mouse brain development using in situ hybridization, from E9.5 to maturity at P21. At embryonic stages, expression is prominent in ventricular zones of stem cell proliferation. This abates during the postnatal period in parallel to the depopulation of ventricular zones. In addition, the Cxcr4 gene is expressed in some differentiating neuronal populations at E12.5, E14.5 and E17.5, such as scattered cells in the reticular formation, cranial nerve nuclei, peripheral ganglia, cerebellar external granule cells, zona incerta, ventral lateral geniculate thalamic nuclei, olfactory glomerular layer, hippocampal primordium and telencephalic preplate. High levels of expression are detected in preplate derivatives in all sectors of the marginal zone (MZ) of the telencephalic vesicles, including Cajal-Retzius (CR) cells, other MZ cells and subplate neurons. Cxcr4 expression is progressively downregulated postnatally, but remains significantly associated in the adult with Bergman glia in the cerebellum, the subgranular layer of the dentate gyrus, and the olfactory glomerular layer. In contrast, expression of Sdf1 mRNA is confined to the meninges and, in embryos, to the telencephalic intermediate zone. This expression pattern suggests that Sdf1 and its receptor Cxcr4 may exert trophic influences on precursor cell proliferation and some neuronal targets that remain to be identified and studied further.     

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.     

Abnormal reorganization of preplate neurons and their associated extracellular matrix: An early manifestation of altered neocortical development in the reeler mutant mouse

The formation of the distinct layers of the cerebral cortex begins when cortical plate neurons take up positions within the extracellular matrix (ECM)-rich preplate, dividing it into the marginal zone above and the subplate below. We have analyzed this process in the reeler mutant mouse, in which cortical lamination is severely disrupted. The recent observation that the product of the reeler gene is an ECM-like protein that is expressed by cells of the marginal zone indicates a critical role for ECM in cortical lamination. We have found that preplate cells in normal cortex that are tagged during their terminal division with bromodeoxyuridine (BrdU) are closely associated with chondroitin sulfate proteoglycans (CSPGs), which were identified by immunolabeling; this association is maintained in the marginal zone and subplate after the preplate is divided by cortical plate formation. Cortical plate cells do not aggregate within the preplate in reeler; instead, preplate cells remain as an undivided superficial layer containing abundant CSPGs, and cortical plate neurons accumulate below them. These findings indicate that preplate cells are responsible for the formation of a localized ECM, because the association of CSPGs with preplate cells is maintained even when these cells are in abnormal positions. The failure of cortical plate neurons to aggregate within the framework of the preplate and its associated ECM and to divide it is one of the earliest structural abnormalities detectable in reeler cortex, suggesting that this step is important for the subsequent formation of cortical layers. J. Comp. Neurol. 378:173–179, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of G protein-coupled receptor 56 protein expression in the mouse developing neocortex

GPR56, one of the adhesion G-protein-coupled receptors (GPCRs), plays an important role in the development of the cerebral cortex. Mutations in GPR56 cause a severe human cortical malformation called bilateral frontoparietal polymicrogyria (BFPP), characterized by a global malformation of the cerebral cortex that most severely affects the frontal and parietal regions. To characterize the expression pattern of GPR56 in the developing cerebral cortex, we developed a mouse monoclonal antibody against mouse GPR56. We revealed that GPR56 is expressed in multiple cell types in the preplate, marginal zone, subventricular zone (SVZ), and ventricular zone (VZ). Most interestingly, the expression of GPR56 in preplate neurons showed an anterior-to-posterior gradient at embryonic day (E) 10.5-11.5. In contrast, the expression pattern of the GPR56 ligand, collagen III, revealed no visible gradient pattern. With the widespread expression of GPR56 in the developing cortex, it is difficult to draw a specific conclusion as to which of the GPR56-expressing cells are critical for human brain development. However, the correlation between GPR56 expression in neurons at E10.5-E11.5 and the anatomic distribution of the cortical malformation in both humans and mice suggests that its function in preplate neurons is indispensible.     

Changes in the distribution of extracellular matrix components accompany early morphogenetic events of mammalian cortical development.

As a step in defining the molecular environment for development of the mammalian cerebral cortex, we have used immunohistochemistry to analyze the distribution and remodeling of three major extracellular matrix (ECM) components, fibronectin, chondroitin sulfate proteoglycan (CSPG), and tenascin, during embryonic and early postnatal stages in the mouse. Fibronectin and CSPG are distributed throughout the proliferative zone that initially comprises the thin wall of the telencephalic vesicle, but their distribution changes as newly generated cells form the preplate just beneath the pia. Immunolabeling for CSPG becomes most prominent in the preplate, and fibronectin becomes restricted to that layer. Just after this change occurs, processes of preplate neurons, visualized with antibodies to neurofilaments, become evident within the matrix-rich preplate zone. The association of fibronectin and CSPG with preplate cells persists as cortical plate neurons divide the preplate; both ECM components are now most prominent in the marginal zone and subplate, the layers above and below the cortical plate that are preplate derived. Within the preplate and its derivatives, immunolabeling of fibronectin is punctate and closely associated with radial glial processes, while labeling of CSPG is more intense and diffuse. Labeling of fibronectin and CSPG declines rapidly as the cortical plate begins to differentiate into cortex; labeling for tenascin first appears at this stage in the most mature layers, the marginal zone and subplate, then gradually becomes widespread throughout all of cortex and subcortical white matter. In early postnatal life, tenascin is eliminated from the hollows of the vibrissal barrels in the somatosensory region; it then declines rapidly throughout cortex. The association of both fibronectin and CSPG with preplate cells and the distribution of fibronectin along radial glia during early cortical development suggest that one or both of these transient cell types might produce specific ECM components or induce their local deposition. The spatial and temporal distribution of fibronectin and CSPG suggests a role in defining a destination for migrating neurons that form the cortical plate and in delineating the pathway for early axonal extension. In contrast, the relatively late appearance of tenascin correlates best with the formation of astrocytes and their processes rather than with the establishment of cortical layers or major axonal pathways. These events are well underway before labeling of tenascin is evident.     

Receptor-like protein tyrosine phosphatase zeta/RPTP beta is expressed on tangentially aligned neurons in early mouse neocortex

Protein tyrosine phosphatase zeta (PTPzeta)/RPTPbeta is a chondroitin sulfate proteoglycan predominantly expressed in the brain. In this study, we examined immunohistochemical localisation of PTPzeta in the mouse telencephalon from embryonic day 9.5 (E9.5) to E15.5. During E10.5-E12.5, immunoreactivities for PTPzeta are specifically observed on the tangentially aligned neurons at the preplate (PP) of the neocortex, as well as on the neurons at the mantle layer (ML) of the ganglionic eminences (GEs). Likewise, neurons immunoreactive for CR50, a marker for Cajal-Retzius neurons, are aligned from the ML of the ganglionic eminences to the PP of the neocortex and co-express PTPzeta. During E13.5-E15.5, PTPzeta-positive neurons are present at the subplate (SP) as well as at the marginal zone (MZ) of the neocortex. These results indicate that PTPzeta is a useful marker for early-generated neocortical neurons in mice: Cajal-Retzius neurons as well as the subplate neurons.     

α2A-adrenergic receptors are expressed by diverse cell types in the fetal primate cerebral wall

The cellular elements of the fetal monkey cerebral wall expressing α2A, the most common subtype of the α2 receptor class, were examined by using nonisotopic in situ hybridization and immunohistochemistry with double-labeling for cell type-specific markers. At the three embryonic ages examined, E70, E90, and E120, α2A receptors were expressed throughout the embryonic cerebral wall. In the E70 and E90 fetuses, α2A receptors were observed in most cells of the proliferative zones. Some α2A-positive cells also expressed a proliferation-associated antigen, Ki-67, suggesting that the receptors are present in dividing cells. Furthermore, at E90, α2A receptors were detected on fibers passing between the ventricular and subventricular proliferative zones. At all ages studied, α2A receptors were expressed by migrating neurons in the intermediate zone, characterized by a spindle-like shape, radial alignment, and close association with radial glia. α2A receptors were also expressed by postmigrational microtubule-associated protein-2-positive neurons of the intermediate and subplate zones and the cortical plate. In the marginal zone, α2A receptors were present in the Cajal-Retzius neurons. Finally, α2A receptors were seen in the glial fibrillary acidic protein-positive cells at all ages studied. In addition, dopamine-β-hydroxylase immunohistochemistry, employed to determine the potential source of noradrenaline in the embryonic cerebral wall, revealed noradrenergic innervation in the marginal, subplate, and intermediate zones of the monkey occipital lobe as early as E70. Based on our observations and available data on α2A signal transduction pathways, we propose that these receptors are involved in regulating the generation, migration, and maturation of cerebral cortical cells. J. Comp. Neurol. 378:493–507, 1997. © 1997 Wiley-Liss, Inc.     

The earliest-generated neurons of the cat cerebral cortex: characterization by MAP2 and neurotransmitter immunohistochemistry during fetal life

The earliest-generated neurons of the cat cerebral cortex have been studied here during development using a combination of 3H-thymidine birthdating with immunohistochemistry for the neuron-specific protein MAP2 or for several neuropeptides/transmitters. These neurons are the first postmitotic cells of the cortex, with birthdates during the 1-week period preceding the genesis of cells of the adult cerebral cortex (Luskin and Shatz, 1985a; Chun et al., 1987). However, they are transient and the majority disappear by adulthood (Luskin and Shatz, 1985a; Chun and Shatz, 1989). When autoradiographic birthdating is combined with MAP2 immunostaining during fetal life, the entire population of these early-generated neurons appears to be stained, resulting in labeled bands above and below the cortical plate. The band above the cortical plate (in the marginal zone) contains early-generated neurons with horizontal morphologies, while the thicker band beneath the cortical plate (within the intermediate zone) contains the somata of early-generated neurons and their elaborate processes that are frequently directed towards the ventricular surface. In view of the correspondence between the location of the early-generated neurons and the MAP2-immunostained band beneath the cortical plate, we suggest that this combined approach can be used to define accurately the subdivision of the intermediate zone known as the subplate. The early-generated neurons are also immunoreactive for GABA, neuropeptide Y (NPY), somatostatin (SRIF), and cholecystokinin (CCK) during fetal life. While GABA, NPY, and SRIF immunostaining could be detected by embryonic day 50 (E50), that for CCK was not found until E60. Moreover, there is a relationship between neuropeptide immunoreactivity and location within the cerebral wall. The marginal-zone neurons are immunoreactive only for CCK. The subplate neurons are immunoreactive for CCK, SRIF, and NPY. Most of those immunoreactive for SRIF tend to be clustered within the upper part of the subplate, while those immunoreactive for NPY tend to be located more deeply. Cells immunoreactive for GABA are more uniformly distributed throughout the cerebral wall. These observations demonstrate directly that the marginal zone and subplate contain peptide- and GABA-immunoreactive neurons that belong to the earliest-generated cell population of the cerebral cortex. The presence of these early-generated neurons, which achieve a remarkable degree of maturity during fetal life, suggests that they perform an essential, yet transient, role in the development of the cerebral cortex.     

Differential localization of high- and low-molecular-weight variants of microtubule-associated protein 2 in the developing rat telencephalon

Microtubule-associated protein 2 (MAP2) occurs in developing mammalian neuronal tissue as both high- and low-molecular-weight forms with temporally regulated expression. We studied the MAP2 expression in the developing rat telencephalon with monoclonal antibodies that recognized both the high- and low-molecular-weight forms of MAP2 variants or that specifically recognized high-molecular-weight forms of MAP2 variants. Differences in the staining patterns of these antibodies reflected differences in the distribution of the high- and low-molecular-weight MAP2s. The immunoreactive sites of high- and low-molecular-weight MAP2 had a more widespread distribution in the embryonic telencephalon than those of high-molecular-weight MAP2. Many bipolar cells in the ganglionic eminence (GE) and in the intermediate zone (IZ) of the neocortex showed low-molecular-weight MAP2 immunoreactivity, but they showed weak or no high-molecular-weight MAP2 immunoreactivity. Expression of mRNA containing exons common to high- and low-molecular-weight MAP2 was detected in the tangentially ellipsoidal cells in the IZ, but expression of mRNA containing an exon specific to high-molecular-weight MAP2 was not detected in these cells by in situ hybridization. We interpreted these observations as indicating that the bipolar cells contained MAP2c preferentially, but contained MAP2a and MAP2b (MAP2a/b) at a very low or negligible level. The cells that expressed MAP2c preferentially among the MAP2 splicing variants composed 50% of the preplate cells, most of the MAP2-positive cells in the hippocampus and the corpus callosum. Double labeling by DiI staining and Dlx2 immunohistochemistry, or by Dlx2 and MAP2 immunohistochemistry, revealed that most of the Dlx2-positive cells in the IZ expressed MAP2c preferentially at embryonic day 16. Another double-labeling study revealed that most GAD-positive cells in the preplate were MAP2a/b positive, whereas most GAD-positive cells in the IZ expressed MAP2c preferentially, with only a negligible level of MAP2a/b immunoreactivity. We conclude that MAP2 immunoreactivity in the IZ was localized in the tangentially migrating neurons. The tangentially migrating neurons seemed to acquire MAP2a/b immunoreactivity as they entered the preplate or cortical plate and developed into mature neurons. Radially migrating neurons in the IZ were MAP2 negative. After entering to the preplate or the cortical plate, they became MAP2a/b positive as they developed into mature neurons.