Subplate Cells Research Articles

Supernumerary neurons within the cerebral cortical subplate in autism spectrum disorders

Autism spectrum disorders (ASDs) involve alterations to cortical connectivity that manifest as reduced coordinated activity between cortical regions. The neurons of the cortical subplate are a major contributor to establishing thalamocortical, corticothalamic and corticocortical long-range connections and only a subset of this cell population survives into adulthood. Previous reports of an indistinct gray-white matter boundary in subjects with ASD suggest that the adjacent subplate may also show organizational abnormalities. Frozen human postmortem tissue samples from the parietal lobe (BA7) were used to evaluate white-matter neuron densities adjacent to layer VI with an antibody to NeuN. In addition, fixed postmortem tissue samples from frontal (BA9), parietal (BA7) and temporal lobe (BA21) locations, were stained with a Golgi-Kopsch procedure, and used to examine the morphology of these neuronal profiles. Relative to control cases, ASD subjects showed a large average density increase of NeuN-positive profiles of 44.7 percent. The morphologies of these neurons were consistent with subplate cells of the fusiform, polymorphic and pyramidal cell types. Lower ratios of fusiform to other cell types are found early in development and although adult ASD subjects showed consistently lower ratios, these differences were not significant. The increased number of retained subplate profiles, along with cell type ratios redolent of earlier developmental stages, suggests either an abnormal initial population or a partial failure of the apoptosis seen in neurotypical development. These results indicate abnormalities within a neuron population that plays multiple roles in the developing and mature cerebral cortex, including the establishment of long-range cortical connections.

In search of common developmental and evolutionary origin of the claustrum and subplate.

The human claustrum, a major hub of widespread neocortical connections, is a thin, bilateral sheet of gray matter located between the insular cortex and the striatum. The subplate is a largely transient cortical structure that contains some of the earliest generated neurons of the cerebral cortex and has important developmental functions to establish intra- and extracortical connections. In human and macaque some subplate cells undergo regulated cell death, but some remain as interstitial white matter cells. In mouse and rat brains a compact layer is formed, Layer 6b, and it remains underneath the cortex, adjacent to the white matter. Whether Layer 6b in rodents is homologous to primate subplate or interstitial white matter cells is still debated. Gene expression patterns, such as those of Nurr1/Nr4a2, have suggested that the rodent subplate and the persistent subplate cells in Layer 6b and the claustrum might have similar origins. Moreover, the birthdates of the claustrum and Layer 6b are similarly precocious in mice. These observations prompted our speculations on the common developmental and evolutionary origin of the claustrum and the subplate. Here we systematically compare the currently available data on cytoarchitecture, evolutionary origin, gene expression, cell types, birthdates, neurogenesis, lineage and migration, circuit connectivity, and cell death of the neurons that contribute to the claustrum and subplate. Based on their similarities and differences we propose a partially common early evolutionary origin of the cells that become claustrum and subplate, a likely scenario that is shared in these cell populations across all amniotes.

Long-range projections from sparse populations of GABAergic neurons in murine subplate.

The murine subplate contains some of the earliest generated populations of neurons in the cerebral cortex, which play an important role in the maturation of cortical inhibition. Here we present multiple lines of evidence, that the subplate itself is only very sparsely populated with GABAergic neurons at postnatal day (P)8. We used three different transgenic mouse lines, each of which labels a subset of GABAergic, ganglionic eminence derived neurons. Dlx5/6‐eGFP labels the most neurons in cortex (on average 11% of NEUN+ cells across all layers at P8) whereas CGE‐derived Lhx6‐Cre::Dlx1‐Venusfl cells are the sparsest (2% of NEUN+ cells across all layers at P8). There is significant variability in the layer distribution of labeled interneurons, with Dlx5/6‐eGFP and Lhx6‐Cre::R26R‐YFP being expressed most abundantly in Layer 5, whereas CGE‐derived Lhx6‐Cre::Dlx1‐Venusfl cells are least abundant in that layer. All three lines label at most 3% of NEUN+ neurons in the subplate, in contrast to L5, in which up to 30% of neurons are GFP+ in Dlx5/6‐eGFP. We assessed all three GABAergic populations for expression of the subplate neuron marker connective tissue growth factor (CTGF). CTGF labels up to two‐thirds of NEUN+ cells in the subplate, but was never found to colocalize with labeled GABAergic neurons in any of the three transgenic strains. Despite the GABAergic neuronal population in the subplate being sparse, long‐distance axonal connection tracing with carbocyanine dyes revealed that some Gad65‐GFP+ subplate cells form long‐range axonal projections to the internal capsule or callosum.

Developmental gene expression in the mouse clarifies the organization of the claustrum and related endopiriform nuclei.

Studies on gene expression in the developing claustrum of the mouse have clarified the relationships and identity of the claustrum proper and related endopiriform nuclei. The cells of the claustrum primordium express Nr4a2; they are formed in combination with the Nr4a2-labeled subplate cells in the lateral pallium at the site of the future insular cortex. The insular cortex cells, which are born later and which are Nr4a2-negative, migrate through the claustrum toward the pial surface to form layers (2-6a) of the insular cortex. The claustrum is made up of distinct deep (subplate-like) and superficial (principal) parts. The cells of the dorsal endopiriform nucleus (which are also Nr4a2-positive) are formed in the deep part of the claustrum primordium in the lateral pallium, but they migrate ventrally to reach the ventral pallium deep to the piriform cortex at E14.5 in the mouse. On the other hand, the ventral endopiriform nucleus is formed by radially migrating Nr4a2-negative cells in the ventral pallium; it is therefore developmentally distinct from the Nr4a2-postive dorsal endopiriform nucleus, which is a lateral pallial derivative. J. Comp. Neurol. 525:1499-1508, 2017. © 2016 Wiley Periodicals, Inc.

Ectopic clustering of Cajal-Retzius and subplate cells is an initial pathological feature in Pomgnt2-knockout mice, a model of dystroglycanopathy.

Aberrant glycosylation of dystroglycan causes congenital muscular dystrophies associated with cobblestone lissencephaly, classified as dystroglycanopathy. However, pathological features in the onset of brain malformations, including the precise timing and primary cause of the pial basement membrane disruption and abnormalities in the migration of pyramidal neurons, remain unexplored. Using the Pomgnt2-knockout (KO) mouse as a dystroglycanopathy model, we show that breaches of the pial basement membrane appeared at embryonic day 11.5, coinciding with the ectopic clustering of Cajal–Retzius cells and subplate neurons and prior to the migration onset of pyramidal neurons. Furthermore, in the Pomgnt2-KO cerebral cortex, preplate splitting failure likely occurred due to the aggregation of Cajal–Retzius and subplate cells, and migrating pyramidal neurons lost polarity and radial orientation. Our findings demonstrate the initial pathological events in dystroglycanopathy mice and contribute to our understanding of how dystroglycan dysfunction affects brain development and progresses to cobblestone lissencephaly.

Secretory function in subplate neurons during cortical development.

Subplate cells are among the first generated neurons in the mammalian cerebral cortex and have been implicated in the establishment of cortical wiring. In rodents some subplate neurons persist into adulthood. Here we would like to highlight several converging findings which suggest a novel secretory function of subplate neurons during cortical development. Throughout the postnatal period in rodents, subplate neurons have highly developed rough endoplasmic reticulum (ER) and are under an ER stress condition. By comparing gene expression between subplate and layer 6, we found that several genes encoding secreted proteins are highly expressed in subplate neurons. One of these secreted proteins, neuroserpin, encoded by the serpini1 gene, is localized to the ER in subplate cells. We propose that subplate might influence cortical circuit formation through a transient secretory function.

Development, evolution and pathology of neocortical subplate neurons.

Subplate neurons have an essential role in cortical circuit formation. They are among the earliest formed neurons of the cerebral cortex, are located at the junction of white and grey matter, and are necessary for correct thalamocortical axon ingrowth. Recent transcriptomic studies have provided opportunities for monitoring and modulating selected subpopulations of these cells. Analyses of mouse lines expressing reporter genes have demonstrated novel, extracortical subplate neurogenesis and have shown how subplate cells are integrated under the influence of sensory activity into cortical and extracortical circuits. Recent studies have revealed that the subplate is involved in neurosecretion and modification of the extracellular milieu.

Subplate in a rat model of preterm hypoxia-ischemia.

ObjectiveHypoxia–ischemia (HI) in preterm infants primarily leads to injuries in the cerebral white matter. However, there is growing evidence that perinatal injury in preterms can also involve other zones including the cortical gray matter. In a neonatal rat model of HI, selective vulnerability of subplate has been suggested using BrdU birth-dating methods. In this study, we aimed to investigate the neuropathological changes of the subplate and deep layers of the cortex following cerebral HI in neonatal rats with specific cell markers.MethodsP2 rats underwent permanent occlusion of the right common carotid artery followed by a period of hypoxia. P8 rats were analyzed using immunohistochemistry; subplate and deep layers cells were quantified and compared with sham-operated case.ResultsA large variability in the extent of the cerebral injury was apparent. For the three analyzed subplate populations (Nurr1+, Cplx3+, and Ctgf+ cells), no significant cell reduction was observed in mild and moderate cases. Only in severe cases, subplate cells were strongly affected, but these injuries were always accompanied by the cell reductions in layers VI and V.InterpretationWe could therefore not confirm a specific vulnerability of subplate cells compared to other deep layers or the white matter in our model.

Extracortical origin of some murine subplate cell populations.

The subplate layer, the deepest cortical layer in mammals, has important roles in cerebral cortical development. The subplate contains heterogeneous cell populations that are morphologically diverse, with several projection targets. It is currently assumed that these cells are generated in the germinative zone of the earliest cortical neuroepithelium. Here we identify a pallial but extracortical area located in the rostromedial telencephalic wall (RMTW) that gives rise to several cell populations. Postmitotic neurons migrate tangentially from the RMTW toward the cerebral cortex. Most RMTW-derived cells are incorporated into the subplate layer throughout its rostrocaudal extension, with others contributing to the GABAergic interneuron pool of cortical layers V and VI.

Late Maternal Hypothyroidism Alters the Expression of Camk4 in Neocortical Subplate Neurons: A Comparison with Nurr1 Labeling

Maternal thyroid hormones (THs) are essential for normal offspring's neurodevelopment even after onset of fetal thyroid function. This is particularly relevant for preterm children who are deprived of maternal THs following birth, are at risk of suffering hypothyroxinemia, and develop attention-deficit/hyperactivity disorder. Expression of neocortical Ca(2+)/calmodulin kinase IV (Camk4), a genomic target of thyroid hormone, and nuclear receptor-related 1 protein (Nurr1), a postnatal marker of cortical subplate (SP) cells, was studied in euthyroid fetuses and in pups born to dams thyroidectomized in late gestation (LMH group, a model of prematurity), and compared with control and developmentally hypothyroid pups (C and MMI groups, respectively). In LMH pups, the extinction of heavy Camk4 expression in an SP was 1-2 days delayed postnatally compared with C pups. The heavy Camk4 and Nurr1 expression in the SP was prolonged in MMI pups, whereas heavy Camk4 and Nurr1 expression in layer VIb remains at P60. The abnormal expression of Camk4 in the cortical SP and in layer VIb might cause altered cortical connectivity affecting neocortical function.

Expression profiling of mouse subplate reveals a dynamic gene network and disease association with autism and schizophrenia

The subplate zone is a highly dynamic transient sector of the developing cerebral cortex that contains some of the earliest generated neurons and the first functional synapses of the cerebral cortex. Subplate cells have important functions in early establishment and maturation of thalamocortical connections, as well as in the development of inhibitory cortical circuits in sensory areas. So far no role has been identified for cells in the subplate in the mature brain and disease association of the subplate-specific genes has not been analyzed systematically. Here we present gene expression evidence for distinct roles of the mouse subplate across development as well as unique molecular markers to extend the repertoire of subplate labels. Performing systematic comparisons between different ages (embryonic days 15 and 18, postnatal day 8, and adult), we reveal the dynamic and constant features of the markers labeling subplate cells during embryonic and early postnatal development and in the adult. This can be visualized using the online database of subplate gene expression at https://molnar.dpag.ox.ac.uk/subplate/. We also identify embryonic similarities in gene expression between the ventricular zones, intermediate zone, and subplate, and distinct postnatal similarities between subplate, layer 5, and layers 2/3. The genes expressed in a subplate-specific manner at some point during development show a statistically significant enrichment for association with autism spectrum disorders and schizophrenia. Our report emphasizes the importance of the study of transient features of the developing brain to better understand neurodevelopmental disorders.

Fetuin‐A in the developing brain

The serum protein fetuin-A is essential for mineral homeostasis and shows immunomodulatory functions, for example by binding to TGF superfamily proteins. It proved neuroprotective in a rat stroke model and reduced lethality after systemic lipopolysaccharide challenge in mice. Serum fetuin-A concentrations are highest during intrauterine life. Different species show intrauterine cerebral fetuin-A immunoreactivity, suggesting a contribution to brain development. We therefore aimed at specifying fetuin-A immunoreactivity in brains of newborn rats (age P0-P28) and human neonates (20-40 weeks of gestation). In humans and rats, fetuin-A was found in cortex, white matter, subplate, hippocampus, subventricular zone, and ependymal cells which supports a global role for brain function. In rats, overall fetuin-A immunoreactivity decreased with age. At P0 fetuin-A immunoreactivity affected most brain structures. Thereafter, it became increasingly restricted to distinct cells of the hippocampus, cingular gyrus, periventricular stem cell layer, and ependyma. In ependymal cells the staining pattern complied with active transependymal transport from cerebrospinal fluid. Double immunofluorescence studies revealed colocalization with NeuN (mature neurons), beta III tubulin (immature neurons), GFAP (astrocytes), and CD68 (activated microglia). This points to a role of fetuin-A in different brain functional systems. In human neonatal autopsy cases, frequently affected from severe neurological and non-neurological diseases, fetuin-A immunoreactivity was heterogeneous and much less associated with age than in healthy tissues studied earlier, suggesting an impact of exogeneous noxious factors on fetuin-A regulation. Further research on the role of fetuin-A in the neonatal brain during physiological and pathological conditions is recommended.

Maternal Hypothyroidism Decreases Progesterone Receptor Expression in the Cortical Subplate of Foetal Rat Brain

Steroid hormones exert profound effects on the development of brain areas controlling complex cognitive function in adulthood. One class, progestins, may contribute by acting on the progestin receptor (PR), which is transiently expressed in a critical layer of developing cortex: the subplate. PR expression in the subplate coincides with the establishment of ongoing cortical connectivity and may play an important organisational role. Identification of the factor(s) that regulate the precise timing of PR expression within subplate may help elucidate the function of PR. Thyroid hormone may interact with hormone response elements within the PR gene. The present study examined the effects of maternal hypothyroidism on levels of PR immunoreactivity (PR-IR) within the foetal subplate. Pregnant rats were made hypothyroid by the administration of methimazole and potassium perchlorate in drinking water. Maternal hypothyroidism significantly decreased PR-IR within the foetal subplate. Using the incorporation of 5-bromo-2'-deoxyuridine (BrDU) during subplate cell neurogenesis (embryonic day 13.5) to determine subplate cell survival in hypothyroid animals, we found that decreases in PR-IR cannot be attributed to significant subplate cell loss but are more likely the result of altered PR expression. Gestational thyroxine replacement to hypothyroid dams prevented the decrease in PR-IR within the subplate. These results identify thyroid hormone as a potential factor in the regulation of PR expression in the developing brain. These results are consistent with the idea that endocrine cross-talk between progesterone and thyroid hormone may be one mechanism by which maternal hypothyroidism alters normal cortical development.

Subclinical exposure to low-dose endotoxin impairs EEG maturation in preterm fetal sheep

Exposure to chorioamnionitis is strongly associated with neurodevelopmental disability after premature birth; however, it remains unclear whether subclinical infection affects functional EEG maturation. Chronically instrumented 103-104-day-old (0.7 gestational age: term 147 days) fetal sheep in utero were randomized to receive either gram-negative LPS by continuous low-dose infusion (100 ng iv over 24 h, followed by 250 ng/24 h for 4 days; n = 6) or the same volume of normal saline (n = 9). Arterial plasma cortisol, ACTH, and IL-6 were measured. The delta (0-3.9 Hz), theta (4-7.9 Hz), alpha (8-12.9 Hz), and beta (13-22 Hz) components of the EEG were determined by power spectral analysis. Brains were taken after 10 days for histopathology. There were no changes in blood gases, cardiovascular variables, or EEG power during LPS infusion, but a transient rise in plasma cortisol and IL-6 (P < 0.05). LPS infusion was associated with loss of the maturational increase to higher frequency activity, with reduced alpha and beta power, and greater delta power than saline controls from 6 to 10 days (P < 0.05). Histologically, LPS was associated with increased numbers of microglia and TNF-α-positive cells in the periventricular white matter and frontoparietal cortex, increased caspase-3-positive cells in white matter, but no loss of CNPase-positive oligodendrocytes, Nurr-1 subplate cells, or gyral complexity. These data suggest that low-dose endotoxin exposure can impair EEG maturation in preterm fetal sheep in association with neural inflammation but without hemodynamic disturbances or cortical injury.

Gene expression analysis of the embryonic subplate.

The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of thalamocortical axons. At later developmental stages, they are predominantly involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. We aimed to further characterize the embryonic murine subplate population by establishing a gene expression profile at embryonic day (E) 15.5 using laser capture microdissection and microarrays. The microarray identified over 300 transcripts with higher expression in the subplate compared with the cortical plate at this stage. Using quantitative reverse transcription-polymerase chain reaction, in situ hybridization (ISH), and immunohistochemistry (IHC), we have confirmed specific expression in the E15.5 subplate for 13 selected genes, which have not been previously associated with this compartment (Abca8a, Cdh10, Cdh18, Csmd3, Gabra5, Kcnt2, Ogfrl1, Pls3, Rcan2, Sv2b, Slc8a2, Unc5c, and Zdhhc2). In the reeler mutant, the expression of the majority of these genes (9 of 13) was shifted in accordance with the altered position of subplate. These genes belong to several functional groups and likely contribute to synapse formation and axonal growth and guidance in subplate cells.

Molecular Diversity of Early-Born Subplate Neurons

Subplate cells in the mouse are generally defined as cells located in the subplate layer between the white matter and layer 6a. They are some of the earliest born and maturing cells of the cerebral cortex. The postnatal subplate layer in mouse contains neurons with expression of the presynaptic protein complexin 3 (Cplx3), connective tissue growth factor (CTGF), the orphan nuclear receptor Nr4a2 (Nurr1), and the G-protein-coupled lysophosphatidic acid receptor 1 (Lpar1/Edg2). All 4 of these molecular markers show layer 6b-restricted expression at young postnatal ages, with CTGF expression being the most widespread in the young postnatal subplate. However, all 4 markers overlap in their expression pattern to varying degrees. Here we demonstrate with bromodeoxyuridine birthdating that cells labeled with any 1 of these molecular subplate markers are indeed generated at E11.5 or E12.5 in the mouse. Furthermore, we demonstrate a correlation between gene expression and cell birthdates. Lpar1-GFP cells are preferentially generated on E11.5, whereas Cplx3 or Nurr1-positive cells are equally generated during the 2-day peak of subplate neurogenesis (E11.5-E12.5). Our study also demonstrates that early-born subplate neurons labeled by Cplx3, Nurr1, and Lpar1-GFP survive preferentially after the first postnatal week compared with other subplate neurons.

Cortical GABAergic Neurons: Stretching It

In the cerebellum and basal ganglia, projection neurons are GABAergic; but in the cerebral cortex, there has been a historically strong dichotomy between glutamatergic projection neurons and GABAergic local circuit neurons. While this dichotomy is overwhelmingly valid, it is now clear that a small population of long-distance projecting GABAergic neurons occurs in primates, as well as in cats and rodents. Beyond their well-documented existence, however, the functional significance, ontogeny, and connectivity of this intriguing subpopulation remain obscure. This volume brings together nine articles that are intended to provide a summary of some of the current thinking regarding cortical GABAergic neurons with long-distance connections. These cover issues of nomenclature, subtype heterogeneity, species differences, and functional importance, in the adult brain, and in development. A final article (Clancy et al.) is organized as a series of extracts and commentaries on the individual articles, each section of which is followed by general comments and discussion. The order of articles in the present volume parallels the order in this final article. The topics in the other eight articles are briefly described below. The first article (Clancy et al.) presents a database of neural developmental events in three commonly used experimental species: rat, mouse, and macaque, using data from the online database www.translatingtime.net, as well as GABAergic and subplate developmental data from the empirical literature. Striking similarities between the two rodent populations permit the identification of developmental dates for GABAergic and subplate neural events in rats that were previously identified only in mice, and proposed timing of development events in mouse, previously identified in rats. The authors also exploit their model to produce estimates of dates for GABAergic and subplate neural events that have not yet been, or cannot be determined empirically in humans. The next two articles focus on projections and neurochemical profiles of long-distance projecting GABAergic neurons in several cortical areas (Higo et al.) and in the hippocampal system (Jinno). The authors use a variety of techniques to visualize and characterize in detail these scattered, hard to localize subpopulations: Golgi-like filling by NADPH-d (Higo et al.), GFP-labeling of GABAergic neurons in GAD67-GFP Cre-reporter mice (Higo et al.), and combined molecular, electrophysiological, and retrograde tracer approaches (Jinno). A cluster of three articles address the identity of white matter neurons, their role in development, and variations across species (Kanold; Luhmann et al.; Petanjek et al.). Subplate neurons are a largely transient subpopulation, morphologically, and neurochemically heterogeneous, that resides in the neonatal cortical white matter. They play a key role in the segregation and functional maturation of thalamocortical and intracortical circuits and, especially as discussed in Luhmann et al., are postulated not only to serve as a transient relay station for afferent inputs, but also as an active element amplifying the afferent and intracortical activity. Local and long-distance projections arise from subpopulations of glutamatergic and GABAergic subplate neurons. Projections to more distant neocortical regions are thought to form a corticocortical synaptic network. Clinical evidence suggests that subplate cells in the mature cortex contribute to the manifestation of abnormal neuronal circuits, pathological activity, and long-term neurological deficits. Espinoza et al. propose a novel classification of non-subplate pioneer neurons, with distinctive neurochemical phenotypes, projections, and localization within the preplate before its partition. They report the two populations as recognizable in rat and mouse, but with species-specific neurochemical features. Finally, Zhang et al. report a subpopulation of immature, interneuron-like cells in the association cortex and amygdala of non-human primates, and infer that these are involved in a life-long role, at least partly associated with plasticity. In summary, these articles present a range of data and issues that are under active investigation. Even since the short time since 2009 (the original publication of these articles), technical advances have occurred that promise significant new approaches to unraveling the structure and function of this subpopulation, not only as relates to basic cortical circuitry and function but also in relation to developmental and psychiatric disorders.

Morphology of mouse subplate cells with identified projection targets changes with age

During embryonic and early postnatal development subplate neurons integrate into the developing intra- and extracortical circuitry in a dynamic fashion. They extend long-range projections to adjacent cortical regions, the contralateral hemisphere through the corpus callosum, and to subcortical structures through the internal capsule. Here we studied the developmental changes of the somatodendritic morphology of subplate neurons with specified projection target in the mouse. To do so we used carbocyanine dye tracing from the callosum, the internal capsule, or the primary somatosensory cortex. The morphology of subplate cells with projections to any of these targets is very diverse and includes pyramidal, multipolar, and neurogliaform cells. Here we demonstrate that a subpopulation of subplate cells in the mouse cortex undergoes significant changes in somatodendritic morphology during the critical period for experimental modification of the cytoarchitectonic development of the barrel cortex. Between P2 and P7 the mean maximal extent of the primary dendrite decreases significantly for subplate cells with an axon projecting through the internal capsule. Moreover, at P2 some subplate cells extend a primary dendrite to the marginal zone, whereas all dendrites of P7 subplate cells end in or below layer 4. Additionally, by tracing connections from multiple targets with different carbocyanine dyes we identified subplate cells with multiple long-range projections.

Early history of subplate and interstitial neurons: from Theodor Meynert (1867) to the discovery of the subplate zone (1974).

In this historical review, we trace the early history of research on the fetal subplate zone, subplate neurons and interstitial neurons in the white matter of the adult nervous system. We arrive at several general conclusions. First, a century of research clearly testifies that interstitial neurons, subplate neurons and the subplate zone were first observed and variously described in the human brain - or, in more general terms, in large brains of gyrencephalic mammals, characterized by an abundant white matter and slow and protracted prenatal and postnatal development. Secondly, the subplate zone cannot be meaningfully defined using a single criterion - be it a specific population of cells, fibres or a specific molecular or genetic marker. The subplate zone is a highly dynamic architectonic compartment and its size and cellular composition do not remain constant during development. Thirdly, it is important to make a clear distinction between the subplate zone and the subplate (and interstitial) neurons. The transient existence of the subplate zone (as a specific architectonic compartment of the fetal telencephalic wall) should not be equated with the putative transient existence of subplate neurons. It is clear that in rodents, and to an even greater extent in humans and monkeys, a significant number of subplate cells survive and remain functional throughout life.

Reelin Promotes Neuronal Orientation and Dendritogenesis during Preplate Splitting

The secreted ligand Reelin is thought to regulate the translocation and positioning of prospective layer 6 (L6) neurons into the preplate, a plexus of neurons overlying the ventricular zone. We examined wild type and Reelin-deficient cortices and found that L6 neurons were equivalently positioned beneath the pia during the period of preplate splitting and initial cortical plate (CP) formation. The absence of detectable L6 ectopia in "reeler" cortices at this developmental time point indicates that Reelin-signaling might not regulate L6 neuron migration or gross positioning during preplate splitting. To explore the acute response of L6 neurons to Reelin, subpial injections of Reelin were made into Reelin-deficient explants. Reelin injection caused L6 neurons to orient their nuclei and polarize their Golgi toward the pia while initiating exuberant dendritic (MAP2+) outgrowth within 4 h. This rapid Reelin-dependent neuronal orientation and alignment created CP-like histology without any significant change in the mean position of the population of L6 neurons. Conversely, subplate cells and chondroitin sulfate proteoglycan immunoreactivity were found at significantly deeper positions from the pial surface after injection, suggesting that Reelin partially rescues preplate splitting within 4 h. Thus, Reelin has a direct role in promoting rapid morphological differentation and orientation of L6 neurons during preplate splitting.