Two modes of radial migration in early development of the cerebral cortex.

Layer formation in the developing cerebral cortex requires the movement of neurons from their site of origin to their final laminar position. We demonstrate, using time-lapse imaging of acute cortical slices, that two distinct forms of cell movement, locomotion and somal translocation, are responsible for the radial migration of cortical neurons. These modes are distinguished by their dynamic properties and morphological features. Locomotion and translocation are not cell-type specific; although at early ages some cells may move by translocation only, locomoting cells also translocate once their leading process reaches the marginal zone. The existence of two modes of radial migration may account for the differential effects of certain genetic mutations on cortical development.

New directions for neuronal migration.

Analysis of genetic mutations that lead to abnormal migration and layer formation in the developing cerebral cortex of mice and humans has led to important new discoveries regarding the molecular mechanisms that underlie these processes. Genetic manipulation and experimental analysis have demonstrated significant tangential migrations of cortical neurons, some arriving from very distant noncortical sites.

Brain magnetic resonance diffusion abnormalities in Creutzfeldt-Jakob disease.

BACKGROUND: Magnetic resonance imaging of the brain has been of limited usefulness in the diagnosis of Creutzfeldt-Jakob disease. Abnormalities on T2-weighted images have been described, but these are neither highly sensitive nor specific. OBJECTIVE: To determine whether diffusion-weighted magnetic resonance images might be useful in the evaluation of Creutzfeldt-Jakob disease. CASE PRESENTATION: A 61-year-old woman with rapidly progressive dementia was referred for cranial magnetic resonance imaging. Diffusion-weighted images were obtained as part of the examination. Brain biopsy confirmed the diagnosis of Creutzfeldt-Jakob disease histologically. FINDINGS AND CONCLUSIONS: The diffusion-weighted magnetic resonance brain images demonstrated bilaterally symmetrical marked increase in signal intensity in the caudate nuclei, putamina, thalami, cingulate gyri, and right inferior frontal cortex. The apparent diffusion coefficient map showed abnormally low diffusion in these regions (as low as 40% of normal in the caudate head). This suggests that there is restricted diffusion in these regions. The T2-weighted images demonstrated slightly increased signal bilaterally in the caudate nuclei and putamina. These findings indicate that diffusion magnetic resonance imaging might be a sensitive means of imaging the abnormalities seen in Creutzfeldt-Jakob disease.

Developmental expression of keratan sulfate-like immunoreactivity distinguishes thalamic nuclei and cortical domains.

Proteoglycans influence axonal outgrowth in several experimental paradigms, and their distribution during development suggests a role in axon guidance. We have used a monoclonal antibody, 5D4, that recognizes an epitope on sulfated keratans (KS), to define the distribution of keratan sulfate proteoglycans (KSPGs) in the developing thalamus and cortex of the rat. During development, 5D4 immunolabeling is present on thalamic axons as they grow through the internal capsule and subplate but is not present in the adjacent pathway for cortical efferent axons. Individual thalamic nuclei differ markedly in their expression of KSPGs; these distinctions persist throughout the period of developmentally regulated expression. Major cortical domains also differ in their expression of KSPGs, which are expressed throughout medial (cingulate and retrosplenial) cortex well before neocortex. Immunolabeling for KSPGs diminishes 2 weeks after birth; in the adult it is associated with small glia. The 5D4 epitope is present on several KSPGs (320, 220, and 160 kD) on Western blots during development but only in a broad 200-kD band in adult brain. Immunolabeling is degraded on sections and Western blots by keratanase II but not by keratanase I or chondroitinase ABC, confirming that the antibody recognizes KS. Bands identified by 5D4 on Western blots differ from those identified by antibodies to known KSPGs (aggrecan, claustrin, SV2, ABAKAN, phosphacan-KS), indicating that 5D4 is labeling KSPGs not previously described in the brain. The selective expression of KSPGs during development suggests that they may be a part of the molecular identity of thalamic nuclei and cortical domains that defines their connectivity.

Neuronal heterotopias in the developing cerebral cortex produced by neurotrophin-4.

The marginal zone (MZ) of embryonic neocortex is crucial to its normal development. We report that neurotrophin-4 (but not NT3 or NGF), applied to embryonic rodent cortex in vitro or in vivo, produces heterotopic accumulations of neurons in the MZ. Although heterotopia production is TrkB mediated, BDNF is >10-fold less effective than NT4. Heterotopic neurons have the same birth date and phenotype as normal MZ neurons; they are not the result of NT4-induced proliferation or rescue from apoptosis. We suggest that NT4 causes excess neurons to migrate into the MZ and thus may play a role in normal MZ formation as well as in the pathogenesis of certain human cortical dysplasias.

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.

Automated recognition and mapping of immunolabelled neurons in the developing brain.

The cerebral cortex is distinguished by layers of neurons of different morphologies and densities. The layers are formed by the migration of newly generated neurons from the ventricular zone to the cortical plate near the outer (pial) boundary of the cortex, along radial paths approximately perpendicular to the cortical surface. Immunochemical labelling makes these cells' patterns visible in brightfield microscopy so that layer formation can be studied. We developed a suite of programs that automatically digitize the entire cortex, identify the labelled cells and compute cell densities along local radial paths. Cell identification used supervised classification on all the significantly stained objects corresponding to maxima in lowpass filtered versions of the digital microgrphs. Classification of all the stained objects as cells or noncell objects was made by a decision rule based on morphometric and grey-level texture features, including features based on Gabor functions. Detection sensitivity and classification accuracy were jointly maximized on training data consisting of about 3000 expert-identified neurons in micrographs. Total program performance was tested on a separate (test) set of labelled neurons the same size as the training data set. The program detected 85% of the cells in the test set with a total error of 0.19. The identified cells' locations were used to compute population densities along normals to the cortical layers, and these densities served as a measure of neuronal migration. Transcortical density profiles obtained by computation and by manual cell counting were very similar. The cell identification program was built on well-established methods in statistical pattern recognition and image analysis and should generalize readily to other histological preparations.

Extracellular matrix in early cortical development.

Studies of the distribution and production of ECM components during development of the cerebral cortex have suggested several hypotheses regarding their functional role. In the earliest stages of cortical development, fibronectin is produced by cells in the ventricular zone throughout the telencephalic vesicle, where it may serve as a part of the local environment that supports cell division and determines cell fate. Fibronectin is also distributed along radial glial processes. It is closely associated with preplate neurons, as are chondroitin sulfate proteoglycans and several other ECM components. This association continues as preplate cells are divided into the marginal zone and subplate by the invasion of cortical plate neurons, suggesting that ECM, preplate cells and radial glia serve as a scaffold for cortical plate formation. Fibronectin is also produced by migrating neurons, but only by those moving into specific cortical domains, suggesting that it may help neurons destined for specific targets discriminate between adjacent glial guides. A recently defined ECM-like protein, reelin, is absent or abnormal in the reeler mutant mouse in which cortical neurons are severely malpositioned. Reelin is produced by marginal zone cells and is therefore appropriately located to serve as a stop signal for migrating neurons. Axons leaving the cortical plate cross the CSPG-rich subplate, then turn to follow a path containing much less CSPG. In contrast, the cortical trajectory of thalamic axons is centered on the subplate, indicating that CSPGs in the subplate are not a barrier to axon outgrowth and may instead be serving as guidance cues that distinguish afferent from efferent pathways. Neurocan, a CNS-specific CSPG with many molecular features that indicate roles in cell-cell and cell-substrate interactions, is the only CSPG defined to date whose distribution supports a role in distinguishing afferent from efferent pathways.

Neuronal production of fibronectin in the cerebral cortex during migration and layer formation is unique to specific cortical domains.

The distribution of fibronectin (FN) changes rapidly during early development of the cerebral cortex, but its cellular source is not known. With in situ hybridization we find two spatially and temporally distinct periods of FN mRNA expression in the embryonic and early postnatal cortex of the mouse. Before and during formation of the preplate by the first postmitotic neurons, FN mRNA levels are high throughout the telencephalic vesicle, deep in the neuroepithelial proliferative zone that contains dividing cells and the cell bodies of radial glia; expression in the cortical proliferative zone is limited to the period of neurogenesis. Just after the cortical plate is formed within the preplate, FN mRNA is expressed in the intermediate zone, which contains migrating neurons, and in the cortical plate, where neurons migrate past their predecessors to form layers. Brefeldin A treatment of an organotypic slice preparation demonstrates FN production in the intermediate zone and cortical plate, in locations that correspond exactly to the distribution of FN mRNA by in situ hybridization. FN-producing cells immunolabel with neuron-specific markers; in the intermediate zone and lower cortical plate they have morphological features characteristic of migrating neurons and are closely apposed to radial glia. FN mRNA expression and protein production continue in neurons of the cortical plate through the period of layer formation and then are downregulated. Examination of dissociated cortical cells by laser confocal microscopy confirms that FN accumulation after brefeldin A treatment is intracellular in neurons as well as in glia. Neuroepithelial expression of FN mRNA takes place throughout the telencephalon; FN produced by neurons is restricted to cells migrating toward and into specific cortical domains that include neocortex, insular and perirhinal cortex, and subiculum. Thus FN may be involved initially in supporting the cell division and fate determination that takes place in the neuroepithelium; later production by migrating neurons may play a role in the selection of radial glial pathways that lead to specific cortical regions, and in interactions between neurons as they form cortical layers within these regions.

Chondroitin sulfate proteoglycans in the developing cerebral cortex: the distribution of neurocan distinguishes forming afferent and efferent axonal pathways.

The first thalamocortical axons to arrive in the developing cerebral cortex traverse a pathway that is separate from the adjacent intracortical pathway for early efferents, suggesting that different molecular signals guide their growth. We previously demonstrated that the intracortical pathway for thalamic axons is centered on the subplate (Bicknese et al. [1994] J. Neurosci. 14:3500-3510), which is rich in chondroitin sulfate proteoglycans (CSPGs; Sheppard et al. [1991] J. Neurosci. 11:3928-3942), whereas efferent axons cross the subplate to exit in a zone containing much less CSPG. To define the molecular composition of the subplate further, we used antibodies against CSPG core proteins and chondroitin sulfate disaccharides in an immunohistochemical analysis of their distribution in the developing neocortex of the rat. Immunolabeling for neurocan, a central nervous system-specific CSPG (Rauch et al. [1992] J. Biol. Chem. 267:19537-19547), and for chondroitin 6-sulfate and unsulfated chondroitin becomes prominent in the subplate before the arrival of thalamic afferents. Immunolabeling is initially sparse in the cortical plate but appears later in maturing cortical layers. A postnatal decline in immunolabeling occurs uniformly for most proteoglycans, but, in the somatosensory cortex, labeling for neurocan, phosphacan, and chondroitin 4- and 6-sulfate declines in the centers of the whisker barrels before the walls. In contrast to neurocan, immunolabeling for other proteoglycans is either uniformly distributed (syndecan-1, N-syndecan, 5F3, phosphacan, chondroitin 4-sulfate), restricted to axons (PGM1), distributed exclusively on nonneuronal elements (2D6, NG2, and CD44), or undetectable (9.2.27, aggrecan, decorin). Thus, neurocan is a candidate molecule for delineating the intracortical pathway of thalamocortical axons and distinguishing it from that of cortical efferents.

Thalamocortical axons extend along a chondroitin sulfate proteoglycan-enriched pathway coincident with the neocortical subplate and distinct from the efferent path.

The distinct axonal tracts of the mature nervous system are defined during development by sets of substrate-bound and diffusible molecular signals that promote or restrict axonal elongation. In the adult cerebral cortex, efferent and afferent axons are segregated within the white matter. To define the relationship of growing efferent and afferent axons in the developing murine cortex to chondroitin sulfate proteoglycans (CSPGs) in the pericellular and extracellular matrix, we used the fluorescent tracer Dil to determine axonal trajectories and immunolabeling to disclose the distribution of CSPGs. Axons of neurons in the preplate are the first to leave the cortex; they arise in the CSPG-rich preplate and extend obliquely across it to enter the CSPG-poor intermediate zone. Slightly later, axons of cortical plate neurons extend directly across the CSPG-rich subplate, and then turn abruptly to run in the upper intermediate zone. In contrast, once afferent axons from the thalamus reach the developing cortical wall, their intracortical trajectory is centered on the CSPG-rich subplate, above the path taken by efferent axons. Our findings demonstrate a molecular difference between the adjacent but distinct efferent and afferent pathways in developing neocortex. Early efferents cross the subplate and follow a pathway that contains very little CSPG, while afferents preferentially travel more superficially within the CSPG-rich subplate. Thus, CSPGs and associated extracellular matrix (ECM) components in the preplate/subplate do not form a barrier to axonal initiation or outgrowth in the neocortex as they may in other locations. Instead, their distribution suggests a role in defining discrete axonal pathways during early cortical development.

EMA: a developmentally regulated cell-surface glycoprotein of CNS neurons that is concentrated at the leading edge of growth cones.

To identify cell-surface molecules that mediate interactions between neurons and their environment during neural development, we used monoclonal antibody techniques to define a developmentally regulated antigen in the central nervous system of the mouse. The antibody we produced (2A1) immunolabels cells throughout the central nervous system; we analyzed its distribution in the developing cerebral cortex, where it is expressed on cells very soon after they complete mitosis and leave the periventricular proliferative zone. Expression continues into adult life. The antibody also labels the epithelium of the choroid plexus and the renal proximal tubules, but does not label neurons of the peripheral nervous system in the dorsal root ganglia. In dissociated cell culture of embryonic cerebral cortex, 2A1 labels the surface of neurons but not glia. Immunolabeling of neurons in tissue culture is particularly prominent on the edge of growth cones, including filopodia and the leading edge of lamellipodia, when observed with either immunofluorescence or freeze-etch immunoelectron microscopy. Immunopurification with 2A1 of a CHAPS-extracted membrane preparation from brains of neonatal mice produces a broad (32-36 kD) electrophoretic band and a less prominent 70 kD band that are sensitive to N-glycosidase but not endoglycosidase H. Thus the 2A1 antibody recognizes a developmentally regulated, neuronal cell surface glycoprotein (or glycoproteins) with complex N-linked oligosaccharide side chains. We have termed the glycoprotein antigen EMA because of its prominence on the edge membrane of growth cones. EMA is similar to the M6 antigen (Lagenaur et al: J. Neurobiol. 23:71-88, 1992) in apparent molecular weight, distribution in tissue sections, and immunoreactivity on Western blots, suggesting that the two antigens are similar or identical. Expression of EMA is a very early manifestation of neuronal differentiation; its distribution on growth cones suggests a role in mediating the interactions between growth cones and the external cues that guide them.

Extension of filopodia by motor-dependent actin assembly.

A variety of mechanisms have been proposed to explain the forward extension of cytoplasm in advancing cells and axonal growth cones, including actin polymerization and osmotic swelling. Based on our observations of the filopodia of cultured neuronal growth cones, we propose a mechanism involving motor-induced extension and retraction. We observed that filopodia (actin-based protrusions 0.2-0.5 mu in diameter) extend and retract from growth cone lamellae at the same rate. Further, force is generated at the tips of filopodia which is sufficient to produce compressive buckling of the proximal portion of the filopodium. From our analysis of these movements we suggest that a motor protein powers both the extension and retraction of filopodia.

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.

Concentration of membrane antigens by forward transport and trapping in neuronal growth cones.

Formation of the nervous system requires that neuronal growth cones follow specific paths and then stop at recognition signals, sensed at the growth cone's leading edge. We used antibody-coated gold particles viewed by video-enhanced differential interference contrast microscopy to observe the distribution and movement of two cell surface molecules, N-CAM and the 2A1 antigen, on growth cones of cultured cortical neurons. Gold particles are occasionally transported forward at 1-2 microns/s to the leading edge where they are trapped but continue to move. Concentration at the edge persists after cytochalasin D treatment or ATP depletion, but active movements to and along edges cease. We also observed a novel outward movement of small cytoplasmic aggregates at 1.8 microns/s in filopodia. We suggest that active forward transport and trapping involve reversible attachment of antigens to and transport along cytoskeletal elements localized to edges of growth cones.

Cortical radial glia: identification in tissue culture and evidence for their transformation to astrocytes.

Radial glia are transiently present in the developing cerebral cortex, where they are thought to guide the migration of neurons from the proliferative zone to the forming cortical plate. To provide a framework for experimental studies of radial glia, we have defined morphological and immunocytochemical criteria to identify them in primary cultures of cortical cells obtained at embryonic day 13 in the mouse. Cortical radial glia in culture for 1-2 d resemble radial glia in vivo: they have a long, thin, unbranched process extending from one or both ends of the elongated cell body and are labeled with the monoclonal antibody RC1 but not with antibodies to glial fibrillary acidic protein (abGFAP). We tested the specificity of RC1 by double-labeling with a panel of cell-type specific antibodies, and found that it labels radial glia, astrocytes, and fibroblast-like cells, but not neurons. Fibroblasts are easily distinguished from glia by morphology and by labeling with antibodies to fibronectin. To test the hypothesis that radial glia become astrocytes when their developmental role is complete, we examined their morphological and immunocytochemical development in culture. After 3-4 d in vitro radial glia develop several branched processes; in this transitional stage they are labeled by both RC1 and abGFAP. Many radial glia lose RC1 immunoreactivity as they become increasingly branched and immunoreactive to abGFAP. In areas of the cultures that have few neurons and in cultures depleted of neurons by washing, flat, nonprocess-bearing glia predominate. These cells do not lose immunoreactivity to RC1 during the 9-d period of observation even though they acquire GFAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell lineage in the cerebral cortex of the mouse studied in vivo and in vitro with a recombinant retrovirus.

To analyze cell lineage in the murine cerebral cortex, we infected progenitor cells with a recombinant retrovirus, then used the retroviral gene product to identify the descendants of infected cells. Cortices were infected on E12-E14 either in vivo or following dissociation and culture. In both cases, nearly all clones contained either neurons or glia, but not both. Thus, neuronal and glial lineages appear to diverge early in cortical development. To analyze the distribution of clonally related cells in vivo, clonal boundaries were reconstructed from serial sections. Perinatally (E18-PN0), clonally related cells were radially arrayed as they migrated to the cortical plate. Thus, clonal cohorts traverse a similar radial path. Following migration (PN7-PN23), neuronal clones generally remained radially arrayed, while glial clones were variable in orientation, suggesting that these two cell types accumulate in different ways. Neuronal clones sometimes spanned the full thickness of the cortex. Thus, a single progenitor can contribute neurons to several laminae.

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)

Toward a unified monitoring system during anesthesia.

A conceptual framework is proposed for the selection of monitored parameters during anesthesia, and a new device for monitoring the parameters in a unified manner is briefly presented. A 'basic set' of 6 parameters is proposed to cover the needs of most routine anesthesia: Blood Pressure, ECG/Heart Rate, Temperature, FiO2, FetCO2, and, Cortical Activity (by EEG spectral analysis). Additional parameters are added in accordance with specified factors such as patient status and complexity of the surgical procedure. An initial version of a new monitor, 'Cerebro Trac', designed for neurosurgery and cardiovascular surgery, is briefly presented, along with planned future capabilities and directions for its use.