Lower Intermediate Zone Research Articles

Adenosine A2A Receptors Contribute to the Radial Migration of Cortical Projection Neurons through the Regulation of Neuronal Polarization and Axon Formation.

Cortical interneurons born in the subpallium reach the cortex through tangential migration, whereas pyramidal cells reach their final position by radial migration. Purinergic signaling via P2Y1 receptors controls the migration of intermediate precursor cells from the ventricular zone to the subventricular zone. It was also reported that the blockade of A2A receptors (A2AR) controls the tangential migration of somatostatin+ interneurons. Here we found that A2AR control radial migration of cortical projection neurons. In A2AR-knockout (KO) mouse embryos or naïve mouse embryos exposed to an A2AR antagonist, we observed an accumulation of early-born migrating neurons in the lower intermediate zone at late embryogenesis. In utero knockdown of A2AR also caused an accumulation of neurons at the lower intermediate zone before birth. This entails the presently identified ability of A2AR to promote multipolar-bipolar transition and axon formation, critical for the transition of migrating neurons from the intermediate zone to the cortical plate. This effect seems to require extracellular ATP-derived adenosine since a similar accumulation of neurons at the lower intermediate zone was observed in mice lacking ecto-5'-nucleotidase (CD73-KO). These findings frame adenosine as a fine-tune regulator of the wiring of cortical inhibitory and excitatory networks.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

Cdk5 regulates N-cadherin-dependent neuronal migration during cortical development

Cyclin-dependent kinase 5 (Cdk5) controls neuronal migration in the developing cortex when multipolar newborn neurons transform to become bipolar. However, by which mechanisms Cdk5 controls cell adhesion in migrating neurons are not fully understood. In this study, we examined the functional interaction between Cdk5 and N-cadherin (Ncad) in newborn neurons when they undergo the multipolar to bipolar transition in the intermediate zone (IZ). Detailed expression analysis revealed that both Cdk5 and Ncad were present in GFP-electroporated migrating neurons in the IZ. Misexpression of dominant negative Cdk5 into the embryonic brains stalled neuronal locomotion in the lower IZ in which arrested cells were round or multipolar. When Ncad was co-introduced with Cdk5DN, however, cells continue to migrate into the cortical plate (CP) and migrating neurons acquired typical bipolar morphology with a pia-directed leading process. Similarly, downregulation of CDK5 resulted in lesser aggregation ability, reversed by the expression of Ncad in vitro. Down-regulation of activity or protein level of CDK5 did not alter the total amount of NCAD proteins but lowered its surface expression in cells. Lastly, expression of CDK5 and NCAD overlapped in the IZ of the human fetal cortex, indicating that the role of Cdk5 and Ncad in neuronal migration is evolutionarily conserved.

Human Cytomegalovirus IE2 Protein Disturbs Brain Development by the Dysregulation of Neural Stem Cell Maintenance and the Polarization of Migrating Neurons.

Despite the high incidence of severe defects in the central nervous system caused by human cytomegalovirus (HCMV) congenital infection, the mechanism of HCMV neuropathogenesis and the roles of individual viral genes have not yet been fully determined. In this study, we show that the immediate-early 2 (IE2) protein may play a key role in HCMV-caused neurodevelopmental disorders. IE2-transduced neural progenitor cells gave rise to neurospheres with a lower frequency and produced smaller neurospheres than control cells in vitro, indicating reduction of self-renewal and expansion of neural progenitors by IE2. At 2 days after in utero electroporation into the ventricle of the developing brain, a dramatically lower percentage of IE2-expressing cells was detected in the ventricular zone (VZ) and cortical plate (CP) compared to control cells, suggesting that IE2 concurrently dysregulates neural stem cell maintenance in the VZ and neuronal migration to the CP. In addition, most IE2+ cells in the lower intermediate zone either showed multipolar morphology with short neurites or possessed nonradially oriented processes, whereas control cells had long, radially oriented monopolar or bipolar neurites. IE2+ callosal axons also failed to cross the midline to form the corpus callosum. Furthermore, we provide molecular evidence that the cell cycle arrest and DNA binding activities of IE2 appear to be responsible for the increased neural stem cell exit from the VZ and cortical migrational defects, respectively. Collectively, our results demonstrate that IE2 disrupts the orderly process of brain development in a stepwise manner to further our understanding of neurodevelopmental HCMV pathogenesis.IMPORTANCE HCMV brain pathogenesis has been studied in limited experimental settings, such as in vitro HCMV infection of neural progenitor cells or in vivo murine CMV infection of the mouse brain. Here, we show that IE2 is a pivotal factor that contributes to HCMV-induced abnormalities in the context of the embryonic brain using an in utero gene transfer tool. Surprisingly, IE2, but not HCMV IE1 or murine CMV ie3, interferes pleiotropically with key neurodevelopmental processes, including neural stem cell regulation, proper positioning of migrating neurons, and the callosal axon projections important for communication between the hemispheres. Our data suggest that the wide spectrum of clinical outcomes, ranging from mental retardation to microcephaly, caused by congenital HCMV infection can be sufficiently explained in terms of IE2 action alone.

C3G/Rapgef1 Is Required in Multipolar Neurons for the Transition to a Bipolar Morphology during Cortical Development.

The establishment of a polarized morphology is essential for the development and function of neurons. During the development of the mammalian neocortex, neurons arise in the ventricular zone (VZ) from radial glia cells (RGCs) and leave the VZ to generate the cortical plate (CP). During their migration, newborn neurons first assume a multipolar morphology in the subventricular zone (SVZ) and lower intermediate zone (IZ). Subsequently, they undergo a multi-to-bipolar (MTB) transition to become bipolar in the upper IZ by developing a leading process and a trailing axon. The small GTPases Rap1A and Rap1B act as master regulators of neural cell polarity in the developing mouse neocortex. They are required for maintaining the polarity of RGCs and directing the MTB transition of multipolar neurons. Here we show that the Rap1 guanine nucleotide exchange factor (GEF) C3G (encoded by the Rapgef1 gene) is a crucial regulator of the MTB transition in vivo by conditionally inactivating the Rapgef1 gene in the developing mouse cortex at different time points during neuronal development. Inactivation of C3G results in defects in neuronal migration, axon formation and cortical lamination. Live cell imaging shows that C3G is required in cortical neurons for both the specification of an axon and the initiation of radial migration by forming a leading process.

Pioneering Axons Regulate Neuronal Polarization in the Developing Cerebral Cortex

The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.

The Suppression of CRMP2 Expression by Bone Morphogenetic Protein (BMP)-SMAD Gradient Signaling Controls Multiple Stages of Neuronal Development

The formation of the functional mammalian cerebral cortex requires a concerted control of neurogenesis, neuronal migration, and neuronal morphogenesis. However, molecular mechanisms that control these processes are not well understood. We have found that the BMP signaling downstream transcription factor SMAD1 and CRMP2 (collapsin response mediator protein-2) are inversely and complementarily expressed in the developing neocortex. BMPs can suppress CRMP2 expression in cortical cells. Our ChIP assay demonstrates that both SMAD1 and -4 bind to CRMP2 promoter in the neocortex, and overexpression of SMAD1 and 4 in vivo suppresses CRMP2 expression. RNA interference of CRMP2 and overexpression of dominant negative forms of CRMP2 in utero cause accumulation of multipolar cells in the ventricular, subventricular, and intermediate zones and suppresses neurite outgrowth, suggesting that CRMP2 is required for multipolar to bipolar transition for directional neuronal migration and neurite outgrowth. Thus, our study reveals a novel mechanism that the BMP-SMAD signaling pathway controls neuronal migration and neurite outgrowth by suppressing the transcription of CRMP2.

Migration of GABAergic interneurons in the cortex: multidirectional migration and roundabout migratory pathway

Event Abstract Back to Event Migration of GABAergic interneurons in the cortex: multidirectional migration and roundabout migratory pathway Fujio Murakami1* 1 Osaka University, Graduate School of Frontier Biosciences, Japan It is well documented that cortical interneurons originate from the basal forebrain and tangentially migrate to the cortex. However, relatively little is known about their migration after their arrival at the cortex. To elucidate the route and mode of intracortical interneuron migration, we performed real-time analysis by utilizing glutamate decarboxylase 67-green fluorescence protein knock-in mice and an electroporation-based gene transfer of dsRed into the ganglionic eminence (GE) of mouse embryos. In coronal slices, ventrolateral-to-dorsomedial migration predominantly occurs in the lower-intermediate zone. Observations of the marginal zone (MZ) or the ventricular zone in flat-mounted cortex from the pial or the ventricular surface, respectively, reveal that that the interneurons tangentially migrate in all directions. Medial GE-derived interneurons visualized by dsRed electroporation exhibit a diverse range of behaviors in terms of the rate and direction of migration. Curiously, a predominant population of these neurons repeatedly change their direction of migration in an unpredictable manner. Trajectories of migration vary from one neuron to another. The migration of individual cells lasts for long periods, sometimes up to two days. Theoretical analyses reveal that these behaviors can be modeled by a random walk. Furthermore, MZ cells migrate from the cortical subventricular zone to the cortical plate, transiently accumulating in the MZ. These results suggest that MGE-derived cortical interneurons, once arriving at the MZ, are released from regulation by guidance cues and initiate random walk movement, which potentially contributes to their dispersion throughout the cortex. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Oral Presentation Topic: Plenary Lectures Citation: Murakami F (2010). Migration of GABAergic interneurons in the cortex: multidirectional migration and roundabout migratory pathway. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00205 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 May 2010; Published Online: 04 May 2010. * Correspondence: Fujio Murakami, Osaka University, Graduate School of Frontier Biosciences, Osaka, Japan, murakami@fbs.osaka-u.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Fujio Murakami Google Fujio Murakami Google Scholar Fujio Murakami PubMed Fujio Murakami Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Oversulfated Chondroitin Sulfate Plays Critical Roles in the Neuronal Migration in the Cerebral Cortex

Chondroitin sulfate (CS) proteoglycans bind with various proteins through CS chains in a CS structure-dependent manner, in which oversulfated structures, such as iB (IdoA(2-O-sulfate)alpha1-3GalNAc(4-O-sulfate)), D (GlcA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate)), and E (GlcAbeta1-3GalNAc(4,6-O-disulfate)) units constitute the critical functional module. In this study, we examined the expression and function of three CS sulfotransferases in the developing neocortex: uronyl 2-O-sulfotransferase (UST), N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (4,6-ST) and dermatan 4-O-sulfotransferase-1 (D4-ST), which are responsible for the synthesis of oversulfated structures. The CS chains of the neocortex of mouse embryos contained significant amounts of D and E units that are generated by UST and 4,6-ST, respectively. UST and 4,6-ST mRNAs were expressed in the ventricular and subventricular zones, and their expression increased during late embryonic development. In utero electroporation experiments indicated that knockdown of UST and 4,6-ST resulted in the disturbed migration of cortical neurons. The neurons electroporated with the short hairpin RNA constructs of UST and 4,6-ST accumulated in the lower intermediate zone and in the subventricular zone, showing a multipolar morphology. The cDNA constructs of UST and 4,6-ST rescued the defects caused by the RNA interference, and the neurons were able to migrate radially. On the other hand, knockdown of D4-ST, which is involved in the biosynthesis of the iB unit, caused no migratory defects. These results revealed that specific oversulfated structures in CS chains play critical roles in the migration of neuronal precursors during cortical development.

The Oral Cavity of the Adult Zebrafish (Danio rerio)

The zebrafish is a common model for developmental studies including those regarding tooth, palate and tongue. Nevertheless, little information is available about the morphology of the oral cavity in this teleost, especially in adult animals. In this study we used light, scanning and transmission electron microscopy to describe in detail the morphology of the oral cavity of adult zebrafish. The oral cavity could be divided into three different zones: the outer containing the lips, the intermediate corresponding to the internal valves and the internal that corresponds to the tongue. In the upper and lower intermediate zones, there were semilunar shaped valves, more prominent in the upper part. The internal lower zones correspond to the palate and the tongue, which is an individualized structure filled with numerous transversal ridges. Both the intermediate and internal zones were covered by a stratified epithelium containing numerous mucous and rodlet cells. Present data provide the first description of the morphology and structure of the oral cavity in the adult zebrafish and might serve as a baseline for developmental studies of the oral cavity using this teleost as a model.

Morphological and Immunohistochemical Study of the Zebrafish (Danio rerio) Oral Cavity

The oral cavity in vertebrates plays a very important role in food intake mechanics and also contains chemical sensors for food and environmental water. Comparative studies revealed morphological and functional inter‐specific differences that can be correlated to evolutionary events and might represent the adaptation to environmental conditions present in different habitats (Iwasaki, 2002). Particularly these events are strictly dependent on the food intake mechanisms determining the success of the adaptation of vertebrates to their environment and therefore their survival (Roth and Wake, 1989). Since data about the morphology of the zebrafish oral cavity are scarce the aim of the present research was to study it by light, scanning and transmission electron microscopy and immunohistochemistry for some growth factor receptors, i.e. trkA and trkB. The zebrafish was chosen because as a vertebrate it has valuable features as a model for study of vertebrate development (Withfield, 2002). Adult zebrafishes were anesthesized with MS222 and the samples were taken and routinely processed for light and electron microscopy and for indirect peroxidase immunohistochemistry. The topographical and ultrastructural study demonstrated the presence of three different zones. The outer zone is characterized orally by the presence of a papilla and by an epithelium with numerous taste buds that are chemosensory endorgans consisting of modified epithelial cells. Their function is to sample potential food and selecting it according to its edibility (Hansen et al., 2002). Both in upper and lower intermediate zones, the presence of internal valves with a semi‐lunar shape was evident, probably able to regulate the mouth closure. The most internal lower zone corresponds to the tongue that in zebrafish is only a thickening of the pavement mucosa with a stratified epithelium. Immunohistochemistry demonstrated both in upper and lower zones the presence of numerous taste buds that were immunoreactive to TrkA and TrkB‐like neurotrophin receptors, confirming the role of these receptors in taste buds sensory cells (Germanà et al., 2004). The present results give a contribution to the morphological knowledge of the zebrafish oral cavity, an animal model commonly used for developmental studies of vertebrates.

Phosphorylation of Neurogenin2 Specifies the Migration Properties and the Dendritic Morphology of Pyramidal Neurons in the Neocortex

The molecular mechanisms specifying the dendritic morphology of different neuronal subtypes are poorly understood. Here we demonstrate that the bHLH transcription factor Neurogenin2 (Ngn2) is both necessary and sufficient for specifying the dendritic morphology of pyramidal neurons in vivo by specifying the polarity of its leading process during the initiation of radial migration. The ability of Ngn2 to promote a polarized leading process outgrowth requires the phosphorylation of a single tyrosine residue at position 241, an event that is neither involved in Ngn2 direct transactivation properties nor its proneural function. Interestingly, the migration defect observed in the Ngn2 knockout mouse and in progenitors expressing the Ngn2(Y241F) mutation can be rescued by inhibiting the activity of the small-GTPase RhoA in cortical progenitors. Our results demonstrate that Ngn2 coordinates the acquisition of the radial migration properties and the unipolar dendritic morphology characterizing pyramidal neurons through molecular mechanisms distinct from those mediating its proneural activity.

Multimodal tangential migration of neocortical GABAergic neurons independent of GPI-anchored proteins.

Neuronal migration is crucial for the construction of neuronal architecture such as layers and nuclei. Most inhibitory interneurons in the neocortex derive from the basal forebrain and migrate tangentially; however, little is known about the mode of migration of these neurons in the cortex. We used glutamate decarboxylase (Gad)67-green fluorescent protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)-ergic neurons and performed time-lapse analysis. In coronal slices, many GFP-positive neurons in the lower intermediate zone (IZ) and subventricular zone (SVZ) showed robust tangential migration from lateral to medial cortex, while others showed radial and non-radial migration mostly towards the pial surface. In flat-mount preparations, GFP-positive neurons of the marginal zone (MZ) showed multidirectional tangential migration. Some of these neurons descended toward the cortical plate (CP). Intracortical migration of these neurons was largely unaffected by a treatment that cleaves glycosylphosphatidylinositol (GPI) anchors. These findings suggest that tangential migration of cortical interneurons from lateral to medial cortex predominantly occurs in the IZ/SVZ and raise the possibility that a part of the pial surface-directed neurons in the IZ/SVZ reach the MZ, whereby they spread into the whole area of the cortex. At least a part of these neurons may descend toward the CP. Our results also suggest that intracortical migration of GABAergic neurons occurs independent of GPI-anchored proteins.

Blockade of GABA(B) receptors alters the tangential migration of cortical neurons.

To better understand the role of neurotransmitter receptors in neuronal differentiation and maturation a detailed knowledge of their identity, location and function in the plasma membrane of specific neuronal populations during development is required. Combining pre-embedding immunocytochemistry with cell tracking in embryonic brain slice cultures we show that virtually all neurons (approximately 98%) migrating through the lower intermediate zone (LIZ) on their way from the medial ganglionic eminence to the cerebral cortex, express GABA(B)R1. Blockade of GABA(B)Rs with a specific antagonist, CGP52432, resulted in a concentration-dependent accumulation of these tangentially migrating neurons in the ventricular/sub-ventricular zones (VZ/SVZ) of the cortex and fewer cells were observed in the cortical plate/marginal zone (CP/MZ) and LIZ. Moreover, they had significantly shorter leading processes compared with similar migrating cells in control slices. Electrophysiological recording in LIZ and CP cells revealed no direct effect of either CGP52432 or the GABA(B)R agonist, baclofen, on resting membrane properties suggesting that the effect of CGP52432 on migration might be mediated through a metabotropic action or the regulation of release of factors controlling migration. These results suggest that GABA(B)Rs have an important modulatory role in the migration of cortical interneurons.

Evidence that Sema3A and Sema3F regulate the migration of GABAergic neurons in the developing neocortex.

The ganglionic eminence (GE) supplies neurons containing gamma-aminobutyric acid (GABA) to the pallium of the telencephalon. We investigated the molecular guidance mechanisms of GE cell migration in the neocortex and found neuropilin-1 (Npn-1) or neuropilin-2 (Npn-2) on the GE cells. Ectopic Sema3A or -3F expression by COS1 cell clusters placed on embryo neocortical slices reduced the cell migration but did not block it completely. However, the cell migration was almost completely blocked by COS1 cell clusters expressing both Sema3A and -3F. The direction of cell migration could be reversed by placing Sema3A- and -3F-coexpressing COS1 cell clusters at the distal cut end of the neocortical slices. Further slice experiments revealed that migration of half of the GE cells in the neocortex was regulated by Sema3A and that migration of the other half of the GE cells in the neocortex was regulated by Sema3F. When the cells responding to Sema3A were diverted by ectopic Sema3A expression in vivo, Dlx2-positive cells were found predominantly in the lower intermediate zone (IZ). When the cells responding to Sema3F were diverted by ectopic Sema3F expression in vivo, Dlx2-positive cells were found predominantly in the upper IZ. It was speculated that the semaphorin-neuropilin interactions distribute the GABAergic GE cells evenly in the neocortex as well as guide the GE cells from the GE to the neocortex. The Sema3A expression site under the subplate extended dorsally as the embryo developed. The Sema3A expression seemed to block the Npn-1-positive GE cells in the neocortex from entering the cortical plate (CP) and guide them to the dorsal cortex and the hippocampus. Sema3F expression in the CP continued through the embryonic stages. The expression seemed to block Npn-2-positive GE cells in the neocortex from entering the CP and make them migrate into the lower IZ. Finally, the semaphorin-neuropilin interactions sorted GABAergic inteneurons into the CP and white matter neurons into the IZ.

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

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

Managing the assessment of neonatal jaundice: importance of timing.

In view of the limitations in the accurate visual assessment of jaundice and its potential role as a predictive vector for serious neurologic sequelae, we propose that a universal screening of bilirubin be considered concurrent to the routine pre-discharge metabolic screening. Universal bilirubin screening in the term and near-term newborns when plotted on "Hour-specific Bilirubin Nomogram" in lieu of the usual "day-specific" value will predict the high-risk and the low-risk groups and facilitate cost-effective and individualized follow-up of those babies at risk. A percentile based bilirubin nomogram for the first week of age was constructed from hour-specific pre- and post-discharge bilirubin values of 2840 healthy term and near-term babies. The accuracy of the pre-discharge bilirubin values was determined as a predictive vector. Pre-discharge (18-72 hours age), 6.1% of the study population had bilirubin values in the high-risk zone (> 95th percentile). Of these, 39.5% remained in that zone (likelihood ratio ¿LR¿ = 14.08). Pre-discharge, 32.1% of the study population had bilirubin values in the intermediate risk zone (40-75th percentiles). In a clinically significant minority of these babies (6.4%), the post-discharge values moved to the high-risk zone (L-R = 3.2 for the move from the upper-intermediate zone and 0.48 from the lower-intermediate zone). In the remainder 61.8% of the population who were identified to be at low risk, there was no measurable risk for significant hyperbilirubinemia (L-R = 0). The bilirubin nomogram can predict which infant is at high, intermediate, and low risk for subsequent excessive hyperbilirubinemia and allows for the individualized follow-up of these high-risk babies with particular attention to those who may need evaluation and intervention. Whereas, identification of the low risk group allows for a less intense bilirubin follow-up and in whom a visual check by an experienced care-provider may suffice.

Developmental history of the subplate and developing white matter in the murine neocortex. Neuronal organization and relationship with the main afferent systems at embryonic and perinatal stages.

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

The Medial Ganglionic Eminence Gives Rise to a Population of Early Neurons in the Developing Cerebral Cortex

During development of the neocortex, the marginal zone (layer I) and the subplate (layer VII) are the first layers to form from a primordial plexiform neoropil. The cortical plate (layers II-VI) is subsequently established between these superficial and deep components of the primordial plexiform neuropil. Neurons in the early zones are thought to play important roles in the formation of the cortex: the Cajal-Retzius cells of the marginal zone are instrumental in neuronal migration and laminar formation, and cells of the subplate are involved in the formation of cortical connections. Using the fluorescent tracer 1,1'-dioctodecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), we have shown here that a substantial proportion of neurons of the marginal zone, including cells with features of Cajal-Retzius cells, and of the subplate and lower intermediate zone are not born in the ventricular neuroepithelium but instead originate in the medial ganglionic eminence (MGE), the pallidal primordium. These neurons follow a tangential migratory route to their positions in the developing cortex. They express the neurotransmitter GABA but seem to lack the calcium binding protein calretinin; some migrating cells found in the marginal zone express reelin. In addition, migrating cells express the LIM-homeobox gene Lhx6, a characteristic marker of the MGE. It is suggested that this gene uniquely or in combination with other transcription factors may be involved in the decision of MGE cells to differentiate in situ or migrate to the neocortex.

Intermediate zone and intermediate zone neurons. Do IZ neurons acts as pioneers for layer V neurons in rats?

It is assumed that pioneer neurons will predominantly show path finding ability, and that other neurons only follow the pioneer fibers. Here, tracers were injected into the newborn rat spinal cord followed by a search for labeled cells other than those in layer V. In all cases, neurons in the lower intermediate zone (IZ) of the dorsomedial cortex were retrogradely labeled. The IZ neurons have several characteristics required for pioneer neurons and project to the spinal cord first. Thus, the IZ neurons may function as pioneer neurons of the corticospinal projection and help in the formation of the permanent tract in rats.