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Sonic Hedgehog (Shh) plays an instrumental role in brain development, fine-tuning processes such as cell proliferation, patterning, and fate specification. Although, mutations in the SHH pathway in humans are associated with various neurodevelopmental disorders, ranging from holoprosencephaly to schizophrenia, its expression pattern in the developing human brain is not well established. We now determined the previously not reported wide expression of SHH in the human fetal cerebral cortex during most of the gestation period (10–40 gestational weeks). This spatiotemporal distribution puts Shh in a position to influence the fundamental processes involved in corticogenesis. SHH expression increased during development, shifting from progenitor cells in the proliferative zones to neurons, both glutamatergic and GABAergic, and astrocytes in upper cortical compartments. Importantly, the expression of its downstream effectors and complementary receptors revealed evolutionary differences in SHH-pathway gene expression between humans and rodents. Electronic supplementary material The online version of this article (10.1007/s00429-018-1621-5) contains supplementary material, which is available to authorized users.

The efficient production of human neocortical neurons from human embryonic stem cells (hESC) is the primary requirement for studying early stages of human cortical development. We used hESC to obtain radial glial cells (hESC-RG) and then compared them with RG cells isolated from human fetal forebrain. Fate of hESC-RG cells critically depends on intrinsic and extrinsic factors. The expression of Pax6 (intrinsic factor) has a similar neurogenic effect on hESC-RG differentiation as reported for human fetal RG cells. Factors from the microenvironment also play a significant role in determining hESC-RG cell fate. In contrast to control cultures, wherein hESC-RG generate mainly astroglia and far fewer neurons, in co-cultures with human fetal forebrain cells, the reverse was found to be true. This neurogenic effect was partly due to soluble factors from human fetal brain cultures. The detected shift towards neurogenesis has significance for developing future efficient neuro-differentiation protocols. Importantly, we established that hESC-RG cells are similar in many respects to human fetal RG cells, including their proliferative capacity, neurogenic potential, and ability to generate various cortical neuronal sub-types. Unlike fetal RG cells, the hESC-RG cells are readily available and can be standardized, features that have considerable practical advantages in research and clinics.

The oxygen (O2) concentration is a vital parameter for controlling the survival, proliferation, and differentiation of neural stem cells. A prenatal reduction of O2 levels (hypoxia) often leads to cognitive and behavioral defects, attributable to altered neural development. In this study, we analyzed the effects of O2 levels on human cortical progenitors, the radial glia cells (RGCs), during active neurogenesis, corresponding to the second trimester of gestation. Small changes in O2 levels profoundly affected RGC survival, proliferation, and differentiation. Physiological hypoxia (3% O2) promoted neurogenesis, whereas anoxia (

An important feature of the cerebral cortex is its layered organization, which is modulated in an area-specific manner. We found that the transcription factor AP2gamma regulates laminar fate in a region-specific manner. Deletion of AP2gamma (also known as Tcfap2c) during development resulted in a specific reduction of upper layer neurons in the occipital cortex, leading to impaired function and enhanced plasticity of the adult visual cortex. AP2gamma functions in apical progenitors, and its absence resulted in mis-specification of basal progenitors in the occipital cortex at the time at which upper layer neurons were generated. AP2gamma directly regulated the basal progenitor fate determinants Math3 (also known as Neurod4) and Tbr2, and its overexpression promoted the generation of layer II/III neurons in a time- and region-specific manner. Thus, AP2gamma acts as a regulator of basal progenitor fate, linking regional and laminar specification in the mouse developing cerebral cortex.

Information about development of the human cerebral cortex (proliferation, migration, and differentiation of neurons) is largely based on postmortem histology. Physiological properties of developing human cortical neurons are difficult to access experimentally and therefore remain largely unexplored. Animal studies have shown that information about the arousal of electrical activity in individual cells within fundamental cortical zones (subventricular zone [SVZ], intermediate zone, subplate [SP], and cortical plate [CP]) is necessary for understanding normal brain development. Here we ask where, in what cortical zone, and when, in what gestational week (gw), human neurons acquire the ability to generate nerve impulses (action potentials [APs]). We performed electrical recordings from individual cells in acute brain slices harvested postmortem from the human fetal cerebral cortex (16--22 gw). Tetrodotoxin-sensitive Na 1 current occurs more frequently among CP cells and with significantly greater peak amplitudes than in SVZ. As early as 16 gw, a relatively small population of CP neurons (27%) was able to generate sodium APs upon direct current injection. Neurons located in the SP exhibited the highest level of cellular differentiation, as judged by their ability to fire repetitive APs. At 19 gw, a fraction of human CP and SP neurons possess bIV spectrin--positive axon initial segments populated with voltage-gated sodium channels (PanNav). These results yield the first physiological characterization of developing human fetal cortical neurons with preserved morphologies in intact surrounding brain tissue.

Bacterial infections during pregnancy often result in premature birth and neonatal white matter damage. During these infections, microglia, the resident immune cells of the CNS, undergo activation and contribute to further neuronal damage of the CNS. Minocycline, a second-generation tetracycline antibiotic, inhibits microglial activation and protects neurons in rodents but data about its effects on human cells are limited. We studied the mechanism of the neuroprotective effect of minocycline in either purified cell cultures or co-cultures of microglia and neurons from human fetal brain during inflammation induced by lipopolysaccharide (LPS). In neuron/microglial co-cultures, minocycline treatment prevented activation and proliferation of microglia and protected neurons as demonstrated by decreased neuronal cell death and a shift of Bcl-2 family proteins toward anti-apoptotic ratio. Notably, neither minocycline nor LPS had an effect on neurons in purified neuronal cultures. The ability of minocycline to regulate activation of human fetal microglia might be relevant in therapies used towards treating neonatal CNS infections.

The polysialic acid (PSA) modification of neural cell adhesion molecule, which reduces neural cell adhesion molecule (NCAM) - mediated cell adhesion, is involved in several developmental processes, such as cell migration, axonal growth, path finding, and synaptic plasticity. It has been suggested that PSA-NCAM expression may inhibit myelination. To clarify the relationship between myelination and the expression of PSA-NCAM we systematically investigated its expression in the human forebrain from embryonic stage to midgestation (19-24 gestation weeks, gw). Immunofluorescence on cryosections showed that PSA-NCAM is expressed at the earliest stage studied (5.5 gw) in the primordial plexiform layer of the telencephalon, which mainly consists of neuronal processes. At midgestation, cortical axonal tracts in the emerging white matter were PSA-NCAM+, but they were not yet myelinated, based on the lack of myelin basic protein (MBP) immunoreaction. To follow the progression of myelination we developed organotypic slice cultures that included the subventricular and intermediate zones of the fetal forebrain. In freshly prepared slices, similar to cryosections, axonal tracts were PSA-NCAM+ but did not express MBP. After 5 days in culture there was a dramatic increase in MBP expression around the axons of the intermediate zone, which suggested the onset of myelination. Simultaneously with MBP up-regulation PSA-NCAM expression in axons was completely lost, as demonstrated both with immunofluorescence and Western blot analysis. These results support the idea that in the human fetal forebrain axonal PSA-NCAM expression is inversely related to primary myelination.

Chemokines, small proinflammatory cytokines, are involved in migration of inflammatory cells, but also have a role in normal central nervous system development. One chemokine, growth-related oncogene-alpha (GRO-alpha) and its receptor CXCR2, are involved in proliferation and migration of oligodendrocyte progenitors in rats. Here we studied the regional and cell type-specific expression of GRO-alpha and CXCR2 in the human telencephalon at midgestation, the time that oligodendrocytes are being generated in the human brain. Our results showed that both GRO-alpha and CXCR2 are predominately expressed by oligodendrocyte progenitors and activated microglial cells in the highly proliferative subventricular zone. This cellular and regional localization suggests that GRO-alpha/CXCR2 may play a role in human oligodendrocyte proliferation and subsequent migration. We also studied the expression of GRO-alpha and CXCR2 in brain sections of multiple sclerosis (MS) patients. Consistent with their role in the inflammatory process of MS, both GRO-alpha and CXCR2 were expressed in activated microglia localized on the border of MS lesions. However, neither GRO-alpha nor CXCR2 were present in early oligodendrocyte progenitors, a finding that may partially explain why remyelination is not more efficient in MS.

Oligodendrocytes, the myelin-producing cells in the central nervous system, represent a large portion of the total number of cells in the human brain. Using cell-specific markers and antibodies to ventral homeodomain transcription factors, NKX2.1 and DLX2, we show here that a subpopulation of early oligodendrocyte progenitor cells (OPCs) in the human telencephalon may originate in the ganglionic eminence (GE). DLX2-labeled OPCs form a well-delineated stream of cells connecting the GE subventricular zone (SVZ) to the cortical intermediate zone through the anterior cortical SVZ. This population of cells is labeled by early OPCs markers, PDGFRalpha, Olig1, and NG2, and not with either neuronal, astrocyte, or late OPCs markers. Intriguingly, numerous CD68(+) microglia/macrophages, nestin(+) neural stem cells, and CD34(+) hematopoietic stem cells (HSCs) are also present in both the GE stream and the cortical SVZ. These cells could be colabeled with DLX2 as well as early OPCs markers. A separate subpopulation of early OPCs, present in the GE and cortical SVZ, did not express either DLX2 or CD68. These findings suggest that different subpopulations of early OPCs, characterized with different sets of transcription factors and cell-specific markers, are present in human forebrain. These subpopulations may have different origins; one may originate in the cortical SVZ, while others may come from the GE and/or outside the CNS as hematopoietic stem cells.

Before the human cortex is able to process sensory information, young postmitotic neurons must maintain occasional bursts of action-potential firing to attract and keep synaptic contacts, to drive gene expression, and to transition to mature membrane properties. Before birth, human subplate (SP) neurons are spontaneously active, displaying bursts of electrical activity (plateau depolarizations with action potentials). Using whole-cell recordings in acute cortical slices, we investigated the source of this early activity. The spontaneous depolarizations in human SP neurons at midgestation (17-23 gestational weeks) were not completely eliminated by tetrodotoxin--a drug that blocks action potential firing and network activity--or by antagonists of glutamatergic, GABAergic, or glycinergic synaptic transmission. We then turned our focus away from standard chemical synapses to connexin-based gap junctions and hemichannels. PCR and immunohistochemical analysis identified the presence of connexins (Cx26/Cx32/Cx36) in the human fetal cortex. However, the connexin-positive cells were not found in clusters but, rather, were dispersed in the SP zone. Also, gap junction-permeable dyes did not diffuse to neighboring cells, suggesting that SP neurons were not strongly coupled to other cells at this age. Application of the gap junction and hemichannel inhibitors octanol, flufenamic acid, and carbenoxolone significantly blocked spontaneous activity. The putative hemichannel antagonist lanthanum alone was a potent inhibitor of the spontaneous activity. Together, these data suggest that connexin hemichannels contribute to spontaneous depolarizations in the human fetal cortex during the second trimester of gestation.

Premature infants exhibit neurodevelopmental delay and reduced growth of the cerebral cortex. However, the underlying mechanisms have remained elusive. Therefore, we hypothesized that neurogenesis in the ventricular and subventricular zones of the cerebral cortex would continue in the third trimester of pregnancy and that preterm birth would suppress neurogenesis. To test our hypotheses, we evaluated autopsy materials from human fetuses and preterm infants of 16–35 gestational weeks (gw). We noted that both cycling and noncycling Sox2+radial glial cells and Tbr2+intermediate progenitors were abundant in human preterm infants until 28 gw. However, their densities consistently decreased from 16 through 28 gw. To determine the effect of premature birth on neurogenesis, we used a rabbit model and compared preterm [embryonic day 29 (E29), 3 d old] and term (E32, +intermediate progenitors and the increased number of Sox2+radial glia. Additionally, hypoxia-inducible factor-1α, vascular endothelial growth factor, and erythropoietin were higher in term than preterm pups, reflecting the hypoxic intrauterine environment of just-born term pups. Proneural genes, including Pax6 and Neurogenin-1 and -2, were higher in preterm rabbit pups compared with term pups. Importantly, neurogenesis and associated factors were restored in preterm pups by treatment with dimethyloxallyl glycine, a hypoxia mimetic agent. Hence, glutamatergic neurogenesis continues in the premature infants, preterm birth suppresses neurogenesis, and hypoxia-mimetic agents might restore neurogenesis, enhance cortical growth, and improve neurodevelopmental outcome of premature infants.

The cathecholaminergic (CA) systems have been described as appearing early in the development of the mammalian central nervous system (CNS), but their exact distribution in humans has been studied only following gestational week (g.w.) 13. Furthermore, it is not known when CA fibers initially penetrate the developing cerebral cortex. In this study, the CA cells groups and fibers are described in the human central nervous system from 6 to 13 g.w. as revealed with immunocytochemical techniques, with antibodies raised against three synthetic enzymes of the catecholamine (CA) pathway: tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). At 6 g.w., TH-like immunoreactive (TH-IR) cell groups were widespread through the caudorostral extension of the CNS corresponding to the different dopaminergic mesencephalic and hypothalamic groups. Noradrenergic groups also were labeled in the medulla oblongata and in the locus coeruleus as well as in other areas in the pons. Additional TH-IR cell groups might represent a transient developmental expression of TH similar to that observed in the rat. DBH immunoreactivity labeled primarily the noradrenergic pontic cell groups and, to a lesser extent, groups located in the medulla oblongata. Rare PNMT-IR neurons were detected in the medulla oblongata only at 13 g.w. The main CA bundles described in the adult were also observed in human embryos and fetuses. At 6 g.w., TH-IR pathways extended caudorostrally within the central tegmental tract and the dorsal tegmental bundle, the latter merging with the dopaminergic mesotelencephalic pathway giving rise to the medial forebrain bundle in the basal forebrain. At 7-8 g.w., TH-IR fibers extended to the basal ganglia and the telencephalic wall. The first TH-IR and, to a much lesser extent, DBH-IR fibers penetrated the frontal lateral cortical anlage through the intermediate zone and sparsely through the marginal zone but not through the thin cortical plate. A second stream entered the telencephalic anlage frontomedially, ventral to the septal area. At 11 g.w., numerous TH-IR fibers invaded the subplate layer, but they penetrated the cortical plate only at 13 g.w. At that time, TH-IR and DBH-IR fibers had reached the occipital cortex in a rostrocaudal gradient. The appearance of well-organized CA system already in embryonic stages in humans could be of great importance for normal shaping of the nervous system as well as for development of cortical circuitry.

Radial glial (RG) cells, in the neocortical ventricular/subventricular zone (VZ/SVZ), generate cortical projection neurons both in rodents and humans, but whether they can also generate cortical interneurons is not clear. We demonstrated both on cryosections and in cell cultures that in the human VZ/SVZ, cells can be double labeled with RG markers and calretinin (CalR) and GABA, markers that suggest interneuronal lineage. We examined in more detail the cell fate of human RG cells isolated from the VZ/SVZ at midterm. After 24 h, no CalR+or GABA+cells were seen in cultures, whereas 5–10% cells expressed Nkx2.1 and Dlx, two ventral transcription factors. CalR+and GABA+cells were apparent for the first time after 3 din vitro, and their number increased in subsequent days, consistent with the gradual transition of RG cells into CalR+or GABA+cells. Indeed, the progeny of genetically labeled RG cells could be immunolabeled with antibodies to CalR and GABA or ventral transcription factors (Nkx2.1+, Dlx+). In contrast to humans, in the embryonic mouse, similar experiments showed that only RG cells isolated from the subpallium (ganglionic eminence) generate CalR+or GABA+cells, whereas this was not the case with RG cells isolated from the pallium. These findings support the idea that human, but not mouse, dorsal RG cells have the potential to generate various subtypes of neocortical interneurons. Multiple progenitors and sites of cortical interneuron origin in human might be an evolutionary adaptation underlying brain expansion and the increased complexity of cortical circuitry in humans.

Our knowledge about the developing human cerebral cortex is based on the analysis of fixed postmortem material. Here we use electrical recordings from unfixed human postmortem tissue to characterize the synaptic physiology and spontaneous network activity of pioneer cortical neurons (“subplate neurons”). Our electrophysiological experiments show that functional glutamate or GABA ionotropic receptors are expressed on human subplate (SP) neurons as early as 20 gestational weeks. Extracellular (synaptic) stimulations evoked postsynaptic potentials in a very small fraction of SP neurons, suggesting that functional synaptic contacts are rare at midgestation. Although synaptic inputs were scarce, we regularly observed spontaneous (unprovoked) electrical activity among human SP neurons, comprised of sustained plateau depolarizations and bursts of action potential firing, which resembled cortical UP and DOWN states in the adult neocortex. Plateau depolarizations and bursts of action potential firing are thought to depend on the mature morphology and physiology of adult cortical network. However, our current data reveal that similar cortical rhythm is generated by a very immature ensemble of human fetal neurons. In the relative absence of sensory inputs, as in developmentin utero, or in slow-wave sleep (i.e., throughout the entire lifespan), the spontaneous slow oscillatory pattern (UP and DOWN states) is a fundamental aspect of human cortical physiology.

Cortical γ-aminobutyric acid (GABA)ergic interneurons in rodents originate mainly in ventrally positioned ganglionic eminences (GEs), but their origin in primates is still debated. We studied human fetal forebrains during the first half of gestation (5-23 gestational weeks, gw) for the expression of ventral transcription factors, Nkx2.1, Dlx1,2, Lhx6, and Mash1, important for development of neocortical interneurons. In embryonic (5-8 gw) human forebrain, these factors were expressed in the GE but also dorsally in the neocortical ventricular/subventricular zones (VZ/SVZ). Furthermore, their expression was retained in cells of all fetal cortical layers up to midgestation (20 gw). Nkx2.1 continued to be expressed not only in the GE but also in a subpopulation of neocortical interneurons. Moreover, proliferation marker Ki67 revealed that calretinin(+), Mash1(+), and Nkx2.1(+) cells proliferate in the neocortical VZ/SVZ at midgestation. At least some of the Mash1(+) progenitors in the neocortical SVZ could be colabeled with GABA, whereas others were oligodendrocyte progenitors, indicating a link between the 2 lineages. Taken together, these results suggest the existence of several categories of dorsal interneuronal progenitors in the human neocortical VZ/SVZ, in addition to ventrally derived cortical interneurons described in rodents. These human-specific developmental events may underlie human brain's higher complexity and capacity to process information.

Qualitative and quantitative parameters of the early neuronal development of the thalamus were examined in human embryos and fetuses ranging in age from 5-16 gestational weeks (g.w.) using Nissl stained and Golgi impregnated tissue. In the earliest period analyzed, from 5-6 weeks of gestation, dorsal and ventral parts of the human thalamus were poorly differentiated structures forming the sides of the third ventricle. At 6 g.w., the prospective thalamic anlage consisted of the ventricular (VZ), the intermediate (IZ) and the marginal zones (MZ). The neurons in the VZ were bipolar and radially arranged to the ventricular surface. From 6-8 gestational weeks detached bipolar or branched bipolar cells with horizontal or oblique orientation were encountered in the IZ and occasionally in the MZ. In the period between 10-14 weeks of gestation, segregation of neurons into different thalamic nuclei began. The lateral geniculate nucleus, centrum medianum and mediodorsal nucleus were the first to be distinguished. In comparison with the previous period, the neurons throughout the rostro-caudal axis of the thalamus were more differentiated, with a higher number of primary dendrites that were often bifurcated. This resulted in various shapes of immature neurons: simple uni- and bipolar (type 1), branched uni- and bipolar (type 2) and young multipolar (type 3). In addition, the first spines were frequently observed on dendrites at 12 weeks of gestation. At 14-16 weeks of gestation, segregation of the neurons into thalamic nuclei continued. The neurons exhibited further dendritic differentiation that followed the caudo rostral gradient. For the first time, multipolar neurons, especially in the caudal part, could be subdivided into two classes: multipolar with a 'bushy' appearance, the result of many primary dendrites with several bifurcations and 'simpler' multipolar neurons with fewer and less branched primary dendrites. Neuronal differentiation was further expressed by an increase in the total dendritic length and somatic surface, up to six and three times, respectively, from the earliest period analyzed to 16 weeks of gestation. Dendritic length increased initially by the formation of new primary branches and their elongation and secondly by formation of higher branch orders. At 16 g.w., a group of immature cells were observed dorsally, on the frontal sections through the rostral thalamus. Its ganglionic eminence origin is discussed.

The time course and rate of synaptogenesis were studied in the somatosensory cortex (Brodmann's areas 1 and 3b) of 27 rhesus monkeys ranging in age from embryonic day 41 to 20 years. Two to four vertical probes, each consisting of a series of overlapping electron micrographs and extending from the pial surface to the interface of the cortex with the white matter, were made from sections cut across the postcentral gyrus in the region of the upper limb representation. We found that the density of synapses per unit volume of cortex as well as per unit volume of neuropil increases steadily throughout the late fetal ages and early infancy. A density of 70/100 microns 3 of neuropil was reached by the second postnatal month; thereafter, between 1 and 3 years a slightly lower density of 50-60/100 microns 3 was maintained. At around puberty, the decrease in concentration of synapses appears to be accelerated. Thus, the average synaptic density of a group of 10 adult animals composed of monkeys over 4 years of age was 30-40 synapses per 100 microns 3 of neuropil. This value is significantly lower than that of the group of 11 infant and juvenile animals below 4 years of age. Since synaptic density per unit volume of neuropil is not affected by changes in other parameters of cortical growth, these numbers reflect an actual overproduction of synapses in infancy followed by their elimination during adolescence. The decline in the number of synapses is due primarily to elimination of asymmetrical junctions located on dendritic spines while symmetrical synapses on dendritic shafts and cell bodies remained relatively constant during postnatal life. The course of synapse formation recorded in the present study coincides with the course of overproduction and elimination of neurotransmitter receptors (Lidow et al., 1991) and the developmental schedule of synaptogenesis in other neocortical areas (Rakic et al., 1986). The timing of synaptogenesis and synaptic elimination in the postcentral gyrus may account for the maturation and plasticity of various aspects of somatosensory function during post-natal life.

Understanding the molecular and physiological determinants of cortical neuronal progenitor cells is essential for understanding the development of the human brain in health and in disease. We used surface marker fucoseN-acetyl lactosamine (LeX) (also known as CD15) to isolate progenitor cells from the cortical ventricular/subventricular zone of human fetal brain at the second trimester of gestation and to study their progenyin vitro. LeX+cells had typical bipolar morphology, radial orientation, and antigen profiles, characterizing them as a subtype of radial glia (RG) cells. Four complementary experimental techniques (clonal analysis, immunofluorescence, transfection experiments, and patch-clamp recordings) indicated that this subtype of RG generates mainly astrocytes but also a small number of cortical neurons. The neurogenic capabilities of RGs were both region and stage dependent. Present results provide the first direct evidence that RGs in the human cerebral cortex serve as neuronal progenitors. Simultaneously, another progenitor subtype was identified as proliferating cells labeled with neuronal (β-III-tubulin and doublecortin) but not RG markers [GFAP, vimentin, and BLBP (brain lipid-binding protein)]. Proliferative and antigenic characteristics of these cells suggested their neuron-restricted progenitor status. In summary, ourin vitrostudy suggests that diverse populations of cortical progenitor cells, including multipotent RGs and neuron-restricted progenitors, contribute differentially to cortical neurogenesis at the second trimester of gestation in human cerebral cortex.

The transcription factors Olig1 and Olig2 are closely associated with the development of oligodendrocyte (OL) lineage in the vertebrate nervous system, but little is known about their role in the human developing CNS. To test the hypothesis that they contribute to initial OL specification in humans, we studied the expression of Olig1 and Olig2 in human fetuses at 5-24 gestational weeks (GW). Both transcription factors were present in well outlined regions of the ventral neuroepithelium at 5 GW, several weeks before oligodendrogenesis. Spatial differences in the expression of Olig1 and Olig2 along the neuronal axis suggest that they specify different subpopulations of progenitor cells. Olig1 was distributed rostrally, from the basal forebrain to the hindbrain, whereas Olig2 was also found in the ventral spinal cord. Furthermore, at 5 GW, Olig1 was coexpressed with vimentin, and Olig2 was coexpressed with a neuronal marker, microtubule-associated protein 2. With the progression of development at 15 GW, both proteins were present throughout the spinal cord and the ventricular-subventricular zone of the ganglionic eminences, whereas at midgestation (20 GW), they were also expressed in the telencephalic proliferative zones and the emerging white matter. Double-labeling studies revealed that early OL progenitor cells and radial glia expressed Olig1, whereas Olig2 was localized predominantly in mature OLs and a subset of neural progenitor cells and mature neurons. Thus, Olig1 and Olig2 transcription factors in the human CNS are important not only for differentiation of the OL lineage, but they may also have a role in neural cell specification.

In this study we examine possible origins and migratory routes of human cortical neurons, with special emphasis on the preplate and layer I. In embryonic stages, two main cell types, Cajal-Retzius cells, and cells labeled with interneuron markers (calretinin, calbindin and GABA), were present in the preplate layer. In addition, a number of preplate GABAergic cells co-expressed either Nkx2.1 or Dlx transcription factors, findings consistent with their origin in the ganglionic eminence and subsequent tangential migration to the layer I. The orientation of the leading process indicates that some of these cells descend to the cortical plate. However, the finding of radially oriented GABAergic, NKX2.1(+) and DLX+ cells in the cortical ventricular zone, argues that, unlike in rodents, a significant subpopulation of these cells originates in the cortical ventricular zone. In embryonic stages, expression of Reelin in Cajal-Retzius cells as well as Reelin/DLX2(+) cells in the embryonic ganglionic eminence and the olfactory region, suggest that these cells in human may have diverse origins. In later fetal stages in human (17-22 gestational weeks) layer I and the newly formed subpial granular layer, contained a population of small interneurons that originated mainly in the lateral ganglionic eminence, since the majority of these cells were double-labeled with DLX/GABA, and rarely with NKX2.1/GABA. Therefore, neurons in the human cortical layer I are heterogeneous, with more complex origin and migratory routes than in rodents. In addition to the ganglionic eminence, both the expended subventricular zone and subpial granular layer, contribute to the neuronal population of the developing layer I and underlining cortical plate.

The myelin basic protein gene (Mbp) encodes for the major myelin structural proteins and it is included in the Golli-Mbp gene complex. Previously, we observed MBP-like proteins in the human central nervous system (CNS) at developmental stages preceding myelination. In an effort to distinguish between Golli (HOG5 and HOG7) and MBP mRNAs and to determine their spatiotemporal distribution, we performed in situ hybridization using two human Golli specific probes: one corresponding to exon 5a absent from all MBP transcripts, and the other corresponding to exon 5c specific for HOG5. HOG7 transcript was observed first, in 5 gestational week-old embryos, whereas both Golli transcripts were detected at 6-7 weeks gestation in the proliferative zones of the entire CNS. Golli proteins immunoreactivity was observed in microglia and early neurons of the developing telencephalon. During midgestation (17-22 weeks gestation), at the onset of myelination, MBP and Golli mRNAs were observed in the telencephalic subventricular zone and occasionally in the future cerebral cortex. Developmental expression of the human Golli-Mbp indicates that the two Golli proteins have different onset of expression, distribution and possibly function. These results support the hypothesis that at least one of them, HOG7, may be involved in the regulation of early neurogenesis, while both may have additional, still undefined function at the onset of myelination. (C) 2002 Wiley-Liss, Inc.

A subset of tyrosine-hydroxylase (TH) neurons of the substantia nigra (A9) containing calbindin D28K (CaBP) appeared to be less vulnerable to cell death induced by Parkinson's disease than the subset containing dopamine (DA) alone. Because grafting procedures of fetal human neurons are increasingly used in the therapy of Parkinson's disease, it is important to study the development of DA neurons coexpressing CaBP. In humans, the genesis of TH immunoreactivity of A9, of the ventral tegmental area (A10), and of the retrorubral area (A8) occurred during a 2-week period from the 4. 5th gestational week (g.w.) in the ventricular zone of the floor plate and the contiguous basal plate of the mesencephalon and diencephalon, i.e., the prosomeres p1-p3. Double-immunolabeled TH-CaBP neurons were detected from 5.5 g.w. on, in the first wave of DA neuron's migration, and were observed in their final residence in the dorsal A9 by 10.5 g.w. Calretinin immunoreactivity was expressed in TH-immunoreactive (IR) neurons from 10.5 g.w. on. Ascending TH-CaBP-IR axons were observed toward the telencephalon from 6-7 g.w. , reaching the anlage of the nucleus accumbens and amygdaloid complex at 10.5 g.w., but were not detected in the ganglionic eminence at this latter stage. Dopaminergic patches were detected at 13 g.w. in the anlage of the putamen, but no TH-CaBP-IR fibers were observed in the matrix at this stage. In conclusion, even if CaBP immunoreactivity was detected in TH-IR cell bodies during the embryonic period, the TH-CaBP-IR axonal terminal was observed earlier in some limbic-related areas than in the matrix compartment of the basal ganglia in humans.

In a previous work, mapping early tyrosine hydroxylase (TH) expressing primordia in human embryos, the tegmental origin of the substantia nigra (SN) and ventral tegmental area (VTA) was located across several neuromeric domains: prosomeres 1–3, midbrain, and isthmus (Puelles and Verney, [1998] J. Comp. Neurol. 394:283–308). The present study examines in detail the architecture of the neural wall along this tegmental continuum in 6–7 week human embryos, to better define the development of the SN and VTA. TH-immunoreactive (TH-IR) structures were mapped relative to longitudinal subdivisions (floor plate, basal plate, alar plate), as well as to radially superposed strata of the neural wall (periventricular, intermediate, and superficial strata). These morphologic entities were delineated at each relevant segmental level by using Nissl-stained sections and immunocytochemical mapping of calbindin, calretinin, and GABA in adjacent sagittal or frontal sections. A numerous and varied neuronal population originates in the floor plate area, and some of its derivatives become related through lateral tangential migration with other neuronal populations born in distinct medial and lateral portions of the basal plate and in a transition zone at the border with the alar plate. Some structural differences characterize each segmental domain within this common schema. The TH-IR neuroblasts arise predominantly within the ventricular zone of the floor plate and, more sparsely, within the adjacent medial part of the basal plate. They first migrate radially from the ventricular zone to the pia and then apparently move laterally and slightly rostralward, crossing the superficial stratum of the basal plate. Several GABA-IR cell populations are present in this region. One of them, which might represent the anlage of the SN pars reticulata, is generated in the lateral part of the basal plate. J. Comp. Neurol. 429:22–44, 2001. © 2000 Wiley-Liss, Inc.

We correlated neuroanatomical developmental parameters with sequential ultrasonography scans to reveal the structural basis of functional recovery after early focal hypoxic lesions of the human frontal lobe in premature infants. We studied the transient fetal subplate zone in the premotor and prefrontal cortex in premature, newborn, infant, and young adult brains by acetylcholinesterase (AChE) histochemical, Golgi, and immunocytochemical methods. The structural in vivo rearrangements of the cerebral wall after perinatal lesions were studied on serial real-time sector scans (5-MHz transducer). The subplate zone contains "waiting" axons and randomly oriented fetal neurons, its developmental peak is between 22 and 34 weeks of gestation, and it is present in the frontal cortex of newborns and disappears after the sixth postnatal month, but individual subplate-like neurons remain until adulthood. Ultrasonography revealed remarkable structural rearrangements of the cerebral wall when the hypoxic lesion occurred during the developmental peak of the subplate zone: anechoic cavities ("cysts") develop rapidly (within 3 weeks) in premature brains, the rebuilding of these lesions continues after birth, and cavities disappear around the 11th month. We propose that the transient population of "waiting" axons and cells of the subplate zone participate in the structural and functional plasticity of the human cerebral cortex after perinatal brain damage.

The differentiation of Purkinje cells and their relationship to other components of the developing cerebellar cortex were analyzed by the Golgi impregnation method and by electron microscopy in human specimens of various pre- and postnatal ages. The three stages of Purkinje cell maturation that have been previously recognized in other species are also evident in man: the first stage occupies primarily the fourth fetal month (12-16 weeks); the second stage lasts through the fifth, sixth and seventh feta months (16-28 weeks); the third stage extends throughout the remaining period of intrauterine life and the first postnatal year and continues at a slow rate thereafter. During the first stage, Purkinje cells are distributed in a layer, several rows deep. Their bipolar somas are relatively smooth and have only a few processes at the apical and basal cell poles. In the 3-month period of the second stage, Purkinje cells become gradually organized into a single row. Their somas become invested with additional randomly oriented dendritic processes and numerous somatic spines (pseudopodia). The first morphologically well-defined synapses appear on the Purkinje cell somatic spines and on their immature dendritic shafts at the beginning of the second stage and become more prominent during the period from 18 to 24 weeks. In the third stage, the dendritic arbor becomes flattened in the plane transverse to the folium and somatic spines disappear. Spines appear on the secondary and tertiary dendrites between the twenty-fourth and twenty-eighth fetal weeks and continue to increase in number during the entire third stage as new dendritic branches develop. These observations indicate that cellular maturation and synaptogenesis in the primate cerebellum differ from these events in non-primate species, with respect to time of birth, in the relative duration of each phase and in the total time necessary for neuronal differentiation. The protracted time of differentiation and the slow growth of Purkinje cell dendrites in man may be due to the numerically complex relationships existing between granule and Purkinje cells. It is probably not simply a reflection of the larger size of human Purkinje cells and their dendrites.