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The coordination of progenitor self-renewal, neuronal production, and migration is essential to the normal development and evolution of the cerebral cortex. Numerous studies have shown that the Notch, Wnt/beta-catenin, and Neurogenin pathways contribute separately to progenitor expansion, neurogenesis, and neuronal migration, but it is unknown how these signals are coordinated. In vitro studies suggested that the mastermind-like 1 (MAML1) gene, homologue of the Drosophila mastermind, plays a role in coordinating the aforementioned signaling pathways, yet its role during cortical development remains largely unknown. Here we show that ectopic expression of dominant-negative MAML (dnMAML) causes exuberant neuronal production in the mouse cortex without disrupting neuronal migration. Comparing the transcriptional consequences of dnMAML and Neurog2 ectopic expression revealed a complex genetic network controlling the balance of progenitor expansion versus neuronal production. Manipulation of MAML and Neurog2 in cultured human cerebral stem cells exposed interactions with the same set of signaling pathways. Thus, our data suggest that evolutionary changes that affect the timing, tempo, and density of successive neuronal layers of the small lissencephalic rodent and large convoluted primate cerebral cortex depend on similar molecular mechanisms that act from the earliest developmental stages.

The paper explores the notion of home in the stratiot poetry authored by 16th-century Venetian comedy author and composer Antonio Molino. Rooted in the life of the stratiots and their struggles against the Ottomans, this poetry allegedly reflects the diasporic memory of Greek stratiots, although it is apparently framed by the genre and preferences of Molino’s theatre audience. By analysing the memoryscape of notions of home which Molino linked to the Greek stratiots of his time, the paper reveals that his poetry projects, in fact, were the more tangible political ambitions of the Republic of Venice to convert the stratiots from Orthodox Christianity to Roman Catholicism, reinforce their Venetian identity and use their memory in its own aspiration to the Byzantine imperial legacy – all highly topical on the eve of the Venetian conflict with the Ottomans over Cyprus (1571–1573)., U ovome članku razmatra se pojam doma u stratiotskom pjesništvu mletačkog komediografa i skladatelja Antonija Molina iz 16. stoljeća. Ukorijenjeno u životu stratiota i njihovim bitkama protiv Osmanlija, to pjesništvo navodno odražava iseljenička sjećanja grčkih stratiota, premda je očito da je oblikovano samim žanrom te sklonostima Molinove kazališne publike. Ispitujući prostor sjećanja kojim su obuhvaćena poimanja doma što ih je Molino povezivao s grčkim stratiotima kao svojim suvremenicima, ovaj članak razotkriva da se u njegovome pjesništvu zapravo ogledaju opipljivije političke težnje Mletačke Republike da se stratiote s pravoslavlja preobrati na rimokatoličanstvo, ojača njihov mletački identitet, a njihovo sjećanje iskoristi za potrebe vlastitog stremljenja prema bizantskoj imperijalnoj ostavštini, što je sve bilo veoma aktualno uoči mletačkog sukoba s Osmanlijama oko Cipra (1571-1573).

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.

One of the main characteristics of the developing brain is that all neurons and the majority of macroglia originate first in the ventricular zone (VZ), next to the lumen of the cerebral ventricles, and later on in a secondary germinal area above the VZ, the subventricular zone (SVZ). The SVZ is a transient compartment mitotically active in humans for several gestational months. It serves as a major source of cortical projection neurons as well as an additional source of glial cells and potentially some interneuron subpopulations. The SVZ is subdivided into the smaller inner (iSVZ) and the expanded outer SVZ (oSVZ). The enlargement of the SVZ and, in particular, the emergence of the oSVZ are evolutionary adaptations that were critical to the expansion and unique cellular composition of the primate cerebral cortex. In this review, we discuss the cell types and organization of the human SVZ during the first half of the 40 weeks of gestation that comprise intrauterine development. We focus on this period as it is when the bulk of neurogenesis in the human cerebral cortex takes place. We consider how the survival and fate of SVZ cells depend on environmental influences, by analyzing the results from in vitro experiments with human cortical progenitor cells. This in vitro model is a powerful tool to better understand human neocortex formation and the etiology of neurodevelopmental disorders, which in turn will facilitate the design of targeted preventive and/or therapeutic strategies.

The classical tradition in Bosnia‐Herzegovina and Serbia illustrates the complex historical development of this entire region that for centuries served as a demarcation line between different civilizations. This chapter elaborates upon similarities and differences through which the classical tradition was received and used in both countries. In Serbia, it was the result of direct Byzantine Greek influence, while in Bosnia‐Herzegovina it was shaped by the Latin West. Then, in Bosnia‐Herzegovina, this tradition served as the key tool for the local Franciscans in their affirmation of the Order and the local Catholic identity, while in late Ottoman and post‐Ottoman Serbia it reflected general intellectual trends of the Enlightenment era and the influence of the Habsburg Monarchy. Justifying the “origins” of nineteenth‐century nations in Latin antiquity, the Franciscan Latinist and neo‐Latinist discourse in Bosnia‐Herzegovina was greatly marginalized after the Austro‐Hungarian occupation (later also by the Communists), while in Serbia, regardless of communist limitations, both Greek and Latin classical traditions were received in literature and studied in modern academia through the curricula in classical languages, literature, art, and history.

This chapter surveys the key historiography works created by the Franciscans of the eighteenth‐century province Bosnia Argentina. In many segments, these works grafted upon late humanist and early neoclassical traditions and their reception of classical antiquity. The chapter examines the details of these influences and attempts to explain the trends of their use by the Bosnian Franciscans, focusing on sources and themes that appear in these works, as well as literary, language, and historiography tools that exploit or evoke classical antiquity, particularly its Roman Latin tradition. Contextualizing these trends with the important circumstances of the local history—Ottoman rule, Franciscan divisions within the province Bosnia Argentea, and the close links of the Order with their local congregation—shows how references to the Latin West helped shape the national identity and perceptions of the local Catholics as Croats. The implanting of the Roman Latin tradition into scholarly study that resulted from the arrival of the Austro‐Hungarians (1878) is discussed, as well as the banishment and degradation of this scholarship, both secular and religious, during the communist regime and the destructions of the conflict of the 1990s.

Summary Evolutionary elaboration of tissues starts with changes in the genome and location of the stem cells. For example, GABAergic interneurons of the mammalian neocortex are generated in the ventral telencephalon and migrate tangentially to the neocortex, in contrast to the projection neurons originating in the ventricular/subventricular zone (VZ/SVZ) of the dorsal telencephalon. In human and nonhuman primates, evidence suggests that an additional subset of neocortical GABAergic interneurons is generated in the cortical VZ and a proliferative niche, the outer SVZ. The origin, magnitude, and significance of this species-specific difference are not known. We use a battery of assays applicable to the human, monkey, and mouse organotypic cultures and supravital tissue to identify neuronal progenitors in the cortical VZ/SVZ niche that produce a subset of GABAergic interneurons. Our findings suggest that these progenitors constitute an evolutionary novelty contributing to the elaboration of higher cognitive functions in primates.

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.

Kynurenic acid (KYNA), a neuroactive metabolite of tryptophan degradation, acts as an endogenous N-methyl-D-aspartate receptor (NMDAR) antagonist. Elevated levels of KYNA have been observed in pregnant women after viral infections and are considered to play a role in neurodevelopmental disorders. However, the consequences of KYNA-induced NMDAR blockade in human cortical development still remain elusive. To study the potential impact of KYNA on human neurodevelopment, we used an in vitro system of multipotent cortical progenitors, i.e., radial glia cells (RGCs), enriched from human cerebral cortex at mid-gestation (16–19 gestational weeks). KYNA treatment significantly decreased RGCs proliferation and survival by antagonizing NMDAR. This alteration resulted in a reduced number of cortical progenitors and neurons while number and activation of astrocytes increased. KYNA treatment reduced differentiation of RGCs into GABAergic neurons, while differentiation into glutamatergic neurons was relatively spared. Furthermore, in mixed cortical cultures KYNA triggered an inflammatory response as evidenced by increased levels of the pro-inflammatory cytokine IL-6. In conclusion, elevated levels of KYNA play a significant role in human RGC fate determination by antagonizing NMDARs and by activating an inflammatory response. The altered cell composition observed in cell culture following exposure to elevated KYNA levels suggests a mechanism for impairment of cortical circuitry formation in the fetal brain after viral infection, as seen in neurodevelopmental disorders such as schizophrenia.

N-methyl d-aspartate receptors (NMDARs), a subtype of glutamate receptor, have important functional roles in cellular activity and neuronal development. They are well-studied in rodent and adult human brains, but limited information is available about their distribution in the human fetal cerebral cortex. Here we show that 3 NMDAR subunits, NR1, NR2A, and NR2B, are expressed in the human cerebral cortex during the second trimester of gestation, a period of intense neurogenesis and synaptogenesis. With increasing fetal age, expression of the NMDAR-encoding genes Grin1 (NR1) and Grin2a (NR2A) increased while Grin2b (NR2B) expression decreased. The protein levels of all 3 subunits paralleled the changes in gene expression. On cryosections, all 3 subunits were expressed in proliferative ventricular and subventricular zones, in radial glia, and in intermediate progenitor cells, consistent with their role in the proliferation of cortical progenitor cells and in the determination of their respective fates. The detection of NR1, NR2A, and NR2B in both glutamatergic and GABAergic neurons of the cortical plate suggests the involvement of NMDARs in the maturation of human cortical neurons and in early synapse formation. Our results and previous studies in rodents suggest that NMDAR expression in the developing human brain is evolutionarily conserved.

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 (

Cortical interneurons play a crucial role in the functioning of cortical microcircuitry as they provide inhibitory input to projection (pyramidal) neurons. Despite their involvement in various neurological and psychiatric disorders, our knowledge about their development in human cerebral cortex is still incomplete. Here we demonstrate that at the beginning of corticogenesis, at embryonic 5 gestation weeks (gw, Carnegie stage 16) in human, early neurons could be labeled with calretinin, calbindin, and GABA antibodies. These immunolabeled cells show a gradient from the ganglionic eminences (GE) toward the neocortex, suggesting that GE is a well conserved source of early born cortical interneurons from rodents to human. At mid-term (20 gw), however, a subset of calretinin+ cells proliferates in the cortical subventricular zone (SVZ), suggesting a second set of interneuron progenitors that have neocortical origin. Neuropeptide Y, somatostatin, or parvalbumin cells are sparse in mid-term cerebral cortex. In addition to the early source of cortical interneurons in the GE and later in the neocortical SVZ, other regions, such as the subpial granular layer, may also contribute to the population of human cortical interneurons. In conclusion, our findings from cryosections and previous in vitro results suggest that cortical interneuron progenitor population is more complex in humans relative to rodents. The increased complexity of progenitors is probably evolutionary adaptation necessary for development of the higher brain functions characteristic to humans. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 18–33, 2011

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.

Several L1-related adhesion molecules, expressed in a well-coordinated temporospatial pattern during development, are important for fine tuning of specific cerebellar circuitries. We tested the hypothesis that CHL1, the close homologue of L1, abundantly expressed in the developing and adult cerebellum, is also required for normal cerebellar histogenesis. We found that constitutive ablation of CHL1 in mice caused significant loss (20–23%) of Purkinje and granule cells in the mature 2-month-old cerebellum. The ratio of stellate/basket interneurons to Purkinje cells was abnormally high (+38%) in CHL1-deficient (CHL1−/−) mice compared with wild-type (CHL1+/+) littermates, but the γ-aminobutyric acid (GABA)ergic synaptic inputs to Purkinje cell bodies and dendrites were normal, as were numbers of Golgi interneurons, microglia, astrocytes, and Bergmann glia. Purkinje cell loss occurred before the first postnatal week and was associated with enhanced apoptosis, presumably as a consequence of CHL1 deficiency in afferent axons. In contrast, generation of granule cells, as indicated by in vivo analyses of cell proliferation and death, was unaffected in 1-week-old CHL1−/− mice, but numbers of migrating granule cells in the molecular layer were increased. This increase was likely related to retarded cell migration because CHL1−/− granule cells migrated more slowly than CHL1+/+ cells in vitro, and Bergmann glial processes guiding migration in vivo expressed CHL1 in wild-type mice. Granule cell deficiency in adult CHL1−/− mice appeared to result from decreased precursor cell proliferation after the first postnatal week. Our results indicate that CHL1 promotes Purkinje and granule cell survival and granule cell migration during cerebellar development. J. Comp. Neurol. 513:496–510, 2009. © 2009 Wiley-Liss, Inc.

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.

Transcription factor Pax6 plays an important role in fate determination of neural progenitor cells in animal models, yet, its distribution and role in the human developing brain have not been reported. Here we demonstrated that Pax6 was strongly expressed in dorsal and ventral proliferative zones, mainly in proliferating radial glia (RG) cells, some neuronal and intermediate progenitors, and sporadic deep cortical plate neurons. In contrast to reports in rodents, Pax6 in the human fetal brain occasionally colocalized with ventral transcription factor Olig2 in progenitor cells. Transfection with short interfering RNA abolished Pax6 expression in the cell cultures of human fetal RG, and significantly decreased the number of neurons generated from Pax6 knock-down cells. Hence, Pax6 has a critical role in neurogenic regulation of RG cells in the human forebrain, similar to reports in rodents. What is different in human forebrain is that Pax6 seems to regulate not only the genesis of cortical pyramidal neurons, but also a subpopulation of interneurons from both dorsal and ventral sources. Thus, regional distribution, colocalization with Olig2, and the role of Pax6 in neurogenesis of both projection and interneurons, suggest that developmental regulation by transcription factors may differ in primates and nonprimate mammals.

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.

Intracellular protein calretinin (CR) acquired its name based on structural similarity to calbindin D28k and the site of first detection—retina (Rogers, 1987). Soon after this discovery, a basic description of the distribution of CR in rodent brain emerged from the work of several authors (reviewed by Baimbridge et al., 1992; Andressen et al., 1993). In the upcoming years, calcium-binding proteins (CaBP) calretinin, parvalbumin (PV), and calbindin (CB) could be shown to be expressed in largely non-overlapping interneuronal populations. The usefulness of utilizing PV, CB, and CR for categorization of interneuronal subpopulations was validated by gene cluster analysis (Toledo-Rodriguez et al., 2004). While extensive literature on PV expressing (PV+) interneurons' physiological functions and changes in various pathological conditions have been published, we still have a limited view about the CR+ (and CB+) interneurons. This is quite disappointing, considering their increasing number in primates and various unique features they possess. In the Research Topic “At the top of the interneuronal pyramid—calretinin expressing cortical interneurons,” we intended to provide a summary of current knowledge on CR+ cortical neurons and especially to emphasize and discuss those “unique features” of CR+ neurons mentioned above. In the first article of this compilation, the story begins with a mini-review on protein calretinin from Beat Schwaller (Schwaller, 2014). The Author not only offers a summary of what is known about the expression and physiological role of calretinin in cellular homeostasis, but, to mention one of many interesting aspects, the reader also learns why calretinin “may be a potential therapeutic target for treatment of Huntington disease” (see Dong et al., 2012 for original research report). Further two original research papers focus on the development of CR+ neurons during the ontogenesis of human cerebral cortex. Miriam Gonzales-Gomez and Gundela Meyer (Gonzalez-Gomez and Meyer, 2014) put emphasis on the expression of CR in neuronal progenitors during the early period (between 6 and 14 gestational weeks, gw) of human corticogenesis. At the early embryonic time, the expression of CR is unrelated to the GABAergic neurons and could be seen also in glutamatergic neurons of the preplate and “pioneer” cortical plate. Complementary to this paper, Nevena V. Radonjic with colleagues (Radonjic et al., 2014) describes the pattern of expression of a transcription factor (Gsx2) and its two downstream effectors, (Ascl1 and Sp8) proposed to be involved in the CR cell lineage, in the human fetal cortex at later stadium of corticogenesis, at midgestation (20 gw). The authors reported the co-expression of these factors with CR+ cells that were dividing in the cortical ventricular and subventricular zones (VZ/SVZ). Their results support the hypothesis that the VZ/SVZ, besides the ganglionic eminences in subpallium, is an additional source of interneurons in the primate developing cortex. However, alternative hypothesis have been suggested (Hansen et al., 2013; Ma et al., 2013). In the next—4th article of our compilation, review paper from Ana Hladnik and colleagues (Hladnik et al., 2014), authors broadly discuss this controversial topic. They propose that contradictory results about the origin of cortical CR+ interneurons in primates might be partially explained by regional variations in the site of CR+ neurons origin. According to their hypothesis, the main source of CR+ interneurons for the frontal lobe might be the local pallial VZ/SVZ, while subpallial ganglionic eminences supply CR+ interneurons for posterior and lateral regions of primate cerebral cortex. One fact, which indirectly supports the concept of an additional source of CR+ interneurons in primate cortex, is the significantly higher proportion of CR+ cells compared to non-primate mammals. Another two main classes of interneurons, those containing PV and CB (or somatostatin) do not show such a robust increase in proportion during evolution. In the 5th article of this Research Topic, mini review from Domagoj Džaja and colleagues (Džaja et al., 2014), authors address this topic and the significance of the 5-fold increase of CR+ neurons in the layers II/III of primate cortex in relation to rodents, for the function of neocortical neuronal circuits in primate brain. Importantly, they open and discuss the question of the role of those additional CR+ interneurons in formation of neuronal assemblies. In the 6th article, Daniela Camillo with colleagues (Camillo et al., 2014) studies the visual response properties of neurons that express or have transiently expressed CR during their development in mouse primary visual cortex. Besides other interesting findings, their results again accentuate the (functionally not well understood) high extent of transient CR expression in pyramidal neuron precursors during corticogenesis. Although the terms “interneurons” and “local circuits neurons” are often used synonymously, the CR+ interneurons were also shown to participate on long range cortical connectivity: by being targeted via connections from remote cortical areas (Gonchar and Burkhalter, 2003) as well as by sending long range cortico-cortical axons (Tomioka and Rockland, 2007). In the 7th article of our Research Topic, Rastislav Druga with colleagues (Druga et al., 2014) describes the distribution of CR+ neurons in the claustrum of the rat. Interestingly, a small portion of those neurons could be found to send dendrites in the neighboring telencephalic structures (striatum) or the insular cortex. This pattern of interconnectivity is consistent with the concept of claustrum as a global processing and synchrony detection system (Smythies et al., 2014), and implies that CR+ neurons could be directly involved in this function. In the 8th article of our Research Topic, Bruno Cauli with colleagues (Cauli et al., 2014) provides a closing comprehensive summary of what is known about the anatomy and physiology of bipolar cortical CR+ interneurons. Besides the themes elaborated also in the individual articles of this compilation, additional exciting topics as for example the involvement of CR+ neurons in neurovascular coupling, are reviewed here. Summarizing the available evidence, authors state that probably the bipolar CR+ interneurons are amongst the most complex cells in the cerebral cortex because they seem to be involved in every major mechanism that supports successful neuronal computation. Hence, from this point of view, the heterogeneous population of CR expressing neurons really seems to stand “at the top of the interneuronal pyramid.” And finally, in the last, 9th article of this research topic, a “window” to an important research field—function and malfunction of CR+ interneurons in various neuropsychiatric disorders—is being opened. In this review paper, Kinga Toth and Zsofia Magloczky (Toth and Magloczky, 2014) discuss the vulnerability of CR+ hippocampal interneurons to epilepsy. They summarize that the sensitivity of CR+ interneurons to epileptic activity plays an important role in the impairment of dendritic inhibition in epilepsy, with potential significant involvement in epileptogenesis. A different situation, with preservation of CR+ interneurons and loss of PV+ and/or CB+ interneurons could be found in other conditions as schizophrenia, bipolar disorder, and major depression (Beasley et al., 2002; Cotter et al., 2002; Sakai et al., 2008). It remains an exciting task for the future to establish a more detailed view of the role of CR+ interneurons in various pathological states. All the aforementioned contributions are collected also in a single e-book. We hope this may be useful for researchers approaching this research field. More than anything else, we hope to inspire further research on this intriguing neuronal population and on its function and malfunction in neural circuits.

The marriage between Leonardo III ? the last member of the Tocco family who ruled the Heptanese (1448-1479) ? and Milica Brankovic, the daughter of the Serbian Despot Lazar Brankovic and Helen Palaiologos, concluded in Dubrovnik on May 1 1463, is an intriguing issue: it was concluded under unusual circumstances, and its significance was variously presented by the couple's earlier and later contemporaries. An analysis of several historical sources (mainly those of documentary character, also some of a narrative nature) shows that, despite the belittlement of its significance made by some of the pair's contemporaries, the marriage was generally seen as a prospective alliance, designed not only to satisfy the existential needs of the couple but also to enhance the interests of various important political factors of the time: Thomas Palaiologos, the authorities of Dubrovnik, the Roman Curia and Cardinal Bessarion. As widely known, the political prospectives opened by this marriage were not fully accomplished due to the short duration of this relationship (Milica died soon after the wedding). Yet this conjugal alliance proved useful for the Tocco party even after its conclusion. In the time following Leonardo's flight from the Ottomans to Naples (after 1479), he referred to the lineage with Milica as an alliance with the Byzantine imperial family, supposedly in order to achieve certain benefices from the Italian environment where he repatriated. In parallel to the analysis of the sources about the motives and significance of the first marriage of the last Tocco despot, in this paper I also deal with several pro-sopographic and topographic details of regional importance, mentioned on the occasion of the Tocco-Brankovic wedding ceremony (e.g. Bishop of Bologna Blasius Constantii Paliki, etc). .

Radial glial (RG) cells have been demonstrated to be a major neural progenitor cell type, but in the human fetal brain, neither their molecular nor their spatiotemporal characteristics are well known. We used glial and neuronal-specific antibodies to determine the antigen characteristics and distribution of RG cells and other neuronal progenitors in the human brain during the first half of intrauterine development. Proliferating RG (4A4+) cells in the ventricular zone (VZ) showed clear caudorostral and ventrodorsal gradients, spreading from the spinal cord to the ventral rhombencephalon, at embryonic stages (4.5–5.5 gestational weeks [gw]). However, in the same embryo, other dividing cells expressed the neuronal marker SMI-31 and were present throughout the entire CNS, including the rostral prosencephalon. At the beginning of cortical neurogenesis (6 gw), proliferating VZ cells labeled either with neuronal markers (SMI-31, MAP2, β-III-tubulin), double-labeled 4A4+/SMI-31+ cells, or cells not labeled with these antibodies, were in close proximity to each other. At midgestation (17–24 gw), RG divisions were less frequent, but were spread throughout the entire cerebral cortex, including the subventricular and intermediate zones and the subpial granular layer. Several subtypes of RG were co-labeled with vimentin and other glial markers (BLBP, GFAP, or GLAST) and quantified in vitro. In conclusion, the diversity of cortical progenitors in the human brain may, in part, explain the unique complexity of the human cerebral cortex. © 2005 Wiley-Liss, Inc.

Understanding the factors that govern human forebrain regionalization along the dorsal-ventral and left-right (L-R) axes is likely to be relevant to a wide variety of neurodevelopmental and neuropsychiatric conditions. Recent work in lower vertebrates has identified several critical signaling molecules involved in embryonic patterning along these axes. Among these are the Wingless-Int (WNT) proteins, involved in the formation of dorsal central nervous system (CNS) structures, as well as in visceral L-R asymmetry. We examined the expression of WNT2b and WNT7b in the human brain, because these genes have highly distinctive expression patterns in the embryonic mouse forebrain. In the human fetal telencephalon, WNT2b expression appears to define the cortical hem, a dorsal signaling center previously characterized in mouse, which is also confirmed by BMP7 expression. In diencephalon, WNT2b expression is restricted to medial dorsal structures, including the developing pineal gland and habenular nucleus, both implicated in CNS L-R asymmetry in lower organisms. At 5 weeks gestation, WNT7b is expressed in cerebral cortical and diencephalic progenitor cells. As the cortical plate develops, WNT7b expression shifts, demarcating deep layer neurons of the neocortex and the hippocampal formation. Spatial and temporal expression patterns show startling similarity between human and mouse, suggesting that the developmental roles of these WNT genes may be highly conserved, despite the far greater size and complexity of the human forebrain.

The Genoese citizenship, granted to Carlo I Tocco and his regent mother Magdalene by the authorities of the Republic of Genova (December 2, 1389) is a document the existence of which is widely accepted in the scholarly circles despite the fact that the details of its contents have still remained largely unknown. Attempting to contribute to a better understanding of the circumstances under which the grant was issued, the first part of this paper brings the transcription of the entire document, as well as an analysis of its political and legal context. The paper's second part deals with the document's palaeographic, diplomatic, and sigillographie features as well as with its prosopographic and topographic details.

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.

Schizophrenia is a disease of abnormal brain development. Considerable evidence now indicates that environmental factors have a causative role in schizophrenia. Elevated incidence of the disease has been linked to a wide range of disturbances in the prenatal environment and to social factors and drug intake during adolescence. Here we examine neurodevelopment of the prefrontal cortex in the first trimester of gestation and during adolescence to gain further insight into the neurodevelopmental processes that may be vulnerable in schizophrenia. Early embryonic development of the prefrontal cortex is characterized by cell proliferation, including renewal of progenitor cells, generation of early transient cell populations and neurogenesis of subcortical populations. Animal models show that curtailing early gestational cell proliferation produces schizophrenia-like pathology in the prefrontal cortex and mimics key behavioral and cognitive symptoms of the disease. At the other end of the spectrum, elimination of excitatory synapses is the fundamental process occurring during adolescent maturation in the prefrontal cortex. Adverse social situations that elevate stress increase dopamine stimulation of the mesocortical pathway and may lead to exaggerated synaptic pruning during adolescence. In a non-human primate model, dopamine hyperstimulation has been shown to decrease prefrontal pyramidal cell spine density and to be associated with profound cognitive dysfunction. Development of the prefrontal cortex in its earliest stage in gestation and in its final stage in adolescence represents two critical periods of vulnerability for schizophrenia in which cell proliferation and synaptic elimination, respectively, may be influenced by environmental factors.

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.

It has been suggested that Golli proteins, structurally related to myelin basic proteins (MBPs), have a role in autoimmune processes. We studied the expression of these proteins in multiple sclerosis (MS) and determined that the number of Golli-immunoreactive (ir) cells was significantly higher around lesions of chronic MS than in control white matter. Golli proteins were expressed in the adult oligodendrocyte precursor cells (OPCs), activated microglia/macrophages, and some demyelinated axons around MS lesions. Their expression in adult OPCs indicates remyelination attempts, whereas the expression in the subpopulation of microglia/macrophages suggests roles in the immune processes of MS. In addition, Golli proteins may be markers of axonal transection, which is characteristic for MS.

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.

Impaired sonic hedgehog (Shh) signaling is involved in the pathology of cortical formation found in neuropsychiatric disorders. However, its role in the specification of human cortical progenitors is not known. Here, we report that Shh is expressed in the human developing cortex at mid-gestation by radial glia cells (RGCs) and cortical neurons. We used RGC cultures, established from the dorsal (cortical) telencephalon of human brain at mid-gestation to study the effect of Shh signaling. Cortical RGCs in vitro maintained their regional characteristics, expressed components of Shh signaling, and differentiated into Nkx2.1, Lhx6, and calretinin-positive (CalR(+)) cells, potential cortical interneuron progenitors. Treatment with exogenous Shh increased the pool of Nkx2.1(+) progenitors, decreased Lhx6 expression, and suppressed the generation of CalR(+) cells. The blockade of endogenous Shh signaling increased the number of CalR(+) cells, but did not affect Nkx2.1 expression, implying the existence of parallel Shh-independent pathways for cortical Nkx2.1 regulation. These results support the idea that, during human brain development, Shh plays an important role in the specification of cortical progenitors. Since direct functional studies in humans are limited, the in vitro system that we established here could be of great interest for modeling the development of human cortical progenitors.

This study examined glutamate-activated current responses of mouse and human Cajal-Retzius (C-R) cells. Thin cortical slices were prepared from the brains of mice 4–6 days after birth and from those of midgestational human fetuses. Both human and mouse C-R cells displayed glutamate-induced whole-cell current responses that were voltage-dependent and included an N-methyl-d-aspartate (NMDA) receptor–mediated component that was differentially sensitive to blockade by the NMDA receptor antagonists 2-amino-5-phosphonovaleric acid and ifenprodil. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a non-NMDA glutamate receptor agonist, induced current responses in human but not in mouse C-R cells. These results, taken together, lead us to conclude that human C-R cells express both NMDA and AMPA types of glutamate receptors very early during development of the cortex. In contrast, mouse C-R cells express only the NMDA type of glutamate receptor. Thus we demonstrate a species-dependent sensitivity of C-R cells to glutamate and postulate that this differential sensitivity may account in part for a species-dependent difference in the persistence of C-R cells during cortical development.

Layer I, which plays an important role in the development of the cerebral cortex, expands in size and diversity in primates. We found that, unlike in rodents, in the macaque monkey, neurons of this layer are generated during the entire 2 month period of corticogenesis, within the middle of the 165-d-long gestation. The large, classical Cajal-Retzius cells, immunoreactive to reelin and calretinin but not to GABA, are generated first [embryonic day 38 (E38)-E50], with the peak of [(3)H]thymidine ([(3)H]TdR) labeling at E43. Ultrastructural analysis revealed that processes of these cells form a stereotyped, rectangular network oriented parallel to the pial surface. Genesis of smaller, GABAergic neurons begins slightly later (E43), reaches a peak of [(3)H]TdR labeling between E54 and E70, and continues until the completion of corticogenesis (E94). These late-generated layer I cells are imported from outside sources such as the olfactory primordium and ganglionic eminence and via a massive subpial granular layer that may also supply some GABAergic interneurons to the subjacent cortical plate. The ratio of large-to-small layer I neurons changes differentially, indicating that each class is produced and/or eliminated at a different rate and suggesting that their roles in primates are diverse.

This paper deals with a familial strategy of "using" kinsmen in keeping power and prominence as seen from the case of the family of the Tocco, the rulers of the Ionian islands (XIV-XV c). A widely-known family narrative and some less-known documentary evidence reveal details on several Italian Tocco who were close to Carlo I (b. c. 1375-1429) in spatial terms, and who, more importantly, played a considerable role in securing useful political alliances for their mighty kinsman of the "Greek" family branch. In addition, regarding new information about one Italian Tocco (Gioanella, the daughter of Carluccio), the paper suggests some more definite conclusions about her genealogical position and conjugal affiliation.

Programmed cell death (PCD) in the form of apoptosis is recognized as one of the central events in the development of the central nervous system. To study the time of onset, extent and distribution of PCD in the human telencephalon, embryos and fetuses from 4.5 to 27 gestational weeks (g.w.) were examined using the TUNEL (TdT-mediated dUTP-biotin nick-end labelling) in situ method. At 4.5 g.w. sparse TUNEL(+) nuclei were observed in the ventricular zone of the neural tube. With the formation of the cortical plate at 7-8 g.w. , TUNEL(+) nuclei were seen in all developmental layers of the cortical anlage, as well as in the subcortical regions such as the ganglionic eminence and the internal capsule. The proliferative zones (the ventricular zone, the subventricular zone and the ganglionic eminence) contained the majority of all apoptotic nuclei observed in each specimen. However, the apoptotic index was highest in the subplate zone and in layer I. Double-labelling experiments suggested that neuronal precursors were the main population of cells undergoing PCD in the first trimester of gestation, whereas glial cells probably start dying around midgestation. The onset of labelling of microglial cells and apoptotic nuclei were synchronous, indicating the involvement of microglia in PCD. In conclusion, two distinct types of PCD were observed during human telencephalic development: embryonic apoptosis, which was synchronous with proliferation and migration of neuronal cells and probably not related to establishment of neuronal circuitry, and fetal apoptosis, which coincided with differentiation and synaptogenesis, and therefore may be related to the development of axonal-target connectivity.

The early expression of reelin, calcium-binding proteins (calretinin, calbindin, and parvalbumin), and neurofilament proteins have been explored in the developing central nervous system of human embryos and fetuses during the first trimester of gestation. Our objective has been to determine further the nature, developmental roles, and contributions of the early neurons and fibers of the original subpial neuropil, i.e., the primordial plexiform layer (PPL). In young embryos (4-5 weeks old), neurofilament protein-labeled fibers run through the subpial neuropil of the caudal portion of the neural tube, reaching the mesencephalon rostrally. At this age, calretinin-immunoreactive and calbindin-immunoreactive neurons are also found among cells already detached from the ventricular zone. The expression of neurofilament protein, calretinin, and calbindin follows an ascending caudorostral gradient, reaching the cerebral vesicles by the 6th-7th week of gestation. In the cerebral cortex, this timing coincides with the initial expression of reelin in the PPL. The reelin immunoreactivity throughout the most superficial cellular population of the cortical PPL supports the early genesis of Cajal-Retzius cells, around the 6th week of gestation. After the splitting of the PPL by the formation of the cortical plate (7-8 weeks of gestation), reelin-immunoreactive cells remain only in the newly established layer I. This study proposes that an initial PPL may be a universal feature of the developing central nervous system.

A fundamental question in brain development is how neurons make the precise topographic connections necessary for function. The hypothesis that transient expression of calcium (Ca2+) signaling molecules may have a role in this process was tested by studying human cerebella at midgestation. In addition, four adult brains, two controls and two from patients with ataxia, were studied as well. The temporal and spatial distribution of intracellular Ca2+ channel/receptors, inositol trisphosphate receptor type 1 (IP3R1) and ryanodine receptor (RyR) and three Ca2+ binding proteins were examined with immunocytochemical methods. A positive immune reaction with all markers of Ca2+ signaling was found in the Purkinje cell layer starting from 17 g.w. (gestational weeks), the youngest age studied. The immune reactions were not homogeneous throughout the extent of the Purkinje cell layer, but instead displayed a 'patchy' appearance in all intrauterine stages. In the adult cerebellum the expression of Ca2+ signaling molecules was homogenous. In the two cerebella obtained from patients suffering from ataxia, a several-fold reduction of immunostaining with IP3R1 was found. Our findings suggest that transient and differential mobilization of intracellular Ca2+ in seemingly homogenous neuronal types may play a role in development of highly organized projection maps of the cerebellar cortex. Moreover, lack of IP3R1 in the diseased brains suggests that internal stores of Ca2+ play an important role in normal function of the cerebellum.

The development of microglia and macrophages was studied in 14 human embryos and fetuses ranging in age from 4.5–13.5 gestational weeks (g.w.), using lectins, Ricinus communis agglutinin-1 [RCA-1], and Lycopersicon esculentum, tomato lectin (TL), which recognize macrophages and microglia, and antibodies for the macrophage antigen CD68. Lectin-positive (+) cells were observed at 4.5 g.w., the youngest age examined. They were detected in the leptomeninges around the neural tube, and only rarely were observed in the CNS parenchyma. At 5.5 g.w., lectin+ cells were present throughout the CNS parenchyma, and a portion of these cells could also be labeled with antibody to CD68. In subsequent weeks, both types of cells, lectin+ and CD68+/lectin+ cells co-existed and progressively developed typical microglial morphology. In addition, in double label experiments, an antibody that labels CD14 antigen present on monocytes, hematogenous precursors of tissue macrophages, did not label either lectin+ or CD68+/lectin+ cells in CNS parenchyma. Additional immunocytochemical studies with appropriate markers excluded the possibility that any of the cells described here were either astrocytes, oligodendrocytes, endothelial cells or neurons. Our finding that one class of cells can be labeled early only with lectins, while another can be labeled with both lectins and CD68 macrophage antibody, may reflect a different origin of microglia in the early embryonic CNS compared to the fetal stages. This subdivision appears to be maintained in the adult brains as well.

Few recent data are available on the development of the precise projection maps of the cerebellar cortex in humans. To address this topic, we studied temporal and spatial distribution of several antigens involved in calcium (Ca)-dependent processes: the intracellular Ca receptors, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and ryanodine receptor (RyR); the Ca-binding proteins, calbindin D-28k (CB), parvalbumin (PV), and synaptophysin; and phosphorylated (SMI 31) and nonphosphorylated (SMI 32) forms of neurofilament protein. All antigens were studied in the human cerebellum during intrauterine development. The results of this study show that immunocytochemical markers appeared in the following sequence: CB and both forms of neurofilament protein were observed at 4–5 gestational weeks (g.w.), PV appeared in the external granular layer and in a few Purkinje cells at 11 g.w., a diffuse immunostaining for IP3R1 and synaptophysin were observed at 13 g.w., whereas RyR was observed at 17–18 g.w. From 24 g.w. on, Purkinje cells expressed all four examined markers of intracellular Ca signaling as well as two forms of neurofilament protein. At the same time, compartmentation of the Purkinje cell layer was detected with three intracellular Ca-signaling molecules (IP3R1, CB, and PV) and with SMI 32. These results indicate that the developmentally regulated expression of antigens studied here may play a role in establishing a highly regular organization of terminal fields in the human cerebellar cortex. Moreover, the initial expression of these antigens is correlated temporally with other developmental processes in the cerebellum, such as cellular maturation, revealed by the immunoreaction to cytoskeletal protein, and synaptogenesis, revealed by immunoreaction to synaptophysin. J. Comp. Neurol. 396:442–460, 1998. © 1998 Wiley-Liss, Inc.

Myelin basic protein (MBP) is a major myelin constituent produced by oligodendrocytes in the central nervous system (CNS). Expression of MBP was considered to be a marker for oligodendrocyte differentiation and myelination in the developing CNS. In this study, expression of myelin basic protein (MBP) and its messenger RNA (mRNA) was examined in human embryos and fetuses ranging in age from 5 to 20 gestational weeks (g.w.). We were able to demonstrate that MBP antibody labels cells in both human nervous and non-nervous tissues beginning from early embryonic life (5-6 g.w.). MBP positive (MBP+) cells were rounded, with either no cell processes or only 1-2 short processes, and were located in caudal regions of the CNS. MBP+ cells were also observed in the non-nervous tissue, such as leptomeninges, choroid plexus, and connective tissues. A number of MBP+ cells in nervous and non-nervous tissues were morphologically similar to macrophages and showed a positive reaction to macrophage-microglia markers: lectin (RCA-1) and the monoclonal antibody (EBM-11) to human macrophage antigen CD68, whereas they were negative for neuronal, astroglial, or marker for oligodendrocyte progenitors. At the same embryonic age, 5 g.w. and onward, the MBP mRNA was observed in the CNS by in situ hybridization. The results of this study show that MBP immune reaction is spread in a large area of the CNS prior to myelin appearance. In addition, for the first time it has been demonstrated that the same population of cells could be labelled with both MBP and macrophage markers. These results indicate that MBP, or MBP-related proteins, could represent a link between the immune and nervous system during early development. Thus, besides the well established role in myelination, these proteins might have an additional and still unknown function in development.

The development of cortical cells immunoreactive for γ-aminobutyric acid (GABA) was studied in human cerebral cortex in the first trimester of gestation (from 4 to 13 gestational weeks; g.w.). The first GABA-immunoreactive (IR) cells were observed at 6.5 g.w., i.e., before the appearance of the cortical plate, which gives rise to a majority of the adult cortical layers. GABA-IR cells were found initially in the telencephalic wall, where a lateromedial gradient in the density of GABA-positive cells was observed at this early developmental time point, but not at later stages. At 7 g.w., as the cortical plate emerged in the ventrolateral region of the cerebral vesicle, GABA-immunoreactive cells were found dorsal and ventral to the developing cortical plate. At this stage, immunoreactivity was also observed in the other transient developmental zones of the cortical anlage: in the subplate layer and in the intermediate, subventricular and ventricular zones. From 8 to 9 g.w. and continuing throughout the end of the studied period (13 g.w.), GABA-IR cells were distributed throughout the full width of the telencephalic wall, and, at 13 g.w., the newly formed subpial granular layer contained GABA-immunoreactive cells, as well. However, the predominant sites for GABA immunoreactivity remained the prospective layer I and the subplate. The population of GABA-positive cells described here was not immunoreactive for glial fibrillary acidic protein (GFAP) at any gestational age examined and, therefore, probably represents GABA-containing neurons. The observation that GABA-IR neurons appear in human developing cortex slightly before the cortical plate formation and beginning of synaptogenesis (6.5 g.w.) suggests that GABA plays an important role in the initial organization of the developing human cerebral cortex. J. Comp. Neurol. 380:495–506, 1997. © 1997 Wiley-Liss, Inc.

Function of oligodendrocytes (OLs), myelin forming cells in the CNS, is disrupted in demyelinating diseases such as periventricular leukomalacia or multiple sclerosis. It is, thus, important to better understand factors that can affect generation or differentiation of human OLs. In rodents, Sonic hedgehog (Shh) is influencing expression of Olig2, a helix-loop-helix transcription factor required for development of OLs. In humans, Olig2 is present in cortical progenitors at midgestation, however the role of Shh in the specification of human OLs, including Olig2 positive (Olig2+) progenitors, is not fully understood. Here we studied in vitro effects of Shh signaling on proliferation and specification of human cortical Olig2+ progenitors at midgestation. First, we established that the spatiotemporal pattern of Olig2 expression in the human developing CNS, described on cryosections, was preserved in mixed and enriched radial glia cell (RGC) cultures. Next, we demonstrated that in vitro treatment with Shh induced an increase in the number of Olig2+ progenitors. Shh treatment increased the density of early oligodendrocyte progenitors (OPCs) at the expense of radial glia cells, while the number of late OPCs, did not change. However, inhibition of endogenous Shh with cyclopamine did not reduce the density of Olig2+ cells, implying the presence of an additional Shh-independent mechanism for OLs generation in vitro. These results suggest that the primary role of Shh signaling in the human dorsal oligodendrogenesis is the expansion and specification of multipotent radial glia progenitors into Olig2+ early OPCs. These results obtained in vitro are relevant to understand primary myelination during CNS development, as well as remyelination in demyelinating diseases.

The temporal and spatial distribution of GAP-43 mRNA in early human development, from 6 to 23 gestational weeks (g.w.), was examined by in situ hybridization histochemistry. GAP-43 mRNA was expressed as early as 6 g.w. in all regions of developing nervous system, the spinal cord, brainstem, cerebellum, diencephalic and telencephalic regions. Although the pronounced level of expression persisted during the entire examined period, the intensity of expression varied along the spatial axis over time. Analysis at the cellular level revealed that early on in development (6 g.w.) GAP-43 mRNA was expressed in the entire neuroblast population. With the onset of differentiation, at 13-23 g.w., GAP-43 mRNA expression had switched to the neurons that are in the process outgrowth. The highest level of GAP-43 mRNA expression was localized in the regions consisting of differentiating neurons, such as the cortical plate and intermediate zone of the telencephalic wall, and several delineated subcortical and thalamic nuclei. The spatial and temporal pattern of GAP-43 mRNA expression obtained suggests a possible dual role of GAP-43 in the development of the human nervous system: in the embryonic brain it could be involved in fundamental processes underlying cell proliferation; in the fetal brain its expression is specifically correlated with differentiation and the outgrowth of axons.

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.