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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.

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.

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

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.

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). .

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.

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.

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.

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.

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.

Transcription factor COUP-TFII in rodents is important for migration of cortical interneurons from caudal ganglionic eminence (CGE) to the neocortex. Since in human, unlike in rodents, cortical interneurons have both ganglionic eminence (GE) and dorsal cortical origin, we studied the distribution of COUP-TFII in the human developing neocortex from 9 to 22 gestational weeks. COUP-TFII is expressed at all stages studied in the GE and in various cortical zones, from the proliferative ventricular/subventricular zone (VZ/SVZ) to layer I. Gradients of COUP-TFII expression are present in the GE, with peak expression in the CGE, and in the neocortex, from high expression in the temporal and occipital cortex to moderate in the frontal and dorsal cortex. Double immunofluorescence with γ-aminobutyric acid (GABA), calretinin, or calbindin, established that subpopulations of interneurons express COUP-TFII. A small fraction of COUP-TFII(+) cells are progenitor cells that proliferate in the CGE (3.4 ± 0.3%) and in the cortical VZ/SVZ (1.7 ± 0.1%). In summary, COUP-TFII is expressed in the human fetal forebrain in GABAergic cells, according to its possible role in migration of cortical interneurons. The source of these cells seems to be the CGE and, to a smaller extent, the cortical VZ/SVZ.

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.

Oligodendrocytes are cells that myelinate axons, providing saltatory conduction of action potentials and proper function of the central nervous system. Myelination begins prenatally in the human, and the sequence of oligodendrocyte development and the onset of myelination are not thoroughly investigated. This knowledge is important to better understand human diseases, such as periventricular leukomalacia, one of the leading causes of motor deficit in premature babies, and demyelinating disorders such as multiple sclerosis (MS). In this review we discuss the spatial and temporal progression of oligodendrocyte lineage characterized by the expression of specific markers and transcription factors in the human fetal brain from the early embryonic period (5 gestational weeks, gw) until midgestation (24 gw). Our in vitro evidence indicated that a subpopulation of human oligodendrocytes may have dorsal origin, from cortical radial glia cells, in addition to their ventral telencephalic origin. Furthermore, we demonstrated that the regulation of myelination in the human fetal brain includes positive and negative regulators. Chemokines, such as CXCL1, abundant in proliferative zones during brain development and in regions of remyelination in adult, are discussed in the view of their potential roles in stimulating oligodendrocyte development. Other signals are inhibitory and may include, but are not limited to, polysialic acid modification of the neural cell adhesion molecule on axons. Overall, important differences in temporal and spatial distribution and regulatory signals for oligodendrocyte differentiation exist between human and rodent brains. Those differences may underlie the unique susceptibility of humans to demyelinating diseases, such as MS.

Limited knowledge about human oligodendrogenesis prompted us to explore the lineage relationship between cortical radial glia (RG) cells and oligodendrocytes (OLs) in the human fetal forebrain. RG cells were isolated from cortical ventricular/subventricular zone and their progeny was followed in vitro. One portion of RG cells differentiated into cells of OL lineage identified by cell-type specific antibodies, including platelet-derived growth factor receptor-alpha (PDGFRα), NG2, O4, myelin basic protein, and myelin oligodendrocyte glycoprotein. Moreover, using Cre Lox fate mapping (brain lipid binding protein-Cre/Floxed-yellow fluorescent protein) we established a direct link between RG cells and OL progenitors. In vitro generation of RG-derived O4+ OL progenitors was enhanced by addition of sonic hedgehog (SHH) and reduced by the SHH inhibitor, cyclopamine, suggesting the role of SHH signaling in this process. In summary, our in vitro experiments revealed that a portion of cortical RG cells isolated from human forebrain at the second trimester of gestation generates OL progenitors and this suggests a role of SHH in this process. © 2008 Wiley-Liss, Inc.

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.

The cortical subventricular zone (SVZ), a proliferative compartment in the forebrain, has a uniquely important role during the second half of intrauterine development in human. This is best observed in numerous neonatal pathologies that result from prenatal SVZ damage. These conditions highlight a need to better understand the contribution of the SVZ to the development of the human cerebral cortex. With this goal in mind, we analyzed histological organization, cell proliferation, and molecular diversity in the human fetal SVZ from 7-27 gestational weeks (gw) using light and electron microscopy, immunohistochemistry, and in vitro methods. Complex histological organization distinguishes human cortical SVZ from that of other mammals. In vitro quantification showed that approximately 50% of cells in the VZ/SVZ region are neurons, 30% are astroglia, 15% are nestin+ cells, with other cell types representing smaller fractions. Immunolabeling with BrdU showed that a considerable number of cells ( approximately 10%) are generated in the human cortical SVZ during midgestation (18-24 gw) under in vitro conditions. Immunofluorescence with cell type-specific markers and BrdU revealed that all major cell types, neural precursors (nestin+), astroglia including radial glia (GFAP+, vimentin+), and oligodendrocyte progenitors (PDGFR-alpha+) were proliferating. An increase in the ratio of the size of the SVZ to VZ, protracted period of cell proliferation, as well as cellular and histological complexity of the human fetal SVZ are directly related to the evolutionary expansion of the human cerebral cortex.

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.

Nat. Neurosci. 12, 1229–1237 (2009); published online 13 September 2009; corrected after print 25 September 2009 In the version of this article initially published, one of the corresponding authors' email addresses was misspelled. It should be luisapinto@ecsaude.uminho.pt. In addition, errors occurred in some of the numbers listed in the last subsection of the Results section.

Synapses develop concurrently and at identical rates in different layers of the visual, somatosensory, motor, and prefrontal areas of the primate cerebral cortex. This isochronic course of synaptogenesis in anatomically and functionally diverse regions indicates that the entire cerebral cortex develops as a whole and that the establishment of cell-to-cell communication in this structure may be orchestrated by a single genetic or humoral signal. This is in contrast to the traditional view of hierarchical development of the cortical regions and provides new insight into the maturation of cortical functions.