Coordination of Neuron Production in Mouse and Human Cerebral Cortex by the Homolog of Drosophila Mastermind Protein.

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.

N-Methyl d-Aspartate Receptor Expression Patterns in the Human Fetal Cerebral Cortex.

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.

Oxygen Levels Regulate the Development of Human Cortical Radial Glia Cells.

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 (<1% O2) and severe hypoxia (1% O2) arrested the differentiation of human RGCs, mainly by altering the generation of glutamatergic neurons. The in vitro activation of Wnt-ß-catenin signaling rescued the proliferation and neuronal differentiation of RGCs subjected to anoxia. Pathologic hypoxia (≤1% O2) also exerted negative effects on gliogenesis, by decreasing the number of O4+ preoligodendrocytes and increasing the number of reactive astrocytes derived from cortical RGCs. O2-dependent alterations in glutamatergic neurogenesis and oligodendrogenesis can lead to significant changes in cortical circuitry formation. A better understanding of the cellular effects caused by changes in O2 levels during human cortical development is essential to elucidating the etiology of numerous neurodevelopmental disorders.

The Role of Sonic Hedgehog in the Specification of Human Cortical Progenitors In Vitro.

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.

Connexin hemichannels contribute to spontaneous electrical activity in the human fetal cortex.

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.

Neurogenesis continues in the third trimester of pregnancy and is suppressed by premature birth.

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, <2 h old) pups at an equivalent postconceptional age. Glutamatergic neurogenesis was suppressed in preterm rabbits, as indicated by the reduced number of Tbr2(+) 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.

COUP-TFII expressing interneurons in human fetal forebrain.

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.

Dorsal radial glial cells have the potential to generate cortical interneurons in human but not in mouse brain.

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 d in 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.

Spontaneous electrical activity in the human fetal cortex in vitro.

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 development in 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.

Enforced Pax6 expression rescues alcohol-induced defects of neuronal differentiation in cultures of human cortical progenitor cells.

BACKGROUND: Alcohol is the most widely consumed substance of abuse, and its use during pregnancy can lead to serious disorders of brain development. The precise molecular action of alcohol on human brain development, however, is still unknown. We previously enriched multipotent progenitor cells, radial glia (RG) cells, from human fetal forebrain and demonstrated that they express transcription factor Pax6 that is necessary for their neurogenic fate. METHODS: Enriched human fetal RG cells were maintained in vitro as either control or Pax6-expressing retrovirus infected cells. Cultures were treated with increasing doses of alcohol to evaluate Pax6 expression, proliferation, and differentiation of RG cells by immunocytochemistry, Western blot, and RT-PCR methods. RESULTS: In vitro treatment with alcohol reduced the expression of transcription factor Pax6 and proliferation of RG cells, which decreased neurogenesis. Consistent with this finding, the overexpression of Pax6 in RG cells under alcohol treatment rescued cell proliferation and restored the generation of neurons. In contrast to this effect on neurogenesis, the overexpression of Pax6 inhibits the generation of astroglia regardless of alcohol treatment, implying lineage-specific effects. CONCLUSIONS: These findings suggest that the effect of alcohol on human neurogenesis is partially due to the reduced expression of transcription factor Pax6 in RG cells.

Multiple origins of human neocortical interneurons are supported by distinct expression of transcription factors.

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.

Electrical excitability of early neurons in the human cerebral cortex during the second trimester of gestation.

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(+) 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 beta IV 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.

Human fetal radial glia cells generate oligodendrocytes in vitro.

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 (PDGFRalpha), 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.

Is Pax6 critical for neurogenesis in the human fetal brain?

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.

Human cortical neurons originate from radial glia and neuron-restricted progenitors.

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 fucose N-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 progeny in 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 (beta-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, our in vitro study 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.

Radial glia cells in the developing human brain.

Human radial glia (RG) share many of the features described in rodents, but also have a number of characteristics unique to the human brain. Results obtained from different mammalian species including human and non-human primates reveal differences in the involvement of RG in neurogenesis and oligodendrogenesis and in the timing of the initial expression of typical RG immunomarkers. A common problem in studying the human brain is that experimental procedures using modern molecular and genetic methods, such as in vivo transduction with retroviruses or creation of knockout or transgenic mutants, are not possible. Nevertheless, abundant and valuable information about the development of the human brain has been revealed using postmortem human material. Additionally, a combination and spectrum of in vitro techniques are used to gain knowledge about normal developmental processes in the human brain, including better understanding of RG as progenitor cells. Molecular and functional characterization of multipotent progenitors, such as RG, is important for future cell replacement therapies in neurological and psychiatric disorders, which are often resistant to conventional treatments. The protracted time of development and larger size of the human brain could provide insight into processes that may go unnoticed in the much smaller rodent cortex, which develops over a much shorter period. With that in mind, we summarize results on the role of RG in the human fetal brain.

Multiple roles of Sonic Hedgehog in the developing human cortex are suggested by its widespread distribution.

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.

The Subventricular Zone: A Key Player in Human Neocortical Development.

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.

Olig transcription factors are expressed in oligodendrocyte and neuronal cells in human fetal CNS.

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.

N-Methyl D-Aspartate Receptor Antagonist Kynurenic Acid Affects Human Cortical Development.

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.