Nada Zecevic

Changes in synaptic density in motor cortex of rhesus monkey during fetal and postnatal life

The density and proportion of synaptic contacts in the primate motor cortex (Brodmann area 4) were determined in 21 rhesus monkeys ranging in age from embryonic day 41 (E41) to 20 years. Two to 4 vertical electron microscopic probes, each consisting of 150-250 overlapping micrographs traversing the thickness of the cortex, were prepared for each specimen. Synapses were categorized according to their morphology (symmetrical or asymmetrical), cellular location (on spines, shafts or soma), number, and ratio of laminar distribution. The density of synapses was expressed per unit area and volume of neuropil (excluding neuronal and glia cell bodies, myelin sheath, blood vessels and extracellular space). The first synapse in the area of the emerging motor cortex were observed at E53 in the marginal zone (prospective layer I) and in the transient subplate zone situated beneath the developing cortical plate. Around midgestation (E89) synapses were observed over the entire width of the cortical plate, and their density was about 5/100 microns 3 of neuropil. During the last two months of gestation synaptic density increased 8-fold across all layers to reach about 40/100 microns 3 at the time of birth (E165). Synaptic production continued postnatally and by the end of the second postnatal month attained a level of 60/100 microns 3 neuropil which is two times higher than in the adults. This level decreased at a slow rate until sexual maturity (3 years of age) and then more rapidly to the adult level which is characterized by relative stability of about 30/100 microns 3. The decline in synaptic density after the peak in infancy occurs predominantly at the expense of asymmetric synapses situated on dendritic spines; the population of symmetric synapses on dendritic shafts remains relatively constant. The development of synaptic connections in the motor cortex of non-human primates involves initial overproduction followed by selective elimination and structural alterations.

Development of Layer I Neurons in the Primate Cerebral Cortex

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 [3H]thymidine ([3H]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 [3H]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.

Programmed cell death in the developing human telencephalon

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.

Oligodendrocyte development and the onset of myelination in the human fetal brain

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.

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 (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, 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.

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.

Early oligodendrocyte progenitor cells in the human fetal telencephalon

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, PDGFRα, 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. GLIA 41:117–127, 2003.

Specific characteristic of radial glia in the human fetal telencephalon

Phenotypic characteristics of cells in the developing human telencephalic wall were analyzed using electron microscopy and immunocytochemistry with various glial and neuronal cell markers. The results suggest that multiple defined cell types emerge in the neocortical proliferative zones and are differentially regulated during embryonic development. At 5–6 weeks gestation, three major cell types are observed. Most proliferating ventricular zone (VZ) cells are labeled with radial glial (RG) markers such as vimentin, glial fibrillary acidic protein (GFAP), and glutamate astrocyte‐specific transporter (GLAST) antibodies. A subpopulation of these RG cells also express the neuronal markers β III‐tubulin, MAP‐2, and phosphorylated neurofilament SMI‐31, in addition to the stem cell marker nestin, indicating their multipotential capacity. In addition, the presence of VZ cells that immunoreact only with neuronal markers indicates the emergence of restricted neuronal progenitors. The number of multipotential progenitors in the VZ gradually decreases, whereas the number of more restricted progenitors increases systematically during the 3‐month course of human corticogenesis. These results suggest that multipotential progenitors coexist with restricted neuronal progenitors and RG cells during initial corticogenesis in the human telencephalon. Since the multipotential VZ cells disappear during the major wave of neocortical neurogenesis, the RG and restricted neuronal progenitors appear to serve as the main sources of cortical neurons. Thus, the diversification of cells in human VZ and overlying subventricular zone (SVZ) begins earlier and is more pronounced than in rodents.

GRO-α and CXCR2 in the Human Fetal Brain and Multiple Sclerosis Lesions

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-α (GRO-α) 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-α 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-α 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-α/CXCR2 may play a role in human oligodendrocyte proliferation and subsequent migration. We also studied the expression of GRO-α and CXCR2 in brain sections of multiple sclerosis (MS) patients. Consistent with their role in the inflammatory process of MS, both GRO-α and CXCR2 were expressed in activated microglia localized on the border of MS lesions. However, neither GRO-α nor CXCR2 were present in early oligodendrocyte progenitors, a finding that may partially explain why remyelination is not more efficient in MS.

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 brains higher complexity and capacity to process information.