Neocortical Ventricular Zone Research Articles

Origin of the Neocortical Subependymal Cells Speculated by Emx1 and GAD67 Expression

More than a decade has passed since adult neurogenesis in the murine telencephalon was described and accepted widely. The adult neurogenesis has been attributed to the progenitors in the subgranular layer in the dentate gyrus and the subependymal layer of the telencephalon. However, the developmental origin of the progenitors in the dentate gyrus and the subependymal layer is poorly understood. It is well known that the neurogenesis in the embryo brains occurs in the ventricular zone (VZ). Recently it has been proposed that the progenitors in the VZ of the telencephalon turn into the subventricular zone (SVZ) astrocytes (Tramontin et al., 2003). The subependymal layer of the neocortex has been regarded as a source of the granule cells and periglomerular cells in the adult olfactory bulb. The subependymal layer produces neurons that migrate to the olfactory bulb as the rostral migratory stream, and the newly produced neurons are going to be GABAergic inhibitory neurons in the olfactory bulb. However, we felt discontinuity in the idea that the neocortical VZ cells turn into SVZ astrocytes and then produce GABAergic neurons. By now, it is well known that most of the neocortical GABAergic neurons were supplied by tangential cell migration originating in the subpallial structures (Anderson et al., 1997; Tamamaki et al., 1997). It is also speculated that the neocortical VZ almost lack the ability to produce GABAergic neurons. Under these circumstances, we speculated that the GABAergic neurons for the olfactory bulb may be produced by progenitors derived from other than the neocortex. In this paper we will show that the subependymal cells in the adult neocortex are heterogeneous and may have different origin.

Citron kinase is a regulator of mitosis and neurogenic cytokinesis in the neocortical ventricular zone.

Successful cell division in neural progenitors in the neocortical ventricular zone (VZ), as in all dividing cells, depends critically upon coordinating chromosome segregation during mitosis with cytokinesis. This coordination further suggests that common molecular regulators may link events in mitosis with those in cytokinesis. Recent genetic evidence indicates that cytokinesis in CNS neuronal progenitors, but not in most other cell types of the body, requires the function of citron kinase. In neocortex, citron kinase is most critical for neurogenic cytokinesis. In citron kinase null mutants, a large proportion of neuronal cells within neocortex are binucleate; however, very few glial cells are binucleate. In addition, confocal time-lapse imaging of mitoses at the VZ surface shows that citron kinase is also necessary for phases of the cell cycle just prior to cytokinesis. Deficits in mitosis seen in mutants indicate aberrant mitotic spindle function, and like deficits in cytokinesis, occur in some but not all cells at the VZ surface. Citron kinase is therefore an essential multifunctional regulator of cell divisions in the VZ, and may serve to coordinate chromosome segregation with cytokinesis in neuronal precursors.

Impaired cell cycle control of neuronal precursor cells in the neocortical primordium of presenilin-1-deficient mice.

Recent studies have implicated presenilin-1 (PS-1) in the processing of the amyloid precursor protein and Notch-1. We show that PS-1 has biological effects on differentiation and cell cycle control of neuronal precursor cells in vivo using PS-1-deficient mice. The expression of Class III beta-tubulin was upregulated throughout the neocortical primordia of PS-1-deficient E14 embryos, especially on the ventricular surface. The increased speed of migration of the immature neurons from the ventricular zone outward in the PS-1-deficient neocortical primordia was indicated by an in vivo bromodeoxyuridine (BrdU)-labeling assay and a DiI-labeling assay in slice culture. Furthermore, we investigated the cell cycle of neuronal precursor cells in the neocortical ventricular zone using an in vivo cumulative BrdU-labeling assay. The length of the cell cycle in the neocortical precursor cells of wild-type mice was 11.4 hr whereas that of the PS-1-deficient mice was 15.4 hr. Among all phases of the cell cycle, S-phase exhibited the most prominent change in length, increasing from 2.4 hr in the wild-type mice to 7.4 hr in the mutant mice. The distribution of beta-catenin was specifically affected in the ventricular zone of the PS-1-deficient mice. These findings suggest that PS-1 is involved in the differentiation and the cell cycle control of neuronal precursor cells in the ventricular proliferating zone of the neocortical primordium.

Origin of GABAergic neurons in the human neocortex.

The mammalian neocortex contains two major classes of neurons, projection and local circuit neurons. Projection neurons contain the excitatory neurotransmitter glutamate, while local circuit neurons are inhibitory, containing GABA. The complex function of neocortical circuitry depends on the number and diversity of GABAergic (gamma-aminobutyric-acid-releasing) local circuit neurons. Using retroviral labelling in organotypic slice cultures of the embryonic human forebrain, we demonstrate the existence of two distinct lineages of neocortical GABAergic neurons. One lineage expresses Dlx1/2 and Mash1 transcription factors, represents 65% of neocortical GABAergic neurons in humans, and originates from Mash1-expressing progenitors of the neocortical ventricular and subventricular zone of the dorsal forebrain. The second lineage, characterized by the expression of Dlx1/2 but not Mash1, forms around 35% of the GABAergic neurons and originates from the ganglionic eminence of the ventral forebrain. We suggest that modifications in the expression pattern of transcription factors in the forebrain may underlie species-specific programmes for the generation of neocortical local circuit neurons and that distinct lineages of cortical interneurons may be differentially affected in genetic and acquired diseases of the human brain.

Regulation of area identity in the mammalian neocortex by Emx2 and Pax6.

The contribution of extrinsic and genetic mechanisms in determining areas of the mammalian neocortex has been a contested issue. This study analyzes the roles of the regulatory genes Emx2 and Pax6, which are expressed in opposing gradients in the neocortical ventricular zone, in specifying areas. Changes in the patterning of molecular markers and area-specific connections between the cortex and thalamus suggest that arealization of the neocortex is disproportionately altered in Emx2 and Pax6 mutant mice in opposing manners predicted from their countergradients of expression: rostral areas expand and caudal areas contract in Emx2 mutants, whereas the opposite effect is seen in Pax6 mutants. These findings suggest that Emx2 and Pax6 cooperate to regulate arealization of the neocortex and to confer area identity to cortical cells.

Noggin Is a Negative Regulator of Neuronal Differentiation in Developing Neocortex

Bone morphogenetic proteins (BMPs) trigger neuronal differentiation of neocortical precursors within the ventricular zone (VZ) [Li et al. (1998): J Neurosci 18:8853–8862]. BMP-2/4 protein is concentrated at the VZ surface and BMPs rapidly promote the differentiation of neocortical precursors in both dissociated cell and explant cultures. Noggin binds to BMP-2/4 with high affinity, and prevents binding to cell surface receptors. In the present study, we used human recombinant noggin protein to determine whether endogenous BMP-2/4 triggers neuronal differentiation in dissociated cell culture. We find that noggin inhibits the differentiation of neocortical neurons: noggin decreases the number of MAP-2- and TUJ1-positive cells after 24 h of treatment, yet has no effect on either proliferation or cell survival. Noggin also significantly decreases neurite growth of MAP-2-positive cells. In addition, using Western blot analysis we show that noggin protein is present in developing cortex at E15. These results are consistent with previous results showing that endogenous BMPs trigger neuronal differentiation in the neocortical VZ and also indicate that a balance of noggin and BMP may regulate the differentiation of neocortical neurons in vivo.

Growth factor influences on the production and migration of cortical neurons.

Production of neurons from progenitor cells is the first step in building the complex, six-layered cerebral cortex. The neurons that will populate the mature cortex are produced during development in an active proliferative neuroepithelium, the ventricular zone (VZ), adjacent to the cerebral ventricle (Rakic 1975; McConnell 1995; Caviness et al. 1996; Ross 1996). The first postmitotic neurons move out of the ventricular zone to form the preplate, just beneath the pia. Subsequent neuronal cohorts, generated in the neo-cortical ventricular zone, move into the preplate to form the cortical plate, which will eventually become layers 2 through 6 of cortex. At the earliest stages of cortical plate formation, preplate neurons are divided into two layers, the marginal zone, above the cortical plate, and the subplate, below it (Marin-Padilla 1971; Luskin and Shatz 1985; Allendoerfer and Shatz 1994). Many neurons move into the cortical plate under the guidance of the processes of radial glia (Hatten 1990; Rakic et al. 1994), but others, arising in distant proliferative zones, migrate tangentially into cortex, guided by cues that have not been defined (reviewed in Pearlman et al. 1998).

Changing properties of GABA(A) receptor-mediated signaling during early neocortical development.

Evidence from several brain regions suggests gamma-aminobutyric acid (GABA) can exert a trophic influence during development, expanding the role of this amino acid beyond its function as an inhibitory neurotransmitter. Proliferating precursor cells in the neocortical ventricular zone (VZ) express functional GABA(A) receptors as do immature postmigratory neurons in the developing cortical plate (CP); however, GABA(A) receptor properties in these distinct cell populations have not been compared. Using electrophysiological techniques in embryonic and early postnatal neocortex, we find that GABA(A) receptors expressed by VZ cells have a higher apparent affinity for GABA and are relatively insensitive to receptor desensitization compared with neurons in the CP. GABA-induced current magnitude increases with maturation with the smallest responses found in recordings from precursor cells in the VZ. No evidence was found that GABA(A) receptors on VZ cells are activated synaptically, consistent with previous data suggesting that these receptors are activated in a paracrine fashion by nonsynaptically released ligand. After neurons are born and migrate to the CP, they begin to demonstrate spontaneous synaptic activity, the majority of which is GABA(A) mediated. These spontaneous GABA(A) postsynaptic currents (sPSCs) first were detected at embryonic day 18 (E18). At birth, approximately 50% of recordings from cortical neurons demonstrated GABA(A)-mediated sPSCs, and this value increased with age. GABA(A)-mediated sPSCs were action potential dependent and arose from local GABAergic interneurons. GABA application could evoke action potential-dependent PSCs in neonatal cortical neurons, suggesting that during the first few postnatal days, GABA can act as an excitatory neurotransmitter. Finally, N-methyl-D-aspartate (NMDA)- but not non-NMDA-mediated sPSCs were also present in early postnatal neurons. These events were not observed in cells voltage clamped at negative holding potentials (-60 to -70 mV) but were evident when the holding potential was set at positive values (+30 to +60 mV). Together these results provide evidence for the early maturation of GABAergic communication in the neocortex and a functional change in GABA(A)-receptor properties between precursor cells and early postmitotic neurons. The change in GABA(A)-receptor properties may reflect the shift from paracrine to synaptic receptor activation.

Neuronal Differentiation of Precursors in the Neocortical Ventricular Zone Is Triggered by BMP

Neocortical neurons begin to differentiate soon after they are generated by mitoses at the surface of the ventricular zone (VZ). We provide evidence here that bone morphogenetic protein (BMP) triggers neuronal differentiation of neocortical precursors within the VZ. In cultures of dissociated neocortical neuroepithelial cells, BMPs increase the number of MAP-2- and TUJ1-positive cells within 24 hr of treatment. In explant cultures, BMP-4 treatment leads to an increase in the number of TUJ1-positive cells within the ventricular zone. Furthermore, truncated, dominant-negative, BMP type I receptor, introduced into neocortical precursors by retrovirus-mediated gene transfer, blocks neurite elaboration and migration out of the VZ. Finally, immunocytochemistry indicates that BMP protein is present at the VZ surface. Together, these results indicate that BMP protein is present within the VZ, that BMP is capable of promoting neuronal differentiation, and that signaling through BMP receptors triggers neuronal precursors to differentiate and migrate out of the VZ.

Lysophosphatidic acid receptor genevzg-1/lpA1/edg-2 is expressed by mature oligodendrocytes during myelination in the postnatal murine brain

The growth-factor-like phospholipid lysophosphatidic acid (LPA) mediates a wide variety of biological functions. We recently reported the cloning of the first G-protein-coupled receptor for LPA, called ventricular zone gene-1 (vzg-1/lpA1/edg-2) because its embryonic central nervous system (CNS) expression is restricted to the neocortical ventricular zone (Hecht et al. [1996] J. Cell Biol. 135:1071-1083). Vzg-1 neural expression diminishes at the end of the cortical neurogenetic period, just before birth. Here, we have investigated the subsequent reappearance of vzg-1 expression in the postnatal murine brain, by using in situ hybridization and northern blot analyses. Vzg-1 expression was undetectable by in situ hybridization at birth, but reappeared in the hindbrain during the 1st postnatal week. Subsequently, expression expanded from caudal to rostral, with peak expression observed around postnatal day 18. At all postnatal ages, vzg-1 expression was concentrated in and around developing white matter tracts, and its expansion, peak, and subsequent downregulation closely paralleled the progress of myelination. Double-label in situ hybridization studies demonstrated that vzg-1-expressing cells co-expressed mRNA encoding proteolipid protein (PLP), a mature oligodendrocyte marker, but not glial fibrillary acidic protein (GFAP), an astrocyte marker. Consistent with this, vzg-1 mRNA expression was reduced by 40% in the brains of jimpy mice, which exhibit aberrant oligodendrocyte differentiation and cell death. Together with our characterization of vzg-1 during cortical neurogenesis, these data suggest distinct pre- and postnatal roles for LPA in the development of neurons and oligodendrocytes and implicate lysophospholipid signaling as a potential regulator of myelination.

Lysophosphatidic acid receptor gene vzg1lpA1edg2 is expressed by mature oligodendrocytes during myelination in the postnatal murine brain

The growth-factor–like phospholipid lysophosphatidic acid (LPA) mediates a wide variety of biological functions. We recently reported the cloning of the first G-protein–coupled receptor for LPA, called ventricular zone gene-1 (vzg-1/lpA1/edg-2) because its embryonic central nervous system (CNS) expression is restricted to the neocortical ventricular zone (Hecht et al. [1996] J. Cell Biol. 135:1071–1083). Vzg-1 neural expression diminishes at the end of the cortical neurogenetic period, just before birth. Here, we have investigated the subsequent reappearance of vzg-1 expression in the postnatal murine brain, by using in situ hybridization and northern blot analyses. Vzg-1 expression was undetectable by in situ hybridization at birth, but reappeared in the hindbrain during the 1st postnatal week. Subsequently, expression expanded from caudal to rostral, with peak expression observed around postnatal day 18. At all postnatal ages, vzg-1 expression was concentrated in and around developing white matter tracts, and its expansion, peak, and subsequent downregulation closely paralleled the progress of myelination. Double-label in situ hybridization studies demonstrated that vzg-1–expressing cells co-expressed mRNA encoding proteolipid protein (PLP), a mature oligodendrocyte marker, but not glial fibrillary acidic protein (GFAP), an astrocyte marker. Consistent with this, vzg-1 mRNA expression was reduced by 40% in the brains of jimpy mice, which exhibit aberrant oligodendrocyte differentiation and cell death. Together with our characterization of vzg-1 during cortical neurogenesis, these data suggest distinct pre- and postnatal roles for LPA in the development of neurons and oligodendrocytes and implicate lysophospholipid signaling as a potential regulator of myelination. J. Comp. Neurol. 398:587–598, 1998. © 1998 Wiley-Liss, Inc.

Patterns of Intracellular Calcium Fluctuation in Precursor Cells of the Neocortical Ventricular Zone

Changes in intracellular free calcium concentration ([Ca2+]i) are known to influence a variety of events in developing neurons. Although spontaneous changes of [Ca2+]i have been examined in immature cortical neurons, the calcium dynamics of cortical precursor cells have received less attention. Using an intact cortical mantle and confocal laser microscopy, we examined the spatiotemporal patterns of spontaneous [Ca2+]i fluctuations in neocortical ventricular zone (VZ) cells in situ. The majority of activity consisted of single cells that displayed independent [Ca2+]i fluctuations. These events occurred in cells throughout the depth of the VZ. Immunohistochemical staining confirmed that these events occurred primarily in precursor cells rather than in postmitotic neurons. When imaging near the ventricular surface, synchronous spontaneous [Ca2+]i increases were frequently observed in pairs of adjacent cells. Cellular morphology, time-lapse imaging, and nuclear staining demonstrated that this activity occurred in mitotically active cells. A third and infrequently encountered pattern of activity consisted of coordinated spontaneous increases in [Ca2+]i in groups of neighboring VZ cells. The morphological characteristics of these cells and immunohistochemical staining suggested that the coordinated events occurred in gap junction-coupled precursor cells. All three patterns of activity were dependent on the release of Ca2+ from intracellular stores. These results demonstrate distinct patterns of spontaneous [Ca2+]i change in cortical precursor cells and raise the possibility that these dynamics may contribute to the regulation of neurogenesis.

Decreased serum vitamin E levels are associated with age-related macular degeneration

The neocortical ventricular zone is composed of a desynchronized population of proliferating cells. These cells give rise to neurons in the infragranular laminae of neocortex. The present study documents a diurnal rhythmicity of cell proliferation in the ventricular zone and examines the effects of ethanol on this biological clock. Pregnant rats were fed one of 3 diets. They were provided an ethanol-containing (6.7% v/v) liquid diet ad libitum between gestational day (G) 6 and G18, pair-fed an isocalorir liquid control diet, or fed chow and water. Throughout the experiments, the rats were fed either at 08.00 h (E.S.T.) or at 17.00 h (lights on 06.00 to 18.00 h). Rats were given a single injections of bromodeoxyuridine (BrdU) on G17 at one point during a 24 h period (03.00, 06.00, 09.00 h, etc.). The fraction of ventricular cells that incorporated the BrdU was determined using quantitative immunohistochemical methods. Pair-fed control rats (fed at 08.00 or 17.00 h) consumed their food within 4 h of presentation. The ratio of cells passing through the S-phase of the cell cycle changed diurnally; the ratio was highest during the day (0.52 ± 0.01 at 12.00 h) and lowest during the night (0.40 ± 0.02 at 03.00 h). In contrast, the ethanol-fed rats grazed on their food throughout the dark cycle regardless of when the food was presented. The mean peak blood ethanol concentration was 142 ± 13 mg/dl during the dark phase and less than 25 mg/dl during the light phase. Prenatal exposure to ethanol eliminates the fetal circadian rhythm in cell proliferation (mean labeling index of 0.45 ± 0.03). This ethanol-induced alteration demonstrates the resiliency of the developing system; its ability to compensate for traumas that alter the epigenetic control of development.

Chronic alcohol treatment promotes oxidative stress in rat peripheral nerve. Beneficial effects of antioxidants

The neocortical ventricular zone is composed of a desynchronized population of proliferating cells. These cells give rise to neurons in the infragranular laminae of neocortex. The present study documents a diurnal rhythmicity of cell proliferation in the ventricular zone and examines the effects of ethanol on this biological clock. Pregnant rats were fed one of 3 diets. They were provided an ethanol-containing (6.7% v/v) liquid diet ad libitum between gestational day (G) 6 and G18, pair-fed an isocalorir liquid control diet, or fed chow and water. Throughout the experiments, the rats were fed either at 08.00 h (E.S.T.) or at 17.00 h (lights on 06.00 to 18.00 h). Rats were given a single injections of bromodeoxyuridine (BrdU) on G17 at one point during a 24 h period (03.00, 06.00, 09.00 h, etc.). The fraction of ventricular cells that incorporated the BrdU was determined using quantitative immunohistochemical methods. Pair-fed control rats (fed at 08.00 or 17.00 h) consumed their food within 4 h of presentation. The ratio of cells passing through the S-phase of the cell cycle changed diurnally; the ratio was highest during the day (0.52 ± 0.01 at 12.00 h) and lowest during the night (0.40 ± 0.02 at 03.00 h). In contrast, the ethanol-fed rats grazed on their food throughout the dark cycle regardless of when the food was presented. The mean peak blood ethanol concentration was 142 ± 13 mg/dl during the dark phase and less than 25 mg/dl during the light phase. Prenatal exposure to ethanol eliminates the fetal circadian rhythm in cell proliferation (mean labeling index of 0.45 ± 0.03). This ethanol-induced alteration demonstrates the resiliency of the developing system; its ability to compensate for traumas that alter the epigenetic control of development.

Circadian rhythm of cell proliferation in the telencephalic ventricular zone: effect of in utero exposure to ethanol

The neocortical ventricular zone is composed of a desynchronized population of proliferating cells. These cells give rise to neurons in the infragranular laminae of neocortex. The present study documents a diurnal rhythmicity of cell proliferation in the ventricular zone and examines the effects of ethanol on this biological clock. Pregnant rats were fed one of 3 diets. They were provided an ethanol-containing (6.7% v/v) liquid diet ad libitum between gestational day (G) 6 and G18, pair-fed an isocalorir liquid control diet, or fed chow and water. Throughout the experiments, the rats were fed either at 08.00 h (E.S.T.) or at 17.00 h (lights on 06.00 to 18.00 h). Rats were given a single injections of bromodeoxyuridine (BrdU) on G17 at one point during a 24 h period (03.00, 06.00, 09.00 h, etc.). The fraction of ventricular cells that incorporated the BrdU was determined using quantitative immunohistochemical methods. Pair-fed control rats (fed at 08.00 or 17.00 h) consumed their food within 4 h of presentation. The ratio of cells passing through the S-phase of the cell cycle changed diurnally; the ratio was highest during the day (0.52 ± 0.01 at 12.00 h) and lowest during the night (0.40 ± 0.02 at 03.00 h). In contrast, the ethanol-fed rats grazed on their food throughout the dark cycle regardless of when the food was presented. The mean peak blood ethanol concentration was 142 ± 13 mg/dl during the dark phase and less than 25 mg/dl during the light phase. Prenatal exposure to ethanol eliminates the fetal circadian rhythm in cell proliferation (mean labeling index of 0.45 ± 0.03). This ethanol-induced alteration demonstrates the resiliency of the developing system; its ability to compensate for traumas that alter the epigenetic control of development.

Cell migration in the rat embryonic neocortex

Three-dimensional reconstructions of the normal rat embryonic (E) neocortex on days E15, E17, E19, and E21, using Skandha (software designed by J. Prothero, University of Washington, Seattle), show that the neocortical ventricular zone shrinks rapidly in the medial direction during cortical morphogenesis. [3H]thymidine autoradiography indicates that the shrinkage of the ventricular zone occurs before neurons in lateral and ventrolateral parts of layers IV-II are generated. Consequently, most of these neurons originate 400-1000 microns medial to their settling sites in the cortical plate. Embryos killed at daily intervals up to E21 after a single injection of [3H]thymidine on either E17 or E18 revealed the presence of a prominent migratory path, the lateral cortical stream, used by neurons migrating to the lateral and ventrolateral cortical plate; neurons migrating to the dorsal cortical plate follow a direct radial path. Arrival times of neurons in the cortical plate depend on the migratory path and are proportional to the overall distance travelled. Neurons that migrate only radially arrive in the dorsal cortical plate in two days (shortest route). Neurons that migrate laterally arrive in the lateral cortical plate in 3 days (longer route) and in the ventrolateral cortical plate in 4 days (longest route). [3H]thymidine autoradiography also shows that cells generated in the neocortical ventricular zone migrate in the lateral cortical stream for 5 or more days and accumulate in a reservoir. Cells leave the reservoir to enter the piriform cortex and destinations (as yet undetermined) in the basal telencephalon. The lateral cortical stream is found wherever the neocortical primordium surrounds the basal ganglia and is absent behind the basal ganglia. A computer analysis of nuclear orientation in anterior and posterior parts of the intermediate zone in the dorsal neocortex between days E17 and E22 shows that horizontally oriented nuclei are more common anteriorly where many cells are migrating laterally than posteriorly where most cells are migrating radially.