Cdc42 and Gsk3 modulate the dynamics of radial glial growth, inter-radial glial interactions and polarity in the developing cerebral cortex

Polarized radial glia are crucial to the formation of the cerebral cortex. They serve as neural progenitors and as guides for neuronal placement in the developing cerebral cortex. The maintenance of polarized morphology is essential for radial glial functions, but the extent to which the polarized radial glial scaffold is static or dynamic during corticogenesis remains an open question. The developmental dynamics of radial glial morphology, inter-radial glial interactions during corticogenesis, and the role of the cell polarity complexes in these activities remain undefined. Here, using real-time imaging of cohorts of mouse radial glia cells, we show that the radial glial scaffold, upon which the cortex is constructed, is highly dynamic. Radial glial cells within the scaffold constantly interact with one another. These interactions are mediated by growth cone-like endfeet and filopodia-like protrusions. Polarized expression of the cell polarity regulator Cdc42 in radial glia regulates glial endfeet activities and inter-radial glial interactions. Furthermore, appropriate regulation of Gsk3 activity is required to maintain the overall polarity of the radial glia scaffold. These findings reveal dynamism and interactions among radial glia that appear to be crucial contributors to the formation of the cerebral cortex. Related cell polarity determinants (Cdc42, Gsk3) differentially influence radial glial activities within the evolving radial glia scaffold to coordinate the formation of cerebral cortex.     

Calcium waves rule and divide radial glia

Radial glial proliferation is a critical step in the construction of cerebral cortex. In this issue of Neuron, Weissman and colleagues use time-lapse calcium imaging techniques to demonstrate that spontaneous calcium waves sweeping through cohorts of radial glia in the ventricular zone can modulate their proliferation during cerebral cortical development.     

Calcium signalling in glial cells

Calcium signals are the universal way of glial responses to the various types of stimulation. Glial cells express numerous receptors and ion channels linked to the generation of complex cytoplasmic calcium responses. The glial calcium signals are able to propagate within glial cells and to create a spreading intercellular Ca2+ wave which allow information exchange within the glial networks. These propagating Ca2+ waves are primarily mediated by intracellular excitable media formed by intracellular calcium storage organelles. The glial calcium signals could be evoked by neuronal activity and vice versa they may initiate electrical and Ca2+ responses in adjacent neurones. Thus glial calcium signals could integrate glial and neuronal compartments being therefore involved in the information processing in the brain.     

Calcium signalling in glial cells

Various electrical, mechanical, and chemical stimuli, including the influences of neurotrasmitters, neuromodulators, and hormones, trigger complex changes in [Ca²⁺] _i in all types of glial cells. Glial [Ca²⁺] _i responses are controlled by coordinated activity of several molecular cascades. The initiation of [Ca²⁺] _i signal in glial cells results from activation of either plasmalemmal, or intracellular Ca²⁺ permeable channels. The interplay of different molecular cascades enables the development of agonist-specific patterns of Ca²⁺ responses. Such agonist specificity may provide the means for intracellular and intercellular information coding. Furthermore, glial [Ca²⁺] _i signals can travel with no decrement within glial networks. These intercellular Ca²⁺ waves can be regarded as a substrate for information exchange between the glial cells. Neuronal activity can trigger [Ca²⁺] _i signals in neighboring glial cells and, moreover, there is some evidence that glial [Ca²⁺] _i waves can activate neuronal electrical and/or [Ca²⁺] _i , responses. Glial Ca²⁺ signalling can be regarded as a form of glial excitability.     

Postnatal morphological changes in neurons and glial cells in neocortex of mice developing at the background of serotonin deficit

It has been shown that deficit of serotonin during embryogenesis in rodents is accompanied by changes of morphological characteristics of neurons and glial cells at the period of postnatal development. A characteristic peculiarity of these changes is cell vacuolization that is of different expression in various cortical layers. In the experimental animals as compared with control ones, neurons of all neocortex layers have changed nuclei and a reduced volume of the cytoplasm. In neurons of upper layers, nuclei and cytoplasm contain occasional small vacuoles. In deep layers, vacuolization both of nuclei and of the cytoplasm is expressed to the much greater degree and vacuoles of large size are predominant. Results of immunocytochemical study have shown that in animals developing at the background of serotonin deficit there takes place a delay of the rates of formation and differentiation of astrocytic glia.     

Postnatal changes of morphology of nerve and glial cells in neocortex of mice developing on the background of serotonin deficit

It has been shown that deficit of serotonin during embryogenesis in rodents is accompanied by changes of morphological characteristics of neurons and glial cells at the period of postnatal development. A characteristic peculiarity of these changes is cell vacuolization that is of different expression in various cortical layers. In the experimental animals as compared with control ones, neurons of all neocortex layers have changed nuclei and a reduced volume of the cytoplasm. In neurons of upper layers, nuclei and cytoplasm contain occasional small vacuoles. In deep layers, vacuolization both of nuclei and of the cytoplasm is expressed to the much greater degree and vacuoles of large size are predominant. Results of immunocytochemical study have shown that in animals developing on the background of serotonin deficit there takes place a delay of the rates of formation and differentiation of astrocytic glia.     

Axo-glial synapses and GABA-accumulating glial cells in the embryonic neocortex of the rat

Summary On embryonic day 18, synapse-like contacts are found on certain non-neuronal cells appearing in clusters in lamina I (LI) of the parieto-occipital cortex of the rat. The structural criteria of these cells resemble those of immature glial cells: (1) The elongated nuclei containing dispersed chromatin are enclosed by a membrane showing narrow folds. (2) The cytoplasm contains many free ribosomes and a few dilated cisterns of the rough endoplasmic reticulum with granular or filamentous contents. (3) The plasma membrane forms concave adaptations toward neighboring neuronal processes. (4) At least one of the processes makes contact with the basal lamina of a vessel wall. The presynaptic elements contain a varying number of synaptic vesicles, and the pre- and postsynaptic membranes show densifications. Certain neurons and glial cells of the neocortex have the capability to accumulate GABA at day 16 of embryonic life. Only the more differentiated glial cells accumulate GABA. Many of these elements closely resemble the glial cells receiving synapse-like contacts, e.g., with respect to their cytological characteristics, clustering, and laminar position. According to recent experiments with adult ganglion cells, GABA released from glial cells might promote synaptogenesis by increasing the number of postsynaptic thickenings on the surrounding neurons. Thus, it cannot be excluded that transitory axo-glial synapses, by inducing GABA release, play a specific role in the earliest stages of synaptogenesis.     

O-GlcNAc modification of radial glial vimentin filaments in the developing chick brain

We examined the post-translational modification of intracellular proteins by β-O-linked N-acetylglucosamine (O-GlcNAc) with regard to neurofilament phosphorylation in the developing chick optic tectum. A regulated developmental pattern of O-GlcNAcylation was discovered in the developing brain. Most notably, discernible staining occurs along radial glial filaments but not along neuronal filaments in vivo. Immunohistochemical analyses in sections of progressive stages of development suggest upregulation of O-GlcNAc in the ependyma, tectofugal neuron bodies, and radial glial processes, but not in axons. In contrast, double-label immunostaining of monolayer cultures made from dissociated embryonic day (E) 7 optic tecta revealed O-GlcNAcylation of most axons. Labeling of brain sections together with Western blot analyses showed O-GlcNAc modification of a few discrete proteins throughout development, and suggested vimentin as the protein in radial glia. Immunoprecipitation of vimentin from E9 whole brain lysates confirmed O-GlcNAcylation of vimentin in development. These results indicate a regulated pattern of O-GlcNAc modification of vimentin filaments, which in turn suggests a role for O-GlcNAc-modified intermediate filaments in radial glia, but not in neurons during brain development. The control mechanisms that regulate this pattern in vivo, however, are disrupted when cells are placed in vitro.     

20-hydroxyecdysone stimulates proliferation of glial cells in the developing brain of the moth Manduca sexta

The steroid hormone 20-hydroxyecdysone (20-HE) controls diverse aspects of neuronal differentiation during metamorphosis in the hawkmoth Manduca sexta. In the present study we have examined the effect of 20-HE on glial cells of the brain during the metamorphic period. The antennal (olfactory) lobe of Manduca provides an ideal system in which to study effects of hormones on glial cells, since three known classes of glial cells participate in its development, and at least one type is critically important for establishment of normal neuronal morphology. These glial cells, associated with the neuropil, form boundaries for developing olfactory glomeruli as a result of proliferation and migration. We determined whether glial cells proliferate in response to 20-HE by injecting a pulse of 20-HE into the hemolymph at different stages of development and monitoring proliferation of all three types of glial cells. Hormone injections at the beginning and end of metamorphic development, when hormone titers are normally low, did not stimulate proliferation of neuropil-associated glial cells. Injections during the period when hormone titers are normally rising produced significant increases in their proliferation. Injections when hormone titers are normally high were ineffective at enhancing their proliferation. One other class of glial cells, the perineurial cells, also proliferate in response to 20-HE. Thus, glial proliferation in the brain is under the control of steroid hormones during metamorphic development. © 1995 John Wiley & Sons, Inc.     

Calcium signals activated by ghrelin and D-Lys-GHRP-6 ghrelin antagonist in developing dorsal root ganglion glial cells

Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys³-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca²⁺]_i followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca²⁺ entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys³-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca²⁺ activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys³-GHRP-6 ghrelin antagonist features ghrelin independent activities.     

Interneurons in the developing human neocortex

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.     

Telencephalic and diencephalic origin of radial glial processes in the developing preoptic area/anterior hypothalamus

Neuronal birth-dating sudies using [³H]thymidine have indicated that neurons in the preoptic area/anterior hypothalamus (POA/AH) are derived primarily from progenitors in proliferative zones surrounding the third ventricle. Radial glial processes are potential guides for neuronal migration, and their presence and orientation during development may provide further information about the origin of cells in the POA/AH. In addition to determining the orientation of radial glial fibers, we examined the relationship of neurons with identified birth dates to radial glial processes in the developing POA/AH of ferrets. Neuronal birth dates were determined by injecting ferret fetuses with bromodeoxyuridine (BrdU) at several different gestational ages; brains were taken from ferret kits at subsequent prenatal ages. Sections were processed for immunocytochemistry to reveal vimentin or glial fibrillary acidic protein in radial glia, or BrdU-labeled cell nuclei. Numerous radial glial processes extended from the lateral ventricles through ventral portions of the septal region to the pial surface of the POA/AH. These fibers both encapsulated and coursed ventrally through and around the anterior commissure of ferret, rat, and mouse fetuses. These ventrally directed fibers were less evident at older ages. In double-labeled sections from ferrets, BrdU-labeled cells in the dorsal POA/AH were often aligned in the same dorsal-ventral orientation as adjacent radial glial fibers. We suggest that a subset of neurons, originating in telencephalic proliferative zones, migrates ventrally along radial glial guides into the dorsal POA/AH. © 1995 John Wiley & Sons, Inc.     

Intercellular calcium waves in glial cells with bistable dynamics

A two-dimensional model is proposed for intercellular calcium (Ca(2 +)) waves with Ca(2 +)-induced IP(3) regeneration and the diffusion of IP(3) through gap junctions. Many experimental observations in glial cells, i.e. responding to local mechanical stimulation, glutamate application, mechanical stimulation followed by ACh application, and glutamate followed by mechanical stimulation, are reproduced and classified by the model. We show that a glial cell model with bistable dynamics, i.e. a Ca(2 +) oscillation state coexisting with a fixed point, can cause a prolonged plateau of Ca(2 +) signals in the cells nearby the stimulated cell when the cell network responds to the local mechanical stimulation.     

Insulin and oxidative stress modulate proliferation of rat ovarian theca-interstitial cells through diverse signal transduction pathways

Insulin and moderate oxidative stress stimulate proliferation of ovarian theca-interstitial cells. The effects of these agents on selected signal transduction pathways were examined. PD98059 (inhibitor of MAP2K1, also known as MEK-1, upstream of extracellular signal-regulated protein kinases MAPK3/1, also known as ERK1/2), wortmannin (inhibitor of PIK3C2A, also known as PI3K), and rapamycin (inhibitor of FRAP1, also known as mTOR, upstream of RPS6KB1) each significantly decreased insulin and oxidative stress-induced proliferation of theca-interstitial cells. The greatest inhibition was observed in the presence of rapamycin; this effect occurred without a significant change in cell viability. Phosphorylation of AKT was stimulated by insulin only, while phosphorylation of MAPK3/1 and RPS6KB1 was increased by insulin and oxidative stress. Insulin-induced and oxidative stress-induced phosphorylation of RPS6KB1 was partly inhibited by wortmannin and partly by PD98059; the greatest inhibition was observed in the presence of a combination of wortmannin plus PD98059. Effects of insulin and oxidative stress on phosphorylation of RPS6KB1 were confirmed by kinase activity assays. These findings indicate that actions of insulin and oxidative stress converge on MAPK3/1 and RPS6KB1. Furthermore, we speculate that activation of RPS6KB1 may be in part induced via the MAPK3/1 pathway.     

Calcium fluxes modulate the radiation-induced bystander responses in targeted glioma and fibroblast cells

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. This study investigated the role of changes in calcium levels in bystander responses leading to chromosomal damage in nonirradiated T98G glioma cells and AG01522 fibroblasts that had been either exposed to conditioned medium from irradiated cells or co-cultured with a population where a fraction of cells were individually targeted through the nucleus or cytoplasm with a precise number of microbeam helium-3 particles. After the recipient cells were treated with conditioned medium from T98G or AG01522 cells that had been irradiated through either nucleus or cytoplasm, rapid calcium fluxes were monitored in the nonirradiated recipient cells. Their characteristics were dependent on the source of the conditioned medium but had no dependence on radiation dose. When recipient cells were co-cultured with an irradiated population of either T98G or AG01522 cells, micronuclei were induced in the nonirradiated cells, but this response was eliminated by treating the cells with calcicludine (CaC), a potent blocker of Ca(2+) channels. Moreover, both the calcium fluxes and the bystander effect were inhibited when the irradiated T98G cells were treated with aminoguanidine, an inhibitor of nitric oxide synthase (NOS), and when the irradiated AG01522 cells were treated with DMSO, a scavenger of reactive oxygen species (ROS), which indicates that NO and ROS were involved in the bystander responses generated from irradiated T98G and AG01522 cells, respectively. Our findings indicate that calcium signaling may be an early response in radiation-induced bystander effects leading to chromosome damage.     

Proliferation and migration of glial precursor cells in the developing rat spinal cord

The mechanisms that control the production and differentiation of glial cells during development are difficult to unravel because of displacement of precursor cells from their sites of origin to their permanent location. The two main neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. Considerable evidence supports the view that oligodendrocytes in the spinal cord are derived from a region of the ventral ventricular zone (VZ). Some astrocytes, at least, may arise from radial glia. In this study a 5-Bromo-2′-deoxyuridine (BrdU) incorporation assay was used to identify proliferating cells and examine the location of proliferating glial precursor cells in the embryonic spinal cord at different times post BrdU incorporation. In this way the migration of proliferating cells into spinal cord white matter could be followed. At E14, most of the proliferating cells in the periventricular region were located dorsally and these cells were probably proliferating neuronal precursors. At E16 and E18, the majority of the proliferating cells in the periventricular region were located ventrally. In the white matter the number of proliferating cells increased as the animals increased in age and much of this proliferation occurred locally. BrdU labelling showed that glial precursor cells migrate from their ventral and dorsal VZ birth sites to peripheral regions of the cord. Furthermore although the majority of proliferating cells in the spinal cord at E16 and E18 were located in the ventral periventricular region, some proliferating cells remained in the dorsal VZ region of the cord.     

Valproic acid stimulates proliferation of glial precursors during cortical gliogenesis in developing rat

Valproic acid (VPA) is a neurotherapeutic drug prescribed for seizures, bipolar disorder, and migraine, including women of reproductive age. VPA is a well‐known teratogen that produces congenital malformations in many organs including the nervous system, as well as later neurodevelopmental disorders, including mental retardation and autism. In developing brain, few studies have examined VPA effects on glial cells, particularly astrocytes. To investigate effects on primary glial precursors, we developed new cell culture and in vivo models using frontal cerebral cortex of postnatal day (P2) rat. In vitro, VPA exposure elicited dose‐dependent, biphasic effects on DNA synthesis and proliferation. In vivo VPA (300 mg/kg) exposure from P2 to P4 increased both DNA synthesis and cell proliferation, affecting primarily astrocyte precursors, as >75% of mitotic cells expressed brain lipid‐binding protein. Significantly, the consequence of early VPA exposure was increased astrocytes, as both S100‐β+ cells and glial fibrillary acidic protein were increased in adolescent brain. Molecularly, VPA served as an HDAC inhibitor in vitro and in vivo as enhanced proliferation was accompanied by increased histone acetylation, whereas it elicited changes in culture in cell‐cycle regulators, including cyclin D1 and E, and cyclin‐dependent kinase (CDK) inhibitors, p21 and p27. Collectively, these data suggest clinically relevant VPA exposures stimulate glial precursor proliferation, though at higher doses can elicit inhibition through differential regulation of CDK inhibitors. Because changes in glial cell functions are proposed as mechanisms contributing to neuropsychiatric disorders, these observations suggest that VPA teratogenic actions may be mediated through changes in astrocyte generation during development. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 780–798, 2016