Emília Madarász

GABA signalling during development: new data and old questions.

Abstract. In addition to being the major inhibitory neurotransmitter, γ-aminobutyric acid (GABA) is thought to play a morphogenetic role in embryonic development. During the last decade, considerable progress has been made in elucidating the molecular mechanisms involved in GABA synthesis and biological action. The present review is an attempt to summarise recent results on the ontogeny of the different components of embryonic GABA signalling with an emphasis on the synthesis of GABA by different molecular forms of glutamic acid decarboxylase (GAD).

Proliferative and migratory responses of astrocytes to in vitro injury

An in vitro “scratch‐wound” model was used to evoke and investigate some astroglial responses to mechanical injury. The changes in the morphology, locomotion, and proliferation of injured astrocytes were analysed under culture conditions devoid of blood‐derived cells responsible for activating the inflammatory cascade. The rate of proliferation was determined by immunocytochemical detection of BrdU‐incorporating cells located next to or far from the wound. The motility of individual cells and the mass‐advancement of cell‐assemblies were monitored by computer controlled video‐microscopy both in injured monolayers and in preparations of single cells or aggregates of astrocytes. The large sets of digitalized data allowed a reliable statistical evaluation of changes in cell positions providing a quantitative approach for studies on dynamics of cell locomotion. The results indicated that cultivated astrocytes respond to injury (1) with enhanced nestin immunoreactivity at the expanding processes, (2) with increased mitotic activity exceeding the rate caused by the liberation from contact inhibition, but (3) without specific, injury‐induced activation of cell locomotion. Some advantages and drawbacks of “scratch‐wound” models of astrocytic responses to mechanical injury are presented and discussed. J. Neurosci. Res. 61:421–429, 2000.

Supersensitivity of P2X7 receptors in cerebrocortical cell cultures after in vitro ischemia

Neuronally enriched primary cerebrocortical cultures were exposed to glucose‐free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline‐associated protein 2 (MAP2)‐IR neurons or of glial fibrillary acidic protein (GFAP)‐IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP‐ and 2′‐3′‐O‐(4‐benzoylbenzoyl)‐adenosine 5′‐triphosphate (BzATP)‐induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G‐sensitive, P2X7 receptor‐mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP‐ and BzATP‐induced small inward currents recorded from a subset of non‐pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol‐induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre‐incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non‐pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor‐mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways.

Astroglia-derived retinoic acid is a key factor in glia-induced neurogenesis

Astroglial cells are essential components of the neurogenic niches within the central nervous system. Emerging evidence suggests that they are among the key regulators of postnatal neurogenesis. Although astrocytes have been demonstrated to possess the potential to instruct stem cells to adopt a neuronal fate, little is known about the nature of the glia‐derived instructive signals. Here we propose that all‐trans reti‐noic acid, one of the most powerful morphogenic molecules regulating neuronal cell fate commitment, may be one of the glia‐derived factors directing astro‐glia‐induced neurogenesis. According to data obtained from several complementary approaches, we show that cultured astrocytes express the key enzyme mRNAs of retinoic acid biosynthesis and actively produce all‐trans retinoic acid. We show that blockage of retinoic acid signaling by the pan‐RAR antagonist AGN193109 prevents glia‐induced neuron formation by noncommitted stem cells. Therefore, we provide strong in vitro evidence for retinoic acid action in astroglia‐induced neuronal differentiation.—Környei, Z., Gócza, E., Rühl, R., Vörös, E., Orsolits, B., Szabo, B., Vágovits, B., Madarász, E. Astroglia‐derived retinoic acid is a key factor in glia‐induced neurogenesis. FASEB J. 21, 2496–2509 (2007)

Retinoic acid induced neural differentiation in a neuroectodermal cell line immortalized by p53 deficiency.

Neuroepithelial cell lines were established from cerebral vesicles of 9‐day‐old mouse embryos lacking functional p53 genes (Livingstone et al: Cell 70:923–935, 1992). All‐trans retinoic acid (RA) induced bulk formation of neurons both in several p53‐deficient neuroepithelial cell lines and in wild‐type neural cells derived from early embryonic (E9–E12) forebrain vesicles. Forty‐eight‐hour treatment with 10−6 MRA was necessary and sufficient to initiate neuron formation by p53‐/‐‐progenitors, but neuronal characteristics appeared with a delay of 3–4 days. The first appearance of cells with astroglial features followed that of neurons with a further delay of 4–5 days. The establishment of neuronal phenotypes involved minimally three rounds of cell cycle. Future neurons were sorted out from substrate‐attached cells and were characterized by a specific rearrangement of nestin‐immunoreactive filaments. The formation of neuronal phenotypes was not synchronized within the RA‐treated cell populations. The data indicate that RA, which promotes the initiation of neural differentiation, cannot function as a direct regulator of cell‐fate decisions made by neural progenitor cells. J. Neurosci. Res. 47:405–415, 1997.

Humoral and contact interactions in astroglia/stem cell co-cultures in the course of glia-induced neurogenesis.

Astroglial cells support or restrict the migration and differentiation of neural stem cells depending on the developmental stage of the progenitors and the physiological state of the astrocytes. In the present study, we show that astroglial cells instruct noncommitted, immortalized neuroectodermal stem cells to adopt a neuronal fate, while they fail to induce neuronal differentiation of embryonic stem cells under similar culture conditions. Astrocytes induce neuron formation by neuroectodermal progenitors both through direct cell‐to‐cell contacts and via short‐range acting humoral factors. Neuron formation takes place inside compact stem cell assemblies formed 30– 60 h after the onset of glial induction. Statistical analyses of time‐lapse microscopic recordings show that direct contacts with astrocytes hinder the migration of neuroectodermal progenitors, while astroglia‐derived humoral factors increase their motility. In non‐contact co‐cultures with astrocytes, altered adhesiveness prevents the separation of frequently colliding neural stem cells. By contrast, in contact co‐cultures with astrocytes, the restricted migration on glial surfaces keeps the cell progenies together, resulting in the formation of clonally proliferating stem cell aggregates. The data indicate that in vitro maintained parenchymal astrocytes (1) secrete factors, which initiate neuronal differentiation of neuroectodermal stem cells; and (2) provide a cellular microenvironment where stem cell/stem cell interactions can develop and the sorting out of the future neurons can proceed. In contrast to noncommitted progenitors, postmitotic neuronal precursors leave the stem cell clusters, indicating that astroglial cells selectively support the migration of maturing neurons as well as the elongation of neurites.

Cultured astrocytes react to LPS with increased cyclooxygenase activity and phagocytosis

Phagocytosis and prostaglandin E(2) production were investigated in purified cultures of perinatal rat forebrain astrocytes. Light and electron microscopic data indicated that astrocytes respond to bacterial endotoxin, lipopolysaccharide (LPS) by increased phagocytosis and by activating the cyclooxygenase enzyme-pathway. LPS-inducible phagocytosis of astrocytes was demonstrated by electron microscopic studies on colloidal gold uptake and by photometric determination of fluorescent bead ingestion. The internalisation of fragments of the plasma membrane was shown by histochemical detection of membrane-bound ecto-ATPase activity within intracellular vesicles. Activation of the cyclooxygenase pathway, a characteristic reaction of immune cells under inflammatory conditions, was also detected in astroglial cells upon treatment with LPS. The increased prostaglandin E(2) (PGE(2)) production by astrocytes in response to LPS was reduced by the non-steroid anti-inflammatory drug, indomethacin. Our data indicate that astrocytes display some tissue-protective reactions in response to inflammation inducing factors, even in the absence of peripheral immune cells or central microglia. The role of inducible astrocytic phagocytosis in a non-immune protection-pathway is discussed.

Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis

NE‐7C2 neuroectodermal cells derived from forebrain vesicles of p53‐deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all‐trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT‐PCR studies revealed that GluRε1 and GluRε4 subunit mRNAs were transcribed by both non‐induced and neuronally differentiated cells. GluRε3 subunit mRNAs were not synthesized by NE‐7C2 cells and increased numbers of messages from the GluRε2 gene were detected only after neural network formation. The presence of the GluRζ1 protein was detected throughout neural induction, whereas retinoic acid‐induced neuron formation elevated the amount of exon 21 (C1)‐ and exon 22 (C2)‐containing GluRζ1 mRNAs and resulted in the appearance of exon 5 (N1)‐containing transcripts. NMDA‐elicited Ca2+‐signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin‐I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE‐7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.

Atomic force microscopy of height fluctuations of fibroblast cells

We investigated the nanometer scale height fluctuations of 3T3 fibroblast cells with the atomic force microscope under physiological conditions. A correlation between these fluctuations and lateral cellular motility can be observed. Fluctuations measured on leading edges appear to be predominantly related to actin polymerization-depolymerization processes. We found fast (5 Hz) pulsatory behavior with 1-2 nm amplitude on a cell with low motility showing emphasized structure of stress fibers. Myosin driven contractions of stress fibers are thought to induce this pulsation.

In vitro pattern formation during neurogenesis in neuroectodermal progenitor cells immortalized by p53-deficiency

In vitro neural differentiation was induced in a p53‐deficient immortalized neuroectodermal progenitor cell line, NE‐4C, by treatment with retinoic acid [K. Schlett and E. Madarász (1997) J. Neurosci. Res.47, 405–416]. Rearrangement of nestin filaments was an early marker of neuron‐formation. The increase in neurofilament protein content was accompanied by a decrease in the expression of nestin filaments in induced precursors. Cells with astroglial features appeared with a delay of 4–5 days compared to the appearence of neurons.