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Histone deacetylase (HDAC) inhibition mediated by small molecule HDAC inhibitors (HDACi) has demonstrated divergent effects including toxicity towards transformed cell lines, neuroprotection in neurological disease models, and inhibition of oligodendrocyte precursor cell (OPC) differentiation to mature oligodendrocytes (OL). However, it remains unknown if transient HDAC inhibition may promote OPC survival. Using mouse cortical OPC primary cultures, we investigated the effects of the FDA approved pan-HDACi suberoylanilide hydroxamic acid (SAHA) on OPC survival. Initial studies showed differences in the HDAC expression pattern of multiple HDAC isoforms in OPCs relative to their terminally differentiated progeny cells, OLs and astrocytes. Treatment of OPCs with SAHA for up to 72h using a maximum concentration either at or lower than those necessary for cytotoxicity in most transformed cell lines resulted in over 67% reduction in viability relative to vehicle-treated OPCs. This was at least partly due to increased apoptosis as SAHA-treated cells displayed activated caspase 3 and were protected by the general caspase inhibitor Q-VD-OPH. Additionally, SAHA treatment of whole mice at postnatal day 5 induced apoptosis of cortical OPCs. These results suggest that SAHA negatively impacts OPC survival and may be detrimental to the myelinating brain and spinal cord. Such toxicity may be relevant in a clinical context as SAHA is currently involved in numerous clinical trials and is in consideration for use in the treatment of psychiatric and neurodegenerative conditions.

In the central nervous system, NG2-glia represent a neural cell population that is distinct from neurons, astrocytes and oligodendrocytes. While in the past the main role ascribed to these cells was that of progenitors for oligodendrocytes, in the last years it has become more obvious that they have further functions in the brain. Here, we will discuss some of the most current and highly debated issues regarding NG2-glia: Do these cells represent a heterogeneous population? Can they give rise to different progenies, and does this change under pathological conditions? How do they respond to injury or pathology? What is the role of neurotransmitter signaling between neurons and NG2-glia? We will first give an overview on the developmental origin of NG2-glia, and then discuss whether their distinct properties in different brain regions are the result of environmental influences, or are due to intrinsic differences. We will then review and discuss their in vitro differentiation potential and in vivo lineage under physiological and pathological conditions, together with their electrophysiological properties in distinct brain regions and at different developmental stages. Finally, we will focus on their potential to be used as therapeutic targets in demyelinating and neurodegenerative diseases. Therefore, this review article will highlight the importance of NG2-glia not only in the healthy, but also in the diseased brain.

Amongst neurological diseases, multiple sclerosis (MS) presents an attractive target for regenerative medicine. This is because the primary pathology, the loss of myelin-forming oligodendrocytes, can be followed by a spontaneous and efficient regenerative process called remyelination. While cell transplantation approaches have been explored as a means of replacing lost oligodendrocytes, more recently therapeutic approaches that target the endogenous regenerative process have been favored. This is in large part due to our increasing understanding of (1) the cell types within the adult brain that are able to generate new oligodendrocytes, (2) the mechanisms and pathways by which this achieved, and (3) an emerging awareness of the reasons why remyelination efficiency eventually fails. Here we review some of these advances and also highlight areas where questions remain to be answered in both the biology and translational potential of this important regenerative process.

We have previously demonstrated that Sox17 expression is prominent at developmental stages corresponding to oligodendrocyte progenitor cell (OPC) cycle exit and onset of differentiation, and that Sox17 promotes initiation of OPC differentiation. In this study, we examined Sox17 expression and regulation under pathological conditions, particularly in two animal models of demyelination/remyelination and in post-mortem multiple sclerosis (MS) brain lesions. We found that the number of Sox17 expressing cells was significantly increased in lysolecithin (LPC)-induced lesions of the mouse spinal cord between 7 and 30 days post-injection, as compared with controls. Sox17 immunoreactivity was predominantly detected in Olig2(+) and CC1(+) oligodendrocytes and rarely in NG2(+) OPCs. The highest density of Sox17(+) oligodendrocytes was observed at 2 weeks after LPC injection, coinciding with OPC differentiation. Consistent with these findings, in cuprizone-treated mice, Sox17 expression was highest in newly generated and in maturing CC1(+) oligodendrocytes, but low in NG2(+) OPCs during the demyelination and remyelination phases. In MS tissue, Sox17 was primarily detected in actively demyelinating lesions and periplaque white matter. Sox17 immunoreactivity was co-localized with NOGO-A+ post-mitotic oligodendrocytes both in active MS lesions and periplaque white matter. Taken together, our data: (i) demonstrate that Sox17 expression is highest in newly generated oligodendrocytes under pathological conditions and could be used as a marker of oligodendrocyte regeneration, and (ii) are suggestive of Sox17 playing a critical role in oligodendrocyte differentiation and lesion repair.

The general view of development consists of the acquisition of committed/differentiated phenotypes following a period of self-renewal and progenitor expansion. Lineage specification and progression are phenomena of antagonistic events, silencing tissue-specific gene expression in precursors to allow self-renewal and multipotentiality, and subsequently suppressing proliferation and embryonic gene expression to promote the restricted expression of tissue-specific genes during maturation. The high mobility group-containing Sox family of transcription factors constitutes one of the earliest classes of genes to be expressed during embryonic development. These proteins are not only indispensable for progenitor cell specification, but are also critical for terminal differentiation of multiple cell types in a wide variety of lineages. Sox transcription factors are now known to induce or repress progenitor cell characteristics and cell proliferation, or activate the expression of tissue-specific genes. Sox proteins fulfill their diverse functions in developmental regulation by distinct molecular mechanisms. Not surprisingly, in addition to DNA binding and bending, Sox transcription factors also interact with different protein partners to function as co-activators or co-repressors of downstream target genes. In this article, we seek to provide an overview of the current knowledge of Sox gene functional mechanisms, in an effort to understand their roles in both development and pathology.

Aims/Background: An impairment of baroreceptor sensitivity has been found in liver cirrhosis. Noninvasive and spontaneous estimates of baroreflex sensitivity are obtained from beat-to-beat blood pressure and heart rate recordings by means of cross-spectrum analysis and calculation of alpha-index (as a measure of baroreflex gain). The aim of the present study was to investigate the function of the spontaneous baroreflex sensitivity related to clinical Child score in liver cirrhosis. Methods: The alpha-index was evaluated in 40 cirrhotic patients (18 with and 22 without ascites) and 17 healthy subjects by analysing finger arterial pressure recorded noninvasively with the Portapres device. Results: Baroreflex sensitivity was significantly lower in cirrhotic patients with and without ascites compared with healthy subjects (p

Progenitor cells expressing the proteoglycan NG2 represent approximately 5% of the total cells in the adult brain, and are found both in grey and white matter regions where they give rise to oligodendrocytes. The finding that these cells receive synaptic contacts from excitatory and inhibitory neurons has not only raised major interest in the possible roles of these synapses, but also stimulated further research on the developmental and cellular functions of NG2-expressing (NG2+) progenitors themselves in the context of neural circuit physiology. Here we review recent findings on the functional properties of the synapses on NG2+ cells in grey and white matter regions of the brain. In this review article we make an attempt to integrate current knowledge on the cellular and developmental properties of NG2+ progenitors with the functional attributes of their synapses, in order to understand the physiological relevance of neuron–NG2+ progenitor signal transmission. We propose that, although NG2+ progenitors receive synaptic contact in all brain regions where they are found, their synapses might have different developmental and functional roles, probably reflecting the distinct functions of NG2+ progenitors in the brain.

Acute hyperammonemia (HA) causes cerebral edema and brain damage in children with urea cycle disorders (UCDs) and in patients in acute liver failure. Chronic HA is associated with developmental delay and mental retardation in children with UCDs, and with neuropsychiatric symptoms in patients with chronic liver failure. Astrocytes are a major cellular target of hyperammonemic encephalopathy, and changes occurring in these cells are thought to be causally related to the brain edema of acute HA. To study the effect of HA on astrocytes in vivo, we crossed the Otc(spf) mouse, a mouse with the X-linked UCD ornithine transcarbamylase (OTC) deficiency, with the hGFAP-EGFP mouse, a mouse selectively expressing green fluorescent protein in astrocytes. We used FACS to purify astrocytes from the brains of hyperammonemic and healthy Otcspf/GFAP-EGFP mice. RNA isolated from these astrocytes was used in microarray expression analyses and qRT-PCR. When compared with healthy littermates, we observed a significant downregulation of the gap-junction channel connexin 43 (Cx43) the water channel aquaporin 4 (Aqp4) genes, and the astrocytic inward-rectifying potassium channel (Kir) genes Kir4.1 and Kir5.1 in hyperammonemic mice. Aqp4, Cx43, and Kir4.1/Kir5.1 are co-localized to astrocytic end-feet at the brain vasculature, where they regulate potassium and water transport. Since, NH4+ ions can permeate water and K+-channels, downregulation of these three channels may be a direct effect of elevated blood ammonia levels. Our results suggest that alterations in astrocyte-mediated water and potassium homeostasis in brain may be key to the development of the brain edema.

While the central nervous system (CNS) is generally thought of as an immunopriviledged site, immune-mediated CNS white matter damage can occur in both the perinatal period and in adults, and can result in severe and persistent neurological deficits. Periventricular leukomalacia (PVL) is an inflammatory white matter disease of premature infants that frequently results in cerebral palsy (CP). Clinical and experimental studies show that both hypoxic/ischemic and innate immune mechanisms contribute to the destruction of immature oligodendroglia and of axons in the deep cerebral white matter in PVL. No data are yet available as to whether there is any genetic predisposition to PVL or to its neurological sequelae. Multiple sclerosis (MS) is an inflammatory white matter disease that often begins in young adulthood, causes multifocal destruction of mature oligodendroglia and of axons, and eventually leads to substantial cumulative neurological disability. Certain genetic polymorphisms contribute to susceptibility to MS, and adaptive immune responses to myelin-associated self antigens, or to exogenous antigens that mimic these self antigens, play a central role in the pathophysiology of this disease. MRDD Research Reviews 2006;12:141–146. © 2006 Wiley-Liss, Inc.

A prominent feature of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is the accumulation of enlarged, multipolar glial fibrillary acidic protein (GFAP) and brain lipid binding protein (BLBP) immunoreactive astroglia within and at the margins of the inflammatory demyelinative lesions. Whether this astrogliosis is due to both astroglial hyperplasia and hypertrophy or solely to astroglial hypertrophy is controversial. We now report that coincident with the first appearance of inflammation and clinical deficits in mice with myelin oligodendrocyte glycoprotein peptide (MOG peptide)-induced EAE, the radially oriented, bipolar, GFAP, and BLBP positive cells (adult radial glia) present in normal spinal cord white matter undergo mitosis and phenotypic transformation to hypertrophic astroglia. To facilitate visualization of relationships between these hypertrophic astroglia and dying and regenerating oligodendroglia, we used mice that express enhanced green fluorescent protein (EGFP) in cells of the oligodendroglial lineage. During the first week after onset of illness, markedly swollen EGFP+ cells without processes were seen within lesions, whereas EGFP+ cells that expressed immunoreactive cleaved caspase-3 were uncommon. These observations support the hypothesis that necrosis contributes to oligodendroglial loss early in the course of EAE. Later in the illness, EGFP+ cells accumulated amongst hypertrophic astroglia at the margins of the lesions, while the lesions themselves remained depleted of oligodendroglia, suggesting that migration of oligodendroglial lineage cells into the lesions was retarded by the intense perilesional gliosis.

Cells that express the NG2 proteoglycan are the largest proliferative progenitor population in the postnatal central nervous system (CNS). Although this entire population has long been considered to be oligodendrocyte progenitors, numerous NG2+ cells are present in the cerebral cortex, where relatively little myelination occurs, and also persist long after myelination is complete in the CNS. Several studies have alluded to the presence of distinct NG2+ cell subtypes based on marker expression, but no experimentally derived hypotheses about the physiological role of these subtypes has been proposed. In the current study, whole-cell patch-clamp data from acutely isolated slices demonstrate that subcortical white matter and cortical NG2+ cells display distinct membrane properties in addition to possessing differing K+- and Na+-channel expression profiles. A striking observation is that a subpopulation of cortical, but not white matter NG2+ cells, elicit depolarization-induced spikes that are akin to immature action potentials. Our data demonstrate that a population of cortical NG2+ cells display physiological properties that differ from their white matter counterparts.

Both the immediate insult and delayed apoptosis contribute to functional deficits after brain injury. Secondary, delayed apoptotic death is more rapid in immature than in adult CNS neurons, suggesting the presence of age-dependent protective factors. To understand the molecular pathobiology of secondary injury in the context of brain development, we identified changes in expression of oxidative stress response genes during postnatal development and target deprivation-induced neurodegeneration. The antioxidants metallothionein I and II (MT I/II) were increased markedly in the thalamus of adult C57BL/6 mice compared to mice

Reversible tyrosine phosphorylation is integral to the process of oligodendrocyte differentiation. To interfere with the subset of the phosphorylation cycle overseen by protein tyrosine phosphatase epsilon (PTP epsilon) in oligodendrocytes, we applied a substrate-trapping approach in the development of transgenic mice overexpressing a catalytically inactive, transmembrane PTP epsilon-hemaglutinin (tm-PTP epsilon-HA) from the dual promoter element of the gene encoding the myelin protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). Transgene expression peaked during the active myelinating period, at 2-3 weeks postnatal. Two tyrosine phosphoproteins, alpha-enolase and beta-actin, were phosphorylated to a greater degree in transgenic mice. Despite a high degree of tm-PTP epsilon-HA expression, myelin was grossly normal in nearly all axonal tracts. Phenotypic abnormalities were limited to optic nerve, where a decrease in the degree of myelination was reflected by reduced levels of myelin proteins on postnatal day 21 (PND21), as well as a decrease in the density of differentiated oligodendrocytes. The optic chiasm was reduced in thickness in transgenic mice; optic nerves similarly exhibited a reduction in transverse width. Further analyses of the optic pathway demonstrated that transgenic protein was unexpectedly present in retinal ganglion cells, whose axons are the targets of myelination by optic nerve oligodendrocytes. On PND28, transgenic protein declined dramatically in both oligodendrocytes and retinal ganglion cells contributing to the recovery of optic nerve myelination. Thus, delayed myelination arises only when tm-PTP epsilon-HA is simultaneously expressed in myelin-forming glia and their neuronal targets. While tm-PTP epsilon related signaling pathways may figure in axon-glial interactions, interfering with tm-PTP epsilon activity does not perceptibly affect the development or myelinating capacity of most oligodendrocytes.

We generated a transgenic mouse expressing the enhanced green fluorescent protein (EGFP) under the control of the 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter. EGFP(+) cells were visualized in live tissue throughout embryonic and postnatal development. Immunohistochemical analysis in brain tissue and in sciatic nerve demonstrated that EGFP expression was restricted to cells of the oligodendrocyte and Schwann cell lineages. EGFP was also strongly expressed in "adult" oligodendrocyte progenitors (OPs) and in gray matter oligodendrocytes. Fluorescence-activated cell sorting allowed high-yield purification of EGFP(+) oligodendrocyte-lineage cells from transgenic brains. Electrophysiological patch clamp recordings of EGFP(+) cells in situ demonstrated that OP cells displayed large outward tetraethylammonium (TEA)-sensitive K(+) currents and very small inward currents, whereas mature oligodendrocytes were characterized by expression of large inward currents and small outward K(+) currents. The proliferation rate of EGFP(+) cells in developing white matter decreased with the age of the animals and was strongly inhibited by TEA. Oligodendrocyte development and physiology can be studied in live tissue of CNP-EGFP transgenic mice, which represent a source of pure EGFP(+) oligodendrocyte-lineage cells throughout development.

To investigate LIM gene function in the rat cerebellar system, we analyzed expression and regulation of the rat homologue of frog Xlim-1 (rlim-1) in vivo and in cultured cells. In developing cerebellum, peak levels of rlim-1 mRNA at postnatal day 8 (p8) are coincident with the peak period of granule cell proliferation. Analysis of rlim-1 protein with a specific antibody showed that expression was also maximal at p8. In situ hybridization showed that at p8 rlim-1 mRNA was expressed in Purkinje and granule cells. Both the proliferative and the premigratory granule cells in the external germinal zone displayed high levels of rlim-1 mRNA expression. Immunocytochemical staining demonstrated that at p8 rlim-1 protein was also present in proliferative and premigratory granule cells. In adult cerebellum (p30), rlim-1 mRNA and protein expression in granule cells was strongly attenuated. The down-regulation of rlim-1 mRNA occurred in granule cells just after the time of final division, coinciding with the onset of their migration. rlim-1 protein was detected in migratory granule neurons. The developmental decrease in rlim-1 mRNA and protein found in vivo was reproduced in pure cerebellar granule cell cultures. In these cultures, granule neurons were postmitotic 1 day after plating but still displayed high levels of rlim-1 protein expression up to 3 days in vitro. Our findings indicate that 1) rlim-1 is likely to act in concert with other genes to specify granule cell fate, 2) rlim-1 expression in granule neurons is regulated autonomously, and 3) rlim-1 protein may also play an important role in granule neuron differentiation and survival. Published 2001 Wiley-Liss, Inc.

Previous electrophysiological evidence has indicated that astrocytes and oligodendrocytes express inwardly rectifying K(+) channels both in vitro and in vivo. Here, for the first time, we have undertaken light microscopic immunohistochemical studies demonstrating the location of one such channel, Kir4.1, in both cell types in regions of the rat CNS. Some astrocytes such as those in the deep cerebellar nuclei, Bergmann glia, retinal Muller cells, and a subset in hippocampus express Kir4.1 immunoreactivity, but not others including those in white matter. Oligodendrocytes also express this protein, strongly in perikarya and to a lesser extent in their processes. Expression of Kir4.1 in astrocytes and oligodendrocytes would enable these cells to clear extracellular K(+) through this channel, whereas nonexpressors might use other mechanisms.

We analyzed the expression and regulation of glutamate receptor subunits in the rat neural tube (10 day embryos) and in cell cultures derived from this tissue. In the cultures, all cells were stained with antibodies against the neural progenitor marker nestin. More than 50% of the cells were also stained by the monoclonal antibodies LB1 or A2B5, which bind to neuronal and glial progenitors. Approximately 6% of the cells were stained with antibodies for the low affinity NGF receptor, a neural crest cell marker. A small percentage of cells differentiated to neurons or astrocytes, as determined by staining with anti-neurofilament and anti-GFAP antibodies, respectively. RT-PCR analysis of neural tube tissue and culture mRNAs demonstrated that the AMPA receptor subunits GluR3 and 4 and the kainate receptor subunits GluR6, 7, KA1 and KA2 were detectable at E10. The kainate receptor subunits GluR6 and KA2 were upregulated by culture conditions which stimulated cell differentiation, as determined by concomitant downregulation of nestin mRNA. Both in neural tube tissue and in cultured cells, GluR6 was 100% unedited. Finally, both GluR6 and KA2 proteins could be detected in subpopulations of neural progenitors and differentiated neurons. Our data indicate that kainate receptor genes are expressed in undifferentiated progenitor cells of the neural tube at E10, and are upregulated during neural cell differentiation.

Intracellular calcium signals triggered by glutamate receptor activation were studied in primary cortical oligodendrocyte lineage cells and in the oligodendrocyte cell line CG-4. Glutamate, kainate, and AMPA (30-300 microM) increased [Ca2+]i in both types of cells at the stage of oligodendrocyte progenitors (O-2A; GD3+) or pro-oligodendroblasts (O4+). The peak amplitude of Ca2+ responses to glutamate receptor agonists was significantly larger in cortical cells. In CG-4 and in cortical cells, the majority (more than 90%) of bipolar GD3+ or multipolar O4+ cells responded to kainate. In all the cells analyzed, kainate was more efficacious than AMPA and glutamate. The percentage of bipolar or multipolar cells responding to glutamate was significantly lower in the CG-4 cell line than in primary cultures. Cellular responses typical of metabotropic glutamate receptor activation were observed in 20% of the cortical O-2A progenitors, but in none of the CG-4 cells. The AMPA-selective antagonist GYKI 52466 blocked kainate-induced Ca2+ responses in cortical O-2A cells. The selective AMPA receptor modulator cyclothiazide (30 microM) greatly potentiated the effects of AMPA (30-100 microM) on [Ca2+]i in cortical and CG-4 cells. Our findings indicate that Ca2+ responses in cells of the oligodendrocyte lineage are primarily shaped by functional AMPA receptors.

What makes quiescent glial cells in the CNS convert into the most aggressive of all glial cell-derived tumours? Contrary to assumptions long past, CNS cells are exquisitely responsive to changes in their physiological surroundings during development and disease. Far from being a settled environment, the intricate cerebral landscape continuously bombards cells with signals to run or grow or migrate, significantly altering cellular behaviour. Some signals even accelerate cell migration in ways that prove fatal. Glioblastoma multiforme (GBM), also classified as grade 4 astrocytoma, is a rapidly growing glioma of the CNS that originates predominantly from astrocytes, the most numerous macroglia in the brain. Glioblastoma multiforme is most often found in the cerebral hemispheres, either frontal or temporal lobes. Several tumour characteristics pose exceptional treatment challenges; as a result, patients typically die 15 months or less after diagnosis. Glioblastoma multiforme is by far the most invasive glial cell-derived tumour, being characterized by fast growth and rapid invasion of surrounding brain tissue. Tumour resection is not curative, firstly, because neoplastic cells can migrate quickly into brain regions near the original tumour, forming secondary tumours; secondly, GBMs are not homogeneous (Holland, 2000) but are composed of diverse cell types (mostly derived from astrocytes and oligodendrocytes) with properties differing from the cells of origin. This is particularly true for GBM that evolves from low-grade astrocytomas or oligodendrogliomas. The tumour environment is also not optimal for drug treatment. Blood supply is usually disrupted in GBM, reducing the efficacy of drug delivery, and capillary leakage causes peritumoural fluid accumulation and intracranial hypertension. Additional therapeutic obstacles include the intrinsic resistance of GBM to conventional cancer therapy and the neurotoxic effects of many treatments targeting these tumours. Invasion of the brain tissue and vasculature by GBM is a crucial and early step in tumour metastasis. Thus, defining the cellular and physiological mechanisms involved in metastasis is fundamental for developing therapeutic approaches to prevent GBM cell migration and metastasis. Despite the heterogeneity of GBM, it would be vital to determine whether the tumours display shared properties that could be harnessed to reduce their migration potential. Identifying common cellular signals and elucidating their mechanisms of action in GBM cell migration would enhance the efficacy of therapeutic interventions. The study in this issue of The Journal of Physiology by Seifert & Sontheimer (2014) identifies a cellular signal that promotes glioma migration, illuminates its mechanism of action and defines the process leading to enhanced neoplastic cell migration. The authors combine in vitro and in vivo approaches to demonstrate that the neuropeptide bradykinin guides glioma cell migration and significantly changes the physiological properties of the cells. Bradykinin induces the formation of irregular bulges (called blebs) in the plasma membrane through a B2 receptor-mediated increase in intracellular Ca2+. This triggers cytoskeletal contraction, cytoplasmic flow and activation of specific Ca2+-dependent K+ and Cl− channels, which then participate in regulating bleb formation. Finally, and most importantly, Seifert & Sontheimer (2014) analysed gliomas formed by human cell lines in mouse brain in vivo and demonstrated that bradykinin treatment stimulates growth and significantly expands tumour size by accelerating cell migration at the tumour periphery. In vivo treatment with blebbistatin, which blocks myosin kinase II and bleb retraction, prevents glioma migration. Why are these findings original and important? Firstly, the cellular factors promoting glioma cell migration are largely unknown, but this study identifies bradykinin as one factor (presumably of many). Sontheimer and his team previously demonstrated that this neuropeptide promotes chemotaxis of glioma cells in vitro (Montana & Sontheimer, 2011), but the present study shows that bradykinin also enhances tumour cell migration in situ. Secondly and more importantly, Seifert and Sontheimer's experimental analysis illuminates a long-standing controversy about how tumour cells, including GBM, migrate through the brain's densely packed extracellular environment. Tumour cells were believed to migrate primarily through a mechanism called mesenchymal migration, involving the release of enzymes that enable the tumour cells themselves to degrade the extracellular matrix (Wolf et al. 2003). Yet, more recent studies highlight a different and pervasive mechanism called amoeboid migration that is specifically associated with bleb formation. This mechanism is common to many tumour cell types and involves activating several cytoskeletal proteins. Some have proposed that cytoskeleton-dependent bleb formation could be the cellular mechanism responsible for directed cell migration towards chemoattractants (Montana & Sontheimer, 2011), which would be consistent with the mechanism of action of bradykinin and its effects on glioma migration. Bradykinin is present in brain vasculature and chemotactically attracts glioma cells to blood vessels (Montana & Sontheimer, 2011), which supports the notion that bradykinin might guide the invasion of glioma cells in vivo through blood vessels or white matter tracts. This observation raises a crucial question. Do relatively less invasive GBMs, with longer than average patient survival (3–5% of patients surviving more than 3 years), originate in brain regions where tumour cells are exposed to lower levels of bradykinin or perhaps in regions with defective bradykinin signalling? Comparing tumours from these patients and from the majority of individuals with shorter postdiagnosis survival has the potential to uncover associations between bradykinin signalling and tumour lethality. Another issue worth investigating is whether bradykinin is a crucial cellular signal regulating GBM growth, migration and invasion in the human brain. Seifert and Sontheimer's analysis was performed in human tumour cell lines, but the therapeutic potential of interfering with bradykinin signalling remains largely undefined. Thus, targeting bradykinin signalling through the B2 receptor might be a beneficial therapeutic approach, in conjunction with other treatments (Huse & Holland, 2010), for preventing the growth of GBM and other similarly aggressive brain tumours.

Kainate (KA), quisqualate (QA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulated gamma-aminobutyric acid [3H]gamma-aminobutyric acid (GABA) release from cultured cerebellar type 2 astrocytes and from their bipotential precursors. The evoked release was prevented by the antagonist 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX). AMPA and QA applied together with KA at concentrations around or above their EC50S (20-50 microM) antagonized the stimulatory effect of KA on [3H]GABA release. On the other hand, the releasing action of KA was potentiated by concentrations of QA in the low micromolar range (2-5 microM), particularly when the concentration of KA was at the borderline of effectiveness (10 microM). KA and QA did not elevate intracellular cyclic GMP levels in astrocyte cultures, although guanylate cyclase was present in both type 2 and type 1 astrocytes. The inability of KA to elevate cyclic GMP levels in astrocytes was the only major difference in the behavior of this glutamate agonist between astroglial and neuronal cultures. The GABA transport inhibitor nipecotic acid or replacement of NaCl with LiCl abolished [3H]GABA uptake and also KA- and QA-induced release of preaccumulated [3H]GABA. Therefore, [3H]GABA was released from type 2 astrocytes and their progenitors through its Na(+)-dependent transport system, operating in an outward direction when the cells were depolarized by non-NMDA receptor agonists.

Which signals make a precursor cell become a neuron? Once identified, will we be able to use those signals to amplify certain types of neurons and direct them to areas where they are needed? While many extrinsic and intrinsic control pathways have been elucidated, it appears that one major mechanism occurs via neural progenitor depolarization by amino acid neurotransmitters. It is well established that the resting membrane potential of glial and neuronal progenitors can be modified by the activation of ligand-gated ionic channels before cells reach their final anatomical destination and differentiate. Among the extrasynaptic signals that can activate these membrane channels, the neurotransmitter γ-aminobutyric acid (GABA) has attracted considerable attention for two main reasons. First, the nature of the GABA-mediated response in neural cells changes during development. In embryonic and neonatal neurons, GABA provides an excitatory drive, whereas in the adult brain GABA is inhibitory (reviewed in Ben-Ari, 2002; Owens & Kriegstein, 2002). This is due to significant changes in [Cl−]i that occur as a consequence of nascent expression of the Na+,K+,Cl−-cotransporter around the end of the first postnatal week (Ben-Ari, 2002; Owens & Kriegstein, 2002). Second, GABA alters the proliferation and migration kinetics of prenatal neuronal progenitors (Behar et al. 1998; Owens & Kriegstein, 2002), and exerts trophic effects on neurons, promoting neurite outgrowth and synapse formation (reviewed in Ben-Ari, 2002; Owens & Kriegstein, 2002). In the postnatal brain, the subventricular zone (SVZ) and the rostral migratory stream (RMS) are the major areas exhibiting simultaneous proliferation and migration of progenitors and newly minted neurons. In an article in this issue of The Journal of Physiology, Wang et al. demonstrate that dividing neuronal progenitors in the SVZ and RMS synthesize GABA and are depolarized through the activation of GABA-gated ionic channels. The electrophysiological and anatomical analysis was conducted using a tissue slice preparation that maintained a relatively intact anatomical structure, preserving interactions between neighbouring cells. These findings invite the question as to the functional role of GABA-mediated depolarization in postnatal cells. Recent descriptions of GABA-induced modulation of prenatal progenitors may inform us as to the role of GABA in postnatal progenitor cells. Prenatal progenitor responses to amino acid neurotransmitters elucidate rapid intrinsic differentiation steps; more primitive VZ cells respond to GABA by increasing proliferation, while later-generated SVZ progenitors respond by proliferating more slowly (Haydar et al. 2000). Similar to the findings in the prenatal studies, Wang et al. (2003) report that expression of functional GABAA receptor channels is detectable when postnatal progenitors are still far from displaying action potentials and becoming synaptically integrated. In a recent patch-clamp study of migrating adult olfactory bulb neurons, Carleton et al. (2003) detected GABAA receptor-mediated depolarizing responses in immature neurons, well before spontaneous synaptic and spiking activities had developed. Thus, like prenatal progenitors, precursors and newly generated neurons in the postnatal and adult brain acquire physiological properties in a specific temporal sequence. It appears that expression of functional ligand-gated channels in neuronal progenitors and the cellular response initiated after their activation are part of an intrinsic programme which is independent of the formation of synaptic connections. As suggested by the prenatal studies, this intrinsic programme could serve to modulate proliferation and migration during postnatal progenitor amplification. While cells proliferate in transit from the SVZ through the RMS to the olfactory bulb, a balance between proliferation and migration must exist for proper numbers and timely ingress of new neurons. If the postnatal SVZ cells respond to GABA like their prenatal cousins, GABA should inhibit proliferation and promote migration. Thus, the proper ‘GABA tone’ may be required for balancing progenitor amplification and mobilization. The resurgence of interest in GABA-mediated signalling in neuronal progenitors during pre- and postnatal development opens fascinating avenues for future research. To further investigate the functional role of this transmitter and its receptors in postnatal and adult neurogenesis, the subunit composition of GABA-activated channels in neuronal progenitors will have to be determined. Then, GABA responses could be modified by using gain- and loss-of-function strategies, testing the hypothesis that targeted molecular perturbations of specific GABA receptor subunits will affect neuronal precursor development. In vivo electroporation of dominant negative subunits, or targeted mutations in the neuronal progenitor populations combined with live imaging, will define whether GABA-induced depolarization affects cell proliferation and/or migration. Another area of future exploration relates to the finding that SVZ neuronal progenitors themselves synthesize and store GABA. It will be important to define whether this neurotransmitter is released by neuronal precursors in a targeted fashion, as in a synapse, or as a paracrine signal. Altogether, recent advances describing the role of GABA depolarization in neuronal progenitor development could lead to novel strategies for repair and repopulation of the damaged and aging brain.

— Several parameters of GABA Auxes across the synaptosomal membrane were studied using synaptosomes prepared from the brain of immature (8-day-old) rats. The following aspects of GABA carrier-mediated transport were similar in immature and mature synaptosomes: (1) magnitude of [3H]GABA accumulation; (2) GABA homoexchange in normal ionic conditions; (3) GABA homoexchange in the presence of cationic fluxes (Na+ and Ca2+ influx, K+ efflux) characteristic of physiological depolarization. As in adult synaptosomes (Levi & Raiteri, 1978), in these conditions the stoichiometry of GABA homoexchange was in the direction of net outward transport (efflux > influx). The essential differences between the behaviour of 8-day-old and adult synaptosomes were the following: (1) β-alanine (a glial uptake inhibitor) inhibited GABA uptake in immature synaptosomes (the inhibition being greater in crude than in purified preparations) and was without a significant effect in adult synaptosomes. DABA and ACHC (two neuronal uptake inhibitors) depressed GABA uptake more efficiently in purified than in crude immature synaptosomes, but were as effective in crude and purified nerve endings from adult animals. The data suggest a greater uptake of GABA in the‘gliosomes’contaminating the synaptosomal preparations from immature animals. (2) In immature synaptosomes prelabelled with [3H]GABA the specific radioactivity of the GABA released spontaneously or by heteroexchange (with 300 μm-OH-GABA) was the same as that present in synaptosomes, while in adult synaptosomes OH-GABA released GABA with increased specific radioactivity. The data suggest a homogeneous distribution of the [3H]GABA taken up within the endogenous GABA pool in immature, but not in mature synaptosomes. (3) In immature synaptosomes the release of GABA (radioactive and endogenous) induced by depolarization with high KC was not potentiated by Ca2+, unless the synaptosomes had been previously depleted of Na+ These data suggest that, although a Ca2+ sensitive pool of GABA may be present, this pool is not susceptible to being released in normal conditions, probably because the high intrasynaptosomal Na+ level prevents a sufficient depolarization. The possible significance of these findings in terms of functional activity of GABAergic neurotransmission in the immature brain is discussed.

In order to better characterize the presynaptic GABA receptors regulating glutamate release in the cerebellum [Levi and Gallo, 1981], a number of GABA agonists and GABA transport inhibitors were tested for their ability to potentiate the depolarization-induced release of the glutamate analog D-[3H]aspartate from superfused cerebellar synaptosomes. Of all the compounds tested, only those which are known to interact specifically with GABA receptors were effective when tested on synaptosomal preparations. The order of effectiveness found was the following: muscimol congruent to 3-APS greater than or equal to P4S greater than isoguvacine greater than THIP. GABA uptake inhibitors were unable to enhance D-[3H]aspartate evoked release from synaptosomes, but were effective when tested in cerebellar slices; in the latter case, the activation of the GABA receptors may be achieved indirectly, through an increase of the extracellular GABA concentrations. The substrate specificity of the presynaptic GABA receptors regulating cerebellar acidic amino acid release appears to be similar to that reported for GABA receptors in radioligand binding studies and for GABA autoreceptors. Studies on synaptosomes from immature cerebella suggested that the presence of the potentiating effect on the acidic amino acid release by GABA agonists is correlated with the development of the parallel fiber terminals, which are believed to be the main site from which glutamate is released in the adult cerebellum.

We present data on the expression of Concanavalin A (ConA) binding glycoproteins by granule cell enriched cultures derived from 8 day postnatal rat cerebellum. Time course studies were conducted over a 12 day culture period. ConA binding glycoproteins were localized on the cell bodies and fibres of the granule neurons using fluorescence microscopy. The fluorescence intensity increased between 4 and 12 days in vitro. Quantitative studies on the capacity of live cells to bind 125I-iodinated ConA showed that there was a significant increase in the amount of lectin bound between 4 and 8 days in vitro. However, in contrast to previous results on the developing cerebellum in vivo [Zanetta et al. (1978) Brain Res.142, 301–319.], there was no decline in binding capacity after 8 days in culture. Glycoproteins expressed by these cells were analysed by staining SDS polyacrylamide gels with [125I]ConA. A large number of lectin binding proteins were observed which spread over a wide range of molecular weights. Only minor changes were detected in the profile of [125I]ConA binding glycoproteins with the maturation of the cells in culture. The comparison of the findings on granule cells developing in culture and in vivo suggested that an interaction between granule cell axons and their normal target neurons is involved in the regulation of the ConA binding protein content in the cerebellum.

We studied some aspects of the differentiation of rat cerebellar neural cells obtained from 8-day postnatal animals and cultured in a serum-free, chemically defined medium (CDM). The ability of the cells to take up radioactive transmitter amino acids was analyzed autoradiographically. The L-glutamate analogue 3H-D-aspartate was taken up by astroglial cells, but not by granule neurons, even in late cultures (20 days in vitro). This is in agreement with the lack of depolarization-induced release of 3H-D-aspartate previously observed in this type of culture. In contrast, 3H-(GABA) was scarcely accumulated by glial-fibrillary-acidic-protein (GFAP)-positive astrocytes, but taken up by glutamate-decarboxylase-positive inhibitory interneurons and was released in a Ca2+-dependent way upon depolarization: 3H-GABA evoked release progressively increased with time in culture. Interestingly, the expression of the vesicle-associated protein synapsin I was much reduced in granule cells cultured in CDM as compared to those maintained in the presence of serum. These data would indicate that in CDM the differentiation of granule neurons is not complete, while that of GABAergic neurons is not greatly affected. Whether the diminished differentiation of granule cells must be attributed only to serum deprivation or also to other differences in the composition of the culture medium remains to be established. 3H-GABA was avidly taken up also by a population of cells which were not recognized by antibodies raised against GFAP, glutamate decarboxylase, and microtubule-associated protein 2. These cells exhibited a stellate morphology, were stained by the monoclonal antibody A2B5, and have been characterized elsewhere [Levi et al, 1986] as bipotential precursors of oligodendrocytes and of a subpopulation of astrocytes bearing a stellate shape and capable of high-affinity 3H-GABA uptake.

We have used postnatal rat cerebellar astrocyte-enriched cultures to study the excitatory amino acid receptors present on these cells. In the cultures used, type-2 astrocytes (recognized by the monoclonal antibodies A2B5 and LB1) selectively took up gamma-[3H]aminobutyric acid ([3H]GABA) and released it when incubated in the presence of micromolar concentrations of kainic and quisqualic acids. The releasing effect of kainic acid was concentration dependent in the range of 5-100 microM. Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (approximately 50 microM). N-Methyl-D-aspartic acid and dihydrokainate (100 microM) did not stimulate [3H]GABA release from cultured astrocytes. L-Glutamic acid (20-100 microM) stimulated [3H]GABA release as effectively as kainate. The stimulatory effects of kainate and quisqualate on [3H]GABA release were completely Na+ dependent; that of kainate was also partially Ca2+ dependent. Kynurenic acid (50-200 microM) selectively antagonized the releasing effects of kainic acid and also that of L-glutamate; quisqualate was unaffected. Quisqualic acid inhibited the releasing effects of kainic acid when both agonists were used at equimolar concentrations (50 microM). D-[3H]aspartate was taken up by both type-1 and type-2 astrocytes, but only type-2 astrocytes released it in the presence of kainic acid. Excitatory amino acid receptors with a pharmacology similar to that of the receptors present in type-2 astrocytes were also expressed by the immature, bipotential progenitors of type-2 astrocytes and oligodendrocytes.