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The brain consumes an enormous amount of energy (20%) that is disproportionate to its small size (2%). It relies on the constant delivery of oxygen and glucose to produce adenosine triphosphate, the cellular energy unit required to maintain brain cells’ functioning. Even brief loss of blood flow as a result of blockage or rupture of a cerebral blood vessel, called ischemia, leads to a sudden appearance of neurological symptoms. Stroke usually affects only one side of the brain, and the perfusion field of the affected blood vessel defines the specific pattern of sensory or motor symptoms produced. In a minority of patients (10%) blood flow is disrupted because of a vessel rupture, causing a hemorrhagic stroke. The vast majority of patients have vessel blockage as a consequence of atherosclerotic plaque, narrowing cerebral blood vessels, or distant plaque, giving rise to floating fragments called emboli that can lodge in cerebral blood vessels. The most effective treatment for stroke involves the rapid opening of an occluded vessel either mechanically or using tissue plasminogen activator (tPA), a clot-busting chemical. Time is of the essence because neurons die quickly in the absence of blood flow and tPA becomes almost ineffective 3 h after a stroke. The cellular and molecular processes underlying the ischemic cascade that culminates in neuronal cell death are well understood, particularly the importance of the excitatory neurotransmitter glutamate (Glu), which is elevated to toxic concentrations after stroke. Its binding to neuronal Glu receptors triggers a process called excitotoxicity, which causes rapid necrotic and slow apoptotic neuronal death. Approximately 800,000 new stroke cases are diagnosed annually in the United States and 200,000 patients die from it. In spite of excellent poststroke physical and occupational therapy, many of the 4 million stroke survivors live with permanent disability at a huge cost to the individual, their families, and society.

Schizophrenia is a neuropsychiatric disorder without a known cause. It is characterized by psychosis along with apathy and poor mentation. The disease affects approximately 1% of the world’s population, and recognizable symptoms typically begin at ages 18–24. Antipsychotic drugs that reduce the activity of dopamine receptors in the midbrain rarely cure patients, and most require life-long support or supervision and are typically unable to work. Patients with schizophrenia occupy 50% of all patient beds in US hospitals, and one-third of homeless people have the disorder. While the condition has long been thought to be a disorder of dopaminergic neurons, new research is challenging this view. Schizophrenia is more likely a neurodevelopmental disorder that begins in early childhood. Genetic changes, many of which are still obscure, cause too many synapses to be eliminated in the forebrain. This results in reduced synaptic activity along with reduced dendritic connectivity between neurons that can be visualized as a thinning of the cortex on magnetic resonance imaging. Developmental abnormalities affect predominantly glutamatergic and GABAergic synapses, which together regulate dopamine release. Reduced dopamine concentrations cause an overall lack of energy, social withdrawal, and loss of interest in pleasurable activities (anhedonia), while reduced glutamatergic signaling causes psychosis. Recreational drugs such as ketamine and phencyclidine, which block glutamate receptors, can cause similar psychotic symptoms in otherwise unaffected people. Current treatment effectively controls psychosis in many patients but provides no relief for apathy and anhedonia. Up to 10% of patients with schizophrenia end their own life; others die prematurely due to other comorbidities such as diabetes and heart and lung disease.

In most individuals, aging is a normal, healthy process and typically does not overtly impair cognition and memory function. However, aging is often feared, since it is the greatest risk factor for developing dementia and Alzheimer disease (AD). Affecting over 35 million people worldwide, AD is the single most common neurodegenerative disease. This number is expected to increase further, as average life expectancy is increasing, particularly in the developing world. The pathological hallmarks of AD are neurofibrillary tangles composed of phosphorylated tau protein and amyloid plaques resulting from aggregated β-amyloid. These are usually identified on autopsy, although recent novel imaging techniques now allow detection of amyloid plaques long before patients become symptomatic. Recent studies suggest that an accumulation of misfolded proteins precedes the onset of cognitive symptoms and dementia by at least a decade. Whether and how plaques and tangles are causal of disease or the consequence thereof remains unknown. Genetic studies show up to a 15-fold increased incidence for sporadic AD in people who carry the e4 allele of apolipoprotein E. Rare, early-onset familiar forms of AD are often associated with mutations in the amyloid precursor protein (APP) or the genes for the APP cleaving enzymes presenilin-1 and 2. These findings suggest that protein deposits are likely to be disease causing. AD is currently incurable and the majority of research efforts are focused on identifying preventive strategies to delay the onset of disease, ideally pushing it past the normal human life span.

Over 50 million people worldwide live with the consequences of brain and spinal cord injury. Unfortunately, nervous system tissue is very fragile and its protection by the bones of skull and vertebral column is insufficient to withstand the forces encountered in motor vehicle accidents or during contact sports. Though our understanding of primary and secondary nervous system injury is excellent, effective therapeutics to enhance a patient’s recovery are largely absent. At best, rehabilitation and robotic assist devices make disease more manageable. The major hurdles are the inability of most neurons to replicate after injury, and the active, multipronged suppression of axonal regeneration by myelin-associated molecules and glial scars. Exciting progress has been made in overcoming these hurdles in preclinical studies, and clinical trials show promise with the implantation of autologous stem cells. Increased public awareness of the danger associated with repeated mild injuries in sporting activities is having positive effects on the use of protective measures in athletics.

Parkinson disease (PD) is the world's second-leading neurodegenerative disorder. It typically presents after age 60 with the following four cardinal motor symptoms: rigidity, tremor, poverty of movement (bradykinesia), and problems with balance. These symptoms are caused by the progressive loss of dopaminergic projections in the midbrain from the substantia nigra to the striatum. Patients also present with apathy, anxiety, and depression, which are due to diffuse loss of monoamine-expressing neurons. Rare cases of familial PD occur at a much younger age and are caused by genetic mutations in one of several genes. Of these, α-synuclein, LRRK2, Parkin, and Pink1 are the most well studied. A common histopathological hallmark of PD is aggregation of misfolded α-synuclein protein called Lewy bodies. In contrast to the rare familial forms of PD, the cause of sporadic PD is unknown. However, exposure to certain chemicals and pesticides that poison mitochondrial function mimic the cardinal symptoms of PD, suggesting that mitochondrial health is pivotal to the etiology of PD. Therefore, it appears that PD is a multifactorial disease involving many as yet unrecognized factors. Genetically established susceptibility, when paired with the wrong environmental influences, such as exposure to toxins, can cause progressive neuronal loss. Fortunately, in most patients, motor symptoms can be effectively treated for many years through dopamine replacement therapy and/or deep brain stimulation. However, the effectiveness of this treatment wears off over time and none of the currently available dopamine replacement therapies corrects the nonmotor symptoms.

Major depression and bipolar disorder can rob a person of the potential to enjoy life and to participate productively in society. Although disease-causing genes have yet to be identified, depression is heritable but likely requires environmental triggers to manifest. Depression presents with structural and functional brain changes that include atrophy of the hippocampus, enlargement of the amygdala, and reduced signaling via the monoamine transmitters serotonin and norepinephrine. Changes in neurotransmission are the result of increased glutamate release from the hippocampus onto neurons in the limbic system. This is accompanied by long-lasting structural changes in dendrites and synapses, including reduced hippocampal neurogenesis. Current treatments inhibit monoamine degradation or reuptake and are no more effective than placebo or behavioral therapies in many cases. The recent finding that the anesthetic ketamine is highly effective in treating the acute phase of treatment-resistant depression provides hope that new, more effective drugs targeting glutamate as opposed to monoamines may be on the horizon.

Many cells, including neuronal and glial progenitor cells, stem cells and microglial cells, have the capacity to move through the extracellular spaces of the developing and mature brain. This is particularly pronounced in astrocyte-derived tumors, gliomas, which diffusely infiltrate the normal brain. Although a significant body of literature exists regarding signals that are involved in the guidance of cells and their processes, little attention has been paid to cell-shape and cell-volume changes of migratory cells. However, extracellular spaces in the brain are very narrow and represent a major obstacle that requires cells to dynamically regulate their volume. Recent studies in glioma cells show that this involves the secretion of Cl− and K+ with water. Pharmacological inhibition of Cl− channels impairs their ability to migrate and limits tumor progression in experimental tumor models. One Cl−-channel inhibitor, chlorotoxin, is currently in Phase II clinical trials to treat malignant glioma. This article reviews our current knowledge of cell-volume changes and the role of ion channels during the migration of glioma cells. It also discusses evidence that supports the importance of channel-mediated cell-volume changes in the migration of immature neurons and progenitor cells during development. New unpublished data is presented, which demonstrates that Cl− and K+ channels involved in cell shrinkage localize to lipid-raft domains on the invadipodia of glioma cells and that their presence might be regulated by trafficking of these proteins in and out of lipid rafts.

Glial cells play an important role in sequestering neuronally released glutamate via Na+-dependent transporters. Surprisingly, these transporters are not operational in glial-derived tumors (gliomas). Instead, gliomas release glutamate, causing excitotoxic death of neurons in the vicinity of the tumor. We now show that glutamate release from glioma cells is an obligatory by-product of cellular cystine uptake via system\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{x}_{\mathrm{c}}^{-}\) \end{document}, an electroneutral cystine-glutamate exchanger. Cystine is an essential precursor for the biosynthesis of glutathione, a major redox regulatory molecule that protects cells from endogenously produced reactive oxygen species (ROS). Glioma cells, but not neurons or astrocytes, rely primarily on cystine uptake via system\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{x}_{\mathrm{c}}^{-}\) \end{document}for their glutathione synthesis. Inhibition of system\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{x}_{\mathrm{c}}^{-}\) \end{document}causes a rapid depletion of glutathione, and the resulting loss of ROS defense causes caspase-mediated apoptosis. Glioma cells can be rescued if glutathione status is experimentally restored or if glutathione is substituted by alternate cellular antioxidants, confirming that ROS are indeed mediators of cell death. We describe two potent drugs that permit pharmacological inhibition of system\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{x}_{\mathrm{c}}^{-}\) \end{document}. One of these drugs, sulfasalazine, is clinically used to treat inflammatory bowel disease and rheumatoid arthritis. Sulfasalazine was able to reduce glutathione levels in tumor tissue and slow tumor growthin vivoin a commonly used intracranial xenograft animal model for human gliomas when administered by intraperitoneal injection. These data suggest that inhibition of cystine uptake into glioma cells through the pharmacological inhibition of system\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{x}_{\mathrm{c}}^{-}\) \end{document}may be a viable therapeutic strategy with a Food and Drug Administration-approved drug already in hand.

Malignant astrocytic gliomas, referred to as astrocytomas, represent the most commonly diagnosed adult primary brain tumor. These tumors are characterized by unrelenting growth that is often resistant to chemotherapy and radiation therapy. Tumor expansion into the healthy surrounding brain tissue produces severe and often fatal consequences. In this study, we examine the potential for the neuregulin-1/erbB receptor signaling cascade to contribute to this process by modulating glioma cell growth. Using antibodies specific for the erbB receptors, we demonstrate the expression patterns for the erbB2, erbB3, and erbB4 receptors in human glioma biopsy samples. We then verify receptor expression in a panel of human glioma cell lines. Next, we investigate the status of the erbB2 and erbB3 receptors in the human glioma cell lines and find that they are constitutively tyrosine-phosphorylated and heterodimerized. Subsequently, we demonstrate that theses same cell lines express membrane bound and released forms of neuregulins, the erbB receptor ligands, suggesting a possible autocrine or paracrine signaling network. Furthermore, we show that exogenous activation of erbB2 and erbB3 receptors in U251 glioma cells by recombinant Nrg-1beta results in enhanced glioma cell growth under conditions of serum-deprivation. This enhancement is due to an increase in cell survival rather than an increase in cell proliferation and is dependent on the activation of erbB2 and phosphatidylinositol-3 kinase (PI3K). Moreover, Nrg-1beta activates an inhibitor of apoptosis, Akt, implying a possible role for this kinase in mediating Nrg-1beta effects in gliomas. This data suggests that glioma cells may use autocrine or paracrine neuregulin-1/erbB receptor signaling to enhance cell survival under conditions where growth would otherwise be limited.

Inwardly rectifying potassium (K(ir)) channels are a prominent feature of mature, postmitotic astrocytes. These channels are believed to set the resting membrane potential near the potassium equilibrium potential (E(K)) and are implicated in potassium buffering. A number of previous studies suggest that K(ir) channel expression is indicative of cell differentiation. We therefore set out to examine K(ir) channel expression in malignant glia, which are incapable of differentiation. We used two established and widely used glioma cell lines, D54MG (a WHO grade 4 glioma) and STTG-1 (a WHO grade 3 glioma), and compared them to immature and differentiated astrocytes. Both glioma cell lines were characterized by large outward K(+) currents, depolarized resting membrane potentials (V(m)) (-38.5 +/- 4.2 mV, D54 and -28.1 +/- 3.5 mV, STTG1), and relatively high input resistances (R(m)) (260.6 +/- 64.7 MOmega, D54 and 687.2 +/- 160.3 MOmega, STTG1). These features were reminiscent of immature astrocytes, which also displayed large outward K(+) currents, had a mean V(m) of -51.1 +/- 3.7 and a mean R(m) value of 627.5 +/- 164 MOmega. In contrast, mature astrocytes had a significantly more negative resting membrane potential (-75.2 +/- 0.56 mV), and a mean R(m) of 25.4 +/- 7.4 MOmega. Barium (Ba(2+)) sensitive K(ir) currents were >20-fold larger in mature astrocytes (4.06 +/- 1.1 nS/pF) than in glioma cells (0.169 +/- 0.033 nS/pF D54, 0.244 +/- 0.04 nS/pF STTG1), which had current densities closer to those of dividing, immature astrocytes (0.474 +/- 0.12 nS/pF). Surprisingly, Western blot analysis shows expression of several K(ir) channel subunits in glioma cells (K(ir)2.3, 3.1, and 4.1). However, while in astrocytes these channels localize diffusely throughout the cell, in glioma cells they are found almost exclusively in either the cell nucleus (K(ir)2.3 and 4.1) or ER/Golgi (3.1). These data suggest that mislocalization of K(ir) channel proteins to intracellular compartments is responsible for a lack of appreciable K(ir) currents in glioma cells.

Voltage-dependent large-conductance Ca(2+)-activated K(+) channels, often referred to as BK channels, are a unique class of ion channels coupling intracellular chemical signaling to electrical signaling. BK channel expression has been shown to be up-regulated in human glioma biopsies, and expression levels correlate positively with the malignancy grade of the tumor. Glioma BK channels (gBK) are a splice variant of the hslo gene, are characterized by enhanced sensitivity to [Ca(2+)](i), and are the target of modulation by growth factors. By using the selective pharmacological BK channel inhibitor iberiotoxin, we examined the potential role of these channels in tumor growth. Cell survival assays examined the ability of glioma cells to grow in nominally serum-free medium. Under such conditions, BK channel inhibition by iberiotoxin caused a dose- and time-dependent decrease in cell number discernible as early as 72 hr after exposure and maximal growth inhibition after 4-5 days. FACS analysis shows that IbTX treatment arrests glioma cells in S phase of the cell cycle, whereupon cells undergo cell death. Interestingly, IbTX effects were nullified when cells were maintained in 7% fetal calf serum. Electrophysiological analysis, in conjunction with biotinylation studies, demonstrates that serum starvation caused a significant translocation of BK channel protein to the plasma membrane, corresponding to a two- to threefold increase in whole-cell conductance, but without a change in total gBK protein. Hence, expression of functional gBK channels appears to be regulated in a growth-factor-dependent manner, with enhanced surface expression promoting tumor cell growth under conditions of growth factor deprivation as might occur under in vivo conditions.

Rat cortical astrocytes regulate their cell volume in response to hypotonic challenge. This regulation is believed to depend largely on the release of chloride or organic osmolytes through anion channels. Using whole-cell recordings, we identified weakly outwardly rectifying chloride currents that could be activated in response to hypotonic challenge. These currents exhibited the following permeability sequence upon replacement of chloride in the bathing solution with various anions: I- > NO3- > Cl- > Gluc- > or = MeS- > Ise-. Interestingly, extracellular I-, albeit showing the greatest permeability, blocked the currents with an IC50 of approximately 50 mM. Currents were almost completely inhibited by 123 microM NPPB and partially inhibited by 200 microM niflumic acid or 200 microM DIDS. Additionally, the total number of Cl- ions effluxed through the hypotonically activated channels was markedly similar to the total solute efflux during volume regulation. We therefore propose the hypotonically activated chloride channel as a major contributor to volume regulation of astrocytes. To examine potential candidate chloride channel genes expressed by astrocytes, we employed RT-PCR to demonstrate the presence of transcripts for ClC-2, 3, 4, 5, and 7, as well as for VDAC and CFTR in cultured astrocytes. Moreover, we performed immunostaining with antibodies against each of these channels and showed the strongest expression of ClC-2 and ClC-3, strong expression of ClC-5 and VDAC, weak expression of ClC-7 and very weak expression of ClC-4 and CFTR. Intriguingly, although we found at least seven Cl- channel proteins from three different gene families in astrocytes, none appeared to be active in resting cells.

The malignant transformation of astrocytes, oligodendrocytes or their progenitor cells gives rise to tumors that are collectively called gliomas. Although the vast majority of these tumors are believed to be of astrocytic origin, the actual cell of origin remains unknown. And whereas gliomas present with some genetic alterations in tumor-suppressor genes or oncogenes that are common among cancers, their biology differs significantly from other neoplasms. Most notably, they grow in a confined space defined by the bones of the skull and spinal column, and they spread by active cell migration rather than by passive, humorous spread. Recent research suggests that astrocyte-derived tumors co-opt neurotransmitters, their transporters and ion channels to support their unusual growth and invasive migration.

Voltage-gated chloride channels have recently been implicated as being important for cell proliferation and invasive cell migration of primary brain tumors cells. In the present study we provide several lines of evidence that glioma Cl–currents are primarily mediated by ClC-2 and ClC-3, two genes that belong to the ClC superfamily. Transcripts for ClC-2 thru ClC-7 were detected in a human glioma cell line by PCR, whereas only ClC-2, ClC-3, and ClC-5 protein could be identified by Western blot. Prominent ClC-2, -3, and -5 channel expression was also detected in acute patient biopsies from low- and high-grade malignant gliomas. Immunogold electron microscopic studies as well as digital confocal imaging localized a portion of these ClC channels to the plasma membrane. Whole-cell patch-clamp recordings show the presence of two pharmacologically and biophysically distinct Cl–currents that could be specifically reduced by 48 hr exposure of cells to channel-specific antisense oligonucleotides. ClC-3 antisense selectively and significantly reduced the expression of outwardly rectifying current with pronounced voltage-dependent inactivation. Such currents were sensitive to DIDS (200–500 μm) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (165 μm). ClC-2 antisense significantly reduced expression of inwardly rectifying currents, which were potentiated by hyperpolarizing prepulses and inhibited by Cd2+(200–500 μm). Currents that were mediated by ClC-5 could not be demonstrated. We suggest that ClC-2 and ClC-3 channels are specifically upregulated in glioma membranes and endow glioma cells with an enhanced ability to transport Cl–. This may in turn facilitate rapid changes in cell size and shape as cells divide or invade through tortuous extracellular brain spaces.

Glial glutamate transport plays a pivotal role in maintaining glutamate homeostasis in the central nervous system. Expression of glutamate transporters is highly regulated during brain development, and a number of pathological conditions are associated with deficits in expression and/or function of glutamate transports. While several soluble factors have been shown to regulate the expression of glutamate transporter, the contribution of cell-cell interaction and cell-environmental interaction in the regulation of glutamate transport is unknown. Extracellular matrix (ECM) molecules are essential components in cell-cell and cell-environmental interactions, and the ECM has been shown to play critical role in normal development and during brain pathogenesis. We, therefore, investigated the possibility that ECM molecules may regulate astrocytic glutamate transport. Therefore, we cultured rat cortical astrocytes with different ECMs and determined expression levels of the two astrocytic glutamate transporters GLT-1 and GLAST by Western Blot and determined transporter activity through measurements of 3H-D-aspartate uptake. Astrocytes grown on poly-ornithine or poly-D/L-lysine showed approximately two-fold higher GLT-1 expression than sister cells grown on plastic dishes without ECM. Naturally occurring ECM's, including laminin and collagen, showed a dose-dependent regulation of GLT-1 protein expression. These effects were specific for GLT-1 as GLAST expression was unaffected by different ECMs. Surprisingly, however, none of the examined ECMs altered the apparent glutamate uptake activity. In probing blots side-by-side for expression of Na(+)/K(+)-ATPase, we found that ECMs affected expression of Na(+)/K(+)-ATPase and GLT-1 in a reciprocal fashion. Poly-ornithine, for example, enhanced GLT-1 expression, but reduced expression of Na(+)/K(+)-ATPase. Na(+) transport may, thus, be a limiting factor for glutamate uptake.

We have previously demonstrated the expression of large-conductance, calcium-activated potassium (BK) channels in human glioma cells. In the present study, we characterized the calcium sensitivity of glioma BK channels in excised membrane patches. Channels in inside-out patches were activated at -60 mV by 2.1 x 10(-6) M cytosolic Ca(2+), were highly K(+)-selective, and had a slope conductance of approximately iqual 210 pS. We characterized the Ca(2+) sensitivity of these channels in detail by isolating BK currents in outside-out patches with different free [Ca(2+)](i). The half-maximal voltage for channel activation, V(0.5), of glioma BK currents in outside-out patches was +138 mV with 0 Ca(2+)/10 EGTA. V(0.5) was shifted to +81 mV and -14 mV with free [Ca(2+)](i) of 1.5 x 10(-7) M and 2.1 x 10(-6) M, respectively. These results suggest that glioma BK channels have a higher Ca(2+) sensitivity than that described in many other human preparations. Data obtained from a cloned BK channel (hbr5) expressed in HEK cells support the conclusion that glioma BK channels have an unusually high sensitivity to calcium. In addition, the sensitivity of glioma BK channels to the BK inhibitor tetrandrine suggests the expression of BK channel auxiliary beta-subunits by glioma cells. Expression of the auxiliary beta-subunit of BK channels by glioma cells may relate to the high Ca(2+) sensitivity of glioma BK channels.

Voltage-dependent large-conductance Ca 2+ -activated K + channels (BK channels) are widely expressed in excitable and nonexcitable cells. BK channels exhibit diverse electrophysiological properties, which are attributable in part to alternative splicing of their α-subunits. BK currents have been implicated in the growth control of glial cells, and BK channels with novel biophysical properties have recently been characterized in human glioma cells. Here we report the isolation, cloning, and functional characterization of glioma BK (gBK), a novel splice isoform of hSlo , the gene that encodes the α-subunits of human BK channels. The primary sequence of gBK is 97% identical to its closest homolog hbr5, but it contains an additional 34-amino-acid exon at splice site 2 in the C-terminal tail of BK channels. hSlo transcripts containing this novel exon are expressed ubiquitously in various normal tissues as well as in neoplasmic samples, suggesting that the novel exon may modulate important physiological functions of BK channels. Expression of gBK in Xenopus oocytes gives rise to iberiotoxin-sensitive (IbTX) currents, with an IC 50 for IbTX of 5.7 nm and a Hill coefficient of 0.76. Single gBK channels have a unitary conductance of ∼250 pS, and the currents show significantly slower activation and higher Ca 2+ sensitivity than hbr5. Ca 2+ sensitivity was enhanced specifically at physiologically relevant [Ca 2+ ] i (100–500 nm). Examination of biopsies from patients with malignant gliomas has revealed specific overexpression of BK channels in gliomas compared with nonmalignant human cortical tissues. Importantly, tumor malignancy grades have correlated positively with BK channel expression, suggesting an important role for the gBK channel in glioma biology.

The molecular and cellular mechanisms governing cell motility and directed migration in response to the neuropeptide bradykinin are largely unknown. Here, we demonstrate that human glioma cells whose migration is guided by bradykinin generate bleb-like protrusions. We found that activation of the B2 receptor leads to a rise in free Ca(2+) from internal stores that activates actomyosin contraction and subsequent cytoplasmic flow into protrusions forming membrane blebs. Furthermore Ca(2+) activates Ca(2+)-dependent K(+) and Cl(-) channels, which participate in bleb regulation. Treatment of gliomas with bradykinin in situ increased glioma growth by increasing the speed of cell migration at the periphery of the tumour mass. To test if bleb formation is related to bradykinin-promoted glioma invasion we blocked glioma migration with blebbistatin, a blocker of myosin kinase II, which is necessary for proper bleb retraction. Our findings suggest a pivotal role of bradykinin during glioma invasion by stimulating amoeboid migration of glioma cells.

Astrocytic endfeet cover the entire cerebral vasculature and serve as exchange sites for ions, metabolites and energy substrates from the blood to the brain. They maintain endothelial tight junctions that form the blood-brain barrier (BBB) and release vasoactive molecules that regulate vascular tone. Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumour form. Here we use a clinically relevant mouse model of glioma and find that glioma cells, as they populate the perivascular space of preexisting vessels, displace astrocytic endfeet from endothelial or vascular smooth muscle cells. This causes a focal breach in the BBB. Furthermore, astrocyte-mediated gliovascular coupling is lost, and glioma cells seize control over the regulation of vascular tone through Ca(2+)-dependent release of K(+). These findings have important clinical implications regarding blood flow in the tumour-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

Current treatment regimens for malignant gliomas are inadequate. Characterized by aggressive cell proliferation and migration, gliomas lead to a median patient survival of about 1 year despite medical intervention, including surgery, chemotherapy, and radiation. Numerous clinical trials are attempting to make headway; intraoperative visualization techniques, intensity-modulated radiation therapy, and inhibitors of growth factor pathways and angiogenesis are making incremental, although valuable, progress in our management of this devastating disease. Both current treatments and most clinical trials modulate traditional oncological features of cancers, such as DNA replication, cell proliferation, neovascularization, and necrosis. However, in this chapter we argue that for a sea-change in treatment to occur, unique characteristics of gliomas must be targeted. Blocking the association of glioma cells with the vasculature could contain tumor spread. Inhibiting ion channels on glioma cells prevents the shape and volume changes necessary for cell migration. And preventing assiduous glutamate release from glioma cells decreases excitotoxic neuronal death. To make a substantive impact on clinical management, novel therapies should focus on these neurodegenerative aspects of glioma biology.

Gap junctions are an important means for intercellular communication during development, processes of tissue differentiation, and in maintenance of adult tissue homeostasis. We investigated the expression levels and distribution of connexin-43 (Cx-43), the most abundant astrocytic gap junction protein, in acutely isolated astrocytes and glioma cells from biopsy tissue obtained from patients diagnosed with glioblastoma multiforme (GBM), low-grade astrocytomas (LGAs), or mesial temporal lobe epilepsy. Western blot and immunohistochemical analyses indicated an inverse correlation between the amount of Cx-43 protein and tumor malignancy grade, as assessed by calculating tissue mitotic indexes (MI) obtained using anti-Ki-67 nuclear antigen staining. Samples from epilepsy patients had a low MI and were intensely positive for Cx-43 staining, while LGA tissue samples exhibited moderate staining for Cx-43 and average MI, and GBM biopsies showed significantly lower levels of Cx-43 and high MI. Functional coupling was assayed using fluorescence recovery after photobleach (FRAP). We found that cells from glioma cell lines and primary cultures of human astrocytes from GBM tissues displayed significantly lower degrees of gap junction intercellular communication (GJIC) as indicated by longer and less complete recovery from photobleaching. Mean recovery values were GBM 23.8% +/- 11.4%, LGA 49.4% +/- 47%, and nontumor astrocytes 67.2% +/- 8.4%. Western blot analysis of several human glioma cell lines and tissue biopsies showed variable expression levels of Cx-43, which correlated negatively with the extent of recovery in the same samples. Taken together, our findings suggest that high-grade brain tumors show reduced intercellular communication and a decrease in connexin-43 protein levels.

Injury and diseases of the nervous system can induce astrocytes to form tenacious glial scars. We induced focal cortical freeze-lesions in neonatal rats and examined scars histologically and electrophysiologically in tissue slices isolated 2-3 weeks after lesioning. Lesions displayed marked gliosis, characterized by upregulation of GFAP labeling. Reactive astrocytes surrounding the scar showed marked hypertrophy, enlarged cell bodies and extended processes frequently terminating with endfeet-like structures on blood vessels. These reactive astrocytes showed enhanced expression of inwardly rectifying K + (K IR ) channels, widely believed to be an important pathway for astrocytic K + buffering. These results suggest that a subpopulation of reactive astrocytes along a glial scar might be instrumental in buffering K + away from the lesion.

Using biophysical techniques, we previously have implicated outwardly rectifying potassium currents in the proliferation of cultured spinal cord astrocytes and have demonstrated that delayed rectifier potassium currents (IKd), in particular, are upregulated on entry into the cell cycle and downregulated with cell cycle exit and differentiation. In the present study, using specific antibodies and antisense oligodeoxynucleotides, we show that this proliferation-dependent potassium current is mediated by theShakerpotassium channel Kv1.5. Downregulation of Kv1.5 protein by antisense oligodeoxynucleotides reduces astrocyte proliferation by ∼50%, although no observed changes occur in Kv1.5 protein expression during spontaneous differentiation in culture. Tyrosine phosphorylation of Kv1.5, however, is downregulated markedly in differentiated cells but unaltered on cell cycle arrest. Using immunoprecipitation, we show that Kv1.5 is associated with Src family protein tyrosine kinases and that this association does not change with cell differentiation. Inhibition of kinase activity with the Src-specific inhibitor PP2 decreases Kv1.5 phosphorylation, reducesIKd, and inhibits astrocyte proliferation, specifically in the G0/G1phase of cell cycle. Conversely,IKdare potentiated when active Src is present in the pipette. Transfection of quiescent astrocytes with constitutively active Src (Src Y529F) causes marked upregulation of astrocyte proliferation. These data suggest that Kv1.5 is phosphorylated constitutively by Src kinases during growth and that downregulation of Src activity may underlie both astrocyte differentiation and the accompanying changes in delayed rectifier potassium channel activity.

Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca2+]i) associated with periodic Ca2+ oscillations during prolonged ACh applications. The early transient rise in [Ca2+]i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca2+ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca2+]i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca2+-sensitive K+ currents. These K+ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca2+ from intracellular stores which in turn activate Ca2+-dependent (BK-type) K+ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration.

Arrest of spinal cord astrocytes at defined stages of the cell cycle clock causes significant changes in the expression of voltage-activated Na+ and K+ currents. Arrest of actively proliferating astrocytes in G1/G0 by all-trans-retinoic acid induces premature expression of inwardly rectifying K+ currents (IKIR) typically expressed only in differentiated astrocytes. By contrast, arrest in S phase by ara-C or Aphidicolin leads to a greater than twofold increase in “delayed” outwardly rectifying currents (IKD) and a concomitant decrease in IKIR. Pharmacological blockade of IKD by TEA and 4AP caused proliferating astrocytes to arrest in G0/G1, suggesting that activity of these channels is required for G1/S checkpoint progression. Conversely, in quiescent astrocytes, inhibition of IKIR by 30 μM BaCl2 led to an increase in astrocyte proliferation and to an increase in the number of cells in S phase from 5% to 26%. These data suggest that a downregulation of KIR promotes cell cycle progression through the G1/S checkpoint. Blockade of IKIR in actively proliferating cells, however, leads to an accumulation in G2/M, suggesting that reappearance of this current may be critical for progression beyond DNA synthesis. Interestingly, Na+ currents (INa+) are increased greater than fourfold in S phase-arrested cells, yet their pharmacological blockade by TTX has no effect on cell cycle progression. However, the resting membrane potential of S phase-arrested cells increases profoundly, and manipulation of membrane potential by the application of low concentrations of ouabain, or reduction of extracellular potassium, induces the accumulation of quiescent astrocytes in S phase of the cell cycle, suggesting that either depolarization or intracellular sodium, or both, play an important role in promoting astrocyte proliferation. GLIA 30:39–48, 2000. © 2000 Wiley-Liss, Inc.

We have studied the effects of the bioactive phospholipid lysophosphatidic acid (LPA) on cell lines derived from highly invasive human glioblastoma multiforme (GBM). Using transwell migration assays, we show that LPA stimulates both chemokinetic and chemotactic migration of glioma cells. Blood brain barrier breakdown and leakage of serum components that most likely include LPA are common features of GBM. Therefore, the effects of LPA on glioma cell motility are intriguing given the fact that, in vivo, GBM cells often migrate great distances from the main tumor, rendering successful therapy extremely difficult. We show here that LPA initiates a variety of signaling cascades in glioma cells. LPA-enhanced transwell migration was sensitive to pertussis toxin (PTX) treatment suggesting an important role for Gi subtype of G proteins. LPA also stimulated Ca2+ fluctuations and activation of extracellular signal-regulated kinases (ERKS) 1 and 2, although blocking either pathway had little effect on glioma cell migration. Exposure of glioma cells to LPA resulted in phosphorylation of the regulatory light chain (RLC) of myosin II and the formation of stress fibers and focal adhesions. These effects were blocked by Y-27632, an inhibitor of Rho-activated ROCK kinases. Time-lapse video microscopy revealed that Y-27632-treatment caused cells to assume long thin morphologies that suggested deficiencies in the contractile apparatus. Furthermore, many cells exhibited a conspicuous extension of processes when Rho/ROCK kinase cascades were inhibited. The above results suggest that LPA/Rho signaling cascades play important roles in glioma cell motility and that exposure of tumor cells to LPA in vivo may contribute to their invasive phenotype. Cell Motil. Cytoskeleton 45:185–199, 2000 © 2000 Wiley-Liss, Inc.

Human malignant gliomas are highly invasive tumors. Mechanisms that allow glioma cells to disseminate, migrating through the narrow extracellular brain spaces are poorly understood. We recently demonstrated expression of large voltage-dependent chloride (Cl−) currents, selectively expressed by human glioma cellsin vitroandin situ(Ullrich et al., 1998). Currents are sensitive to several Cl−channel blockers, including chlorotoxin (Ctx), (Ullrich and Sontheimer, 1996; Ullrich et al., 1996), tetraethylammonium chloride (TEA), and tamoxifen (Ransom and Sontheimer, 1998). Using Transwell migration assays, we show that blockade of glioma Cl−channels specifically inhibits tumor cell migration in a dose-dependent manner. Ctx (5 μm), tamoxifen (10 μm), and TEA (1 mm) also prevented invasion of human glioma cells into fetal rat brain aggregates, used as anin vitromodel to assess tumor invasiveness. Anion replacement studies suggest that permeation of chloride ions through glioma chloride channel is obligatory for cell migration. Osmotically induced cell swelling and subsequent regulatory volume decrease (RVD) in cultured glioma cells were reversibly prevented by 1 mmTEA, 10 μmtamoxifen, and irreversibly blocked by 5 μmCtx added to the hypotonic media. Cl−fluxes associated with adaptive shape changes elicited by cell swelling and RVD in glioma cells were inhibited by 5 μmCtx, 10 μmtamoxifen, and 1 mmTEA, as determined using the Cl−-sensitive fluorescent dye 6-methoxy-N-ethylquinolinium iodide. Collectively, these data suggest that chloride channels in glioma cells may enable tumor invasiveness, presumably by facilitating cell shape and cell volume changes that are more conducive to migration and invasion.

Astrocytes are thought to control extracellular glutamate concentrations ([Glu]o) in the brain, thereby protecting neurons from excitotoxic injury. We investigated the effects of metabotropic glutamate receptor (mGluR) agonists on glutamate transport and [Glu]o in primary hippocampal astrocytic cultures. Acute or chronic exposure of astrocytes to the mGluR agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) or its active isomer 1S,3R-ACPD reduced [Glu]o in a time- and dose-dependent manner (44.5 ± 3.6% reductions of [Glu]o in astrocytes from P0–P10 rats and 65.9 ± 4.1 % from rats P20 by 100 μM 1S,3R-ACPD, EC50 ∼ 5 μM). 1S,3R-ACPD effects developed slowly (median effective at ∼60 min) and persisted for several hours after agonist removal. ACPD-pretreated astrocytes established lower steady-state [Glu]o levels. ACPD effects persisted in the presence of the glutamate uptake inhibitors D,L-threo-β-hydroxyaspartate (THA) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC) but were impaired by disruption of the transmembrane Na+, K+, or H+ gradients. In addition, 1S,3R-ACPD had no effects on intracellular glutamate content and did not directly block glutamate transport. Furthermore, ACPD effects could be mimicked by glutamate per se and several other compounds presumed to be mGluR agonists, although (S)-3,5-dihydroxyphenylglycine (DHPG), (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) were without effect. These data suggest that glutamate and certain mGluR agonists may regulate [Glu]o by modulating the transmembrane equilibrium of glutamate transport, especially by attenuating glutamate release. GLIA 25:270–281, 1999. © 1999 Wiley-Liss, Inc.

Astrocytes that are recorded in acute tissue slices of rat hippocampus using whole-cell patch-clamp, commonly exhibit voltage-activated Na+ and K+ currents. Some reports have described astrocytes that appear to lack voltage-activated currents and proposed that these cells constitute a subpopulation of electrophysiologically passive astrocytes. We show here that these cells can spontaneously change during a recording unmasking expression of previously suppressed voltage-activated currents, suggesting that such cells do not represent a subpopulation of passive astrocytes. Superfusion of a low Ca2+/EGTA solution was able to reversibly suppress voltage-activated K+ currents in cultured astrocytes. Currents were restored upon addition of normal bath Ca2+. These effects of Ca2+ on both outward and inward K+ currents were dose- and time-dependent, with increasing concentrations of Ca2+ (from 0 to 800 micrometers) leading to a gradual unmasking of voltage-dependent outward and inward K+ currents. The transition from an apparently passive cell to one exhibiting prominent voltage-activated currents was not associated with any changes in membrane capacitance or access resistance. By contrast, in cells in which low access resistance or poor seal accounted for the absence of voltage-activated currents, improvement of cell access was always accompanied by changes in series resistance and membrane capacitance. We propose that spillage of pipette solution containing low Ca2+/EGTA during cell approach in slice recordings and/or poor cell access, lead to a transient masking of voltage-activated currents even in astrocytes that express prominent voltage-activated currents. These cells, however, do not constitute a subpopulation of electrophysiologically passive astrocytes.

Lysophosphatidic acid (LPA) is an extracellular signaling molecule that can enter the central nervous system following injury or diseases that disrupt the blood-brain-barrier. Using a combination of time-lapse microscopy, immunocytochemistry, and biochemical techniques, we demonstrate that LPA stimulates profound changes in astrocyte morphology that are due to effects on the actomyosin cytoskeleton. Flat astrocytes in primary culture display prominent actin stress fibers. Treatment with the myosin light chain kinase inhibitor, ML-9, causes stress fiber dissolution and dramatic morphology changes including rounding of the cell body and the formation of processes. LPA can stabilize actin stress fibers and inhibit the morphology changes in ML-9-treated cells. Furthermore, this activity is dependent upon activation of the GTP-binding protein Rho as evidenced by the ability of C3 exoenzyme, a specific inhibitor of Rho, to block the effect. Phosphorylation of the regulatory light (RLC) chain initiates conformational changes in myosin II that result in the formation of myosin filaments and the recruitment of actin into contractile stress fibers. LPA-induced stabilization of stress fibers is accompanied by increases in phosphorylation of the RLC of myosin. Furthermore, astrocytes grown on flexible silicone undergo rapid contraction in response to LPA treatment. The forces generated by these cells manifest themselves as increased wrinkling in the silicone. The observed contraction and accompanying increases in regulatory light chain phosphorylation suggest that LPA-induced signaling cascades in astrocytes regulate actin/myosin interactions.

Bordey, Angélique and Harald Sontheimer. Electrophysiological properties of human astrocytic tumor cells in situ: enigma of spiking glial cells. J. Neurophysiol. 79: 2782–2793, 1998. To better understand physiological changes that accompany the neoplastic transition of astrocytes to become astrocytoma cells, we studied biopsies of low-grade, pilocytic astrocytomas. This group of tumors is most prevalent in children and the tumor cells maintain most antigenic features typical of astrocytes. Astrocytoma cells were studied with the use of whole cell patch-clamp recordings in acute biopsy slices from 4-mo- to 14-yr-old pediatric patients. Recordings from 53 cells in six cases of low-grade astrocytomas were compared to either noncancerous peritumoral astrocytes or astrocytes obtained from other surgeries. Astrocytoma cells almost exclusively displayed slowly activating, sustained, tetraethylammonium (TEA)-sensitive outward potassium currents (delayed rectifying potassium currents; IDR) and transient, tetrodotoxin (TTX)-sensitive sodium currents ( INa). By contrast, comparison glial cells from peritumoral regions or other surgeries showed IDRand INa, but in addition these cells also expressed transient “A”-type K+currents and inwardly rectifying K+currents ( IIR), both of which were absent in astrocytoma cells. IIRconstituted the predominant conductance in comparison astrocytes and was responsible for a high-resting K+conductance in these cells. Voltage-activated Na+currents were observed in 37 of 53 astrocytoma cells. Na+current densities in astrocytoma cells, on average, were three- to fivefold larger than in comparison astrocytes. Astrocytoma cells expressing INacould be induced to generate slow action potential-like responses (spikes) by current injections. The threshold for generating such spikes was −34 mV (from a holding potential of −70 mV). The spike amplitude and time width were 52.5 mV and 12 ms, respectively. No spikes could be elicited in comparison astrocytes, although some of them expressed Na+currents of similar size. Comparison of astrocytes to astrocytoma cells suggests that the apparent lack of IIR, which leads to high-input resistance (>500 MΩ), allows glioma cells to be sufficiently depolarized to generate Na+spikes, whereas the high resting K+conductance in astrocytes prevents their depolarization and thus generation of spikes. Consistent with this notion, Na+spikes could be induced in spinal cord astrocytes in culture when IIRwas experimentally blocked by 10 μM Ba2+, suggesting that the absence of IIRin astrocytoma cells is primarily responsible for the unusual spiking behavior seen in these glial tumor cells. It is unlikely that such glial spikes ever occur in vivo.

MacFarlane, Stacey Nee and Harald Sontheimer. Spinal cord astrocytes display a switch from TTX-sensitive to TTX-resistant sodium currents after injury-induced gliosis in vitro. J. Neurophysiol. 79: 2222–2226, 1998. Two distinct morphological subtypes of astrocytes have been shown to express Na+ currents that differ biophysically and pharmacologically. Using an in vitro model for reactive gliosis, we recently reported marked changes in Na+ and K+ channel expression by astrocytes induced to proliferate. Using this in vitro assay in which a confluent monolayer of astrocytes is mechanically scarred to induce gliosis, we now demonstrate that sodium currents of scar-associated cells, in addition to doubling in current density, also switch from being tetrodotoxin-sensitive(TTX-S, IC50 8 nM) to being ∼40-fold more TTX-resistant (TTX-R,IC50 314 nM). These changes occurred within 6 h after injury and were not associated with any notable changes in cell morphology. Changes in biophysical properties were analyzed for the two current types. The activation curve for TTX-R currents demonstrated a significant depolarized shift versus that of TTX-S currents ( P ≤ 0.003), and TTX-R currents have more depolarized V1/2 of activation (−33 vs. −23 mV). The V1/2 of inactivation was slightly, but not significantly, more depolarized for TTX-R currents as compared to TTX-S (−63 vs. −68 mV). Most notably, TTX-R currents showed significantly slower inactivation kinetics at depolarized voltage potentials than TTX-S sodium currents (0.76 vs. 1.128 ms, at −10 mV; P < 0.0004).

Summary: Purpose: We studied Na+ channel expression and the ability to generate action potential (AP)-like responses in primary cultures of human astrocytes by whole cell patchclamp recording techniques. Methods: Tissue samples from 22 patients with various classifications of temporal lobe epilepsy (TLE) were plated to form separate astrocyte cultures from three regions; the hippocampus, parahippocampus, and anterolateral temporal neocortex. Results: The resting membrane potential of seizure focus astiocytes (MTLE, mesial TLE) was significantly depolarized (approximately -55 mV) as compared with cortical astrocytes (-80 mV). Hippocampal astrocytes from other substrates for TLE (MaTLE, mass-associated TLE; PTLE, paradoxical TLE) in which the hippocampus is not the seizure focus displayed resting membrane potentials similar to those of neocortical astrocytes (approximately -75 mV). Astrocytes from the seizure focus (MTLE) displayed much larger tetrodotoxin (TTX)- sensitive Na+ currents with -66-fold higher Na+ channel density (113.5 ± 17.41 pA/pf) than that of comparison neocortical astrocytes (1.7 ± 3.7 pA/pf) or than that of the hippocampal and parahippocampal astrocytes of the MaTLE and PTLE groups. As a consequence of this higher channel density, seizure focus astrocytes were capable of generating AP-like responses. However, at the resting potential, most Na+ channels are inactive and no spontaneous firing was observed. In contrast, astrocytes in the comparison neocortex from all groups and the hippocampus and parahippocampus from the MaTLE and PTLE groups could not fire AP-like responses even after large current injections. Conclusions: The function of Na+ channels in these astrocytes is unclear. However, the marked differences in seizure focus astrocytes as compared with cortical and nonseizure focus hippocampal astrocytes suggest a more active role for astrocytes associated with hyperexcitable neurons at a seizures focus.

Serum is used widely for culturing neurons and glial cells, and is thought to provide essential, albeit undefined, factors such as hormones, growth factors, and trace elements that promote the growth of cells in vitro. Moreover, serum can have profound effects on cell proliferation, differentiation, and cell morphology, and may even influence cell fate decisions. Despite the overall growth-promoting influence of serum on cell culture, frequent media changes have been shown to be detrimental to neuronal cultures, significantly reducing the yield of viable neurons. The reason for this loss of neurons by frequent media changes has been puzzling. We demonstrate that bovine and horse sera, the most popular serum complements for CNS cell culture, are a significant source for glutamate, supplying glutamate at concentrations sufficient to kill primary cultured hippocampal neurons. By using the bioluminescence detection method, we determined the glutamate concentration [Glu] in several batches of fetal bovine (calf) sera (FBS) to be close to 1 mM, and that of horse sera to be ∼0.3 mM. Thus 10% serum supplement to culture media results in [Glu] of 30–100 μM due to serum alone. We subsequently produced glutamate depleted media (GDM) by using primary cultures of hippocampal astrocytes to absorb glutamate from media containing 10% FBS. Within 3 h, astrocytes reduced the [Glu] in the medium from ∼90 μM to less than 1 μM. Sister cultures of hippocampal neuron that underwent frequent media changes with GDM or GDM + partial untreated media demonstrated that GDM significantly increase neuronal survival (10-fold at 21 DIV). Subsequent exposure to glutamate provided by either untreated serum or by equivalent doses of exogenous glutamate added to GDM led to dose-dependent neuronal cell death. The relative sensitivity of hippocampal neurons to glutamate increased with increasing culture age from initial ED50 values of > 100 μM (< 6 DIV) to ∼6 μM in cultures maintained for 3 weeks or longer. The relative sensitivity to exogenous glutamate was at least 2-fold higher in neurons cultured in GDM than in sister cultures maintained in media containing untreated serum. The death of neurons exposed to untreated media was blocked by the NMDA receptor antagonist MK-801. These experiments suggest that the vulnerability of neurons to media changes can be solely explained by excitotoxicity resulting from serum-borne glutamate. Moreover, we propose that use of GDM may be advantageous for culturing hippocampal neurons and may eliminate the possible selection for glutamate resistant neurons. The use of GDM could be particularly important for studies of excitotoxicity; our study predicts that the ED50 for neuronal culture with regular serum will be artificially high and may not adequately reflect the in vivo state. GLIA 22:237–248, 1998. © 1998 Wiley-Liss, Inc.

Many studies have demonstrated that astrocytes respond with fluctuations in intracellular calcium concentration ([Ca 2+ ] i ) and membrane potential following the application of a number of ligands. Moreover, calcium (Ca 2+ ) waves that spread through astrocytic syncitia have been described in numerous reports. We had the rare opportunity to study Ca 2+ responses in astrocytes obtained from a patient diagnosed with Rasmussen's encephalitis, a rare form of intractable epilepsy. Using the ratiometric fluorescent indicator fura-2, we observed large spontaneous [Ca 2+ ] i oscillations. The mean time between initial rise in [Ca 2+ ] i and the return to baseline was 5.1 ± 0.19 minutes (SEM; n = 201) and [Ca 2+ ] i increased to a mean level of 271 ± 8 nM (SEM; n = 201) from a baseline of 136 ± 6 nM (SEM; n = 201). Removal of Ca 2+ from the perfusion solution combined with the addition of the Ca 2+ chelator EGTA (2 mM) completely but reversibly eliminated all oscillations suggesting the fluctuations were dependent on Ca 2+ flux across the membrane. The percentage of cells undergoing spontaneous changes in [Ca 2+ ] i decreased over time in culture. At 10-11 days post-surgery, approximately 70% of the cells were exhibiting this behavior, and by day 23 transients were no longer observed. We did not observe comparable spontaneous [Ca 2+ ] i oscillations in rat cortical astrocytes. The potential that the spontaneous [Ca 2+ ] i oscillations observed may be a unique feature of epileptic tissues is discussed.

We report that lysophosphatidic acid (LPA) stimulates dynamic calcium (Ca2+) fluctuations and morphological rearrangements in astrocytes derived from neonatal rat spinal cord. Addition of 10 microM LPA elicited single Ca2+ transients, or biphasic oscillations and sustained increases in intracellular Ca2+ ([Ca2+]i). The biphasic Ca2+ response consisted of an initial release from intracellular stores, often followed by a sustained elevation or continued oscillations that required Ca2+ flux across the cell membrane. The type of Ca2+ response, but not the overall magnitude, was dependent on LPA concentration. Higher concentrations (> 10 microM) often elicited sustained increases in [Ca2+]i, while lower concentrations stimulated oscillations or single Ca2+ transients. It has previously been established that agents that elevate cyclic adenosine monophosphate (cAMP) induce flat astrocytes to adopt a more stellate morphology. LPA can completely reverse this morphological change at a half-maximal concentration of 215 nM. Inhibiting LPA-induced [Ca2+]i fluctuations using BAPTA-AM to buffer [Ca2+]i and EGTA in the bath to prevent transmembrane flux had little effect on the ability of LPA to reverse stellation. LPA is found bound to serum albumin, in which crude preparations have been shown to induce various physiological responses in a number of cell types. Many of the activities have been attributed to albumin-associated lipid factors including LPA. We show that lipid factors associated with BSA can mimic the effect of LPA in both Ca2+ mobilization and reversal of cAMP-induced stellation.

Since the seminal studies of Otto Warburg in the 1920s, it has been widely recognized that cancers grow glycolytically, even in the presence of oxygen. This generates an abundance of protons in a gradient across most solid tumors with an acidic core and an alkaline rim. Whether and how this proton gradient may also serve in an autocrine fashion in these tumors is unclear. We demonstrate that human glioma cells form spheroids that act as a viable three-dimensional tumor model, forming physiologically relevant extracellular pH (pHe) and cell proliferation gradients. Using fluorescent cell cycle trackers, we determined that the rate of cell proliferation is directly dependent on pHe and that cells adjust their growth rate according to their position within the pH gradient. We further show that glioma cells sense pH via H+-sensitive K+ channels, which translate changes in pH into changes in membrane voltage. These channels are tonically active and blocked by acidic pHe, quinine, and ruthenium red. Blockade of this K+ conductance by acidic pHe or drug inhibition depolarized glioma cells and tumor spheroids and prevented their passage through the hyperpolarization-dependent G1-to-S phase cell cycle checkpoint, thereby inhibiting cell division. In this way, pHe directly determines the proliferative state of glioma cells.

Gliomas, primary brain cancers, are characterized by remarkable invasiveness and fast growth. While they share many qualities with other solid tumors, gliomas have developed special mechanisms to convert the cramped brain space and other limitations afforded by the privileged central nervous system into pathophysiological advantages. In this review we discuss gliomas and other primary brain cancers in the context of acid-base regulation and interstitial acidification; namely, how the altered proton (H+) content surrounding these brain tumors influences tumor development in both autocrine and paracrine manners. As proton movement is directly coupled to movement of other ions, pH serves as both a regulator of cell activity as well as an indirect readout of other cellular functions. In the case of brain tumors, these processes result in pathophysiology unique to the central nervous system. We will highlight what is known about pH-sensitive processes in brain tumors in addition to gleaning insight from other solid tumors.

Malignant gliomas are relentless tumors that offer a dismal clinical prognosis. They develop many biological advantages that allow them to grow and survive in the unique environment of the brain. The glutamate transporters system x c (-) and excitatory amino acid transporters (EAAT) are emerging as key players in the biology and malignancy of these tumors. Gliomas manipulate glutamate transporter expression and function to alter glutamate homeostasis in the brain, which supports their own growth, invasion, and survival. As a consequence, malignant cells are able to quickly destroy and invade surrounding normal brain. Recent findings are painting a larger picture of these transporters in glioma biology, and as such are providing opportunities for clinical intervention for patients. This review will detail the current understanding of glutamate transporters in the biology of malignant gliomas and highlight some of the unique aspects of these tumors that make them so devastating and difficult to treat.

From the subventricular zone (SVZ), neuronal precursor cells (NPCs), called neuroblasts, migrate through the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). Ion channels regulate neuronal migration during development, yet their role in migration through the adult RMS is unknown. To address this question, we utilized Nestin-CreER(T2)/R26R-YFP mice to fluorescently label neuroblasts in the adult. Patch-clamp recordings from neuroblasts reveal K(+) currents that are sensitive to intracellular Ca(2+) levels and blocked by clotrimazole and TRAM-34, inhibitors of intermediate conductance Ca(2+)-activated K(+) (KCa3.1) channels. Immunolabeling and electrophysiology show KCa3.1 expression restricted to neuroblasts in the SVZ and RMS, but absent in OB neurons. Time-lapse confocal microscopy in situ showed inhibiting KCa3.1 prolonged the stationary phase of neuroblasts' saltatory migration, reducing migration speed by over 50%. Both migration and KCa3.1 currents could also be inhibited by blocking Ca(2+) influx via transient receptor potential (TRP) channels, which, together with positive immunostaining for transient receptor potential canonical 1 (TRPC1), suggest that TRP channels are an important Ca(2+) source modulating KCa3.1 activity. Finally, injecting TRAM-34 into Nestin-CreER(T2)/R26R-YFP mice significantly reduced the number of neuroblasts that reached the OB, suggesting an important role for KCa3.1 in vivo. These studies describe a previously unrecognized protein in migration of adult NPCs.

Previous reports demonstrate that cell migration in the nervous system is associated with stereotypic changes in intracellular calcium concentration ([Ca2+]i), yet the target of these changes are essentially unknown. We examined chemotactic migration/invasion of human gliomas to study how [Ca2+]iregulates cellular movement and to identify downstream targets. Gliomas are primary brain cancers that spread exclusively within the brain, frequently migrating along blood vessels to which they are chemotactically attracted by bradykinin. Using simultaneous fura-2 Ca2+imaging and amphotericin B perforated patch-clamp electrophysiology, we find that bradykinin raises [Ca2+]iand induces a biphasic voltage response. This voltage response is mediated by the coordinated activation of Ca2+-dependent, TRAM-34-sensitive KCa3.1 channels, and Ca2+-dependent, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS)-sensitive and gluconate-sensitive Cl−channels. A significant portion of these Cl−currents can be attributed to Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation of ClC-3, a voltage-gated Cl−channel/transporter, because pharmacological inhibition of CaMKII or shRNA-mediated knockdown of ClC-3 inhibited Ca2+-activated Cl−currents. Western blots show that KCa3.1 and ClC-3 are expressed in tissue samples obtained from patients diagnosed with grade IV gliomas. Both KCa3.1 and ClC-3 colocalize to the invading processes of glioma cells. Importantly, inhibition of either channel abrogates bradykinin-induced chemotaxis and reduces tumor expansion in mouse brain slicesin situ. These channels should be further explored as future targets for anti-invasive drugs. Furthermore, these data elucidate a novel mechanism placing cation and anion channels downstream of ligand-mediated [Ca2+]iincreases, which likely play similar roles in other migratory cells in the nervous system.

Glial cells have been shown to express several biophysically and pharmacology distinct potassium channel types. However, the molecular identity of most glial K+ channels is unknown. We have developed an antibody specific for the Shaker type potassium channel Kv1.5 protein, and demonstrate by immunohistochemistry the presence of this channel in glial cells of adult rat hippocampal and cerebellar slices, as well as in cultured spinal cord astrocytes. Immunoreactivity was particularly intense in the endfoot processes of astrocytes surrounding the microvasculature of the hippocampus. The specific contribution of this channel protein to the delayed rectifying K+ current of spinal cord astrocytes was determined by incubating these cells with antisense oligodeoxynucleotides complementary to the mRNA coding for Kv1.5 protein. Such treatment reduced delayed rectifier current density and shifted the potassium current steady-state inactivation, without altering current activation, cell capacitance, or cell resting potential. The tetraethylammonium acetate (TEA) sensitivity of astrocytic delayed rectifier current was enhanced following antisense oligodeoxynucleotide treatment, suggesting that Kv1.5 channel protein may provide a significant component of the TEA-insensitive current in this preparation. Our results suggest that Kv1.5 is widely expressed in glial cells of brain and spinal cord and that delayed rectifying K+ currents in astrocytes are largely mediated by Kv1.5 channel protein. © 1996 Wiley-Liss, Inc.

Glial cells are nervous-system cells that have classically been considered to be inexcitable. Despite their lack of electrical excitability, they can express voltage-activated Na + channels with properties similar to the Na + channels used by excitable cells to generate action potentials. The functional role that these voltage-activated Na + channels play in glia is unclear. Three functions have been proposed: (1) glial cells might synthesize Na + channels and donate them to adjacent neurons, thereby reducing the biosynthetic load of neurons; (2) Na + channels might endow glial cells with the ability to sense electric activity of neighboring neurons, and might thus play a role in neuro-glial communication; and (3) Na + influx through voltage-gated Na + channels could be important to fuel the glial (Na + ,K + )-ATPase, thereby facilitating and possibly modulating K + uptake from the extracellular space. Trends Neurosci. (1996) 19, 325–331

Throughout the nervous system, neurons are closely surrounded by glial cells, leaving only a 20-nm wide extracellular space filled with interstitial fluid. Ions, transmitters, hormones, nutrients, and waste products all share this narrow diffusion pathway. Because the interstitial space occupies only a small volume, neuronal activity can lead to appreciable changes in the extracellular concentration of ions, protons, and neurotrans mitters. These changes can affect neuronal activity and are believed to be influenced by glial cells. The proximity of glial processes to synapses and axons make glial cells ideal partners to sequester ions and transmitters released by neurons. The failure of glial cells to perform such essential homeostatic functions can have profound effects, and these homeostatic activities may constitute one way in which glial cells can influence neuronal signaling. In addition, glial cells, which, unlike most neurons, are coupled to each other through gap-junctions, communicate with each other and possibly also with adjacent neurons through prop agated intracellular Ca2+waves. The importance of such interglial signaling is not understood. Additionally, glial cells and neurons mutually modulate their expression of ion channels, most likely through factors re leased into the extracellular space. The range of responses observed in glial cells and their intimate anatomical relationship with neurons suggest a broader role for glia than is currently appreciated. It also emphasizes the importance of a better understanding of glial-neuronal interactions to an understanding of brain function. The Neuroscientist 1:328-337, 1995

Fluorescence Recovery After Photobleach (FRAP) was used to quantify astrocyte gap junction coupling from tissues surgically resected from medically intractable epilepsy patients. Mesial temporal lobe cases provided hippocampus, surrounding hyperexcitable parahippocampus and normal cortex for culture. Cortical tumor cases yielded astrocytoma proper, cortex margins with normal EEG activity, and hyperexcitable cortex. Cells isolated from cortex surrounding astrocytomas and the parahippocampus surrounding the hippocampus showed an increase in glutamate-induced Ca2+ oscillations and intercellular Ca2+ waves. Gap-junction coupling was more pronounced in cells isolated from hyperexcitable tissue than from normal tissues as judged by their faster and more complete fluorescence recovery from laser bleach [FRAP]. This data suggests that intractable epilepsy may be associated with alterations in glial gap junction coupling.

Since their discovery in the late nineteenth century, glial cells have taken a backstage role to neurons in our investigations of the nervous system. Yet, in the mammalian brain, glial cells outnumber neurons and comprise 50% of the brain volume. A number of proteins, including important synthetic enzymes, are located almost exclusively in glia, suggesting that glial cells may serve functions beyond providing structural support. Speculations of more active roles for glial cells in brain function began at the turn of the century, at which time it had been suggested that they serve nutritive functions (Golgi, 1885), insulate nerve fibers (Ramon y Cajal, 1909), phagocytose dying neurons (Marinesco, 1896), secrete fac tors (Achucarro, 1915), and remove neurotransmitters (the substances by which one nerve cell excites another) from the synaptic cleft (Lugaro, 1907). Many of these early hypotheses have subsequently gained experimental support; however, our understanding of glial roles in brain function is still limited. The Neuroscientist 1:123-126, 1995

Ion channels no longer belong to students of the neuron. The development of the patch- clamp technique has triggered an avalanche of ion channel studies extending far beyond the initial investigations that tended to focus on neuronal excitability. Studies of basic cell properties, even in cells other than neurons, now routinely include the evaluation of a cell's electrophysiological features and have yielded a large and growing database con cerning the electrophysiological properties of inexcitable cells. These include such cells as fibroblasts, macrophages, glial cells, bone cells, epithelial cells, and even plant cells, to name but a few, and the electrophysiological properties of these cells are as wide ranging as their cell functions and tissue origins. The Neuroscientist 1:64-67, 1995

Malignant gliomas are highly invasive brain cancers that carry a dismal prognosis. Recent studies indicate that Cl(-) channels facilitate glioma cell invasion by promoting hydrodynamic cell shape and volume changes. Here we asked how Cl(-) channels are regulated in the context of migration. Using patch-clamp recordings we show Cl(-) currents are activated by physiological increases of [Ca(2+)]i to 65 and 180nM. Cl(-) currents appear to be mediated by ClC-3, a voltage-gated, CaMKII-regulated Cl(-) channel highly expressed by glioma cells. ClC-3 channels colocalized with TRPC1 on caveolar lipid rafts on glioma cell processes. Using perforated-patch electrophysiological recordings, we demonstrate that inducible knockdown of TRPC1 expression with shRNA significantly inhibited glioma Cl(-) currents in a Ca(2+)-dependent fashion, placing Cl(-) channels under the regulation of Ca(2+) entry via TRPC1. In chemotaxis assays epidermal growth factor (EGF)-induced invasion was inhibition by TRPC1 knockdown to the same extent as pharmacological block of Cl(-) channels. Thus endogenous glioma Cl(-) channels are regulated by TRPC1. Cl(-) channels could be an important downstream target of TRPC1 in many other cells types, coupling elevations in [Ca(2+)]i to the shape and volume changes associated with migrating cells.

Astrocytes are coupled to each other via gap-junctions both in vivo and in vitro. Gap-junction coupling is essential to a number of astrocyte functions including the spatial buffering of extracellular K+ and the propagation of Ca2+ waves. Using fluorescence recovery after photo-bleach, we quantitatively assayed and compared the coupling of astrocytes cultured from six different central nervous system (CNS) regions in the rat: spinal cord, cortex, hypothalamus, hippocampus, optic nerve, and cerebellum. The degree of fluorescence recovery (% recovery) and time constant of recovery (tau) served as quantitative indicators of coupling strength. Gap-junction coupling differed markedly between CNS regions. Coupling was weakest in astrocytes derived from spinal cord (43% recovery, tau approximately 400 s) and strongest in astrocytes from optic nerve (91% recovery, tau approximately 226 s) and cerebellum (95% recovery, tau approximately 100 s). As indicated by the degree of recovery, coupling strength among CNS regions could be ranked as follows: spinal cordcortexhypothalamushippocampus = optic nerve = cerebellum. Gap-junction coupling also differed between CNS regions with respect to its sensitivity to inhibition by the uncoupling agent octanol. Kd values for 50% inhibition by octanol ranged from 188 microM in spinal cord astrocytes to 654 microM in hippocampal astrocytes. Sensitivity of gap-junctions to octanol could be ranked as follows: spinal cord = cortex = hypothalamuscerebellumoptic nervehippocampus. The observed differences in coupling indicate differences in the number of gap-junction connections in astrocytes cultured from the six CNS regions. These differences may reflect the adaptation of astrocytes to varying functional requirements in different CNS regions.