Glutamate release promotes growth of malignant gliomas.

Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative diseases. Although recent data show that cultured glioma cells secrete glutamate, the growth potential of brain tumors has not yet been linked to an excitotoxic mechanism. Using bioluminescence detection of glutamate release from freshly prepared brain slices, we show that implanted glioma cells continue to secrete glutamate. Moreover, gliomas with high glutamate release have a distinct growth advantage in host brain that is not present in vitro. Treatment with the NMDA receptor antagonists MK801 or memantine slowed the growth of glutamate-secreting tumors in situ, suggesting that activation of NMDA receptors facilitates tumor expansion. These findings support a new approach for therapy of brain tumors, based upon antagonizing glutamate secretion or its target receptors.

In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus.

Neurogenesis persists in the adult mammalian hippocampus. To identify and isolate neuronal progenitor cells of the adult human hippocampus, we transfected ventricular zone-free dissociates of surgically-excised dentate gyrus with DNA encoding humanized green fluorescent protein (hGFP), placed under the control of either the nestin enhancer (E/nestin) or the Talpha1 tubulin promoter (P/Talpha1), two regulatory regions that direct transcription in neural progenitor cells. The resultant P/Talpha1:hGFP+ and E/nestin:enhanced (E)GFP+ cells expressed betaIII-tubulin or microtubule-associated protein-2; many incorporated bromodeoxyuridine, indicating their genesis in vitro. Using fluorescence-activated cell sorting, the E/nestin:EGFP+ and P/Talpha1:hGFP+ cells were isolated to near purity, and matured antigenically and physiologically as neurons. Thus, the adult human hippocampus contains mitotically competent neuronal progenitors that can be selectively extracted. The isolation of these cells may provide a cellular substrate for re-populating the damaged or degenerated adult hippocampus.

Direct signaling from astrocytes to neurons in cultures of mammalian brain cells.

Although astrocytes have been considered to be supportive, rather than transmissive, in the adult nervous system, recent studies have challenged this assumption by demonstrating that astrocytes possess functional neurotransmitter receptors. Astrocytes are now shown to directly modulate the free cytosolic calcium, and hence transmission characteristics, of neighboring neurons. When a focal electric field potential was applied to single astrocytes in mixed cultures of rat forebrain astrocytes and neurons, a prompt elevation of calcium occurred in the target cell. This in turn triggered a wave of calcium increase, which propagated from astrocyte to astrocyte. Neurons resting on these astrocytes responded with large increases in their concentration of cytosolic calcium. The gap junction blocker octanol attenuated the neuronal response, which suggests that the astrocytic-neuronal signaling is mediated through intercellular connections rather than synaptically. This neuronal response to local astrocytic stimulation may mediate local intercellular communication within the brain.

A kainate receptor increases the efficacy of GABAergic synapses.

Brain functions are based on the dynamic interaction of excitatory and inhibitory inputs. Spillover of glutamate from excitatory synapses may diffuse to and modulate nearby inhibitory synapses. By recording unitary inhibitory postsynaptic currents (uIPSCs) from cell pairs in CA1 of the hippocampus, we demonstrated that low concentrations of Kainate receptor (KAR) agonists increased the success rate (P(s)) of uIPSCs, whereas high concentrations of KAR agonists depressed GABAergic synapses. Ambient glutamate released by basal activities or stimulation of the stratum radiatum increases the efficacy of GABAergic synapses by activating presynaptic KARs, which facilitate Ca(2+)-dependent GABA release. The results suggest that glutamate released from excitatory synapses may also function as an intermediary between excitatory and inhibitory synapses to protect overexcitation of local circuits.

Adhesive properties of connexin hemichannels.

Gap junctions are intercellular channels formed by hemichannels (or connexons) from two neighboring cells. Hemichannels, which are composed of proteins called connexins, can function as conduits of ATP and glutamate, and interact with adhesion molecules and other signaling elements. As a result, their functional repertoire is expanding into other roles, such as control of cell growth or cell migration. Here we further elucidate the involvement of hemichannels in cell-cell adhesion by analyzing how connexins regulate cell adhesion without the need of gap junction formation. Using a short-term aggregation assay with C6-glioma and HeLa cells stably transfected with connexin (Cx) 43 or Cx32, we found that the connexin type dictates the ability of these cells to aggregate, even though these two cell types do not usually adhere to each other. We have also found that high expression of Cx43, but not Cx32 hemichannels, can drive adhesion of cells expressing low levels of Cx43. Aggregation was not dependent on high levels of extracellular Ca(2+), as Ca(2+) removal did not change the aggregation of Cx43-expressing cells. Our data confirm that connexin hemichannels can establish adhesive interactions without the need for functional gap junctions, and support the concept that connexins act as adhesion molecules independently of channel formation.

Astrocyte-mediated potentiation of inhibitory synaptic transmission.

We investigated the role of astrocytes in activity-dependent modulation of inhibitory synaptic transmission in hippocampal slices. Repetitive firing of an interneuron decreased the probability of synaptic failures in spike-evoked inhibitory postsynaptic currents (unitary IPSCs) in CA1 pyramidal neurons. The GABAB-receptor antagonist CGP55845A abolished this effect. Direct stimulation of astrocytes, or application of the GABAB-receptor agonist baclofen, potentiated miniature inhibitory postsynaptic currents (mIPSCs) in pyramidal neurons. These effects were blocked by inhibition of astrocytic calcium signaling with the calcium chelator BAPTA or by antagonists of the ionotropic glutamate receptors. These observations suggest that interneuronal firing elicits a GABAB-receptor-mediated elevation of calcium in surrounding astrocytes, which in turn potentiates inhibitory transmission. Astrocytes may therefore be a necessary intermediary in activity-dependent modulation of inhibitory synapses in the hippocampus.

Gap-junction-mediated propagation and amplification of cell injury.

Gap junctions are conductive channels that connect the interiors of coupled cells. We determined whether gap junctions propagate transcellular signals during metabolic stress and whether such signaling exacerbates cell injury. Although overexpression of the human proto-oncogene bcl2 in C6 glioma cells normally increased their resistance to injury, the relative resistance of bcl2+ cells to calcium overload, oxidative stress and metabolic inhibition was compromised when they formed gap junctions with more vulnerable cells. The likelihood of death was in direct proportion to the number and density of gap junctions with their less resistant neighbors. Thus, dying glia killed neighboring cells that would otherwise have escaped injury. This process of glial 'fratricide' may provide a basis for the secondary propagation of brain injury in cerebral ischemia.

Tamoxifen-induced enhancement of calcium signaling in glioma and MCF-7 breast cancer cells.

The antiestrogen tamoxifen is commonly used to treat breast cancer, but it also has therapeutic activity in several other types of cancer. Many of these tumors, including malignant gliomas, are estrogen receptor negative. Nonetheless, high concentrations of tamoxifen can directly reduce cell proliferation in some of these tumors and induce apoptosis. In this study, the role of tamoxifen in calcium signaling and calcium-induced cell death was studied in both malignant glioma cell lines and MCF-7 breast cancer cells. Tamoxifen potently increased the spatial expansion of calcium waves by 30-150% while significantly enhancing and prolonging agonist-induced calcium elevations. Furthermore, tamoxifen pretreatment accelerated calcium ionophore-induced death by more than 20 min, suggesting that tamoxifen lowered cellular resistance to calcium loads. In contrast to its potentiating of calcium signaling in tumors, tamoxifen had no significant effect on calcium signaling in cultures of primary astrocytes from either human or rat brain. This study demonstrates the existence of calcium signaling in breast cancer and glioma cells and identifies tamoxifen as a potential modulator of tumor-associated calcium signaling.

Direct gap junction communication between malignant glioma cells and astrocytes.

Gap junctions are intercellular channels that connect the interiors of coupled cells. We sought to determine the extent to which malignant glioma cells form gap junction channels with astrocytes from either adult human brain or rat forebrain. The astrocytic gap junction protein, connexin 43 (Cx43), was identified in immunoreactive plaques at areas of cell-to-cell contact between cocultured glioma cells and astrocytes. These gap junction plaques were composed of functional channels, because extensive dye coupling was evident between the glioma cells and astrocytes from both human and rat brain. Calcium signaling was also readily transmitted from glioma cells to astrocytes and vice versa. In live rat brain, injection of glioma cells prelabeled with the gap junction tracer, dicarboxy-dichlorofluorescein, revealed extensive dye transfer to host cells, demonstrating that malignant glioma cells directly couple with normal brain cells. These observations suggest that intercellular communication via gap junctions may play a role in regulating cellular interactions during tumor invasion. In fact, the presence of gap junctions between astrocytes and glioma cells was sufficient to induce a transformation of astrocytic phenotype. Astrocytes cocultured with C6 glioma cells overexpressing Cx43 were significantly smaller and expressed a lower level of glial fibrillary acidic protein than astrocytes cocultured with otherwise identical mock-transfected, gap junction-deficient C6 cells. Thus, direct cellular coupling with glioma cells result in a phenotypic transformation of astrocytes that may contribute to the susceptibility of surrounding tissue to glioma invasion.

Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis.

Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca(2+)-triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca(2+)-regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte--Syt XI--suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair.

ATP-mediated glia signaling.

Glia calcium signaling has recently been identified as a potent modulator of synaptic transmission. We show here that the spatial expansion of calcium waves is mediated by ATP and subsequent activation of purinergic receptors. Ectopic expression of gap junction proteins, connexins (Cxs), leads to an increase in both ATP release and the radius of calcium wave propagation. Cx expression was also associated with a phenotypic transformation, and cortical neurons extended longer neurites when co-cultured with Cx-expressing than with Cx-deficient cells. Purinergic receptor activation mediated both these effects, because treatment with receptor antagonists restored the glia phenotype and slowed neurite outgrowth. These results identify a key role of ATP in both short-term calcium signaling events and in long-term differentiation regulated by glia.

Regional brain glucose metabolism and blood flow in streptozocin-induced diabetic rats.

Brain regional glucose metabolism and regional blood flow were measured from autoradiographs by the uptake of [3H]-2-deoxy-D-glucose and [14C]iodoantipyrine in streptozocin-induced diabetic (STZ-D) rats. After 2 days of diabetes, glucose metabolism in the neocortex, basal ganglia, and white matter increased by 34, 37, and 8%, respectively, whereas blood flow was unchanged. After 4 mo, glucose metabolism in the same three regions was decreased by 32, 43, and 60%. This reduction was paralleled by a statistically nonsignificant reduction in blood flow in neocortex and basal ganglia. It is suggested that the decrease of brain glucose metabolism in STZ-D reflects increased ketone body oxidation and reduction of electrochemical work.

External Dentin Stimulation Induces ATP Release in Human Teeth.

ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain.

Infarct rim: effect of hyperglycemia on direct current potential and [14C]2-deoxyglucose phosphorylation.

Focal ischemia was produced by occlusion of the right middle cerebral artery (MCA) in normo- and hyperglycemic rats. In the cortical infarct rim, regional [14C]2-deoxyglucose [( 14C]2-DG) phosphorylation was correlated to spontaneous transient changes in extracellular potassium recorded as direct current (DC) potential deflections. In normoglycemic rats the DC potential showed transient but recurrent deflections in the first hours following MCA occlusion. The 2-DG phosphorylation was elevated by 200% in the same area. In contrast, hyperglycemic rats had no, or a single, deflection of the DC potential in the rim, and the 2-DG phosphorylation remained normal. The same pattern was obtained by application of 3 M KCl to the exposed cortex. In normoglycemia potassium application resulted in recurrent deflections of the DC potential, and 2-DG phosphorylation increased in most parts of the hemisphere. Hyperglycemic animals had a nearly stable DC potential, and 2-DG phosphorylation increased only in the tissue area situated directly below the site of potassium application. The results indicate that metabolism in the cortical infarct rim is stimulated by spontaneous and recurrent changes in extracellular potassium--a phenomenon that may be related to spreading depression--and that the metabolism remained normal in the same area in hyperglycemic animals owing to an inhibition of transient increases of extracellular potassium.

Experimental cerebral ischemia: barbiturate resistant increase in regional glucose utilization.

During the first hours after experimental occlusion of the middle cerebral artery (MCA) cerebral glucose utilization increases in the tissue adjacent to ischemic focus. To test whether the increased glucose utilization was a consequence of increased neuronal activity, the effect of preocclusion pentobarbital administration was investigated. Rats in barbiturate-induced coma showed a metabolic response to MCA occlusion similar to those seen with light halothane anesthesia. This indicates that the enhanced glucose utilization adjacent to the ischemic core is not a result of increased neuronal activity.

Characterization of cortical depolarizations evoked in focal cerebral ischemia.

Cortical tissue surrounding acute ischemic infarcts undergoes repetitive spontaneous depolarizations. It is unknown whether these events are episodes of spreading depression (SD) elicited by the elevated interstitial K+ ([K+]e) in the ischemic core or whether they are evoked by transient decreases of the local blood flow. Electrophysiologically, depolarization caused by SD or by ischemia (ID) can be distinguished by their characteristic patterns of [K+]e rise: During SD, [K+]e rises abruptly, while in ID, this fast rate of increase is preceded by a slow rate lasting minutes. To characterize the depolarizations, we occluded the right middle cerebral artery (MCA) in rats and inserted two K(+)-sensitive microelectrodes into the cortex surrounding the evolving infarct. Repeated increases in [K+]e arose spontaneously following MCA occlusion. [K+]e increased during these transients from a resting level of 3-6 to 60 mM. One-third of these transient increases in [K+]e were biphasic, consisting of a slow initial increase to 10-12 mM, which lasted for minutes, followed by an abrupt increase, a pattern characteristic of ID. The remaining two-thirds exhibited a steep monotonic increase in [K+]e (< 10 s), characteristic of SD. The duration of the transients was a function of the pattern of [K+]e increase: ID-like transients lasted an average 10.7 +/- 5.1 min, whereas the duration of SD-like transients was 5.7 +/- 3.4 min. Both types of K+ transients occurred in an apparently random fashion in individual animals. A K+ transient was never observed solely at one electrode.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic determination of cerebral glucose content, blood flow, and glucose utilization in focal ischemia of the rat brain: influence of the plasma glucose concentration.

Focal cerebral ischemia was produced by occlusion of the middle cerebral artery in rats. Cerebral blood flow measured with [14C]iodoantipyrine was severely reduced in the lateral portion of neostriatum. This area of dense ischemia was sharply demarcated against the surroundings. The adjacent cortex was perfused at one-third of normal, whereas blood flow in the medial neostriatum was only slightly reduced. This pattern of perfusion was independent of the plasma glucose concentration of the animal. In contrast, the glucose utilization calculated from the 2-[3H]deoxyglucose accumulation depended on the plasma glucose concentration. Enhanced glucose utilization was evident in the border areas surrounding the ischemic focus in normoglycemic animals. Neither acutely nor chronically diabetic animals had such an increase of metabolism in the borderzone. Moderately hyperglycemic rats had a narrow rim of enhanced glucose utilization immediately surrounding the ischemic core, whereas animals with plasma glucose values above 22 mmol/L had no such rim. In mild hypoglycemia (2-4 mmol/L), the glucose utilization was slightly enhanced in the border areas, but during severe hypoglycemia (less than 2.5 mmol/L), the glucose utilization declined gradually toward the ischemic core. Glucose content, and thereby the lumped constant (measured by 3-0-[14C]methylglucose) showed little regional variation, except in the ischemic core. These findings indicate that blood flow alterations after occlusion of the middle cerebral artery in rats are not influenced by the plasma glucose utilizations. In contrast, glucose utilization depends on a combination of plasma glucose concentration and blood flow instead of blood flow per se.

Focal ischemia of the rat brain: autoradiographic determination of cerebral glucose utilization, glucose content, and blood flow.

Focal cerebral ischemia was induced in rats by occlusion of the middle cerebral artery. By a triple-tracer technique, cerebral glucose utilization, glucose content, and blood flow were simultaneously determined. Computer-assisted autoradiography revealed a core of dense ischemia in the lateral two-thirds of the striatum. A border zone of increased 2-deoxy-D-glucose (DG) uptake surrounded the ischemic insult in the acute stage. The lumped constant was increased only moderately in the border zone. Therefore, the enhanced DG uptake reflected increased glucose consumption. CBF was reduced to 20-30% in the cortical border, while minor depression and in some animals hyperemia were evident in the striate border. Six hours after the insult, the border zones of increased glucose consumption had disappeared in half the animals. In no animals examined after 20 h was glucose consumption enhanced. The study indicated a stable metabolic response to a reproducible focal insult. We conclude that continued enhancement of glucose consumption in marginally perfused areas indicates neuronal damage.

The neurobiology of glia in the context of water and ion homeostasis.

Astrocytes are highly complex cells that respond to a variety of external stimulations. One of the chief functions of astrocytes is to optimize the interstitial space for synaptic transmission by tight control of water and ionic homeostasis. Several lines of work have, over the past decade, expanded the role of astrocytes and it is now clear that astrocytes are active participants in the tri-partite synapse and modulate synaptic activity in hippocampus, cortex, and hypothalamus. Thus, the emerging concept of astrocytes includes both supportive functions as well as active modulation of neuronal output. Glutamate plays a central role in astrocytic-neuronal interactions. This excitatory amino acid is cleared from the neuronal synapses by astrocytes via glutamate transporters, and is converted into glutamine, which is released and in turn taken up by neurons. Furthermore, metabotropic glutamate receptor activation on astrocytes triggers via increases in cytosolic Ca(2+) a variety of responses. For example, calcium-dependent glutamate release from the astrocytes modulates the activity of both excitatory and inhibitory synapses. In vivo studies have identified the astrocytic end-foot processes enveloping the vessel walls as the center for astrocytic Ca(2+) signaling and it is possible that Ca(2+) signaling events in the cellular component of the blood-brain barrier are instrumental in modulation of local blood flow as well as substrate transport. The hormonal regulation of water and ionic homeostasis is achieved by the opposing effects of vasopressin and atrial natriuretic peptide on astroglial water and chloride uptake. In conjuncture, the brain appears to have a distinct astrocytic perivascular system, involving several potassium channels as well as aquaporin 4, a membrane water channel, which has been localized to astrocytic endfeet and mediate water fluxes within the brain. The multitask functions of astrocytes are essential for higher brain function. One of the major challenges for future studies is to link receptor-mediated signaling events in astrocytes to their roles in metabolism, ion, and water homeostasis.

Transient focal ischemia in hyperglycemic rats is associated with increased cerebral infarction.

To study whether transient ischemia is influenced by hyperglycemia, the middle cerebral artery was occluded for 5, 10 and 15 min in normo- and hyperglycemic rats. Five-minute ischemia induced minor lesions in both groups. After 10-min ischemia a significant greater infarct volume was found in hyperglycemia compared with normoglycemia (29 +/- 9 mm3 vs 4 +/- 4 mm3, P less than 0.001). Fifteen-minute artery occlusion induced even more damage in both hyper- and normoglycemia (63 +/- 20 mm3 vs 13 +/- 12 mm3, P less than 0.006). The lateral part of striatum was infarcted in all hyperglycemic animals exposed to 10 or 15 min of ischemia. In the same area selective neuronal injury occurred in 6 out of 9 normoglycemic animals. The findings show that hyperglycemia increases brain damage during transient ischemia by conversion of selective neuronal injury into cerebral infarction.