Role of astrocytes and altered regulation of spinal glutamatergic neurotransmission in stress-induced visceral hyperalgesia in rats

Glutamate (Glu) is the primary excitatory neurotransmitter in the central nervous system and plays a critical role in the neuroplasticity of nociceptive networks. We aimed to examine the role of spinal astroglia in the modulation of glutamatergic neurotransmission in a model of chronic psychological stress-induced visceral hyperalgesia in male Wistar rats. We assessed the effect of chronic stress on different glial Glu control mechanisms in the spinal cord including N-methyl-d-aspartate receptors (NMDARs), glial Glu transporters (GLT1 and GLAST), the Glu conversion enzyme glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP). We also tested the effect of pharmacological inhibition of NMDAR activation, of extracellular Glu reuptake, and of astrocyte function on visceral nociceptive response in naive and stressed rats. We observed stress-induced decreased expression of spinal GLT1, GFAP, and GS, whereas GLAST expression was upregulated. Although visceral hyperalgesia was blocked by pharmacological inhibition of spinal NMDARs, we observed no stress effects on NMDAR subunit expression or phosphorylation. The glial modulating agent propentofylline blocked stress-induced visceral hyperalgesia, and blockade of GLT1 function in control rats resulted in enhanced visceral nociceptive response. These findings provide evidence for stress-induced modulation of glia-controlled spinal Glu-ergic neurotransmission and its involvement in chronic stress-induced visceral hyperalgesia. The findings reported in this study demonstrate a unique pattern of stress-induced changes in spinal Glu signaling and metabolism associated with enhanced responses to visceral distension.     

Role of astrocytes in trimethyltin neurotoxicity

Although the neurotoxicity of trimethyltin (TMT) is well known, mechanisms are still not clear. Glia have been proposed to mediate the toxic action of TMT on nerve cells. Accordingly, the effects of TMT were tested in primary neuronal cultures from rat cerebellum and compared to effects in astrocytes and mixed cultures. Neuronal damage observed following TMT exposure was less in the presence of astrocytes and astrocytes alone were resistant to TMT. Thus, astrocytes have a protective effect against TMT-induced neurotoxicity. TMT caused an oxidative stress in granule cell cultures involving a variety of oxidative species (O2)*-, H2O2, NO), but astrocytes were less sensitive to TMT-induced oxidative species generation. Antioxidants, glutathione and 7-nitroindazole attenuated neuronal cell death induced by TMT. It appears that oxidative stress mediates a large part of the destructive action of TMT in neuronal cultures. The presence of astrocytes appears to modulate TMT-induced oxidative stress so that TMT causes only a small increase in lipid peroxidation in mouse brain after systemic administration. Thus, TMT induces a pronounced oxidative stress in cultured neurons, but when astrocytes are present, oxidative species play a lesser role in the neurotoxic action of TMT.     

Role of astrocytes in neurovascular coupling

Neural activity is intimately tied to blood flow in the brain. This coupling is specific enough in space and time that modern imaging methods use local hemodynamics as a measure of brain activity. In this review, we discuss recent evidence indicating that neuronal activity is coupled to local blood flow changes through an intermediary, the astrocyte. We highlight unresolved issues regarding the role of astrocytes and propose ways to address them using novel techniques. Our focus is on cellular level analysis in?vivo, but we also relate mechanistic insights gained from ex?vivo experiments to native tissue. We also review some strategies to harness advances in optical and genetic methods to study neurovascular coupling in the intact brain.     

Perivascular nerves and the regulation of cerebrovascular tone.

Brain perfusion is tightly coupled to neuronal activity, is commonly used to monitor normal or pathological brain function, and is a direct reflection of the interactions that occur between neuronal signals and blood vessels. Cerebral blood vessels at the surface and within the brain are surrounded by nerve fibers that originate, respectively, from peripheral nerve ganglia and intrinsic brain neurons. Although of different origin and targeting distinct vascular beds, these "perivascular nerves" fulfill similar roles related to cerebrovascular functions, a major one being to regulate their tone and, therein, brain perfusion. This utmost function, which underlies the signals used in functional neuroimaging techniques and which can be jeopardized in pathologies such as Alzheimer's disease, stroke, and migraine headache, is thus regulated at several levels. Recently, new insights into our understanding of how neural input regulate cerebrovascular tone resulted in the rediscovery of the functional "neurovascular unit." These remarkable advances suggest that neuron-driven changes in vascular tone result from interactions that involve all components of the neurovascular unit, transducing neuronal signals into vasomotor responses not only through direct interaction between neurons and vessels but also indirectly via the perivascular astrocytes. Neurovascular coupling is thus determined by chemical signals released from activated perivascular nerves and astrocytes that alter vascular tone to locally adjust perfusion to the spatial and temporal changes in brain activity.     

Differential effects of cAMP in neurons and astrocytes. Role of B-raf.

Mitogen-activated protein kinase (MAPK) activation provides cell type-specific signals important for cellular differentiation, proliferation, and survival. Cyclic AMP (cAMP) has divergent effects on MAPK activity depending on whether signaling is through Ras/Raf-1 or Rap1/B-raf. We found that central nervous system-derived neurons, but not astrocytes, express B-raf. In neurons, cAMP activated MAPK in a Rap1/B-raf-dependent manner, while in astrocytes, cAMP decreased MAPK activity. Inhibition of MAPK in neurons decreased neuronal growth factor-mediated survival, and activation of MAPK by cAMP analogues rescued neurons from death. Furthermore, constitutive expression of B-raf in astrocytoma cells increased MAPK activation, as seen in neurons, and enhanced proliferation. These data provide the first experimental evidence that B-raf is the molecular switch which dominantly permits differential cAMP-dependent regulation of MAPK in neurons versus astrocytes, with important implications for both survival and proliferation.     

Substrate regulation of ascorbate transport activity in astrocytes

Astrocytes possess a concentrativel-ascorbate (vitamin C) uptake mechanism involving a Na⁺-dependentl-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellularl-ascorbate on the activity of this transport system. Initial rates ofl-ascorbate uptake were measured by incubating primary cultures of rat astrocytes withl-[¹⁴C]ascorbate for 1 min at 37°C. We observed that the apparent maximal rate of uptake (V _max) increased rapidly (<1 h) when cultured cells were deprived ofl-ascorbate. In contrast, there was no change in the apparent affinity of the transport system forl-[¹⁴C]ascorbate. The increase inV _max was reversed by addition ofl-ascorbate, but notD-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures withl-ascorbate did not affect uptake of 2-deoxy-D-[³H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.     

Cell volume regulation of cerebrovascular endothelium in vitro

Regulation of cell volume as a fundamental cellular function of high biological priority was studied in cultured cerebrovascular endothelium. The use of a multiparameter flow cytometric system allowed simultaneous measurements of cell volume, viability, and membrane potential or intracellular pH. Endothelium, the cellular constituent of the blood-brain barrier (BBB), swells immediately on exposure to low osmolality. This is associated with membrane depolarization and a fall of intracellular pH. Within 30-60 min, cell volume and membrane potential recover completely, although the extracellular osmolality is kept low. Intracellular pH does not normalize fully. Measurements of intracellular K+ and Na+ concentrations reveal their involvement in the regulatory process. The findings strongly suggest that the cerebrovascular endothelium has a highly effective built-in capacity for homeostatic control essential for normal BBB function.     

Energy-dependent volume regulation in primary cultured cerebral astrocytes

Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40-50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na,K)-ATPase-coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na,K)-ATPase ion pump. This process may be important in the energy-dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo.     

Impaired cell volume regulation in taurine deficient cultured astrocytes

Taurine concentration was reduced by 40 and 65%, respectively in rat cerebellar astrocytes grown in a chemically defined medium or in culture medium containing a blocker of taurine transport (GES). Cell volume in these taurine deficient cells was 10%–16% higher than in controls. When challenged by hyposmotic conditions, astrocytes release taurine and this efflux contributes to the volume regulatory decrease observed in these cells. Taurine deficient astrocytes showed a less efficient volume recovery as compared to controls with normal taurine levels. Exposed to 50% hyposmotic medium, astrocytes with normal taurine concentration recovered 60% of their original volume whereas taurine deficient cells recovered only 30–35%. Similarly, in 30% hyposmotic medium, taurine deficient astrocytes recovered only 40% as compared to 75% in controls. No compensatory increases in the efflux of other osmolytes (free amino acids or potassium) were observed during regulatory volume decrease in taurine deficient astrocytes.     

Differential regulation of estrogen in iron metabolism in astrocytes and neurons

Previous studies have demonstrated an effect of estrogen on iron metabolism in peripheral tissues. The role of estrogen on brain iron metabolism is currently unknown. In this study, we investigated the effect and mechanism of estrogen on iron transport proteins. We demonstrated that the iron exporter ferroportin 1 (FPN1) and iron importer divalent metal transporter 1 (DMT1) were upregulated and iron content was decreased after estrogen treatment for 12 hr in primary cultured astrocytes. Hypoxia-inducible factor-1 alpha (HIF-1α) was upregulated, but HIF-2α remained unchanged after estrogen treatment for 12 hr in primary cultured astrocytes. In primary cultured neurons, DMT1 was downregulated, FPN1 was upregulated, iron content decreased, iron regulatory protein (IRP1) was downregulated, but HIF-1α and HIF-2α remained unchanged after estrogen treatment for 12 hr. These results suggest that the regulation of iron metabolism by estrogen in astrocytes and neurons is different. Estrogen increases FPN1 and DMT1 expression by inducing HIF-1α in astrocytes, whereas decreased expression of IRP1 may account for the decreased DMT1 and increased FPN1 expression in neurons.