Regulation of glial inflammatory mediators synthesis: possible role of endothelins

Endothelins are well known as modulators of inflammation in the periphery, but little is known about their possible role in brain inflammation. Stimulation of astrocyte prostaglandin, an inflammatory mediator, synthesis was shown so far only by endothelin 3 (ET-3). By contrast, several studies showed no change or slight decrease of basal nitric oxide synthesis after treatment of astrocytes with endothelin 1 (ET-1) and ET-3. However, a significant increase in astrocytic and microglial nitric oxide synthase (NOS) was observed after exposure to ET-1 and ET-3 in a model of forebrain ischaemia. Here we demonstrate that all three endothelins (ET-1, ET-2, ET-3) significantly enhanced the synthesis of prostaglandin E(2) and nitric oxide in glial cells. Each of the selective antagonists for ETA and ETB receptors (BQ123 and BQ788 respectively), significantly inhibited endothelins-induced production of both nitric oxide and prostaglandin E(2). These results suggest a regulatory mechanism of endothelins, interacting with both endothelin receptors, on glial inflammation. Therefore, inhibition of endothelin receptors may have a therapeutic potential in pathological conditions of the brain, when an uncontrolled inflammatory response is involved.     

Nitric oxide and peroxynitrite cause irreversible increases in the Km for oxygen of mitochondrial cytochrome oxidase: in vitro and in vivo studies

Mitochondrial cytochrome oxidase is competitively and reversibly inhibited by inhibitors that bind to ferrous heme, such as carbon monoxide and nitric oxide. In the case of nitric oxide, nanomolar levels inhibit cytochrome oxidase by competing with oxygen at the enzyme's heme-copper active site. This raises the K_m for cellular respiration into the physiological range. This effect is readily reversible and may be a physiological control mechanism. Here we show that a number of in vitro and in vivo conditions result in an irreversible increase in the oxygen K_m. These include: treatment of the purified enzyme with peroxynitrite or high (μM) levels of nitric oxide; treatment of the endothelial-derived cell line, b.End5, with NO; activation of astrocytes by cytokines; reperfusion injury in the gerbil brain. Studies of cell respiration that fail to vary the oxygen concentration systematically are therefore likely to significantly underestimate the degree of irreversible damage to cytochrome oxidase.     

Increased β<Subscript>2</Subscript>-Adrenergic Receptor Activity by Thyroid Hormone Possibly Leads to Differentiation and Maturation of Astrocytes in Culture

Summary (1) Our earlier studies indicate a downsteam regulatory role of the β-adrenergic receptor (β-AR) system in thyroid hormone induced differentiation and maturation of astrocytes. In the present study we have investigated the contributions of the subtypes of β-AR in the above phenomenon. (2) Primary astrocyte cultures were grown under thyroid hormone deficient as well as under euthyroid conditions. [¹²⁵I]Pindolol ([¹²⁵I]PIN) binding studies showed a gradual increase in the specific binding to β₂-AR when observed at 5, 10, 15, and 20 days under both cultural conditions. Thyroid hormone caused an increase in binding of [¹²⁵I]PIN to β₂-AR compared to thyroid hormone deficient controls at all ages of astrocyte culture. (3) Saturation studies using [¹²⁵I]PIN in astrocyte membranes prepared from 20-day-old cultures showed a significant increase in the affinity of the receptors (K _D) in the thyroid hormone treated cells without any change in receptor number (B _max). (4) β₂-AR mRNA levels were measured by real-time PCR during ontogenic development as well as during exposure of 10-day-old hypothyroid cultures to normal levels of thyroid hormone for 2, 6, 12, and 24 h. None of the conditions caused any significant change in the β₂-adrenergic receptor mRNA levels when compared with corresponding hypothyroid controls. (5) Over expression of β₂-AR cDNA in hypothyroid astrocytes caused morphological transformation in spite of the absence of thyroid hormone in the medium. (6) Taken together, results suggest thyroid hormone causes a selective increase in [¹²⁵I]PIN binding to β₂-AR due to increase in receptor affinity, which may lead to maturation of astrocytes.     

Activation of cultured astrocytes by amphotericin B: Stimulation of NO and cytokines production and changes in neurotrophic factors production

Amphotericin B (AmB) is a polyene antibiotic and reported to be one of a few reagents having therapeutic effects on prion diseases, such as the delay in the appearing of the clinical signs and the prolongation of the survival time. In prion diseases, glial cells have been suggested to play important roles by proliferating and producing various factors such as nitric oxide, proinflammatory cytokines, and neurotrophic factors. However, the therapeutic mechanism of AmB on prion diseases remains elusive. We have previously reported that AmB changed the expression of neurotoxic and neurotrophic factors in microglia (Motoyoshi et al., 2008, Neurochem. Int. 52, 1290–1296). In the present study, we examined the effects of AmB on cellular functions of rat cultured astrocytes. We found that AmB could activate astrocytes to produce nitric oxide via inducible nitric oxide synthase induction. AmB also induced mRNA expression of interleukin-1β and tumor necrosis factor-α, and productions of their proteins in astrocytes. Moreover, AmB changed levels of neurotrophic factor mRNAs and proteins. Among three neurotrophic factors examined here, neurotrophin-3 mRNA expression and its protein production in the cells were down-regulated by AmB stimulation. On the other hand, AmB significantly enhanced the amounts of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor proteins in the cells and the medium. These results suggest that AmB might show therapeutic effects on prion diseases by controlling the expression and production of such mediators in astrocytes.     

Nitric oxide-enhanced caspase-3 and acidic sphingomyelinase interaction: a novel mechanism by which airway epithelial cells escape ceramide-induced apoptosis

Reactive nitrogen species (RNS) are implicated in the pathophysiology of inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease. The molecular mechanisms and signaling events involved in lung cell injury by RNS are still poorly understood. In the current study, we observe a novel anti-apoptotic response to nitric oxide (NO) exposure (via the NO donors 3-morpholine-syndnonimine (SIN1) or papa-NONOate) of human airway epithelial (HAE) cells. NO exposure via the NO donors increased cellular ceramide levels via ceramide synthase but did not trigger an apoptotic response. Rather, exposure to the NO donors promoted an increase in the protein-protein interaction between acidic sphingomyelinase (aSMase) and caspase-3, with aSMase sequestering caspase-3 and preventing its cleavage. In contrast, when aSMase was silenced in HAE cells or was knocked out in mice, an increase in cleaved caspase-3 was observed. This elevated caspase-3 cleavage was further augmented upon NO exposure (via SIN1 or papa-NONOate) of HAE cells and could be prevented by an inhibitor to ceramide synthase. These results demonstrate a novel mechanism of NO modulation of apoptosis, in which HAE cells exposed to NO via an NO donor induces ceramide generation via ceramide synthase. However, this ceramide induction does not lead to apoptosis unless aSMase is knocked down, allowing the release of caspase-3, its activation and execution of apoptosis.     

Tobacco carcinogen induces microglial activation and subsequent neuronal damage

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific procarcinogen. We have investigated whether NNK causes inflammatory upheaval in the brain by activation of resident microglia and astrocyte and result in bystander neuronal damage. We have carried out the work in both in vitro and in vivo models. We have found that treatment with NNK causes significant activation of mouse microglial (BV2) cell line as evident by increase in reactive oxygen species and nitric oxide level. Western blot analysis has showed increase in proinflammatory signaling proteins, proinflammatory effector proteins, and other stress-related proteins. Interestingly, increased levels of proinflammatory cytokines like interleukin (IL)-6, tumor necrosis factor-α, monocyte chemoattractant protein 1 (MCP1), and IL-12p70 are also detected. Work from our in vivo studies has demonstrated similar increase in proinflammatory signaling and effector molecules along with the proinflammatory cytokine levels, following NNK treatment. Immunohistochemical staining of the brain sections of NNK-treated mice reveals massive microglial and astrocyte activation along with distinct foci of neuronal damage. Both in vitro and in vivo results provide strong indication that NNK causes significant upheaval of the inflammatory condition of brain and inflicts subsequent neuronal damage.     

15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone modulate Staphylococcus aureus-dependent astrocyte activation primarily through a PPAR-gamma-independent pathway

Brain abscesses arise from a focal parenchymal infection by various pathogens, particularly Staphylococcus aureus. We have shown that astrocytes are activated upon exposure to S. aureus and may contribute to the excessive tissue damage characteristic of brain abscess. Therefore, modulating astrocyte activation may facilitate a reduction in brain abscess severity. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists are potent inhibitors of microglial activation; however, the effects of these compounds on S. aureus-dependent astrocyte activation have not yet been examined. Here, we demonstrate that two chemically distinct PPAR-gamma agonists, 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, suppress the production of several pro-inflammatory molecules in S. aureus-stimulated astrocytes including interleukin-1beta and nitric oxide (NO). Interestingly, 15d-PGJ2 attenuated Toll-like receptor 2 (TLR2) and inducible nitric oxide synthase expression, but failed to modulate macrophage inflammatory protein-2 (MIP-2/CXCL2) production, suggesting that 15d-PGJ2 is not a global inhibitor of astrocyte activation. Another novel finding of this study was the fact that both 15d-PGJ2 and ciglitazone were capable of attenuating pre-existing astrocyte activation, indicating their potential benefit in a therapeutic setting. Importantly, 15d-PGJ2 and ciglitazone were still capable of inhibiting S. aureus-induced pro-inflammatory mediator release in PPAR-gamma-deficient astrocytes, supporting PPAR-gamma-independent effects of these compounds. Collectively, these results suggest that 15d-PGJ2 and ciglitazone exert their anti-inflammatory actions on astrocytes primarily independent of the PPAR-gamma pathway.     

Effects of IL-1β on Receptor-Mediated Poly-Phosphoinositide Signaling Pathway in Immortalized Astrocytes (DITNC)

Astrocytes are known to play multi-functional roles in support of many homeostatic mechanisms in the central nervous system including host defense mechanisms. Despite the ability of cytokines to alter gene expression and cellular activity, their effect on receptor-mediated poly-phosphoinositide (poly-PI) signaling pathway has not been examined in detail. In this study, an immortalized astrocyte cell line (DITNC) was used to test the effect of IL-1 exposure on the poly-PI signaling pathway. Similar to primary astrocytes, DITNC cells exhibit P₂-purinergic receptor response to ATP and UTP leading to transient increases in inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and intracellular calcium concentration, [Ca²⁺]i. Upon exposure of DITNC cells to IL-1 (100U/ml) for 24 hrs, an increased response to the poly-PI agonists was observed. The increase in ATP-mediated Ins(1,4,5)P₃ release could not be attributed to a shift in the ATP dose or an alteration of the time profile for the release of Ins(1,4,5)P₃. Since the increase in response required a lag time of 4 hr after IL-1 exposure, it is unlikely that this effect was due to a direct interaction of IL-1 with the purinergic receptor. On the other hand, an increase in ATP response could be observed in DITNC cells exposed to conditioned medium obtained after IL-1 treatment. It can be concluded that exposure of astrocytes to cytokines may lead to an increase in receptor-mediated poly-PI signaling activity and this may involve compounds secreted into the culture medium, e.g., the secretory phospholipase A₂.     

Tetrandrine suppresses LPS-induced astrocyte activation via modulating IKKs-IκBα-NF-κB signaling pathway

Astrocyte activation has been implicated in the pathogenesis of many neurological diseases. These reactive astrocytes are capable of producing a variety of proinflammatory mediators and potentially neurotoxic compounds, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1beta (IL-1beta). In this study, we examined the suppressive effects of Tetrandrine (TET) on astrocyte activation induced by lipopolysaccharide (LPS) in vitro. We found that TET decreased the release of NO, TNF-alpha, IL-6 and IL-1beta in LPS-activated astrocytes. Also mRNA expression levels of inducible nitric oxide synthase (iNOS), macrophage inflammatory protein-1alpha (MIP-1alpha) and vascular cell adhesion molecule-1 (VCAM-1) were inhibited in TET pretreated astrocytes. Such suppressive effects might be resulted from the inhibition of nuclear factor kappa B (NF-kappaB) activation through downregulating IkappaB kinases (IKKs) phosphoration, which decreased inhibitor of nuclear factor-kappaB-alpha (IkappaBalpha) phosphoration and degradation. Our results suggest that TET acted to regulate astrocyte activation through inhibiting IKKs-IkappaBalpha-NF-kappaB signaling pathway.     

pPKCα mediated-HIF-1α activation related to the morphological modifications occurring in neonatal myocardial tissue in response to severe and mild hyperoxia

In premature babies birth an high oxygen level exposure can occur and newborn hyperoxia exposure can be associated with free radical oxygen release with impairment of myocardial function, while in adult animal models short exposure to hyperoxia seems to protect heart against ischemic injury. Thus, the mechanisms and consequences which take place after hyperoxia exposure are different and related to animals age. The aim of our work has been to analyze the role played by HIF-1α in the occurrence of the morphological modifications upon hyperoxia exposure in neonatal rat heart. Hyperoxia exposure induces connective compartment increase which seems to allow enhanced blood vessels growth. An increased hypoxia inducible factor-1α (HIF-1α) translocation and vascular endothelial growth factor (VEGF) expression has been found upon 95% oxygen exposure to induce morphological modifications. Upstream pPKC-α expression increase in newborn rats exposed to 95% oxygen can suggest PKC involvement in HIF-1α activation. Since nitric oxide synthase (NOS) are involved in heart vascular regulation, endothelial NOS (e-NOS) and inducible NOS (i-NOS) expression has been investigated: a lower eNOS and an higher iNOS expression has been found in newborn rats exposed to 95% oxygen related to the evidence that hyperoxia provokes a systemic vasoconstriction and to the iNOS pro-apoptotic action, respectively. The occurrence of apoptotic events, evaluated by TUNEL and Bax expression analyses, seems more evident in sample exposed to severe hyperoxia. All in all such results suggest that in newborn rats hyperoxia can trigger oxygen free radical mediated membrane injury through a pPKCα mediated HIF-1α signalling system, even though specificity of such response could be obtained by in vivo administration to the rats of specific inhibitors of PKCα. This intracellular signalling can switch molecular events leading to blood vessels development in parallel to pro-apoptotic events due to an immature anti-oxidant defensive system in newborn rat hearts.     

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents

Upregulation and activation of phospholipases A₂ (PLA₂) and cyclooxygenases (COX) leading to prostaglandin E₂(PGE₂) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE₂ production in primary rat astrocytes in response to agents that activate PLA₂ including pro-inflammatory cytokines (IL-1β, TNFα and IFNγ), the P2 nucleotide receptor agonist ATP, and oxidants (H₂O₂ and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE₂ production that was marked by increased expression of secretory sPLA₂ and COX-2, but not COX-1 and cytosolic cPLA₂. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA₂ phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE₂. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H₂O₂ or menadione, markedly increased PGE₂ production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE₂ production and may contribute to chronic inflammation seen in Alzheimer's disease.     

Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus

Astrocytes play an important role in initiating and regulating CNS immune responses through the release of proinflammatory cytokines and chemokines. Here we demonstrate that primary astrocytes are capable of recognizing the Gram-positive bacterium Staphylococcus aureus and its cell wall product peptidoglycan (PGN) and respond by producing numerous proinflammatory mediators including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1beta (MIP-1beta), MIP-2, and monocyte chemoattractant protein (MCP-1). Astrocytes have recently been shown to express Toll-like receptor 2 (TLR2), a pattern recognition receptor important for recognizing structural components of various Gram-positive bacteria, fungi, and protozoa. However, the functional significance of TLR2 in mediating astrocyte activation remains unknown. Primary astrocytes from TLR2 knockout mice were used to evaluate the role of TLR2 in astrocyte responses to S. aureus and PGN. The results demonstrate that TLR2 is essential for maximal proinflammatory cytokine and chemokine production, but not phagocytosis, in primary astrocytes following S. aureus and PGN exposure. In addition, both stimuli led to a significant increase in TLR2 mRNA expression in wild-type astrocytes as assessed by real-time quantitative RT-PCR. These findings suggest that astrocytes may play a key role in the initial antibacterial immune response in the CNS through engagement of TLR2.     

Nitric oxide, mitochondria and neurological disease

Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis of a range of neurological disorders, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, stroke and amyotrophic lateral sclerosis. There is also a growing body of evidence to implicate excessive or inappropriate generation of nitric oxide (NO) in these disorders. It is now well documented that NO and its toxic metabolite, peroxynitrite (ONOO-), can inhibit components of the mitochondrial respiratory chain leading, if damage is severe enough, to a cellular energy deficiency state. Within the brain, the susceptibility of different brain cell types to NO and ONOO- exposure may be dependent on factors such as the intracellular reduced glutathione (GSH) concentration and an ability to increase glycolytic flux in the face of mitochondrial damage. Thus neurones, in contrast to astrocytes, appear particularly vulnerable to the action of these molecules. Following cytokine exposure, astrocytes can increase NO generation, due to de novo synthesis of the inducible form of nitric oxide synthase (NOS). Whilst the NO/ONOO- so formed may not affect astrocyte survival, these molecules may diffuse out to cause mitochondrial damage, and possibly cell death, to other cells, such as neurones, in close proximity. Evidence is now available to support this scenario for neurological disorders, such as multiple sclerosis. In other conditions, such as ischaemia, increased availability of glutamate may lead to an activation of a calcium-dependent nitric oxide synthase associated with neurones. Such increased/inappropriate NO formation may contribute to energy depletion and neuronal cell death. The evidence available for NO/ONOO--mediated mitochondrial damage in various neurological disorders is considered and potential therapeutic strategies are proposed.     

Role of astrocytes in cerebrovascular regulation.

Astrocytes send processes to synapses and blood vessels, communicate with other astrocytes through gap junctions and by release of ATP, and thus are an integral component of the neurovascular unit. Electrical field stimulations in brain slices demonstrate an increase in intracellular calcium in astrocyte cell bodies transmitted to perivascular end-feet, followed by a decrease in vascular smooth muscle calcium oscillations and arteriolar dilation. The increase in astrocyte calcium after neuronal activation is mediated, in part, by activation of metabotropic glutamate receptors. Calcium signaling in vitro can also be influenced by adenosine acting on A2B receptors and by epoxyeicosatrienoic acids (EETs) shown to be synthesized in astrocytes. Prostaglandins, EETs, arachidonic acid, and potassium ions are candidate mediators of communication between astrocyte end-feet and vascular smooth muscle. In vivo evidence supports a role for cyclooxygenase-2 metabolites, EETs, adenosine, and neuronally derived nitric oxide in the coupling of increased blood flow to increased neuronal activity. Combined inhibition of the EETs, nitric oxide, and adenosine pathways indicates that signaling is not by parallel, independent pathways. Indirect pharmacological results are consistent with astrocytes acting as intermediaries in neurovascular signaling within the neurovascular unit. For specific stimuli, astrocytes are also capable of transmitting signals to pial arterioles on the brain surface for ensuring adequate inflow pressure to parenchymal feeding arterioles. Therefore, evidence from brain slices and indirect evidence in vivo with pharmacological approaches suggest that astrocytes play a pivotal role in regulating the fundamental physiological response coupling dynamic changes in cerebral blood flow to neuronal synaptic activity. Future work using in vivo imaging and genetic manipulation will be required to provide more direct evidence for a role of astrocytes in neurovascular coupling.     

Transient acidification and subsequent proinflammatory cytokine stimulation of astrocytes induce distinct activation phenotypes

The foot processes of astrocytes cover over 60% of the surface of brain microvascular endothelial cells, regulating tight junction integrity. Retraction of astrocyte foot processes has been postulated to be a key mechanism in pathology. Therefore, movement of an astrocyte in response to a proinflammatory cytokine or even limited retraction of processes would result in leaky junctions between endothelial cells. Astrocytes lie at the gateway to the CNS and are instrumental in controlling leukocyte entry. Cultured astrocytes typically have a polygonal morphology until stimulated. We hypothesized that cultured astrocytes which were induced to stellate would have an activated phenotype compared with polygonal cells. We investigated the activation of astrocytes derived from adult macaques to the cytokine TNF‐α under resting and stellated conditions by four parameters: morphology, intermediate filament expression, adhesion, and cytokine secretion. Astrocytes were stellated following transient acidification; resulting in increased expression of GFAP and vimentin. Stellation was accompanied by decreased adhesion that could be recovered with proinflammatory cytokine treatment. Surprisingly, there was decreased secretion of proinflammatory cytokines by stellated astrocytes compared with polygonal cells. These results suggest that astrocytes are capable of multiple phenotypes depending on the stimulus and the order stimuli are applied. J. Cell. Physiol. 228: 1284–1294, 2013. © 2012 Wiley Periodicals, Inc.     

TRPM7 Activates m-Calpain by Stress-Dependent Stimulation of p38 MAPK and c-Jun N-Terminal Kinase

TRPM7 is a Ca²⁺-permeant and Mg²⁺-permeant ion channel in possession of its own kinase domain. In a previous study, we showed that overexpression of the channel-kinase in HEK-293 cells produced cell rounding and loss of adhesion, which was dependent on the Ca²⁺-dependent protease m-calpain. The TRPM7-elicited change in cell morphology was channel-dependent and occurred without any significant increase in cytosolic Ca²⁺. Here we demonstrate that overexpression of TRPM7 increased levels of cellular reactive oxygen species (ROS) and nitric oxide, causing the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Application of inhibitors of p38 MAPK and JNK blocked TRPM7-induced cell rounding and activation of m-calpain, without affecting the phosphorylation state of the protease. Overexpression of TRPM7 increased intracellular Mg²⁺; however, when the concentration of either external Ca²⁺ or Mg²⁺ was increased to favor the permeation of one divalent cation over the other, a similar increase in cell rounding and calpain activity was detected, indicating that TRPM7-mediated activation of m-calpain is not dependent on the nature of the divalent conducted by the channel. Application of inhibitors of nitric oxide synthase and mitochondrial-derived ROS reduced TRPM7-induced increases in nitric oxide and ROS production, blocked the change in cell morphology, and reduced cellular calpain activity. Collectively, our data reveal that excessive TRPM7 channel activity causes oxidative and nitrosative stresses, producing cell rounding mediated by p38 MAPK/JNK-dependent activation of m-calpain.     

Astrocytic energetics during excitatory neurotransmission: What are contributions of glutamate oxidation and glycolysis?

Astrocytic energetics of excitatory neurotransmission is controversial due to discrepant findings in different experimental systems in vitro and in vivo. The energy requirements of glutamate uptake are believed by some researchers to be satisfied by glycolysis coupled with shuttling of lactate to neurons for oxidation. However, astrocytes increase glycogenolysis and oxidative metabolism during sensory stimulation in vivo, indicating that other sources of energy are used by astrocytes during brain activation. Furthermore, glutamate uptake into cultured astrocytes stimulates glutamate oxidation and oxygen consumption, and glutamate maintains respiration as well as glucose. The neurotransmitter pool of glutamate is associated with the faster component of total glutamate turnover in vivo, and use of neurotransmitter glutamate to fuel its own uptake by oxidation-competent perisynaptic processes has two advantages, substrate is supplied concomitant with demand, and glutamate spares glucose for use by neurons and astrocytes. Some, but not all, perisynaptic processes of astrocytes in adult rodent brain contain mitochondria, and oxidation of only a small fraction of the neurotransmitter glutamate taken up into these structures would be sufficient to supply the ATP required for sodium extrusion and conversion of glutamate to glutamine. Glycolysis would, however, be required in perisynaptic processes lacking oxidative capacity. Three lines of evidence indicate that critical cornerstones of the astrocyte-to-neuron lactate shuttle model are not established and normal brain does not need lactate as supplemental fuel: (i) rapid onset of hemodynamic responses to activation delivers oxygen and glucose in excess of demand, (ii) total glucose utilization greatly exceeds glucose oxidation in awake rodents during activation, indicating that the lactate generated is released, not locally oxidized, and (iii) glutamate-induced glycolysis is not a robust phenotype of all astrocyte cultures. Various metabolic pathways, including glutamate oxidation and glycolysis with lactate release, contribute to cellular energy demands of excitatory neurotransmission.