Astrocyte activation in vivo during graded photic stimulation

Astrocytes have important roles in control of extracellular environment, de novo synthesis of neurotransmitters, and regulation of neurotransmission and blood flow. All of these functions require energy, suggesting that astrocytic metabolism should rise and fall with changes in neuronal activity and that brain imaging can be used to visualize and quantify astrocytic activation in vivo . A unilateral photic stimulation paradigm was used to test the hypothesis that graded sensory stimuli cause progressive increases in the uptake coefficient of [2-¹⁴C]acetate, a substrate preferentially oxidized by astrocytes. The acetate uptake coefficient fell in deafferented visual structures and it rose in intact tissue during photic stimulation of conscious rats; the increase was highest in structures with monosynaptic input from the eye and was much smaller in magnitude than the change in glucose utilization (CMR_glc) by all cells. The acetate uptake coefficient was not proportional to stimulus rate and did not correlate with CMR_glc in resting or activated structures. Simulation studies support the conclusions that acetate uptake coefficients represent mainly metabolism and respond to changes in metabolism rate, with a lower response at high rates. A model portraying regulation of acetate oxidation illustrates complex relationships among functional activation, cation levels, and astrocytic metabolism.     

A glycogen phosphorylase inhibitor selectively enhances local rates of glucose utilization in brain during sensory stimulation of conscious rats: implications for glycogen turnover

Glycogen is degraded during brain activation but its role and contribution to functional energetics in normal activated brain have not been established. In the present study, glycogen utilization in brain of normal conscious rats during sensory stimulation was assessed by three approaches, change in concentration, release of (14)C from pre-labeled glycogen and compensatory increase in utilization of blood glucose (CMR(glc)) evoked by treatment with a glycogen phosphorylase inhibitor. Glycogen level fell in cortex, (14)C release increased in three structures and inhibitor treatment caused regionally selective compensatory increases in CMR(glc) over and above the activation-induced rise in vehicle-treated rats. The compensatory rise in CMR(glc) was highest in sensory-parietal cortex where it corresponded to about half of the stimulus-induced rise in CMR(glcf) in vehicle-treated rats; this response did not correlate with metabolic rate, stimulus-induced rise in CMR(glc) or sequential station in sensory pathway. Thus, glycogen is an active fuel for specific structures in normal activated brain, not simply an emergency fuel depot and flux-generated pyruvate greatly exceeded net accumulation of lactate or net consumption of glycogen during activation. The metabolic fate of glycogen is unknown, but adding glycogen to the fuel consumed during activation would contribute to a fall in CMR(O2)/CMR(glc) ratio.     

Increased cerebral uptake and oxidation of exogenous betaHB improves ATP following traumatic brain injury in adult rats

There is growing evidence of the brain's ability to increase its reliance on alternative metabolic substrates under conditions of energy stress such as starvation, hypoxia and ischemia. We hypothesized that following traumatic brain injury (TBI), which results in immediate changes in energy metabolism, the adult brain increases uptake and oxidation of the alternative substrate beta-hydroxybutyrate (betaHB). Arterio-venous differences were used to determine global cerebral uptake of betaHB and production of 14CO2 from [14C]3-betaHB 3 h after controlled cortical impact (CCI) injury. Quantitative bioluminescence was used to assess regional changes in ATP concentration. As expected, adult sham and CCI animals with only endogenously available betaHB showed no significant increase in cerebral uptake of betaHB or 14CO2 production. Increasing arterial betaHB concentrations 2.9-fold with 3 h of betaHB infusion failed to increase cerebral uptake of betaHB or 14CO2 production in adult sham animals. Only CCI animals that received a 3-h betaHB infusion showed an 8.5-fold increase in cerebral uptake of betaHB and greater than 10.7-fold increase in 14CO2 production relative to sham betaHB-infused animals. The TBI-induced 20% decrease in ipsilateral cortical ATP concentration was alleviated by 3 h of betaHB infusion beginning immediately after CCI injury.     

Measurement of β-hydroxybutyrate in acute hyperketonemia in human brain

We report the measurement of D-beta-hydroxybutyrate (BHB) in the brains of six normal adult subjects during acute infusions of BHB. We used high field in vivo (1)H magnetic resonance (MR) spectroscopy in the occipital lobe in conjunction with an acute infusion protocol to elevate plasma BHB levels from overnight fasted levels (0.20 +/- 0.10 mM) to a steady state value of 2.12 +/- 0.30 mM. At this level of hyperketonemia, we determined a tissue BHB level of 0.24 +/- 0.04 mM. No increases in brain lactate levels were seen in these data. The concentrations of BHB and lactate were both considerably lower in comparison with previous data acquired in fasted adult subjects. This suggests that up-regulation of the monocarboxylic acid transporter occurs with fasting.     

Chloramphenicol decreases brain glucose utilization and modifies the sleep-wake cycle architecture in rats

We studied the effects of chloramphenicol on brain glucose utilization and sleep-wake cycles in rat. After slightly anaesthetized animals were injected with [18F]fluoro-2-deoxy-D-glucose, we acquired time-concentration curves from three radiosensitive beta microprobes inserted into the right and left frontal cortices and the cerebellum, and applied a three-compartment model to calculate the cerebral metabolic rates for glucose. The sleep-wake cycle architecture was analysed in anaesthetic-free rats by recording electroencephalographic and electromyographic signals. Although chloramphenicol is a well-established inhibitor of oxidative phosphorylation, no compensatory increase in glucose utilization was detected in frontal cortex. Instead, chloramphenicol induced a significant 23% decrease in the regional cerebral metabolic rate for glucose. Such a metabolic response indicates a potential mismatch between energy supply and neuronal activity induced by chloramphenicol administration. Regarding sleep-wake states, chloramphenicol treatment was followed by a 64% increase in waking, a 20% decrease in slow-wave sleep, and a marked 59% loss in paradoxical sleep. Spectral analysis of the electroencephalogram indicates that chloramphenicol induces long-lasting modifications of delta-band power during slow-wave sleep.     

Interleukin-1β-Mediated Regulation of μ-Opioid Receptor mRNA in Primary Astrocyte-Enriched Cultures

Abstract: Opioids have been found to modulate the immune system by regulating the function of immunocompetent cells. Several studies suggest that the interaction between immune and opioid systems is not unidirectional, but rather reciprocal, in nature. In the CNS, one cellular target of immune system activation is the astrocytes. These glial cells have been shown to produce the opioid peptide, proenkephalin, to express the μ-, δ-, and κ-opioid receptors, and to respond to the immune factor interleukin-1β (IL1β) with an increased proenkephalin synthesis. To characterize more completely the astrocytic opioid response to immune factor stimulation, we examined the effect of IL1β (1 ng/ml) on the μ-receptor mRNA expression in primary astrocyte-enriched cultures derived from rat (postnatal day 1–2) cortex, striatum, cerebellum, hippocampus, and hypothalamus. A 24-h treatment with IL1β produced a 70–80% increase in the μ-receptor mRNA expression in the striatal, cerebellar, and hippocampal cultures but had no effect on this expression in the cortical and hypothalamic cultures. This observation represents one of the few demonstrated increases in levels of the μ-receptor mRNA in vitro or in vivo, since the cloning of the receptor. The enhanced μ-receptor mRNA expression, together with the previous observation that IL1β stimulates proenkephalin synthesis in astrocytes, supports the IL1β-mediated regulation of an astroglial opioid peptide and receptor in vitro, a phenomenon that may be significant in the modulation of the gliotic response to neuronal damage. Therefore, the astroglial opioid "system" may be important in the IL1β-initiated, coordinated response to CNS infection, trauma, or injury.     

Leucine-nitrogen metabolism in the brain of conscious rats: its role as a nitrogen carrier in glutamate synthesis in glial and neuronal metabolic compartments

The source of nitrogen (N) for the de novo synthesis of brain glutamate, glutamine and GABA remains controversial. Because leucine is readily transported into the brain and the brain contains high activities of branched-chain aminotransferase (BCAT), we hypothesized that leucine is the predominant N-precursor for brain glutamate synthesis. Conscious and unstressed rats administered with [U-13C] and/or [15N]leucine as additions to the diet were killed at 0-9 h of continuous feeding. Plasma and brain leucine equilibrated rapidly and the brain leucine-N turnover was more than 100%/min. The isotopic dilution of [U-13C]leucine (brain/plasma ratio 0.61 +/- 0.06) and [15N]leucine (0.23 +/- 0.06) differed markedly, suggesting that 15% of cerebral leucine-N turnover derived from proteolysis and 62% from leucine synthesis via reverse transamination. The rate of glutamate synthesis from leucine was 5 micro mol/g/h and at least 50% of glutamate-N originally derived from leucine. The enrichment of [5-15N]glutamine was higher than [15N]ammonia in the brain, indicating glial ammonia generation from leucine via glutamate. The enrichment of [15N]GABA, [15N]aspartate, [15N]glutamate greater than [2-15N]glutamine suggests direct incorporation of leucine-N into both glial and neuronal glutamate. These findings provide a new insight for the role of leucine as N-carrier from the plasma pool and within the cerebral compartments.     

Interaction between interferon gamma and insulin-like growth factor-1 in hippocampus impacts on the ability of rats to sustain long-term potentiation

There is compelling evidence to suggest that inflammation significantly contributes to neurodegenerative changes. Consistent with this is the observation that several neurodegenerative disorders are accompanied by an increase in the concentration of interleukin (IL)-1beta. IL-1beta has a negative impact on synaptic plasticity and therefore an increased concentration of IL-1beta, such as that in the hippocampus of the aged rat, is associated with a deficit in long-term potentiation (LTP). IL-1beta is derived mainly from activated microglia but the trigger leading to this activation, specifically in the aged brain, remains to be identified. Here we examined the possibility that interferon (IFN)gamma may stimulate microglial activation and increase IL-1beta concentration, thereby inhibiting LTP. The IFNgamma concentration was increased in hippocampus prepared from aged, compared with young, rats and inversely correlated with the ability of rats to sustain LTP. Intracerebroventricular injection of IFNgamma inhibited LTP, and increased microglial activation was observed in both IFNgamma-injected and aged rats. The age-related increase in IFNgamma was accompanied by a decrease in the hippocampal concentration of insulin-like growth factor (IGF)-1. The evidence presented suggests that IGF-1 acts to antagonize the IFNgamma-induced microglial activation, the accompanying increase in IL-1beta concentration and the consequent deficit in LTP.     

The in vivo neuron-to-astrocyte lactate shuttle in human brain: evidence from modeling of measured lactate levels during visual stimulation

Functional magnetic resonance spectroscopy (fMRS) allows the non-invasive measurement of metabolite concentrations in the human brain, including changes induced by variations in neurotransmission activity. However, the limited spatial and temporal resolution of fMRS does not allow specific measurements of metabolites in different cell types. Thus, the analysis of fMRS data in the context of compartmentalized metabolism requires the formulation and application of mathematical models. In the present study we utilized the mathematical model introduced by Simpson et al . (2007) to gain insights into compartmentalized metabolism in vivo from the fMRS data obtained in humans at ultra high magnetic field by Mangia et al . (2007a) . This model simulates brain glucose and lactate levels in a theoretical cortical slice. Using experimentally determined concentrations and catalytic activities for the respective transporter proteins, we calculate inflow and export of glucose and lactate in endothelium, astrocytes, and neurons. We then vary neuronal and astrocytic glucose and lactate utilization capacities until close correspondence is observed between in vivo and simulated glucose and lactate levels. The results of the simulations indicate that, when literature values of glucose transport capacity are utilized, the fMRS data are consistent with export of lactate by neurons and import of lactate by astrocytes, a mechanism that can be referred to as a neuron-to-astrocyte lactate shuttle. A shuttle of lactate from astrocytes to neurons could be simulated, but this required the astrocytic glucose transport capacity to be increased by 12-fold, and required that neurons not respond to activation with increased glycolysis, two conditions that are not supported by current literature.     

Effects of intrahippocampal CT105, a carboxyl terminal fragment of beta-amyloid precursor protein,alone/with inflammatory cytokines on working memory in rats

In this study, we examined the effects of a 105 amino acid carboxyl terminal fragment of beta-amyloid precursor protein (CT105) and inflammatory cytokines on working memory in rats, by using a three-panel runway set-up. CT105 at 10 nmol/side significantly impaired working memory when it was administered bilaterally into the hippocampus. Furthermore, to elucidate the interaction of CT105 with inflammatory cytokines, we co-administered tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in combination with CT105. Concurrent injections of CT105 (1.0 nmol/side) and TNF-alpha (100 ng/side) produced a synergistic deficit of working memory, whereas IL-1beta (100 ng/side) combined with CT105 (1.0 nmol/side) did not affect the working memory performance. These results indicate that the CT105-induced impairment of working memory is strongly aggravated by an increase in the level of the inflammatory cytokine TNF-alpha, which may occur in the brains of patients with Alzheimer's disease.     

Pathways of neurosteroid biosynthesis in cell lines from human brain: regulation of dehydroepiandrosterone formation by oxidative stress and beta-amyloid peptide

Neurosteroids in rodents can originate from peripheral tissues or be locally synthesized in specific brain areas. There is, as yet, no information about the synthesis and regulation of neurosteroids in human brain. We examined the ability of human brain cells to synthesize steroids from a radiolabeled precursor and the mRNA and protein expression of key components of peripheral steroidogenic machinery. Oligodendrocytes are the source of pregnenolone in human brain. Human astrocytes do not synthesize radiolabeled pregnenolone, nor do human neurons. There is potential for all three cell types to metabolize pregnenolone to other neurosteroids, including dehydroepiandrosterone. mRNA and protein for cytochrome P450 17alpha-hydroxylase were found in all cell types, although no activity could be demonstrated. We examined the ability of the cells to make dehydroepiandrosterone via an alternative pathway induced by treatment with Fe2+. Oligodendrocytes and astrocytes make dehydroepiandrosterone via this pathway, but neurons do not. In searching for a natural regulator of dehydroepiandrosterone formation, we observed that treating oligodendrocytes with beta-amyloid, which increases reactive oxygen species, also increased dehydroepiandrosterone formation. These effects of beta-amyloid were blocked by vitamin E. These results indicate that human brain makes steroids in a cell-specific manner and suggest that dehydroepiandrosterone synthesis can be regulated by intracellular free radicals.     

Activation of the heat shock response in plants by chlorophenols: transgenic Physcomitrella patens as a sensitive biosensor for organic pollutants

The ability to detect early molecular responses to various chemicals is central to the understanding of biological impact of pollutants in a context of varying environmental cues. To monitor stress responses in a model plant, we used transgenic moss Physcomitrella patens expressing the beta-glucuronidase reporter (GUS) under the control of the stress-inducible promoter hsp17.3B. Following exposure to pollutants from the dye and paper industry, GUS activity was measured by monitoring a fluorescent product. Chlorophenols, heavy metals and sulphonated anthraquinones were found to specifically activate the hsp17.3B promoter (within hours) in correlation with long-term toxicity effects (within days). At mildly elevated physiological temperatures, the chemical activation of this promoter was strongly amplified, which considerably increased the sensitivity of the bioassay. Together with the activation of hsp17.3B promoter, chlorophenols induced endogenous chaperones that transiently protected a recombinant thermolabile luciferase (LUC) from severe heat denaturation. This sensitive bioassay provides an early warning molecular sensor to industrial pollutants under varying environments, in anticipation to long-term toxic effects in plants. Because of the strong cross-talk between abiotic and chemical stresses that we find, this P. patens line is more likely to serve as a direct toxicity bioassay for pollutants combined with environmental cues, than as an indicator of absolute toxicity thresholds for various pollutants. It is also a powerful tool to study the role of heat shock proteins (HSPs) in plants exposed to combined chemical and environmental stresses.     

Multifunctional roles of enolase in Alzheimer's disease brain: beyond altered glucose metabolism

Enolase enzymes are abundantly expressed, cytosolic carbon-oxygen lyases known for their role in glucose metabolism. Recently, enolase has been shown to possess a variety of different regulatory functions, beyond glycolysis and gluconeogenesis, associated with hypoxia, ischemia, and Alzheimer's disease (AD). AD is an age-associated neurodegenerative disorder characterized pathologically by elevated oxidative stress and subsequent damage to proteins, lipids, and nucleic acids, appearance of neurofibrillary tangles and senile plaques, and loss of synapse and neuronal cells. It is unclear if development of a hypometabolic environment is a consequence of or contributes to AD pathology, as there is not only a significant decline in brain glucose levels in AD, but also there is an increase in proteomics identified oxidatively modified glycolytic enzymes that are rendered inactive, including enolase. Previously, our laboratory identified α-enolase as one the most frequently up-regulated and oxidatively modified proteins in amnestic mild cognitive impairment (MCI), early-onset AD, and AD. However, the glycolytic conversion of 2-phosphoglycerate to phosphoenolpyruvate catalyzed by enolase does not directly produce ATP or NADH; therefore it is surprising that, among all glycolytic enzymes, α-enolase was one of only two glycolytic enzymes consistently up-regulated from MCI to AD. These findings suggest enolase is involved with more than glucose metabolism in AD brain, but may possess other functions, normally necessary to preserve brain function. This review examines potential altered function(s) of brain enolase in MCI, early-onset AD, and AD, alterations that may contribute to the biochemical, pathological, clinical characteristics, and progression of this dementing disorder.     

Synergistic Stimulation of Interleukin 6 Release and Gene Expression by Phorbol Esters and Interleukin 1β in Rat Cortical Astrocytes: Role of Protein Kinase C Activation and Blockade

Abstract: The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12-myristate 13-acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.     

Acute exposure of rats to air ions: effects on the regional concentration and utilization of serotonin in brain

Exposure to electrically charged gas molecules (air ions) has been reported to influence physiological and behavioral functions in animals and humans although there is controversy as to whether these findings are valid. A popular hypothesis concerning the reported effects of air ions is that alterations in serotonin (5HT) metabolism, particularly in the brain, are involved. We measured the concentration and turnover of 5HT in rats exposed to 5.0 X 10(5) ions/cm3 for up to 66 hours. Contrary to previous reports of other investigators, we were unable to demonstrate any effect of exposure to air ions or associated DC electric fields on the concentration or turnover of 5HT in rats under carefully controlled and characterized exposure conditions.