Short-term memory impairment following recovery from systemic inflammation induced by lipopolysaccharide in mice.

The relationship between neuroinflammation and mental disorders has been recognized and investigated for over 30 years. Diseases of systemic or peripheral inflammation, such as sepsis, peritonitis, and infection, are associated with increased risk of mental disorders with neuroinflammation. To elucidate the pathogenesis, systemic administration of lipopolysaccharide (LPS) in mice is often used. LPS-injected mice exhibit behavioral abnormalities with glial activation. However, these studies are unlikely to recapitulate the clinical pathophysiology of human patients, as most studies focus on the acute inflammatory response with systemic symptoms occurring within 24 h of LPS injection. In this study, we focus on the effects of LPS on behavioral abnormalities following recovery from systemic symptoms and investigate the mechanisms of pathogenesis. Several behavioral tests were performed in LPS-injected mice, and to assess neuroinflammation, the time course of the morphological change and expression of inflammatory factors in neurons, astrocytes, and microglia were investigated. At 7 days post-LPS injection, mice exhibited short-term memory impairment accompanied by the suppression of neuronal activity and increases in morphologically immature spines. Glial cells were transiently activated in the hippocampus concomitant with upregulation of the microglial phagocytosis marker CD68 3 days after injection. Here we show that transient glial cell activation in the acute response phase affects neuronal activity and behavior following recovery from systemic symptoms. These findings provide novel insights for studies using the LPS-induced inflammation model and that will contribute to the development of treatments for mental disorders of this etiology.

Dopamine regulates astrocytic IL-6 expression and process formation via dopamine receptors and adrenoceptors.

Dopamine levels in the central nervous system change under pathological conditions such as Parkinson's disease, Huntington's disease, and addiction. Under those pathological conditions, astrocytes become reactive astrocytes characterized by morphological changes and the release of inflammatory cytokines involved in pathogenesis. However, it remains unclear whether dopamine regulates astrocytic morphology and functions. Elucidating these issues will help us to understand the pathogenesis of neurodegenerative diseases caused by abnormal dopamine signaling. In this study, we investigated the effects of dopamine on IL-6 expression and process formation in rat primary cultured astrocytes and acute hippocampal slices. Dopamine increased IL-6 expression in a concentration-dependent manner, and this was accompanied by CREB phosphorylation. The effects of a low dopamine concentration (1 µM) were inhibited by a D1-like receptor antagonist, whereas the effects of a high dopamine concentration (100 µM) were inhibited by a ß-antagonist and enhanced by a D2-like receptor antagonist. Furthermore, dopamine (100 µM) promoted process formation, which was inhibited by a ß-antagonist and enhanced by both an α-antagonist and a D2-like receptor antagonist. In acute hippocampal slices, both a D1-like receptor agonist and ß-agonist changed astrocytic morphology. Together, these results indicate that dopamine promotes IL-6 expression and process formation via D1-like receptors and ß-adrenoceptors. Furthermore, bidirectional regulation exists; namely, the effects of D1-like receptors and ß-adrenoceptors were negatively regulated by D2-like receptors and α2-adrenoceptors.

Glu333 in rabies virus glycoprotein is involved in virus attenuation through astrocyte infection and interferon responses.

The amino acid residue at position 333 of the rabies virus (RABV) glycoprotein (G333) is a major determinant of RABV pathogenicity. Virulent RABV strains possess Arg333, whereas the attenuated strain HEP-Flury (HEP) possesses Glu333. To investigate the potential attenuation mechanism dependent on a single amino acid at G333, comparative analysis was performed between HEP and HEP333R mutant with Arg333. We examined their respective tropism for astrocytes and the subsequent immune responses in astrocytes. Virus replication and subsequent interferon (IFN) responses in astrocytes infected with HEP were increased compared with HEP333R both in vitro and in vivo. Furthermore, involvement of IFN in the avirulency of HEP was demonstrated in IFN-receptor knockout mice. These results indicate that Glu333 contributes to RABV attenuation by determining the ability of the virus to infect astrocytes and stimulate subsequent IFN responses.

Alpha and beta adrenoceptors activate interleukin-6 transcription through different pathways in cultured astrocytes from rat spinal cord.

In brain astrocytes, noradrenaline (NA) has been shown to up-regulate IL-6 production via ß-adrenoceptors (ARs). However, the underlying intracellular mechanisms for this regulation are not clear, and it remains unknown whether α-ARs are involved. In this study, we investigated the AR-mediated regulation of IL-6 mRNA levels in the cultured astrocytes from rat spinal cord. NA, the α1-agonist phenylephrine, and the ß-agonist isoproterenol increased IL-6 mRNA levels. The phenylephrine-induced IL-6 increase was accompanied by an increase in ERK phosphorylation, and these effects were blocked by inhibitors of PKC and ERK. The isoproterenol-induced IL-6 increase was accompanied by an increase in CREB phosphorylation, and these effects were blocked by a PKA inhibitor. Our results indicate that IL-6 increases by α1- and ß-ARs are mediated via the PKC/ERK and cAMP/PKA/CREB pathways, respectively. Moreover, conditioned medium collected from astrocytes treated with the α2-AR agonist dexmedetomidine, increased IL-6 mRNA in other astrocytes. In this study, we elucidate that α1- and α2-ARs, in addition to ß-ARs, promote IL-6 transcription through different pathways in spinal cord astrocytes.

Opposing functions of α- and ß-adrenoceptors in the formation of processes by cultured astrocytes.

Astrocytes are glial cells with numerous fine processes which are important for the functions of the central nervous system. The activation of ß-adrenoceptors induces process formation of astrocytes via cyclic AMP (cAMP) signaling. However, the role of α-adrenoceptors in the astrocyte morphology has not been elucidated. Here, we examined it by using cultured astrocytes from neonatal rat spinal cords and cortices. Exposure of these cells to noradrenaline and the ß-adrenoceptor agonist isoproterenol increased intracellular cAMP levels and induced the formation of processes. Noradrenaline-induced process formation was enhanced with the α1-adrenoceptor antagonist prazosin and α2-adrenoceptor antagonist atipamezole. Atipamezole also enhanced noradrenaline-induced cAMP elevation. Isoproterenol-induced process formation was not inhibited by the α1-adrenoceptor agonist phenylephrine but was inhibited by the α2-adrenoceptor agonist dexmedetomidine. Dexmedetomidine also inhibited process formation induced by the adenylate cyclase activator forskolin and the membrane-permeable cAMP analog dibutyryl-cAMP. Moreover, dexmedetomidine inhibited cAMP-independent process formation induced by adenosine or the Rho-associated kinase inhibitor Y27632. In the presence of propranolol, noradrenaline inhibited Y27632-induced process formation, which was abolished by prazosin or atipamezole. These results demonstrate that α-adrenoceptors inhibit both cAMP-dependent and -independent astrocytic process formation.

Hydrogen sulfide induces Ca2+ release from intracellular Ca2+ stores and stimulates lactate production in spinal cord astrocytes.

Hydrogen sulfide (H2S) is a well-known inhibitor of the mitochondrial electron transport chain (ETC). H2S also increases intracellular Ca2+ levels in astrocytes, which are glial cells and that supply lactate as an energy substrate to neurons. Here, we examined the relationship between H2S-induced metabolic changes and Ca2+ responses in spinal cord astrocytes. Na2S (150 µM), an H2S donor, increased the intracellular Ca2+ concentration, which was inhibited by an ETC inhibitor and an uncoupler of mitochondrial oxidative phosphorylation. Na2S also increased the accumulation of extracellular lactate. Na2S alone did not change intracellular ATP content, but decreased it when glycolysis was inhibited. The Na2S-induced Ca2+ increase and accumulation of extracellular lactate were inhibited by emetine, an inhibitor of translocon complex, which mediates Ca2+ leak from the endoplasmic reticulum (ER). Furthermore, an inhibitor of the Ca2+-sensitive NADH shuttle decreased Na2S-mediated accumulation of lactate. We conclude that inhibition of the mitochondrial ETC by H2S induces Ca2+ release from mitochondria and the ER in spinal cord astrocytes, which increases lactate production. H2S may promote glycolysis by activating the Ca2+-sensitive NADH shuttle and facilitating the supply of lactate from astrocytes to neurons.

Hydrogen sulfide induces Ca2+ release from the endoplasmic reticulum and suppresses ATP-induced Ca2+ signaling in rat spinal cord astrocytes.

Hydrogen sulfide (H2S) has a variety of physiological functions. H2S reportedly increases intracellular Ca2+ concentration ([Ca2+]i) in astrocytes. However, the precise mechanism and functional role of this increase are not known. Here, we examined the effects of H2S on [Ca2+]i in astrocytes from the rat spinal cord and whether H2S affects ATP-induced Ca2+ signaling, which is known to be involved in synaptic function. Na2S (150 µM), an H2S donor, produced a nontoxic increase in [Ca2+]i. The [Ca2+]i increase by Na2S was inhibited by Ca2+ depletion in the endoplasmic reticulum (ER) but not by removal of extracellular Ca2+, indicating that H2S releases Ca2+ from the ER. On the other hand, Na2S inhibited ATP-induced [Ca2+]i increase when Na2S clearly increased [Ca2+]i in the astrocytes, which was not suppressed by a reducing agent. In addition, Na2S had no effect on intracellular cyclic AMP (cAMP) level. These results indicate that oxidative post-translational modification of proteins and cAMP are not involved in the inhibitory effect of H2S on ATP-induced Ca2+ signaling. We conclude that H2S indirectly inhibits ATP-induced Ca2+ signaling by decreasing Ca2+ content in the ER in astrocytes. In this way, H2S may influence intercellular communication between astrocytes and neurons, thereby contributing to neuronal signaling in the nervous system.

Fibroblast growth factor 2 upregulates ecto-5'-nucleotidase and adenosine deaminase via MAPK pathways in cultured rat spinal cord astrocytes.

Adenosine triphosphate (ATP) and adenosine are neurotransmitters and neuromodulators in the central nervous system. Astrocytes regulate extracellular concentration of purines via ATP release and its metabolisms via ecto-enzymes. The expression and activity of purine metabolic enzymes in astrocytes are increased under pathological conditions. We previously showed that fibroblast growth factor 2 (FGF2) upregulates the expression and activity of the enzymes ecto-5'-nucleotidase (CD73) and adenosine deaminase (ADA). Here, we further demonstrate that this occurs in concentration- and time-dependent manners in cultured rat spinal cord astrocytes and is suppressed by inhibitors of the FGF receptor as well as the mitogen-activated protein kinases (MAPKs). We also found that FGF2 increased the phosphorylation of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK, leading to the increased expression and activity of CD73 and ADA. Our findings reveal the involvement of FGF2/MAPK pathways in the regulation of purine metabolic enzymes in astrocytes. These pathways may contribute to the control of extracellular purine concentrations under physiological and pathological conditions.

Bidirectional modulation of TNF-α transcription via α- and ß-adrenoceptors in cultured astrocytes from rat spinal cord.

Noradrenaline (NA) suppresses TNF-α production via ß-adrenoceptors (ARs) in brain astrocytes. However, the downstream pathways from ß-ARs, and the involvement of α-ARs, remains unknown. In this study, we investigated the AR-mediated regulation of TNF-α mRNA levels in cultured astrocytes from rat spinal cord. NA, the α1-agonist phenylephrine, and the ß-agonist isoproterenol decreased the TNF-α mRNA level, while the α2-agonist dexmedetomidine increased it. The isoproterenol-induced TNF-α mRNA decrease was accompanied by a decrease in ERK phosphorylation. An adenylyl cyclase activator and an ERK inhibitor mimicked these effects. These results indicate that the transcriptional regulation of TNF-α by ß-ARs is mediated via cAMP pathways followed by the ERK pathway inhibition. The dexmedetomidine-induced TNF-α mRNA increase was accompanied by phosphorylation of JNK and ERK, which was blocked by a JNK inhibitor. Furthermore, the LPS-induced increase in the TNF-α mRNA level was accompanied by NF-κB nuclear translocation, and both these effects were blocked by phenylephrine. An NF-κB inhibitor suppressed the LPS-induced increase in the TNF-α mRNA level. These results suggest that α1-ARs suppress the LPS-induced increase in the TNF-α mRNA level via inhibition of NF-κB nuclear translocation. Taken together, our study reveals that both α- and ß-ARs are involved in the transcriptional regulation of TNF-α in astrocytes.

Fibroblast growth factor 2 modulates extracellular purine metabolism by upregulating ecto-5'-nucleotidase and adenosine deaminase in cultured rat spinal cord astrocytes.

Purinergic signaling via ATP and adenosine produced by astrocytes is one pathway underlying neuron-glia interactions in the central nervous system (CNS). In production of purines, extracellular metabolism of released purines via ecto-enzymes is important. The expression and activities of these enzymes are altered under pathological conditions. Production of fibroblast growth factor 2 (FGF2) is increased under pathological conditions, and this has various effects on astrocytes. Here, we investigated the effects of FGF2 on purine metabolism in cultured rat spinal cord astrocytes. Astrocytes rapidly metabolized purines added to the extracellular solution. FGF2 increased extracellular metabolism of AMP to adenosine and of adenosine to inosine by upregulating ecto-5'-nucleotidase and adenosine deaminase (ADA), respectively. ADA activity and protein were detected both in the cytosol and external solution of astrocytes, and their levels were markedly increased by FGF2. FGF2 also increased metabolism of endogenously released ATP, resulting in a transient increase in adenosine and substantial accumulation of extracellular inosine. Moreover, FGF2 increased ATP release by upregulating the activity of gap junction hemichannels. These data show that FGF2 regulates purine production in astrocytes and suggest that extracellular ADA released by astrocytes plays an important role in extracellular purine metabolism in the CNS.

IL-1ß augments H2S-induced increase in intracellular Ca2+ through polysulfides generated from H2S/NO interaction.

H2S has excitatory and inhibitory effects on Ca2+ signals via transient receptor potential ankyrin 1 (TRPA1) and ATP-sensitive K+ channels, respectively. H2S converts intracellularly to polysulfides, which are more potent agonists for TRPA1 than H2S. Under inflammatory conditions, changes in the expression and activity of these H2S target channels and/or the conversion of H2S to polysulfides may modulate H2S effects. Effects of proinflammatory cytokines on H2S-induced Ca2+ signals and polysulfide production in RIN14B cells were examined using fluorescence imaging with fura-2 and SSP4, respectively. Na2S, a H2S donor, induced 1) the inhibition of spontaneous Ca2+ signals, 2) inhibition followed by [Ca2+]i increase, and 3) rapid [Ca2+]i increase without inhibition in 50% (23/46), 22% (10/46), and 17% (8/46) of cells tested, respectively. IL-1ß augmented H2S-induced [Ca2+]i increases, which were inhibited by TRPA1 and voltage-dependent L-type Ca2+ channel blockers. However, IL-1ß treatment did not affect [Ca2+]i increases evoked by a TRPA1 agonist or high concentration of KCl. Na2S increased intracellular polysulfide levels, which were enhanced by IL-1ß treatment. A NOS inhibitor suppressed the increased polysulfide production and [Ca2+]i increase in IL-1ß-treated cells. These results suggest that IL-1ß augments H2S-induced [Ca2+]i increases via the conversion of H2S to polysulfides through NO synthesis, but not via changes in the activity and expression of target channels. Polysulfides may play an important role in the effects of H2S during inflammation.

Different mechanisms of extracellular adenosine accumulation by reduction of the external Ca(2+) concentration and inhibition of adenosine metabolism in spinal astrocytes.

Extracellular adenosine is a neuromodulator in the central nervous system. Astrocytes mainly participate in adenosine production, and extracellular adenosine accumulates under physiological and pathophysiological conditions. Inhibition of intracellular adenosine metabolism and reduction of the external Ca(2+) concentration ([Ca(2+)]e) participate in adenosine accumulation, but the precise mechanisms remain unclear. This study investigated the mechanisms underlying extracellular adenosine accumulation in cultured rat spinal astrocytes. The combination of adenosine kinase and deaminase (ADK/ADA) inhibition and a reduced [Ca(2+)]e increased the extracellular adenosine level. ADK/ADA inhibitors increased the level of extracellular adenosine but not of adenine nucleotides, which was suppressed by inhibition of equilibrative nucleoside transporter (ENT) 2. Unlike ADK/ADA inhibition, a reduced [Ca(2+)]e increased the extracellular level not only of adenosine but also of ATP. This adenosine increase was enhanced by ENT2 inhibition, and suppressed by sodium polyoxotungstate (ecto-nucleoside triphosphate diphosphohydrolase inhibitor). Gap junction inhibitors suppressed the increases in adenosine and adenine nucleotide levels by reduction of [Ca(2+)]e. These results indicate that extracellular adenosine accumulation by ADK/ADA inhibition is due to the adenosine release via ENT2, while that by reduction of [Ca(2+)]e is due to breakdown of ATP released via gap junction hemichannels, after which ENT2 incorporates adenosine into the cells.

Usefulness of the tri-axial accelerometer for assessing balance function in children.

BACKGROUND: The aim of this study was to verify whether the tri-axial accelerometer can be used for quantitatively evaluating balance function in children. METHODS: In total, 198 participants, including 172 healthy children aged 3-11 years (87 boys, 85 girls) and 26 young adults aged 21-24 years (seven men, 19 women), were enrolled in this study. The participants undertook three types of balance tasks: quiet standing with eyes open and closed, one-leg standing on the dominant leg and non-dominant leg, and walking on the floor and a balance beam. We derived the root mean square from participants' accelerations measured by the tri-axial accelerometer. RESULTS: We found that for quiet standing, one-leg standing, and walking tasks, postural sway decreased with age. Girls controlled their posture better than boys of the same age on all tasks. There was a significant sex difference in quiet standing for children aged 8-9 years. Furthermore, sex differences existed in one-leg standing for children aged 5-11 years. A mild positive correlation was observed between static and dynamic balance. CONCLUSIONS: The tri-axial accelerometer is a useful quantitative tool for evaluating both static and dynamic balance function in children. Thus, it has the potential to be used clinically for diagnosis and rehabilitation.