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Microglia as a defensive arm of the nervous system emerged early in evolution. The surveilling microglia with motile and ramified processes are the main phenotype in the healthy CNS; the surveilling microglial patrol neuronal somata, dendrites, dendritic spines and axons. Increasing evidence suggests that microglia play fundamental roles in development, maturation and ageing of the brain, as well as contribute to a variety of physiological brain processes including sleep and circadian rhythm. Physiological state of microglia is tightly regulated by brain microenvironment and controlled by a sophisticated system of receptors and signalling cascades including ionotropic and metabotropic purinoceptors, pattern-recognition receptors, and receptors for chemokines and cytokines. Microglia also utilise ion channels and transporters in regulating ionic homeostasis and various aspects of microglial function. The major ion transporters expressed by microglia include Na + /H + exchanger 1 and Na + /Ca 2+ exchangers, which are involved in regulation of pH i and Ca 2+ homeostasis during microglial physiological responses. Microglial cells control development, maturation and plasticity of neuronal ensembles through controlled physiological phagocytosis of synapses or synaptic fragments - processes known as synaptic pruning and trogocytosis. This special issue on "Physiological roles of microglia" is an assembly of papers written by the leading experts in this research field. We start this special issue with this snapshot of microglial physiological functions as a prelude to the indepth discussion of microglia in physiological processes in the nervous system., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Microglial cells in normal mature brains have long been considered to be cells that are resting until pathological events take place, activating the microglial cells. However, it is currently well known that the microglia that have resting ramified morphology in normal mature brains move actively in the brain parenchyma and phagocytose synapses, thus forming and maintaining neural circuits. This review summarizes recent findings on the roles of microglia in mature brains, with special reference to phagocytosis of synapses and higher brain functions. Phagocytic elimination of synapses by microglia may affect the balance between excitatory and inhibitory synaptic transmission, termed the E/I balance. When impaired synaptic elimination by microglia leads to disturbed E/I balance, various problems may follow in brain functions: in memory and cognitive functions, sleep, movement, social behaviors, and thinking. In addition to the roles of microglia in normal developing and mature brains, impaired microglial phagocytosis functions also correlate with disturbances to these higher brain functions that are caused by neurological, mental, and developmental disorders; Parkinson's and Alzheimer's diseases, autism spectrum disorder, attention deficit/hyperactivity disorder, and schizophrenia., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

To investigate the effects of suppressors of cytokine signaling 3 (SOCS3) on neural stem cell fate, stem cells were infected with an adenoviral vector expressing SOCS3. Three days later, western blot analysis and immunocytochemical analysis revealed that the protein level of MAP2 and the number of MAP2-positive cells were significantly increased in SOCS3-transfected cells, whereas the protein level of GFAP and the number of GFAP-positive cells were significantly decreased. Furthermore, promoter assay revealed a significant reduction in the transcriptional level of signal transducer and activator of transcription 3 (Stat3) in the transfected cells. In addition, the mRNA levels of Notch family member (notch1) and inhibitory basic helix-loop-helix (bHLH) factors (hes5 and id3) were significantly up-regulated 1 day after overexpression of SOCS3. Three days after transfection, the mRNA level of hes5 was significantly decreased, whereas that of notch1 was still up-regulated. Moreover, all of SOCS3-positive cells expressed Nestin protein but did not express MAP2 or GFAP proteins. These data indicate that overexpression of SOCS3 induced neurogenesis and inhibited astrogliogenesis in neural stem cells. Our data also show that SOCS3 promoted maintenance of neural stem cells.

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb(1) (gRb(1)) (C(54)H(92)O(23), molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb(1)-induced expression of gene products responsible for neuronal death or survival, we showed that gRb(1) stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-x(L) in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb(1) treatment. Ginsenoside Rb(1) appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.

To investigate the effects of signal transducer and activator of transcription 3 (Stat3) on neural stem cell fate, stem cells were inoculated with an adenovirus vector expressing dominant negative form of Stat3 (Stat3F). One day later, a promoter assay revealed significant reduction of the transcriptional level in the transfected cells. Three days later, Western blot analysis and immunocytochemical analysis revealed that the protein level of microtubule-associated protein (MAP)2 and the number of MAP2-positive cells were increased significantly in the transfected cells whereas the protein level of glial fibrillary acidic protein (GFAP) and the number of GFAP-positive cells were decreased significantly. In addition, mRNA levels of Notch family members (Notch1, 2, and 3) and of inhibitory basic helix-loop-helix (bHLH) factors (Hes5, Id2, and Id3) were significantly downregulated at 3 days after viral inoculation with Stat3F; however, mRNA levels of bHLH determination factors (Math1 and Neurogenin3) and bHLH differentiation factors (NeuroD1 and NeuroD2) were significantly upregulated. These data indicated that suppression of Stat3 directly induced neurogenesis and inhibited astrogliogenesis in neural stem cells.

Microglia are considered the only cell population of mesodermal origin in the brain, although their role is not fully understood. The present study demonstrated that rat primary microglial cells expressed nestin, A2B5, and O4 antigens, which are markers for oligodendrocyte precursor cells. Based on these findings, we investigated whether microglial cells generated neurons or macroglial cells. Purified microglial cells were cultured in the presence of 10% fetal bovine serum for 3 days, followed by culture in the presence of 70% serum for 2 days. During the two-step culture, microglial cells became highly proliferative and strongly expressed inhibitor of DNA binding (Id) genes, indicative of dedifferentiation of the cells. The dedifferentiated cells also expressed transcription factors that promote differentiation into neurons or macroglial cells. When the dedifferentiated cells were transferred into serum-free medium on poly-L-lysine-coated substrate, a substantial number of the cells rapidly turned into long process-bearing cells, which expressed microtubule-associated protein 2, synapsin I, neurofilament proteins, glial fibrillary acidic protein, or galactocerebroside. When microglial cells were fluorescently labeled through acetylated low-density lipoprotein (LDL) receptors or by a phagocytosis-dependent mechanism, fluorescence-bearing neurons, astrocytes, or oligodendrocytes were observed. Neurospheres, aggregates of neural stem cells, expressed Musashi 1 and epidermal growth factor receptor, but the microglia-derived cells did not. These results suggest a novel role of microglia as multipotential stem cells to give rise to neurons, astrocytes, or oligodendrocytes., (Copyright 2003 Wiley-Liss, Inc.)

Recent research indicates that the novel multimodular adaptor protein EHSH1 plays an important role in a partnership between clathrin-mediated endocytosis and intracellular signaling pathways, including the MAPK pathway, receptor-tyrosine kinase/ras-mediated pathway, and the rho family of the GTPase-dependent pathway. We report the detailed expression pattern of EHSH1 in the rat CNS, using separate cultures of neurons, astrocytes, and microglia, and biochemical and immunohistochemical analyses. Cultured neurons from the cortex express primarily the long isoform EHSH1-l, as well as a small amount of the short isoform EHSH1-s. Cultured astrocytes express EHSH1-s, at a level similar to neurons, and a trace of EHSH1-l. Cultured microglia express only EHSH1-s. Double immunofluorescent staining of cortical sections showed that EHSH1 is expressed predominantly in neurons. These results suggest that EHSH1-l is a primary isoform and that EHSH1-l is highly enriched in neurons in the rat adult CNS. Immunohistochemistry of a series of brain sections revealed widespread distribution of EHSH1 throughout the brain. Particularly intense immunoreactivity was observed in the somatodendritic region of neurons in Layer III of the neocortex, hippocampus, globus pallidus, subthalamic nucleus, and substantia nigra. Interestingly, all pyramidal neurons in Layer III of the neocortex and hippocampus did not necessarily exhibit equal levels of immunostaining. In contrast, little EHSH1 immunoreactivity was detected in septofimbrial nucleus and subfornical organ of the septal region, and solitary tract and external cuneate nuclei of the medulla. Variety in the expression of EHSH1 in neurons of different regions may reflect different conditions in clathrin-mediated endocytosis and the following signal transduction., (Copyright 2002 Wiley-Liss, Inc.)

Erythropoietin (EPO) promotes neuronal survival after cerebral ischemia in vivo and after hypoxia in vitro. However, the mechanisms underlying the protective effects of EPO on ischemic/hypoxic neurons are not fully understood. The present in vitro experiments showed that EPO attenuated neuronal damage caused by chemical hypoxia at lower extracellular concentrations (10(- 4)-10(-2) U/ml) than were previously considered. Moreover, EPO at a concentration of 10(-3) U/ml up-regulated Bcl-xL mRNA and protein expressions in cultured neurons. Subsequent in vivo study focused on whether EPO rescued hippocampal CA1 neurons from lethal ischemic damage and up-regulated the expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils. EPO was infused into the cerebroventricles of gerbils immediately after 3 min of ischemia for 28 days. Infusion of EPO at a dose of 5 U/day prevented the occurrence of ischemia-induced learning disability. Subsequent light microscopic examinations showed that pyramidal neurons in the hippocampal CA1 field were significantly more numerous in ischemic gerbils infused with EPO (5 U/day) than in those receiving vehicle infusion. The same dose of EPO infusion caused significantly more intense expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils than did vehicle infusion. These findings suggest that EPO prevents delayed neuronal death in the hippocampal CA1 field, possibly through up-regulation of Bcl-xL, which is known to facilitate neuron survival.

Febrile seizures (FS) is the most common convulsive disorder in children, but there have been no clinical and experimental studies of the possible treatment of FS with herbal medicines, which are widely used in Asian countries. Paeoniflorin (PF) is a major bioactive component of Radix Paeoniae alba, and PF- containing herbal medicines have been used for neuromuscular, neuropsychiatric, and neurodegenerative disorders. In this study, we analyzed the anticonvulsive effect of PF and Keishikashakuyaku-to (KS; a PF-containing herbal medicine) for hyperthermia-induced seizures in immature rats as a model of human FS. When immature (P5) male rats were administered PF or KS for 10 days, hyperthermia- induced seizures were significantly suppressed compared to control rats. In cultured hippocampal neurons, PF suppressed glutamate-induced elevation of intracellular Ca2+ ([Ca2+]i), glutamate receptor-mediated membrane depolarization, and glutamate-induced neuronal death. In addition, PF partially suppressed the elevation in [Ca2+]i induced by activation of the metabotropic glutamate receptor 5 (mGluR5), but not that mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. However, PF did not affect production or release of γ-aminobutyric acid (GABA) in hippocampal neurons. These results suggest that PF or PF-containing herbal medicines exert anticonvulsive effects at least in part by preventing mGluR5-dependent [Ca2+]i elevations. Thus, it could be a possible candidate for the treatment of FS in children. [ABSTRACT FROM AUTHOR]

To investigate the effects of suppressors of cytokine signaling 3 (SOCS3) on neural stem cell fate, stem cells were infected with an adenoviral vector expressing SOCS3. Three days later, western blot analysis and immunocytochemical analysis revealed that the protein level of MAP2 and the number of MAP2-positive cells were significantly increased in SOCS3-transfected cells, whereas the protein level of GFAP and the number of GFAP-positive cells were significantly decreased. Furthermore, promoter assay revealed a significant reduction in the transcriptional level of signal transducer and activator of transcription 3 (Stat3) in the transfected cells. In addition, the mRNA levels of Notch family member (notch1) and inhibitory basic helix-loop-helix (bHLH) factors (hes5 and id3) were significantly up-regulated 1 day after overexpression of SOCS3. Three days after transfection, the mRNA level of hes5 was significantly decreased, whereas that of notch1 was still up-regulated. Moreover, all of SOCS3-positive cells expressed Nestin protein but did not express MAP2 or GFAP proteins. These data indicate that overexpression of SOCS3 induced neurogenesis and inhibited astrogliogenesis in neural stem cells. Our data also show that SOCS3 promoted maintenance of neural stem cells. [ABSTRACT FROM AUTHOR]

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb1 (gRb1) (C54H92O23, molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb1-induced expression of gene products responsible for neuronal death or survival, we showed that gRb1 stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-xL in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb1 treatment. Ginsenoside Rb1 appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.Journal of Cerebral Blood Flow & Metabolism (2006) 26, 708–721. doi:10.1038/sj.jcbfm.9600225; published online 14 September 2005 [ABSTRACT FROM AUTHOR]

Abstract Parkinson's disease (PD) is a frequent neurodegenerative disease causing bradykinesia, tremor, muscle rigidity and postural instability. Although its main pathology is progressive dopaminergic (DArgic) neuron loss in the substantia nigra, motor deficits are thought not to become apparent until most DArgic neurons are lost, probably due to compensatory mechanisms that overcome the decline of DA level in the striatum. Even in animal PD models, it is difficult to detect motor deficits when most DArgic neurons are functional. In this study, we performed various behavioral tests (apomorphine-induced rotation, cylinder, forepaw adjustment steps (FAS), beam walking, rota-rod, and open-field), using 6-hydroxydopamine (OHDA) and lipopolysaccharide (LPS)-induced hemi-PD model rats with various striatal DA levels, to find the best way to predict the DA level from earlier disease stages. Different from the 6-OHDA-induced model, reduction in the striatal DA levels in the LPS-model was less significant. Among the behavioral tests, data from cylinder and FAS tests, which evaluate forelimb movements, best correlated with decline of the DA level. They also correlated well with decreased body weight gain. The beam and apomorphine tests showed less significant correlation than the cylinder and FAS tests. Open-field and rota-rod tests were not useful. Expressional levels of mRNA encoding tyrosine hydroxylase (TH), a marker of DArgic neurons, correlated well with the DA level. Metabotropic glutamate receptor 4 mRNA expression correlated with the striatal DA level and may be related to compensatory mechanisms. These results suggest that motor impairments of PD should be evaluated by forelimb movements, or hands and forearms in clinical settings, rather than movement of the body or large joints. The combination of cylinder and FAS tests may be the best to evaluate the rat PD models, in which many DArgic neurons survive. Graphical abstract Image 1 Highlights • The 6-OHDA- and LPS-induced Parkinson's disease (PD) rat models were used. • Cylinder and FAS tests are suitable to evaluate motor deficits of forelimbs. • Forelimb impairments correlated linearly with striatal dopamine (DA) decline. • Apomorphine-induced rotation was apparent when DArgic neurons were totally lost. • For early detection of PD, examination of hand and forearm movements is suitable. [ABSTRACT FROM AUTHOR]