Physiology of microglial cells

Microglial cells in culture and in situ express a defined pattern of K⁺ channels, which is distinct from that of other glial cells and neurons. This pattern undergoes defined changes with microglial activation. As expected for a cell with immunological properties, microglia express a variety of cytokine and chemokine receptors, which are linked to the mobilization of Ca²⁺ (cytosolic free calcium) from internal stores. Microglial cells also have the capacity to respond to neuronal activity: they express receptors for the major excitatory receptor glutamate and the main inhibitory receptor GABA (γ-amino butyric acid). By expressing purinergic receptors, microglia can sense astrocyte activity in the form of Ca²⁺ waves. Activation of transmitter receptors can affect cytokine release which is a potential means as to how brain activity can affect immune function.     

Induction of resting microglia in culture medium devoid of glycine and serine

Cultured microglial cells usually exhibit ameboid morphology and peripheral macrophage-like properties, which are distinct from those observed in the normal mature brain. This might be caused by the inappropriate culture of microglial cells in high concentrations (∼200–400 μM) of Gly and Ser, although the concentrations of the amino acids in extracellular spaces of the brain parenchyma are quite low (∼5 μM). In the present study, we focused on the concentration-dependent effects of glycine (Gly) and serine (Ser) on microglial morphology and function. Under Gly/Ser-free and serum-free condition, the majority of rat microglial cells displayed round morphology, whereas in the presence of 5 μM Gly and 25 μM Ser, which correspond to the concentrations of Gly and Ser in the cerebrospinal fluid, they extended multiple branched processes and formed clusters of rough endoplasmic reticulum. On the other hand, Gly and Ser did not affect morphology of astrocytes. The viability of microglia was not affected by the changes in the concentrations of Gly and Ser. Metabolic activity, activities of acid phosphatase and inducible nitric oxide synthase, and superoxide anion (O₂^-) generation were all strongly suppressed in Gly/Ser-free medium or in medium containing physiological concentrations of both amino acids. Such activities were all enhanced in harmony with increases in the concentrations of Gly and Ser. Thus, microglial cells cultured in Gly/Ser-free medium, even though exhibiting ameboid morphology, appears to be in the functionally resting state. Furthermore, once the resting state was achieved, the microglial cells remained inactive even after the subsequent 24 h culture in serum-supplemented medium containing 400 μM of both amino acids. The medium conditioned by microglial cells that were cultured in the presence of 400 μM of Gly and Ser was toxic to cortical neurons, whereas the microglia-conditioned medium obtained in the absence of both amino acids facilitated the survival of cortical neurons. Therefore, microglial cells in the resting state, which was induced in the Gly/Ser-free condition, are likely to support neurons. Microglial cells could ramify on glass coverslips coated with astrocyte-derived extracellular matrix or on coverslips coated thinly with fibronectin and/or laminin even under the Gly/Ser-free condition. The ramified cells as induced in this way kept suppressed O₂^- generating activity. These findings suggest that resting ramified microglial cells with a neurotrophic activity can be induced with the combination of Gly/Ser-free medium and small amounts of extracellular matrix proteins, and that the resting state is rather stable. GLIA 24:198–215, 1998. © 1998 Wiley-Liss, Inc.     

Differential regulation of microglial activation by propentofylline via cAMP signaling

A pathological microglial activation is believed to contribute to progressive neuronal damage in neurodegenerative diseases by the release of potentially toxic agents and by triggering reactive astrocytic changes. Using cultured microglia from neonatal rat brains, we investigated the mode of propentofylline action in strengthening cAMP-dependent intracellular signaling. We compared this action with the effects of dibutyryl-cAMP, a cell-permeable cAMP analog. Propentofylline inhibited lipopolysaccharide (LPS)-induced release of both tumor necrosis factor (TNF)-α and interleukin (IL)-1β in a dose-dependent manner within the therapeutic low micromolar range. However, LPS-induced release of IL-6 and NO were not affected by propentofylline. All these differential effects of propentofylline on LPS-induced microglial release were mimicked by the addition of dibutyryl-cAMP. Microglial proliferation and phorbol myristate acetate (PMA)-induced O₂⁻ release were also dose-dependently inhibited by propentofylline as well as dibutyryl-cAMP. These results suggest that propentofylline, probably via reinforcement of cAMP intracellular signaling, alters the profile of the newly adopted immune properties in a way that it inhibits potentially neurotoxic functions while maintaining beneficial functions. This differential regulation of microglial activation may explain the neuroprotective mechanism exerted by propentofylline.     

Microglial proliferation and monocyte infiltration contribute to microgliosis following status epilepticus

Microglial activation has been recognized as a major contributor to inflammation of the epileptic brain. Seizures are commonly accompanied by remarkable microgliosis and loss of neurons. In this study, we utilize the CX3CR1^GFP/+ CCR2^RFP/+ genetic mouse model, in which CX3CR1⁺ resident microglia and CCR2⁺ monocytes are labeled with GFP and RFP, respectively. Using a combination of time-lapse two-photon imaging and whole-cell patch clamp recording, we determined the distinct morphological, dynamic, and electrophysiological characteristics of infiltrated monocytes and resident microglia, and the evolution of their behavior at different time points following kainic acid-induced seizures. Seizure activated microglia presented enlarged somas with less ramified processes, whereas, infiltrated monocytes were smaller, highly motile cells that lacked processes. Moreover, resident microglia, but not infiltrated monocytes, proliferate locally in the hippocampus after seizure. Microglial proliferation was dependent on the colony-stimulating factor 1 receptor (CSF-1R) pathway. Pharmacological inhibition of CSF-1R reduced seizure-induced microglial proliferation, which correlated with attenuation of neuronal death without altering acute seizure behaviors. Taken together, we demonstrated that proliferation of activated resident microglia contributes to neuronal death in the hippocampus via CSF-1R after status epilepticus, providing potential therapeutic targets for neuroprotection in epilepsy.     

Sialic acid on the neuronal glycocalyx prevents complement C1 binding and complement receptor-3-mediated removal by microglia

Microglial cells are professional phagocytes of the CNS responsible for clearance of unwanted structures. Neuronal processes are marked by complement C1 before they are removed in development or during disease processes. Target molecules involved in C1 binding and mechanisms of clearance are still unclear. Here we show that the terminal sugar residue sialic acid of the mouse neuronal glycocalyx determines complement C1 binding and microglial-mediated clearance function. Several early components of the classical complement cascade including C1q, C1r, C1s, and C3 were produced by cultured mouse microglia. The opsonin C1q was binding to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx. Desialylated neurites, but not neurites with intact sialic acid caps, were cleared and taken up by cocultured microglial cells. The removal of the desialylated neurites was mediated via the complement receptor-3 (CR3; CD11b/CD18). Data demonstrate that mouse microglial cells via CR3 recognize and remove neuronal structures with an altered neuronal glycocalyx lacking terminal sialic acid.     

Decreased neuronal CD200 expression in IL-4-deficient mice results in increased neuroinflammation in response to lipopolysaccharide

Maintenance of the balance between pro- and anti-inflammatory cytokines in the brain, which is affected by the activation state of microglia, is important for maintenance of neuronal function. Evidence has suggested that IL-4 plays an important neuromodulatory role and has the ability to decrease lipopolysaccharide-induced microglial activation and the production of IL-1β. We have also demonstrated that CD200–CD200R interaction is involved in immune homeostasis in the brain. Here, we investigated the anti-inflammatory role of IL-4 and, using in vitro and in vivo analysis, established that the effect of lipopolysaccharide was more profound in IL-4^−/−, compared with wildtype, mice. Intraperitoneal injection of lipopolysaccharide exerted a greater inhibitory effect on exploratory behaviour in IL-4^−/−, compared with wildtype, mice and this was associated with evidence of microglial activation. We demonstrate that the increase in microglial activation is inversely related to CD200 expression. Furthermore, CD200 was decreased in neurons prepared from IL-4^−/− mice, whereas stimulation with IL-4 enhanced CD200 expression. Importantly, neurons prepared from wildtype, but not from IL-4^−/−, mice attenuated the lipopolysaccharide-induced increase in pro-inflammatory cytokine production by glia. These findings suggest that the neuromodulatory effect of IL-4, and in particular its capacity to maintain microglia in a quiescent state, may result from its ability to upregulate CD200 expression on neurons.     

Neuroprotective and toxic changes in microglia in neurodegenerative disease

Microglia are macrophage-like cells in the CNS. As macrophages, activated microglia remove potentially deleterious debris and promote tissue repair. However, they can release potentially cytotoxic substances in vitro. So-called fully activated microglia, observed at the injury site in many neurodegenerative conditions, are neurotoxic. This suggests that some factor(s) may contribute to change microglial phenotype from protective to toxic, but details are not clear. Recently, we generated HIV-derived Nef protein-transduced microglia. They increase the potential to produce O^?₂ and MPO-like peroxidase activity, resulting in neurotoxicity. Therefore, the target protein(s) of Nef might be involved in the control of microglial neurotoxicity.     

一种高产量的小胶质细胞培养及其鉴定方法

目的建立一种简易高产量的原代小胶质细胞体外纯化培养方法.方法在小胶质细胞原代培养过程中以McCarthy等的经典培养方法为基础,通过提高初次接种密度,减少细胞离心过滤过程,进行不换液的营养缺失培养等重要改进,并使用低浓度胰酶-EDTA消化法分离纯化小胶质细胞,使用MAC-1抗体免疫化学染色标记.结果取得了高纯度和高产量的小胶质细胞.结论小胶质细胞培养过程中联合使用营养缺失法和低浓度胰酶-EDTA消化法分离纯化可有效提高培养产量.     

Resting microglial cells in vitro: Analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures

Neurons in organotypic hippocampal slice cultures (OHSCs) are known to preserve morphological and physiological features of the in vivo situation; however, little is known about the properties of microglial cells under these in vitro conditions. In this study, we addressed the question whether microglial cells in OHSCs are initially activated following explantation but return to a resting state during in vitro cultivation. Thus, we analyzed a) microglial cell morphology, b) microglial cell distribution, and c) expression of integrin adhesion molecules as putative markers of microglial activation. Hippocampal slices fixed immediately following explantation showed only resting microglial cells, mainly located in the paraventricular regions. After 3 days in vitro (div) OHSC surfaces were covered by activated microglia, whereas intermediate layers contained fewer microglial cells, giving the slices a sandwich-like appearance with the intact hippocampal formation being surrounded by glial tissue. After 3 div, microglial cells in intermediate layers of OHSCs showed activated morphology with ovaloid cytoplasm and no or merely few cytoplasmic processes; after 6 div, however, an increasing degree of ramification could be observed. After 9 div, microglia in intermediate layers had almost regained the morphological appearance of resting cells with filigrane cytoplasmic processes extended in all directions. The integrin adhesion molecules LFA-1 (α and β chains) and VLA-4 were expressed on most microglial cells with activated morphology, as verified by co-localization with double immunofluorescence labeling for LFA-1 or VLA-4 and Griffonia simplicifolia isolectin B₄ (GFS-B₄). In contrast, only low levels of integrin adhesion molecule expression were also found on reactive astrocytes along slice surfaces. However, LFA-1 or VLA-4 were never found on ramified microglial cells, and double immunofluorescence labeling of LFA-1 or VLA-4 with ramified GFS-B₄⁺ microglia never occurred. We conclude that a) originally resting microglial cells activated in an early phase of in vitro culture but regain a resting status after at least 6 div; and b) integrin adhesion molecules LFA1 and VLA-4 are potential markers of microglial activation, as they were found on activated but never on resting microglial cells. This enables further investigations on immunological and electrophysiological features of resting and activated microglial cells under in vitro conditions. © 1996 Wiley-Liss, Inc.     

Serotonin stimulates secretion of exosomes from microglia cells

Microglia are resident immune cells in the brain and exert important functions in the regulation of inflammatory processes during infection or cellular damage. Upon activation, microglia undergo complex morphological and functional transitions, including increased motility, phagocytosis and cytokine secretion. Recent findings indicate that exosomes, small vesicles that derive from fusion of multivesicular bodies with the plasma membrane, are involved in secretion of certain cytokines. The presence of specific receptors on the surface of microglia suggests communication with neurons by neurotransmitters. Here, we demonstrate expression of serotonin receptors, including 5‐HT_2a,b and 5‐HT₄ in microglial cells and their functional involvement in the modulation of exosome release by serotonin. Our data demonstrate the involvement of cAMP and Ca²⁺ dependent signaling pathways in the regulation of exosome secretion. Co‐culture of microglia with embryonic stem cell‐derived serotonergic neurons further demonstrated functional signaling between neurons and microglia. Together, these data provide evidence for neurotransmitter‐dependent signaling pathways in microglial cells that regulate exosome release. GLIA 2015;63:626–634     

Vitamin E suppression of microglial activation is neuroprotective

Neurotoxic microglial-neuronal interactions have been implicated in the pathogenesis of various neurodegenerative diseases such as Alzheimer's disease, and vitamin E has been shown to have direct neuroprotective effects. To determine whether vitamin E also has indirect neuroprotective effects through suppression of microglial activation, we used a microglial-neuronal coculture. Lipopolysaccharide (LPS) treatment of a microglial cell line (N9) induced a time-dependent activation of both p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappaB (NFkappaB), with consequent increases in interleukin-1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production. Differentiated neuronal cells (PC12 cells treated with nerve growth factor) exhibited marked loss of processes and decreased survival when cocultured with LPS-activated microglia. Preincubation of microglia with vitamin E diminished this neurotoxic effect, independently of direct effects of the antioxidant on the neuronal cells. Microglial NO production and the induction of IL-1alpha and TNFalpha expression also were attenuated by vitamin E. Such antiinflammatory effects of vitamin E were correlated with suppression of p38 MAPK and NFkappaB activation and were mimicked by an inhibition of either p38 MAPK (by SB203580) or NFkappaB (by decoy oligonucleotides). These results suggest that, in addition to the beneficial effects of providing direct antioxidant protection to neurons reported by others, vitamin E may provide neuroprotection in vivo through suppression of signaling events necessary for microglial activation.     

The effect of microglia on embryonic dopaminergic neuronal survival in vitro: diffusible signals from neurons and glia change microglia from neurotoxic to neuroprotective

When embryonic dopaminergic neurons are transplanted into the adult brain, approximately 95% die within a few days. To assess whether microglia activated during transplantation might be responsible for this rapid death, we examined the effect of microglia on rat embryonic dopaminergic neurons in vitro. Conditioned medium from 7-day-old microglia was found to decrease the number of dopamine neurons surviving in primary culture, but activation of the microglia with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or Zymosan A did not increase the toxicity of the conditioned medium. We next tested the effect of coculturing microglia and dopaminergic neurons by placing microglia in semipermeable well inserts over the neuronal cultures. The presence of microglia now increased dopaminergic neuronal survival, microglial activation again having no effect. To increase yet further the possible interactions between microglia and neurons, the mesencephalic cells and microglia were mixed together and placed as a tissue in three-dimensional culture, and here again the presence of microglia increased dopaminergic neuronal survival with no effect of activation. Contact of microglia with the mesencephalic cells therefore converted them from being toxic to dopaminergic neurons to promoting their survival. The change in microglial effect from toxic to protective was caused by soluble molecules secreted by cells in the neuronal cultures, as conditioned medium derived from microglia-neuronal cocultures also had a dopaminergic neuron survival effect, indicating that microglia in cocultures behave differently from microglia removed from neuronal and glial influence. Microglia cocultured with either neurons or astrocytes downregulated inducible nitric oxide synthase (iNOS), indicating a decrease in the production of nitric oxide and possibly other toxic molecules. These findings indicate that in their natural environment, microglia are likely to be beneficial for the survival of embryonic dopaminergic grafts.     

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1β secretion. Both caspase-1 and IL-1β contribute to disease progression in the mouse SOD1^G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1^G93A mice microglia do not express NLRP3, and SOD1^G93A protein generated IL-1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1^G93A mice. We show that both aggregated and soluble SOD1^G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1β cleavage, ASC speck formation, and the secretion of IL-1β in a dose- and time-dependent manner. Importantly, SOD1^G93A was unable to induce IL-1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1β secretion. Microglial NLRP3 upregulation was also observed in the TDP-43^Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1^G93A-mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.     

Neuronal gamma oscillations and activity-dependent potassium transients remain regular after depletion of microglia in postnatal cortex tissue

Microglial cells (resident macrophages) feature rapid activation in CNS disease and can acquire multiple phenotypes exerting neuroprotection or neurotoxicity. The functional impact of surveying (“resting”) microglia on neural excitability and neurotransmission in physiology is widely unknown, however. We addressed this issue in male rat hippocampal slice cultures (in situ) by pharmacological microglial ablation within days and by characterizing neuronal gamma-band oscillations (30–70 Hz) that are highly sensitive to neuromodulators and disturbances in ion and energy regulation. Gamma oscillations support action potential timing and synaptic plasticity, associate with higher brain functions like perception and memory, and require precise communication between excitatory pyramidal cells and inhibitory (GABAergic) interneurons. The slice cultures featured well-preserved hippocampal cytoarchitecture and parvalbumin-positive interneuron networks, microglia with ramified morphology, and low basal levels of IL-6, TNF-α, and nitric oxide (NO). Stimulation of slice cultures with the pro-inflammatory cytokine IFN-γ or bacterial LPS serving as positive controls for microglial reactivity induced MHC-II expression and increased cytokine and NO release. Chronic exposure of slice cultures to liposome-encapsulated clodronate reduced the microglial cell population by about 96%, whereas neuronal structures, astrocyte GFAP expression, and basal levels of cytokines and NO were unchanged. Notably, the properties of gamma oscillations reflecting frequency, number and synchronization of synapse activity were regular after microglial depletion. Also, electrical stimulus-induced transients of the extracellular potassium concentration ([K⁺]_o) reflecting cellular K⁺ efflux, clearance and buffering were unchanged. This suggests that nonreactive microglia are dispensable for neuronal homeostasis and neuromodulation underlying network signaling and rhythm generation in cortical tissue.     

Localization and characterization of epidermal growth-factor receptors in the developing rat medial septal area in culture

The presence and binding properties of epidermal growth-factor receptors (EGF-Rs) in different cell types purified from the rat medial septal area in culture were investigated. We report that astrocytes, oligodendrocytes and neurons from this area possess EGF-Rs while microglia do not. EGF-binding sites are detectable on astrocytes derived from the medial septum of both embryonic and neonatal rats. Scatchard analysis of the data for astrocytes from the fetal rats show that EGF specifically binds to both high- (K_d = 7.21 × 10⁻¹⁰ M, B_max = 3602 receptors/cell) and low-affinity (K_d = 3.99 × 10⁻⁸ 10⁻⁸ M, B_max = 6,265 receptors/cell) receptors on these cells. On the other hand, astrocytes purified from neonatal tissue possess a greater number of high-affinity receptors (B_max = 10,938 receptors/cell) when compared with the embryonic astroglia. With time in culture, the number of both types of receptors on neonatal astrocytes decreases. Oligodendrocytes also possess high- and low-affinity EGF-Rs with dissociation constants of 3.25 × 10⁻¹⁰ M and 3.85 × 10⁻⁸ M, respectively. The number of receptors on oligodendrocytes is significantly lower than those on neonatal astrocytes (B_max = 1185 and 25,081 receptors/cell for high- and low-affinity binding sites, respectively). Finally, neurons from this area also exhibit two different EGF-R types with dissociation constants similar to those described for astrocytes. As the number of receptors/neuron (B_max = 136 and 1159 receptors/cell for high- and low-affinity binding sites, respectively) appears to be extremely low, it is possible that EGF specifically binds only to a subpopulation of neurons from this area. These studies demonstrate which cell types in the developing medial sepal area posses EGF-Rs and provide a detailed characterization of these binding sites. These EGF-R-bearing cells may be potential targets for this growth factor or for transforming growth factor α in this brain area.     

The A1 adenosine receptor as a new player in microglia physiology

The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P₂X_4,7 and P₂Y₁₂ receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2_A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca²⁺ influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive‐specific neuron before and after application of resting or ATP‐treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone. GLIA 2014;62:122–132     

Microglia activation influences dye coupling and Cx43 expression of the astrocytic network

Under inflammatory conditions, activated microglia are capable of producing proinflammatory cytokines that are reported to influence cell-to-cell communication. The present study was performed to evaluate the influence of microglial activation on the coupling efficiency of the astroglial network. Primary astrocyte cultures of newborn rats were cocultured with either 5% (M5) or 30% (M30) microglia. Microglial activation (rounded phagocytotic phenotype) was investigated using the monoclonal anti-ED1 antibody, and immunofluorescence with a polyclonal anti-Cx43 antibody was used to study astroglial Cx43 expression and distribution. Functional coupling of astrocytes was evaluated by monitoring the transfer of microinjected Lucifer yellow into neighboring cells. The data obtained can be summarized as follows: astroglia/M30 cocultures contained significantly fewer resting microglia and significantly more activated microglia than the M5 cocultures; significantly reduced astroglial Cx43 staining was found in M30 cocultures concurrently with a reduced number of dye coupled astrocytes; and the positive correlation of percent activated microglia with reduced astroglial Cx43 expression was highly significant, indicating that the degree of intercellular communication in the astroglial network may be modulated by the activation of microglia under in vitro conditions.     

Blood monocytes and spleen macrophages differentiate into microglia-like cells on monolayers of astrocytes: Membrane currents

Microglia, the resident macrophages of the central nervous system (CNS), can be distinguished from most other cells of the myelomonocytic lineage by a distinct pattern of memberane currents. In the accompanying paper we have shown that the characteristic morphological feature of microglia, ramification, develops both in microglia and other myelomonocytic cells when they are cocultured with astrocytes. We therefore propose that the electrophysiological properties of microglia also develop under the influence of astrocytes, and moreover, that these properties can also be induced in other cells of the myelomonocytic lineage. Microglia cultured on poly-d-lysine or on a monolayer of fibroblasts possess an inwardly rectifying K⁺ -current only, which is of composite nature. In single-channel recordings two types of K⁺ -channels are found: (i) a non-inactivating channel with a conductance of 43pS, and (ii) an inactivating channel with 32pS. Microglia cultured on a monolayer of astrocytes additionally develop an outward K⁺ -current and a Na⁺ -current. The electric parameters of activation and inactivation of the microglial Na⁺ -current are identical to those of the neuronal Na⁺ -current. Monocytes from peripheral blood and macrophages from spleen exhibit no inward currents. However, when these cells are cocultured with astrocytes they develop microglia-like membrane currents, including the inward and outward K⁺ -rectifyer and the Na⁺ -current. By contrast, on fibroblasts they retain their macrophage current profile. The expression of the microglia-like membrane currents in the monounclear phagocytes is induced by a diffusible factor released from the astrocytes into the culture medium, since monocytes and microglia develop the mature microglial current profile, when cultured in astrocyte conditioned medium. These findings show that the current profile of microglia develops only, when they are in association with astrocytes, and that it is induced in myelomonocytic cells from blood and spleen when these also are associated with astrocytes. These findings add to the growing body of evidence that microglia are derived from the myelmonocytic lineage and indicate that the astrocytes are involved in regulating their morphological and functional properties.     

Purification of mouse schwann cells using neurite-induced proliferation in serum-free monolayer culture

We have recently reported that neonatal mouse dorsal root ganglionic Schwann cells will (i) survive and assume characteristic morphologies in a serum-free, fully defined culture medium (N1 medium), (ii) proliferate extensively in the same N1 medium if neurons are also present and maintained by nerve growth factor, and (iii) display a strong proliferative response to serum even in the absence of neuronal elements, while also undergoing marked changes in their morphology and their associative behavior toward neurites. In this report, we present a detailed procedure, based upon these earlier observations, which yields purified cultures of either neurons plus associated Schwann cells or Schwann cells in the absence of neurons. The procedure utilizes the neuritic mitogen for selective expansion of Schwann cell numbers in serum-free primary cultures, and a secondary culture step involving neuronal removal and additional Schwann cell expansion using the serum mitogen. The procedure requires 9 days for the generation of3−4 × 10⁶ Schwann cells from 12 newborn mice (with a Schwann cell to neuron ratio of 10) and an additional 6–7 days for the generation of a neuron-free secondary population of40 × 10⁶ Schwann cells with less than 3% contamination by identifiable ganglionic fibroblasts.     

Calcium ion influx in microglial cells: Physiological and therapeutic significance

Microglial cells, the immunocompetent cells of the central nervous system (CNS), exhibit a resting phenotype under healthy conditions. In response to injury, however, they transform into an activated state, which is a hallmark feature of many CNS diseases. Factors or agents released from the neurons, blood vessels, and/or astrocytes could activate these cells, leading to their functional and structural modifications. Microglial cells are well equipped to sense environmental changes within the brain under both physiological and pathological conditions. Entry of calcium ions (Ca²⁺) plays a critical role in the process of microglial transformation; several channels and receptors have been identified on the surface of microglial cells. These include store‐operated channel, Orai1, and its sensor protein, stromal interaction molecule 1 (STIM1), in microglial cells, and their functions are modulated under pathological stimulations. Transient receptor potential (TRP) channels and voltage‐ and ligand‐gated channels (ionotropic and metabotropic receptors) are also responsible for Ca²⁺ influx into the microglial cells. An elevation of intracellular Ca²⁺ concentration subsequently regulates microglial cell functions by activating a diverse array of Ca²⁺‐sensitive signaling cascades. Perturbed Ca²⁺ homeostasis contributes to the progression of a number of CNS disorders. Thus, regulation of Ca²⁺ entry into microglial cells could be a pharmacological target for several CNS‐related pathological conditions. This Review addresses the recent insights into microglial cell Ca²⁺ influx mechanisms, their roles in the regulation of functions, and alterations of Ca²⁺ entry in specific CNS disorders. © 2014 Wiley Periodicals, Inc.