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Neuroimmune-Mediated Alcohol Effects on Ventral Tegmental Area NeuronsWilliams, Stephanie Bair 01 April 2018 (has links)
Dopamine (DA) transmission is a key player in the rewarding aspects of ethanol as well as ethanol dependence. The current dogma is that DA transmission is increased during ethanol via the inhibition of ventral tegmental area (VTA) GABA neurons and that excitation of VTA GABA neurons during withdrawal results in decreased DA transmission. Microglia, the major neuroimmune effector in the brain, may be a key mediator in this process by releasing cytokines following activation. We evaluated the effect of ethanol on cytokine concentrations in the VTA and NAc using a cytometric bead array, and found that low dose ethanol (1.0 g/kg) decreased interleukin (IL)-10 levels, but high dose ethanol increased IL-10 levels (4.0 g/kg). We also used standard cell-attached mode electrophysiological techniques to evaluate the effects of select cytokines on VTA neuron firing rate in vitro. We found no change in firing rate in response to IL-6, but an increase in firing rate in VTA DA neurons response to IL-10. Consistent with the changes in firing rate, optically-evoked IPSCs were also found to be decreased in response to IL-10. Ex vivo voltammetry and in vivo microdialysis were done to determine whether IL-10 can directly result in an increase in DA release. Although ex vivo voltammetry showed no change in DA release, IL-10 increased DA release in vivo. These findings suggest that the rewarding and/or addictive effects of ethanol are mediated by cytokines, specifically the anti-inflammatory cytokine IL-10.
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Neuroimmune-Mediated Alcohol Effects on Ventral Tegmental Area Microglia and Infiltrating LeukocytesClarke, Travis Jonathan 01 August 2018 (has links)
Microglia are the primary immune cell in the central nervous system and are known as the “resident” macrophages in the central nervous system (CNS). While microglia are classically known as the immune cells of the CNS, their role has more recently been shown to extend far beyond immunity. The effects of ethanol on the brain are closely linked to neuroimmune responses mediated by microglia that are present in the healthy brain from the time of development. Though microglia have been classified as the “resident” immune cells of the CNS, new research suggests that other immune cells may be implicated in the immune response. Normally, the blood-brain barrier (BBB) prevents the infiltration of cells and foreign pathogens from crossing from the periphery into the CNS. However, peripheral monocytes are known to infiltrate the CNS in response to seizures, traumatic brain injury, infection, and multiple sclerosis. Whether or not these cells engraft and become microglia is still a topic of debate. The aim of this study was to determine the effect of acute ethanol on microglia activation and monocyte infiltration into the CNS. We hypothesized that acute EtOH would lead to an increase in neuroinflammation by activating “resident” microglia to an inflammatory polarization and induce the infiltration of macrophages across the BBB. Using the Macrophage FAS-Induced Apoptosis (MaFIA) mouse model (GFP+ on Csf1r promoter), fluorescent microscopy, and flow cytometry we assessed the presence and phenotype of microglia and infiltrating macrophages following 1, 2, and 4 g/kg ethanol at .5, 1, and 2 hours post-injection. By measuring volume/surface area of microglia in the VTA and NAc following EtOH, we found that EtOH caused microglia activation in these areas, and that the microglia are shifting toward an M1 polarization. However, some of our findings were counter to our hypothesis. We found that EtOH, decreases the number of infiltrating monocytes in the VTA and NAc. It is possible that other cells like T and B cells are recruited across the BBB. These findings suggest a neuroimmune connection for acute ethanol use and challenge the dogma that ethanol has exclusively central effects on DA neuronal activity and release. Further research is being performed to examine the implications of this effect, and what effects a conditional knockdown of monocytes has on ethanol intoxication and reward.
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Amyloid plaque deposition accelerates tau propagation via activation of microglia in a humanized app mouse modelClayton, Kevin A. 17 June 2021 (has links)
Alzheimer’s disease is characterized by the formation of two major pathological hallmarks: amyloid plaques and neurofibrillary tangles. Although there have been many studies to understand the role of microglia in Alzheimer’s disease, it is not yet known how microglia can promote disease progression while actively phagocytosing amyloid plaques or phosphorylated tau (p-tau). Through stereotaxic injection of adeno-associated virus expressing mutant P301L tau (AAV-P301L-tau) into the medial entorhinal cortex (MEC) of both wild-type (WT) and APPNL-G-F mice, we demonstrate how amyloid plaques exacerbate p-tau propagation to the granule cell layer (GCL) of the hippocampus. However, in mice receiving the colony-stimulating factor 1 receptor inhibitor (PLX5622), ~95% of microglia were depleted, which dramatically reduced p-tau propagation to the GCL. Although microglia depletion curtailed p-tau propagation, it also led to reduced plaque compaction and an increase in overall amyloid-beta (Aβ) plaque presence. Additionally, we found microglia depletion resulted in greater p-tau aggregation in dystrophic neurites surrounding amyloid plaques. We investigated neurodegenerative microglia (MGnD), which are activated in response to amyloid plaques, for their propensity to release extracellular vesicles in comparison to homeostatic microglia. We discovered that MGnD, identified by Clec7a or Mac2 staining, strongly express Tumor susceptibility gene 101 (Tsg101), which is an ESCRT-1 protein and a marker for extracellular vesicles (EVs). To further investigate EV release and MGnD, a novel lentivirus expressing fluorescent mEmerald conjugated to CD9 (mE-CD9) was constructed and injected into the MEC of both WT and APPNL-G-F mice which allowed for visualization of mE-CD9+ puncta around individual microglia. CD9 is a tetraspanin and also a marker for EVs. We observed that the number of mEmerald+ particles surrounding MGnD was three-fold higher compared to non-diseased, homeostatic microglia. Sequential injection of mE-CD9 and AAV-P301L-tau into the MEC revealed that microglia-derived EVs encapsulate pathologic p-tau, which is augmented by the MGnD phenotype. Taken together, these data provide strong evidence that MGnD exhibit increased secretion of tau-containing EVs, providing a possible mechanism for how amyloid deposition indirectly exacerbates tau propagation.
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Memory-Like Responses of Brain Microglia Are Controlled by Developmental State and Pathogen DoseLajqi, Trim, Stojiljkovic, Milan, Williams, David L., Hudalla, Hannes, Bauer, Michael, Witte, Otto W., Wetzker, Reinhard, Bauer, Reinhard, Schmeer, Christian 25 September 2020 (has links)
Microglia, the innate immune cells of the central nervous system, feature adaptive immune memory with implications for brain homeostasis and pathologies. However, factors involved in the emergence and regulation of these opposing responses in microglia have not been fully addressed. Recently, we showed that microglia from the newborn brain display features of trained immunity and immune tolerance after repeated contact with pathogens in a dose-dependent manner. Here, we evaluate the impact of developmental stage on adaptive immune responses of brain microglia after repeated challenge with ultra-low (1 fg/ml) and high (100 ng/ml) doses of the endotoxin LPS in vitro. We find that priming of naïve microglia derived from newborn but not mature and aged murine brain with ultra-low LPS significantly increased levels of pro-inflammatory mediators TNF-α, IL-6, IL-1β, MMP-9, and iNOS as well as neurotrophic factors indicating induction of trained immunity (p < 0.05). In contrast, stimulation with high doses of LPS led to a robust downregulation of pro-inflammatory cytokines and iNOS independent of the developmental state, indicating induced immune tolerance. Furthermore, high-dose priming with LPS upregulated anti-inflammatory mediators IL-10, Arg-1, TGF- β, MSR1, and IL-4 in newborn microglia (p < 0.05). Our data indicate pronounced plasticity of the immune response of neonate microglia compared with microglia derived from mature and aged mouse brain. Induced trained immunity after priming with ultra-low LPS doses may be responsible for enhanced neuro-inflammatory susceptibility of immature brain. In contrast, the immunosuppressed phenotype following high-dose LPS priming might be prone to attenuate excessive damage after recurrent systemic inflammation.
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Memory-Like Inflammatory Responses of Microglia to Rising Doses of LPS: Key Role of PI3KγLajqi, Trim, Lang, Guang Ping, Haas, Fabienne, Williams, David L., Hudalla, Hannes, Bauer, Michael, Groth, Marco, Wetzker, Reinhard, Bauer, Reinhard 08 November 2019 (has links)
Trained immunity and immune tolerance have been identified as long-term response patterns of the innate immune system. The causes of these opposing reactions remain elusive. Here, we report about differential inflammatory responses of microglial cells derived from neonatal mouse brain to increasing doses of the endotoxin LPS. Prolonged priming with ultra-low LPS doses provokes trained immunity, i.e., increased production of pro-inflammatory mediators in comparison to the unprimed control. In contrast, priming with high doses of LPS induces immune tolerance, implying decreased production of inflammatory mediators and pronounced release of anti-inflammatory cytokines. Investigation of the signaling processes and cell functions involved in these memory-like immune responses reveals the essential role of phosphoinositide 3-kinase γ (PI3Kγ), one of the phosphoinositide 3-kinase species highly expressed in innate immune cells. Together, our data suggest profound influence of preceding contacts with pathogens on the immune response of microglia. The impact of these interactions—trained immunity or immune tolerance—appears to be shaped by pathogen dose.
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Glycogen Synthase Kinase-3 and p38 MAPK Are Required for Opioid-Induced Microglia ApoptosisXie, Nanchang, Li, Hui, Wei, Dailin, LeSage, Gene, Chen, Lin, Wang, Shengjun, Zhang, Yi, Chi, Lingyi, Ferslew, Kenneth, He, Lei, Chi, Zhaofu, Yin, Deling 01 November 2010 (has links)
Opioids have been widely applied in clinics as one of the most potent pain relievers for centuries, but their abuse has deleterious physiological effects beyond addiction. We previously reported that opioids inhibit cell growth and trigger apoptosis in lymphocytes. However, the underlying mechanism by which microglia apoptosis in response to opioids is not yet known. In this study, we show that morphine induces microglia apoptosis and caspase-3 activation in an opioid-receptor dependent manner. Morphine decreased the levels of microglia phosphorylated Akt (p-Akt) and p-GSK-3β (glycogen synthase kinase-3 beta) in an opioid-receptor dependent manner. More interestingly, GSK-3β inhibitor SB216763 significantly increases morphine-induced apoptosis in both BV-2 microglia and mouse primary microglial cells. Moreover, co-treatment of microglia with SB216763 and morphine led to a significant synergistic effect on the level of phospho-p38 mitogen-activated protein kinase (MAPK). In addition, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly inhibited morphine-induced apoptosis and caspase-3 activation. Taken together, our data clearly demonstrates that morphine-induced apoptosis in microglial cells, which is mediated via GSK-3β and p38 MAPK pathways.
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The Role of p38 MAPK in Valproic Acid Induced Microglia ApoptosisXie, Nanchang, Wang, Cui, Lin, Youting, Li, Hui, Chen, Lin, Zhang, Tongxia, Sun, Yong, Zhang, Yi, Yin, Deling, Chi, Zhaofu 01 September 2010 (has links)
Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, induces apoptosis in microglia, but the underlying mechanism by which microglia apoptosis in response to VPA is not yet known. In this study, we found that the mitochondrial pathway played an important role in VPA-induced apoptosis in both BV-2 microglia and mouse primary microglial cells. In addition, VPA increased the level of phospho-p38 mitogen-activated protein kinase (MAPK), but had no effects on phospho-ERK and phospho-JNK MAPKs. Moreover, p38 inhibitor SB203580 strongly inhibited VPA-induced apoptosis and caspase-3 activation. Taken together, our results clearly demonstrated that VPA could induce apoptosis of microglia via p38 MAPK and mitochondrial apoptosis pathway.
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Investigating the role of microglia in neural development and synaptic maintenanceYeh, Hana 04 February 2022 (has links)
Maternal immune activation (MIA) disrupts the central innate immune system during a critical neurodevelopmental period. Microglia are the primary innate immune cells in the brain and can mediate neurodevelopment, but the direct influence of microglia on the MIA phenotype remains largely unknown. Here, we show that MIA can lead to long-lasting effects on microglial phenotype, neuronal circuitry, and behaviors. Transcriptomic analysis revealed aberrant expression of neurogenic genes in MIA microglia. We found that microglia repopulation by colony-stimulating factor receptor 1 (CSF1R) inhibition reversed MIA-induced social deficits and corrected expression of the newly identified MIA-associated neuritogenic molecules in microglia. In vitro whole-cell patch-clamp recording and immunohistochemistry revealed that microglia repopulation restored MIA-induced changes in intrinsic excitability, dendritic spine density, and microglia-neuron interactions of layer V intrinsically bursting pyramidal neurons in the prefrontal cortex. Maternal inflammation therefore alters microglial phenotypes and changes neuronal functions by mediating microglia-neuron interactions. We found that Wingless-related MMTV integration site 5a (WNT5a) is a critical regulator of this microglia-neuron communication. Studies have shown that the neurotrophic factor WNT5a plays a critical role in neurodevelopment, and here we demonstrate that WNT5a is one of the neuritogenic genes significantly upregulated in embryonic MIA microglia. We showed using microarray analysis that the microglial secretome can promote neural stem cell differentiation through various pathways, including Wnt pathways. Live imaging of neuron-microglia co-culture demonstrated that microglia enhanced neurite development and dendritic spine density and that this was diminished by microglial Wnt5a silencing using siRNA transfection. Multi-electrode array recordings revealed that microglia co-culture increased spontaneous neuronal firing rate. Thus, microglia can secrete WNT5a and regulate dendritic spine development, maintenance, and neural circuitry. These results indicate that altered expression of microglial WNT5a due to pathogenic states such as inflammation can lead to abnormal neuronal activity. To further elucidate microglia biology, we developed an inducible immortalized murine microglial cell line using a tetracycline expression system. The addition of doxycycline can induce rapid cell proliferation for the expansion of cell colonies. Upon withdrawal of doxycycline, this monoclonal microglial cell line can differentiate and resemble in vivo microglia physiology as assessed by expression of microglial genes, innate immune response, chemotaxis, and phagocytic capabilities. This cell line becomes a convenient and useful method to study microglia in vitro. / 2024-02-03T00:00:00Z
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Investigating Microglia-Vascular Interactions in the Developing and Adult Central Nervous SystemMondo, Erica 26 August 2020 (has links)
Microglia, the resident macrophages of the central nervous system (CNS), are dynamic cells, constantly extending and retracting their processes as they contact and functionally regulate neurons and other glial cells. There is far less known about how microglia interact with the CNS vasculature, particularly under healthy steady-state conditions. Here, I provide the first extensive characterization of juxtavascular microglia in the healthy, postnatal brain and identify a molecular mechanism regulating the timing of these interactions during development. Using the mouse cerebral cortex, I show that microglia are intimately associated with the vasculature in the CNS, directly contacting the basal lamina in vascular sites that are devoid of astrocyte endfeet. I demonstrate a high percentage of microglia are associated with the vasculature during the first week of postnatal development, which is concomitant with a peak in microglial colonization of the cortex and recruitment to synapses. I find that as microglia colonize the cortex, juxtavascular microglia are highly motile along vessels and become largely stationary as the brain matures. 2-photon live imaging in adult mice reveals that these vascular-associated microglia in the mature brain are stable and stationary for several weeks. Further, a decrease in microglia motility along the vasculature is tightly correlated with the expansion of astrocyte endfeet along the vasculature. Finally, I provide evidence that the timing of these microglia-vascular interactions during development is regulated by the microglial fractalkine receptor (CX3CR1). Together, these data support a model by which microglia use the vasculature as a scaffold to migrate and colonize the developing brain and the timing of these associations is modulated by CX3CR1. This migration along the vasculature becomes restricted as astrocyte vascular endfoot territory expands and, upon maturation, vascular-associated microglia become largely stationary.
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Involvement of Wnt/β-catenin signaling in the development of neuropathic pain / 神経因性疼痛の発症にWnt/βカテニンシグナルが関与するItokazu, Takahide 24 March 2014 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18149号 / 医博第3869号 / 新制||医||1002(附属図書館) / 31007 / 京都大学大学院医学研究科医学専攻 / (主査)教授 福田 和彦, 教授 渡邉 大, 教授 河野 憲二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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