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1.
J Neuroinflammation ; 21(1): 175, 2024 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-39020359

RESUMEN

BACKGROUND: Key functions of Ca2+ signaling in rodent microglia include monitoring the brain state as well as the surrounding neuronal activity and sensing the danger or damage in their vicinity. Microglial Ca2+ dyshomeostasis is a disease hallmark in many mouse models of neurological disorders but the Ca2+ signal properties of human microglia remain unknown. METHODS: We developed a novel genetically-encoded ratiometric Ca2+ indicator, targeting microglial cells in the freshly resected human tissue, organotypically cultured tissue slices and analyzed in situ ongoing Ca2+ signaling of decades-old microglia dwelling in their native microenvironment. RESULTS: The data revealed marked compartmentalization of Ca2+ signals, with signal properties differing across the compartments and resident morphotypes. The basal Ca2+ levels were low in ramified and high in ameboid microglia. The fraction of cells with ongoing Ca2+ signaling, the fraction and the amplitude of process Ca2+ signals and the duration of somatic Ca2+ signals decreased when moving from ramified via hypertrophic to ameboid microglia. In contrast, the size of active compartments, the fraction and amplitude of somatic Ca2+ signals and the duration of process Ca2+ signals increased along this pathway.


Asunto(s)
Señalización del Calcio , Calcio , Microglía , Microglía/metabolismo , Humanos , Señalización del Calcio/fisiología , Calcio/metabolismo , Masculino , Femenino , Células Cultivadas
2.
Glia ; 71(12): 2884-2901, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37596829

RESUMEN

Microglia, the major immune cells of the brain, are functionally heterogeneous but in vivo functional properties of these cells are rarely studied at single-cell resolution. By using microRNA-9 regulated viral vectors for multicolor labeling and longitudinal in vivo monitoring of individual microglia, we followed their fate in the cortex of healthy adult mice and at the onset of amyloidosis in a mouse model of Alzheimer's disease. In wild-type mice, microglia were rather mobile (16% of the cells migrated at least once in 10-20 days) but had a low turnover as documented by low division and death rates. Half of the migratory events were tightly associated with blood vessels. Surprisingly, basic migration properties of microglia (i.e., fraction of migrating cells, saltatory migration pattern, speed of migration, translocation distance, and strong association with blood vessels) were preserved in amyloid-depositing brains, despite amyloid plaques becoming the major destination of migration. Besides, amyloid deposition significantly increased microglial division and death rates. Moreover, the plaque vicinity became a hotspot of microglial turnover, harboring 33% of all migration, 70% of death and 54% of division events.

3.
Brain Behav Immun ; 96: 113-126, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34052361

RESUMEN

Peripheral inflammation is known to impact brain function, resulting in lethargy, loss of appetite and impaired cognitive abilities. However, the channels for information transfer from the periphery to the brain, the corresponding signaling molecules and the inflammation-induced interaction between microglia and neurons remain obscure. Here, we used longitudinal in vivo two-photon Ca2+ imaging to monitor neuronal activity in the mouse cortex throughout the early (initiation) and late (resolution) phases of peripheral inflammation. Single peripheral lipopolysaccharide injection induced a substantial but transient increase in ongoing neuronal activity, restricted to the initiation phase, whereas the impairment of visual processing was selectively observed during the resolution phase of systemic inflammation. In the frontal/motor cortex, the initiation phase-specific cortical hyperactivity was seen in the deep (layer 5) and superficial (layer 2/3) pyramidal neurons but not in the axons coming from the somatosensory cortex, and was accompanied by reduced activity of layer 2/3 cortical interneurons. Moreover, the hyperactivity was preserved after depletion of microglia and in NLRP3-/- mice but absent in TNF-α-/- mice. Together, these data identify microglia-independent and TNF-α-mediated reduction of cortical inhibition as a likely cause of the initiation phase-specific cortical hyperactivity and reveal the resolution phase-specific impairment of sensory processing, presumably caused by activated microglia.


Asunto(s)
Inflamación , Microglía , Animales , Ratones , Neuronas , Células Piramidales , Corteza Somatosensorial
4.
Proc Natl Acad Sci U S A ; 115(6): E1279-E1288, 2018 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-29358403

RESUMEN

Neuronal hyperactivity is the emerging functional hallmark of Alzheimer's disease (AD) in both humans and different mouse models, mediating an impairment of memory and cognition. The mechanisms underlying neuronal hyperactivity remain, however, elusive. In vivo Ca2+ imaging of somatic, dendritic, and axonal activity patterns of cortical neurons revealed that both healthy aging and AD-related mutations augment neuronal hyperactivity. The AD-related enhancement occurred even without amyloid deposition and neuroinflammation, mainly due to presenilin-mediated dysfunction of intracellular Ca2+ stores in presynaptic boutons, likely causing more frequent activation of synaptic NMDA receptors. In mutant but not wild-type mice, store emptying reduced both the frequency and amplitude of presynaptic Ca2+ transients and, most importantly, normalized neuronal network activity. Postsynaptically, the store dysfunction was minor and largely restricted to hyperactive cells. These findings identify presynaptic Ca2+ stores as a key element controlling AD-related neuronal hyperactivity and as a target for disease-modifying treatments.


Asunto(s)
Enfermedad de Alzheimer/patología , Canales de Calcio/metabolismo , Calcio/metabolismo , Modelos Animales de Enfermedad , Inflamación/patología , Neuronas/patología , Presenilina-1/fisiología , Envejecimiento , Enfermedad de Alzheimer/metabolismo , Animales , Humanos , Inflamación/metabolismo , Ratones , Neuronas/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patología , Terminales Presinápticos/metabolismo , Terminales Presinápticos/patología , Transducción de Señal
5.
Int J Mol Sci ; 22(3)2021 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-33499206

RESUMEN

Microglia, the innate immune cells of the brain, are commonly perceived as resident macrophages of the central nervous system (CNS). This definition, however, requires further specification, as under healthy homeostatic conditions, neither morphological nor functional properties of microglia mirror those of classical macrophages. Indeed, microglia adapt exceptionally well to their microenvironment, becoming a legitimate member of the cellular brain architecture. The ramified or surveillant microglia in the young adult brain are characterized by specific morphology (small cell body and long, thin motile processes) and physiology (a unique pattern of Ca2+ signaling, responsiveness to various neurotransmitters and hormones, in addition to classic "immune" stimuli). Their numerous physiological functions far exceed and complement their immune capabilities. As the brain ages, the respective changes in the microglial microenvironment impact the functional properties of microglia, triggering further rounds of adaptation. In this review, we discuss the recent data showing how functional properties of microglia adapt to age-related changes in brain parenchyma in a sex-specific manner, with a specific focus on early changes occurring at middle age as well as some strategies counteracting the aging of microglia.


Asunto(s)
Envejecimiento , Encéfalo/fisiología , Microglía/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio , Restricción Calórica , Sistema Nervioso Central/citología , Femenino , Humanos , Macrófagos/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Neuronas/fisiología , Neurotransmisores/metabolismo , Fagocitosis , Fenotipo , Factores Sexuales , Transducción de Señal , Transcripción Genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo
6.
Brain Behav Immun ; 87: 243-255, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-31837418

RESUMEN

Peripheral inflammation is known to trigger a mirror inflammatory response in the brain, involving brain's innate immune cells - microglia. However, the functional phenotypes, which these cells adopt in the course of peripheral inflammation, remain obscure. In vivo two-photon imaging of microglial Ca2+ signaling as well as process motility reveals two distinct functional states of cortical microglia during a lipopolysaccharide-induced peripheral inflammation: an early "sensor state" characterized by dramatically increased intracellular Ca2+ signaling but ramified morphology and a later "effector state" characterized by slow normalization of intracellular Ca2+ signaling but hypertrophic morphology, substantial IL-1ß production in a subset of cells as well as increased velocity of directed process extension and loss of coordination between individual processes. Thus, lipopolysaccharide-induced microglial Ca2+ signaling might represent the central element connecting receptive and executive functions of microglia.


Asunto(s)
Inflamación , Microglía , Encéfalo , Humanos , Lipopolisacáridos , Transducción de Señal
7.
Neurobiol Dis ; 121: 315-326, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30366066

RESUMEN

Besides deficits in memory and cognition, impaired visual processing is common for Alzheimer's disease (AD) patients and mouse models of AD but underlying mechanisms still remain unclear. Using in vivo Ca2+ imaging of the mouse primary visual cortex (V1) we tested whether such impairment is caused by neuronal hyperactivity, an emerging functional hallmark of AD. Profound neuronal hyperactivity was indeed found in V1 of APPSWE/PS1G384A and even of PS1G384A mice, presenting neither with plaque accumulation nor with neuroinflammation. This hyperactivity was accompanied by over-responsiveness to visual stimuli and impaired visual tuning properties of individual neurons, largely caused by insufficient suppression of responses to non-preferred orientation/direction stimuli. Moreover, visual stimulation robustly suppressed the ongoing spontaneous activity in WT but not in APPSWE/PS1G384A mice. Emptying intracellular Ca2+ stores significantly reduced neuronal hyperactivity and the pathological over-responsiveness to visual stimuli, but could not rescue stimulus-induced suppression of spontaneous activity and impaired tuning properties of individual cells. Thus, our data identify the AD-mediated dysfunction of intracellular Ca2+ stores as a main cause of pathologically increased visual responsiveness in APPSWE/PS1G384A mice. At the same time, the impairment of visual tuning and the stimulus-induced suppression of spontaneous activity, identified in this study, are likely caused by different mechanisms as, for example, dysfunction of local interneurons.


Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Señalización del Calcio , Neuronas/fisiología , Corteza Visual/fisiología , Percepción Visual/fisiología , Animales , Modelos Animales de Enfermedad , Potenciales Evocados Visuales , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Estimulación Luminosa
8.
Proc Natl Acad Sci U S A ; 109(44): 18150-5, 2012 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-23071306

RESUMEN

Under most physiological circumstances, monocytes are excluded from parenchymal CNS tissues. When widespread monocyte entry occurs, their numbers decrease shortly after engraftment in the presence of microglia. However, some disease processes lead to focal and selective loss, or dysfunction, of microglia, and microglial senescence typifies the aged brain. In this regard, the long-term engraftment of monocytes in the microglia-depleted brain remains unknown. Here, we report a model in which a niche for myeloid cells was created through microglia depletion. We show that microglia-depleted brain regions of CD11b-HSVTK transgenic mice are repopulated with new Iba-1-positive cells within 2 wk. The engrafted cells expressed high levels of CD45 and CCR2 and appeared in a wave-like pattern frequently associated with blood vessels, suggesting the engrafted cells were peripheral monocytes. Although two times more numerous and morphologically distinct from resident microglia up to 27 wk after initial engraftment, the overall distribution of the engrafted cells was remarkably similar to that of microglia. Two-photon in vivo imaging revealed that the engrafted myeloid cells extended their processes toward an ATP source and displayed intracellular calcium transients. Moreover, the engrafted cells migrated toward areas of kainic acid-induced neuronal death. These data provide evidence that circulating monocytes have the potential to occupy the adult CNS myeloid niche normally inhabited by microglia and identify a strong homeostatic drive to maintain the myeloid component in the mature brain.


Asunto(s)
Sistema Nervioso Central/citología , Homeostasis , Microglía/citología , Adenosina Trifosfato/metabolismo , Animales , Sistema Nervioso Central/metabolismo , Ratones , Microglía/metabolismo , Timidina Quinasa/genética
9.
Cell Tissue Res ; 357(2): 427-38, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24553999

RESUMEN

Dysregulation of intracellular Ca(2+) homeostasis has been proposed as a common proximal cause of neural dysfunction during aging and Alzheimer's disease (AD). In this context, aberrant Ca(2+) signaling has been viewed as a neuronal phenomenon mostly related to the dysfunction of intracellular Ca(2+) stores. However, recent data suggest that, in AD, Ca(2+) dyshomeostasis is not restricted to neurons but represents a global phenomenon affecting virtually all cells in the brain. AD-related aberrant Ca(2+) signaling in astrocytes and microglia, which is activated during the disease, probably contributes profoundly to an inflammatory response that, in turn, impacts neuronal Ca(2+) homeostasis and brain function. Based on recent data obtained in vivo and in vitro, we propose that bidirectional interactions between the inflammatory responses of glial cells and aberrant Ca(2+) signaling represent a vicious cycle accelerating disease progression.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Señalización del Calcio , Homeostasis , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/fisiopatología , Animales , Astrocitos/inmunología , Astrocitos/metabolismo , Astrocitos/patología , Calcio/inmunología , Calcio/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/fisiopatología , Microglía/inmunología , Microglía/metabolismo , Microglía/patología , Neuronas/inmunología , Neuronas/metabolismo , Neuronas/patología
10.
Acta Neuropathol ; 127(4): 495-505, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24407428

RESUMEN

Neuroinflammation is a hallmark of Alzheimer's disease (AD) both in man and in multiple mouse models, and epidemiological studies link the use of anti-inflammatory drugs with a reduced risk of developing the disease. AD-related neuroinflammation is largely mediated by microglia, the main immune cells of the central nervous system. In vitro, executive functions of microglia are regulated by intracellular Ca(2+) signals, but little is known about microglial Ca(2+) signaling in vivo. Here we analyze in vivo properties of these cells in two mouse models of AD. In both strains plaque-associated microglia had hypertrophic/amoeboid morphology and were strongly positive for markers of activation such as CD11b and CD68. Activated microglia failed to respond reliably to extracellular release of adenosine triphosphate (ATP, mimicking tissue damage) and showed an increased incidence of spontaneous intracellular Ca(2+) transients. These Ca(2+) transients required activation of ATP receptors and Ca(2+) release from the intracellular Ca(2+) stores, and were not induced by neuronal or astrocytic hyperactivity. Neuronal silencing, however, selectively increased the frequency of Ca(2+) transients in plaque-associated microglia. Thus, our in vivo data reveal substantial dysfunction of plaque-associated microglia and identify a novel Ca(2+) signal possibly triggering a Ca(2+)-dependent release of toxic species in the plaque vicinity.


Asunto(s)
Enfermedad de Alzheimer/patología , Señalización del Calcio/fisiología , Calcio/metabolismo , Corteza Cerebral/patología , Neuroglía/metabolismo , Placa Amiloide/patología , Factores de Edad , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Regulación de la Expresión Génica/genética , Humanos , Indoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroglía/efectos de los fármacos , Neuroglía/patología , Presenilina-1/genética , Bloqueadores de los Canales de Sodio/farmacología
11.
Biochim Biophys Acta ; 1813(5): 1014-24, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21056596

RESUMEN

In the healthy adult brain microglia, the main immune-competent cells of the CNS, have a distinct (so-called resting or surveying) phenotype. Resting microglia can only be studied in vivo since any isolation of brain tissue inevitably triggers microglial activation. Here we used in vivo two-photon imaging to obtain a first insight into Ca(2+) signaling in resting cortical microglia. The majority (80%) of microglial cells showed no spontaneous Ca(2+) transients at rest and in conditions of strong neuronal activity. However, they reliably responded with large, generalized Ca(2+) transients to damage of an individual neuron. These damage-induced responses had a short latency (0.4-4s) and were localized to the immediate vicinity of the damaged neuron (< 50 µm cell body-to-cell body distance). They were occluded by the application of ATPγS as well as UDP and 2-MeSADP, the agonists of metabotropic P2Y receptors, and they required Ca(2+) release from the intracellular Ca(2+) stores. Thus, our in vivo data suggest that microglial Ca(2+) signals occur mostly under pathological conditions and identify a Ca(2+) store-operated signal, which represents a very sensitive, rapid, and highly localized response of microglial cells to brain damage. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.


Asunto(s)
Señalización del Calcio , Microglía/metabolismo , Neuronas/metabolismo , Neuronas/patología , Animales , Imagenología Tridimensional , Ratones
12.
EBioMedicine ; 60: 102989, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32920368

RESUMEN

BACKGROUND: Stem cells` (SC) functional heterogeneity and its poorly understood aetiology impedes clinical development of cell-based therapies in regenerative medicine and oncology. Recent studies suggest a strong correlation between the SC migration potential and their therapeutic efficacy in humans. Designating SC migration as a denominator of functional SC heterogeneity, we sought to identify highly migrating subpopulations within different SC classes and evaluate their therapeutic properties in comparison to the parental non-selected cells. METHODS: We selected highly migrating subpopulations from mesenchymal and neural SC (sMSC and sNSC), characterized their features including but not limited to migratory potential, trophic factor release and transcriptomic signature. To assess lesion-targeted migration and therapeutic properties of isolated subpopulations in vivo, surgical transplantation and intranasal administration of MSCs in mouse models of glioblastoma and Alzheimer's disease respectively were performed. FINDINGS: Comparison of parental non-selected cells with isolated subpopulations revealed superior motility and migratory potential of sMSC and sNSC in vitro. We identified podoplanin as a major regulator of migratory features of sMSC/sNSC. Podoplanin engineering improved oncovirolytic activity of virus-loaded NSC on distantly located glioblastoma cells. Finally, sMSC displayed more targeted migration to the tumour site in a mouse glioblastoma model and remarkably higher potency to reduce pathological hallmarks and memory deficits in transgenic Alzheimer's disease mice. INTERPRETATION: Functional heterogeneity of SC is associated with their motility and migration potential which can serve as predictors of SC therapeutic efficacy. FUNDING: This work was supported in part by the Robert Bosch Stiftung (Stuttgart, Germany) and by the IZEPHA grant.


Asunto(s)
Movimiento Celular , Células Madre/fisiología , Enfermedad de Alzheimer/terapia , Animales , Biomarcadores , Supervivencia Celular , Rastreo Celular/métodos , Células Cultivadas , Modelos Animales de Enfermedad , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Transgénicos , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Viroterapia Oncolítica , Trasplante de Células Madre , Células Madre/citología , Resultado del Tratamiento
13.
Methods Mol Biol ; 2034: 231-241, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31392689

RESUMEN

Similar to many nonexcitable cells, microglia utilize intracellular Ca2+ signaling for the communication with each other as well as neurons and astrocytes and for triggering a magnitude of their executive functions. However, in vivo measurements of the intracellular Ca2+ dynamics in microglia have been challenging due to technical reasons. Here, we describe an approach utilizing a single-cell electroporation technique to facilitate the study of microglial Ca2+ signaling in the living brain.


Asunto(s)
Astrocitos , Señalización del Calcio , Calcio/metabolismo , Electroporación , Microglía , Neuronas , Animales , Astrocitos/citología , Astrocitos/metabolismo , Ratones , Microglía/citología , Microglía/metabolismo , Neuronas/citología , Neuronas/metabolismo
14.
Methods Mol Biol ; 2034: 165-175, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31392684

RESUMEN

Plant lectins bind to carbohydrates, which are found on the surface of different immune and endothelial cells including microglia. Using commercially available conjugates of lectins with different fluorophores allows one-step detection and visualization of microglia in vivo. Here, we describe a protocol enabling the use of a specific plant lectin isolated from Lycopersicon esculentum. Tomato lectin enables high-quality labeling of microglial cells in vivo and is applicable in any mouse strain at any age of the experimental animal without the need of genetic labeling, which is associated with time- and resource-consuming procedures.


Asunto(s)
Colorantes Fluorescentes/química , Histocitoquímica/métodos , Microglía , Lectinas de Plantas/química , Coloración y Etiquetado/métodos , Animales , Ratones , Microglía/citología , Microglía/metabolismo
16.
Neurobiol Aging ; 61: 187-197, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29107186

RESUMEN

Functioning at the interface between the nervous and immune systems, in the amyloid-depositing brain, astrocytes become hypertrophic and accumulate around senile plaques. Moreover, hippocampal astrocytes upregulate their γ-aminobutyric acid (GABA) content and enhance tonic inhibition, likely causing local circuit imbalance. It remains, however, unclear whether this effect is hippocampus specific and how it is regulated during disease progression. Here, we studied changes in astrocytic morphology and GABA content in the frontal cortex and dentate gyrus of control and amyloid-depositing mice. Healthy aging was accompanied by a transient increase in astrocytic GABA content at middle age and region-specific alterations of soma size. In contrast, amyloid deposition caused a gradual cortex-accentuated increase in soma size. Importantly, our data uncovered a bell-shaped relationship between the mouse age and astrocytic GABA content in both brain regions. Moreover, in mice carrying an Alzheimer's disease-related mutation in presenilin 1, astrocytes accumulated GABA even in the absence of amyloidosis. These data question the proposed inhibition of astrocytic GABA synthesis as a universal strategy for treating network dysfunction in Alzheimer's disease.


Asunto(s)
Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Proteínas Amiloidogénicas/metabolismo , Amiloidosis/metabolismo , Astrocitos/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Amiloidosis/patología , Animales , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Presenilina-1/genética
17.
Cell Calcium ; 64: 109-117, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28359543

RESUMEN

Microglia, the innate immune cells of the brain, are becoming increasingly recognized as an important player both in the context of physiological brain function and brain pathology. To fulfill their executive functions microglia can modify their morphology, migrate or move their processes in a directed fashion, and modify the intracellular Ca2+ dynamics leading to modifications in gene expression, phagocytosis, release of cytokines and other inflammation markers, etc. Here we describe the recently developed tools enabling in vivo monitoring of morphology and Ca2+ signaling of microglia and show how these techniques may be used for examining microglial function in healthy and diseased brain.


Asunto(s)
Corteza Cerebral/citología , Microglía/fisiología , Imagen Molecular/métodos , Envejecimiento/fisiología , Animales , Señalización del Calcio , Humanos
18.
Sci Rep ; 7(1): 6030, 2017 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-28729628

RESUMEN

Microglia, resident immune cells of the brain, react to the presence of pathogens/danger signals with a large repertoire of functional responses including morphological changes, proliferation, chemotaxis, production/release of cytokines, and phagocytosis. In vitro studies suggest that many of these effector functions are Ca2+-dependent, but our knowledge about in vivo Ca2+ signalling in microglia is rudimentary. This is mostly due to technical reasons, as microglia largely resisted all attempts of in vivo labelling with Ca2+ indicators. Here, we introduce a novel approach, utilizing a microglia-specific microRNA-9-regulated viral vector, enabling the expression of a genetically-encoded ratiometric Ca2+ sensor Twitch-2B in microglia. The Twitch-2B-assisted in vivo imaging enables recording of spontaneous and evoked microglial Ca2+ signals and allows for the first time to monitor the steady state intracellular Ca2+ levels in microglia. Intact in vivo microglia show very homogenous and low steady state intracellular Ca2+ levels. However, the levels increase significantly after acute slice preparation and cell culturing along with an increase in the expression of activation markers CD68 and IL-1ß. These data identify the steady state intracellular Ca2+ level as a versatile microglial activation marker, which is highly sensitive to the cell's environment.


Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Microglía/metabolismo , Imagen Molecular , Animales , Línea Celular , Células Cultivadas , Expresión Génica , Orden Génico , Genes Reporteros , Vectores Genéticos , Humanos , Ratones , Imagen Molecular/métodos
19.
Cell Calcium ; 53(3): 159-69, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23395344

RESUMEN

Microglial cells are the resident immune cells of the CNS. They mediate innate immune response of the brain to injury, inflammation and neurodegenerative diseases. Apart from their role in disease they are critically involved in the development and plasticity-driven reorganization of neuronal networks and the homeostatic maintenance of brain tissue. Accumulating in vitro evidence suggests that executive functions of microglia are coupled to the intracellular Ca(2+) signaling of these cells. So far, however, very little is known about microglial Ca(2+) signaling in situ or in vivo, both in the healthy and in the diseased brain. Here, we summarize the recent in vivo/in situ findings and compare the properties of surveillant microglia in these preparations with those of microglia in vitro. The data suggest that surveillant microglia rarely show spontaneous Ca(2+) transients, express fewer functional receptors directly coupled to changes in the intracellular free Ca(2+) concentration on their surface, but vividly respond with Ca(2+) transients to cell or tissue damage in their microenvironment. Interestingly, some of these properties microglia share with monocytes engrafting in the brain under pathological conditions.


Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Señalización del Calcio , Calcio/metabolismo , Microglía/metabolismo , Animales , Humanos , Microglía/citología
20.
Brain Res Bull ; 81(4-5): 484-90, 2010 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-19854245

RESUMEN

Reactive oxygen species (ROS), formed during normal aerobic metabolism, are involved in signal transduction and cognitive functions, but highly increased ROS concentrations may also have detrimental effects. The aim of the present study was to investigate whether aging and cognitive functions are associated with ROS generation in human neocortex obtained from neurosurgical patients. ROS formation in mitochondria from fresh and re-thawed neocortical specimens was measured by monitoring ROS-mediated conversion of dihydrorhodamine 123 to fluorescent rhodamine 123. The validity of this technique was characterized in rat brain mitochondria. The increase in the concentration-response curve of the complex I inhibitor rotenone on ROS generation, as measured by rhodamine 123 (Rh123) fluorescence, was much more pronounced than that of rotenone on mitochondrial [(3)H]-choline uptake [which indicates changes in the mitochondrial membrane potential (DeltaPsi(M))]. Thus, mitochondrial ROS generation can be shown by Rh123 fluorescence although this fluorescence may also reflect changes in DeltaPsi(M) to some extent. ROS formation in human brain mitochondria positively correlated with the age of patients. Moreover, an age-corrected positive correlation of ROS formation with presurgical cognitive performance was observed. Our data suggest a mild increase in ROS formation with aging possibly reflecting a physiological compensation of mitochondrial function. Furthermore, higher cognitive performances in tests of executive functions may be paralleled by slightly increased ROS levels.


Asunto(s)
Envejecimiento/metabolismo , Cognición/fisiología , Mitocondrias/metabolismo , Neocórtex/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Adolescente , Adulto , Anciano , Animales , Niño , Preescolar , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo I de Transporte de Electrón/metabolismo , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Neocórtex/efectos de los fármacos , Neocórtex/cirugía , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Rotenona/farmacología , Adulto Joven
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