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1.
Methods Mol Biol ; 2034: 111-125, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31392681

RESUMO

Although microglial cells are not electrically excitable, they express a large repertoire of ion channels that are activated by voltage, stretch, extracellular ligands, or intracellular pathways (e.g. Ca2+, G-proteins). The patch-clamp technique is the electrophysiological method of choice to study these channels whose expression varies largely in pathological conditions but also during normal development and aging. This chapter focuses on protocols allowing the recording and the analysis of these channels in acute brain slices, with a particular emphasis on the study of channels activated by extracellular ligands.


Assuntos
Encéfalo , Potenciais da Membrana , Microglia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Humanos , Microglia/citologia , Microglia/metabolismo , Técnicas de Patch-Clamp
2.
Neuroscience ; 406: 186-201, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30872165

RESUMO

In the developing brain, microglial cells play an important role in shaping neuronal circuits. These immune cells communicate with neurons through fractalkine (CX3CL1), a neuronal cytokine that acts on microglial CX3CR1 receptor. Among various functions, this signaling pathway has been implicated in the postnatal maturation of glutamatergic synapses. Although microglial cells are present in the neonate hippocampus when GABA receptor-mediated synaptic transmission and synchronized oscillatory events take place, it remains unknown whether microglial cells tune the establishment of these activities. Using CX3CR1-deficient mice and electrophysiological means, we investigated in CA3 pyramidal neurons the role of the fractalkine signaling in the maturation of GABAA receptor-mediated synaptic currents and giant depolarizing potentials (GDPs), a network activity important for shaping synaptic connections. In CX3CR1-deficient mice, GABAergic currents were slightly altered, whereas the developmental changes of these currents were comparable with wild-type animals. Despite these minor changes in GABAergic transmission, the GDP frequency was strikingly reduced in CX3CR1-deficient mice compared to wild-type, with no change in the GDP shape and ending period. Collectively, it emerges that, in the neonate hippocampus, the fractalkine signaling pathway tunes GDP activities and is marginally involved in the maturation of GABAergic synapses, suggesting that microglial cells have distinct impact on maturing GABAergic, glutamatergic, and network functions.


Assuntos
Receptor 1 de Quimiocina CX3C/fisiologia , Neurônios GABAérgicos/fisiologia , Hipocampo/crescimento & desenvolvimento , Rede Nervosa/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos
3.
Sci Rep ; 6: 32422, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27604518

RESUMO

Recently microglia, the resident immune cells of the brain, have been recognized as multi-tasking talents that are not only essential in the diseased brain, but also actively contribute to synaptic circuit remodeling during normal brain development. It is well established that microglia dynamically scan their environment and thereby establish transient physical contacts with neuronal synapses, which may allow them to sense and influence synaptic function. However, it is unknown whether and how the morphological dynamics of microglia and their physical interactions with synapses are affected by the induction of synaptic plasticity in the adult brain. To this end, we characterized the morphological dynamics of microglia and their interactions with synapses before and after the induction of synaptic plasticity (LTP) in the hippocampus by time-lapse two-photon imaging and electrophysiological recordings in acute brain slices. We demonstrate that during hippocampal LTP microglia alter their morphological dynamics by increasing the number of their processes and by prolonging their physical contacts with dendritic spines. These effects were absent in the presence of an NMDA receptor antagonist. Taken together, this altered behavior could reflect an active microglial involvement in circuit remodeling during activity-dependent synaptic plasticity in the healthy adult brain.


Assuntos
Espinhas Dendríticas/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Microglia/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Comunicação Celular , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/ultraestrutura , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Expressão Gênica , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Microglia/citologia , Microglia/efeitos dos fármacos , Microtomia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Imagem com Lapso de Tempo/métodos , Técnicas de Cultura de Tecidos , Valina/análogos & derivados , Valina/farmacologia
4.
J Neuroinflammation ; 12: 202, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26538404

RESUMO

BACKGROUND: Microglia cells are the resident macrophages of the central nervous system and are considered its first line of defense. In the normal brain, their ramified processes are highly motile, constantly scanning the surrounding brain tissue and rapidly moving towards sites of acute injury or danger signals. These microglial dynamics are thought to be critical for brain homeostasis. Under pathological conditions, microglial cells undergo "activation," which modifies many of their molecular and morphological properties. Investigations of the effects of activation on motility are limited and have given mixed results. In particular, little is known about how microglial motility is altered in epilepsy, which is characterized by a strong inflammatory reaction and microglial activation. METHODS: We used a mouse model of status epilepticus induced by kainate injections and time-lapse two-photon microscopy to image GFP-labeled microglia in acute hippocampal brain slices. We studied how microglial activation affected the motility of microglial processes, including basal motility, and their responses to local triggering stimuli. RESULTS: Our study reveals that microglial motility was largely preserved in kainate-treated animals, despite clear signs of microglial activation. In addition, whereas the velocities of microglial processes during basal scanning and towards a laser lesion were unaltered 48 h after status epilepticus, we observed an increase in the size of the territory scanned by single microglial processes during basal motility and an elevated directional velocity towards a pipette containing a purinergic agonist. CONCLUSIONS: Microglial activation differentially impacted the dynamic scanning behavior of microglia in response to specific acute noxious stimuli, which may be an important feature of the adaptive behavior of microglia during pathophysiological conditions.


Assuntos
Microglia/patologia , Estado Epiléptico/patologia , Animais , Receptor 1 de Quimiocina CX3C , Movimento Celular , Agonistas de Aminoácidos Excitatórios , Hipocampo/patologia , Técnicas In Vitro , Inflamação/patologia , Ácido Caínico , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptores de Quimiocinas/genética , Receptores Purinérgicos P2Y12 , Estado Epiléptico/induzido quimicamente
5.
Glia ; 61(8): 1306-19, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23828736

RESUMO

Within the central nervous system, functions of the ATP-gated receptor-channel P2X4 (P2X4R) are still poorly understood, yet P2X4R activation in neurons and microglia coincides with high or pathological neuronal activities. In this study, we investigated the potential involvement of P2X4R in microglial functions in a model of kainate (KA)-induced status epilepticus (SE). We found that SE was associated with an induction of P2X4R expression in the hippocampus, mostly localized in activated microglial cells. In P2X4R-deficient mice, behavioral responses during KA-induced SE were unaltered. However, 48h post SE specific features of microglial activation, such as cell recruitment and upregulation of voltage-dependent potassium channels were impaired in P2X4R-deficient mice, whereas the expression and function of other microglial purinergic receptors remained unaffected. Consistent with the role of P2X4R in activity-dependent degenerative processes, the CA1 area was partially protected from SE-induced neuronal death in P2X4R-deficient mice compared with wild-type animals. Our findings demonstrate that P2X4Rs are brought into play during neuronal hyperexcitability and that they control specific aspects of microglial activation. Our results also suggest that P2X4Rs contribute to excitotoxic damages by regulating microglial activation.


Assuntos
Hipocampo/metabolismo , Microglia/metabolismo , Receptores Purinérgicos P2X4/fisiologia , Estado Epiléptico/metabolismo , Animais , Proliferação de Células , Hipocampo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Técnicas de Cultura de Órgãos , Receptores Purinérgicos P2X4/biossíntese , Receptores Purinérgicos P2X4/deficiência , Estado Epiléptico/patologia , Regulação para Cima/fisiologia
6.
Glia ; 61(10): 1582-94, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23893820

RESUMO

Accumulative evidence indicates that microglial cells influence the normal development of central nervous system (CNS) synapses. Yet, the functional properties of microglia in relation with synapse development remain unclear. We recently showed that in layer 4 of the whisker-related barrel field of the mouse somatosensory cortex, microglial cells are recruited only after postnatal day (P)5 in the center of the barrels where thalamo-cortical synapses are concentrated and begin their maturation. In the present study, we analyzed the phenotype of microglia during this developmental process. We show that between P5 and P7 microglial cells acquire a more ramified morphology with a smaller soma, they express classical markers of microglia (Iba1, CD11b, and CD68) but never markers of activation (Mac-2 and MHCII) and rarely the proliferation marker Ki67. Electrophysiological recordings in acute cortical slices showed that at P5 a proportion of layer 4 microglia transiently express voltage-dependant potassium currents of the delayed rectifier family, mostly mediated by Kv1.3 subunits, which are usually expressed by activated microglia under pathological conditions. This proportion of cells with rectifying properties doubles between P5 and P6, in concomitance with the beginning of microglia invasion of the barrel centers. Finally, analysis of the responses mediated by purinergic receptors indicated that a higher percentage of rectifying microglia expressed functional P2Y6 and P2Y12 receptors, as compared with nonrectifying cells, whereas all cells expressed functional P2X7 receptors. Our results indicate that during normal cortical development distinct microglia properties mature differentially, some of them being exquisitely influenced by the local environment of the maturating neuronal network.


Assuntos
Adaptação Fisiológica/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Microglia/fisiologia , Córtex Somatossensorial/citologia , Córtex Somatossensorial/crescimento & desenvolvimento , Adaptação Fisiológica/genética , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígeno CD11b/metabolismo , Receptor 1 de Quimiocina CX3C , Proteínas de Ligação ao Cálcio/metabolismo , Estimulação Elétrica , Galectina 3/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Antígeno Ki-67/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Bloqueadores dos Canais de Potássio/farmacologia , Receptores de Quimiocinas/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Tetraetilamônio/farmacologia , Difosfato de Uridina/farmacologia
7.
Biophys J ; 104(4): 778-85, 2013 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-23442956

RESUMO

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ~350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.


Assuntos
Encéfalo/citologia , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Espinhas Dendríticas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal
8.
J Neurosci ; 32(43): 15106-11, 2012 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-23100431

RESUMO

Accumulative evidence indicates that microglial cells influence the normal development of brain synapses. Yet, the mechanisms by which these immune cells target maturating synapses and influence their functional development at early postnatal stages remain poorly understood. Here, we analyzed the role of CX3CR1, a microglial receptor activated by the neuronal chemokine CX3CL1 (or fractalkine) which controls key functions of microglial cells. In the whisker-related barrel field of the mouse somatosensory cortex, we show that the recruitment of microglia to the sites where developing thalamocortical synapses are concentrated (i.e., the barrel centers) occurs only after postnatal day 5 and is controlled by the fractalkine/CX3CR1 signaling pathway. Indeed, at this developmental stage fractalkine is overexpressed within the barrels and CX3CR1 deficiency delays microglial cell recruitment into the barrel centers. Functional analysis of thalamocortical synapses shows that CX3CR1 deficiency also delays the functional maturation of postsynaptic glutamate receptors which normally occurs at these synapses between the first and second postnatal week. These results show that reciprocal interactions between neurons and microglial cells control the functional maturation of cortical synapses.


Assuntos
Deficiências do Desenvolvimento/patologia , Receptores de Quimiocinas/deficiência , Córtex Somatossensorial/patologia , Sinapses/patologia , Tálamo/patologia , Fatores Etários , Animais , Animais Recém-Nascidos , Receptor 1 de Quimiocina CX3C , Quimiocina CX3CL1/metabolismo , Deficiências do Desenvolvimento/genética , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Antagonistas GABAérgicos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Receptores de Quimiocinas/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Estatísticas não Paramétricas
9.
PLoS One ; 4(8): e6770, 2009 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-19707551

RESUMO

BACKGROUND: Growing evidence indicates that the functional state of microglial cells differs according to the pathological conditions that trigger their activation. In particular, activated microglial cells can express sets of Kv subunits which sustain delayed rectifying potassium currents (Kdr) and modulate differently microglia proliferation and ability to release mediators. We recently reported that hippocampal microglia is in a particular activation state after a status epilepticus (SE) and the present study aimed at identifying which of the Kv channels are functionally expressed by microglia in this model. METHODOLOGY/PRINCIPAL FINDINGS: SE was induced by systemic injection of kainate in CX3CR1(eGFP/+) mice and whole cell recordings of fluorescent microglia were performed in acute hippocampal slices prepared 48 h after SE. Microglia expressed Kdr currents which were characterized by a potential of half-maximal activation near -25 mV, prominent steady-state and cumulative inactivations. Kdr currents were almost abolished by the broad spectrum antagonist 4-Aminopyridine (1 mM). In contrast, tetraethylammonium (TEA) at a concentration of 1 mM, known to block Kv3.1, Kv1.1 and 1.2 subunits, only weakly reduced Kdr currents. However, at a concentration of 5 mM which should also affect Kv1.3 and 1.6, TEA inhibited about 30% of the Kdr conductance. Alpha-dendrotoxin, which selectively inhibits Kv1.1, 1.2 and 1.6, reduced only weakly Kdr currents, indicating that channels formed by homomeric assemblies of these subunits are not important contributors of Kdr currents. Finally, agitoxin-2 and margatoxin strongly inhibited the current. CONCLUSIONS/SIGNIFICANCE: These results indicate that Kv1.3 containing channels predominantly determined Kdr currents in activated microglia after SE.


Assuntos
Hipocampo/fisiopatologia , Canal de Potássio Kv1.3/fisiologia , Microglia/fisiologia , Estado Epiléptico/fisiopatologia , Animais , Camundongos , Camundongos Endogâmicos C57BL
10.
J Neurosci ; 28(37): 9133-44, 2008 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-18784294

RESUMO

Microglia cells are the resident macrophages of the CNS, and their activation plays a critical role in inflammatory reactions associated with many brain disorders, including ischemia, Alzheimer's and Parkinson's diseases, and epilepsy. However, the changes of microglia functional properties in epilepsy have rarely been studied. Here, we used a model of status epilepticus (SE) induced by intraperitoneal kainate injections to characterize the properties of microglial cells in hippocampal slices from CX3CR1(eGFP/+) mice. SE induced within 3 h an increased expression of inflammatory mediators in the hippocampus, followed by a modification of microglia morphology, a microglia proliferation, and a significant neurodegeneration in CA1. Changes in electrophysiological intrinsic membrane properties of hippocampal microglia were detected at 24-48 h after SE with, in particular, the appearance of new voltage-activated potassium currents. Consistent with the observation of an upregulation of purinergic receptor mRNAs in the hippocampus, we also provide pharmacological evidence that microglia membrane currents mediated by the activation of P2 receptors, including P2X(7), P2Y(6), and P2Y(12), were increased 48 h after SE. As a functional consequence of this modification of purinergic signaling, motility of microglia processes toward a source of P2Y(12) receptor agonist was twice as fast in the epileptic hippocampus. This study is the first functional description of microglia activation in an in vivo model of inflammation and provides evidence for the existence of a particular microglial activation state after a status epilepticus.


Assuntos
Microglia/fisiologia , Purinas/metabolismo , Transdução de Sinais/fisiologia , Estado Epiléptico/patologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Receptor 1 de Quimiocina CX3C , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Eletroencefalografia , Fluoresceínas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Técnicas In Vitro , Ácido Caínico , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Compostos Orgânicos , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Receptores de Quimiocinas/genética , Receptores Purinérgicos/fisiologia , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/fisiopatologia , Tionucleotídeos/farmacologia , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos
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