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1.
J Neurosci ; 36(26): 7002-13, 2016 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-27358457

RESUMEN

UNLABELLED: Astrocytes can both sense and shape the evolution of neuronal network activity and are known to possess unique ion regulatory mechanisms. Here we explore the relationship between astrocytic intracellular pH dynamics and the synchronous network activity that occurs during seizure-like activity. By combining confocal and two-photon imaging of genetically encoded pH reporters with simultaneous electrophysiological recordings, we perform pH measurements in defined cell populations and relate these to ongoing network activity. This approach reveals marked differences in the intracellular pH dynamics between hippocampal astrocytes and neighboring pyramidal neurons in rodent in vitro models of epilepsy. With three different genetically encoded pH reporters, astrocytes are observed to alkalinize during epileptiform activity, whereas neurons are observed to acidify. In addition to the direction of pH change, the kinetics of epileptiform-associated intracellular pH transients are found to differ between the two cell types, with astrocytes displaying significantly more rapid changes in pH. The astrocytic alkalinization is shown to be highly correlated with astrocytic membrane potential changes during seizure-like events and mediated by an electrogenic Na(+)/HCO3 (-) cotransporter. Finally, comparisons across different cell-pair combinations reveal that astrocytic pH dynamics are more closely related to network activity than are neuronal pH dynamics. This work demonstrates that astrocytes exhibit distinct pH dynamics during periods of epileptiform activity, which has relevance to multiple processes including neurometabolic coupling and the control of network excitability. SIGNIFICANCE STATEMENT: Dynamic changes in intracellular ion concentrations are central to the initiation and progression of epileptic seizures. However, it is not known how changes in intracellular H(+) concentration (ie, pH) differ between different cell types during seizures. Using recently developed pH-sensitive proteins, we demonstrate that astrocytes undergo rapid alkalinization during periods of seizure-like activity, which is in stark contrast to the acidification that occurs in neighboring neurons. Rapid astrocytic pH changes are highly temporally correlated with seizure activity, are mediated by an electrogenic Na(+)/HCO3- cotransporter, and are more tightly coupled to network activity than are neuronal pH changes. As pH has profound effects on signaling in the nervous system, this work has implications for our understanding of seizure dynamics.


Asunto(s)
Astrocitos/metabolismo , Epilepsia/patología , Hipocampo/citología , Membranas Intracelulares/metabolismo , Simportadores de Sodio-Bicarbonato/genética , Uniones Estrechas/fisiología , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Epilepsia/etiología , Epilepsia/fisiopatología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Microscopía Confocal , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Transducción Genética
2.
J Neurosci ; 34(46): 15208-22, 2014 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-25392490

RESUMEN

Epileptic seizures are characterized by periods of hypersynchronous, hyperexcitability within brain networks. Most seizures involve two stages: an initial tonic phase, followed by a longer clonic phase that is characterized by rhythmic bouts of synchronized network activity called afterdischarges (ADs). Here we investigate the cellular and network mechanisms underlying hippocampal ADs in an effort to understand how they maintain seizure activity. Using in vitro hippocampal slice models from rats and mice, we performed electrophysiological recordings from CA3 pyramidal neurons to monitor network activity and changes in GABAergic signaling during epileptiform activity. First, we show that the highest synchrony occurs during clonic ADs, consistent with the idea that specific circuit dynamics underlie this phase of the epileptiform activity. We then show that ADs require intact GABAergic synaptic transmission, which becomes excitatory as a result of a transient collapse in the chloride (Cl(-)) reversal potential. The depolarizing effects of GABA are strongest at the soma of pyramidal neurons, which implicates somatic-targeting interneurons in AD activity. To test this, we used optogenetic techniques to selectively control the activity of somatic-targeting parvalbumin-expressing (PV(+)) interneurons. Channelrhodopsin-2-mediated activation of PV(+) interneurons during the clonic phase generated excitatory GABAergic responses in pyramidal neurons, which were sufficient to elicit and entrain synchronous AD activity across the network. Finally, archaerhodopsin-mediated selective silencing of PV(+) interneurons reduced the occurrence of ADs during the clonic phase. Therefore, we propose that activity-dependent Cl(-) accumulation subverts the actions of PV(+) interneurons to perpetuate rather than terminate pathological network hyperexcitability during the clonic phase of seizures.


Asunto(s)
Región CA3 Hipocampal/fisiopatología , Neuronas GABAérgicas/fisiología , Interneuronas/fisiología , Parvalbúminas/metabolismo , Células Piramidales/fisiopatología , Convulsiones/fisiopatología , Potenciales de Acción/fisiología , Animales , Ratones , Modelos Neurológicos , Optogenética , Ratas
3.
Circ Cardiovasc Imaging ; 10(3)2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28292859

RESUMEN

BACKGROUND: Combined positron emission tomography (PET) and computed tomography (CT) can assess both anatomy and biology of carotid atherosclerosis. We sought to assess whether 18F-fluoride or 18F-fluorodeoxyglucose can identify culprit and high-risk carotid plaque. METHODS AND RESULTS: We performed 18F-fluoride and 18F-fluorodeoxyglucose PET/CT in 26 patients after recent transient ischemic attack or minor ischemic stroke: 18 patients with culprit carotid stenosis awaiting carotid endarterectomy and 8 controls without culprit carotid atheroma. We compared standardized uptake values in the clinically adjudicated culprit to the contralateral asymptomatic artery, and assessed the relationship between radiotracer uptake and plaque phenotype or predicted cardiovascular risk (ASSIGN score [Assessing Cardiovascular Risk Using SIGN Guidelines to Assign Preventive Treatment]). We also performed micro PET/CT and histological analysis of excised plaque. On histological and micro PET/CT analysis, 18F-fluoride selectively highlighted microcalcification. Carotid 18F-fluoride uptake was increased in clinically adjudicated culprit plaques compared with asymptomatic contralateral plaques (log10standardized uptake valuemean 0.29±0.10 versus 0.23±0.11, P=0.001) and compared with control patients (log10standardized uptake valuemean 0.29±0.10 versus 0.12±0.11, P=0.001). 18F-Fluoride uptake correlated with high-risk plaque features (remodeling index [r=0.53, P=0.003], plaque burden [r=0.51, P=0.004]), and predicted cardiovascular risk [r=0.65, P=0.002]). Carotid 18F-fluorodeoxyglucose uptake appeared to be increased in 7 of 16 culprit plaques, but no overall differences in uptake were observed in culprit versus contralateral plaques or control patients. However, 18F-fluorodeoxyglucose did correlate with predicted cardiovascular risk (r=0.53, P=0.019), but not with plaque phenotype. CONCLUSIONS: 18F-Fluoride PET/CT highlights culprit and phenotypically high-risk carotid plaque. This has the potential to improve risk stratification and selection of patients who may benefit from intervention.


Asunto(s)
Arteria Carótida Interna/diagnóstico por imagen , Estenosis Carotídea/diagnóstico por imagen , Fluoruros/administración & dosificación , Radioisótopos de Flúor/administración & dosificación , Fluorodesoxiglucosa F18/administración & dosificación , Ataque Isquémico Transitorio/etiología , Placa Aterosclerótica , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radiofármacos/administración & dosificación , Accidente Cerebrovascular/etiología , Anciano , Anciano de 80 o más Años , Arteria Carótida Interna/cirugía , Estenosis Carotídea/complicaciones , Estenosis Carotídea/cirugía , Estudios de Casos y Controles , Endarterectomía Carotidea , Femenino , Humanos , Ataque Isquémico Transitorio/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Fenotipo , Proyectos Piloto , Valor Predictivo de las Pruebas , Factores de Riesgo , Índice de Severidad de la Enfermedad , Accidente Cerebrovascular/diagnóstico por imagen , Microtomografía por Rayos X
4.
Nat Commun ; 6: 7495, 2015 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-26151378

RESUMEN

Vascular calcification is a complex biological process that is a hallmark of atherosclerosis. While macrocalcification confers plaque stability, microcalcification is a key feature of high-risk atheroma and is associated with increased morbidity and mortality. Positron emission tomography and X-ray computed tomography (PET/CT) imaging of atherosclerosis using (18)F-sodium fluoride ((18)F-NaF) has the potential to identify pathologically high-risk nascent microcalcification. However, the precise molecular mechanism of (18)F-NaF vascular uptake is still unknown. Here we use electron microscopy, autoradiography, histology and preclinical and clinical PET/CT to analyse (18)F-NaF binding. We show that (18)F-NaF adsorbs to calcified deposits within plaque with high affinity and is selective and specific. (18)F-NaF PET/CT imaging can distinguish between areas of macro- and microcalcification. This is the only currently available clinical imaging platform that can non-invasively detect microcalcification in active unstable atherosclerosis. The use of (18)F-NaF may foster new approaches to developing treatments for vascular calcification.


Asunto(s)
Aterosclerosis/diagnóstico , Arterias Carótidas/patología , Tomografía de Emisión de Positrones/métodos , Fluoruro de Sodio/química , Calcificación Vascular/diagnóstico , Anciano , Aterosclerosis/patología , Femenino , Radioisótopos de Flúor , Humanos , Masculino
5.
Front Mol Neurosci ; 5: 68, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22666186

RESUMEN

The regulation of hydrogen ion concentration (pH) is fundamental to cell viability, metabolism, and enzymatic function. Within the nervous system, the control of pH is also involved in diverse and dynamic processes including development, synaptic transmission, and the control of network excitability. As pH affects neuronal activity, and can also itself be altered by neuronal activity, the existence of tools to accurately measure hydrogen ion fluctuations is important for understanding the role pH plays under physiological and pathological conditions. Outside of their use as a marker of synaptic release, genetically encoded pH sensors have not been utilized to study hydrogen ion fluxes associated with network activity. By combining whole-cell patch clamp with simultaneous two-photon or confocal imaging, we quantified the amplitude and time course of neuronal, intracellular, acidic transients evoked by epileptiform activity in two separate in vitro models of temporal lobe epilepsy. In doing so, we demonstrate the suitability of three genetically encoded pH sensors: deGFP4, E(2)GFP, and Cl-sensor for investigating activity-dependent pH changes at the level of single neurons.

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