RESUMO
Cytotoxic brain edema triggered by neuronal swelling is the chief cause of mortality following brain trauma and cerebral infarct. Using fluorescence lifetime imaging to analyze contributions of intracellular ionic changes in brain slices, we find that intense Na(+) entry triggers a secondary increase in intracellular Cl(-) that is required for neuronal swelling and death. Pharmacological and siRNA-mediated knockdown screening identified the ion exchanger SLC26A11 unexpectedly acting as a voltage-gated Cl(-) channel that is activated upon neuronal depolarization to membrane potentials lower than -20 mV. Blockade of SLC26A11 activity attenuates both neuronal swelling and cell death. Therefore cytotoxic neuronal edema occurs when sufficient Na(+) influx and depolarization is followed by Cl(-) entry via SLC26A11. The resultant NaCl accumulation causes subsequent neuronal swelling leading to neuronal death. These findings shed light on unique elements of volume control in excitable cells and lay the ground for the development of specific treatments for brain edema.
Assuntos
Edema Encefálico/patologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Neurônios/metabolismo , Animais , Edema Encefálico/metabolismo , Morte Celular , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato/química , Humanos , Técnicas In Vitro , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Neurônios/patologia , Ratos , Sódio/metabolismo , Transportadores de SulfatoRESUMO
OBJECTIVE: Genetic alterations have been identified in the CACNA1H gene, encoding the CaV 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of CaV 3.2 calcium channels, and we investigated whether a gain-of-function mutation in the Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) contributes to seizure propagation and pacemaking in the TRN. METHODS: Pathophysiological contributions of CaV 3.2 calcium channels to burst firing and absence seizures were assessed in vitro using acute brain slice electrophysiology and quantitative real-time polymerase chain reaction (PCR) and in vivo using free-moving electrocorticography recordings. RESULTS: TRN neurons from GAERS display sustained oscillatory burst-firing that is both age- and frequency-dependent, occurring only in the frequencies overlapping with GAERS SWDs and correlating with the expression of a CaV 3.2 mutation-sensitive splice variant. In vivo knock-down of CaV 3.2 using direct thalamic injection of lipid nanoparticles containing CaV 3.2 dicer small interfering (Dsi) RNA normalized TRN burst-firing, and in free-moving GAERS significantly shortened seizures. SIGNIFICANCE: This supports a role for TRN CaV 3.2 T-type channels in propagating thalamocortical network seizures and setting the pacemaking frequency of SWDs.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo T/fisiologia , Epilepsia Tipo Ausência/fisiopatologia , Neurônios/fisiologia , Convulsões/fisiopatologia , Tálamo/fisiopatologia , Animais , Eletroencefalografia/métodos , Epilepsia Tipo Ausência/genética , Feminino , Masculino , Ratos , Ratos Transgênicos , Convulsões/genéticaRESUMO
Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca2+ signaling was only examined in soma and thick primary processes of astrocytes where Ca2+ -sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca2+ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two-photon imaging of Ca2+ transients in adult CA1 hippocampal astrocytes using a new single-cell patch-loading strategy to image Ca2+ -sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca2+ transients whereas astrocyte soma rarely showed spontaneous Ca2+ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L- or T-type voltage-gated calcium channels. Removal of extracellular Ca2+ almost completely and reversibly abolished the spontaneous signals while IP3 R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca2+ . The Ca2+ influx dependency of the spontaneous signals in patch-loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca2+ influx pathways in astrocytes. The mechanisms underlying these localized Ca2+ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093-2103.
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Hipocampo/citologia , Potenciais de Ação/efeitos dos fármacos , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbenoxolona/farmacologia , Cromonas/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Transportador 1 de Aminoácido Excitatório/genética , Transportador 1 de Aminoácido Excitatório/metabolismo , Feminino , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Piridinas/farmacologia , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/metabolismoRESUMO
Microglia are morphologically dynamic cells that rapidly extend their processes in response to various stimuli including extracellular ATP. In this study, we tested the hypothesis that stimulation of neuronal NMDARs trigger ATP release leading to communication with microglia. We used acute mouse hippocampal brain slices and two-photon laser scanning microscopy to study microglial dynamics and developed a novel protocol for fixation and immunolabeling of microglia processes. Similar to direct topical ATP application in vivo, short multiple applications of NMDA triggered transient microglia process outgrowth that was reversible and repeatable indicating that this was not due to excitotoxic damage. Stimulation of NMDAR was required as NMDAR antagonists, but not blockers of AMPA/kainate receptors or voltage-gated sodium channels, prevented microglial outgrowth. We report that ATP release, secondary to NMDAR activation, was the key mediator of this neuron-microglia communication as both blocking purinergic receptors and inhibiting hydrolysis of ATP to prevent locally generated gradients abolished outgrowth. Pharmacological and genetic analyses showed that the NMDA-triggered microglia process extension was independent of Pannexin 1, the ATP releasing channels, ATP release from astrocytes via connexins, and nitric oxide generation. Finally, using whole-cell patch clamping we demonstrate that activation of dendritic NMDAR on single neurons is sufficient to trigger microglia process outgrowth. Our results suggest that dendritic neuronal NMDAR activation triggers ATP release via a Pannexin 1-independent manner that induces outgrowth of microglia processes. This represents a novel uncharacterized form of neuron-microglial communication mediated by ATP.
Assuntos
Trifosfato de Adenosina/metabolismo , Encéfalo/citologia , Microglia/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Feminino , Técnicas In Vitro , Magnésio/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Receptores Purinérgicos P2Y12/metabolismo , Fatores de TempoRESUMO
Calcium signalling in astrocytes couples changes in neural activity to alterations in cerebral blood flow by eliciting vasoconstriction or vasodilation of arterioles. However, the mechanism for how these opposite astrocyte influences provide appropriate changes in vessel tone within an environment that has dynamic metabolic requirements remains unclear. Here we show that the ability of astrocytes to induce vasodilations over vasoconstrictions relies on the metabolic state of the rat brain tissue. When oxygen availability is lowered and astrocyte calcium concentration is elevated, astrocyte glycolysis and lactate release are maximized. External lactate attenuates transporter-mediated uptake from the extracellular space of prostaglandin E(2), leading to accumulation and subsequent vasodilation. In conditions of low oxygen concentration extracellular adenosine also increases, which blocks astrocyte-mediated constriction, facilitating dilation. These data reveal the role of metabolic substrates in regulating brain blood flow and provide a mechanism for differential astrocyte control over cerebrovascular diameter during different states of brain activation.
Assuntos
Arteríolas/metabolismo , Astrócitos/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Vasoconstrição/fisiologia , Vasodilatação/fisiologia , Adenosina/metabolismo , Adenosina/farmacologia , Animais , Arteríolas/efeitos dos fármacos , Dinoprostona/metabolismo , Glicólise , Ácido Láctico/metabolismo , Masculino , Transportadores de Ânions Orgânicos/metabolismo , Oxigênio/metabolismo , Pressão , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Neurovascular coupling links brain activity to local changes in blood flow, forming the basis for non-invasive brain mapping. Using multiscale imaging, we investigated how vascular activity spatially relates to neuronal activity elicited by single whiskers across different columns and layers of mouse cortex. Here we show that mesoscopic hemodynamic signals quantitatively reflect neuronal activity across space but are composed of a highly heterogeneous pattern of responses across individual vessel segments that is poorly predicted by local neuronal activity. Rather, this heterogeneity is dependent on vessel directionality, specifically in thalamocortical input layer 4, where capillaries respond preferentially to neuronal activity patterns along their downstream perfusion domain. Thus, capillaries fine-tune blood flow based on distant activity and encode laminar-specific activity patterns. These findings imply that vascular anatomy sets a resolution limit on functional imaging signals, where individual blood vessels inaccurately report neuronal activity in their immediate vicinity but, instead, integrate activity patterns along the vascular arbor.
RESUMO
Ultrasound Localization Microscopy (ULM) can map microvessels at a resolution of a few micrometers ( [Formula: see text]). Transcranial ULM remains challenging in presence of aberrations caused by the skull, which lead to localization errors. Herein, we propose a deep learning approach based on recently introduced complex-valued convolutional neural networks (CV-CNNs) to retrieve the aberration function, which can then be used to form enhanced images using standard delay-and-sum beamforming. CV-CNNs were selected as they can apply time delays through multiplication with in-phase quadrature input data. Predicting the aberration function rather than corrected images also confers enhanced explainability to the network. In addition, 3D spatiotemporal convolutions were used for the network to leverage entire microbubble tracks. For training and validation, we used an anatomically and hemodynamically realistic mouse brain microvascular network model to simulate the flow of microbubbles in presence of aberration. The proposed CV-CNN performance was compared to the coherence-based method by using microbubble tracks. We then confirmed the capability of the proposed network to generalize to transcranial in vivo data in the mouse brain (n=3). Vascular reconstructions using a locally predicted aberration function included additional and sharper vessels. The CV-CNN was more robust than the coherence-based method and could perform aberration correction in a 6-month-old mouse. After correction, we measured a resolution of [Formula: see text] for younger mice, representing an improvement of 25.8%, while the resolution was improved by 13.9% for the 6-month-old mouse. This work leads to different applications for complex-valued convolutions in biomedical imaging and strategies to perform transcranial ULM.
Assuntos
Microscopia , Redes Neurais de Computação , Camundongos , Animais , Microscopia/métodos , Encéfalo/diagnóstico por imagem , Encéfalo/irrigação sanguínea , Crânio/diagnóstico por imagem , Ultrassonografia/métodos , MicrobolhasRESUMO
The dynamics, computational power, and strength of neural circuits are essential for encoding and processing information in the CNS and rely on short and long forms of synaptic plasticity. In a model system, residual calcium (Ca(2+)) in presynaptic terminals can act through neuronal Ca(2+) sensor proteins to cause Ca(2+)-dependent facilitation (CDF) of P/Q-type channels and induce short-term synaptic facilitation. However, whether this is a general mechanism of plasticity at intact central synapses and whether mutations associated with human disease affect this process have not been described to our knowledge. In this report, we find that, in both exogenous and native preparations, gain-of-function missense mutations underlying Familial Hemiplegic Migraine type 1 (FHM-1) occlude CDF of P/Q-type Ca(2+) channels. In FHM-1 mutant mice, the alteration of P/Q-type channel CDF correlates with reduced short-term synaptic facilitation at cerebellar parallel fiber-to-Purkinje cell synapses. Two-photon imaging suggests that P/Q-type channels at parallel fiber terminals in FHM-1 mice are in a basally facilitated state. Overall, the results provide evidence that FHM-1 mutations directly affect both P/Q-type channel CDF and synaptic plasticity and that together likely contribute toward the pathophysiology underlying FHM-1. The findings also suggest that P/Q-type channel CDF is an important mechanism required for normal synaptic plasticity at a fast synapse in the mammalian CNS.
Assuntos
Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/fisiologia , Sinalização do Cálcio/fisiologia , Enxaqueca com Aura/genética , Enxaqueca com Aura/fisiopatologia , Plasticidade Neuronal/fisiologia , Animais , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Sinalização do Cálcio/genética , Linhagem Celular , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Mutantes , Modelos Neurológicos , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Mutação de Sentido Incorreto , Células de Purkinje/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologiaRESUMO
Pericytes are multifunctional cells of the vasculature that are vital to brain homeostasis, yet many of their fundamental physiological properties, such as Ca2+ signaling pathways, remain unexplored. We performed pharmacological and ion substitution experiments to investigate the mechanisms underlying pericyte Ca2+ signaling in acute cortical brain slices of PDGFRß-Cre::GCaMP6f mice. We report that mid-capillary pericyte Ca2+ signalling differs from ensheathing type pericytes in that it is largely independent of L- and T-type voltage-gated calcium channels. Instead, Ca2+ signals in mid-capillary pericytes were inhibited by multiple Orai channel blockers, which also inhibited Ca2+ entry triggered by endoplasmic reticulum (ER) store depletion. An investigation into store release pathways indicated that Ca2+ transients in mid-capillary pericytes occur through a combination of IP3R and RyR activation, and that Orai store-operated calcium entry (SOCE) is required to sustain and amplify intracellular Ca2+ increases evoked by the GqGPCR agonist endothelin-1. These results suggest that Ca2+ influx via Orai channels reciprocally regulates IP3R and RyR release pathways in the ER, which together generate spontaneous Ca2+ transients and amplify Gq-coupled Ca2+ elevations in mid-capillary pericytes. Thus, SOCE is a major regulator of pericyte Ca2+ and a target for manipulating their function in health and disease.
Assuntos
Sinalização do Cálcio , Pericitos , Camundongos , Animais , Pericitos/metabolismo , Capilares , Retículo Endoplasmático/metabolismo , EncéfaloRESUMO
Neuronal swelling during cytotoxic edema is triggered by Na+ and Cl- entry and is Ca2+ independent. However, the causes of neuronal death during swelling are unknown. Here, we investigate the role of large-conductance Pannexin-1 (Panx1) channels in neuronal death during cytotoxic edema. Panx1 channel inhibitors reduce and delay neuronal death in swelling triggered by voltage-gated Na+ entry with veratridine. Neuronal swelling causes downstream production of reactive oxygen species (ROS) that opens Panx1 channels. We confirm that ROS activates Panx1 currents with whole-cell electrophysiology and find scavenging ROS is neuroprotective. Panx1 opening and subsequent ATP release attract microglial processes to contact swelling neurons. Depleting microglia using the CSF1 receptor antagonist PLX3397 or blocking P2Y12 receptors exacerbates neuronal death, suggesting that the Panx1-ATP-dependent microglia contacts are neuroprotective. We conclude that cytotoxic edema triggers oxidative stress in neurons that opens Panx1 to trigger death but also initiates neuroprotective feedback mediated by microglia contacts.
Assuntos
Conexinas , Microglia , Microglia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Conexinas/metabolismo , Morte Celular , Trifosfato de Adenosina/metabolismoRESUMO
The spatial-temporal sequence of cerebral blood flow (CBF), cerebral blood volume (CBV) and blood velocity changes triggered by neuronal activation is critical for understanding functional brain imaging. This sequence follows a stereotypic pattern of changes across different zones of the vasculature in the olfactory bulb, the first relay of olfaction. However, in the cerebral cortex, where most human brain mapping studies are performed, the timing of activity evoked vascular events remains controversial. Here we utilized a single whisker stimulation model to map out functional hyperemia along vascular arbours from layer II/III to the surface of primary somatosensory cortex, in anesthetized and awake Thy1-GCaMP6 mice. We demonstrate that sensory stimulation triggers an increase in blood velocity within the mid-capillary bed and a dilation of upstream large capillaries, and the penetrating and pial arterioles. We report that under physiological stimulation, response onset times are highly variable across compartments of different vascular arbours. Furthermore, generating transfer functions (TFs) between neuronal Ca2+ and vascular dynamics across different brain states demonstrates that anesthesia decelerates neurovascular coupling (NVC). This spatial-temporal pattern of vascular events demonstrates functional diversity not only between different brain regions but also at the level of different vascular arbours within supragranular layers of the cerebral cortex.
Assuntos
Encéfalo/fisiologia , Córtex Cerebral/fisiologia , Circulação Cerebrovascular/fisiologia , Acoplamento Neurovascular/fisiologia , Córtex Somatossensorial/fisiologia , Animais , Encéfalo/irrigação sanguínea , Mapeamento Encefálico/métodos , Capilares/fisiologia , Córtex Cerebral/irrigação sanguínea , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neuroimagem/métodos , Neurônios/fisiologia , Bulbo Olfatório/irrigação sanguínea , Bulbo Olfatório/fisiologia , Córtex Somatossensorial/irrigação sanguínea , Vibrissas/fisiologia , Vigília/fisiologiaRESUMO
Functional ultrasound imaging (fUS) is an emerging technique that detects changes of cerebral blood volume triggered by brain activation. Here, we investigate the extent to which fUS faithfully reports local neuronal activation by combining fUS and two-photon microscopy (2PM) in a co-registered single voxel brain volume. Using a machine-learning approach, we compute and validate transfer functions between dendritic calcium signals of specific neurons and vascular signals measured at both microscopic (2PM) and mesoscopic (fUS) levels. We find that transfer functions are robust across a wide range of stimulation paradigms and animals, and reveal a second vascular component of neurovascular coupling upon very strong stimulation. We propose that transfer functions can be considered as reliable quantitative reporters to follow neurovascular coupling dynamics.
Assuntos
Cálcio/metabolismo , Ebolavirus/patogenicidade , Neurônios/metabolismo , Western Blotting , Proteínas de Transporte/metabolismo , Sobrevivência Celular/fisiologia , Proteínas do Citoesqueleto , Ebolavirus/genética , Células HEK293 , Células HeLa , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Imunoprecipitação , Interferons/metabolismo , Cinética , UltrassonografiaRESUMO
Perivascular spaces include a variety of passageways around arterioles, capillaries and venules in the brain, along which a range of substances can move. Although perivascular spaces were first identified over 150 years ago, they have come to prominence recently owing to advances in knowledge of their roles in clearance of interstitial fluid and waste from the brain, particularly during sleep, and in the pathogenesis of small vessel disease, Alzheimer disease and other neurodegenerative and inflammatory disorders. Experimental advances have facilitated in vivo studies of perivascular space function in intact rodent models during wakefulness and sleep, and MRI in humans has enabled perivascular space morphology to be related to cognitive function, vascular risk factors, vascular and neurodegenerative brain lesions, sleep patterns and cerebral haemodynamics. Many questions about perivascular spaces remain, but what is now clear is that normal perivascular space function is important for maintaining brain health. Here, we review perivascular space anatomy, physiology and pathology, particularly as seen with MRI in humans, and consider translation from models to humans to highlight knowns, unknowns, controversies and clinical relevance.
Assuntos
Encefalopatias , Sistema Glinfático/anatomia & histologia , Sistema Glinfático/diagnóstico por imagem , Sistema Glinfático/fisiologia , Animais , Encefalopatias/diagnóstico por imagem , Encefalopatias/patologia , Encefalopatias/fisiopatologia , HumanosRESUMO
Previously, we reported the first oxygen partial pressure (Po2) measurements in the brain of awake mice, by performing two-photon phosphorescence lifetime microscopy at micrometer resolution (Lyons et al., 2016). However, this study disregarded that imaging through a cranial window lowers brain temperature, an effect capable of affecting cerebral blood flow, the properties of the oxygen sensors and thus Po2 measurements. Here, we show that in awake mice chronically implanted with a glass window over a craniotomy or a thinned-skull surface, the postsurgical decrease of brain temperature recovers within a few days. However, upon imaging with a water immersion objective at room temperature, brain temperature decreases by ~2-3°C, causing drops in resting capillary blood flow, capillary Po2, hemoglobin saturation, and tissue Po2. These adverse effects are corrected by heating the immersion objective or avoided by imaging through a dry air objective, thereby revealing the physiological values of brain oxygenation.
Assuntos
Gasometria/métodos , Encéfalo/fisiologia , Craniotomia/métodos , Microscopia Intravital/métodos , Oxigênio/análise , Animais , Circulação Cerebrovascular , Camundongos , TemperaturaRESUMO
Imaging based on blood flow dynamics is widely used to study sensory processing. Here we investigated the extent to which local neuronal and capillary responses (two-photon microscopy) are correlated to mesoscopic responses detected with fast ultrasound (fUS) and BOLD-fMRI. Using a specialized chronic olfactory bulb preparation, we report that sequential imaging of the same mouse allows quantitative comparison of odour responses, imaged at both microscopic and mesoscopic scales. Under these conditions, functional hyperaemia occurred at the threshold of neuronal activation and fUS-CBV signals could be detected at the level of single voxels with activation maps varying according to blood velocity. Both neuronal and vascular responses increase non-linearly as a function of odour concentration, whereas both microscopic and mesoscopic vascular responses are linearly correlated to local neuronal calcium. These data establish strengths and limits of mesoscopic imaging techniques to report neural activity.
Assuntos
Bulbo Olfatório/diagnóstico por imagem , Bulbo Olfatório/fisiologia , Animais , Velocidade do Fluxo Sanguíneo , Mapeamento Encefálico , Sinalização do Cálcio , Circulação Cerebrovascular , Feminino , Neuroimagem Funcional , Hiperemia/diagnóstico por imagem , Hiperemia/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Odorantes , Bulbo Olfatório/irrigação sanguínea , Olfato/fisiologia , UltrassonografiaRESUMO
Functional hyperemia, a regional increase of blood flow triggered by local neural activation, is used to map brain activity in health and disease. However, the spatial-temporal dynamics of functional hyperemia remain unclear. Two-photon imaging of the entire vascular arbor in NG2-creERT2;GCaMP6f mice shows that local synaptic activation, measured via oligodendrocyte precursor cell (OPC) Ca2+ signaling, generates a synchronous Ca2+ drop in pericytes and smooth muscle cells (SMCs) enwrapping all upstream vessels feeding the activated synapses. Surprisingly, the onset timing, direction, and amplitude of vessel diameter and blood velocity changes vary dramatically from juxta-synaptic capillaries back to the pial arteriole. These results establish a precise spatial-temporal sequence of vascular changes triggered by neural activity and essential for the interpretation of blood-flow-based imaging techniques such as BOLD-fMRI.
Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/fisiologia , Hiperemia/fisiopatologia , Pia-Máter/irrigação sanguínea , Pia-Máter/fisiologia , Sinapses/fisiologia , Animais , Química Encefálica/fisiologia , Hiperemia/diagnóstico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal/métodos , Músculo Liso Vascular/química , Músculo Liso Vascular/fisiologia , Pericitos/química , Pericitos/fisiologia , Pia-Máter/química , Sinapses/químicaRESUMO
Optogenetics is increasingly used to map brain activation using techniques that rely on functional hyperaemia, such as opto-fMRI. Here we test whether light stimulation protocols similar to those commonly used in opto-fMRI or to study neurovascular coupling modulate blood flow in mice that do not express light sensitive proteins. Combining two-photon laser scanning microscopy and ultrafast functional ultrasound imaging, we report that in the naive mouse brain, light per se causes a calcium decrease in arteriolar smooth muscle cells, leading to pronounced vasodilation, without excitation of neurons and astrocytes. This photodilation is reversible, reproducible and energy-dependent, appearing at about 0.5 mJ. These results impose careful consideration on the use of photo-activation in studies involving blood flow regulation, as well as in studies requiring prolonged and repetitive stimulations to correct cellular defects in pathological models. They also suggest that light could be used to locally increase blood flow in a controlled fashion.
Assuntos
Encéfalo/efeitos da radiação , Circulação Cerebrovascular/efeitos da radiação , Microscopia Confocal/métodos , Neuroimagem/métodos , Ultrassonografia/métodos , Animais , Astrócitos/fisiologia , Astrócitos/efeitos da radiação , Astrócitos/ultraestrutura , Encéfalo/diagnóstico por imagem , Cálcio/metabolismo , Circulação Cerebrovascular/fisiologia , Feminino , Luz , Imageamento por Ressonância Magnética/instrumentação , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Microscopia Confocal/instrumentação , Neuroimagem/instrumentação , Neurônios/fisiologia , Neurônios/efeitos da radiação , Neurônios/ultraestrutura , Optogenética/instrumentação , Optogenética/métodos , Ultrassonografia/instrumentação , Vasodilatação/efeitos da radiaçãoRESUMO
Spreading depression (SD) is a slowly propagating neuronal depolarization that underlies certain neurologic conditions. The wave-like pattern of its propagation suggests that SD arises from an unusual form of neuronal communication. We used enzyme-based glutamate electrodes to show that during SD induced by transiently raising extracellular K(+) concentrations ([K(+)]o) in rat brain slices, there was a rapid increase in the extracellular glutamate concentration that required vesicular exocytosis but unlike fast synaptic transmission, still occurred when voltage-gated sodium and calcium channels (VGSC and VGCC) were blocked. Instead, presynaptic N-methyl-D-aspartate (NMDA) receptors (NMDARs) were activated during SD and could generate substantial glutamate release to support regenerative glutamate release and propagating waves when VGSCs and VGCCs were blocked. In calcium-free solutions, high [K(+)]o still triggered SD-like waves and glutamate efflux. Under such a condition, glutamate release was blocked by mitochondrial Na(+)/Ca(2+) exchanger inhibitors that likely blocked calcium release from mitochondria secondary to NMDA-induced Na(+) influx. Therefore presynaptic NMDA receptor activation is sufficient for triggering vesicular glutamate release during SD via both calcium entry and release from mitochondria by mitochondrial Na(+)/Ca(2+) exchanger. Our observations suggest that presynaptic NMDARs contribute to a cycle of glutamate-induced glutamate release that mediate high [K(+)]o-triggered SD.
Assuntos
Encéfalo/metabolismo , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Ácido Glutâmico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Pré-Sinápticos/metabolismo , Animais , Técnicas Biossensoriais , Encéfalo/fisiopatologia , Cálcio/metabolismo , Eletrodos , Eletrofisiologia , Feminino , Técnicas In Vitro , Masculino , Potássio/administração & dosagem , Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Trocador de Sódio e Cálcio/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Manipulation of gene expression in the brain is fundamental for understanding the function of proteins involved in neuronal processes. In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection. We show that neurons accumulate these LNPs in an apolipoprotein E-dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, intracortical or intracerebroventricular (ICV) siRNA-LNP injections resulted in knockdown of target genes either in discrete regions around the injection site or in more widespread areas following ICV injections with no apparent toxicity or immune reactions from the LNPs. Effective targeted knockdown was demonstrated by showing that intracortical delivery of siRNA against GRIN1 (encoding GluN1 subunit of the NMDA receptor (NMDAR)) selectively reduced synaptic NMDAR currents in vivo as compared with synaptic AMPA receptor currents. Therefore, LNP delivery of siRNA rapidly manipulates expression of proteins involved in neuronal processes in vivo, possibly enabling the development of gene therapies for neurological disorders.Molecular Therapy-Nucleic Acids (2013) 2, e136; doi:10.1038/mtna.2013.65; published online 3 December 2013.