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Oxygen is critical for neural metabolism, but under most physiological conditions, oxygen levels in the brain are far more than are required. Oxygen levels can be dynamically increased by increases in respiration rate that are tied to the arousal state of the brain and cognition, and not necessarily linked to exertion by the body. Why these changes in respiration occur when oxygen is already adequate has been a long-standing puzzle. In humans, performance on cognitive tasks can be affected by very high or very low oxygen levels, but whether the physiological changes in blood oxygenation produced by respiration have an appreciable effect is an open question. Oxygen has direct effects on potassium channels, increases the degradation rate of nitric oxide, and is rate limiting for the synthesis of some neuromodulators. We discuss whether oxygenation changes due to respiration contribute to neural dynamics associated with attention and arousal.
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Oxigênio , Respiração , Humanos , Oxigênio/metabolismo , Consumo de Oxigênio/fisiologia , Encéfalo/metabolismo , Óxido Nítrico/metabolismoRESUMO
Significance: All functional brain imaging methods have technical drawbacks and specific spatial and temporal resolution limitations. Unraveling brain function requires bridging the data acquired with cellular and mesoscopic functional imaging. This imposes the access to animal preparations, allowing longitudinal and multiscale investigations of brain function in anesthetized and awake animals. Such preparations are optimal to study normal and pathological brain functions while reducing the number of animals used. Aim: To fulfill these needs, we developed a chronic and stable preparation for a broad set of imaging modalities and experimental design. Approach: We describe the detailed protocol for a chronic cranial window, transparent to light and ultrasound, devoid of BOLD functional magnetic resonance imaging (fMRI) artifact and allowing stable and longitudinal multimodal imaging of the entire mouse cortex. Results: The inexpensive, transparent, and curved polymethylpentene cranial window preparation gives access to the entire mouse cortex. It is compatible with standard microscopic and mesoscopic neuroimaging methods. We present examples of data on the neurovascular unit and its activation using two-photon, functional ultrasound imaging, and BOLD fMRI. Conclusion: This preparation is ideal for multimodal imaging in the same animal.
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Functional ultrasound (fUS) is an emerging technique that measures blood flow to report brain activity. In this issue of Neuron, Nunez-Elizalde et al. (2022) use simultaneous electrophysiological and fUS measurements to quantify the relationship between firing and fUS signals in awake mice.
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Neurônios , Som , Animais , Fenômenos Eletrofisiológicos , Camundongos , VigíliaRESUMO
Laser scanning microscopy is widely used to measure blood hemodynamics with line-scans in physiological and pathological vessels. With scans of broken lines, i.e., lines made of several segments with different orientations, it also allows simultaneous monitoring of vessel diameter dynamics or the activity of specific cells. Analysis of red blood cell (RBC) velocity from line-scans requires specific image-processing algorithms, as angle measurements, Line-Scanning Particle Image Velocimetry (LSPIV) or Fourier transformation of line-scan images. The conditions under which these image-processing algorithms give accurate measurements have not been fully characterized although the accuracy of measurements vary according to specific experimental parameters: the vessel type, the RBC velocity, the scanning parameters, and the image signal to noise ratio. Here, we developed mathematical models for the three previously mentioned line-scan image-processing algorithms. Our models predict the experimental conditions in which RBC velocity measurements are accurate. We illustrate the case of different vessel types and give the parameter space available for each of them. Last, we developed a software generating artificial line-scan images and used it to validate our models.
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An ongoing controversy in brain metabolism is whether increases in neural activity cause a local and rapid decrease in oxygen concentration (i.e., the "initial dip") preceding functional hyperemia. This initial dip has been suggested to cause a transient increase in vascular deoxyhemoglobin with several imaging techniques and stimulation paradigms, but not consistently. Here, we investigate contributors to this initial dip in a distinct neuronal network, an olfactory bulb (OB) glomerulus most sensitive to a specific odorant (ethyl tiglate [ET]) and a site of strong activation and energy consumption upon ET stimulation. Combining two-photon fluorescence and phosphorescence lifetime microscopy, and calcium, blood flow, and pO2 measurements, we characterized this initial dip in pO2 in mice chronically implanted with a glass cranial window, during both awake and anesthetized conditions. In anesthetized mice, a transient dip in vascular pO2 was detected in this glomerulus when functional hyperemia was slightly delayed, but its amplitude was minute (0.3 SD of resting baseline). This vascular pO2 dip was not observed in other glomeruli responding nonspecifically to ET, and it was poorly influenced by resting pO2. In awake mice, the dip in pO2 was absent in capillaries as well as, surprisingly, in the neuropil. These high-resolution pO2 measurements demonstrate that in awake mice recovered from brain surgery, neurovascular coupling was too fast and efficient to reveal an initial dip in pO2.
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Encéfalo , Acoplamento Neurovascular , Oxigênio , Vigília , Animais , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Camundongos , Rede Nervosa , Neurônios/fisiologia , Bulbo Olfatório/metabolismo , Oxigênio/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.
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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
Understanding the relationships between biological processes is paramount to unravel pathophysiological mechanisms. These relationships can be modeled with Transfer Functions (TFs), with no need of a priori hypotheses as to the shape of the transfer function. Here we present Iliski, a software dedicated to TFs computation between two signals. It includes different pre-treatment routines and TF computation processes: deconvolution, deterministic and non-deterministic optimization algorithms that are adapted to disparate datasets. We apply Iliski to data on neurovascular coupling, an ensemble of cellular mechanisms that link neuronal activity to local changes of blood flow, highlighting the software benefits and caveats in the computation and evaluation of TFs. We also propose a workflow that will help users to choose the best computation according to the dataset. Iliski is available under the open-source license CC BY 4.0 on GitHub (https://github.com/alike-aydin/Iliski) and can be used on the most common operating systems, either within the MATLAB environment, or as a standalone application.
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Software , Algoritmos , Biologia Computacional/métodos , Fluxo de TrabalhoRESUMO
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.
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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.
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Encefalopatias , Sistema Glinfático/anatomia & histologia , Sistema Glinfático/diagnóstico por imagem , Sistema Glinfático/fisiologia , Animais , Encefalopatias/diagnóstico por imagem , Encefalopatias/patologia , Encefalopatias/fisiopatologia , HumanosRESUMO
In the brain, increased neural activity is correlated with increases of cerebral blood flow and tissue oxygenation. However, how cerebral oxygen dynamics are controlled in the behaving animal remains unclear. We investigated to what extent cerebral oxygenation varies during locomotion. We measured oxygen levels in the cortex of awake, head-fixed mice during locomotion using polarography, spectroscopy, and two-photon phosphorescence lifetime measurements of oxygen sensors. We find that locomotion significantly and globally increases cerebral oxygenation, specifically in areas involved in locomotion, as well as in the frontal cortex and the olfactory bulb. The oxygenation increase persists when neural activity and functional hyperemia are blocked, occurred both in the tissue and in arteries feeding the brain, and is tightly correlated with respiration rate and the phase of respiration cycle. Thus, breathing rate is a key modulator of cerebral oxygenation and should be monitored during hemodynamic imaging, such as in BOLD fMRI.
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Encéfalo/fisiologia , Circulação Cerebrovascular/fisiologia , Locomoção/fisiologia , Oxigênio/metabolismo , Respiração , Animais , Encéfalo/metabolismo , Feminino , Hemodinâmica/fisiologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos Endogâmicos C57BL , Bulbo Olfatório/metabolismo , Bulbo Olfatório/fisiologia , Oxigênio/sangue , Vigília/fisiologiaRESUMO
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.
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Gasometria/métodos , Encéfalo/fisiologia , Craniotomia/métodos , Microscopia Intravital/métodos , Oxigênio/análise , Animais , Circulação Cerebrovascular , Camundongos , TemperaturaRESUMO
Two-photon laser scanning microscopy is widely used to measure blood hemodynamics in brain blood vessels. Still, the algorithms used so far to extract red blood cell (RBC) size and velocity from line-scan acquisitions have ignored the extent to which scanning speed influences the measurements. Here, we used a theoretical approach that takes into account the velocity and direction of both scanning mirrors and RBCs during acquisition to provide an algorithm that measures the real RBC size and velocity. We validate our approach in brain vessels of anesthetized mice, and demonstrate that it corrects online measurement errors that can reach several 10s of percent as well as data previously acquired. To conclude, our analysis allows unbiased comparisons of blood hemodynamic parameters from brain capillaries and large vessels in control and pathological animal models.
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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.
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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.
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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
Small vessel diseases (SVDs) are a group of disorders that result from pathological alteration of the small blood vessels in the brain, including the small arteries, capillaries and veins. Of the 35-36 million people that are estimated to suffer from dementia worldwide, up to 65% have an SVD component. Furthermore, SVD causes 20-25% of strokes, worsens outcome after stroke and is a leading cause of disability, cognitive impairment and poor mobility. Yet the underlying cause(s) of SVD are not fully understood. Magnetic resonance imaging has confirmed enlarged perivascular spaces (PVS) as a hallmark feature of SVD. In healthy tissue, these spaces are proposed to form part of a complex brain fluid drainage system which supports interstitial fluid exchange and may also facilitate clearance of waste products from the brain. The pathophysiological signature of PVS and what this infers about their function and interaction with cerebral microcirculation, plus subsequent downstream effects on lesion development in the brain has not been established. Here we discuss the potential of enlarged PVS to be a unique biomarker for SVD and related brain disorders with a vascular component. We propose that widening of PVS suggests presence of peri-vascular cell debris and other waste products that form part of a vicious cycle involving impaired cerebrovascular reactivity, blood-brain barrier dysfunction, perivascular inflammation and ultimately impaired clearance of waste proteins from the interstitial fluid space, leading to accumulation of toxins, hypoxia, and tissue damage. Here, we outline current knowledge, questions and hypotheses regarding understanding the brain fluid dynamics underpinning dementia and stroke through the common denominator of SVD.
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Doenças de Pequenos Vasos Cerebrais/fisiopatologia , Sistema Glinfático/fisiopatologia , Microvasos/fisiopatologia , Animais , Barreira Hematoencefálica/fisiopatologia , Doenças de Pequenos Vasos Cerebrais/diagnóstico por imagem , Doenças de Pequenos Vasos Cerebrais/patologia , Sistema Glinfático/diagnóstico por imagem , Sistema Glinfático/patologia , Humanos , Imageamento por Ressonância Magnética , Microvasos/diagnóstico por imagem , Microvasos/patologia , PrognósticoRESUMO
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.
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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
Although critical for brain function, the physiological values of cerebral oxygen concentration have remained elusive because high-resolution measurements have only been performed during anesthesia, which affects two major parameters modulating tissue oxygenation: neuronal activity and blood flow. Using measurements of capillary erythrocyte-associated transients, fluctuations of oxygen partial pressure (Po2) associated with individual erythrocytes, to infer Po2 in the nearby neuropil, we report the first non-invasive micron-scale mapping of cerebral Po2 in awake, resting mice. Interstitial Po2 has similar values in the olfactory bulb glomerular layer and the somatosensory cortex, whereas there are large capillary hematocrit and erythrocyte flux differences. Awake tissue Po2 is about half that under isoflurane anesthesia, and within the cortex, vascular and interstitial Po2 values display layer-specific differences which dramatically contrast with those recorded under anesthesia. Our findings emphasize the importance of measuring energy parameters non-invasively in physiological conditions to precisely quantify and model brain metabolism.