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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.

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.

International audience; 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.

Functional ultrasound imaging (fUSI) is an appealing method for measuring blood flow and thus infer brain activity, but it relies on the physiology of neurovascular coupling and requires extensive signal processing. To establish to what degree its trial-by-trial signals reflect neural activity, we performed simultaneous fUSI and neural recordings with Neuropixels probes in awake mice. fUSI signals strongly correlated with the slow (

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., Author summary Iliski is a software helping the user to find the relationship between two sets of data, namely transfer functions. Although transfer functions are widely used in many scientific fields to link two signals, their computation can be tricky due to data features such as multisource noise, or to specific shape requirements imposed by the nature of the signals, e.g. in biological data. Iliski offers a user-friendly graphical interface to ease the computation of transfer functions for both experienced and users with no coding skills. It proposes several signal pre-processing methods and allows rapid testing of different computing approaches, either based on deconvolution or on optimization of multi-parametric functions. This article, combined with a User Manual, provides a detailed description of Iliski functionalities and a thorough description of the advantages and drawbacks of each computing method using experimental biological data. In the era of Big Data, scientists strive to find new models for patho-physiological mechanisms, and Iliski fulfils the requirements of rigorous, flexible, and fast data driven hypothesis testing.

Laser scanning microscopy is widely used to measure blood hemodynamics with line-scans. Analysis of red blood cell (RBC) velocity involves two types of algorithms. The algorithms of the first kind are the image-processing algorithms such as the shear [1], the LSPIV [2], the radon-transform [3] and the Fourier transform [4, 5] algorithms. Each of them provides the apparent velocity (VRBCapp) of RBCs in the image, which does not consider the scanning conditions. The algorithms of the second kind give the real RBC velocity from VRBC app and the scanning conditions [6]. Here we developed a predictive model that provide the accuracy of one image-processing algorithms. We find that the accuracy of the image-processing algorithm depends on several parameters such as the design of the scanned line, the scanning velocity and the velocity of RBCs. Last we validate our model by testing various types of artificial linescan images. The same strategy will be followed for various image-processing algorithms. Overall, our analysis will allow accurate comparisons of blood velocity from all vessel types in control and pathological animal models.

Small vessel diseases 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 (MRI) 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 forms part of a vicious cycle involving impaired cerebrovascular reactivity (CVR), blood-brain barrier (BBB) dysfunction, perivascular inflammation and ultimately impaired clearance of waste proteins from the interstitial fluid (ISF) 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.

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.

The activity of mitral and tufted cells, the principal neurons of the olfactory bulb, is modulated by several classes of interneurons. Among them, diverse periglomerular (PG) cell types interact with the apical dendrites of mitral and tufted cells inside glomeruli at the first stage of olfactory processing. We used paired recording in olfactory bulb slices and two-photon targeted patch-clamp recordingin vivoto characterize the properties and connections of a genetically identified population of PG cells expressing enhanced yellow fluorescent protein (EYFP) under the control of the Kv3.1 potassium channel promoter. Kv3.1–EYFP+PG cells are axonless and monoglomerular neurons that constitute ∼30% of all PG cells and include calbindin-expressing neurons. They respond to an olfactory nerve stimulation with a short barrage of excitatory inputs mediated by mitral, tufted, and external tufted cells, and, in turn, they indiscriminately release GABA onto principal neurons. They are activated by even the weakest olfactory nerve input or by the discharge of a single principal neuron in slices and at each respiration cycle in anesthetized mice. They participate in a fast-onset intraglomerular lateral inhibition between principal neurons from the same glomerulus, a circuit that reduces the firing rate and promotes spike timing variability in mitral cells. Recordings in other PG cell subtypes suggest that this pathway predominates in generating glomerular inhibition. Intraglomerular lateral inhibition may play a key role in olfactory processing by reducing the similarity of principal cells discharge in response to the same incoming input.

During synaptic activation, the function of astrocyte endfeet depends on the vascular target: at the capillary, but not at the arteriole, a newly described P2X1R–phospholipase D2 pathway modulates prostaglandin E2 release and vessel dilation.

Summary 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., Highlights • Odor triggers rapid Ca2+ elevations in OPC process that are input specific • All pericyte subtypes and SMCs respond to downstream synaptic activation • Synchronous mural cell activation is associated with heterogeneous local hemodynamics • The arteriole and first-order capillary dilate first and form the primary functional unit, Rungta et al. perform in vivo two-photon calcium imaging of neuron, oligodendrocyte precursor cell, pericyte, and smooth muscle cell responses to olfactory sensory stimulation in combination with vessel diameter and red blood cell velocity measurements along the entire vascular arbor.

Previous studies have shown that layer V pyramidal neurons projecting either to subcortical structures or the contralateral cortex undergo different morphological and electrophysiological patterns of development during the first three postnatal weeks. To isolate the determinants of this differential maturation, we analyzed the gene expression and intrinsic membrane properties of layer V pyramidal neurons projecting either to the superior colliculus (SC cells) or the contralateral cortex (CC cells) by combining whole cell recordings and single-cell RT-PCR in acute slices prepared from postnatal day (P) 5–7 or P21–30 old mice. Among the 24 genes tested, the calcium channel subunits α1B and α1C, the protease Nexin 1, and the calcium-binding protein calbindin were differentially expressed in adult SC and CC cells and the potassium channel subunit Kv4.3 was expressed preferentially in CC cells at both stages of development. Intrinsic membrane properties, including input resistance, amplitude of the hyperpolarization-activated current, and action potential threshold, differed quantitatively between the two populations as early as from the first postnatal week and persisted throughout adulthood. However, the two cell types had similar regular action potential firing behaviors at all developmental stages. Surprisingly, when we increased the duration of anesthesia with ketamine–xylazine or pentobarbital before decapitation, a proportion of mature SC cells, but not CC cells, fired bursts of action potentials. Together these results indicate that the two populations of layer V pyramidal neurons already start to differ during the first postnatal week and exhibit different firing capabilities after anesthesia.

The great demand for long-wavelength and high signal-to-noise Ca2+ indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments. DOI: http://dx.doi.org/10.7554/eLife.05808.001, eLife digest The movement of calcium ions within cells controls many vital biological processes, ranging from cell growth to muscle contraction and brain activity. These calcium signals are triggered by stimuli, such as nerve impulses, which drive calcium entry into cells or release calcium from internal stores. These changes in calcium levels can span several orders of magnitude, and can be either localized to very small parts of the cell or span the entire cell. Scientists have developed numerous indicators or ‘probes’ that can detect even very low levels of calcium. One common method uses proteins that fluoresce when viewed under a fluorescence microscope each time the protein senses increases of calcium. Most of these probes fluoresce green, and so to view a second signal that occurs in the cell at the same time it's easier to use a probe that fluoresces with a different color, such as red. However, the red-shifted probes that are currently available either produce unreliable results because they tend to leak through cell membranes, or are not very sensitive to calcium ions. New types of red-shifted probes are therefore urgently needed. In 2012, researchers developed a family of red fluorescent probes known as Calcium Ruby (CaRuby for short) that were more versatile than earlier red probes. Now, Collot, Wilms et al.—including several of the researchers involved in the 2012 research—have enhanced the properties of CaRuby by modifying the chemical structure of the probes. This increased the ability of CaRuby to bind calcium ions, making it more sensitive to small calcium changes. Testing the usefulness of the newly developed probes—called CaRuby Nano—in mouse nerve cells revealed the probes are highly sensitive and can even detect the calcium signal resulting from a single nerve impulse. Collot, Wilms et al. then went on to demonstrate that CaRuby-Nano can be used alongside a green-fluorescing probe to record two signals at the same time. In one experiment, the release of chemical messengers known as neurotransmitters was stimulated, which caused calcium ions to flow into the observed nerve cells. The researchers succeeded in simultaneously detecting a green signal indicating an increase in neurotransmitter levels and a red signal produced by the corresponding release of calcium. Such dual-color imaging was not possible with previous probes. Finally, it was shown that CaRuby-Nano can also be used to produce dual-color images of the brain activity of live mice. In summary, these results demonstrate that CaRuby-Nano is a highly sensitive and versatile indicator and can be used together with other probes to observe two simultaneous events in cells. DOI: http://dx.doi.org/10.7554/eLife.05808.002

International audience; Enhanced neuronal activity in the brain triggers a local increase in blood flow, termed functional hyperemia, via several mechanisms, including calcium (Ca2+) signaling in astrocytes. However, recent in vivo studies have questioned the role of astrocytes in functional hyperemia because of the slow and sparse dynamics of their somatic Ca2+ signals and the absence of glutamate metabotropic receptor 5 in adults. Here, we reexamined their role in neurovascular coupling by selectively expressing a genetically encoded Ca2+ sensor in astrocytes of the olfactory bulb. We show that in anesthetized mice, the physiological activation of olfactory sensory neuron (OSN) terminals reliably triggers Ca2+ increases in astrocyte processes but not in somata. These Ca2+ increases systematically precede the onset of functional hyperemia by 1-2 s, reestablishing astrocytes as potential regulators of neurovascular coupling.

BOLD-fMRI is the preferred technique to study brain activity in humans. Recently, functional ultrasound (fUS) was introduced as a novel technique to record blood volume changes while overcoming the poor spatiotemporal resolution of BOLD-fMRI. BOLD-fMRI and fUS both report signals related to changes in blood flow to determine activated brain areas, although through different physical mechanisms. Indeed, neuronal activation systematically triggers an increase in blood flow, through a cascade of cellular and molecular reactions named neurovascular coupling. Thus, the interpretation of vascular-based functional imaging techniques requires a thorough understanding of neurovascular coupling to recover the spatiotemporal dynamics of neuronal activation from blood flow changes. During my PhD, I linked microscopic measurements of neuronal and vascular responses to odor to the mesoscopic signal detected with functional ultrasound imaging in the mouse. My first project evaluates the correspondence between neuronal activation in the olfactory bulb measured with two-photon microscopy and the fUS output in the same co-registered 100x100x200 µm volume, through a variety of odorant stimuli. This study also allowed me to model the neurovascular coupling, between neuronal activation and the increase of red blood cell velocity in nearby capillaries. I showed that, at high stimulus strength, neurovascular coupling is no longer linear with the appearance of a delayed vascular component independent of neural activity. To achieve these results, I developed 'Iliski', an analysis software that is provided in a GitHub repository and is described in detail in a second article. The second part of my thesis focuses on a long-lasting controversy in the BOLD-fMRI field: do neuronally activated areas generate a local transient decrease in brain oxygenation that can be measured inside vessels before the increase in oxygenated blood flow? It has been hypothesized that this oxygen decrease would be spatially more specific than the oxygen increase, and allow a more precise interpretation of the BOLD-fMRI output. Previous studies reported this "initial dip" in oxygenation, but it was mainly measured in acute animal preparations, where neurovascular coupling is impaired by anesthesia and the invasiveness of the preparation. Here we report that the initial dip cannot be seen in chronic animals, whether anesthetized or awake. Overall, this corpus of work helps to better interpret functional imaging techniques based on blood flow changes by shedding light on the microscopic mechanisms underlying the mesoscopic signals., L'IRM fonctionnelle BOLD est aujourd'hui la technique de prédilection pour étudier l'activité cérébrale chez l'être humain. Récemment, l'échographie fonctionnelle ultrasonore (fUS) a été décrite comme une nouvelle technique d'imagerie fonctionnelle pouvant supplanter l'imagerie BOLD. Ces deux approches ne mesurent pas directement l'activité neuronale mais l'augmentation réflexe du flux sanguin qui en résulte et que l'on appelle l'hyperémie fonctionnelle. Ce réflexe vasculaire met en jeu le couplage neurovasculaire, un ensemble complexe de voies de signalisation et de types cellulaires. L'imagerie fUS ou IRMf-BOLD nécessite une très bonne compréhension de la dynamique spatio-temporelle de l'hyperémie fonctionnelle pour correctement construire les cartes d'activation cérébrale. Pendant ma thèse, j'ai voulu mieux comprendre la relation entre les variations microscopiques du flux sanguin déclenchées par une activation sensorielle et les variations mésoscopiques mesurées par ces techniques. Dans mon premier projet, j'ai étudié le lien entre l'activation neuronale déclenchée par un stimulus odorant dans le bulbe olfactif de la souris et mesurée en microscopie biphotonique, avec les réponses vasculaires mesurées en fUS dans le même volume cérébral, un voxel de 100x100x200 µm. J'ai ensuite modélisé une fonction de transfert permettant de prédire l'hyperémie fonctionnelle à partir d'activation neuronale. Cela m'a permis de montrer qu'à de hautes concentrations d'odeur, l'hyperémie fonctionnelle n'est plus linéaire et qu'apparaît une deuxième composante vasculaire indépendante de l'activité neuronale. Pour ce travail théorique, j'ai développé 'Iliski', un logiciel d'analyse qui est disponible sur GitHub et est décrit dans un second article. La deuxième partie de ma thèse s'est concentrée sur une controverse du domaine de l'IRMf-BOLD : peut-on mesurer une diminution rapide et localisée de l'oxygénation vasculaire au cours d'une activation neuronale proche ? Hypothétiquement, cette diminution, connue sous l'appellation « initial dip », serait spatialement plus spécifique que l'hyperémie fonctionnelle, ce qui permettrait d'améliorer les cartes d'activations d'IRMf-BOLD. Cette diminution a été rapportée précédemment à une échelle microscopique mais uniquement chez des modèles animaux préparés de façon aiguë, c'est-à-dire au cours de chirurgies invasives qui impacte l'hyperémie fonctionnelle. Ici, je montre que la diminution n'est pas détectable chez les animaux préparés de manière chronique, qu'ils soient anesthésiés ou éveillés. Mes résultats soulignent qu'une meilleure compréhension des données microscopiques permet d'améliorer l'interprétation des signaux détectés avec les techniques mésoscopique d'imagerie fonctionnelle cérébrale.

Two-photon phosphorescence lifetime microscopy allows depth-resolved micron-scale measurements of oxygen partial pressure (Po2) in the brain. The spatiotemporal resolution of these measurements has shown that the portion of capillary plasma in the vicinity of red blood cells (RBCs) has a higher Po2(Po2RBC) than that distant from RBCs(Po2InterRBC). Our group has shown that Po2InterRBC equilibrates with neuropil Po2 and can thus be used to non-invasively measure tissue Po2 (Parpaleix et al., 2013). The relevance of reported high-resolution Po2 values remains uncertain as measurements have only been performed during anaesthesia, which affects both neuronal activity and cerebral blood flow, and thus brain Po2.I measured Po2 at rest, in the awake, unstressed mouse in two brain regions, the olfactory bulb glomerular layer (GL) and the somatosensory cortex. The first section of my research, conducted in the GL, produced the first measurements of blood flow and Po2 parameters in the cerebral microvasculature in physiological conditions. I determined mean Po2 levels of values of 60.6 mmHg for Po2RBC, 23 mmHg for tissue Po2. In the cortex Po2 values show differences between the superficial layers. Furthermore the relationships of RBC Po2 and tissue Po2 to the blood flow parameters differed between the cortical layers, and also in comparison to the olfactory bulb GL.I compared both vascular and tissue Po2 between the awake and anaesthetised states, and observed that anaesthetics can dramatically change Po2 at the microvascular scale. This finding emphasises the importance of measuring these values in the physiologically normal brain.; Notre laboratoire a récemment développé une méthode d'imagerie bi-photonique de phosphorescence en profondeur, pour mesurer la Po2 in vivo, avec une résolution micrométrique dans le cerveau de rongeurs anesthésiés (Parpaleix et al. 2013). Le laboratoire a également fait l'observation que la valeur de la Po2 à mi-distance entre deux érythrocytes d'un capillaire, rapporte indirectement la Po2 dans le tissu voisin. Mon projet de thèse a été de mettre au point une approche permettant de mesurer la Po2 cérébrale chez l'animal éveillé non-stressé. Pour ce faire, j'ai développé une approche chirurgicale permettant d'observer en microscopie bi-photonique, le bulbe olfactif et le cortex somato-sensoriel de souris éveillées. J'ai ensuite mis au point une technique d'entrainement permettant à ces souris d'être maintenues en contention en l'absence de stress. La première partie de mon travail, menée dans la couche glomérulaire, a permis de déterminer une Po2 érythrocytaire de 60.6 mm Hg et une Po2 tissulaire de 23 mm Hg, un flux érythrocytaire moyen de 30.6 cellules/s et un hématocrite moyen de 34.6 %.Dans un deuxième temps, j'ai reproduit ces mesures dans le cortex somato-sensoriel et observé des différences régionales touchant la Po2 tissulaire moyenne et les paramètres vasculaires, tels l'hématocrite et le flux érythrocytaire. Enfin, j'ai analysé combien l'anesthésie change l'état d'oxygénation cérébrale. Mes données, obtenues dans des conditions vraiment physiologiques, permettront d'améliorer les modèles de diffusion de l'oxygène, ainsi que l'analyse quantitative du métabolisme cérébral et l'interprétation de la nature des signaux mesurés en imagerie cérébrale humaine.