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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.

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., Neurovascular coupling refers to changes in cerebral blood flow in response to neuronal stimulation, but to what extent this change can report neuronal activation is not known. Here the authors develop transfer functions between neural calcium signals and functional ultrasound changes in blood volume in co-registered single voxel brain volumes.

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.

Odors synchronize the activity of olfactory bulb mitral cells that project to the same glomerulus.In vitro, a slow rhythmic excitation intrinsic to the glomerular network persists, even in the absence of afferent input. We show here that a subpopulation of juxtaglomerular cells, external tufted (ET) cells, may trigger this rhythmic activity. We used paired whole-cell recording and Ca2+imaging in bulb slices from wild-type and transgenic mice expressing the fluorescent Ca2+indicator protein GCaMP-2. Slow, periodic population bursts in mitral cells were synchronized with spontaneous discharges in ET cells. Moreover, activation of a single ET cell was sufficient to evoke population bursts in mitral cells within the same glomerulus. Stimulation of the olfactory nerve induced similar population bursts and activated ET cells at a lower threshold than mitral cells, suggesting that ET cells mediate feedforward excitation of mitral cells. We propose that ET cells act as essential drivers of glomerular output to the olfactory cortex.

The amino acid gamma-aminobutiric acid (GABA) is a major inhibitory transmitter in the vertebrate central nervous system (CNS) where it can be released by neurons and by glial cells. Neuronal GABAergic signaling is well characterized: the mechanisms of GABA release, the receptors it targets and the functional consequences of their activation have been extensively studied. In contrast, the corresponding features of glial GABAergic signaling have attracted less attention. In this review, we first discuss evidence from the literature for GABA accumulation, production and release by glial cells. We then review the results of recent experiments that point toward functional roles of GABA as a "gliotransmitter".

Survival and differentiation of myogenic cells grafted into infarcted myocardium have raised the hope that cell transplantation becomes a new therapy for cardiovascular diseases. The approach was further supported by transplantation of skeletal myoblasts, which was shown to improve cardiac performance in several animal species. Despite the success of myoblast transplantation and its recent trial in human, the mechanism responsible for the functional improvement remains unclear. Here, we used intracellular recordings coupled to video and fluorescence microscopy to establish whether myoblasts, genetically labeled with enhanced GFP and transplanted into rat infarcted myocardium, retain excitable and contractile properties, and participate actively to cardiac function. Our results indicate that grafted myoblasts differentiate into peculiar hyperexcitable myotubes with a contractile activity fully independent of neighboring cardiomyocytes. We conclude that mechanisms other than electromechanical coupling between grafted and host cells are involved in the improvement of cardiac function.

International audience

International audience; By focusing a pulsed laser beam into a sample, harmonic up-conversion can be generated as well as multi-photon excited fluorescence. Whereas multi-photon excited fluorescence microscopy is well established, the use of multi-harmonic generation for three-dimensional image contrast is very recent. Both techniques can provide similar resolution and, for adequate radiating source density, comparable signal levels, allowing them to be combined in a single versatile instrument. However, harmonic generation differs fundamentally from fluorescence generation in that it is coherent and produces radiation patterns that are highly sensitive to phase. As such, multi-harmonic generation microscopy provides a unique window into molecular spatial organization that is inaccessible to fluorescence.

Mitral cell dendrites do more than passively integrate and convey synaptic potentials to the soma, they release transmitter onto local interneurones to mediate recurrent and lateral inhibition. Several mechanisms may control the level of dendritic intracellular calcium ([Ca 2+ ]) and define timing for dendritic release. Here we investigated in vivo , how odor controls calcium dynamics in mitral cell dendrites by combining intracellular recording and two-photon microscopy imaging of [Ca 2+ ]. During odor stimulation, two types of [Ca 2+ ] changes accompany membrane potential oscillations that are phase-locked with the respiratory cycle: ( i ) one is graded and parallels the membrane potential, even below the threshold for action potential firing; ( ii ) a second is transient, triggered by sodium action potentials that invade the entire dendritic tree. These results indicate that mitral cell dendritic compartments are synchronized by action potentials and suggest that the efficacy of dendritic synapses is finely tuned by odor-evoked graded changes in [Ca 2+ ].

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.

The relationship between metabolism of neuronal activity, microvascular organization and blood flow dynamics is critical for interpreting functional brain imaging. Here we used the rat dorsal olfactory bulb as a model to determine in vivo the correlation between action potential propagation, synaptic transmission, oxygen consumption and capillary density during odor stimulation. We find that capillaries are extremely dense in layers of intense synaptic interactions like the glomerular layer while it is very low in the overlying nerve layer, devoid of capillaries. In glomeruli, odor triggers a local early decrease in tissue oxygen partial pressure that results principally from dendritic activation rather than from firing of axon terminals, transmitter release or astrocyte activation. In the nerve layer, action potential propagation does not generate local changes in tissue oxygen partial pressure. We also show that under strong peripheral stimulation, i.e. high odor concentration, glomerular neuronal and metabolic responses are shaped by peripheral adaptation of olfactory sensory neurons. Eventually, we show the importance of oxygen diffusion processes from brain capillaries to neuronal tissue. We characterize how transient changes in blood flow are precisely influencing the oxygen tissue level., Les techniques modernes d'imagerie fonctionnelle du cerveau utilisent le métabolisme cérébral comme marqueur d'activité neuronale. En effet le cerveau dépend intimement des apports sanguins en métabolites pour son fonctionnement. Cependant les mécanismes de régulation du métabolisme sont encore mal connus. Dans cette étude nous avons utilisé le modèle du bulbe olfactif chez le rat anesthésié pour caractériser la consommation d'oxygène en réponse à une stimulation physiologique. La quantification précise de la vascularisation du bulbe olfactif a pu mettre en évidence que la couche glomérulaire, très dense en synapses, est l'une des zones les plus vascularisées du cerveau. Cette couche est aussi le lieu d'une intense consommation d'oxygène lors du traitement de l'information olfactive. Par contraste, la couche du nerf, complètement dénuée d'interactions synaptiques et très peu vascularisée, consomme peu d'oxygène. L'étude pharmacologique de ces réponses métaboliques nous a permis de montrer que le compartiment post-synaptique du glomérule est le siège de cette intense activité métabolique. Cette dernière est aussi dépendante du traitement de l'information olfactive qui est effectué à la fois dans le bulbe olfactif et à la périphérie, dans la cavité nasale. Ceci nous a permis de caractériser l'effet de l'adaptation périphérique sur la consommation d'oxygène et le traitement local de l'information olfactive. Enfin, nous avons décrit en détail l'importance des phénomènes de diffusion au niveau du réseau microvasculaire dans le rééquilibrage transitoire du taux d'oxygène local.

Les astrocytes sont capables de sécréter des gliotransmetteurs en réponse à une stimulation qui engendre l'augmentation de la concentration calcique intra-cellulaire. Différents mécanismes de sécrétion ont été proposés, parmi lesquels l'exocytose régulée. Mais les expériences menées dans le but d'observer la fusion d'organelles individuels dans des astrocytes en culture ont conduit à des résultats contradictoires, notamment en terme d'identité des vésicules libérables. Nos expériences préliminaires nous ont convaincus que les conflits sur l'identité des organelles libérables sont dus à de fausses colocalisations à cause du recouvrement spectral des marqueurs fluorescents utilisés et de la présence d'autofluorescence dans les astrocytes en culture. Nous avons donc adapté la décomposition spectrale à l'identification rigoureuse d'organelles individuels et au suivi de leur exocytose. La décomposition spectrale permet la séparation de sources de fluorescence mal séparées et ainsi l'étude de l'expression et de la colocalisation de protéines fluorescentes, même en présence d'autofluorescence. Nous avons à cette occasion introduit un intervalle de confiance du résultat de l'estimation des quantités de colorants. Appliquée au marquage des organelles astrocytaires avec la EGFP et l'acridine orange, cette méthode a montré que l'apparente colocalisation entre ces marqueurs reflète en fait la présence d'acridine orange plus intense que la EGFP et coexistant dans les mêmes organelles sous deux formes verte et rouge. A l'aide de la décomposition spectrale et de la microscopie à onde évanescente, nous avons ensuite montré que les organelles autofluorescents dans les astrocytes sont en majorité des lysosomes capables de fusionner lors d'une stimu\-lation qui engendre l'augmentation du calcium intra-cellulaire. Ces lysosomes sont peut-être les organelles majoritairement responsables de l'exocytose dans les astrocytes en culture., Non disponible