Publications by authors named "Bruno Weber"

95 Publications

Diversity of neurovascular coupling dynamics along vascular arbors in layer II/III somatosensory cortex.

Commun Biol 2021 07 9;4(1):855. Epub 2021 Jul 9.

INSERM U1128, Laboratory of Neurophysiology and New Microscopy, Université Paris Descartes, Paris, France.

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 Ca 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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http://dx.doi.org/10.1038/s42003-021-02382-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8270975PMC
July 2021

Distinct signatures of calcium activity in brain mural cells.

Elife 2021 07 6;10. Epub 2021 Jul 6.

Institute of Pharmacology and Toxicology, University of Zurich, Zürich, Switzerland.

Pericytes have been implicated in various neuropathologies, yet little is known about their function and signaling pathways in health. Here, we characterized calcium dynamics of cortical mural cells in anesthetized or awake -CreERT2;Rosa26< LSL-GCaMP6s > mice and in acute brain slices. Smooth muscle cells (SMCs) and ensheathing pericytes (EPs), also named as terminal vascular SMCs, revealed similar calcium dynamics in vivo. In contrast, calcium signals in capillary pericytes (CPs) were irregular, higher in frequency, and occurred in cellular microdomains. In the absence of the vessel constricting agent U46619 in acute slices, SMCs and EPs revealed only sparse calcium signals, whereas CPs retained their spontaneous calcium activity. Interestingly, chemogenetic activation of neurons in vivo and acute elevations of extracellular potassium in brain slices strongly decreased calcium activity in CPs. We propose that neuronal activation and an extracellular increase in potassium suppress calcium activity in CPs, likely mediated by Kir2.2 and K channels.
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http://dx.doi.org/10.7554/eLife.70591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294852PMC
July 2021

Shear-stress sensing by PIEZO1 regulates tendon stiffness in rodents and influences jumping performance in humans.

Nat Biomed Eng 2021 May 24. Epub 2021 May 24.

University Hospital Balgrist, University of Zurich, Zurich, Switzerland.

Athletic performance relies on tendons, which enable movement by transferring forces from muscles to the skeleton. Yet, how load-bearing structures in tendons sense and adapt to physical demands is not understood. Here, by performing calcium (Ca) imaging in mechanically loaded tendon explants from rats and in primary tendon cells from rats and humans, we show that tenocytes detect mechanical forces through the mechanosensitive ion channel PIEZO1, which senses shear stresses induced by collagen-fibre sliding. Through tenocyte-targeted loss-of-function and gain-of-function experiments in rodents, we show that reduced PIEZO1 activity decreased tendon stiffness and that elevated PIEZO1 mechanosignalling increased tendon stiffness and strength, seemingly through upregulated collagen cross-linking. We also show that humans carrying the PIEZO1 E756del gain-of-function mutation display a 13.2% average increase in normalized jumping height, presumably due to a higher rate of force generation or to the release of a larger amount of stored elastic energy. Further understanding of the PIEZO1-mediated mechanoregulation of tendon stiffness should aid research on musculoskeletal medicine and on sports performance.
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http://dx.doi.org/10.1038/s41551-021-00716-xDOI Listing
May 2021

The severity of microstrokes depends on local vascular topology and baseline perfusion.

Elife 2021 05 18;10. Epub 2021 May 18.

Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.

Cortical microinfarcts are linked to pathologies like cerebral amyloid angiopathy and dementia. Despite their relevance for disease progression, microinfarcts often remain undetected and the smallest scale of blood flow disturbance has not yet been identified. We employed blood flow simulations in realistic microvascular networks from the mouse cortex to quantify the impact of single-capillary occlusions. Our simulations reveal that the severity of a microstroke is strongly affected by the local vascular topology and the baseline flow rate in the occluded capillary. The largest changes in perfusion are observed in capillaries with two inflows and two outflows. This specific topological configuration only occurs with a frequency of 8%. The majority of capillaries have one inflow and one outflow and is likely designed to efficiently supply oxygen and nutrients. Taken together, microstrokes bear potential to induce a cascade of local disturbances in the surrounding tissue, which might accumulate and impair energy supply locally.
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http://dx.doi.org/10.7554/eLife.60208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8421069PMC
May 2021

Author Correction: A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents.

Nat Protoc 2021 Aug;16(8):4108

Laboratory of Molecular and Behavioral Neuroscience, Institute for Neuroscience, Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland.

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http://dx.doi.org/10.1038/s41596-021-00493-6DOI Listing
August 2021

DeepVesselNet: Vessel Segmentation, Centerline Prediction, and Bifurcation Detection in 3-D Angiographic Volumes.

Front Neurosci 2020 8;14:592352. Epub 2020 Dec 8.

Department of Computer Science, TU München, München, Germany.

We present DeepVesselNet, an architecture tailored to the challenges faced when extracting vessel trees and networks and corresponding features in 3-D angiographic volumes using deep learning. We discuss the problems of low execution speed and high memory requirements associated with full 3-D networks, high-class imbalance arising from the low percentage (<3%) of vessel voxels, and unavailability of accurately annotated 3-D training data-and offer solutions as the building blocks of DeepVesselNet. First, we formulate 2-D orthogonal cross-hair filters which make use of 3-D context information at a reduced computational burden. Second, we introduce a class balancing cross-entropy loss function with false-positive rate correction to handle the high-class imbalance and high false positive rate problems associated with existing loss functions. Finally, we generate a synthetic dataset using a computational angiogenesis model capable of simulating vascular tree growth under physiological constraints on local network structure and topology and use these data for transfer learning. We demonstrate the performance on a range of angiographic volumes at different spatial scales including clinical MRA data of the human brain, as well as CTA microscopy scans of the rat brain. Our results show that cross-hair filters achieve over 23% improvement in speed, lower memory footprint, lower network complexity which prevents overfitting and comparable accuracy that does not differ from full 3-D filters. Our class balancing metric is crucial for training the network, and transfer learning with synthetic data is an efficient, robust, and very generalizable approach leading to a network that excels in a variety of angiography segmentation tasks. We observe that sub-sampling and max pooling layers may lead to a drop in performance in tasks that involve voxel-sized structures. To this end, the DeepVesselNet architecture does not use any form of sub-sampling layer and works well for vessel segmentation, centerline prediction, and bifurcation detection. We make our synthetic training data publicly available, fostering future research, and serving as one of the first public datasets for brain vessel tree segmentation and analysis.
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http://dx.doi.org/10.3389/fnins.2020.592352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753013PMC
December 2020

Predicting Vessel Diameter Changes to Up-Regulate Biphasic Blood Flow During Activation in Realistic Microvascular Networks.

Front Physiol 2020 16;11:566303. Epub 2020 Oct 16.

Institute of Fluid Dynamics, ETH Zurich, Zurich, Switzerland.

A dense network of blood vessels distributes blood to different regions of the brain. To meet the temporarily and spatially varying energy demand resulting from changes in neuronal activity, the vasculature is able to locally up-regulate the blood supply. However, to which extent diameter changes of different vessel types contribute to the up-regulation, as well as the spatial and temporal characteristics of their changes, are currently unknown. Here, we present a new simulation method, which solves an inverse problem to calculate diameter changes of individual blood vessels needed to achieve predefined blood flow distributions in microvascular networks. This allows us to systematically compare the impact of different vessel types in various regulation scenarios. Moreover, the method offers the advantage that it handles the stochastic nature of blood flow originating from tracking the movement of individual red blood cells. Since the inverse problem is formulated for time-averaged pressures and flow rates, a deterministic approach for calculating the diameter changes is used, which allows us to apply the method for large realistic microvascular networks with high-dimensional parameter spaces. Our results obtained in both artificial and realistic microvascular networks reveal that diameter changes at the level of capillaries enable a very localized regulation of blood flow. In scenarios where only larger vessels, i.e., arterioles, are allowed to adapt, the flow increase cannot be confined to a specific activated region and flow changes spread into neighboring regions. Furthermore, relatively small dilations and constrictions of all vessel types can lead to substantial changes of capillary blood flow distributions. This suggests that small scale regulation is necessary to obtain a localized increase in blood flow.
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http://dx.doi.org/10.3389/fphys.2020.566303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596696PMC
October 2020

Neutrophils Obstructing Brain Capillaries Are a Major Cause of No-Reflow in Ischemic Stroke.

Cell Rep 2020 10;33(2):108260

Department of Neurology, University Hospital and University of Zurich, and Zurich Neuroscience Center, Zurich, Switzerland. Electronic address:

Despite successful clot retrieval in large vessel occlusion stroke, ∼50% of patients have an unfavorable clinical outcome. The mechanisms underlying this functional reperfusion failure remain unknown, and therapeutic options are lacking. In the thrombin-model of middle cerebral artery (MCA) stroke in mice, we show that, despite successful thrombolytic recanalization of the proximal MCA, cortical blood flow does not fully recover. Using in vivo two-photon imaging, we demonstrate that this is due to microvascular obstruction of ∼20%-30% of capillaries in the infarct core and penumbra by neutrophils adhering to distal capillary segments. Depletion of circulating neutrophils using an anti-Ly6G antibody restores microvascular perfusion without increasing the rate of hemorrhagic complications. Strikingly, infarct size and functional deficits are smaller in mice treated with anti-Ly6G. Thus, we propose neutrophil stalling of brain capillaries to contribute to reperfusion failure, which offers promising therapeutic avenues for ischemic stroke.
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http://dx.doi.org/10.1016/j.celrep.2020.108260DOI Listing
October 2020

Arousal-induced cortical activity triggers lactate release from astrocytes.

Nat Metab 2020 02 17;2(2):179-191. Epub 2020 Feb 17.

Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.

It has been suggested that, in states of arousal, release of noradrenaline and β-adrenergic signalling affect long-term memory formation by stimulating astrocytic lactate production from glycogen. However, the temporal relationship between cortical activity and cellular lactate fluctuations upon changes in arousal remains to be fully established. Also, the role of β-adrenergic signalling and brain glycogen metabolism on neural lactate dynamics in vivo is still unknown. Here, we show that an arousal-induced increase in cortical activity triggers lactate release into the extracellular space, and this correlates with a fast and prominent lactate dip in astrocytes. The immediate drop in astrocytic lactate concentration and the parallel increase in extracellular lactate levels underline an activity-dependent lactate release from astrocytes. Moreover, when β-adrenergic signalling is blocked or the brain is depleted of glycogen, the arousal-evoked cellular lactate surges are significantly reduced. We provide in vivo evidence that cortical activation upon arousal triggers lactate release from astrocytes, a rise in intracellular lactate levels mediated by β-adrenergic signalling and the mobilization of lactate from glycogen stores.
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http://dx.doi.org/10.1038/s42255-020-0170-4DOI Listing
February 2020

A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents.

Nat Protoc 2020 08 6;15(8):2301-2320. Epub 2020 Jul 6.

Laboratory of Molecular and Behavioral Neuroscience, Institute for Neuroscience, Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland.

The locus coeruleus (LC) is a region in the brainstem that produces noradrenaline and is involved in both normal and pathological brain function. Pupillometry, the measurement of pupil diameter, provides a powerful readout of LC activity in rodents, primates and humans. The protocol detailed here describes a miniaturized setup that can screen LC activity in rodents in real-time and can be established within 1-2 d. Using low-cost Raspberry Pi computers and cameras, the complete custom-built system costs only ~300 euros, is compatible with stereotaxic surgery frames and seamlessly integrates into complex experimental setups. Tools for pupil tracking and a user-friendly Pupillometry App allow quantification, analysis and visualization of pupil size. Pupillometry can discriminate between different, physiologically relevant firing patterns of the LC and can accurately report LC activation as measured by noradrenaline turnover. Pupillometry provides a rapid, non-invasive readout that can be used to verify accurate placement of electrodes/fibers in vivo, thus allowing decisions about the inclusion/exclusion of individual animals before experiments begin.
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http://dx.doi.org/10.1038/s41596-020-0324-6DOI Listing
August 2020

Author Correction: Structural basis of astrocytic Ca signals at tripartite synapses.

Nat Commun 2020 05 18;11(1):2541. Epub 2020 May 18.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-16453-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235234PMC
May 2020

Structural basis of astrocytic Ca signals at tripartite synapses.

Nat Commun 2020 04 20;11(1):1906. Epub 2020 Apr 20.

University of Bordeaux, Bordeaux, France.

Astrocytic Ca signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca imaging showed that nodes were biochemical compartments and Ca microdomains. Mapping astrocytic Ca signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.
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http://dx.doi.org/10.1038/s41467-020-15648-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170846PMC
April 2020

A Bright and Colorful Future for G-Protein Coupled Receptor Sensors.

Front Cell Neurosci 2020 20;14:67. Epub 2020 Mar 20.

Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.

Neurochemicals have a large impact on brain states and animal behavior but are notoriously hard to detect accurately in the living brain. Recently developed genetically encoded sensors obtained from engineering a circularly permuted green fluorescent protein into G-protein coupled receptors (GPCR) provided a vital boost to neuroscience, by innovating the way we monitor neural communication. These new probes are becoming widely successful due to their flexible combination with state of the art optogenetic tools and imaging techniques, mainly fiber photometry and 2-photon microscopy, to dissect dynamic changes in brain chemicals with unprecedented spatial and temporal resolution. Here, we highlight current approaches and challenges as well as novel insights in the process of GPCR sensor development, and discuss possible future directions of the field.
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http://dx.doi.org/10.3389/fncel.2020.00067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098945PMC
March 2020

Red blood cells stabilize flow in brain microvascular networks.

PLoS Comput Biol 2019 08 30;15(8):e1007231. Epub 2019 Aug 30.

Institute of Fluid Dynamics, ETH Zurich, Sonneggstrasse 3, Zurich, Switzerland.

Capillaries are the prime location for oxygen and nutrient exchange in all tissues. Despite their fundamental role, our knowledge of perfusion and flow regulation in cortical capillary beds is still limited. Here, we use in vivo measurements and blood flow simulations in anatomically accurate microvascular network to investigate the impact of red blood cells (RBCs) on microvascular flow. Based on these in vivo and in silico experiments, we show that the impact of RBCs leads to a bias toward equating the values of the outflow velocities at divergent capillary bifurcations, for which we coin the term "well-balanced bifurcations". Our simulation results further reveal that hematocrit heterogeneity is directly caused by the RBC dynamics, i.e. by their unequal partitioning at bifurcations and their effect on vessel resistance. These results provide the first in vivo evidence of the impact of RBC dynamics on the flow field in the cortical microvasculature. By structural and functional analyses of our blood flow simulations we show that capillary diameter changes locally alter flow and RBC distribution. A dilation of 10% along a vessel length of 100 μm increases the flow on average by 21% in the dilated vessel downstream a well-balanced bifurcation. The number of RBCs rises on average by 27%. Importantly, RBC up-regulation proves to be more effective the more balanced the outflow velocities at the upstream bifurcation are. Taken together, we conclude that diameter changes at capillary level bear potential to locally change the flow field and the RBC distribution. Moreover, our results suggest that the balancing of outflow velocities contributes to the robustness of perfusion. Based on our in silico results, we anticipate that the bi-phasic nature of blood and small-scale regulations are essential for a well-adjusted oxygen and energy substrate supply.
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http://dx.doi.org/10.1371/journal.pcbi.1007231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750893PMC
August 2019

In vivo imaging with a water immersion objective affects brain temperature, blood flow and oxygenation.

Elife 2019 08 9;8. Epub 2019 Aug 9.

Laboratory of Neurophysiology and New Microscopy, INSERM U1128, Université Paris Descartes, Paris, France.

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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http://dx.doi.org/10.7554/eLife.47324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707784PMC
August 2019

Rapid Reconfiguration of the Functional Connectome after Chemogenetic Locus Coeruleus Activation.

Neuron 2019 08 18;103(4):702-718.e5. Epub 2019 Jun 18.

Laboratory of Molecular and Behavioral Neuroscience, Institute for Neuroscience, Department of Health Sciences and Technology, ETH Zürich, Zürich, Switzerland; Neuroscience Center Zürich, ETH Zürich and University of Zürich, Zürich, Switzerland. Electronic address:

The locus coeruleus (LC) supplies norepinephrine (NE) to the entire forebrain and regulates many fundamental brain functions. Studies in humans have suggested that strong LC activation might shift network connectivity to favor salience processing. To causally test this hypothesis, we use a mouse model to study the effect of LC stimulation on large-scale functional connectivity by combining chemogenetic activation of the LC with resting-state fMRI, an approach we term "chemo-connectomics." We show that LC activation rapidly interrupts ongoing behavior and strongly increases brain-wide connectivity, with the most profound effects in the salience and amygdala networks. Functional connectivity changes strongly correlate with transcript levels of alpha-1 and beta-1 adrenergic receptors across the brain, and functional network connectivity correlates with NE turnover within select brain regions. We propose that these changes in large-scale network connectivity are critical for optimizing neural processing in the context of increased vigilance and threat detection.
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http://dx.doi.org/10.1016/j.neuron.2019.05.034DOI Listing
August 2019

Oxyphor 2P: A High-Performance Probe for Deep-Tissue Longitudinal Oxygen Imaging.

Cell Metab 2019 03 24;29(3):736-744.e7. Epub 2019 Jan 24.

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Quantitative imaging of oxygen distributions in tissue can provide invaluable information about metabolism in normal and diseased states. Two-photon phosphorescence lifetime microscopy (2PLM) has been developed to perform measurements of oxygen in vivo with micron-scale resolution in 3D; however, the method's potential has not yet been fully realized due to the limitations of current phosphorescent probe technology. Here, we report a new sensor, Oxyphor 2P, that enables oxygen microscopy twice as deep (up to 600 μm below the tissue surface) and with ∼60 times higher speed than previously possible. Oxyphor 2P allows longitudinal oxygen measurements without having to inject the probe directly into the imaged region. As proof of principle, we monitored oxygen dynamics for days following micro-stroke induced by occlusion of a single capillary in the mouse brain. Oxyphor 2P opens up new possibilities for studies of tissue metabolic states using 2PLM in a wide range of biomedical research areas.
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http://dx.doi.org/10.1016/j.cmet.2018.12.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402963PMC
March 2019

Non-Canonical Control of Neuronal Energy Status by the Na Pump.

Cell Metab 2019 03 6;29(3):668-680.e4. Epub 2018 Dec 6.

Centro de Estudios Científicos (CECs), Casilla 1469, 5110466 Valdivia, Chile. Electronic address:

Neurons have limited intracellular energy stores but experience acute and unpredictable increases in energy demand. To better understand how these cells repeatedly transit from a resting to active state without undergoing metabolic stress, we monitored their early metabolic response to neurotransmission using ion-sensitive probes and FRET sensors in vitro and in vivo. A short theta burst triggered immediate Na entry, followed by a delayed stimulation of the Na/K ATPase pump. Unexpectedly, cytosolic ATP and ADP levels were unperturbed across a wide range of physiological workloads, revealing strict flux coupling between the Na pump and mitochondria. Metabolic flux measurements revealed a "priming" phase of mitochondrial energization by pyruvate, whereas glucose consumption coincided with delayed Na pump stimulation. Experiments revealed that the Na pump plays a permissive role for mitochondrial ATP production and glycolysis. We conclude that neuronal energy homeostasis is not mediated by adenine nucleotides or by Ca, but by a mechanism commanded by the Na pump.
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http://dx.doi.org/10.1016/j.cmet.2018.11.005DOI Listing
March 2019

Intravitreal AAV-Delivery of Genetically Encoded Sensors Enabling Simultaneous Two-Photon Imaging and Electrophysiology of Optic Nerve Axons.

Front Cell Neurosci 2018 23;12:377. Epub 2018 Oct 23.

Institute of Pharmacology & Toxicology, University of Zurich, Zurich, Switzerland.

Myelination of axons by oligodendrocytes is a key feature of the remarkably fast operating CNS. Oligodendrocytes not only tune axonal conduction speed but are also suggested to maintain long-term axonal integrity by providing metabolic support to the axons they ensheath. However, how myelinating oligodendrocytes impact axonal energy homeostasis remains poorly understood and difficult to investigate. Here, we provide a method of how to study electrically active myelinated axons expressing genetically encoded sensors by combining electrophysiology and two-photon imaging of acutely isolated optic nerves. We show that intravitreal adeno-associated viral (AAV) vector delivery is an efficient tool to achieve functional sensor expression in optic nerve axons, which is demonstrated by measuring axonal ATP dynamics following AAV-mediated sensor expression. This novel approach allows for fast expression of any optical sensor of interest to be studied in optic nerve axons without the need to go through the laborious process of producing new transgenic mouse lines. Viral-mediated biosensor expression in myelinated axons and the subsequent combination of nerve recordings and sensor imaging outlines a powerful method to investigate oligodendroglial support functions and to further interrogate cellular mechanisms governing axonal energy homeostasis under physiological and pathological conditions.
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http://dx.doi.org/10.3389/fncel.2018.00377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205974PMC
October 2018

The Relation Between Capillary Transit Times and Hemoglobin Saturation Heterogeneity. Part 2: Capillary Networks.

Front Physiol 2018 21;9:1296. Epub 2018 Sep 21.

Department of Mechanical and Process Engineering, Institute of Fluid Dynamics, ETH Zürich, Zurich, Switzerland.

Brain metabolism is highly dependent on continuous oxygen supply. Cortical microvascular networks exhibit heterogeneous blood flow, leading to non-uniform tissue oxygenation and capillary hemoglobin saturation. We recently proposed capillary outflow saturation heterogeneity (COSH) to represent effects of heterogeneity on oxygen supply to tissue regions most vulnerable to hypoxia, and showed that diffusive oxygen exchange among red blood cells within capillaries and among capillaries (diffusive interaction) significantly reduces COSH in simplified geometrical configurations. Here, numerical simulations of oxygen transport in capillary network geometries derived from mouse somatosensory cortex are presented. Diffusive interaction was found to reduce COSH by 41 to 62% compared to simulations where diffusive interaction was excluded. Hemoglobin saturation drop across the microvascular network is strongly correlated with red blood cell transit time, but the coefficient of variation of saturation drop is approximately one third lower. Unexpectedly, the radius of the tissue cylinder supplied by a capillary correlates weakly with the anatomical tissue cylinder radius, but strongly with hemoglobin saturation. Thus, diffusive interaction contributes greatly to the microcirculation's ability to achieve tissue oxygenation, despite heterogeneous capillary transit time and hematocrit distribution. These findings provide insight into the effects of cerebral small vessel disease on tissue oxygenation and brain function.
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http://dx.doi.org/10.3389/fphys.2018.01296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160581PMC
September 2018

The Relation Between Capillary Transit Times and Hemoglobin Saturation Heterogeneity. Part 1: Theoretical Models.

Front Physiol 2018 26;9:420. Epub 2018 Apr 26.

Department of Mechanical and Process Engineering, Institute of Fluid Dynamics, ETH Zurich, Zurich, Switzerland.

Capillary dysfunction impairs oxygen supply to parenchymal cells and often occurs in Alzheimer's disease, diabetes and aging. Disturbed capillary flow patterns have been shown to limit the efficacy of oxygen extraction and can be quantified using capillary transit time heterogeneity (CTH). However, the transit time of red blood cells (RBCs) through the microvasculature is not a direct measure of their capacity for oxygen delivery. Here we examine the relation between CTH and capillary outflow saturation heterogeneity (COSH), which is the heterogeneity of blood oxygen content at the venous end of capillaries. Models for the evolution of hemoglobin saturation heterogeneity (HSH) in capillary networks were developed and validated using a computational model with moving RBCs. Two representative situations were selected: a Krogh cylinder geometry with heterogeneous hemoglobin saturation (HS) at the inflow, and a parallel array of four capillaries. The heterogeneity of HS after converging capillary bifurcations was found to exponentially decrease with a time scale of 0.15-0.21 s due to diffusive interaction between RBCs. Similarly, the HS difference between parallel capillaries also drops exponentially with a time scale of 0.12-0.19 s. These decay times are substantially smaller than measured RBC transit times and only weakly depend on the distance between microvessels. This work shows that diffusive interaction strongly reduces COSH on a small spatial scale. Therefore, we conclude that CTH influences COSH yet does not determine it. The second part of this study will focus on simulations in microvascular networks from the rodent cerebral cortex. Actual estimates of COSH and CTH will then be given.
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http://dx.doi.org/10.3389/fphys.2018.00420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932636PMC
April 2018

Cortical Circuit Activity Evokes Rapid Astrocyte Calcium Signals on a Similar Timescale to Neurons.

Neuron 2018 05 26;98(4):726-735.e4. Epub 2018 Apr 26.

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; Neuroscience Center, University and ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Electronic address:

Sensory stimulation evokes intracellular calcium signals in astrocytes; however, the timing of these signals is disputed. Here, we used novel combinations of genetically encoded calcium indicators for concurrent two-photon imaging of cortical astrocytes and neurons in awake mice during whisker deflection. We identified calcium responses in both astrocyte processes and endfeet that rapidly followed neuronal events (∼120 ms after). These fast astrocyte responses were largely independent of IPR2-mediated signaling and known neuromodulator activity (acetylcholine, serotonin, and norepinephrine), suggesting that they are evoked by local synaptic activity. The existence of such rapid signals implies that astrocytes are fast enough to play a role in synaptic modulation and neurovascular coupling. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.neuron.2018.03.050DOI Listing
May 2018

Fiber-optic implant for simultaneous fluorescence-based calcium recordings and BOLD fMRI in mice.

Nat Protoc 2018 05 29;13(5):840-855. Epub 2018 Mar 29.

Institute for Biomedical Engineering, University of Zurich and ETH Zurich, Zurich, Switzerland.

Despite the growing popularity of blood oxygen level-dependent (BOLD) functional MRI (fMRI), understanding of its underlying principles is still limited. This protocol describes a technique for simultaneous measurement of neural activity using fluorescent calcium indicators together with the corresponding hemodynamic BOLD fMRI response in the mouse brain. Our early work using small-molecule fluorophores in rats gave encouraging results but was limited to acute measurements using synthetic dyes. Our latest procedure combines fMRI with optical detection of cell-type-specific virally delivered GCaMP6, a genetically encoded calcium indicator (GECI). GCaMP6 fluorescence, which increases upon calcium binding, is collected by a chronically implanted optical fiber, allowing longitudinal studies in mice. The chronic implant, placed horizontally on the skull, has an angulated tip that reflects light into the brain and is connected via fiber optics to a remote optical setup. The technique allows access to the neocortex and does not require adaptations of commercial MRI hardware. The hybrid approach permits fiber-optic calcium recordings with simultaneous artifact-free BOLD fMRI with full brain coverage and 1-s temporal resolution using standard gradient-echo echo-planar imaging (GE-EPI) sequences. The method provides robust, cell-type-specific readouts to link neural activity to BOLD signals, as emonstrated for task-free ('resting-state') conditions and in response to hind-paw stimulation. These results highlight the power of fiber photometry combined with fMRI, which we aim to further advance in this protocol. The approach can be easily adapted to study other molecular processes using suitable fluorescent indicators.
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http://dx.doi.org/10.1038/nprot.2018.003DOI Listing
May 2018

DCC Is Required for the Development of Nociceptive Topognosis in Mice and Humans.

Cell Rep 2018 01;22(5):1105-1114

Neural Circuit Development Laboratory, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, QC, Canada; Integrated Program in Neuroscience, McGill University, Montreal, QC, Canada; Department of Anatomy and Cell Biology, Division of Experimental Medicine, McGill University, Montreal, QC, Canada. Electronic address:

Avoidance of environmental dangers depends on nociceptive topognosis, or the ability to localize painful stimuli. This is proposed to rely on somatotopic maps arising from topographically organized point-to-point connections between the body surface and the CNS. To determine the role of topographic organization of spinal ascending projections in nociceptive topognosis, we generated a conditional knockout mouse lacking expression of the netrin1 receptor DCC in the spinal cord. These mice have an increased number of ipsilateral spinothalamic connections and exhibit aberrant activation of the somatosensory cortex in response to unilateral stimulation. Furthermore, spinal cord-specific Dcc knockout animals displayed mislocalized licking responses to formalin injection, indicating impaired topognosis. Similarly, humans with DCC mutations experience bilateral sensation evoked by unilateral somatosensory stimulation. Collectively, our results constitute functional evidence of the importance of topographic organization of spinofugal connections for nociceptive topognosis.
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http://dx.doi.org/10.1016/j.celrep.2018.01.004DOI Listing
January 2018

Current technical approaches to brain energy metabolism.

Glia 2018 06 7;66(6):1138-1159. Epub 2017 Nov 7.

Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.

Neuroscience is a technology-driven discipline and brain energy metabolism is no exception. Once satisfied with mapping metabolic pathways at organ level, we are now looking to learn what it is exactly that metabolic enzymes and transporters do and when, where do they reside, how are they regulated, and how do they relate to the specific functions of neurons, glial cells, and their subcellular domains and organelles, in different areas of the brain. Moreover, we aim to quantify the fluxes of metabolites within and between cells. Energy metabolism is not just a necessity for proper cell function and viability but plays specific roles in higher brain functions such as memory processing and behavior, whose mechanisms need to be understood at all hierarchical levels, from isolated proteins to whole subjects, in both health and disease. To this aim, the field takes advantage of diverse disciplines including anatomy, histology, physiology, biochemistry, bioenergetics, cellular biology, molecular biology, developmental biology, neurology, and mathematical modeling. This article presents a well-referenced synopsis of the technical side of brain energy metabolism research. Detail and jargon are avoided whenever possible and emphasis is given to comparative strengths, limitations, and weaknesses, information that is often not available in regular articles.
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http://dx.doi.org/10.1002/glia.23248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5903992PMC
June 2018

CHIPS: an Extensible Toolbox for Cellular and Hemodynamic Two-Photon Image Analysis.

Neuroinformatics 2018 01;16(1):145-147

Institute of Pharmacology and Toxicology, University of Zurich and Neuroscience Center Zurich, Winterthurerstrasse 190, 8057, Zürich, Switzerland.

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http://dx.doi.org/10.1007/s12021-017-9344-yDOI Listing
January 2018

Long-term In Vivo Calcium Imaging of Astrocytes Reveals Distinct Cellular Compartment Responses to Sensory Stimulation.

Cereb Cortex 2018 01;28(1):184-198

Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich, Switzerland.

Localized, heterogeneous calcium transients occur throughout astrocytes, but the characteristics and long-term stability of these signals, particularly in response to sensory stimulation, remain unknown. Here, we used a genetically encoded calcium indicator and an activity-based image analysis scheme to monitor astrocyte calcium activity in vivo. We found that different subcellular compartments (processes, somata, and endfeet) displayed distinct signaling characteristics. Closer examination of individual signals showed that sensory stimulation elevated the number of specific types of calcium peaks within astrocyte processes and somata, in a cortical layer-dependent manner, and that the signals became more synchronous upon sensory stimulation. Although mice genetically lacking astrocytic IP3R-dependent calcium signaling (Ip3r2-/-) had fewer signal peaks, the response to sensory stimulation was sustained, suggesting other calcium pathways are also involved. Long-term imaging of astrocyte populations revealed that all compartments reliably responded to stimulation over several months, but that the location of the response within processes may vary. These previously unknown characteristics of subcellular astrocyte calcium signals provide new insights into how astrocytes may encode local neuronal circuit activity.
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http://dx.doi.org/10.1093/cercor/bhw366DOI Listing
January 2018

Stabilizing g-States in Centrosymmetric Tetrapyrroles: Two-Photon-Absorbing Porphyrins with Bright Phosphorescence.

J Phys Chem A 2017 Aug 15;121(33):6243-6255. Epub 2017 Aug 15.

NanoScience Technology Center, Department of Chemistry and Department of Physics, University of Central Florida , Orlando, Florida United States.

Using time-dependent density functional theory (TDDFT) and sum-overstates (SOS) formalism, we predicted significant stabilization of 2P-active g-states in a compact fully symmetric porphyrin, in which all four pyrrolic fragments are fused with phathalimide residues via the β-carbon positions. The synthesis of a soluble, nonaggregating meso-unsubstituted tetraarylphthalimidoporphyrin (TAPIP) was then developed, and the spectroscopic measurements confirmed that a strongly 2P-active state in this porphyrin is stabilized below the B (Soret) state level. Single-crystal X-ray analysis revealed near-ideally planar geometry of the TAPIP macrocycle, while its tetra-meso-arylated analogue (meso-ArTAPIP) was found to be highly saddled. Consistent with these structural features, Pt meso-ArTAPIP phosphoresces rather weakly (ϕ = 0.05 in DMF at 22 °C), while both Pt and Pd complexes of TAPIP are highly phosphorescent (ϕ = 0.45 and 0.23, respectively). In addition PdTAPIP exhibits non-negligible thermally activated (E-type) delayed fluorescence (ϕ(d) ∼ 0.012). Taken together, these photophysical properties make metal complexes of meso-unsubstituted tetaarylphthalimidoporphyrins the brightest 2P-absorbing phosphorescent chromophores known to date.
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http://dx.doi.org/10.1021/acs.jpca.7b04333DOI Listing
August 2017

Vascular density and distribution in neocortex.

Neuroimage 2019 08 29;197:792-805. Epub 2017 Jun 29.

Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland; Neuroscience Center, University and ETH Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.

An amazingly wide range of complex behavior emerges from the cerebral cortex. Much of the information processing that leads to these behaviors is performed in neocortical circuits that span throughout the six layers of the cortex. Maintaining this circuit activity requires substantial quantities of oxygen and energy substrates, which are delivered by the complex yet well-organized and tightly-regulated vascular system. In this review, we provide a detailed characterization of the most relevant anatomical and functional features of the cortical vasculature. This includes a compilation of the available data on laminar variation of vascular density and the topological aspects of the microvascular system. We also review the spatio-temporal dynamics of cortical blood flow regulation and oxygenation, many aspects of which remain poorly understood. Finally, we discuss some of the important implications of vascular density, distribution, oxygenation and blood flow regulation for (laminar) fMRI.
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http://dx.doi.org/10.1016/j.neuroimage.2017.06.046DOI Listing
August 2019

Depth-dependent flow and pressure characteristics in cortical microvascular networks.

PLoS Comput Biol 2017 02 14;13(2):e1005392. Epub 2017 Feb 14.

Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.

A better knowledge of the flow and pressure distribution in realistic microvascular networks is needed for improving our understanding of neurovascular coupling mechanisms and the related measurement techniques. Here, numerical simulations with discrete tracking of red blood cells (RBCs) are performed in three realistic microvascular networks from the mouse cerebral cortex. Our analysis is based on trajectories of individual RBCs and focuses on layer-specific flow phenomena until a cortical depth of 1 mm. The individual RBC trajectories reveal that in the capillary bed RBCs preferentially move in plane. Hence, the capillary flow field shows laminar patterns and a layer-specific analysis is valid. We demonstrate that for RBCs entering the capillary bed close to the cortical surface (< 400 μm) the largest pressure drop takes place in the capillaries (37%), while for deeper regions arterioles are responsible for 61% of the total pressure drop. Further flow characteristics, such as capillary transit time or RBC velocity, also vary significantly over cortical depth. Comparison of purely topological characteristics with flow-based ones shows that a combined interpretation of topology and flow is indispensable. Our results provide evidence that it is crucial to consider layer-specific differences for all investigations related to the flow and pressure distribution in the cortical vasculature. These findings support the hypothesis that for an efficient oxygen up-regulation at least two regulation mechanisms must be playing hand in hand, namely cerebral blood flow increase and microvascular flow homogenization. However, the contribution of both regulation mechanisms to oxygen up-regulation likely varies over depth.
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http://dx.doi.org/10.1371/journal.pcbi.1005392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347440PMC
February 2017
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