Cortex-wide transcranial localization microscopy with fluorescently labeled red blood cells.

Large-scale imaging of brain activity with high spatio-temporal resolution is crucial for advancing our understanding of brain function. The existing neuroimaging techniques are largely limited by restricted field of view, slow imaging speed, or otherwise do not have the adequate spatial resolution to capture brain activities on a capillary and cellular level. To address these limitations, we introduce fluorescence localization microscopy aided with sparsely-labeled red blood cells for cortex-wide morphological and functional cerebral angiography with 4.9 µm spatial resolution and 1 s temporal resolution. When combined with fluorescence calcium imaging, the proposed method enables extended recordings of stimulus-evoked neuro-vascular changes in the murine brain while providing simultaneous multiparametric readings of intracellular neuronal activity, blood flow velocity/direction/volume, and vessel diameter. Owing to its simplicity and versatility, the proposed approach will become an invaluable tool for deciphering the regulation of cortical microcirculation and neurovascular coupling in health and disease.

The Astrocyte: Metabolic Hub of the Brain.

Astrocytic metabolism has taken center stage. Interposed between the neuron and the vasculature, astrocytes exert control over the fluxes of energy and building blocks required for neuronal activity and plasticity. They are also key to local detoxification and waste recycling. Whereas neurons are metabolically rigid, astrocytes can switch between different metabolic profiles according to local demand and the nutritional state of the organism. Their metabolic state even seems to be instructive for peripheral nutrient mobilization and has been implicated in information processing and behavior. Here, we summarize recent progress in our understanding of astrocytic metabolism and its effects on metabolic homeostasis and cognition.

Leptomeningeal collaterals regulate reperfusion in ischemic stroke and rescue the brain from futile recanalization.

Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet-unknown functions. We applied laser speckle imaging, ultrafast ultrasound, and two-photon microscopy in a thrombin-based mouse model of stroke and fibrinolytic treatment to show that LMCs maintain cerebral autoregulation and allow for gradual reperfusion, resulting in small infarcts. In mice with poor LMCs, distal arterial segments collapse, and deleterious hyperemia causes hemorrhage and mortality after recanalization. In silico analyses confirm the relevance of LMCs for preserving perfusion in the ischemic region. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion and preventing futile recanalization after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for beneficial reperfusion after stroke.

The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation.

Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.

Cortical astrocyte N-methyl-D-aspartate receptors influence whisker barrel activity and sensory discrimination in mice.

Astrocytes express ionotropic receptors, including N-methyl-D-aspartate receptors (NMDARs). However, the contribution of NMDARs to astrocyte-neuron interactions, particularly in vivo, has not been elucidated. Here we show that a knockdown approach to selectively reduce NMDARs in mouse cortical astrocytes decreases astrocyte Ca2+ transients evoked by sensory stimulation. Astrocyte NMDAR knockdown also impairs nearby neuronal circuits by elevating spontaneous neuron activity and limiting neuronal recruitment, synchronization, and adaptation during sensory stimulation. Furthermore, this compromises the optimal processing of sensory information since the sensory acuity of the mice is reduced during a whisker-dependent tactile discrimination task. Lastly, we rescue the effects of astrocyte NMDAR knockdown on neurons and improve the tactile acuity of the animal by supplying exogenous ATP. Overall, our findings show that astrocytes can respond to nearby neuronal activity via their NMDAR, and that these receptors are an important component for purinergic signaling that regulate astrocyte-neuron interactions and cortical sensory discrimination in vivo.

Oligodendrocyte-axon metabolic coupling is mediated by extracellular K+ and maintains axonal health.

The integrity of myelinated axons relies on homeostatic support from oligodendrocytes (OLs). To determine how OLs detect axonal spiking and how rapid axon-OL metabolic coupling is regulated in the white matter, we studied activity-dependent calcium (Ca2+) and metabolite fluxes in the mouse optic nerve. We show that fast axonal spiking triggers Ca2+ signaling and glycolysis in OLs. OLs detect axonal activity through increases in extracellular potassium (K+) concentrations and activation of Kir4.1 channels, thereby regulating metabolite supply to axons. Both pharmacological inhibition and OL-specific inactivation of Kir4.1 reduce the activity-induced axonal lactate surge. Mice lacking oligodendroglial Kir4.1 exhibit lower resting lactate levels and altered glucose metabolism in axons. These early deficits in axonal energy metabolism are associated with late-onset axonopathy. Our findings reveal that OLs detect fast axonal spiking through K+ signaling, making acute metabolic coupling possible and adjusting the axon-OL metabolic unit to promote axonal health.

SAFIR-I: first NEMA NU 4-2008-based performance characterization.

BACKGROUND: Small Animal Fast Insert for MRI detector I (SAFIR-I) is a novel Positron Emission Tomography insert for a [Formula: see text] Bruker BioSpec 70/30 Ultra Shield Refrigerated Magnetic Resonance Imaging (MRI) system. It facilitates truly simultaneous quantitative imaging in mice and rats at injected activities as high as [Formula: see text]. Exploitation of the resulting high count rates enables quick image formation at few seconds per frame. In this investigation, key performance parameters of SAFIR-I have been determined according to the evaluations outlined in the National Electrical Manufacturers Association (NEMA) Standards Publication NU 4-2008 (NEMA-NU4) protocol. RESULTS: Using an energy window of 391 to [Formula: see text] and a Coincidence Timing Window of [Formula: see text], the following performance was observed: The average spatial resolution at [Formula: see text] radial offset (Full Width at Half Maximum) is [Formula: see text] when using Filtered Backprojection, 3D Reprojection reconstruction. For the mouse- and rat-like phantoms, the maximal Noise-Equivalent Count Rates (NECRs) are [Formula: see text] at the highest tested average effective concentration of [Formula: see text], and [Formula: see text] at the highest tested average effective concentration of [Formula: see text], respectively. The NECR peak is not yet reached for either of these cases. The peak sensitivity is [Formula: see text]. The Image Quality phantom uniformity standard deviation is [Formula: see text]. The Recovery Coefficient for the [Formula: see text] rod is [Formula: see text]. The Spill-Over Ratios are [Formula: see text] and [Formula: see text], for the water- and air-filled cylinder, respectively. An accuracy of [Formula: see text] was achieved for the quantitative calibration of reconstructed voxel values. CONCLUSIONS: The measured performance parameters indicate that the various design goals have been achieved. SAFIR-I offers excellent performance, especially at the high activities it was designed for. This facilitates planned experiments with fast tracer kinetics in small animals. Ways to potentially improve performance can still be explored. Simultaneously, further performance gains can be expected for a forthcoming insert featuring 2.7 times longer axial coverage named Small Animal Fast Insert for MRI detector II (SAFIR-II).

The role of leptomeningeal collaterals in redistributing blood flow during stroke.

Leptomeningeal collaterals (LMCs) connect the main cerebral arteries and provide alternative pathways for blood flow during ischaemic stroke. This is beneficial for reducing infarct size and reperfusion success after treatment. However, a better understanding of how LMCs affect blood flow distribution is indispensable to improve therapeutic strategies. Here, we present a novel in silico approach that incorporates case-specific in vivo data into a computational model to simulate blood flow in large semi-realistic microvascular networks from two different mouse strains, characterised by having many and almost no LMCs between middle and anterior cerebral artery (MCA, ACA) territories. This framework is unique because our simulations are directly aligned with in vivo data. Moreover, it allows us to analyse perfusion characteristics quantitatively across all vessel types and for networks with no, few and many LMCs. We show that the occlusion of the MCA directly caused a redistribution of blood that was characterised by increased flow in LMCs. Interestingly, the improved perfusion of MCA-sided microvessels after dilating LMCs came at the cost of a reduced blood supply in other brain areas. This effect was enhanced in regions close to the watershed line and when the number of LMCs was increased. Additional dilations of surface and penetrating arteries after stroke improved perfusion across the entire vasculature and partially recovered flow in the obstructed region, especially in networks with many LMCs, which further underlines the role of LMCs during stroke.

Ultrasound trapping and navigation of microrobots in the mouse brain vasculature.

The intricate and delicate anatomy of the brain poses significant challenges for the treatment of cerebrovascular and neurodegenerative diseases. Thus, precise local drug delivery in hard-to-reach brain regions remains an urgent medical need. Microrobots offer potential solutions; however, their functionality in the brain remains restricted by limited imaging capabilities and complications within blood vessels, such as high blood flows, osmotic pressures, and cellular responses. Here, we introduce ultrasound-activated microrobots for in vivo navigation in brain vasculature. Our microrobots consist of lipid-shelled microbubbles that autonomously aggregate and propel under ultrasound irradiation. We investigate their capacities in vitro within microfluidic-based vasculatures and in vivo within vessels of a living mouse brain. These microrobots self-assemble and execute upstream motion in brain vasculature, achieving velocities up to 1.5 µm/s and moving against blood flows of ~10 mm/s. This work represents a substantial advance towards the therapeutic application of microrobots within the complex brain vasculature.

Sensitive multicolor indicators for monitoring norepinephrine in vivo.

Genetically encoded indicators engineered from G-protein-coupled receptors are important tools that enable high-resolution in vivo neuromodulator imaging. Here, we introduce a family of sensitive multicolor norepinephrine (NE) indicators, which includes nLightG (green) and nLightR (red). These tools report endogenous NE release in vitro, ex vivo and in vivo with improved sensitivity, ligand selectivity and kinetics, as well as a distinct pharmacological profile compared with previous state-of-the-art GRABNE indicators. Using in vivo multisite fiber photometry recordings of nLightG, we could simultaneously monitor optogenetically evoked NE release in the mouse locus coeruleus and hippocampus. Two-photon imaging of nLightG revealed locomotion and reward-related NE transients in the dorsal CA1 area of the hippocampus. Thus, the sensitive NE indicators introduced here represent an important addition to the current repertoire of indicators and provide the means for a thorough investigation of the NE system.

Development of a genetically-encoded sensor for probing endogenous nociceptin opioid peptide release.

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fiber photometry enabled a direct recording of binding by N/OFQ receptor ligands, as well as the detection of natural or chemogenetically-evoked endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA). In summary, we show that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely-behaving animals.

Deep optoacoustic localization microangiography of ischemic stroke in mice.

Super-resolution optoacoustic imaging of microvascular structures deep in mammalian tissues has so far been impeded by strong absorption from densely-packed red blood cells. Here we devised 5 µm biocompatible dichloromethane-based microdroplets exhibiting several orders of magnitude higher optical absorption than red blood cells at near-infrared wavelengths, thus enabling single-particle detection in vivo. We demonstrate non-invasive three-dimensional microangiography of the mouse brain beyond the acoustic diffraction limit (<20 µm resolution). Blood flow velocity quantification in microvascular networks and light fluence mapping was also accomplished. In mice affected by acute ischemic stroke, the multi-parametric multi-scale observations enabled by super-resolution and spectroscopic optoacoustic imaging revealed significant differences in microvascular density, flow and oxygen saturation in ipsi- and contra-lateral brain hemispheres. Given the sensitivity of optoacoustics to functional, metabolic and molecular events in living tissues, the new approach paves the way for non-invasive microscopic observations with unrivaled resolution, contrast and speed.

Deficits in mitochondrial TCA cycle and OXPHOS precede rod photoreceptor degeneration during chronic HIF activation.

BACKGROUND: Major retinal degenerative diseases, including age-related macular degeneration, diabetic retinopathy and retinal detachment, are associated with a local decrease in oxygen availability causing the formation of hypoxic areas affecting the photoreceptor (PR) cells. Here, we addressed the underlying pathological mechanisms of PR degeneration by focusing on energy metabolism during chronic activation of hypoxia-inducible factors (HIFs) in rod PR. METHODS: We used two-photon laser scanning microscopy (TPLSM) of genetically encoded biosensors delivered by adeno-associated viruses (AAV) to determine lactate and glucose dynamics in PR and inner retinal cells. Retinal layer-specific proteomics, in situ enzymatic assays and immunofluorescence studies were used to analyse mitochondrial metabolism in rod PRs during chronic HIF activation. RESULTS: PRs exhibited remarkably higher glycolytic flux through the hexokinases than neurons of the inner retina. Chronic HIF activation in rods did not cause overt change in glucose dynamics but an increase in lactate production nonetheless. Furthermore, dysregulation of the oxidative phosphorylation pathway (OXPHOS) and tricarboxylic acid (TCA) cycle in rods with an activated hypoxic response decelerated cellular anabolism causing shortening of rod photoreceptor outer segments (OS) before onset of cell degeneration. Interestingly, rods with deficient OXPHOS but an intact TCA cycle did not exhibit these early signs of anabolic dysregulation and showed a slower course of degeneration. CONCLUSION: Together, these data indicate an exceeding high glycolytic flux in rods and highlight the importance of mitochondrial metabolism and especially of the TCA cycle for PR survival in conditions of increased HIF activity.

Simultaneous dynamic glucose-enhanced (DGE) MRI and fiber photometry measurements of glucose in the healthy mouse brain.

Glucose is the main energy source in the brain and its regulated uptake and utilization are important biomarkers of pathological brain function. Glucose Chemical Exchange Saturation Transfer (GlucoCEST) and its time-resolved version Dynamic Glucose-Enhanced MRI (DGE) are promising approaches to monitor glucose and detect tumors, since they are radioactivity-free, do not require 13C labeling and are is easily translatable to the clinics. The main principle of DGE is clear. However, what remains to be established is to which extent the signal reflects vascular, extracellular or intracellular glucose. To elucidate the compartmental contributions to the DGE signal, we coupled it with FRET-based fiber photometry of genetically encoded sensors, a technique that combines quantitative glucose readout with cellular specificity. The glucose sensor FLIIP was used with fiber photometry to measure astrocytic and neuronal glucose changes upon injection of D-glucose, 3OMG and L-glucose, in the anaesthetized murine brain. By correlating the kinetic profiles of the techniques, we demonstrate the presence of a vascular contribution to the signal, especially at early time points after injection. Furthermore, we show that, in the case of the commonly used contrast agent 3OMG, the DGE signal actually anticorrelates with the glucose concentration in neurons and astrocytes.

Altered hemodynamics and vascular reactivity in a mouse model with severe pericyte deficiency.

Pericytes are the mural cells of the microvascular network that are in close contact with underlying endothelial cells. Endothelial-secreted PDGFB leads to recruitment of pericytes to the vessel wall, but this is disrupted in Pdgfbret/ret mice when the PDGFB retention motif is deleted. This results in severely reduced pericyte coverage on blood vessels. In this study, we investigated vascular abnormalities and hemodynamics in Pdgfbret/ret mice throughout the cerebrovascular network and in different cortical layers by in vivo two-photon microscopy. We confirmed that Pdgfbret/ret mice are severely deficient in pericytes throughout the vascular network, with enlarged brain blood vessels and a reduced number of vessel branches. Red blood cell velocity, linear density, and tube hematocrit were reduced in Pdgfbret/ret mice, which may impair oxygen delivery to the tissue. We also measured intravascular PO2 and found that concentrations were higher in cortical Layer 2/3 in Pdgfbret/ret mice, indicative of reduced blood oxygen extraction. Finally, we found that Pdgfbret/ret mice had a reduced capacity for vasodilation in response to an acetazolamide challenge during functional MRI imaging. Taken together, these results suggest that severe pericyte deficiency can lead to vascular abnormalities and altered cerebral blood flow, reminiscent of pathologies such as arteriovenous malformations.

Three-dimensional wide-field fluorescence microscopy for transcranial mapping of cortical microcirculation.

Wide-field fluorescence imaging is an indispensable tool for studying large-scale biodynamics. Limited space-bandwidth product and strong light diffusion make conventional implementations incapable of high-resolution mapping of fluorescence biodistribution in three dimensions. We introduce a volumetric wide-field fluorescence microscopy based on optical astigmatism combined with fluorescence source localization, covering 5.6×5.6×0.6 mm3 imaging volume. Two alternative configurations are proposed exploiting multifocal illumination or sparse localization of point emitters, which are herein seamlessly integrated in one system. We demonstrate real-time volumetric mapping of the murine cortical microcirculation at capillary resolution without employing cranial windows, thus simultaneously delivering quantitative perfusion information across both brain hemispheres. Morphological and functional changes of cerebral vascular networks are further investigated after an acute ischemic stroke, enabling cortex-wide observation of concurrent collateral recruitment events occurring on a sub-second scale. The reported technique thus offers a wealth of unmatched possibilities for non- or minimally invasive imaging of biodynamics across scales.

Pericyte remodeling is deficient in the aged brain and contributes to impaired capillary flow and structure.

Deterioration of brain capillary flow and architecture is a hallmark of aging and dementia. It remains unclear how loss of brain pericytes in these conditions contributes to capillary dysfunction. Here, we conduct cause-and-effect studies by optically ablating pericytes in adult and aged mice in vivo. Focal pericyte loss induces capillary dilation without blood-brain barrier disruption. These abnormal dilations are exacerbated in the aged brain, and result in increased flow heterogeneity in capillary networks. A subset of affected capillaries experience reduced perfusion due to flow steal. Some capillaries stall in flow and regress, leading to loss of capillary connectivity. Remodeling of neighboring pericytes restores endothelial coverage and vascular tone within days. Pericyte remodeling is slower in the aged brain, resulting in regions of persistent capillary dilation. These findings link pericyte loss to disruption of capillary flow and structure. They also identify pericyte remodeling as a therapeutic target to preserve capillary flow dynamics.

Measurement of cerebral oxygen pressure in living mice by two-photon phosphorescence lifetime microscopy.

The ability to quantify partial pressure of oxygen (pO2) is of primary importance for studies of metabolic processes in health and disease. Here, we present a protocol for imaging of oxygen distributions in tissue and vasculature of the cerebral cortex of anesthetized and awake mice. We describe in vivo two-photon phosphorescence lifetime microscopy (2PLM) of oxygen using the probe Oxyphor 2P. This minimally invasive protocol outperforms existing approaches in terms of accuracy, resolution, and imaging depth. For complete details on the use and execution of this protocol, please refer to Esipova et al. (2019).

Direct vascular contact is a hallmark of cerebral astrocytes.

Astrocytes establish extensive networks via gap junctions that allow each astrocyte to connect indirectly to the vasculature. However, the proportion of astrocytes directly associated with blood vessels is unknown. Here, we quantify structural contacts of cortical astrocytes with the vasculature in vivo. We show that all cortical astrocytes are connected to at least one blood vessel. Moreover, astrocytes contact more vessels in deeper cortical layers where vessel density is known to be higher. Further examination of different brain regions reveals that only the hippocampus, which has the lowest vessel density of all investigated brain regions, harbors single astrocytes with no apparent vascular connection. In summary, we show that almost all gray matter astrocytes have direct contact to the vasculature. In addition to the glial network, a direct vascular access may represent a complementary pathway for metabolite uptake and distribution.