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.

Highlights d A high-performance phosphorescent probe for measuring oxygen in biological systems d Two-photon imaging as deep as 600 mm at 60 times higher speed than using existing methods d A probe delivery approach that avoids local tissue damage in the brain d The ability to perform longitudinal oxygen imaging over many-day periods SUMMARY 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 mm 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.

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.

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. AbstractLocalized, heterogeneous calcium transients occur throughout astrocytes, but the characteristics and

The purpose of this study was to determine the effects of inhibitors of Rho kinase (ROK) and matrix metalloproteinases (MMPs) on angiogenesis and tumor growth and to evaluate ROK activity in human prostate cancer PC3 cells and endothelial cells (HUVECs). Vacuolation by endothelial cells and lumen formation, the earliest detectable stages of angiogenesis, were inhibited by the ROK inhibitor Wf-536. Combining Wf-536 with the MMP inhibitor Marimastat greatly enhanced in vitro inhibition of endothelial vacuolation, lumen and cord formation, and VEGF- and HGF-stimulated endothelial sprout formation from aorta. Inhibition of sprout formation by the two inhibitors was synergistic. Both agents inhibited migration of HUVECs. The regulatory subunit (MYPT1) of the myosin phosphatase was phosphorylated in PC3 cells and HUVECs, and phosphorylation of MYPT1 and the myosin regulatory light chain was reduced by Wf-536, providing direct evidence of ROK activity. Early treatment of immuno-incompetent mice bearing xenotransplants of PC3 cells with a combination of Wf-536 plus Marimastat with or without Paclitaxel, significantly inhibited tumor growth, prevented tumor growth escape after discontinuation of Paclitaxel, and increased survival.

Two-photon microscopy is able to produce exquisite and informative functional images of cells and blood vessels in living animals or tissue,1 especially when combined with genetically-encoded indicators of cellular activity.2 Since these imaging techniques typically produce too much data to analyse manually, a number of automated or semi-automated image analysis approaches have been developed. However, even aside from the algorithms themselves, many factors can complicate analysis workflows.

Abstract:We present a cost-effective in vivo two-photon microscope with a highly flexible frontend for in vivo research. Our design ensures fast and reproducible access to the area of interest, including rotation of imaging plane, and maximizes space for auxiliary experimental equipment in the vicinity of the animal. Mechanical flexibility is achieved with large motorized linear stages that move the objective in the X, Y, and Z directions up to 130 mm. 360° rotation of the frontend (rotational freedom for one axis) is achieved with the combination of a motorized high precision bearing and gearing. Additionally, the modular design of the frontend, based on commercially available optomechanical parts, allows straightforward updates to future scanning technologies. The design exceeds the mobility of previous movable microscope designs while maintaining high optical performance.

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.