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Background and Purpose: The TRPV4 ion channels are Ca 2+ permeable, non-selective cation channels that mediate large, but highly localized, Ca 2+ signals in the endothelium. The mechanisms that permit highly localized Ca 2+ changes to evoke cell-wide activity are incompletely understood. Here, we tested the hypothesis that TRPV4-mediated Ca 2+ influx activates Ca 2+ release from internal Ca 2+ stores to generate widespread effects. Experimental Approach: Ca 2+ signals in large numbers (~100) of endothelial cells in intact arteries were imaged and analysed separately. Key Results: Responses to the TRPV4 channel agonist GSK1016790A were heterogeneous across the endothelium. In activated cells, Ca 2+ responses comprised localized Ca 2+ changes leading to slow, persistent, global increases in Ca 2+ followed by large propagating Ca 2+ waves that moved within and between cells. To examine the mechanisms underlying each component, we developed methods to separate slow persistent Ca 2+ rise from the propagating Ca 2+ waves in each cell. TRPV4-mediated Ca 2+ entry was required for the slow persistent global rise and propagating Ca 2+ signals. The propagating waves were inhibited by depleting internal Ca 2+ stores, inhibiting PLC or blocking IP 3 receptors. Ca 2+ release from stores was tightly controlled by TRPV4-mediated Ca 2+ influx and ceased when influx was terminated. Furthermore, Ca 2+ release from internal stores was essential for TRPV4-mediated control of vascular tone. Conclusions and Implications: Ca 2+ influx via TRPV4 channels is amplified by Ca 2+-induced Ca 2+ release acting at IP 3 receptors to generate propagating Ca 2+ waves and provide a large-scale endothelial communication system. TRPV4-mediated control of vascular tone requires Ca 2+ release from the internal store.

Key points Age is proposed to be associated with altered structure and function of mitochondria; however, in fully‐differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel‐by‐pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during ‘flickers’ of mitochondrial membrane potential.Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats.A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly‐elongated.Some mitochondria in myocytes from younger, but not aged, animals are highly‐motile.Mitochondria that are motile are located more peripherally in the cell than non‐motile mitochondria. Abstract Mitochondrial function, motility and architecture are each central to cell function. Age‐associated mitochondrial dysfunction may contribute to vascular disease. However, mitochondrial changes in ageing remain ill‐defined because of the challenges of imaging in native cells. We determined the structure of mitochondria in live native cells, demarcating boundaries of individual organelles by inducing stochastic ‘flickers’ of membrane potential, recorded as fluctuations in potentiometric fluorophore intensity (flicker‐assisted localization microscopy; FaLM). In freshly‐isolated myocytes from rat cerebral resistance arteries, FaLM showed a range of mitochondrial X‐Y areas in both young adult (3 months; 0.05–6.58 μm2) and aged rats (18 months; 0.05–13.4 μm2). In cells from young animals, most mitochondria were small (mode area 0.051 μm2) compared to aged animals (0.710 μm2). Cells from older animals contained a subpopulation of highly‐elongated mitochondria (5.3% were >2 μm long, 4.2% had a length:width ratio >3) that was rare in younger animals (0.15% of mitochondria >2 μm long, 0.4% had length:width ratio >3). The extent of mitochondrial motility also varied. 1/811 mitochondria observed moved slightly (∼0.5 μm) in myocytes from older animals, whereas, in the younger animals, directed and Brownian‐like motility occurred regularly (215 of 1135 mitochondria moved within 10 min, up to distance of 12 μm). Mitochondria positioned closer to the cell periphery showed a greater tendency to move. In conclusion, cerebral vascular myocytes from young rats contained small, motile mitochondria. In aged rats, mitochondria were larger, immobile and could be highly‐elongated. These age‐associated alterations in mitochondrial behaviour may contribute to alterations in cell signalling, energy supply or the onset of proliferation., Key points Age is proposed to be associated with altered structure and function of mitochondria; however, in fully‐differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel‐by‐pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during ‘flickers’ of mitochondrial membrane potential.Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats.A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly‐elongated.Some mitochondria in myocytes from younger, but not aged, animals are highly‐motile.Mitochondria that are motile are located more peripherally in the cell than non‐motile mitochondria.

Endothelial cells are reported to be glycolytic and to minimally rely on mitochondria for ATP generation. Rather than providing energy, mitochondria in endothelial cells may act as signaling organelles that control cytosolic Ca2+ signaling or modify reactive oxygen species (ROS). To control Ca2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca2+ signaling by mitochondria in large numbers of endothelial cells (~50 per field) in intact arteries from rats. We observed that mitochondria were mostly spherical or short-rod structures and were distributed widely throughout the cytoplasm. The density of these organelles did not increase near contact sites with smooth muscle cells. However, local inositol trisphosphate (IP3)-mediated Ca2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca2+ signals occurred even when mitochondria were far from Ca2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP3-mediated Ca2+ release events. ROS buffering did not significantly alter local Ca2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP3-evoked local Ca2+ release in intact endothelium

Blood flow, blood clotting, angiogenesis, vascular permeability and vascular remodeling are each controlled by a large number of variable, noisy and interacting chemical inputs to the endothelium. The endothelium processes the entire chemical composition to which the cardiovascular system is exposed, carrying out sophisticated computations to determine physiological output. A major challenge faced by the endothelium is the requirement to process an enormous quantity of information held in the overall chemical environment to which the vascular system is exposed. We analyzed hundreds of endothelial cells and show that the endothelium segregates the chemical environment into small components of complementary information streams which are processed in parallel. Chemical stimuli arriving at the endothelium are mapped to different clusters of cells which each generate unique signal patterns. When there is more than one stimulus, cells communicate and combine inputs across information streams to generate new distinct signals. Our results establish the endothelium is a structured,collaborative, sensory network which simplifies the complex environment using separate cell clusters concerned with small distinct aspects of the overall information. These clusters interactively compute signals from the diverse but interrelated chemical inputs. These features permit the endothelium to selectively process separate signals and perform multiple computations in an environment that is noisy and variable

Graded refractive index lenses are inherent to advanced visual systems in animals. By understanding their formation and local optical properties, significant potential for improved ocular healthcare may be realized. We report a novel technique measuring the developing optical power of the eye lens, in a living animal, by exploiting the orthogonal imaging modality of a selective plane illumination microscope (SPIM). We have quantified the maturation of the lenticular refractive index at three different visible wavelengths using a combined imaging and ray tracing approach. We demonstrate that the method can be used with transgenic and vital dye labeling as well as with both fixed and living animals. Using a key eye lens morphogen and its inhibitor, we have measured their effects both on lens size and on refractive index. Our technique provides insights into the mechanisms involved in the development of this natural graded index micro-lens and its associated optical properties.

Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise, and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca2+ concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca2+ signals spanning one order of magnitude. Interestingly however, intercellular response variation extended over three orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially-restricted clusters of comparably-sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca2+ signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate population-wide inputs and enable robust noise filtering for efficient high-fidelity signaling.

Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca(2+) concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca(2+) signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca(2+) signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.-Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

Mitochondrial morphology is central to normal physiology and disease development. However, in many live cells and tissues, complex mitochondrial structures exist and morphology has been difficult to quantify. We have measured the shape of electrically-discrete mitochondria, imaging them individually to restore detail hidden in clusters and demarcate functional boundaries. Stochastic “flickers” of mitochondrial membrane potential were visualized with a rapidly-partitioning fluorophore and the pixel-by-pixel covariance of spatio-temporal fluorescence changes analyzed. This Flicker-assisted Localization Microscopy (FaLM) requires only an epifluorescence microscope and sensitive camera. In vascular myocytes, the apparent variation in mitochondrial size was partly explained by densely-packed small mitochondria. In normotensive animals, mitochondria were small spheres or rods. In hypertension, mitochondria were larger, occupied more of the cell volume and were more densely clustered. FaLM provides a convenient tool for increased discrimination of mitochondrial architecture and has revealed mitochondrial alterations that may contribute to hypertension.

In vivo bioluminescent imaging (BLI) is a sensitive and reliable technique for studying gene expression, although experiments must be controlled tightly to obtain reproducible and quantitative measurements. The luciferase reaction depends on the availability of the reaction substrate, oxygen, and ATP, the distribution of which can vary markedly in different tissues. Here we used in vivo fiber optic technology, combined with stereotaxis-assisted surgery, to assess luciferase reaction kinetics in response to 2 anesthetic regimens, isoflurane and ketamine-xylazine. Transgenic rats that expressed luciferase under the control of the human prolactin promoter were used as a model organism. Anesthesia had a marked effect on luciferase reaction kinetics. The rise time to peak emission differed by 20 min between isoflurane and ketamine-xylazine. Optical imaging using a charge-coupled-device camera confirmed this delay. These results demonstrate that different anesthetics can have substantial effects on luciferase reaction kinetics and suggest that the timing of image acquisition after substrate injection should be optimized in regard to experimental conditions and the tissues of interest.