Alexey R. Brazhe

Rapid stimulus-evoked astrocyte Ca2+ elevations and hemodynamic responses in mouse somatosensory cortex in vivo

Significance The morphology of astrocytes places them as likely contributors to communication between nerve cells and blood vessels. They are reported to respond with few and slow Ca2+ elevations, which exclude them as possible participants in initiation of blood flow responses or synapse communication. We establish that astrocytes have fast responses in addition to the slow. These rapid, brief Ca2+ responses were present in a large proportion of astrocytes. We were able to observe these changes due to a signal enhancement analysis, which is useful when responses are small compared with baseline activity. Our findings indicate a higher sensitivity than generally believed of astrocytes. Increased neuron and astrocyte activity triggers increased brain blood flow, but controversy exists over whether stimulation-induced changes in astrocyte activity are rapid and widespread enough to contribute to brain blood flow control. Here, we provide evidence for stimulus-evoked Ca2+ elevations with rapid onset and short duration in a large proportion of cortical astrocytes in the adult mouse somatosensory cortex. Our improved detection of the fast Ca2+ signals is due to a signal-enhancing analysis of the Ca2+ activity. The rapid stimulation-evoked Ca2+ increases identified in astrocyte somas, processes, and end-feet preceded local vasodilatation. Fast Ca2+ responses in both neurons and astrocytes correlated with synaptic activity, but only the astrocytic responses correlated with the hemodynamic shifts. These data establish that a large proportion of cortical astrocytes have brief Ca2+ responses with a rapid onset in vivo, fast enough to initiate hemodynamic responses or influence synaptic activity.

Dynamical patterns of calcium signaling in a functional model of neuron–astrocyte networks

We propose a functional mathematical model for neuron-astrocyte networks. The model incorporates elements of the tripartite synapse and the spatial branching structure of coupled astrocytes. We consider glutamate-induced calcium signaling as a specific mode of excitability and transmission in astrocytic–neuronal networks. We reproduce local and global dynamical patterns observed experimentally.

New Insight into Erythrocyte through In Vivo Surface-Enhanced Raman Spectroscopy

The article presents a noninvasive approach to the study of erythrocyte properties by means of a comparative analysis of signals obtained by surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RS). We report step-by-step the procedure for preparing experimental samples containing erythrocytes in their normal physiological environment in a mixture of colloid solution with silver nanoparticles and the procedure for the optimization of SERS conditions to achieve high signal enhancement without affecting the properties of living erythrocytes. By means of three independent techniques, we demonstrate that under the proposed conditions a colloid solution of silver nanoparticles does not affect the properties of erythrocytes. For the first time to our knowledge, we describe how to use the SERS-RS approach to study two populations of hemoglobin molecules inside an intact living erythrocyte: submembrane and cytosolic hemoglobin (Hb(sm) and Hb(c)). We show that the conformation of Hb(sm) differs from the conformation of Hb(c). This finding has an important application, as the comparative study of Hb(sm) and Hb(c) could be successfully used in biomedical research and diagnostic tests.

Mapping of Redox State of Mitochondrial Cytochromes in Live Cardiomyocytes Using Raman Microspectroscopy

This paper presents a nonivasive approach to study redox state of reduced cytochromes , and of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach we perform studies of rod- and round-shaped cardiomyocytes, representing different morphological and functional states. Raman mapping and cluster analysis reveal that these cardiomyocytes differ in the amounts of reduced cytochromes , and . The rod-shaped cardiomyocytes possess uneven distribution of reduced cytochromes , and in cell center and periphery. Moreover, by means of Raman spectroscopy we demonstrated the decrease in the relative amounts of reduced cytochromes , and in the rod-shaped cardiomyocytes caused by H2O2-induced oxidative stress before any visible changes. Results of Raman mapping and time-dependent study of reduced cytochromes of complexes II and III and cytochrome in cardiomyocytes are in a good agreement with our fluorescence indicator studies and other published data.

Planar SERS nanostructures with stochastic silver ring morphology for biosensor chips

Surface-enhanced Raman spectroscopy (SERS) of living cells has rapidly become a powerful trend in biomedical diagnostics. It is a common belief that highly ordered, artificially engineered substrates are the best future decision in this field. This paper, however, describes an alternative successful solution, a new effortless chemical approach to the design of nanostructured silver and heterometallic continuous coatings with a stochastic “coffee ring” morphology. The coatings are formed from an ultrasonic mist of aqueous diamminesilver hydroxide, free of reducing agents and nonvolatile pollutants, under mild conditions, at about 200–270 °C in air. They consist of 30–100 micrometer wide and 100–400 nm high silver rings composed, in turn, of a porous silver matrix with 10–50 nm silver grains decorating the sponge. This hierarchic structure originates from ultrasonic droplet evaporation, contact-line motion, silver(I) oxide decomposition and evolution of a growing ensemble of silver rings. The fabricated substrates are a remarkable example of a new scalable and low cost material suitable for SERS analyses of living cells. They evoke no hemolysis and reduce erythrocyte lateral mobility due to suitable “coffee ring” sizes and a tight contact with the silver nanostructure. A high SERS enhancement, characteristic of pure silver rings, made it possible to record Raman scattering spectra from submembrane hemoglobin in its natural cellular environment inside single living erythrocytes, thus making the substrates promising for various biosensor chips.

Activity-dependent Increases in Local Oxygen Consumption Correlate with Postsynaptic Currents in the Mouse Cerebellum In Vivo

Evoked neural activity correlates strongly with rises in cerebral metabolic rate of oxygen (CMRO2) and cerebral blood flow (CBF). Activity-dependent rises in CMRO2 fluctuate with ATP turnover due to ion pumping. In vitro studies suggest that increases in cytosolic Ca2+ stimulate oxidative metabolism via mitochondrial signaling, but whether this also occurs in the intact brain is unknown. Here we applied a pharmacological approach to dissect the effects of ionic currents and cytosolic Ca2+ rises of neuronal origin on activity-dependent rises in CMRO2. We used two-photon microscopy and current source density analysis to study real-time Ca2+ dynamics and transmembrane ionic currents in relation to CMRO2 in the mouse cerebellar cortex in vivo. We report a direct correlation between CMRO2 and summed (i.e., the sum of excitatory, negative currents during the whole stimulation period) field EPSCs (∑fEPSCs) in Purkinje cells (PCs) in response to stimulation of the climbing fiber (CF) pathway. Blocking stimulus-evoked rises in cytosolic Ca2+ in PCs with the P/Q-type channel blocker ω-agatoxin-IVA (ω-AGA), or the GABAA receptor agonist muscimol, did not lead to a time-locked reduction in CMRO2, and excitatory synaptic or action potential currents. During stimulation, neither ω-AGA or (μ-oxo)-bis-(trans-formatotetramine-ruthenium) (Ru360), a mitochondrial Ca2+ uniporter inhibitor, affected the ratio of CMRO2 to fEPSCs or evoked local field potentials. However, baseline CBF and CMRO2 decreased gradually with Ru360. Our data suggest that in vivo activity-dependent rises in CMRO2 are correlated with synaptic currents and postsynaptic spiking in PCs. Our study did not reveal a unique role of neuronal cytosolic Ca2+ signals in controlling CMRO2 increases during CF stimulation.

Laser interference microscopy in erythrocyte study

With the laser interference microscopy (LIM) technique, one can measure phase height of cells—a variable proportional to the cell thickness and the difference in the refractive indices of the cell and the surrounding medium. This makes functional optical cell imaging possible, and estimation of shape, thickness, and area of erythrocytes feasible. In this paper, we studied changes in erythrocyte shape and volume with osmolarity and pH. Obtained from the LIM technique, erythrocyte phase heights and area values, as well as the hematocrit-measured erythrocyte volume, were used to estimate changes in the refractive index with osmolarity and pH. A comparison between the estimated refractive index with the refractive index, calculated in the assumption that it can only depend on the hemoglobin concentration in the cell, indicates that these two estimates are identical in the range of osmolarity (250–1000 mOsm) and pH (4.5–10.0) values. Thus, refractive index changes result exclusively from the changes in hemoglo...

Spontaneous calcium waves in Bergman glia increase with age and hypoxia and may reduce tissue oxygen.

Glial calcium (Ca2+) waves constitute a means to spread signals between glial cells and to neighboring neurons and blood vessels. These waves occur spontaneously in Bergmann glia (BG) of the mouse cerebellar cortex in vivo. Here, we tested three hypotheses: (1) aging and reduced blood oxygen saturation alters wave activity; (2) glial Ca2+ waves change cerebral oxygen metabolism; and (3) neuronal and glial wave activity is correlated. We used two-photon microscopy in the cerebellar cortexes of adult (8- to 15-week-old) and aging (48- to 80-week-old) ketamine-anesthetized mice after bolus loading with OGB-1/AM and SR101. We report that the occurrence of spontaneous waves is 20 times more frequent in the cerebellar cortex of aging as compared with adult mice, which correlated with a reduction in resting brain oxygen tension. In adult mice, spontaneous glial wave activity increased on reducing resting brain oxygen tension, and ATP-evoked glial waves reduced the tissue O2 tension. Finally, although spontaneous Purkinje cell (PC) activity was not associated with increased glia wave activity, spontaneous glial waves did affect intracellular Ca2+ activity in PCs. The increased wave activity during aging, as well as low resting brain oxygen tension, suggests a relationship between glial waves, brain energy homeostasis, and pathology.

Phase-modulation laser interference microscopy: an advance in cell imaging and dynamics study

We describe how phase-modulation laser interference microscopy and wavelet analysis can be applied to noninvasive nonstained visualization and study of the structural and dynamical properties of living cells. We show how phase images of erythrocytes can reveal the difference between various erythrocyte forms and stages of hemolysis and how phase images of neurons reveal their complex intracellular structure. Temporal variations of the refractive index are analyzed to detect cellular rhythmic activity on different time scales as well as to uncover interactions between the cellular processes.

Multinephron dynamics on the renal vascular network

Tubuloglomerular feedback (TGF) and the myogenic mechanism combine in each nephron to regulate blood flow and glomerular filtration rate. Both mechanisms are nonlinear, generate self-sustained oscillations, and interact as their signals converge on arteriolar smooth muscle, forming a regulatory ensemble. Ensembles may synchronize. Smooth muscle cells in the ensemble depolarize periodically, generating electrical signals that propagate along the vascular network. We developed a mathematical model of a nephron-vascular network, with 16 versions of a single nephron model containing representations of both mechanisms in the regulatory ensemble, to examine the effects of network structure on nephron synchronization. Symmetry, as a property of a network, facilitates synchronization. Nephrons received blood from a symmetric electrically conductive vascular tree. Symmetry was created by using identical nephron models at each of the 16 sites and symmetry breaking by varying nephron length. The symmetric model achieved synchronization of all elements in the network. As little as 1% variation in nephron length caused extensive desynchronization, although synchronization was maintained in small nephron clusters. In-phase synchronization predominated among nephrons separated by one or three vascular nodes and antiphase synchronization for five or seven nodes of separation. Nephron dynamics were irregular and contained low-frequency fluctuations. Results are consistent with simultaneous blood flow measurements in multiple nephrons. An interaction between electrical signals propagated through the network to cause synchronization; variation in vascular pressure at vessel bifurcations was a principal cause of desynchronization. The results suggest that the vasculature supplies blood to nephrons but also engages in robust information transfer.