239 research outputs found
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Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.
Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged âŒP80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function
Monthly direct and indirect greenhouse gases emissions from household consumption in the major Japanese cities
Urban household consumption contributes substantially to global greenhouse gases (GHGs) emissions. Urban household emissions encompass both direct and indirect emissions, with the former associated with the direct use of fossil fuels and the latter with the emissions embodied in the consumed goods and services. However, there is a lack of consistent and comprehensive datasets outlining in great detail emissions from urban household consumption. To bridge this data gap, we construct an emission inventory of urban household emissions for 52 major cities in Japan that covers around 500 emission categories. The dataset spans from January 2011 to December 2015 and contains 12,384 data records for direct emissions and 1,543,128 records for indirect emissions. Direct emission intensity is provided in g-CO2/JPY to facilitate both future studies of household emission in Japan, as well as act as a reference for the development of detailed household emission inventories in other countries
Effective Theoretical Approach to Back Reaction of the Dynamical Casimir Effect in 1+1 Dimensions
We present an approach to studying the Casimir effects by means of the
effective theory. An essential point of our approach is replacing the mirror
separation into the size of space S^1 in the adiabatic approximation. It is
natural to identify the size of space S^1 with the scale factor of the
Robertson-Walker-type metric. This replacement simplifies the construction of a
class of effective models to study the Casimir effects. To check the validity
of this replacement we construct a model for a scalar field coupling to the
two-dimensional gravity and calculate the Casimir effects by the effective
action for the variable scale factor. Our effective action consists of the
classical kinetic term of the mirror separation and the quantum correction
derived by the path-integral method. The quantum correction naturally contains
both the Casimir energy term and the back-reaction term of the dynamical
Casimir effect, the latter of which is expressed by the conformal anomaly. The
resultant effective action describes the dynamical vacuum pressure, i.e., the
dynamical Casimir force. We confirm that the force depends on the relative
velocity of the mirrors, and that it is always attractive and stronger than the
static Casimir force within the adiabatic approximation.Comment: Published Version, 16 pages, LaTeX2e with graphics package, 1 figur
High-Throughput Analysis of Calcium Signalling Kinetics in Astrocytes Stimulated with Different Neurotransmitters
Astrocytes express a wide range of receptors for neurotransmitters and hormones that are coupled to increases in intracellular Ca2+ concentration, enabling them to detect activity in both neuronal and vascular networks. There is increasing evidence that astrocytes are able to discriminate between different Ca2+-linked stimuli, as the efficiency of some Ca2+ dependent processes â notably release of gliotransmitters â depends on the stimulus that initiates the Ca2+ signal. The spatiotemporal complexity of Ca2+ signals is substantial, and we here tested the hypothesis that variation in the kinetics of Ca2+ responses could offer a means of selectively engaging downstream targets, if agonists exhibited a âsignature shapeâ in evoked Ca2+ response. To test this, astrocytes were exposed to three different receptor agonists (ATP, glutamate and histamine) and the resultant Ca2+ signals were analysed for systematic differences in kinetics that depended on the initiating stimulus. We found substantial heterogeneity between cells in the time course of Ca2+ responses, but the variation did not correlate with the type or concentration of the stimulus. Using a simple metric to quantify the extent of difference between populations, it was found that the variation between agonists was insufficient to allow signal discrimination. We conclude that the time course of global intracellular Ca2+ signals does not offer the cells a means for distinguishing between different neurotransmitters
Disentangling astroglial physiology with a realistic cell model in silico
Electrically non-excitable astroglia take up neurotransmitters, buffer extracellular K+ and generate Ca2+ signals that release molecular regulators of neural circuitry. The underlying machinery remains enigmatic, mainly because the sponge-like astrocyte morphology has been difficult to access experimentally or explore theoretically. Here, we systematically incorporate multi-scale, tri-dimensional astroglial architecture into a realistic multi-compartmental cell model, which we constrain by empirical tests and integrate into the NEURON computational biophysical environment. This approach is implemented as a flexible astrocyte-model builder ASTRO. As a proof-of-concept, we explore an in silico astrocyte to evaluate basic cell physiology features inaccessible experimentally. Our simulations suggest that currents generated by glutamate transporters or K+ channels have negligible distant effects on membrane voltage and that individual astrocytes can successfully handle extracellular K+ hotspots. We show how intracellular Ca2+ buffers affect Ca2+ waves and why the classical Ca2+ sparks-and-puffs mechanism is theoretically compatible with common readouts of astroglial Ca2+ imaging
Ultrasensitive gold micro-structured electrodes enabling the detection of extra-cellular long-lasting potentials in astrocytes populations
Ultra-sensitive electrodes for extracellular recordings were fabricated and electrically characterized. A signal detection limit defined by a noise level of 0.3-0.4 mu V for a bandwidth of 12.5 Hz was achieved. To obtain this high sensitivity, large area (4 mm(2)) electrodes were used. The electrode surface is also micro-structured with an array of gold mushroom-like shapes to further enhance the active area. In comparison with a flat gold surface, the micro-structured surface increases the capacitance of the electrode/electrolyte interface by 54%. The electrode low impedance and low noise enable the detection of weak and low frequency quasi-periodic signals produced by astrocytes populations that thus far had remained inaccessible using conventional extracellular electrodes. Signals with 5 mu V in amplitude and lasting for 5-10 s were measured, with a peak-to-peak signal-to-noise ratio of 16. The electrodes and the methodology developed here can be used as an ultrasensitive electrophysiological tool to reveal the synchronization dynamics of ultra-slow ionic signalling between non-electrogenic cells.Portuguese Foundation for Science and Technology (FCT), through the project "Implantable organic devices for advanced therapies" (INNOVATE) [PTDC/EEI-AUT/5442/2014]; Instituto de Telecomunicacoes [UID/Multi/04326/2013]; Associated Laboratory - Institute of Nanoscience and Nanotechnology [POCI-01-0145-FEDER-016623]; [PTDC/CTM-NAN/3146/2014
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Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of âGCaMP5â sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.Molecular and Cellular Biolog
Few-layer Nanoplates of Bi2Se3 and Bi2Te3 with Highly Tunable Chemical Potential
Topological insulator (TI) represents an unconventional quantum phase of
matter with insulating bulk bandgap and metallic surface states. Recent
theoretical calculations and photoemission spectroscopy measurements show that
Group V-VI materials Bi2Se3, Bi2Te3 and Sb2Te3 are TI with a single Dirac cone
on the surface. These materials have anisotropic, layered structures, in which
five atomic layers are covalently bonded to form a quintuple layer, and
quintuple layers interact weakly through van der Waals interaction to form the
crystal. A few quintuple layers of these materials are predicted to exhibit
interesting surface properties. Different from our previous nanoribbon study,
here we report the synthesis and characterizations of ultrathin Bi2Te3 and
Bi2Se3 nanoplates with thickness down to 3 nm (3 quintuple layers), via
catalyst-free vapor-solid (VS) growth mechanism. Optical images reveal
thickness-dependant color and contrast for nanoplates grown on oxidized silicon
(300nm SiO2/Si). As a new member of TI nanomaterials, ultrathin TI nanoplates
have an extremely large surface-to-volume ratio and can be electrically gated
more effectively than the bulk form, potentially enhancing surface states
effects in transport measurements. Low temperature transport measurements of a
single nanoplate device, with a high-k dielectric top gate, show decrease in
carrier concentration by several times and large tuning of chemical potential.Comment: 6 figure
P2X7 receptors induce degranulation in human mast cells.
Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αÎČmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated
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Optimization of a GCaMP calcium indicator for neural activity imaging
© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Neuroscience 32 (2012): 13819-13840, doi:10.1523/JNEUROSCI.2601-12.2012.Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of âGCaMP5â sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.A.F. has been supported by a European Molecular Biology Organization long-term fellowship. Work in H.B.âs
laboratory was funded by the National Institutes of Health (NIH) Nanomedicine Development Center âOptical Control
of Biological Function,â and work in S.S.-H.W.âs laboratory was funded by NIH R01 NS045193
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