108 research outputs found
Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes
The brain contains glial cells. Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons. However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions
Astrocyte Calcium Signaling: From Observations to Functions and the Challenges Therein
We provide an overview of recent progress on the study of astrocyte intracellular Ca2+ signaling. We consider the methods that have been used to monitor astrocyte Ca2+ signals, the various types of Ca2+ signals that have been discovered (waves, microdomains, and intrinsic fluctuations), the approaches used to broadly trigger and block Ca2+ signals, and, where possible, the proposed and demonstrated physiological roles for astrocyte Ca2+ signals within neuronal microcircuits. Although important progress has been made, we suggest that further detailed work is needed to explore the biophysics and molecular mechanisms of Ca2+ signaling within entire astrocytes, including their fine distal extensions, such as processes that interact spatially with neurons and blood vessels. Improved methods are also needed to mimic and block molecularly defined types of Ca2+ signals within genetically specified populations of astrocytes. Moreover, it will be essential to study astrocyte Ca2+ activity in vivo to distinguish between pharmacological and physiological activity, and to study Ca2+ activity in situ to rigorously explore mechanisms. Once methods to reliably measure, mimic, and block specific astrocyte Ca2+ signals with high temporal and spatial precision are available, researchers will be able to carefully explore the correlative and causative roles that Ca2+ signals may play in the functions of astrocytes, blood vessels, neurons, and microcircuits in the healthy and diseased brain
Allosteric Control of Gating and Kinetics at P2Xâ‚„ Receptor Channels
The CNS abundantly expresses P2X receptor channels for ATP; of these the most widespread in the brain is the P2X₄ channel. We show that ivermectin (IVM) is a specific positive allosteric effector of heterologously expressed P2X₄ and possibly of heteromeric P2X₄/P2X₆channels, but not of P2X₂, P2X₃, P2X₂/P2X₃, or P2X₇ channels. In the submicromolar range (EC₅₀, ∼250 nM) the action of IVM was rapid and reversible, resulting in increased amplitude and slowed deactivation of P2X₄ channel currents evoked by ATP. IVM also markedly increased the potency of ATP and that of the normally low-potency agonist α,β-methylene-ATP in a use- and voltage-independent manner without changing the ion selectivity of P2X₄ channels. Therefore, IVM evokes a potent pharmacological gain-of-function phenotype that is specific for P2X₄ channels. We also tested whether IVM could modulate endogenously expressed P2X channels in the adult trigeminal mesencephalic nucleus and hippocampal CA1 neurons. Surprisingly, IVM produced no significant effect on the fast ATP-evoked inward currents in either type of neuron, despite the fact that IVM modulated P2X₄ channels heterologously expressed in embryonic hippocampal neurons. These results suggest that homomeric P2X₄ channels are not the primary subtype of P2X receptor in the adult trigeminal mesencephalic nucleus and in hippocampal CA1 neurons
Allosteric Control of Gating and Kinetics at P2Xâ‚„ Receptor Channels
The CNS abundantly expresses P2X receptor channels for ATP; of these the most widespread in the brain is the P2X₄ channel. We show that ivermectin (IVM) is a specific positive allosteric effector of heterologously expressed P2X₄ and possibly of heteromeric P2X₄/P2X₆channels, but not of P2X₂, P2X₃, P2X₂/P2X₃, or P2X₇ channels. In the submicromolar range (EC₅₀, ∼250 nM) the action of IVM was rapid and reversible, resulting in increased amplitude and slowed deactivation of P2X₄ channel currents evoked by ATP. IVM also markedly increased the potency of ATP and that of the normally low-potency agonist α,β-methylene-ATP in a use- and voltage-independent manner without changing the ion selectivity of P2X₄ channels. Therefore, IVM evokes a potent pharmacological gain-of-function phenotype that is specific for P2X₄ channels. We also tested whether IVM could modulate endogenously expressed P2X channels in the adult trigeminal mesencephalic nucleus and hippocampal CA1 neurons. Surprisingly, IVM produced no significant effect on the fast ATP-evoked inward currents in either type of neuron, despite the fact that IVM modulated P2X₄ channels heterologously expressed in embryonic hippocampal neurons. These results suggest that homomeric P2X₄ channels are not the primary subtype of P2X receptor in the adult trigeminal mesencephalic nucleus and in hippocampal CA1 neurons
An Angstrom Scale Interaction between Plasma Membrane ATP-Gated P2X₂ and α₄β₂ Nicotinic Channels Measured with Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Microscopy
Structurally distinct nicotinic and P2X channels interact functionally, such that coactivation results in cross-inhibition of one or both channel types. It is hypothesized, but not yet proven, that nicotinic and P2X channels interact at the plasma membrane. Here, we show that plasma membrane α₄β₂ nicotinic and P2X₂ channels form a molecular scale partnership and also influence each other when coactivated, resulting in nonadditive cross-inhibitory responses. Total internal reflection fluorescence and fluorescence resonance energy transfer microscopy between fluorescently labeled P2X₂ and α₄β₂ nicotinic channels demonstrated close spatial arrangement of the channels in human embryonic kidney cells and in hippocampal neuron membranes. The data suggest that P2X₂ and α₄β₂ channels may form a dimer, with the channels ∼80 Å apart. The measurements also show that P2X₂ subunits interact specifically and robustly with the β₂ subunits in α₄β₂ channels. The data provide direct evidence for the close spatial apposition of full-length P2X₂ and α₄β₂ channels within 100 nm of the plasma membrane of living cells
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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 ∼P80) 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
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