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Trends in Neurosciences May 2016Genetically encoded optical sensors of cell activity are powerful tools that can be targeted to specific cell types. This is especially important in neuroscience because... (Review)
Review
Genetically encoded optical sensors of cell activity are powerful tools that can be targeted to specific cell types. This is especially important in neuroscience because individual brain regions can include a multitude of different cell types. Optical imaging allows for simultaneous recording from numerous neurons or brain regions. Optical signals of membrane potential are useful because membrane potential changes are a direct sign of both synaptic and action potentials. Here we describe recent improvements in the in vitro and in vivo signal size and kinetics of genetically encoded voltage indicators (GEVIs) and discuss their relationship to alternative sensors of neural activity.
Topics: Animals; Brain; Membrane Potentials; Neurons; Voltage-Sensitive Dye Imaging
PubMed: 27130905
DOI: 10.1016/j.tins.2016.02.005 -
International Journal of Molecular... Jan 2023Membrane potential is a fundamental property of biological cells. Changes in membrane potential characterize a vast number of vital biological processes, such as the... (Review)
Review
Membrane potential is a fundamental property of biological cells. Changes in membrane potential characterize a vast number of vital biological processes, such as the activity of neurons and cardiomyocytes, tumorogenesis, cell-cycle progression, etc. A common strategy to record membrane potential changes that occur in the process of interest is to utilize organic dyes or genetically-encoded voltage indicators with voltage-dependent fluorescence. Sensors are introduced into target cells, and alterations of fluorescence intensity are recorded with optical methods. Techniques that allow recording relative changes of membrane potential and do not take into account fluorescence alterations due to factors other than membrane voltage are already widely used in modern biological and biomedical studies. Such techniques have been reviewed previously in many works. However, in order to investigate a number of processes, especially long-term processes, the measured signal must be corrected to exclude the contribution from voltage-independent factors or even absolute values of cell membrane potential have to be evaluated. Techniques that enable such measurements are the subject of this review.
Topics: Membrane Potentials; Cell Membrane; Fluorescent Dyes; Neurons; Optical Imaging
PubMed: 36768759
DOI: 10.3390/ijms24032435 -
Molecules (Basel, Switzerland) Jan 2023In the course of action potential firing, all axons and neurons release K from the intra- cellular compartment into the interstitial space to counteract the depolarizing... (Review)
Review
In the course of action potential firing, all axons and neurons release K from the intra- cellular compartment into the interstitial space to counteract the depolarizing effect of Na influx, which restores the resting membrane potential. This efflux of K from axons results in K accumulation in the interstitial space, causing depolarization of the K reversal potential (E), which can prevent subsequent action potentials. To ensure optimal neuronal function, the K is buffered by astrocytes, an energy-dependent process, which acts as a sink for interstitial K, absorbing it at regions of high concentration and distributing it through the syncytium for release in distant regions. Pathological processes in which energy production is compromised, such as anoxia, ischemia, epilepsy and spreading depression, can lead to excessive interstitial K accumulation, disrupting sensitive trans-membrane ion gradients and attenuating neuronal activity. The changes that occur in interstitial [K] resulting from both physiological and pathological processes can be monitored accurately in real time using K-sensitive microelectrodes, an invaluable tool in electrophysiological studies.
Topics: Microelectrodes; Neurons; Membrane Potentials; Axons; Action Potentials; Potassium
PubMed: 36677581
DOI: 10.3390/molecules28020523 -
Methods in Enzymology 2020Voltage imaging in living cells offers the tantalizing possibility of combining the temporal resolution of electrode-based methods with the spatial resolution of imaging...
Voltage imaging in living cells offers the tantalizing possibility of combining the temporal resolution of electrode-based methods with the spatial resolution of imaging techniques. Our lab has been developing voltage-sensitive fluorophores, or VoltageFluors, that respond to changes in cellular and neuronal membrane potential via a photoinduced electron transfer (PeT)-based mechanism. This unique mechanism enables both the fast response kinetics and high sensitivity required to record action potentials in single trials, across multiple cells without the need for stimuli-triggered averaging. In this chapter, we present a methodology for imaging membrane potential dynamics from dozens of neurons simultaneously in vitro. Using simple, commercially available cameras, illumination sources, and microscope optics in combination with the far-red synthetic voltage-sensitive fluorophore BeRST-1 (Berkeley Red Sensor of Transmembrane potential) provides a readily applied method for monitoring neuronal activity in cultured neurons. We discuss different types of voltage-sensitive dyes, considerations for selecting imaging modalities, and outline procedures for the culture of rat hippocampal neurons and performing voltage imaging experiments with these samples. Finally, we provide an example of how changes to the metabolic input to cultured hippocampal neurons can alter their activity profile.
Topics: Action Potentials; Animals; Fluorescent Dyes; Hippocampus; Membrane Potentials; Neurons; Rats
PubMed: 32560798
DOI: 10.1016/bs.mie.2020.04.028 -
The Journal of Physiology May 2016Activation of neurons not only changes their membrane potential and firing rate but as a secondary action reduces membrane resistance. This loss of resistance, or... (Review)
Review
Activation of neurons not only changes their membrane potential and firing rate but as a secondary action reduces membrane resistance. This loss of resistance, or increase of conductance, may be of central importance in non-invasive magnetic or electric stimulation of the human brain since electrical fields cause larger changes in transmembrane voltage in resting neurons with low membrane conductances than in active neurons with high conductance. This may explain why both the immediate effects and after-effects of brain stimulation are smaller or even reversed during voluntary activity compared with rest. Membrane conductance is also increased during shunting inhibition, which accompanies the classic GABAA IPSP. This short-circuits nearby EPSPs and is suggested here to contribute to the magnitude and time course of short-interval intracortical inhibition and intracortical facilitation.
Topics: Biophysical Phenomena; Excitatory Postsynaptic Potentials; Humans; Inhibitory Postsynaptic Potentials; Membrane Potentials; Neural Inhibition; Synapses
PubMed: 26940751
DOI: 10.1113/JP271452 -
Physiological Reports Jan 2023The purpose of this study was to determine how sensory neurons respond to high-frequency membrane potential alternation between depolarization and hyperpolarization....
The purpose of this study was to determine how sensory neurons respond to high-frequency membrane potential alternation between depolarization and hyperpolarization. Membrane currents were recorded from dissociated dorsal root ganglion (DRG) neurons of adult rats using the whole cell patch clamp technique in voltage clamp mode. Stepwise depolarization of the membrane was applied first to determine the threshold membrane potential for inducing an action potential (AP) current. Then, membrane potential alternation between depolarization (to +20 mV) and hyperpolarization (to -110 mV) was applied to the neuron for 10 s at different frequencies (10 Hz to 1 kHz). The tested DRG neurons had APs of either a long duration (>10 ms) or a short duration (<10 ms). Membrane potential alternation at ≥500 Hz completely disrupted the AP generation, disabled the ion channel gating function, and produced membrane current alternating symmetrically across zero. Replacing extracellular sodium with potassium increased the amplitude of the membrane current response and caused the membrane current to be larger during hyperpolarization than during depolarization. These results support the hypothesis that high-frequency biphasic stimulation blocks axonal conduction by driving the potassium channel open constantly. Understanding neural membrane response to high-frequency membrane potential alternation is important to reveal the possible mechanisms underlying axonal conduction block induced by high-frequency biphasic stimulation.
Topics: Rats; Animals; Membrane Potentials; Ganglia, Spinal; Neurons; Action Potentials; Neurons, Afferent
PubMed: 36695759
DOI: 10.14814/phy2.15582 -
Proceedings of the National Academy of... Apr 2024Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators...
Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.
Topics: Membrane Potentials; Fluorescent Dyes; Action Potentials; Cell Membrane; Microscopy, Fluorescence
PubMed: 38551837
DOI: 10.1073/pnas.2315264121 -
Cells Aug 2022All living cells maintain a charge distribution across their cell membrane (membrane potential) by carefully controlled ion fluxes. These bioelectric signals regulate...
All living cells maintain a charge distribution across their cell membrane (membrane potential) by carefully controlled ion fluxes. These bioelectric signals regulate cell behavior (such as migration, proliferation, differentiation) as well as higher-level tissue and organ patterning. Thus, voltage gradients represent an important parameter for diagnostics as well as a promising target for therapeutic interventions in birth defects, injury, and cancer. However, despite much progress in cell and molecular biology, little is known about bioelectric states in human stem cells. Here, we present simple methods to simultaneously track ion dynamics, membrane voltage, cell morphology, and cell activity (pH and ROS), using fluorescent reporter dyes in living human neurons derived from induced neural stem cells (hiNSC). We developed and tested functional protocols for manipulating ion fluxes, membrane potential, and cell activity, and tracking neural responses to injury and reinnervation in vitro. Finally, using morphology sensor, we tested and quantified the ability of physiological actuators (neurotransmitters and pH) to manipulate nerve repair and reinnervation. These methods are not specific to a particular cell type and should be broadly applicable to the study of bioelectrical controls across a wide range of combinations of models and endpoints.
Topics: Humans; Induced Pluripotent Stem Cells; Membrane Potentials; Neural Stem Cells; Neuronal Outgrowth; Neurons
PubMed: 36010547
DOI: 10.3390/cells11162470 -
Biochimica Et Biophysica Acta 2008Mitochondrial function and dysfunction contributes to a range of important aspects of biomedical research. Consequently there is considerable interest in developing... (Review)
Review
Mitochondrial function and dysfunction contributes to a range of important aspects of biomedical research. Consequently there is considerable interest in developing approaches to modify and report on mitochondria in cells and in vivo. One approach has been to target bioactive molecules to mitochondria by conjugating them to lipophilic cations. Due to the large mitochondrial membrane potential, the cations are accumulated within mitochondria inside cells. This approach had been used to develop mitochondria-targeted antioxidants that selectively block mitochondrial oxidative damage and prevent some types of cell death and also to develop probes of mitochondrial function. Here we outline some of the background to the development of these compounds.
Topics: Adenosine Triphosphate; Antioxidants; Cations; Cell Physiological Phenomena; Cytoplasm; Humans; Membrane Potentials; Mitochondria; Mitochondrial Membranes; Organophosphorus Compounds
PubMed: 18439417
DOI: 10.1016/j.bbabio.2008.03.029 -
Advances in Pharmacology (San Diego,... 2017Potassium channels importantly contribute to the regulation of vascular smooth muscle (VSM) contraction and growth. They are the dominant ion conductance of the VSM cell... (Review)
Review
Potassium channels importantly contribute to the regulation of vascular smooth muscle (VSM) contraction and growth. They are the dominant ion conductance of the VSM cell membrane and importantly determine and regulate membrane potential. Membrane potential, in turn, regulates the open-state probability of voltage-gated Ca channels (VGCC), Ca influx through VGCC, intracellular Ca, and VSM contraction. Membrane potential also affects release of Ca from internal stores and the Ca sensitivity of the contractile machinery such that K channels participate in all aspects of regulation of VSM contraction. Potassium channels also regulate proliferation of VSM cells through membrane potential-dependent and membrane potential-independent mechanisms. VSM cells express multiple isoforms of at least five classes of K channels that contribute to the regulation of contraction and cell proliferation (growth). This review will examine the structure, expression, and function of large conductance, Ca-activated K (BK) channels, intermediate-conductance Ca-activated K (K3.1) channels, multiple isoforms of voltage-gated K (K) channels, ATP-sensitive K (K) channels, and inward-rectifier K (K) channels in both contractile and proliferating VSM cells.
Topics: Animals; Humans; Membrane Potentials; Muscle, Smooth, Vascular; Potassium Channels; Vasoconstriction
PubMed: 28212804
DOI: 10.1016/bs.apha.2016.07.001