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Voltage-sensitive dyes, also known as potentiometric dyes, are dyes which change their spectral properties in response to voltage changes. [1] They are able to provide linear measurements of firing activity of single neurons, large neuronal populations or activity of myocytes. Many physiological processes are accompanied by changes in cell membrane potential which can be detected with voltage sensitive dyes. Measurements may indicate the site of action potential origin, and measurements of action potential velocity and direction may be obtained. [2]
Potentiometric dyes are used to monitor the electrical activity inside cell organelles where it is not possible to insert an electrode, such as the mitochondria and dendritic spine. This technology is especially powerful for the study of patterns of activity in complex multicellular preparations. It also makes possible the measurement of spatial and temporal variations in membrane potential along the surface of single cells.
Fast-response probes: These are amphiphilic membrane staining dyes which usually have a pair of hydrocarbon chains acting as membrane anchors and a hydrophilic group which aligns the chromophore perpendicular to the membrane/aqueous interface. The chromophore is believed to undergo a large electronic charge shift as a result of excitation from the ground to the excited state and this underlies the putative electrochromic mechanism for the sensitivity of these dyes to membrane potential. This molecule (dye) intercalates among the lipophilic part of biological membranes. This orientation assures that the excitation induced charge redistribution will occur parallel to the electric field within the membrane. A change in the voltage across the membrane will therefore cause a spectral shift resulting from a direct interaction between the field and the ground and excited state dipole moments.
New voltage dyes can sense voltage with high speed and sensitivity using photoinduced electron transfer (PeT) through a conjugated molecular wire. [3] [4]
Slow-response probes: These exhibit potential-dependent changes in their transmembrane distribution which are accompanied by a fluorescence change. Typical slow-response probes include cationic carbocyanines and rhodamines, and ionic oxonols.
Commonly used voltage sensitive dyes are substituted aminonaphthylethenylpyridinium (ANEP) dyes, such as di-4-ANEPPS, di-8-ANEPPS, and RH237. Depending on their chemical modifications which change their physical properties they are used for different experimental procedures. [5] They were first described in 1985 by the research group of Leslie Loew. [6] ANNINE-6plus is a voltage sensitive dye with fast response (ns response time) and high sensitivity. It has been applied to measure the action potentials of a single t-tubule of cardiomyocytes by Guixue Bu et al. [7] More recently, a series of fluorinated ANEP dyes was introduced that offer enhanced sensitivity and photostability; they are also available over a wide choice of excitation and emission wavelengths. [8] A recent computational study confirmed that the ANEP dyes are affected only by the electrostatic environment and not by specific molecular interactions. [9] Other structural scaffolds, such as xanthenes, [10] are also successfully used.
The core material for imaging brain activity with voltage-sensitive dyes are the dyes themselves. These voltage-sensitive dyes are lipophilic and preferably localized in membranes with their hydrophobic tails. They are used in applications involving fluorescence or absorption; they are fast acting and are able to provide linear measurements of changes in membrane potential. [11] Voltage sensitive dyes are supplied by many companies who offer fluorescent probes for biological applications. Potentiometric Probes, LLC specializes only in voltage sensitive dyes; they have an exclusive license to distribute the large set of fluorinated VSDs, marketed under the ElectroFluor brand.
A variety of specialized equipment may be used in conjunction with the dyes, and choices in equipment will vary according to the particularities of a preparation. Essentially, equipment will include specialized microscopes and imaging devices, and may include technical lamps or lasers. [11]
Strengths of imaging brain activity with voltage-sensitive dyes include the following abilities:
Weaknesses of imaging brain activity with voltage-sensitive dyes include the following problems:
Voltage-sensitive dyes have been used to measure neural activity in several areas of the nervous system in a variety of organisms, including the squid giant axon, [19] whisker barrels of the rat somatosensory cortex, [20] [21] olfactory bulb of the salamander, [22] [23] [24] visual cortex of the cat, [25] optic tectum of the frog, [26] and the visual cortex of the rhesus monkey. [27] [28]
Many applications in cardiac electrophysiology have been published, including ex vivo mapping of electrical activity in whole hearts from various animal species, [29] [30] subcellular imaging from single cardiomyocytes, [31] and even mapping both sinus rhythms and arrhytmias in open heart in vivo pig, [18] where motion artifacts could be eliminated by dual wavelength ratio imaging of the voltage sensitive dye fluorescence.
A dendrite or dendron is a branched protoplasmic extension of a nerve cell that propagates the electrochemical stimulation received from other neural cells to the cell body, or soma, of the neuron from which the dendrites project. Electrical stimulation is transmitted onto dendrites by upstream neurons via synapses which are located at various points throughout the dendritic tree.
Electrophysiology is the branch of physiology that studies the electrical properties of biological cells and tissues. It involves measurements of voltage changes or electric current or manipulations on a wide variety of scales from single ion channel proteins to whole organs like the heart. In neuroscience, it includes measurements of the electrical activity of neurons, and, in particular, action potential activity. Recordings of large-scale electric signals from the nervous system, such as electroencephalography, may also be referred to as electrophysiological recordings. They are useful for electrodiagnosis and monitoring.
An action potential occurs when the membrane potential of a specific cell rapidly rises and falls. This depolarization then causes adjacent locations to similarly depolarize. Action potentials occur in several types of animal cells, called excitable cells, which include neurons, muscle cells, and in some plant cells. Certain endocrine cells such as pancreatic beta cells, and certain cells of the anterior pituitary gland are also excitable cells.
A dendritic spine is a small membranous protrusion from a neuron's dendrite that typically receives input from a single axon at the synapse. Dendritic spines serve as a storage site for synaptic strength and help transmit electrical signals to the neuron's cell body. Most spines have a bulbous head, and a thin neck that connects the head of the spine to the shaft of the dendrite. The dendrites of a single neuron can contain hundreds to thousands of spines. In addition to spines providing an anatomical substrate for memory storage and synaptic transmission, they may also serve to increase the number of possible contacts between neurons. It has also been suggested that changes in the activity of neurons have a positive effect on spine morphology.
Behavioral neuroscience, also known as biological psychology, biopsychology, or psychobiology, is the application of the principles of biology to the study of physiological, genetic, and developmental mechanisms of behavior in humans and other animals.
Pyramidal cells, or pyramidal neurons, are a type of multipolar neuron found in areas of the brain including the cerebral cortex, the hippocampus, and the amygdala. Pyramidal cells are the primary excitation units of the mammalian prefrontal cortex and the corticospinal tract. Pyramidal neurons are also one of two cell types where the characteristic sign, Negri bodies, are found in post-mortem rabies infection. Pyramidal neurons were first discovered and studied by Santiago Ramón y Cajal. Since then, studies on pyramidal neurons have focused on topics ranging from neuroplasticity to cognition.
A basal dendrite is a dendrite that emerges from the base of a pyramidal cell that receives information from nearby neurons and passes it to the soma, or cell body. Due to their direct attachment to the cell body itself, basal dendrites are able to deliver strong depolarizing currents and therefore have a strong effect on action potential output in neurons. The physical characteristics of basal dendrites vary based on their location and species that they are found in. For example, the basal dendrites of humans are overall found to be the most intricate and spine-dense, as compared to other species such as Macaques. It is also observed that basal dendrites of the prefrontal cortex are larger and more complex in comparison to the smaller and simpler dendrites that can be seen within the visual cortex. Basal dendrites are capable of vast amounts of analog computing, which is responsible for many of the different nonlinear responses of modulating information in the neocortex. Basal dendrites additionally exist in dentate granule cells for a limited time before removal via regulatory factors. This removal usually occurs before the cell reaches adulthood, and is thought to be regulated through both intracellular and extracellular signals. Basal dendrites are part of the more overarching dendritic tree present on pyramidal neurons. They, along with apical dendrites, make up the part of the neuron that receives most of the electrical signaling. Basal dendrites have been found to be involved mostly in neocortical information processing.
Neural oscillations, or brainwaves, are rhythmic or repetitive patterns of neural activity in the central nervous system. Neural tissue can generate oscillatory activity in many ways, driven either by mechanisms within individual neurons or by interactions between neurons. In individual neurons, oscillations can appear either as oscillations in membrane potential or as rhythmic patterns of action potentials, which then produce oscillatory activation of post-synaptic neurons. At the level of neural ensembles, synchronized activity of large numbers of neurons can give rise to macroscopic oscillations, which can be observed in an electroencephalogram. Oscillatory activity in groups of neurons generally arises from feedback connections between the neurons that result in the synchronization of their firing patterns. The interaction between neurons can give rise to oscillations at a different frequency than the firing frequency of individual neurons. A well-known example of macroscopic neural oscillations is alpha activity.
Matteo Carandini is a neuroscientist who studies the visual system. He is currently a professor at University College London, where he co-directs the Cortical Processing Laboratory with Kenneth D Harris.
In neuroscience, single-unit recordings provide a method of measuring the electro-physiological responses of a single neuron using a microelectrode system. When a neuron generates an action potential, the signal propagates down the neuron as a current which flows in and out of the cell through excitable membrane regions in the soma and axon. A microelectrode is inserted into the brain, where it can record the rate of change in voltage with respect to time. These microelectrodes must be fine-tipped, impedance matching; they are primarily glass micro-pipettes, metal microelectrodes made of platinum, tungsten, iridium or even iridium oxide. Microelectrodes can be carefully placed close to the cell membrane, allowing the ability to record extracellularly.
Neural backpropagation is the phenomenon in which, after the action potential of a neuron creates a voltage spike down the axon, another impulse is generated from the soma and propagates towards the apical portions of the dendritic arbor or dendrites. In addition to active backpropagation of the action potential, there is also passive electrotonic spread. While there is ample evidence to prove the existence of backpropagating action potentials, the function of such action potentials and the extent to which they invade the most distal dendrites remain highly controversial.
Biological neuron models, also known as a spiking neuron models, are mathematical descriptions of neurons. In particular, these models describe how the voltage potential across the cell membrane changes over time. In an experimental setting, stimulating neurons with an electrical current generates an action potential, that propagates down the neuron's axon. This axon can branch out and connect to a large number of downstream neurons at sites called synapses. At these synapses, the spike can cause release of a biochemical substance (neurotransmitter), which in turn can change the voltage potential of downstream neurons, potentially leading to spikes in those downstream neurons, thus propagating the signal. As many as 85% of neurons in the neocortex, the outermost layer of the mammalian brain, consists of excitatory pyramidal neurons, and each pyramidal neuron receives tens of thousands of inputs from other neurons. Thus, spiking neurons are a major information processing unit of the nervous system.
Optogenetics is a biological technique to control the activity of neurons or other cell types with light. This is achieved by expression of light-sensitive ion channels, pumps or enzymes specifically in the target cells. On the level of individual cells, light-activated enzymes and transcription factors allow precise control of biochemical signaling pathways. In systems neuroscience, the ability to control the activity of a genetically defined set of neurons has been used to understand their contribution to decision making, learning, fear memory, mating, addiction, feeding, and locomotion. In a first medical application of optogenetic technology, vision was partially restored in a blind patient.
Nonsynaptic plasticity is a form of neuroplasticity that involves modification of ion channel function in the axon, dendrites, and cell body that results in specific changes in the integration of excitatory postsynaptic potentials and inhibitory postsynaptic potentials. Nonsynaptic plasticity is a modification of the intrinsic excitability of the neuron. It interacts with synaptic plasticity, but it is considered a separate entity from synaptic plasticity. Intrinsic modification of the electrical properties of neurons plays a role in many aspects of plasticity from homeostatic plasticity to learning and memory itself. Nonsynaptic plasticity affects synaptic integration, subthreshold propagation, spike generation, and other fundamental mechanisms of neurons at the cellular level. These individual neuronal alterations can result in changes in higher brain function, especially learning and memory. However, as an emerging field in neuroscience, much of the knowledge about nonsynaptic plasticity is uncertain and still requires further investigation to better define its role in brain function and behavior.
ANNINE-6plus is a water soluble voltage sensitive dye. This compound was developed at the Max Planck Institute for Biochemistry in Germany. It is used to optically measure the changes in transmembrane voltage of excitable cells, including neurons, skeletal and cardiac myocytes.
GCaMP is a genetically encoded calcium indicator (GECI) initially developed in 2001 by Junichi Nakai. It is a synthetic fusion of green fluorescent protein (GFP), calmodulin (CaM), and M13, a peptide sequence from myosin light-chain kinase. When bound to Ca2+, GCaMP fluoresces green with a peak excitation wavelength of 480 nm and a peak emission wavelength of 510 nm. It is used in biological research to measure intracellular Ca2+ levels both in vitro and in vivo using virally transfected or transgenic cell and animal lines. The genetic sequence encoding GCaMP can be inserted under the control of promoters exclusive to certain cell types, allowing for cell-type specific expression of GCaMP. Since Ca2+ is a second messenger that contributes to many cellular mechanisms and signaling pathways, GCaMP allows researchers to quantify the activity of Ca2+-based mechanisms and study the role of Ca2+ ions in biological processes of interest.
Ralph Mitchell Siegel, a researcher who studied the neurological underpinnings of vision, was a professor of neuroscience at Rutgers University, Newark, in the Center for Molecular and Behavioral Neuroscience. He died September 2, 2011, at his home following a long illness.
Genetically encoded voltage indicator is a protein that can sense membrane potential in a cell and relate the change in voltage to a form of output, often fluorescent level. It is a promising optogenetic recording tool that enables exporting electrophysiological signals from cultured cells, live animals, and ultimately human brain. Examples of notable GEVIs include ArcLight, ASAP1, ASAP3, Archons, SomArchon, and Ace2N-mNeon.
Brian M. Salzberg is an American neuroscientist, biophysicist and professor. He is Professor of Neuroscience and of Physiology at the Perelman School of Medicine, University of Pennsylvania.
Optogenetics began with methods to alter neuronal activity with light, using e.g. channelrhodopsins. In a broader sense, optogenetic approaches also include the use of genetically encoded biosensors to monitor the activity of neurons or other cell types by measuring fluorescence or bioluminescence. Genetically encoded calcium indicators (GECIs) are used frequently to monitor neuronal activity, but other cellular parameters such as membrane voltage or second messenger activity can also be recorded optically. The use of optogenetic sensors is not restricted to neuroscience, but plays increasingly important roles in immunology, cardiology and cancer research.