Optogenetic methods to record cellular activity

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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.

Contents

History

The first experiments to measure intracellular calcium levels via protein expression were based on aequorin, a bioluminescent protein from the jellyfish Aequorea. To produce light, however, this enzyme needs the 'fuel' compound coelenteracine, which has to be added to the preparation. This is not practical in intact animals, and in addition, the temporal resolution of bioluminescence imaging is relatively poor (seconds-minutes). The first genetically encoded fluorescent calcium indicator (GECI) to be used to image activity in an animal was cameleon, designed by Atsushi Miyawaki, Roger Tsien and coworkers in 1997. [1] Cameleon was first used successfully in an animal by Rex Kerr, William Schafer and coworkers to record from neurons and muscle cells of the nematode C. elegans. [2] Cameleon was subsequently used to record neural activity in flies [3] and zebrafish. [4] In mammals, the first GECI to be used in vivo was GCaMP, [5] first developed by Junichi Nakai and coworkers in 2001. [6] GCaMP has undergone numerous improvements, notably by a team of scientists at the Janelia Farm Research Campus (GENIE project, HHMI), and GCaMP6 [7] in particular has become widely used in neuroscience. Very recently, G protein-coupled receptors have been harnessed to generate a series of highly specific indicators for various neurotransmitters. [8] [9]

Design principles

Genetically encoded sensors are fusion proteins, consisting of a ligand binding domain (sensor) and a fluorescent protein, attached by a short linker (flexible peptide). When the sensor domain binds the correct ligand, it changes conformation. This movement is transferred to the fluorescent protein and the resulting deformation leads to a change in fluorescence. The efficiency of this process depends critically on the length of the linker region, which has to be optimized in a labor-intensive process. The fluorescent protein is often circularly permuted, i.e. new C-terminal and N-terminal ends were created. Single-wavelength sensors are easy to use for qualitative measurements, but difficult to calibrate for quantitative measurements of ligand concentration.

A second class of sensors relies on Förster resonance energy transfer (FRET) between two fluorescent proteins (FP) of different color. The shorter wavelength FP (donor) is excited with blue light from a laser or LED. If the second FP (acceptor) is very close, the energy is transferred to the acceptor, resulting in yellow or red fluorescence. When the acceptor FP moves further away, the donor emits green fluorescence. The sensor domain is typically spliced between the two FPs, resulting in a hinge-type movement upon ligand binding that changes the distance between donor and acceptor. The imaging procedure is more complex for FRET sensors, but the fluorescence ratio can be calibrated to measure the absolute concentration of a ligand. Read-out via fluorescence lifetime imaging (FLIM) of donor fluorescence is also possible, as the FRET process speeds up the fluorescence decay.

Advantages of optogenetic sensors

Drawbacks, limitations

Classes of genetically encoded indicators

The calcium indicator GCaMP in its calcium-bound (top) and calcium-free form (bottom). When Ca-calmodulin (cyan) binds to M13, the conformation changes and the cpGFP barrel closes, enabling green fluorescence. Gcamp.jpg
The calcium indicator GCaMP in its calcium-bound (top) and calcium-free form (bottom). When Ca-calmodulin (cyan) binds to M13, the conformation changes and the cpGFP barrel closes, enabling green fluorescence.

Indicators have been designed to measure ion concentrations, membrane potential, neurotransmitters, and various intracellular signaling molecules. The following list provides only examples for each class; many more have been published.

Intracellular signaling

Neurotransmitters and other extracellular signals

Further reading

A recent review of GPCR-based genetically encoded fluorescent indicators for neuromodulators [9]

Related Research Articles

<span class="mw-page-title-main">Green fluorescent protein</span> Protein that exhibits bright green fluorescence when exposed to ultraviolet light

The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets.

<span class="mw-page-title-main">Fluorescent tag</span>

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.

<span class="mw-page-title-main">Fura-2</span> Chemical compound

Fura-2, an aminopolycarboxylic acid, is a ratiometric fluorescent dye which binds to free intracellular calcium. It was the first widely used dye for calcium imaging, and remains very popular. Fura-2 is excited at 340 nm and 380 nm of light, and the ratio of the emissions at those wavelengths is directly related to the amount of intracellular calcium. Regardless of the presence of calcium, Fura-2 emits at 510 nm of light. The use of the ratio automatically cancels out confounding variables, such as variable dye concentration and cell thickness, making Fura-2 one of the most appreciated tools to quantify calcium levels. The high photon yield of fura-2 allowed the first real time measurements of calcium inside living cells in 1986. More recently, genetically encoded calcium indicators based on spectral variants of the green fluorescent protein, such as Cameleons, have supplemented the use of Fura-2 and other small molecule dyes for calcium imaging, but Fura-2 remains faster.

<span class="mw-page-title-main">Förster resonance energy transfer</span> Photochemical energy transfer mechanism

Förster resonance energy transfer (FRET), fluorescence resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.

<span class="mw-page-title-main">Chemical biology</span> Scientific discipline

Chemical biology is a scientific discipline between the fields of chemistry and biology. The discipline involves the application of chemical techniques, analysis, and often small molecules produced through synthetic chemistry, to the study and manipulation of biological systems. Although often confused with biochemistry, which studies the chemistry of biomolecules and regulation of biochemical pathways within and between cells, chemical biology remains distinct by focusing on the application of chemical tools to address biological questions.

<span class="mw-page-title-main">Two-photon excitation microscopy</span> Fluorescence imaging technique

Two-photon excitation microscopy is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image. Due to the non-linearity of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.

Cameleon is an engineered protein based on variant of green fluorescent protein used to visualize calcium levels in living cells. It is a genetically encoded calcium sensor created by Roger Y. Tsien and coworkers. The name is a conflation of CaM (the common abbreviation of calmodulin) and chameleon to indicate the fact that the sensor protein undergoes a conformation change and radiates at an altered wavelength upon calcium binding to the calmodulin element of the Cameleon. Cameleon was the first genetically encoded calcium sensor that could be used for ratiometric measurements and the first to be used in a transgenic animal to record activity in neurons and muscle cells. Cameleon and other genetically encoded calcium indicators (GECIs) have found many applications in neuroscience and other fields of biology, including understanding the mechanisms of cell signaling by conducting time-resolved Ca2+ activity measurement experiments with endoplasmic reticulum (ER) enzymes. It was created by fusing BFP, calmodulin, calmodulin-binding peptide M13 and EGFP.

<span class="mw-page-title-main">Axon terminal</span> Nerve fiber part

Axon terminals are distal terminations of the branches of an axon. An axon, also called a nerve fiber, is a long, slender projection of a nerve cell that conducts electrical impulses called action potentials away from the neuron's cell body in order to transmit those impulses to other neurons, muscle cells or glands. In the central nervous system, most presynaptic terminals are actually formed along the axons, not at their ends.

<span class="mw-page-title-main">RoGFP</span> Prodified GFP protein that exhibits different fluorescent properties when oxidized and reduced

The reduction-oxidation sensitive green fluorescent protein (roGFP) is a green fluorescent protein engineered to be sensitive to changes in the local redox environment. roGFPs are used as redox-sensitive biosensors.

Fluorescent chloride sensors are used for chemical analysis. The discoveries of chloride (Cl−) participations in physiological processes stimulates the measurements of intracellular Cl− in live cells and the development of fluorescent tools referred below.

<span class="mw-page-title-main">GCaMP</span> Genetically encoded calcium indicator

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.

Calcium imaging is a microscopy technique to optically measure the calcium (Ca2+) status of an isolated cell, tissue or medium. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca2+ ions by fluorescence properties. Two main classes of calcium indicators exist: chemical indicators and genetically encoded calcium indicators (GECI). This technique has allowed studies of calcium signalling in a wide variety of cell types. In neurons, action potential generation is always accompanied by rapid influx of Ca2+ ions. Thus, calcium imaging can be used to monitor the electrical activity in hundreds of neurons in cell culture or in living animals, which has made it possible to observe the activity of neuronal circuits during ongoing behavior.

<span class="mw-page-title-main">SmURFP</span>

Small ultra red fluorescent protein (smURFP) is a class of far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein, α-allophycocyanin. Native α-allophycocyanin requires an exogenous protein, known as a lyase, to attach the chromophore, phycocyanobilin. Phycocyanobilin is not present in mammalian cells. smURFP was evolved to covalently attach phycocyanobilin without a lyase and fluoresce, covalently attach biliverdin and fluoresce, blue-shift fluorescence to match the organic fluorophore, Cy5, and not inhibit E. coli growth. smURFP was found after 12 rounds of random mutagenesis and manually screening 10,000,000 bacterial colonies.

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.

A genetically engineered fluorescent protein that changes its fluorescence when bound to the neurotransmitter glutamate. Glutamate-sensitive fluorescent reporters are used to monitor the activity of presynaptic terminals by fluorescence microscopy. GluSnFRs are a class of optogenetic sensors used in neuroscience research. In brain tissue, two-photon microscopy is typically used to monitor GluSnFR fluorescence.

<span class="mw-page-title-main">William Schafer</span> American geneticist

William Ronald Schafer is a neuroscientist and geneticist who has made important contributions to understanding the molecular and neural basis of behaviour. His work, principally in the nematode C. elegans, has used an interdisciplinary approach to investigate how small groups of neurons generate behavior, and he has pioneered methodological approaches, including optogenetic neuroimaging and automated behavioural phenotyping, that have been widely influential in the broader neuroscience field. He has made significant discoveries on the functional properties of ionotropic receptors in sensory transduction and on the roles of gap junctions and extrasynaptic modulation in neuronal microcircuits. More recently, he has applied theoretical ideas from network science and control theory to investigate the structure and function of simple neuronal connectomes, with the goal of understanding conserved computational principles in larger brains. He is an EMBO member, Welcome Investigator and Fellow of the Academy of Medical Sciences.

Jin Zhang is a Chinese-American biochemist. She is a professor of pharmacology, chemistry and biochemistry, and biomedical engineering at the University of California, San Diego.

FAST is a small, genetically-encoded, protein tag which allows for fluorescence reporting of proteins of interest. Unlike natural fluorescent proteins and derivates such as GFP or mCherry, FAST is not fluorescent by itself. It can bind selectively a fluorogenic chromophore derived from 4-hydroxybenzylidene rhodanine (HBR), which is itself non fluorescent unless bound. Once bound, the pair of molecules goes through a unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, hence providing high labeling selectivity. The FAST-fluorogen reporting system can be used in fluorescence microscopy, flow cytometry and any other fluorometric method to explore the living world: biosensors, protein trafficking.

Fiber photometry is a calcium imaging technique that captures 'bulk' or population-level calcium (Ca2+) activity from specific cell-types within a brain region or functional network in order to study neural circuits Population-level calcium activity can be correlated with behavioral tasks, such as spatial learning, memory recall and goal-directed behaviors. The technique involves the surgical implantation of fiber optics into the brains of living animals. The benefits to researchers are that optical fibers are simpler to implant, less invasive and less expensive than other calcium methods, and there is less weight and stress on the animal, as compared to miniscopes. It also allows for imaging of multiple interacting brain regions and integration with other neuroscience techniques. The limitations of fiber photometry are low cellular and spatial resolution, and the fact that animals must be securely tethered to a rigid fiber bundle, which may impact the naturalistic behavior of smaller mammals such as mice.

Lin Tian is a Chinese-American neuroscientist and biochemist. She is a Scientific Director of the Max Planck Florida Institute for Neuroscience in Jupiter, FL, and was formerly a professor in the Department of Biochemistry and Molecular Medicine at the University of California, Davis. Tian is known for her research in the fields of neuroscience and biochemical engineering. She develops and applies molecular tools to understand brain function and dysfunction at the individual, neuronal level.

References

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