Optogenetics 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 individ ...

began with methods to ''alter'' neuronal activity with light, using e.g.

channelrhodopsin Channelrhodopsins are a subfamily of retinylidene proteins ( rhodopsins) that function as light-gated ion channels. They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis: movement in response to light. Express ...

s. In a broader sense, optogenetic approaches also include the use of genetically encoded

biosensor A biosensor is an analytical device, used for the detection of a chemical substance, that combines a biological component with a physicochemical detector. The ''sensitive biological element'', e.g. tissue, microorganisms, organelles, cell rece ...

s to ''monitor'' the activity of neurons or other cell types by measuring

fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...

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 Neuroscience is the scientific study of the nervous system (the brain, spinal cord, and peripheral nervous system), its functions and disorders. It is a multidisciplinary science that combines physiology, anatomy, molecular biology, developme ...

, but plays increasingly important roles in

immunology Immunology is a branch of medicineImmunology for Medical Students, Roderick Nairn, Matthew Helbert, Mosby, 2007 and biology that covers the medical study of immune systems in humans, animals, plants and sapient species. In such we can see the ...

cardiology Cardiology () is a branch of medicine that deals with disorders of the heart and the cardiovascular system. The field includes medical diagnosis and treatment of congenital heart defects, coronary artery disease, heart failure, valvular h ...

and

cancer research Cancer research is research into cancer to identify causes and develop strategies for prevention, diagnosis, treatment, and cure. Cancer research ranges from epidemiology, molecular bioscience to the performance of clinical trials to evaluate and ...

History

The first experiments to measure intracellular calcium levels via protein expression were based on

aequorin Aequorin is a calcium-activated photoprotein isolated from the hydrozoan ''Aequorea victoria''. Its bioluminescence was studied decades before the protein was isolated from the animal by Osamu Shimomura in 1962. In the animal, the protein occur ...

, 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 Roger Yonchien Tsien (pronounced , "'' CHEN''"'';'' February 1, 1952 – August 24, 2016) was an American biochemist. He was a professor of chemistry and biochemistry at the University of California, San Diego and was awarded the Nobel Prize in ...

and coworkers in 1997. 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''. Cameleon was subsequently used to record neural activity in flies and zebrafish. In mammals, the first GECI to be used ''in vivo'' was GCaMP, first developed by Nakai and coworkers in 2001. GCaMP has undergone numerous improvements, notably by a team of scientists at the

Janelia Farm Research Campus Janelia Research Campus is a scientific research campus of the Howard Hughes Medical Institute that opened in October 2006. The campus is located in Loudoun County, Virginia, near the town of Ashburn. It is known for its scientific research and ...

(GENIE project,

HHMI The Howard Hughes Medical Institute (HHMI) is an American non-profit medical research organization based in Chevy Chase, Maryland. It was founded in 1953 by Howard Hughes, an American business magnate, investor, record-setting pilot, engineer, fi ...

), and GCaMP6 in particular has become widely used in neuroscience. Very recently,

G protein-coupled receptor G protein-coupled receptors (GPCRs), also known as seven-(pass)-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors (GPLR), form a large group of evolutionarily-related p ...

s have been harnessed to generate a series of highly specific indicators for various neurotransmitters.

Advantages of optogenetic sensors

* can be targeted to specific classes of cells (e.g.

astrocyte Astrocytes (from Ancient Greek , , "star" + , , "cavity", "cell"), also known collectively as astroglia, are characteristic star-shaped glial cells in the brain and spinal cord. They perform many functions, including biochemical control of e ...

s or pyramidal cells). This allows for optical read-out without spatial resolution, e.g.

fiber photometry 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 activi ...

from deep brain areas. * can be targeted to sub-cellular compartments (e.g. synapses, organelles, nucleus) by fusing the indicator protein with specific anchoring domains, retention signals or intrabodies. * work in a variety of species ( nematodes, insects, fish, mammals) and in cell culture systems ( FLIPR assay) * can be delivered by viral vectors (e.g. rAAV) *can be used to record the activity of thousands of neurons at the same time

Drawbacks, limitations

* will buffer the measured ion or protein, potentially interfering with cellular signaling * are subject to

photobleaching In optics, photobleaching (sometimes termed fading) is the photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between ...

, compromising long-term measurements * can be toxic when expressed at very high concentration * require highly sensitive cameras or laser scanning microscopes * some

GPCR G protein-coupled receptors (GPCRs), also known as seven-(pass)-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors (GPLR), form a large group of evolutionarily-related p ...

-based sensors are sensitive to polarization * most indicators are green fluorescent, making it difficult to measure several cellular parameters simultaneously ( multiplexing).

Classes of genetically encoded indicators

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. * Genetically encoded calcium indicators (GECI): A large class of tools, based on natural calcium binding proteins ( calmodulin,

troponin image:Troponin Ribbon Diagram.png, 400px, Ribbon representation of the human cardiac troponin core complex (52 kDa core) in the calcium-saturated form. Blue = troponin C; green = troponin I; magenta = troponin T.; ; rendered with PyMOL Troponin, ...

). Different affinities, kinetics, and colors (green, red) available. Read-out via fluorescence intensity (single wavelength indicators), FRET or

BRET Bret or BRET may refer to: People and fictional characters * Bret (given name), a personal name, including a list of people and fictional characters * Bret (surname), a list of people Other uses * a regional name for either the brill or the ...

. Have been targeted to various organelles. Current version: JGCaMP8 * Genetically encoded

chloride The chloride ion is the anion (negatively charged ion) Cl−. It is formed when the element chlorine (a halogen) gains an electron or when a compound such as hydrogen chloride is dissolved in water or other polar solvents. Chloride sa ...

indicators: Clomeleon * Genetically encoded indicators for intracellular pH (GEPhI): CypHer *

Genetically encoded voltage indicator Genetically encoded voltage indicator (or GEVI) 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 exp ...

s (GEVI): ArcLight * Genetically encoded vesicle fusion sensors:

Synapto-pHluorin Synapto-pHluorin is a genetically encoded optical indicator of vesicle release and recycling. It is used in neuroscience to study transmitter release. It consists of a pH-sensitive form of green fluorescent protein (GFP) fused to the luminal side of ...

, Synaptophysin-pHluorin * Genetically encoded glutamate sensors: GluSnFR * Genetically encoded GABA sensors: iGABASnFR * Genetically encoded dopamine sensors: dLight1, GRAB-DA * Genetically encoded serotonin sensors: GRAB_5-HT, sDarken *Genetically encoded

norepinephrine Norepinephrine (NE), also called noradrenaline (NA) or noradrenalin, is an organic chemical in the catecholamine family that functions in the brain and body as both a hormone and neurotransmitter. The name "noradrenaline" (from Latin '' ad' ...

sensors: GRAB_NE * Genetically encoded ATP sensors: QUEEN-37C * Genetically encoded cAMP sensors: EPAC * Genetically encoded kinase activity sensors: CaMui, SmURFP * Genetically encoded Small G-protein sensors: FRas *Genetically encoded sensor for

endocannabinoid Cannabinoids () are several structural classes of compounds found in the cannabis plant primarily and most animal organisms (although insects lack such receptors) or as synthetic compounds. The most notable cannabinoid is the phytocannabinoid tet ...

activity: GRAB_eCB2.0 *Genetically encoded sensor for orexin/hypocretin neuropeptides: OxLight1

External links

Fluorescent Biosensor Database
a fairly complete searchable list of published sensors and their basic properties, maintained by Jin Zhang's lab at UCSD.

References

{{Optogenetics Neuroscience Biological techniques and tools Optics