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 individu ...
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 recep ...
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
Bioluminescence is the production and emission of light by living organisms. It is a form of chemiluminescence. Bioluminescence occurs widely in marine vertebrates and invertebrates, as well as in some fungi, microorganisms including some b ...
. 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, development ...
, 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 there ...
,
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 heart d ...
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
neurotransmitter
A neurotransmitter is a signaling molecule secreted by a neuron to affect another cell across a synapse. The cell receiving the signal, any main body part or target cell, may be another neuron, but could also be a gland or muscle cell.
Neuro ...
s.
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 endo ...
s or
pyramidal cell
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 neurons are the primary excitation units of the mammalian prefrontal cor ...
s). 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,
organelle
In cell biology, an organelle is a specialized subunit, usually within a cell, that has a specific function. The name ''organelle'' comes from the idea that these structures are parts of cells, as organs are to the body, hence ''organelle,'' the ...
s, nucleus) by fusing the indicator protein with specific anchoring domains, retention signals or
intrabodies.
* work in a variety of species (
nematode
The nematodes ( or grc-gre, Νηματώδη; la, Nematoda) or roundworms constitute the phylum Nematoda (also called Nemathelminthes), with plant-Parasitism, parasitic nematodes also known as eelworms. They are a diverse animal phylum inhab ...
s, insects, fish, mammals) and in
cell culture
Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This te ...
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
In telecommunications and computer networking, multiplexing (sometimes contracted to muxing) is a method by which multiple analog or digital signals are combined into one signal over a shared medium. The aim is to share a scarce resource - a ...
).
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
Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. It is an intracellular target of the secondary messenger Ca2+, and the bind ...
,
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
A fret is any of the thin strips of material, usually metal wire, inserted laterally at specific positions along the neck or fretboard of a stringed instrument. Frets usually extend across the full width of the neck. On some historical instrume ...
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 salts ...
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
Dopamine (DA, a contraction of 3,4-dihydroxyphenethylamine) is a neuromodulatory molecule that plays several important roles in cells. It is an organic compound, organic chemical of the catecholamine and phenethylamine families. Dopamine const ...
sensors: dLight1, GRAB-DA
* Genetically encoded
serotonin
Serotonin () or 5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter. Its biological function is complex and multifaceted, modulating mood, cognition, reward, learning, memory, and numerous physiological processes such as vomiting and vas ...
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
In biochemistry, a kinase () is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the high-energy ATP molecule don ...
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