Optogenetics Guide
Optogenetics (or photogenetics) is a technology that involves the use of light to manipulate cell activities with high temporal and spatial accuracy in vitro or in vivo. Activities of the chosen cell can be turned on and off on a biologically relevant time scale of a few milliseconds. The field of optogenetics integrates optics and genetic engineering to measure and manipulate cells (usually neurons) and the corresponding molecular processes. Optogenetics provides a far greater degree of specificity and control of individual cell than that of drugs and lesions.
In 2010, Nature declared optogenetics to be their “method of the year”, while Science classed it as one of the breakthroughs of the last decade.
Accessory of optogenetics technology can be broadly classified based on their functions into actuators and sensors group. Actuators group genetically-encoded tools for light-activated control of proteins like microbial opsins and optical switches. Sensors group genetically-encoded reporters of molecular signals like calcium indicators.
Microbial Opsins
Opsins are optically gated ion channel or pump, which can absorb light of specific wavelength and respond by opening or closing, so as to guide ion flow in and out of cells. A variety of naturally occurring microbial opsins have been identified, including Channelrhodopsins, Halorhodopsins, Archaerhodopsins and Wild-Type Leptoshpaeria schematic (Mac).
Researchers have used genetic engineering to improve these natural opsins by inducing point mutations to alter the absorption spectrum or adding trafficking signals to localize opsins to the cell membrane.
| Microbial Opsin |
Description |
Variant |
Response spectra |
| Channelrhodopsins |
Channelrhodopsins is a basic photogenetic tool - they usually allow rapid depolarization of neurons exposed to light by directly stimulating ion channels. Rhodopsin was found naturally in the green algae Chlamydomonas reinhardtii. Channelrhodopsin-1 (chr1) is excited by blue light and allows nonspecific cations to flow into cells when stimulated. |
ChR2, ChR2/H134, ChETA, ChR/T159C, SFO/SSFO, ReaChR |
450 mm-590 mm |
| Halorhodopsins |
Halorhodopsins is an optically gated inward chloride pump isolated from salt bacteria. The wild-type salt rhodopsin called NpHR (from natronomonas pharaoni) can cause cell hyperpolarization (inhibition) when triggered by yellow light, thereby inhibiting the function of neurons. |
Jaws, Halo/NpHR, eNpHR 3.0 |
566 mm (Halo/NpHR, eNpHR 3.0), 632 mm (Jaws) |
| Archaerhodopsins |
Arch is a light activated outward proton pump that hyperpolarizes (inhibits) cells when triggered by green and yellow light. |
Arch, eArch 3.0, ArchT, eArchT |
566 mm |
| Leptosphaeria rhodopsins |
Leptosphaeria rhodopsin (MAC) is a blue-green light activated proton pump derived from the fungus Leptosphaeria maculans. Mac and its variants allow blue-green light to inhibit neurons. |
3.0 Mac, eMac 3.0 |
540 mm |
Optogenetics Switches
A variety of protein systems using plant and bacterial photoreceptors have been developed to create light controlled protein systems. These "light controlled" proteins provide precise spatial and temporal control of protein activity. See Comparison of Each Photosensitive Proteins →
Available Optogenetics Switches at Creative BioMart.
Basic Principle of Optogenetics Experiment
One of the advantages of photogenetics is its flexibility. The technology can be used to up regulate or down regulate cell activity in multiple brain regions (and more and more other tissues) of many species in vitro or in vivo. All of these applications require the same basic steps:
The genes encoding opsin (such as ChR2, but there are many others now, each with different characteristics) must be put under the control of the promoter to motivate its expression at the required time and place.
For in vitro experiments, this can be achieved by transfection (by electroporation, calcium phosphate, or liposomes). For in vivo work, constructs are usually packaged as viruses and then injected into the target region (virus transduction). Alternatively, transgenic mice expressing opsin under the control of a specific promoter may be generated (or, in some cases, purchased).
For in vivo experiments, this is usually coupling an LED or laser to a fiber optic cable to transmit light to a precise area.
- Measure the effect of manipulation on cell activity.
The method selected may include techniques such as electrophysiological recording, calcium imaging, optical functional magnetic resonance imaging, molecular biology or behavior testing, depend on the specific objectives of the experiment.
Plan your Optogenetics Experiment
When designing optogenetics experiments, you need to select opsin and delivery system at the same time. Here are some key factors to consider
- Photogenetic excitation or photogenetic inhibition.
Do you want to turn on or off in your experiment? According to your answer, you will choose excitatory or inhibitory opsin respectively.
- Activate the color of the light.
There are many different activation wavelengths, ranging from blue to yellow to red. Red light shows better tissue permeability, which allows you to place optical fibers outside the brain, thus reducing the invasiveness of the experimental process. Different activation wavelengths also make it possible to combine multiple opsins in the same experiment.
Time accuracy is the key of optogenetics experiment. Your experimental design will determine whether you need short-term or long-term neuronal activation / inactivation. These can range from milliseconds (hChR2) to "long-acting", such as seconds to minutes with stable step function opsin (SSFO).
Two factors determine which population of neurons to manipulate in a given experiment: opsin expression and the illuminated region. There are several different ways to control opsin expression.
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References
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Parr-Brownlie LC, Bosch-Bouju C, Schoderboeck L, Sizemore RJ, Abraham WC, Hughes SM. Lentiviral vectors as tools to understand central nervous system biology in mammalian model organisms. Front Mol Neurosci. 2015 May 18;8:14.
