Optogenetics |
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Optogenetic approaches promise to revolutionize neuroscience by using light to manipulate neural activity in genetically or functionally defined neurons with millisecond precision. It is a neuromodulation technique facilitating monitoring and control of individual neurons in living organisms in real time. Harnessing the full potential of optogenetic tools, however, requires light to be targeted to the right neurons at the right time.
Physical delivery of virus to a given anatomical location can exploit or uncover circuit connectivity patterns either by making use of axonal projections or by using viruses that are able to cross one or more synapses. Cell types can be addressed if the cell type of interest has a known genetic identity. Directing the illumination source to a given set of cells or even individual neurons and processes is useful when the targets of interest are separated in space relative to the spatial resolution of the technique used.
As the study of neuroscience moves from dish to whole animal, new techniques are needed to study individual cellular responses within a broader functional context. Optogenetics is a technique that allows researchers to modulate the activity of target neurons using specific wavelengths of light. What makes the technique so useful is its ability to control neural activity so precisely even in free moving organisms. This is accomplished by coupling genetic expression of light-sensitive channel proteins within targeted cells to precision light exposure and measurement.
Optogenetic components
Although the concept of using light to control cellular behavior is well over 30 years old, obtaining the right optics, light source, photoactivatable protein, genetic construction tools was only accomplished first by the Karl Deisseroth laboratory in 2005.Light sensitive proteins
The key reagents used in optogenetics are light-sensitive proteins. Several types of microbial opsins, are currently used to modulate neuronal function. Each type is sensitive to a different range of wavelengths and responds by altering different ion conductances. Blue light is commonly used to stimulate channelrhodopsin-2 (ChR2), a nonselective cation channel, resulting in potassium, sodium, and calcium flow which depolarizes the neuron to threshold. Other channelrhodopsin variants are now available that differ in depolarization and wavelength sensitivity characteristics yielding even finer control over neural modulation. The halorhodopsin (NpHR) class of chloride pumps, which respond to yellow light, are typically used to hyperpolarize neural membranes and induce inhibitory responses.Measuring the effects
The acceptance in the scientific community of optogenetics as an effective tool for modulating neural function or cellular signaling has come from careful verification of effects with traditional, well established tools. Electrodes are routinely used to record neural function during stimulation. So-called optogenetic sensors are also being used to measure calcium, chloride or membrane voltage shifts. Sequencing verifies the construct insertion. Protein presence and downstream protein-protein interactions and cell signaling are measured using antibodies and antibody-based technologies such as Western blotting and immunohistochemistry.Below is a table showing the top published targets interrogated by optogenetics methods. Antibodies to these targets needed to verify protein presence/localization are also listed.
| Gene Name | Cat no. | Description | Key Applications | Species Reactivity |
|---|---|---|---|---|
| LARGE | In Testing | Anti-LARGE Rabbit Polyclonal Antibody | WB, IHC | |
| CHAT | AB143 | Anti-ChAT Rabbit polyclonal Antibody | WB, IHC, IH(P), IP | Mk, H, R, M, Bat, Fe |
| CHAT | AB144 | Anti-ChAT Goat polyclonal Antibody | WB, IHC | R, Gp, M |
| CHAT | AB144P | Anti-ChAT Goat polyclonal Antibody | WB, IHC | Op, Av, Gp, H, M, Mk, R |
| CHAT | AB1582 | Anti-ChAT Sheep polyclonal Antibody | WB, IHC | Rb, Gp, R |
| CHAT | AB5042 | Anti-ChAT Rabbit polyclonal Antibody | IHC | R, Av, GP |
| CHAT | AB5042P | Anti-ChAT Rabbit polyclonal Antibody | IHC, ELISA | R |
| CHAT | AB15468 | Anti-ChAT Chicken polyclonal Antibody | IHC, ICC | M |
| CHAT | MAB5270-100UG | Anti-ChAT Mouse Monoclonal Antibody | WB, ELISA, IHC, IH(P) | R, H, Po |
| CHAT | MAB305 | Anti-ChAT Mouse Monoclonal Antibody | IHC | R, H, Mk |
| CHAT | MAB5350 | Anti-ChAT Mouse Monoclonal Antibody | IHC, WB, ELISA | R, Gp, H, Mk |
| CHAT | MAB5422 | Anti-ChAT Mouse Monoclonal Antibody | WB, ELISA | H |
| ACHE | MAB303 | Anti-Acetylcholinesterase Mouse Monoclonal Antibody | IP, ELISA, IHC | Mk, B, Ca, Ch, Gp, H |
| REST | 07-579 | Anti-REST Rabbit Polyclonal Antibody | WB | R, H, Mk |
| REST | 09-019 | Anti-REST Rabbit Polyclonal Antibody | WB | R, H, M |
| PARK2 | AB5112 | Anti-PARK2(Parkin) Rabbit Polyclonal Antibody | IHC, ELISA, WB | R, H |
| PARK2 | 05-882 | Anti-PARK2(Parkin) Mouse Monoclonal Antibody | IHC, IP, WB, ICC | M, H |
| PARK2 | MAB5512 | Anti-PARK2(Parkin) Mouse Monoclonal Antibody | IHC, IP, WB | M, H |
| THY1 | CBL415 | Anti-Thy-1 Mouse Monoclonal Antibody | FC, IP, IH | H |
| THY1 | CBL1500F | Anti-Thy-1 Mouse Monoclonal Antibody-FITC | ICC, FC | M |
| THY1 | MABF34 | Anti-Thy-1 Rat Monoclonal Antibody | FUNC, WB | M |
| AGRP | AB3402P | Anti-Aouti Related Protein Rabbit Polyclonal Antibody | WB, ELISA | M |
| POMC | AB5087 | Anti-Melanocyte Stimulating Hormone α (POMC) Sheep Polyclonal Antibody | RIA, IHC | H, M, R |
| SLC17A6 | MAB5504 | Anti-Vesicular Glutamate Transporter 2 Mouse Monoclonal Antibody | IHC, WB | H, M, R |
| SLC17A6 | MAB55054A4 | Anti-Vesicular Glutamate Transporter 2 Mouse Monoclonal Antibody-Alexa488 | IHC | H, M, R |
| CRY2 | AB15056 | Anti-Cryptochrome 2 Rabbit Polyclonal Antibody | WB | M |
| HCRT | PC362 | Anti-HCRT/Orexin Rabbit Polyclonal Antibody | EIA, IHC, IF | H, M, R |
| HCRT | PC345 | Anti-HCRT/Orexin Rabbit Polyclonal Antibody | EIA, IHC, IF | H, M, R |
| HCRT | AB3096 | Anti-HCRT/Orexin Rabbit Polyclonal Antibody | EIA, IHC, WB | H, M, R |
| HCRT | AB3098 | Anti-HCRT/Orexin Rabbit Polyclonal Antibody | EIA,WB | Fe, H, M, R |
| HCRT | AB3704 | Anti-HCRT/Orexin Rabbit Polyclonal Antibody | IHC | R |
| HCRT | AB15690 | Anti-HCRT/Orexin Chicken Polyclonal Antibody | EIA | H, M, R |
| KITLG | AP1154 | Anti-KITLG Mouse Monoclonal Antibody | EIA | H |
| RAC1 | 05-389 | Anti-Rac1 Mouse Monoclonal Antibody | WB, IHC, IP | R, H, M |
| RAC1 | 07-1464 | Anti-Rac1 Rabbit Polyclonal Antibody | WB | R, H, M |
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Optogenetics and Cell Signaling
The narrow targeting of specific cell types conferred by Optogenetics may have extensive value in interrogating protein-protein interactions in signaling pathways. Traditional cell signaling research techniques are often limited in spatial or temporal resolution and plagued by off-target effects. The precision offered by optogenetics allows easier delivery, activation, and recovery of cellular function. In using this new approach, nicely reviewed by (Zhang and Cui, Trends Biotechnol. 2015), signaling could be manipulated by either light-induced protein translocation or light-induced protein uncaging. In the first case, light controlled target protein binding could be used to study DNA transcription, protein splicing, catalytic activity, protein inactivation or even protein trafficking and secretion. In the second case, light controlled uncaging could release steric inhibition of signaling members resulting in modulation of downstream pathways. In combination with recently developed genome editing techniques, optogenetic modulation of intracellular signaling could be a valuable tool in tightly controlling genetic manipulation in gene therapy.Zhang, K. and Cui, B. (2015) Optogenetic control of intracellular signaling pathways. Trends Biotechnol. 33:92-100.