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Optogenetics represents a cutting-edge technology that integrates optical methods with genetic engineering to enable precise control over neuronal activity in living animals. This technology allows researchers to manipulate the activity of specific types of neurons within the brain, spinal cord, and peripheral nerves, including those in freely moving animals. The precision of optogenetics extends to both temporal and spatial dimensions, reaching millisecond-level accuracy in timing and single-cell resolution in spatial targeting.
At the heart of optogenetics is introducing light-sensitive genes into specific cell types within the nervous system using advanced genetic engineering techniques. These light-sensitive genes encode proteins such as ChR2, NpHR3.0, Arch, or optoXR, which are integrated into the cell's membrane. These proteins act as ion channels or G-protein-coupled receptors (GPCRs) that respond to different wavelengths of light. This response can selectively regulate the flow of ions such as Cl-, Na+, H+, and K+, thereby altering the membrane potential and modulating cellular activity.
For example, the opsin ChR2 opens in response to blue light, allowing cations to enter the cell, leading to depolarization and activation. Conversely, halorhodopsin (HR) responds to yellow light, causing an influx of anions, hyperpolarization, and inhibition of neuronal activity. Additionally, optoXR channels, when activated by light, modulate intracellular kinase pathways, influencing cell function more indirectly.
1. Selection of Suitable Light-Sensitive Proteins: Light-sensitive proteins come from a wide range of sources, including bacteria, fungi, algae, plants, and vertebrate retinas. The tools used in optogenetics are categorized based on their light response mechanisms into four main types: (i) opsin-based ion channels and pumps, (ii) light-induced conformational changes in proteins, (iii) light-dependent oligomerization or de-oligomerization elements, and (iv) light-sensitive cleavage elements.
Table 1. Common Light-Sensitive Proteins
| Category | Opsin | Excitation wavelength | Characteristics |
| Activating | hChR2(H134R) | 470 nm | A mutant of the most commonly used ChR2; the channel switching speed is also twice as slow as ChR2 |
| hChR2(E123T/T159C) | 470 nm | A mutated ChR2 with a larger photoelectric current and faster kinetics, suitable for high-frequency activation | |
| Achieve(E163A/T199C) | 470 nm | A hybrid of ChR1 and ChR2 can be used for high-frequency stimulation | |
| C1V1(t/t) | 560 nm | A hybrid of ChR1 and VChR1 (mutant of ChR), suitable for specific applications | |
| nChR2(C128S/D156A) | 470–490 nm | Significant stability in the activated state, usable for inducing prolonged depolarization | |
| ChETA | 560 nm | E123T mutation allows neurons to fire spikes at 200 Hz under light stimulation, while other ChR2 channel proteins can only reach 40 Hz | |
| CheRiff | 530 nm | Improved light sensitivity, kinetics, and spectral orthogonality | |
| ChrimsonR | 590 nm | A K176R point mutation of the naturally occurring CnChR1 (Chrimson) | |
| Chronos | 530 nm | Naturally occurring ShChR (Chronos) | |
| ChroME2s | 1030 nm | Fast response, capable of high-frequency neuronal activation; activation intensity (inward current) is greater than the commonly used ChrimsonR | |
| ReaChR | 590–630 nm | Composed of ChEF/ChIEF, VChR1, VChR2 with an added L171I mutation | |
| Inhibitory | eNpHR3.0 | 589 nm | Third-generation light-driven inward chloride pump, improved expression of NpHR on the cell membrane |
| Arch | 566 nm | Improving the localization and uniform distribution of the photosensitive tool on the cell membrane | |
| hGtACR1 | 540 nm | Anion channel mode of channel rhodopsins; usable for rapid and reversible neuronal silencing | |
| eArch3.0 | 590 nm | Larger dynamic range; off-target effects exist when applied to the presynaptic terminal | |
| eOPN3 | 512 nm | Well tolerated in mammalian neurons and does not cause significant light-dependent physiological changes in neuronal excitability | |
| QuasAr2 | 640 nm | Increased brightness and voltage sensitivity, microsecond response time, and does not produce photocurrent | |
| SwiChRca | 475 nm | C1C2 chimera mutation C128A (SwiChRCA) can slow down channel closure, continuously activate chloride ion channels by a single blue light, keeping the cell in a sustained inhibitory state, and close the chloride ion channels after red light irradiation | |
| Jaws | 632 nm | In a red-shifted chloride ion pump, the hyperpolarization current caused by 632-nm light is significantly larger than eHpHR3.0 or ArchT, mainly used for inhibiting target sites with infrared lasers and even noninvasive lighting methods can inhibit Jaw-infected sites | |
| Two-way Regulation | eNPAC2.0 | 590–620 nm (inhibitory) | Contains a dual cis–trans sequence of NpHR and ChR2 Excitation |
| 448 nm (activating) | |||
| BiPOLES | 595/635 nm (activating) | A fusion of blue light-inhibited GtACR2 and red light-activated Chrimson | |
| 460 nm (inhibitory) |
2. Gene Delivery to Target Cells: The genes encoding light-sensitive proteins are introduced into target cells using transfection, viral injection, or the creation of transgenic animals. Due to the complexity, time requirements, and costs associated with creating transgenic animals, viral vectors, particularly adeno-associated viruses (AAVs), are often used for in vivo expression of these proteins.
Creative Biogene's optogenetic technologies allow you to explore the frontiers of neuroscience. Our breakthrough technology seamlessly merges optics and genetics to provide you with unprecedented control and monitoring capabilities over individual neurons, even in freely moving animals. Our skilled technical team is available to help you select and configure the ideal optogenetic reagents and vectors for your specific applications. Enjoy fast turnaround times with worldwide shipping of our optogenetic plasmids and AAVs.
3. Temporal and Spatial Control of Light Signals: Light is delivered to the target area using optical fibers or lasers. Researchers can adjust various parameters, including wavelength, light intensity, frequency, and duty cycle, to control the timing of neuronal activity. Spatial control is achieved by directing light to specific regions of interest, allowing precise manipulation of neuronal activity.
4. Validation of Experimental Methods: The effectiveness of optogenetic manipulation is typically validated using techniques such as patch-clamp recordings to measure changes in membrane potential and behavioral tests to evaluate how neuronal activity influences animal behavior. These methods provide insights into how light stimulation affects neurons, neural circuits, and overall behavior.
1. High Temporal Precision: Optogenetics provides unparalleled temporal precision, achieving millisecond and even sub-millisecond resolution through laser control. This high temporal accuracy is crucial for studying rapid neuronal processes.
2. Precise Stimulus Intensity: The ability to precisely control light intensity allows for accurate modulation of neuronal stimulation. This feature is particularly advantageous for investigating neural circuits that depend on specific stimulus strengths.
3. Tissue and Cell-Specific Targeting: Optogenetics allows for the expression of light-sensitive proteins in specific cell populations or subgroups within particular brain regions. This targeting capability enables detailed regulation of neuronal activity within complex tissue structures.
4. Cellular Projection Specificity: Light-sensitive proteins can extend along the axons of infected neurons and even across synapses, facilitating the study of direct projections between brain regions and the functional connections between upstream and downstream neurons.
5. Visualization of Cellular Activity: By coupling light-sensitive proteins with fluorescent reporter genes, researchers can visualize and record neuronal activity in real time while performing specific tasks or functions.
6. Diverse Tools: Various newly engineered light-sensitive channels with different temporal characteristics and light activation requirements are available, offering flexibility to meet specific experimental needs.
7. Direct Action: Unlike DREADDs technology, which relies on metabolic processes, optogenetics directly modulates cellular activity through laser control, providing a more straightforward approach to manipulating neuronal function.
In neurobiology, optogenetics is used to achieve precise control over neuronal activity, reducing neural toxicity and enhancing stimulation accuracy compared to traditional electrical stimulation methods. This precision helps uncover the functions and mechanisms of neural circuits.
Case Study
On May 29, 2024, Nature Methods featured a study introducing an innovative optogenetic tool— the bistable inhibitory optoGPCR. This cutting-edge tool, derived from the ciliary opsin (PdCO) of Platynereis dumerilii, allows for highly precise control of neural circuits. PdCO demonstrates exceptional performance in inhibiting synaptic transmission with high temporal precision and supports spectral multiplexing with other optogenetic tools. Its unique properties enable detailed functional mapping of neural circuits and open new possibilities for studying specific neural pathways and developing treatments for neurodegenerative and neuropsychiatric disorders.
Figure. 1 Evaluation of Candidate Inhibitory OptoGPCRs. (Wietek J, et al., 2024)
Research Directions:
In the field of cancer treatment, optogenetics is being explored for its potential in light-controlled immunotherapy and targeted cell killing. This approach aims to enhance the specificity and effectiveness of cancer immunotherapy.
Case Study
While surgical removal is a primary method for treating solid tumors, its high recurrence rate poses a challenge. The researchers explore a novel strategy combining surgical tumor removal with advanced immunotherapy to enhance cancer treatment. Traditional surgery often faces challenges with tumor recurrence, while immunotherapy, although promising, struggles with side effects like cytokine storms. To address this, researchers developed a Far-red Light Controlled Immune-modulating Cell (FLIC) that uses far-red light to precisely release immune cytokines (mIFN-β, mTNF-α, mIL-12). In mouse models, FLICs embedded in hydrogels at tumor resection sites, activated by LED far-red light, effectively triggered an anti-tumor immune response, reduced residual cancer cells, and improved long-term protection against recurrence. This approach proved more effective than soluble cytokine injections, showing significant promise for enhancing post-surgical cancer therapies.
Figure 2. FLICs-loaded hydrogel implants activated by far-red light to release cytokines and prevent tumor relapse after melanoma surgery in mice. (Yu Y, et al., 2024)
Research Directions:
Optogenetics is applied to develop novel treatment strategies for cardiovascular conditions.
Case Study
Cardiac function is under neural regulation. Using retrograde trans-synaptic viral tracing, researchers identified excitatory neurons in the primary motor cortex (M1) that influence cardiac function in mice. Optogenetic activation of these neurons increased heart rate, ejection fraction, and blood pressure, while inhibition decreased these parameters and reduced tyrosine hydroxylase (TH) expression in the heart. The median raphe nucleus (MnR) was identified as a key relay region in this circuit. In a mouse model of myocardial infarction (MI), activating M1 neurons impaired cardiac function, whereas ablation improved it by decreasing norepinephrine and TH expression. These findings elucidate the role of M1 neurons in regulating cardiac function and blood pressure, enhancing understanding of the neural mechanisms underlying cardiovascular regulation.
Figure 3. A look at whole-brain imaging using cardiac-derived pseudorabies virus tracing. (Bo W, et al., 2024)
Research Directions:
In diabetes management, optogenetics is used to regulate the expression of insulin and GLP-1, which play crucial roles in glucose metabolism and insulin secretion.
Case Study
Regulating β-cell proliferation is a key goal in diabetes research, potentially benefiting many diabetics. Junta Imai's team published a new approach in Nature Biomedical Engineering using optogenetics to control insulin secretion. They focused on the links between liver, neural signals, lipid metabolism, pancreatic β-cells, and diabetes treatment. The team developed two optogenetic methods for vagus nerve stimulation, enhancing glucose-stimulated insulin secretion and promoting β-cell proliferation in ChAT-ChR2 mice. One method involves sub-diaphragmatic optical fiber implants to stimulate cholinergic neurons. The other uses blue-light-emitting lanthanide particles in the pancreatic duct with near-infrared irradiation to activate vagus nerve fibers. Both methods show that vagus nerve signals, especially those innervating the pancreas, can regulate insulin secretion and β-cell proliferation.
Figure 4. Graphical abstract of two optogenetic vagus nerve stimulation (oVNS) methods (Kawana Y, et al., 2023)
Research Directions:
Optogenetics is applied to regulate the metabolism and colonization of gut microbiota in the treatment of gut diseases.
Case Study
Researchers from Baylor College of Medicine and Rice University recently published a study in eLife revealing that green light can induce Escherichia coli to produce colanic acid, which protects intestinal cells from stress-induced damage and extends nematode lifespan. This suggests that optogenetics could become a novel method for enhancing gut health. In related research, macrophages can adopt different metabolic states, such as the pro-inflammatory M1 phenotype, which relies on glycolysis, and the anti-inflammatory M2 phenotype, which depends on oxidative phosphorylation. These metabolic pathways impact macrophage function and may serve as therapeutic targets to improve immune responses in sepsis survivors.
Figure 5. The study explores optogenetic control of gut bacterial gene expression in C. elegans. (Hartsough LA, et al., 2020)
Research Directions:
Optogenetics provides a precise means for spatial and temporal control in gene editing and transcription regulation. For instance, light-controlled Cre recombinase systems can precisely regulate gene recombination, offering new tools for gene function research and therapeutic applications.
Case Study
The researchers developed a blue light-activated base editor (BLBE) that enables precise nucleotide conversions in genomic DNA without double-strand breaks. By using light-responsive magnetic switches, BLBE achieves efficient A-to-G and C-to-T base editing with minimal off-target effects in both prokaryotic and eukaryotic cells. This technology demonstrates the potential of optogenetics to enhance base editing for genomic engineering.
Figure 6. The mechanisms of BLABE and BLCBE systems, where blue light activates split DNA deaminases to convert adenine to inosine or cytosine to uracil. (Sun Y, et al., 2024)
Research Directions:
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