Optogenetic Indicators

The complexity of the mammalian brain has no comparison: dozens of billions of interconnected neurons, with complex morphology and circuit interaction, capable of exchanging electrical signals with a precise temporal scan in the order of milliseconds. A great challenge is to develop the ability to control only one type of cell in the brain without affecting others. Electrical, physical, pharmacological, and genetic methods are traditionally used to manipulate cells and synapses. However, all these methods lack temporal and spatial resolution and can cause stimulation, inhibition, or inactivation of off-target cells and processes. To overcome these limitations, new genetic tools referred to as "optogenetics" have been developed.

Overview of Optogenetic

The term optogenetics indicates a methodology that allows controlling the activity of specific neurons within intact neuronal circuits. The idea of using light as a tool to control neuronal function was originally put forward by Francis Crick. In the 1970s, biologists discovered that some microorganisms generate proteins that, in response to visible light, regulate the flow of charges across the membranes. These proteins, termed opsins, are photosensitive trans-membrane proteins that, when illuminated at defined frequencies, induce transmembrane ion fluxes and, thereby, changes in the electrical activity of the cell. Through viral vectors, the gene coding for an opsin can be integrated into target neurons, leading to the expression of the opsin protein on the membrane. A nearby source of light, set on the right wavelength and frequency, can then interact with it, activating or inhibiting neuronal activity. The introduction of mutations to existing opsin variants allowed to overcome certain problems associated with light delivery.

Examples of Optogenetic Indicators

• Channelrhodopsin (ChR): A blue light-activated cation-channel from Chlamydomonas reinhardtii, used to excite neurons. Channelrhodopsin2 (ChR2) identified from green alga is a blue light-gated nonselective cation channel that offers multiple ideal features for in vitro and in vivo neural stimulation.
• Halorhodopsin (NpHR): a yellow light-activated chloride pump from Natronomonas pharaonic, used to inhibit neurons.
• Arch/ArchT: Arch/ArchT are proton pumps from Halorubrum sodomense and Halorubrum strain TP009, respectively, are widely used for inhibitory optogenetics.

Fig.1 Summary of most available opsins for optogenetic studies.¹

Advantages of Optogenetic

A big advantage of optogenetic tools compared to conventional neuromodulatory approaches (e.g., electrical, surgical, pharmacological, thermal, etc.) is the compatibility with genetics. In model organisms such as fruit fly and mice, microbial opsins can be easily delivered to target neuron populations using a variety of genetic approaches to achieve cell-type-specific manipulation. Diverse transgenic fly and mouse models exist and are publicly available.

Applications of Optogenetic Indicators in Diseases

The use of optogenetics as therapeutic tools for neurological disorders has been investigated in PD, AD, and epilepsy. Enhanced excitation of pyramidal neurons is a common feature of many forms of epilepsy, and its control may lead to therapeutic effects. NpHR delivery into these neurons promptly and dramatically reduced seizures upon light stimulation. Enhancing the inhibitory activity of interneurons via transfection of the excitatory opsin ChR also resulted in reduced seizure frequency and severity upon light stimulation. Optical inhibition of the subthalamic nucleus in PD models significantly improved akinesia and ameliorated levodopa-induced dyskinesia.

Services at Creative Biolabs

Optogenetic indicators are undoubtedly useful neuroscience research tools with a wide range of applications. As a professional in neuroscience, Creative Biolabs has rich experience in the application of optogenetic indicators in neuroscience. With an advanced technology platform and extensive expertise accumulated from practice, we have absolute confidence in the quality of our service. If you are interested in optogenetic indicators or looking for neuroscience research tools and relevantly custom productions, please don't hesitate to contact us for more information.

We offer a variety of optogenetic indicators, including genetically encoded calcium indicators (GECIs) and voltage indicators, that can be used to study neural activity with high spatial and temporal resolution. These indicators are compatible with a wide range of experimental models, from cell cultures to whole organisms, and can be easily integrated into existing optogenetic setups.

Our services include custom design and optimization of optogenetic indicators to meet the specific needs of each research project. We work closely with researchers to develop indicators that are tailored to their experimental requirements, ensuring optimal performance and data quality.

By rendering this service and related services, we aim to accelerate research and drug discovery in neurodegenerative diseases, including but not limited to:

Published Data

The ability to optically image cellular transmembrane voltages with millisecond resolution can provide unprecedented insights into in vivo brain function in behavioral animals. Ahmed S. Abdelfattah et al. described a point mutation that increases the sensitivity of the Ace2 retinoid-based voltage indicator. They used the mutation to develop Voltron2, an improved chemogenetic voltage indicator.

In their experiments, they performed a large-scale point mutation screen to find an improved version of Voltron. Ultimately, it was found that the introduction of the A122D mutation increased the sensitivity of Voltron, especially in the subthreshold range, without affecting kinetics, membrane transport, or photobleaching. Overall, they found a generalizable mutation that significantly increased the sensitivity of Ace2 retinoid-based sensors, thereby improving their voltage-reporting capabilities.

Fig. 2 High-throughput screening of Voltron mutants.²

Applications of Optogenetic Indicators in Neuroscience

• Calcium Imaging
• Voltage Imaging
• Neurotransmitter Sensing
• Functional Circuit Mapping
• In Vivo Imaging
• High-Throughput Screening

We provide customized design services for developing optogenetic indicators tailored to specific research needs. Whether you need a new indicator for an understudied neurotransmitter or optimization for improved kinetics, our team can engineer and validate specialized indicators.

FAQs

Q: What kind of support do you offer during the experimental setup and data analysis phases?

A: We provide comprehensive support throughout the entire process, from experimental setup to data analysis. Our team of experts is available to assist you with the initial design of your experiments, ensuring that the optogenetic indicators are used effectively in your specific research context. We also offer troubleshooting support during the setup phase, helping you optimize the expression and performance of the indicators in your system. Additionally, we provide guidance on data acquisition and analysis, offering tools and protocols to help you interpret the results accurately. Our goal is to ensure that you get the most out of our services, and we're here to support you at every step.

Q: Are your optogenetic indicators suitable for long-term studies, and how stable are they over time?

A: Our optogenetic indicators are designed for both short-term and long-term studies. We select and engineer indicators with high stability to ensure consistent performance over extended periods. The viral vectors we use for delivery are optimized for long-term expression, and the indicators themselves are tested for resistance to photobleaching and other forms of degradation. We can provide recommendations on experimental conditions that help preserve the integrity of the indicators throughout your study.

Q: What are the turnaround times for your services?

A: Turnaround times vary depending on the level of customization and the specific indicators requested. For standard indicators with minimal customization, delivery can take between 4 to 6 weeks. For highly customized or novel indicator designs, timelines may extend to 8 to 12 weeks, considering production, validation, and quality testing. During the consultation phase, we provide a clear timeline and update you regularly on the progress of your project to ensure timely delivery.

Q: How do you ensure that the delivered optogenetic indicators are ready for immediate use in experiments?

A: We ensure our optogenetic indicators are fully ready for immediate use by packaging them with all the necessary reagents, protocols, and validation data. Our indicators are delivered in stable, ready-to-inject formats with pre-tested titers and detailed instructions for application. We also offer pre-packaged injection kits, vector formulations, and real-time support for initial set-up. Additionally, each package includes step-by-step guides for vector handling, injection protocols, and troubleshooting tips to help you seamlessly integrate the indicators into your experiments.

Scientific Resources

Integration of Neuronal Activity Monitoring with Optogenetics

References

1. Chang, Rui B. "Optogenetic control of the peripheral nervous system." Cold Spring Harbor Perspectives in Medicine 9.12 (2019): a034397.
2. Abdelfattah, Ahmed S., et al. "Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator." Neuron 111.10 (2023): 1547-1563.

For Research Use Only. Not For Clinical Use.