Lighting up the Brain: The Power of Optogenetics

Introduction

In order to grasp the role of a specific set of neurons in the behavior of a living organism, one could activate or deactivate the gene that dictates their activity and observe the changes. Researchers have had the capability to dampen or augment the expression of genes for decades. But the procedure to bring about this change takes hours, days or even months with conventional genetic methods, making it hard to measure or even see the resulting neurological change.  Alternatively, electrical stimulation provides some level of stimulation or inhibition of neurons on much shorter timescales and can be directed to a specific region however it cannot specifically target individual cell types.

Optogenetics integrates light and genetics to enable direct control of electric and biochemical processes, like firing or inhibiting neurons, induced by direct light activation. This is done through the introduction of light-gated channels of proteins, referred to as Opsins, into the cells of choice using targeted genetic methods. After installation, these channels enable manipulation of neurons on timescales of milliseconds instead of months. The outcome is a method that can be employed to study the function of neurons as well as manipulate the behavior of an organism, either in vivo or in vitro.

• In-vivo, devices to regulate neuron behavior and observe neuron activity can be inserted into the brain of any freely-moving animal from a fruit fly to a primate.

• In-vitro, light stimulation may be coupled with optical and electrophysiological methods to monitor the consequences on a cellular level. 

The resultant methods do not merely give us useful information about the regular function of brain tissue but also about neurological disorders like Parkinson's disease, Alzheimer's disease and epilepsy. Additionally the optically controllable cells themselves can be used to cure disorders in the future.

Optogenetic Control

There are three objectives for studies using optogenetics:

• Evoking the discharge of a neuron (Activation)

• Inhibiting its response (Inhibition)

• Biasing its response to ensure spontaneous discharge more likely

All of these objectives need a distinct class of opsins. The most prevalent opsins that induce the firing of the neuron are the opsins causing it to fire, and there are dozens of options available that can be stimulated with a wide range of wavelengths of light and with a wide range of response rates from milliseconds to minutes. The most commonly used opsins are CHR2, NpHR, ReaChR, Chrimson, SFO, VChR1 and iC1C2.

Studies have revealed that Proteins that can suppress neuronal activity using light are less common however, Wietek et al. and Berndt et al. have each designed channel-rhodopsins that can suppress action potentials (Wietek et al. 2014; Berndt et al. 2014). A more covert method is to not directly make the neuron fire with a particular stimulus, and instead place the neuron in an 'excited state' in which firing is more probable. This can be done using a 'step-function opsin' where the neuron stays excited for many seconds after the light is turned off, so that the pulse optical fibre delivering the pulse can now be switched off, releasing the animal to move freely. Advances in step-function opsins have lead to the development of neurons with the capability to demonstrate up to 30 minutes of biased activity following a single 10ms blue light pulse, and have been observed to get switched off using a pulse of yellow light (Yizhar et al. 2011).  

Preparation of Samples

The proteins for optogenetic control are generally introduced into neuron cells through a virus vector. Viruses offer rapid and flexible insertion, with a high percentage of target cells infected, offering stable expression. There are several methods of attaining cell specificity, including utilizing special promoters, spatial targeting of the virus injection, and accurate targeting of the light delivery. Nevertheless, the majority of viral vectors have limited capacity for carrying genetic information. Another method of delivering the target proteins is to utilize genetic 'knock-in' methods to raise animals that code for the proteins as their own genes, thus bypassing the virus's limited carrying capacity. But it takes more time, effort and expense to deliver opsins in this manner (Fenno et al., 2011).

Control and Observation during Experiments

• The in-vivo experiments are able to observe the macroscopic behavior of an organism being subjected to optogenetic control whereas the in-vitro experiments can offer control and observation at the level of individual cells. 

• For in-vitro experiments, cells are usually cultured neuronal cells and are simply illuminated with filtered light from mercury arc lamps or LEDs in a microscope setup. 

• In-vivo illumination is slightly more complex in that normally the animal would still have to be capable of movement with normal behavior, but involves implanting a flexible optical fibre into the brain, with laser or high-power LED light being employed for illumination. Though this method is relatively invasive and limits movement of the animal.

• In 2013, Bruchas and Rogers built a wireless, ultrathin needle-based system of control and observation that can be injected deep into soft tissues and provides a greater freedom to animal (Kim et al. 2013). The device incorporates 8.5 μm thick micro-LEDs, a microelectrode, an integrated photo-detector and a temperature sensor in order to optically stimulate and observe the brain of a mobile animal.

• Both in-vivo and in-vitro experiments need to image deep within thick tissues and a way to do this is two-photon microscopy with a pulsed laser. This enables control and observation simultaneously, with extremely good spatial and temporal resolution (Andrasfalvy et al. 2010). In-vivo, 2-photon calcium imaging is able to penetrate deeper into tissues in living systems to monitor the effect of optogenetic stimulation in real-time. However, these techniques require the animal to be fixed, e.g., on a stationary treadmill (Kim et al. 2017).

Imaging procedures in Optogenetics

• The requirements for camera imaging in optogenetics are extremely wide, ranging from the macroscopic observation of organisms as big as primates to high-speed, high-magnification microscopic observation of single cells.

• In-vivo experiments usually monitor animal behavior as a primary experimental result. Video or time-lapse imaging of the freely moving animal in its environment has been commonly employed.

• At the microscopic level, fluorescence imaging of the target cells can be an essential tool in confirming the validity of cell-type targeting strategies. For experiments in vitro, the methods of optogenetics tend to accompany those of electrophysiology in general like patch-clamping and calcium imaging.

Path-breaking Results

• In order to observe the effects of optogenetic control at both the microscopic level and the macroscopic 'behavioral' level, electrophysiological methods are commonly employed simultaneously, along with control and observation equipment built into a unified device. 

• In-vitro studies have revealed that microscopic methods such as the observation of beating heart muscle cell that is under optogenetic control are also possible (Bruegmann et al. 2010). This enabled the scientists to stimulate, examine and control heart muscle without the harmful effects of electrical stimulation, offering a means of pace-making research. Further, fluorescence imaging has validated the cell specificity of the light-sensitive proteins and the electrical response of the heart tissues to light.

• ·A recent in vivo experiment in mice employed optogenetics to identify brain pathways that control 'reward-seeking' behavior (Kim et al. 2017). By optogenetically boosting these pathways, the researchers made a mouse, less inclined to press a lever that had a chance of getting either a bite of food or a weak shock. This could open the door to psychiatric illness treatments that increase reward-seeking like drug addiction, or depression.

Future Challenges

• One of the main concerns for any behaviorological experiment is to guarantee that the experimental equipment, and any genetic alteration to the organism, doesn't affect behavior. For optogenetics, this usually means having parallel experiments with and without cranially-implanted devices, and with and without genetic alteration.

• Additionally, to prepare a sample for optogenetic control or observation with cell-type specificity there is need of difficult and advanced techniques, careful validation, especially check avoidance non-targeted populations. 

Conclusions

• For the LED and laser light microscopists, having available the facility for using electrophysiological approaches, the potential for controlling neuronal cells with cell-type specificity and millisecond temporal resolution provides unprecedented access to insight into the brain's cellular-level dynamics.

• Optogenetics is a wonderful addition to the neuroscientist's toolkit, and the abundance of research involving these methods in everything from small-scale in vitro experiments to human clinical trials of medical treatments is testimony to its new path breaking possibilities.

References

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2. Berndt, Andre, Soo Yeun Lee, Charu Ramakrishnan, and Karl Deisseroth. 2014. “Structure-Guided Transformation of Channelrhodopsin into a Light-Activated Chloride Channel.” Science 344 (6182). American Association for the Advancement of Science:420–24.

3. Bruegmann, Tobias, Daniela Malan, Michael Hesse, Thomas Beiert, Christopher J Fuegemann, Bernd K Fleischmann, and Philipp Sasse. 2010. “Optogenetic Control of Heart Muscle in Vitro and in Vivo.” Nature Methods 7 (11):897– 900. https://doi.org/10.1038/nmeth.1512.

4. Fenno, Lief, Ofer Yizhar, and Karl Deisseroth. 2011. “The Development and Application of Optogenetics.” Annual Review of Neuroscience 34 (1):389–412. https://doi.org/10.1146/annurev-neuro-061010-113817.

5. Kim, Christina K., Li Ye, Joshua H. Jennings, Nandini Pichamoorthy, Daniel D. Tang, Ai Chi W. Yoo, Charu Ramakrishnan, and Karl Deisseroth. 2017. “Molecular and Circuit-Dynamical Identification of Top-Down Neural Mechanisms for Restraint of Reward Seeking.” Cell 170 (5):1013–1027.e14. https://doi.org/10.1016/j.cell.2017.07.020.

6. Kim, T.-i., J. G. McCall, Y. H. Jung, X. Huang, E. R. Siuda, Y. Li, J. Song, et al. 2013. “Injectable, Cellular-Scale Optoelectronics with Applications for Wireless Optogenetics.” Science 340 (6129):211–16. https://doi.org/10.1126/science.1232437.

7. Wietek, Jonas, J Simon Wiegert, Nona Adeishvili, Franziska Schneider, Hiroshi Watanabe, Satoshi P Tsunoda, Arend Vogt, Marcus Elstner, Thomas G Oertner, and Peter Hegemann. 2014. “Conversion of Channelrhodopsin into a Light-Gated Chloride Channel.” Science 344 (6182). American Association for the Advancement of Science:409– 12.

8. Yizhar, Ofer, Lief E Fenno, Matthias Prigge, Franziska Schneider, Thomas J Davidson, Daniel J O'Shea, Vikaas S Sohal, et al. 2011. “Neocortical Excitation/inhibition Balance in Information Processing and Social Dysfunction.” Nature 477 (7363). Nature Research:171–78.