Back in 2005, the landscape of neuroscience was very different. Given the brain’s complexity, the questions that are being asked – and most of all answered – are extremely limited by technology. The activity of individual neurons could be recorded using electrodes and imaging techniques could look at the entirety of the brain. However, the brain isn’t like the heart or the lungs where it has one function and has few cell types that fulfill that function. Neurons that are next to each other could function in completely different ways (Namburi et al., 2015) giving way to an increasing number of psychiatric, degenerative, developmental and social conditions. Therefore, trying to find therapies and to understand the underlying physiology behind these conditions requires way more than understanding the function of pockets of neurons or seeing structural activation. As such, in order to even start to understand the brain, it needs to be possible to artificially control one type of cell just like they are naturally manipulated, while leaving all of the other types unaltered. Put non-scientifically, we need to develop an “on/off” button to activate and deactivate neurons.

Neither the aforementioned electrical stimulation, nor drugs are specific enough to do this. Drugs operate too slowly and while electrical currents can be applied to mimic the natural activation of cells, all of the cells within a region get activated. However, photostimulation is a promising alternative to electrical stimulation. Why? Light can be delivered in time pulses and at specific locations. Thus, in 2005, the birth of optogenetics revolutionized neuroscience (Boyden et al., (2005). This relatively new technology is a combination of “optics” and “genetics” and is a neuromodulation technique using light to manipulate activity within the brain. At its simplest level, the process is undergone as follows: (1) neurons, which are not light-sensitive, are first genetically engineered to express light-sensitive proteins – hence the involvement of “genetics,” and (2) the neuron will be illuminated with light – “optics” – and depending on the properties of the light-sensitive protein that was introduced, certain changes will occur. This technique has both high spatial and temporal resolution.

Light-Activated Proteins

At the crux of optogenetics are light-activated proteins, “optogenetic actuators,” that have the ability to modify the cell in which they are expressed when that cell is exposed to light (Guru et al., 2015). They have the ability to activate or inhibit the neuron’s activity.

The most famous type of light-activated proteins is opsins – naturally occurring light-sensitive transmembrane proteins. There are two types of opsins (Guru et al., 2015) – one of which occurs in vertebrates and is involved in vision and the second of which is found in microbial organisms such as algae and bacteria. In their native organisms, these opsins are involved in a variety of activities, including navigation towards energy sources (Nagel et al., 2002). In vertebrates, they were first introduced as a part of an optogenetic experiment (Boyden et al., 2005).

Neurons are surrounded by membranes that have proteins embedded within them. Put simply, these proteins govern the flow of ions so that when positive ions rush into the cell, equivalent to a metaphorical “on” signal, the cell is activated, and when negative ions come into the cell, they act as an “off” signal and the cell is deactivated.The opsins found in microorganisms are made up of membrane-bound protein components that function as a pump or channel that controls for a variety of ions. Furthermore, these membrane-bound proteins are activated by light so that when light hits them, the pumps or channels will open or close leading to either cell activation (depolarization) or cell inhibition (hyperpolarization). It should be emphasized that there are many types of opsins, collected from different organisms, which have various relevant properties and are employed based on the focus of the optogenetic research.

Genetic Engineering- Introducing Optogenetic Actuators into Neurons

Primates and rodents, the main organisms that are studied in neuroscience research, do not express these light-sensitive protein channels. Therefore, it was necessary to find a way in which to efficiently deliver and express these genes into these organisms. This ability – previously deemed an impossible feat – is credited to Boyden et al., (2005), a study conducted in the Deisseroth lab, which has spearheaded optogenetic research and will be mentioned frequently throughout the rest of this article. The introduction of these genes can be done in multiple ways.

One method, and the first used, involves the use of viral vector targeting systems. In this mechanism, an engineered virus that has the opsin gene and a promotor – an “on” switch – is introduced into the cells. A promotor is selective and makes sure that only the kinds of neurons with the relevant characteristics that are within the experiment will make the encoded proteins. However, viruses are limited by the amount of genetic material they are able to carry, which limits the size and ability of the promoter. Most cell types that are specifically targeted would need more genetic material (Guru et al., 2015). Although some viral vectors have been developed to target specific cell classes, in order to have the requisite specificity of modern experiments, more genetic material is needed than this approach permits (Guru et al., 2015).

However, transgenic animals – animals that have DNA that has been artificially introduced – can express an opsin without being limited by the size of the genetic material. In these cases the optogenetic actuator gene is introduced with a promoter in the zygotes of the mice. For instance, Zhao et al., (2008), members of the Deisseroth team, were able to create transgenic mouse lines that expressed a specific type of opsin in specific cell types. It should be noted, however, that unlike the viral vector method, transgenic animals lack spatial localization and are much more difficult to use given that a new mouse line needs to be created for each type of opsin.

A more optimal method has been manufactured that combines viral vectors and transgenic animals and has both high genetic and high spatial targeting. Within this method, viral vectors are used in combination with Cre recombinase-based mouse lines i.e. mice that express Cre recombinase in specific cell types. The Cre protein – “cre” standing for “causes recombination”– is an enzyme that modifies gene expression between two sites that surround the gene of interest. Therefore, when the Cre is present, this will affect the gene expression in the targeted specific cell types and when it is not present, no changes will occur. As such, when a viral vector with the optogenetic actuator gene is introduced, although it will infect multiple cells, the opsin will only become functional in the cells that have Cre (Guru et al., 2015). As noted, for transgenic animals that are born with the opsin gene, only one type of opsin can be experimented with. With Cre recombinase-based mice, any opsin gene can be introduced with the virus.

Any of these methods will make neurons artificially sensitive to light, but each approach comes with its own limitations.

Optogenetic Control

The brain consists of numerous types of excitatory and inhibitory neurons and each cell type has a functional role in the development of various diseases or physiological and psychological states. Therefore, optogenetic technology has been developed to artificially induce all of these changes within the cells.

The first time opsin was introduced to modulated neuronal activity was with a type of light-gated ion channel called channelrhodopsin-2 (ChR2) that was discovered in green algae (Nagel et al., 2002; Boyden et al., 2005). A pulse of light is able to induce a single action potential (activity) in neurons that expressed the green algae opsin, demonstrating that ChR2, when activated by light, is a sort of “on” switch for the cell. On the other hand, another type of opsin – halorhodopsin (NpHR) – is a sort of “off” switch for the cell (Zhanget al., 2007).

Interestingly, different opsins respond to different wavelengths of light i.e. different colors; one color can command some opsins and another color can command others. ChR2 responds to blue light and NpHR responds to yellow light. Therefore, if both Chr2 and NpHR were introduced into the same cell, the presence of one type of light versus another would either activate or suppress the activity of the cell (Zhang et al., 2007). Each pulse of blue light has the potential to drive activity while each pulse of yellow light suppresses the cell’s activity (Fig. 1). Essentially, colored light would act like a remote control over these cells.



Figure 1: Shows how the blue light activates neuronal activity while the yellow light blocks activity (Zhang et al., 2007).

Limitations in the Technique

In addition to the limitations noted with each method of introducing the optogenetic actuator, as with any technology, there are some crucial concerns. Guru et al. (2015) introduce three main limitations. The expression of the optogenetic actuator gene is not expected to be uniform across all of the light-sensitive neurons and neither will the light delivery to each neuron, meaning that there will be variety in the activation patterns. Second of all, the activation and suppression of cell activity may be opening channels that might not be activated during normal cell activity. For instance, stimulation might turn “on” the cells too much so that they begin to operate outside their normal range. Finally, when all of the opsin-expressing neurons are bathed in light, they will all become activated – synchronizing their activity in an unnatural manner, hiding the individual cell properties. Given the importance of keeping research within ecologically sound conditions in order to draw relevant conclusions, both of these limitations are major concerns given that the purpose of optogenetics is to understand the underlying properties of individual cells. Moreover, the expression of foreign proteins, in general, has been shown to cause alteration within the host animal, which is a major concern with opsins (Zalocusky, Fenno & Deisseroth, 2013). With that in mind, given that the opsins are being introduced artificially, it is imperative to ensure that the kinetics of the channels fall within the range of what is natural (Zalocusky, Fenno & Deisseroth, 2013).

Evidently, the largest limitation is the extent to which optogenetic techniques will be ethically used on humans. However, the following section, underlining the key advantages of the technique, will highlight how it may be used.

Advantages

Despite these challenges, optogenetics has completely revolutionized neuroscience research. Fundamentally, optogenetics functions as a remote control that is able to turn on or off specific cells within live organisms, yielding insights into physiology and into possible treatments for disease. Its importance as a research tool, especially in conjunction with other technology, is still growing.

At the core of optogenetics is Karl Diesseroth’s lab, whose work has been cited throughout this article. He, a psychiatrist and bioengineer, developed optogenetics as a solution to unanswered questions for neuropsychiatric disorders, considering the stigma associated with psychiatric disease to be due to a lack of knowledge of the underpinning physiology. Diesseroth thinks that the discussion behind mental illnesses needs to be shifted beyond the chemical imbalance paradigm (Diesseroth, 2010). Certain treatments for neuropsychiatric or neurodegenerative disorders such as depression or Parkinson’s include electrode-based deep brain stimulation or vagus nerve stimulation and while these have been shown to improve symptoms, they also have huge side effects. Why? Both of these techniques stimulate huge areas of the brain as opposed to localized regions and given the heterogeneity of brain tissues (different structures have multiple functions and neurons that are next to each other could be involved in vastly different activities) these stimulation tools might be impacting relevant neurons as well as irrelevant neurons, thus causing horrible side effects (Gradinaru et al, 2009). Optogenetics and its ability to modulate specific types of neurons is able to bypass this concern.

For example, historically, Parkinson’s patients have been treated with deep brain stimulation. However, Gradinaru et al. (2009), used optogenetics to understand the circuitry underlying Parkinson’s; they introduced ChR2-NpHR into target cells in order to be able to activate and inhibit them and systematically investigated different elements of the disease circuit until they found that stimulation would be improved if it applied to the connections between cells as opposed to the cells themselves. Their findings demonstrate how optogenetics can be employed to understand different disease circuits.

Given that neurons underlie function, researchers have found that the tool is able to turn behaviors “on” and “off,” as well. For example, Adhikari et al., (2015) looked at the pathways involved in anxiety and found that the optogenetic activation of a specific type of amygdalar cell using ChR2 was able to decrease anxiety while the inhibition of the same cell type using NpHR increased anxiety. Similarly, addictive behaviors can be “turned off” or “on.” Chen et al., (2013) used cocaine-addicted rodents and found that the optogenetic stimulation of neurons in the prelimbic cortex would prevent the cocaine-administering behavior, while inhibiting the same neurons increased it. Evidently, this work is done in non-human animals and these findings are not revealing the entirety of the physiology behind such complex behaviors, but such findings denote how optogenetics can eventually be used as therapy. The same investigations described here are being applied to vastly other forms of addiction such as alcoholism (Davis, 2013) or other neuropsychiatric conditions.

The most promising application of optogenetics to humans right now and the one most likely to get approved, is to explore an area that already responds to light: the retina. Many forms of blindness are due to a dysfunction or disease in the photoreceptors that take light and generate neural signals in response to it. However, there are other cells that usually get input from the photoreceptors (lower on the visual pathway) such as bipolar cells and ganglion cells that could be optogenetically stimulated to respond and to counter blindness. Similar work has been done in mouse models already (Caporale et al., 2011).

Outside the clinic, optogenetics has been used to improve other technologies. Functional magnetic resonance imaging (fMRI) is a commonplace technique in neuroscience that scans brains and allows us to image responses to various stimuli. It is well established that although fMRIs tell us which brain regions are involved in what response, they do not provide temporal information about when this activation may occur. Therefore, the introduction of optogenetics – which has both high spatial and temporal attributes – is able to bypass this limitation. Even more can be accomplished once fMRIs are combined with optogenetics – ofMRI (Lee et al., 2010). Another enduring concern was the magnitude of the signal that was needed for an fMRI machine to detect it; it is established that localized activity occurs within the brain and it was unclear whether or not this activity was visible on an fMRI. If it were not, then this would introduce a severe constraint to a multitude of studies that used the technique. Lee et al., (2010) activated local cells using optogenetics and then tested the readouts with an fMRI to see that there were positive signals, validating years of fMRI research. Given that this new tool – ofMRI – stimulates specific neurons, it is able to provide causal information about how activity patterns work. Thus, the technique could potentially improve how current circuit analysis is being done.

Conclusions

Optogenetic findings yield incredible insights into the fundamental aspects of physiology and dysfunction. In terms of treatment, we can see that there are two sides to optogenetic research: taking established therapies and improving them or introducing the potential for new therapies. Now that we are able to turn cells “off” and “on” using light, the possibilities of answering research questions and developing new applications has expanded enormously. Optogenetics is still a relatively novel technique, but given its birth as a quest to try to find viable solutions to psychiatric diseases and its applicability, it seems indisputable that it will eventually start being used for human patients.

Literature Cited

Adhikari, A.,T.N. Lerner, J. Finkelstein, S. Pak, J.H.Jennings, T.J. Davidson, E. Ferenczi, L.A. Gunaydin, J.J. Mirzabekov, L. Ye, et al. Basomedial amygdala mediates top-down control of anxiety and fear. Nature, 527 (2015), pp. 179-185

Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K.Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).

Caporale N, Kolstad KD, Lee T, Tochitsky I, et al. LiGluR restores visual responses in rodent models of inherited blindness. Mol Ther. 2011;19:1212–9.

Chen, B. T. et al. Rescuing cocaine-induced prefrontal cortex hypoactivity prevents compulsive cocaine seeking. Nature 496, 359–362 (2013).

Davis, Bonnie : “Researchers Study Alcohol Addiction Using Optogenetics.” Wake Forest Baptist Health , 16 Dec. 2013, www.wakehealth.edu/News-Releases/2013/Researchers_Study_Alcohol_Addiction_Using_Optogenetics.htm.

Deisseroth, Karl. “Optogenetics: Controlling the Brain with Light [Extended Version].” Scientific American, 20 Oct. 2010, www.scientificamerican.com/article/optogenetics-controlling/.

Gradinaru, V., M. Mogri, K.R. Thompson, J.M. Henderson, K. Deisseroth.Optical deconstruction of parkinsonian neural circuitry. Science, 324 (2009), pp. 354-359

Guru A, Post RJ, Ho Y-Y, Warden MR. Making sense of optogenetics. Int J Neuropsychopharmacol.2015;18:yv079.

Nagel G, Ollig D, Fuhrmann M, Kateriya S, Musti A.M, Bamberg E, Hegemann P. Channelrhodopsin-1: A light-gated proton channel in green algae. Science. (2002);296:2395–2398.

Namburi, P.,A. Beyeler, S. Yorozu, G.G. Calhoon, S.A. Halbert, R.Wichmann, S.S. Holden, K.L. Mertens, M. Anahtar, A.C. Felix-Ortiz, I.R. Wickersham, J.M. Gray, K.M. Tye. A circuit mechanism for differentiating positive and negative associations. Nature, 520 (7549) (2015), pp. 675-678, 10.1038/nature14366

Lee, J. H., Durand, R., Gradinaru, V., Zhang, F., Goshen, I., Kim, D.-S., … Deisseroth, K. (2010). Global and local fMRI signals driven by neurons defined optogenetically by type and wiring. Nature, 465(7299), 788–792. http://doi.org/10.1038/nature09108

Zalocusky, K.A., Fenno, L., and Deisseroth, K. (2013). Current Challenges in Optogenetics. In Optogenetics – Dahlem workshop reports, P. Hegemann and S. Sigrist, eds. (Walter de Gruyter).

Zhang, F., Aravanis, A. M, Adamantidis, A., de Lecea, L. & Deisseroth, K. Circuit-breakers: optical technologies for probing neural signals and systems. Nature Rev. Neurosci.8, 577–581 (2007).

Zhao, S. C. Cunha, F. Zhang, Q. Liu, B. Gloss, K. Deisseroth, G.J.Augustine, G. Feng.Improved expression of halorhodopsin for light-induced silencing of neuronal activity. Brain Cell Biol., 36 (2008), pp. 141-154