Elbert Mets

To better understand behavior and cognitive function, neuroscientists have long sought the ability to activate specific neurons within the brain. For much of the field’s history, neuroscience researchers have had limited tools to selectively manipulate neurons in vivo (in a live animal) and conclusively determine their functions.

At best, scientists were able to stimulate one or several neurons with a sharp electrode and observe the subsequent neural responses by recording electrical activity at another site in the brain, or by monitoring an animal’s behavior. Alternatively, the study of neurons in vitro (in culture) limits scientists’ ability to draw definitive conclusions about specific neurons in a living brain.

Feeling constrained by existing research methods, Stanford scientists Karl Deisseroth and Edward Boyden began seeking novel methods to selectively stimulate neurons in vivo to identify their functions in the early 2000s.1 Researchers have previously studied neural activity in response to stimulation with light, but existing technology’s limited ability to quickly activate brain cells was incongruent with research questions that demanded a finer timescale.2

Utilizing a light-related technique, Boyden and Deisseroth began experimenting with channelrhodopsin-2 (ChR2), a cell membrane-bound ion channel that, in response to blue light, activates the neuron containing the channel.¹ They also experimented with other opsins (light-activated ion channels), namely halorhodopsin (Halo/NpHR), which deactivates neurons in response to light.¹

Boyden’s initial experiments demonstrated that rapid pulses of blue light could be used to control the activity of ChR2-containing neurons on a millisecond timescale, and his subsequent work established the technique’s viability in mammals.1-3

Shortly after the publication of Deisseroth and Boyden’s work, the technique of using light to control neuronal activity in vivo became known as optogenetics.  Thousands of research groups have since adopted this technique.1,4 The primary advantage of optogenetics over previously existing, coarser methods to control neurons using light is its specificity. Because opsins are encoded by genes, they can be selectively introduced into different types of neurons, many with distinct functions, and characteristics.1

Existing treatments for epilepsy, which affects 65 million people worldwide, are limited in their effectiveness and often have adverse side effects.5 A growing body of work has used optogenetic techniques to better understand the brain basis of epilepsy, which could serve as the foundation for research into novel treatments for the disorder.5-9

Studies in mouse models of epilepsy have shown that optogenetic inhibition of excitatory cells (which normally promote seizure activity) or activation of inhibitory neurons (which normally prevent seizures), are both effective in halting an epileptic epsiode.7-9

Desearchers have also developed optogenetic techniques that monitor animals’ brain activity using an electroencephalogram (EEG). The information feeds back to a light source, which shines light onto opsin-containing cells to modulate neural activity as desired.8 In one such system, when the EEG readout shows increased brain activity consistent with a seizure, light is shone onto specific neurons in the thalamus, which stops the seizure.8

Another exciting application of optogenetics is in the study of strokes.10 Strokes occur when a blood vessel becomes blocked and a section of the brain is deprived of nutrients and oxygen (ischemic stroke), or when one of the brain’s blood vessel ruptures, damaging the surrounding brain cells (hemorrhagic stroke). These vascular changes result in neuronal death in the area surrounding the dead tissue, which can impair language, movement, or thought depending on the location and severity of the lesion.

However, patients who have suffered from strokes can recover some of their lost neural function, a phenomenon thought to result from the regrowth of synapses between neurons around the injured region.10 The formation of new synapses or connections between neurons is driven by activity in parts of the brain directly surrounding the infarct (dead tissue), which is associated with increased growth factor expression and blood flow near the injury site.10

Since neural activity near the damaged neurons is important in driving recovery following a stroke, it is fitting that new treatments aim to increase activity in areas around the stroke lesion.10

Previous methods to stimulate neurons around a stroke have been nonspecific and associated with side effects such as impaired speech.10 However, in one study, researchers demonstrated that optogenetic activation of neurons in the primary motor cortex (the brain structure responsible for movement) in mice that sustained a stroke led to increased expression of neuronal growth factors. This was then associated with improved sensory and motor behavior.10 These findings suggest that the activation of cortical motor neurons following a stroke improves recovery, which could present a novel target for drugs or treatment with optogenetics in humans.10

In addition to its use in the study of epilepsy and strokes, optogenetics has also been used in research surrounding the treatment of chronic pain.11

Neuronal activity in the anterior cingulate cortex (ACC) is responsible for pain sensation in both mice and humans.11 Because excitatory and inhibitory neurons are closely packed together and interspersed with one another in the ACC, past attempts to activate inhibitory (and not excitatory) ACC neurons with electrodes have had limited success.11

However, because ChR2 (the ion channel that activates a neuron when stimulated with blue light) is genetically encoded, it can be experimentally placed into specific inhibitory neurons within the ACC. This allows researchers to selectively activate these neurons, thereby decreasing overall ACC activity.11 In mouse models, the result of decreased ACC activity is reduced pain-related behavior.11 A similar intervention may hold promise for people suffering from debilitating chronic pain.

Optogenetics’ utility is not limited to addressing questions about the brain. In addition to identifying new ways to treat neurological conditions, researchers have used optogenetic techniques to characterize cellular pathology underlying type 2 diabetes in the pancreas.12

Additionally, in the field of cardiovascular medicine, researchers have introduced opsins into heart cells in vitro.13 The ultimate goal of this technique is to develop a benign method of defibrillating the heart.13 However, in vivo manipulation of heart cells faces a number of challenges. Namely the method of delivering opsins to cells and how to effectively illuminate cells deep in the heart (cardiac tissue prevents blue light from traveling far into the heart; a similar issue arises in the human brain).4,13

To introduce opsins into heart cells, viral vectors can be “painted” onto the heart surface, or donor cells expressing opsins can be grafted onto the heart, though both methods have limitations.13

Although neuroscientists can target opsin expression to specific types of neurons, their ability to introduce opsins into individual neurons remains limited.4 Consequently, although optogenetics has improved the capacity to stimulate specific groups of neurons rather than activating neurons nondiscriminantly.4

More broadly, optogenetics researcher and Oxford University professor Gero Miesenböck, cites “the lack of a theoretical underpinning for much of neuroscience” as a challenge facing the technique.4 Furthermore, “we don’t understand most neural systems well enough to articulate and test clear hypotheses.”4

The salient improvements seen in animal models of human neurological diseases point to the use of optogenetics in clinical medicine. The fact that opsins have been successfully introduced into, and maintained in, neurons in the brains of nonhuman primates without complication over a period of several months suggests the technique could also be a safe therapeutic tool for humans.3

It should be noted that although animal models are a powerful tool in understanding the functions of the human brain, research findings in animals – even those evolutionarily close to people – do not always translate exactly to humans. This is particularly true in brain research since the human brain is substantially more complex than that of even our closest evolutionary relatives.

Since light-activated ion channels are not naturally expressed in mammals, one clear hurdle for the use of optogenetic therapies in humans is the targeted delivery of genes coding for opsin proteins into patients’ brain cells.

Transgenic animals (animals bred with specific proteins encoded into their genome) are often used in optogenetic studies. This is not practical for treating humans, as it would require introducing genes for opsins into the patient’s genome before they developed the condition being treated, and likely before they were born.

Alternatively, opsins can be introduced into the neurons of a living animal using a viral vector.4 Using this method, a gene coding for a protein of interest (here, an opsin) is inserted into the genome of a virus, which will then ‘infect’ specific human host cells. The virus has its illness-causing components removed, and serves instead as a template that will deliver the gene of interest. Once inside the body, the virus enters the host’s cells and incorporates its genome into the host cell’s genome.

Subsequently, the host cell transcribes and translates the viral genome to express the viral proteins of interest – here, an opsin such as ChR2. Despite the efficacy of viral vectors in animal research, the approach poses obstacles in humans.4

Although work in Boyden’s group has demonstrated the safety of ChR2 delivered by a viral vector in the brain of a non-human primate, there are concerns about the safety of such a therapy and the stability of the opsins over the years.4

A further challenge to clinical use is the portability of optogenetic systems. Classically, optogenetic setups involve an optrode (laser light port) implanted into an area of an animal’s skull such that laser light, fed through an optic fiber, can shine directly onto the neurons of interest.

Clearly, such a set up impedes movement, and a less conspicuous method of light delivery would be preferable in humans.4 To address this concern, researchers have recently developed a soft, implantable device that allows for inconspicuous optogenetic stimulation in the absence of a clumsy optrode.14

Optogenetics presents a novel tool in neuroscience research, and perhaps ultimately in clinical medicine. To date, optogenetics has allowed researchers to characterize the function of populations of neurons and their role in neurological conditions.

Still in its infancy and evolving rapidly, optogenetics promises to continue to improve our understanding of the human brain and may ultimately present an effective method of alleviating patients’ suffering from brain-based neurological illnesses.

References:

1. Boyden ES. A history of optogenetics: the development of tools for controlling

brain circuits with light. F1000 Biology Reports 2011;3.

2. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. Millisecond-timescale, genetically targeted optical control

of neural activity. Nature Neuroscience 2005;8:1263-8.

3. Han X, Qian X, Bernstein JG, et al. Millisecond-timescale optical control of neural dynamics in the nonhuman primate brain. Neuron 2009;62:191-8.

4. Adamantidis A, Arber S, Bains JS, et al. Optogenetics: 10 years after ChR2 in

neurons—views from the community. Nature Neuroscience 2015;18:1202-12.

5. Krook-Magnuson E, Soltesz I. Beyond the hammer and the scalpel: selective circuit control for the epilepsies. Nat Neurosci 2015;18:331-8.

6. Krook-Magnuson E, Armstrong C, Oijala M, Soltesz I. On-demand optogenetic control of spontaneous seizures in temporal lobe epilepsy. Nat Commun 2013;4:1376.

7. Krook-Magnuson E, Szabo GG, Armstrong C, Oijala M, Soltesz I. Cerebellar Directed Optogenetic Intervention Inhibits Spontaneous Hippocampal Seizures in a Mouse Model of Temporal Lobe Epilepsy. eNeuro 2014;1.

8. Paz JT, Davidson TJ, Frechette ES, et al. Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury. Nat Neurosci 2013;16:64-70.

9. Berglind F, Ledri M, Sorensen AT, et al. Optogenetic inhibition of chemically induced hypersynchronized bursting in mice. Neurobiol Dis 2014;65:133-41.

10. Cheng MY, Wang EH, Woodson WJ, et al. Optogenetic neuronal stimulation promotes functional recovery after stroke. Proc Natl Acad Sci U S A 2014;111:12913-8.

11. Gu L, Uhelski ML, Anand S, et al. Pain inhibition by optogenetic activation of specific anterior cingulate cortical neurons. PLoS One 2015;10:e0117746.

12. Reinbothe TM, Safi F, Axelsson AS, Mollet IG, Rosengren AH. Optogenetic control of insulin secretion in intact pancreatic islets with β-cell-specific expression of Channelrhodopsin-2. Islets 2014;6:e28095.

13. Boyle PM, Karathanos TV, Trayanova NA. “Beauty is a light in the heart”: the transformative potential of optogenetics for clinical applications in cardiovascular medicine. Trends Cardiovasc Med 2015;25:73-81.

14. Park SI, Brenner DS, Shin G, et al. Soft, stretchable, fully implantable miniaturized optoelectronic systems for wireless optogenetics. Nat Biotechnol 2015;33:1280-6.

15. Russell, Lloyd, and Häusser Lab. Optogenetics. N.d. University College London, London.

16. Boyden, Ed. Sputnik Animation. N.d. McGovern Institute for Brain Research at MIT, Cambridge.

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