Optogenetics is a technique used to alter neurons so they can be activated by light. Though it sounds a bit like science fiction, its tools are grounded firmly here on earth, and it all started with pond scum.
In fact, Ed Boyden, associate professor of biological engineering and brain and cognitive sciences at the Massachusetts Institute of Technology (MIT), and leader of their Synthetic Neurobiology Group, says that one reason he and collaborators developed optogenetics a few years ago (1) was a desire for more precise control over neurons—rather than bathing all of the neurons in pharmacological compounds, for instance. Today, the potential for optogenetic applications to treat debilitating brain disorders like schizophrenia, epilepsy, and post-traumatic stress disorder, is tantalizing, but how close are recent developments bringing us to that possibility?
New Solar Panels
The original channelrhodopsin is activated by blue light—but soon after this discovery, Boyden’s group identified inhibitory counterparts to channelrhodopsin: halorhodopsin (called Halo) is a light-activated inward chloride pump, and archaerhodopsin (called Arch) is a light-activated outward proton pump. When neurons express these molecules, a flash of yellow-red light suppresses neuronal firing by hyperpolarizing the neurons.
Boyden’s group hasn’t stopped searching for new light-sensitive channels, especially those gated by different colors of light. Newer inhibitory opsins include a proton pump from the fungus Leptosphaeria maculans—called Mac—that is activated by blue-green light. Two inhibitory opsins with different activation profiles offer new and exciting possibilities for experiments requiring precise control in neural circuits.
For example, blue light would suppress the activity of a Mac-expressing neuron, but not a Halo-expressing neuron. Meanwhile, red light would suppress the activity of a Halo-expressing neuron, but not a Mac-expressing neuron. Neuronal silencers like Halo can be driven by yellow or red light, while one also uses blue light to activate other neurons expressing channelrhodopsin. In fact, one could even express a combination of activators and silencers in the same neuron—for example, channelrhodopsin and Halo. Such double targeting of neurons with two colors of light (in this example, blue and yellow) would give you the power to depolarize and hyperpolarize individual neurons in intact animals merely with flashes of light.
To expand their molecular toolkit, Boyden’s lab keeps searching for different light-sensitive opsins. “Our lab is developing a number of new molecules, including red-shifted molecules that enable noninvasive neural perturbation, especially neural silencing with red light,” says Boyden. Using red light is desirable because it scatters less, and the longer wavelengths can penetrate more deeply into brain tissue.
Particularly, they are interested in “color-shifted molecules that enable multi-color control of neurons using multiple channelrhodopsins,” says Boyden. These types of molecular tools will allow Boyden and colleagues to target distinct groups of neurons independently according to the color of light used to activate them. A collaboration with a group from the University of Alberta is helping the team to search plant genomes for new light-activated opsins. They are looking for opsins stimulated by light that will let them activate (i.e. depolarize) neurons with two distinct colors.
Shine the Light
A rainbow of light-sensitive proteins would yield sophisticated tools to allow control of neuronal excitability, and examine neural network interactions. Years ago, Boyden and others solved the problem of how to deliver a flash of one-color light to an intact brain in an awake and freely moving animal. But how would you stimulate many different types of neurons with multiple colors of light? How do you get all of that light in there?
Typically, the activation of neurons using optogenetics requires bathing a large number of neurons in the same color and pattern of light. But this simply isn’t realistic given the complex, 3D structure of the brain. “In reality, most of the neurons fire at different times with respect to each other,” says Boyden. “Accordingly, it would be great to be able to stimulate many individual neurons, in good temporal registration with one another.” His group is addressing this need by building a device capable of delivering multiple light stimuli, and that can be implanted in the brain (2). “Recently we have been developing probes that can deliver light to many individual points in the brain,” says Boyden.
Such a probe consists of microfabricated waveguides, or a set of tiny optical fibers. The device has the potential to introduce arrays of hundreds or thousands of sources of light that can be used to construct complex activation patterns that might more closely resemble real neuronal codes in the brain. “These devices enable light to be independently delivered to many individual points in the brain, thus enabling cells at those points to be controlled to fire independent patterns,” says Boyden. Such a device could, indeed, shuttle many colors and patterns of light into the brain.
Meanwhile, Kotaro Kimura, associate professor in biological sciences at Osaka University, is trying to understand similar principles of neural activation without the complications of working with mammalian brains. So he uses optogenetics in a simpler system that permits easier light delivery, the invertebrate nematode worm Caenorhabditis (3). In a recent paper published by Neuroscience Research, Kimura’s group described “a very simple optogenetic system for small animals such as C. elegans, Drosophila larvae, and maybe zebrafish,” says Kimura. “Because it consists of just a USB camera and a couple of ring LEDs and a PC, it can be introduced to many laboratories that are interested to try out optogenetics.”
With their optogenetic system, Kimura’s group modulates the activity of specific neurons, to shed light on basic principles of neural network function. “We can now measure neural activities that are associated with sensory behaviors as well as learning and memory of C. elegans,” says Kimura. “If we can mimic the neural activity without the sensory stimulus or learning by optogenetic techniques and ‘operate’ the worms' behavior as we expect, I think we can say that we really understand the function of the neuron.” Kimura hopes in future work to integrate optogenetic manipulation with sensory stimuli and calcium imaging.
A Goal in Sight
Will prosthetic light sources and an expanding palette of photoactive molecules power up the therapeutic toolbox?
Recent work indicates that indeed, the gap between research lab and clinic is beginning to close. For example, Boyden’s group, in collaboration with Alan Horsager of the University of Southern California and others, published work last year showing that optogenetic tools could be used to restore vision to blind mice (4).
In that experiment, the researchers stably expressed channelrhodopsin in ON bipolar cells of a strain of blind mice with retinitis pigmentosa, using a recombinant adeno-associated viral vector. They found that the channelrhodopsin expression resulted in ON responses in postsynaptic retinal ganglion cells, and that the mice showed restored vision for months following the treatment. The fact that such a dramatically positive result was achieved with no evidence of side effects from the viral vector, retinal damage from bright light exposure, or immune response from expression of the foreign, algal-derived protein, holds great therapeutic promise for late-stage retinal degeneration in humans. “Many groups are seeking to treat blindness with optogenetics,” says Boyden. “It's a very intriguing area.”
“Intriguing” describes all future applications of optogenetics. Boyden suggests that implanting a brain prosthetic device to deliver complex light stimuli to the brain at different times could help to treat otherwise intractable neurological conditions such as schizophrenia or epilepsy. For example, epileptic seizure activity in the brain might be dampened by light activation of silencing opsins like Halo. Implanted brain devices are already in use, treating hearing loss with cochlear implants, and Parkinson’s symptoms with deep brain stimulators. One main difference, however, is that the optogenetic implant requires a molecular partner in light-activated channels. If the first human trials demonstrate safety, there will likely be an explosion of optogenetic therapies for humans.
1. Boyden, E. S., F. Zhang, E. Bamberg, G. Nagel, and K. Deisseroth. 2005. Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8(9):1263-1268.
2. Zorzos, A. N., E. S. Boyden, and C. G. Fonstad. 2010. Multiwaveguide implantable probe for light delivery to sets of distributed brain targets. Opt. Lett. 35(24):4133-4135.
3. Kawazoe, Y., H. Yawo, and K. D. Kimura. 2012. A simple optogenetic system for behavioral analysis of freely moving small animals. Neuroscience Research (May).
4. Doroudchi, M. M., K. P. Greenberg, J. Liu, K. A. Silka, E. S. Boyden, J. A. Lockridge, A. C. Arman, R. Janani, S. E. Boye, S. L. Boye, G. M. Gordon, B. C. Matteo, A. P. Sampath, W. W. Hauswirth, and A. Horsager. 2011. Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness. Molecular Therapy 19(7):1220-1229.