Lights, Opsins, Action!
Optogenetics brings complex neuronal circuits into sharper focus

by Sandra A. Swanson

December 16, 2009

As neuroscientists unravel the brain’s tangled mysteries, they’ve recruited an unlikely assistant: pond scum.

The single-celled green algae isn’t much to look at, but Chlamydomonas reinhardtii has a useful attribute: light-sensitive proteins called opsins. When the opsin channelrhodopsin-2 (ChR2) is exposed to blue light, it makes algae move. When ChR2 is embedded in a cell membrane and then bathed with blue light, it can also open channels that let positively-charged ions rush in, creating electrical signals.

“That happens to be neural code for ‘on,’ ” says Karl Deisseroth of Stanford University. Deisseroth is a pioneer in the field of optogenetics, genetically altering neurons with light-sensitive proteins and then using fiber optics to illuminate them. The light is delivered via implants in animals’ brains; different colors affect different opsins. Some (like ChR2) prompt neurons to fire, while others pull the plug on electrical signals.

Using these techniques, researchers can study cells with greater precision than had been possible before. They also can stimulate specific cells without disturbing their neuronal neighbors—a challenge that’s plagued neuroscience for decades. “This is a technology which may have utility for understanding the neural circuit basis of normal function, but also of disease mechanism and disease treatment,” Deisseroth said at the recent Society for Neuroscience (SfN) meeting in Chicago.

In fact, there was no shortage of researchers outlining optogenetics techniques at the meeting. Whether it’s plumbing the depths of addiction or memory circuitry, their work gives new meaning to the term “blue light special.”

Marking memory

For example, how many neurons are needed to reactivate a memory? Michael Hausser and his team at University College London are addressing this question by using the opsin ChR2. The researchers focused on neurons in the dentate gyrus, part of the hippocampus. First they targeted a specific subset of cells involved in memory formation by linking ChR2 expression to a c-Fos promoter—a gene commonly used to label neurons involved in memory formation. Researchers created fear memories in mice by pairing a tone with an electric shock. The mice quickly learned that “tone = ouch,” and the sound alone caused them to freeze in their tracks.

One day after that fear-conditioning session, the researchers illuminated the animals’ dentate gyrus region with blue light, which stimulated only the small set of cells that had expressed the gene c-Fos. That blue light stimulus mirrored the effect of the tone—the mice froze in fear. When researchers performed the test with a random assortment of cells in the dentate gyrus, the light did not reactivate the fear memory.

Labeling the ChR2-expressing cells, the scientists found that the fear memories were triggered by only about 200 cells. Matteo Rizzi, one of the lead authors of the poster presented at the SfN meeting, was surprised to see so few neurons involved. “The dentate gyrus is highly populated—it’s home to about a million granule cells, so that’s a tiny proportion,” he says.

On and off

To make strides in optogenetics research, the field needs new tools and techniques. That includes a broader array of traffic lights to control ion flow through cell membranes. When halorhodopsin is present, yellow light inhibits neurons (by allowing negatively-charged ions to enter instead of the positive ions that induce a nerve signal). With ChR2, blue light activates neurons. Now researchers Deisseroth’s Stanford lab have identified a new light-sensitive opsin, called Guillardia theta Rhodopsin 3 (GtR3). It’s the polar opposite of ChR2 --- shine a blue light on GtR3, and it will inhibit neurons instead of activating them. “We can use [GtR3] in conjunction with yellow-light inhibitory halorhodopsin, and introduce them into different cell populations and selectively turn off one or the other, or both,” says Feng Zhang, the lead author of the yet-unpublished work.

Altering behavior

At Mount Sinai School of Medicine, meanwhile, researchers used optogenetics to learn more about the role of the prefrontal cortex in stress. The scientists took individual mice and paired them with a bully—a more aggressive, larger mouse. The bullied mice became unwilling to interact with other animals, and the researchers wondered if they could reverse that effect. To make the stressed-out mice more social, they stimulated the prefrontal cortex (injected with ChR2) by using precise flashes of blue light, specifically a 100-hertz, 40 millisecond burst every three seconds. As predicted, the mice increased their level of social interaction.

“By trying to mimic the normal patterns of activity in the prefrontal cortex, we wanted to see if we could drive an anti-depressant-like response,” says Herbert Covington, lead author of the study. A better understanding of prefrontal cortex activity in stressed animals might lead to improved treatments for depression in humans, he notes.

By isolating specific cell types, Mount Sinai researcher Mary Kay Lobo is studying cocaine addiction in mice. Using ChR2, Lobo compared the roles of two specific neuron types in the nucleus accumbens: those that express or contain D1 or D2. They are most noted for the differences in their dopamine receptors, but Lobo emphasizes that the yet-unpublished study was not designed to compare that aspect.

Rather, by activating D1 and D2 with blue light, she has shown that the two neuron subsets affect addiction behavior in distinctly different ways. When D1 neurons fire, they boosted the animals’ preference for cocaine. But when D2 neurons are switched on in mice, cocaine addiction behavior ceased.

If one of these neuron subsets was turned off, would it produce an opposite effect? Lobo plans to find out, with the help of halorhodopsin—the opsin that inhibits cells when exposed to yellow light. “That will allow us to develop better therapeutic techniques, aimed at one cell type versus another,” she said.

Seeking route to addiction

What brain connections drive addictive behaviors? Like Lobo and her colleagues, researchers at the University of California, San Francisco, studied the nucleus accumbens—a brain region known for its involvement in reward-driven behavior.

“What’s not known so well is the connectivity within the nucleus accumbens,” says Garret Stuber, the lead author of the study. To tackle that knowledge gap, Stuber and his colleagues looked at two synaptic subsets within that brain region: one connecting to the basolateral amygdala, and the other to the prefrontal cortex.

They injected the animals’ basolateral amygdala with a virus carrying ChR2. The researchers then triggered a blue-light stimulation whenever the animals poked their noses in a tiny hole within their cages. Those animals performed the nose pokes repeatedly, as long as the light stimulation persisted. When researchers shut off the laser, the animals lost interest in nose poking.

Then the researchers ran the same experiment with the other pathway—the prefrontal cortex-to-nucleus accumbens. When those mice received the blue light, they did not turn into obsessive nose pokers. “We made sure all of the animals sampled the nose poke, so they had the opportunity to learn to receive optical stimulation,” says Stuber, whose earlier results appeared online April 23 in Science. The number of nose pokes performed by the basolateral amygdala mice was significantly higher than the prefrontal cortex mice, he says.

Building on that research, Stuber is now exploring what happens to natural, goal-driven behavior (such as the need for food) when specific neuron subtypes are shut down.  “Just having a better understanding of the neural circuitry is going to be very important for coming up with better treatment for addiction disorders,” he says.

Stuber also notes that prior to optogenetics, researchers lacked the ability to parse out the complex connections between neurons. “It’s still very much in its infancy, but in my mind, [optogenetics] is probably the most exciting thing to come along in neuroscience in 30 or 40 years,” he says.

That enthusiasm is spreading. “There are probably more than 500 laboratories using this technology,” Deisseroth told SfN attendees at his optogenetics talk. As more researchers follow suit, optogenetic techniques will continue to illuminate brain function—both literally and figuratively.