ARTICLE IN BRIEF
Karl Deisseroth, MD, PhD, reviews the history of optogenetics and its most recent applications.
HONOLULU — “Breakthrough” is an over‐worked word in research, but after the journal Science called optogenetics one of the “Breakthroughs of the Decade,” and Nature Methods named it the Method of the Year, it doesn't seem over‐reaching to call the technique a true breakthrough in understanding the brain.
Developed less than a decade ago, optogenetics is now in use in over 1000 laboratories worldwide because of its ability to shed light, both literally and figuratively, on the full range of questions about the function of neural circuits.
“The essence of optogenetics can be defined very simply as the integration of genetic and optics to achieve gain or loss of function, not of a gene, but of well‐defined events within targeted cells, even within living organisms,” said the technique's inventor, Karl Deisseroth, MD, PhD, associate professor of psychiatry and bioengineering at Stanford University.
Dr. Deisseroth reviewed the history of the technique and its most recent applications in a plenary session here at the AAN annual meeting.
Optogenetics exploits the unique characteristics of light‐sensitive bacterial ion channels: when exposed to light of the correct frequency, the channel opens. So‐called channelrhodopsins are cation channels, and are sensitive to blue light, while the halorhodopsins are chloride pumps activated by red light. When embedded in the plasma membrane of a neuron, a channelrhodopsin will allow sodium ions to enter, depolarizing the membrane and causing the neuron to fire. A halorhodopsin will hyperpolarize the membrane and suppress firing.
The genes for these channels can be delivered into animal models by viral vectors, and their expression can be restricted to the target cell type by including a cell‐specific promoter in the gene package. This is a major advantage over microelectrode stimulation. “An electrode cannot resolve an individual cell within a complex circuit,” Dr. Deisseroth said.
The light needed to activate the channels is delivered by an optical fiber 200 microns in diameter, thinner than the electrode used for deep brain stimulation.
It sounds simple, and essentially it is, but there were several hurdles to overcome in developing optogenetics for use in mammalian cells, including getting the proteins to embed in the plasma membrane. Bacteria don't have the same trafficking machinery as eukaryotic cells, and the molecular signals eukaryotic proteins include that tell them where to go in the cell are absent from the bacterial genes. “So we added in an engineered motif for trafficking to solve this problem,” Dr. Deisseroth said. Further tinkering led to channel proteins that are transported to the ends of axons.
APPLICATIONS: MAMMALIAN BRAIN CIRCUITS
These improvements have led to recent insights in the circuitry of drug addiction, Parkinson disease, and anxiety, among others. “We sought to understand the role of cholinergic interneurons in the nucleus acumbens. This is an interesting population,” Dr. Deisseroth said. They are quite sparsely distributed, accounting for less than 1 percent of the neurons in the nucleus, and they are hypothesized to play a role in reward. To explore that role, Dr. Deisseroth developed rats with light‐sensitive channels in the cholinergic neurons within the nucleus acumbens, and exposed them to cocaine in one chamber of an experimental apparatus, which they began to prefer because of its association with a cocaine reward. But when the cholinergic cells were inhibited during cocaine exposure, “you no longer see that preference. They do not prefer to spend time in the cocaine chamber,” indicating a central role for these neurons in mediating cocaine‐based reward. “This was the first demonstration of a causal necessity for a specific population of cells for a mammalian behavior,” Dr. Deisseroth said.
In Parkinson disease, the challenge was to understand the mechanism of deep brain stimulation. Within the basal ganglia, the so‐called direct pathway is believed to facilitate movement, while the indirect pathway inhibits it. “This had been hypothesized, but it was very hard to show this directly with high precision,” Dr. Deisseroth explained. His lab explored this question in parkinsonian rats, using channelrhodopsin delivered to medium spiny neurons. Excitation of indirect pathway neurons induced parkinsonism, while direct pathway stimulation relieved it, supporting the standard model of the basal ganglia circuitry.
The latest application of optogenetics, called “projection targeting,” delivers the channels to one set of neurons, and illuminates another set elsewhere in the brain, to determine if the two sets of cells are linked in a circuit. “This works because the opsins are being trafficked down the axons,” Dr. Deisseroth said. If illumination of the opsin proteins causes the neuron to change its activity, that establishes that the neuron connects to the illuminated region.
The technique has most recently been used to explore unconditioned anxiety in rodents, with a focus on the amygdala. The basolateral amygdala (BLA) receives much of the information coming into the amygdala, including from the cortex. It is believed that the central nucleus (CeA) receives information from the BLA, and delivers inhibitory signals to the main output structure, the central medial nucleus.
Output from this center gives rise to anxiety, Dr. Deisseroth explained, suggesting that reducing its output would reduce anxiety. “This is a complex disorder, and not much is known about its circuitry implementation. We hypothesized we could control this major anxiolytic pathway,” which has been impossible to deconstruct with other methods.
To map out the circuitry, the researchers introduced channelrhodopsins into the BLA, and illuminated the downstream structure, the CeA. Delivery of light to the CeA triggered channelrhodopsin activity in the BLA, and increased BLA firing. This caused the rats to increase the amount of a typical low‐anxiety behavior, spending more time in the center of an open‐field arena. Conversely, triggering halorhodopsin activity reduced BLA firing, and increased high‐anxiety behavior, with the rats spending more time at the periphery of the arena. The change from one behavior to another was immediate, literally with the flick of a switch.
“It seems anxiety‐related behaviors are regulated in real time, continuously, and can be reversed by activation of this particular pathway from the basolateral nucleus of the amygdala.”
The potential applications of optogenetics are legion, limited mainly by the imagination of researchers. “We see the most important application as a research tool,” Dr. Deisseroth said.
That said, these devices are relatively simple, he noted, and they are smaller than DBS electrodes. “There are no fundamental barriers” to use in therapy. The channels could be delivered by adeno‐associated virus gene therapy, and light could be supplied by an implanted optical fiber. “I would imagine there would be direct therapeutic applications,” he said.
“This field is amazing,” said Tim Pedley, MD, professor of neurology at Columbia University Medical Center. “It's early days yet, but this is a technique that has almost unlimited potential.” Dr. Pedley noted optogenetics has a direct application to understanding circuits involved in epilepsy. “This is a natural target” for the technique, he said.