Remote controlled brains: a neuroscience revolution.

The worm is not a toy or a robot but a living creature. It has been engineered so that its nerves and muscles can be controlled with light. With each flash of blue its neurons fire electric pulses, causing the muscles they control to clench. A flash of yellow stops the nerves firing, relaxing the worm’s muscles and lengthening its body once again.

The worm is in the vanguard of a revolution in brain science the most spectacular application yet of a technology that allows scientists to turn individual brain cells on and off at will. "It’s really changing the whole field of neuroscience," says the worm’s developer, neurobiologist Alexander Gottschalk at the University of Frankfurt.

One possibility is that the technology, coupled with a method of getting light into the human skull, could create a Brave New World of neuro-modification in which conditions such as depression or Parkinson’s disease are treated not with sledgehammer drugs or electrodes, but with delicate pinpricks of light. In the long term it is even possible that such treatments could be modified to enhance normal brain function, for example improving memory or alertness.

The technology could also lead to spectacular advances in basic neuroscience, allowing researchers to tease apart the neural circuits that control everything from reflexes to consciousness with unprecedented accuracy. "We’ll be able to understand how specific cell types in the brain give rise to fuzzy concepts like hope and motivation," predicts Karl Deisseroth, a psychiatrist at Stanford University in California, who is spearheading some of the work.

These new possibilities materialised when neuroscientists finally cracked a long-standing problem in their field: how to take control of individual neurons.

A nerve cell is an electrical entity. Its membrane is normally charged like a battery, to about a tenth of a volt. Nerve cells communicate using electric pulses, which arise when the voltage across the membrane briefly leaps from ?0.07 volts to around +0.04 volts. That spike of excitation races down the tendrils of the neuron until it reaches the ends, where it jumps across synapses to set up new waves of excitement in neighbouring cells.

We’ve known since the 18th century that zapping neurons with a wire electrode triggers an electrical spike. This approach has provided much of our knowledge of brain function: scientist have inserted electrodes into the brains of flies, mice and monkeys, zapped away, and observed what happened.

Neurologists also use electrodes to treat medical disorders. Depressed patients who don’t respond to medication are sometimes given electroconvulsive therapy via scalp electrodes, or vagal nerve stimulation using a device implanted in the neck. Tremor disorders such as Parkinson’s disease are sometimes treated with deep brain stimulation using an electrode permanently implanted at a specific spot in the brain. DBS is considered to be a more targeted treatment than standard drug therapy, because it directly affects only a small piece of the brain whereas most drugs act throughout the brain.

But electrodes leave much to be desired, both for basic science and therapy, because even where they only stimulate a few cubic millimetres of brain tissue their targeting is still crude. A cubic millimetre of brain tissue contains tens of thousands of neurons tangled like spaghetti. Some are excitatory when they fire they cause their neighbours to fire too while others are inhibitory, quieting their neighbours down. The ultimate aim for many researchers is to target only one of these two types, as precisely as possible.

"If you just jam an electrode into the brain you stimulate all the cell types," says Deisseroth. "You get serious side effects because you get the cell types you want, but also the cell types you don’t want."

Electroconvulsive therapy has the worst reputation. It cures some patients, but can erase memories in a few unfortunate souls. The author Ernest Hemingway lost much of his capacity for writing after receiving electroshock therapy for depression in 1961. He committed suicide shortly afterwards.

Vagal nerve stimulation, meanwhile, causes side effects such as pain, hoarseness and rapid breathing. And then there’s DBS for Parkinson’s disease, in which an electrode implanted in a spot called the subthalamic nucleus (STN) quiets the chaotic activity there. Turning on the electrode can cause strange effects the first time around. It electrifies a few patients’ funny bones, causing them to laugh uncontrollably. It plunged one patient into hopelessness, causing her to weep until the electrode was switched off moments later.

These hiccups can be remedied on the spot by adjusting the electrode’s position, but others are more problematic. Some studies suggest that a few DBS patients become vulnerable to mood swings and suicide over time. Various explanations have been offered, one of which is that the mood changes are caused by the electrode stimulating not only the right cells but also a few of the wrong ones too.

At least one brain imaging study has shown that electrical stimulation of the STN can accidentally turn on nearby nerve fibres that connect to the limbic system, a network of brain areas that controls mood. DBS can cause the whole limbic system to light up with patterns of brain activity that are eerily similar to those seen in depression.

Precision instrumentFor years, neuroscientists and neurologists have wanted something better. If they could turn on nerve cells one at a time, leaving everything else alone, they’d be well on their way to targeted therapies, as well as decoding the function of the neural circuits that control complex behaviours. "The goal was to modify a subset of neurons and make them sensitive to light," says Gero Miesenbck, a neurobiologist at Yale University. "By shining light, you can then activate only one type of neuron at a time, while leaving the others alone."

Miesenbck took his first steps towards that goal in 2002 (Neuron

, vol 33, p 15) and by 2003 was closing in fast. He inserted a rat gene into fruit flies that caused them to make a membrane protein called P2X2 in certain brain cells. When P2X2 binds to a molecule called ATP, it makes the neuron fire as if zapped by an electrode. Miesenbck made the P2X2 neurons light-sensitive by injecting the flies with a form of ATP that is only activated by exposure to light of a specific wavelength.

He demonstrated the power of his technique using a strain of flies engineered to make P2X2 in just two of the 125,000 nerve cells in their brains the so-called giant fibre cells, which are known to trigger escape responses. When Miesenbck flashed the correct wavelength of light at the flies, the giant fibre cells fired and, sure enough, the flies jumped and fluttered their wings as though taking flight. Flies with no P2X2 did not respond to the flashes.

When Miesenbck’s results appeared in April 2005 they caused an instant sensation (Cell

, vol 121, p 141). Within an hour of the paper appearing online, Miesenbck’s phone rang: it was the US Defense Advanced Research Projects Agency wanting to know if his work had possible military applications (he now works with them). A few days later, Jay Leno spoofed the work on the Tonight Show , pretending to steer a buzzing fly by remote control into the open mouth of President George W. Bush.

For all its power, Miesenbck’s approach suffered a killer limitation: the need to inject light-sensitive ATP into the animals’ brains. This would complicate efforts to use the technology in humans, but around the time that Miesenbck was making flies jump, another team was already on its way to solving that problem.

Deisseroth, then finishing his medical residency at Stanford, also wanted to find a way to activate neurons selectively. As a practising psychiatrist, he had seen first-hand the dark side of electrode therapy. He had administered electroconvulsive therapy over 200 times and had also treated patients for depression using vagal nerve stimulation. On a weekly basis he saw the shortcomings: patients who were still depressed but couldn’t have their stimulation level increased for fear of side effects. "It’s not very satisfying for someone who wants to engineer precise therapies," says Deisseroth. "So the motivation was enormous."

His opportunity arrived in November 2003 when a team led by Ernst Bamberg at the Max Planck Institute for Biophysics in Frankfurt identified a protein that produces electric pulses in response to blue light. Called channelrhodopsin-2 (ChR2), the protein came from single-celled pond algae. ChR2 allows the algae to detect sunlight, which they need to manufacture sugars. Unlike P2X2, ChR2 is inherently sensitive to light, which meant researchers could probably use it in animals’ brains without having to inject them with a light-sensitive compound first.

Deisseroth contacted the German team and asked to collaborate. They agreed and by September 2005 they had managed to express the ChR2

gene in nerve cells in a Petri dish. Flashes of blue light stimulated the neurons to crank out electric spikes as though they had been zapped by an electrode.

The next piece of the puzzle fell into place when the team came across another light-sensitive membrane protein, called halorhodopsin (NpHR), which had been isolated from a desert-dwelling microbe years before. Crucially, NpHR does the exact opposite of ChR2: when exposed to yellow light, nerve cells containing the protein are effectively paralysed and briefly prevented from firing. With both ChR2 and NpHR in their tool kit, neuroscientists could at last turn specific neurons on and off at will. All they had to do was decide which neurons to target, and then genetically engineer the animal so that those cells manufactured the light-sensitive proteins.

Gottschalk, who collaborated with Deisseroth, was the first to try it. He chose Caenorhabditis elegans as his subject a species of roundworm 1 millimetre long. He engineered the worms to manufacture both ChR2 and NpHR in the muscles along the sides of their bodies and also in the nerves that control those muscles. By flashing a worm with alternating blue and yellow lights, he caused its body to shorten and lengthen in synchrony with the lights the dancing worm (Nature , vol 446, p 633).

"It’s a proof of concept," says Gottschalk. "In principle, any behaviour controlled by neurons could be mimicked by turning exactly the right cells on and off using light."

By turning on neurons that sense touch, Gottschalk has also used the technology to make a swimming worm react as if it has bumped into an obstacle, causing it to change direction. And Andr Fiala at the University of Wrzburg in Germany has trained fly larvae to like some odours and dislike others by switching on neurons in the reward centres and aversion centres of their brains.

Nobody is out to create an army of remote-controlled zombie worms, however. The hope is that by learning to induce behaviours and sensations, the research will shed light on the specific neural circuits that control them.

For example, Karel Svoboda at the Howard Hughes Medical Institute in Ashburn, Virginia, is trying to crack the code that brains use to transmit physical sensations. He inserts ChR2 into neurons in a rat’s barrel cortex a brain area that processes electrical signals or "action potentials" received from the base of the animal’s whiskers. He then makes the rats turn either right or left by making them "feel" sensations through their whiskers. These feelings are created by pulses of light flashed into the barrel cortex through a small hole in the skull from an LED mounted on the rodent’s head.

"We can turn down the light knob and tweak the number of action potentials and really get at the fundamental question of how many action potentials, in what pattern, can be perceived by an animal," says Svoboda.

Mating and fightingIn another set of experiments, Miesenbck is searching for the neurons that control key behaviours in fruit flies such as mating, grooming and fighting. He is using "off-the-shelf" strains of fly that have been engineered to make it easy to insert a foreign gene into a specific subset of their brain cells as little as a few dozen of the full complement of 125,000. Miesenbck has tried several fly strains, engineering them to express P2X2 and testing whether flashing them with light leads to noteworthy behaviours. Results so far have been mixed. "Most of the behaviours that you get are a sort of uncoordinated seizure," says Miesenbck, suggesting that too many neurons were reacting to the light to produce a coordinated response.

This illustrates a major obstacle to using light for studying behaviour: the need to improve genetic engineering so that researchers can target exactly the right neurons with their light-sensitive proteins. That goal is being hotly pursued by Deisseroth, Miesenbck and others.

Meeting this challenge will be key to using the technology to treat brain disorders. The most obvious medical goal would be to restore vision in people blinded by diseases such as retinitis pigmentosa, which kills light-detecting cells in the retina. Zhuo-Hua Pan, a visual neuroscientist at Wayne State University in Detroit, Michigan, has spent several years tackling the problem. Working in a strain of blind mice, Pan inserted the gene for ChR2 into surviving cells in the mice’s retina in the hope of converting them into new light-detecting cells.

Amazingly, it seems to work. In his early experiments, published in 2006, Pan found that once the gene for ChR2 had been inserted, retinal cells responded to light by emitting electric pulses (Neuron , vol 50, p 23). Pan monitored the animals’ brains and confirmed that the light signal was transmitted through the optic nerve to the visual centres in the brain. "The signal goes to the visual cortex," he says. "The only question is how well the brain integrates this signal." Pan plans to carry out behavioural experiments to determine whether the mice are actually gaining some sort of useful vision.

Deisseroth, meanwhile, hopes to use light to treat Parkinson’s disease and depression. His team is preparing to experiment with the ChR2/NpHR combination in rats with a brain disorder analogous to Parkinson’s. They are also gearing up to use it in rats that are considered to be a good model for clinical depression owing to their lethargy, poor sleep, reduced eating and a distinct lack of excitement when offered the finer things in a lab rat’s life, such as sugar water. The goal of these studies will be to determine not only which brain area but also which cell types need to be switched on or off to produce a therapeutic response.

Deisseroth won’t say exactly which brain area he is targeting for depression, but one likely suspect is a sliver of the limbic system called area 25. This is overactive in depressed people and can sometimes be dampened down using antidepressants. In a clinical trial in 2005, stimulating area 25 with an electrode quieted its activity and improved mood in four out of six patients. Deisseroth now wants to work out which cells are involved and target them with light.

Unfortunately, unlike worms and fly larvae, humans and rats have opaque heads, so getting light into their brains presents a challenge. Alex Aravanis, a member of Deisseroth’s team, has created a device that delivers light to the brain using hair-thin optical fibres. As a proof of concept, he used it to regulate the twitching of a rat’s whiskers (Journal of Neural Engineering, vol p S143).

The biggest barrier may not be getting light inside the skull, however, but the need for gene therapy. Whether you’re restoring light sensitivity to a damaged retina or silencing cells in area 25, you first have to get those cells to manufacture a light-sensing protein. This means inserting a foreign gene into the brain. Though clinical trials have dabbled in gene therapy for serious inherited conditions like cystic fibrosis, the technique still hasn’t received approval for routine use. Inserting genes into the brain, of all organs, would require powerful justification.

Exactly how powerful is currently being explored. In a recent trial, 12 patients underwent gene therapy for Parkinson’s disease. The researchers used a modified human cold virus to insert a foreign gene into the neurons of the subthalamic nucleus, aiming to muffle their disordered chitter-chatter. All 12 patients experienced a reduction in their tremors, according to results presented at the October 2006 Society for Neuroscience meeting in Atlanta, Georgia. The work suggests that genes for ChR2 or NpHR could also be delivered deep into the brain, targeted to locations such as the subthalamic nucleus for Parkinson’s disease or area 25 for depression.

Neurologist Helen Mayberg at Emory University in Atlanta, Georgia, who conducted the first clinical trial of deep brain stimulation in depressed patients, is optimistic about Deisseroth’s work. "We have to keep safety in mind, but this could have absolutely unbelievable implications," she says. "Time will tell."

Deisseroth firmly believes that the technology will lead to better therapies in humans. In the short term he remains focused on goals that he believes can be reached through animal studies, such as hunting down the neural bases of airy concepts like fear and motivation. "They probably boil down to the activity of a specific kind of cell in a specific part of the brain," he says. Deisseroth hopes that his tiny spotlights of blue and yellow will one day illuminate those elusive neurons and take mind control into a whole new dimension.

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