Literal Window into the Brain Shows How to Induce Illusions in Mice

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Stanford scientists, using direct brain stimulation, reproduced both the brain dynamics and the behavioral response of mice taught to discriminate between two different images. [Steve Fisch]

Research in mice by neuroscientists at Stanford University School of Medicine has found that stimulating a remarkably few neurons can trigger the perception of something that isn’t really there—a hallucination, effectively. The findings, reported in Science, beg the question not so much why some people do occasionally experience hallucinations, but why more of us don’t hallucinate more of the time.

The researchers used a technique called optogenetics to stimulate individual neurons in the visual cortex of mice, and induce an illusory image. They were surprised that stimulating just 20 or sometimes fewer neurons was enough to generate a perception that caused the animals to behave in a predicted way.

“Back in 2012, we had described the ability to control the activity of individually selected neurons in an awake, alert animal,” said research head Karl Deisseroth, MD, PhD, professor of bioengineering and of psychiatry and behavioral sciences. “Now, for the first time, we’ve been able to advance this capability to control multiple individually specified cells at once, and make an animal perceive something specific that in fact is not really there—and behave accordingly.” Deisseroth, who is a Howard Hughes Medical Institute investigator and holds the D. H. Chen professorship, is the study’s senior author. Lead authors include James Marshel, PhD, and Sean Quirin, PhD, together with graduate student Yoon Seok Kim and postdoctoral scholar Timothy Machado, PhD.

Their published paper, titled, “Cortical layer-specific critical dynamics triggering perception,” provides new insights that could lead to a better understanding of how the brain processes information, both in healthy people and in individuals with psychiatric disorders such as schizophrenia, and other neuropsychiatric symptoms that involve hallucinations or delusions. The study results also hint at the possibility of designing neural prosthetic devices with single-cell resolution.

Visual perception in mammals is correlated with neural circuitry in the visual cortex region of the brain. In both mice and humans, the visual cortex is responsible for processing information relayed from the retina. What scientists don’t know is why some activity patterns result in “perceptual experiences”, and others do not, the authors wrote. “While visual cortex could play a causal role initiating percepts, it has not been technologically possible to causally test the precise influence on perceptually-driven behavior of groups of individually-specified cells—either stimulated sequentially or as synchronously-activated multi-neuron ensembles distributed across anatomical layers or volumes.”

Deisseroth is a pioneer of optogenetics, a technology that uses pulses of light to stimulate individual neurons. For their studies in mice, the authors were able to use the technology to stimulate neurons in freely moving animals, and observe the effects on their brain function and behavior. Deisseroth and his colleagues inserted a combination of two genes into large numbers of neurons in the visual cortex of lab mice. One gene encoded a light-sensitive protein that caused the neuron to fire in response to a pulse of laser light in the infrared spectrum. The other gene encoded a fluorescent protein that glowed green whenever the neuron was active.

The scientists created cranial windows in the animals’ skulls, exposing part of visual cortex, and protected these windows with glass. They then used a device developed for the study to project holograms—3D configurations of targeted photos—onto and into the visual cortex. These photons landed at precise spots along specific neurons. The researchers were then able to monitor the resulting activity of nearly all of the several thousand individual neurons in two distinct layers of the cerebral cortex spanning about 1 mm2.

To carry out the tests the mice were placed with their heads fixed in comfortable positions, and were shown random series of horizontal and vertical bars displayed on a screen. The researchers observed and recorded which neurons in the exposed part of the visual cortex were activated by one or other orientation of the bar projections. From these results, the scientists were able to identify dispersed populations of individual neurons that were responsive to either horizontal or vertical visual displays.

The investigators then effectively played back these recordings in the form of holograms that generated spots of infrared light on just those neurons that were tuned to the horizontal, or to the vertical bars. The resulting downstream neuronal activity, even at locations relatively distant to the stimulated neurons, was similar to that observed when the natural stimulus of a black horizontal or vertical bar on a white background was displayed on the screen.

The mice were trained to lick the end of a nearby tube for water when they saw a vertical bar, but not when they saw a horizontal bar or neither bar. Over the course of several days, as the animals’ became better at discriminating between horizontal and vertical bars, the scientists gradually reduced the black-white contrast to make the task increasingly more difficult. They found that the animals’ performance perked up if the visual displays were supplemented using simultaneous optogenetic stimulation.

So, if an animal’s performance started to deteriorate as a result of reduced contrast, its ability to discriminate between the visual cues could be boosted by stimulating neurons previously identified as preferentially disposed to fire in response to the horizontal or vertical bar being visually displayed. The improvements only occurred when the optogenetic stimulation was consistent with the visual stimulation, so a vertical bar visual display plus stimulation of neurons that had previously been identified as likely to fire in response to a vertically oriented projection.

The researchers then confirmed that they could remove the visual display completely. Optogenetic stimulation of just small numbers of the vertical bar-responsive neurons was enough to get mice to exhibit licking behavior. The mice wouldn’t lick the tube if the pattern for stimulating “horizontal bar” neurons was projected instead. “Not only is the animal doing the same thing, but the brain is, too,” Deisseroth said. “So we know we’re either recreating the natural perception or creating something a whole lot like it.”

Surprisingly, optogenetic stimulation of only about 20 neurons—or sometimes even fewer— that were responsive to the right orientation was all that was needed to produce the same neuronal activity and animal behavior that was triggered by a visual display. “It’s quite remarkable how few neurons you need to specifically stimulate in an animal to generate a perception,” Deisseroth said. “A mouse brain has millions of neurons; a human brain has many billions. If just 20 or so can create a perception, then why are we not hallucinating all the time, due to spurious random activity? Our study shows that the mammalian cortex is somehow poised to be responsive to an amazingly low number of cells without causing spurious perceptions in response to noise.”

“Here we have developed and applied tools suitable for fast optogenetic control over ensembles of many neurons spanning large volumes of cortex during visually-guided behavior in mice, finding that natural dynamics and associated behavior can be elicited by optogenetic recruitment of a critical number of individually defined percept-specific neurons,” the authors concluded. “Studying specific sensory experiences with ensemble stimulation under different conditions may help advance development of therapeutic strategies, for neural prosthetics as well as for neuropsychiatric symptoms such as those involving hallucinations or delusions. More broadly, the ability to track and control large cellular-resolution ensembles over time during learning, and to selectively link new cells and ensembles together into behaviorally relevant circuitry, may have important implications for studying and leveraging plasticity underlying learning and memory in health and disease.

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