Mapping the brain with light
Light receptors in the brains of transgenic mice can reveal neuron interactions
David L. Shenkenberg
Like listening to people talk in a crowded restaurant, it can be difficult to determine which neurons are communicating with each other, and it can be even harder to understand what they are saying. However, researchers recently have made sense of the noise, thanks to genetically modified mice that have light receptors in their brains.
The scientists — from Duke University in Durham, N.C., from the University of Tokyo, and from Stanford University in Palo Alto, Calif. — used the receptors to map the communications of neurons.
Because many neurological and psychiatric disorders result from too much or too little synaptic activity, the light receptors someday may be used to treat those disorders, said principal investigator George J. Augustine. For example, the receptors could be delivered to a patient’s brain, and shining a light on the receptors could turn these synaptic connections on or off. “It’s an optical version of deep-brain stimulation,” he said. He also noted that the mice could be used to screen for drugs that target synapses.
Through genetics, the light receptors can be strongly or weakly expressed in neurons of the transgenic mice, and they can be expressed in specific or broad areas. Because they are light-activated, they can be excited across an area or in discrete spots.
The light receptors are channelrhodopsin-2 proteins, which are part of the photosynthesis machinery of a type of green algae, Chlamydomonas reinhardtii. Once stimulated with light, the channel proteins allow ions to enter the cell. In the brain, this can trigger a measurable electrical signal.
Because the scientists did not know which neurons would express channelrhodopsin-2, the light receptor was expressed with yellow fluorescent protein. To identify neurons expressing the receptor, they excited brain slices with 465- to 495-nm light and examined the resulting fluorescence with a Nikon epifluorescence microscope and a progressive-scan CCD camera from Photometrics of Tucson, Ariz. (Figure 1).
Figure 1. Researchers used light receptors to map the function of neurons. As shown above, they expressed the light receptors with yellow fluorescent protein to locate where they were being expressed. Images reprinted with permission from PNAS.
In the neurons that expressed channelrhodopsin-2, the researchers first studied the relationship of photostimulation to neuronal electrical output, and they subsequently performed optical mapping. They performed these experiments by focusing a laser on the cerebral cortex of the mice and by measuring the resulting electrical activity with a patch-clamp amplifier.
The samples were excited at either 488 or 458 nm with a Coherent argon-ion laser. To view the samples, the researchers employed an Olympus laser-scanning microscope equipped with a water-immersion objective. Because the microscope is computer-controlled, they could make a pattern of light stimulation, avoiding excitation or scattering in unwanted regions. The pattern they made ensured an interpixel distance of >270 μm with a 100-ms time interval.
Stimulating with light
The researchers found that photostimulation induced an electrical current in the brains of transgenic mice but not in those of wild-type mice. Thus, optical excitation of the light receptors caused a current, and nothing else did. The scientists also observed the largest responses when the light spot was near the cell body, a fact that they attributed to the larger surface area of that portion of the neuron.
The investigators discovered that the amplitude of the current increased with the brightness of the laser and that the currents quickly reached a peak and subsequently disappeared during prolonged illumination. These currents were sufficient to generate action potentials, which varied in proportion to light intensity (Figure 2 left). These results correspond with a classical equation used in photosynthesis studies (the Hill equation), convenient when using these receptors for optical mapping and not surprising given the origin of channelrhodopsin-2.
Figure 2. Focusing a laser on neurons of mouse brain slices triggered action potentials (left). Researchers created this optical map of a brain slice from a transgenic mouse. It shows where neuron interactions occur (right).
During optical mapping, the researchers stimulated neurons expressing channelrhodopsin-2 while monitoring electrical responses in neurons that did not express the channel protein. Importantly, their system detected inhibitory interactions as well as excitatory interactions. They created 2-D maps of neuron interactions and found that the spatial distribution of these interactions varied greatly between various types of neurons (Figure 2 right). The scientists reported this work in the May 8 issue of PNAS.
Now the function of individual neurons in specific regions can be determined, said principal investigator Guoping Feng. He and Augustine said that determining the pattern and frequency of stimulation is important because those parameters greatly influence the brain’s responses.
In addition to demonstrating the efficacy of the mice as a research tool, Augustine said that the maps have intrinsic value because they have better resolution than maps generated by other techniques, and because they show what is happening in the microcircuitry of the brain.
The researchers plan to make mice that express channelrhodopsin-2 in different types of neurons and to explore how to turn off the cells, Augustine said. Feng noted that they also may modify the technique to study neurons below the cortex.
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