A short pulse of ultraviolet light followed by green light triggers burst swimming in a genetically engineered zebrafish larva expressing a light-gated channel in a specific cell type: the KA neuron. This demonstration proves that these cells, whose function had remained unknown for decades, are responsible of inducing spontaneous swimming activity.

UC BERKELEY (US)—A new way to select and switch on one cell type in an organism using light has helped answer a long-standing question about the function of one class of enigmatic nerve cells in the spinal cord.

Researchers at the University of California, Berkeley and University of California, San Francisco say the strategy could be generalized to noninvasively study all types of neurons, such as those in the smell, vision, touch, and hearing centers of the brain.

Using targeted insertion of light-sensitive switches into the nerve cells of awake zebrafish larvae, the scientists found that the cells trigger the periodic tail twitching typical of larvae.

Because mammals have similar cells protruding into the spinal fluid, the finding could have implications for humans, researchers say.

The discovery also highlights the power of new techniques that employ light-gated ion channels called photoswitches and gene targeting to noninvasively turn on small populations of cells as easily as flipping a light switch.

“With these optogenetic tools, we can activate single neurons in awake behaving animals and directly demonstrate the consequence of neuron activation on behavior,” explains Claire Wyart, postdoctoral fellow at UC Berkeley’s department of molecular and cell biology. “This optogenetic approach enabled us to learn something important about spinal circuits.”

“Optogenetics opens up a new and extremely exciting area of study, singling out one type of cell and finding out what it’s doing,” she says.

The approach is a “new way to do neuroscience” says coauthor Herwig Baier, professor of physiology at UCSF. “Instead of sticking electrodes into the brain to record and monitor activity in the nervous system, what we are doing is manipulating the function of neurons noninvasively with light, the gentlest way to make a manipulation.”

“With these optically sensitive channels, it becomes possible to play back to the nervous system its normal innate activity and see what behavior results,” adds coauthor Ehud Isacoff, professor of molecular and cell biology at Berkeley.

Wyart and her colleagues applied the technique to search for a behaviorally relevant cell and found a previously unknown function for the Kolmer-Agduhr (KA) cells in the spinal cord.

The KA cells aren’t standard relay neurons with dendrites and axons, but sensory neurons with cilia, small, movable hairs, that protrude into the spinal fluid, plus long axons extending up the spinal cord. The scientists could see the KA cells were sensing something, but didn’t know exactly what.

They then produced 10 strains of zebrafish with photoswitches inserted in specific spinal cord nerve cell populations. When light was shined on the fish with photoswitches in their KA neurons, the fish waggled their tails in a manner that exactly mirrored spontaneous slow forward swimming.

Placing the transparent zebrafish larvae under a microscope, Wyart used a Digital Micromirror Device (DMD) to strongly focus light onto a small number of KA neurons, successfully switching on only a few KA cells at a time. She found that she had to switch on about 10 of the KA neurons to trigger the swimming motion.

Knocking out these cells greatly reduced burst swimming, but did not eliminate it, suggesting that the KA neurons may be lowering the threshold for triggering reflex swimming.

“It came as a great surprise that these neurons played a role in locomotion at all,” says Isacoff. “There is an apparent homologue of the KA neuron in mammals, so this may be a general modulatory principle for vertebrate locomotion, although it may change from positive drive early in development to negative drive later.”

Wyart continues to explore the role of KA neurons, but hopes to exploit optogenetic and gene targeting techniques to discover the roles of other types of neurons in the spinal cord.

“Optogenetic targeting is a powerful approach, and we have really only started the work,” Baier adds. “We still have to learn how the KA neurons are connected to drive the muscles. Really, there is no way we could have done this experiment other than with optogenetics.”

Researchers from the University of Munich and the University of Queensland in Brisbane, Australia contributed to the study, which appears in a recent issue of Nature, and was supported by the National Institutes of Health.

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