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Activating neurons “on the fly”

Scientists have found a way to switch specific neurons on and off with a laser beam. An international research team used this technique to study how the zebrafish brain responds when external stimuli trigger a certain behavior. This work, which has been published in Nature Methods, will be continued at the MDC.

When zebrafish sense they are being swept away by a current, they begin to swim against it. This behavior is innate and can be elicited at will in the laboratory – even if there isn’t an actual current, but only a projected image passing over the aquarium floor. 

Dr. Nikita Vladimirov, the lead author of the study published in the journal Nature Methods, wanted to find out how the swimming response originates in the zebrafish brain and which neurons are involved in this optomotor reflex. This illustrates a fundamental problem in the neurosciences: How does one identify the complete set of neurons that underlie any given animal behavior? “So far we know very little about how the brain transforms external stimuli into certain behavioral patterns,” he says. “I hope my experiments will help change that.”

When the neurons fire, they fluoresce very brightly

In early 2017 Vladimirov joined the Max Delbrück Center for Molecular Medicine (MDC), where he has been a member of the BIMSB research group led by Dr. Stephan Preibisch. While at the Janelia Research Campus of the Howard Hughes Medical Institute (HHMI) in Ashburn, Virginia, Vladimirov developed in Dr. Misha Ahrens’ lab a special microscope with which one can essentially observe the zebrafish brain at work. Zebrafish just a few days old turned out to be the ideal model animal for his studies. They are only three to four millimeters long and nearly translucent, making them particularly suitable for microscopy,” the researcher explains. 

What’s more, the brains of baby zebrafish have only around 100,000 neurons, a reasonably manageable number compared with other animals. The zebrafish used by Vladimirov were also genetically altered to carry a fluorescent protein in their neurons. This protein lights up more brightly when the neurons become active. “This allows us to observe which neurons across the entire brain start firing when the fish perform a certain behavior,” Vladimirov explains.

© Nikita Vladimirov, MDC

When paired with high performance computing, three-dimensional ‘brain-activity maps’ of larval zebrafish allow researchers to monitor changes in animal behavior and brain activity. The nuclei of the fish neurons are marked with GCaMP6f fluorescent protein. Colors indicate neuronal response tuning to swimming, forward stimulus and backward stimulus.

More brain regions become active than previously thought

While working in the United States, Vladimirov built a “Zebrascope” for his experiments. This light-sheet microscope, which is connected to a high-performance computer, enables high-precision imaging of the entire zebrafish brain. “Although the optical stimulus and corresponding response were very simple, the neural activity of the fish was remarkably complex,” reports Vladimirov, adding that numerous, often far-flung, regions of the brain became active when presented with a forward-moving visual stimulus. “And when the fish started swimming, even more regions began to fire,” the researcher explains.

In order to figure out which neurons are responsible for triggering the swimming response, Vladimirov also equipped his microscope with a computerized laser device. Its laser beam is so fine that it allows him to selectively turn off single neurons.

The zebrafish brain is remarkably resilient

“I assumed that the ‘master switch’ for optomotor response would be located in the most active brain regions,” says Vladimirov. To his great surprise he found that wasn’t the case. Although turning off highly active regions of the brain changed the duration and amplitude of the swimming movement, it did not abolish swimming completely. It was also possible to see how neurons in other brain regions began increasingly to fire.

“All of this shows us how little we actually knew about the workings of the brain – even when it comes to such a simple organism as the zebrafish and such simple behavior as swimming,” says Vladimirov. He believes there must be deeper reasons to explain the complexity of the fish nervous system that he and his team discovered, reasons that are still unknown to us. “But at least now,” Vladimirov emphasizes, “we have the tools needed to carry out further research in this direction.”

Nematode worms will be studied next

Vladimirov’s plans at the MDC now include investigating the brain of the tiny nematode worm C. elegans, in collaboration with Stephan Preibisch, who was also involved in the current study. The nematode brain only contains a few hundred neurons. “Nematode worms are capable of surviving bad times – such as foodless periods – in a sort of resting stage,” says Preibisch. With the help of a modified light-sheet microscope, his lab now wants to find out how the nervous system initiates the worm’s further development once the food situation improves.

Further Information

Press release by Janelia Research Campus

Citation

Nikita Vladimirov et al. (2018): “Brain-wide circuit interrogation at the cellular level guided by online analysis of neuronal function.” Nature Methods. doi: 10.1038/s41592-018-0221-x