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After color-coding all 302 neurons in the worm C. elegans, scientists have uncovered a vast “wireless” communication network.
Investigator, Columbia University
After color-coding all 302 neurons in the worm C. elegans, scientists have uncovered a vast “wireless” communication network.

Scientists can identify the neurons in this C. elegans worm by their color and position, thanks to a new genetic technique called NeuroPAL that labels each neuron with a colored fluorescent protein. Credit: E. Yemini et al./Cell 2021

Using a technique that lets them paint every neuron in a tiny worm, scientists can now spy on communication among nerve cells in ways never before possible.

Earlier this year, Howard Hughes Medical Institute (HHMI) Investigator Oliver Hobert and his colleagues reported a tool called NeuroPAL that color-codes individual neuronsexternal link, opens in a new tab – allowing identification of all 302 neurons in living Caenorhabditis elegans worms. The team has now used the tool to reveal an extensive wireless communication network operating in the worm’s nervous systemexternal link, opens in a new tab, Hobert and his colleagues report July 7, 2021, in the journal Cell.

Neuroscientists have always dreamed of getting a glimpse of the brain in action, says Hobert, a molecular biologist at Columbia University’s biological sciences department. His team’s “painting” tool is already giving researchers their clearest look yet into a worm’s mind.

“It’s a really marvelous piece of work,” says Erik Jorgensen, a neuroscientist and HHMI Investigator at the University of Utah, who was not involved in the study. “We don’t understand connectivity. We don’t understand how a signal passes through a nervous system.” But by studying the relatively simple nervous system of C. elegans, he says, we can learn much more about brain function.

In the future, Jorgensen says, tools such as NeuroPAL will help scientists understand brain activity in more complex organisms, likely starting with fruit flies but ultimately in mammals such as mice.

A new color palette

Scientists from HHMI and other institutes have been using fluorescent compounds to see neurons in living animals for years – yet this gave an incomplete picture of brain activity. Such techniques can distinguish between neighboring neurons but can’t identify different types of cells to see which one is doing what. It’s like hearing 100 people talking at a party, Hobert says. “You hear every word that is spoken, but you don’t know who’s saying the words and who is listening.”

With the new technique, scientists can now see each neuron in genetically modified worms and also identify what type of neuron each one is. Hobert’s team created a transgene called NeuroPAL (Neuronal Polychromatic Atlas of Landmarks) that expresses different combinations of fluorescent proteins in different neuron types. By engineering this transgene into a worm’s nervous system, the scientists can identify individual neurons based on their color and position within the worm’s body, the team reported on January 7, 2021, in the journal Cell.

This feat had eluded neuroscientists until now. “A lot of people didn’t believe it was possible,” says Eviatar Yemini, a computational neurobiologist on the Columbia team. To distinguish nearby neurons from one another, the team needed enough colors to label every neuron in a nerve cluster.

But Yemini’s color palette was limited by the number of fluorescent proteins that could be incorporated into a live worm’s genome at once without irreparably harming the worm. Color-coding each neuron requires two main components, he explains: fluorescent proteins and DNA instructions for where the proteins should be built. Those instuctions tell some neurons to make blue proteins, for example, and others to make red ones.

Yemini tested more than 130 different instruction sets and 15 different fluorescent proteins. He discovered a combo that could be safely inserted into a living worm and used to determine each neuron’s identity. That let Yemini create a complete, full-color atlas of the worm nervous system and use colorized worms in experiments. And a molecular sensor called GCaMP6 which was developed at HHMI’s Janelia Research Campus, let him measure each neuron’s activity.

Listening to neurons’ chatter

With the new tool in hand, the team wanted to track communication within the worm’s nervous system. Neurons can communicate in different ways, akin to speaking in different languages. Neuroscientists have focused largely on mapping the physical connections, or wiring, of the brain. This network of connected neurons uses five main chemicals, called neurotransmitters, to send messages from cell to cell, like links in a chain.

Flashing white circles show the activity of neurons in the head of a C. elegans worm whose neurons have been labeled in a rainbow of colors. Scientists can use these colors to identify neuron types. Video has been sped up 12-fold. Credit: E. Yemini et al./Cell 2021

But neurons can also broadcast molecular messages to unconnected cells, and these messages have been much more difficult to trace. Teaming with other colleagues, Hobert and Yemini aimed to eavesdrop on part of this communication system. Using NeuroPAL to visualize the entire C. elegans nervous system, they also monitored gene activity in the nerve cells, looking for signs that these types of messages are being sent and received.

The team mapped gene expression to individual nerve cells, which revealed a vast wireless communication network separate from the physical wiring of the worm’s nervous system. Worm neurons communicate by sending and receiving dozens of chemical signals – neurotransmitters and neuropeptides. But the extent of such communication between non-neighboring neurons, especially messages involving neuropeptides, far exceeded what was known, the team discovered. Those results indicate that wireless communication likely plays a major role in nervous-system communications.

“We were living in a world of five languages” based on the five major neurotransmitters, says Hobert. “Now we know that every neuron is multilingual.” The extent to which wireless communication is important in animals other than C. elegans remains to be determined.

The team is making its software and methods freely availableexternal link, opens in a new tab so that other researchers can use them in their own experiments, whether in worms or in other animals. “It’s brilliant,” says Jorgensen, who had been working on a similar technique but now plans to adopt NeuroPAL in his own research. “You know,” he says, “getting scooped by somebody who does it better than you is wonderful.”



Eviatar Yemini et al. “NeuroPAL: A Multicolor Atlas for Whole-Brain Neuronal Identification in C. elegansexternal link, opens in a new tab.” Cell. January 7, 2021. doi: 10.1016/j.cell.2020.12.012

Seth R. Taylor et al. “Molecular topography of an entire nervous systemexternal link, opens in a new tab.” Cell. Published online  July 7, 2021. doi: 10.1101/2020.12.15.422897