Red fluorescent protein helps view live brain function

Researchers have identified a red fluorescent protein that will make it possible for live neurons to glow red when activated.

Red voltage indicator VARNAM expressed in the motor cortex of a mouse brain; the inset shows a magnified view of a cortical pyramidal neuron showing VARNAM expression at the membrane.
Red voltage indicator VARNAM expressed in the motor cortex of a mouse brain; the inset shows a magnified view of a cortical pyramidal neuron showing VARNAM expression at the membrane.
Image by Madhuvanthi Kannan and Ganesh Vasan; composite by Michael Helfenbein

Researchers at the John B. Pierce Laboratory and the Yale School of Medicine (New Haven, CT), together with collaborators at Stanford University (Stanford, CA), have identified a red fluorescent protein that will make it possible for live neurons to glow red when activated.

The Voltage-Activated Red Neuronal Activity Monitor (VARNAM; which means "hue" in Sanskrit) is part of a family of proteins called genetically encoded voltage indicators (GEVIs), which change their fluorescence intensity when an electrical impulse shoots through a live neuron.

"While green fluorescent proteins have been worked on for over 50-60 years and tailored for a plethora of applications, red fluorescent proteins are newer, fewer, and notorious for folding poorly inside living cells," explains Madhuvanthi Kannan, co-first author and associate research scientist. "We went through several different red fluorescent proteins to find one with optimal photophysical properties that could express and perform well in many different living systems."

Ganesh Vasan, co-first author and associate research scientist, says that the automated screening approach he established allowed them to identify VARNAM. The high-throughput approach made it possible to screen thousands of potential red fluorescent proteins and select the most voltage-sensitive among them—the brightest and fastest to appear when a neuron is activated—in an efficient manner.

Previously, red fluorescent indicators had been too slow or too dull to be useful for researchers who are observing the functioning brain. Additionally, when experimenters wanted to watch two different groups or types of cells in action, they could not tag them with more than one color GEVI at the same time. The team not only demonstrated that VARNAM works in multiple systems, both in vitro and in vivo, but also used VARNAM to capture brain activity spikes in live flies in two hues—green and red. Now, scientists will be able to monitor the full read-write process of live neurons accurately and easily, the researchers say.

"The development of this probe represents another triumph in the field of directed evolution, honored this year with the Nobel Prize for Chemistry," says senior author Vincent Pieribone, professor of cellular and molecular physiology and of neuroscience at Yale. "VARNAM represents a significant advance in our ability to directly monitor the electrical activity of brain cells in a less invasive and more comprehensive manner. This opens the door to ultimately understanding how complex nervous systems produce the elaborate behaviors that are the hallmark of advanced human and animal life."

Full details of the work appear in the journal Nature Methods.

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