Whereas existing tagging techniques require hours to activate, the FLiCRE tagging process takes just minutes. The researchers also designed FLiCRE so that they can use standard genetic sequencing to find the cells in which FLiCRE activated. This allows them to study tens of thousands of cells at once, while other techniques tend to require the analysis of multiple microscopic images that each contain hundreds of cells.

In one series of experiments, the researchers injected FLiCRE into cells in the nucleus accumbens and used an opsin to activate a neural pathway associated with avoidance behavior in the mice. Once the calcium in FLiCRE-containing cells spiked – the cellular indication that the mouse is avoiding something – the cells glowed a permanent red that was visible through a microscope. The researchers also sequenced the RNA of the cells to see which ones contained the fluorescent protein, producing a cell-by-cell record of neural activity.

“One goal was to map how brain regions are connected to each other in living animals, which is a really hard problem,” said Christina Kim, a postdoctoral scholar in genetics at Stanford and co-lead author of the paper. “The beauty of FLiCRE is that we can pulse and activate neurons in one region and then record all of the connected downstream neurons. It is a really cool way to look at long-range brain activity connections.”

In the next experiments, the researchers used the cellular activity map from the first experiments. They also adjusted FLiCRE so that the protein expressed the opsin protein, which can be controlled by orange light to alter neuronal activity. After activating FLiCRE in the cells, the researchers sent orange light through the fiber optic implant whenever the mice would enter a certain room. In response, the mice steered clear of that room, indicating that FLiCRE had indeed located cells in the brain that drive avoidant behavior.

A dream project

The development and testing of FLiCRE combined chemistry, genetics, biology and neuroscience, and many specialties within those disciplines. As a result, the tool has a wide range of possible applications, including in cells outside the brain, the researchers say.

“I moved to Stanford in 2016 with the hope of being able to carry out extremely interdisciplinary and collaborative projects such as this,” said Ting. “This project has been one of the most rewarding aspects of my move to Stanford – seeing something this challenging and ambitious actually work out.”

The researchers are now working on additional versions of FLiCRE, with a goal of streamlining the process. They are hoping to simplify its structure and also make it capable of working with other biochemical events, such as protein interactions or neurotransmitter release.

Mateo Sánchez, a former postdoctoral scholar in the Ting lab, is also co-lead author of the paper. Additional authors are Paul Hoerbelt, Lief E. Fenno and Robert Malenka, the Pritzker Professor of Psychiatry and Behavioral Sciences, Director of the Nancy Pritzker Laboratory and Deputy Director of the Wu Tsai Neurosciences Institute. Deisseroth, is the D. H. Chen Professor, and a professor of bioengineering and of psychiatry and behavioral sciences; and a member of Stanford Bio-X and the Wu Tsai Neurosciences Institute. Ting is a member of Stanford Bio-X, the Maternal & Child Health Research Institute (MCHRI), the Stanford Cancer Institute and the Wu Tsai Neurosciences Institute, and a faculty fellow of Stanford ChEM-H.

This research was funded by the Walter V. and Idun Berry Postdoctoral Fellowship Program, the EMBO long-term postdoctoral fellowship, the National Institute of Mental Health, Stanford Psychiatry, the Wu Tsai Neurosciences Institute, the National Institute on Drug Abuse, the Defense Advanced Research Projects Agency Neuro-FAST program, the NOMIS Foundation, the Wiegers Family Fund, the Nancy and James Grosfeld Foundation, the H. L. Snyder Medical Foundation, the Samuel and Betsy Reeves Fund, the Gatsby Foundation, the AE Foundation, the Fresenius Foundation and the Chan Zuckerberg Biohub.

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