Researchers at the Howard Hughes Medical Institute Janelia Research Campus have developed a new tool to permanently demarcate neurons that are active at a particular time in their development. The researchers designed a fluorescent protein, called CaMPARI (calcium-modulated photoactivatable ratiometric integrator), which changes from green to red as calcium enters the neuronal tissue as the cells fire, thus allowing scientists to observe cellular activity in real-time.  

Results from this study were published today in Science through an article entitled “Labeling of active neural circuits in vivo with designed calcium integrators”.

Calcium-sensitive fluorescent markers have been a staple of scientists studying neuronal firing for a number of years, however the signals are fleeting, and useful images can be lost if researchers happen to miss the appropriate field of view through the microscope. CaMPARI allows researchers to visualize neuronal activity in large cross-sections of brain tissue. 

“The most enabling thing about this technology may be that you don't have to have your organism under a microscope during your experiment,” said Loren Looger, Ph.D., group leader at Janelia and co-author on the study. “So we can now visualize neural activity in fly larvae crawling on a plate or fish swimming in a dish.”

In order to make CaMPARI, the Janelia team began with a photoactivatable fluorescent protein called Eos, which emits green fluorescence until it is exposed to light in the violet wavelength spectrum. The violet light causes Eos to permanently change its protein configuration converting its fluorescence to red.  

“That was the perfect starting place,” says Eric Schreiter, Ph.D., senior scientist at Janelia and senior author on the study.  “That conversion from green to red gives us a permanent signal. So we just needed a way to couple that conversion to the activity that's going on in the cell.” Dr. Schreiter and the team accomplished this by incorporating the calcium-sensitive protein calmodulin into the structure of Eos. This allows the color change of CaMPARI to be dependent on the flood of calcium that accompanies neural activity. 

The researchers screened tens of thousands of potential candidate before they found the one that behaved in a manner that was physiologically relevant. Then they spent a year tweaking the protein for increased brightness and responsiveness to calcium, in order to insure that it would be applicable to cells from an array of species, such as fruit flies, zebrafish, and mice.

“Ideally, we can flip the light switch on while an animal is doing the behavior that we care about, then flip the switch off as soon as the animal stops doing the behavior,” explained Dr. Schreiter. “Then we're capturing a snapshot of only the activity that occurs while the animal is doing that behavior.”
Dr. Schreiter and the team were able to demonstrate CaMPARI’s effectiveness in a series of experiments using live zebrafish. They captured the neuronal activity of the entire brain during a ten-second period where the fish swam within a dish.  

Dr. Looger believes that in the near future they will be able to make CaMPARI more sensitive and even more reliable than the current version. He feels it is important to allow other scientists to experiment now with the tool they have developed. “The idea is probably more powerful than the tool, as it stands right now. We will definitely benefit from a couple hundred—hopefully a thousand—labs taking CaMPARI and seeing what they can do with it,” Dr. Looger concluded.