Bioluminescent sensor, paired with optogenetics, tracks hundreds of neurons


A team of researchers at Vanderbilt University (Nashville, TN) has developed a bioluminescent sensor that causes individual neurons (brain cells) to glow in the dark. The sensor is a genetically modified form of luciferase, the enzyme that a number of species—such as fireflies—use to produce light. When combined with optogenetics (a technique that uses light to control cells in living tissue), scientists could have the ability to track the interactions within large neural networks in the brain.



Related: Optogenetics helps untangle ambiguity in neural circuits

Using electrical techniques to record neural activity are very good at monitoring individual neurons, but are limited to small numbers of them, explains Carl Johnson, Stevenson Professor of Biological Sciences, who led the work.
By contrast, optical techniques could enable scientists to record the activity of hundreds of neurons at the same time, Johnson says. However, most current efforts in optical recording use fluorescence, which requires a strong external light source that can cause the tissue to heat up and can interfere with some biological processes, particularly those that are light-sensitive, he adds.

Based on their research on bioluminescence in the green alga Chlamydomonas, Johnson and his colleagues realized that if they could combine luminescence (which works in the dark) rather than fluorescence with optogenetics, they could create a powerful new tool for studying brain activity.

Johnson and his collaborators genetically modified a type of luciferase obtained from a luminescent species of shrimp so that it would light up when exposed to calcium ions. Then, they hijacked a virus that infects neurons and attached it to their sensor molecule so that the sensors are inserted into the cell interior.



The researchers picked calcium ions because they are involved in neuron activation. Although calcium levels are high in the surrounding area, normally they are very low inside the neurons. However, the internal calcium level spikes briefly when a neuron receives an impulse from one of its neighbors.

They tested their new calcium sensor with one of the optogenetic probes (channelrhodopsin) that causes the calcium ion channels in the neuron's outer membrane to open, flooding the cell with calcium. Using neurons grown in culture, they found that the luminescent enzyme reacted visibly to the influx of calcium produced when the probe was stimulated by brief light flashes of visible light.

To determine how well their sensor works with larger numbers of neurons, they inserted it into brain slices from the mouse hippocampus that contain thousands of neurons. In this case, they flooded the slices with an increased concentration of potassium ions, which causes the cell's ion channels to open. Again, they found that the sensor responded to the variations in calcium concentrations by brightening and dimming.

The next step, Johnson says, is to determine exactly how sensitive their approach is.