It’s cool and dark, and a multitude of lights twinkle and dim. Fireflies in a meadow? No, neurons in a petri dish. In this case, the neurons are aglow thanks to a new, genetically encoded calcium sensor. Unlike other sensors that require a strong external light source in order to fluoresce, the new sensor is bioluminescent. So it not only stays cool, it also does so without the sort of light stimulation that can interfere with optogenetic probes, activating them at inopportune times and disturbing neuroscientific investigation. Such light stimulation can even heat tissue samples or trigger autofluorescence.
The new sensor was devised by a team of Vanderbilt University researchers led by Carl Johnson, Ph.D., a professor of biological sciences. The team published its results October 27 in the journal Nature Communications, in an article entitled, “Coupling Optogenetic Stimulation with NanoLuc-Based Luminescence (BRET) Ca++ Sensing.”
According to the article, the new bioluminescent probe is part of a new and improved method for recording the activity of neurons. “For a long time, neuroscientists relied on electrical techniques for recording the activity of neurons,” noted Dr. Johnson. “These are very good at monitoring individual neurons but are limited to small numbers of neurons. The new wave is to use optical techniques to record the activity of hundreds of neurons at the same time.”
Ideally, optical sensors would disturb tissue samples little or not at all, while being compatible with other neuroscientific tools such as optogenetic probes. Accordingly, optical sensors may work better if they rely on luminescence rather than fluorescence.
“Using a new bright luciferase, we here develop a genetically encoded Ca++ sensor that is ratiometric by virtue of bioluminescence resonance energy transfer (BRET),” wrote the authors of the Nature Communications article. “This sensor has a large dynamic range and partners optimally with optogenetic probes. Ca++ fluxes that are elicited by brief pulses of light to cultured cells expressing melanopsin and to neurons-expressing channelrhodopsin are quantified and imaged with the BRET Ca++ sensor in darkness, thereby avoiding undesirable consequences of fluorescence irradiation.”
Dr. 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.
Calcium ions were selected 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.
The Vanderbilt team tested the 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, the Vanderbilt researchers 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.
“We've shown that the approach works,” Dr. Johnson said. “Now we have to determine how sensitive it is. We have some indications that it is sensitive enough to detect the firing of individual neurons, but we have to run more tests to determine if it actually has this capability.”