In his search for research experience to help him decide on a graduate school path, Ben Fosque became captivated by the idea of visualizing the activity of neurons with fluorescence. To understand how the brain works, scientists must be able to study neural activity on all scales – from a single neuron to large, complicated networks. Techniques have been engineered to help scientists see this activity, but they have not been able to mark large populations of active neurons at specific moments in time.
After receiving his undergraduate degree from MIT, Fosque, now a first year graduate student in the Department of Biochemistry and Molecular Biology, found an opportunity to work on a project focused on improving these technologies. Under the guidance of Eric Schreiter, PhD, Doug Kim, PhD and Loren Looger, PhD at the Howard Hughes Medical Institute’s (HHMI) Janelia Research Campus, Fosque worked on developing a new tool that would allow scientists to permanently mark all the neurons that are active at a particular time in live animals with a flash of light – a literal snapshot of neural activity.
In a paper published Feb 13 in Science, they described the results of their work – a fluorescent protein called CaMPARI.
“The ability to take a high resolution ‘snapshot’ of neuronal activity across several hundred thousand neurons is a new and potentially transformative development for neuronal imaging,” said Fosque, co-first author on the study. “We imagine it could find broad application in correlating behavior with neural activity with single-cell resolution.”
Fosque and his mentors built upon two traditional methods: genetically encoded calcium indicators, which can only tag active neurons in a limited field of view, and immediate early genes, which extend the field of view but only over long time scales.
Their new protein sensor, CaMPARI – which stands for calcium-modulated photoactivatable ratiometric integrator – switches from green to red when the concentration of calcium inside a neuron changes, indicating its activation. But only when the protein is illuminated with violet light, giving experimenters precise control over the time period during which neural activity is tracked.
The team spent more than a year tweaking their protein – making it brighter and more responsive to calcium and ensuring that it would work in cells and then in living mice, fruit flies and zebrafish. By applying pulses of violet light to zebrafish larvae tagged with CaMPARI, for example, the researchers were able to generate neuronal activity snapshots in large volumes of brain tissue. They took these snapshots while the zebrafish larvae were hot, cold, and placed in turbulent water, comparing the animals’ differing neuronal activity under each condition.
Video: neurons labeled by CaMPARI are shown in purple.
In fruit flies, the team used the tool to identify neurons that were activated in response to specific odors. CaMPARI indicated that different odors activated distinct sets of neurons in the flies’ antennal lobes. In a subsequent set of experiments, the researchers experimentally activated the neurons that directly responded to the odors, then looked for neurons elsewhere in the brain that subsequently turned red.
The technique could eventually help researchers understand the brain circuitry involved in more complex behaviors. “If you were interested in studying decision making processes, for example, you could point the fiber light source at a part of the brain thought to be involved in decisions, and then trigger it when the subject is at a decision point between two paths in a maze,” Fosque said. “Our tool would permanently label all neurons that were active precisely at that moment.”
With ongoing development, the scientists expect future versions of CaMPARI will be more sensitive and reliable than the current tool. But it is important to get CaMPARI into the hands of neuroscientists right away, according to the authors. “The idea is probably more powerful than the tool, as it stands right now,” said Looger, study co-author and group leader at HHMI. “We will definitely benefit from a couple hundred—hopefully a thousand—labs taking CaMPARI and seeing what they can do with it.”