Princeton University researchers have captured some of the first near-whole-brain recordings of 3-D neural activity of a free-moving animal, and at single-neuron resolution. They studied the nematode Caenorhabditis elegans, a worm species 1 millimeter long with a nervous system containing just 302 neurons.

The three-dimensional recordings could provide scientists with a better understanding of how neurons coordinate action and perception in animals.

As the researchers report in the journal Proceedings of the National Academy of Sciences, their technique allowed them to record the activity of 77 neurons from the animal’s nervous system, focusing on specific behaviors such as backward or forward motion and turning.



Andrew Leifer/Lewis-Sigler Institute for Integrative Genomics | This video — displayed in quarter-time — shows the four simultaneous video feeds the Princeton researchers used to capture the nematodes’ neural activity. Upper left: the position of the nuclei in all the neurons in an animal’s brain. Upper right: recorded neural activity, indicated by a fluorescent calcium indicator. Lower left: the animal’s posture on the microscope plate, which automatically adjusted to keep the animal within the cameras’ view. Bottom right: a low-magnification fluorescent image of a nematode brain, which contains 302 neurons.

Most previous research on brain activity has focused on small subregions of the brain or is based on observations of organisms that are unconscious or somehow limited in mobility, explained corresponding author Andrew Leifer, an associate research scholar in Princeton’s Lewis-Sigler Institute for Integrative Genomics.

“This system is exciting because it provides the most detailed picture yet of brain-wide neural activity with single-neuron resolution in the brain of an animal that is free to move around,” Leifer said. “Neuroscience is at the beginning of a transition towards larger-scale recordings of neural activity and towards studying animals under more natural conditions,” he said. “This work helps push the field forward on both fronts.”

A current focus in neuroscience is understanding how networks of neurons coordinate to produce behavior. “The technology to record from numerous neurons as an animal goes about its normal activities, however, has been slow to develop,” Leifer said.



Andrew Leifer, Lewis-Sigler Institute for Integrative Genomics | Nematode neural nuclei in 3-D, showing the location of brain-cell nuclei in a nematode’s head.

The simpler nervous system of C. elegans provided the researchers with a more manageable testing ground, but could also reveal information about how neurons work together, which applies to more complex organisms, Leifer said. For instance, the researchers were surprised by the number of neurons involved in the seemingly simple act of turning around.

“One reason we were successful was that we chose to work with a very simple organism,” Leifer said. “It would be immensely more difficult to perform whole-brain recordings in humans. The technology needed to perform similar recordings in humans is many years away. By studying how the brain works in a simple animal like the worm, however, we hope to gain insights into how collections of neurons work that are universal for all brains, even humans.”

The researchers designed an instrument that captures calcium levels in brain cells as they communicate with one another. The level of calcium in each brain cell tells the researchers how active that cell is in its communication with other cells in the nervous system. They induced the nemotodes’ brain cells to generate a protein known as a “calcium indicator” that becomes fluorescent when it comes in contact with calcium.

The researchers used a special type of microscope to record the nematodes’ free movements and also neuron-level calcium activity for more than four minutes and in 3-D. Special software the researchers designed monitored the position of an animal’s head in real time as a motorized platform automatically adjusted to keep the animal within the field of view of a series of cameras.



Andrew Leifer, Lewis-Sigler Institute for Integrative Genomics | A visualization of neural activity in the nematode brain. Upper-left: Each colored sphere represents a neuron, and its location in the drawing shows the position of that neuron in the worm’s head. Upper-right: The size and color of a sphere indicates the level of neural activity (purple spheres: the least amount of activity; large yellow spheres: most significant). By watching neurons that grow and shrink, the viewer can get an impression of the range of neural activity in the worm. Lower left and right panels: The worm’s movement in real time and the worm’s location plotted on a graph.

Leifer said these recordings are very large and the researchers have only begun the process of carefully mining all of the data.

“An exciting next step is to use correlations in our recordings to build mathematical and computer models of how the brain functions,” he said. “We can use these models to generate hypotheses about how neural activity generates behavior. We plan to then test these hypotheses, for example, by stimulating specific neurons in an organism and observing the resulting behavior.”

Abstract of Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal’s position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal’s position and orientation. Custom software tracks the 3D position of the animal’s head in real time and two feedback loops adjust a motorized stage and objective to keep the animal’s head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal’s behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.