A fluorescence microscopy technique has yielded 3D footage of brain activity in unrestrained worms, offering insight into how populations of neurons generate animal behavior. Researchers from Princeton University developed an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of C. elegans, a species of worm also known as nematodes, while simultaneously recording the animal’s position, posture and locomotion. Nematodes are 1 mm long with a nervous system containing just 302 neurons. The team found that, across different specimens, multiple neurons showed significant correlations with modes of behavior corresponding to forward, backward and turning locomotion. A visualization of neural activity in the nematode brain (upper left panel). Each colored sphere represents a neuron, and its location in the drawing above shows the position of that neuron in the worm's head. The size and color of a sphere (upper right) indicates the level of neural activity. The worm's movement is shown in real time (lower left) and plotted on a graph (lower right). Courtesy of Andrew Leifer/Lewis-Sigler Institute for Integrative Genomics. A spinning-disk confocal microscope captured 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes per second. The setup included a suite of three cameras to monitor neuronal fluorescence, as well as the worm's position and orientation. Custom software tracked the 3D position of the worm's head in real time, and two feedback loops adjusted a motorized stage and objective to keep the head within the field of view as the worm roamed freely. The researchers observed calcium transients from up to 77 neurons for over four minutes and correlated this activity with the worm's behavior. "Neuroscience is at the beginning of a transition towards larger-scale recordings of neural activity and towards studying animals under more natural conditions," said Princeton fellow and lecturer Andrew Leifer, who led the work. "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, Leifer said. The technology to observe numerous neurons as an animal performs normal activities, however, has been slow to develop because neural networks are infinitesimal arrangements of chemical signals and electrical impulses that can include, as in humans, billions of cells. The simpler nervous system of the nematodes provided the researchers with a manageable testing ground for their instrument. It also could reveal information about how neurons work together that applies to more complex organisms, the researchers said. For instance, they were surprised by the number of nematode neurons involved in the seemingly simple act of turning around. Results of the study were published in Proceedings of the National Academy of Sciences (doi: 10.1073/pnas.1507110112).