To understand the full relationship between brain activity and behavior, scientists need a way to map this relationship for all of the neurons across a whole brain, something that has so far remained an unsolved challenge. Researchers at the Picower Institute for Learning and Memory at MIT have now developed technologies that can record high-fidelity brain wide activity in the model organism Caenorhabditis elegans, and devised a mathematical model to help interpret how each neuron in the tiny worm encodes behavior.

Applying that model specifically to each cell, the team produced an atlas of how most of the brain cells, and the circuits they take part in, encode the animal’s essential behaviors, such as movement and feeding. The resulting atlas effectively outlines the underlying “logic” of how the worm’s brain produces a sophisticated and flexible repertoire of behaviors, even as its environmental circumstances change.

“This study provides a global map of how the animal’s nervous system is organized to control behavior,” said Steven Flavell, associate professor in MIT’s Department of Brain and Cognitive Sciences. “It shows how the many defined nodes that make up the animal’s nervous system encode precise behavioral features, and how this depends on factors like the animal’s recent experience and current state.” Flavell is senior author of the team’s published paper in Cell, which is titled “Brain-wide representations of behavior spanning multiple timescales and states in C. elegans.” The team has made its data, and the findings of their model and atlas, available at the WormWideWeb.

Credit: Picower Institute

Changes in an animal’s behavior and internal state are accompanied by widespread changes in activity across its brain, the authors wrote, and while the neural circuits that control these behaviors are distributed across the brain, how neurons encode behavior, and how this encoding is impacted by state isn’t well understood. “Animals must adapt their behavior to a constantly changing environment,” they pointed out. However, given the vast number of cell types in mammals that may be involved in behavior, and their broad spatial distributions in the brain, characterizing this entire system has not been tractable, the team further stated.  “… it is challenging to record activity across the brain of a freely moving animal and relate brain-wide activity to comprehensive behavioral information. For this reason, it has remained unclear how neurons and circuits across entire nervous systems represent an animal’s varied behavioral repertoire and how this flexibly changes depending on context or state.”

In contrast to the complexity in mammals, C. elegans may represent a model system that could allow investigators to better study these relationships. The C. elegans nervous system comprises just 302 neurons with known connectivity. The animal exhibits a well-defined repertoire of motor functions, from locomotion, to feeding, head oscillation, defecation, egg-laying, and postural changes. C. elegans also expresses different behaviors as it switches states, the investigators continued. For example, the organism enters sleep-like states after intense stress, while awake animals exhibit different foraging states, and aversive stimuli trigger sustained states of heightened arousal. “In C. elegans, it may be feasible to decipher how behavior is encoded across an entire nervous system and how this can flexibly change across behavioral states,” the researchers suggested. The results of previous studies, including brain recordings in immobilized animals, have indicated that many neurons carry behavioral information in the worm, but, as the team stated, “we still lack an understanding of how quantitative behavioral features are encoded by most C. elegans neurons.”

To make the measurements needed to develop their model, Flavell’s lab developed a new type of microscope and software system that automatically tracks almost all behaviors of the worm—movement, feeding, sleeping, egg-laying, etc.—and the activity of every neuron in its head, using a fluorescence system in which the cells are engineered to flash when calcium ions build up. “We built a microscopy platform for brain-wide calcium imaging in freely moving animals and wrote software to automate processing of these recordings,” the team stated.

Reliably distinguishing and tracking separate neurons as the worms moved or bent also required writing custom software, utilizing the latest tools from machine learning. “We also wrote software that extracts behavioral variables from the brightfield images: velocity, body posture, feeding (or pharyngeal pumping), angular velocity, and head curvature (bending of the head, associated with steering).”

The team confirmed the platform to be 99.7% accurate in sampling the activity of individual neurons, with greatly improved signal-to-noise compared to previous systems. The team then used the system to record simultaneous behavior and neural data from more than 60 worms as they moved freely about their environment.

Data analysis revealed three novel observations about neural activity in the worm: that neurons track behavior not only of the present moment but also the recent past; that neurons also tuned their encoding of behaviors, such as motion, based on a surprising variety of factors; and that that many neurons simultaneously encode multiple behaviors.

For example, while the behavior of wriggling around a lab dish might seem like a very simple act, neurons represented factors such as speed, steering, and whether the worm was eating or not. In some cases they represented the animal’s motion spanning back in time by about a minute. “Most neurons primarily encoded current behavior, but a sizable subset weighed past behavior,” the team stated.  By encoding recent, rather than just current motion, these neurons could then help the worm compute how its past actions influenced its current outcome. Many neurons also combined behavioral information to execute more complex maneuvers. Akin to a human driver remembering to steer the car in the opposite way when going in reverse, compared with when going forwards, certain neurons in the worm’s brain integrated the animal’s direction of motion and steering direction.

By carefully analyzing these kinds of patterns of how neural activity correlated with behaviors the scientists developed the C. elegans Probabilistic Neural Encoding Model (CePNEM). The model, encapsulated in a single equation, accounts for how each neuron represents various factors to accurately predict whether and how the neural activity reflects behavior. “ … we constructed an encoding model that uses behavioral features to predict each neuron’s activity,” the team further explained. “This model provides a quantitative explanation of how each neuron’s activity is related to behavior … In contrast to decoding analyses, which reveal the presence of behavioral information in groups of neurons, an encoding model can provide precise information about how each neuron’s dynamics relate to behavior.”

In fitting the model, the research team used a probabilistic modeling approach that allowed them to understand how certain they were about each fit model parameter, an approach pioneered by co-author Vikash Mansinghka, PhD, a principal research scientist who leads MIT’s Probabilistic Computing Project.

In creating a model that could quantify and predict how any brain cell would represent behavior, the team initially gathered data from neurons without tracking the cells’ specific identities. But a key goal of studying the worms is to understand how each cell and circuit contributes to behavior. So to apply the model’s capability to each of the worm’s specific neurons, which have all been previously mapped out, the team’s next step was to relate neural activity and behavior for each cell on the map. Doing that required labeling each neuron with a unique color so that its activity could be associated with its identity. The team did this in dozens of freely-moving animals, which provided them with information of how almost all of the defined neurons in the worm’s head related to the animal’s behavior.

They found that 58.6% of the neurons in the worm’s head indeed accounted for at least one behavior, “… with approximately one-third of these conjunctively encoding multiple behaviors.”

The atlas resulting from this work revealed many insights, more fully mapping out the neural circuits that control each of the animal’s behaviors Another major outcome of the team’s work was the finding that while most neurons always obeyed the predictions of the model, a smaller set of neurons in the C. elegans brain—about 30% of those that encode behavior—was able to flexibly remap their behavior encoding, essentially taking on new jobs. The neurons in this group were reliably similar across animals, and were well connected with one another in the worm’s synaptic wiring diagram. “”Under the environmental conditions explored here, we observed that 30% of the worm’s nervous system can flexibly remap,” the authors noted. “Neurons changed encoding in different ways: some changed which behaviors they encoded; others showed gains or losses of encoding; and others showed subtle changes in tuning … This suggests that some neurons in the C. elegans connectome are variably coupled to behavioral circuits and remap how they couple to these circuits over time.”

Theoretically these remapping events could occur for any number of reasons, so the team ran further experiments to see if they could cause neurons to remap. As the worms wriggled around their dishes, the researchers applied a quick laser zap that heated the agar around the worm’s head. The heat was harmless but enough to annoy the worms for a while, inducing a change in the animal’s behavior state that lasted for minutes. From these recordings the team was able to see that many neurons remapped their behavioral encoding right as animals switched behavioral states. Interestingly, some neurons displayed transient responses to the temperature-related stimulus, others displayed minutes-long responses, while some neurons demonstrated persistent changes in activity that lasted for the rest of the recordings after the stimulus. “The neurons that changed encoding were stereotyped across animals, especially the neurons related to feeding, which is the behavior most robustly altered by the heat stimulus,” the scientists added. “Overall, these results show how changes in behavioral state are accompanied by persistent activity changes and alterations in how neural activity is functionally coupled to behavior.”

“Behavioral information is richly expressed across the brain in many different forms – with distinct tunings, timescales, and levels of flexibility – that map onto the defined neuron classes of the C. elegans connectome,” the authors concluded. “Our results provide a global map of how the cell types across an animal’s brain encode its behavior.”

The new findings will enable a more holistic understanding of how these behaviors are controlled, Flavell said. “It allowed us to complete the circuits,” he said. “Our hope is that as our colleagues study aspects of neural circuit function, they can refer to this atlas to obtain a fairly complete view of the key neurons involved.”

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