Wednesday, March 22, 2023

As the worm turns: New twists in behavioral association theories

The researchers conducted experiments on C. elegans, a roundworm with just 300 neurons, that offers a simple laboratory model for studying how an animal learns.

By Carol Clark

Physicists have developed a dynamical model of animal behavior that may explain some mysteries surrounding associative learning going back to Pavlov’s dogs. The Proceedings of the National Academy of Sciences (PNAS) published the findings, based on experiments on a common laboratory organism, the roundworm C. elegans

“We showed how learned associations are not mediated by just the strength of an association, but by multiple, nearly independent pathways — at least in the worms,” says Ilya Nemenman, an Emory professor of physics and biology whose lab led the theoretical analyses for the paper. “We expect that similar results will hold for larger animals as well, including maybe in humans.” 

“Our model is dynamical and multi-dimensional,” adds William Ryu, an associate professor of physics at the Donnelly Centre at the University of Toronto, whose lab led the experimental work. “It explains why this example of associative learning is not as simple as forming a single positive memory. Instead, it’s a continuous interplay between positive and negative associations that are happening at the same time.” 

First author of the paper is Ahmed Roman, who worked on the project as an Emory graduate student and is now a postdoctoral fellow at the Broad Institute. Konstaintine Palanski, a former graduate student at the University of Toronto, is also an author. 

The conditioned reflex

More than 100 years ago, Ivan Pavlov discovered the “conditioned reflex” in animals through his experiments on dogs. For example, after a dog was trained to associate a sound with the subsequent arrival of food, the dog would start to salivate when it heard the sound, even before the food appeared. 

About 70 years later, psychologists built on Pavlov’s insights to develop the Rescorla-Wagner model of classical conditioning. This mathematical model describes conditioned associations by their time-dependent strength. That strength increases when the conditioned stimulus (in Pavlov dog’s case the sound) can be used by the animal to decrease the surprise in the arrival of the unconditioned response (the food). 

Such insights helped set the stage for modern theories of reinforcement learning in animals, which in turn enabled reinforcement learning algorithms in artificial intelligence systems. But many mysteries remain, including some related to Pavlov’s original experiments. 

After Pavlov trained dogs to associate the sound of a bell with food he would then repeatedly expose them to the bell without food. During the first few trials without food, the dogs continued to salivate when the bell rang. If the trials continued long enough, the dogs “unlearned” and stopped salivating in response to the bell. The association was said to be “extinguished.” 

Pavlov discovered, however, that if he waited a while and then retested the dogs, they would once again salivate in response to the bell, even if no food was present. Neither Pavlov nor more recent associative-learning theories could accurately explain or mathematically model this spontaneous recovery of an extinguished association. 

Teasing out the puzzle

Researchers have explored such mysteries through experiments with C. elegans. The one-millimeter roundworm only has about 1,000 cells and 300 of them are neurons. That simplicity provides scientists with a simple system to test how the animal learns. At the same time, C. elegans’ neural circuitry is just complicated enough to connect some of the insights gained from studying its behavior to more complex systems. 

Earlier experiments have established that C. elegans can be trained to prefer a cooler or warmer temperature by conditioning it at a certain temperature with food. In a typical experiment, the worms are placed in a petri dish with a gradient of temperatures but no food. Those trained to prefer a cooler temperature will move to the cooler side of the dish, while the worms trained to prefer a warmer temperature go to the warmer side. 

But what exactly do these result mean? Some believe that the worms crawl toward a particular temperature in expectation of food. Others argue that the worms simply become habituated to that temperature, so they prefer to hang out there even without a food reward. 

The puzzle could not be resolved due to a major limitation of many of these experiments — the lengthy amount of time it takes for a worm to traverse a nine-centimeter petri dish in search of the preferred temperature. 

Measuring how learning changes over time

Nemenman and Ryu sought to overcome this limitation. They wanted to develop a practical way to precisely measure the dynamics of learning, or how learning changes over time. 

Ryu’s lab used a microfluidic device to shrink the experimental model of nine-centimeter petri dishes into four-millimeter droplets. The researchers could rapidly run experiments on hundreds of worms, each worm encased within its individual droplet. 

“We could observe in real time how a worm moved across a linear gradient of temperatures,” Ryu says. “Instead of waiting for it to crawl for 30 minutes or an hour, we could much more quickly see which side of the droplet, the cold side or the warm side, that the worm preferred. And we could also follow how its preferences changed with time.” 

Their experiments confirmed that if a worm is trained to associate food with a cooler temperature it will move to the cooler side of the droplet. Over time, however, with no food present, this memory preference seemingly decays. 

“We found that suddenly the worms wanted to spend more time on the warm side of the droplet,” Ryu says. “That’s surprising because why would the worms develop a different preference and even avoidance of the temperature they had come to associate with food?” 

Eventually the worm begins moving back and forth between the cooler and warmer temperatures. The researchers hypothesized that the worm does not simply forget the positive memory of food associated with cooler temperatures but instead starts to negatively associate the cooler side with no food. That spurs it to head for the warmer side. Then as more time passes, it begins to form a negative association of no food with the warmer temperature, which combined with the residual positive association to the cold, makes it migrate back to the cooler one. 

“The worm is always learning, all the time,” Ryu explains. “There is an interplay between the drive of a positive association and a negative association that causes it to start oscillating between cold and warm.” 

'It's like when you lose your keys'

Nemenman’s team developed theoretical equations to describe the interactions over time between the two independent variables — the positive, or excitatory, association that drives a worm toward one temperature and the negative, or inhibitory, association that drives it away from that temperature. 

“The side that the worm gravitates toward depends on when exactly you take the measurements,” Nemenman explains. “It’s like when you lose your keys you may check the desk where you usually keep them first. If you don’t see them there right away, you run around different places looking for them. If you still don’t find them, you go back to the original desk figuring you just didn’t look hard enough.” 

The researchers repeated the experiments under different conditions. They trained the worms at different starting temperatures and starved them for different durations before testing their temperature preference, and the worms’ behaviors were correctly predicted by the equations. 

They also tested their hypothesis by genetically modifying the worms, knocking out the insulin-like signaling pathway known to serve as a negative association pathway. 

“We perturbed the biology in specific ways and when we ran the experiments, the worm’s behavior changed as predicted by our theoretical model,” Nemenman says. “That gives us more confidence that the model reflects the underlying biology of learning, at least in C. elegans.” 

The researchers hope that others will test their model in studies of larger animals across species. 

“Our model provides an alternative quantitative model of learning that is multi-dimensional,” Ryu says. “It explains results that are difficult, or in some cases impossible, for other theories of classical conditioning to explain.” 


Physicists develop theoretical model for neural activity of mouse brain

Machine learning used to understand and predict dynamics of worm behavior

Tuesday, March 21, 2023

Hidden 'super spreaders' spur dengue fever transmission

A NASA satellite image shows Iquitos, Peru, nestled in the Amazon Basin, on the banks of the Amazon River and surrounded by smaller rivers, lakes and lagoons.

By Carol Clark

For mosquito-borne diseases such as dengue fever, the abundance of the insects in places where people gather has long served as the main barometer for infection risk. A new study, however, suggests that the number of “hidden” infections tied to a place, or cases of infected people who show no symptoms, is the key indicator for dengue risk. 

PNAS Nexus published the research led by scientists at Emory University, which drew from six years of data collected in the Amazonian city of Iquitos, Peru. 

The results found that 8% of human activity spaces in the study accounted for more than half of infections during a dengue outbreak. And these “super spreader” spaces were associated with a predominance of asymptomatic cases, or 74% of all infections. 

“Our findings show that any public health intervention that focuses on responding to symptomatic cases of dengue is going to fail to control an outbreak,” says Gonzalo Vazquez-Prokopec, first author of the study and an Emory associate professor of environmental sciences. “Symptomatic cases represent only the tip of the iceberg.” 

Co-authors of the research include Uriel Kitron, Emory professor of environmental sciences; Lance Waller, professor of biostatistics and bioinformatics at Emory’s Rollins School of Public Health; and scientists from University of California-Davis, Tulane University, San Diego State University, University of Notre Dame, North Carolina State University and the U.S. Naval Medical Research Unit in Lima, Peru. 

'What matters is where you went'

Dengue fever is caused by a virus transmitted by the bite of a female Aedes aegypti mosquito. When the insect takes a blood meal from a human infected with dengue, the virus begins replicating within the mosquito. The virus may then spread to another person that the mosquito bites days later. 

This species of mosquito feeds exclusively on human blood, has a limited flight range of about 100 meters and thrives in sprawling urban areas of the tropics and subtropics. Its preferred habitat is inside homes, where it rests on the backs of furniture and at the bases of walls. Even the little bit of water held by an upturned bottle cap can serve as a nursery for its larvae. 

Vazquez-Prokopec is pioneering new mosquito-borne disease interventions, including tapping spatiotemporal data to track, predict and control outbreaks of pathogens transmitted by Aedes aegypti. The mosquito spreads the Zika, chikungunya and yellow fever viruses in addition to dengue. 

Around 500,000 cases of dengue occur annually around the world, according to the World Health Organization. The disease is caused by four distinct but closely related serotypes of the dengue virus. Infected people may have some immunity that prevents them from experiencing any noticeable effects while others may be severely debilitated for a week or more by symptoms such as extreme aches and pains, vomiting and rashes. Dengue hemorrhagic fever, the most severe form of the disease, causes an estimated 25,000 deaths annually worldwide. 

Iquitos, a city of nearly 500,000 people on the edge of the Amazon rainforest in Peru, is a dengue hotspot. For more than a decade, Vazquez-Prokopec and colleagues have mapped patterns of human mobility and dengue spread in Iquitos. 

“For diseases that are directly spread from one person to another, like COVID-19, what matters is who you were near,” he says. “But in the case of dengue, what matters most is where you went.” 

Tracking hidden cases

For the current study, the researchers wanted to determine the role of asymptomatic cases. People without symptoms may continue to go about their daily routines, unknowingly infecting any mosquitoes that bite them, which can then later spread the virus to more people. 

The study involved 4,600 people in two different neighborhoods. They were surveyed three times a week about their mobility. This data was used to map “activity spaces,” such as residences, churches and schools. 

The study participants were also regularly surveilled to determine if they experienced any dengue symptoms. Blood analyses confirmed a total of 257 symptomatic cases of dengue during the six-year study period. That led to investigations of other participants whose activity spaces overlapped with the symptomatic cases. More than 2,000 of these location-based contacts were confirmed by blood tests to have dengue infections and more than half of them reported not having any noticeable symptoms. 

A cascade of circumstances

The results pinpointed the role of asymptomatic “super spreaders” in a dengue outbreak. A small number of the activity spaces, or 8%, were linked to more than half of the infections and most of the cases associated with those places were asymptomatic. 

The comprehensive, one-of-a-kind study broke down the virus infections by serotype and measured the amount of mosquitoes in the activity spaces. 

“We found that the mosquito numbers in a location alone is not a predictor of the risk of infection,” Vazquez-Prokopec says. 

Instead, risk prediction for a location requires a cascade of circumstances: a high number of asymptomatic cases frequenting the location combined with high levels of mosquitos and high numbers of people who are not immune to the particular serotype of dengue virus that is circulating. 

“That’s the complicated nature of this virus,” Vazquez-Prokopec says. “We have underestimated the role of asymptomatic cases in spreading dengue.” 

Generally, about 50 to 70% of dengue cases are asymptomatic, making detection by public health officials impractical, and the current study reveals that asymptomatic cases are tied to a third of transmission. 

“The lesson is that we need to focus on prevention of dengue outbreaks,” Vazquez-Prokopec says. “The interventions for dengue for decades have been reactive. Simply reacting by closing a net around reported cases of the disease, however, will fail to contain an outbreak because that’s missing the super spreaders.” 

The study was funded by the U.S. National Institute of Allergy and Infectious Diseases, Bill and Melinda Gates Foundation, University of Notre Dame, Defense Threat Reduction Agency, Military Infectious Disease Research Program and the Armed Forces Health Surveillance Branch Global Emerging Infections Systems research program. 


Tapping big data to target a tiny predator 

Mapping dengue hot spots pinpoints risks for Zika and chikungunya 

Mutant mosquitoes make insecticide-resistance monitoring key to control Zika

Wednesday, March 8, 2023

Atlanta Science Festival expands your horizons

Rae Wynn-Grant admires a bear cub after tagging it. "I hope that I can play a small role in helping people see that science is a space where anyone can find belonging," she says.

"Science has quite literally taken me around the world," says Rae Wynn-Grant, an Emory alumna and wildlife biologist whose field research has spanned six continents.

"But you don't have to physically travel to be a great scientist," she adds. "I want people to know that there are many different ways that science can expand your horizons." 

Wynn-Grant returns to Atlanta as a featured speaker to launch this year's Atlanta Science Festival, set for March 10-25. The festival is bigger and more expansive than ever with more than 150 events and an overarching theme: Where will science take you?

Read more about the festival here.