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.” 

Related:

Physicists develop theoretical model for neural activity of mouse brain

Machine learning used to understand and predict dynamics of worm behavior

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. 

Related:

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

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.

Study shows long-term trend of undocumented Mexican immigrants most at risk for deportation from U.S.

No matter the U.S. political climate, young, single and less educated men seemed to be at higher risk for deportation than other undocumented Mexican immigrants from 2001 to 2019, a study led by Emory University finds. 

The Proceedings of the National Academy of Sciences (PNAS) published the study analyzing deportation and voluntary return migration data encompassing the administrations of U.S. Presidents George W. Bush, Barack Obama and Donald Trump. 

“Even through the Trump administration’s anti-immigrant rhetoric advocated deporting all undocumented immigrants, particularly from Mexico, the characteristics of Mexican immigrants deported during the Trump years were not dramatically different from previous administrations,” says Heeju Sohn, Emory assistant professor of sociology and lead author of the study. 

Co-authors include Anne Pebley and Amanda Landrian Gonzalez from the University of California Los Angeles and Noreen Goldman of Princeton University. 

The researchers examined trends in socio-demographic characteristics of undocumented Mexican immigrants deported by the U.S. along with those who chose to return to Mexico. 

While the study does not predict or offer any absolute probabilities, it provides insight into relative potential risks. 

On average, each administration annually deported about 893,000 people with the majority of them Mexican citizens. 

“Despite each administration’s differing approach and rhetoric, who was actually being deported or deciding to leave didn’t change all that much,” Sohn said. “Just because an undocumented person voluntary leaves the U.S. doesn’t always mean they felt they had a choice in that decision either.” 

Fewer immigrants were deported annually during the Trump administration than under Obama or Bush who had the highest number of deportations. During Obama’s first term, there was an increase in deportation of Mexican immigrants with criminal convictions but that percentage decreased in the last two years of his presidency. 

While Trump’s administration prioritized all undocumented immigrants for deportation, the result shows deportation focused more on young adults and those with less education, groups which already face higher deportation risks. 

"Policy makers and the public need to understand the consequences of the immigration policies that are implemented – whether they work or not,” says Pebley, a UCLA California Center for Population Research faculty fellow. “While the Trump administration's anti-immigrant rhetoric and policies had many negative effects on immigrants and Americans, they did not do what they were apparently intended to in terms of deporting a larger and more diverse group of undocumented immigrants.” 

The Trump administration’s anti-immigrant rhetoric and heightened enforcement didn’t appear to motivate a more diverse group of undocumented immigrants to leave voluntarily. Rather, voluntary return migration to Mexico was a trend that began early in the Obama administration after the great recession of 2007-2009, according to the study. 

“People who are leaving or being deported do not exist in a vacuum,” Sohn says. “You can’t isolate them separately from the social and family connections they have interwoven in U.S. society. So, what happens to undocumented people that society has neglected has a direct effect on the well-being of U.S. citizens. We have a duty to not discriminate and there is a need for additional research.” 

The experiences of undocumented children living in the U.S. is a blind spot in national data; the youngest age group in this study is 18 to 31. 

“Moving across countries is a disruptive life event,” Sohn says. “This is an age group where people take major steps as adults—finding a partner, having children or establishing a career. This can have reverberating consequences for the rest of their lives.” 

For the study, Sohn and the other researchers combined deportees’ and voluntary returnees’ data from both sides of the border—the Migration Survey on the Borders of Mexico-North (EMIF-N) and U.S. Current Population Survey’s Annual Social and Economic Supplement (ASEC). It’s the first time these two major sets of data were combined for research purposes and studied in a novel way. 

“It was critical that we understood the nuances of the data and sampling strategy. We took a lot of timand effort making sure our method accounted for the differences,” Sohn says. 

“This is part of a bigger desire to make sure the lives of underrepresented groups have adequate representation,” she adds. “A lot of the research in social sciences are based on large data sets that don’t put much focus on the smaller groups or ones that are harder to measure. I hope getting this important topic published will get visibility to a wider audience.” 

Funding for the story was provided by the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (ROOHDO96322).

Genomic study reveals signs of TB adaptation in ancient Andeans

"Human-pathogen co-evolution is an understudied area that has a huge bearing on modern-day public health," says Sophie Joseph, first author of the study and an Emory graduate student in anthropology.

By Carol Clark

People have inhabited the Andes mountains of South America for more than 9,000 years, adapting to the scarce oxygen available at high altitudes, along with cold temperatures and intense ultraviolet radiation. A new genomic study suggests that Indigenous populations in present-day Ecuador also adapted to the tuberculosis bacterium, thousands of years before the arrival of Europeans. 

The journal iScience published the findings, led by scientists at Emory University. 

“We found that selection for genes involved in TB-response pathways started to uptick a little over 3,000 years ago,” says Sophie Joseph, first author of the paper and an Emory graduate student in anthropology. “That’s an interesting time because it was when agriculture began proliferating in the region. The development of agriculture leads to more densely populated societies that are better at spreading a respiratory pathogen like TB.” 

The investigators had originally set out to investigate how the Indigenous people of Ecuador adapted to living at high altitude. 

“We were surprised to find that the strongest genetic signals of positive selection were not associated with high altitude but for the immune response to tuberculosis,” says John Lindo, Emory assistant professor of anthropology and senior author of the study. “Our results bring up more questions regarding the prevalence of tuberculosis in the Andes prior to European contact.” 

The Lindo lab specializes in mapping little-explored human lineages of the Americas. 

Previously published research found evidence of the tuberculosis bacterium in the skeletal material of 1,400-year-old Andean mummies, contradicting some theories that TB did not exist in South America until the arrival of Europeans 500 years ago. 

The current paper provides the first evidence for a human immune-system response to TB in ancient Andeans and gives clues to when and how their genomes may have adapted to that exposure. 

A graphic from the iScience paper
 

“Human-pathogen co-evolution is an understudied area that has a huge bearing on modern-day public health,” Joseph says. “Understanding how pathogens and humans have been linked and affecting each other over time may give insights into novel treatments for any number of infectious diseases.” 

Co-authors of the paper include scientists at Central University of Ecuador, Technical University of Manabi in Ecuador, University of Pavia in Italy, University of Iowa and Florida Atlantic University. 

The researchers sequenced whole genomes using blood samples from 15 present-day Indigenous individuals living at altitudes above 2,500 meters in several different Ecuadorian provinces. They performed a series of scans to look for signatures of positive selection for genes in their ancestral past. 

“Computational techniques for sequencing genomes and modeling ancestral selection keep improving,” Joseph says. “The genomes of people living today give us a window into the past.” 

Among the strongest signals detected were for biomarkers that are switched on in modern humans during an active TB infection. The researchers modeled the timing of selection for several of the genes involved in the TB-response pathways. 

Although they were not as strong as for exposure to TB, some signals were also detected for biomarkers related to adaptation to hypoxia, or low levels of oxygen in the blood that result from living at high altitude. 

Previous research has revealed stark differences in how high-altitude populations in Tibet, Ethiopia and the Peruvian Andes adapted to hypoxia. 

“For the Ecuadorean samples, we did see a couple of overlaps with studies from the Peruvian Andes in the overarching genes involved in the selection for hypoxia, although the variants were slightly different,” Joseph says. “To me, that suggests that there may have been independent adaptations within even small populations, at the community level. It shows the robustness of the genome to solve adaptive problems through different pathways.” 

Joseph plans a career focused on mapping ancestral data for Indigenous populations from the Americas. 

“South America has far fewer genomic studies and publications compared to Europe and I’d like to help close that gap,” she says. 

“I want to understand human evolution and health from an integrated biological perspective,” Joseph adds. “The genome can reveal many fascinating things and yet it is just one aspect of a human being. You also have to consider the environment and social-cultural aspects.” 

Related:

Genomic mapping of little-explored human lineages

Ancient DNA analyses add new complexity to South America settlement

 Ancient DNA from Sudan shines new light on Nile Valley past