A clock that works in the dark

Physics, biology, and technology come together to reveal the circadian rhythms of a subterranean rodent

Imagem: PATRICIA TACHINARDI / USP Digging: one of the rare moments when tuco-tucos appear on the surfaceImagem: PATRICIA TACHINARDI / USP

In nature, conditions can be inconsistent. Food may or may not be available, predators are more abundant in certain situations, and at times the climate can be a challenge. Anticipating some of these conditions allows animals to act at more productive or safer times; this is where the biological clock comes into play. Over the last 30 years, the genetic control of this internal mechanism has been revealed, and was recognized in this year’s Nobel Prize in Physiology or Medicine. This ability to plan ahead is especially valuable for animals that do not have easy access to environmental information, which is the case for the burrowing rodents known as tuco-tucos. They live in extreme environment with regard to light, one which also applies to animals that live at the poles, in deep underwater trenches, or caves, and consequently is very important for studies of chronobiology. “We are comparing biological rhythms in nature and in the laboratory,” says physicist Gisele Oda, coordinator of the Chronobiology Laboratory at the Institute of Biosciences of the University of São Paulo (IB-USP). “This field work is unusual in chronobiology, which is traditionally studied in controlled environments,” she says. Oda came to biology by way of physics, studying oscillations in dynamic systems.

The comparison between the two situations soon presented an additional question when the tuco-tucos, which are active during the day in the wild, became nocturnal in the laboratory. This change had already been observed in other animals, including Chilean relatives of tuco-tucos known as coruros. The biologist Patricia Tachinardi, who is part of Oda’s group, focused on this aspect and concluded that it benefits the animals by allowing them to save energy, as reported in an article published last year in Physiological and Biochemical Zoology.

As part of the doctorate she defended this year, Tachinardi placed tuco-tucos into chambers that measure their metabolic activity through the oxygen they consume during respiration. From these experiments, she determined that temperatures between 23 and 33 degrees Celsius are ideal for these animals, and estimated how much energy they save by being active during the day or night. The American physiologist Loren Buck of Northern Arizona University, an expert in energy balance under natural conditions, also participated in the study. A surprising setback occurred: three of the nine animals in the experiment, which were nocturnal in the laboratory, instantly became active during the day when placed into these chambers. “The humidity increases and the oxygen concentration is lower than in normal lab conditions; we need to control these conditions to investigate what encourages this change in activity.”

As it turns out, tuco-tucos in the wild need to be more active in winter, when the warmth of the underground nest would be welcome. They need to eat more in order to obtain enough energy to face the cold, and food is scarcer at this time of year. It consequently becomes crucial for the animals to restrict their activity to those hours when the sun raises the temperature. To study energy consumption in the field in detail, Oda’s group faced technical limitations. “The accelerometers that we use are still too large to be attached to the animals roaming free,” says the physicist. She refers to devices which are implanted or attached to collars, precisely recording the animals’ movements and energy expenditures.

Energy budget
The animals’ ability to adapt their active times, as if the environmental conditions had thrown a switch that instantly changed the pattern, was a curious observation. “Being nocturnal or diurnal tends to be associated with the identity of each species, like an unchangeable label,” notes Oda. The observations fit the “working for food” model proposed by the Dutch chronobiologist Roelof Hut of the University of Groningen, where Tachinardi spent some time during her doctorate. Hut showed that mice transform from nocturnal to diurnal as they have more difficulty obtaining food. “The more they need to work to eat, the more diurnal they become,” explains Tachinardi. According to this hypothesis, this was what happened when the tuco-tucos needed to dig tunnels to find sparse foliage. “The energy budget in nature is tight, while in the lab they have as much food as they want.” Eating the diet they are fed in captivity, these animals go back to being nocturnal, which is thought to be the ancestral standard of this species.

From the beginning, evaluating the tuco-tucos’ activity was not an easy task. One problem is that they spend most of their time in underground galleries in the desert soils near Anillaco, Argentina, a town of 1,400 better known as the birthplace of former president Carlos Menem. In 1998, during his administration, a Regional Center for Scientific Research and Technological Transfer (CRILAR) was founded in the city as part of an initiative to establish scientific centers in distant areas. It is the ideal setting for studying these rodents, which may be a species not yet described by science. Although their taxonomy is not yet defined, the researchers identified them as Ctenomys knighti, a species known in the region. “The Menem garden is full of them, and they’re considered pests by the winemakers on the property neighboring the research center,” says Oda. Even so, the greatest evidence of when the tuco-tucos are active were the vocalizations they emit all day from within their dens.

Oda began studying these animals eight years ago, in partnership with Argentinean biologist Verónica Valentinuzzi of CRILAR, who she met during a post-doctorate internship at IB-USP. From the beginning, she had the help of Tachinardi, Danilo Flôres, and Barbara Tomotani, who at the time were undergraduate biology students. In order to monitor the tuco-tucos, they had to capture the animals, get sensors to monitor them without disrupting their activities, and also develop semi-natural areas for experiments where they could not escape by digging, among other challenges. After some trial and error, the researchers built underground concrete walls below the fences, installed collars with light and movement sensors on the animals, and implanted temperature sensors in their bodies.

Using this equipment in eight animals during the winters of 2014 and 2015, the researchers found that all were exposed to sunlight for brief periods, while removing dirt during tunnel excavations, looking for leaves to eat, or pausing at the tunnel entrance, perhaps warming themselves. Exposure to light occurs at least once but generally several times per day, as shown in an article published in 2016 in Scientific Reports as part of Flôres’s doctoral thesis, which was completed in 2016.

Imagem: PATRICIA TACHINARDI / USP Fences installed at the research center permit experiments in semi-natural conditionsImagem: PATRICIA TACHINARDI / USP

Day in the lab
The group then sought to understand how the circadian cycle is regulated. If the tuco-tucos are left in permanent darkness in the laboratory, the activity recorded by an exercise wheel (identical to those commonly used in pet rodent cages) shows that the animals maintain a pace of activity that corresponds to 25 hours, a common deviation in other animals. A pulse of light lasting only a few seconds per day, even at variable times, is enough to synchronize the biological clock to the 24 hours of Earth’s rotation, according to a computational model developed by the USP group which simulates a circadian oscillator, the central mechanism that controls the biological clock. Flôres was surprised by this prediction and conducted the test in real life. With only an hour of light per day, the time to perform tasks such as feeding the animals and cleaning their habitat in the laboratory, nine tuco-tucos began to show activity consistent with a 24-hour day. This happened when the light was on randomly (within the limits of daylight hours outside the laboratory) or at predetermined times.

The results, which unite experiments in the laboratory, the natural environment, and measurements in the field, help define the factors which are most central to the circadian cycle. They also mediate the theoretical discussion on how this regulation occurs. In what is known as the parametric model, light has a continuous effect in synchronization. “It is as if a swing were being continuously pushed,” explains Oda. In the non-parametric model, this swing remains in motion thanks to periodic pushes. These pushes, in the case of the biological clock, are the abrupt changes in light that happen at dawn and at dusk. The tuco-tucos tell another story, a hybrid between the two models, in which the pushes can happen at any point during the swing’s trajectory.

The focus on mathematical modeling also guided the discovery of the molecular mechanisms behind circadian rhythms, for which this year’s Nobel Prize in Physiology or Medicine was awarded to the Americans Jeffrey Hall, Michael Rosbash, and Michael Young. “The important finding for the prize was identifying which elements were involved in regulating the 24-hour rhythm,” says Oda. The PER protein, which is produced by the period gene identified in the 1980s, accumulates in the nuclei of the cells and inhibits the action of this gene. “But the model indicated that the biochemical steps in this process did not reach 24 hours, so it was mathematically established that there should be some element of delay.” This happens thanks to another protein that degrades PER, slowing its accumulation. Pulses of light also act to degrade another protein, leading to a synchronization explained by mathematical modeling.

“The dynamics of the system that oscillates are governed by differential equations,” says Gisele, predicting that the system revealed by underground model will work for rats and even for people. “The oscillator is the same for everyone,” she concludes with her physicist’s perspective.

Chronobiology of subterranean South American rodents in the lab and in the field (No. 14/20671-0); Grant Mechanism Regular Research Grant; Principal Investigator Gisele Akemi Oda (USP); Investment R$162,935.51.

Scientific articles
FLÔRES, D. E. F. L. et al. Entrainment of circadian rhythms to irregular light/dark cycles: A subterranean perspective. Scientific Reports. V. 6, 34264. Oct. 4, 2016.
TACHINARDI, P. et al. The interplay of energy balance and daily timing of activity in a subterranean rodent: A laboratory and field approach. Physiological and Biochemical Zoology. V. 90, No. 5, p. 546–52. Sept.–Oct. 2017.
TACHINARDI, P. et al. Nocturnal to diurnal switches with spontaneous suppression of wheel-running behavior in a subterranean rodent. PLOS ONE. V. 10, No. 10, e0140500. Oct. 13, 2015.