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The hidden rhythm that can confuse studies with mice
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The hidden rhythm that can confuse studies with mice
by Nature Research and Tecniplast
The activity and physiology of the mice vary throughout the day, but the researchers have learned to control them. Now, a seasonal oscillation may make it difficult to reproduce the experiments with mice.
Life has its rhythms, and this is true for both laboratory mice and humans. Mice tend to sleep all day and feed, run, mate, and explore at night. The time of day can affect frequently studied mouse behaviors, such as running and feeding, and can even affect how drug compounds are absorbed or how the compounds interact with target receptors. For these reasons, a study in mice conducted in the morning may yield different results than one conducted in the afternoon.
Brun Ulfhake, a neuroscientist at the Karolinska Institute in Stockholm, knew that circadian rhythms, which are tuned to the 24-hour day/night cycle, could cause such variability. In this way, they regulate a number of biological functions, including sleep, metabolism, hormone release, and digestion.
Recently, Ulphace’s team identified a hidden biological rhythm in lab mice that oscillates over months rather than hours, and appears to make mice much more active than usual. “These variations were totally unexpected,” Ulfhake says. And the new pace could affect the reproducibility of mouse experiments everywhere.
Seeking reproducibility
Circadian rhythms originate from signals in the hypothalamus and act on circuits expressed by almost every cell in the body. As the researchers learned more about these rhythms, they sought to control for circadian variability. Drug researchers now consider circadian rhythms and their influences on treatment efficacy and side effects when developing dosing regimens, says Marcello Raspa, a pharmacologist at the Italian National Research Council in Rome. Other researchers take measurements at the same time of day and make sure collaborators keep their animals at the same light-dark times, says Ryan Logan, a neuroscientist at Boston University.
Ulfhake further elevated this quest for reproducibility by monitoring activity levels in 64 animals, day and night, for 19 months. His team housed the animals in a monitoring system developed by Tecniplast, an Italian manufacturer of equipment for laboratory animals. Equipped with 12 floor-mounted electrodes, the Digital Ventilated Cage (DVC®) collects motion data multiple times per second. In this way, it generates a digital marker of activity in real time and reveals unusual patterns that would otherwise be undetectable, according to Guido Gottardo, digital product manager at Tecniplast.
During those 19 months, the animals’ environment remained the same – light cycles, temperature, and feeding routines remained constant. However, their activity levels would rise, stagnate, and suddenly fall. During these cyclical episodes, which lasted two to four months at a time, the mice were two-thirds more active than their corresponding baseline values. “This is a magnitude that can affect physiological readings,” Ulfhake says.
“Clearly, there’s a persistent pattern here that came from somewhere,” says Benjamin Smarr, a neurobiologist at the University of California, San Diego, who specializes in biological rhythms and who was not involved in the study. Biological rhythms are of concern for research in laboratory animals, as they generate significant experimental variability, says Smarr. Gene expression, for example, varies at different points in a rhythmic cycle. “So you’re asking how much of the effect you observe is due to your intervention or the effects of a biological rhythm that you may not be aware of.”
The C57BL/6 mice used in the study experienced an infradian rhythm, although they were unable to produce melatonin (shown), which a previous study suggested was necessary. The researchers say this may point to an unknown biological mechanism.
Seasonal synchronization
Biological rhythms longer than 24 hours are technically called infradian rhythms and can range from days to years. Certain bird species molt at the same time every year, even under controlled experimental conditions, during which they are exposed to the same daily intervals of light and dark. Bears hibernate in an infradian cycle. The human menstrual cycle is infradian.
Ulfhake suspects that his team detected a seasonal infradian rhythm in laboratory mice. However, he acknowledges that this conclusion is controversial. Typically, animals with endogenous seasonal clocks are also exposed to environmental signals, such as day length, that push their clocks forward or backward to synchronize them with external changes, but laboratory mice are housed indoors and do not receive these signals.
What’s more, the C57BL/6 mice studied by Ulfhake are deficient in melatonin, a hormone that plays a key role in seasonal rhythms. During a recent study, German researchers compared seasonal variations in C57B1 mice with C3H laboratory mice that are not deficient in melatonin. The mice were housed in outdoor cages for a year and monitored with detectors that collected data at ten-minute intervals. But only the C3H mice showed clear seasonal differences in activity patterns, which suggests that melatonin is what allows the mice to respond to environmental stimuli.
However, Ulfhake and his co-authors believe that the rhythm they detected is endogenous. In their study, they used monitoring data from the automated cages to generate heat maps of the mice’s activity. These revealed that mice in different cages showed high activity that started at different times and varied in duration. For Ulfhake and his co-authors, this suggests that the rhythm was generated by some kind of internal clock, rather than an external signal, which would have produced a synchronized response.
The investigation continues
After years of research, biologists understand a lot about circadian rhythms. But seasonal rhythms are comparatively more mysterious, and the ways to control them are not obvious. “We’re investigating oscillators and temporal patterns in physiology and behavior that are outside the bounds of circadian regulation,” Logan says. “But it’s very difficult to isolate and discern what those mechanisms are.”
Scientists who study seasonal phenomena often attribute them to thyroid signaling and melatonin regulation, Logan says. But because the C57BL/6 mice studied by Ulfhake produce very little circulating melatonin, it has to be something else. “It may originate in the thyroid, or perhaps in some other seasonal signaling mechanism that has not yet been discovered,” Logan argues. Ulfhake, in turn, speculates that an endogenous oscillator located in the nervous system controls the rhythm.
Ulfhake acknowledges that the results, published in Scientific Reports , have met with some resistance, which he says is to be expected given the controversies over seasonal rhythms in laboratory rodents. But the burden of proof now falls on those who deny the existence of the rhythm, who must come up with an alternative explanation for the unbiased DVC recordings, he says.
Meanwhile, Ulfhake is investigating further. “We still have a lot of work to do to validate our results and extend our observations to other mouse species,” he says. “And most importantly, we have to find the oscillator.”
Source: https://www.nature.com/articles/d42473-021-00213-4
References:
Pernold K., Rullman E. & Ulfhake B. Sci Rep. 11 (1):4961 (2021)
Metzger J. et al. J. Biol. Rhythms 35 (1): 58–71 (2020)
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