ost circadian clock research since the 1970s has looked exclusively at male model organisms. Females were more challenging to study, the old logic goes, because they may have an estrus cycle, or lay eggs, complicating experimental protocols. But in recent years, female organisms, and their contrasts with males, have gotten more attention.
A recent paper now suggests that female fruit flies are more resilient to disturbances of their circadian system than males. When study researchers disrupted the expression of a key peptide in circadian clock neurons, males’ sleep–wake and activity cycles became erratic during the experiment, but females maintained more regularity. “We saw females were not as affected as males,” says senior author Maria de la Paz Fernandez, a neurobiologist at Indiana University Bloomington. The work, published in PLoS Biology, supports other recent studies in mice that highlight sex differences in the sensitivity of the circadian system.
It’s been a big ongoing question in recent years: Is what’s true of males’ circadian clocks also true of females? “We can’t just assume everything done in males will be the same for females,” Fernandez says, especially because there are certain behaviors—for instance, aggression or displays during mating—that are sex-specific.
To explore how the clock controls behavior in males versus females, Fernandez and coauthors eliminated the expression of a key peptide, Pigment Dispersing Factor (PDF), in circadian clock cells in fly brains. PDF keeps circadian neurons firing in rhythm, helping to orchestrate a cascade of molecular reactions that set the duration and timing of sleep–wake cycles. In the absence of PDF, males are known to become arrhythmic. But what about females?
To find out, Fernandez placed hundreds of experimental and control flies into individual glass tubes, with a ray of infrared light bisecting each tube. The flies couldn’t see the light, but a digital monitor recorded their activity levels by noting how often they walked across the beam. Both experimental and control flies acted relatively normally, with discrete periods of activity and sleep, when they were exposed to 24-hour light and dark cycles.
But when the flies were kept in total darkness, such that their circadian systems alone should cue the timing of behaviors, most of the experimental males could no longer maintain rhythmic sleep–wake cycles without PDF. “The fly is just running around the tube all the time,” Fernandez says, without consolidated periods of activity versus sleep. Experimental females, however, maintained some rhythm in their sleep–wake cycles even without PDF, though they weren’t as consistent as controls.
Next, Fernandez modified the pacemaker neurons, which release PDF, in fly brains. She made these neurons in both males and females run fast, on an 18-hour cycle rather than a 24-hour one. Once again, males were more obviously affected. “We have a group of neurons that run the show, and those have more control over male behavior than over female behavior,” Fernandez says. Males’ active period shifted from a 24-hour day to an 18- to 20-hour one. Some females seemed unaffected, keeping their 24-hour periodicity. All this suggests that females may use different signaling to communicate between clock cells in the brain and might be less reliant on PDF, or any small subset of neurons, compared to males.
It’s a strong study, which “fits into a general picture of female resilience to perturbation of their circadian systems,” says physician scientist Garret FitzGerald, at the University of Pennsylvania in Philadelphia. FitzGerald points in particular to the study’s identifying some of the circuitry involved, by examining the role of PDF. The authors were able to track the geospatial importance of specific signaling pathways, he notes. FitzGerald has studied circadian sex differences in mice and is the senior author of a review, in preprint, summarizing known sex differences in the circadian systems of male and female model organisms.
One next question, Fernandez says, is how exactly circadian clock neurons are organized differently across the sexes and how they may communicate differently using signaling cascades. Her lab plans, for instance, to use live imaging of neurons in fruit flies, to see how different groups of neural cells respond to stimulation how that stimulation may have an impact on other cells downstream, and how different groups of clock neurons communicate with each other.
Clearly, there are sex differences in the circadian circuitry of the flies and other organisms. “But we don’t know why,” Fernandez says. “Understanding the mechanism,” she says, “will help us understand the system better.”
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