Fruitfly Study Identifies Brain Circuit that Drives Daily Cycles of Rest, Activity

Amita Sehgal, PhD, a professor of Neuroscience at the Perelman School of Medicine, University of Pennsylvania, describes in Cell a circuit in the brain of fruit flies that controls their daily, rhythmic behavior of rest and activity. The new study also found that the fly version of the human brain protein known as corticotrophin releasing factor (CRF) is a major coordinating molecule in this circuit. Fly CRF, called DH44, is required for rest/activity cycles and is produced in cells that receive input from the clock cells in the fly brain. In mammals, CRF is secreted rhythmically and it drives the expression of glucocorticoids such as cortisol and is associated with stress and anxiety.

Animal models like flies are helping to fill gaps in current knowledge about how the brain works, notes Sehgal. Indeed, she says, the Brain Research through Advancing Innovative Neurotechnologies (BRAIN), initiative, a project of the National Institutes of Health, includes the study of simple animal models, which are expected to provide more detailed insight into brain function.

Though much is known about the cellular and molecular components of the clock, the connections that link clock cells to overt behaviors, such as rest/activity behavior, have not been identified. “This study is essentially a map-of-the-circuitry experiment,” says Sehgal, who is also an investigator with the Howard Hughes Medical Institute (HHMI). Like humans, flies are active during the day — walking, flying, feeding and mating — and spend most of the night asleep.

“We conducted a screen for circadian-relevant neurons in the flybrain and found that cells of the pars intercerebralis — the fly version of the mammalian hypothalamus — comprise an important component of the circadian output pathway for rest/activity rhythms in flies,” Sehgal says. The mammalian hypothalamus is a neuroendocrine structure that regulates sleep, circadian rhythms, feeding and, metabolism.   

The Penn team did a random targeting of cells, activating neuronal firing with a transgene designed for this purpose, to see which cells are important in the rest/active behavior. They found that cells in the pars intercerebralis (PI) are essential for rhythmic behavior, and PI cells are connected to the clock cells through a circuit of at least two synapses.

Molecular profiling of PI cells identified the fly version of DH44 as a circadian molecule that is specifically expressed by PI neurons and required for normal rest/activity rhythms in flies. And, when the scientists selectively activated or removed just six PI cells positive for DH44, the fly’s activity cycles became irregular. In other words, the flies no longer restricted their sleep to the dark and their activity to the light, but instead showed more random distribution of these behaviors

Researchers find link between sleep and immune function in fruit flies

When we get sick it feels natural to try to hasten our recovery by getting some extra shuteye. Researchers from the Perelman School of Medicine at the University of Pennsylvania found that this response has a definite purpose, in fruitflies: enhancing immune system response and recovery to infection. Their findings appear online in two related papers in the journal Sleep, in advance of print editions in May and June.

"It’s an intuitive response to want to sleep when you get sick," notes Center for Sleep and Circadian Neurobiology research associate Julie A. Williams, PhD. "Many studies have used sleep deprivation as a means to understand how sleep contributes to recovery, if it does at all, but there is surprisingly little experimental evidence that supports the notion that more sleep helps us to recover. We used a fruitfly model to answer these questions." Along with post-doctoral fellow, Tzu-Hsing Kuo, PhD, Williams conducted two related studies to directly examine the effects of sleep on recovery from and survival after an infection.

In the first paper, they took a conventional approach by subjecting fruit flies to sleep deprivation before infecting them with either Serratia marcescens or Pseudomonas aeruginosa bacteria. Both the sleep-deprived flies and a non-sleep-deprived control group displayed increased sleep after infection, what the experimenters call an “acute sleep response.”

Unexpectedly, the pre-infection, sleep-deprived flies had a better survival rate. “To our surprise they actually survived longer after the infection than the ones who were not sleep-deprived,” notes Williams. The Penn team found that prior sleep deprivation made the flies sleep for a longer period after infection as compared to the undisturbed controls. They slept longer and they lived longer during the infection. Inducing sleep deprivation after infection rather than before made little difference, as long as the infected flies then got adequate recovery sleep. “We deprived flies of sleep after infection with the idea that if we blocked this sleep, things would get worse in terms of survival,” Williams explains. “Instead they got better, but not until after they had experienced more sleep.”

Sleep deprivation increases activity of an NFkB transcription factor, Relish, which is also needed for fighting infection. Flies without the Relish gene do not experience an acute sleep response and very quickly succumb to infection. But, when these mutants are sleep-deprived before infection, they displayed increased sleep and survival rates after infection. The team then evaluated mutant flies that lacked two varieties of NFkB (Relish and Dif). When flies lacked both types of NFkB genes, sleep deprivation had no effect on the acute sleep response, and the effect on survival was abolished. Flies from both sleep-deprived and undisturbed groups succumbed to infection at equal rates within hours.

"Taken together, all of these data support the idea that post-infection sleep helps to improve survival," Williams says.

In the second study, the researchers manipulated sleep through a genetic approach. They used the drug RU486 to induce expression of ion channels to alter neuronal activity in the mushroom body of the fly brain, and thereby regulate sleep patterns. Compared to a control group, flies that were induced to sleep more, and for longer periods of time for up to two days before infection, showed substantially greater survival rates. The flies with more sleep also showed faster and more efficient rates of clearing the bacteria from their bodies. “Again, increased sleep somehow helps to facilitate the immune response by increasing resistance to infection and survival after infection,” notes Williams.

Because the genetic factors investigated by the Penn team, such as the NFkB pathway, are preserved in mammals, the relative simplicity of the Drosophila model provides an ideal avenue to explore basic functions like sleep. “Investigators have been working on questions about sleep and immunity for more than 40 years, but by narrowing down the questions in the fly we’re now in a good position to identify potentially novel genes and mechanisms that may be involved in this process that are difficult to see in higher animals,” explains Williams.

"These studies provide new evidence of the direct and functional effects of sleep on immune response and of the underlying mechanisms at work. The take-home message from these papers is that when you get sick, you should sleep as much as you can — we now have the data that supports this idea," she concludes.

For resetting circadian rhythms, neural cooperation is key

Fruit flies are pretty predictable when it comes to scheduling their days, with peaks of activity at dawn and dusk and rest times in between. Now, researchers reporting in the Cell Press journal Cell Reports on April 17th have found that the clusters of brain cells responsible for each of those activity peaks—known as the morning and evening oscillators, respectively—don’t work alone. For flies’ internal clocks to follow the sun, cooperation is key.

"Without proper synchronization, circadian clocks are useless or can even be deleterious to organisms," said Patrick Emery from the University of Massachusetts Medical School. "In addition, most organisms have to detect changes in day length to adapt their rhythms to seasons.

"Our work clearly shows that light is detected by individual neurons that then communicate with each other to properly define the phase of circadian behavior," he added. "This emphasizes the importance of neural interaction in the generation of properly phased circadian rhythms."

In the brains of Drosophila fruit flies, there are approximately 150 circadian neurons, explained Emery and coauthor Yong Zhang, including a small group of morning oscillators that promote activity early in the day and another group of evening oscillators that promote activity later. Morning oscillators also set the pace of molecular rhythms in other parts of the brain, and hence the phase of circadian behavior. Scientists had thought they did this by relying heavily on their own sensitivity to light—what Emery calls “cell-autonomous photoreception.” Indeed, these cells do express fruit flies’ dedicated photoreceptor Cryptochrome (CRY). But recent evidence suggested that something was missing from that simple view.

In the new study, the researchers manipulated CRY’s ability to function through another clock component, known as JET (short for Jetlag), in different circadian neurons and watched what happened. The studies show that light detection by the morning oscillators isn’t enough to keep flies going about their business in a timely way. They need those evening oscillators too.

JET’s role is bigger than expected as well. In addition to enabling cell-autonomous light sensing, the protein also allows distinct circadian neurons to talk to each other in rapid fashion after light exposure, although the researchers don’t yet know how.

The new model also suggests that flies and mammals have more similarities than had been appreciated when it comes to synchronizing their activities to the sun, the researchers say. In mammals, specific neurons of the circadian pacemaker of the brain (known as the Suprachiasmatic Nucleus or SCN) receive light input from the retina. Those cells then communicate with pacemaker neurons, which resets the circadian network as a whole.

Study Connects Sleep Deficits Among Young Fruitflies to Disruption in Mating Later in Life

Mom always said you need your sleep, and it turns out, she was right. According to a new study published in Science this week from researchers at the Perelman School of Medicine at the University of Pennsylvania, lack of sleep in young fruit flies profoundly diminishes their ability to do one thing they do really, really well – make more flies.

The study, led by Amita Sehgal PhD, professor of Neuroscience and a Howard Hughes Medical Institute (HHMI) Investigator, links sleep disruption in newborn fruit flies with a critical adult behavior: courtship and mating.

The team, addressed sleep in the very youngest of flies. “These flies sleep considerably more than adults and that behavior repeats across the animal kingdom,” Sehgal says. “Infant humans, rats, and flies, they all sleep a lot.”

Co-author Matthew Kayser, MD, PhD, in the Department of Psychiatry and Center for Sleep and Circadian Neurobiology, whose research centers on the link between sleep disruption and human neuropsychiatric diseases, used the fly – which is far more genetically pliant than mammals — to ask two basic questions: Why do young animals sleep so much? And, what is the implication of altering those patterns?

The team used genetically manipulated flies to show that young flies normally produce relatively little dopamine – a wake-promoting neurotransmitter — in certain neural circuits that feed into the sleep-promoting brain region called the dorsal fan-shaped body (dFSB). Premature activation of those circuits profoundly inhibits the dFSB, reducing sleep.

That answers the first question, Sehgal explains: Young flies make less dopamine, which keeps the dFSB active and sleep levels high. These animals sleep more than adults and are harder to rouse from sleep.

Some clues to the second question – what is the consequence of sleep loss – came from Kayser’s finding that increased dopamine in young flies not only causes sleep loss, but also affects their ability to court when they’re older. “The flies spend less time courting, and those that do usually don’t make it all the way to the end,” Sehgal says.

To address whether sleep loss in young flies affects development of courtship circuits, the team investigated a group of neurons implicated in courtship. One particular subset of those neurons, localized in a specific brain region called VA1v, was smaller in sleep-deprived animals than normal flies, suggesting a possible mechanism for how sleep deprivation can lead to altered courting behavior.

That sleep-deprived flies have altered behavior is not itself a novel finding, Sehgal notes. Earlier studies from her lab and others used mechanical disruption to alter sleep patterns, but in the current study, Sehgal’s team was able to drill down to the specific neural network that is affected. “We identified the circuit that is less active in young flies. If you activate that circuit, you disrupt courtship by impairing the development of a different, courtship-relevant circuit.”

The question now is how these findings relate to human behavior – Kayser’s original question. Though no direct lines can be drawn, the study “does provide the first mechanistic link between sleep in early life and adult behavior,” says Sehgal.

Existence of new neuron repair pathway discovered

Most of your neurons can’t be replaced.

Other parts of your body – such as skin and bone – can be replaced by the body growing new cells, but when you injure your neurons, you can’t just grow new ones; instead, the existing cells have to repair themselves.

In the case of axon injury, the neuron is able to repair or sometimes even fully regenerate its axon. But neurons have two sides – the axon (which sends signals to other cells) and the dendrite (which receives signals from other cells).

Melissa Rolls, an associate professor of biochemistry and molecular biology at Penn State and director of the Huck Institutes’ Center for Cellular Dynamics, has done extensive comparisons of axons and dendrites – culminating recently in a paper published in Cell Reports.

“We know that the axon side can repair itself,” says Rolls, “and we know a bunch of the molecular players. But we really didn’t know whether neurons have the same capacity to regenerate their dendrites, and so that’s what we set out to find in this study.”

“Our lab uses a Drosophila model system, where the dendrites are very accessible to manipulation,” she says, “so we decided that we would start by removing all the dendrites from the neurons to see if they could regenerate. We didn’t start with anything subtle, like taking off just a few dendrites. We said ‘Let’s just push the system to its maximum and see if this is even possible.’ And we were surprised because we found that not only is it possible, it’s actually much faster than axon regeneration: at least in the cells that we’re using, axon regeneration takes a day or two to initiate, while dendrite regeneration typically initiates within four to six hours and it works really well. All the cells where we removed the dendrites grew new dendrites – none of them died; so it’s clear that these cells have a way to both detect dendrite injury and initiate regrowth of the injured part.”

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Fruit flies, fighter jets use similar nimble tactics when under attack

When startled by predators, tiny fruit flies respond like fighter jets – employing screaming-fast banked turns to evade attacks.

Researchers at the University of Washington used an array of high-speed video cameras operating at 7,500 frames a second to capture the wing and body motion of flies after they encountered a looming image of an approaching predator.

“Although they have been described as swimming through the air, tiny flies actually roll their bodies just like aircraft in a banked turn to maneuver away from impending threats,” said Michael Dickinson, UW professor of biology and co-author of a paper on the findings in the April 11 issue of Science. “We discovered that fruit flies alter course in less than one one-hundredth of a second, 50 times faster than we blink our eyes, and which is faster than we ever imagined.”

In the midst of a banked turn, the flies can roll on their sides 90 degrees or more, almost flying upside down at times, said Florian Muijres, a UW postdoctoral researcher and lead author of the paper.

“These flies normally flap their wings 200 times a second and, in almost a single wing beat, the animal can reorient its body to generate a force away from the threatening stimulus and then continues to accelerate,” he said.

The fruit flies, a species called Drosophila hydei that are about the size of a sesame seed, rely on a fast visual system to detect approaching predators.

“The brain of the fly performs a very sophisticated calculation, in a very short amount of time, to determine where the danger lies and exactly how to bank for the best escape, doing something different if the threat is to the side, straight ahead or behind,” Dickinson said.

“How can such a small brain generate so many remarkable behaviors? A fly with a brain the size of a salt grain has the behavioral repertoire nearly as complex as a much larger animal such as a mouse. That’s a super interesting problem from an engineering perspective,” Dickinson said.

The researchers synchronized three high-speed cameras each able to capture 7,500 frames per second, or 40 frames per wing beat. The cameras were focused on a small region in the middle of a cylindrical flight arena where 40 to 50 fruit flies flitted about. When a fly passed through the intersection of two laser beams at the exact center of the arena, it triggered an expanding shadow that caused the fly to take evasive action to avoid a collision or being eaten.

With the camera shutters opening and closing every one thirty-thousandth of a second, the researchers needed to flood the space with very bright light, Muijres said. Because flies rely on their vision and would be blinded by regular light, the arena was ringed with very bright infrared lights to overcome the problem. Neither humans nor fruit flies register infrared light.

How the fly’s brain and muscles control these remarkably fast and accurate evasive maneuvers is the next thing researchers would like to investigate, Dickinson said.

Moonwalker Flies Backing Up

Most land animals walk forward by default, but can switch to backward walking when they sense an obstacle or danger in the path ahead. The impulse to change walking direction is likely to be transmitted by descending neurons of the brain that control local motor circuits within the central nervous system. This neuronal input can change walking direction by adjusting the order or timing of individual leg movements.

Screening for flies with altered walking patterns

In the current study, Dickson and his team aimed to understand the fly’s change in walking direction at the cellular level. Using a novel technology known as thermogenetics, they were able to identify the neurons in the brain that cause a change in locomotion. Their studies involved screening large numbers of flies with it which specific neurons were activated by heat, producing certain behaviors only when warmed to 30°C, but not at 24°C . Analysing several thousand flies, the researchers looked for strains that exhibited altered walking patterns compared to control animals.

Moonwalker-neurons control backward walking

Using the thermogenetic screen, the IMP-researchers isolated four lines of flies that walked backward on heat activation. They were able to track down these changes to specific nerve cells in the fly brain which they dubbed “moonwalker neurons”. They could also show that silencing the activity of these neurons using tetanus toxin rendered the flies unable to walk backward.

Among the moonwalker neurons, the activity of descending MDN-neurons is required for flies to walk backward when they encounter an obstacle. Input from MDN brain cells is sufficient to induce backward walking in flies that would otherwise walk forward. Ascending moonwalker neurons (MAN) promote persistent backward walking, possibly by inhibiting forward walking.

“This is the first identification of specific neurons that carry the command for the switch in walking direction of an insect”, says Salil Bidaye, lead author of the study. “Our findings provide a great entry point into the entire walking circuit of the fly. “
Although there are obvious differences in how insects and humans walk, it is likely that there are functional analogies at a neural circuit level. Insights into the neural basis of insect walking could also generate applications in the field of robotics. To date, none of the engineered robots that are used for rescue or exploration missions can walk as robustly as animals. Understanding how insects change their walking direction at a neuronal level would reveal the mechanistic basis of achieving such robust walking behavior.

(Image credit)

Study in Fruit Flies Shows that Epilepsy Drug Target May Have Implications for Brain Disorder Sleep Disruption

A new study in a mutant fruitfly called sleepless (sss) confirmed that the enzyme GABA transaminase, which is the target of some epilepsy drugs, contributes to sleep loss. The findings, published online in Molecular Psychiatry, were led by Amita Sehgal, PhD, head of the Chronobiology Program at the University of Pennsylvania’s Perelman School of Medicine. The findings shed light on mechanisms that may be shared between sleep disruption and some neurological disorders. A better understanding of this connection could enable treatments that target both types of symptoms and perhaps provide better therapeutic efficacy.

“Epilepsy is essentially an increase-in-firing disorder of the brain and maybe a decrease in activity of the neurotransmitter GABA, too,” says Sehgal, who is also a professor of Neuroscience and an investigator with the Howard Hughes Medical Institute (HHMI). “This connects our work to drugs that inhibit GABA transaminase. Changes in GABA transaminase activity are implicated in epilepsy and some other psychiatric disorders, which may account for some of the associated sleep problems.”

The team looked at the proteomics of the sss mutant brain – a large-scale study of the structure and function of related proteins — and found that GABA transaminase is increased in the sss brain compared to controls. This enzyme breaks down GABA, so GABA is decreased in the sss brain. Because GABA promotes sleep, there is a decrease in sleep in the sss mutant fly, as the name implies.  

The relationship between the SSS protein and GABA is not fully understood. The SSS protein controls neural activity, and its absence results in increased neural firing, which likely uses up a lot of energy, says Sehgal. GABA transaminase works in the mitochondria, the energy-production organelle in the glial cells of the brain, which provide fuel for neurons. The large energy demand created by the increased neural firing in sss brains probably alters mitochondrial metabolism, including GABA transaminase function in glia.

In the sss mutant fly, there is a stream of connections that leads to its signature loss of sleep: The sss mutant has increased neuron firing caused by downregulation of a potassium channel protein called Shaker. Recently, the Sehgal lab showed that SSS also affects activity of acetylcholine receptors. Both of these actions may directly cause an inability to sleep. In addition, increased energy demands on glia, which increase GABA transaminase and decrease GABA, may further contribute to sleep loss. On the other hand, if GABA is increased, then sleep is increased, as in flies that lack GABA transaminase.

The circadian clock is like an orchestra with many conductors

You’ve switched to the night shift and your weight skyrockets, or you wake at 7 a.m. on weekdays but sleep until noon on weekends—a social jet lag that can fog your Saturday and Sunday.

Life runs on rhythms driven by circadian clocks, and disruption of these cycles is associated with serious physical and emotional problems, says Orie Shafer, a University of Michigan assistant professor of molecular, cellular and developmental biology.

Now, new findings from Shafer and U-M doctoral student Zepeng Yao challenge the prevailing wisdom about how our body clocks are organized, and suggest that interactions among neurons that govern circadian rhythms are more complex than originally thought.

Yao and Shafer looked at the circadian clock neuron network in fruit flies, which is functionally similar to that of mammals, but at only 150 clock neurons is much simpler. Previously, scientists thought that a master group of eight clock neurons acted as pacemaker for the remaining 142 clock neurons—think of a conductor leading an orchestra—thus imposing the rhythm for the fruit fly circadian clock. It is thought that the same principle applies to mammals.

Interactions among clock neurons determine the strength and speed of circadian rhythms, Yao says. So, when researchers genetically changed the clock speeds of only the group of eight master pacemakers they could examine how well the conductor alone governed the orchestra. They found that without the environmental cues, the orchestra didn’t follow the conductor as closely as previously thought.

Some of the fruit flies completely lost sense of time, and others simultaneously demonstrated two different sleep cycles, one following the group of eight neurons and the other following some other set of neurons.

"The finding shows that instead of the entire orchestra following a single conductor, part of the orchestra is following a different conductor or not listening at all," Shafer said.

The findings suggest that instead of a group of master pacemaker neurons, the clock network consists of many independent clocks, each of which drives rhythms in activity. Shafer and Yao suspect that a similar organization will be found in mammals, as well.

"A better understanding of the circadian clock mechanisms will be critical for attempts to alleviate the adverse effects associated with circadian disorders," Yao said.

Disrupting the circadian clock through shift work is associated with diabetes, obesity, stress, heart disease, mood disorders and cancer, among other disorders, Yao says. The International Agency for Research on Cancer classified shift work that disrupts circadian rhythms as a human carcinogen equal to cancer-causing ultraviolet radiation.