Waking up to the new science of sleep

Like many children from ambitious south Indian families, Sridhar Jagannathan studied engineering at Anna University due to parental pressure. His electronics degree got him a job at the German company Bosch, but Jagannathan moved to the Netherlands after four years of work to study biomedical engineering and cognitive neuroscience. Then, halfway into a PhD programme at the University of Cambridge, he made a small but critical switch in his choice of research topic.

Jagannathan had joined his PhD programme with a grounding in engineering, neuroscience, and some clinical medicine. He wanted to use this knowledge to study the human brain.

Specifically, he had questions about the process of falling asleep. How do people lose consciousness as they drift into sleep? Can this process be described in mathematical terms? He started his PhD in 2015 with hopes of using his expertise to model transitions of consciousness in human beings.

As he advanced through the programme, Jagannathan realised that the human brain was too complex to deconstruct using available technology. Why not study smaller organisms, he thought, and then move on to humans? Jagannathan told his PhD advisor Tristan Bekinschtein about his changing ideas, and Bekinschtein asked him to go to the lab of Bruno van Swinderen at the Queensland Brain Institute (QBI) in Australia.

Van Swinderen is an experimental neurobiologist with a significant body of work on sleep in fruit flies (see 'The brain knows what kind of sleep it needs'). Jagannathan spent a few months in the Australian lab just before COVID-19 started ravaging the world. During this time, scientists in the lab, along with Jagannathan, established that electrical activity in the sleeping fly brain was not uniform, and that insects, like humans, had different stages of sleep.

They accomplished this with an experiment of exquisite precision: by inserting 16 electrodes in the tiny brain of a fly, which survived long enough to give them cues about at least four different sleep stages.

For at least a century, fruit flies have been ideal models for understanding several aspects of biology. Such research has resulted in many breakthroughs and five Nobel Prizes. Fruit flies share with humans 75% of the genes that cause disease. However, sleep researchers had not been able to monitor the brain of the sleeping fly in fine detail. This changed when van Swinderen and his colleagues inserted an electrode every 25 micrometres (one-fortieth of a millimetre) of the fly brain, thereby covering the entire organ. The experiment helped them gather electrophysiological data continuously for 20 hours.

Sleep has been one of biology's greatest mysteries. During sleep, the brain undergoes a complex set of processes to ensure that the organism wakes up feeling refreshed. Although sleep is known to consolidate memory and repair tissues, apart from clearing waste in the brain its exact purpose is still a puzzle. Sleep experiments involving human beings are not easy. With animals increasingly used to study sleep, researchers now expect an explosion of data — and hence knowledge — on sleep.

It took the Australian scientists two years to analyse 20 hours' data from the fruit fly. Their analysis showed that flies have four distinct sleep stages: pre-sleep, early sleep, mid-sleep, and late sleep. The fly is active during pre-sleep, but recordings show a step-down in neuronal activity as the insect prepares itself for sleep. The other three are light to deep sleep stages. During a bout of five minutes of sleep, each stage lasts 0-2 minutes. The mid-sleep stage is like Rapid Eye Movement (REM) sleep in humans, with brain signatures resembling the waking periods.

Such results cannot be translated directly into the human situation; the same phenomenon can have different purposes in different animals. However, accumulating evidence now points to the universality of sleep in animals, raising the possibility that studying animals can improve the understanding of human sleep physiology.

"We still do not know the molecular mechanism behind anaesthesia," says Jagannathan, now at the Institute of Neurophysiology, CharitéCenter for Basic Medicine, in Berlin. "One of the reasons why Bruno's lab is studying sleep is to deepen our understanding of anaesthesia."

Some recent results on animals point to the possibilities ahead. Van Swinderen, Jagannathan and others have shown that the four stages of sleep are present in insects, and not just in mammals. In June 2023, two German groups — the Max Planck Institute for Biological Intelligence and Ruhr University Bochum — showed that birds and mammals have remarkably similar sleep phases. Their work, which appeared in the journal Nature Communications (go.nature.com/457HWJt), showed for the first time that bird species, too, have a waste clearance system for the brain, which gets proactive during deep sleep. Earlier, this was seen only in mammals.

In March 2023, researchers from the Max Planck Institute for Brain Research (MPIBR) in Frankfurt found coordination as well as competition between the right and left hemispheres of the brain in Australian bearded dragons, a lizard species, during REM sleep. "It could reflect a competition between hemispheres linked to visual competition between the two eyes... in animals with mostly monocular vision," says Gilles Laurent, MPIBR Director and lead author of a study which appeared in Nature (go.nature.com/44uUF94). Many reptiles and mammals, and most birds, have monocular vision.

"That was a cool paper," says Krishna Melnattur, Assistant Professor of Psychology and Biology at Ashoka University. "It is very interesting that the two hemispheres are apparently in competition. We will only appreciate it when we have more information."

Melnattur is among several scientists to have moved recently into sleep research from other fields. He finished his PhD in neural development and then did post-doctoral research on colour vision at the U.S. National Institutes of Health. He then set up his lab at Ashoka University to work on his true interest: sleep in fruit flies. In a broader sense, he is using fruit flies to understand how sleep and the environment affect each other.

Until recently, sleep research was being done only in a few academic labs in the country, the prominent ones being at the Jawaharlal Nehru University (JNU) in Delhi, the Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) in Bengaluru, and the National Brain Research Centre (NBRC) near Gurugram. This list has grown in the last decade as young scientists returned from abroad and older ones moved to new institutions.

Dhandapany Perundurai started his lab in 2016 at the Institute for Stem Cell Science and Regenerative Medicine (InStem) in Bengaluru. Among other things, he researches the connection between sleep, heart disease and stroke. In 2021, sleep researcher Birendra Nath Mallick moved from JNU to Amity University in Noida. Melnattur set up his lab at Ashoka University in 2022. In the same year, Nitin Chouhan, a neuroscientist by training, set up a sleep lab at the Tata Institute of Fundamental Research in Mumbai to study the link between sleep and cognitive function. Earlier this year, after a PhD from JNCASR, post-doctoral research from Johns Hopkins University and working briefly at the Central University of Punjab at Bathinda, Shahnaz Rahman Lone moved to GITAM University in Visakhapatnam to set up a sleep lab.

According to the PubMed database, the number of sleep research papers has increased nine times in the last 25 years, up from 2,763 in 1997 to 24,569 in 2022. Part of the reason is the explosive growth in research itself, especially in subjects related to health. However, it is also due to the development of sleep studies as an independent scientific discipline.

Most human beings spend one-third of their lives sleeping. This was seen as inactivity, a period in which the body and brain were resting, and so not important from a scientific point of view. The only scientific interest in sleep till the 19th century was in the interpretation of dreams.

This outlook changed in the mid-19th century. In 1845, British physician John Davy studied body temperature changes during sleep. A major technique for sleep research was developed in the 1920s when German psychiatrist Hans Berger developed electroencephalogram (EEG) and recorded brain waves during surgery.

In the same decade, physiologist Nathaniel Kleitman opened the first sleep lab at the University of Chicago. His work on sleep deprivation and health earned him the title 'father of sleep medicine'. Kleitman and his students also discovered REM sleep in 1952. This discovery was a milestone in sleep research. In fact, many sleep researchers consider the discovery of REM sleep as the beginning of real sleep medicine.

Sleep medicine continued to advance through the second half of the 20th century, with labs worldwide studying obstructive sleep apnoea, the world's most common sleep disorder. The field expanded through the 21st century, with the recognition of deep connections between sleep and metabolic and neurodegenerative diseases.

Through all these developments, sleep research remained a branch of medicine. Mainstream biologists became interested in sleep only by the end of the 20th century. The mindset changed when scientists gradually realised, over a few decades, that sleep was not rest but a phase of high activity.

"Many basic researchers considered it a boring problem to study even though they knew sleep deprivation has adverse effects clinically," says Melnattur. "Their perception then was that these clinical outcomes were because people were not getting adequate rest."

Lack of access made the scientific study of the human brain always difficult. It was not possible to put electrodes through the brains of people and record the activity of different parts of the brain. It was even more difficult to understand sleep at a fundamental level, using current technology, by studying sleeping human beings. For a long time, the recording of the electrical activity of a sleeping person used to be the main input for understanding sleep. This technique has limitations as very little data can be gathered from electrodes placed externally on the skull.

"It is important to record brain activity from different regions of the brain, not just surface recording," says sleep researcher Sheeba Vasu, a Professor at JNCASR.

Techniques such as actigraphy, which monitors physical movements with wristwatches and other devices and then deduces sleep patterns, also have limitations. However, it has been used widely and provided valuable data in the last decade. Correlating the two methods — EEG and actigraphy — could provide useful information compared to either acting alone, but the process requires advanced computational tools that are still being developed. Analyses of blood and cellular markers are needed in parallel. Such techniques are not used widely yet and are in their early stages of development. Scientists learn about sleep also when looking at why it goes wrong. However, people lose sleep for a variety of reasons, which are not always easy to deconstruct and synthesise.

Using animals can remove some of these limitations, especially when behaviour is used to define sleep. In flies, for instance, scientists use sleep monitors, with each one able to monitor 20-30 flies at a time. Since a typical lab will have several dozens of them, scientists can study the sleep habits of 1,000-1,500 flies at a time. Such high throughput data is impossible to get in human beings.

Similarly, the technique of electrode insertion into the brain has improved significantly in recent years. Scientists can now pinpoint a single neuron with an electrode inserted in the brain. New optical methods can measure the electrical activity of a large number of neurons (See 'The Golden Age of Neuroscience', bit.ly/neuro-gold). Also being developed are precise molecular genetic techniques that can manipulate single neurons in the animal brain.

All these techniques have brought a new level of capability to animal sleep researchers, resulting in a spate of discoveries around the world. Though a late entrant, Indian institutions have also begun to be part of this movement.

Vasu started her PhD work in chronobiology at JNCASR late in the 20th century by working with evolutionary biologist Amitabh Joshi and chronobiologist M.K. Chandrashekaran, a pioneer of the discipline in the country. Chronobiology is the study of rhythms in living organisms and their timing processes, sleep being one of the processes.

After setting up a lab at the Madurai Kamaraj University in the 1980s, Chandrashekaran discovered the social synchronisation of circadian rhythms in bats. He had also worked on fruit flies, where he had discovered the clock that determines the hatching of eggs.

Vasu finished her PhD in chronobiology at JNCASR in 2002, working on fruit flies. She had shown at that time how flies retain their time cues even when deprived of normal light-dark cycles. She took flies that had not seen darkness for 600 generations and put them through normal light-dark cycles once again. They regained their time cues easily. "It seemed to be hard-wired in these organisms," Vasu says. "It was almost as if they would lose some critical life processes if they lost their time cues."

Vasu returned to JNCASR in 2009, after post-doctoral stints in New York University and University of California in Irvine. While in the U.S., her attention shifted to the neurobiology of circadian rhythms, such as neural circuits and neurotransmitters involved in time-keeping. Over the next decade, she focused on the neurobiology of sleep in fruit flies.

In a study published in 2018, her team characterised a neuropeptide called Pigment Dispersing Factor, produced in the circadian pace-making neurons, and studied its role in keeping organisms awake during the day. In her most recent study, published in June this year, Vasu and her colleagues showed how sleep and circadian rhythms could be restored in Huntington's Disease, which involves sleep disorders, among other things (see 'Eyes wide shut').

In a study published in 2021 (bit.ly/rock-sleep), Lone at GITAM University, in collaboration with Vasu, unravelled how signals relating to sleep from limbs of insects, which are rocked to slumber, are despatched to a part of the brain and which are then collectively transmitted to the sleep centre in the brain. Such studies have proved that animals share many of the sleep features of human beings.

Examining sleep in animals can have direct applications as well. Vasu, for example, is trying to use such insights in fruit flies to develop sleep-modulating substances in humans. Melnattur's recent work, mostly done with collaborators abroad before he returned to India, has shown how sleep can restore learning deficits in flies.

Melnattur had worked at Washington University in St. Louis with Paul Shaw, a Professor of Neuroscience at the university, who had been studying the relationship between sleep and cognition among animals. One of his research projects is to use fruit flies to understand how sleep influences neural plasticity. In an experiment in which Melnattur was also involved, the university scientists used a thermal maze — a grid of 64 tiles where most tiles were kept warm while a few were cold. In the experiment, the flies had to find and move to the cooler ones after learning from visual cues provided initially and subsequently withdrawn. Flies that learnt the visual cues associated with the location of the cooled spot consistently and quickly got there first, whereas flies that failed to learn the visual cues took significantly longer to perform the task.

Older flies and those who lacked a gene for an enzyme called rutabaga — which mediates synaptic plasticity — were not able to perform this spatial learning assay well. But performance levels improved in the older and mutant flies when deep sleep was induced using an experimental sleep-aid drug.

"What it showed was that sleep is not only important for consolidating memories in a healthy brain, but it can also be vital for restoring functions to a brain that is impaired," says Melnattur, who is setting up a similar infrastructure at Ashoka University to study sleep's role in improving spatial memory.

Universality is one of life's guiding principles. All life is organised around a few kinds of molecules, such as nucleic acids (DNA and RNA), proteins and fats. The genetic code for synthesising proteins remains the same throughout all organisms, plants and animals.

All animals and plants are made of cells. At a higher level, living beings grow and change, respond to the environment, and pass their genes to the next generation. It now turns out that most animals, if not all, sleep.

This fact was not known or fully appreciated till recently when animal experiments began to bring new insights. "For long, sleep was viewed from a human and mammalian-centric point of view," Melnattur says. "That changed in the last 20-odd years."

Some of the results have been truly surprising. Till 2017, the widespread scientific view was that only animals with a brain would sleep. But this notion was shattered when Caltech researchers — graduate students Ravi Nath, Claire Bedbrook and Michael Abrams in the labs of Paul Sternberg, Viviana Gradinaru and Lea Goentoro — serendipitously discovered that jellyfish, which has a distributed nervous system with no central command, also sleeps. This finding implied that sleep was an ancient behaviour, largely untouched by millennia of evolution.

Animals and plants have a phenomenon called exaptation, a process by which a phenomenon acquires purposes for which it was not originally designed.

Sleep can be no different. It is possible that some purposes of sleep in animals are different from those in human beings. And yet, there are many places where they overlap, and they have applications for a sleep-deprived society.

See also:

Eyes wide shut

'The brain knows what kind of sleep it needs'