The intersection of circadian rhythms and the sleep-wake cycle

Most people feel refreshed by a good night's sleep. Sleep is vital for human and animal health, and lack of sleep leads to mental distress, poor cognitive ability and mood, and in the long term, to mental illness. In the body, a lack of sleep can produce immune and metabolic disorders. In fact, it seems that sleep is so important that no animal has yet been discovered, whether insect, fish or mammal, that does not sleep at some point in the 24 hour cycle. Yet, we do not know either why, or how, we or animals sleep. We wish to study the mechanisms used in the brain that cause us to sleep. For many years, researchers in this area have been proposing that two urges or drives cause humans and animals to sleep. These are the "sleep homeostasis" (from the Greek, hómoios, "similar", and stásis, "standing still") and "circadian" (Latin for "about a day") drives. Sleep homeostasis is a process that may allow repair of the cellular "wear and tear" on the brain that builds up while the animal is awake. We all know the feeling that the longer we have been kept awake the worse we feel until we reach a point where we can no longer stay awake. This feeling has been called the homeostatic drive. It is envisaged as negative feedback in proportion to the amount of time awake, and it is assumed, although not proven, to be restorative. After sleep deprivation, we and animals sleep proportionately longer, and when we enter sleep, we also sleep proportionately deeper. The idea is that the longer we are awake, some metabolite(s) builds up in the brain in proportion to how long we have been awake. After a time, this metabolite (it might be adenosine or a prostaglandin) is at high concentrations in the brain and induces sleep. While asleep the metabolite is degraded, and the process then starts over again during waking hours. On the other hand, and on top of this homeostatic drive to sleep, the circadian drive ensures that, even if we have had enough sleep, we still always feel awake or sleepy at certain regular times (which vary with individuals and species). It might be 10 pm for one individual, midnight for another, or 4 AM for a mouse, but at these times we have an urge to sleep independent of how much sleep we have already had. Various other physiological functions also have different rates depending on the time of day, such that temperature, reaction time, thinking ability, metabolic activity of muscle, liver and heart all cycle, and have some optimal point(s) during the day or night. For sleep, the classical proposal is that the circadian and homeostatic drives add or subtract from each other to determine the overall amount and depth of sleep. These two things converge with shift work (working at the wrong time) and sleep loss. It has been widely accepted that the two sleep drives are controlled by separate mechanisms. However recently, it has been found that mice with changed circadian drives also have altered homeostatic sleep drives. Researchers are beginning to question the independence of these two sleep processes. It has also been discovered that most cell types in the brain and body have their own circadian clocks. We wish to examine one cell type in mice where the classical circadian and homeostatic sleep drives seem to converge: these are specialized neurons that release histamine (an excitatory neurotransmitter) and gamma-aminobutyric acid (an inhibitory neurotransmitter) during the day, but not the night, and which help keep us and animals awake. We are going to use several genetic methods to disrupt the circadian process only in histamine neurons, and not other types of cell, and then examine the consequences of this using mild sleep deprivation tests. We predict the homeostatic sleep process might be disrupted, because, in fact, the two components previously regarded as distinct mechanisms (homeostatic and circadian), may not really be separate. This work will be a step in helping us understand more about how we sleep.

Mammalian sleep is hypothesized to involve two independent drives: circadian and homeostatic. These drives are thought to arise by distinct biochemical mechanisms. For the circadian drive, the master circadian clock is in the suprachiasmatic nuclei, and determines when during the 24-hour cycle an animal or human is active or asleep. The core circadian clock contains the transcription factor Bmal1. Mice without Bmal1 have no circadian rhythm. Sleep homeostasis means that after sleep deprivation, there is a recovery of sleep debt, with higher delta power in non-REM sleep and longer sleep times. The currently accepted model is that at sleep onset, and throughout sleep, a sustained GABAergic drive onto arousal neurons, such as histaminergic neurons, stops them firing. Previously, however, we found that the GABA drive onto histamine neurons is dispensable for sleep-wake control in mice. In preliminary work for this application, we found that histaminergic neurons express the circadian Bmal1 and clock control proteins, whose levels oscillate daily, and that mRNA levels of the histamine synthesising enzyme histidine decarboxylase (HDC) also have a daily cycle; further, we have confirmed earlier work that histaminergic neurons are GABAergic, and have found that GAD67 mRNA levels oscillate daily in these cells. We hypothesize that the sleep-wake cycle and homeostatic regulation of sleep partly depends, not on GABAergic input, but is cell-intrinsic under the control of Bmal1. Using conditional Cre-lox histaminergic-selective knockouts of Bmal1 (clock removal) and casein kinase 1 tau (clock speed), and selective knockdown of the vesicular GABA transporter (clock coherence?), we will test the significance of the local circadian clock in histaminergic neurons for the sleep-wake cycle. Our work might reveal how a local circadian clock influences brain function.

1. Who might benefit form this research? In the long term our proposed work might impact on: the wider public; life scientists (see Academic Beneficiaries in the above box); the life science industry.
2. How might they benefit from this research? "Sleep is the most important predictor of how long you will live - perhaps more important than smoking, exercise or high blood pressure". This quote is widely attributed to Prof. William C. Dement (Stanford School of Medicine, Stanford University), the scientist co-credited with discovering REM sleep. Whether one agrees with Dement's statement or not, there is no doubting that we humans, our pets and our farm stock all require sleep for full health. Yet, scientifically, and surprisingly, there is a great deal that we don't know about the sleep process. Prof. Clifford Saper (Harvard Medical School) thinks that "the way in which sleep is restorative and why brain function is impaired in its absence remain among the most enduring mysteries of neuroscience" [Saper, C.B. et al. Neuron 68, 1023 (2010)]. Thus understanding how sleep is caused and maintained is an important research endeavour, whose outcome will eventually underpin improved health and healthy ageing. Our work will also enhance the research capacity and knowledge of private sector organizations. In a recent report in the New York Times (May 6th, 2012) Tom Brady wrote: "10 to 20% of the world's population uses sleeping pills or tranquilizers, according to Global Industry Analysts, a worldwide market research firm. It estimates the global market for sleep aids will be worth $9 billion by 2015". Additionally, there is much interest in developing drugs that promote wakefulness or alertness. Thus any parts of the Life Science Industry developing sleep medicines and drugs to aid sleep or wakefulness (e.g. for shift work) would find the knowledge we discover potentially useful for drug discovery and development.
3. What will be done to ensure that beneficiaries have the opportunity to engage with this research?
The general public and other academic scientists: We will publish in peer-reviewed open-access journals, and present at conferences (national, British Neuroscience Association and the "Clock Club") and international (Society for Neuroscience, Federation European Neuroscience Associations, European Biological Rhythm Society). We will issue press releases to explain our peer-reviewed publications - Imperial College and LMB are strong in this activity and their websites are daily advertising new research from the many researchers. MHH has been on the radio talking about circadian rhythms, and has organized a Royal Society exhibition on this topic, and NFP's work has often been featured in the media. Imperial College holds an annual Festival weekend (in May) at South Kensington (next to the Natural History and Science Museums) where members of the public come into the College for interactive and fun scientific displays in the campus grounds. Last year some members of WW's and NPF's lab took part, and we will plan to do a "sleep stall" in future years. By Googling "Imperial College" and "Festival" you can see a movie with highlights of this year's festival events. The Natural History Museum also runs late ("party") nights for the public, and the museum staff often ask Imperial to provide installations. We would aim to do a "sleep installation" at one of these events.
Industry: Franks and Wisden supervise a PhD student (BBSRC CASE studentship) with Keith Wafford at Ely Lilly (Surrey, UK); the student is working on histamine neurons, although not on the work proposed here. At our regular meetings with Dr Wafford, we will update him on the progress of the work proposed in the current application.