How does light control the activity and electrical properties of neurons integrating arousal behaviour, circadian rhythms, and sleep?

Life on our planet is exposed to the regular changes of night and day. As a consequence animals have evolved a 24 hour timing mechanism, the so-called 'circadian clock', which tunes our behaviour and physiology to the day-night cycle (e.g. sleep-wake cycles, metabolism). An animal's clock still ticks when it lives in continuous darkness (i.e. a cave). Circadian clocks work similar to a normal clock, e.g. they run at a steady pace (with a 24hr period) and can be reset if they go wrong. In nature this resetting is caused by the environment, i.e. the regular changes of light and dark. As a consequence, circadian rhythms are synchronized with the environment. Famous examples for both the independence of circadian clocks and their ability to communicate with the environment are jetlag (caused by travel across time zones) or shift work. If you are jetlagged, your circadian clock is still ticking according to the time where you boarded your plane and is telling you to be awake in the middle of the night. Gradually though, your internal clock will adjust (synchronize) to the new time zone and you will feel comfortable again.

Circadian clocks consist of clock genes that switch on and then switch themselves off every 24hrs in the clock neurons of the brain. Classical genetic experiments mostly performed using the fruit fly Drosophila identified nearly all the clock genes and the clock mechanism that was later found to be similar in humans. However, it is still not known how all the parts are integrated to form a working clock sensitive to light and able to orchestrate the physiology and behaviour of the whole animal: this is the overall aim of the current proposal.

The fly clock consists of 150 neurons that communicate with each other via electrical and chemical signals that are thought to synchronise rhythms between these neurons generating the overall circadian behaviour. The number and frequency of these impulses (called electrical activity) controls the flow of information in the clock circuit, much like a computer. We wish to study the proteins involved in relaying these signals (called channels, co-transporters and receptors) between the clock neurons, which in turn control circadian behaviour and sleep. We know that the clock is exquisitely sensitive to light so we are interested in the proteins that transmit information about the light conditions to and around the clock. We previously isolated a light sensitive clock protein called Cryptochrome (Cry) in flies, which later was shown to have important circadian functions from plants to humans. Again we have used the power of fly genetics and discovered another key light sensor called Qsm, a channel called Shaw, a receptor called GABAA and co-transporter called NKCC that all influence the clock. In this proposal we want to work out how they fit together to control circadian behaviour, arousal and sleep. This will be achieved by answering the following experimental questions:
1) What is the mechanism by which light activates Qsm and causes molecular and behavioural synchronization of the clock? We will go on to see if the human version of Qsm can also function in the clock.
2) How do Qsm, Shaw and NKCC interact with each other in the clock?
3) How does Shaw generate electrical signals in the clock neurons and how are these affected by light and Qsm? This will involve placing small electrodes on the clock neurons in the fly's brain and recording their electrical signals under different light conditions.
4) How do Qsm, NKCC and GABAA receptors act together in the clock to control arousal and sleep?

This research will help us to better understand the effect of light on circadian clocks and sleep identifying new potential targets for treatment of sleep disorders and jetlag. Research into circadian clocks and sleep is important as they profoundly affect our health, productivity and quality of life.

Synchronization of circadian clocks with the environment is important for optimal performance of animals. Our proposal addresses the question how light synchronizes clocks but also affects fly behaviour directly. We focus on the clock regulated gene qsm, previously isolated in our lab. Qsm acutely responds to light via a Cry-independent mechanism, defining a potential new light-input to the clock. Our preliminary data suggest that Qsm interacts with the Kv channel Shaw, involved in regulating rhythmic electrical properties in fly and mammalian clock neurons. Qsm also interacts with a Na+ K+ 2Cl- co-transporter (NKCC), whose mammalian homologue is expressed in clock neurons and circadianly regulates GABA responses. This potential new way of signalling light information to the clock (light > Qsm > Shaw/NKCC > neuronal activation and intracellular signalling) will be the focus of the proposal.

In particular we will study Qsm and the role of Shaw and NKCC in relation to two clock neuron groups, (i) the large-LNv, which integrate arousal, sleep, and clock input, and (ii) the DN, known to mediate Qsm-dependent light input to the clock. We will (1) study the function of modified Qsm proteins and that of its putative human homologues. Are they able to respond to light, and to signal this information to the clock? Which domains of Qsm are important for this? We will then (2) verify the interactions of Qsm with Shaw and NKCC and determine how they contribute to Qsm function and the effects of light on behaviour. These molecular and genetic interaction studies will be extended to a mechanic level by (3) performing a detailed electrophysiological characterization of the l-LNv and DN. How do neuronal electrical properties respond to light changes and how are they influenced by Qsm, Shaw, and NKCC? Since we have evidence for a role of Qsm and NKCC in arousal and GABA signalling, we will (4) elucidate their contribution to light-dependent changes of [Cl-]i in the l-LNv and DN.

The project is relevant for a general understanding of circadian clocks, which also control numerous physiological and behavioural processes in humans. Proper circadian clock function and synchronization with the natural environment contributes to our well being as dysfunction (or desynchronization) can cause severe sleep disorders and depression.

Research on body clocks and sleep are of great interest to the general public and specifically the large and ever-increasing proportion (about a third) of the population who have sleep problems. Generally sleep disorders are poorly treated therefore our research on clock endogenous channels (N.B. a third of all drugs in development target channels) will provide excellent targets for novel drugs to treat these disorders. The outcome of this research will impact on the pharmaceutical industry that focuses on this area of research. Furthermore by studying light input mechanisms to the clock, it might be possible to design behavioural therapies to improve sleep i.e. light exposure to treat seasonally affected depression (SAD) or darkening a person's room to help them to sleep. By studying the genetic basis of these chronotypes it will be possible in future to sequence a person's genome (~£1000) and work out the best treatment for their sleep disorder (personalized medicine). Circadian rhythms and sleep become weaker and more fragmented with age. With an aging population more and more people will suffer the consequences of poor rhythms and sleep, therefore circadian research influences "Ageing research: lifelong health and wellbeing" (BBSRC strategic priority), a better understanding of changes in these processes over time will inform doctors of how to assess the implications of circadian rhythms on the treatment options for their patients.

Detailed and basic understanding of circadian clock synchronization is also important to better adjust the problems related to shift work (~20% of the working population), jet lag and 24/7 culture both in terms of creating 'clock-friendly' work environments (e.g. sufficient illumination, designed time-tables), as well as treatment of people who suffer from it (e.g. light-therapy). Increased knowledge in this area will impact the health and safety recommendations of government organisations regarding workplace requirements that need to provide a healthy working environment.

Moreover, environmental disruption of body clocks alone or compounded by clock gene mutations can lead to sleep disorders, obesity, cancer, psychiatric disorders such as SAD, bipolar disorder and addiction which increasingly is becoming a burden on the NHS. Circadian rhythms influence the symptoms an individual experiences such as migraine, pain and asthma. While heart attacks are more likely to occur in the morning. Furthermore drug efficacy varies with circadian time i.e. chemotherapy is given at certain times of day. In addition, in Morvan's Syndrome, patients exhibit marked sleeplessness associated with Kv channel dysfunction which if untreated leads to death. Therefore this fundamental circadian research will help us understand how disruption of clocks can have negative consequences for the health and wellbeing of individuals and will inform various areas of biomedical research that are extrinsically link to the body clock. The potential impact of realizing the benefits of working with a healthy circadian regime is evident increasing the productivity of the public sector, industry, business, general public and schools. Workers and children alike do better during their working day after a good night's sleep therefore circadian and sleep research can generally inform and improve UK economic productivity and societal health and well-being. This could be brought about by our public engagement and internship activities that help influence public policy and legislation bring about operational and organizational change.