Genetically defined neurons at the intersection of the sleep and circadian systems

Sleep is a universal animal behaviour. Its occurrence is tightly interwoven with the body's circadian clock, which in turn, is a natural adaptation that accounts for planet Earth's rotation, or in other words: daylight and darkness. Sleep is a fascinating phenomenon, because of its similarities with unconsciousness and for the appearance of dreams. Mammalian sleep is a complex behaviour that can be divided into two main types, non-rapid-eye movement sleep (NREM) and rapid eye movement sleep (REM). The functional significance of sleep in its many forms is being heavily investigated but remains poorly understood. Nonetheless, it is accepted that the brain benefits the most from sleep: NREM sleep provides the conditions for cellular recovery, neural circuit maintenance and consolidation of memories. At organism level, extended periods of continuous brain activity result in a "need" for sleep, which is proportional to the time spent awake. It is also intuitively clear that there is a propensity to restrict sleep to the night, or to the day in nocturnal animals. Nocturnal sleep is referred to as the "circadian" component of sleep, whilst the need for sleep as the "homeostatic" component of sleep. Our aim is to identify the points of contact where the sleep and the circadian systems interact. By investigating the existence and architecture of synaptic connectivity among genetically defined neuronal populations, we hope to understand how the body clock influences sleep and how external cues about circadian time interact with clock and sleep circuits; similarly, we hope to identify the structural underpinnings allowing for the need for sleep to reset the phase of the body clock. We have recently discovered a gene that is required to maintain a normal balance between the animal's activity and rest periods. Mice lacking expression of the gene display an early onset of the active phase of the circadian rhythm and a significant loss of rest-time. The gene is only expressed by a small fraction of neurons and at the time when these take up their final position in the brain and establish connections with other neuron types. Interestingly, we also noticed that major sleep and circadian regulatory areas in the brain do not express the gene, but, we hypothesize, they may require information from neurons that do. We are therefore confident that we now have the right tools and experimental conditions to discover novel, genetically defined, neuronal cell types required to maintain the balance between sleep, activity and their coordination with circadian time. As human lifespan increases, efforts should be made so that "healthspan" increases too. The normal process of ageing is very often accompanied by a decline in sleep quality and anticipation of the circadian rest-activity rhythm, which both contribute to the deterioration of life quality in elderly people: an intriguing correlation with our mutant mouse behaviour. More generally, social jet lag and sleep hygiene are terms increasingly used to indicate the cause and remedy to a variety of modern age human ailments, which include obesity, cardiovascular disorders and depression. Yet, there are cases when the causes of a disrupted circadian rhythm, or sleep, are not found in an individuals' life style, but in their neurons' connectivity and function. A disruption of the circadian and sleep systems is very often observed in common psychiatric disorders, including schizophrenia, bipolar disorder and Alzheimer's. This correlation is so significant that many scientists and clinicians now hypothesize that the same neuronal circuits that are affected in these psychiatric disorders are also involved with regulation of sleep and circadian rhythms. Hence the importance of basic research into the neural networks regulating circadian behaviours.

Despite it being acknowledged, a neuroanatomical substrate for the interaction of the sleep and circadian systems has not been found yet. Our aim is to identify genetically defined neurons in circuits that support the alternation of sleep and wake and its coordination with the circadian clock. A significant recent discovery in circadian neuroscience is the identification of intrinsically photosensitive retinal ganglion cells (ipRGCs), which sense light via a melanopsin photoreceptor and are required to entrain the phase of the endogenous circadian clock at the suprachiasmatic nucleus (SCN). Sleep regulation by ipRGCs has been proposed, but the underling brain circuitry remains unknown. While many ipRGCs target the SCN directly, some also project to other brain regions. We hypothesize that non-SCN ipRGC-targets may integrate inputs that are important for the sleep-wake cycle and act to modulate the activity of known sleep centres and the SCN. Many of these non-SCN targets express the Sox14 gene. We studied the consequences of Sox14 inactivation on robustness of photoentrainment of circadian rhythms. We have then made preliminary observations that are suggestive of disrupted sleep in these mutant mice. A strength of this proposal lies in the availability of a new Sox14cre/+ knock-in mouse line that allows for the specific targeting of the neurons that are ultimately responsible for the observed phenotypes with new viral tools for circuit tracing and manipulation. Changes in sleep and circadian behaviour will be monitored with electrophysiological recordings and correlated with localized, genetically driven cell ablation, in freely behaving mutant mice. In conclusion, we think that recent discoveries, including our own, and availability of new tools give us the opportunity to progress further in the understanding of regulatory networks underlying biological rhythms and sleep in particular.

Up to a quarter of the population in the western world reports some kind of sleep disturbance. Reported cases of sleep disturbances increase to more than 40% in the elderly population (Foley et al. 1995; Sleep. pp: 425-32. Sleep complaints among elderly persons: an epidemiologic study of three communities.), becoming the norm in people suffering from neurodegenerative Alzheimer's and Parkinson's diseases. The roots of this are multiple and can be found in an increased disconnection between the biological circadian clock and socially driven daily activities, or in underlying genetic allelic types or in comorbid psychiatric and neurodegenerative conditions. Whatever the causes, the costs to society are high, as reported in a recent study (Institute of Medicine. Washington, DC: The National Academies Press; 2006 Sleep Disorders and Sleep Deprivation: An Unmet Public Health Problem.). The dissection of the neuronal circuitries that link circadian rhythms, sleep and the environment (i.e. environmental light), for which we hereby seek BBSRC funding, will contribute to increase awareness in general practitioners and consultants in the Health System about the deleterious consequences that disconnection of circadian rhythms and sleep have on well-being. The precedent exists of basic research on light regulation of circadian rhythms, which rapidly contributed to the implementation of cost effective light-therapy protocols to ameliorate symptoms ranging from poor sleep quality to depression. The introduction to the common language of the term "sleep hygiene" is an example of where sleep research has been rapidly translated in a cost effective and easily implementable way to improve life quality. Notably, drug development was not required to translate this type of research into therapy. The government can benefit from our proposed research too: policy makers often face the challenge of informing the public of the validity and returns of research conducted in part with government's tax revenue. We believe that research on sleep and circadian rhythms has the inherent property of being easily relatable to by the general public: we have all heard of grandparents complaining of how early in the morning they wake up, or read the distressing news of an accident happening because of "tiredness" of those involved. This is also reflected by the ease at which daily newspapers and television channels report on scientific discoveries on sleep and circadian rhythms. We feel therefore that our research can provide a case to help policy makers and government agencies in feeding back to the public examples of publicly funded basic research with a clear impact on everyday life. A further step towards public engagement will occur via my affiliation with the Society of Biology, a UK charity with the aim of influencing government policy on science and public engagement. We foresee a direct benefit towards maintaining and enhancing the international competitiveness of UK's academic institutions (KCL in particular): our research is centred on the application of a new technique based on the modified rabies virus to trace and manipulate neuronal connectivity in vivo. Academic research in the UK has so far lagged behind other countries in implementing this technological development. I have gained in-depth knowledge on this technique as an EMBO-funded visiting scientist in the lab of Prof. Roska, at the FMI, Basel, CH, at the forefront of research into new virus-based circuit-tracing technology. King's College London and the Institute of Psychiatry are committed to introduce the rabies-based technology to their research community, which I shall coordinate. Funding from the BBSRC will support the transfer of knowledge and tools from the collaborator (Prof. Botond Roska, FMI, Basel, CH) to the Institute of Psychiatry at KCL.