Distinct forebrain system regulating arousal

Lead Research Organisation: University of Manchester
Department Name: School of Medical Sciences

Abstract

Insomnia affects up to 30% of the adult population in the UK, reducing the beneficial effects of sleep and increasing the incidence of daytime drowsiness. Regular lack of sleep has profound consequences for physical and mental health, shortens life expectancy and puts individuals at risk of serious medical conditions, including obesity, diabetes and heart disease. Furthermore, as people age, they tend to have a harder time falling asleep and more trouble staying asleep. Many older adults report being less satisfied with sleep and more tired during the day. There are effective drugs to help people to get to sleep at night, or alternatively to help them stay awake during the day. However, long-term use of these drugs can be dangerous as can cause drowsiness or they may be abused. Thus, it is imperative that we are able to understand and, ultimately, better regulate our sleep either by lifestyle changes or with safer drugs. This project will study a recently described protein, called QRFP that acts in the brain as a messenger to regulate arousal.
Mice are nocturnal animals, meaning that they are normally asleep during the day. We have found that when mice are given QRFP during the daytime they become aroused from their sleep. Conversely, we have bred a mouse that does not produce QRFP and which displays more sleep: it appears to find it difficult to wake up at the beginning of the night, when mice are usually their most active. There is a possibility that, in the future, we may be able to develop drugs which mimic the effects of QRFP and help humans stay awake, or perhaps drugs which block the action of QRFP and help humans get more sleep. As QRFP is a natural messenger and seems to be relatively selective in its effects, it may be possible to produce drugs which are less dangerous than those currently available. However, before that can happen, we must get a better understanding of how QRFP functions in the brain.
We already know about some of the complex circuits in the brain that control sleep and wakefulness. So, we want to learn how the cells (neurones) which produce QRFP fit into these circuits. We have bred another type of mouse which allows us to control QRFP neurones. Firstly, this means we can make QRFP neurones "shine" fluorescently so that we can cut slices of brain, see where the cells are and make recordings of their electrical activity. We have found that QRFP neurones are located exclusively in a small area of the brain, called the hypothalamus, where they intermingle with other cells which have an established role in affecting arousal. Also, QRFP neurones send long fibres to distant parts of the brain that control wakefulness. Thus, they would appear to be well placed. However, just because they send fibres to these other parts of the brain does not mean that they are functionally connected. To test this we can make QRFP neurones express a special light-sensitive receptor, similar to that which is found in the human eye. By shining a blue light on the cell bodies we can make QRFP neurones start firing and measure what affect this has on sleep and arousal. Moreover, we can also activate QRFP-containing fibres by shining the blue light in specific target regions of the brain. If this, in turn, switches on other types of neurone in the target regions then we can be sure that they a functionally connected and regulated by QRFP neurones. Finally, we can record from QRFP neurones in brain slices and measure how they respond to different hormones and drugs which are already known to affect sleep and wakefulness. Together this information will teach us about the physiology of QRFP that will underpin future development of QRFP as a potential drug for use in humans.

Technical Summary

Insomnia affects up to 30% of the adult population in the UK, reducing the beneficial effects of sleep and increasing the incidence of daytime drowsiness. Regular lack of sleep has profound consequences for physical and mental health, shortens life expectancy and puts individuals at risk of serious medical conditions, including obesity, diabetes and heart disease. Furthermore, as people age, they tend to have a harder time falling asleep and more trouble staying asleep. The long-term use if benzodiazepines to help people to get to sleep, amphetamines to help them stay awake during the day, or other similar drugs, can be dangerous as they may be abused. Dual-orexin receptor antagonists, are the first new class of drug affecting sleep to be developed in ten years, but these too can have unwanted side effects.
We have recently described the action of the RFamide, QRFP, to increase arousal in mice, while knock-out of the Qrfp gene causes a reduction in night-time activity. Using a cross between a Qrfp-cre mouse and channel rhodopsin (ChR2)-EYFP reporter, we have described the location of QRFP neurones, exclusively in the baso-lateral hypothalamus, intermingled with orexin-containing cells. Furthermore, like orexin-, QRFP neurones project to monoaminergic cell groups which constitute the ascending, reticular arousal system (RAS). We will record from Qrfp-cre::EYFP neurones in vitro and see how they interact with orexin and other factors known to affect sleep-wakefulness. We will stimulate QRFP cells bodies with ChR2 or designer receptors and measure both EEG and behaviour. Then we will use ChR2-assisted circuit mapping, both in vitro and in vivo, to define functional connections between QRFP and neurones of the RAS, and to determine which aspects of sleep/arousal are controlled by different projections. This information will underpin future potential development of QRFP as a target for new pharmaceutical interventions.

Planned Impact

Insomnia affects up to 30% of the adult population in the UK, reducing the beneficial effects of sleep and increasing the incidence of daytime drowsiness (Mental Health Foundation, Sleep Report 2011). Regular lack of sleep has profound consequences for physical and mental health, shortens life expectancy and puts individuals at risk of serious medical conditions, including obesity, diabetes and heart disease. Furthermore, as people age, they tend to have a harder time falling asleep and more trouble staying asleep. Many older adults report being less satisfied with sleep and more tired during the day. Thus, it is imperative that we are able to understand and, ultimately, better regulate our sleep. This project will study a distinct neuronal system which we have found modulates arousal. Our laboratory is well placed to make a major impact on understanding the physiology of this system, as we have available a number of animal models and the genetic tools to interrogate them. Our findings will be disseminated to our academic and clinical colleagues at international conferences and by publication in high-impact journals during the grant's duration. Following publication, each of the mouse models we develop will be made freely available.
Conservative commercial estimates suggest the global market for sleep-aid products (pharmaceuticals, specialist equipment, etc.) will grow to $77 billion by 2019. This project will help guide future treatments and the development of drugs, especially as co-therapies for lifestyle changes. The PI has been involved previously in successful collaborative projects with a number of industrial partners, providing evidence for several novel targets for drug development. Before the end of the project, we may be in a position to approach a company which may be interested in making peptide mimetics, which could provide composition of matter filings on novel therapeutics comprising long-lasting peptide derivatives.
During the lifetime of the grant, the basic research will be discussed at meetings organised as forums for colleagues, psychologists, clinicians, community nurses and other health professionals, patient group representatives and policy makers. Outreach work will be encouraged at all levels within the laboratory. Over the three years, the applicant will lecture at two local schools and at a local Café Scientifique-type meeting. The PDRA will be strongly encouraged to follow the example set by previous lab members, to tutor for the Manchester Access and STEM programmes (aimed at helping under-privileged children into further education), and to complete both a Wellcome Trust Researchers in Residence Scheme and a UK GRADschool.
This project will provide strong training in both in vivo skills and specialist techniques in electrophysiology, chemogenetics and optogenetics, metabolic and behavioural research. The applicant has supervised 15 post-doctoral associates, 14 PhD students and 17 masters students, the vast majority of whom have remained in science (some have their own independent research groups and others have moved into the commercial sector). The PI directs a cross-University IMB initiative to promote and expand research and training in in vivo biology. This problematic area is crucial to the UK economy and to the ambitions of Manchester to be a world-leading university. He is external examiner on other integrative masters courses.

Publications

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Description We have identified a novel pathway inn the brain that affects arousal and other behaviours. We have made three transgenic mice which will help us to unravel the biology of this system. We have presented some of our data at national and international meetings. We are preparing manuscripts for publication.
Exploitation Route This system might be manipulated to regulate arousal, e.g. safer drugs to regulate sleep.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Title Transgenic mouse loxSTOPlox-Qrfp 
Description Transgenic mouse loxSTOPlox-Qrfp 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2019 
Provided To Others? Yes  
Impact Increased knowledge. High-impact publications 
 
Description Industrial partnership with Eli Lilly, Indiannapolis, USA 
Organisation Eli Lilly & Company Ltd
Country United Kingdom 
Sector Private 
PI Contribution Exchange of data and staff.
Collaborator Contribution Direct funding of 10% of three Industrial Partnership Awards (IPAs with the BBSRC), plus research materials. Intellectual input and processing of samples.
Impact Papers. Target identificatin and validation.
Start Year 2010