Tonic GABAA transmission and regulation of activity-dependent changes in neurotransmission across sleep-wake cycles

Sleep is considered to be essential for brain function, but what this function is and which molecular mechanisms regulate sleep, continues to be unknown. Sleep is subdivided in two stages: rapid eye movement (REM) sleep and nonREM sleep. The latter is better known as slow wave sleep, because during this sleep stage the scalp electroencephalogram (EEG) is dominated by low frequency waves. There is considerable interest in slow wave sleep because it is regulated very accurately in response to how long we have been awake and asleep. Furthermore, it has been established that the slow waves in the EEG are a reflection of the simultaneous occurrence of slow oscillations in large networks of neurons in the cortex and thalamus. Traditionally, the regulation of the alternation between sleep and wakefulness was considered to be a global brain process, regulated by the biological (circadian) clock in interaction with the homeostatic process, which keeps track of how long we have been awake and asleep. More recently, data have emerged supporting the view that sleep is regulated, in part, at a local level in a use-dependent manner. According to these data, those brain regions that are most activated during wakefulness show more slow waves during subsequent sleep. The mechanisms underlying this local variation in slow wave sleep remain largely unidentified. One hypothesis is that local variation in slow wave sleep reflects changes in excitability of local neuronal networks. Brain excitability depends on the balance between excitatory and inhibitory transmission. Recent developments emphasise the importance of 'tonic' inhibitory transmission mediated by extrasynaptic delta-GABAA receptors. This novel type of transmission plays a key-role in maintaining the excitability of brain circuits. Interestingly, drugs enhancing tonic inhibitory transmission induce slow waves in the EEG. The aim of this proposal is to investigate the contribution of tonic inhibitory transmission to the local regulation of slow wave sleep. We will use an established model of local sleep regulation in mice. In this model, whiskers are stimulated unilaterally. This is known to specifically induce changes in brain circuits (barrel cortex), which are then reflected in slow waves during sleep. We will be able to assess whether components of tonic inhibitory transmission (delta-GABAA, alpha4-GABAA, GABAB, TASK-1, GABA transporters) are altered in brain regions activated following this stimulation during wakefulness. We will also assess whether these changes are reversed during subsequent sleep. We will further perform sleep studies to assess whether the detected changes in components of tonic inhibitory transmission are correlated with sleep slow waves. In addition, we will investigate whether drug-induced slow waves, by activation of the tonic transmission, can alter the sleep EEG and sleep regulation. Although according to the present hypothesis local changes in excitability should primarily be use-dependent, it is well known that the circadian clock also plays an important role in sleep regulation. To evaluate whether tonic inhibitory transmission is part of the mechanisms underlying this circadian control, we will use an established protocol to separate use-dependent (sleep-wake dependent) and circadian contributions to sleep regulation. The proposed research will be conducted in C57BL/6 mice and mice showing a greatly reduced tonic inhibitory transmission (mice lacking delta-GABAA). The research represents a multidisciplinary approach in which a wide range of methodologies used in in-vivo sleep research, biochemistry and neuroanatomy will be applied. The research will contribute to our understanding of the mechanisms underlying our daily sleep-wake cycle. Ultimately, this may lead to a better understanding of the contribution of sleep to brain function, sleep-disorders as well as the development of new treatments for these disorders.

Sleep is considered fundamental for brain function, but the mechanisms underlying sleep regulation remain poorly understood. It has recently emerged that slow wave sleep is regulated, at least in part, locally. It has been hypothesised that local regulation of slow waves reflects wake-activity-induced changes in excitability and connectivity in neuronal networks, and that slow wave sleep reverses these wake-dependent changes thereby maintaining functional neuronal networks. Which neurotransmitters systems contribute to these changes, is largely unknown. Tonic transmission, mediated by extrasynaptic delta-GABAA receptors, has emerged as a potent mechanism to establish baseline excitability levels of neuronal networks and has been shown to affect slow waves. The aim of the proposed research is to investigate the contribution of GABAA tonic transmission to the local, activity-dependent changes of neuronal networks across sleep-wake cycles. I will first investigate whether the local activation of the somatosensory cortex during wakefulness leads to changes in slow waves and markers of tonic transmission (i.e. number and distribution of delta-GABAA, alpha4-GABAA, GABAB, TASK-1, GABA transporters), and whether these changes are reversed during sleep. I will next investigate whether the activity-dependent changes in slow waves and markers of tonic GABAA transmission remain correlated in C57BL/6 mice in which tonic GABAA transmission has been enhanced pharmacologically, and in mice lacking delta-GABAA receptors. Finally, I will investigate whether the changes in markers of tonic GABAA transmission are exclusively dependent on sleep-wake alteration, or are also modulated directly by circadian rhythmicity. This research project will contribute to the understanding of the role of tonic GABAA transmission in local and activity-dependent aspects of sleep regulation, as well as its role in the circadian control of sleep.

In order to engage users and beneficiaries from the private sector, I will maintain and expand the current research network of the Surrey Sleep Research Centre and Surrey Clinical Research Centre with industry. There are very few animal sleep research groups in the UK and Europe. Only a minority has the expertise for long-term EEG recordings combined with quantitative EEG analysis in mice, as will be used in the proposal research. I have already been approached for advice in relation to this expertise. I will attempt to establish formal collaborations with industry in the near future. Communication of my research, not only to the academic community but also to the beneficiaries outside this academic community, will be also achieved by attending conferences in the field of Sleep and Circadian Rhythms. These meetings are frequently sponsored and attended by numerous industrial partners, thereby providing a unique opportunity to communicate my profile as an academic animal sleep researcher with a keen interest to collaborate with industry. The results of our research will be communicated ultimately through academic publications and where appropriate, in open access journals. Our research will also be accessible to the public via the annual Festival of Research from the University of Surrey. Sleep researchers at the University of Surrey are frequently approached by the media. I will attempt to make use of this way to communicate the proposed research to a wider audience. The proposed research aims at deciphering the basic mechanisms underlying the daily regulation of sleep-wake cycles, with an emphasis on the contribution of tonic GABAergic transmission. By assessing the alterations of tonic transmission across the sleep-wake cycles at the cellular, electrophysiological and behavioural levels, we will further characterise its role in neural processes underlying sleep and complex behaviours. Our assessment of the role of tonic GABAergic transmission in sleep regulation is important for several reasons. The interest in the contribution of sleep to brain function is increasing rapidly. There is a new sense of excitement in the field of sleep research due to the current focus on the study of sleep in the context of local network connectivity/excitability. It is felt that this provides a new and fruitful approach to the study of sleep regulation and function. Slow wave sleep is of particular interest, not only because of its accurate homeostatic regulation, but also because of its role in plasticity and sleep-dependent memory consolidation. Finally, sleep and slow wave sleep in particular, decline both in quality and quantity with ageing. Overall, results from the proposed research on the contribution of tonic transmission to slow wave sleep regulation will be valuable for users in many disciplines investigating basic cellular mechanisms underlying neural functions and age-related changes. In addition, the proposed research would also contribute to the basic knowledge necessary to predict the effects of pharmacological compounds acting at receptors mediating GABAergic tonic transmission. This knowledge would thus greatly benefit industry developing new sedative-hypnotics. Most currently used hypnotics enhance phasic GABAergic transmission and reduce slow wave sleep. By contrast, manipulation of GABAergic tonic transmission is a powerful tool to enhance slow wave sleep. Thereby, this form of transmission remains an interesting target for the treatment of disorders of sleep-wake cycles and deficiencies in slow wave sleep. The impact activities will be undertaken primarily by the PI. A webpage for my group has already been activated on the website of the University of Surrey. Publications from this proposal will be advertised on this webpage and thereby will be accessible to the public.