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

Lead Research Organisation: University of Surrey
Department Name: Biochemistry & Physiology


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.

Technical Summary

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.

Planned Impact

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.
Description We identified the role of a novel inhibitory (GABAergic) transmission, mediated by delta-GABAA receptors, in local and activity-dependent aspects of sleep regulation.
In terms of achievement to date to assess the role of this type of transmission on brain function (i.e., behavioura and sleep regulation), behavioural experiments have been completed and the levels of markers for this neural transmission have also been measured. In addition, we completed the sleep-electroencephalogram recordings in mice lacking delta-GABAA receptors and in control mice, using a naturalistic behavioural paradigm and a drug to pharmacologically induce sleep slow waves (i.e., double-blind crossover design with 2 doses versus placebo), according to plan. Data collection and manual scoring of the vigilance states are finalised, as well as quantitative analyses of the EEG data. We are currently performing secondary analyses to further assess the role of extrasynaptic delta-GABAA receptors in the regulation of local activity-dependent generation of slow wave sleep. In addition, performance on several behavioural and memory tasks were assessed in the experimental mice included in the sleep-EEG studies mentioned above. The analysis of the behavioural data is completed.
The PDRA was invited to present her behavioural data and preliminary sleep analyses at the International Scientific Group of Circadian Rhythms Experts in April 2013 (Viareggio, Italy) sponsored by Industry, as well as at the 2014 European Sleep Research Society Conference. A manuscript is in preparation. A PhD student has also performed secondary analyses of the sleep data by developing non-linear analyses assessing the emergence of new pattern in the EEG. This has led to a publication in 2017. The developed algorithms have been shared with colleagues performing clinical studies and this led to an additional publication to further assess the effects of ageing and gender on brain activity and the sleep-wake cycle (
Moreover, some of the findings identified in this grant, including the role of extrasynaptic delta-GABAA receptors in sleep promotion and in cognitive functions has led to a collaboration to use an alternative model to provide further proof-of-concept that manipulating sleep via these receptors could revert memory deficits. The model used was the fruit fly (in order to replace the use of rodents). Through this collaboration, it was demonstrated that indeed sleep enhancement using a drug acting at the extrasynaptic GABAA receptors can improve short-term and long-term memory in several fly memory mutants, as well as fly models of Alzheimer's disease. Some of the underlying mechanisms of action at the molecular were also identified. These results led to the publications of two scientific papers in 2015 and 2017 (doi: 10.1016/j.cub.2015.03.027.;
Exploitation Route This research award contributes to a better understanding of a novel type of brain transmission (i.e., tonic GABAergic) and of its contribution to the basic mechanisms underlying the daily regulation of sleep-wake, which is the optimal basis to develop new compounds to treat sleep disorders.
Interestingly, GABAergic tonic transmission displays a very selective pharmacological profile. Thereby, this form of transmission could be an interesting target for the treatment of brain disorders and in particular sleep disorders.
Thus, the outcome of this research award will be useful not only to a broad range of academics in the neuroscience/pharmacology field, but also to industry.

In addition, the electrophysiological datasets were shared with colleagues in medical engineering to identify novel analysis methods to identify the emergence of new patterns in brain activity. A PhD research student performed the corresponding analyses which led to 2 publications and was shared with industrial collaborators. This would be useful to a number of academics in the sleep/neuroscience and pharmacology fields, as well as clinicians.
We are using the proof-of-concept knowledge obtained using the fruit fly model within a collaboration to further investigate the role of sleep in dementia.
Sectors Pharmaceuticals and Medical Biotechnology

Description This basic research grant has generated some findings that have been useful for UK/US based pharmaceutical industry collaborators who are developing drug compounds to improve symptoms of Alzheimer's disease and other types of dementia such as frontotemporal dementia. This will ultimately contribute to improve the quality of life of patients and their carers. The data obtained in the BBSRC award contributed to the use of a specific GABA agonist in a study characterising the effects of sleep in memory using Drosphila models of Alzheimer's disease. The Drosophila study led to the following publication (doi: 10.1016/j.nbscr.2016.09.001.)
First Year Of Impact 2015
Sector Pharmaceuticals and Medical Biotechnology
Title Development of algorithms using non linear methods to analyse the electroencephalogram 
Description Developed algorithms (in Matlab) to apply novel methods such as non-linear analysis methods based on complexity and/or entropy measures to further characterise the impact of sleep loss and the effects of drugs on the electroencephalogram. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2017 
Provided To Others? Yes  
Impact This methodology proved to provide additional information and be complementary to the gold standard (fast fourier transform) used in sleep and neuroscience research to analyse the EEG. While we developed and assessed the value of these methods on a preclinical study sponsored by the BBSRC, we have now shared our programs and the data from clinical studies are currently being analyses to further assessed the effect of aging, gender and pharmacotherapies. 
Title Behavioural characterisation of GABAA delta-subunit-containing receptor knockout mice compared to wild-type controls 
Description Behavioural characterisation of a specific transgenic line, in both sexes for behaviour and working memory. Data collection and analyses (including sex differences) are finalised. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Further understanding of the role of GABAergic tonic transmission in mediating different behaviours relevant to mood disorders in humans. Differences between the transgenic group and control littermates were identified for some of the behavioural tasks. This is of relevance in the context of developing new medications targeting this transmitter system and their impact on mood disorders, taking into account sex differences. 
Title Biochemical characterisation of sleep deprivation in the GABAA delta-subunit-containing knockout and control mice 
Description Collection of tissue samples in GABAA delta-subunit-containing receptor knockout mice versus wild-type controls for biochemical and neuromorphological analyses. Western blotting protocols were optimised for several antibodies and experiments performed. Data analysis is completed. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Characterisation of the effects of sleep deprivation on GABAergic tonic transmission which provides insight into the identification and potential therapeutic value of pharmacological compounds acting on this transmitter system. 
Title Sleep study in GABAA delta-subunit-containing receptor knockout mice and wild-type controls 
Description Collection of a comprehensive dataset from a sleep and pharmacology study in male and female knockout mice versus wild-type controls: Electroencephalogram/Electromyogram (EEG/EMG) recordings for 24-h baseline, 6-h sleep deprivation and subsequent recovery period with placebo or an anticonvulsant administration. We completed the electroencephalogram recordings in mice lacking delta-GABAA receptors and in the control C57BL/6 mice, using a naturalistic behavioural paradigm and a drug enhancing tonic GABAergic inhibition to pharmacologically induce sleep slow waves (i.e., double-blind crossover design with 2 doses versus placebo). Data collection and manual scoring of the vigilance states is finalised. Quantitative analyses of the EEG data are well underway and will allow us to further assess the role of extrasynaptic delta-GABAA receptors in the regulation of local activity-dependent generation of slow wave sleep. A manuscript is in preparation. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Provide insight into the role of GABAergic tonic transmission in the regulation of the 24-h sleep-wake cycle; Characterise the effects of a compound indirectly enhancing GABAergic tonic transmission, on sleep and the EEG following sleep deprivation, i.e., to evaluate the potential therapeutic value of the pharmacological manipulation of GABAergic tonic transmission to address sleep disorders and in particular insomnia. Dataset available to investigate the value of non-linear analysis algorithm to further characterise the effects of drugs/medications on the 24-h sleep-wake cycle, the EEG and brain activity. 
Description Biomedical Engineering 
Organisation University of Surrey
Department Department of Mechanical Engineering Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I provided electrophysiological datasets and supervision to a PhD student in Biomedical Engiennering to develop new algorithm to explore the emergence of new signals in brain activity (measure by electroencephalogram)
Collaborator Contribution The PhD student and supervisor in the field of Engineering reviewed the various methods that could be used and the PhD student developed algorithms for 3 non-linear analysis methods
Impact Multidisciplinary collaboration (Neuroscience/Biomedical Engineering) which has led so far to two publications:
Start Year 2013