Metabotropic Glutamate Receptors of Subtype 5 (mGluR5) and Sleep Homeostasis: Effects of Gene Knock-out and of Selective Negative Allosteric Modulation on EEG, Behavioral and Molecular Variables in Mice

Abstract

Sleep is a universal behavior observed across all species of the animal kingdom. One characteristic of sleep is its homeostatic regulation, in that rebound of sleep length and intensity follow periods of prolonged wakefulness. A widely accepted marker of sleep intensity and thereby sleep need is electroencephalographic (EEG) delta power (0.75 – 4 Hz), which accumulates with time spent awake and dissipates during sleep. The molecular mechanisms underlying sleep homeostasis are poorly understood. In this work, I focused on the contribution of the metabotropic glutamate receptor of subtype 5 (mGluR5) to the regulation of sleep (Chapter 1). The ultimate function(s) of sleep are not known. However, sleep is necessary for optimal functioning and acute and chronic sleep loss, poor sleep quality or timing entail risks for wellbeing, performance and health. Psychiatric and mood disorders are typically comorbid with disturbed sleep and already partial sleep deprivation (SD) has a pronounced impact on mood states, leading to confusion, loss of vigor, reduced frustration tolerance and coping capacities, amongst others. Also emotional processing is affected, in that ‘bidirectional affective imbalance’ is observed as increased response towards positive and negative stimuli after SD. Among neurobehavioral deficits caused by sleep deprivation, subjective and behavioral alertness, i.e. sustained attention, and cognitive processing capabilities, i.e. working memory, are the most pronounced. In addition, certain higher order cognitive functions, mainly relying on divergent flexible thinking, are impaired, such as decision-making, risk assessment and expectation of reward. Changed activity and connectivity of several areas of the brain including amygdala, the mesolimbic reward network and prefrontal cortical areas seem to contribute to the observed behavioral alternations upon sleep loss. The exact role of sleep in the various kinds and aspects of memory remains to be clarified. It has been shown repeatedly, that sleep supports memory consolidation. Local and use-dependent aspects of homeostatic sleep regulation and memory consolidation have been proposed, in that memory reactivation represents an ‘active system consolidation’. Other hypotheses suggest that sleep serves to erase certain memories, and in the ‘synaptic homeostasis hypothesis’ it is proposed that sleep serves the downscaling of neuronal synaptic strength, which has been potentiated by the encoding of experiences made during the preceding period of wakefulness (Chapter 2). 4 Since early twin studies it became apparent that genetic factors contribute to several aspects of sleep. Genetic factors influence many sleep characteristics such as sleep duration and quality, sleep efficiency, sleep architecture, and the neurobehavioral response to sleep loss. For several primary sleep disorders, including insomnia, narcolepsy, sleep talking and sleep walking amongst others, contributing genetic factors were found. However, these are partly modulated by gender and age, as for sleep efficiency and sleep talking, respectively. Tremendous research efforts yielded considerable knowledge about the influence of genetic variants and neurotransmitter systems on sleep characteristics. Adenosinergic, dopaminergic, serotonergic and GABAergic transmission, as well as monoamine oxidase, brain-derived neurotrophic factor (BDNF), tumor necrosis factor (TNF) alpha, prion protein, orexin/hypocreting and circadian ‘clock’ genes were found to affect various aspects of sleep (Chapter 3). To investigate the role of mGluR5 in sleep homeostasis, mGluR5 knock-out (KO), heterozygous (HT) and wild-type (WT) mice were studied regarding their sleep-wake distribution and the time course of EEG delta power during two days of undisturbed baseline conditions and 18 hours of recovery after 6 hours of sleep deprivation. While all mice showed a similar sleep-wake distribution during baseline, mGluR5 importantly affected the behavioral response to sleep loss, as sleep rebound was missing in the dark phase of recovery. Impaired build-up of sleep pressure during spontaneous and enforced wakefulness in mGluR5 KO mice, and during sleep deprivation in mice carrying only one functional copy of the mGluR5 gene provides strong evidence that mGluR5 is a crucial element in the molecular network underlying sleep homeostasis. We found, that sleep deprivation has sustained effects on behavioral adaptation to a novel challenge, even though presented after recovery sleep. Moreover, mGluR5 might benefit habituation capacities after sleep loss, a competence frequently compromised in psychiatric diseases (Chapter 4). In a second recovery day after sleep deprivation, mGluR5 KO mice still showed aberrant behavior, in form of increased wakefulness during the dark phase. This indicates that the lack of mGluR5 not only affects the immediate response to sleep loss, but also changes the sleep-wake distribution beyond recovery. Therefore, mGluR5 seem to be necessary to avoid enduring behavioral changes triggered by sleep loss and to return to baseline behavior. The observed sleep phenotype cannot be attributed to altered Homer1a expression, which was consistently increased by sleep deprivation in cortex, hippocampus and striatum, 5 independently of mGluR5 genotype. Further, increased availability of mGluR5 after sleep loss observed in humans may not originate from altered mGluR5 gene expression, since no differences were observed between control and sleep deprived wild-type mice. In a sleep deprivation study, we administered a novel negative allosteric modulator (NAM) of mGluR5 at the beginning or the end of prolonged wakefulness. NREM sleep was increased on the expense of wakefulness during the light phase of recovery when the compound was injected at the end of SD, whereas REM sleep was reduced after both, injection at the beginning or the end of sleep deprivation (Chapter 5). This thesis provides evidence that mGluR5 is an important player in the complex molecular network regulating sleep-wake distribution and the homeostatic regulation of sleep. Moreover, mGluR5 might confer resilience to detrimental effects of sleep loss in terms of behavioral habituation to a novel challenge. Impaired habituation together with disturbed sleep is frequently observed in psychiatric diseases such a schizophrenia, autism spectrum and anxiety disorders, and the findings might help to untangle the complex interplay of sleep disturbances and the etiology of psychiatric disorders. Notably, the effects caused by lack of mGluR5 are triggered by sleep loss, but endure beyond recovery in terms of sleep rebound and dissipation of accumulated EEG delta power. We found a modulatory effect of a novel mGluR5 NAM on sleep-wake distribution after sleep loss. The pronounced impact on REM sleep in terms of long-lasting reduction despite increased sleep pressure could make this compound interesting for the treatment of diseases with typically dysregulated REM sleep such as depression and narcolepsy. The mechanisms, however, by which mGluR5 exerts its functions on sleep regulation and behavioral habituation remain to be determined. The observed sleep phenotype in mGluR5 knock-out animals cannot be attributed to altered Homer1a expression. Moreover, the induction of increased Homer1a expression with time spent awake is exerted by mechanisms that do not rely on functional mGluR5. In addition, the observed increase of mGluR5 availability after sleep deprivation in humans seems to be due to mechanism different from mGluR5 gene expression. Future studies will need to address by which mechanisms the lack of mGluR5 affects the homeostatic build-up of sleep pressure and leads to the sustained behavioral aberrations after sleep loss. The mGluR5 NAM we used for the first time in a sleep deprivation study, should be further characterized for its potential to alleviate the detrimental effects of sleep loss and to correct dysregulated REM sleep patterns (Chapter 6).