Giulio Tononi - US grants

The functions of sleep constitute a genuine mystery in present day biology. Such functions can be investigated by preventing sleep from occurring. In rats, prolonged total or REM sleep deprivation lead to extreme sleepiness, weight loss, increase in food intake and metabolic rate, and death after 2-5 weeks. Decades of investigation have failed to reveal any substantial abnormality in peripheral organs or in the brain. Over the past few years, molecular approaches have been employed the to study the cellular correlates of sleep and waking. Several compounds have been shown to accumulate during waking and after short periods of sleep derivation and to return to basal levels during sleep, in line with homoeostatic models of sleep regulation. To evaluate the functional significance of these findings in the context of sleep homeostasis, new gene screening methods such as microarrays and differential display have been used to examine gene expression after both short-and long-term sleep deprivation. While most genes screened (approximately 10000) were not modified, the levels of brain arylsulfotransferase (AST) were increased by short periods of waking and even more so by prolonged sleep derivation. This enzyme is responsible for the inactivation of noradrenaline, which is released in the brain during waking and much less during NREM and REM sleep. The induction of AST may thus constitute a first indication of a homoeostatic response by the brain to the uninterrupted activity of the central noradrenergic system. This hypothesis will be tested by examining whether AST mRNA (1) progressively increases with the length of total sleep deprivation, increases after selective REM sleep derivation, and returns to normal after recovery sleep; (2) is accompanied by indices of increased central noradrenaline turnover; and (3) is reduced in animals with lesions of the central noradrenergic system. Finally, it will be established whether lesioned animals are more resistant to the harmful effects of sleep deprivation. These studies should provide insights into the molecular consequences of prolonged sleep deprivation and chronic insomnia and suggest new therapeutic approaches.

Abstract Not Provided

[unreadable] DESCRIPTION (provided by applicant): We spend a third of our life asleep, and even partial sleep deprivation has serious consequences on cognition, mood, and health, suggesting that sleep must serve some fundamental functions. Unfortunately, we lack a neurobiological understanding of what these functions might be. However, we know that sleep is tightly regulated as a function of prior wakefulness, suggesting that it may be needed to reverse some changes that take place when we are awake. We also know that the organ that has the greatest need for sleep is the brain, although we still do not know why. The overall goal of this project is to test a recent, comprehensive hypothesis about the function of non-rapid eye movement sleep - the synaptic homeostasis hypothesis. According to the hypothesis, the brain needs to sleep because of the progressive strengthening of neural circuits that occurs during wakefulness and the associated energy and performance costs. We will test some key prediction of the hypothesis by using a novel method for performing high-density sleep electroencephalography (hd-EEG, 256 channels). We will take advantage of the well-known fact that sleep need is reflected by the amount of slow wave activity (SWA) in the sleep EEG. Specifically, we will test the prediction that procedures leading to local increases in synaptic strength, such as learning tasks involving particular brain regions, should lead to a local increase in sleep SWA. We will use both an implicit learning task (rotation adaptation) involving right parietal cortex, and an explicit learning task involving prefrontal cortex (motor sequence learning). We will then test the converse prediction that procedures leading to a local depression of synaptic circuits, such as arm immobilization, should lead to a local decrease in SWA during subsequent sleep. If these predictions are confirmed, they will provide strong evidence that sleep SWA is regulated at a local level, that its regulation is tied to plastic changes in cortical circuits, and that the level of local SWA has important consequences on performance after sleep. The results provided by this project will lend strong support to the synaptic homeostasis hypothesis and greatly advance our understanding of the functions of sleep at the fundamental level. There is overwhelming evidence that good, restorative sleep is exceedingly important to human health, that sleep deprivation and sleep restriction have enormous social costs, that sleep disorders are extremely common, and that they are frequently associated with psychiatric and neurological disorders. By tying brain plasticity to local sleep regulation, the results of these investigations will provide a novel, rational basis for designing therapeutic approaches aimed at enhancing the restorative value of sleep in health and disease. Thus, they are highly relevant to the mission of NINDS, NIMH, and NHLBI. [unreadable] [unreadable] [unreadable]

The problem I intend to address is the function of sleep, a biological puzzle that is still unsolved. Specifically, I intend to test a comprehensive, novel hypothesis about what sleep is for, called the synaptic homeostasis hypothesis (Tononi and Cirelli, Brain Res Bull 2003, Sleep Medicine Rev., 2005). The hypothesis accounts for many facts about sleep and its regulation, from behavioral and phylogenetic evidence to electrophysiological and molecular observations, and has fundamental implications for health and disease. The hypothesis makes intriguing predictions that are relevant for both basic and clinical neuroscience, and I propose to test such predictions using several complementary approaches. If the hypothesis survives the tests, we may have for the first time a solid scientific explanation of why we need to sleep. Understanding the function of sleep is obviously important both scientifically and from the perspective of human health. Sleep is a pervasive, universal, and fundamental behavior: It occupies a third of our life, and an even larger proportion in infants;it is present in every animal species where it has been studied, from fruit flies to humans;it is tightly regulated, as indicated by the irresistible mounting of sleep pressure after prolonged wakefulness;and even partial deprivation of sleep has serious consequences on cognition, mood, and health. While all available evidence indicates that sleep is of the brain, by the brain, and for the brain, the function of sleep remains unknown despite decades of intensive research. No comprehensive theory had been advanced so far, which is why the synaptic homeostasis hypothesis, if corroborated, may constitute a much needed breakthrough. In fact, the lack of understanding of why we need to sleep is problematic not only from a scientific viewpoint, but also because of its vast implications for public health. Millions of people complain of sleep problems, from insomnia to excessive daytime sleepiness, from chronic fatigue to irritability associated with unsatisfactory sleep. Sleep problems are an important aspect of several psychiatric disorders, notably mood disorders and anxiety disorders. Finally, sleep deprivation has high social costs, from driving and work-related accidents to chronically poor performance. A large segment of the population is therefore treated routinely with drugs aimed at improving sleep, or at maintaining wakefulness in the face of sleep pressure. However, such treatments are hampered by our ignorance concerning the functions of sleep. Which sleep disturbances should be taken seriously because they reflect a functional impairment and which, if any, do not interfere significantly with the functions carried out by sleep? Which abnormalities of sleep are likely to have neurobiological consequences that can lead to psychiatric disorders such as depression? And finally, what aspect of sleep should be enhanced by pharmacological or behavioral treatments, and what indices should we consider to determine their effectiveness?

Slow wave activity in the sleep electroencephalogram (EEC) is a marker of sleep need, increasing with the duration of prior wakefulness and decreasing exponentially during sleep. Unfortunately, we do not know which biological process is responsible for the increase of sleep slow waves as a function of wakefulness, or what function they may serve. According to a recent hypothesis - the synaptic homeostasis hypothesis of sleep function - plastic processes occurring during wakefulness result in a net increase in synaptic strength in many cortical circuits. As a consequence, when cortical neurons begin oscillating at low frequencies during sleep, they become strongly synchronized, leading to EEG slow waves of high amplitude. These slow waves, in turn, are responsible for the renormalization of synaptic strength and have beneficial effects on energy metabolism and performance. Recent work has shown that, consistent with the hypothesis, a visuomotor learning task that involves a specific cortical area leads to a local increase in slow wave activity during subsequent sleep. This work has also shown that performance is enhanced after sleep, and this enhancement is correlated with the local increase in slow wave activity. Building upon these results, this project will test two further, crucial predictions of the synaptic homeostasis hypothesis: that sleep slow waves i) are necessary for the renormalization of cortical circuits after learning;and ii) are necessary for the enhancement of performance after sleep. TO do so, sleep slow waves will be suppressed using mild acoustic stimuli that do not interrupt sleep. The specific aims are thus designed to evaluate whether, as predicted by the hypothesis, learning leaves a local EEG trace that is renormalized after sleep, and whether the selective deprivation of sleep slow waves leads to a persistence of such EEG traces.and to a suppression of postsleep performance enhancement. If these predictions are confirmed, they will lend strong support to the synaptic homeostasis hypothesis of sleep function and aid in the interpretation of the results of Projects I, III, and IV.

DESCRIPTION (Provided by applicant): This application requests four years of funding to establish a Conte Center to Develop Collaborative Neuroscience Research. Through the Center, we intend to test a comprehensive, novel hypothesis about the function of sleep - the synaptic homeostasis hypothesis. The hypothesis states that plastic processes during wakefulness result in a net increase in synaptic strength in many brain circuits, leading to increased metabolic consumption. Strengthened brain circuits then lead to larger slow waves during subsequent sleep. In turn, sleep slow waves renormalize synaptic strength to a baseline level that is energetically sustainable and beneficial for memory and performance. Sleep is therefore the price we pay for plasticity, and its function is the homeostatic regulation of the total synaptic weight impinging on neurons. The hypothesis accounts for many facts about sleep and its regulation and makes intriguing predictions that are relevant for both basic and clinical neuroscience. We propose to test such predictions through four tightly linked and complementary projects, to be carried out jointly at the University of Wisconsin and at Washington University. Project I (PI Cirelli) employs a combined molecular / electrophysiological approach in an animal model to establish a relationship between synaptic potentiation during waking, an increase in sleep slow waves, and the resulting synaptic renormalization;Project II (PI Tononi) employs behavioral / high-density (hd)-EEG paradigms in healthy human subjects to determine whether learning leaves a local EEC trace in both wakefulness and sleep, and to determine whether sleep slow waves are necessary to renormalize this trace;Project III (PI Raichle) employs the same behavioral paradigms in conjunction with PET and fMRI to investigate whether learning leaves a local metabolic trace that is renormalized by sleep;and Project IV (PI Benca) employs the same behavioral / hd-EEG paradigms in patients with major depression to evaluate a predicted relationship between preserved slow wave homeostasis and therapeutic response to sleep deprivation. If the hypothesis survives these combined tests, it will provide a scientific explanation of why we need to sleep that ranges all the way from molecular and cellular function to systems neurophysiology and neuroimaging. Given the central role of sleep in the life of every organism, at every age, we expect that the results of our program will have major implications for many aspects of human health and disease.

DESCRIPTION (provided by applicant): During development, brain circuits undergo extensive remodeling, involving both synaptogenesis and pruning. Adolescence, in particular, is thought to be a sensitive period for synaptic pruning in cortical circuits involved in cognitive functions and emotional regulation. Adolescence is also a sensitive period for the pathophysiology of many psychiatric disorders, presumably due to this extensive synaptic remodeling. Thus, it would be useful to be able to track changes in the number and efficacy of synapses longitudinally and non-invasively during adolescence. New evidence suggests that changes in sleep slow wave activity (SWA), which can be assessed longitudinally and non-invasively using electroencephalography (EEG), may parallel neurodevelopmental changes in cortical synaptic density. To develop SWA as a potential marker of synaptic function during developmental sensitive periods requires an animal model in which: i) anatomical, molecular, and physiological changes in cortical synapses can be evaluated directly; ii) a point- to-point, intra-subject correlation can be established between sleep SWA and direct measures of synaptic number/molecular composition/efficacy. In Aim 1 of this project, we will pursue these goals by performing both chronic EEG recordings and repeated in vivo imaging with two-photon microscopy in transgenic mice that express yellow fluorescent protein in cortical neurons. Moreover, we will measure molecular and electrophysiological markers of synaptic strength in these mice throughout development. In addition to monitoring synaptic remodeling in vivo, it is important to begin investigating which factors can influence it during the sensitive period of adolescence. Since major changes in synaptogenesis/pruning during development are correlated with major changes in sleep/wake patterns, it has been hypothesized that changes in behavioral state may not only reflect, but also affect synaptic remodeling. Consistent with this notion, new evidence in animals and humans shows that, in the adult brain, waking is associated with a net increase in synaptic strength, and sleep with a net decrease, and that SWA reflects molecular and physiological changes in synaptic function brought about by wake and sleep. Aim 2 of this proposal will test the hypothesis that sleep/wake behavior affect synaptic structure/function also during development. Specifically, we will determine whether sleep and waking differentially affect synaptogenesis and synaptic pruning, consistent with their effects on synaptic strength in adults. If successful, Aim 1 will lead the foundation for EEG monitoring of synaptic efficacy during neurodevelopment in human subjects at risk or patient populations as an essential aid for both diagnosis and therapy. Aim 2 will open the way to preventive/therapeutic approaches for influencing synaptogenesis/pruning by stabilizing/adjusting sleep/wake patterns in children.

DESCRIPTION (provided by applicant): Synaptic plasticity is a fundamental feature of the nervous system that underlies neural development, adaptation and learning. There is growing evidence that deficits in the mechanisms of synaptic plasticity are involved in the pathophysiology of many psychiatric disorders, from schizophrenia to mood disorders. For this reason, NIMH has established as one of his strategic research priorities the study of brain plasticity at the cellular, synaptic, circuit, and behavioral level, with the final goal of determining the neurobiological bases of these processes. This proposal will study humans and three animal models (flies, mice, rats) to test the novel and provocative idea that synaptic plasticity is adaptive up to a point, but beyond that point, or in vulnerable individuals, it can become maladaptive. The cost of synaptic plasticity is not often considered but may be crucial in the pathophysiology of psychiatric disorders, and will be assessed at the ultrastructural, cellular, circuit, and behavioral level. Our previous NIMH-funded work has established that the overall result of wake plasticity is a net increase in synaptic strength, which is renormalized by sleep. But what happens when plasticity is excessive, for instance because it is extended beyond the physiological range without intervening sleep? Based on preliminary results obtained in both animals and humans, we hypothesize that extended plasticity can lead to negative consequences on neuronal activity (OFF periods, performance deficits) and on cellular function/integrity (cellular stress, ultrastructural abnormalities). Aim 1 will use rats to test whether plasticity-dependent synaptic overload leads to the occurrence of neuronal OFF periods, local EEG slowing during wake, and performance impairment. It will also establish to what extent these effects are a region- specific consequence of plasticity, rather than a general effect of prolonged wake. Aim 2 will use high density (hd) EEG in humans to ask whether the local increase in EEG theta waves, which occurs during wake as a result of extended plasticity in specific brain circuits, leads to local performance deficits, locally increased sleep need, and o sleep-dependent restoration of function. Aim 3 will use flies and mice to test whether extending plasticity by prolonging wakefulness leads to cellular stress and subcellular damage, and whether doing so chronically under sleep restriction conditions leads to lasting cellular damage and cognitive deficits. Plasticity plays a central role in the life of every organism, but its coston neural structure and function may be substantial especially at vulnerable developmental times, such as adolescence, or in vulnerable populations, such as psychiatric patients. Demonstrating the cost of plasticity at the cellular and systems level will have clear practical implications forthe prevention and treatment of mental disorders.

SUMMARY The Administrative Core will coordinate all aspects of this Program Project, and will provide the framework to assist each project towards overall productivity. All Center activities will be directed by Dr. Giulio Tononi, who will oversee Ms Pfister-Genskow. This Core will serve the following functions: 1) Facilitate exchange of scientific information between the Center sites at the University of Wisconsin-Madison (UW), CUNY, and the National Center for Microscopy and Imaging Research (NCMIR) in San Diego by organizing regular scientific and administrative meetings, and by fostering interactions among members of the three laboratories; 2) Establish an external Advisory Committee and organize meetings of its members at UW every 12-18 months; 3) Develop and monitor the outreach and educational initiatives of the Center. The administrative team will also maintain the already existent UW website and work with a webmaster to insure that the site is timely and up-to- date; 4) Coordinate training within the Center by identifying opportunities in individual projects and helping to identify suitable undergraduate, graduate, and postdoctoral candidates for these opportunities.

? DESCRIPTION (provided by applicant): Sleep is pervasive, universal, irresistible, tightly regulated, and its loss impairs performance and cognition. Sleep is thought to perform essential restorative functions for the brain, and increasing evidence suggests that sleep may ultimately reflect a local need for homeostasis that is triggered by the cellular consequences of wake, primarily related to the costs of synaptic plasticity associated with learning. This evidence is consistent with the hypothesis that sleep and wake may occur, be regulated, and perform their functions at the level of individual neurons. Recently, using multi-array recordings in freely moving rats, we obtained direct evidence that sleep can occur locally within a group of cortical neurons, while the rest of the brain remains awake, and that such local sleep increases with the duration of wake. If so, does local sleep occur in different species, and what are the underlying mechanisms? Are they similar to those governing the regulation of sleep at the systems level? And do they reflect cellular needs for restoration triggered by the progressive accumulation of plastic changes due to learning in wake (neuronal tiredness) or merely by intense activity (fatigue)? Moreover, are there cellular/ultrastructural signatures of the cost o wake plasticity for brain cells and, conversely, that sleep has occurred and restored homeostasis? Finally, does the occurrence of local sleep during wake lead to performance impairments, which could explain the performance/cognitive deficits observed in sleep-restricted humans and in some neuropsychiatric disorders? These are fascinating and long-standing questions, and it is finally becoming possible to address them directly by taking advantage of several technological breakthroughs that permit single cell analysis and causal manipulations, including multi-array recordings, high-density (hd) EEG, in vivo calcium imaging, thermo/opto/pharmacogenetics, and serial block- face scanning electron microscopy (SBF-SEM). This proposal includes three highly integrated and complementary projects employing flies (Project I), mice (Project II), and humans (Project III) to address three related questions: . Does local sleep occur in different species, and is it triggered by mechanisms similar to those that trigger sleep proper, or merely by fatigue? 2. What are the cellular/ultrastructural signature of wake plasticity and, conversely, of the restorative functions of sleep? 3. What are the behavioral/cognitive consequences of local sleep in an awake brain?

SUMMARY Sleep is pervasive, universal, irresistible, tightly regulated, and its loss impairs performance and cognition. Sleep is thought to perform essential restorative functions for the brain, and increasing evidence suggests that a key function may be to rebalance cellular changes triggered by plasticity during wake. This evidence is consistent with the hypothesis that sleep and wake may occur, be regulated, and perform their functions at the level of individual neurons. Recently, using multi-array recordings in freely moving rats, we have obtained direct evidence that sleep can occur locally within a group of cortical neurons, while the rest of the brain remains awake, and that such local sleep increases with the duration of wake. In this proposal, we will use multi-array recordings to establish whether OFF periods during wake (local sleep) occur in mice, whether it increases with wake duration and whether it is associated with slow/theta waves in the local EEG and impaired performance, providing the rationale for the use of high density EEG in humans in Project III. We will then test whether local sleep, like sleep proper, is regulated by intense synaptic plasticity (tiredness) or instead by mere activity (fatigue). To do so we will first establish if local sleep increases with locally induced intense plasticity. We will induce local tiredness in one sensorimotor cortex using both a learning task (reaching) and synaptic potentiation through electrical stimulation, and compare the effects on local sleep on the ipsi- vs. contralateral side. Next, we will determine the effects on local sleep of local opto-pharmacogenetic activation (intense activity with little plasticity), expected to lead to fatigue, during both wake and sleep, again comparing the results to the contralateral side. Finally, we will use SBF-SEM to test whether there are ultrastructural signatures that distinguish between neurons that have been kept awake and those that have slept, and between intense plasticity (tiredness) and intense activity (fatigue). Altogether, these studies in mice will complement those in flies in Project I, which use similar methods. Together, they will establish if sleep and wake are regulated homeostatically at the single neuron level, and if they leave ultrastructural signatures that reflect their consequences and functions for individual cells.

SUMMARY The EEG Analysis Core will be directed by Dr. Giulio Tononi and will provide the hardware, software, and technical personnel resources to assist Projects II (mice) and III (humans) in the analysis of electrophysiological and EEG data.

? DESCRIPTION (provided by applicant): We have proposed that general anesthetics, irrespective of their precise mechanism of action, induce loss of consciousness when they bring about the breakdown of information integration within the corticothalamic system. Here, we will test this proposal using animal models in which we can investigate the role of different neuronal populations in anesthetic unconsciousness. Specifically, it is unclear whether anesthetic loss/recovery of consciousness (LOC/ROC) relies on a thalamic switch or a direct action on cortical cells. Within cortex, it is not known whether anesthetic LOC/ROC is a global phenomenon or is due to specific neural populations and fiber pathways. We hypothesize that pyramidal cells in supragranular (SG) layers, which form a highly integrated network both within and across cortical areas, are ideally poised to support information integration and thereby consciousness. The roles of different thalamic populations in modulating cortical interactions are also unknown. We hypothesize that thalamic matrix cells, with their widespread cortical projections focused especially in SG layers, enable cortical information integration. In addition t actions on specific cell types, we propose that anesthetics target specific synaptic pathways, suppressing cortico-cortical (CC) and matrix thalamo-cortical (TC) synaptic connections, while leaving core TC connections intact. To test these hypotheses, we will take advantage of recent developments in laminar multiunit recordings in freely- moving rodents to examine the neural correlates of LOC/ROC, and recordings of network activity in brain slices to investigate the cellular and circuit mechanisms of these correlates. Moreover, we will use optogenetic and pharmacogenetic methods to transiently activate/inactivate specific cortical layers and thalamic populations and explore their causal involvement in LOC/ROC.