Marcos G. Frank - US grants

DESCRIPTION (provided by applicant): This application proposes neurophysiological studies to determine the role of sleep in the development and plasticity of the mammalian visual cortex. Our previous research has demonstrated a role for sleep in the plasticity elicited by monocular deprivation (MD) during the critical period for visual development in the cat. These findings suggest an important role for sleep in the developing brain and have important implications for our understanding of cortical plasticity, learning and memory, and the function of sleep. In addition to providing insights into these areas of study, the proposed studies should provide important information regarding the role of sleep in human neural development, and the long-term consequences of sleep loss on the developing brain. The overall goal of the proposed studies is to determine the contribution of rapid-eye-movement (REM) and nonREM sleep to two forms of developmental plasticity in visual cortex. Short periods of MD during the cat critical period trigger rapid remodeling of thalamocortical circuits in primary visual cortex in favor of the open eye. A related form of synaptic plasticity, elicited by opening the previously closed eye and closing the previously open eye (reverse-MD) can produce recovery of cortical function. We will first determine the contribution of each sleep state to the cortical plasticity elicited by MD. We will then determine if the effects of sleep on cortical plasticity are mediated by the patterns of neural activity present in REM or nonREM sleep. We will then investigate the role of sleep in the cortical plasticity elicited by reverse-MD.

Description (provided by applicant): Consolidation refers to time-dependent processes that convert labile forms of brain plasticity or memory into more permanent forms. This has been best described in the hippocampus, but similar processes occur in the developing visual cortex and may be essential for the maturation of visual circuits. Our goal is to identify the cellular mechanisms governing the consolidation of a classic model of experience-dependent cortical plasticity (ocular dominance plasticity (ODP). To achieve this goal we will use our established methods of triggering ODP in the visual cortex combined with behavioral state monitoring, intracortical infusion, optical imaging of intrinsic cortical signals, immunohistochemistry and single-neuron electrophysiology in vivo. These techniques are combined in a simple experimental design that allows us to examine the role of kinase, translational and post-transcriptional pathways that may be necessary for long-lasting modifications of cortical circuitry. We propose the following specific aims: 1. Determine the role of protein kinase A (PKA) in the consolidation of ODP. In this specific aim, we will test the hypothesis that PKA signaling is required for the consolidation of cortical plasticity. This will be accomplished by a) inhibiting PKA activity b) activating PKA activity and c) inhibiting PKA-AKAP binding in the visual cortex during the consolidation phase of ODP. 2. Determine the role of protein synthesis in the consolidation of ODP. In this specific aim, we will test the hypothesis that protein synthesis is required for the consolidation of cortical plasticity. This will be accomplished by reversibly inhibiting dendritic and global protein synthesis in the visual cortex during the consolidation phase of ODP. 3. Determine the role of transcriptional regulation in the consolidation of ODP. In this specific aim, we will test the hypothesis that chromatin modification is required for the consolidation of ODP. This will be accomplished by measuring changes in histone acetylation and by increasing or decreasing histone acetylation in the visual cortex during the consolidation phase of ODP. PUBLIC HEALTH RELEVANCE The research in this proposal will provide important new insights into how endogenous brain activation leads to long-lasting changes in cortical circuits. This will improve our understanding of normal and pathological brain development and memory formation.

DESCRIPTION (provided by applicant): Ocular dominance plasticity is a canonical form of synaptic plasticity in vivo triggered by changes in binocular vision. It has provided crucial insights into how cortical circuits develop and remodel in early life. We have shown that ocular dominance plasticity is consolidated via cellular mechanisms similar to those mediating long-term synaptic potentiation (LTP). Our goal is to more completely identify these mechanisms and the brain states in which they occur. To achieve this goal we will use our established methods of eliciting ocular dominance plasticity combined with behavioral state monitoring, inactivation of intracortical enzymes, optical imaging of intrinsic cortical signals, acute single-neuron electrophysiology in vivo, chronic tetrode recording of single-neurons in freely-behaving animals, and Western blot measurements of cortical proteins. These techniques are combined in a simple experimental design that allows us to determine how different brain states and intracellular signaling pathways promote long-lasting modifications of cortical circuitry. More specifically, we will test the following hypotheses a) rapid-eye-movement (REM) sleep is necessary for the consolidation of ocular dominance plasticity b) this is mediated by protein kinases (Ca2+/calmodulin- dependent protein kinase [CaMKII] and extracellular regulated kinase [ERK]) activated in REM sleep. We propose the following Specific Aims: 1. Determine the role of REM sleep in the consolidation of ocular dominance plasticity. In this Aim, we will examine the effects of different amounts of REM sleep on two different types of cortical plasticity that require strengthening of visual circuits. This will be accomplished by quantitatively measuring and manipulating vigilance states and binocular visual input to primary visual cortex in the freely-behaving animal. This is followed by three independent and objective measures of cortical plasticity in vivo (acute and chronic single-neuron electrophysiology and optical imaging of intrinsic cortical signals). 2. Determine the role of CaMKII and ERK in the consolidation of ocular dominance plasticity. In this Aim, we will examine the role of CaMKII and ERK signaling in the strengthening of cortical circuits that occurs during sleep. This will be achieved by a) measuring total and phosphorylated CaMKII and ERK proteins in the primary visual cortices of animals that are sacrificed after different amounts of visual experience and rapid- eye-movement sleep b) determining the effects of intracortical pan-CaMK, selective CaMKII and ERK inhibition on the consolidation of ocular dominance plasticity c) mimicry and occlusion experiments are then used to determine if the effects of REM sleep are mediated by CaMK, CaMKII or ERK kinase activity. The results of our investigations will provide new insights into how experience and endogenous brain activity guide cortical circuit development and plasticity. They will also provide new information about how normal and abnormal sleep impacts mammalian brain development.

DESCRIPTION (provided by applicant): Sleep problems such as excessive daytime sleepiness and insomnia are common in the United States. They are found in many psychiatric and neurological disorders and cause deficits in attention, learning and memory. The cellular mechanism that makes animals sleepy and causes cognitive deficits in sleepy animals is unknown. Chemical signaling between glia and neurons (i.e. gliotransmission) may be an important part of this mechanism. Glial astrocytes are brain cells that are electrically silent (relative to neurons) and for many decades were thought to serve purely supportive functions in the brain (e.g. ion buffering). More recent findings indicate that astrocytes are important partner in synaptic neurotransmission. Astrocytes surround synapses and respond to neurotransmitters by secreting their own chemical messengers (gliotransmitters), which in turn regulate neuronal excitability and synaptic transmission. The role of gliotransmission in mammalian behavior is only now beginning to be explored. In mammals astrocytes are densely concentrated in brain regions critical for arousal, sleep and higher cognitive function. We hypothesize that gliotransmission in these regions mediates not only sleepiness, but cognitive deficits associated with sleep loss. We will test our hypothesis by quantitatively measuring sleep regulation in mice with an inducible (conditional) mutation that inhibits astrocytic gliotransmission in vivo. We will also use these mice test the role of gliotransmission in learning and attention deficits caused by sleep loss. In this revised application, we have added two new sets of experiments in response to initial review. First, we now address the role of regional gliotransmission within brain areas implicated in sleep regulation (e.g. the basal forebrain and pre-optic area of the hypothalamus) or memory (hippocampus). This is accomplished by regionally expressing transgenes in the brain in vivo. Second, we also have added a new cognitive task, which is sensitive to sleep loss, but never before examined with respect to gliotransmission (T-maze reference reversal). Our revised application now far exceeds our previous explorations of these phenomena. Our findings will thus provide new insights into the cellular basis of sleep need and the function of non-neuronal cells in animal behavior.

Summary Ocular dominance plasticity (ODP) is a canonical form of synaptic plasticity in vivo triggered by monocular deprivation (MD). It has provided crucial insights into how visual circuits develop and remodel in early life. We have shown that ODP in cats is enhanced by sleep. Our goal is to more completely identify the underlying mechanisms and the brain states in which they occur. To achieve this goal we will develop a new mouse- based model of sleep-dependent ODP. MD in mice triggers physiological and morphological changes in cortical circuits similar to those described in carnivore species. However, unlike carnivore species, molecular techniques used to visualize neuronal activity and measure mRNA are widely used in mice. There are currently no studies that have exploited this model system to identify how experience and sleep influence ODP. In this exploratory proposal, we will use a novel combination of polysomnography and calcium- fluorescence based microscopy in vivo to record plastic changes in visual cortical neurons across sleep and wakefulness. We will also use Translating Ribosome Affinity Purification (TRAP) technology combined with microarray and RNAseq to isolate changes in the transcriptome that occur in the sleeping, remodeling visual cortex. The results of these investigations will provide the foundation for a larger investigation of how experience and sleep shape the developing brain.

Summary The neural growth factors known as neurotrophins are hypothesized to play central roles in sleep homeostasis; an enigmatic process that regulates sleep need. Brain neurotrophin mRNA and protein levels increase with wakefulness and administration of exogenous neurotrophin increases sleep time and indices of sleep intensity. Neurotrophins are secreted by active neurons where they can act globally throughout the brain, or locally in a use-dependent fashion. Neurotrophins also govern cellular processes linked to sleep function including brain cell survival and synaptic plasticity. Nevertheless, the precise role of neurotrophins in sleep and sleep regulation is unclear. This is because previous investigations have relied on non-selective pharmacological agents, non-physiological neurotrophin concentrations, or are principally correlational in nature. We will more precisely determine the role of neurotrophins in sleep regulation using a novel chemo-genetic technique that allows for rapid and reversible inhibition of all three neurotrophin-TrK receptors in vivo. This is accomplished using transgenic mice with knock-in TrKA, B or C receptors that are potently inhibited in the presence of a small, brain permeable molecule. We will use this model to determine the role of each TrK receptor in sleep homeostasis and genome-wide changes in mRNA expression that accompany the accumulation and discharge of sleep need.

Summary Sleep problems such as excessive daytime sleepiness and insomnia are common in the United States. They are found in many psychiatric and neurological disorders and cause deficits in attention, learning and memory. Some sleep problems may be caused by disrupted circadian rhythms, but others may reflect changes in sleep homeostasis; an enigmatic regulatory mechanism that increases sleep drive, sleep amounts and sleep intensity as a function of prior time awake. The cellular mechanisms of sleep homeostasis are incompletely described but have traditionally thought to be neuronal. We, however, have shown that glial astrocytes are part of this mechanism. More specifically, we propose that sleep homeostasis arises from interactions between astrocytes and neurons. We therefore hypothesize that the normal compensatory response to sleep loss involves intracellular and molecular changes in astrocytes. This A1 submission has been extensively revised in accordance with initial review. New experiments and preliminary data are included (indicated by red font). We will test this overall hypothesis with three innovative approaches in vivo. In Aim 1, we combine genetically encoded Ca2+ indicator (GECI) astrocyte imaging with simultaneous polysomnographic recording in unanesthetized mice in vivo. This allows us to measure astrocyte Ca2+ dynamics in natural states of rapid- eye-movement (REM) sleep, non(N)REM sleep and wakefulness using both 2-photon and epiflorescent microscopy. We also more directly test the necessity of intracellular Ca2+ in sleep homeostasis by inducibly reducing this signal in vivo and measuring changes in sleep expression and homeostasis. In Aim 2, we use inducible molecular techniques to alter the major signaling pathways known to exist in astrocytes (i.e. Gq, Gi and Gs proteins) and examine the resulting changes in sleep expression and homeostasis. In Aim 3, we use next generation sequencing technology (single-cell RNA sequencing (scRNA-seq)) to isolate additional (but currently unknown) signaling pathways that are involved in astrocyte-mediated sleep homeostasis. Mammalian astrocytes are highly diverse based on morphology, cell-specific markers (e.g. GFAP+), ion channels, glutamate transporters and metabolic substrates. The relative contribution of these different astrocytes to sleep is unknown. scRNA-seq provides a new and powerful method to address this problem. Impact: Our characterization of a novel glial sleep mechanism will provide new insights into the etiology of abnormal sleep and arousal. Our experiments will also provide new insights into the function of non-neuronal brain cells. This in turn can lead to the development of new therapeutics that target glia, rather than neurons.