A genetically encoded sensor for in vivo imaging of orexin neuropeptides.

Orexins (also called hypocretins) are hypothalamic neuropeptides that carry out essential functions in the central nervous system; however, little is known about their release and range of action in vivo owing to the limited resolution of current detection technologies. Here we developed a genetically encoded orexin sensor (OxLight1) based on the engineering of circularly permutated green fluorescent protein into the human type-2 orexin receptor. In mice OxLight1 detects optogenetically evoked release of endogenous orexins in vivo with high sensitivity. Photometry recordings of OxLight1 in mice show rapid orexin release associated with spontaneous running behavior, acute stress and sleep-to-wake transitions in different brain areas. Moreover, two-photon imaging of OxLight1 reveals orexin release in layer 2/3 of the mouse somatosensory cortex during emergence from anesthesia. Thus, OxLight1 enables sensitive and direct optical detection of orexin neuropeptides with high spatiotemporal resolution in living animals.

Oscillating circuitries in the sleeping brain.

Brain activity during sleep is characterized by circuit-specific oscillations, including slow waves, spindles and theta waves, which are nested in thalamocortical or hippocampal networks. A major challenge is to determine the relationships between these oscillatory activities and the identified networks of sleep-promoting and wake-promoting neurons distributed throughout the brain. Improved understanding of the neurobiological mechanisms that orchestrate sleep-related oscillatory activities, both in time and space, is expected to generate further insight into the delineation of sleep states and their functions.

REM sleep stabilizes hypothalamic representation of feeding behavior.

During rapid eye movement (REM) sleep, behavioral unresponsiveness contrasts strongly with intense brain-wide neural network dynamics. Yet, the physiological functions of this cellular activation remain unclear. Using in vivo calcium imaging in freely behaving mice, we found that inhibitory neurons in the lateral hypothalamus (LHvgat) show unique activity patterns during feeding that are reactivated during REM, but not non-REM, sleep. REM sleep-specific optogenetic silencing of LHvgat cells induced a reorganization of these activity patterns during subsequent feeding behaviors accompanied by decreased food intake. Our findings provide evidence for a role for REM sleep in the maintenance of cellular representations of feeding behavior.

Reciprocal Lateral Hypothalamic and Raphe GABAergic Projections Promote Wakefulness.

The lateral hypothalamus (LH), together with multiple neuromodulatory systems of the brain, such as the dorsal raphe nucleus (DR), is implicated in arousal, yet interactions between these systems are just beginning to be explored. Using a combination of viral tracing, circuit mapping, electrophysiological recordings from identified neurons, and combinatorial optogenetics in mice, we show that GABAergic neurons in the LH selectively inhibit GABAergic neurons in the DR, resulting in increased firing of a substantial fraction of its neurons that ultimately promotes arousal. These DRGABA neurons are wake active and project to multiple brain areas involved in the control of arousal, including the LH, where their specific activation potently influences local network activity leading to arousal from sleep. Our results show how mutual inhibitory projections between the LH and the DR promote wakefulness and suggest a complex arousal control by intimate interactions between long-range connections and local circuit dynamics.SIGNIFICANCE STATEMENT: Multiple brain systems including the lateral hypothalamus and raphe serotonergic system are involved in the regulation of the sleep/wake cycle, yet the interaction between these systems have remained elusive. Here we show that mutual disinhibition mediated by long range inhibitory projections between these brain areas can promote wakefulness. The main importance of this work relies in revealing the interaction between a brain area involved in autonomic regulation and another in controlling higher brain functions including reward, patience, mood and sensory coding.

Loss of Snord116 alters cortical neuronal activity in mice: a preclinical investigation of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder that is characterized by metabolic alteration and sleep abnormalities mostly related to rapid eye movement (REM) sleep disturbances. The disease is caused by genomic imprinting defects that are inherited through the paternal line. Among the genes located in the PWS region on chromosome 15 (15q11-q13), small nucleolar RNA 116 (Snord116) has been previously associated with intrusions of REM sleep into wakefulness in humans and mice. Here, we further explore sleep regulation of PWS by reporting a study with PWScrm+/p- mouse line, which carries a paternal deletion of Snord116. We focused our study on both macrostructural electrophysiological components of sleep, distributed among REMs and nonrapid eye movements. Of note, here, we study a novel electroencephalography (EEG) graphoelements of sleep for mouse studies, the well-known spindles. EEG biomarkers are often linked to the functional properties of cortical neurons and can be instrumental in translational studies. Thus, to better understand specific properties, we isolated and characterized the intrinsic activity of cortical neurons using in vitro microelectrode array. Our results confirm that the loss of Snord116 gene in mice influences specific properties of REM sleep, such as theta rhythms and, for the first time, the organization of REM episodes throughout sleep-wake cycles. Moreover, the analysis of sleep spindles present novel specific phenotype in PWS mice, indicating that a new catalog of sleep biomarkers can be informative in preclinical studies of PWS.

Slow Waves Promote Sleep-Dependent Plasticity and Functional Recovery after Stroke.

Functional recovery after stroke is associated with a remapping of neural circuits. This reorganization is often associated with low-frequency, high-amplitude oscillations in the peri-infarct zone in both rodents and humans. These oscillations are reminiscent of sleep slow waves (SW) and suggestive of a role for sleep in brain plasticity that occur during stroke recovery; however, direct evidence is missing. Using a stroke model in male mice, we showed that stroke was followed by a transient increase in NREM sleep accompanied by reduced amplitude and slope of ipsilateral NREM sleep SW. We next used 5 ms optical activation of Channelrhodopsin 2-expressing pyramidal neurons, or 200 ms silencing of Archeorhodopsin T-expressing pyramidal neurons, to generate local cortical UP, or DOWN, states, respectively, both sharing similarities with spontaneous NREM SW in freely moving mice. Importantly, we found that single optogenetically evoked SW (SWopto) in the peri-infarct zone, randomly distributed during sleep, significantly improved fine motor movements of the limb corresponding to the sensorimotor stroke lesion site compared with spontaneous recovery and control conditions, while motor strength remained unchanged. In contrast, SWopto during wakefulness had no effect. Furthermore, chronic SWopto during sleep were associated with local axonal sprouting as revealed by the increase of anatomic presynaptic and postsynaptic markers in the peri-infarct zone and corresponding contralesional areas to cortical circuit reorganization during stroke recovery. These results support a role for sleep SW in cortical circuit plasticity and sensorimotor recovery after stroke and provide a clinically relevant framework for rehabilitation strategies using neuromodulation during sleep.SIGNIFICANCE STATEMENT Brain stroke is one of the leading causes of death and major disabilities in the elderly worldwide. A better understanding of the pathophysiological mechanisms underlying spontaneous brain plasticity after stroke, together with an optimization of rehabilitative strategies, are essential to improve stroke treatments. Here, we investigate the role of optogenetically induced sleep slow waves in an animal model of ischemic stroke and identify sleep as a window for poststroke intervention that promotes neuroplasticity and facilitates sensorimotor recovery.

Neural substrates of awakening probed with optogenetic control of hypocretin neurons.

The neural underpinnings of sleep involve interactions between sleep-promoting areas such as the anterior hypothalamus, and arousal systems located in the posterior hypothalamus, the basal forebrain and the brainstem. Hypocretin (Hcrt, also known as orexin)-producing neurons in the lateral hypothalamus are important for arousal stability, and loss of Hcrt function has been linked to narcolepsy. However, it is unknown whether electrical activity arising from Hcrt neurons is sufficient to drive awakening from sleep states or is simply correlated with it. Here we directly probed the impact of Hcrt neuron activity on sleep state transitions with in vivo neural photostimulation, genetically targeting channelrhodopsin-2 to Hcrt cells and using an optical fibre to deliver light deep in the brain, directly into the lateral hypothalamus, of freely moving mice. We found that direct, selective, optogenetic photostimulation of Hcrt neurons increased the probability of transition to wakefulness from either slow wave sleep or rapid eye movement sleep. Notably, photostimulation using 5-30 Hz light pulse trains reduced latency to wakefulness, whereas 1 Hz trains did not. This study establishes a causal relationship between frequency-dependent activity of a genetically defined neural cell type and a specific mammalian behaviour central to clinical conditions and neurobehavioural physiology.

Sleep and the hypothalamus.

Neural substrates of wakefulness, rapid eye movement sleep (REMS), and non-REMS (NREMS) in the mammalian hypothalamus overlap both anatomically and functionally with cellular networks that support physiological and behavioral homeostasis. Here, we review the roles of sleep neurons of the hypothalamus in the homeostatic control of thermoregulation or goal-oriented behaviors during wakefulness. We address how hypothalamic circuits involved in opposing behaviors such as core body temperature and sleep compute conflicting information and provide a coherent vigilance state. Finally, we highlight some of the key unresolved questions and challenges, and the promise of a more granular view of the cellular and molecular diversity underlying the integrative role of the hypothalamus in physiological and behavioral homeostasis.

Optogenetic interrogation of dopaminergic modulation of the multiple phases of reward-seeking behavior.

Phasic activation of dopaminergic neurons is associated with reward-predicting cues and supports learning during behavioral adaptation. While noncontingent activation of dopaminergic neurons in the ventral tegmental are (VTA) is sufficient for passive behavioral conditioning, it remains unknown whether the phasic dopaminergic signal is truly reinforcing. In this study, we first targeted the expression of channelrhodopsin-2 to dopaminergic neurons of the VTA and optimized optogenetically evoked dopamine transients. Second, we showed that phasic activation of dopaminergic neurons in freely moving mice causally enhances positive reinforcing actions in a food-seeking operant task. Interestingly, such effect was not found in the absence of food reward. We further found that phasic activation of dopaminergic neurons is sufficient to reactivate previously extinguished food-seeking behavior in the absence of external cues. This was also confirmed using a single-session reversal paradigm. Collectively, these data suggest that activation of dopaminergic neurons facilitates the development of positive reinforcement during reward-seeking and behavioral flexibility.

How REM sleep shapes hypothalamic computations for feeding behavior.

The electrical activity of diverse brain cells is modulated across states of vigilance, namely wakefulness, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Enhanced activity of neuronal circuits during NREM sleep impacts on subsequent awake behaviors, yet the significance of their activation, or lack thereof, during REM sleep remains unclear. This review focuses on feeding-promoting cells in the lateral hypothalamus (LH) that express the vesicular GABA and glycine transporter (vgat) as a model to further understand the impact of REM sleep on neural encoding of goal-directed behavior. It emphasizes both spatial and temporal aspects of hypothalamic cell dynamics across awake behaviors and REM sleep, and discusses a role for REM sleep in brain plasticity underlying energy homeostasis and behavioral optimization.

Sleep and Metabolism: Implication of Lateral Hypothalamic Neurons.

During the last decade, optogenetic-based circuit mapping has become one of the most common approaches to systems neuroscience, and amassing studies have expanded our understanding of brain structures causally involved in the regulation of sleep-wake cycles. Recent imaging technologies enable the functional mapping of cellular activity, from population down to single-cell resolution, across a broad repertoire of behaviors and physiological processes, including sleep-wake states. This chapter summarizes experimental evidence implicating hypocretins/orexins, melanin-concentrating hormone, and inhibitory neurons from the lateral hypothalamus (LH) in forming an intricate network involved in regulating sleep and metabolism, including feeding behaviors. It further confirms the dual sleep-metabolic functions of LH cells, and sheds light on a possible mechanism underlying brain plasticity during sleep and metabolic disorders.

A role for spindles in the onset of rapid eye movement sleep.

Sleep spindle generation classically relies on an interplay between the thalamic reticular nucleus (TRN), thalamo-cortical (TC) relay cells and cortico-thalamic (CT) feedback during non-rapid eye movement (NREM) sleep. Spindles are hypothesized to stabilize sleep, gate sensory processing and consolidate memory. However, the contribution of non-sensory thalamic nuclei in spindle generation and the role of spindles in sleep-state regulation remain unclear. Using multisite thalamic and cortical LFP/unit recordings in freely behaving mice, we show that spike-field coupling within centromedial and anterodorsal (AD) thalamic nuclei is as strong as for TRN during detected spindles. We found that spindle rate significantly increases before the onset of rapid eye movement (REM) sleep, but not wakefulness. The latter observation is consistent with our finding that enhancing spontaneous activity of TRN cells or TRN-AD projections using optogenetics increase spindle rate and transitions to REM sleep. Together, our results extend the classical TRN-TC-CT spindle pathway to include non-sensory thalamic nuclei and implicate spindles in the onset of REM sleep.

Rapid fast-delta decay following prolonged wakefulness marks a phase of wake-inertia in NREM sleep.

Sleep-wake driven changes in non-rapid-eye-movement sleep (NREM) sleep (NREMS) EEG delta (δ-)power are widely used as proxy for a sleep homeostatic process. Here, we noted frequency increases in δ-waves in sleep-deprived mice, prompting us to re-evaluate how slow-wave characteristics relate to prior sleep-wake history. We identified two classes of δ-waves; one responding to sleep deprivation with high initial power and fast, discontinuous decay during recovery sleep (δ2) and another unrelated to time-spent-awake with slow, linear decay (δ1). Reanalysis of previously published datasets demonstrates that δ-band heterogeneity after sleep deprivation is also present in human subjects. Similar to sleep deprivation, silencing of centromedial thalamus neurons boosted subsequent δ2-waves, specifically. δ2-dynamics paralleled that of temperature, muscle tone, heart rate, and neuronal ON-/OFF-state lengths, all reverting to characteristic NREMS levels within the first recovery hour. Thus, prolonged waking seems to necessitate a physiological recalibration before typical NREMS can be reinstated.

Sleep-wake control and the thalamus.

Sleep is an essential component of animal behavior, controlled by both circadian and homeostatic processes. Typical brain oscillations for sleep and wake states are distinctive and reflect recurrent activity amongst neural circuits spanning localized to global brain regions. Since the original discovery of hypothalamic centers controlling both sleep and wakefulness, current views now implicate networks of neuronal and non-neuronal cells distributed brain-wide. Yet the mechanisms of sleep-wake control remain unclear. In light of recent studies, here we review experimental evidence from lesional, correlational, pharmacological and genetics studies, which support a role for the thalamus in several aspects of sleep-wake states. How these thalamo-cortical network mechanisms contribute to other executive functions such as memory consolidation and cognition, remains an open question with direct implications for neuro-psychiatric diseases and stands as a future challenge for basic science and healthcare research.

Thalamic dual control of sleep and wakefulness.

Slow waves (0.5-4 Hz) predominate in the cortical electroencephalogram during non-rapid eye movement (NREM) sleep in mammals. They reflect the synchronization of large neuronal ensembles alternating between active (UP) and quiescent (Down) states and propagating along the neocortex. The thalamic contribution to cortical UP states and sleep modulation remains unclear. Here we show that spontaneous firing of centromedial thalamus (CMT) neurons in mice is phase-advanced to global cortical UP states and NREM-wake transitions. Tonic optogenetic activation of CMT neurons induces NREM-wake transitions, whereas burst activation mimics UP states in the cingulate cortex and enhances brain-wide synchrony of cortical slow waves during sleep, through a relay in the anterodorsal thalamus. Finally, we demonstrate that CMT and anterodorsal thalamus relay neurons promote sleep recovery. These findings suggest that the tonic and/or burst firing pattern of CMT neurons can modulate brain-wide cortical activity during sleep and provides dual control of sleep-wake states.

Monoaminergic control of brain states and sensory processing: Existing knowledge and recent insights obtained with optogenetics.

Monoamines are key neuromodulators involved in a variety of physiological and pathological brain functions. Classical studies using physiological and pharmacological tools have revealed several essential aspects of monoaminergic involvement in regulating the sleep-wake cycle and influencing sensory responses but many features have remained elusive due to technical limitations. The application of optogenetic tools led to the ability of monitoring and controlling neuronal populations with unprecedented temporal precision and neurochemical specificity. Here, we focus on recent advances in revealing the roles of some monoamines in brain state control and sensory information processing. We summarize the central position of monoamines in integrating sensory processing across sleep-wake states with an emphasis on research conducted using optogenetic techniques. Finally, we discuss the limitations and perspectives of new integrated experimental approaches in understanding the modulatory mechanisms of monoaminergic systems in the mammalian brain.

Sleep: the sound of a local alarm clock.

Besides the master clock located in the suprachiasmatic nucleus (SCN) of the brain, additional clocks are distributed across the central nervous system and the body. The role of these 'secondary' clocks remains unclear. A new study shows that the lack of an internal clock in histamine neurons profoundly perturbs sleep.