Three classes of propofol binding sites on GABAA receptors.

Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator (PAM) of gamma-aminobutyric acid type A (GABAA) receptors. Several potential propofol binding sites that may mediate this effect have been identified using propofol-analogue photoaffinity labeling. o-PD labels ß-H267, a pore-lining residue, whereas AziPm labels residues ß-M286, ß-M227 and α-I239 in the two membrane-facing interfaces (ß(+)/α(-) and α(+)/ß(-)) between α and ß subunits. This study used photoaffinity labeling of α1ß3 GABAA receptors to reconcile the apparently conflicting results obtained with AziPm and o-PD labeling, focusing on whether ß3-H267 identifies specific propofol binding site(s). The results show that propofol, but not AziPm protects ß3-H267 from labeling by o-PD, whereas both propofol and o-PD protect against AziPm labeling of ß3-M286, ß3-M227 and α1I239. These data indicate that there are three distinct classes of propofol binding sites, with AziPm binding to two of the classes and o-PD to all three. Analysis of binding stoichiometry using native mass spectrometry in ß3 homomeric receptors, demonstrated a minimum of five AziPm labeled residues and three o-PD labeled residues per pentamer, suggesting that there are two distinct propofol binding sites per ß-subunit. The native MS data, coupled with photolabeling performed in the presence of zinc, indicate that the binding site(s) identified by o-PD are adjacent to, but not within the channel pore, since the pore at the 17' H267 residue can accommodate only one propofol molecule. These data validate the existence of three classes of specific propofol binding sites on α1ß3 GABAA receptors.

A REM-active basal ganglia circuit that regulates anxiety.

Rapid eye movement (REM) sleep has been hypothesized to promote emotional resilience, but any neuronal circuits mediating this have not been identified. We find that in mice, somatostatin (Som) neurons in the entopeduncular nucleus (EPSom)/internal globus pallidus are predominantly active during REM sleep. This unique REM activity is both necessary and sufficient for maintaining normal REM sleep. Inhibiting or exciting EPSom neurons reduced or increased REM sleep duration, respectively. Activation of the sole downstream target of EPSom neurons, Vglut2 cells in the lateral habenula (LHb), increased sleep via the ventral tegmental area (VTA). A simple chemogenetic scheme to periodically inhibit the LHb over 4 days selectively removed a significant amount of cumulative REM sleep. Chronic, but not acute, REM reduction correlated with mice becoming anxious and more sensitive to aversive stimuli. Therefore, we suggest that cumulative REM sleep, in part generated by the EP → LHb → VTA circuit identified here, could contribute to stabilizing reactions to habitual aversive stimuli.

Brain clearance is reduced during sleep and anesthesia.

It has been suggested that the function of sleep is to actively clear metabolites and toxins from the brain. Enhanced clearance is also said to occur during anesthesia. Here, we measure clearance and movement of fluorescent molecules in the brains of male mice and show that movement is, in fact, independent of sleep and wake or anesthesia. Moreover, we show that brain clearance is markedly reduced, not increased, during sleep and anesthesia.

Somatostatin neurons in prefrontal cortex initiate sleep-preparatory behavior and sleep via the preoptic and lateral hypothalamus.

The prefrontal cortex (PFC) enables mammals to respond to situations, including internal states, with appropriate actions. One such internal state could be 'tiredness'. Here, using activity tagging in the mouse PFC, we identified particularly excitable, fast-spiking, somatostatin-expressing, γ-aminobutyric acid (GABA) (PFCSst-GABA) cells that responded to sleep deprivation. These cells projected to the lateral preoptic (LPO) hypothalamus and the lateral hypothalamus (LH). Stimulating PFCSst-GABA terminals in the LPO hypothalamus caused sleep-preparatory behavior (nesting, elevated theta power and elevated temperature), and stimulating PFCSst-GABA terminals in the LH mimicked recovery sleep (non-rapid eye-movement sleep with higher delta power and lower body temperature). PFCSst-GABA terminals had enhanced activity during nesting and sleep, inducing inhibitory postsynaptic currents on diverse cells in the LPO hypothalamus and the LH. The PFC also might feature in deciding sleep location in the absence of excessive fatigue. These findings suggest that the PFC instructs the hypothalamus to ensure that optimal sleep takes place in a suitable place.

Understanding Sleep Regulation in Normal and Pathological Conditions, and Why It Matters.

Sleep occupies a peculiar place in our lives and in science, being both eminently familiar and profoundly enigmatic. Historically, philosophers, scientists and artists questioned the meaning and purpose of sleep. If Shakespeare's verses from MacBeth depicting "Sleep that soothes away all our worries" and "relieves the weary laborer and heals hurt minds" perfectly epitomize the alleviating benefits of sleep, it is only during the last two decades that the growing understanding of the sophisticated sleep regulatory mechanisms allows us to glimpse putative biological functions of sleep. Sleep control brings into play various brain-wide processes occurring at the molecular, cellular, circuit, and system levels, some of them overlapping with a number of disease-signaling pathways. Pathogenic processes, including mood disorders (e.g., major depression) and neurodegenerative illnesses such Huntington's or Alzheimer's diseases, can therefore affect sleep-modulating networks which disrupt the sleep-wake architecture, whereas sleep disturbances may also trigger various brain disorders. In this review, we describe the mechanisms underlying sleep regulation and the main hypotheses drawn about its functions. Comprehending sleep physiological orchestration and functions could ultimately help deliver better treatments for people living with neurodegenerative diseases.

NMDA Receptors in the Lateral Preoptic Hypothalamus Are Essential for Sustaining NREM and REM Sleep.

The lateral preoptic (LPO) hypothalamus is a center for NREM and REM sleep induction and NREM sleep homeostasis. Although LPO is needed for NREM sleep, we found that calcium signals were, surprisingly, highest in REM sleep. Furthermore, and equally surprising, NMDA receptors in LPO were the main drivers of excitation. Deleting the NMDA receptor GluN1 subunit from LPO abolished calcium signals in all cells and produced insomnia. Mice of both sexes had highly fragmented NREM sleep-wake patterns and could not generate conventionally classified REM sleep. The sleep phenotype produced by deleting NMDA receptors depended on where in the hypothalamus the receptors were deleted. Deleting receptors from the anterior hypothalamic area (AHA) did not influence sleep-wake states. The sleep fragmentation originated from NMDA receptors on GABA neurons in LPO. Sleep fragmentation could be transiently overcome with sleeping medication (zolpidem) or sedatives (dexmedetomidine; Dex). By contrast, fragmentation persisted under high sleep pressure produced by sleep deprivation (SD), mice had a high propensity to sleep but woke up. By analyzing changes in δ power, sleep homeostasis (also referred to as "sleep drive") remained intact after NMDA receptor ablation. We suggest NMDA glutamate receptor activation stabilizes firing of sleep-on neurons and that mechanisms of sleep maintenance differ from that of the sleep drive itself.SIGNIFICANCE STATEMENT Insomnia is a common affliction. Most insomniacs feel that they do not get enough sleep, but in fact, often have good amounts of sleep. Their sleep, however, is fragmented, and sufferers wake up feeling unrefreshed. It is unknown how sleep is maintained once initiated. We find that in mice, NMDA-type glutamate receptors in the hypothalamus are the main drivers of excitation and are required for a range of sleep properties: they are, in fact, needed for both sustained NREM sleep periods, and REM sleep generation. When NMDA receptors are selectively reduced from inhibitory preoptic (PO) neurons, mice have normal total amounts of sleep but high sleep-wake fragmentation, providing a model for studying intractable insomnia.

A specific circuit in the midbrain detects stress and induces restorative sleep.

In mice, social defeat stress (SDS), an ethological model for psychosocial stress, induces sleep. Such sleep could enable resilience, but how stress promotes sleep is unclear. Activity-dependent tagging revealed a subset of ventral tegmental area γ-aminobutyric acid (GABA)-somatostatin (VTAVgat-Sst) cells that sense stress and drive non-rapid eye movement (NREM) and REM sleep through the lateral hypothalamus and also inhibit corticotropin-releasing factor (CRF) release in the paraventricular hypothalamus. Transient stress enhances the activity of VTAVgat-Sst cells for several hours, allowing them to exert their sleep effects persistently. Lesioning of VTAVgat-Sst cells abolished SDS-induced sleep; without it, anxiety and corticosterone concentrations remained increased after stress. Thus, a specific circuit allows animals to restore mental and body functions by sleeping, potentially providing a refined route for treating anxiety disorders.

Nitric Oxide Synthase Neurons in the Preoptic Hypothalamus Are NREM and REM Sleep-Active and Lower Body Temperature.

When mice are exposed to external warmth, nitric oxide synthase (NOS1) neurons in the median and medial preoptic (MnPO/MPO) hypothalamus induce sleep and concomitant body cooling. However, how these neurons regulate baseline sleep and body temperature is unknown. Using calcium photometry, we show that NOS1 neurons in MnPO/MPO are predominantly NREM and REM active, especially at the boundary of wake to NREM transitions, and in the later parts of REM bouts, with lower activity during wakefulness. In addition to releasing nitric oxide, NOS1 neurons in MnPO/MPO can release GABA, glutamate and peptides. We expressed tetanus-toxin light-chain in MnPO/MPO NOS1 cells to reduce vesicular release of transmitters. This induced changes in sleep structure: over 24 h, mice had less NREM sleep in their dark (active) phase, and more NREM sleep in their light (sleep) phase. REM sleep episodes in the dark phase were longer, and there were fewer REM transitions between other vigilance states. REM sleep had less theta power. Mice with synaptically blocked MnPO/MPO NOS1 neurons were also warmer than control mice at the dark-light transition (ZT0), as well as during the dark phase siesta (ZT16-20), where there is usually a body temperature dip. Also, at this siesta point of cooled body temperature, mice usually have more NREM, but mice with synaptically blocked MnPO/MPO NOS1 cells showed reduced NREM sleep at this time. Overall, MnPO/MPO NOS1 neurons promote both NREM and REM sleep and contribute to chronically lowering body temperature, particularly at transitions where the mice normally enter NREM sleep.

Brain Clocks, Sleep, and Mood.

The suprachiasmatic nucleus houses the master clock, but the genes which encode the circadian clock components are also expressed throughout the brain. Here, we review how circadian clock transcription factors regulate neuromodulator systems such as histamine, dopamine, and orexin that promote arousal. These circadian transcription factors all lead to repression of the histamine, dopamine, and orexin systems during the sleep period, so ensuring integration with the ecology of the animal. If these transcription factors are deleted or mutated, in addition to the global disturbances in circadian rhythms, this causes a chronic up-regulation of neuromodulators leading to hyperactivity, elevated mood, and reduced sleep, which have been suggested to be states resembling mania.

The inescapable drive to sleep: Overlapping mechanisms of sleep and sedation.

Common human experience is that a long period without sleep is unsustainable, and it is also detrimental to health and behavior. The powerful and primal urge to sleep after sleep deprivation is intense and seems inescapable. The longer we stay awake, the more we feel the need to sleep, and however much we resist, we will inevitably succumb. Although it is obvious what benefits derive from other common and strong physiological drives, such as hunger, sex, and thirst, it is less obvious what drives us to sleep and what benefits accrue. Understanding the biochemical or circuit basis for the sleep drive could enable the benefits of sleep to be artificially stimulated with a new generation of sedative drugs.

Dysfunction of ventral tegmental area GABA neurons causes mania-like behavior.

The ventral tegmental area (VTA), an important source of dopamine, regulates goal- and reward-directed and social behaviors, wakefulness, and sleep. Hyperactivation of dopamine neurons generates behavioral pathologies. But any roles of non-dopamine VTA neurons in psychiatric illness have been little explored. Lesioning or chemogenetically inhibiting VTA GABAergic (VTAVgat) neurons generated persistent wakefulness with mania-like qualities: locomotor activity was increased; sensitivity to D-amphetamine was heightened; immobility times decreased on the tail suspension and forced swim tests; and sucrose preference increased. Furthermore, after sleep deprivation, mice with lesioned VTAVgat neurons did not catch up on lost sleep, even though they were starting from a sleep-deprived baseline, suggesting that sleep homeostasis was bypassed. The mania-like behaviors, including the sleep loss, were reversed by valproate, and re-emerged when treatment was stopped. Lithium salts and lamotrigine, however, had no effect. Low doses of diazepam partially reduced the hyperlocomotion and fully recovered the immobility time during tail suspension. The mania like-behaviors mostly depended on dopamine, because giving D1/D2/D3 receptor antagonists reduced these behaviors, but also partially on VTAVgat projections to the lateral hypothalamus (LH). Optically or chemogenetically inhibiting VTAVgat terminals in the LH elevated locomotion and decreased immobility time during the tail suspension and forced swimming tests. VTAVgat neurons help set an animal's (and perhaps human's) mental and physical activity levels. Inputs inhibiting VTAVgat neurons intensify wakefulness (increased activity, enhanced alertness and motivation), qualities useful for acute survival. In the extreme, however, decreased or failed inhibition from VTAVgat neurons produces mania-like qualities (hyperactivity, hedonia, decreased sleep).

Xenon treatment after severe traumatic brain injury improves locomotor outcome, reduces acute neuronal loss and enhances early beneficial neuroinflammation: a randomized, blinded, controlled animal study.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of morbidity and mortality, but there are no clinically proven treatments that specifically target neuronal loss and secondary injury development following TBI. In this study, we evaluate the effect of xenon treatment on functional outcome, lesion volume, neuronal loss and neuroinflammation after severe TBI in rats. METHODS: Young adult male Sprague Dawley rats were subjected to controlled cortical impact (CCI) brain trauma or sham surgery followed by treatment with either 50% xenon:25% oxygen balance nitrogen, or control gas 75% nitrogen:25% oxygen. Locomotor function was assessed using Catwalk-XT automated gait analysis at baseline and 24 h after injury. Histological outcomes were assessed following perfusion fixation at 15 min or 24 h after injury or sham procedure. RESULTS: Xenon treatment reduced lesion volume, reduced early locomotor deficits, and attenuated neuronal loss in clinically relevant cortical and subcortical areas. Xenon treatment resulted in significant increases in Iba1-positive microglia and GFAP-positive reactive astrocytes that was associated with neuronal preservation. CONCLUSIONS: Our findings demonstrate that xenon improves functional outcome and reduces neuronal loss after brain trauma in rats. Neuronal preservation was associated with a xenon-induced enhancement of microglial cell numbers and astrocyte activation, consistent with a role for early beneficial neuroinflammation in xenon's neuroprotective effect. These findings suggest that xenon may be a first-line clinical treatment for brain trauma.

Sleep and thermoregulation.

In homeothermic animals sleep preparatory behaviours often promote thermal efficiency, including warmth-seeking, adopting particular postures (curling up, head tucking) and nest building, all promoting warmer skin microclimates. Skin warmth induces NREM sleep and body cooling via circuitry that connects skin sensation to the preoptic hypothalamus. Coupling sleep induction and lower body temperature could serve to minimise energy expenditure or allow energy reallocation. Cooling during NREM sleep may also induce transcriptional changes in genes whose products facilitate housekeeping functions or measure the time spent sleeping.

Sleep deprivation and stress: a reciprocal relationship.

Sleep is highly conserved across evolution, suggesting vital biological functions that are yet to be fully understood. Animals and humans experiencing partial sleep restriction usually exhibit detrimental physiological responses, while total and prolonged sleep loss could lead to death. The perturbation of sleep homeostasis is usually accompanied by an increase in hypothalamic-pituitary-adrenal (HPA) axis activity, leading to a rise in circulating levels of stress hormones (e.g. cortisol in humans, corticosterone in rodents). Such hormones follow a circadian release pattern under undisturbed conditions and participate in the regulation of sleep. The investigation of the consequences of sleep deprivation, from molecular changes to behavioural alterations, has been used to study the fundamental functions of sleep. However, the reciprocal relationship between sleep and the activity of the HPA axis is problematic when investigating sleep using traditional sleep-deprivation protocols that can induce stress per se. This is especially true in studies using rodents in which sleep deprivation is achieved by exogenous, and potentially stressful, sensory-motor stimulations that can undoubtedly confuse their conclusions. While more research is needed to explore the mechanisms underlying sleep loss and health, avoiding stress as a confounding factor in sleep-deprivation studies is therefore crucial. This review examines the evidence of the intricate links between sleep and stress in the context of experimental sleep deprivation, and proposes a more sophisticated research framework for sleep-deprivation procedures that could benefit from recent progress in biotechnological tools for precise neuromodulation, such as chemogenetics and optogenetics, as well as improved automated real-time sleep-scoring algorithms.

Noble gas neuroprotection: xenon and argon protect against hypoxic-ischaemic injury in rat hippocampus in vitro via distinct mechanisms.

BACKGROUND: Noble gases may provide novel treatments for neurological injuries such as ischaemic and traumatic brain injury. Few studies have evaluated the complete series of noble gases under identical conditions in the same model. METHODS: We used an in vitro model of hypoxia-ischaemia to evaluate the neuroprotective properties of the series of noble gases, helium, neon, argon, krypton, and xenon. Organotypic hippocampal brain slices from mice were subjected to oxygen-glucose deprivation, and injury was quantified using propidium iodide fluorescence. RESULTS: Both xenon and argon were equally effective neuroprotectants, with 0.5 atm of xenon or argon reducing injury by 96% (P<0.0001), whereas helium, neon, and krypton were devoid of any protective effect. Neuroprotection by xenon, but not argon, was reversed by elevated glycine. CONCLUSIONS: Xenon and argon are equally effective as neuroprotectants against hypoxia-ischaemia in vitro, with both gases preventing injury development. Although xenon's neuroprotective effect may be mediated by inhibition of the N-methyl-d-aspartate receptor at the glycine site, argon acts via a different mechanism. These findings may have important implications for their clinical use as neuroprotectants.

Galanin Neurons Unite Sleep Homeostasis and α2-Adrenergic Sedation.

Our urge to sleep increases with time spent awake, until sleep becomes inescapable. The sleep following sleep deprivation is longer and deeper, with an increased power of delta (0.5-4 Hz) oscillations, a phenomenon termed sleep homeostasis [1-4]. Although widely expressed genes regulate sleep homeostasis [1, 4-10] and the process is tracked by somnogens and phosphorylation [1, 3, 7, 11-14], at the circuit level sleep homeostasis has remained mysterious. Previously, we found that sedation induced with α2-adrenergic agonists (e.g., dexmedetomidine) and sleep homeostasis both depend on the preoptic (PO) hypothalamus [15, 16]. Dexmedetomidine, increasingly used for long-term sedation in intensive care units [17], induces a non-rapid-eye-movement (NREM)-like sleep but with undesirable hypothermia [18, 19]. Within the PO, various neuronal subtypes (e.g., GABA/galanin and glutamate/NOS1) induce NREM sleep [20-22] and concomitant body cooling [21, 22]. This could be because NREM sleep's restorative effects depend on lower body temperature [23, 24]. Here, we show that mice with lesioned PO galanin neurons have reduced sleep homeostasis: in the recovery sleep following sleep deprivation there is a diminished increase in delta power, and the mice catch up little on lost sleep. Furthermore, dexmedetomidine cannot induce high-power delta oscillations or sustained hypothermia. Some hours after dexmedetomidine administration to wild-type mice there is a rebound in delta power when they enter normal NREM sleep, reminiscent of emergence from torpor. This delta rebound is reduced in mice lacking PO galanin neurons. Thus, sleep homeostasis and dexmedetomidine-induced sedation require PO galanin neurons and likely share common mechanisms.