Moderate cooling after birth asphyxia is associated with substantial reductions in death and disability, but additional therapies might provide further benefit. We assessed whether the addition of xenon gas, a promising novel therapy, after the initiation of hypothermia for birth asphyxia would result in further improvement.

Acute chemogenetic inhibition of histamine (HA) neurons in adult mice induced nonrapid eye movement (NREM) sleep with an increased delta power. By contrast, selective genetic lesioning of HA neurons with caspase in adult mice exhibited a normal sleep-wake cycle overall, except at the diurnal start of the lights-off period, when they remained sleepier. The amount of time spent in NREM sleep and in the wake state in mice with lesioned HA neurons was unchanged over 24 hr, but the sleep-wake cycle was more fragmented. Both the delayed increase in wakefulness at the start of the night and the sleep-wake fragmentation are similar phenotypes to histidine decarboxylase knockout mice, which cannot synthesize HA. Chronic loss of HA neurons did not affect sleep homeostasis after sleep deprivation. However, the chronic loss of HA neurons or chemogenetic inhibition of HA neurons did notably reduce the ability of the wake-promoting compound modafinil to sustain wakefulness. Thus, part of modafinil's wake-promoting actions arise through the HA system.

How external stimuli prevent the onset of sleep has been little studied. This is usually considered to be a non-specific type of phenomenon. However, the hypnotic drug dexmedetomidine, an agonist at α2 adrenergic receptors, has unusual properties that make it useful for investigating this question. Dexmedetomidine is considered to produce an 'arousable' sleep-like state, so that patients or animals given dexmedetomidine become alert following modest stimulation. We hypothesized that it might be more difficult to make mice unconscious with dexmedetomidine if there was a sufficient external stimulus. Employing a motorized rotating cylinder, which provided a continuous and controlled arousal stimulus, we quantitatively measured the ability of such a stimulus to prevent dexmedetomidine loss of righting reflex in two inbred strains of mice (C57BL/6 and 129X1). We found that whereas the C57BL/6 strain required a strong stimulus to prevent dexmedetomidine-induced hypnosis, the 129X1 strain stayed awake even with minimal stimuli. Remarkably, this could be calibrated as a simple threshold trait, i.e. a binary 'yes-no' response, which after crossing the two mouse strains behaved as a dominant-like trait. We carried out a genome-wide linkage analysis on the F2 progeny to determine if the ability of a stimulus to prevent dexmedetomidine hypnosis could be mapped to one or more chromosomal regions. We identified a locus on chromosome 4 with an associated Logarithm of Odds score exceeding the pre-established threshold level. These results show that complex traits, such as the ability of a stimulus to reverse drug-induced hypnosis, may have precise genetic determinants.

Cell-type specific differences in the kinetics of inhibitory postsynaptic conductance changes (IPSCs) are believed to impact upon network dynamics throughout the brain. Much attention has focused on how GABAA receptor (GABAAR) α and β subunit diversity will influence IPSC kinetics, but less is known about the influence of the γ subunit. We have examined whether GABAAR γ subunit heterogeneity influences IPSC properties in the thalamus. The γ2 subunit gene was deleted from GABAARs selectively in the dorsal lateral geniculate nucleus (dLGN). The removal of the γ2 subunit from the dLGN reduced the overall spontaneous IPSC (sIPSC) frequency across all relay cells and produced an absence of IPSCs in a subset of relay neurons. The remaining slower IPSCs were both insensitive to diazepam and zinc indicating the absence of the γ2 subunit. Because these slower IPSCs were potentiated by methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), we propose these IPSCs involve γ1 subunit-containing GABAAR activation. Therefore, γ subunit heterogeneity appears to influence the kinetics of GABAAR-mediated synaptic transmission in the visual thalamus in a cell-selective manner. We suggest that activation of γ1 subunit-containing GABAARs give rise to slower IPSCs in general, while faster IPSCs tend to be mediated by γ2 subunit-containing GABAARs.

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).

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.

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.

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.

Mammals, including humans, prepare for sleep by nesting and/or curling up, creating microclimates of skin warmth. To address whether external warmth induces sleep through defined circuitry, we used c-Fos-dependent activity tagging, which captures populations of activated cells and allows them to be reactivated to test their physiological role. External warming tagged two principal groups of neurons in the median preoptic (MnPO)/medial preoptic (MPO) hypothalamic area. GABA neurons located mainly in MPO produced non-rapid eye movement (NREM) sleep but no body temperature decrease. Nitrergic-glutamatergic neurons in MnPO-MPO induced both body cooling and NREM sleep. This circuitry explains how skin warming induces sleep and why the maximal rate of core body cooling positively correlates with sleep onset. Thus, the pathways that promote NREM sleep, reduced energy expenditure, and body cooling are inextricably linked, commanded by the same neurons. This implies that one function of NREM sleep is to lower brain temperature and/or conserve energy.

Zolpidem, a GABAA receptor-positive modulator, is the gold-standard drug for treating insomnia. Zolpidem prolongs IPSCs to decrease sleep latency and increase sleep time, effects that depend on α2 and/or α3 subunit-containing receptors. Compared with natural NREM sleep, zolpidem also decreases the EEG power, an effect that depends on α1 subunit-containing receptors, and which may make zolpidem-induced sleep less optimal. In this paper, we investigate whether zolpidem needs to potentiate only particular GABAergic pathways to induce sleep without reducing EEG power. Mice with a knock-in F77I mutation in the GABAA receptor γ2 subunit gene are zolpidem-insensitive. Using these mice, GABAA receptors in the frontal motor neocortex and hypothalamic (tuberomammillary nucleus) histaminergic-neurons of γ2I77 mice were made selectively sensitive to zolpidem by genetically swapping the γ2I77 subunits with γ2F77 subunits. When histamine neurons were made selectively zolpidem-sensitive, systemic administration of zolpidem shortened sleep latency and increased sleep time. But in contrast to the effect of zolpidem on wild-type mice, the power in the EEG spectra of NREM sleep was not decreased, suggesting that these EEG power-reducing effects of zolpidem do not depend on reduced histamine release. Selective potentiation of GABAA receptors in the frontal cortex by systemic zolpidem administration also reduced sleep latency, but less so than for histamine neurons. These results could help with the design of new sedatives that induce a more natural sleep.

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.

Histaminergic neurons in the tuberomammilary nucleus (TMN) of the hypothalamus form a widely projecting, wake-active network that sustains arousal. Yet most histaminergic neurons contain GABA. Selective siRNA knockdown of the vesicular GABA transporter (vgat, SLC32A1) in histaminergic neurons produced hyperactive mice with an exceptional amount of sustained wakefulness. Ablation of the vgat gene throughout the TMN further sharpened this phenotype. Optogenetic stimulation in the caudate-putamen and neocortex of "histaminergic" axonal projections from the TMN evoked tonic (extrasynaptic) GABAA receptor Cl(-) currents onto medium spiny neurons and pyramidal neurons. These currents were abolished following vgat gene removal from the TMN area. Thus wake-active histaminergic neurons generate a paracrine GABAergic signal that serves to provide a brake on overactivation from histamine, but could also increase the precision of neocortical processing. The long range of histamine-GABA axonal projections suggests that extrasynaptic inhibition will be coordinated over large neocortical and striatal areas.

Do sedatives engage natural sleep pathways? It is usually assumed that anesthetic-induced sedation and loss of righting reflex (LORR) arise by influencing the same circuitry to lesser or greater extents. For the α2 adrenergic receptor agonist dexmedetomidine, we found that sedation and LORR were in fact distinct states, requiring different brain areas: the preoptic hypothalamic area and locus coeruleus (LC), respectively. Selective knockdown of α2A adrenergic receptors from the LC abolished dexmedetomidine-induced LORR, but not sedation. Instead, we found that dexmedetomidine-induced sedation resembled the deep recovery sleep that follows sleep deprivation. We used TetTag pharmacogenetics in mice to functionally mark neurons activated in the preoptic hypothalamus during dexmedetomidine-induced sedation or recovery sleep. The neuronal ensembles could then be selectively reactivated. In both cases, non-rapid eye movement sleep, with the accompanying drop in body temperature, was recapitulated. Thus, α2 adrenergic receptor-induced sedation and recovery sleep share hypothalamic circuitry sufficient for producing these behavioral states.

Xenon is a noble gas with neuroprotective properties that can improve short and long-term outcomes in young adult mice after controlled cortical impact. This follow-up study investigates the effects of xenon on very long-term outcomes and survival.

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.

Glycine receptors are important players in pain perception and movement disorders and therefore important therapeutic targets. Glycine receptors can be modulated by the intravenous anaesthetic propofol (2,6-diisopropylphenol). However, the drug is more potent, by at least one order of magnitude, on GABAA receptors. It has been proposed that halogenation of the propofol molecule generates compounds with selective enhancement of glycinergic modulatory properties.

In this study, we explored the possibility that two-pore domain potassium (K2P) channels are sufficient to support action potential (AP) generation in the absence of conventional voltage-gated potassium (KV) channels. Hodgkin-Huxley parameters were used to mimic the presence of voltage-gated sodium (NaV) channels in HEK-293 cells. Recombinant expression of either TREK-1 or TASK-3 channels was then used to generate a hyperpolarised resting membrane potential (RMP) leading to the characteristic non-linear current-voltage relationship expected of a K2P-mediated conductance. During conductance simulation experiments, both TASK-3 and TREK-1 channels were able to repolarise the membrane once AP threshold was reached, and at physiologically relevant current densities, this K2P-mediated conductance supported sustained AP firing. Moreover, the magnitude of the conductance correlated with the speed of the AP rise in a manner predicted from our computational studies. We discuss the physiological impact of axonal K2P channels and speculate on the possible clinical relevance of K2P channel modulation when considering the actions of general and local anaesthetics.

Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA (γ-aminobutyric acid type A) receptors, but where it binds to this receptor is not known and has been a matter of some debate. We synthesized a new propofol analog photolabeling reagent whose biological activity is very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin that have been identified using X-ray crystallography. Using a combination of protiated and deuterated versions of the reagent to label mammalian receptors in intact membranes, we identified a new binding site for propofol in GABAA receptors consisting of both β3 homopentamers and α1β3 heteropentamers. The binding site is located within the β subunit at the interface between the transmembrane domains and the extracellular domain and lies close to known determinants of anesthetic sensitivity in the transmembrane segments TM1 and TM2.

The lateral habenula has been widely studied for its contribution in generating reward-related behaviors [1, 2]. We have found that this nucleus plays an unexpected role in the sedative actions of the general anesthetic propofol. The lateral habenula is a glutamatergic, excitatory hub that projects to multiple targets throughout the brain, including GABAergic and aminergic nuclei that control arousal [3-5]. When glutamate release from the lateral habenula in mice was genetically blocked, the ability of propofol to induce sedation was greatly diminished. In addition to this reduced sensitivity to propofol, blocking output from the lateral habenula caused natural non-rapid eye movement (NREM) sleep to become highly fragmented, especially during the rest ("lights on") period. This fragmentation was largely reversed by the dual orexinergic antagonist almorexant. We conclude that the glutamatergic output from the lateral habenula is permissive for the sedative actions of propofol and is also necessary for the consolidation of natural sleep.

GABA neurons in the VTA and SNc play key roles in reward and aversion through their local inhibitory control of dopamine neuron activity and through long-range projections to several target regions including the nucleus accumbens. It is not clear whether some of these GABA neurons are dedicated local interneurons or if they all collateralize and send projections externally as well as making local synaptic connections. Testing between these possibilities has been challenging in the absence of interneuron-specific molecular markers. We hypothesized that one potential candidate might be neuronal nitric oxide synthase (nNOS), a common interneuronal marker in other brain regions. To test this, we used a combination of immunolabelling (including antibodies for nNOS that we validated in tissue from nNOS-deficient mice) and cell type-specific virus-based anterograde tracing in mice. We found that nNOS-expressing neurons, in the parabrachial pigmented (PBP) part of the VTA and the SNc were GABAergic and did not make detectable projections, suggesting they may be interneurons. In contrast, nNOS-expressing neurons in the rostral linear nucleus (RLi) were mostly glutamatergic and projected to a number of regions, including the lateral hypothalamus (LH), the ventral pallidum (VP), and the median raphe (MnR) nucleus. Taken together, these findings indicate that nNOS is expressed by neurochemically- and anatomically-distinct neuronal sub-groups in a sub-region-specific manner in the VTA and SNc.