Cholinergic stimulation of prefrontal cortex (PFC) can reverse anesthesia. Conversely, inactivation of PFC can delay emergence from anesthesia. PFC receives cholinergic projections from basal forebrain, which contains wake-promoting neurons. However, the role of basal forebrain cholinergic neurons in arousal from the anesthetized state requires refinement, and it is currently unknown whether the arousal-promoting effect of basal forebrain is mediated through PFC. To address these gaps in knowledge, we implemented a novel approach to the use of chemogenetic stimulation and tested the role of basal forebrain cholinergic neurons in behavioral arousal during sevoflurane anesthesia. Next, we investigated the effect of tetrodotoxin-mediated inactivation of PFC on behavioral arousal produced by electrical stimulation of basal forebrain during sevoflurane anesthesia.

Studies aimed at investigating brain regions involved in arousal state control have been traditionally limited to subcortical structures. In the current study, we tested the hypothesis that inactivation of prefrontal cortex, but not two subregions within parietal cortex-somatosensory barrel field and medial/lateral parietal association cortex-would suppress arousal, as measured by an increase in anesthetic sensitivity. Male and female Sprague Dawley rats were surgically prepared for recording electroencephalogram and bilateral infusion into prefrontal cortex (N = 13), somatosensory barrel field (N = 10), or medial/lateral parietal association cortex (N = 9). After at least 10 days of post-surgical recovery, 156 μM tetrodotoxin or saline was microinjected into one of the cortical sites. Ninety minutes after injection, rats were anesthetized with 2.5% sevoflurane and the time to loss of righting reflex, a surrogate for loss of consciousness, was measured. Sevoflurane was stopped after 45 min and the time to return of righting reflex, a surrogate for return of consciousness, was measured. Tetrodotoxin-mediated inactivation of all three cortical sites decreased (p < 0.05) the time to loss of righting reflex. By contrast, only inactivation of prefrontal cortex, but not somatosensory barrel field or medial/lateral parietal association cortex, increased (p < 0.001) the time to return of righting reflex. Burst suppression ratio was not altered following inactivation of any of the cortical sites, suggesting that there was no global effect due to pharmacologic lesion. These findings demonstrate that prefrontal cortex plays a causal role in emergence from anesthesia and behavioral arousal.

The role of the brainstem cholinergic system in the regulation of sleep-wake states has been studied extensively but relatively little is known about the role of cholinergic mechanisms in prefrontal cortex in the regulation of sleep-wake states. In a recent study, we showed that prefrontal cholinergic stimulation in anesthetized rat can reverse the traits associated with anesthesia and restore a wake-like state, thereby providing evidence for a causal role for prefrontal cholinergic mechanisms in modulating level of arousal. However, the effect of increase in prefrontal cholinergic tone on spontaneous sleep-wake states has yet to be demonstrated. Therefore, in this study, we tested the hypothesis that delivery of cholinergic agonists - carbachol or nicotine - into prefrontal cortex of rat during slow wave sleep (SWS) would produce behavioral arousal and increase the time spent in wake state. We show that unilateral microinjection (200 nL) of carbachol (1 mM) or nicotine (100 mM) into prefrontal cortex during SWS decreased the latency to the onset of wake state (p = 0.03 for carbachol, p = 0.03 for nicotine) and increased the latency to the onset of rapid eye movement sleep (p = 0.008 for carbachol, p = 0.006 for nicotine). Although the infusion of 1 mM carbachol increased the time spent in wake state (p = 0.01) and decreased the time spent in SWS (p = 0.01), infusion of 10 or 100 mM nicotine did not produce any statistically significant change in sleep-wake architecture. These data demonstrate a differential role of prefrontal cholinergic receptors in modulating spontaneous sleep-wake states.

Conscious access to sensory information is likely gated at an intermediate site between primary sensory and transmodal association cortices, but the structure responsible remains unknown. We perform functional neuroimaging to determine the neural correlates of conscious access using a volitional mental imagery task, a report paradigm not confounded by motor behavior. Titrating propofol to loss of behavioral responsiveness in healthy volunteers creates dysfunction of the anterior insular cortex (AIC) in association with an impairment of dynamic transitions of default-mode and dorsal attention networks. Candidate subcortical regions mediating sensory gating or arousal (thalamus, basal forebrain) fail to show this association. The gating role of the AIC is consistent with findings in awake participants, whose conscious access is predicted by pre-stimulus AIC activity near perceptual threshold. These data support the hypothesis that AIC, situated at an intermediate position of the cortical hierarchy, regulates brain network transitions that gate conscious access.

Nav1.6 is a major voltage-gated sodium channel in the central and peripheral nervous systems. Within neurons, the channel protein is concentrated at the axon initial segment and nodes of Ranvier, where it functions in initiation and propagation of action potentials. We examined the role of Nav1.6 in general anesthesia using two mouse mutants with reduced activity of Nav1.6, Scn8amedJ/medJ and Scn8a9J/9J. The mice were exposed to the general anesthetics isoflurane and sevoflurane in step-wise increments; the concentration required to produce loss of righting reflex, a surrogate for anesthetic-induced unconsciousness in rodents, was determined. Mice homozygous for these mutations exhibited increased sensitivity to both isoflurane and sevoflurane. The increased sensitivity was observed during induction of unconsciousness but not during the recovery phase, suggesting that the effect is not attributable to compromised systemic physiology. Electroencephalographic theta power during baseline waking was lower in mutants, suggesting decreased arousal and reduced neuronal excitability. This is the first report linking reduced activity of a specific voltage-gated sodium channel to increased sensitivity to general anesthetics in vivo.

Recent data from our laboratory demonstrate that high-frequency gamma connectivity across the cortex is present during consciousness and depressed during unconsciousness. However, these data were derived from static and well-defined states of arousal rather than during transitions that would suggest functional relevance. We also recently found that subanesthetic ketamine administered during isoflurane anesthesia accelerates recovery upon discontinuation of the primary anesthetic and increases gamma power during emergence. In the current study we re-analyzed electroencephalogram (EEG) data to test the hypothesis that functional cortical connectivity between anterior and posterior cortical regions would be increased during accelerated recovery induced by ketamine when compared to saline-treated controls. Rodents were instrumented with intracranial EEG electrodes and general anesthesia was induced with isoflurane anesthesia. After 37.5 min of continuous isoflurane anesthesia, a subanesthetic dose of ketamine (25 mg/kg intraperitoneal) was administered, with evidence of a 44% reduction in emergence time. In this study, we analyzed gamma and theta coherence (measure of undirected functional connectivity) and normalized symbolic transfer entropy (measure of directed functional connectivity) between frontal and parietal cortices during various levels of consciousness, with a focus on emergence from isoflurane anesthesia. During accelerated emergence in the ketamine-treated group, there was increased frontal-parietal coherence {p = 0.005, 0.05-0.23 [95% confidence interval (CI)]} and normalized symbolic transfer entropy [frontal to parietal: p < 0.001, 0.010-0.026 (95% CI); parietal to frontal: p < 0.001, 0.009-0.025 (95% CI)] in high-frequency gamma bandwidth as compared with the saline-treated group. Surrogates of cortical information exchange in high-frequency gamma are increased in association with accelerated recovery from anesthesia. This finding adds evidence suggesting a functional significance of high-gamma information transfer in consciousness.

Clinical and experimental data from the last nine decades indicate that the preoptic area of the hypothalamus is a critical node in a brain network that controls sleep onset and homeostasis. By contrast, we recently reported that a group of glutamatergic neurons in the lateral and medial preoptic area increases wakefulness, challenging the long-standing notion in sleep neurobiology that the preoptic area is exclusively somnogenic. However, the precise role of these subcortical neurons in the control of behavioral state transitions and cortical dynamics remains unknown. Therefore, in this study, we used conditional expression of excitatory hM3Dq receptors in these preoptic glutamatergic (Vglut2+) neurons and show that their activation initiates wakefulness, decreases non-rapid eye movement (NREM) sleep, and causes a persistent suppression of rapid eye movement (REM) sleep. We also demonstrate, for the first time, that activation of these preoptic glutamatergic neurons causes a high degree of NREM sleep fragmentation, promotes state instability with frequent arousals from sleep, decreases body temperature, and shifts cortical dynamics (including oscillations, connectivity, and complexity) to a more wake-like state. We conclude that a subset of preoptic glutamatergic neurons can initiate, but not maintain, arousals from sleep, and their inactivation may be required for NREM stability and REM sleep generation. Further, these data provide novel empirical evidence supporting the hypothesis that the preoptic area causally contributes to the regulation of both sleep and wakefulness.SIGNIFICANCE STATEMENT Historically, the preoptic area of the hypothalamus has been considered a key site for sleep generation. However, emerging modeling and empirical data suggest that this region might play a dual role in sleep-wake control. We demonstrate that chemogenetic stimulation of preoptic glutamatergic neurons produces brief arousals that fragment sleep, persistently suppresses REM sleep, causes hypothermia, and shifts EEG patterns toward a "lighter" NREM sleep state. We propose that preoptic glutamatergic neurons can initiate, but not maintain, arousal from sleep and gate REM sleep generation, possibly to block REM-like intrusions during NREM-to-wake transitions. In contrast to the long-standing notion in sleep neurobiology that the preoptic area is exclusively somnogenic, we provide further evidence that preoptic neurons also generate wakefulness.