Sigrid C. Veasey

An essential role for orexins in emergence from general anesthesia

The neural mechanisms through which the state of anesthesia arises and dissipates remain unknown. One common belief is that emergence from anesthesia is the inverse process of induction, brought about by elimination of anesthetic drugs from their CNS site(s) of action. Anesthetic-induced unconsciousness may result from specific interactions of anesthetics with the neural circuits regulating sleep and wakefulness. Orexinergic agonists and antagonists have the potential to alter the stability of the anesthetized state. In this report, we refine the role of the endogenous orexin system in impacting emergence from, but not entry into the anesthetized state, and in doing so, we distinguish mechanisms of induction from those of emergence. We demonstrate that isoflurane and sevoflurane, two commonly used general anesthetics, inhibit c-Fos expression in orexinergic but not adjacent melanin-concentrating hormone (MCH) neurons; suggesting that wake-active orexinergic neurons are inhibited by these anesthetics. Genetic ablation of orexinergic neurons, which causes acquired murine narcolepsy, delays emergence from anesthesia, without changing anesthetic induction. Pharmacologic studies with a selective orexin-1 receptor antagonist confirm a specific orexin effect on anesthetic emergence without an associated change in induction. We conclude that there are important differences in the neural substrates mediating induction and emergence. These findings support the concept that emergence depends, in part, on recruitment and stabilization of wake-active regions of brain.

Selective loss of catecholaminergic wake active neurons in a murine sleep apnea model.

The presence of refractory wake impairments in many individuals with severe sleep apnea led us to hypothesize that the hypoxia/reoxygenation events in sleep apnea permanently damage wake-active neurons. We now confirm that long-term exposure to hypoxia/reoxygenation in adult mice results in irreversible wake impairments. Functionality and injury were next assessed in major wake-active neural groups. Hypoxia/reoxygenation exposure for 8 weeks resulted in vacuolization in the perikarya and dendrites and markedly impaired c-fos activation response to enforced wakefulness in both noradrenergic locus ceruleus and dopaminergic ventral periaqueductal gray wake neurons. In contrast, cholinergic, histaminergic, orexinergic, and serotonergic wake neurons appeared unperturbed. Six month exposure to hypoxia/reoxygenation resulted in a 40% loss of catecholaminergic wake neurons. Having previously identified NADPH oxidase as a major contributor to wake impairments in hypoxia/reoxygenation, the role of NADPH oxidase in catecholaminergic vulnerability was next addressed. NADPH oxidase catalytic and cytosolic subunits were evident in catecholaminergic wake neurons, where hypoxia/reoxygenation resulted in translocation of p67phox to mitochondria, endoplasmic reticulum, and membranes. Treatment with a NADPH oxidase inhibitor, apocynin, throughout hypoxia/reoxygenation exposures conferred protection of catecholaminergic neurons. Collectively, these data show that select wake neurons, specifically the two catecholaminergic groups, can be rendered persistently impaired after long-term exposure to hypoxia/reoxygenation, modeling sleep apnea; wake impairments are irreversible; catecholaminergic neurons are lost; and neuronal NADPH oxidase contributes to this injury. It is anticipated that severe obstructive sleep apnea in humans destroys catecholaminergic wake neurons.

Single-unit responses of serotonergic dorsal raphe neurons to specific motor challenges in freely moving cats

Serotonin has been hypothesized to play an important role in the central control of motor function. Consistent with this hypothesis, virtually all serotonergic neurons within the medullary nuclei raphe obscurus and raphe pallidus in cats are activated in response to specific motor challenges. To determine whether the response profile of serotonergic neurons in the midbrain is similar to that observed in the medulla, the single-unit activity of serotonergic dorsal raphe nucleus cells was studied during three specific motor activities: treadmill-induced locomotion, hypercarbia-induced ventilatory response and spontaneous feeding. In contrast to the results obtained for medullary raphe cells, none of the serotonergic dorsal raphe cells studied (n=26) demonstrated increased firing during treadmill-induced locomotion. A subset of serotonergic dorsal raphe cells (8/36) responded to the hypercarbic ventilatory challenge with increased firing rates that were directly related to the fraction of inspired carbon dioxide, and a non-overlapping subset of cells (6/31) was activated during feeding. All feeding-on cells demonstrated a rapid activation and de-activation coincident with feeding onset and offset, respectively. Although the proportions of serotonergic cells activated by hypercarbia or feeding in the dorsal raphe nucleus were similar to those found in the medullary raphe, there were several major distinctions in the response characteristics for the two cell groups. In contrast to the medullary serotonergic neurons, only a minority of dorsal raphe nucleus serotonergic neurons responded to a motor challenge. Overall, the above results suggest very different roles for the midbrain and medullary serotonergic neurons in response to motor activities.

Mutations in Rab3a alter circadian period and homeostatic response to sleep loss in the mouse.

Rab3a is the most abundant Rab (ras-associated binding) protein in the brain and has a regulatory role in synaptic vesicle trafficking. Mice with a targeted loss-of-function mutation in Rab3a have defects in Ca2+-dependent synaptic transmission: the number of vesicles released in response to an action potential is greater than in wildtype mice, resulting in greater synaptic depression and the abolishment of CA3 mossy-fiber long term potentiation. The effect of these changes on behavior is unknown. In a screen for mouse mutants with abnormal rest–activity and sleep patterns, we identified a semidominant mutation, called earlybird, that shortens the circadian period of locomotor activity. Sequence analysis of Rab3a identified a point mutation in the conserved amino acid (Asp77Gly) within the GTP-binding domain of this protein in earlybird mutants, resulting in significantly reduced levels of Rab3a protein. Phenotypic assessment of earlybird mice and a null allele of Rab3a revealed anomalies in circadian period and sleep homeostasis, providing evidence that Rab3a-mediated synaptic transmission is involved in these behaviors.

Daytime Sleepiness in Obesity: Mechanisms Beyond Obstructive Sleep Apnea—A Review

Increasing numbers of overweight children and adults are presenting to sleep medicine clinics for evaluation and treatment of sleepiness. Sleepiness negatively affects quality of life, mental health, productivity, and safety. Thus, it is essential to comprehensively address all potential causes of sleepiness. While many obese individuals presenting with hypersomnolence will be diagnosed with obstructive sleep apnea and their sleepiness will improve with effective therapy for sleep apnea, a significant proportion of patients will continue to have hypersomnolence. Clinical studies demonstrate that obesity without sleep apnea is also associated with a higher prevalence of hypersomnolence and that bariatric surgery can markedly improve hypersomnolence before resolution of obstructive sleep apnea. High fat diet in both humans and animals is associated with hypersomnolence. This review critically examines the relationships between sleepiness, feeding, obesity, and sleep apnea and then discusses the hormonal, metabolic, and inflammatory mechanisms potentially contributing to hypersomnolence in obesity, independent of sleep apnea and other established causes of excessive daytime sleepiness.

Obstructive sleep apnea: a stand-alone risk factor for chronic kidney disease

BACKGROUND Previous studies have found an association between obstructive sleep apnea (OSA) and chronic kidney disease (CKD). However, subjects with confounding factors such as diabetes and hypertension were not excluded. The purpose of the present study was to determine whether patients with OSA without meeting criteria for diabetes or hypertension would also show increased likelihood of CKD. METHODS We prospectively enrolled adult patients with a chief complaint of habitual snoring. Overnight polysomnography, fasting blood triglyceride, cholesterol, glucose, insulin, creatinine, albumin and hemoglobin A1c, and first voiding urine albumin and creatinine were examined. Estimated glomerular filtration rate (eGFR), urine albumin-to-creatinine ratio (UACR), homeostatic model assessment-insulin resistance and percentage of CKD were calculated. RESULTS The final analyses involved 40 patients who were middle-aged [44.8 (8.6) years] predominantly male (83%), obese [body mass index, 28.2 (5.1) kg/m(2)] and more severe OSA, with an apnea-hypopnea index (AHI) of 51.6 (39.2)/h. The mean eGFR and UACR were 85.4 (18.3) mL/min/1.73m(2) and 13.4 (23.4) mg/g, respectively. The prevalence of CKD in severe OSA subjects is 18%. With stepwise multivariate linear regression analysis, AHI and desaturation index were the only independent predictor of UACR (β = 0.26, P = 0.01, R(2) = 0.17) and eGFR (β = 0.32, P < 0.01, R(2) = 0.32), respectively. CONCLUSIONS High prevalence of CKD is present in severe OSA patients without hypertension or diabetes. Significantly positive correlations were found between severity of OSA and renal function impairment.

Direct Activation of Sleep-Promoting VLPO Neurons by Volatile Anesthetics Contributes to Anesthetic Hypnosis

BACKGROUND Despite seventeen decades of continuous clinical use, the neuronal mechanisms through which volatile anesthetics act to produce unconsciousness remain obscure. One emerging possibility is that anesthetics exert their hypnotic effects by hijacking endogenous arousal circuits. A key sleep-promoting component of this circuitry is the ventrolateral preoptic nucleus (VLPO), a hypothalamic region containing both state-independent neurons and neurons that preferentially fire during natural sleep. RESULTS Using c-Fos immunohistochemistry as a biomarker for antecedent neuronal activity, we show that isoflurane and halothane increase the number of active neurons in the VLPO, but only when mice are sedated or unconscious. Destroying VLPO neurons produces an acute resistance to isoflurane-induced hypnosis. Electrophysiological studies prove that the neurons depolarized by isoflurane belong to the subpopulation of VLPO neurons responsible for promoting natural sleep, whereas neighboring non-sleep-active VLPO neurons are unaffected by isoflurane. Finally, we show that this anesthetic-induced depolarization is not solely due to a presynaptic inhibition of wake-active neurons as previously hypothesized but rather is due to a direct postsynaptic effect on VLPO neurons themselves arising from the closing of a background potassium conductance. CONCLUSIONS Cumulatively, this work demonstrates that anesthetics are capable of directly activating endogenous sleep-promoting networks and that such actions contribute to their hypnotic properties.

mTOR is required for pulmonary arterial vascular smooth muscle cell proliferation under chronic hypoxia

Pulmonary arterial vascular smooth muscle (PAVSM) cell proliferation is a key pathophysi‐ological component of vascular remodeling in pulmonary arterial hypertension (PAH) for which cellular and molecular mechanisms are poorly understood. The goal of our study was to determine the role of mammalian target of rapamycin (mTOR) in PAVSM cell proliferation, a major pathological manifestation of vascular remodeling in PAH. Our data demonstrate that chronic hypoxia promoted mTOR(Ser‐2481) phosphorylation, an indicator of mTOR intrinsic catalytic activity, mTORC1‐specific S6 and mTORC2‐specific Akt (Ser‐473) phosphorylation, and proliferation of human and rat PAVSM cells that was inhibited by siRNA mTOR PAVSM cells derived from rats exposed to chronic hypoxia (VSM‐H cells) retained increased mTOR(Ser‐2481), S6, Akt (Ser‐473) phosphorylation, and DNA synthesis compared to cells from nor‐moxia‐exposed rats. Suppression of mTORC2 signaling with siRNA rictor, or inhibition of mTORC1 signaling with rapamycin and metformin, while having little effect on other complex activities, inhibited VSM‐H and chronic hypoxia‐induced human and rat PAVSM cell proliferation. Collectively, our data demonstrate that up‐regulation of mTOR activity and activation of both mTORC1 and mTORC2 are required for PAVSM cell proliferation induced by in vitro and in vivo chronic hypoxia and suggest that mTOR may serve as a potential therapeutic target to inhibit vascular remodeling in PAH.—Krymskaya, V. P., Snow, J., Cesarone, G., Khavin, I., Goncharov, D. A., Lim, P. N., Veasey, S. C, Ihida‐Stansbury, K., Jones, P. L., Goncharova, E. A. mTOR is required for pulmonary arterial vascular smooth muscle cell proliferation under chronic hypoxia. FASEB J. 25, 1922‐1933 (2011). www.fasebj.org

Extended Wakefulness: Compromised Metabolics in and Degeneration of Locus Ceruleus Neurons

Modern society enables a shortening of sleep times, yet long-term consequences of extended wakefulness on the brain are largely unknown. Essential for optimal alertness, locus ceruleus neurons (LCns) are metabolically active neurons that fire at increased rates across sustained wakefulness. We hypothesized that wakefulness is a metabolic stressor to LCns and that, with extended wakefulness, adaptive mitochondrial metabolic responses fail and injury ensues. The nicotinamide adenine dinucleotide-dependent deacetylase sirtuin type 3 (SirT3) coordinates mitochondrial energy production and redox homeostasis. We find that brief wakefulness upregulates SirT3 and antioxidants in LCns, protecting metabolic homeostasis. Strikingly, mice lacking SirT3 lose the adaptive antioxidant response and incur oxidative injury in LCns across brief wakefulness. When wakefulness is extended for longer durations in wild-type mice, SirT3 protein declines in LCns, while oxidative stress and acetylation of mitochondrial proteins, including electron transport chain complex I proteins, increase. In parallel with metabolic dyshomeostasis, apoptosis is activated and LCns are lost. This work identifies mitochondrial stress in LCns upon wakefulness, highlights an essential role for SirT3 activation in maintaining metabolic homeostasis in LCns across wakefulness, and demonstrates that extended wakefulness results in reduced SirT3 activity and, ultimately, degeneration of LCns.

Impaired rapid eye movement sleep in the Tg2576 APP murine model of Alzheimer's disease with injury to pedunculopontine cholinergic neurons.

Impaired rapid eye movement sleep (REMS) is commonly observed in Alzheimers disease, suggesting injury to mesopontine cholinergic neurons. We sought to determine whether abnormal beta-amyloid peptides impair REMS and injure mesopontine cholinergic neurons in transgenic (hAPP695.SWE) mice (Tg2576) that model brain amyloid pathologies. Tg2576 mice and wild-type littermates were studied at 2, 6, and 12 months by using sleep recordings, contextual fear conditioning, and immunohistochemistry. At 2 months of age, REMS was indistinguishable by genotype but was reduced in Tg2576 mice at 6 and 12 months. Choline acetyltransferase-positive neurons in the pedunculopontine tegmentum of Tg2576 mice at 2 months evidenced activated caspase-3 immunoreactivity, and at 6 and 12 months the numbers of pedunculopontine tegmentum choline acetyltransferase-positive neurons were reduced in the Tg2576 mice. Other cholinergic groups involved in REMS were unperturbed. At 12 months, Tg2576 mice demonstrated increased 3-nitrotyrosine immunoreactivity in cholinergic projection sites but not in cholinergic soma. We have identified a population of selectively compromised cholinergic neurons in young Tg2576 mice that manifest early onset REMS impairment. The differential vulnerability of these cholinergic neurons to Abeta injury provides an invaluable tool with which to understand mechanisms of sleep/wake perturbations in Alzheimers disease.