Giancarlo Vanini

γ-Aminobutyric acid-mediated neurotransmission in the pontine reticular formation modulates hypnosis, immobility, and breathing during isoflurane anesthesia

Background:Many general anesthetics are thought to produce a loss of wakefulness, in part, by enhancing γ-aminobutyric acid (GABA) neurotransmission. However, GABAergic neurotransmission in the pontine reticular formation promotes wakefulness. This study tested the hypotheses that (1) relative to wakefulness, isoflurane decreases GABA levels in the pontine reticular formation; and (2) pontine reticular formation administration of drugs that increase or decrease GABA levels increases or decreases, respectively, isoflurane induction time. Methods:To test hypothesis 1, cats (n = 5) received a craniotomy and permanent electrodes for recording the electroencephalogram and electromyogram. Dialysis samples were collected from the pontine reticular formation during isoflurane anesthesia and wakefulness. GABA levels were quantified using high-performance liquid chromatography. For hypothesis 2, rats (n = 10) were implanted with a guide cannula aimed for the pontine reticular formation. Each rat received microinjections of Ringers (vehicle control), the GABA uptake inhibitor nipecotic acid, and the GABA synthesis inhibitor 3-mercaptopropionic acid. Rats were then anesthetized with isoflurane, and induction time was quantified as loss of righting reflex. Breathing rate was also measured. Results:Relative to wakefulness, GABA levels were significantly decreased by isoflurane. Increased power in the electroencephalogram and decreased activity in the electromyogram caused by isoflurane covaried with pontine reticular formation GABA levels. Nipecotic acid and 3-mercaptopropionic acid significantly increased and decreased, respectively, isoflurane induction time. Nipecotic acid also increased breathing rate. Conclusion:Decreasing pontine reticular formation GABA levels comprises one mechanism by which isoflurane causes loss of consciousness, altered cortical excitability, muscular hypotonia, and decreased respiratory rate.

Functional and Topological Conditions for Explosive Synchronization Develop in Human Brain Networks with the Onset of Anesthetic-Induced Unconsciousness

Sleep, anesthesia, and coma share a number of neural features but the recovery profiles are radically different. To understand the mechanisms of reversibility of unconsciousness at the network level, we studied the conditions for gradual and abrupt transitions in conscious and anesthetized states. We hypothesized that the conditions for explosive synchronization (ES) in human brain networks would be present in the anesthetized brain just over the threshold of unconsciousness. To test this hypothesis, functional brain networks were constructed from multi-channel electroencephalogram (EEG) recordings in seven healthy subjects across conscious, unconscious, and recovery states. We analyzed four variables that are involved in facilitating ES in generic, non-biological networks: (1) correlation between node degree and frequency, (2) disassortativity (i.e., the tendency of highly-connected nodes to link with less-connected nodes, or vice versa), (3) frequency difference of coupled nodes, and (4) an inequality relationship between local and global network properties, which is referred to as the suppressive rule. We observed that the four network conditions for ES were satisfied in the unconscious state. Conditions for ES in the human brain suggest a potential mechanism for rapid recovery from the lightly-anesthetized state. This study demonstrates for the first time that the network conditions for ES, formerly shown in generic networks only, are present in empirically-derived functional brain networks. Further investigations with deep anesthesia, sleep, and coma could provide insight into the underlying causes of variability in recovery profiles of these unconscious states.

Neurophysiological correlates of sevoflurane-induced unconsciousness.

Background:Recent studies of anesthetic-induced unconsciousness in humans have focused predominantly on the intravenous drug propofol and have identified anterior dominance of alpha rhythms and frontal phase–amplitude coupling patterns as neurophysiological markers. However, it is unclear whether the correlates of propofol-induced unconsciousness are generalizable to inhaled anesthetics, which have distinct molecular targets and which are used more commonly in clinical practice. Methods:The authors recorded 64-channel electroencephalograms in healthy human participants during consciousness, sevoflurane-induced unconsciousness, and recovery (n = 10; n = 7 suitable for analysis). Spectrograms and scalp distributions of low-frequency (1 Hz) and alpha (10 Hz) power were analyzed, and phase–amplitude modulation between these two frequencies was calculated in frontal and parietal regions. Phase lag index was used to assess phase relationships across the cortex. Results:At concentrations sufficient for unconsciousness, sevoflurane did not result in a consistent anteriorization of alpha power; the relationship between low-frequency phase and alpha amplitude in the frontal cortex did not undergo characteristic transitions. By contrast, there was significant cross-frequency coupling in the parietal region during consciousness that was not observed after loss of consciousness. Furthermore, a reversible disruption of anterior–posterior phase relationships in the alpha bandwidth was identified as a correlate of sevoflurane-induced unconsciousness. Conclusion:In humans, sevoflurane-induced unconsciousness is not correlated with anteriorization of alpha and related cross-frequency patterns, but rather by a disruption of phase–amplitude coupling in the parietal region and phase–phase relationships across the cortex.

Endogenous GABA Levels in the Pontine Reticular Formation Are Greater during Wakefulness than during Rapid Eye Movement Sleep

Studies using drugs that increase or decrease GABAergic transmission suggest that GABA in the pontine reticular formation (PRF) promotes wakefulness and inhibits rapid eye movement (REM) sleep. Cholinergic transmission in the PRF promotes REM sleep, and levels of endogenous acetylcholine (ACh) in the PRF are significantly greater during REM sleep than during wakefulness or non-REM (NREM) sleep. No previous studies have determined whether levels of endogenous GABA in the PRF vary as a function of sleep and wakefulness. This study tested the hypothesis that GABA levels in cat PRF are greatest during wakefulness and lowest during REM sleep. Extracellular GABA levels were measured during wakefulness, NREM sleep, REM sleep, and the REM sleep-like state (REMNeo) caused by microinjecting neostigmine into the PRF. GABA levels varied significantly as a function of sleep and wakefulness, and decreased significantly below waking levels during REM sleep (−42%) and REMNeo (−63%). The decrease in GABA levels during NREM sleep (22% below waking levels) was not statistically significant. Compared with NREM sleep, GABA levels decreased significantly during REM sleep (−27%) and REMNeo (−52%). Comparisons of REM sleep and REMNeo revealed no differences in GABA levels or cortical EEG power. GABA levels did not vary significantly as a function of dialysis site within the PRF. The inverse relationship between changes in PRF levels of GABA and ACh during REM sleep indicates that low GABAergic tone combined with high cholinergic tone in the PRF contributes to the generation of REM sleep.

Extrasynaptic GABAA receptors in rat pontine reticular formation increase wakefulness.

STUDY OBJECTIVES Gamma-aminobutyric acid (GABA) causes phasic inhibition via synaptic GABAA receptors and tonic inhibition via extrasynaptic GABAA receptors. GABA levels in the extracellular space regulate arousal state and cognition by volume transmission via extrasynaptic GABAA receptors. GABAergic transmission in the pontine reticular formation promotes wakefulness. No previous studies have determined whether an agonist at extrasynaptic GABAA receptors administered into the pontine reticular formation alters sleep and wakefulness. Therefore, this study used gaboxadol (THIP; agonist at extrasynaptic GABAA receptors that contain a δ subunit) to test the hypothesis that extrasynaptic GABAA receptors within the pontine reticular formation modulate sleep and wakefulness. DESIGN Within/between subjects. SETTING University of Michigan. PATIENTS OR PARTICIPANTS Adult male Crl:CD*(SD) (Sprague-Dawley) rats (n = 10). INTERVENTIONS Microinjection of gaboxadol, the nonsubtype selective GABAA receptor agonist muscimol (positive control), and saline (negative control) into the rostral pontine reticular formation. MEASUREMENTS AND RESULTS Gaboxadol significantly increased wakefulness and decreased both nonrapid eye movement sleep and rapid eye movement sleep in a concentration-dependent manner. Relative to saline, gaboxadol did not alter electroencephalogram power. Microinjection of muscimol into the pontine reticular formation of the same rats that received gaboxadol increased wakefulness and decreased sleep. CONCLUSION Tonic inhibition via extrasynaptic GABAA receptors that contain a δ subunit may be one mechanism by which the extracellular pool of endogenous GABA in the rostral pontine reticular formation promotes wakefulness. CITATION Vanini G; Baghdoyan HA. Extrasynaptic GABAA receptors in rat pontine reticular formation increase wakefulness. SLEEP 2013;36(3):337-343.

Dexmedetomidine-induced sedation does not mimic the neurobehavioral phenotypes of sleep in Sprague Dawley rat.

STUDY OBJECTIVES Dexmedetomidine is used clinically to induce states of sedation that have been described as homologous to nonrapid eye movement (NREM) sleep. A better understanding of the similarities and differences between NREM sleep and dexmedetomidine-induced sedation is essential for efforts to clarify the relationship between these two states. This study tested the hypothesis that dexmedetomidine-induced sedation is homologous to sleep. DESIGN This study used between-groups and within-groups designs. SETTING University of Michigan. PARTICIPANTS Adult male Sprague Dawley rats (n = 40). INTERVENTIONS Independent variables were administration of dexmedetomidine and saline or Ringers solution (control). Dependent variables included time spent in states of wakefulness, sleep, and sedation, electroencephalographic (EEG) power, adenosine levels in the substantia innominata (SI), and activation of pCREB and c-Fos in sleep related forebrain regions. MEASUREMENTS AND RESULTS Dexmedetomidine significantly decreased time spent in wakefulness (-49%), increased duration of sedation (1995%), increased EEG delta power (546%), and eliminated the rapid eye movement (REM) phase of sleep for 16 h. Sedation was followed by a rebound increase in NREM and REM sleep. Systemically administered dexmedetomidine significantly decreased (-39%) SI adenosine levels. Dialysis delivery of dexmedetomidine into SI did not decrease adenosine level. Systemic delivery of dexmedetomidine did not alter c-Fos or pCREB expression in the horizontal diagonal band, or ventrolateral, median, and medial preoptic areas of the hypothalamus. CONCLUSIONS Dexmedetomidine significantly altered normal sleep phenotypes, and the dexmedetomidine-induced state did not compensate for sleep need. Thus, in the Sprague Dawley rat, dexmedetomidine-induced sedation is characterized by behavioral, electrographic, and immunohistochemical phenotypes that are distinctly different from similar measures obtained during sleep.

GABAergic transmission in rat pontine reticular formation regulates the induction phase of anesthesia and modulates hyperalgesia caused by sleep deprivation

The oral part of the pontine reticular formation (PnO) contributes to the regulation of sleep, anesthesia and pain. The role of PnO γ‐aminobutyric acid (GABA) in modulating these states remains incompletely understood. The present study used time to loss and time to resumption of righting response (LoRR and RoRR) as surrogate measures of loss and resumption of consciousness. This study tested three hypotheses: (i) pharmacologically manipulating GABA levels in rat PnO alters LoRR, RoRR and nociception; (ii) propofol decreases GABA levels in the PnO; and (iii) inhibiting GABA synthesis in the PnO blocks hyperalgesia caused by sleep deprivation. Administering a GABA synthesis inhibitor [3‐mercaptopropionic acid (3‐MPA)] or a GABA uptake inhibitor [nipecotic acid (NPA)] into rat PnO significantly altered LoRR caused by propofol. 3‐MPA significantly decreased LoRR for propofol (−18%). NPA significantly increased LoRR during administration of propofol (36%). Neither 3‐MPA nor NPA altered RoRR following cessation of propofol or isoflurane delivery. The finding that LoRR was decreased by 3‐MPA and increased by NPA is consistent with measures showing that extracellular GABA levels in the PnO were decreased (41%) by propofol. Thermal nociception was significantly decreased by 3‐MPA and increased by NPA, and 3‐MPA blocked the hyperalgesia caused by sleep deprivation. The results demonstrate that GABA levels in the PnO regulate the time for loss of consciousness caused by propofol, extend the concept that anesthetic induction and emergence are not inverse processes, and suggest that GABAergic transmission in the PnO mediates hyperalgesia caused by sleep loss.

Relevance of sleep neurobiology for cognitive neuroscience and anesthesiology

Although general anesthetics are used for approximately 21 million patients per year in the United States,1 the molecular and cellular mechanisms by which anesthetics produce loss of waking consciousness are poorly understood. The complexity of consciousness and relatively imprecise clinical signs that are used to evaluate the depth of anesthesia are significant limitations for the study of consciousness. Recent advances in sleep neurobiology continue to enhance the understanding of different physiological traits that define altered states of consciousness. The original hypothesis2 that neural networks that evolved to generate sleep are preferentially modulated by anesthetic drugs has been supported by multiple lines of evidence.3–9 These data demonstrate that sleep neurobiology can contribute to understanding the mechanisms by which anesthetics cause loss of consciousness. The goal of this chapter is to selectively review the neurobiology of sleep and wakefulness in relation to anesthesia-induced loss of consciousness.

Network efficiency and posterior alpha patterns are markers of recovery from general anesthesia: A high-density electroencephalography study in healthy volunteers

Recent studies have investigated local oscillations, long-range connectivity, and global network patterns to identify neural changes associated with anesthetic-induced unconsciousness. These studies typically employ anesthetic protocols that either just cross the threshold of unconsciousness, or induce deep unconsciousness for a brief period of time—neither of which models general anesthesia for major surgery. To study neural patterns of unconsciousness and recovery in a clinically-relevant context, we used a realistic anesthetic regimen to induce and maintain unconsciousness in eight healthy participants for 3 h. High-density electroencephalogram (EEG) was acquired throughout and for another 3 h after emergence. Seven epochs of 5-min eyes-closed resting states were extracted from the data at baseline as well as 30, 60, 90, 120, 150, and 180-min post-emergence. Additionally, 5-min epochs were extracted during induction, unconsciousness, and immediately prior to recovery of consciousness, for a total of 10 analysis epochs. The EEG data in each epoch were analyzed using source-localized spectral analysis, phase-lag index, and graph theoretical techniques. Posterior alpha power was significantly depressed during unconsciousness, and gradually approached baseline levels over the 3 h recovery period. Phase-lag index did not distinguish between states of consciousness or stages of recovery. Network efficiency was significantly depressed and network clustering coefficient was significantly increased during unconsciousness; these graph theoretical measures returned to baseline during the 3 h recovery period. Posterior alpha power may be a potential biomarker for normal recovery of functional brain networks after general anesthesia.

Estimating the Integrated Information Measure Phi from High-Density Electroencephalography during States of Consciousness in Humans

The integrated information theory (IIT) proposes a quantitative measure, denoted as Φ, of the amount of integrated information in a physical system, which is postulated to have an identity relationship with consciousness. IIT predicts that the value of Φ estimated from brain activities represents the level of consciousness across phylogeny and functional states. Practical limitations, such as the explosive computational demands required to estimate Φ for real systems, have hindered its application to the brain and raised questions about the utility of IIT in general. To achieve practical relevance for studying the human brain, it will be beneficial to establish the reliable estimation of Φ from multichannel electroencephalogram (EEG) and define the relationship of Φ to EEG properties conventionally used to define states of consciousness. In this study, we introduce a practical method to estimate Φ from high-density (128-channel) EEG and determine the contribution of each channel to Φ. We examine the correlation of power, frequency, functional connectivity, and modularity of EEG with regional Φ in various states of consciousness as modulated by diverse anesthetics. We find that our approximation of Φ alone is insufficient to discriminate certain states of anesthesia. However, a multi-dimensional parameter space extended by four parameters related to Φ and EEG connectivity is able to differentiate all states of consciousness. The association of Φ with EEG connectivity during clinically defined anesthetic states represents a new practical approach to the application of IIT, which may be used to characterize various physiological (sleep), pharmacological (anesthesia), and pathological (coma) states of consciousness in the human brain.